| 1. |
- Hendrikse, Natalie
(författare)
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Engineering enzymes towards biotherapeutic applications using ancestral sequence reconstruction
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Enzymes are versatile biocatalysts that fulfill essential functions in all forms of life and, therefore, play an important role in health and disease. One specific application of enzymes in life science is their use as biopharmaceuticals, which typically benefits from high catalytic activity and stability. Increased stability and activity are both desirable properties for biopharmaceuticals as they are directly related to dosage, which in turn affects administration time, cost of production and potency of a drug. The aim of the work presented in this thesis is to enhance the therapeutic potential of enzymes by means of enzyme engineering, in particular using ancestral sequence reconstruction. In Paper I, we established the utility of this method in a model system and obtained ancestral terpene cyclases with increased activity, stability and substrate scope. In Paper II, we described the successful crystallization of the most stable ancestral terpene cyclase, which allowed for rational design of substrate specificity. Finally, we applied the method to two therapeutically relevant enzyme families associated with rare metabolic disorders. We obtained ancestral phenylalanine/tyrosine ammonia-lyases with substantially enhanced thermostability and long-term stability in Paper III and ancestral iduronate-2-sulfatases with increased activity in Paper IV. In summary, the results presented herein highlight the potential of ancestral sequence reconstruction as a method to obtain stable enzyme scaffolds for further engineering and to enhance therapeutic properties of enzymes.
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| 2. |
- Mebrahtu, Aman
(författare)
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Platforms and strategies harnessing signaling pathways of multifactorial diseases by multispecific antibodies
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Abstract [ENG]Proteins govern a multitude of biological functions vital to sustain life. The ability to withstand diseases development and harboring a defense against foreign pathogens is attributed to the wonders of the immune system and the proteins and cells its comprised of. Antibodies arguably stands as one of the most important protein classes involved in conferring immunity, able to recognize and engage, i.e., bind to pathogenic agents and exert their function. With the rise of engineered antibodies, the last three decades have ushered in an age of targeted therapeutics to address complex diseases with a more favorably efficacy and safety profile. However, an array of these diseases are governed by multifactorial variables often in interplay with each other, demanding a more broadened therapeutic strategy. Hence, significant efforts have been invested in the engineering of antibodies, expanding beyond a single speci- ficity to bi or multispecific molecules able to recognize more than one antigen within one and the same molecule. Naturally, this opens up for a multitude of functions i.e. mode of actions and harnessing of numerous diseases relevant pathways a multispe- cific potential drug compounds could carry out for a therapeutically beneficial out- come. The increased drug design complexity is accompanied with developability chal- lenges related to optimal drug design between the two or more binding specificities to achieve intended effect in a particular diseases’ biology setting. Moreover, the de- velopability profile of multispecific affinity proteins with regards to product yields and quality have long hampered the true translation-ability of these novel drug com- pounds into a clinical and industrial setting. The herein presented thesis aimed at highlighting the importance of a harmonized drug development pipeline taking into account aforementioned challenges and introducing toolboxes, platforms and work- flows to screen and optimize a variety of bispecific drug formats. Furthermore, we exploratively developed four unique bispecific constructs carrying out different mode- of-actions inhabiting partly rationally tailored design with respect to targeted dis- eases setting in the field of autoimmune diseases and oncology. The first bispecific molecule developed herein presented in paper I, aimed at target- ing the diseases condition in SLE from multiple fronts, with a dual blocking mode of action targeting two related ligands driving diseases progression. A lead bispecific AffiMab format chosen post screening of several candidates in molecular and in vitro systems was evaluated in ex vivo whole blood model assay demonstrating a signifi- cant effect by the dual blocking strategy to actively decrease the levels of the target ligands. The molecule warrants further evaluation in approriate in vivo models and ii ex vivo whole blood assay with patient derived material given the potential beneficial effect of the proposed therapeutic strategy based on the fundmenetal biology of the diseases and clinical observations. In paper II, a novel bispecific format able to de- liver cargo antigens to antigen presenting cells in a modular fashion was developed. Moreover, the bispecific exerts agonistic downstream signaling of targeted cells via CD40 engagement, synergistically priming immune cell activation whilst delivering the cargo antigen simultaneously. The delivery is based on an affinity interaction between a static peptide stretch synthesized with the antigen peptide sequence and a single chain attached to the structure of an anti-CD40 agonistic antibody. Employ- ment of the established adaptable drug affinity conjugate platform (ADAC) enabled the delivery of antigen cargo strictly dependent on the affinity interaction, inducing a significant anti-tumor response by expansion of antigen specific CD8+ T cells demonstrated in vivo. In paper III, we explore a HER2 and EGFR dual blocking strategy employing bis- pecific AffiMabs. The bispecifics demonstrated a significantly greater effect in an