Generation of enantiospecific monoclonal antibodies against (2R,6R)-hydroxynorketamine

Antibodies against small psychoactive molecules have been developed for applications ranging from substance detection and overdose protection to mechanistic understanding of the actions of complex substance mixtures. In this study, we describe the design, synthesis, formulation, and animal testing of an initial immunogenic bioconjugate, as well as subsequent isolation of enantiospecific monoclonal antibodies against (2R,6R)-hydroxynorketamine, a putatively active metabolite of the dissociative-anesthetic and rapidly-acting antidepressant ketamine. Following pharmacophore synthesis, hapten generation was achieved through installation of 6-aminohexanoic acid linkers using reductive amination. Bioconjugation to the carrier protein, cross-reactive material 197 (CRM) was achieved via amide coupling. Upon administration to mice in combination with alum and CpG oligodeoxynucleotide 1826, (2R,6R)-hydroxynorketamine-CRM generated equivalent antibody titers to a comparator racemic 6-hydroxynorketamine-CRM hapten. Following creation of hybridomas arising from B-cells responsive to (2R,6R)-hydroxynorketamine-CRM exposure and subsequent screening, subcloning, sequencing, and production, we were able to identify a monoclonal antibody, 6F11-HC₁-LC₂, which yielded antibodies strongly responsive to (2R,6R)-hydroxynorketamine, but showed no responsiveness to (2S,6S)-hydroxynorketamine in a competitive binding enzyme-linked immunosorbent assay format. Surface plasmon resonance analysis of this monoclonal species demonstrated sub-nanomolar (0.4 nM) antibody affinity for (2R,6R)-hydroxynorketamine-BSA bioconjugates and > 150-fold selectivity in comparison to ketamine-BSA bioconjugates. Overall, these results demonstrate successful production of monoclonal antibodies capable of robustly distinguishing between hydroxynorketamine enantiomers, enabling their use in future in vivo studies to better understand their relative contributions to the rapidly-acting antidepressant properties of ketamine.