Autoantibodies against structural proteins of the basement membrane zone (BMZ) cause dermal-epidermal adhesion loss in pemphigoid diseases. Biopsies usually show IgG and C3 at the BMZ, indicating complement (C) activation that facilitates attraction and activation of inflammatory cells and blister formation. We generated 23 anti BP180 antibody (ab) clones from two bullous pemphigoid (BP) patients by phage display. All ab clones were validated by ELISA and immunofluorescence (IF). Unexpectedly, our monovalent scFv abs displaced patient IgG from immobilized antigen in vitro. When injected into human skin organ culture, anti BP180 scFvs were non pathogenic, because for pathogenicity bivalent abs with cross linking and C fixing properties are required. As a proof of concept we exploited this non pathogenicity by designing proteins that allow targeted C inhibition at the BMZ: An anti BP180 scFv was fused with a C1s inhibiting domain and tested for targeted inhibition of the classical C activation pathway at the BMZ, in an ex vivo assay. Our recombinant molecule bound to normal human skin sections preincubated with BP sera and efficiently inhibited C3 fixation and C5a anaphylatoxin liberation. Another fusion protein designed for targeted inhibition of the alternative pathway was not effective in our model, illustrating the dependence on the classical C activation pathway in human BP. Because BP180 is expressed at the BMZ of skin, mucous membranes, and the retina, this innovative approach may be translated to various C dependent diseases affecting these tissues, e.g., mucous membrane pemphigoid, epidermolysis bullosa acquisita, or age related macular degeneration. Alternatively, the compound domain may be exchanged for other pharmaceutically active drugs, allowing for targeted delivery to the BMZ in a broad range of other diseases of the skin and mucous membranes.

ABSTRACT Despite the central role of IFNγ in vitiligo pathogenesis, systemic IFNγ neutralization is an impractical treatment option due to strong immunosuppression. However, most vitiligo patients present with less than 20% affected body surface area, which provides an opportunity for localized treatments that avoid systemic side effects. After identifying keratinocytes as key cells that amplify IFNγ signaling during vitiligo, we hypothesized that tethering an IFNγ neutralizing antibody to keratinocytes would limit anti-IFNγ effects to the treated skin for the localized treatment. To that end, we developed a bispecific antibody (BsAb) capable of blocking IFNγ signaling while binding to desmoglein expressed by keratinocytes. We characterized the effect of the BsAb in vitro , ex vivo , and in a mouse model of vitiligo. SPECT/CT biodistribution and serum assays after local footpad injection revealed that the BsAb had improved skin retention, faster elimination from the blood, and less systemic IFNγ inhibition than the non-tethered version. Furthermore, the BsAb conferred localized protection almost exclusively to the treated footpad during vitiligo that was not possible by local injection of the non-tethered anti-IFNγ antibody. Thus, keratinocyte-tethering proved effective while significantly diminishing off-tissue effects of IFNγ blockade, offering a new treatment strategy for localized skin diseases, including vitiligo.

Anti-desmoglein (Dsg) 1 and Dsg3 IgG autoantibodies in pemphigus foliaceus and pemphigus vulgaris cause blisters through loss of desmosomal adhesion. It is controversial whether blister formation is due to direct inhibition of Dsg, intracellular signaling events causing desmosome destabilization, or both. Recent studies show that heterophilic binding between Dsg and desmocollin (Dsc) is the fundamental adhesive unit of desmosomes. To eliminate cellular contributions to potential pathogenicity of pemphigus antibodies, bead assays coated with recombinant Dsg1, Dsc1, Dsg3, or Dsc3 ectodomains were developed. A mixture of Dsg beads and Dsc beads formed large aggregates, confirming that the heterophilic binding is dominant. The pathogenic anti-Dsg1 and anti-Dsg3 mAbs, which bind the transadhesive interface, blocked the aggregation of Dsg1/Dsc1 and Dsg3/Dsc3 beads, respectively, whereas nonpathogenic mAbs did not. All sera tested from eight patients with pemphigus foliaceus and eight patients with mucosal pemphigus vulgaris with active disease inhibited the adhesion of Dsg1/Dsc1 and Dsg3/Dsc3 beads, respectively. When paired sera obtained from seven patients with pemphigus foliaceus and six patients with pemphigus vulgaris in active disease and remission were compared, the former inhibited aggregation better than the latter. These findings strongly suggest that steric hindrance of heterophilic transinteraction between Dsg and Dsc is important for disease pathology in both pemphigus foliaceus and pemphigus vulgaris.

