Persistent splenic-derived IgMs preferentially recognize factor VIII A2 and C2 domain epitopes but do not alter antibody production

The most significant treatment complication for patients with hemophilia A is the development of neutralizing immunoglobins (Igs) G, termed inhibitors, against factor VIII (FVIII), which prevent FVIII replacement therapy. Low titers of FVIII-specific IgMs have been identified in hemophilia A patients with and without inhibitors, as well as in healthy individuals. However, the duration and influence of IgMs on the immune response to FVIII remains unclear. To characterize the binding interactions of persistently secreted FVIII-specific IgMs in hemophilia A mice and assess their effect on IgG antibody development. Splenic-derived monoclonal antibodies (mAbs) from immunized FVIII knockout mice were isolated and purified using hybridoma technology. Binding interactions were assessed utilizing a novel fluid-phase enzyme-linked immunosorbent assay and computational modeling with High Ambiguity-Driven protein-protein DOCKing to account for weak IgM binding. Sixteen porcine cross-reactive and noninhibitory FVIII-specific IgM mAbs were identified. RNA sequencing of FVIII-specific IgMs revealed 13 unique variable, diversity, and joining (VDJ)/variable and joining (VJ) sequences indicating derivation from 13 unique B cell clones. The IgMs demonstrated polyclonal and polyreactive binding to FVIII in vitro and in silico. Molecular docking studies with reconstructed IgM variable, diversity, and joining/variable and joining regions identified frequent IgM interactions with amino acid residues K376, T381, K437, R2215, or K2249 within the FVIII A2 and C2 domains. Injections of individual IgMs prior to FVIII exposure and co-injection of FVIII/IgM immune complexes did not affect de novo FVIII antibody production. Persistent FVIII-specific IgMs are polyclonal but preferentially bind the A2 and C2 domains. FVIII/IgM immune complex formation does not significantly alter inhibitor development.