Updated project metadata. Many archetypal and emerging classes of small-molecule therapeutics form covalent 38 protein adducts. In vivo, both the resulting conjugates and their off-target side conjugates have the potential to elicit antibodies, with implications for allergy and drug sequestration. Although β-lactam antibiotics are a drug class long associated with these immunological phenomena, the molecular underpinnings of off-target drug protein conjugation and consequent drug-specific immune responses remain incomplete. Here, using the classical β-lactam penicillin G (PenG) we have now probed the B and T cell determinants of drug-specific IgG responses to such conjugates in mice. Deep clonotyping reveals a dominant murine clonal antibody class encompassing phylogenetically-related IGHV1, IGHV5 and IGHV10 subgroup gene segments. Protein NMR and x-ray structural analyses reveal that these drive structurally convergent binding modes in adduct-specific antibody clones. Their common primary recognition mechanisms of the penicillin side-chain moiety (phenylacetamide in PenG)—regardless of CDRH3 length—limits cross-reactivity against other β-lactam antibiotics. This immunogenetics-guided discovery of the limited binding solutions available to antibodies against side products of an archetypal covalent inhibitor now suggests future potential strategies for the ‘germline-guided reverse engineering’ of such drugs away from unwanted immune responses.