Bullous pemphigoid induced by IgG targeting type XVII collagen non‐NC16A/NC15A extracellular domains is driven by Fc gamma receptor‐ and complement‐mediated effector mechanisms and is ameliorated by neonatal Fc receptor blockade

Bullous pemphigoid (BP) is an autoimmune blistering disease characterized by autoantibodies targeting type XVII collagen (Col17) with the noncollagenous 16A (NC16A) ectodomain representing the immunodominant site. The role of additional extracellular targets of Col17 outside NC16A has not been unequivocally demonstrated. In this study, we showed that Col17 ectodomain‐reactive patient sera depleted in NC16A IgG induced dermal–epidermal separation in a cryosection model indicating the pathogenic potential of anti‐Col17 non‐NC16A extracellular IgG. Moreover, injection of IgG targeting the murine Col17 NC14–1 domains (downstream of NC15A, the murine homologue of human NC16A) into C57BL/6J mice resulted in erythematous skin lesions and erosions. Clinical findings were accompanied by IgG/C3 deposits along the basement membrane and subepidermal blistering with inflammatory infiltrates. Disease development was significantly reduced in either Fc‐gamma receptor (FcγR)‐ or complement‐5a receptor‐1 (C5aR1)‐deficient mice. Inhibition of the neonatal FcR (FcRn), an atypical FcγR regulating IgG homeostasis, with the murine Fc fragment IgG2c‐ABDEG, a derivative of efgartigimod, reduced anti‐NC14–1 IgG levels, resulting in ameliorated skin inflammation compared with isotype‐treated controls. These data demonstrate that the pathogenic effects of IgG targeting the Col17 domain outside human NC16A/murine NC15A are partly attributable to antibody‐mediated FcγR‐ and C5aR1 effector mechanisms while pharmacological inhibition of the FcRn represents a promising treatment for BP. The mouse model of BP will be instrumental in further investigating the role of Col17 non‐NC16A/NC15A extracellular epitopes and validating new therapies for this disease. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.


Introduction
Bullous pemphigoid (BP), the most common autoimmune blistering disease, presents with tense blisters on normal and/or erythematosus skin and intense pruritus [1].Mucosal involvement occurs in a minority of patients.The immunopathological characteristics of BP patients' skin include IgG and/or C3 deposition at the dermal-epidermal junction, subepidermal cleavage, and a variable inflammatory cell infiltrate [2].Pathogenic autoantibodies are directed against type XVII collagen (Col17), also known as BP antigen 180 (BP180).
In addition, autoantibody responses targeting BP230 (BPAG1e) have also been implicated in BP etiology [3][4][5].Col17 is a 180 kDa homotrimeric type II transmembrane glycoprotein and a structural component of the hemidesmosomes that mediates adhesion of basal keratinocytes to the underlying basement membrane zone (BMZ) in epithelial tissues [6,7].It is composed of three alpha 1 chains, each consisting of an intracellular N-terminal domain, a short transmembrane domain, and an extracellular domain with 15 collagenous repeats flanked by 16 noncollagenous (NC) segments [6].The extracellular stretch of the 16th NC domain (NC16A), which is adjacent to the cellular membrane, represents the immunodominant region in BP patients; 80-90% of BP sera contain anti-NC16A IgG [8].In addition, antibodies against epitopes of the intracellular domain and ectodomain outside of the NC16A domain have been detected in BP sera [9][10][11][12][13][14][15][16][17].Anti-Col17 antibodies targeting intra-and/or extracellular domains were also identified in other pemphigoid diseases, including mucous membrane pemphigoid (MMP), linear IgA dermatosis, and pemphigoid gestationis [1].The pathogenic relevance of anti-Col17 NC16A autoantibodies was proposed based on the correlation of their serum levels with disease activity in BP patients [18,19] and has irrefutably been shown in various experimental models [20][21][22][23][24][25][26][27][28][29][30][31][32].In contrast, the role of autoantibodies targeting additional extracellular epitopes of Col17 downstream of NC16A and the murine homolog NC15A (UniProt ID Q07563, isoform 1) has not yet been explicitly proven.In this study, we investigated the pathogenicity of antibodies targeting the human Col17 extracellular domains outside NC16A using immunoaffinity-purified patient IgG against the Col17 ectodomain ex vivo as well as in vivo using rabbit IgG directed against a fusion peptide consisting of the NC14-1 domains of mouse Col17.

