| 1. |
- Anukam, Anthony, et al.
(författare)
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A review of the mechanism of bonding in densified biomass pellets
- 2021
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Ingår i: Renewable & sustainable energy reviews. - : Elsevier. - 1364-0321 .- 1879-0690. ; 148
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Tidskriftsartikel (refereegranskat)abstract
- The production of durable biomass pellets have always been challenged by several factors including the lack of understanding of the mechanism involved in how particles combine to form pellets under standard conditions of the pellet press. This is because contributing factors span several molecular, microscopic, and even nanoscopic levels as biomass undergoes pelleting. The characteristics of the bonds formed between the combining particles and their relevance to the quality of pellets remains vague, no matter how quality is defined. However, even though few researchers have attempted to explain the mechanism of bonding in densified biomass pellets using different theories, none of their hypotheses supports particle bonding from a structural chemistry perspective. There are still no clear explanations which consider the role of molecular structure and the interactions of substances as milled biomass undergo pelleting. In view of these arguments therefore, this review presents an in-depth analysis of a structural chemistry perspective of the mechanism of bonding and the use of additives in densified biomass pellets and helps identify research areas needed to facilitate better understanding of bonding in densified biomass pellets. The status of current research in biomass pelleting, types of materials suitable as additives and their structural characteristics, as well as the current technical specifications of using additives are also discussed.
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| 2. |
- Assenhöj, Maria, et al.
(författare)
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Protein interaction, monocyte toxicity and immunogenic properties of cerium oxide crystals with 5% or 14% gadolinium, cobalt oxide and iron oxide nanoparticles–an interdisciplinary approach
- 2021
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Ingår i: Nanotoxicology. - : Taylor and Francis Ltd.. - 1743-5390 .- 1743-5404. ; 15:8, s. 1035-1038
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Tidskriftsartikel (refereegranskat)abstract
- Metal oxide nanoparticles are widely used in both consumer products and medical applications, but the knowledge regarding exposure-related health effects is limited. However, it is challenging to investigate nanoparticle interaction processes with biological systems. The overall aim of this project was to improve the possibility to predict exposure-related health effects of metal oxide nanoparticles through interdisciplinary collaboration by combining workflows from the pharmaceutical industry, nanomaterial sciences, and occupational medicine. Specific aims were to investigate nanoparticle-protein interactions and possible adverse immune reactions. Four different metal oxide nanoparticles; CeOx nanocrystals with 5% or 14% Gd, Co3O4, and Fe2O3, were characterized by dynamic light scattering and high-resolution transmission electron microscopy. Nanoparticle-binding proteins were identified and screened for HLA-binding peptides in silico. Monocyte interaction with nanoparticle–protein complexes was assessed in vitro. Herein, for the first time, immunogenic properties of nanoparticle-binding proteins have been characterized. The present study indicates that especially Co3O4-protein complexes can induce both ‘danger signals’, verified by the production of inflammatory cytokines and simultaneously bind autologous proteins, which can be presented as immunogenic epitopes by MHC class II. The clinical relevance of these findings should be further evaluated to investigate the role of metal oxide nanoparticles in the development of autoimmune disease. The general workflow identified experimental difficulties, such as nanoparticle aggregate formation and a lack of protein-free buffers suitable for particle characterization, protein analyses, as well as for cell studies. This confirms the importance of future interdisciplinary collaborations. © 2021 The Author(s).
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| 3. |
- Berghel, Jonas, 1966-, et al.
(författare)
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A comparison of relevant data and results from single pellet press research is Mission Impossible : A review
- 2022
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Ingår i: Bioresource Technology Reports. - : Elsevier. - 2589-014X. ; 18
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Tidskriftsartikel (refereegranskat)abstract
- A global increase in the wood fuel pellet market requires knowledge of new biomasses pelleting abilities. As large-scale industrial tests of new materials are costly, tests in e.g., a single pellet press (SPP) are desirable. SPPs have many different configurations and it typically produces one pellet at a time and can give results of its pelletability. This review has surveyed the research that has been carried out of SPPs to ascertain the feasibility of comparing their obtained data and the results. The results show that it is almost impossible to compare the data and results of the various different SPP studies, e.g., some information from the data used was missing, resulting in that only 27 out of 70 papers were comparable. One solution could be the introduction of a common SPP testing method using a determined set of data that enables a reference pellet to be produced in every study.
