Characterization of a high-affinity human antibody with a disulfide bridge in the third complementarity-determining region of the heavy chain
Characterization of a high-affinity human antibody with a disulfide bridge in the third complementarity-determining region of the heavy chain
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DOI：
10.1002/jmr.1168
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发表时间：
2012-03-01
影响因子：
2.7
通讯作者：
Fransson, Johan
中科院分区：
文献类型：
--
作者：
Almagro, Juan Carlos;Raghunathan, Gopalan;Fransson, Johan
二硫键在鲨鱼（新型抗原受体）和骆驼（单可变重链结构域，VHH）的抗原结合位点中很常见，它们赋予了结构多样性和结构域稳定性。在人类抗体中，重链第三互补决定区（CDR - H3）中的半胱氨酸残基很少见，但在IGHD种系基因中自然编码。在此，通过用基于人种系基因和合成的CDR - H3区域设计的噬菌体展示文库针对一种人类细胞因子进行淘选，我们鉴定出一种在CDR - H3中含有两个半胱氨酸残基的抗体（M3）。它以高亲和力（0.4纳米）结合该细胞因子，识别抗原上的一个独特表位，并且与从该文库中选出的所有其他抗体相比，具有独特的中和特性。通过质谱肽图分析确定这两个半胱氨酸残基形成一个二硫键。用丙氨酸替代半胱氨酸并没有改变单克隆抗体的溶解性和稳定性，但与抗原的结合显著受损。采用三维建模和动态模拟来探究二硫键如何影响CDR - H3的构象以及与抗原的结合。基于这些结果，我们设想设计包含CDR内或CDR间二硫键的人类组合抗体文库可能会导致鉴定出具有独特结合特性的人类抗体。版权所有（c）2012约翰威立父子有限公司
Disulfide bridges are common in the antigen-binding site from sharks (new antigen receptor) and camels (single variable heavy-chain domain, VHH), in which they confer both structural diversity and domain stability. In human antibodies, cysteine residues in the third complementarity-determining region of the heavy chain (CDR-H3) are rare but naturally encoded in the IGHD germline genes. Here, by panning a phage display library designed based on human germline genes and synthetic CDR-H3 regions against a human cytokine, we identified an antibody (M3) containing two cysteine residues in the CDR-H3. It binds the cytokine with high affinity (0.4?nm), recognizes a unique epitope on the antigen, and has a distinct neutralization profile as compared with all other antibodies selected from the library. The two cysteine residues form a disulfide bridge as determined by mass spectrometric peptide mapping. Replacing the cysteines with alanines did not change the solubility and stability of the monoclonal antibody, but binding to the antigen was significantly impaired. Three-dimensional modeling and dynamic simulations were employed to explore how the disulfide bridge influences the conformation of CDR-H3 and binding to the antigen. On the basis of these results, we envision that designing human combinatorial antibody libraries to contain intra-CDR or inter-CDR disulfide bridges could lead to identification of human antibodies with unique binding profiles. Copyright (c) 2012 John Wiley & Sons, Ltd.