Characterization of anti-fentanyl antibodies as antagonists of ultra-toxic, clinically relevant or endogenous opioids

The ultra-potent synthetic opioid carfentanil acts lethally by potently activating µ opioid receptors (µOR). Treatment based on competitive antagonists is of limited use due to the ultra-high affinity of the carfentanil/µOR complex. Thus, preventing formation of this complex by car neutralizing antibodies might be a promising alternative strategy. We tested nine antibodies raised against fentanyl (ab-fen) in receptor-ligand binding and receptor activation assays using µOR expressing HEK293 cells in the presence of fen or car. When high antibody concentrations (500 nM) were pre-incubated with opioids, seven ab-fen significantly inhibited fen binding to the µOR completely and four car binding up to 90 %. None of the tested antibodies affected remifentanil, morphine or endomorphine-1. Concentration-response curves revealed IC50-values of ab-fen between 25 and 74 nM against fentanyl and between 121 and 900 nM against carfentanil. Hill-slopes against fen were way above one (2.7–6.0), indicating extremely high positive cooperativity of ab-fen, which was not observed against carfentanil. Furthermore, low antibody concentrations (1.0 nM) enhanced fentanyl- and to a lesser extent carfentanil-induced µOR activation, indicating bi-functional actions of ab-fen. Moreover, when lethal carfentanil concentrations were first added to cells, ab-fen also disrupted the µOR/carfentanil complex with carfentanil still being present. Overall, ab-fen maybe able to stop and reverse carfentanil intoxications in vivo and their effects on opioid efficacy at low ab-fen concentrations suggest that they might be used in a new way to improve opioid-based pain therapy. Our findings might pave the way for future antibody development and refinement.