Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, 428 Church St, Ann Arbor, MI 48109, United States.
Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, 428 Church St, Ann Arbor, MI 48109, United States.
Int J Pharm. 2023 Mar 25;635:122646. doi: 10.1016/j.ijpharm.2023.122646. Epub 2023 Jan 26.
FDA-approved anti-TNFα biopharmaceuticals are successful in treating a range of autoimmune diseases. However, not all anti-TNFα products are identical in their patient outcomes, suggesting that there may be product-specific differences stemming from protein structural differences, doses and routes of administration. In this work, we focus only on structural and functional differences across three full-length anti-TNFα mAbs (Humira®, Remicade®, and Simponi Aria®) to better understand the implications of such differences on the products' efficacy. For structural characterization, we quantified N-glycans using mass spectrometry and fluorescence labeling. From these studies, we observed that Remicade® had the highest percent of afucosylated glycans (15.5 ± 1.3 %) and the largest number of unique glycans, 28. While Humira® had the fewest unique glycans, 15, and 11.4 ± 0.8 % of afucosylated, high-mannose glycans. For the functional studies we tested TNFα binding via ELISA, FcγRIIIa binding via AlphaLISA and effector function using an ADCC bioreporter assay. Humira® had a significantly lower EC50 (1.9 ± 0.1 pM) for ELISA and IC50 (10.5 ± 1.1 nM) for AlphaLISA, suggesting that Humira® has higher TNFα and FcγRIIIa binding affinity than Remicade® and Simponi Aria®. Humira® was also the most potent in the bioreporter assay with an EC50 value of 0.55 ± 0.03 nM compared to Remicade® (0.64 ± 0.04 nM) and Simponi Aria® (0.67 ± 0.03 nM). This comparison is significant as it highlights functional differences between mAbs with shared mechanisms of action when examined in a single laboratory and under one set of conditions.
美国食品药品监督管理局批准的抗 TNFα 生物制药在治疗一系列自身免疫性疾病方面取得了成功。然而，并非所有的抗 TNFα 产品在患者疗效方面都完全相同，这表明可能存在由于蛋白质结构差异、剂量和给药途径导致的产品特异性差异。在这项工作中，我们仅关注三种全长抗 TNFα mAb（修美乐®、类克®和欣普尼®）之间的结构和功能差异，以更好地理解这些差异对产品疗效的影响。为了进行结构表征，我们使用质谱和荧光标记定量测定 N-糖基化。从这些研究中，我们观察到类克®具有最高比例的去岩藻糖基化聚糖（15.5±1.3%）和最多的 28 种独特聚糖。而修美乐®具有最少的独特聚糖，为 15 种，以及 11.4±0.8%的去岩藻糖基化高甘露糖聚糖。对于功能研究，我们通过 ELISA 测试 TNFα 结合，通过 AlphaLISA 测试 FcγRIIIa 结合，通过 ADCC 生物报告器测定测试效应功能。修美乐®在 ELISA 中的 EC50（1.9±0.1pM）和 AlphaLISA 中的 IC50（10.5±1.1nM）均显著较低，表明修美乐®与类克®和欣普尼®相比，对 TNFα 和 FcγRIIIa 的结合亲和力更高。在生物报告器测定中，修美乐®也最有效，EC50 值为 0.55±0.03nM，而类克®为 0.64±0.04nM，欣普尼®为 0.67±0.03nM。这种比较很重要，因为它突出了在单个实验室和一组条件下检查时具有相同作用机制的 mAb 之间的功能差异。
