From Antigenic Determinants to Dendrimeric Antigens: A MAT-Based Study of Structure–Activity Relationships in Clavulanic Acid Allergy

Background/Objectives: Drug–protein conjugates are crucial for effector cell activation in allergic reactions; however, the structural requirements for drugs like clavulanic acid (CLV) remain poorly understood. We developed synthetic models (Dendrimeric Antigen (DeAn) conjugates displaying CLV-determinants multivalently and monomeric analogues) mimicking drug–protein conjugates to investigate how scaffold size, epitope density, and determinant reactivity influence IgE-mediated effector cell activation. Methods: Synthetic monovalent determinants and multivalent DeAn of six generations (G0–G5) were designed based on the structure of Clav, an acetal-protected mimetic of the CLV-derived N-carrier-3-oxopropanamide antigenic determinant. Purified compounds were characterized by Nuclear Magnetic Resonance. Passive mast cell activation tests (MAT) were performed using humanized RBL-2H3 cells sensitized with sera from 8 CLV-allergic patients and 10 tolerant controls, stimulated with these compounds, and degranulation was quantified by β-hexosaminidase release. Results: CLV determinants and Clav-DeAn, displaying 4–128 peripheral determinants, were obtained. Only the larger G4 and G5 Clav-DeAn, CLV, and the reactive low-molecular-weight analogue induced significant degranulation with CLV-allergic sera, whereas lower-generation DeAn conjugates (G0–G3) and unreactive monovalent determinants did not. No degranulation was observed with tolerant-control sera. Conclusions: IgE-mediated effector cell degranulation in CLV allergy is influenced by overall antigen architecture and determinant reactivity to enable FcεRI receptor cross-linking, when using the acetal-protected analogue as a mimetic for the N-carrier-3-oxopropanamide determinant. Clav-DeAn provides standardized tools for investigating structure–activity relationships in drug allergy and may support improved in vitro diagnostic approaches.