If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
Chronic skin inflammation, subepidermal blistering, and severe itching are the clinical hallmarks of bullous pemphigoid (BP). The disease is caused by autoantibodies against type XVII collagen (COL17, BP180), more specifically, the extracellular fraction of the 16th noncollagenous domain of the protein (NC16A) (
). Two pathways are thought to drive BP pathogenesis. First, autoantibody binding to COL17 leads to activation of the complement cascade, evidenced by the detection of complement deposits along the dermal-epidermal junction in patients with BP (
). Therefore, treatments maintaining the initial therapeutic response, or at least reducing the steroid dose, are urgently needed. Yet, with the exception of the anti-C5 antibody eculizumab, no complement-targeting biologicals have been approved for clinical use. In addition, eculizumab inhibits the activation of the terminal cascade driven by all three complement pathways. As BP pathology has been linked specifically to classical complement pathway (CP) activity, its selective blockade would maintain full functionality of the alternative and lectin complement pathways to mediate innate humoral immunity. Furthermore, targeting upstream of C5 in the CP would also prevent the production of upstream anaphylatoxins such as C4a and C3a that may induce migration and activation of effector immune cells to the site of complement activation.
To assess the role of the CP in driving BP autoantibody-mediated complement activation, we used TNT003, a mouse monoclonal IgG2a antibody that inhibits activation of C1s, a CP-specific serine protease (
) using human biomaterial as approved by the Institutional Review Board at the University of Lübeck and after written informed consent. In this assay, cryosections of human skin are incubated with the serum of patients with BP and a complement source, leading to the deposition of complement along the dermal-epidermal junction of the skin section. We selected this model based on previous data in animal models of BP, hinting toward a prominent role of CP activation in BP pathogenesis (
), and so far missing data on the role of complement activation in human models of the disease.
Although the deposition of complement at the dermal-epidermal junction is well established, no data on the concentration of complement components in the serum of patients with BP are available. To test if the complement activation in BP is restricted to the skin or (as reported for certain cytokines) is also “systemically” present, we first analyzed the concentration of several complement components (C1s, C1q, C1s-C1INH, C3a, C4, C4a, and C5) (Supplementary Figure S1 online and Figure 1) in the plasma of patients with BP (Supplementary Table S1 online). The concentrations of all above-mentioned anaphylatoxins (C3a, C4a, C5a) were similar between newly diagnosed patients with BP and age-and sex-matched controls (Figure 1a). Furthermore, all anaphylatoxin concentrations did not change after treatment (Figure 1b) and did not correlate with the concentration of BP180-NC16A serum autoantibodies (Figure 1c). Hence, in BP, complement activation seems to be locally restricted to the skin compartment, as the anaphylatoxins in the plasma were at similar levels compared with controls.
To investigate the effect of TNT003 on complement activation, we next evaluated if TNT003 can modulate C3 deposition at the dermal-epidermal junction and anaphylatoxin formation in the complement activation assay (
). For this, complement-inactivated serum from patients with BP (Supplementary Table S2 online) was first incubated on skin cryosections from healthy donors followed by the addition of normal human plasma as a complement source in the absence or presence of TNT003. Interestingly, we observed only C3 deposits in 32 of 91 tested sera from patients with BP, despite the presence of C3 deposits in many of the patients at diagnosis (Supplementary Table S2). This significantly lower number of patients with complement-fixing BP might result from differences in the assay protocols. For example, in this study patient sera were more diluted and unspecific complement activation in patient sera was inhibited by the addition of EDTA. When examining 18 of these 32 complement-fixing samples, blockade of C1s by TNT003 dose-dependently (≥10 μg/ml) alleviated C3 deposition at the dermal-epidermal junction in all 18 tested samples that had C3 deposits (Figure 2a). In addition, TNT003, but not TNT001 (isotype control), reduced C4a and C5a concentrations to baseline levels (defined as concentrations in the presence of EDTA) in the assay supernatants. Levels of C3a were unaffected by TNT003 or TNT001 (Figure 2b), which could be caused by the nonclassical pathway C3c deposition observed at the stratum corneum. We also observed a similar degree of inhibition of anaphylatoxin generation when sections were incubated with normal human serum that might be due to unspecific complement activation mechanisms like binding of naturally occurring autoantibodies (
Furthermore, because C1s blockade hampered anaphylatoxin formation, we also investigated its relevance on neutrophil functionality. For this, a chemotaxis assay was employed, using supernatants of the complement activation assay as chemoattractant. In line with the previous results, neutrophil chemoattraction is reduced by TNT003-dependent complement inhibition (Supplementary Figure S2).
Collectively, TNT003 is capable of completely blocking CP pathway activation, evidenced by the reduction of C4a and C5a production induced by incubation of sera from patients with BP on cryosections of human skin, and the reduction of C3 deposition in the complement activation test. Although only one-third of our patients demonstrated complement-fixing capacity, all 91 patients had C3 deposits at the dermal-epidermal junction. Thus, serum titers of complement-fixing antibodies do not reflect the local situation in skin, which is also supported by unchanged levels of complement factors in patient plasma. Consequently, the impact of complement inhibitor TNT003 on inflammation and blistering in BP needs to be evaluated in a clinical study. TNT009, the recently developed humanized IgG4 mAb version of TNT003, is currently being tested in a phase I clinical trial in patients with CP-mediated diseases, including BP (NCT02502903). Given favorable data from this phase I study, phase II clinical trials using TNT009 would be warranted in patients with BP.
Conflict of Interest
SP, ELR, and SH are employees and shareholders of the company True North Therapeutics that also financed parts of this study.
We thank Claudia Kauderer and Cindy Hass for excellent technical assistance as well as Ana Luiza Lima and Vanessa Krull for the management of human material.
Bullous pemphigoid is a potentially life-threatening autoantibody-mediated dermatosis characterized by blister formation. Experimental mouse models of bullous pemphigoid feature complement-induced inflammation and tissue damage. Kasprick et al. now provide preclinical data that utilize ex vivo human skin assays and support testing of complement inhibition as a therapeutic strategy in human bullous pemphigoid.