Combinatorial libraries against libraries for selecting neoepitope activation-specific antibodies
Combinatorial libraries against libraries for selecting neoepitope activation-specific antibodies
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DOI：
10.1073/pnas.0914358107
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发表时间：
2010-04-06
影响因子：
11.1
通讯作者：
Jin, Moonsoo M.
中科院分区：
文献类型：
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作者：
Hu, Xuebo;Kang, Sungkwon;Jin, Moonsoo M.
发现构象特异性抗体或识别信号分子和酶中激活诱导的新表位的系统方法将成为开发用于基础科学和治疗的抗体的强大工具。在这里，我们报告了抗体拮抗剂的分离，该抗体拮抗剂优先结合活化的整合素 Mac-1 (alpha(M)beta(2))，并能有效阻止中性粒细胞粘附和迁移。为此任务开发了一种新策略，包括 Mac-1 插入 (I) 结构域文库的酵母表面展示、定向进化以分离 I 结构域的活性突变体，以及针对酵母中活性 I 结构域筛选人抗体库的噬菌体展示。然后将富集的抗体文库引入酵母表面双杂交系统中，以便从单体抗原抗体相互作用中最终定量选择抗体。这导致了中等到高亲和力抗体的高效分离，这些抗体优先与活性 I 结构域反应，拮抗 I 结构域与细胞间粘附分子 (ICAM)-1、补体 C3 片段 iC3b 和纤连蛋白的结合，并有效抑制中性粒细胞在纤维蛋白原上的迁移。本文证明的策略可广泛适用于开发针对在非活性和活性构象之间切换的模块结构域的抗体，特别是在治疗和诊断应用中发现抗体拮抗剂。
A systematic approach to the discovery of conformation-specific antibodies or those that recognize activation-induced neoepitopes in signaling molecules and enzymes will be a powerful tool in developing antibodies for basic science and therapy. Here, we report the isolation of antibody antagonists that preferentially bind activated integrin Mac-1 (alpha(M)beta(2)) and are potent in blocking neutrophil adhesion and migration. A novel strategy was developed for this task, consisting of yeast surface display of Mac-1 inserted (I) domain library, directed evolution to isolate active mutants of the I domain, and screening of phage display of human antibody library against the active I domain in yeast. Enriched antibody library was then introduced into yeast surface two-hybrid system for final quantitative selection of antibodies from monomeric antigen antibody interaction. This led to highly efficient isolation of intermediate to high affinity antibodies, which preferentially reacted with the active I domain, antagonized the I domain binding to intercellular adhesion molecule (ICAM)-1, complement C3 fragment iC3b, and fibronectin, and potently inhibited neutrophil migration on fibrinogen. The strategy demonstrated herein can be broadly applicable to developing antibodies against modular domains that switch between inactive and active conformations, particularly toward the discovery of antibody antagonists in therapeutic and diagnostic applications.