Synthetic antibody libraries focused towards peptide ligands
Synthetic antibody libraries focused towards peptide ligands
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DOI：
10.1016/j.jmb.2008.02.037
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发表时间：
2008-05-02
影响因子：
5.6
通讯作者：
Georgiou, George
中科院分区：
文献类型：
--
作者：
Cobaugh, Christian W.;Almagro, Juan C.;Georgiou, George
合成抗体文库已被证明对于从头分离抗体非常有用，无需动物免疫。最近，设计用于识别特定类别配体（例如半抗原或蛋白质）的重点文库已被用来促进高亲和力抗体的选择。使用编码典型结构组合的 V 区构建聚焦文库，这些典型结构类似于结合所需类别配体的抗体的结构特征，并且在通常参与识别的残基处引入序列多样性。在这里，我们描述了两种不同的单链抗体可变片段库的生成和实验验证，这些库有效地生成肽的结合物，肽是一类已被证明是抗体生成的困难靶标的分子。首先，通过使支架多样化来构建人抗肽文库：人可变重链（V-H）种系基因3-23，其与人可变轻链NO种系基因A27的变体融合，其中L1被修饰以编码抗肽抗体中发现的规范结构。仅将序列多样性引入到 3-23 (V-H) 中，针对与蛋白质和肽抗原接触时常见的多样化残基。其次，使用抗体 26-10 生成了小鼠文库，该抗体最初是基于其与半抗原地高辛的亲和力而分离的，但也结合肽并表现出抗肽抗体典型的规范结构模式。仅使用在经常接触肽抗原的位置处发现的氨基酸谱在V-H中引入多样性。通过溶液噬菌体淘选筛选后，两个文库都产生了两种模型肽（血管紧张素和神经肽 Y）的结合物。尽管只有 V-H 进行了多样化，但小鼠文库产生的抗体对两个靶标的亲和力均低于 20 nM。 (C) 2008 Elsevier Ltd. 保留所有权利。
Synthetic antibody libraries have proven immensely useful for the de novo isolation of antibodies without the need for animal immunization. Recently, focused libraries designed to recognize particular classes of ligands, such as haptens or proteins, have been employed to facilitate the selection of high-affinity antibodies. Focused libraries are built using V regions encoding combinations of canonical structures that resemble the structural features of antibodies that bind the desired class of ligands and sequence diversity is introduced at residues typically involved in recognition. Here we describe the generation and experimental validation of two different single-chain antibody variable fragment libraries that efficiently generate binders to peptides, a class of molecules that has proven to be a difficult target for antibody generation. First, a human anti-peptide library was constructed by diversifying a scaffold: the human variable heavy chain (V-H) germ line gene 3-23, which was fused to a variant of the human variable light chain NO germ line gene A27, in which L1 was modified to encode the canonical structure found in anti-peptide antibodies. The sequence diversity was introduced into 3-23 (V-H) only, targeting for diversification residues commonly found in contact with protein and peptide antigens. Second, a murine library was generated using the antibody 26-10, which was initially isolated based on its affinity to the hapten digoxin, but also binds peptides and exhibits a canonical structure pattern typical of anti-peptide antibodies. Diversity was introduced in the V-H only using the profile of amino acids found at positions that frequently contact peptide antigens. Both libraries yielded binders to two model peptides, angiotensin and neuropeptide Y, following screening by solution phage panning. The mouse library yielded antibodies with affinities below 20 nM to both targets, although only the V-H had been subjected to diversification. (C) 2008 Elsevier Ltd. All rights reserved.