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| 4. |
- Bhalla, Nayanika, et al.
(författare)
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Spatial transcriptomics of human placentas reveal distinct RNA patterns associated with morphology and preeclampsia
- 2023
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Ingår i: Placenta. - : Elsevier BV. - 0143-4004 .- 1532-3102. ; 139, s. 213-216
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Tidskriftsartikel (refereegranskat)abstract
- Spatial transcriptomics (ST) maps RNA level patterns within a tissue. This technology has not been previously applied to human placental tissue. We demonstrate analysis of human placental samples with ST. Unsupervised clustering revealed that distinct RNA patterns were found corresponding to different morphological structures. Additionally, when focusing upon terminal villi and hemoglobin associated structures, RNA levels differed between placentas from full term healthy pregnancies and those complicated by preeclampsia. The results from this study can provide a benchmark for future ST studies in placenta.
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| 5. |
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| 6. |
- Cheung, Pierre, et al.
(författare)
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Preclinical evaluation of Affibody molecule for PET imaging of human pancreatic islets derived from stem cells
- 2023
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Ingår i: EJNMMI Research. - : Springer Nature. - 2191-219X. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Beta-cell replacement methods such as transplantation of isolated donor islets have been proposed as a curative treatment of type 1 diabetes, but widespread application is challenging due to shortages of donor tissue and the need for continuous immunosuppressive treatments. Stem-cell-derived islets have been suggested as an alternative source of beta cells, but face transplantation protocols optimization difficulties, mainly due to a lack of available methods and markers to directly monitor grafts survival, as well as their localization and function. Molecular imaging techniques and particularly positron emission tomography has been suggested as a tool for monitoring the fate of islets after clinical transplantation. The integral membrane protein DGCR2 has been demonstrated to be a potential pancreatic islet biomarker, with specific expression on insulin-positive human embryonic stem-cell-derived pancreatic progenitor cells. The candidate Affibody molecule ZDGCR2:AM106 was radiolabeled with fluorine-18 using a novel click chemistry-based approach. The resulting positron emission tomography tracer [18F]ZDGCR2:AM106 was evaluated for binding to recombinant human DGCR2 and cryosections of stem-cell-derived islets, as well as in vivo using an immune-deficient mouse model transplanted with stem-cell-derived islets. Biodistribution of the [18F]ZDGCR2:AM106 was also assessed in healthy rats and pigs. Results: [18F]ZDGCR2:AM106 was successfully synthesized with high radiochemical purity and yield via a pretargeting approach. [18F]ZDGCR2:AM106 retained binding to recombinant human DCGR2 as well as to cryosectioned stem-cell-derived islets, but in vivo binding to native pancreatic tissue in both rat and pig was low. However, in vivo uptake of [18F]ZDGCR2:AM106 in stem-cell-derived islets transplanted in the immunodeficient mice was observed, albeit only within the early imaging frames after injection of the radiotracer. Conclusion: Targeting of DGCR2 is a promising approach for in vivo detection of stem-cell-derived islets grafts by molecular imaging. The synthesis of [18F]ZDGCR2:AM106 was successfully performed via a pretargeting method to label a site-specific covalently bonded fluorine-18 to the Affibody molecule. However, the rapid washout of [18F]ZDGCR2:AM106 from the stem-cell-derived islets graft indicates that dissociation kinetics can be improved. Further studies using alternative binders of similar classes with improved binding potential are warranted.
