title: Anti-collagen XVII single-chain Fv antibody blocks the autoimmune reaction mediated by pathogenic autoantibodies in bullous pemphigoid.
abstract: BACKGROUND
  Pathogenic autoantibodies in bullous pemphigoid (BP) recognize the non-collagenous 16A domain (NC16A) of collagen XVII (COL17), a hemidesmosomal component at the skin membrane. This immune inflammation involves activation of the complement cascade via the classical pathway. With similar antigen binding activity, Fab and single-chain variable fragments (scFv) of pathogenic anti-COL17 antibodies can interfere with COL17 binding of autoantibodies, blocking subsequent complement activation and granulocyte activation.
  
  
  OBJECTIVE
  To characterize the biological functions of human anti-COL17 scFv antibody.
  
  
  METHODS
  We constructed scFv antibodies against the corresponding antigen from parental Fab by expression in Escherichia coli. IgG autoantibodies against COL17 were purified by affinity chromatography from serum of BP patients. The inhibitory effects of anti-COL17 scFv on binding of BP autoantibodies to the NC16A domain of human COL17 antigen were observed by inhibition ELISA, immunofluorescence, and inhibition of complement activation. Reactive oxygen production assay and BP cryosection model were performed to assess the inhibitory effect of scFv on granulocyte activation and then the dermal-epidermal separation.
  
  
  RESULTS
  ELISA and Western blot showed specific binding of scFv to COL17. We found that anti-COL17 scFv can inhibit the binding of intact IgG purified from BP parents to the corresponding COL17 antigen and then subsequent C1q and C3 activation and granulocyte activation in vitro. Most importantly, we confirmed that recombinant scFv can inhibit BP-IgG induced dermal-epidermal separation by BP cryosection model.
  
  
  CONCLUSIONS
  The anti-COL17 scFv antibody can inhibit the binding of BP-IgG autoantibodies to COL17, thereby affecting subsequent complement activation and granulocyte activation in vitro. Our results suggest that blocking pathogenic epitopes using engineered scFv is an efficient BP therapy.
authors.affiliation: Department of Dermatology, Xijing Hospital, Fourth Military Medical University, 127 Changlexi Road, Xi'an 710032, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wu
authors.forename: Na-Na
authors.initials: NN
authors.lastname: Sun
authors.forename: Er-Le
authors.initials: EL
authors.lastname: Dang
authors.forename: Liang
authors.initials: L
authors.lastname: Jin
authors.forename: Zhen-Feng
authors.initials: ZF
authors.lastname: Liu
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Lu-Ting
authors.initials: LT
authors.lastname: Yang
authors.forename: Gang
authors.initials: G
authors.lastname: Wang
mesh_terms: D019138:Antibodies, Blocking / Q000008:administration & dosage* / Q000235:genetics; D000937:Antigen-Antibody Reactions; D001323:Autoantibodies / Q000276:immunology; D001324:Autoantigens / Q000276:immunology*; D015551:Autoimmunity; D003167:Complement Activation; D006098:Granulocytes / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007167:Immunotherapy; D024041:Non-Fibrillar Collagens / Q000037:antagonists & inhibitors* / Q000276:immunology*; D010391:Pemphigoid, Bullous / Q000276:immunology* / Q000628:therapy*; D015202:Protein Engineering; D057127:Single-Chain Antibodies / Q000008:administration & dosage* / Q000235:genetics; D000097805:Collagen Type XVII
chemical_list: D019138:Antibodies, Blocking; D001323:Autoantibodies; D001324:Autoantigens; D007074:Immunoglobulin G; D024041:Non-Fibrillar Collagens; D057127:Single-Chain Antibodies; D000097805:Collagen Type XVII
keywords: Autoantibodies; Bullous pemphigoid; Complement; Single-chain Fv; Therapy