Synthetic Glycopeptides Allow for the Quantitation of Scarce Nonfucosylated IgG Fc N-Glycans of Therapeutic Antibody,ACS Medicinal Chemistry Letters

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Synthetic Glycopeptides Allow for the Quantitation of Scarce Nonfucosylated IgG Fc N-Glycans of Therapeutic Antibody
ACS Medicinal Chemistry Letters ( IF 3.5 ) Pub Date : 2018-08-10 00:00:00 , DOI: 10.1021/acsmedchemlett.8b00127
Kazuki Hammura ₁ , Akari Ishikawa ₁ , Ravi Kumar H. V. ₁ , Risho Miyoshi ₂ , Yasuhiro Yokoi ₁ , Masakazu Tanaka ₂ , Hiroshi Hinou _{1,

2} , Shin-Ichiro Nishimura _{1,

2}

Affiliation

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 N-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 N-glycans expected to show much higher ADCC/CDC than abundant IgG N-glycans, and their application to the SRM-based quantitative glycoproteomics. Use of a key intermediate, phenyl (2-O-benzyl-4,6-O-benzylidine-β-d-mannopyranosyl)-(1 → 4)-3,6-di-O-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 trans-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.

中文翻译：

合成糖肽可以定量治疗抗体的稀少的非岩藻糖基化IgG Fc N-聚糖

连接至IgG Fc结构域的聚糖强烈影响生物学活性，例如治疗性抗体的抗体依赖性细胞毒性（ADCC）和补体依赖性细胞毒性（CDC）。然而，IgG的糖基依赖性功能调节的分子机制仍然难以捉摸。本研究表明，胰蛋白酶IgG Fc糖肽的基于反应监测（SRM）的选择测定是一种有潜力的表征抗体的方法，当与结构集中的合成Fc肽结合使用时，该Fc肽具有聚焦的N-糖型作为校准标准。我们描述了一种新的合成方法，将人IgG1 Fc肽分为两等分的十糖及其非等分的对应化合物，涉及具有罕见N的Fc结构域的抗体的签名-聚糖期望显示出比大量IgG N-聚糖高得多的ADCC / CDC ，并将其应用于基于SRM的定量糖蛋白组学中。关键中间体，苯基的使用（2- ø -苄基-4,6- ö -benzylidine-β- d -mannopyranosyl） - （1→4）-3,6-二- ø -苄基-2-叠氮基-2-刺槐豆胶半乳甘露聚糖衍生的-deoxy -1-thio-β- d - glucopyranoside极大地促进了二等分九糖的合成，这是酶促反式所需的稳定的恶唑啉衍生物的前体用在Asn297残基上带有GlcNAc的Fc九肽进行β-糖基化，而突变型end-M-N175Q催化的偶联反应进行得非常缓慢。令人惊讶的是，使用合成Fc糖肽作为校准标准品的SRM分析揭示了在158的胰蛋白酶消化物中携带非岩藻糖基化和一分为二（315 fmol，0.20％）及其未二等分的对应物（1154 fmol，0.73％）的目标IgG1 Fc片段的存在pmol抗癌抗体赫赛汀（trastuzumab）。结果表明异常糖基化的IgG Fc变体可能有助于治疗性抗体的总生物学活性。

更新日期：2018-08-10

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