Agonist antibody to MuSK protects mice from MuSK myasthenia gravis

Abstract

Myasthenia gravis (MG) is a chronic and severe disease of the skeletal neuromuscular junction (NMJ) in which the effects of neurotransmitters are attenuated, leading to muscle weakness. In the most common forms of autoimmune MG, antibodies attack components of the postsynaptic membrane, including the acetylcholine receptor (AChR) or muscle-specific kinase (MuSK). MuSK, a master regulator of NMJ development, associates with the low-density lipoprotein-related receptor 4 (Lrp4) to form the signaling receptor for neuronal Agrin, a nerve-derived synaptic organizer. Pathogenic antibodies to MuSK interfere with binding between MuSK and Lrp4, inhibiting the differentiation and maintenance of the NMJ. MuSK MG can be debilitating and refractory to treatments that are effective for AChR MG. We show here that recombinant antibodies, derived from MuSK MG patients, cause severe neuromuscular disease in mice. The disease can be prevented by a MuSK agonist antibody, presented either prophylactically or after disease onset. These findings suggest a therapeutic alternative to generalized immunosuppression for treating MuSK MG by selectively and directly targeting the disease mechanism.

Keywords: autoimmune disease; myasthenia gravis; neuromuscular; synapse; therapeutic antibody.

Fig. 1.

Monovalent 3F6C and 3B5 antibodies cause neuromuscular disease. (A) Mice reached disease onset when O₂ consumption was reduced by 10% on two consecutive days, as shown for a single control mouse and a single mouse injected with 5 mg/kg either 3F6C or 3B5 (Left). The scatter plot (Right) shows the mean ± SEM levels of O₂ consumption at disease onset (n = 8 control mice and eight mice injected with either 3F6C or 3B5). The mean values on day 1 were set as 100%. (B) At disease onset, CO₂ production and energy expenditure were reduced by 20 to 30%. (C) At disease onset, synaptic size and AChR density were reduced by 60 to 70% (AChRs, red; axons and nerve terminals, green). (Scale bar, 10 m (micrometers)) The scatter plot shows the mean ± SEM values for >50 synapses/mouse in ≥8 mice. The mean values for control mice were set at 100%. (D, E) At disease onset, grip strength and performance on a rotarod were reduced (n = 8 mice injected with 3F6C or 3B5 and n = 8 noninjected control mice). A Mann–Whitney test was used in panels A, B, D, and E.

Fig. 2.

ARGX-119 reverses inhibition of Agrin-stimulated MuSK phosphorylation caused by 3F6C in cultures myotubes. C2C12 myotubes were treated with neuronal Agrin (1 nM), neuronal Agrin and monovalent 3F6C (10 nM), or neuronal Agrin and one-armed 3F6C, followed 30 min later by either motavizumab (10 nM) or ARGX-119 (10 nM). MuSK was immunoprecipitated, and Western blots were probed with antibodies to MuSK or p-Tyr (4G10) (28). MuSK phosphorylation was normalized to MuSK expression. 3F6C reduced Agrin-stimulated MuSK phosphorylation by 10-fold; subsequent treatment with ARGX-119 increased MuSK phosphorylation by eightfold. The scatter plot shows the mean (±SEM) levels of Agrin-stimulated MuSK phosphorylation, normalized to the level of MuSK expression, from three separate experiments.

Fig. 3.

Prophylactic treatment with ARGX-119 protects mice from neuromuscular disease caused by 3F6C. (A) Mice were injected with 3F6C and 1 d later with either motavizumab or ARGX-119. (B) All eight motavizumab-treated mice reached disease endpoint. 5/8 ARGX-119-treated mice survived to study endpoint. Log-rank (Mantel-Cox) test (**P < 0.005). (C) Within a week after motavizumab injection, O₂ consumption, CO₂ production, and lower energy expenditure were diminished, and all eight mice reached disease endpoint by 2 wk. Most (5/8) ARGX-119-treated mice failed to develop signs of disease. (D) AChRs (red) motor axons and nerve terminals (green) were stained at disease endpoint for motavizumab-treated mice and at study endpoint for surviving ARGX-119-treated mice. (Scale bar, 10 m (micrometers)) Synaptic size and AChR density were reduced by 77% and 70%, respectively, at disease endpoint in motavizumab-treated mice and by 50% at study endpoint in ARGX-treated mice. (E) Motor performance declined in motavizumab-treated mice, whereas ARGX-119-treatment fully protected mice from the debilitating effects of 3F6C.

