Following the blueprint of plasma cells to design a yeast IgG factory

IgG antibodies are powerful biotherapeutics that are used in the treatment of several severediseases, for example cancer and autoimmune diseases. Specialized cells in the human immunesystem, plasma cells, naturally produce antibodies with high efficiency. However, biotechnologicalproduction methods based on mammalian cell cultures remain inadequate and expensive. The expanding market of antibody biotherapeutics has spurred the aspiration to develop alternativeproduction methods. One potential platform for antibody production is the yeast Saccharomycescerevisiae, which has been successfully engineered to produce a range of products by utilizing the versatile genetic toolkit available for modifying this organism. Plasma cell differentiation depicts a comprehensive molecular model of cellular transformation into an efficient antibody factory, and this model provides a blueprint for genetic engineering. In this thesis, we studied whether the key elements from plasma cells would improve IgG secretion in yeast. First, we modified the yeast ER to mimic the plasma cell ER morphology. Both an increase inER size and an altered shape, achieved by deletion of OPI1 and shape determinant genes, respectively, increased IgG secretion at least 2.4-fold. In addition, these mutants displayed a reduced stress response related to antibody production. We selected the strain with the OPI1 gene deletion for engineering of protein folding, along with the wild-type production strain. Relying on principles of synthetic biology, we created a modular plasmid library of mammalian folding factors shown to interact with IgG. To aid plasmid library creation, we established a new high-throughput cloning method to complement the available synthetic biology tools. Screening of the plasmid library led us to identify GRP170, BiP and FKBP2 as the most potent enhancers of IgG folding and secretion in yeast. We concluded that upregulation of ER-localized PPIase activity is critical for improving IgG titers in yeast. Additionally, we explored transcriptomics data from plasma cell differentiation to find genetargets which would otherwise be overlooked in engineering approaches. Through this datadrivenapproach, we selected seven novel genetic modifications to analyze in yeast. Two of these seven modifications, the overexpression of the genes GOT1 and IRE1 led to significant improvements in IgG secretion, resulting in a 1.6- and a 3.5-fold increase in specific product yields, respectively. However, in the future the emphasis should be in improving the quality of the secreted antibody. This thesis demonstrates that plasma cells are useful cellular models for antibody secretion,also when applied to an evolutionary distant species, such as the yeast S. cerevisiae. The IgGtiters were increased from 40 ng/ml to up to 160 ng/ml, confirming that this yeast is a promisingplatform for future applications of the biotherapeutics industry.