Ethics approvals
Studies using clinical samples were approved by the ethics committee of the University of Lübeck  and were conducted according to principle of the Declaration of Helsinki.All animal experiments were approved by the Schleswig-Holstein Ministry of Energy Transition, Agriculture, Environment, Nature and Digitalization (16-3/20, 17-3/20, 16-2/21).

Patient material
Skin samples used for the cryosection model were obtained from patients undergoing breast reduction surgery after informed consent.Sera of patients with BP and healthy donors were collected at the Department of Dermatology, Allergology and Venerology, University of Lübeck in Lübeck, Germany, after informed consent was given.Diagnosis of BP was made according to national and international guidelines [33,34] and was based on a compatible clinical picture, circulating autoantibodies labeling the epidermal side of human salt-split skin and reactivity of IgG and/or C3 along the cutaneous BMZ by direct immunofluorescence microscopy and with NC16A by ELISA (Euroimmun, Lübeck, Germany).
The protein sequence of NC14-1 (supplementary material, Table S1) was transcribed into the corresponding DNA sequence using Geneious Prime software (Geneious, Boston, MA, USA).The NC14-1 cDNA was generated via gene synthesis (Eurofins MWG Operon, Ebersberg, Germany), optimized for E. coli codon usage, and cloned into the pET24d-N expression vector.The NC14-1 fragment was expressed as a histidine-fusion protein in E. coli and purified by affinity chromatography using TALON ® metal affinity resin (TaKaRa, San Jose, CA, USA) according to the manufacturer's instructions.

Additional methods
Immobilization of recombinant proteins, affinity purification of total and antigen-specific IgG, preparation of leukocytes from human blood, cryosection assay for evaluation of dermal-epidermal separation, generation of murine extracellular matrix extracts, SDS-PAGE and immunoblotting, direct and indirect immunofluorescence staining, histological analysis, ELISA for detection of circulating autoantibodies, flow cytometry, and statistical analyses are described in Supplementary materials and methods.

Col17 ectodomain-reactive sera depleted of NC16A IgG induced dermal-epidermal separation in cryosections of human skin
To explore the pathogenic potential of antibodies targeting the Col17 non-NC16A extracellular domains (Figure 1A), we analyzed serum samples obtained from 21 BP patients (supplementary material, Table S2).All patients showed reactivity to Col17 as determined by an anti-NC16A ELISA.Eight of 21 sera (38%) additionally showed BP230 reactivity in an anti-BP230C3 ELISA.We first adsorbed anti-NC16A IgG from all sera and confirmed full depletion of anti-NC16A reactivity using an anti-NC16A ELISA.Subsequently, the pathogenic potential of BP sera before and after depletion of NC16A reactivity was analyzed in an ex vivo model of autoantibody-mediated neutrophil activation and dermal-epidermal separation using cryosections of human skin [39,40].All 21 BP sera induced split formation at the dermal-epidermal junction (Figure 1B), while sera from healthy volunteers did not.Six of the 21 patient sera (29%) depleted of anti-NC16A reactivity lost their split-inducing ability (all without BP230 reactivity).Fifteen of 21 BP sera (71%) remained pathogenic after depletion of NC16A specific antibodies, and the collected fractions depleted of anti-NC16A reactivity still harbored the potential to induce subepidermal splitting (Figure 1C,D).Hence, the pathogenicity in the tested BP sera was not the sole contributor to their anti-NC16A reactivity and indicated that in a substantial number of BP patients, autoantibodies against epitopes outside the immunodominant NC16A domain of Col17 may be of pathogenic relevance.To further support this view, we affinity-purified four of the 21 NC16A-depleted sera (including two sera with BP230 reactivity) against the entire human Col17 ectodomain as well as the C-terminal portion of BP230 (BP230C3).In a cryosection assay, the Col17 ectodomain/BP230C3-depleted sera now failed to induce leukocyte-dependent dermal-epidermal separation in frozen sections of human skin.However, only one of the four ectodomain-specific IgG fractions (depleted of anti-NC16A reactivity) led to subepidermal cleavage (Figure 1E,F), which might be due to the low quantities/ concentrations of the IgG fractions following Col17 ectodomain/BP230C3 IgG depletion.Neither of the two BP230C3 IgG fractions alone showed any split formation.The role of other autoantibodies targeting different Col17 epitopes, including conformational changes within NC16A or neoepitopes generated by shedding of the Col17 ectodomain, or even other autoantigens, cannot be fully excluded here, yet these results indicate that autoantibodies targeting the Col17 extracellular domain excluding NC16A can lead to subepidermal split formation ex vivo.

