Cancer immunotherapy, where a patient's immune system is harnessed to eradicate cancer cells selectively, is a leading strategy for cancer treatment. However, successes with immune checkpoint inhibitors (ICI) are hampered by reported systemic and organ-specific toxicities and by two-thirds of the patients being non-responders or subsequently acquiring resistance to approved ICIs. Hence substantial efforts are invested in discovering novel targeted immunotherapies aimed at reduced side-effects and improved potency. One way is utilizing the dual targeting feature of bispecific antibodies, which have made them increasingly popular for cancer immunotherapy. Easy and predictive screening methods for activation ranking of candidate drugs in tumor contra non-tumor environments are however lacking. Herein, we present a cell-based assay mimicking the tumor microenvironment by co-culturing B cells with engineered human embryonic kidney 293 T cells (HEK293T), presenting a controllable density of platelet-derived growth factor receptor β (PDGFRβ). A target density panel with three different surface protein levels on HEK293T cells was established by genetic constructs carrying regulatory elements limiting RNA translation of PDGFRβ. We employed a bispecific antibody-affibody construct called an AffiMab capable of binding PDGFRβ on cancer cells and CD40 expressed by B cells as a model. Specific activation of CD40-mediated signaling of immune cells was demonstrated with the two highest receptor-expressing cell lines, Level 2/3 and Level 4, while low-to-none in the low-expressing cell lines. The concept of receptor tuning and the presented co-culture protocol may be of general utility for assessing and developing novel bi-specific antibodies for immuno-oncology applications.

Rare diseases are, despite their name, collectively common and millions of people are affected daily of conditions where treatment often is unavailable. Sulfatases are a large family of activating enzymes related to several of these diseases. Heritable genetic variations in sulfatases may lead to impaired activity and a reduced macromolecular breakdown within the lysosome, with several severe and lethal conditions as a consequence. While therapeutic options are scarce, treatment for some sulfatase deficiencies by recombinant enzyme replacement are available. The recombinant production of such sulfatases suffers greatly from both low product activity and yield, further limiting accessibility for patient groups. To mitigate the low product activity, we have investigated cellular properties through computational evaluation of cultures with varying media conditions and comparison of two CHO clones with different levels of one active sulfatase variant. Transcriptome analysis identified 18 genes in secretory pathways correlating with increased sulfatase production. Experimental validation by upregulation of a set of three key genes improved the specific enzymatic activity at varying degree up to 150-fold in another sulfatase variant, broadcasting general production benefits. We also identified a correlation between product mRNA levels and sulfatase activity that generated an increase in sulfatase activity when expressed with a weaker promoter. Furthermore, we suggest that our proposed workflow for resolving bottlenecks in cellular machineries, to be useful for improvements of cell factories for other biologics as well.

Aggregation of therapeutic bispecific antibodies negatively affects the yield, shelf-life, efficacy and safety of these products. Pairs of stable Chinese hamster ovary (CHO) cell lines produced two difficult-to-express bispecific antibodies with different levels of aggregated product (10-75% aggregate) in a miniaturised bioreactor system. Here, transcriptome analysis was used to interpret the biological causes for the aggregation and to identify strategies to improve product yield and quality. Differential expression-and gene set analysis revealed upregulated proteasomal degradation, unfolded protein response and autophagy processes to be correlated with reduced protein aggregation. Fourteen candidate genes with the potential to reduce aggregation were co expressed in the stable clones for validation. Of these, HSP90B1, DDIT3, AKT1S1, and ATG16L1, were found to significantly lower aggregation in the stable producers and two (HSP90B1 and DNAJC3) increased titres of the anti-HER2 monoclonal antibody trastuzumab by 50% during transient expression. It is suggested that this approach could be of general use for defining aggregation bottlenecks in CHO cells.

Biologics represent the fastest growing group of therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here, we identify metabolic engineering targets limiting expression of recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293 during recombinant expression. In an expression comparison of 24 difficult to express proteins, one third of the challenging human proteins displayed improved secretion upon host cell swapping from CHO to HEK293. Guided by a comprehensive transcriptomics comparison between cell lines, especially highlighting differences in secretory pathway utilization, a co-expression screening of 21 secretory pathway components validated ATF4, SRP9, JUN, PDIA3 and HSPA8 as productivity boosters in CHO. Moreover, more heavily glycosylated products benefitted more from the elevated activities of the N- and O-glycosyltransferases found in HEK293. Collectively, our results demonstrate the utilization of HEK293 for expression rescue of human proteins and suggest a methodology for identification of secretory pathway components for metabolic engineering of HEK293 and CHO.

