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. 2020 May;177(10):2381-2392.
doi: 10.1111/bph.14986. Epub 2020 Mar 6.

Treatment with anti-neonatal Fc receptor (FcRn) antibody ameliorates experimental epidermolysis bullosa acquisita in mice

Anika Kasprick  1 , Maxi Hofrichter  1 , Bryan Smith  2 , Penelope Ward  3 , Katja Bieber  1 , Anthony Shock  2 , Ralf J Ludwig  1 , Enno Schmidt  1
Affiliations

Treatment with anti-neonatal Fc receptor (FcRn) antibody ameliorates experimental epidermolysis bullosa acquisita in mice

Anika Kasprick et al. Br J Pharmacol. 2020 May.

Abstract

Background and purpose: Pemphigus and pemphigoid diseases are characterized and caused predominantly by IgG autoantibodies targeting structural proteins of the skin. Their current treatment relies on general and prolonged immunosuppression that causes severe adverse events, including death. Hence, novel safe and more effective treatments are urgently needed. Due to its' physiological functions, the neonatal Fc receptor (FcRn) has emerged as a potential therapeutic target for pemphigus and pemphigoid, primarily because IgG is protected from proteolysis after uptake into endothelial cells. Thus, blockade of FcRn would reduce circulating autoantibody concentrations. However, long-term effects of pharmacological FcRn inhibition in therapeutic settings of autoimmune diseases are unknown.

Experimental approach: Therapeutic effects of FcRn blockade were investigated in a murine model of the prototypical autoantibody-mediated pemphigoid disease, epidermolysis bullosa acquisita (EBA). B6.SJL-H2s C3c/1CyJ mice with clinically active disease were randomized to receive either an anti-FcRn monoclonal antibody (4470) or an isotype control over 4 weeks.

Key results: While clinical disease continued to worsen in isotype control-treated mice, overall disease severity continuously decreased in mice injected with 4470, leading to almost complete remission in over 25% of treated mice. These clinical findings were paralleled by a reduction of autoantibody concentrations. Reduction of autoantibody concentrations, rather than modulating neutrophil activation, was responsible for the observed therapeutic effects.

Conclusion and implications: The clinical efficacy of anti-FcRn treatment in this prototypical autoantibody-mediated disease encourages further development of anti-FcRn antibodies for clinical use in pemphigoid diseases and potentially in other autoantibody mediated diseases.

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Conflict of interest statement

