Therapeutic targeting of alternative pathway and C5 but not C5a protects from disease development in a preclinical model of autoimmune blistering dermatosis

Abstract

Introduction: Epidermolysis Bullosa Acquisita (EBA) is an autoimmune blistering dermatosis characterized by autoantibodies (AAbs) against type VII collagen (COL7) located at the dermal epidermal junction (DEJ). Local complement activation drives C5a generation associated with neutrophil recruitment and activation resulting in skin lesions and inflammation. Here we tested the impact of C5a/C5adesArg, C5 or combined C5 and alternative pathway (AP) targeting on disease development and skin inflammation in a preclinical mouse model mimicking the effector phase of EBA.

Methods: C57BL/6 mice were treated subcutaneously with purified rabbit anti-mouse-COL7 IgG in the presence of IgG1 mAbs directed against murine C5a/C5adesArg (M031), C5 (mBB5.1), a bifunctional protein comprising mBB5.1 fused to an active fragment of the AP inhibitor factor H (M014) or an IgG1 isotype control mAb. Formation of skin lesions was evaluated 12 days every other day. On day 12, DEJ separation, IgG AAb and C3b deposition and neutrophil infiltration was assessed.

Results: Isotype IgG1-treated mice developed first skin lesions on day 4 peaking on day 12. Prophylactic treatment with either M031 or M014 markedly reduced the development of skin lesions, the dermal/epidermal separation and neutrophil recruitment. Surprisingly, C5 or combined AP/C5 inhibition by M014 but not C5a/C5adesArg-targeting by M031 reduced the development of skin lesions and dermal/epidermal separation in the setting of therapeutic treatment. IgG and C3b deposition was not affected by either treatment. Importantly, direct comparison of isolated C5 targeting by mBB5.1 vs. combined AP/C5 inhibition by M014 revealed that M014 reduced the development of skin lesions earlier and more pronounced than mBB5.1.

Discussion: Our findings identify combined C5/AP targeting as a novel therapeutic option for autoimmune blistering dermatoses.

Keywords: C5a; EBA; alternative pathway; bullous pemphigoid; complement.

Figure 1

Prophylactic inhibition of C5a/C5adesArg by M031 or combined targeting of AP/C5 by M014 reduces the formation of skin lesions. (A) Representative picture of mice on day 12 after prophylactic treatment with anti-HEL control IgG1 mAb, M031 or M014 on days -1, 2, 5 and 8. Red arrows point toward skin lesions. (B) (Left panel) Cumulative disease scores benchmarked as percentage of the total body surface area affected by skin lesions (ABSA) of mice treated with anti-HEL mAb (blue), M031 (black) or M014 (red). Blue arrows show the time point when COL7-specific IgG AAbs were injected. Black (M031) or red (M014) arrows show the time points when complement inhibitors were administered. Results were pooled from 3 independent experiments. Statistical differences between groups were determined by One way ANOVA with Holm-Šídák’s posthoc multiple-comparisons test. **p < 0.01, ***p < 0.001. ****p < 0.0001 for M031-treated compared with HEL-IgG1 treated mice and ^#p<0.05, ^## p < 0.01, ^### p < 0.001. ^#### p < 0.0001 for M014-treated compared with HEL-IgG1-treated mice.; (right panel) peak value of ABSA assessed for each mouse. Scatter plots show the mean ± SEM (n = 12 mice per group). ****p<0.0001. (C) (Left panel) Histopathologic evaluation of dermal-epidermal separation. Shown are presentative pictures of skin sections from mice treated with anti-HEL IgG1, M031 or M014 on day 12. Red arrows indicate subepidermal clefts; (right panel) percentage of dermal-epidermal separation determined individually for each mouse treated with anti-HEL IgG1 (blue), M031 (black) or M014 (red). Results were pooled from 3 independent experiments. The scatter plots show the mean ± SEM (n = 12 mice per group). Statistical differences between the treatment groups were determined by One-way ANOVA with Holm-Šídák’s posthoc multiple-comparisons test. ***p <0.001, ****p < 0.0001.

Figure 2

Impact of prophylactic C5a/C5adesArg- or combined AP/C5-targeting on neutrophil infiltration, IgG and C3b deposition in the skin. (A) Representative immunofluorescence pictures of ear skin sections from C57BL/6 mice treated with anti-HEL, M031 or M014 on day 12. Blue = DAPI; Red = Ly6G⁺ neutrophils; Green = MPO⁺ cells. The inserts show magnifications of the areas marked by the white rectangles (B) Quantitative evaluation of Ly6G⁺ or MPO⁺ neutrophils per μm² in ear sections from mice treated with anti-HEL IgG1 (blue), M031 (black) or M014 (red). (C) Representative immunofluorescence pictures of ear skin sections from mice treated with Anti-HEL IgG1, M031 or M014 on day 12. Blue = DAPI; Red = IgG AAb deposition; Green = C3b deposition. (D) Quantitative evaluation of C3b or IgG AAb deposition per μm² in ear sections from mice treated with anti-HEL IgG1 (blue), M031 (black) or M014 (red). Microscopic pictures were analyzed via Keyence analyzer software. Results in (B, D) were pooled from 3 independent experiments. Scatter plots show the mean ± SEM (n =10–12 mice per group). Data were analyzed using One-way ANOVA with Holm-Šídák’s posthoc multiple-comparisons test. *p < 0.05, **p < 0.01.