Proteins are the building blocks of all living organisms enabling us to function and survive. There are more than 100,000 different proteins in the human body performing a variety of vital tasks. Examples of essential proteins are antibodies defending our body against foreign invaders and hemoglobulin responsible for importing oxygen to our cells and exporting carbon dioxide out from our cells. Consequently, mutations leading to dysfunctional proteins is the cause of many known diseases. Fortunately, the advancement of modern medicine has enabled proteins also to be employed as therapeutics to treat and cure various conditions. For instance, human insulin is recombinantly produced in the bacterium E. coli and is used as a biopharmaceutical to treat patients with Diabetes. The increased knowledge about diseases, their cause, and what cellular pathway to target has led to the discovery of many novel and complex biologics. Hence, the manufacturing of biopharmaceuticals is a rapidly emerging field that enables the production of complex molecules that are target-specific, effective, and highly active in the human body. Mammalian cell lines are often the preferred cell factories for manufacturing biologics since they generate proteins with human-like post-translational modifications, which are often essential features to obtain functional, safe, and effective therapeutics. Unfortunately, these life-saving biologics are costly, making them affordable for a fraction of patients worldwide. Therefore, one of the goals of the biotech industry is to make accessible biologics for everyone who needs it regardless of financial background. One way to achieve this goal is to engineer mammalian cell factories to improve the quantity and quality of biopharmaceuticals while reducing the production cost.

The results presented in this thesis are the outcome of five different studies aiming to improve and balance the expression levels of recombinant proteins in mammalian cell lines. In the first study, we investigated the productivity differences between mammalian cell lines from different origins. In the second and third projects, by utilizing transcriptomic analysis, helper genes were identified for improving the quantity and quality of two difficult-to-express biologics. The fourth study generated an easy-to-use toolbox for balancing the expression levels of recombinant proteins in mammalian cell lines. In the final project, the toolbox from the fourth project was employed to develop an in vitro cell-based cancer assay which is a crucial tool in cancer research and drug discovery.

In summary, this thesis provides strategies to improve the production process of biologics in mammalian cell lines and thereby contributes to the goal of offering safe, effective, and affordable medicine to patients in every part of this world.

Proteiner är livets byggstenar och därav nödvändiga för vår överlevnad. Det finns mer än 100,000 olika proteiner i människokroppen som utför åtskilliga och livsviktiga funktioner. Två exempel på viktiga och allmänkända proteiner är antikroppar, kroppens soldater som förvarar oss mot främmande mikroorganismer och hemoglobin som transporterar syre till kroppens olika organ och för bort den giftiga koldioxiden från cellerna. Följaktligen är mutationer som leder till dysfunktionella proteiner den främsta orsaken till majoriteten av kända sjukdomar. Lyckligtvis har den stora framgången inom forskning och medicin möjliggjort användandet av proteiner som läkemedel för behandling av olika sjukdomar. Till exempel är insulin som ett protein och används som läkemedel för diabetiker och som produceras rekombinant i bakterien E. coli. Den ökade kunskapen om sjukdomar, hur de uppkommit och vilka cellulära mekanismer som är viktiga för deras utveckling, har lett till upptäckten av flera nya och komplexa biologiska läkemedel. Detta har lett till att tillverkningen av bioläkemedel har blivit ett snabbt växande område som möjliggör produktion av komplexa molekyler som är målspecifika, effektiva och mycket aktiva i människokroppen. Däggdjurscellinjer är ofta det mest förekommande typen av cellfabriker för tillverkning av biologiska läkemedel då de är kapabla att generera proteiner med modifieringar som liknar det humana och som ofta är väsentliga för att erhålla funktionella, säkra och effektiva läkemedel. Tyvärr är dessa livräddande biologiska läkemedel mycket dyra, vilket gör dem tillgängliga för endast en bråkdel av patienter över hela världen. Därför är ett av målen för bioteknikindustrin att göra biologiska läkemedel tillgängliga för alla som behöver det oavsett ekonomisk bakgrund. Ett sätt att uppnå detta mål är att framställa effektivare däggdjurscellfabriker för att förbättra mängden och kvalitén på bioläkemedel och samtidigt reducera produktionskostnaden.

Resultaten som presenteras i denna avhandling är skörden av fem distinkta studier, som syftar till att förbättra och balansera uttrycksnivåerna av rekombinanta proteiner i däggdjurscellinjer. I den första studien undersökte vi skillnaderna i produktivitet mellan två däggdjurscellinjer från olika ursprung. I det andra och tredje projektet, genom att använda transkriptom-analys, identifierades hjälpargener för att förbättra kvantiteten och kvaliteten på två svåruttryckbara biologiska läkemedel. Den fjärde studien genererade en lättanvänd verktygslåda för att balansera uttrycksnivåerna av rekombinanta proteiner i däggdjurscellinjer. I det sista projektet användes verktygslådan från den fjärde studien för att utveckla en i vitro cellbaserad canceranalys-plattform som är ett verktyg för cancerforskning och upptäckter av nya läkemedel.

Sammanfattningsvis, presenterar denna avhandling verktyg för att kunna förbättra produktionsprocessen av biologiska läkemedel i däggdjurscellinjer och därmed bidrar till målet att erbjuda säkert, effektivt och överkomligt läkemedel till alla patienter i världen.

