EA7501 Groupe Innovation et Ciblage Cellulaire, Equipe Fc Récepteurs, Anticorps et MicroEnvironnement, Université de Tours, Tours, France.
Laboratoire d'Immunologie, CHRU de Tours, Tours, France.
Front Immunol. 2020 Feb 11;11:168. doi: 10.3389/fimmu.2020.00168. eCollection 2020.
The hinge region of immunoglobulin G (IgG) is involved in C1q and FcγRIIIA-expressing natural killer (NK) cell recruitment. Both heavy chains (HCs) of the hinge region can be cleaved sequentially by several proteases of the tumor/inflammatory/infectious microenvironment, including matrix metalloproteinase 12 (MMP12), or immunoglobulin-degrading enzyme from (IdeS), impairing Fc-mediated functions. The cleavage of therapeutic monoclonal antibodies (TmAbs), which are based on a human IgG1, IgG2 or IgG4 structure, has been poorly investigated, although it may represent an escape mechanism to these treatments. Therefore, we used non-reducing SDS-PAGE to compare the cleavage kinetics of five IgG1 TmAbs (trastuzumab, rituximab, cetuximab, infliximab, ipilimumab), one IgG2 TmAb (panitumumab), and two IgG4 TmAbs (nivolumab and pembrolizumab) by MMP12 and IdeS, which were found to cleave the first and second HCs with different kinetics. Panitumumab was more protease-resistant than IgG1 and IgG4 TmAbs. The latter were usually more protease-sensitive, whereas IgG1 TmAbs were usually cleaved with intermediate kinetics. However, we observed intra-subclass variability among IgG4 and IgG1 TmAbs. Nivolumab and pembrolizumab were cleaved similarly by MMP12, whereas pembrolizumab was more IdeS-resistant. Ipilimumab was more IdeS-sensitive and MMP12-resistant than the other IgG1 TmAbs, regardless of G1m allotype. In addition the Fc fragment of IgG1 TmAbs were highly resistant to cleavage by MMP12, whereas their cleavage kinetic by IdeS was very similar to that observed with the intact forms (excluding ipilimumab). Importantly, the cleavage kinetic of ipilimumab Fc fragment by IdeS was superimposable to that of trastuzumab, cetuximab and infliximab Fc fragment, showing that the variability observed for intact ipilimumab is unrelated to its Fc portion. We propose that the variability in the cleavage sensitivity/resistance balance among TmAbs of IgG1 and IgG4 subclasses results partially, from TmAb characteristics related to and/or located in the Fab region. Finally, with ELISA and flow cytometry, we observed that a single cleavage of IgG1 TmAbs greatly decreased their affinity for FcγRIIIA and C1q and their ability to induce FcγRIIIA-dependent functional responses of NK cells. Overall, our results indicate that the cleavage of the hinge region should be considered with TmAbs treatment and in the development of new molecules.
免疫球蛋白 G（IgG）的铰链区参与 C1q 和表达 FcγRIIIA 的自然杀伤（NK）细胞的募集。铰链区的两条重链（HCs）都可以被肿瘤/炎症/感染微环境中的几种蛋白酶（包括基质金属蛋白酶 12（MMP12）或源自金黄色葡萄球菌的免疫球蛋白降解酶（IdeS））依次切割，从而损害 Fc 介导的功能。尽管可能代表对这些治疗的逃避机制，但基于人 IgG1、IgG2 或 IgG4 结构的治疗性单克隆抗体（TmAb）的切割尚未得到充分研究。因此，我们使用非还原 SDS-PAGE 比较了五种 IgG1 TmAb（曲妥珠单抗、利妥昔单抗、西妥昔单抗、英夫利昔单抗、伊匹单抗）、一种 IgG2 TmAb（帕尼单抗）和两种 IgG4 TmAb（纳武单抗和派姆单抗）由 MMP12 和 IdeS 切割，发现它们以不同的动力学切割第一和第二 HCs。帕尼单抗比 IgG1 和 IgG4 TmAb 更具蛋白酶抗性。后者通常更易被蛋白酶切割，而 IgG1 TmAb 通常以中等动力学被切割。然而，我们观察到 IgG4 和 IgG1 TmAb 之间存在亚类内变异性。纳武单抗和派姆单抗被 MMP12 切割相似，而派姆单抗对 IdeS 的抗性更强。伊匹单抗对 IdeS 的敏感性和 MMP12 的抗性均高于其他 IgG1 TmAb，与 G1m 同种型无关。此外，IgG1 TmAb 的 Fc 片段对 MMP12 的切割具有高度抗性，而其 IdeS 切割动力学与完整形式非常相似（不包括伊匹单抗）。重要的是，伊匹单抗 Fc 片段被 IdeS 切割的动力学与曲妥珠单抗、西妥昔单抗和英夫利昔单抗 Fc 片段的动力学可叠加，表明对完整伊匹单抗的变异性与其 Fc 部分无关。我们提出，IgG1 和 IgG4 亚类的 TmAb 之间的切割敏感性/抗性平衡的变异性部分源于与 Fab 区域相关和/或位于 Fab 区域的 TmAb 特征。最后，通过 ELISA 和流式细胞术，我们观察到 IgG1 TmAb 的单次切割大大降低了它们与 FcγRIIIA 和 C1q 的亲和力以及诱导 NK 细胞 FcγRIIIA 依赖性功能反应的能力。总体而言，我们的结果表明，应考虑在 TmAb 治疗和新型分子的开发中考虑铰链区的切割。
