Pathogenic Anti-Desmoglein 3 mAbs Cloned from a Paraneoplastic Pemphigus Patient by Phage Display

Abstract

Paraneoplastic pemphigus (PNP) is an autoimmune blistering disease associated with lymphoproliferative neoplasms and characterized by antibodies against plakins and desmoglein 3 (Dsg3). Anti-Dsg3 antibodies have a primary role in blister formation in PNP. In this study, we used phage display to clone monoclonal anti-Dsg3 antibodies from a PNP patient to further characterize their pathogenicity. We isolated 20 unique Dsg3-reactive mAbs, which we classified into four groups according to the heavy-chain complementarity-determining region 3 (CDR3) region. Genetic analyses demonstrated that three antibody groups used the VH1-46 gene (18 clones) and one group used the VH1-02 gene (2 clones). The results of an in vitro keratinocyte dissociation assay and a human skin organ culture injection assay showed that three antibodies displayed pathogenic activity in blister formation with different potencies. Epitope mapping using domain-swapped Dsg3/Dsg2 showed that these pathogenic mAbs bound Ca(2+)-dependent conformational epitopes in the middle portion of the extracellular region of Dsg3 (EC2 and EC3 domains), in contrast to most previously characterized pathogenic pemphigus vulgaris antibodies, which bound to the EC1 domain of Dsg3. These mAbs reflect the unique polyclonal nature of anti-Dsg3 antibodies in PNP and represent an important tool for detailing the pathophysiological mechanisms of blister formation in PNP.

Desmosomes in acquired disease

“…Autoantibodies in PNP are directed against multiple desmosomal proteins, including Dsgs, Dscs, desmoplakin and plakophilins (Futei, et al, 2003, Gallo, et al, 2014, Hashimoto, et al, 1995a, Lambert, et al, 2010, Seishima, et al, 2004). Similar to other forms of pemphigus, blistering in this disease can be attributed to autoantibodies directed against Dsgs (Amagai, et al, 1998, Nishifuji, et al, 2007, Saleh, et al, 2012), particularly the Dsg3 EC2-3 domains (Saleh, et al, 2012). However, autoantibodies directed against the plakin family including envoplakin and periplakin are useful diagnostic markers that help to distinguish PNP from other forms of pemphigus (Amagai, 1999, Amagai, et al, 1998, Mahoney, et al, 1998, Poot, et al, 2013, Probst, et al, 2009).…”

Shared VH1-46 gene usage by pemphigus vulgaris autoantibodies indicates common humoral immune responses among patients

“…However, mutation of the tryptophan blocked mAb pathogenicity, and mutation of all 5 residues in the motif abolished Dsg binding. Subsequent studies of pathogenic PV or paraneoplastic pemphigus anti-Dsg3 mAbs did not identify shared CDR3 motifs 10, 29 . Our current study indicates that acidic amino acid residues throughout the heavy chain CDRs of VH1-46 PV mAbs are necessary for Dsg binding (Figure 4).…”

“…Phage display previously identified VH5-51 gene usage by transglutaminase autoAbs in celiac disease patients, which was subsequently validated by single-cell PCR 27, 28 ; several Abs cloned using the two different techniques even demonstrated the same heavy and light chain variable region gene pairings. A prior study of one paraneoplastic pemphigus patient using phage display identified VH1-46 anti-Dsg3 mAbs that induced suprabasal blisters in human skin explants 29 . Paraneoplastic pemphigus is a different disease with autoAbs to multiple antigenic targets, but nevertheless this study supports our finding that VH1-46 is commonly used among anti-Dsg3 Abs.…”

Pemphigus

“…In pemphigus vulgaris and paraneoplastic pemphigus, V H 1-46 antibody heavy chain gene usage by desmoglein 3-reactive B cells is commonly observed 83,84 . Surprisingly, anti-desmoglein 3-secreting B cells that use V H 1-46 have relatively few somatic mutations and require few to none of these mutations to bind to desmoglein 3, in contrast to B cells using other V H genes, which are highly mutated and require somatic mutations to maintain desmoglein 3 autoreactivity 60,83 .…”