Department of Neurology (M.G.H., Y.E.F.-G., I.E.v.E., J.J.P., J.J.V.), Department of Human Genetics (M.G.H., D.L.V., Y.F.-G., I.E.v.E., S.M.v.d.M.), Department of Hematology (M.T.K., H.V.), and Department of Rheumatology (L.M.S.), Leiden University Medical Center, The Netherlands.
Neurol Neuroimmunol Neuroinflamm. 2019 Feb 21;6(3):e547. doi: 10.1212/NXI.0000000000000547. eCollection 2019 May.
To isolate and characterize muscle-specific kinase (MuSK) monoclonal antibodies from patients with MuSK myasthenia gravis (MG) on a genetic and functional level.
We generated recombinant MuSK antibodies from patient-derived clonal MuSK-specific B cells and produced monovalent Fab fragments from them. Both the antibodies and Fab fragments were tested for their effects on neural agrin-induced MuSK phosphorylation and acetylcholine receptor (AChR) clustering in myotube cultures.
The isolated MuSK monoclonal antibody sequences included IgG1, IgG3, and IgG4 that had undergone high levels of affinity maturation, consistent with antigenic selection. We confirmed their specificity for the MuSK Ig-like 1 domain and binding to neuromuscular junctions. Monovalent MuSK Fab, mimicking functionally monovalent MuSK MG patient Fab-arm exchanged serum IgG4, abolished agrin-induced MuSK phosphorylation and AChR clustering. Surprisingly, bivalent monospecific MuSK antibodies instead activated MuSK phosphorylation and partially induced AChR clustering, independent of agrin.
Patient-derived MuSK antibodies can act either as MuSK agonist or MuSK antagonist, depending on the number of MuSK binding sites. Functional monovalency, induced by Fab-arm exchange in patient serum, makes MuSK IgG4 antibodies pathogenic.
从肌特异性激酶（MuSK）重症肌无力（MG）患者中分离和鉴定 MuSK 单克隆抗体，并在遗传和功能水平上对其进行分析。
我们从患者来源的克隆 MuSK 特异性 B 细胞中产生重组 MuSK 抗体，并从中制备单价 Fab 片段。我们测试了这些抗体和 Fab 片段对神经调节素诱导的 MuSK 磷酸化和乙酰胆碱受体（AChR）在肌管培养物中的聚集的影响。
分离出的 MuSK 单克隆抗体序列包括 IgG1、IgG3 和 IgG4，它们经历了高水平的亲和力成熟，与抗原选择一致。我们证实了它们对 MuSK Ig 样 1 结构域的特异性和对神经肌肉接头的结合。单价 MuSK Fab，模拟功能上单价的 MuSK MG 患者 Fab 臂交换的 IgG4，可消除神经调节素诱导的 MuSK 磷酸化和 AChR 聚集。令人惊讶的是，单价的特异性 MuSK 抗体反而激活了 MuSK 磷酸化，并部分诱导了 AChR 聚集，这与神经调节素无关。
患者来源的 MuSK 抗体可根据 MuSK 结合位点的数量，作为 MuSK 激动剂或 MuSK 拮抗剂发挥作用。在患者血清中 Fab 臂交换诱导的功能性单价，使 MuSK IgG4 抗体具有致病性。
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