Hinge-deleted IgG4 blocker therapy for acetylcholine receptor myasthenia gravis in rhesus monkeys

Abstract

Autoantibodies against ion channels are the cause of numerous neurologic autoimmune disorders. Frequently, such pathogenic autoantibodies have a restricted epitope-specificity. In such cases, competing antibody formats devoid of pathogenic effector functions (blocker antibodies) have the potential to treat disease by displacing autoantibodies from their target. Here, we have used a model of the neuromuscular autoimmune disease myasthenia gravis in rhesus monkeys (Macaca mulatta) to test the therapeutic potential of a new blocker antibody: MG was induced by passive transfer of pathogenic acetylcholine receptor-specific monoclonal antibody IgG1-637. The effect of the blocker antibody (IgG4Δhinge-637, the hinge-deleted IgG4 version of IgG1-637) was assessed using decrement measurements and single-fiber electromyography. Three daily doses of 1.7 mg/kg IgG1-637 (cumulative dose 5 mg/kg) induced impairment of neuromuscular transmission, as demonstrated by significantly increased jitter, synaptic transmission failures (blockings) and a decrease in the amplitude of the compound muscle action potentials during repeated stimulations (decrement), without showing overt symptoms of muscle weakness. Treatment with three daily doses of 10 mg/kg IgG4Δhinge-637 significantly reduced the IgG1-637-induced increase in jitter, blockings and decrement. Together, these results represent proof-of principle data for therapy of acetylcholine receptor-myasthenia gravis with a monovalent antibody format that blocks binding of pathogenic autoantibodies.

Figure 1

Design and in vitro characterization of IgG4Δhinge. (a) Sequence alignment of residues 205–234 (EU-numbering convention) of human IgG1, IgG4 and IgG4Δhinge. (b) Purified human IgG1, IgG4 and IgG4Δhinge variants of mAb 637 analyzed by non-reducing SDS-PAGE (image is cropped to show relevant bands, the complete gel is shown in supplementary Fig. 5). (c) In silico model of IgG4Δhinge based on crystal structures 1ADQ (CH2-CH3 domains), 1MCO (Δhinge region) and 1HZH (Fab region). HCΔhinge depicted in green, LC depicted in pale green. (d) AChR surface down-modulation on TE671 cells by purified variants of mAb 637 as indicated. Pooled intravenous immunoglobulin (IVIg) is included as negative control (e) Inhibition of (auto-) antibody-mediated AChR surface down-modulation on TE671 cells by IgG4Δhinge-637. Different concentrations of the challenge autoantibody (IgG1-637) or a negative control (IVIG) were tested in combination with IgG4Δhinge-637; each curve corresponds to a single concentration of challenge antibody (0–10 nM) as shown in the key on the right.

Figure 2

Electrophysiological evaluation of IgG4Δhinge-637 treated rhesus monkeys. (a) Treatment schedule. Monkeys were treated with PBS or IgG4Δhinge-637 (IgG4Δhinge) on three consecutive days (T1-T3), 6 hours prior to challenge (Ch1-Ch3) with PBS or IgG1-637 (IgG1). Intercostal muscle biopsies, single fiber electromyography (SFMG) and compound muscle action potential (CMAP) measurements during repetitive nerve stimulation were performed before (pre) and 7 days after start of treatment. Red arrowheads indicate blood sampling. Mean consecutive difference (MCD) of the delay between motor nerve stimulation and muscle fiber action potential (Jitter) (b) and neuromuscular transmission failures (Blockings) (c) in the orbicularis oculi muscle in different treatment groups. Decrement amplitude (d) and area (e) in the CMAP of the extensor digitorum brevis muscle in different treatment groups. Reference values were recorded before start of treatment (Pre). Data represent mean ± SEM.

Figure 3

Electron microscopic analyses of intercostal neuromuscular junctions in rhesus monkeys. Transmission electron micrographs showing representative nerve boutons of 3 animals, either before (‘Prescreening’, a,b,c) or seven days after challenge with IgG4Δhinge-637 (d), IgG1-637 (e) or the combination of IgG4Δhinge-637 and IgG1-637 (f). Each micrograph has the dimension of 5 × 6 µm. Asterisks indicate nerve terminals/boutons and arrowheads point at the (intact) primary synaptic clefts. Arrows point to normal secondary postsynaptic clefts/folds; the daggers in panel e indicate widening of the primary synaptic cleft, where the presynaptic and the postsynaptic membrane were separated from each other. (g) The folding index (length of postsynaptic membrane/length of the corresponding presynaptic membrane), a measure of the degree of postsynaptic folding. (h) Blinded scoring of the normal versus widened synaptic clefts.

Figure 4

Quantitative Immunofluorescent analysis of NMJ endplates in rhesus monkey intercostal biopsies. Biopsies were obtained from each animal before (“Pre”) or 7 days after antibody challenge with IgG4Δhinge-637, IgG1-637 or the combination of both. (a) Representative photomicrographs from the four groups showing staining of the AChR (detected by alpha-bungarotoxin fluorescence), IgG (IgG + IgG4Δhinge), the membrane attack complex (MAC) and a merged image to show colocalization. Relative fluorescence intensities (RFU) of (b) MAC staining and (c) human IgG staining was normalized with AChR expression in individual endplates. Intensities were quantitated in a total of 589 endplates (5-158 endplates per biopsy) and averaged per biopsy. The number n indicates the number of animals/biopsies analyzed for each condition. Data represent means ± SEM.