Anti-murine Col17 extracellular NC14-1 IgG produces tissue damage in vitro and in vivo
To further corroborate our in vitro findings using BP patient sera, we aimed to explore the pathogenic potential of IgG against the Col17 non-NC15A extracellular stretch in a new antibody-transfer mouse model of BP.To ensure a high immunogenicity, we generated a fusion peptide consisting of all 14 noncollagenous domains (NC14-1) of the murine Col17 ectodomain outside NC15A (Figure 2A and supplementary material, Figure S1A) instead of the entire murine Col17 ectodomain.Serum obtained from rabbits after immunization with the NC14-1 antigen as well as affinity-purified rabbit anti-NC14-1 IgG generated against recombinant histidinetagged murine NC14-1 but not IgG from preimmune rabbit sera recognized NC14-1 as well as full-length murine Col17 in extracellular matrix derived from normal murine C5N keratinocytes by immunoblotting and labeled the BMZ (Figure 2B and supplementary material, Figure S1B).In addition, salt-split skin obtained from healthy control mice displayed anti-NC14-1 IgG binding at the epidermal side, whereas anti-murine lamininalpha-3 IgG, which are implicated in MMP [38], bound along the dermal side of murine salt-split skin (Figure 2C).
The in vitro pathogenicity of the anti-NC14-1 IgG was demonstrated using the cryosection assay model, where sections of normal murine tail skin were incubated with either anti-NC14-1 IgG, anti-type VII collagen IgG, or normal rabbit IgG, followed by incubation with freshly harvested leukocytes from healthy human volunteers.Previously, it was demonstrated that rabbit anti-murine type VII collagen IgG induced dermal-epidermal separation in the presence of humanderived granulocytes [41] and was used as positive control in this study.Both anti-NC14-1 IgG and anti-type VII collagen induced subepidermal splitting, while normal rabbit IgG did not (Figure 2D).
Following in vitro validation, anti-NC14-1 IgG was passively transferred to adult C57BL/6J WT mice.Repeated s.c.injection of 10 mg anti-NC14-1 every Pathogenicity of Col17 non-NC16A/NC15A extracellular domain-specific IgG other day (Days 0, 2, 4, 6, 8, and 10) over a period of 12 days led to the development of erythematous lesions, erosions, and crusts, particularly on the head and neck, as well as forepaws, snout, periorbital areas, and base of the ears, recapitulating a BP-like phenotype (Figure 2E,F).Lesions were already present at Day 4, and an increase in the extent and severity of the ABSA was observed up to Day 12 (Figure 2G).Subepidermal cleavage with inflammatory infiltrates in the upper dermis as well as linear IgG and C3 deposits along the BMZ were apparent in lesional and perilesional skin, respectively (Figure 2H,I).
Since anti-Col17 antibodies are associated with mucosal lesions in patients with pemphigoid disease, we also assessed the involvement of mucosal membranes.While IgG binding was detected in various mucosal tissues, including conjunctiva, tongue, and esophagus, neither subepithelial split formation nor signs of inflammation were apparent in anti-NC14-1 IgG-treated mice (supplementary material, Figure S2A-C).In addition, endoscopy showed no signs of oral mucosal involvement (supplementary material, Figure 2D).No changes in weight were observed over the 12-day treatment period.

