A high‐affinity anti‐cocaine monoclonal antibody, designated h2E2, is entering phase 1 clinical trials for cocaine abuse therapy. To gain insight into the molecular details of its structure that are important for binding cocaine and cocaine metabolites, the Fab fragment was generated and crystallized with and without ligand. Structures of the unliganded Fab and the Fab fragment bound to benzoylecgonine were determined, and were compared with each other and with other crystallized anti‐cocaine antibodies. The affinity of the h2E2 antibody for cocaine is 4 nM, while that of the cocaine metabolite benzoylecgonine is 20 nM. Both are higher than the reported affinity for cocaine of the two previously crystallized anti‐cocaine antibodies. Consistent with cocaine fluorescent quenching binding studies for the h2E2 mAb, four aromatic residues in the CDR regions of the Fab (TyrL32, TyrL96, TrpL91 and TrpH33) were found to be involved in ligand binding. The aromatic side chains surround and trap the tropane moiety of the ligand in the complex structure, forming significant van der Waals interactions which may account for the higher affinity observed for the h2E2 antibody. A water molecule mediates hydrogen bonding between the antibody and the carbonyl group of the benzoyl ester. The affinity of binding to h2E2 of benzoylecgonine differs only by a factor of five compared with that of cocaine; therefore, it is suggested that h2E2 would bind cocaine in the same way as observed in the Fab–benzoylecgonine complex, with minor rearrangements of some hypervariable segments of the antibody.

中文翻译：

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高亲和力抗可卡因抗体 h2E2 的 Fab 片段的游离形式和配体形式的结构分析。


一种高亲和力抗可卡因单克隆抗体，称为 h2E2，正在进入可卡因滥用治疗的 1 期临床试验。为了深入了解其结构的分子细节（对于结合可卡因和可卡因代谢物非常重要），生成了 Fab 片段并在有或没有配体的情况下结晶。确定了未配体的 Fab 和与苯甲酰爱康宁结合的 Fab 片段的结构，并进行了相互比较以及与其他结晶抗可卡因抗体的比较。 h2E2 抗体对可卡因的亲和力为 4 n M ，而可卡因代谢物苯甲酰爱康宁的亲和力为 20 n M 。两者均高于先前报道的两种结晶抗可卡因抗体对可卡因的亲和力。与 h2E2 mAb 的可卡因荧光猝灭结合研究一致，发现 Fab CDR 区域的四个芳香族残基（TyrL32、TyrL96、TrpL91 和 TrpH33）参与配体结合。芳香族侧链围绕并捕获复杂结构中配体的托烷部分，形成显着的范德华相互作用，这可能是观察到 h2E2 抗体具有更高亲和力的原因。水分子介导抗体和苯甲酰酯的羰基之间的氢键。与可卡因相比，苯甲酰爱康宁与 h2E2 的结合亲和力仅相差五分之一；因此，表明 h2E2 会以与 Fab-苯甲酰爱康宁复合物中观察到的相同方式结合可卡因，但抗体的一些高变片段会发生轻微重排。