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首页> 外文期刊>Journal of Molecular Biology >High-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries
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High-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries

机译：从功能强大的极简噬菌体展示文库高通量生成合成抗体

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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.
机译：我们以前已经通过使用噬菌体展示的框架来建立抗体工程的极简方法，以支持限于酪氨酸和丝氨酸二进制编码的互补决定区（CDR）多样性。在这里，我们通过附加的多样性级别系统地扩展了原始二进制库并检查了效果。通过将轻链增加多样性，使可能影响CDR构象的非对位残基多样化并增加其他分步，逐步扩展最简单的文库的多样性（在该文库中，仅重链CDR位置由二进制代码随机化）以逐步方式扩展CDR-H3的化学多样性。额外的多样性逐渐提高了针对人血管内皮生长因子的抗体的亲和力，并且抗体-抗原复合物的结构表明酪氨酸侧链足以介导大多数与抗原的相互作用，但CDR-H3中的甘氨酸残基对于提供适合于高亲和力结合的构象至关重要。使用新的高通量方法和最复杂的文库，我们生产了多种高亲和力抗体，其解离常数在单位纳摩尔范围内，可抵抗多种蛋白质抗原。因此，这种完全合成的，极简的文库已实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，因为它们最大程度地减少了阻碍建立高通量程序的天然抗体的内在复杂性。此外，对源自这些定义明确且简单的文库的大量抗体的分析使我们得以发现CDR序列的统计学显着趋势，从而为了解抗体文库设计和控制蛋白质与蛋白质相互作用的因素提供了宝贵的见识。 （C）2007年由Elsevier Ltd.出版

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