in vitro cell based assay compared to the combination treatment with the two monospe- cific molecules targeting respective antigen, indicating a potential synergistic effect conferred by the format. However, the effect of the molecule and potential benefit versus the monospecific or combination treatment need to be further evaluated in vivo. Paper IV aimed at harnessing the CD40 dendritic cell activation axis by a bispecific immune cell engager AffiMab, governing CD40 mediated activation depen- dent on the engagement with a stroma antigen upregulated in the tumor microenvi- ronment, platelet growth factor receptor B (PDGFRβ). The AffiMab demonstrated the intended mode of action in in vitro cell based model assays, and with isolated antigen presenting cells and B cells from healthy donor blood, albeit room for format optimization should be taken into consideration. The study warrants further investi- gation in appropriate in vivo models for treatment of solid tumors. In paper V we developed a modular platform to fine tune protein expression in mammalian cells on a translational level utilizing 5’UTR hairpin structure, herein coined as Regulatory elements (RgEs). Hypothesizing that “less is more” wherein a balanced expression of a proteins subunits was demonstrated to be of greater impor- tance than a maximum expression output of each component to apprehend correctly assembled protein product. The developed tool box holds possibility for multifaceted applications, and was extended in paper VI to the use in the establishment of an in vitro culture system to fine tune receptor densities on the cell surface of a defined iii cell line. The applications end-use would be functioning as an integrative part in the high throughput screening pipeline of bispecific immune cell engagers for early eval- uation and ranking of formats and access to target antigens impact on the function- ality of screened constructs. In summary, the herein presented work exploratively evaluated mode of actions, de- sign, format, and engineering of bispecific molecules to address both challenges in terms of achieving intended effect but equally important considerations and solutions to improve and evaluate product manufacturability early on in the drug development pipeline.
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| 3. |
- Moradi Barzadd, Mona, 1987-
(författare)
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Strategies to improve and balance the expression levels of recombinant proteins in mammalian cell lines
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 4. |
- Thalén, Niklas, 1985-
(författare)
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Mammalian protein expression and characterization tools for next generation biologics
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 5. |
- Aniander, Gustav
(författare)
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Improved candidate screening through tailored co-culture assays and precise tuning of protein expression
- 2024
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The field of biopharmaceuticals is a rapidly growing one. In the last ten years the number of approved biopharmaceuticals has more than doubled. A major hurdle to overcome for increased availability of all the new, effective biopharmaceuticals is the cost of treatment. Much of this can be attributed to the sheer time required for their development. Owing to this, interest in improvements to the biopharmaceuticals and their development process has also rapidly increased. As costs increase the further into development a drug candidate progresses, increasing the fidelity of screening at early stages could alleviate some of the exorbitant costs of development.In paper I, we showcase a novel way of targeting the tumor microenvironment (TME) to allow for TMElocalized CD40 activation. This is of interest as CD40 agonists have shown great potential for immune activation, but with systemic activation leading to severe adverse effects. The localized activation is achieved through the construction of an affinity fusion protein termed an AffiMab through fusion of a platelet derived growth factor receptor beta (PDGFRβ) targeting affibody to the heavy chain of a CD40 agonistic monoclonal antibody (mAb). We demonstrate PDGFRβ-dependent activation in a variety of assays, showing that the approach merits further investigation.Building on the activation assays set up in paper I, we aim to generate an in vitro screening platform for immune cell engagers in paper II. Screening candidates for on-target off-tumor activation is essential, as such activation would lead to adverse effects and be a doselimiting factor. To screen for this, we construct a series of plasmids which upon transfecting cells allow for different levels of a cell-surface target protein to be expressed, a so-called target density panel. This is achieved through the use of hairpin forming elements in the 5’ untranslated region of the mRNA dubbed regulatory elements (RgEs). Through use of different RgEs, we show that a target density panel can be generated and validate it in activation assays with the AffiMab developed in paper I. The platforms’ uniform cell surface background due to all different levels of target being expressed in the same host cell line and tunability through use of different RgEs are features that make it interesting for further research.Finally in paper III, we construct and test an improved translation initiation site (TIS) sequence. Using previous studies on the impact of the nucleotides in the sequence on the efficacy of the TIS, we constructed a novel sequence, TISNOV. This sequence enhanced titer and quality for recombinant production of IgG1 and IgG4 in both stable and transient settings. Further research into other TIS sequences and their uses in regulating protein expression, as well as usage of the TISNOV to improve expression of difficult to express proteins such as bispecifics remain interesting.In conclusion this thesis focuses on different manners to improve and hasten development of new biopharmaceuticals through usage of new workflows, platforms, and genetic engineering strategies.
