药学学报, 2019, 54(1): 122-129
引用本文:
于传飞, 曹兴军, 王文波, 倪海晴, GUO Amy Y, 王兰. LC-MS法对抗PD-1信迪利单抗药物分子铰链区序列改构稳定性的研究[J]. 药学学报, 2019, 54(1): 122-129.
YU Chuan-fei, CAO Xing-jun, WANG Wen-bo, NI Hai-qing, GUO Amy Y, WANG Lan. LC-MS analysis of sintilimab as an anti-PD-1 therapeutic mab for its improved hinge stability study[J]. Acta Pharmaceutica Sinica, 2019, 54(1): 122-129.

LC-MS法对抗PD-1信迪利单抗药物分子铰链区序列改构稳定性的研究
于传飞1, 曹兴军2, 王文波1, 倪海晴2, GUO Amy Y2, 王兰1
1. 中国食品药品检定研究院, 北京 102629;
2. 信达生物制药(苏州)有限公司, 江苏 苏州 215123
摘要:
抗体药物目前已成为治疗自身免疫性疾病及肿瘤等多种疾病的重要创新药物。以IgG4为框架开发的抗体药物面临的挑战之一是野生型IgG4的铰链区不稳定,容易发生铰链区Fab臂交换现象。将IgG4分子的铰链区序列-CPSC-(野生型IgG4)改构成-CPPC-(改构型IgG4)是降低铰链区交换和提高IgG4类分子稳定性的有效手段。信迪利单抗(sintilimab)是用于肿瘤免疫治疗的全人源抗PD-1抗体,采用IgG4分子框架并对其铰链区序列进行了改构(S228P)。利用LC-MS检测方法从体外(PBS和人血清)到动物体内(SCID小鼠)对改构后的铰链区稳定性进行了评估。结果表明,LC-MS作为一种简便快速的方法可以有效地检测体外和体内IgG4分子的Fab臂交换反应,经IgG4分子改构后的信迪利单抗结构稳定,避免了Fab臂的交换现象。
关键词:    IgG4      Fab臂交换      LC-MS      PD-1      信迪利单抗     
LC-MS analysis of sintilimab as an anti-PD-1 therapeutic mab for its improved hinge stability study
YU Chuan-fei1, CAO Xing-jun2, WANG Wen-bo1, NI Hai-qing2, GUO Amy Y2, WANG Lan1
1. National Institutes for Food and Drug Control, Beijing 102629, China;
2. Innovent Biologics Suzhou Co. Ltd, Suzhou 215123, China
Abstract:
Monoclonal antibodies (mAbs) have been widely used as therapeutic drugs for treating diseases such as cancers and auto-immune diseases. When using an IgG4 isotype, one of the challenges is the instability of its hinge which is prone to Fab-arm exchange (FAE). The hinge sequence of a wild type IgG4 is -CPSC-, however, a single point mutation S228P from -CPSC-to -CPPC-can effectively diminish FAE, thereby improving hinge stability of the IgG4 molecule. Sintilimab is the fully human anti-PD-1 monoclonal antibody designed and developed for immuno-oncology, in which serine 228 in the hinge was engineered to proline to mitigate FAE. In this study, LC-MS is used to study hinge stability of sintilimab in both in vitro (PBS and human serum) and in vivo (SCID mouse) studies. The studies demonstrate that LC-MS is a fast and simple way to monitor for the occurrence of FAE in vitro and in vivo, and FAE can be eliminated by antibody engineering with a single point mutation.
Key words:    IgG4    Fab-arm exchange    LC-MS    PD-1    sintilimab   
收稿日期: 2018-06-21
DOI: 10.16438/j.0513-4870.2018-0574
基金项目: 中国医学科学院中央级公益性科研院所课题(2017PT31041);重大新药创制科技重大专项-创新生物技术药评价及标准化关键技术研究(2018ZX09101001-004).
