Extending Mass Spectrometry Contribution to Therapeutic Monoclonal Antibody Lead Optimization: Characterization of Immune Complexes Using Noncovalent ESI-MS
Extending Mass Spectrometry Contribution to Therapeutic Monoclonal Antibody Lead Optimization: Characterization of Immune Complexes Using Noncovalent ESI-MS
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DOI：
10.1021/ac9007557
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发表时间：
2009-08-01
影响因子：
7.4
通讯作者：
Sanglier-Cianferani, Sarah
中科院分区：
文献类型：
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作者：
Atmanene, Cedric;Wagner-Rousset, Elsa;Sanglier-Cianferani, Sarah
单克隆抗体（mAb）对于治疗各种疾病（包括癌症、免疫性病症和其他病理学）已经具有越来越重要的意义。这些大的生物分子显示出特定的结构特征，这会影响它们的效率，因此需要使用灵敏的正交分析技术进行广泛的表征。其中，质谱（MS）已成为研究mAb氨基酸序列及其翻译后修饰的首选方法。在目前的工作中，最近的非共价MS技术，包括自动芯片的纳米电喷雾MS和行波离子迁移率MS首次用于表征免疫复合物，涉及鼠和人源化mAb 6 F4针对人JAM-A，一种新发现的抗原蛋白（Ag）在肿瘤细胞中过表达。基于MS的结构见解证明，重组JAM-A的异质二硫键配对既不改变其天然结构，也不改变mAb 6 F4识别特性。针对mAb：Ag复合物的研究显示，与鼠mAb类似，人源化mAb 6 F4以相似的亲和力选择性结合多达四种抗原分子，以这种方式证实了人源化过程的可靠性。非共价质谱似乎是治疗性mAb先导化合物表征和开发的额外支持技术。
Monoclonal antibodies (mAbs) have taken on an increasing importance for the treatment of various diseases including cancers, immunological disorders, and other pathologies. These large biomolecules display specific structural features, which affect their efficiency and need, therefore, to be extensively characterized using sensitive and orthogonal analytical techniques. Among them, mass spectrometry (MS) has become the method of choice to study mAb amino acid sequences as well as their post-translational modifications. In the present work, recent noncovalent MS-technologies including automated chip-based nanoelectrospray MS and traveling wave ion mobility MS were used for the first time to characterize immune complexes involving both murine and humanized mAb 6F4 directed against human JAM-A, a newly identified antigenic protein (Ag) overexpressed in tumor cells. MS-based structural insights evidenced that heterogeneous disulfide bridge pairings of recombinant JAM-A alter neither its native structure nor mAbs 6F4 recognition properties. Investigations focused on mAb:Ag complexes revealed that, similarly to murine mAb, humanized mAb 6F4 binds selectively up to four antigen molecules with a similar affinity, confirming in this way the reliability of the humanization process. Noncovalent MS appears as an additional supporting technique for therapeutic mAbs lead characterization and development.