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在糖基化工程 CHO 细胞中高水平表达野生型和氧化抗性重组α-1-抗胰蛋白酶。

High-level production of wild-type and oxidation-resistant recombinant alpha-1-antitrypsin in glycoengineered CHO cells.

机构信息

Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, Quebec, Canada.

Life Sciences, Human Health Therapeutics Research Centre, Building Montreal-Royalmount, National Research Council Canada, Montréal, Quebec, Canada.

出版信息

Biotechnol Bioeng. 2022 Sep;119(9):2331-2344. doi: 10.1002/bit.28129. Epub 2022 May 25.

Abstract

Alpha-1-antitrypsin (A1AT) is a serine protease inhibitor which blocks the activity of serum proteases including neutrophil elastase to protect the lungs. Its deficiency is known to increase the risk of pulmonary emphysema as well as chronic obstructive pulmonary disease. Currently, the only treatment for patients with A1AT deficiency is weekly injection of plasma-purified A1AT. There is still today no commercial source of therapeutic recombinant A1AT, likely due to significant differences in expression host-specific glycosylation profile and/or high costs associated with the huge therapeutic dose needed. Accordingly, we aimed to produce high levels of recombinant wild-type A1AT, as well as a mutated protein (mutein) version for increased oxidation resistance, with N-glycans analogous to human plasma-derived A1AT. To achieve this, we disrupted two endogenous glycosyltransferase genes controlling core α-1,6-fucosylation (Fut8) and α-2,3-sialylation (ST3Gal4) in CHO cells using CRISPR/Cas9 technology, followed by overexpression of human α-2,6-sialyltransferase (ST6Gal1) using a cumate-inducible expression system. Volumetric A1AT productivity obtained from stable CHO pools was 2.5- to 6.5-fold higher with the cumate-inducible CR5 promoter compared to five strong constitutive promoters. Using the CR5 promoter, glycoengineered stable CHO pools were able to produce over 2.1 and 2.8 g/L of wild-type and mutein forms of A1AT, respectively, with N-glycans analogous to the plasma-derived clinical product Prolastin-C. Supplementation of N-acetylmannosamine to the cell culture media during production increased the overall sialylation of A1AT as well as the proportion of bi-antennary and disialylated A2G2S2 N-glycans. These purified recombinant A1AT proteins showed in vitro inhibitory activity equivalent to Prolastin-C and substitution of methionine residues 351 and 358 with valines rendered A1AT significantly more resistant to oxidation. The recombinant A1AT mutein bearing an improved oxidation resistance described in this study could represent a viable biobetter drug, offering a safe and more stable alternative for augmentation therapy.

摘要

α-1 抗胰蛋白酶(A1AT)是一种丝氨酸蛋白酶抑制剂,可阻断包括中性粒细胞弹性蛋白酶在内的血清蛋白酶的活性,从而保护肺部。已知其缺乏会增加患肺气肿和慢性阻塞性肺疾病的风险。目前,A1AT 缺乏症患者的唯一治疗方法是每周注射血浆纯化的 A1AT。目前,仍然没有商业来源的治疗性重组 A1AT,可能是由于表达宿主特异性糖基化谱的显著差异和/或与所需巨大治疗剂量相关的高成本。因此,我们旨在生产高水平的重组野生型 A1AT 以及一种突变蛋白(突变体)版本,以提高氧化抗性,其 N-糖基化类似于人血浆衍生的 A1AT。为了实现这一目标,我们使用 CRISPR/Cas9 技术破坏了控制核心 α-1,6-岩藻糖基化(Fut8)和 α-2,3-唾液酸化(ST3Gal4)的两个内源性糖基转移酶基因,然后使用 cumate 诱导表达系统过表达人 α-2,6-唾液酰转移酶(ST6Gal1)。与五个强组成型启动子相比,使用 cumate 诱导型 CR5 启动子可使稳定的 CHO 池中的 A1AT 体积生产率提高 2.5-6.5 倍。使用 CR5 启动子,糖基工程化的稳定 CHO 池能够分别产生超过 2.1 和 2.8 g/L 的野生型和突变体形式的 A1AT,其 N-糖基类似于血浆衍生的临床产品 Prolastin-C。在生产过程中向细胞培养物培养基中添加 N-乙酰甘露糖胺可提高 A1AT 的总体唾液酸化程度以及双天线和二唾液酸化 A2G2S2 N-糖基的比例。这些纯化的重组 A1AT 蛋白在体外显示出与 Prolastin-C 相当的抑制活性,并且将 351 和 358 位的蛋氨酸残基替换为缬氨酸使 A1AT 对氧化的抗性显著提高。本研究中描述的具有改善氧化抗性的重组 A1AT 突变体可能代表一种可行的生物改良药物,为增强疗法提供了一种安全且更稳定的替代方法。

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