通讯作者: 王兰,Tel:86-10-53852159,E-mail:wanglan@nifdc.org.cn
Email: wanglan@nifdc.org.cn
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参考文献:
[1] Jiang XR, Song A, Bergelson S, et al. Advances in the assessment and control of the effector functions of therapeutic antibodies[J]. Nat Rev Drug Discov, 2011, 10:101-111.
[2] Salfeld JG. Isotype selection in antibody engineering[J]. Nat Biotechnol, 2007, 25:1369-1372.
[3] Carter PJ. Potent antibody therapeutics by design[J]. Nat Rev Immunol, 2006, 6:343-357.
[4] Martin WL, West AP Jr, Gan L, et al. Crystal structure at 2.8 A of an FcRn/heterodimeric Fc complex:mechanism of pH-dependent binding[J]. Mol Cell, 2001, 7:867-877.
[5] Topalian SL, Drake CG, Pardoll DM. Targeting the PD-1/B7-H1(PD-L1) pathway to activate anti-tumor immunity[J]. Curr Opin Immunol, 2012, 24:207-212.
[6] Li Y, Li F, Jiang F, et al. A mini-review for cancer immunotherapy:molecular understanding of PD-1/PD-L1 pathway and translational blockade of immune checkpoints[J]. Int J Mol Sci, 2016, 17:1151.
[7] Lee HT, Lee JY, Lim H, et al. Molecular mechanism of PD-1/PD-L1 blockade via anti-PD-L1 antibodies atezolizumab and durvalumab[J]. Sci Rep, 2017, 7:5532.
[8] Rogstad S, Faustino A, Ruth A, et al. A retrospective evaluation of the use of mass spectrometry in FDA biologics license applications[J]. J Am Soc Mass Spectrom, 2017, 28:786-794.
[9] Rispens T, Ooijevaar-de Heer P, Bende O, et al. Mechanism of immunoglobulin G4 Fab-arm exchange[J]. J Am Chem Soc, 2011, 133:10302-10311.
[10] Schuurman J, Perdok GJ, Gorter AD, et al. The inter-heavy chain disulfide bonds of IgG4 are in equilibrium with intra-chain disulfide bonds[J]. Mol Immunol, 2001, 38:1-8.
[11] Angal S, King DJ, Bodmer MW, et al. A single amino acid substitution abolishes the heterogeneity of chimeric mouse/human (IgG4) antibody[J]. Mol Immunol, 1993, 30:105-108.
[12] Labrijn AF, Rispens T, Meesters J, et al. Species-specific determinants in the IgG CH3 domain enable Fab-arm exchange by affecting the noncovalent CH3-CH3 interaction strength[J]. J Immunol, 2011, 187:3238-3246.
[13] Labrijn AF, Buijsse AO, van den Bremer ET, et al. Therapeutic IgG4 antibodies engage in Fab-arm exchange with endogenous human IgG4in vivo[J]. Nat Biotechnol, 2009, 27:767-771.
[14] Beck A, Wurch T, Bailly C, et al. Strategies and challenges for the next generation of therapeutic antibodies[J]. Nat Rev Immunol, 2010, 10:345-352.
[15] Yang X, Zhang Y, Wang F, et al. Analysis and purification of IgG4 bispecific antibodies by a mixed-mode chromatography[J]. Anal Biochem, 2015, 484:173-179.
[16] Rispens T, den Bleker TH, Aalberse RC. Hybrid IgG4/IgG4 Fc antibodies form upon ‘Fab-arm’ exchange as demonstrated by SDS-PAGE or size-exclusion chromatography[J]. Mol Immunol, 2010, 47:1592-1594.
[17] van der Neut Kolfschoten M, Schuurman J, Losen M, et al. Anti-inflammatory activity of human IgG4 antibodies by dynamic Fab arm exchange[J]. Science, 2007, 317:1554-1557.
[18] Yang X, Wang F, Zhang Y, et al. Comprehensive analysis of the therapeutic IgG4 antibody pembrolizumab:hinge modification blocks half molecule exchange in vitro and in vivo[J]. J Pharm Sci, 2015, 104:4002-4014.