Long-range allosteric communication within antibodies affects antigen-binding affinity.

As an essential component of the immune system, antibodies are among the most rigorously studied molecules in the biosciences; they combine antigen recognition with isotype-dependent effector functions. Despite this, allosteric communication within antibodies and the resulting effects on antigen-binding affinities, specifically those conferred by antibody class (isotype) or subclass, remain poorly understood. Using surface plasmon resonance, we performed a comprehensive comparison of IgA1, IgD, IgE, IgG1, IgG4 and IgM Fabs across five distinct antibody-antigen systems. While different CH1 domains produced small, medium-range allosteric differences in affinity, we identified much larger, long-range effects transmitted from the Fc region to the Fab region. Full-length antibodies consistently exhibited higher binding affinities than their Fab counterparts, independent of avidity. Thermodynamic analysis indicates these effects are driven by differences in antibody flexibility and pre-organization. Taken together, these findings demonstrate that allosteric modulation of antigen binding - mediated by the CH1 domain, the Fc region, and Fc-ligand interactions - is a critical determinant of antibody function, offering essential insights for the assessment and engineering of diverse therapeutic formats.