Opioid use disorders (OUD) and overdoses are ever-evolving public health threats that continue to grow in incidence and prevalence in the United States and abroad. Current treatments consist of opioid receptor agonists and antagonists, which are safe and effective but still suffer from some limitations. Murine and humanized monoclonal antibodies (mAb) have emerged as an alternative and complementary strategy to reverse and prevent opioid-induced respiratory depression. To explore antibody applications beyond traditional heavy-light chain mAbs, we identified and biophysically characterized a novel single-domain antibody specific for fentanyl from a camelid variable-heavy-heavy (VHH) domain phage display library. Structural data suggested that VHH binding to fentanyl was facilitated by a unique domain-swapped dimerization mechanism, which accompanied a rearrangement of complementarity-determining region loops leading to the formation of a fentanyl-binding pocket. Structure-guided mutagenesis further identified an amino acid substitution that improved the affinity and relaxed the requirement for dimerization of the VHH in fentanyl binding. Our studies demonstrate VHH engagement of an opioid and inform on how to further engineer a VHH for enhanced stability and efficacy, laying the groundwork for exploring the applications of VHH-based biologics against OUD and overdose.

中文翻译：

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芬太尼特异性新型域交换骆驼抗体的鉴定和生物物理表征


阿片类药物使用障碍 (OUD) 和过量服用是不断变化的公共卫生威胁，在美国和国外的发病率和流行率持续上升。目前的治疗方法包括阿片受体激动剂和拮抗剂，安全有效，但仍存在一些局限性。鼠和人源化单克隆抗体 (mAb) 已成为逆转和预防阿片类药物引起的呼吸抑制的替代和补充策略。为了探索传统重-轻链 mAb 之外的抗体应用，我们从骆驼科动物可变重-重 (VHH) 域噬菌体展示库中鉴定了一种新型芬太尼特异性单域抗体，并进行了生物物理表征。结构数据表明，独特的结构域交换二聚化机制促进了 VHH 与芬太尼的结合，该机制伴随着互补决定区环的重排，导致芬太尼结合袋的形成。结构引导诱变进一步鉴定了氨基酸取代，该氨基酸取代提高了亲和力并放宽了芬太尼结合中 VHH 二聚化的要求。我们的研究证明了 VHH 与阿片类药物的结合，并告知如何进一步设计 VHH 以增强稳定性和功效，为探索基于 VHH 的生物制剂对抗 OUD 和过量的应用奠定了基础。