Control of recombinant monoclonal antibody effector functions by Fc N-glycan remodeling in vitro
Control of recombinant monoclonal antibody effector functions by Fc N-glycan remodeling in vitro
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DOI：
10.1021/bp050228w
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发表时间：
2005-11-01
影响因子：
2.9
通讯作者：
James, DC
中科院分区：
文献类型：
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作者：
Hodoniczky, J;Zheng, YZ;James, DC
IgG分子Fc结构域中Asn（297）处的N-聚糖是Fc受体介导的效应子功能（如抗体依赖性细胞介导的细胞毒性（ADCC）和补体依赖性细胞毒性（CDC））所必需的。在这项研究中，我们用杆状病毒感染的昆虫细胞产生的可溶性重组大鼠β-1，4-N-乙酰葡糖胺转移酶III（rGnTIII）特异性重塑了完整重组IgG（1）治疗性单克隆抗体（Mab）产品Rituxan和Herceptin的Fc N-聚糖。体外N-聚糖重塑允许二等分β 1，4-连接的GlcNAc受控和选择性转移到去半乳糖基化Mab N-聚糖的核心β-连接的甘露糖，以产生二等分GlcNAc：含量从31%变化到85%的Mab。通过正相HPLC和MALDI-MS分析N-聚糖证实了这一点，后者产生了203.2 Da的预期质量增加，没有其他寡糖修饰。分别使用外周血单核细胞作为效应细胞和CD 20（+）（SKW6.4和SU-DHL-4）或Her 2（+）（SKBR-3）靶细胞测定重塑Rituxan和Herceptin Mabs的ADCC。对于具有高（> 80%）二等分GlcNAc含量的两种重塑治疗性Mab，观察到ADCC保守的10倍增加。相比之下，尽管二等分GlcNAc的存在对CDC具有最小的影响，但相对于未修饰的（半乳糖基化的）对照Mab，Rituxan的去半乳糖基化使CDC降低约一半。总之，我们的数据表明，治疗性Mab Fc N-聚糖的体外重塑可用于控制Mab的体内治疗功效，并为重组Mab产品提供更“人源化”的糖型特征。
N-Glycans at Asn(297) in the Fc domain of IgG molecules are required for Fc receptor-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In this study we have specifically remodeled the Fc N-glycans of intact recombinant IgG(1) therapeutic monoclonal antibody (Mab) products, Rituxan and Herceptin, with a soluble recombinant rat beta-1,4-N-acetylglucosaminyltransferase III (rGnTIII) produced by baculovirus-infected insect cells. N-Glycan remodeling in vitro permitted a controlled and selective transfer of a bisecting beta 1,4-linked GlcNAc to the core beta-linked mannose of degalactosylated Mab N-glycans to yield Mabs varying in bisecting GlcNAc: content from 31% to 85%. This was confirmed by analysis of N-glycans by both normal phase HPLC and MALDI-MS, the latter yielding the expected mass increase of 203.2 Da with no other oligosaccharide modifications evident. ADCC of remodeled Rituxan and Herceptin Mabs was determined using peripheral blood mononuclear cells as effectors and either CD20(+) (SKW6.4 and SU-DHL-4) or Her2(+) (SKBR-3) target cells, respectively. A conserved 10-fold increase in ADCC was observed for both remodeled therapeutic Mabs with high (> 80%) bisecting GlcNAc content. In contrast, although the presence of a bisecting GlcNAc had minimal effect on CDC, degalactosylation of Rituxan reduced CDC by approximately half, relative to unmodified (variably galactosylated) control Mab. In summary, our data suggests that in vitro remodeling of therapeutic Mab Fc N-glycans may be utilized to control the therapeutic efficacy of Mabs in vivo and to offer a more "humanized" glycoform profile for recombinant Mab products.