Update on the cloning of monoclonal anti-desmoglein antibodies from human pemphigus patients: implications for targeted therapy.
Autoantibodies in pemphigus foliaceus (PF) and vulgaris (PV) bind to desmoglein (Dsg) 1 and 3, respectively, and cause loss of keratinocyte adhesion. To characterize the pathogenicity and genetics of such antibodies we have used phage display to isolate monoclonal antibodies (mAbs) from patients. PCR is used to clone the heavy and light chain variable region of the peripheral B cells into a vector that creates a phage particle with the antibody expressed on its surface and the cDNA encoding that antibody inside. The library of phage produced from a PF or PV patient are then panned on a plate containing Dsg1 or Dsg3 to isolate clones. The cDNA of each clone is sequenced to characterize the genetics of the expressed mAb. The mAb from each unique clone is tested for pathogenicity either by injecting into normal human skin organ culture or into neonatal mice. Pathogenic antibodies cause typical pemphigus blisters. In both PV and PF patients the heavy chain (VH) genes used for Dsg-binding antibodies are severely restricted. PV and PF patients have both pathogenic and non-pathogenic mAbs. The immunochemical characteristics of the antibodies (including pathogenicity) sort with the VH, not the VL, gene. These monoclonal pathogenic antibodies can be used to screen peptide libraries to find short peptides that block antibody binding. In summary, the antibody response is restricted and, therefore, it may be feasible to target the specific pathogenic antibodies for therapy.
Stanley
John R
JR
University of Pennsylvania School of Medicine, Philadelphia, PA, USA. jrstan@mail.med.upenn.edu
Ishii
Ken
K
Siegel
Don L
DL
Payne
Aimee S
AS
D000911:Antibodies, Monoclonal / Q000276:immunology*; D003001:Cloning, Molecular / Q000379:methods*; D051182:Desmogleins / Q000378:metabolism; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000378:metabolism; D007135:Immunoglobulin Variable Region; D010392:Pemphigus / Q000276:immunology*; D019151:Peptide Library; D011485:Protein Binding
D000911:Antibodies, Monoclonal; D051182:Desmogleins; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library