The proteins secreted by human tissues and blood cells, the secretome, are important both for the basic understanding of human biology and for identification of potential targets for future diagnosis and therapy. Here, a high-throughput mammalian cell factory is presented that was established to create a resource of recombinant full-length proteins covering the majority of those annotated as 'secreted' in humans. The full-length DNA sequences of each of the predicted secreted proteins were generated by gene synthesis, the constructs were transfected into Chinese hamster ovary (CHO) cells and the recombinant proteins were produced, purified and analyzed. Almost 1,300 proteins were successfully generated and proteins predicted to be secreted into the blood were produced with a success rate of 65%, while the success rates for the other categories of secreted proteins were somewhat lower giving an overall one-pass success rate of ca. 58%. The proteins were used to generate targeted proteomics assays and several of the proteins were shown to be active in a phenotypic assay involving pancreatic beta-cell dedifferentiation. Many of the proteins that failed during production in CHO cells could be rescued in human embryonic kidney (HEK 293) cells suggesting that a cell factory of human origin can be an attractive alternative for production in mammalian cells. In conclusion, a high-throughput protein production and purification system has been successfully established to create a unique resource of the human secretome.

Predictably regulating protein expression levels to improve recombinant protein production has become an important tool, but is still rarely applied to engineer mammalian cells. We therefore sought to set-up an easy-to-implement toolbox to facilitate fast and reliable regulation of protein expression in mammalian cells by introducing defined RNA hairpins, termed 'regulation elements (RgE)', in the 5'-untranslated region (UTR) to impact translation efficiency. RgEs varying in thermodynamic stability, GC-content and position were added to the 5'-UTR of a fluorescent reporter gene. Predictable translation dosage over two orders of magnitude in mammalian cell lines of hamster and human origin was confirmed by flow cytometry. Tuning heavy chain expression of an IgG with the RgEs to various levels eventually resulted in up to 3.5-fold increased titers and fewer IgG aggregates and fragments in CHO cells. Co-expression of a therapeutic Arylsulfatase-A with RgE-tuned levels of the required helper factor SUMF1 demonstrated that the maximum specific sulfatase activity was already attained at lower SUMF1 expression levels, while specific production rates steadily decreased with increasing helper expression. In summary, we show that defined 5'-UTR RNA-structures represent a valid tool to systematically tune protein expression levels in mammalian cells and eventually help to optimize recombinant protein expression.

Aggregation of therapeutic bispecific antibodies negatively affects the yield, shelf-life, efficacy and safety of the product. Pairs of stable Chinese hamster ovary cell lines produced two difficult- to-express bispecific antibodies with different levels of aggregated product (10-75% aggregate) in a miniaturized bioreactor system. Here, we analyse the cellular response and link to product aggregation by comparative transcriptome analysis of these CHO cells, to define biological causes and infer strategies to improve yield and quality. Differential expression- and gene set analysis revealed upregulated proteosomal degradation, unfolded protein response and autophagy processes to be correlated with reduction of protein aggregation. Fourteen candidate genes with potential to reduce aggregation were co-expressed in the stable clones for validation. Of these, HSP90B1, DDIT3, AK1S1, and ATG16L1, were found to significantly lower aggregation in the stable producers and two (HSP90B1 and DNAJC3) increased trastuzumab titres by 50% each during transient expression. We suggest our approach to be of general use for defining aggregation bottlenecks in CHO.

Biologics represent the fastest growing group of therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here, we identify bottlenecks limiting expression of recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293 during recombinant overexpression. Surprisingly, one third of the challenging human proteins displayed improved secretion upon host cell swapping from CHO to HEK293. While most components of the secretory machinery showed comparable expression levels in both expression hosts, genes with significant expression variation were identified. Among these, ATF4, SRP9, JUN, PDIA3 and HSPA8 were validated as productivity boosters in CHO. Further, more heavily glycosylated products benefitted more from the elevated activities of the N- and O-glycosyltransferases found in HEK293. Collectively, our results demonstrate the utilization of HEK293 for expression rescue of human proteins and suggest a methodology for identification of secretory pathway components improving recombinant protein yield in HEK293 and CHO.

Rare diseases are, despite their name, collectively common and millions of people are affected daily of conditions where treatment often is unavailable. Sulfatases are a large family of activating enzymes related to several of these diseases. Heritable genetic variations in sulfatases may lead to impaired activity and a reduced macromolecular breakdown within the lysosome, with several severe and lethal conditions as a consequence. While therapeutic options are scarce, treatment for some sulfatase deficiencies by recombinant enzyme replacement are available. However, such recombinant production of sulfatases suffers greatly from low product activity and yield, further limiting accessibility for patient groups. Here, we have addressed this problem by defining key-proteins necessary for active sulfatase secretion by comparison of CHO clones with different levels of production of active sulfatase. Quantitative transcriptomic analysis highlighted 14 key genes associated with sulfatase production, and experimental validation by co-expression improved the sulfatase enzyme activity by up to 150-fold. Furthermore, a correlation between product mRNA levels and sulfatase activity were observed and expression with lower activity promoters showed an increased in sulfatase activity. The workflow devised is general and we propose it to be useful for resolving bottlenecks in cellular machineries for improvement of cell factories for other biologics as well.