Dashivets Tetyana, Thomann Marco, Rueger Petra, Knaupp Alexander, Buchner Johannes, Schlothauer Tilman
Biochemical and Analytical Research, Large Molecule Research, Roche Pharma Research and Early Development (pRED), Roche Innovation Center, Penzberg, Germany.
Center for Integrated Protein Science Munich, Department Chemie, Technische Universität München, 85748, Garching, Germany.
PLoS One. 2015 Dec 11;10(12):e0143520. doi: 10.1371/journal.pone.0143520. eCollection 2015.
Therapeutic performance of recombinant antibodies relies on two independent mechanisms: antigen recognition and Fc-mediated antibody effector functions. Interaction of Fc-fragment with different FcR triggers antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity and determines longevity of the antibody in serum. In context of therapeutic antibodies FcγRs play the most important role. It has been demonstrated that the Fc-attached sugar moiety is essential for IgG effector functionality, dictates its affinity to individual FcγRs and determines binding to different receptor classes: activating or inhibitory. In this study, we systematically analyze effector functions of monoclonal IgG1 and its eight enzymatically engineered glycosylation variants. The analysis of interaction of glycovariants with FcRs was performed for single, as well as for antigen-bound antibodies and IgGs in a form of immune complex. In addition to functional properties we addressed impact of glycosylation on the structural properties of the tested glycovariants. We demonstrate a clear impact of glycosylation pattern on antibody stability and interaction with different FcγRs. Consistent with previous reports, deglycosylated antibodies failed to bind all Fcγ-receptors, with the exception of high affinity FcγRI. The FcγRII and FcγRIIIa binding activity of IgG1 was observed to depend on the galactosylation level, and hypergalactosylated antibodies demonstrated increased receptor interaction. Sialylation did not decrease the FcγR binding of the tested IgGs; in contrast, sialylation of antibodies improved binding to FcγRIIa and IIb. We demonstrate that glycosylation influences to some extent IgG1 interaction with FcRn. However, independent of glycosylation pattern the interaction of IgG1 with a soluble monomeric target surprisingly resulted in an impaired receptor binding. Here, we demonstrate, that immune complexes (IC), induced by multimeric ligand, compensated for the decreased affinity of target bound antibody towards FcRs, showing the importance of the IC-formation for the FcR- mediated effector functions.
抗原识别和Fc介导的抗体效应功能。Fc片段与不同FcR的相互作用引发抗体依赖性细胞毒性和补体依赖性细胞毒性，并决定抗体在血清中的半衰期。在治疗性抗体的背景下，FcγRs发挥着最重要的作用。已经证明，Fc连接的糖基部分对于IgG效应功能至关重要，决定其对各个FcγRs的亲和力，并决定与不同受体类别（激活型或抑制型）的结合。在本研究中，我们系统地分析了单克隆IgG1及其八个酶工程糖基化变体的效应功能。对糖基变体与FcRs的相互作用进行了分析，包括单体形式以及抗原结合抗体和免疫复合物形式的IgG。除了功能特性外，我们还研究了糖基化对测试糖基变体结构特性的影响。我们证明糖基化模式对抗体稳定性和与不同FcγRs的相互作用有明显影响。与先前的报道一致，去糖基化抗体除了高亲和力的FcγRI外，无法结合所有Fcγ受体。观察到IgG1的FcγRII和FcγRIIIa结合活性取决于半乳糖基化水平，高半乳糖基化抗体表现出增强的受体相互作用。唾液酸化并未降低测试IgG与FcγR的结合；相反，抗体的唾液酸化改善了与FcγRIIa和IIb的结合。我们证明糖基化在一定程度上影响IgG1与FcRn的相互作用。然而，与糖基化模式无关，IgG1与可溶性单体靶标的相互作用令人惊讶地导致受体结合受损。在这里，我们证明，由多聚配体诱导的免疫复合物（IC）补偿了靶标结合抗体对FcRs亲和力的降低，显示了IC形成对FcR介导的效应功能的重要性。
