Abstract：
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 (CDR) 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 in vivo applications of VHH-based biologics against OUD and overdose.
摘要：
阿片类药物使用障碍（OUD）和过量是不断发展的公共卫生威胁，在美国和国外的发病率和患病率持续增长。目前的治疗包括阿片受体激动剂和拮抗剂，这是安全和有效的，但仍然受到一些限制。鼠和人源化单克隆抗体（mAb）已成为逆转和预防阿片类药物诱导的呼吸抑制的替代和补充策略。探索超越传统重链-轻链mAb的抗体应用，我们从骆驼科动物可变重链(VHH)结构域噬菌体展示文库中鉴定出一种对芬太尼特异的新型单域抗体,并进行了生物物理鉴定.结构数据表明，VHH与芬太尼的结合是由独特的结构域交换二聚化机制促进的。它伴随着互补决定区（CDR）环的重排，导致芬太尼结合袋的形成。结构指导的诱变进一步鉴定了氨基酸取代,其提高了亲和力并放宽了在芬太尼结合中VHH的二聚化要求。我们的研究证明了阿片类药物的VHH参与，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和过量用药的体内应用奠定基础。
