替代剪接可调整蛋白质亚基的表达比例

IF 5.6 2区 医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL
mAbs Pub Date : 2024-04-22 DOI:10.1080/19420862.2024.2342243
Christel Aebischer-Gumy, Pierre Moretti, Timothee Brunstein Laplace, Jana Frank, Ysaline Grand, Farid Mosbaoui, Emilie Hily, Anna Galea, Megane Peltret, Carole Estoppey, Daniel Ayoub, Roberto Giovannini, Martin Bertschinger
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引用次数: 0

摘要

摘要 以确定且稳定的比例控制两种或多种蛋白质的表达仍然是一项巨大的挑战,尤其是在双特异性和多特异性抗体领域。实现蛋白质亚基的最佳比例可以促进多聚蛋白的高效组装,并最大限度地减少副产品的产生。在本研究中,我们提出了一种基于另类剪接的解决方案,即在单个启动子的控制下,从相同的前核糖核酸(pre-mRNA)中表达可调且预定比例的两条不同的多肽链。这项研究中使用的前 mRNA 包含两个位于不同外显子上的开放阅读框。第一个外显子两侧是两个拷贝的鸡肌钙蛋白内含子 4(cTNT-I4),容易通过替代剪接从前 mRNA 中切除。这种特殊的设计可以通过调整剪接受体的强度来调节剪接比率。为了说明这种方法,我们开发了表达不同比例 GFP 和 dsRED 的构建体,并将其应用扩展到单克隆抗体等多聚蛋白,在 14 天的喂料批处理过程中达到了工业相关的表达水平(大于 1 克/升)。通过液滴数字 PCR,我们在一个培养了 28 天的稳定池中确认了剪接比的稳定性,同时通过完整质量分析评估了产品质量,结果表明不存在因不希望的剪接事件而产生的与产品相关的杂质。此外,我们还通过表达 BEAT® 型双特异性抗体的两个亚基展示了该构建体的多功能性,该抗体总共包含三个不同的亚基。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Alternative splicing for tuneable expression of protein subunits at desired ratios
ABSTRACT The controlled expression of two or more proteins at a defined and stable ratio remains a substantial challenge, particularly in the bi- and multispecific antibody field. Achieving an optimal ratio of protein subunits can facilitate the assembly of multimeric proteins with high efficiency and minimize the production of by-products. In this study, we propose a solution based on alternative splicing, enabling the expression of a tunable and predefined ratio of two distinct polypeptide chains from the same pre-mRNA under the control of a single promoter. The pre-mRNA used in this study contains two open reading frames situated on separate exons. The first exon is flanked by two copies of the chicken troponin intron 4 (cTNT-I4) and is susceptible to excision from the pre-mRNA by means of alternative splicing. This specific design enables the modulation of the splice ratio by adjusting the strength of the splice acceptor. To illustrate this approach, we developed constructs expressing varying ratios of GFP and dsRED and extended their application to multimeric proteins such as monoclonal antibodies, achieving industrially relevant expression levels (>1 g/L) in a 14-day fed-batch process. The stability of the splice ratio was confirmed by droplet digital PCR in a stable pool cultivated over a 28-day period, while product quality was assessed via intact mass analysis, demonstrating absence of product-related impurities resulting from undesired splice events. Furthermore, we showcased the versatility of the construct by expressing two subunits of a bispecific antibody of the BEAT® type, which contains three distinct subunits in total.
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来源期刊
mAbs
mAbs 工程技术-仪器仪表
CiteScore
10.70
自引率
11.30%
发文量
77
审稿时长
6-12 weeks
期刊介绍: mAbs is a multi-disciplinary journal dedicated to the art and science of antibody research and development. The journal has a strong scientific and medical focus, but also strives to serve a broader readership. The articles are thus of interest to scientists, clinical researchers, and physicians, as well as the wider mAb community, including our readers involved in technology transfer, legal issues, investment, strategic planning and the regulation of therapeutics.
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