Importance of the Side Chain at Position 296 of Antibody Fc in Interactions with FcγRIIIa and Other Fcγ Receptors.
Importance of the Side Chain at Position 296 of Antibody Fc in Interactions with FcγRIIIa and Other Fcγ Receptors.
复制标题
DOI：
10.1371/journal.pone.0140120
复制
发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Iida S
中科院分区：
文献类型：
--
作者：
Isoda Y;Yagi H;Satoh T;Shibata-Koyama M;Masuda K;Satoh M;Kato K;Iida S
抗体依赖性细胞毒性（ADCC）是决定治疗性抗体的临床功效的重要效应子功能。从免疫球蛋白G（IgG）Fc部分的N-聚糖中去除核心岩藻糖可提高对Fcγ受体IIIa（FcγRIIIa）的结合亲和力，并显著增强ADCC。我们之前的结构分析显示，IgG 1-Fc的Tyr-296在与FcγRIIIa的相互作用中起关键作用，特别是在增强非岩藻糖基化IgG 1的FcγRIIIa结合中。然而，尚未阐明抗体中Tyr-296残基在与各种Fcγ受体相互作用中的重要性。为了进一步阐明该残基的生物学重要性，我们建立了全面的Tyr-296突变体作为岩藻糖基化和非岩藻糖基化抗CD 20 IgG 1利妥昔单抗变体，并检查了其与重组可溶性人Fcγ受体：shFcγRI、shFc γ RIIa、shFcγRIIIa和shFcγRIIIb的结合。其中一些突变不仅影响抗体与shFcγRIIIa的结合，而且影响抗体与shFcγRIIa和shFcγRIIIb的结合，表明抗体中的Tyr-296残基也参与了与FcγRIIa和FcγRIIIb的相互作用。对于FcγRIIIa结合，几乎所有Tyr-296变体的结合亲和力均低于野生型抗体，无论其核心岩藻糖基化如何，尤其是在Y296 K和Y296 P中。值得注意的是，仅Y296 W突变体显示与FcγRIIIa的结合改善。非岩藻糖基化Y296 W突变体与含有两个N-聚糖的shFcγRIIIa复合物的3.00 nm分辨率晶体结构显示，Tyr-至-Trp取代增加了复合物中潜在接触原子的数量，从而改善了抗体与shFcγRIIIa的结合。相对于非岩藻糖基化野生型IgG 1，非岩藻糖基化Y296 W突变体保留了较高的ADCC活性，并显示出更高的FcγRIIa结合亲和力。我们的数据可能会提高我们对人IgG 1-Fc Tyr-296与各种Fcγ受体相互作用的生物学重要性的理解，并可用于调节治疗性抗体的IgG 1-Fc功能。
Antibody-dependent cellular cytotoxicity (ADCC) is an important effector function determining the clinical efficacy of therapeutic antibodies. Core fucose removal from N-glycans on the Fc portion of immunoglobulin G (IgG) improves the binding affinity for Fcγ receptor IIIa (FcγRIIIa) and dramatically enhances ADCC. Our previous structural analyses revealed that Tyr–296 of IgG1-Fc plays a critical role in the interaction with FcγRIIIa, particularly in the enhanced FcγRIIIa binding of nonfucosylated IgG1. However, the importance of the Tyr–296 residue in the antibody in the interaction with various Fcγ receptors has not yet been elucidated. To further clarify the biological importance of this residue, we established comprehensive Tyr–296 mutants as fucosylated and nonfucosylated anti-CD20 IgG1s rituximab variants and examined their binding to recombinant soluble human Fcγ receptors: shFcγRI, shFcγRIIa, shFcγRIIIa, and shFcγRIIIb. Some of the mutations affected the binding of antibody to not only shFcγRIIIa but also shFcγRIIa and shFcγRIIIb, suggesting that the Tyr–296 residue in the antibody was also involved in interactions with FcγRIIa and FcγRIIIb. For FcγRIIIa binding, almost all Tyr–296 variants showed lower binding affinities than the wild-type antibody, irrespective of their core fucosylation, particularly in Y296K and Y296P. Notably, only the Y296W mutant showed improved binding to FcγRIIIa. The 3.00 Å-resolution crystal structure of the nonfucosylated Y296W mutant in complex with shFcγRIIIa harboring two N-glycans revealed that the Tyr-to-Trp substitution increased the number of potential contact atoms in the complex, thus improving the binding of the antibody to shFcγRIIIa. The nonfucosylated Y296W mutant retained high ADCC activity, relative to the nonfucosylated wild-type IgG1, and showed greater binding affinity for FcγRIIa. Our data may improve our understanding of the biological importance of human IgG1-Fc Tyr–296 in interactions with various Fcγ receptors, and have applications in the modulation of the IgG1-Fc function of therapeutic antibodies.