Chen Tiffany F, Sazinsky Stephen L, Houde Damian, DiLillo David J, Bird Julie, Li Kevin K, Cheng George T, Qiu Huawei, Engen John R, Ravetch Jeffrey V, Wittrup K Dane
Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139, United States; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139, United States.
Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139, United States.
J Mol Biol. 2017 Aug 4;429(16):2528-2541. doi: 10.1016/j.jmb.2017.07.001. Epub 2017 Jul 8.
The binding of human IgG1 to human Fc gamma receptors (hFcγRs) is highly sensitive to the presence of a single N-linked glycosylation site at asparagine 297 of the Fc, with deglycosylation resulting in a complete loss of hFcγR binding. Previously, we demonstrated that aglycosylated human IgG1 Fc variants can engage the human FcγRII class of the low-affinity hFcγRs, demonstrating that N-linked glycosylation of the Fc is not a strict requirement for hFcγR engagement. In the present study, we demonstrate that aglycosylated IgG variants can be engineered to productively engage with FcγRIIIA, as well as the human Fc gamma RII subset. We also assess the biophysical properties and serum half-life of the aglycosylated IgG variants to measure stability. Aglycosylated constructs N297D/S298T (DTT)-K326I/A327Y/L328G (IYG) and N297D/S298A-IYG optimally drove tumor cell phagocytosis. A mathematical model of phagocytosis suggests that hFcγRI and hFcγRIIIA dimers were the main drivers of phagocytosis. In vivo tumor control of B16F10 lung metastases further confirmed the variant DTT-IYG to be the best at restoring wild-type-like properties in prevention of lung metastases. While deuterium incorporation was similar across most of the protein, several peptides within the CH2 domain of DTT-IYG showed differential deuterium uptake in the peptide region of the FG loop as compared to the aglycosylated N297Q. Thus, in this study, we have found an aglycosylated variant that may effectively substitute for wild-type Fc. These aglycosylated variants have the potential to allow therapeutic antibodies to be produced in virtually any expression system and still maintain effector function.
人IgG1与人类Fcγ受体（hFcγRs）的结合对Fc区天冬酰胺297处单个N-连接糖基化位点的存在高度敏感，去糖基化会导致hFcγR结合完全丧失。此前，我们证明无糖基化的人IgG1 Fc变体可与低亲和力hFcγRs的人FcγRII类结合，表明Fc的N-连接糖基化并非hFcγR结合的严格要求。在本研究中，我们证明无糖基化的IgG变体可被设计为能有效与FcγRIIIA以及人类FcγRII亚群结合。我们还评估了无糖基化IgG变体的生物物理性质和血清半衰期以测定稳定性。无糖基化构建体N297D/S298T（DTT）-K326I/A327Y/L328G（IYG）和N297D/S298A-IYG能最佳地驱动肿瘤细胞吞噬作用。吞噬作用的数学模型表明hFcγRI和hFcγRIIIA二聚体是吞噬作用的主要驱动因素。对B16F10肺转移瘤的体内肿瘤控制进一步证实变体DTT-IYG在预防肺转移方面最能恢复类似野生型的特性。虽然大部分蛋白质中的氘掺入情况相似，但与无糖基化的N297Q相比，DTT-IYG的CH2结构域内的几个肽段在FG环的肽区域显示出不同的氘摄取情况。因此，在本研究中，我们发现了一种可能有效替代野生型Fc的无糖基化变体。这些无糖基化变体有可能使治疗性抗体在几乎任何表达系统中产生，并且仍能保持效应器功能。