For many years, The Journal of Investigative Dermatology (JID) has been a leader in our understanding of many aspects of the major autoimmune blistering skin diseases, pemphigus and bullous pemphigoid. The purpose of this review is to highlight and summarize those advances by discussing the respective articles, published in the JID from 2015 to 2019. Seminal articles from elsewhere in the literature that set the stage for those advances, or that are "classics" in the area, are also included to provide context and a more complete picture.

Bullous pemphigoid (BP) and mucous membrane pemphigoid (MMP) are blistering skin diseases in which autoantibodies against basal keratinocyte antigens cause loss of cell adhesion to the basement-membrane zone (BMZ). Biopsies usually show BMZ-bound IgG and C3 in direct immunofluorescence (IF), indicating complement activation that results in attraction and activation of inflammatory cells and subsequently dermal-epidermal separation. We cloned more than 30 anti-BP180/BP230 single chain variable fragment (scFv) monoclonal antibodies (mAbs) from two active BP patients. All mAbs were validated by BP180-NC16A/BP230 ELISA and indirect IF. Unexpectedly, our scFvs displaced bound patient IgG from the immobilized antigen in vitro. Confirming previous data, monovalent anti-BP180-scFvs tested were non-pathogenic when injected into human skin organ culture, because for pathogenicity bivalent IgG abs with crosslinking and complement-binding properties are required. We exploited this non-pathogenicity by designing proteins that allow targeted complement inhibition at the BMZ; fusing an anti-BP180-NC16A scFv with a C1s-inhibiting gigastasin domain we were able to test for targeted inhibition of the classical complement activation pathway at the BMZ, in an BP ex vivo model. This recombinant molecule bound to normal human skin sections pre-incubated with BP sera and inhibited C3 fixation as shown by IF microscopy. This finding was independently confirmed by decreased C5a levels, as detected by ELISA. Another fusion protein designed for inhibition of the alternative pathway at the BMZ was not effective in our model, illustrating the dependence on the classical complement activation pathway in BP. Because BP180-NC16A is expressed at the BMZ of skin, mucous membranes and the retina, this innovative approach could be translated to other complement-dependent diseases affecting these tissues.

BACKGROUND:Bullous pemphigoid (BP) is the most common autoimmune subepidermal blistering skin disease. Two antigens have been identified as targets of circulating autoantibodies (autoAbs) - BP180 and BP230 - with BP180 being a critical transmembrane adhesion protein of basal keratinocytes of the epidermis. The noncollagenous domain 16A (NC16A) of BP180 is the immunodominant epitope in patients with BP, and anti-BP180-NC16A IgG antibodies (Abs) correlate to disease activity. Routine serological testing and follow-up of BP relies on indirect immunofluorescence (IIF) of serum Abs, commonly performed on monkey oesophagus (ME), and/or enzyme-linked immunosorbent assay (ELISA) testing on recombinantly produced fragments of BP180 and BP230 (BP180-NC16A, BP230-C/N).OBJECTIVES:To determine if NC16A epitopes are well represented on ME substrate.METHODS:Sera from different BP cohorts were tested by IIF on ME and normal human skin (NHS). To confirm findings, affinity-purified anti-BP180-NC16A/BP230 polyclonal Abs and recombinant anti-BP180-NC16A/BP230 monoclonal antibodies (mAbs) were used.RESULTS:For sensitive detection of BP180-NC16A-specific IgG Abs, sections of NHS are superior to the widely used ME. Confirmation comes from polyclonal affinity-purified anti-BP180-NC16A/BP230 Abs, and by mAbs cloned from a patient with active BP.CONCLUSIONS:Use of NHS is preferable over ME in routine IIF testing for BP. These results are of clinical relevance because anti-BP180-NC16A IgG titres are correlated to disease activity and detecting them reliably is important for screening, diagnosis and follow-up of patients with BP.

bullous pemphigoid (BP) is an autoimmune blistering skin disease that predominantly occurs in the elderly, often accompanied by neurological disorders and increased mortality. Diagnosis of BP is established by clinical presentation, typical histology with subepidermal blisters and an inflammatory infiltrate containing eosinophils, positive direct immunofluorescence (DIF) microscopy of perilesional skin and serological assays. Almost all patients show linear basement membrane zone (BMZ) deposits of complement component 3 (C3) and IgG in DIF microscopy, implying a critical role of complement for disease. In this regard, formation of the terminal membrane attack complex of complement can be observed in BP patients' epidermal basal cells, potentially resulting in direct cytotoxic effects, and the complement-activating capacity of BP autoantibodies ex vivo has been shown to be correlated to disease activity. This article is protected by copyright. All rights reserved.

