Mechanisms of IgM mediated activation of the complement system

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SUMMARY Immunglobulin µ (IgM) is an evolutionary old class of antibody that is the first antibody produced in response to an infection. Several IgMs are present as natural antibodies and are vital for immunity during the early stages of development. Unlike any other class of antibody, a single copy is sufficient for activating the classical complement system, and several monoclonal IgMs have shown massive potential for the treatment of cancers. The large size, high degree of glycosylation, and structural heterogeneity of IgM has rendered them refractory to structural studies and to date the molecular mechanisms of how antigen binding activates IgM to initiate the complement cascade remain murky. In contrast, a detailed understanding of the structure and various interactions of immunoglobulin g (IgG) have been fundamental to their advancement as the premier molecular platform for biotherapeutics. Accordingly, a similar level of understanding of IgM will be critical for their development as a new class of biotherapeutics. This proposal aims to apply structural mass spectrometry techniques, electron microscopy with complementary structural approaches, and biophysical tools to study the structural changes within IgM that govern activation of the complement cascade. Various types of IgM-antigen complexes will be prepared and analyzed using hydrogen/deuterium exchange and X-ray foot printing with mass spectrometry to track the local structural changes within IgM upon binding different presentations of antigen (Aim 1). Electron microscopy and small angle X-ray scattering will be used to visualize and track large-scale structural transitions in the antigen-IgM complexes (Aim 2). The full combination of techniques will be used to study how IgM recruits and activates the initial component, C1, of the complement cascade (Aim 3). The molecular mechanisms of how IgM recognizes antigen and recruits complement will provide a foundation for modulating the immune system for a new paradigm in biotherapeutics.