Haptenic adducts of β-lactam antibiotics elicit antibody responses with narrow clonality and specificity

Abstract

AbstractMany classes of small-molecule drugs form protein adductsin vivo, which may elicit antibodies via a classical hapten-carrier-type response, with implications for both allergy and drug sequestration. Although β-lactam antibiotics are a drug class long associated with these phenomena, the molecular determinants of drug-protein conjugation and consequent drug-specific immune responses remain incomplete. Here, we interrogated factors influencing penicilloyl adduct formation and immunogenicity, and used penicillin G (PenG) to probe the B and T cell determinants of drug-specific IgG responses in mice. We identify through deep clonotyping a dominant murine penicilloyl-specific clonal antibody class encompassing phylogenetically relatedIGHV1,IGHV5andIGHV10subgroup gene segments. Through protein NMR and x-ray structural analysis, we determined that adduct specific antibody clones—the MIL series—predominantly recognise the variable side-chain moiety (which for PenG is phenylacetamide) via a hydrophobic pocket, while secondary H-bond contacts with both thiazolidine and the adducted lysine residue is made. As a result, the cross-reactivity against other β-lactam antibiotics is limited. These data demonstrate the relationship between the chemistry of protein-reactive drugs such as penicilloyls, and how their predisposition to generating B cell responses can inform the functional implications at the clonal level.Highlights-PenG readily forms immunogenic adducts on lysine sidechains of diverse self- and non-self proteins including complete serum under physiological conditions.-PenG-protein adductionin vitroorin vivois sufficient to elicit penicillin-specific IgG responses.-Murine B cell clonotypic responses are characterised by near-uniform antibody binding modes of similar immunogenetic origin.-The dominant murine PenG-specific clonotype is dominated by benzene ring recognition and correlates with serological cross-reactivity profiles.