Isolation of Pathogenic Monoclonal Anti-Desmoglein 1 Human Antibodies by Phage Display of Pemphigus Foliaceus Autoantibodies

Abstract

Pemphigus foliaceus (PF) is a blistering disease caused by autoantibodies to desmoglein 1 (Dsg1) that cause loss of epidermal cell adhesion. To better understand PF pathophysiology, we used phage display to isolate anti-Dsg1 mAbs as single-chain variable fragments (scFvs) from a PF patient. Initial panning of the library isolated only non-pathogenic scFvs. We then used these scFvs to block non-pathogenic epitopes and were able to isolate two unique scFvs, each of which caused typical PF blisters in mice or human epidermis models, showing that a single mAb can disrupt Dsg1 function to cause disease. Both pathogenic scFvs bound conformational epitopes in the N terminus of Dsg1. Other PF sera showed a major antibody response against the same or nearby epitopes defined by these pathogenic scFvs. Finally, we showed restriction of the heavy-chain gene usage of all anti-Dsg1 clones to only five genes, which determined their immunological properties despite promiscuous light-chain gene usage. These mAbs will be useful for studying Dsg1 function and mechanisms of blister formation in PF and for developing targeted therapies and tools to monitor disease activity.

Homologous regions of autoantibody heavy chain complementarity-determining region 3 (H-CDR3) in patients with pemphigus cause pathogenicity

“…Because these antibodies bind near presumed Dsg adhesion sites (19), Dsgs are in the cadherin cell adhesion family (20)(21)(22), Dsgs are in cell adhesion structures (20,22), and pathogenicity does not require the effector region of IgG and does not require a bivalent antibody (i.e., monovalent Fab′ or scFv antibody fragments are pathogenic) (6-9), it is thought that pathogenicity is, at least in part, due to direct antibody interference with Dsg adhesion. In addition, individual mAbs cloned by phage display from PV and PF patients define epitopes on Dsgs that are shared by IgG from various pemphigus patients (7,8). All these observations show that pathogenic autoantibodies from pemphigus patients bind to closely related functional epitopes on 2 homologous molecules and therefore, may share molecular similarities.…”

“…Shared amino acid sequences in H-CDR3s of pathogenic pemphigus mAbs. We have previously found that epitopes defined by pathogenic mAbs cloned from a PV and a PF patient are shared by sera from various PV and PF patients, respectively (7,8). These data suggest that pathogenic antibodies bind similar sites on Dsg and might, therefore, share common idiotypes.…”

“…For example, polyclonal IgGs from PV and PF patients have been shown to be pathogenic in organ culture of normal human skin (4,5). In addition, the F ab ′ fragment of pemphigus IgG as well as singlechain variable fragments (scFv) of pemphigus IgG, both of which lack the effector constant region, can induce typical PF or PV blisters by passive transfer to neonatal mouse and human skin organ culture (6)(7)(8)(9).…”

“…In this report, we analyze the variable heavy chain gene (VH) usage and heavy chain complementaritydetermining region 3 (H-CDR3) regions of these cloned mAbs to establish whether there are commonalities among pathogenic pemphigus antibodies. We focused on the heavy chain of the pathogenic antibodies because previous studies have shown that pathogenicity and binding of pemphigus antibodies correlates with heavy chain, rather than light chain, gene usage (7,8,13). We focused on the H-CDR3 because, in most antibodies, it is thought to provide the most important variable sequences for antibody specificity (14)(15)(16).…”

Autoantibody-producing plasmablasts after B cell depletion identified in muscle-specific kinase myasthenia gravis

“…This range of binding strengths is in agreement with other studies of human monoclonal rIg derived from patients with autoantibody-mediated diseases. This includes pemphigus (54,55), NMO (56), and AChR MG (44), all of which include rIg reported to exhibit a range of binding capacities. It is important to highlight that it is not known how the strength of binding correlates with pathogenic potential of MuSK-specific rIg.…”