A novel differential scanning fluorimetry analysis of a humanized anticocaine mAb and its ligand binding characteristics
A novel differential scanning fluorimetry analysis of a humanized anticocaine mAb and its ligand binding characteristics
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DOI：
10.1016/j.jim.2019.112676
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发表时间：
2020-01-01
影响因子：
2.2
通讯作者：
Wetzel, Hanna N.
中科院分区：
文献类型：
--
作者：
Kirley, Terence L.;Norman, Andrew B.;Wetzel, Hanna N.
一种名为h2 E2的抗可卡因单克隆抗体（mAb）将很快进入临床试验，用于治疗可卡因滥用障碍。重要的是，该抗体以高亲和力选择性结合可卡因及其活性代谢物古柯乙烯，而以显著较低的亲和力结合非活性代谢物。在这里，我们使用差示扫描荧光法（DSF）来表征该抗体及其可卡因结合Fab片段的稳定性和配体结合特性。通常用于DSF的Sypro橙子染料揭示了mAb和Fab片段的多个重叠的热蛋白质变性转变，使得通过热稳定进行配体结合的定量分析成问题。然而，通过使用“转子”染料，DASPMI（4-（4-（二甲基氨基）苯乙烯基）-N-甲基吡啶碘化物），其测量荧光染料的旋转限制（与测量染料微环境疏水性的Sypro橙子染料相反），对于h2 E2 mAb观察到通过配体结合稳定的简单的两态热变性转变，使得能够对DASPMI DSF mAb可卡因和代谢物结合数据进行玻尔兹曼拟合和定量热力学分析。计算的亲和力与使用其他技术测定的配体结合亲和力一致。因此，这种新的DASPMI DSF方法可以简单，廉价，非常迅速地产生h2 E2 mAb的配体结合常数，尽管存在多个，重叠，所有mAb的特征性的热解折叠蛋白质结构域。这种方法可能适用于目前用于许多研究和治疗应用的其他mAb。
An anti-cocaine monoclonal antibody (mAb) designated h2E2 will soon enter clinical trials for the treatment of cocaine abuse disorders. Importantly, this antibody selectively binds cocaine and its active metabolite, cocaethylene, with high affinity, while binding inactive metabolites with substantially lower affinities. Here, we used differential scanning fluorimetry (DSF) to characterize the stability and ligand binding properties of this antibody and its cocaine-binding Fab fragment. The Sypro orange dye commonly used for DSF revealed multiple overlapping thermal protein denaturation transitions for both the mAb and the Fab fragment, making quantitative analysis of ligand binding by thermal stabilization problematic. However, by using the "rotor" dye, DASPMI (4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide), which measures the rotational restriction of the fluorescent dye (as opposed to the Sypro orange dye which measures the hydrophobicity of the dye microenvironment), a simple two state thermal denaturation transition that is stabilized by ligand binding was observed for the h2E2 mAb, enabling Boltzmann fitting and quantitative thermodynamic analysis of the DASPMI DSF mAb cocaine and metabolite binding data. The computed affinities were consistent with ligand binding affinities determined using other techniques. Thus, this novel DASPMI DSF method can simply, inexpensively, and very rapidly generate ligand binding constants for the h2E2 mAb, despite the presence of multiple, overlapping, thermally unfolding protein domains characteristic of all mAbs. This approach is likely applicable to other mAbs currently in use for many research and therapeutic applications.