Stephen I. Katz, MD, PhD (Figure 1), died suddenly on December 20, 2018. It is a testament to his humble nature that such a giant in our field was universally known simply as Steve or Steve Katz. His influence as a physician-scientist in dermatology and beyond cannot be overstated.

Anti-desmoglein (Dsg) 3 IgG autoantibodies in pemphigus vulgaris (PV) cause blisters through loss of desmosome adhesion. The Dsg-desmocollin (Dsc) interaction has been shown to be the fundamental adhesive unit of desmosomes. Whether PV antibodies (Ab) require cellular signaling to cause pathology is controversial. To completely eliminate all cellular contributions to potential pathogenicity of PV Abs, we developed a bead assay coated with recombinant Dsg3 and Dsc3 ectodomains produced by a mammalian expression system. Beads coated with Dsg3 or Dsc3 alone showed minimal aggregation, while a mixture of Dsg3 and Dsc3 beads formed large aggregates, confirming that the heterophilic binding of Dsg3 and Dsc3 is dominant over homophilic adhesion. To determine if the heterophilic bead assay can detect the pathogenicity of anti-Dsg3 Abs, we first tested anti-Dsg3 mouse monoclonal Abs. The pathogenic AK23 which binds the trans-adhesive interface of Dsg3 blocked the aggregation of Dsg3/Dsc3 beads in a dose-dependent manner. On the other hand, the non-pathogenic AK15 did not inhibit aggregation. These findings indicated that the bead assay is quantitative for a pathogenic Ab and can discriminate it from a non-pathogenic Ab. We then tested sera from 8 mucosal PV patients with active disease. All inhibited the aggregation of Dsg3/Dsc3 beads. Sera from normals did not. Paired sera obtained from 6 PV patients with active disease vs. remission were then tested. Inhibition rate calculated by the size of aggregation (%inhibition= [1-size(X)/size(normal)] X100) correlated well with the disease activity. (Case1, 90% vs 17%; Case2, 76% vs -33%; Case3, 21% vs -5%; Case4, 55% vs -8%; Case5, 32% vs 3%; Case6, 84% vs 30%) These results show that PV autoantibodies cause loss of Dsg3/Dsc3 adhesion by steric hindrance without cellular signaling or other cellular contribution. Furthermore, this bead assay could be used to assess pathogenic strength of PV sera.

Pemphigus foliaceus (PF) is an autoimmune blistering disease caused by autoantibodies against desmoglein 1 (Dsg1). It is controversial whether blister formation is due to direct inhibition of Dsg interaction or intracellular signaling events that cause desmosome destabilization or both. A recent study showed that heterophilic binding of Dsg and desmocollin (Dsc) is important for adhesive unit of desmosomes. Here we analyzed that PF autoantibodies can block the trans-interaction of Dsg1 and Dsc1. Ectodomains of Dsg1 and Dsc1 were produced by a mammalian expression system and then coated on microbeads. Beads coated with Dsg1 alone or Dsc1 alone showed minimal aggregation while a mixture of Dsg1 beads and Dsc1 beads formed large aggregates after 30 min incubation, confirming that the heterophilic binding of Dsg1 and Dsc1 is dominant. The aggregation required a mature form of Dsg1 and Dsc1, consistent with adhesion being a property only of mature, not proprotein. To determine if PF Abs can directly block Dsg/Dsc adhesion, we tested anti-Dsg1 mAbs isolated from a PF patient by phage display. A pathogenic anti-Dsg1 IgG or scFv mAb which binds putative trans-adhesive interface of Dsg1 could block the aggregation of Dsg1 and Dsc1 beads. On the other hand, non-pathogenic Dsg1 IgG or scFv mAbs did not inhibit aggregation. These findings indicated that this in vitro aggregation bead assay can discriminate pathogenic vs. non-pathogenic PF Abs. Furthermore, sera from 8 PF patients with active disease all inhibited the aggregation of Dsg1 and Dsc1 beads. When paired sera obtained from 3 PF patients in active stage and in remission were compared, the former inhibited aggregation much better than the latter, as determined by aggregate size. These findings show direct evidence that PF autoantibodies cause steric hindrance of the heterophilic trans-interaction of Dsg and Dsc that provide adhesion to desmosomes. Furthermore, this assay will be a valuable and simple tool to assess pathogenic strength of PF autoantibodies.