Disease development was significantly abrogated in FcγR-deficient mice
FcR-mediated effector mechanisms, particularly involving the FcγRs, have been shown to contribute significantly to tissue damage and disease development in pemphigoid diseases [24,29,[42][43][44][45].To investigate the pathogenic importance of the FcγRs in the BP mouse model, anti-NC14-1 IgG was injected repeatedly over 12 days in adult C57BL/6J WT mice and FcγR-deficient mice (FcγR À/À ) [46].At Day 12, FcγR À/À mice showed a significant reduction in the ABSA compared to WT mice (p < 0.0001) (Figure 3A,B).FcγR À/À mice treated with normal rabbit IgG displayed no clinical symptoms.The ameliorated skin phenotype in FcγR À/À mice was accompanied by a reduction in skin histological signs characterized by markedly less pronounced crust formation/hyperkeratosis, epidermal and dermal thickening, inflammatory infiltrate, and subepidermal split formation (p < 0.01) (Figure 3C,D).IgG and C3 deposits along the BMZ of perilesional skin were observed in both anti-NC14-1 IgG-treated groups along with unchanged serum anti-NC14-1 IgG levels (Figure 3E-G).Interestingly, the composition of the lesional inflammatory cell infiltrate in FcγR À/À mice treated with anti-NC14-1 IgG resembled the cell composition of site-matched skin obtained from the normal rabbit-IgG-treated control mice (Figure 4A).In the skin of the anti-NC14-1 IgG-treated FcγR À/À mice, we found significantly decreased frequencies of dendritic cells, neutrophils, and T cells, while the frequency of macrophages significantly increased compared to WT mice receiving anti-NC14-1 IgG (p < 0.05) (Figure 4B and supplementary material, Figure S3).Yet, significant differences in cell frequencies, particularly dendritic cells and macrophages, were also apparent between C57BL/6J WT mice receiving NC14-1 IgG and FcγR À/À mice injected with normal rabbit-IgG.No significant changes in eosinophil numbers were observed.

The C5a/C5aR1 axis plays a critical role in tissue destruction
In addition to FcγR-mediated effector mechanisms, complement activation is pivotal in the pathogenesis of autoimmune blistering diseases [47].In particular, the complement-5a (C5a)/C5aR1 axis has been

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M Pigors et al demonstrated to play a crucial role in the development of skin blisters in pemphigoid mouse models; therefore, we also aimed to explore the pathophysiological relevance of complement activation in the NC14-1 BP mouse model using mice deficient in the C5aR1 (C5ar1 À/À ).Following injection of anti-NC14-1 IgG, C5aR1 À/À mice showed a reduction in skin lesions at Day 12 ( p < 0.0001) and skin histological signs (p = 0.053), while tissue-bound IgG and anti-NC14-1 serum IgG levels as well as C3 deposits along the cutaneous BMZ showed no notable changes compared to C57BL/6J WT mice (Figure 5A-G).In addition, reduced signals for Ly6G+ neutrophils were observed in C5ar1 À/À mice compared to C57BL6/J WT mice, while signals for CD3+ T cells were comparable among all three groups (supplementary material, Figure S4).No signs of disease activity were observed in the C5aR1 À/À mice receiving normal rabbit IgG.

Inhibition of FcRn improved disease activity
Antagonizing the interaction between IgG and FcRn represents a promising therapeutic approach to treating autoimmune diseases.Therefore, we tested a murine Fc fragment IgG2c-ABDEG (mABDEG), a derivative of the FcRn inhibitor efgartigimod, in the anti-NC14-1 IgG-induced BP mouse model.C57BL/6J mice were injected with anti-NC14-1 IgG for disease induction (Figure 6A).Additionally, mice were treated i.  Pathogenicity of Col17 non-NC16A/NC15A extracellular domain-specific IgG 167

