Pathogenic Anti-Desmoglein 3 mAbs Cloned from a Paraneoplastic Pemphigus Patient by Phage Display Marwah A. Saleh, Ken Ishii, Jun Yamagami, Yuji Shirakata,

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Pathogenic Anti-Desmoglein 3 mAbs Cloned from a Paraneoplastic Pemphigus Patient by Phage Display Marwah A. Saleh, Ken Ishii, Jun Yamagami, Yuji Shirakata, Koji Hashimoto, Masayuki Amagai Journal of Investigative Dermatology Volume 132, Issue 4, Pages 1141-1148 (April 2012) DOI: 10.1038/jid.2011.449 Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Immunological analysis of anti-desmoglein 3 (Dsg3) mAbs isolated from the paraneoplastic pemphigus (PNP) patient. Indirect immunofluorescence of anti-Dsg3 single-chain variable fragment (scFv) clones in human skin. (a) PNP-A1, (b) PNP-B1, and (c) PNP-C1showed cell surface staining of the keratinocytes, whereas clone (d) PNP-D1 was negative. (e) An scFv isolated from a pemphigus vulgaris (PV) patient (D31)2/28 was used as a positive control. (f) Pretreatment of human skin cryosections with EDTA prevented the cell binding of PNP-C1 to keratinocytes. Bar=100μm. Journal of Investigative Dermatology 2012 132, 1141-1148DOI: (10.1038/jid.2011.449) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 The isolated antibodies bound to the EC2 and EC3 domains of desmoglein 3 (Dsg3) and accounted for some of the polyclonal antibodies in the paraneoplastic pemphigus (PNP) patient (pt.). (a) Schematic diagram for the binding of the anti-Dsg3 antibodies in PNP and pemphigus vulgaris (PV) patients. (b) Epitope mapping of the patient's serum and the isolated single-chain variable fragments (scFvs). Immunoprecipitation/immunoblot analysis of PV, PNP patients’ sera, the isolated scFvs, and anti-E-tag antibody (positive control) was performed using recombinant human Dsg3/Dsg2 domain-swapped molecules. The PNP patient's Dsg3 epitopes were EC1 (weak), EC2, EC3, and EC4. In classic PV patients, the epitopes are mainly EC1 and EC2. (c) Competition ELISA of the four mAbs isolated from the PNP patient's serum in a human Dsg3-coated ELISA plate. Data are shown as the mean±SD. Journal of Investigative Dermatology 2012 132, 1141-1148DOI: (10.1038/jid.2011.449) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Anti-desmoglein 3 (Dsg3) single-chain variable fragments (scFvs) caused the dissociation of cultured human epidermal keratinocytes. Cultured human keratinocytes were incubated with exfoliative toxin A (ETA) with or without scFvs or a mixture of the scFvs at a concentration of 1μgml–1 and then treated with dispase to release cell monolayers. Released cell sheets were exposed to mechanical shear stress to evaluate intercellular adhesion. The dissociation index was calculated from the number of cell sheet fragments (N) for each treatment using the following formula: ((N with mAb–N with ETA only)/(N with AK23–N with ETA only)) × 100. Data are shown as the mean±SD. Top panel shows the photos of the wells. PNP, paraneoplastic pemphigus. Journal of Investigative Dermatology 2012 132, 1141-1148DOI: (10.1038/jid.2011.449) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Pathogenicity assay (injection of single-chain variable fragments (scFvs) into organ cultured human skin). Injection of PNP-C1 (paraneoplastic pemphigus, group C, clone number 1) with low-dose exfoliative toxin A (ETA) caused suprabasal acantholysis. PNP-A1 and PNP-B1 caused focal acantholysis. An scFv isolated from a pemphigus vulgaris (PV) patient (D31)2/28 was used as a positive control. We did not add ETA to the positive control because the positive control inhibits both desmoglein (Dsg)3 and Dsg1 (Payne et al., 2005). Calculation of the degree of acantholysis based on the organ culture acantholysis index (OCAI). Hematoxylin and eosin staining was performed, and the OCAI was calculated after the injection of 150μl of 1μgμl–1 scFv. Data are shown as the mean±SD. Bar=50μm. Journal of Investigative Dermatology 2012 132, 1141-1148DOI: (10.1038/jid.2011.449) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Clinical, histological, and immunofluorescence photographs of the paraneoplastic pemphigus (PNP) patient from whom the phage antibody library was constructed. (a) The patient suffered from severe mucosal and skin erosions. (b) Hematoxylin and eosin staining of the patient's skin biopsy showed suprabasal blistering and vacuolar degeneration. (c) Direct immunofluorescence (DIF) of the patient's skin showed IgG deposition on the surfaces of keratinocytes. (d) Indirect immunofluorescence of the patient's IgG-stained rat bladder transitional epithelium. Bars=100μm. Journal of Investigative Dermatology 2012 132, 1141-1148DOI: (10.1038/jid.2011.449) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions