Autoantibodies against leucine-rich glioma inactivated 1 (LGI1) are found in patients with limbic encephalitis and focal seizures. Here, we generate patient-derived monoclonal antibodies (mAbs) against LGI1. We explore their sequences and binding characteristics, plus their pathogenic potential using transfected HEK293T cells, rodent neuronal preparations, and behavioural and electrophysiological assessments in vivo after mAb injections into the rodent hippocampus. In live cell-based assays, LGI1 epitope recognition was examined with patient sera (n = 31), CSFs (n = 11), longitudinal serum samples (n = 15), and using mAbs (n = 14) generated from peripheral B cells of two patients. All sera and 9/11 CSFs bound both the leucine-rich repeat (LRR) and the epitempin repeat (EPTP) domains of LGI1, with stable ratios of LRR:EPTP antibody levels over time. By contrast, the mAbs derived from both patients recognized either the LRR or EPTP domain. mAbs against both domain specificities showed varied binding strengths, and marked genetic heterogeneity, with high mutation frequencies. LRR-specific mAbs recognized LGI1 docked to its interaction partners, ADAM22 and ADAM23, bound to rodent brain sections, and induced internalization of the LGI1-ADAM22/23 complex in both HEK293T cells and live hippocampal neurons. By contrast, few EPTP-specific mAbs bound to rodent brain sections or ADAM22/23-docked LGI1, but all inhibited the docking of LGI1 to ADAM22/23. After intrahippocampal injection, and by contrast to the LRR-directed mAbs, the EPTP-directed mAbs showed far less avid binding to brain tissue and were consistently detected in the serum. Post-injection, both domain-specific mAbs abrogated long-term potentiation induction, and LRR-directed antibodies with higher binding strengths induced memory impairment. Taken together, two largely dichotomous populations of LGI1 mAbs with distinct domain binding characteristics exist in the affinity matured peripheral autoantigen-specific memory pools of individuals, both of which have pathogenic potential. In human autoantibody-mediated diseases, the detailed characterization of patient mAbs provides a valuable method to dissect the molecular mechanisms within polyclonal populations.

中文翻译：

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人单克隆 LGI1 自身抗体的独特结合特性决定了致病机制。


在患有边缘脑炎和局灶性癫痫发作的患者中发现了针对富含亮氨酸的神经胶质瘤失活 1 (LGI1) 的自身抗体。在这里，我们生成了源自患者的针对 LGI1 的单克隆抗体 (mAb)。我们利用转染的 HEK293T 细胞、啮齿动物神经元制剂以及将 mAb 注射到啮齿动物海马后的体内行为和电生理学评估来探索它们的序列和结合特征，以及它们的致病潜力。在基于活细胞的检测中，使用患者血清 ( n = 31)、脑脊液 ( n = 11)、纵向血清样本 ( n = 15) 以及从外周 B 细胞产生的单克隆抗体 ( n = 14) 检查 LGI1 表位识别两个病人的。所有血清和 9/11 CSF 均结合 LGI1 的富含亮氨酸重复序列 (LRR) 和表温蛋白重复序列​​ (EPTP) 结构域，并且随着时间的推移，LRR:EPTP 抗体水平的比率保持稳定。相比之下，来自两名患者的 mAb 均识别 LRR 或 EPTP 结构域。针对这两个域特异性的单克隆抗体表现出不同的结合强度，并具有明显的遗传异质性和高突变频率。 LRR 特异性 mAb 识别与其相互作用伙伴 ADAM22 和 ADAM23 对接的 LGI1，与啮齿动物脑切片结合，并诱导 HEK293T 细胞和活海马神经元中 LGI1-ADAM22/23 复合物的内化。相比之下，很少有 EPTP 特异性单克隆抗体与啮齿动物脑切片或 ADAM22/23 对接的 LGI1 结合，但都抑制 LGI1 与 ADAM22/23 的对接。海马内注射后，与 LRR 导向的 mAb 相比，EPTP 导向的 mAb 与脑组织的结合力要低得多，并且在血清中始终检测到。 注射后，两种结构域特异性单克隆抗体均消除了长时程增强诱导作用，而具有较高结合强度的 LRR 导向抗体则诱导记忆损伤。总而言之，在个体的亲和力成熟的外周自身抗原特异性记忆池中存在具有不同结构域结合特征的两个很大程度上二分的LGI1 mAb群体，两者都具有致病潜力。在人类自身抗体介导的疾病中，患者单克隆抗体的详细表征提供了一种有价值的方法来剖析多克隆群体内的分子机制。