Discussion
Previously published mouse models have provided detailed mechanistic insights into BP etiology and clearly demonstrated the pathogenic role of anti-human Col17 NC16A/anti-mouse Col17 NC15A IgG [48] and also provided evidence for the pathogenic function of anti-BP230 IgG in BP development [4,49].Yet the role of antibodies targeting the Col17 ectodomain downstream of NC16A/ NC15A in skin blister formation has not been demonstrated [27,31,50].However, recent data indicate that non-NC16A extracellular IgG can enhance Col17 depletion and contribute to the pathogenicity of anti-Col17 autoantibodies [51,52].In this study we used both BP patient autoantibodies and rabbit IgG targeting the Col17 extracellular domain lacking NC16A/ NC15A to elicit an inflammatory response and tissue injury.
In the well-established ex vivo cryosection model, antibodies can induce subepidermal splitting after binding to the dermal-epidermal junction of cryosections of normal skin following incubation with human leukocytes from healthy donors.Split formation was shown to depend on the release of granulocyte-derived reactive oxygen species and specific proteases observed upon use of anti-NC16A/NC15A, anti-type VII collagen, and anti-laminin-alpha-3 IgG [29,38,40,50,53].Here, (1) BP patient IgG without anti-BP230 reactivity and depleted of anti-NC16A reactivity, (2) BP patient IgG purified against the Col17 ectodomain and depleted of anti-NC16A reactivity, and (3) rabbit IgG raised against murine Col17 NC14-1 induced dermal-epidermal separation.These results indicate that in a subgroup of BP patients and in the murine model, epitopes not only within but also outside the immunodominant NC16A/ NC15A domain of the Col17 ectodomain can lead to subepidermal split formation ex vivo.
The collagenous domains of the ectodomain harbor highly repetitive triplet amino acid sequences (Gly-X-Y) [54] and are known to be less immunogenic compared to the NC domains [3,7,[55][56][57].As such, rabbit IgG generated against NC14-1, a fusion protein containing all NC domains except the NC15A of murine Col17, were employed in mice.Repeated injections of anti-NC14-1 IgG into C57BL6/J WT mice led to development of a BP-like phenotype with skin lesions, subepidermal split formation, and an inflammatory infiltrate in the upper dermis, as well as IgG and C3 deposits along the BMZ.Of note, no mucosal lesions were apparent,  although antibodies against the Col17 non-NC16A extracellular domains are prevalent in the majority of patients with MMP [2,3].Yet these studies suggest that (1) in addition to autoantibodies of the IgG isotype, IgA are also critical in the pathogenesis of MMP and (2) MMP patient sera preferentially recognize the most distal stretches of the Col17 ectodomain [58][59][60][61][62][63].The latter observation is also supported by immunoelectron microscopy analyses, which demonstrated that MMP autoantibodies map to extracellular stretches of Col17, which extend into the lamina densa [63,64].Failure to induce mucosal lesions in the anti-NC14-1 antibodytransfer mouse model might thus be due to the exclusive administration of antibodies of the IgG class.Moreover, titers of the IgG within the polyclonal anti-NC14-1 antibody pool that targets specifically the most distal extracellular regions might be too low to induce visible lesions in the mucous membranes.
Binding of anti-Col17 autoantibodies to the cutaneous BMZ was shown to trigger inflammatory cascades that can be mediated by Fc-dependent mechanisms, including activation of the complement system and FcγRs [24,38,[42][43][44][45]. FcγRs bind the Fc part of IgG and thereby trigger the activation of effector cells primarily of the myeloid lineage [65,66], which in turn leads to the release of reactive oxygen species and proteases causing the associated tissue damage [29,36,38].In this study, we employed mice deficient in the immunoreceptor tyrosine-based activation motif (ITAM)-bearing γ subunit shared by the activating receptors FcγRI, FcγRIII, and FcγRIV (and also by FcεRI) [46,66,67].Following administration of anti-NC14-1 IgG, the extent of skin lesions was reduced by $40% in FcγR À/À mice compared to the WT group.While the FcγRI, a high-affinity receptor for IgG2a, mainly binds free or monomeric IgG, and FcγR À/À mice still express partially functional FcγRI [68], we presume that its diminished expression in the new BP mouse model will not affect disease progression significantly.Moreover, a limited role of FcγRI was reported in pemphigoid pathogenesis and other disease models [29,66,69].In contrast, FcγRIII and FcγRIV were shown to be crucial drivers of tissue destruction in pemphigoid disease [29,69].FcγRIII is considered a low-affinity receptor binding IgG1, IgG2a, and IgG2b in a multimeric state, e.g., if they are present in an immune complex [66], while FcγRIV is a high-affinity receptor for IgG2a and IgG2b.Both receptors are expressed highly on effector leukocytes, and consequently we observed significant changes in the frequencies of neutrophils in lesional skin of anti-NC14-1 IgG-treated FcγR À/À mice compared with anti-NC14-1 IgG-treated WTs.These findings are consistent with previously published antibody-transfer mouse models of BP [23,26,29], where a predominant lesional infiltration with neutrophils is observed, rather than infiltration of eosinophils, which in addition to lymphocytes are much more frequently encountered in BP patient skin [2,70,71].The composition of the inflammatory cell infiltrates of both FcγR À/À groups (treated with anti-NC14-1 or normal rabbit IgG) were comparable with respect to the proportions of dendritic cells, macrophages, and T cells and significantly different from the infiltrate of anti-NC14-1 IgG-treated WT mice.Since these observations were not consistently evident in BP mice following pharmacological inhibition of the FcRn, it is possible that the changes in the proportion of inflammatory cells, other than neutrophils, can partially also be attributed to the lack of FcγRs, independent of the experimentally induced BP.To this end, previous data implicated that γ chain depletion resulted in the impaired function of effector cells, including macrophages, which lost their phagocytotic activity, and possibly also affects some T-cell populations where the γ subunit is a component of distinct functional T-cell receptor-CD3 complexes [46].While FcγR À/À mice lack FcγRI, FcγRIII, and FcγRIV functions, expression of FcγRII, which was protective in another BP mouse model [29], is retained.These results suggest an additional mechanism for the pathogenesis of the IgG targeting the Col17 ectodomains downstream of NC16A/ NC15A.