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| 7. |
- Dahlsson Leitao, Charles
(författare)
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Affibody-mediated targeting of HER-family receptors for cancer imaging and therapy
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are remarkable molecules with diverse and specialized functions playing essential roles in most biological processes. One such function is protecting us from diseases by the action of antibodies in our immune system that can recognize and mediate the destruction of invading pathogens by binding to foreign epitopes found on non-self proteins. The concept of utilizing specific protein-protein interactions to achieve a therapeutic effect has for several decades been a cornerstone for the development of cancer-directed treatments. While antibodies have formed a basis for the development of such drugs, other protein alternatives may be engineered to complement current antibody-based treatments, and may even prove to possess superior features. This thesis focuses on the engineering of affibody molecules, a small alternative scaffold protein, for design and development of novel cancer-targeting therapeutic and diagnostic drugs. There are many different strategies that have been investigated for inhibiting cancer progression and tumour growth with perhaps one of the most straightforward involving disruption of dysregulated growth-promoting signalling pathways. Members of the human epidermal growth factor receptor (HER) family is prominently expressed in various cancer types and have been shown to be intricately involved in tumorigenesis. One of the members (HER3) often becomes upregulated in cancer and have been shown to mediate acquired resistance to targeted therapies by the mechanism of ligand-induced activation. We have designed five novel affibody-based HER3-targeting molecules able to prevent ligand-binding and consequently activation of HER3. We investigated the targeting properties and biodistribution profiles of these molecules in vivo and subsequently evaluated the anti-tumour efficacy for the most promising variants in direct comparison to a HER3-targeting antibody with a similar inhibitory mechanism. We observed a large influence of design on both the biodistribution properties and the in vivo efficacy of different affibody molecules. Moreover, we demonstrated that two of the affibody-formats were equally effective as the antibody in inhibiting tumour growth and prolonging survival of mice bearing HER3-positive xenografts. The effectiveness of cancer treatments depends on efficient diagnostic approaches that can reliably stratify patients based on these targetable biomarkers, which is possible using radionuclide molecular imaging. We have performed a direct comparison of the diagnostic potential for visualizing HER3-expressing tumours of affibody- and antibody-based imaging probes. We concluded that affibody molecules provide superior imaging quality with higher diagnostic potential and enable early visualization of HER3-expression in tumours. Another member of the HER family that is of interest for cancer therapy is HER1 (or EGFR) but due to substantial expression in healthy tissues, targeted therapies may lead to severe side-effects. One possible solution to this is taking advantage of the distinct milieu of the tumour microenvironment to design EGFR-targeting drugs that become conditionally activated at the tumour site, but not in normal tissues, with the aim of drastically reducing systemic toxicity. We have generated an affibody molecule with anti-idiotypic binding specificity for a previously generated EGFR-binding affibody molecule, which we used to construct an affibody-based prodrug. We were able to show that, in a proof-of-concept format, this anti-idiotypic masking domain was able to block the binding to EGFR until removed by protease-mediated cleavage. We subsequently developed and characterized a more refined version of this prodrug, which we call a pro-affibody, and could show that activation by cancer-associated proteases confers binding to EGFR-expressing cancer cells and enables conditional cytotoxic payload delivery in vitro. The pro-affibody was further evaluated in vivo using tumour-bearing mice to investigate the feasibility for masked uptake in healthy tissues while retaining binding-activity in tumours. We observed a substantial reduction in EGFR-specific liver uptake compared to a control construct without a masking domain, and a strong indication of protease-mediated EGFR-binding in tumours. In conclusion, the experimental work presented in this thesis provides a rationale for designing effective affibody-based cancer therapeutics and diagnostics with different targeting strategies and demonstrates the potential of such drugs from preclinical in vivo data.
