Hammura Kazuki, Ishikawa Akari, H V Ravi Kumar, Miyoshi Risho, Yokoi Yasuhiro, Tanaka Masakazu, Hinou Hiroshi, Nishimura Shin-Ichiro
Division of Drug Discovery Research, Faculty of Advanced Life Science and Graduate School of Life Science, Hokkaido University, N21, W11, Kita-ku, Sapporo 001-0021, Japan.
Medicinal Chemistry Pharmaceuticals, Co., Ltd., N9, W15, Chuo-ku, Sapporo 060-0009, Japan.
ACS Med Chem Lett. 2018 Aug 10;9(9):889-894. doi: 10.1021/acsmedchemlett.8b00127. eCollection 2018 Sep 13.
Glycans attached to the IgG Fc domain affect strongly biological activities such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of therapeutic antibodies. However, molecular mechanism in the glycoform-dependent functional modulation of the IgGs remains elusive. The present study communicates that selected reaction monitoring (SRM)-based assay of tryptic IgG Fc glycopeptides is a promising approach for the characterization of antibodies when combined with structure-defined synthetic Fc peptides having a focused -glycoform as a calibration standard. We describe a novel synthetic approach to the human IgG1 Fc peptide having a bisected decasaccharide and its nonbisected counterpart compound, the signatures of antibodies involving Fc domain with rare -glycans expected to show much higher ADCC/CDC than abundant IgG -glycans, and their application to the SRM-based quantitative glycoproteomics. Use of a key intermediate, phenyl (2--benzyl-4,6--benzylidine-β-d-mannopyranosyl)-(1 → 4)-3,6-di--benzyl-2-azido-2-deoxy-1-thio-β-d-glucopyranoside, derived from locust bean gum galactomannan, facilitated greatly the synthesis of a bisected nonasaccharide as a stable precursor of oxazoline derivative needed for the enzymatic -glycosylation with Fc nonapeptide carrying a GlcNAc at Asn297 residue, while the coupling reaction catalyzed by mutant endo-M-N175Q proceeded very slowly. Strikingly, SRM assay using the synthetic Fc glycopeptides as calibration standards uncovered the occurrence of the targeted IgG1 Fc fragment carrying a nonfucosylated and bisected (315 fmol, 0.20%) and its nonbisected counterpart (1154 fmol, 0.73%) in the tryptic digests from 158 pmol of anticancer antibody Herceptin (trastuzumab). The results suggest that aberrantly glycosylated IgG Fc variants may contribute to the total biological activities of the therapeutic antibodies.
附着于IgG Fc结构域的聚糖强烈影响治疗性抗体的生物活性，如抗体依赖性细胞毒性（ADCC）和补体依赖性细胞毒性（CDC）。然而，IgG糖型依赖性功能调节的分子机制仍不清楚。本研究表明，基于选择反应监测（SRM）的胰蛋白酶IgG Fc糖肽分析是一种很有前景的抗体表征方法，当与具有特定糖型的结构明确的合成Fc肽作为校准标准结合使用时。我们描述了一种合成人IgG1 Fc肽的新方法，该肽具有一个平分的十糖及其未平分的对应化合物，涉及Fc结构域且带有稀有聚糖的抗体特征预计比丰富的IgG聚糖显示出更高的ADCC/CDC，以及它们在基于SRM的定量糖蛋白质组学中的应用。使用一种关键中间体，即由刺槐豆胶半乳甘露聚糖衍生而来的苯基（2 - O - 苄基 - 4,6 - O - 亚苄基 - β - D - 甘露吡喃糖基） - （1→4） - 3,6 - 二 - O - 苄基 - 2 - 叠氮基 - 2 - 脱氧 - 1 - 硫代 - β - D - 葡萄糖吡喃糖苷，极大地促进了平分九糖的合成，该九糖是与在Asn297残基处携带GlcNAc的Fc九肽进行酶促β - 糖基化所需的恶唑啉衍生物的稳定前体，而由突变型内切 - M - N175Q催化的偶联反应进行得非常缓慢。引人注目的是，使用合成Fc糖肽作为校准标准的SRM分析在158 pmol抗癌抗体赫赛汀（曲妥珠单抗）的胰蛋白酶消化物中发现了携带非岩藻糖基化且平分的靶向IgG1 Fc片段（315 fmol，0.20%）及其未平分的对应片段（1154 fmol，0.73%）。结果表明，异常糖基化的IgG Fc变体可能对治疗性抗体的总生物活性有贡献。