In addition to FcγRs, autoimmune responses are largely controlled through the complement system [72].Most notably, terminal complement pathways, particularly the C5a/C5aR1 axis, have been demonstrated to play a role in the development of skin blisters in mouse models of BP [24,42,43], epidermolysis bullosa acquisita [44,45,73], and anti-laminin-332 MMP [38], as opposed to an antithetic regulation by C5aR2, which was shown to be protective of disease development in BP [42] while contributing to disease progression in epidermolysis bullosa acquisita [74].C5aRs are typically co-expressed with FcγRs, and a bidirectional crosstalk between the C5a/C5aR1 axis and FcγR signaling has been shown to play a crucial role in modulating autoimmune responses [75].In line with the markedly reduced disease activity in the anti-NC14-1 IgG-treated FcγR À/À mice, we also demonstrated significantly reduced signs of BP in C5ar1 À/À compared with WT mice on both clinical and histological levels following repeated injections of anti-NC14-1 IgG.The partial disease remission observed in both FcγR À/À and C5ar1 À/À mice is further attributed to FcγR-and complement-independent mechanisms previously reported in BP [3,76,77].These include a direct effect of Fab fragments on Col17, secretion of proinflammatory mediators (e.g., IL-6, IL-8, tissue-type plasminogen activator), which promote inflammation and recruitment of neutrophils, and internalization/ depletion of Col17-antibody complexes by basal keratinocytes via micropinocytosis, resulting in impaired keratinocyte adhesion to the underlying BMZ and dermal-epidermal detachment.While complement-(in)dependent mechanisms seem to play comparable roles in both anti-NC15A and anti-NC14-1 IgG induced BP with partial disease remission observed in both experimental models [42], FcγR-independent mechanisms seem to be integral for autoantibodies against the membrane-distal NC14-1 domains compared with the membrane-proximate NC15A region [29].
Yet, a direct comparison of these different in-vivo BP models has not been performed and should be addressed in future studies.
In the last set of experiments, pharmacological modulation of experimental BP was validated by prophylactic treatment with a murine derivative for FcRn antagonist efgartigimod.The FcRn is an atypical FcγR, which plays an important role in IgG homeostasis as it protects IgG from lysosomal degradation [78,79].Disease activity was abrogated in FcRn À/À mice employed in the neonatal mouse model of BP and in the adult mouse model of epidermolysis bullosa acquisita [80,81].Thus, inhibition of FcRn has the potential to specifically decrease half-life extension and survival of circulating pathogenic IgGs, as opposed to other IgG-modulating therapies such as immunoadsorption and intravenous immunoglobulin, which also lower IgG levels and have successfully been applied in patients with BP [79,[82][83][84][85]. Efgartigimod has already been approved for treatment of generalized myasthenia gravis and is currently in clinical development for various indications, including BP and pemphigus [86,87].Efgartigimod, a human IgG1-derived Fc fragment, has been modified using ABDEG technology to increase its affinity for FcRn at both neutral and acidic pH, blocking FcRn-IgG interaction and, thus, resulting in an enhanced degradation of endogenous IgG [88,89].Consistent with previous preclinical and clinical studies [79,86], we observed a rapid decrease in anti-NC14-1 IgG levels and reduced binding of IgG at the cutaneous BMZ following treatment with the mouse derivative of efgartigimod.The effective treatment with the antibody was further reflected in significantly improved skin lesions and reduced inflammation, which was particularly characterized by lower frequencies of neutrophils in lesional skin biopsies.
Taken together, our data demonstrate the pathogenic effects of IgG targeting the Col17 extracellular domains located outside of NC16A/NC15A, which are in part attributable to both antibody-mediated FcγR-and C5aR1-effector mechanisms.While the antibodytransfer mouse model is limited to characterization of the effector phase of BP and certainly not all features of the disease are mimicked in anti-Col17 IgG-mediated BP, such as lack of eosinophilic skin infiltration, the new BP mouse model will be instrumental in the further characterization of the pathogenic role of antibodies directed against Col17 non-NC16A/NC15A extracellular epitopes and in the evaluation of specific therapeutic approaches for pemphigoid diseases.Altogether, the present data support a pharmacological inhibition of the FcRn as a promising treatment for BP and various other autoantibody-mediated diseases.In addition, other therapeutics targeting complement or FcγRs are currently in clinical trial evaluation [90,91], yet the development of several FcRn antagonists have advanced more rapidly in recent years.FcRn blockers certainly have the advantage that in comparison to anticomplement drugs, they intervene early in disease development and reduce all IgG subclasses, while bypassing the complexity of the Fc-FcγRs interactions, which largely complicates the development of therapeutic modalities targeting specific FcγRs [92,93].

