Anti-MDA5 monoclonal antibodies from patients with dermatomyositis – B cell characteristics and differential targeting of the helicase domains

Autoantibodies targeting melanoma differentiation associated protein 5 (MDA5) are strongly associated with dermatomyositis (DM) and may contribute to its pathogenesis. Here we aimed to investigate MDA5 + B cells, their phenotype and generate MDA5 monoclonal antibodies to assess their epitope specificity. MDA5-reactive B cells were captured from peripheral blood of patients with anti-MDA5 + DM (n = 3) using an MDA5-fluorescent probe. B cell receptor (BCR) sequences were analysed from single-sorted B cells (n = 240). Selected clones were re-expressed as IgG1 monoclonal antibodies (mAbs, n = 23). Reactivity was assessed using recombinant MDA5 protein constructs, peptide epitope mapping, ELISA, western blot and a commercial line blot assay. Of 240 anti-MDA5 + sorted B cells, 23 BCRs were re-expressed as mAbs, two of which showed high reactivity and specificity for MDA5. These antibody sequences originated from one CD19 + IgD − CD27 − CD38 + and one CD19 + IgD − CD27 + CD38 + IgG + B cell with low somatic hypermutation (SHM). Both mAbs had nanomolar apparent affinity and bound to sites within the helicase domains of the MDA5 protein but with distinct epitope recognition. Serology screening confirmed targeting of a linear epitope identified in the mAb studies. Our results show that anti-MDA5 + B cells recognize the helicase domains, which are the enzymatically active domains of the protein. These results have implications for understanding the etiopathology of anti-MDA5 + DM and development of new antigen-specific therapies. • Isolated rare MDA5-specific B cells from blood of patients with dermatomyositis. • Generated the first human derived anti-MDA5 monoclonal antibodies. • Anti-MDA5 antibodies showed low levels of mutations and high binding affinity. • Anti-MDA5 antibodies target distinct epitopes of the MDA5 helicase 2i domain. • Identified a novel linear epitope for anti-MDA5 antibody binding.