IgM anti-GM2 antibodies in patients with multifocal motor neuropathy target Schwann cells and are associated with early onset.

BACKGROUND: Multifocal motor neuropathy (MMN) is a rare, chronic immune-mediated polyneuropathy characterized by asymmetric distal limb weakness. An important feature of MMN is the presence of IgM antibodies against gangliosides, in particular GM1 and less often GM2. Antibodies against GM1 bind to motor neurons (MNs) and cause damage through complement activation. The involvement of Schwann cells (SCs), expressing GM1 and GM2, in the pathogenesis of MMN is unknown. METHODS: Combining the data of our 2007 and 2015 combined cross-sectional and follow-up studies in Dutch patients with MMN, we evaluated the presence of IgM antibodies against GM1 and GM2 in serum from 124 patients with MMN and investigated their binding to SCs and complement-activating properties. We also assessed the relation of IgM binding and complement deposition with clinical characteristics. RESULTS: Thirteen out of 124 patients (10%) had a positive ELISA titer for IgM anti-GM2. Age at onset of symptoms was significantly lower in MMN patients with anti-GM2 IgM. IgM binding to SCs correlated with IgM anti-GM2 titers. We found no correlation between IgM anti-GM2 titers and MN binding or with IgM anti-GM1 titers. IgM binding to SCs decreased upon pre-incubation of serum with soluble GM2, but not with soluble GM1. IgM anti-GM2 binding to SCs correlated with complement activation, as reflected by increased C3 fixation on SCs and C5a formation in the supernatant. CONCLUSION: Circulating IgM anti-GM2 antibodies define a subgroup of patients with MMN that has an earlier onset of disease. These antibodies probably target SCs specifically and activate complement, similarly as IgM anti-GM1 on MNs. Our data indicate that complement activation by IgM antibodies bound to SCs and MNs underlies MMN pathology.

Anti-C2 Antibody ARGX-117 Inhibits Complement in a Disease Model for Multifocal Motor Neuropathy.

BACKGROUND AND OBJECTIVES: To determine the role of complement in the disease pathology of multifocal motor neuropathy (MMN), we investigated complement activation, and inhibition, on binding of MMN patient-derived immunoglobulin M (IgM) antibodies in an induced pluripotent stem cell (iPSC)-derived motor neuron (MN) model for MMN. METHODS: iPSC-derived MNs were characterized for the expression of complement receptors and membrane-bound regulators, for the binding of circulating IgM anti-GM1 from patients with MMN, and for subsequent fixation of C4 and C3 on incubation with fresh serum. The potency of ARGX-117, a novel inhibitory monoclonal antibody targeting C2, to inhibit fixation of complement was assessed. RESULTS: iPSC-derived MNs moderately express the complement regulatory proteins CD46 and CD55 and strongly expressed CD59. Furthermore, MNs express C3aR, C5aR, and complement receptor 1. IgM anti-GM1 antibodies in serum from patients with MMN bind to MNs and induce C3 and C4 fixation on incubation with fresh serum. ARGX-117 inhibits complement activation downstream of C4 induced by patient-derived anti-GM1 antibodies bound to MNs. DISCUSSION: Binding of IgM antibodies from patients with MMN to iPSC-derived MNs induces complement activation. By expressing complement regulatory proteins, particularly CD59, MNs are protected against complement-mediated lysis. Yet, because of expressing C3aR, the function of these cells may be affected by complement activation upstream of membrane attack complex formation. ARGX-117 inhibits complement activation upstream of C3 in this disease model for MMN and therefore represents an intervention strategy to prevent harmful effects of complement in MMN.

ARGX-117, a therapeutic complement inhibiting antibody targeting C2.

BACKGROUND: Activation of the classical and lectin pathway of complement may contribute to tissue damage and organ dysfunction of antibody-mediated diseases and ischemia-reperfusion conditions. Complement factors are being considered as targets for therapeutic intervention. OBJECTIVE: We sought to characterize ARGX-117, a humanized inhibitory monoclonal antibody against complement C2. METHODS: The mode-of-action and binding characteristics of ARGX-117 were investigated in detail. Furthermore, its efficacy was analyzed in in vitro complement cytotoxicity assays. Finally, a pharmacokinetic/pharmacodynamic study was conducted in cynomolgus monkeys. RESULTS: Through binding to the Sushi-2 domain of C2, ARGX-117 prevents the formation of the C3 proconvertase and inhibits classical and lectin pathway activation upstream of C3 activation. As ARGX-117 does not inhibit the alternative pathway, it is expected not to affect the antimicrobial activity of this complement pathway. ARGX-117 prevents complement-mediated cytotoxicity in in vitro models for autoimmune hemolytic anemia and antibody-mediated rejection of organ transplants. ARGX-117 exhibits pH- and calcium-dependent target binding and is Fc-engineered to increase affinity at acidic pH to the neonatal Fc receptor, and to reduce effector functions. In cynomolgus monkeys, ARGX-117 dose-dependently reduces free C2 levels and classical pathway activity. A 2-dose regimen of 80 and 20 mg/kg separated by a week, resulted in profound reduction of classical pathway activity lasting for at least 7 weeks. CONCLUSIONS: ARGX-117 is a promising new complement inhibitor that is uniquely positioned to target both the classical and lectin pathways while leaving the alternative pathway intact.

Comprehensive Landscape of Nrf2 and p53 Pathway Activation Dynamics by Oxidative Stress and DNA Damage.

A quantitative dynamics pathway map of the Nrf2-mediated oxidative stress response and p53-related DNA damage response pathways as well as the cross-talk between these pathways has not systematically been defined. To allow the dynamic single cell evaluation of these pathways, we have used BAC-GFP recombineering to tag for each pathway's three key components: for the oxidative stress response, Keap1-GFP, Nrf2-GFP, and Srxn1-GFP; for the DNA damage response, 53bp1-GFP, p53-GFP, and p21-GFP. The dynamic activation of these individual components was assessed using quantitative high throughput confocal microscopy after treatment with a broad concentration range of diethyl maleate (DEM; to induce oxidative stress) and etoposide (to induce DNA damage). DEM caused a rapid activation of Nrf2, which returned to baseline levels at low concentrations but remained sustained at high concentrations. Srxn1-GFP induction and Keap1-GFP translocation to autophagosomes followed later, with upper boundaries reached at high concentrations, close to the onset of cell death. Etoposide caused rapid accumulation of 53bp1-GFP in DNA damage foci, which was later followed by the concentration dependent nuclear accumulation of p53-GFP and subsequent induction of p21-GFP. While etoposide caused activation of Srxn1-GFP, a modest activation of DNA damage reporters was observed for DEM at high concentrations. Interestingly, Nrf2 knockdown caused an inhibition of the DNA damage response at high concentrations of etoposide, while Keap1 knockdown caused an enhancement of the DNA damage response already at low concentrations of etoposide. Knockdown of p53 did not affect the oxidative stress response. Altogether, the current stress response landscapes provide insight in the time course responses of and cross-talk between oxidative stress and DNA-damage and defines the tipping points where cell injury may switch from adaptation to injury.