014 A novel Pemphigus vulgaris patient-derived antibody with sequence homology to antibodies directed against desmosomal and non-desmosomal targets induces keratinocyte dissociation

Abstract

The predominant autoantigenic targets in the autoimmune blistering skin disease Pemphigus vulgaris (PV) are the keratinocyte associated desmosomal proteins desmoglein (Dsg) 3 and 1. However, the presence of autoantibodies to these targets does not fully explain disease activity and phenotype. We and others have described the presence of numerous non-Dsg antibodies in the context of disease. However, the scope, specificity, and particularly functionality of non-Dsg autoantibodies has not been fully defined. Our group has previously reported the discovery of a patient derived antibody (AtS13) that bears 74% heavy-chain homology to anti-thyroid peroxidase (TPO) antibody and 86% light-chain homology to an anti-desmosome antibody as per BLAST alignment. While this antibody did not bind to Dsg3, -1 or TPO by ELISA and Western Blot and did not stain intercellular regions on monkey esophagus by IIF we did observe binding to a 55-60kDa protein in HaCaT keratinocyte lysates. Additionally, immunofluorescence revealed a cytoplasmic target within HaCaT keratinocytes but no co-localization with the cell membrane or any component thereof, including Dsg3. In order to investigate the functional role of this novel antibody and its potential to induce keratinocyte dissociation, HaCaT keratinocytes were grown to confluence and subjected to treatment with increasing concentrations of Ak23, an established mouse anti-human Dsg3 antibody, and/or AtS13. We show that while AtS13 induces a strong dose-dependent dissociation of keratinocytes in vitro, the rate of fragmentation is lower than that of Ak23 alone. AtS13 in combination with Ak23, however, induces an approximately 3-fold higher fragmentation rate than Ak23 alone, indicating a synergistic effect of these autoAbs in vivo. While the exact epitope target of the AtS13 antibody is yet to be defined, our data suggests a functional activity of this novel patient-derived antibody in the skin with potential disease relevance.