Identifying previously unknown proteins or detecting the presence of known proteins in research samples is critical to many experiments conducted in life sciences, including dermatology. Sensitive protein detection can help elucidate new intervention targets and mechanisms of disease, such as in autoimmune blistering skin diseases, atopic eczema, or other conditions. Historically, peptides from highly purified single proteins were sequenced, with many limitations, by stepwise degradation from the N-terminus to the C-terminus with subsequent identification by UV absorbance spectroscopy of the released amino acids (i.e., Edman degradation). Recently, however, the availability of comprehensive protein databases from different species (derived from high-throughput next-generation sequencing of those organisms' genomes) and sophisticated bioinformatics analysis tools have facilitated the development and use of mass spectrometry for identification and global analysis of proteins, summarized as mass spectrometry-based proteomics. Mass spectrometry is an analytical technique measuring the mass (m)-to-charge (z) ratio of ionized biological molecules such as peptides. Proteins can be identified by correlating peptide-derived experimental mass spectrometry spectra with theoretical spectra predicted from protein databases. Here we briefly describe how this technique works, how it can be used for identification of proteins, and how this knowledge can be applied in elucidating human biology and disease.

In autoantibody-mediated diseases such as pemphigus, serum antibodies lead to disease. Genetic analysis of B cells has allowed characterization of antibody repertoires in such diseases but would be complemented by proteomic analysis of serum autoantibodies. Here, we show using proteomic analysis that the serum autoantibody repertoire in pemphigus is much more polyclonal than that found by genetic studies of B cells. In addition, many B cells encode pemphigus autoantibodies that are not secreted into the serum. Heavy chain variable gene usage of serum autoantibodies is not shared among patients, implying targeting of the coded proteins will not be a useful therapeutic strategy. Analysis of autoantibodies in individual patients over several years indicates that many antibody clones persist but the proportion of each changes. These studies indicate a dynamic and diverse autoantibody response not revealed by genetic studies and explain why similar overall autoantibody titers may give variable disease activity.

Desmoglein (Dsg) 3 is overexpressed in oral squamous cell carcinoma (OSCC). We hypothesized that Dsg3 could be used for targeted therapy and developed a method of targeting an antitumor agent, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), to the OSCC by fusing it with Px44, a non-pathogenic anti-Dsg3 single chain variable fragment (scFv) antibody cloned from a pemphigus patient. TRAIL has been shown to cause apoptosis of rapidly proliferating tumor cells, and we previously showed that Px44TRAIL had stable proapoptotic activity on Jurkat lymphoma cells even after incubation for 48 h at 37 °C and that it did not have toxic effects on normal skin. In this study, we tested the activity of Px44TRAIL on human OSCC cell lines derived from primary tumor and metastatic lymph nodes of 6 OSCC patients. Real time qPCR showed that 11 of the 12 cell lines expressed Dsg3 mRNA. Cells from the metastatic lymph nodes of each patient expressed more Dsg3 than those from the primary tumor of the same patient. Immunofluorescence (IF) detected Px 44TRAIL binding on the cell surface of the 11 cell lines expressing Dsg3, but not on the Dsg3-negative line. TRAIL alone caused apoptosis in 6 of these lines over 16 h, whereas Px44TRAIL targeted for only 2 h (then washed) caused apoptosis of up to 50% of cells in 9 of the 12 cell lines at 16 h. AM3-13TRAIL (in which AM3-13 is an irrelevant scFv) did not bind on the cells or cause apoptosis. Finally, when injected into human SCC organ culture, IF detected Px44TRAIL binding on the invading tumor cells. Our data show the feasibility of targeting TRAIL to SCC, and suggest such therapy may be useful for oral cancer treatment.

