Develop pan-specific antibody against mutant glycyl-tRNA synthetase for treating CMT2D

摘要 Charcot-Marie-Tooth（CMT）疾病2D型是由编码甘氨酰-tRNA的加尔斯中的显性突变引起的 合成酶（GlyRS）。CMT是一种临床和遗传异质性的疾病集合，其中外周血淋巴细胞减少。 运动和感觉轴突退化。这些疾病特别影响周围神经系统， 其特征在于进行性运动神经元变性、肌肉萎缩和感觉丧失。没有CMT 疾病亚型具有靶向治疗，因此CMT仍然是未满足的医疗需求。遗传 异质性使得单一疗法不可能对所有形式的CMT有效。尽管基因 对于单基因疾病，如CMT，大量不同的突变， 参与，以及受每种突变影响的少数患者使经典基因治疗变得繁重， CMT2D。因此，确定适用于加尔斯不同突变的因果治疗策略将是 CMT 2D最有吸引力的治疗方法。同时，能够直接与GlyRS蛋白结合， 这本身就是CMT 2D补救的关键。我们以前的工作已经确定， 引起CMT的突变GlyRS起源于细胞外空间，突变蛋白在细胞外空间异常相互作用 与Neuropilin 1（Nrp 1）受体结合并拮抗对运动神经元维持重要的信号通路。 我们设计了一种新的策略，基于我们的GlyRS突变体的结构洞察力。我们发现， CMT 2D突变导致GlyRS中的共享构象变化，该变化在GlyRS中暴露了新的蛋白质表面。 二聚体与溶液的界面。通过使用来自GlyRS二聚体界面的两种不同肽作为抗原， 通过免疫接种，我们已经成功地获得了两种单克隆抗体（mAb）（每种肽一种）， 小鼠杂交瘤，可以阻断病理性Nrp 1相互作用，并表现出有前途的泛突变体的选择性。 这两种mAb候选物将表征其生物物理性质，并用于进行生物学分析。 在人类疾病的小鼠模型中的药效学、药代动力学和体内功效研究， 评估它们是否具有足够的生物活性以保证进一步开发治疗CMT 2D。

Abstract Charcot-Marie-Tooth (CMT) disease type 2D is caused by dominant mutations in GARS, encoding glycyl-tRNA synthetase (GlyRS). CMT is a clinically and genetically heterogeneous collection of disorders in which peripheral motor and sensory axons degenerate. The diseases specifically affect the peripheral nervous system and are characterized by progressive motor neuron degeneration, muscle atrophy, and sensory loss. None of the CMT disease subtypes have a targeted treatment, thus CMT remains an unmet medical need. The genetic heterogeneity makes it unlikely that a single therapy will be effective for all forms of CMT. Although gene therapies have emerged strongly for monogenic diseases such as CMT, the large number of different mutations involved, and the small number of patients affected by each mutation render classic gene therapy onerous for CMT2D. Identifying a causal treatment strategy applicable to different mutations in GARS would therefore be the most attractive therapeutic approach for CMT2D. Also, being able to directly engage with the GlyRS protein itself is key to CMT2D remediation. Our previous work has established that a major source of the toxicity of the CMT-causing mutant GlyRS is originated from the extracellular space, where mutant proteins aberrantly interact with Neuropilin 1 (Nrp1) receptor and antagonize a signaling pathway important for motor neuron maintenance. We designed a novel strategy based on our structural insight of the GlyRS mutants. We found that different CMT2D mutations caused a shared conformational change in GlyRS that exposes new protein surfaces at the dimer interface to solution. By using two different peptides from the dimer interface of GlyRS as antigens for immunization, we have successfully obtained two monoclonal antibodies (mAbs) (one for each peptide) from mouse hybridomas that can block the pathological Nrp1 interaction and exhibit promising pan-mutant selectivity. These two mAb candidates will be characterized for their biophysical properties, and used for conducting pharmacodynamic, pharmacokinetic, and in vivo efficacy studies in a mouse model of the human disease to evaluate whether they have sufficient biological activity to warrant further development to treat CMT2D.