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| 6. |
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| 7. |
- Jönsson, Malin, et al.
(författare)
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CaRA – A multi-purpose phage display library for selection of calcium-regulated affinity proteins
- 2022
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Ingår i: New Biotechnology. - : Elsevier B.V.. - 1871-6784 .- 1876-4347. ; 72, s. 159-167
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Tidskriftsartikel (refereegranskat)abstract
- Protein activity regulated by interactions with metal ions can be utilized for many different purposes, including biological therapies and bioprocessing, among others. Calcium ions are known to interact with the frequently occurring EF-hand motif, which can alter protein activity upon binding through an induced conformational change. The calcium-binding loop of the EF-hand motif has previously been introduced into a small protein domain derived from staphylococcal Protein A in a successful effort to render antibody binding dependent on calcium. Presented here, is a combinatorial library for calcium-regulated affinity, CaRA, based on this domain. CaRA is the first alternative scaffold library designed to achieve novel target specificities with metal-dependent binding. From this library, several calcium-dependent binders could be isolated through phage display campaigns towards a set of unrelated target proteins (IgE Cε3-Cε4, TNFα, IL23, scFv, tPA, PCSK9 and HER3) useful for distinct applications. Overall, these monomeric CaRA variants showed high stability and target affinities within the nanomolar range. They displayed considerably higher melting temperatures in the presence of 1 mM calcium compared to without calcium. Further, all discovered binders proved to be calcium-dependent, with the great majority showing complete lack of target binding in the absence of calcium. As demonstrated, the CaRA library is highly capable of providing protein-binding domains with calcium-dependent behavior, independent of the type of target protein. These binding domains could subsequently be of great use in gentle protein purification or as novel therapeutic modalities.
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| 8. |
- Malm, Magdalena, 1983-, et al.
(författare)
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Harnessing secretory pathway differences between HEK293 and CHO to rescue production of difficult to express proteins
- 2022
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Ingår i: Metabolic engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 72, s. 171-187
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 9. |
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| 10. |
- Rockberg, Johan, 1979-
(författare)
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Methods for Generation and Characterization of Monospecific Antibodies
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Recent advances in biotechnology have generated possibilities to investigate and measure parts of life previously left for believers to explain. Utilizing the same book of recipes, the genome, our cells produce selections of proteins at a time and thereby niche into a multitude of specialized cell types, tissues and organs comprising our body. Knowledge of the precise protein composition in a given organ at normal and disease condition would be of invaluable importance, both for identification of disease causes and the design of new pharmaceuticals, as well as for a deeper understanding of the processes of life. This doctoral thesis describes the start and progress of a visionary project (HPR) to localize all human proteins in our body, with emphasis on the generation and characterization of antibodies used as protein targeting missiles. To facilitate the identification of one human protein in a complex environment like our body, it is of significant importance to have precise and specific means of detection. The first two papers (I-II), describe software developed for generation of monospecific antibodies satisfying such needs, using a set of rules for antigen optimization. Five years after project start a large amount of antibodies with documented characteristics have been generated. The third paper (III), illustrates an attempt to sieve these antibody characteristics to develop a tool, for further improvement of antigen selection, based on the correlation between antigen sequence and amount of specific antibody generated.Having a panel of protein-specific antibodies is a possession of a great value, not only for localization studies, but also as possible target-directed pharmaceuticals. In such cases, knowledge of the precise epitope recognized by the antibody on its target protein, is an important aid, both for understanding its effect as well as unwanted cross-reactivity. Paper (IV) describes the development of a high-resolution method for epitope mapping of antibodies using staphylococcal display. An application of the method is described in the last paper (V) where it is used to map an anti-HER2 monospecific antibody with growth-inhibiting effects on breast cancer cells. The monospecific antibody was fractionated into separate populations and five novel epitopes related to cancer cell growth-inhibition was determined.Altogether these methods are valuable tools for generation and characterization of monospecific antibodies.
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