Polyclonal anti-desmoglein1 (Dsg1) autoantibodies in pemphigus foliaceus (PF) patients’ sera consist of pathogenic (P) and non-pathogenic (NP) antibodies. We previously showed that injection of a mixture of P and NP anti-Dsg1 IgG monoclonal Abs (mAbs) induced aberrant Dsg1 distribution, Dsg1 clustering, in organ cultured human skin, while a single P mAb did not. In the meanwhile, it has been shown that p38 mitogen-activated protein kinase (MAPK) has a key role for blistering through endocytosis and clustering of Dsg3 in pemphigus vulgaris. We aimed to determine to what extent p38MAPK signaling was involved in this Dsg1 clustering and blister formation in PF. Effects of a single mAb and a mixture of mAbs were compared for the subcellular distribution of Dsg1 and the pathogenic activity in human skin organ culture and in primary keratinocytes. In organ culture skin, inhibition of p38MAPK blocked Dsg1 clustering but did not prevent superficial blisters induced by a mixture of P and NP IgG mAbs. In comparison, a single P IgG mAb induced blisters, not blocked by the inhibitor, without Dsg1 clustering. By using primary keratinocytes, we confirmed that a mixture of P and NP IgG mAbs caused Dsg1 clustering while a single P or NP mAb showed linear staining along cell borders. In addition, we also confirmed that the p38 MAPK inhibitor blocked Dsg1 clustering induced by a mixture of P and NP mAbs. When the pathogenic activity was measured by in vitro dissociation assay, a mixture of P and NP mAbs disrupted keratinocytes adhesion more than the single P mAb. This pathogenic effect was only partially suppressed by the p38MAPK inhibitor. These findings indicate that p38MAPK enhances the pathogenic effect along with Dsg1 clustering but not required for blister formation in PF.

The purpose of this study was to compare QOL in patients with AIBD with other diseases to determine which variables are associated with worse QOL. This was a cross-sectional, questionnaire-based study conducted at the University of Pennsylvania. Subjects had a diagnosis of pemphigus vulgaris (PV, n=30), pemphigus foliaceus (PF, n=11), or bullous pemphigoid (BP, n=9). Each participant was asked to complete a set of questionnaires, including the SF-36 and the Skindex-29. There were 24 (48%) men and 27 (54%) women, predominantly Caucasian (76%). The mean disease severity measured by the PDAI and BPDAI was 5.1 and 15.2, respectively. Of the Skindex-29 subscales, patients with AIBD were most affected by the symptoms and emotions domains with a mean (SD) of 33.3 (26.1) and 31.8 (29.7). They were least affected by the functioning domain [21.4 (27.1)]. Within the symptoms domain, patients were most bothered by their skin being sensitive [44.9(37.8)], itchy [40.3 (32.9)], and irritated [39.6 (33.4)]. Within the emotions domain, patients worried that their skin condition may be serious [45.6 (38.3)] and could get worse [42.9 (37.8)]. Skindex-29 scores in AIBD were compared with those in 11 other skin conditions and to those in patients without skin disease. AIBD patients had significantly worse scores in all three domains compared to those without skin disease (all P < .05). Even with mild disease activity, patients with AIBD had similar QOL burden compared to other skin diseases. SF-36 scores were calculated and AIBD patients had significantly worse RP (how physical health affects work) and RE (how emotional health affects work) compared to those with CHF, depression, recent MI, hypertension, type 2 DM, and the general population (P < .01). These results indicate that even with mild disease, patients with AIBD still exhibit a significantly poor quality of life, especialy as it relates to emotions and symptoms.

Pemphigus foliaceus (PF) is an autoimmune blistering disease caused by anti-desmoglein1 (Dsg1) IgG antibodies (Abs). Pathogenic and non-pathogenic anti-Dsg1 monoclonal Abs (mAbs) were previously isolated as single chain variable fragments (scFvs) by phage display. However, it is unknown how each anti-Dsg1 mAb works for blister formation under polyclonal condition in vivo. In this study, we focused on the difference of a single mAb and a combination of mAbs on blister formation in the aspect of the Dsg1 distribution, desmosomal structure by using organ culture human skin injection assay. At first we created IgG form antibodies from scFv to reflect patient serum condition. A single pathogenic anti-Dsg1 IgG mAb caused subcorneal blister after 22 h injection and immunofluorescence showed IgG mAb deposited and Dsg1 localized linearly at the cell surfaces of keratinocytes. When a pathogenic and a non-pathogenic anti-Dsg1 mAbs were injected together, IgG deposits and Dsg1 localization showed an aberrant granular pattern at basal and spinous layers. This Dsg 1 clustering needed cross-linking of Dsg1 and trans-interaction blocking by a pathogenic IgG Ab. Electron microscopy revealed that a combination of pathogenic and non-pathogenic anti-Dsg1 mAbs shortened desmosome length more than a single mAb at the basal and spinous layers. In addition, dissociation assay showed a combination of pathogenic and non-pathogenic mAbs inhibited cell adhesion of keratinocytes more than a single pathogenic mAb. These findings indicated that a combination of PF mAbs induced Dsg1 clustering which may cause desmosome morphology change and promote loss of cell adhesion of keratinocytes synergistically. In conclusion, polyclonal condition of the anti-Dsg 1 Ab influences the pathogenicity in PF.