Engineering of a human kringle domain into agonistic and antagonistic binding proteins functioning in vitro and in vivo
Engineering of a human kringle domain into agonistic and antagonistic binding proteins functioning in vitro and in vivo
复制标题
将人 kringle 结构域工程化为在体外和体内发挥作用的激动性和拮抗性结合蛋白
DOI：
10.1073/pnas.1001541107
复制
发表时间：
2010
期刊：
影响因子：
--
通讯作者：
Yong
中科院分区：
文献类型：
--
作者：
Chang;Kyung;Eunsil Sung;A. Kim;Jida Choi;Jeong;Soohyun Kim;Myung;Yong
在这里，我们报告了基于 kringle 结构域 (KD)（~80 个残基）的靶标特异性结合蛋白的开发，KD 结构域是真核物种中普遍存在的模块化结构单元。通过利用核心残基高度保守的主链折叠，但使用天然存在的人类 KD 的七个环区域中的广泛序列变异，我们通过随机化人类 KD 模板环中的 45 个残基，在酵母细胞表面生成了合成 KD 文库。我们分离出了与抗癌靶蛋白（例如人类死亡受体 4 (DR4) 和/或 DR5）特异性结合的 KD 变体，并作为激动剂在体外诱导多种癌细胞系中的细胞凋亡，并在小鼠模型中抑制肿瘤进展。与单独变体的治疗相比，在同一靶蛋白上具有不同识别位点的 KD 变体的联合治疗发挥了协同杀肿瘤活性。除了激动剂之外，我们还分离了一种拮抗性 KD 变体，它可以结合人肿瘤坏死因子-α (TNFα)，并在体外和体内有效中和 TNFα 诱导的细胞毒性。 KD 支架具有从中央核心突出的七个柔性环，对环区域的突变具有很强的序列耐受性，为单个结构域上的目标识别提供了不同结合位点的潜在优势。我们的结果表明，KD 支架可用于开发靶标特异性结合蛋白，作为给定靶分子的激动剂或拮抗剂，表明它们作为生物治疗药物的潜在用途。
Here, we report the development of target-specific binding proteins based on the kringle domain (KD) (∼80 residues), a ubiquitous modular structural unit occurring across eukaryotic species. By exploiting the highly conserved backbone folding by core residues, but using extensive sequence variations in the seven loop regions of naturally occurring human KDs, we generated a synthetic KD library on the yeast cell surface by randomizing 45 residues in the loops of a human KD template. We isolated KD variants that specifically bind to anticancer target proteins, such as human death receptor 4 (DR4) and/or DR5, and that function as agonists to induce apoptotic cell death in several cancer cell lines in vitro and inhibit tumor progression in mouse models. Combined treatments with KD variants possessing different recognition sites on the same target protein exerted synergisitic tumoricidal activities, compared to treatment with individual variants. In addition to the agonists, we isolated an antagonistic KD variant that binds human tumor necrosis factor-α (TNFα) and efficiently neutralizes TNFα-induced cytotoxicity in vitro and in vivo. The KD scaffold with seven flexible loops protruding from the central core was strongly sequence-tolerant to mutations in the loop regions, offering a potential advantage of distinct binding sites for target recognition on the single domain. Our results suggest that the KD scaffold can be used to develop target-specific binding proteins that function as agonists or antagonists toward given target molecules, indicative of their potential use as biotherapeutics.
DOI：
10.1073/pnas.0813399106
发表时间：
2009-05-19
影响因子：
11.1
作者：
Schoenfeld, D.;Matschiner, G.;Skerra, A.
通讯作者：
Skerra, A.
影响因子：
5.6
作者：
Rios-Steiner, JL;Schenone, M;Castellino, FJ
通讯作者：
Castellino, FJ
影响因子：
2.9
作者：
Marti, DN;Schaller, J;Llinás, M
通讯作者：
Llinás, M