Probing the binding mechanism and affinity of tanezumab, a recombinant humanized anti-NGF monoclonal antibody, using a repertoire of biosensors
Probing the binding mechanism and affinity of tanezumab, a recombinant humanized anti-NGF monoclonal antibody, using a repertoire of biosensors
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DOI：
10.1110/ps.035402.108
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发表时间：
2008-08-01
期刊：
影响因子：
8
通讯作者：
Pons, Jaume
中科院分区：
文献类型：
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作者：
Abdiche, Yasmina Noubia;Malashock, Dan Stephen;Pons, Jaume
我们描述了使用四种互补生物传感器(Biacore 3000、Octet QK、ProteOn XPR36和KinExA 3000)来表征人神经生长因子(NGF)与人源化NGF中和单抗(tanezumab，以前称为RN624)结合的动力学。Tanezumab是治疗慢性疼痛的临床候选药物。我们的测量结果一致，NGF/tanezumab的结合亲和力比晚上10点更紧密，这是因为形成了一个极其稳定的络合物，估计半衰期超过100小时，这超出了我们任何方法的分辨率。该系统的研究特别具有挑战性，因为NGF是一种专有的同源二聚体，我们描述了各种测定方向和固定方法，这些方法在我们的实验中用于将亲和力降至最低，同时尽可能使NGF处于自然状态。我们还探索了NGF与其天然受体TrkA和P75的相互作用，以及tanezumab如何阻止它们。我们设计的Biacore阻断试验用于量化tanezumab的效力，比目前可用的基于细胞的功能分析更准确和可重复性。
We describe the use of four complementary biosensors ( Biacore 3000, Octet QK, ProteOn XPR36, and KinExA 3000) in characterizing the kinetics of human nerve growth factor ( NGF) binding to a humanized NGF- neutralizing monoclonal antibody ( tanezumab, formerly known as RN624). Tanezumab is a clinical candidate as a therapy for chronic pain. Our measurements were consistent with the NGF/ tanezumab binding affinity being tighter than 10 pM due to the formation of an extremely stable complex that had an estimated half- life exceeding 100 h, which was beyond the resolution of any of our methods. The system was particularly challenging to study because NGF is an obligate homodimer, and we describe various assay orientations and immobilization methods that were used to minimize avidity in our experiments while keeping NGF in as native a state as possible. We also explored the interactions of NGF with its natural receptors, TrkA and P75, and how tanezumab blocks them. The Biacore blocking assay that we designed was used to quantify the potency of tanezumab and is more precise and reproducible than the currently available cell- based functional assays.