IgG2 subclass antibodies have unique properties that include low effector function and a rigid hinge region. Although some IgG2 subclasses have been clinically tested and approved for therapeutic use, they have a higher propensity than IgG1 for aggregation, which can curtail or abolish their biological activity and enhance their immunogenicity. In this regard, acid-induced aggregation of monoclonal antibodies during purification and virus inactivation must be prevented. In the present study, we replaced the constant domain of IgG2 with that of IgG1, using anti-2,4-dinitrophenol (DNP) IgG2 as a model antibody, and investigated whether that would confer greater stability. While the anti-DNP IgG2 antibody showed significant aggregation at low pH, this was reduced for the IgG2 antibody containing the IgG1 CH2 domain. Substituting three amino acids within the CH2 domain-namely, F300Y, V309L, and T339A (IgG2_YLA)-reduced aggregation at low pH and increased CH2 transition temperature, as determined by differential scanning calorimetric analysis. IgG2_YLA exhibited similar antigen-binding capacity to IgG2, low affinity for FcγRIIIa, and low binding ability to C1q. The same YLA substitution also reduced the aggregation of panitumumab, another IgG2 antibody, at low pH. Our engineered human IgG2 antibody showed reduced aggregation during bioprocessing and provides a basis for designing improved IgG2 antibodies for therapeutic applications.

中文翻译：

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工程化在低pH下稳定的人IgG2抗体。

IgG2亚类抗体具有独特的特性，包括低效应子功能和刚性铰链区。尽管某些IgG2亚类已经过临床测试并被批准用于治疗，但是它们比IgG1具有更高的聚集倾向，这可以减少或消除其生物学活性并增强其免疫原性。在这方面，必须防止在纯化和病毒灭活期间酸诱导的单克隆抗体的聚集。在本研究中，我们使用抗-2,4-二硝基苯酚（DNP）IgG2作为模型抗体，将IgG2的恒定结构域替换为IgG1的恒定结构域，并研究了其是否具有更大的稳定性。尽管抗DNP IgG2抗体在低pH下显示出显着的聚集，但对于包含IgG1 CH2域的IgG2抗体却减少了。通过差示扫描量热分析确定，在低pH值下，在CH2域中替换三个氨基酸（即F300Y，V309L和T339A（IgG2_YLA））可减少聚集并增加CH2转变温度。IgG2_YLA表现出与IgG2相似的抗原结合能力，对FcγRIIIa的亲和力低，对C1q的结合能力低。在低pH值下，相同的YLA取代也减少了另一种IgG2抗体panitumumab的聚集。我们的工程人IgG2抗体在生物加工过程中显示出减少的聚集，并为设计用于治疗应用的改良IgG2抗体提供了基础。IgG2_YLA表现出与IgG2相似的抗原结合能力，对FcγRIIIa的亲和力低，对C1q的结合能力低。在低pH值下，相同的YLA取代也减少了另一种IgG2抗体panitumumab的聚集。我们的工程人IgG2抗体在生物加工过程中显示出减少的聚集，并为设计用于治疗应用的改良IgG2抗体提供了基础。IgG2_YLA表现出与IgG2相似的抗原结合能力，对FcγRIIIa的亲和力低，对C1q的结合能力低。在低pH值下，相同的YLA取代也减少了另一种IgG2抗体panitumumab的聚集。我们的工程人IgG2抗体在生物加工过程中显示出减少的聚集，并为设计用于治疗应用的改良IgG2抗体提供了基础。