LINGO-1 is a membrane protein of the central nervous system (CNS) that suppresses myelination of axons. Preclinical studies have revealed that blockade of LINGO-1 function leads to CNS repair in demyelinating animal models. The anti-LINGO-1 antibody Li81 (opicinumab), which blocks LINGO-1 function and shows robust remyelinating activity in animal models, is currently being investigated in a Phase 2 clinical trial as a potential treatment for individuals with relapsing forms of multiple sclerosis (AFFINITY: clinical trial.gov number NCT03222973). Li81 has the unusual feature that it contains two LINGO-1 binding sites: a classical site utilizing its complementarity-determining regions and a cryptic secondary site involving Li81 light chain framework residues that recruits a second LINGO-1 molecule only after engagement of the primary binding site. Concurrent binding at both sites leads to formation of a 2:2 complex of LINGO-1 with the Li81 antigen-binding fragment, and higher order complexes with intact Li81 antibody. To elucidate the role of the secondary binding site, we designed a series of Li81 variant constructs that eliminate it while retaining the classic site contacts. These Li81 mutants retained the high affinity binding to LINGO-1, but lost the antibody-induced oligodendrocyte progenitor cell (OPC) differentiation activity and myelination activity in OPC- dorsal root ganglion neuron cocultures seen with Li81. The mutations also attenuate antibody-induced internalization of LINGO-1 on cultured cortical neurons, OPCs, and cells over-expressing LINGO-1. Together these studies reveal that engagement at both LINGO-1 binding sites of Li81 is critical for robust functional activity of the antibody.

中文翻译：

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抗-LINGO-1抗体奥皮单抗的功能活性需要靶标在二级结合位点参与。

LINGO-1是抑制轴突髓鞘形成的中枢神经系统（CNS）的膜蛋白。临床前研究表明，在脱髓鞘动物模型中，LINGO-1功能的阻断可导致中枢神经系统修复。抗LINGO-1抗体Li81（opicinumab）可以阻断LINGO-1的功能并在动物模型中显示强大的髓鞘再生活性，目前正在2期临床试验中进行研究，作为潜在的复发性多发性硬化症患者的治疗方法（亲和力：Clinical trial.gov编号NCT03222973）。Li81具有一个不寻常的特征，它包含两个LINGO-1结合位点：一个利用其互补决定区的经典位点和一个涉及Li81轻链框架残基的隐秘二级位点，该二级位点仅在初级结合后才募集第二个LINGO-1分子现场。在两个位点的并发结合会导致LINGO-1与Li81抗原结合片段形成2：2的复合物，并与完整的Li81抗体形成更高级的复合物。为了阐明二级结合位点的作用，我们设计了一系列Li81变异构建体，可在保留经典位点接触的同时将其消除。这些Li81突变体保留了与LINGO-1的高亲和力结合，但是在用Li81观察到的OPC背根神经节神经元共培养物中失去了抗体诱导的少突胶质祖细胞（OPC）分化活性和髓鞘形成活性。突变还减弱了抗体诱导的LINGO-1在培养的皮层神经元，OPC和过表达LINGO-1的细胞上的内在化。