UNL, CONICET, FBCB (School of Biochemistry and Biological Sciences), CBL (Biotechnological Center of Litoral), Ciudad Universitaria, Ruta Nacional 168, Km 472.4, C.C. 242, S3000ZAA, Santa Fe, Argentina.
Pharm Res. 2021 Jan;38(1):37-50. doi: 10.1007/s11095-020-02978-7. Epub 2021 Jan 14.
IFN4N is a glycoengineered version of recombinant human interferon alpha 2 (rhIFN-α2) that was modified to exhibit four N-glycosylation sites. It shows reduced in vitro specific biological activity (SBA) mainly due to R23 mutation by N23. However, it has improved pharmacokinetics and led to a high in vivo antitumor activity in mice. In order to prepare a new IFN-based biobetter, this work compares the influence of glycosylation (affecting pharmacokinetics) with the in vitro antiproliferative SBA on the in vivo efficacy.
Based on IFN4N, three groups of muteins were designed, produced, and characterized. Group A: variants with the same glycosylation degree (4N) but higher in vitro antiproliferative SBA (R23 restored); group B: muteins with higher glycosylation degree (5N) but similar in vitro antiproliferative activity; and group C: variants with improved glycosylation (5N and 6N) and in vitro antiproliferative bioactivity.
Glycoengineering was successful for improving pharmacokinetics, and R23 restoration considerably increased in vitro antiproliferative activity of new muteins compared to IFN4N. Hyperglycosylation was able to improve the in vivo efficacy similarly to or even better than R23 restoration. Additionally, the highest glycosylated mutein exhibited the lowest immunogenicity.
Hyperglycosylation constitutes a successful strategy to prepare a novel IFN biobetter.
IFN4N 是一种经过糖基化工程改造的重组人干扰素 α2（rhIFN-α2），经过修饰后表现出四个 N-糖基化位点。它显示出体外特定生物活性（SBA）降低，主要是由于 N23 导致 R23 突变。然而，它改善了药代动力学特性，并在小鼠体内表现出高抗肿瘤活性。为了制备一种新的基于 IFN 的生物类似药，本研究比较了糖基化（影响药代动力学）与体外增殖抑制 SBA 对体内疗效的影响。
基于 IFN4N，设计、生产并表征了三组突变体。组 A：具有相同糖基化程度（4N）但体外增殖抑制 SBA 更高的变体（R23 恢复）；组 B：糖基化程度更高（5N）但体外增殖抑制活性相似的突变体；组 C：具有改善的糖基化（5N 和 6N）和体外增殖生物活性的变体。
糖基化工程成功地改善了药代动力学特性，R23 恢复使新突变体的体外增殖抑制活性明显高于 IFN4N。高糖基化能够提高体内疗效，与 R23 恢复相当，甚至更好。此外，糖基化程度最高的突变体表现出最低的免疫原性。
高糖基化是制备新型 IFN 生物类似药的成功策略。
