Gstöttner Christoph, Lippold Steffen, Hook Michaela, Yang Feng, Haberger Markus, Wuhrer Manfred, Falck David, Schlothauer Tilman, Domínguez-Vega Elena
Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, Netherlands.
Protein Analytical Chemistry, Genentech, A Member of the Roche Group, South San Francisco, CA, United States.
Front Immunol. 2024 Feb 26;15:1347871. doi: 10.3389/fimmu.2024.1347871. eCollection 2024.
The antibody- FcγRIIIa interaction triggers key immunological responses such as antibody dependent cellular cytotoxicity (ADCC), making it highly important for therapeutic mAbs. Due to the direct glycan-glycan interaction with FcγRIIIa receptor, differences in antibody glycosylation can drastically influence the binding affinity. Understanding the differential binding of mAb glycoforms is a very important, yet challenging task due to the co-existence of multiple glycoforms in a sample. Affinity liquid chromatography (AC) and affinity capillary electrophoresis (ACE) hyphenated with mass spectrometry (MS) can provide glycoform-resolved affinity profiles of proteins based on their differences in either dissociation (AC) or equilibrium (ACE) constants. To cross-validate the affinity ranking provided by these complementary novel approaches, both techniques were benchmarked using the same FcγRIIIa constructs. Both approaches were able to assess the mAb - FcγRIIIa interaction in a glycoform selective manner and showed a clear increase in binding for fully versus hemi-fucosylated mAbs. Also, other features, such as increasing affinity with elevated galactosylation or the binding affinity for high mannose glycoforms were consistent. We further applied these approaches to assess the binding towards the F158 allotype of FcγRIIIa, which was not reported before. The FcγRIIIa F158 allotype showed a very similar profile compared to the V158 receptor with the strongest increase in binding due to afucosylation and only a slight increase in binding with additional galactosylation. Both techniques showed a decrease of the binding affinity for high mannose glycoforms for FcγRIIIa F158 compared to the V158 variant. Overall, both approaches provided very comparable results in line with orthogonal methods proving the capabilities of separation-based affinity approaches to study FcγR binding of antibody glycoforms.
抗体与FcγRIIIa的相互作用触发了关键的免疫反应，如抗体依赖性细胞毒性（ADCC），这对治疗性单克隆抗体非常重要。由于聚糖与FcγRIIIa受体之间存在直接的聚糖-聚糖相互作用，抗体糖基化的差异会极大地影响结合亲和力。由于样品中多种糖型共存，了解单克隆抗体糖型的差异结合是一项非常重要但具有挑战性的任务。与质谱（MS）联用的亲和液相色谱（AC）和亲和毛细管电泳（ACE）可以根据蛋白质在解离（AC）或平衡（ACE）常数上的差异，提供糖型解析的蛋白质亲和图谱。为了交叉验证这些互补的新方法提供的亲和力排名，两种技术都使用相同的FcγRIIIa构建体进行了基准测试。两种方法都能够以糖型选择性的方式评估单克隆抗体与FcγRIIIa的相互作用，并显示出完全岩藻糖基化与半岩藻糖基化单克隆抗体相比，结合力明显增加。此外，其他特征，如随着半乳糖基化增加亲和力或对高甘露糖糖型的结合亲和力，也是一致的。我们进一步应用这些方法来评估对FcγRIIIa的F158同种异型的结合，此前尚未有相关报道。与V158受体相比，FcγRIIIa F158同种异型显示出非常相似的图谱，岩藻糖基化导致结合力最强增加，额外半乳糖基化仅使结合力略有增加。与V158变体相比，两种技术都显示FcγRIIIa F158对高甘露糖糖型的结合亲和力降低。总体而言，两种方法提供了非常可比的结果，与正交方法一致，证明了基于分离的亲和方法研究抗体糖型FcγR结合的能力。