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| 8. |
- Ding, Haozhong, 1990-
(författare)
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Targeting HER2-expressing tumors with potent drug conjugates and fusion toxins based on scaffold proteins
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Targeted therapy is an emerging treatment for a variety of cancers. Small- sized scaffold proteins are an alternative to conventional antibody-based targeting molecules. Two small scaffold proteins—the 58-amino-acid protein class, the affibody molecules, and the 46-amino-acid protein class, the Albumin binding domain Derived Affinity Proteins (ADAPTs)—have previously been engineered to bind to a large variety of tumor-associated molecular targets with a high affinity. The human epidermal growth factor receptor 2 (HER2) is a membrane-bound receptor for growth signal transmission. Expression of a high level of HER2 can cause cells to proliferate and may ultimately lead to cancer. It has earlier been shown that HER2 is involved in several different types of cancers, e.g., breast, ovarian, bladder, and gastric cancers. HER2-targeted affibody and ADAPT molecules have previously been developed, such as ZHER2:2891 and ADAPT6 with strong affinity to HER2 with equilibrium dissociation constants of 76 pM and 2.5 nM, respectively. Their small size and high specificity have rendered these two scaffold proteins promising candidates for imaging of HER2-positive breast cancer tumors in clinical trials. Delivery of cytotoxic agents to cancer cells, using a cell-targeting domain, may potentially precisely kill the cancer cells while having very low cytotoxic effects on normal cells. Many cancer-targeted antibody drug conjugates (ADCs) and toxic proteins (immunotoxins) have advanced the field of cancer treatment. Small-sized scaffold proteins hold promise as alternative targeting domains to build novel drug conjugates or fusion toxins for cancer treatment. In this thesis, I first investigated an affibody-based drug conjugate (AffiDC) composed of an anti-HER2 affibody and an anti-mitotic maytansine-derived drug (DM1) for treatment of HER2-overexpressing cells. I studied a variety of targeting domain formats for efficacy optimization. All ZHER2:2891-based AffiDCs showed specific anti-tumor activity on HER2-overexpressing cancer cells in vitro as well as in mouse tumor xenografts. The hepatic uptake of the AffiDCs could be reduced by shielding the hydrophobic DM1 using a poly-glutamic-acid spacer, which might help to reduce potential liver toxicity allowing for administration of higher doses. In addition, tuning the valency of the affibody-targeting domain (ZHER2) from a divalent domain to a monovalent domain showed increased potency and reduced liver uptake. We also investigated the influence of the number of drug payloads on the pharmacokinetic profile of the AffiDCs. An AffiDC bearing three DM1s showed higher delivery of DM1 to the cancer cells in vivo, but fast blood clearance and an elevated liver retention was also observed. With regards to fusion toxin design, we constructed a variety of recombinant toxins. The targeting domains were ZHER2:2891 and/or ADAPT6, which were genetically fused with truncated versions of the highly cytotoxic Pseudomonas Exotoxin A (PE). All fusion toxins we studied showed potent HER2-specific anti-tumor activity. The results suggested that both ZHER2:2891 and ADAPT6 could direct the PE-based cytotoxins specifically to HER2- overexpressing cancer cells. In this work, we have demonstrated the potential of using ZHER2:2891 and ADAPT6 as targeting domains to carry the small molecule drug DM1, or cytotoxic PE-derived peptides to cancer cells. It can be concluded that careful molecular design of the targeting domain may considerably improve the potency and minimize the off-target uptake.
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| 9. |
- Ernholm, Linda, et al.
(författare)
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Understanding the interface between European wild boar (Sus scrofa) and domestic pigs (Sus scrofa domesticus) in Sweden through a questionnaire study
- 2023
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Ingår i: Acta Veterinaria Scandinavica. - 0044-605X .- 1751-0147. ; 65
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Tidskriftsartikel (refereegranskat)abstract
- Background In recent years, the wildlife/livestock interface has attracted increased attention due to disease transmission between wild and domestic animal populations. The ongoing spread of African swine fever (ASF) in European wild boar (Sus scrofa) emphasize the need for further understanding of the wildlife/livestock interface to prevent disease spill-over between the wild and domestic populations. Although wild boar may also act as a potential source for other infectious disease agents, ASF is currently the most severe threat from wild boar to domestic pigs. To gather information on the wild boar situation at commercial pig producing farms in Sweden, a digital questionnaire survey was distributed through the animal health services. Results Most pigs produced for commercial purposes in Sweden are raised without outdoor access. Of the 211 responding pig producers, 80% saw wild boar or signs of wild boar activity in the vicinity of their farm at least once during the year. Observations were significantly correlated with geographical region, but there was no correlation between farm characteristics (farm size, main type of production, outdoor access) and observed wild boar presence or proximity. However, a reported higher frequency of wild boar observations was positively correlated with the observations being made in closer proximity to the farm. Hunting and strategic baiting were the most common mitigation strategies used to keep wild boar at bay. Of the 14 farms raising pigs with outdoor access, 12 responded that these pigs could be raised solely indoors if needed. Pigs with outdoor access are required to be fenced in, but double fencing in these outdoor pig enclosures was not practiced by all. A perimeter fence surrounding any type of pig farm was very rare. More than half of the producers that grew crops with intended use for pigs reported crop damage by wild boar. Conclusion This study shows that although pigs raised for commercial purposes in Sweden are, to a large extent, kept indoors the potential for indirect contact with wild boar exists and must be considered. Variable local situations regarding wild boar abundance may require an adaptive approach regarding biosecurity efforts.
