Structural insights into the activation of the classical complement system
In our research, we focused on understanding the activation of the classical complement pathway and the unique features of IgG3 antibodies. The classical complement pathway is initiated by antigen-bound IgG antibodies, which oligomerize to activate complement through the hexameric C1q complex. Surprisingly, structural data has revealed that it is not essential to engage all six arms of C1q to initiate complement activation. This highlights a symmetry mismatch between C1 and the hexameric IgG complex, which remains unexplained.
To address these questions, we use DNA nanotechnology to engineer specific nanostructures for antigen templating, allowing us to control the valency of IgG. These DNA nano-templated IgG complexes were able to activate complement on cell-mimetic lipid membranes, offering valuable insights into the impact of IgG valency on complement activation without the need to mutate the antibodies. We employed biophysical assays in conjunction with 3D cryo-electron tomography to investigate this phenomenon further. Our findings show that the cleavage of complement component C4 by the C1 complex is proportional to the number of antigens present. Additionally, higher IgG valency correlated with improved activation of the terminal complement pathway and the formation of the membrane attack complex. These results demonstrate how nanopatterning antigen-antibody complexes influence C1 complex activation, suggesting potential routes for modulating complement activation through antibody engineering.
In a parallel line of research, we explored the unique characteristics of IgG3 antibodies. IgG3 stands out among IgG subclasses due to its extended hinge region, allotypic diversity, and superior effector functions, such as highly efficient pathogen neutralization and complement activation. Despite its potential, IgG3 has been underrepresented as an immunotherapeutic candidate, primarily due to a lack of structural information. To bridge this knowledge gap, we use cryoEM to determine the structures of antigen-bound IgG3, both in isolation and in complex with complement components. These structural analyses unveiled a tendency for IgG3-Fab clustering, facilitated by the flexible upper hinge region specific to IgG3, potentially maximizing pathogen neutralization through high-density antibody arrays. IgG3 also forms elevated hexameric Fc platforms that extend above the protein corona, enhancing receptor binding and interactions with the complement C1 complex, which displayed a unique protease conformation in this context, possibly preceding C1 activation. Mass spectrometry results indicated that C1 deposits C4b directly onto specific IgG3 residues in proximity to the Fab domains. This is attributed to the height of the C1-IgG3 complex. This knowledge is valuable for the development and design of future immunotherapeutics based on IgG3, potentially expanding its role in therapeutic applications.