Discovery, Structural Characterization, and Preclinical Evaluation of Monoclonal Antibodies against Xylazine Poisoning.

Xylazine is a nonopioid sedative that has emerged as a major adulterant in the illicit drug supply, contributing to rising fatal overdoses across the United States. Xylazine toxicity is not reversed by naloxone, and there are no current FDA-approved therapeutics to counteract its effects in humans. To address this issue, we developed and characterized monoclonal antibodies (mAbs) capable of sequestering xylazine to prevent or treat acute toxicity. By immunizing mice with conjugated haptens targeting distinct epitopes of the xylazine molecule, we generated two lead candidates, Xy1001 and Xy3001. Comparative kinetic analysis revealed that Xy3001 showed a subnanomolar affinity and was selected for structural characterization. X-ray crystallography of the Xy3001-xylazine complex revealed the structural basis for this affinity, showing the binding of xylazine in a deep pocket anchored by hydrogen bonding. Humanization of Xy3001 resulted in a minor reduction in affinity but demonstrated improved biophysical stability and high specificity with no significant cross-reactivity to off-target opioids or stimulants. In a murine model of toxicity, prophylactic administration of both lead candidates significantly attenuated xylazine-induced antinociception, with Xy3001 demonstrating additional mitigation of respiratory depression and bradycardia. Pharmacokinetic analysis demonstrated that mAb treatment limited xylazine entry into the brain. This study validates peripheral sequestration against xylazine as a therapeutic strategy with future efforts aimed at optimization of these lead candidates to increase translational potential.