Engineering a high-affinity peptide binding site into the anti-CEA mAb M5A.
Engineering a high-affinity peptide binding site into the anti-CEA mAb M5A.
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将高亲和力肽结合位点设计到抗 CEA mAb M5A 中。
DOI：
10.1093/protein/gzx016
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发表时间：
2017
期刊：
影响因子：
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通讯作者：
Williams,JohnC
中科院分区：
文献类型：
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作者：
Zer,Cindy;Avery,KendraN;Meyer,Kassondra;Goodstein,Leah;Bzymek,KrzysztofP;Singh,Gagandeep;Williams,JohnC
我们之前已经确定了一种称为meditope的环状肽，它与西妥昔单抗Fab部分的中心腔结合，并表明该肽结合位点可以移植到曲妥珠单抗上，或“meditope-enabled”。该肽已被证明可作为显像剂非共价附着于美多托普激活抗体的障碍。在此，我们探索将该肽结合位点嫁接到M5A上的过程，M5A是一种抗cea抗体，用于癌症诊断的临床试验。为了探索氨基酸的作用，我们依次在Fab中引入对氨基酸取代，然后在其他取代存在的情况下对关键残基进行反向取代。我们发现轻链中的Pro40Thr、Gly41Asn、Phe83Ile和Thr85Asp足以以个位数的微摩尔亲和力重建M5A的中质粒结合位点。我们发现Pro40在存在其他12个残基取代的情况下可以消除肽结合，并且所有13个取代的存在都不会干扰抗体：抗原识别。总的来说，这些研究为定义和微调抗体中媒介位结合位点的结合亲和力提供了详细的见解。
We have previously identified a cyclic peptide called meditope which binds to the central cavity of the Fab portion of cetuximab and shown that this peptide binding site can be grafted, or ‘meditope-enabled’, onto trastuzumab. This peptide has been shown to act as a hitch for the non-covalent attachment of imaging agents to meditope-enabled antibodies. Herein, we explore the process of grafting this peptide binding site onto M5A, an anti-CEA antibody in clinical trials for cancer diagnostics. In order to explore the contributions of the amino acids, we sequentially introduced pairs of amino acid substitutions into the Fab and then we reverse-substituted key residues in the presence of the other substitutions. We demonstrate that Pro40Thr, Gly41Asn, Phe83Ile and Thr85Asp in the light chain are sufficient to recreate the meditope binding site in M5A with single-digit micromolar affinity. We show that Pro40 abrogates peptide binding in the presence of the other 12 residue substitutions, and that the presence of all 13 substitutions does not interfere with antibody:antigen recognition. Collectively, these studies provide detailed insight for defining and fine-tuning the binding affinity of the meditope binding site within an antibody.