High-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries
High-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries
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DOI：
10.1016/j.jmb.2007.08.005
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发表时间：
2007-11-02
影响因子：
5.6
通讯作者：
Sidhu, Sachdev S.
中科院分区：
文献类型：
--
作者：
Fellouse, Frederic A.;Esaki, Kaori;Sidhu, Sachdev S.
我们之前已经建立了一种极简的抗体工程方法，通过使用噬菌体展示框架来支持仅限于酪氨酸和丝氨酸二进制代码的互补决定区（CDR）多样性。在这里，我们系统地增强了原始二进制库的多样性，并检查了效果。最简单的文库（其中仅重链 CDR 位置通过二进制代码随机化）的多样性通过向轻链添加多样性、通过使可能影响 CDR 构象的非互补位残基多样化以及通过向 CDR-H3 添加额外的化学多样性来逐步扩展。额外的多样性逐渐提高了针对人血管内皮生长因子产生的抗体的亲和力，并且抗体-抗原复合物的结构表明酪氨酸侧链足以介导与抗原的大部分相互作用，但CDR-H3中的甘氨酸残基对于提供适合高亲和力结合的构象至关重要。使用新的高通量程序和最复杂的库，我们生产了多种高亲和力抗体，其针对多种蛋白质抗原的解离常数在个位数纳摩尔范围内。因此，这个完全合成的、简约的文库本质上概括了天然免疫系统产生高亲和力抗体的能力。这种类型的文库对于蛋白质组学应用应该非常有用，因为它们最大限度地减少了阻碍高通量程序建立的天然抗体的固有复杂性。此外，对源自这些明确且简单的文库的大量抗体的分析使我们能够发现 CDR 序列的统计显着趋势，这为抗体文库设计和控制蛋白质-蛋白质相互作用的因素提供了有价值的见解。 (C) 2007 年，爱思唯尔有限公司出版。
We have previously established a minimalist approach to antibody engineering by using a phage-displayed framework to support complementarity determining region (CDR) diversity restricted to a binary code of tyrosine and serine. Here, we systematically augmented the original binary library with additional levels of diversity and examined the effects. The diversity of the simplest library in which only heavy chain CDR positions were randomized by the binary code, was expanded in a stepwise manner by adding diversity to the light chain, by diversifying non-paratope residues that may influence CDR conformations, and by adding additional chemical diversity to CDR-H3. The additional diversity incrementally improved the affinities of antibodies raised against human vascular endoethelial growth factor and the structure of an antibody-antigen complex showed that tyrosine side-chains are sufficient to mediate most of the interactions with antigen, but a glycine residue in CDR-H3 was critical for providing a conformation suitable for high-affinity binding. Using new high-throughput procedures and the most complex library, we produced multiple high-affinity antibodies with dissociation constants in the single-digit nanomolar range against a wide variety of protein antigens. Thus, this fully synthetic, minimalist library has essentially recapitulated the capacity of the natural immune system to generate high-affinity antibodies. Libraries of this type should be highly useful for proteomic applications, as they minimize inherent complexities of natural antibodies that have hindered the establishment of high-throughput procedures. Furthermore, analysis of a large number of antibodies derived from these well-defined and simplistic libraries allowed us to uncover statistically significant trends in CDR sequences, which provide valuable insights into antibody library design and into factors governing protein-protein interactions. (C) 2007 Published by Elsevier Ltd.