| 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. |
- Nilvebrant, Johan, 1982-, et al.
(författare)
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An introduction to epitope mapping
- 2018
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Ingår i: Methods in Molecular Biology. - New York, NY : Humana Press. - 1064-3745 .- 1940-6029. ; 1785, s. 1-10
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies are protein molecules used routinely for therapeutic, diagnostic, and research purposes due to their exquisite ability to selectively recognize and bind a given antigen. The particular area of the antigen recognized by the antibody is called the epitope, and for proteinaceous antigens the epitope can be of complex nature. Information about the binding epitope of an antibody can provide important mechanistic insights and indicate for what applications an antibody might be useful. Therefore, a variety of epitope mapping techniques have been developed to localize such regions. Although the real picture is even more complex, epitopes in protein antigens are broadly grouped into linear or discontinuous epitopes depending on the positioning of the epitope residues in the antigen sequence and the requirement of structure. Specialized methods for mapping of the two different classes of epitopes, using high-throughput or high-resolution methods, have been developed. While different in their detail, all of the experimental methods rely on assessing the binding of the antibody to the antigen or a set of antigen mimics. Early approaches utilizing sets of truncated proteins, small numbers of synthesized peptides, and structural analyses of antibody-antigen complexes have been significantly refined. Current state-of-the-art methods involve combinations of mutational scanning, protein display, and high-throughput screening in conjunction with bioinformatic analyses of large datasets.
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| 3. |
- Rockberg, Johan, et al.
(författare)
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Preface
- 2018
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Ingår i: Methods in Molecular Biology. - : Humana Press. ; , s. v-vi
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)
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| 4. |
- Thalén, Niklas, 1985-, et al.
(författare)
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Systems biology greatly improve activity of secreted therapeutic sulfatase in CHO bioprocess
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Annan publikation (övrigt vetenskapligt/konstnärligt)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. 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.
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| 5. |
- 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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| 6. |
- Anfelt, Josefine, et al.
(författare)
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Genetic and nutrient modulation of acetyl-CoA levels in Synechocystis for n-butanol production
- 2015
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Ingår i: Microbial Cell Factories. - : BioMed Central. - 1475-2859 .- 1475-2859. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- Background: There is a strong interest in using photosynthetic cyanobacteria as production hosts for biofuels and chemicals. Recent work has shown the benefit of pathway engineering, enzyme tolerance, and co-factor usage for improving yields of fermentation products. Results: An n-butanol pathway was inserted into a Synechocystis mutant deficient in polyhydroxybutyrate synthesis. We found that nitrogen starvation increased specific butanol productivity up to threefold, but cessation of cell growth limited total n-butanol titers. Metabolite profiling showed that acetyl-CoA increased twofold during nitrogen starvation. Introduction of a phosphoketolase increased acetyl-CoA levels sixfold at nitrogen replete conditions and increased butanol titers from 22 to 37 mg/L at day 8. Flux balance analysis of photoautotrophic metabolism showed that a Calvin-Benson-Bassham-Phosphoketolase pathway had higher theoretical butanol productivity than CBB-Embden-Meyerhof-Parnas and a reduced butanol ATP demand. Conclusion: These results demonstrate that phosphoketolase overexpression and modulation of nitrogen levels are two attractive routes toward increased production of acetyl-CoA derived products in cyanobacteria and could be implemented with complementary metabolic engineering strategies.
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| 7. |
- Aniander, Gustav, et al.
(författare)
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Enhanced titer and quality of IgGs through alterations at the translation initiation sequence
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Enhancing recombinant expression in mammalian cells remains an important and valuable goal. Much work has been done to develop cell lines, growth conditions, and expression vectors. However, some parts of the expression vector have been comparatively neglected in this quest for improvement. One such part is the translation initiation site (TIS) sequence, which drives translation initiation by increasing ribosomal recognition of the start codon. Using earlier work on what nucleotides could be exchanged for increased expression, we here present a novel TIS sequence. This TIS sequence increased titer in transient settings 4-fold while yielding 13-33 % increase in titer in final selected stable clones. This increase also comes with an increase in quality through reduction in non-paired chains, shift in charged species distribution, whilst maintaining the glycosylation profile. This increase in both titer and quality in transient and stable settings showing that TIS sequence engineering is of great interest for optimizing recombinant production in mammalian cells.
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| 8. |
- 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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| 9. |
- Berglund, Lisa, et al.
(författare)
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A whole-genome bioinformatics approach to selection of antigens for systematic antibody generation
- 2008
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Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 8:14, s. 2832-2839
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Tidskriftsartikel (refereegranskat)abstract
- Here, we present an antigen selection strategy based on a whole-genome bioinformatics approach, which is facilitated by an interactive visualization tool displaying protein features from both public resources and in-house generated data. The web-based bioinformatics platform has been designed for selection of multiple, non-overlapping recombinant protein epitope signature tags by display of predicted information relevant for antigens, including domain- and epitope sized sequence similarities to other proteins, transmembrane regions and signal peptides. The visualization tool also displays shared and exclusive protein regions for genes with multiple splice variants. A genome-wide analysis demonstrates that antigens for approximately 80% of the human protein-coding genes can be selected with this strategy.
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| 10. |
- Buus, S., et al.
(författare)
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High-resolution mapping of linear antibody epitopes using ultrahigh-density peptide microarrays
- 2012
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 11:12, s. 1790-1800
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies empower numerous important scientific, clinical, diagnostic, and industrial applications. Ideally, the epitope(s) targeted by an antibody should be identified and characterized, thereby establishing antibody reactivity, highlighting possible cross-reactivities, and perhaps even warning against unwanted (e.g. autoimmune) reactivities. Antibodies target proteins as either conformational or linear epitopes. The latter are typically probed with peptides, but the cost of peptide screening programs tends to prohibit comprehensive specificity analysis. To perform high-throughput, high-resolution mapping of linear antibody epitopes, we have used ultrahigh-density peptide microarrays generating several hundred thousand different peptides per array. Using exhaustive length and substitution analysis, we have successfully examined the specificity of a panel of polyclonal antibodies raised against linear epitopes of the human proteome and obtained very detailed descriptions of the involved specificities. The epitopes identified ranged from 4 to 12 amino acids in size. In general, the antibodies were of exquisite specificity, frequently disallowing even single conservative substitutions. In several cases, multiple distinct epitopes could be identified for the same target protein, suggesting an efficient approach to the generation of paired antibodies. Two alternative epitope mapping approaches identified similar, although not necessarily identical, epitopes. These results show that ultrahigh-density peptide microarrays can be used for linear epitope mapping. With an upper theoretical limit of 2,000,000 individual peptides per array, these peptide microarrays may even be used for a systematic validation of antibodies at the proteomic level.
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