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| 10. |
- Faresjö, Rebecca, et al.
(författare)
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Transferrin Receptor Binding BBB-Shuttle Facilitates Brain Delivery of Anti-Aβ-Affibodies
- 2022
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Ingår i: Pharmaceutical research. - : Springer Nature. - 0724-8741 .- 1573-904X. ; 39:7, s. 1509-1521
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Tidskriftsartikel (refereegranskat)abstract
- Affibodies targeting amyloid-beta (Aβ) could potentially be used as therapeutic and diagnostic agents in Alzheimer’s disease (AD). Affibodies display suitable characteristics for imaging applications such as high stability and a short biological half-life. The aim of this study was to explore brain delivery and retention of Aβ protofibril-targeted affibodies in wild-type (WT) and AD transgenic mice and to evaluate their potential as imaging agents. Two affibodies, Z5 and Z1, were fused with the blood–brain barrier (BBB) shuttle single-chain variable fragment scFv8D3. In vitro binding of 125I-labeled affibodies with and without scFv8D3 was evaluated by ELISA and autoradiography. Brain uptake and retention of the affibodies at 2 h and 24 h post injection was studied ex vivo in WT and transgenic (tg-Swe and tg-ArcSwe) mice. At 2 h post injection, [125I]I-Z5 and [125I]I-Z1 displayed brain concentrations of 0.37 ± 0.09% and 0.46 ± 0.08% ID/g brain, respectively. [125I]I-scFv8D3-Z5 and [125I]I-scFv8D3-Z1 showed increased brain concentrations of 0.53 ± 0.16% and 1.20 ± 0.35%ID/g brain. At 24 h post injection, brain retention of [125I]I-Z1 and [125I]I-Z5 was low, while [125I]I-scFv8D3-Z1 and [125I]I-scFv8D3-Z5 showed moderate brain retention, with a tendency towards higher retention of [125I]I-scFv8D3-Z5 in AD transgenic mice. Nuclear track emulsion autoradiography showed greater parenchymal distribution of [125I]I-scFv8D3-Z5 and [125I]I-scFv8D3-Z1 compared with the affibodies without scFv8D3, but could not confirm specific affibody accumulation around Aβ deposits. Affibody-scFv8D3 fusions displayed increased brain and parenchymal delivery compared with the non-fused affibodies. However, fast brain washout and a suboptimal balance between Aβ and mTfR1 affinity resulted in low intrabrain retention around Aβ deposits.
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| 11. |
- Frodeson, Stefan, Universitetsadjunkt, 1968-, et al.
(författare)
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Densification of Wood-Influence on Mechanical and Chemical Properties when 11 Naturally Occurring Substances in Wood Are Mixed with Beech and Pine
- 2021
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Ingår i: Energies. - : MDPI AG. - 1996-1073. ; 14:18
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Tidskriftsartikel (refereegranskat)abstract
- The need to increase the use of renewable biomasses for energy supply, such as fuel pellets is significant. However, different types of biomasses have different mechanical properties to be pelletized, which entails a limitation in available raw materials for pellet producers. Within this study eleven different pure substances from biomasses were separately mixed with European beech and Scots pine, to identify its impact on the densification process. Beech and pine pellets were used as control materials against their corresponding pellets mixed with substances representing: cellulose, hemicelluloses, other polysaccharides, lignin, protein, and extractives. The mechanical properties were investigated as well as FT-IR and SEM analyses on the pellets. The results showed that the addition of the substances xylan and galactan created the hardest pellets for both pine and beech and that adding extractives to wood affects pine more than beech in relation to hardness. The FT-IR data could not provide clear explanations as to the variation in hardness and springback behavior through the identification of major functional groups in each pellet. It can be concluded that biomass residues rich in xylan and galactan increase pellet quality in terms of strength and durability without affecting the production process.
