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 1

Options: View larger image (or click on image) Download as PowerPoint
Isolation of monovalent scFv mAbs. (A) ScFv nomenclature. (D3), (D1), or...
Isolation of monovalent scFv mAbs. (A) ScFv nomenclature. (D3), (D1), or (D31) indicates the antigens used to select anti-Dsg mAbs from the scFv phage display library (Dsg3, Dsg1, or both, respectively). This is followed by a unique heavy chain and light chain nucleotide sequence designation. For mAbs that share the same clonal origin (same VDJ rearrangement) but differ in sequence because of somatic hypermutation, lower-case letters indicate unique members of a given clone (see Discussion and legend to Figure 9). (B) Soluble scFv mAbs were purified by nickel-chelation chromatography (see Methods). Two representative scFvs are shown by Coomassie blue staining after SDS-PAGE. (C) Gel-filtration HPLC demonstrates that scFvs are primarily monomeric in solution. Transferrin (Tf, 75 kDa) and carbonic anhydrase (CA, 30 kDa) served as protein standards, with 0.1 M Mops as a marker of the total volume of the column. The percentage of monomeric protein is shown, calculated from the area under the curve.