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.

Precise epitope determination of therapeutic antibodies is of great value as it allows for further comprehension of mechanism of action, therapeutic responsiveness prediction, avoidance of unwanted cross reactivity, and vaccine design. The golden standard for discontinuous epitope determination is the laborious X-ray crystallography method. Here, we present a combinatorial method for rapid mapping of discontinuous epitopes by mammalian antigen display, eliminating the need for protein expression and purification. The method is facilitated by automated workflows and tailored software for antigen analysis and oligonucleotide design. These oligos are used in automated mutagenesis to generate an antigen receptor library displayed on mammalian cells for direct binding analysis by flow cytometry. Through automated analysis of 33930 primers an optimized single condition cloning reaction was defined allowing for mutation of all surface-exposed residues of the receptor binding domain of SARS-CoV-2. All variants were functionally expressed, and two reference binders validated the method. Furthermore, epitopes of three novel therapeutic antibodies were successfully determined followed by evaluation of binding also towards SARS-CoV-2 Omicron BA.2. We find the method to be highly relevant for rapid construction of antigen libraries and determination of antibody epitopes, especially for the development of therapeutic interventions against novel pathogens.

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.

We show the successful application of ancestral sequence reconstruction to enhance the activity of iduronate-2-sulfatase (IDS), thereby increasing its therapeutic potential for the treatment of Hunter syndrome-a lysosomal storage disease caused by impaired function of IDS. Current treatment, enzyme replacement therapy with recombinant human IDS, does not alleviate all symptoms, and an unmet medical need remains. We reconstructed putative ancestral sequences of mammalian IDS and compared them with extant IDS. Some ancestral variants displayed up to 2-fold higher activity than human IDS in in vitro assays and cleared more substrate in ex vivo experiments in patient fibroblasts. This could potentially allow for lower dosage or enhanced therapeutic effect in enzyme replacement therapy, thereby improving treatment outcomes and cost efficiency, as well as reducing treatment burden. In summary, we showed that ancestral sequence reconstruction can be applied to lysosomal enzymes that function in concert with modern enzymes and receptors in cells.

Protein therapeutics are increasingly important for modern medicine. Novel recombinant proteins developed today can bind towards their target with high specificity and with low adverse effect. This has enabled the treatment of diseases that for a few years ago were deemed uncurable. Discovery of therapeutic proteins is driven through protein engineering, a field that is in constant expansion. And, through artificial construction of recombinant proteins, a large array of diseases can be defeated. The function and quality of these protein therapeutics rely on the correct folding, assembly and residue modification that occurs during their production within a living production cell host. Furthermore, producing them in large quantities are essential for accessibility of the best biopharmaceuticals available. Commonly, mammalian cells are the production host of choice when it comes to production of biopharmaceuticals. Mainly, due to the conserved nature of protein expression pathways within its biological class. Although an evergrowing number of biopharmaceuticals are produced in mammalian cells, there is always room for improvement. Development of novel recombinant protein therapeutics rely on accurate production of the protein. And if this is not achieved, a potential biopharmaceutical will never see the light of day. Furthermore, limited production capabilities can hamper product quality, with less efficacy and increased side-effects as a result. This thesis examines several different pathways for improvements on recombinant protein production for pharmaceutical purposes in mammalian cells. First, the basics of recombinant protein technology and mammalian cell function is outlined. Followed by a summary of six scientific articles revolving within expression and characterization tools for mammalian produced proteins. In paper I, utilization of transcriptomics identifies genes involved in protein expression, which enable the production of a difficult-to-express protein with up to a 150-fold greater activity. Furthermore, in paper IV, transcriptomics reveals genomic differences in a novel cell line that exhibit several fold protein expression capabilities. Besides omics technologies, methods for recombinant protein expression and modification are presented that generate more useable product for several different protein families. And, a protocol for the generation of a pre matured split-GFP variant is presented. Lastly, in paper VI, a mammalian cell display method with an optimized setting that enables precise epitope mapping of glycosylated antigens in a high throughput manner is outlined. With this method, the epitope of four neutralizing antibodies against SARS-CoV-2 is determined. For all of the papers involved within the presented thesis, mammalian cell production of recombinant proteins is the common denominator. Exploring the capabilities of mammalian cell production of current and next-generation biopharmaceuticals is of utter importance to continue the struggle against the gruesome nature of human diseases.

