title: Developing A New Mammalian Cell-Based Vaccine Manufacturing Platform and Novel Antibodies for the Treatment of Dengue Fever
Creators: name: Dyball, Laura Elizabeth
Publisher: name: University of Kent
Year: 2024

description: The development of biotherapeutics is an important field in the modern approach to treating and preventing current and emerging global health challenges. Vaccines and monoclonal antibodies are two biotherapeutic modalities that are particularly important in addressing the unmet need for the treatment or prevention of a wide range of diseases and conditions. Alongside the development of such biotherapeutics comes the need to be able to manufacture/produce these complex medicines at a quality and quantity suitable for treating the target population. Chinese hamster ovary (CHO) host cell lines are the gold-standard mammalian expression system used in bioprocessing for the expression of biotherapeutics due to their ability to produce high quality products with human-like post-translational modifications (particularly glycosylation) and a record of producing safe therapies at large scale. Due to these attributes and their ability to tolerate genetic engineering for manipulation of secretion and vesicular pathways, CHO cells are an excellent candidate for the development of a mammalian-cell based vaccine platform for manufacture of recombinant exosomes. Additionally, CHO cells are routinely used for the production of monoclonal antibodies (mAbs) and have been successfully used to produce difficult-to-express (DTE) antibodies. This thesis presents investigations into the development of (1) a 'plug &amp; play' CHO cell-based system for the expression of engineered exosomes which could be adapted for rapid response to outbreaks, and (2) the enhanced expression of a DTE novel anti-dengue antibody (Den54) using the CHO cell expression system via antibody sequence engineering. CHO-S host cells were shown to be a suitable cell host for the production of engineered exosomes to which can be targeted proteins/antigens of interest when fused (tagged) with exosome-targeting proteins. Two exosome targeting strategies were explored, ubiquitin-tagging and tetraspanin-tagging, both of which resulted in the production of engineered exosomes bearing fluorescent reporter proteins GFP and mCherry. However, of the strategies, a CD81 tetraspanin-tagging method resulted in more consistent targeting of cargo (GFP/mCherry) into recombinant exosomes, possibly due to the higher abundance of CD81+ exosome populations found in native CHO-S exosome populations. Further investigation using different SARS-CoV-2 structural proteins to test strategies to target antigens of varied lengths and complexities showed that successful generation of engineered exosomes is also dependent on the antigen targeted to them, with the Spike subunit 2 (S2) being most successfully targeted to recombinant CHO-S cell exosomes. The CHO-S host cell line was also used to investigate enhanced expression of a novel anti-dengue mAb that was DTE. Hybridisation or grafting of the DTE antibody sequences, particularly the variable regions, onto the constant and framework regions of well-characterised mAbs known to express well in CHO cells, trastuzumab and nivolumab, resulted in a marked improvement of secreted anti-dengue antibody. The molecule produced at the greatest yield, Den2, was a hybrid which contained more of the Den54 native sequence than its counterpart, Den4. Further investigation of the expression of these molecules and localisation of the antibody chains within the cells to determine where bottlenecking may be occurring, highlighted a problem with the Fc region of the native molecule resulting in a lack of secretory expression. This may be due to the formation of intra-ER crystals and the absence of the native Den54 Fc in the hybridised molecules allowed for higher secreted mAb amounts to be observed. The combined data described in this thesis provides evidence that CHO-S cells can be a platform for manufacture of engineered exosomes containing target antigens that could be investigated as vaccines and shows that protein engineering of a model mAb can improve the secretory expression of an anti-dengue DTE mAb and identify limitations upon its expression.