| 1. |
- Ding, Mei, et al.
(författare)
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Secretome screening reveals immunomodulating functions of IFNα-7, PAP and GDF-7 on regulatory T-cells
- 2021
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Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; , s. 16767-
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Tidskriftsartikel (refereegranskat)abstract
- Regulatory T cells (Tregs) are the key cells regulating peripheral autoreactive T lymphocytes. Tregs exert their function by suppressing effector T cells. Tregs have been shown to play essential roles in the control of a variety of physiological and pathological immune responses. However, Tregs are unstable and can lose the expression of FOXP3 and suppressive functions as a consequence of outer stimuli. Available literature suggests that secreted proteins regulate Treg functional states, such as differentiation, proliferation and suppressive function. Identification of secreted proteins that affect Treg cell function are highly interesting for both therapeutic and diagnostic purposes in either hyperactive or immunosuppressed populations. Here, we report a phenotypic screening of a human secretome library in human Treg cells utilising a high throughput flow cytometry technology. Screening a library of 575 secreted proteins allowed us to identify proteins stabilising or destabilising the Treg phenotype as suggested by changes in expression of Treg marker proteins FOXP3 and/or CTLA4. Four proteins including GDF-7, IL-10, PAP and IFNα-7 were identified as positive regulators that increased FOXP3 and/or CTLA4 expression. PAP is a phosphatase. A catalytic-dead version of the protein did not induce an increase in FOXP3 expression. Ten interferon proteins were identified as negative regulators that reduced the expression of both CTLA4 and FOXP3, without affecting cell viability. A transcriptomics analysis supported the differential effect on Tregs of IFNα-7 versus other IFNα proteins, indicating differences in JAK/STAT signaling. A conformational model experiment confirmed a tenfold reduction in IFNAR-mediated ISG transcription for IFNα-7 compared to IFNα-10. This further strengthened the theory of a shift in downstream messaging upon external stimulation. As a summary, we have identified four positive regulators of FOXP3 and/or CTLA4 expression. Further exploration of these Treg modulators and their method of action has the potential to aid the discovery of novel therapies for both autoimmune and infectious diseases as well as for cancer.
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| 2. |
- Thalén, Niklas, et al.
(författare)
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Tuning of CHO secretional machinery improve activity of secreted therapeutic sulfatase 150-fold
- 2024
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Ingår i: Metabolic engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 81, s. 157-166
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- 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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| 4. |
- Eisenhut, P., et al.
(författare)
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Systematic use of synthetic 5'-UTR RNA structures to tune protein translation improves yield and quality of complex proteins in mammalian cell factories
- 2020
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 48:20
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Hendrikse, Natalie, et al.
(författare)
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Ancestral lysosomal enzymes with increased activity harbor therapeutic potential for treatment of Hunter syndrome
- 2021
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Ingår i: ISCIENCE. - : Elsevier BV. - 2589-0042. ; 24:3
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- 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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| 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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Evolution from adherent to suspension: systems biology of HEK293 cell line development
- 2020
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- The need for new safe and efficacious therapies has led to an increased focus on biologics produced in mammalian cells. The human cell line HEK293 has bio-synthetic potential for human-like production attributes and is currently used for manufacturing of several therapeutic proteins and viral vectors. Despite the increased popularity of this strain we still have limited knowledge on the genetic composition of its derivatives. Here we present a genomic, transcriptomic and metabolic gene analysis of six of the most widely used HEK293 cell lines. Changes in gene copy and expression between industrial progeny cell lines and the original HEK293 were associated with cellular component organization, cell motility and cell adhesion. Changes in gene expression between adherent and suspension derivatives highlighted switching in cholesterol biosynthesis and expression of five key genes (RARG, ID1, ZIC1, LOX and DHRS3), a pattern validated in 63 human adherent or suspension cell lines of other origin.
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| 9. |
- 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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| 10. |
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