Figure 3

Therapeutic inhibition of AP/C5 targeting by M014 but not inhibition of C5a/C5adesArg by M031 reduces the formation of skin lesions (A) Representative picture of mice on day 12 after therapeutic treatment with anti-HEL IgG1, M031 or M014 on days 5 and 8. Red arrows indicate skin lesions. (B) (Left panel) Cumulative disease scores shown as ABSA of mice treated with anti-HEL IgG1 (blue), M031 (black) or M014 (red). Results were pooled from 3 independent experiments. Scatter plots show the mean ± SEM (n = 12 mice per group). Statistical differences between groups were determined by One way ANOVA with Holm-Šídák’s posthoc multiple-comparisons test. *p < 0.05, **p < 0.01. ***p < 0.001 for M014-treated compared with anti-HEL IgG1-treated mice. Blue arrows show the time point when COL7-specific IgG AAbs were injected. Black (M031) or red (M014) arrows show the time points when complement inhibitors were administered; (right panel) peak value of ABSA assessed for each mouse. ***p<0.0001. (C) (Left panel) Histopathologic evaluation of dermal-epidermal separation. Shown are presentative pictures of skin sections from mice treated with anti-HEL IgG1, M031 or M014 on day 12. Red arrows indicate subepidermal clefts; (right panel) percentage of dermal-epidermal separation determined individually for each mouse treated with anti-HEL IgG1 (blue), M031 (black) or M014 (red). Results were pooled from 3 independent experiments. The scatter plots show the mean ± SEM (n = 12 mice per group). Statistical differences between the treatment groups were determined by One-way ANOVA with Holm-Šídák’s posthoc multiple-comparisons test. *p < 0.05.

Figure 4

Impact of therapeutic C5a/C5adesArg- or combined AP/C5 targeting on neutrophil infiltration, IgG and C3b deposition in the skin. (A) Representative immunofluorescence pictures on day 12 of ear skin sections from mice treated with anti-HEL IgG1, M031 or M014. Blue = DAPI; Red = Ly6G⁺ neutrophils; Green = MPO⁺ cells. (B) Quantitative evaluation of Ly6G⁺ or MPO⁺ neutrophils per μm² in ear sections from mice treated with anti-HEL IgG1 (blue), M031 (black) or M014 (red). (C) Representative immunofluorescence pictures of ear skin sections from mice treated with Anti-HEL IgG1, M031 or M014 on day 12. Blue = DAPI; Red = IgG AAb deposition; Green = C3b deposition. (D) Quantitative evaluation of C3b or IgG AAb deposition per μm² in ear sections from mice treated with anti-HEL IgG1 (blue), M031 (black) or M014 (red). Microscopic pictures were analyzed via Keyence analyzer software. Results in B and D were pooled from 3 independent experiments. Scatter plots show the mean ± SEM (n =10–12 mice per group). Data were analyzed using One-way ANOVA with Holm-Šídák’s posthoc multiple-comparisons test. ns, not signficant.

Figure 5

Side-by-side comparison of the therapeutic effect of M014 and mBB5.1 treatment on the development of skin lesions. (A) Cumulative disease scores shown as ABSA of mice treated with anti-HEL IgG1 (blue), M014 (red; left panel) or mBB51 (purple; right panel). Black arrows show the time points when the different compounds were administered (day 5 and 8). (B) Peak values of ABSA assessed for each mouse on days 8, 10 and 12. Scatter plots show the mean ± SEM (n = 12 mice per group) for the individual scoring days. *p<0.05; **p<0.01; ***p<0.001. (C) Area under the curve (AUC) of ABSA calculated after day 8, 10 or 12 for each mouse. Scatter plots show the mean ± SEM (n = 12 mice per group). *p < 0.05, **p < 0.01. ns, not significant. Data in (B, C) were analyzed using One way ANOVA with Holm-Šídák’s posthoc multiple-comparisons test.

Figure 6

Schematic detailing the proposed mechanisms underlying the AP pathway activation by rabbit anti-COL7 AAbs and the targeting of C5, the AP and C5a/C5adesArg by mAbs mBB5.1, M014 and M031. It is well appreciated that spontaneous initiation of the AP by C3 tickover results in continuous cleavage of C3 into C3a and C3b (43, 44). Such C3b has strong affinity for IgG molecules and can form C3b2–IgG complexes (45). Also, rabbit IgG F(ab’)2 fragments can activate the AP (46). Thus, rabbit anti-COL7 IgG molecules can promote AP activation through their Fc and Fab parts. This can be amplified by C5a-dependent neutrophil activation, resulting in C3, factor B and properdin production (47). The SCR1–5 of factor H in M014 will compete with factor B for the Bb binding site on C3b leading (A) to the displacement of Bb and accelerated decay of the AP C3 convertase. Further, C3b-bound factor H SCR 1–5 forms a binding platform for factor I, resulting in cofactor activity and cleavage of C3b to iC3b (B). Subsequently, factor I can degrade iC3b to C3c and C3dg with complement receptor 1 (CR1) as cofactor. Taken together, M014 will block the generation of C5a at several levels, i.e. upstream of C5 by: (i) the inhibition of the AP amplification loop (A); and (ii) the degradation of C3b to iC3b (B) and directly through the inhibition of C5 cleavage by the C5 convertase of the AP or the classical pathway (C). In contrast, the modified BB5.1 mAb will only prevent the cleavage of C5 by the C5 convertase (C). M031 will neutralize C5a/C5adesArg (D) and inhibit their binding to C5aR1 or C5aR2 and the consecutive activation of neutrophils. Created in BioRender. Köhl, J. (2025) https://BioRender.com/n4q7slv.