Optimization of Heavy Chain and Light Chain Signal Peptides for High Level Expression of Therapeutic Antibodies in CHO Cells

Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig) heavy chain (HC) and kappa light chain (LC) was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells.

新生蛋白通过信号肽（signal peptide）介导从细胞质（cytosol）转位至内质网（ER），是蛋白质分泌过程中的关键步骤。本研究旨在开发一种平台技术，用于优化信号肽以实现在中国仓鼠卵巢细胞（Chinese Hamster Ovary, CHO）中高效生产治疗性抗体。本研究构建了涵盖大量人类免疫球蛋白（immunoglobulin, Ig）重链（heavy chain, HC）与κ轻链（kappa light chain, LC）信号肽的数据库。多数重链信号肽含19个氨基酸，可划分为三个结构域；轻链信号肽则含22个氨基酸。随后，研究人员依据序列相似性对这些信号肽进行聚类分析。基于聚类结果，选取8条重链信号肽与2条轻链信号肽，分析它们对5款顶级畅销治疗性抗体——赫赛汀（Herceptin）、安维汀（Avastin）、英夫利昔单抗（Remicade）、利妥昔单抗（Rituxan）、阿达木单抗（Humira）——表达产量的影响。最终筛选出适配这5款抗体的最优重链与轻链信号肽。适配利妥昔单抗的优化后信号肽，其介导的抗体表达产量较公共数据库中的天然信号肽提升了一倍。针对安维汀优化得到的重链信号肽，若替换其中单个氨基酸，其介导的抗体表达产量会显著降低。质谱（mass spectrometry）分析结果显示，所有优化后的信号肽均可在成熟抗体中被精准切除。本研究结果对于将这5款抗体开发为生物类似药（biosimilar drug）具有重要意义，同时这些优化后的信号肽也有望成为在CHO细胞中生产新型抗体的最优选择。