Fig. 4.

ARGX-119 rescued mice from neuromuscular disease caused by monovalent 3F6C. (A) Mice were injected with 3F6C and either motavizumab or ARGX-119 at disease onset. (B) ARGX-119 rescued mice from lethality: All eight ARGX-119-injected mice survived to study endpoint, whereas all eight motavizumab-treated mice reached disease endpoint within 5 d after disease onset. Plots show the percentage of surviving mice. Log-rank (Mantel-Cox) test (****P < 0.0005; graph). (C) ARGX-119 reversed the decline in O₂ consumption, CO₂ production, and energy expenditure in all eight mice. In contrast, all eight mice treated with motavizumab continued to decline and reached disease endpoint 5 d after disease onset. (D) AChRs (red) and motor axons and nerve terminals (green) were visualized at disease endpoint for motavizumab-treated mice and at study endpoint for ARGX-119-treated mice. (Scale bar, 10 m (micrometers)) Synaptic size and AChR density were reduced by 88% and 90%, respectively, in mice injected with motavizumab. Synaptic deterioration was partially protected by ARGX-119, as synaptic size and synaptic AChR density were reduced by 41% and 55%, respectively. (E) Motavizumab-treated mice reached disease endpoint before motor performance could be assessed. In contrast, ARGX-119-treatment halted the decline in grip strength and improved rotarod performance.

Fig. 5.

ARGX-119 rescued mice from neuromuscular disease caused by monovalent 3B5. (A) Mice were injected with 3B5 and either motavizumab or ARGX-119 at disease onset. (B) ARGX-119 rescued mice from lethality: half (4/8) of the ARGX-119-treated mice survived to study endpoint, whereas all eight motavizumab-treated mice reached disease endpoint within 5 d after disease onset. Plots show the percentage of surviving mice. Log-rank (Mantel-Cox) test (**P < 0.005). (C) ARGX-119 reversed the decline in O₂ consumption, CO₂ production, and energy expenditure in half (4/8) of the mice. In contrast, all eight motavizumab-treated mice continued to decline and reached disease endpoint 5 d after disease onset. (D) AChRs (red) and motor axons and nerve terminals (green) were visualized at disease endpoint for motavizumab-treated mice and at study endpoint for ARGX-119-treated and untreated mice. (Scale bar, 10 m (micrometers)) Synaptic size and AChR density were reduced by 84% and 90%, respectively, in motavizumab-treated mice and by 40% in ARGX-119-treated mice. (E) Motor performance of motavizumab-treated mice declined over the first week, and mice reached disease-endpoint before the beginning of the second week. ARGX-119 halted the initial decline in grip strength and rotarod performance between 1 and 2 wk.

Fig. 6.

3F6C and 3B5 do not compete with ARGX-119 for binding to mouse MuSK. A monoclonal antibody to Strep-tag II was immobilized to a Biacore CM5 chip by amine coupling. (A) The full extracellular region of mouse MuSK, containing a Strep-tag II at the carboxy terminus, was allowed to bind to the monoclonal antibody. Buffer (red), 3F6C (green), or 3B5 (blue), followed by ARGX-119, were passed sequentially over the sensor. 3F6C and 3B5 bind additively with ARGX-119 to the sensor, indicating no competition between ARGX-119 and either 3F6C or 3B5 for binding to MuSK. (B) The Table shows that the difference between the RU value after binding of 3F6C or 3B5 and the RU value after binding of ARGX-119 is nearly identical to the RU value for ARGX-119 alone, indicating no competition between 3F6C or 3B5 and ARGX-119.