Simmons David P, Streltsov Victor A, Dolezal Olan, Hudson Peter J, Coley Andrew M, Foley Michael, Proll David F, Nuttall Stewart D
CSIRO Division of Molecular and Health Technologies, Parkville, Victoria 3052, Australia.
Proteins. 2008 Apr;71(1):119-30. doi: 10.1002/prot.21663.
Mimotopes mimic the three-dimensional topology of an antigen epitope, and are frequently recognized by antibodies with affinities comparable to those obtained for the original antibody-antigen interaction. Peptides and anti-idiotypic antibodies are two classes of protein mimotopes that mimic the topology (but not necessarily the sequence) of the parental antigen. In this study, we combine these two classes by selecting mimotopes based on single domain IgNAR antibodies, which display exceptionally long CDR3 loop regions (analogous to a constrained peptide library) presented in the context of an immunoglobulin framework with adjacent and supporting CDR1 loops. By screening an in vitro phage-display library of IgNAR variable domains (V(NAR)s) against the target antigen monoclonal antibody MAb5G8, we obtained four potential mimotopes. MAb5G8 targets a linear tripeptide epitope (AYP) in the flexible signal sequence of the Plasmodium falciparum Apical Membrane Antigen-1 (AMA1), and this or similar motifs were detected in the CDR loops of all four V(NAR)s. The V(NAR)s, 1-A-2, -7, -11, and -14, were demonstrated to bind specifically to this paratope by competition studies with an artificial peptide and all showed enhanced affinities (3-46 nM) compared to the parental antigen (175 nM). Crystallographic studies of recombinant proteins 1-A-7 and 1-A-11 showed that the SYP motifs on these V(NAR)s presented at the tip of the exposed CDR3 loops, ideally positioned within bulge-like structures to make contact with the MAb5G8 antibody. These loops, in particular in 1-A-11, were further stabilized by inter- and intra- loop disulphide bridges, hydrogen bonds, electrostatic interactions, and aromatic residue packing. We rationalize the higher affinity of the V(NAR)s compared to the parental antigen by suggesting that adjacent CDR1 and framework residues contribute to binding affinity, through interactions with other CDR regions on the antibody, though of course definitive support of this hypothesis will rely on co-crystallographic studies. Alternatively, the selection of mimotopes from a large (<4 x 10(8)) constrained library may have allowed selection of variants with even more favorable epitope topologies than present in the original antigenic structure, illustrating the power of in vivo selection of mimotopes from phage-displayed molecular libraries.
模拟表位可模拟抗原表位的三维拓扑结构，并且常常能被亲和力与原始抗体 - 抗原相互作用所获得的亲和力相当的抗体识别。肽和抗独特型抗体是两类蛋白质模拟表位，它们模拟亲本抗原的拓扑结构（但不一定是序列）。在本研究中，我们基于单域IgNAR抗体选择模拟表位，将这两类结合起来，单域IgNAR抗体展示出异常长的互补决定区3（CDR3）环区域（类似于一个受限肽库），其呈现在具有相邻且起支持作用的互补决定区1（CDR1）环的免疫球蛋白框架背景中。通过针对靶抗原单克隆抗体MAb5G8筛选IgNAR可变区（V(NAR)s）的体外噬菌体展示文库，我们获得了四个潜在的模拟表位。MAb5G8靶向恶性疟原虫顶端膜抗原1（AMA1）柔性信号序列中的线性三肽表位（AYP），并且在所有四个V(NAR)s的CDR环中都检测到了这个或类似的基序。通过与人工肽进行竞争研究，证明V(NAR)s 1 - A - 2、 - 7、 - 11和 - 14能特异性结合该抗原结合位点，并且与亲本抗原（175 nM）相比，它们都显示出增强的亲和力（3 - 46 nM）。重组蛋白1 - A - 7和1 - A - 11的晶体学研究表明，这些V(NAR)s上的SYP基序呈现在暴露的CDR3环的顶端，理想地定位在凸起样结构内以与MAb5G8抗体接触。这些环，特别是在1 - A - 11中，通过环间和环内二硫键、氢键、静电相互作用以及芳香族残基堆积进一步稳定。我们通过提出相邻的CDR1和框架残基通过与抗体上的其他CDR区域相互作用来促进结合亲和力，从而解释了V(NAR)s比亲本抗原具有更高亲和力的原因，当然，这一假设的确切支持将依赖于共晶体学研究。或者，从一个大的（<4×10⁸）受限文库中选择模拟表位可能使得能够选择出具有比原始抗原结构中更有利的表位拓扑结构的变体，这说明了从噬菌体展示分子文库中体内选择模拟表位的强大能力。