Figure 1 .
Figure 1.Type XVII collagen (Col17) ectodomain-reactive sera depleted of NC16A IgG induced dermal-epidermal cleavage in human skin ex vivo.(A) Schematic presentation of human Col17 consisting of an intracellular domain, a short transmembrane (TM) domain, and an extracellular domain with 15 collagenous (C) repeats, which are interspersed with 16 noncollagenous (NC) regions.(B-F) Cryosection assay with a representative picture of each antibody fraction tested.Cryosections of normal human skin showed dermal-epidermal separation (black arrowheads) in the presence of normal human leukocytes following incubation with (B) all 21 BP sera and (C) purified anti-NC16A IgG fractions.In addition, (D) IgG depleted of anti-NC16A reactivity induced split formation in 15/21 patients, whereas (E) ectodomain-specific IgG depleted of anti-NC16A IgG only showed subepidermal cleavage in 1/4 patient sera.In contrast, (F) BP IgG depleted of reactivity against the entire ectodomain did not show split formation in all four tested serum samples.Anti-Col17 NC16A ELISA values from the serum and IgG fractions used for the cryosections assay are shown in blue.Scale bar, 50 μm.
p. with 30 mg/kg mABDEG or murine Fc fragment IgG2c isotype control.Effective blocking of the FcRn was demonstrated by significantly lower serum anti-NC14-1 IgG levels (p < 0.05) and lower levels of endogenous total mouse IgG in mABDEG-versus isotype control-treated mice (p < 0.0001) from Day 6 throughout Day 12 (Figure6B,C).mABDEG-versus isotype control-treated mice showed a significant reduction in the ABSA at Day 12 (p < 0.05) (Figure6D,E).This was paralleled by reductions in tissue-bound IgG (p = 0.0518) and C3 (p < 0.01) binding at the BMZ of perilesional skin in the treatment versus control group (Figure6F-I).Moreover, lesional skin biopsies showed significantly less inflammation, particularly characterized by strongly decreased frequencies of neutrophils in mABDEG-versus isotype control-treated mice (Figure6J,K and supplementary material, FigureS5A,B).

Figure 6 .
Figure 6.Inhibition of neonatal FcR (FcRn) by mouse ABDEG improved disease activity in anti-NC14-1 IgG-induced BP. (A) Study design.Anti-NC14-1 IgG was injected s.c.into C57BL/6J WT mice every other day over 12 days.The affected body surface area (ABSA) was assessed every 4 days.The murine FcRn antagonist (mABDEG) or isotype control was administered i.p. every 3 days, and blood was withdrawn 3 days prior to the start of the experiment as well as 2 h after treatment with mABDEG or the isotype control; a final bleed was performed on Day 12. (B and C) Inhibition of FcRn resulted in significantly lower anti-NC14-1 serum IgG (n = 10/group) and endogenous total mouse IgG levels (n = 16/group) in mABDEG-versus isotype-treated mice.(D and E) mABDEG-versus isotype control-treated mice showed a significant reduction in skin lesions at Day 12 (n = 16/group).(F-K) IgG, C3 binding, and histological scores were markedly lower in mABDEG-versus isotype control-treated skin.Black arrowheads indicate dermal-epidermal separation.Scale bars, 50 μm.****p < 0.0001; ***p < 0.001; **p < 0.01; *p ≤ 0.05.