The S228P Mutation Prevents in Vivo and in Vitro IgG4 Fab-arm Exchange as Demonstrated using a Combination of Novel Quantitative Immunoassays and Physiological Matrix Preparation
The S228P Mutation Prevents in Vivo and in Vitro IgG4 Fab-arm Exchange as Demonstrated using a Combination of Novel Quantitative Immunoassays and Physiological Matrix Preparation
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DOI：
10.1074/jbc.m114.600973
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发表时间：
2015-02-27
影响因子：
4.8
通讯作者：
Kirby, Hishani
中科院分区：
文献类型：
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作者：
Silva, John-Paul;Vetterlein, Olivia;Kirby, Hishani
当需要降低效应子功能时，人免疫球蛋白G同种型4（IgG 4）抗体（Ab）是免疫疗法的潜在候选物。IgG 4 Ab是能够经历称为Fab臂交换（FAE）的过程的动态分子。这导致具有未知特异性的功能性单价双特异性抗体（bsAb），并因此潜在地降低治疗功效。IgG 4 FAE被认为是一种重要的生物学机制，为IgG 4 Ab的抗炎活性提供了基础。迄今为止，FAE的机制尚未完全理解，并且测量离体基质中FAE的研究受到内源性IgG 4野生型（WT）Ab的存在和丰度的阻碍。使用代表性人源化WT IgG 4单克隆抗体，即抗IL-6和抗TNF，以及核心铰链稳定的丝氨酸228至脯氨酸（S228 P）抗IL-6 IgG 4突变体，首次证明了抗IgG 4亲和色谱法如何用于制备生理相关基质，以评估和定量FAE。还报道了一种使用单一MSD免疫测定定量FAE的新方法，并证实了先前的发现，即依赖于氧化还原条件，S228 P突变可以在体外和体内将IgG 4 FAE预防至不可检测的水平。总之，这些发现和新的方法将使研究人员能够监测和量化生理相关基质中他们自己的IgG 4分子的FAE。
Human immunoglobulin G isotype 4 (IgG4) antibodies (Abs) are potential candidates for immunotherapy when reduced effector functions are desirable. IgG4 Abs are dynamic molecules able to undergo a process known as Fab arm exchange (FAE). This results in functionally monovalent, bispecific antibodies (bsAbs) with unknown specificity and hence, potentially, reduced therapeutic efficacy. IgG4 FAE is suggested to be an important biological mechanism that provides the basis for the anti-inflammatory activity attributed to IgG4 Abs. To date, the mechanism of FAE is not entirely understood and studies measuring FAE in ex vivo matrices have been hampered by the presence and abundance of endogenous IgG4 wild-type (WT) Abs. Using representative humanized WT IgG4 monoclonal Abs, namely, anti-IL-6 and anti-TNF, and a core-hinge stabilized serine 228 to proline (S228P) anti-IL-6 IgG4 mutant, it is demonstrated for the first time how anti-IgG4 affinity chromatography can be used to prepare physiologically relevant matrices for assessing and quantifying FAE. A novel method for quantifying FAE using a single MSD immunoassay is also reported and confirms previous findings that, dependent on the redox conditions, the S228P mutation can prevent IgG4 FAE to undetectable levels both in vitro and in vivo. Together, the findings and novel methodologies will allow researchers to monitor and quantify FAE of their own IgG4 molecules in physiologically relevant matrices.