Structure of CD40 ligand in complex with the Fab fragment of a neutralizing humanized antibody

Background: CD40 ligand (CD40L or CD154), a member of the tumor necrosis fa ctor (TNF) family, plays a critical role in both humoral and cellular immun e responses and has been implicated in biological pathways involving epithe lial cells, fibroblasts, and platelets. Such a pathway is T cell-mediated B cell activation, a process that occurs through the interaction of CD40L wi th CD40 receptor expressed on B cells. It results in various B cell respons es, including immunoglobulin isotype switching and B cell differentiation a nd proliferation. These responses can be inhibited by the monoclonal antibo dy 5c8, which binds with high affinity to CD40L. Results: To understand the structural basis of the inhibition, we determine d the crystal structure of the complex of the extracellular domain of GD40L and the Fab fragment of humanized 5c8 antibody. The structure shows that t he complex has the shape of a three-bladed propeller with three Feb fragmen ts bound symmetrically to a CD40L homotrimer, To further study the nature o f the antibody-antigen interface, we assessed the ability of 23 site-direct ed mutants of CD40L to bind to 5c8 and CD40 and analyzed the results in the context of the crystal structure. Finally, we observed via confocal micros copy that 5c8 binding to CD40L on the cell surface results in the formation of patches of clustered complexes. Conclusions: The structure reveals that 5c8 neutralizes CD40L function by s terically blocking CD40 binding. The antigenic epitope is localized in a re gion of the surface that is likely to be structurally perturbed as a result of genetic mutations that cause hyper-IgM syndrome. The symmetric trimeric arrangement of the Fab fragments in the complex results in a geometry that facilitates the formation of large clusters of complexes on the cell surfa ce.