Genetic and functional characterization of human pemphigus vulgaris monoclonal autoantibodies isolated by phage display
Aimee S. Payne, … , John R. Stanley, Don L. Siegel
Aimee S. Payne, … , John R. Stanley, Don L. Siegel
Published April 1, 2005
Citation Information: J Clin Invest. 2005;115(4):888-899. https://doi.org/10.1172/JCI24185.
View: Text | PDF
Article Dermatology

Genetic and functional characterization of human pemphigus vulgaris monoclonal autoantibodies isolated by phage display

Abstract

Pemphigus is a life-threatening blistering disorder of the skin and mucous membranes caused by pathogenic autoantibodies to desmosomal adhesion proteins desmoglein 3 (Dsg3) and Dsg1. Mechanisms of antibody pathogenicity are difficult to characterize using polyclonal patient sera. Using antibody phage display, we have isolated repertoires of human anti-Dsg mAbs as single-chain variable-region fragments (scFvs) from a patient with active mucocutaneous pemphigus vulgaris. ScFv mAbs demonstrated binding to Dsg3 or Dsg1 alone, or both Dsg3 and Dsg1. Inhibition ELISA showed that the epitopes defined by these scFvs are blocked by autoantibodies from multiple pemphigus patients. Injection of scFvs into neonatal mice identified 2 pathogenic scFvs that caused blisters histologically similar to those observed in pemphigus patients. Similarly, these 2 scFvs, but not others, induced cell sheet dissociation of cultured human keratinocytes, indicating that both pathogenic and nonpathogenic antibodies were isolated. Genetic analysis of these mAbs showed restricted patterns of heavy and light chain gene usage, which were distinct for scFvs with different desmoglein-binding specificities. Detailed characterization of these pemphigus mAbs should lead to a better understanding of the immunopathogenesis of disease and to more specifically targeted therapeutic approaches.

Authors

Aimee S. Payne, Ken Ishii, Stephen Kacir, Chenyan Lin, Hong Li, Yasushi Hanakawa, Kazuyuki Tsunoda, Masayuki Amagai, John R. Stanley, Don L. Siegel

×

Figure 9

Options: View larger image (or click on image) Download as PowerPoint
Heavy and light chain restriction of Dsg-panned scFv mAbs. (A) Dendrogra...
Heavy and light chain restriction of Dsg-panned scFv mAbs. (A) Dendrogram and CDR3 alignment of (D3), (D1), and (D31) heavy chain sequences. Heavy chain (HC) sequence analysis of 63 randomly selected mAbs from the PV autoantibody repertoire identified 12 different VDJ families (indicated as VDJ1–VDJ12). Each VDJ family shares a common B cell precursor, defined by a common CDR3 amino acid sequence. The presence of a letter suffix in the heavy chain indicates the presence of somatic mutations in the variable region outside the CDR3, which reflect genetic diversification of the original parental B cell clone. The VH gene family and gene segment usage is indicated for each of the 31 unique heavy chain sequences. (B) Unique pemphigus mAbs show restricted and nonoverlapping usage of heavy and light chain gene segments. The horizontal axis represents unique heavy chains, and the vertical axis represents the unique λ and κ light chains (based on nucleic acid sequence). The heavy chains are restricted into 12 VDJ groupings, whereas the light chain repertoire comprises 30 groupings, defined by a unique light chain junctional region. Of the 26, 24, and 13 randomly screened D3, D1, and D31 mAbs, 16, 22, and 5 unique heavy and light chain combinations, respectively, were identified (represented within the matrix as magenta, blue, and black boxes, respectively). In most cases, antibodies panned against a particular desmoglein bound only that desmoglein substrate. However, 2 mAbs, (D3)3a/9 and (D3)3c/9, although panned only against Dsg3, also weakly bound Dsg1 (indicated by asterisks).