Proteinläkemedel får stadigt en starkare ställning inom den moderna medicinen. Nya rekombinanta proteiner som utvecklas idag kan binda mot sitt mål med hög specificitet och med få sidoeffekter. Detta har möjliggjort behandling av sjukdomar som för bara några år sen var letala. Utvecklandet av terapeutiska proteiner möjliggörs av proteinteknik, ett relativt ungt område som är i konstant utveckling. Där artificiell konstruktion av rekombinanta proteiner möjliggör bekämpandet av en uppsjö av sjukdomar. För att uppnå rätt funktion och kvalité, så behöver terapeutiska proteiner vara korrekt producerade. Detta sker inom en levande produktions cell, där rätt veckning samt modifikation möjliggör dess konstruktion. Utöver detta så behöver även enorma kvantiteter av biofarmaceutiska läkemedel kunna produceras, för att säkerställa tillgången av de bästa läkemedel som finns att erbjuda. För detta ändamål används främst mammalieceller som produktionsvärd då tillvägagångsättet för proteinkonstruktion är konserverat inom den biologiska klassen. Men även om mammalieceller är bäst lämpade för ändamålet, så finns det ett stort utrymme för förbättringar hos dessa. Utvecklingen an nya rekombinanta terapeutiska proteiner är beroende av att tillverkningsprocessen fungerar, och om det ej uppnås så kommer potentiella nya läkemedel aldrig realiseras. Även funktionella tillverkningsprocesser med inneboende begränsningar kan påverka det producerade proteinet negativt, med en lägre och ogynnsam effektivitet som följd. I denna avhandling så undersöks flera olika tillvägagångsätt för att förbättra produktion av rekombinanta proteiner i mammalieceller. Inledningsvis så presenteras det fundamentala inom rekombinant proteinteknik samt mammaliecellers funktion för tillverkande av dessa. Följt av summeringen av sex vetenskapliga artiklar som behandlar metoder för uttryckandet samt karakteriseringen av proteiner tillverkade inom mammalieceller. I artikel I används transkriptomik för identifikation av gener som är involverade i tillverkningen av ett svåruttryckt protein, detta möjliggjorde en 150-faldig ökning av aktivitet hos produkten. Även i artikel IV så identifierades genomiska skillnader kopplade till produktionsökningen hos en ny cellinje med hjälp av transkriptomik. Förutom omik tekniker så presenteras metoder för uttryckandet samt modifieringen av rekombinanta proteiner som genererar mer funktionell produkt för flera olika proteinfamiljer. Även ett protokoll för genererandet av en split-GFP variant där ena delen av molekylen har fått forma fluoroforen i ett tidigare skede presenteras i artikel V. Avslutande så introduceras en optimerad process där ett membran-förankrat antigen möjliggör en detaljrik epitope mappning via mammalieceller. Med denna metod så identifieras inbindningen av fyra antikroppar mot SARS-CoV-2. För samtliga artiklar som presenteras i denna avhandling så är produktion av proteiner inom mammalieceller den gemensamma nämnaren. Utforskandet av möjligheterna inom produktion av rekombinanta proteiner i mammalieceller är av yttersta vikt för att producera funktionella biofarmaceutiska läkemedel både idag samt i framtiden. Vilket möjliggör vidare framgångar i förhindrandet av sjukdomars lidande.

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.

Complementation-dependent fluorescence is a powerful way to study co-localization or interactions between biomolecules. A split-GFP variant, involving the self-associating GFP 1-10 and GFP 11, has previously provided a convenient approach to measure recombinant protein titers in cell supernatants. A limitation of this approach is the slow chromophore formation after complementation. Here, we alleviate this lag in signal generation by allowing the GFP 1-10 chromophore to mature on a solid support containing GFP 11 before applying GFP 1-10 in analyses. The pre-maturated GFP 1-10 provided up to 150-fold faster signal generation compared to the non-maturated version. Moreover, pre-maturated GFP 1-10 significantly improved the ability of discriminating between Chinese hamster ovary (CHO) cell lines secreting GFP 11-tagged erythropoietin protein at varying rates. Its improved kinetics make the pre-maturated GFP 1-10 a suitable reporter molecule for cell biology research in general, especially for ranking individual cell lines based on secretion rates of recombinant proteins.