Development of a CHOK1SV transient expression system for rapid generation of recombinant proteins

生物制药工业依赖于筛选和适应程序来确定能够在限定的介质中生长以产生重组蛋白(RP)的稳定、高效的转染体。当有必要筛选和评估潜在的RP药物靶点和/或更快地产生重组材料将允许更多的分子更快地进入首次人类研究时，这种程序在药物开发流水线的早期不是理想的。因此，瞬时基因表达为这类研究提供了一种快速产生RP的方法。生产生物治疗相关RPS的瞬时表达技术主要使用悬浮培养的人胚胎肾细胞(HEK293细胞)和磷酸钙沉淀法，尽管最近也使用了中国仓鼠卵巢(CHO)细胞。事实上，一些公司正在研究瞬时表达技术，使用非病毒DNA递送系统和在生物反应器中培养的细胞在几天内生产数百毫克的RPS。最近的报告还表明，使用一种系统的方法来提高瞬时过程中的滴度是可能的，包括优化载体和转染条件，使用细胞周期调节器(p18，p21)和成纤维细胞生长因子，以及在培养基中添加丙戊酸。在CHO细胞中，优化重链和轻链基因的比例以及使用补料分批培养产生利用CHO瞬时单抗(MAb)表达的IgG材料已被报道以提高产量，而重链和轻链转录物的基因优化也增加了CHO细胞瞬时表达期间mAb的产生水平。龙沙生物公司拥有一个CHO悬浮适应宿主细胞系(CHOK1SV)，与其他悬浮适应CHO细胞系相比，该细胞系能够达到较高的细胞浓度并保持较长时间的生存能力。龙沙还拥有自己专有的载体表达系统GS系统。在这个项目中，学生将开发一种新型的基于CHOK1SV和GS载体的瞬时表达系统，用于快速生产100‘S毫克蛋白。这将建立在学术和工业实验室的初步工作基础上，这些工作表明操纵瞬变系统可以导致产量增加。测试分子将包括模型单抗(基因优化和非优化)、tPA和rhEPO。首先，学生将研究载体修改后的传递系统和DNA内化到细胞核(例如，通过使用核定位序列和分割元件)。学生还将确定环境条件(如温度、pH值)、介质成分和饲料对产品产量的影响。学生还将研究宿主细胞工程策略，以提高瞬时表达过程的产量和速度。特别是，学生将专注于细胞周期工程(例如p21)和我们在实验室中展示的伴侣和折叠酶，这些研究增加了模型蛋白(例如Torsin A，翻译启动因子)的高水平瞬时表达。最后，痘苗病毒E3L蛋白在CHO株中的表达对mAb的瞬时表达有积极作用。E3L的作用是阻止对病毒感染做出反应的细胞基因的激活，特别是PKR。肯特实验室的工作表明，PKR的活性也可以通过操纵p58来调节，p58参与了阻止或调节这种激酶的活性。因此，学生将研究调节PKR活性对瞬时产量的影响。研究结果：1.新型表达载体CHOK1SV衍生宿主和加工(介质/饲料)技术的发展2.对CHOK1SV细胞瞬时表达RPS的限制

The biopharmaceutical industry relies upon screening and adaptation procedures to identify stable, productive transfectants able to grow in defined media to produce a recombinant protein (rP). This procedure is not ideal early in the drug development pipeline when it is necessary to screen and evaluate potential rP drug targets and/or when the more rapid generation of recombinant material would allow more molecules into first-in-human studies faster. Transient gene expression therefore provides a means for the generation of rapid amounts of rP for such studies. Transient expression technology for the production of biotherapeutic relevant rPs has mostly utilised suspension adapted human embryonic kidney cells (HEK293 cells) and calcium phosphate precipitation although recently Chinese hamster ovary (CHO) cells have been used. Indeed, a number of companies are investigating transient expression technology to produce hundreds of mgs of rPs in a matter of days using non-viral DNA delivery systems and cells cultured in bioreactors. Recent reports also demonstrate the potential to improve titres in transient processes using a systematic approach including optimisation of the vector and transfection conditions, use of cell cycle regulators (p18, p21) and fibroblast growth factor, and addition of valproic acid to the media. The optimisation of the ratio of IgG heavy and light chain genes and the use of fed-batch cultures when generating IgG material using CHO transient monoclonal antibody (mAb) expression have been reported to improve yields whilst gene optimisation of heavy and light chain transcripts also increases mAb production levels during transient expression in CHO cells. The project Lonza Biologics has a CHO suspension adapted host cell line (CHOK1SV) which is able to achieve high cell concentrations and maintain viability for long periods of time compared to other suspension adapted CHO lines. Lonza also has its own proprietary vector expression system, the GS System. In this project the student will develop a novel CHOK1SV and GS vector based (DNA) transient expression system for the rapid production of 100's of mg's of rPs. This will build upon preliminary work from both the academic and industrial labs that show manipulation of transient systems can lead to increased yields. The test molecules will include a model mAb (gene optimised and non-optimised), tPA and rhEPO. Initially the student will investigate delivery systems and internalisation of the DNA to the nucleus after modification of the vector (e.g. via use of nuclear localisation sequences and partitioning elements). The student will also determine the influence of environmental conditions (e.g. temperature, pH) and media composition and feeding on product yield. The student will also investigate host cell engineering strategies to improve yield and speed of the transient expression process. In particular the student will focus upon cell cycle engineering (e.g. p21) and chaperones and foldases that we have shown in the laboratory in transient studies increases the high-level transient expression of model proteins (e.g. Torsin A, translation initiation factors). Finally, expression of the Vaccinia E3L protein in CHO strains has a positive effect upon transient expression of mAb. E3L acts to block the activation of cellular genes that respond to viral infection, in particular PKR. Work in the lab at Kent has shown that PKR activity can also be modulated by manipulation of p58 involved in preventing or modulating the activity of this kinase. The student will therefore investigate the effect of modulating PKR activity on transient yields. The outcomes: 1. Development of novel vector, CHOK1SV derived host and processing (media/feeds) technology for the expression of 100's mg of rPs 2. An understanding of the limitations upon transient expression of rPs from CHOK1SV cells