In the last 3 years, R.J.L. has received research funding from Almirall, True North Therapeutics, UCB Pharma, ArgenX, TxCell, Topadur, Incyte, and Admirx and fees for consulting or speaking from ArgenX, Immunogenetics, Novartis, and Lilly. E.S. has received research funding from Almirall, UCB Pharma, ArgenX, TxCell, Incyte, Novartis, Euroimmun, and Admirx and fees for consulting or speaking from ArgenX, UCB, TxCell, Novartis, Fresenius, and Almirall. A.S. is an employee of UCB, and P.W. and B.S. are former employees of UCB. All other authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Anti‐FcRn treatment improves already clinically manifest experimental EBA. (a) B6.s mice were immunized with mCOL7vWFA2 for induction of experimental EBA. Three to 8 weeks after immunization, mice were weekly clinically evaluated. If, in an individual mouse, 2% or more of the body surface area was affected by EBA lesions, it was randomized to either isotype or anti‐FcRn antibody treatment. Treatments were carried out for 4 weeks, and clinical disease severity, expressed as affected body surface area, was evaluated weekly. (b) At randomization (Week 0), clinical disease severity, expressed as percentage of affected body surface area, was identical in isotype‐ and anti‐FcRn antibody‐treated mice. In isotype‐treated mice, clinical EBA symptoms worsened during the 4‐week treatment period, while it improved in anti‐FcRn treated mice. Starting from Week 2, compared to mice injected with isotype antibody, a significant lower affected body surface area was observed in anti‐FcRn‐treated mice until the end of the experiment. Data shown are the means ± SEM, from 11 mice per group. *P< .05, significantly different as indicated; two‐way ANOVA with Bonferroni t test for pairwise multiple comparisons. (c) Dermal infiltration was semi‐quantitatively evaluated in biopsies from lesional skin at the end of the experiment. No difference in infiltration among the two groups was noted; Student's t test. Data shown are the individual values with means ± SEM, from 11 mice per group. (d) Burden score (excluding EBA skin lesions) of the mice treated with isotype or anti‐FcRn antibody at Week 4 after randomization. The graph shows each score (dots) and the median (line). Data shown are the individual values with medians, from 11 mice per group. *P< .05, significantly different as indicated; Mann–Whitney test. (e) Representative clinical images of the same mice at randomization (Week 0) and at the end of the treatment (Week 4) receiving either isotype or anti‐FcRn antibody. Representative H&E stained skin biopsies from lesional skin, obtained at the end of the treatment period
Figure 2
Figure 2
Anti‐FcRn treatment reduces circulating anti‐COL7 IgG concentrations and decreases IgG deposition in the skin of mice with experimental EBA. Isotype or anti‐FcRn antibody treatment was initiated if, in an individual mouse, 2% or more of the body surface area was affected by EBA lesions. At the start of treatment, as well as 2 and 4 weeks thereafter, serum was obtained for determination of total and COL7‐specific IgG. (a) In both groups, total IgG concentrations increased during the 4‐week treatment period. This increase was less pronounced in mice treated with anti‐FcRn. Data shown are the means ± SEM, from 9‐10 mice per group, after removal of outliers using ROUT, accounting for the unequal n. *P< .05, significantly different from anti‐FcRn; two‐way ANOVA with Bonferroni t test for pairwise multiple comparisons.. (b) Concentrations of COL7‐specfic IgG remained relatively constant in isotype antibody treated mice, while anti‐FcRn antibody treatment reduced the specific autoantibody concentration by approximately 50%. Data shown are the means ± SEM, from 9‐10 mice per group, after removal of outliers using ROUT, accounting for the unequal n. *P< .05, significantly different from anti‐FcRn; two‐way ANOVA with Bonferroni t test for pairwise multiple comparisons. In (c) the deposition of IgG, but not of C3 (d), at the dermal–epidermal junction junction was reduced in mice treated with anti‐FcRn antibody, compared with mice injected with isotype control antibody. Data shown are the individual values with means ± SEM, from 9‐12 mice per group, after removal of outliers using ROUT. *P< .05, significantly different as indicated; t test (IgG) or rank sum test (C3). (e) Representative images of direct IF microscopy for IgG from peri‐lesional skin biopsies obtained at the end of the experiment
Figure 3
Figure 3
Anti‐FcRn treatment does not alter neutrophil CD62L function in vivo. After 4 weeks of treatment with either isotype or anti‐FcRn antibody, indicated organs from mice with experimental EBA were obtained for isolation of neutrophils, followed by flow cytometry. Mice injected with TiterMax® alone served as negative controls. (a–c) Percentage of CD62L− CD45+, CD1b+ Gr‐1+, and Ly6G+ singlet cells was determined. No differences in neutrophil activation was detected in the three investigated organs. Data are shown as individual values in box and whisker plots with medians, quartiles and ranges, from seven to eight mice per group; unequal sample sizes are due to technical issues (i.e., clotting). For normally distributed data, t test was used for statistical analysis, and for non‐equally distributed data, rank sum test was applied. (d–f) Representative FACS images are shown in panels a–c
Figure 4
Figure 4
Blockade of the FcRn did not affect immune complex‐induced neutrophil activation. (a) Neutrophils were stimulated with immune complexes, and their activation was determined by the cumulative release of ROS over time. Data shown are the individual values with means ± SEM, from 6 samples per group. *P< .05, significantly different from isotype; ANOVA with Bonferroni t test for multiple comparisons. (b) Representative results of one ROS‐release experiment. Time is measured in repeats, whereby one repeat corresponds to approximately 1 s. (c) Dermal–epidermal separation was induced on skin cryosections by incubating these with anti‐COL17 IgG and neutrophils. Blockade of the FcRn had no effect on the magnitude of dermal–epidermal separation. If sections were incubated with normal IgG, instead of anti‐COL17 IgG, and neutrophils, dermal–epidermal separation ranged between 0% and 5%. Data shown are the individual values with means ± SEM, from 12 samples per group, with the exception of the 100 μg·ml−1 dose, where n = 6. ANOVA, with Bonferroni t test for multiple comparisons, was used to test for statistical difference from isotype
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