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| 12. |
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| 13. |
- Güler, Rezan
(författare)
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Affibody Molecules Targeting VEGFR2 - Two Turns Off and Four Turns On
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The notion of employing proteins as drugs traces back several decades. As recombinant DNA technology emerged, it became a powerful tool for the tailoring of protein traits via genetic approaches - so-called protein engineering. The application of such tools to develop proteins that bind specific molecular targets has seen remarkable clinical success, and today seven out of the ten top-selling drugs in the world belong to this class of proteins. A well-investigated protein scaffold for generating novel target-binding moieties is the small staphylococcal protein A-derived affibody molecule. This thesis revolves around the engineering of affibody-based binding proteins that aim to influence the signaling network in the biological process of blood vessel formation, so-called angiogenesis. The first study in this thesis describes the engineering of a heterodimeric affibody molecule that targets the principal regulating receptor of angiogenesis, vascular endothelial growth factor receptor-2 (VEGFR2). Two separate affibody molecules that bind adjacent VEGFR2 epitopes were previously fused into one biparatopic construct, leading to a remarkable increase in apparent affinity. Further, the biparatopic protein here demonstrated inhibition of vascular endothelial growth factor A (VEGF-A) binding to VEGFR2, and consequently inhibition of VEGFR2 phosphorylation, proliferation and in vitro sprouting of endothelial cells. In the second study, the aim was to evaluate the biparatopic protein as a molecular imaging probe for in vivo visualization of VEGFR2 expression in a glioblastoma multiforme (GBM) brain tumor-model. The results displayed significantly higher probe uptake in tumor compared to normal brain tissue, with a two-hour post injection tumor-to-brain ratio of 78. In the third study, the goal was to mimic the ability of the natural ligand to agonize VEGFR2 via receptor dimerization, and also simulate presentation as extracellular matrix (ECM) bound factors. To this end, the dimeric antagonist was reformatted into a tetrameric construct, hypothesized to bridge two receptor units, and fused to recombinant spider silk. Interestingly, whereas the tetramer displayed agonistic effects both in soluble and immobilized form, the activity of the dimer shifted from antagonistic to agonistic when immobilized. In the fourth study a combined in silico and directed evolution approach was used to increase the thermal stability and hydrophilicity of the biparatopic protein. The final construct demonstrated an increase in melting temperature of about 15°C, complete refolding after heat-induced denaturation and decreased uptake in normal tissues when evaluated in mouse biodistribution studies. In conclusion, this thesis covers the development, characterization and engineering of VEGFR2-binding affibody molecules, aimed for use in research, therapy and diagnosis.
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| 14. |
- Hande, Liv Nesse, et al.
(författare)
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Effect of N-3 Polyunsaturated Fatty Acids on Lipid Composition in Familial Hypercholesterolemia : A Randomized Crossover Trial
- 2022
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Ingår i: Biomedicines. - : MDPI. - 2227-9059. ; 10:8
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Tidskriftsartikel (refereegranskat)abstract
- Individuals with familial hypercholesterolemia (FH) have an increased risk of cardiovascular disease. Treatment is mainly low-density lipoprotein cholesterol (LDL-C) reduction. How omega-3 polyunsaturated fatty acids (n-3 PUFAs) supplements affect lipoproteins in FH subjects is unknown. We hypothesized that a high-dose n-3 PUFA supplement would reduce atherogenic lipoproteins and influence the high-density lipoprotein cholesterol (HDL-C) function. We performed a randomized, double-blinded crossover study with 34 genetically verified FH individuals (18-75 years, clinically stable, statin treatment > 12 months). Treatment was 4 g n-3 PUFAs (1840 mg eicosapentaenoic acid and 1520 mg docosahexaenoic acid daily) or four capsules of olive oil for three months in a crossover design with a washout period of three months. The defined outcomes were changes in triglycerides, lipoproteins, lipoprotein subfractions, apolipoproteins, and HDL-C function. After treatment with n-3 PUFAs, total cholesterol, LDL-C, and triglycerides were reduced compared to placebo (p <= 0.01 for all). Total HDL-C levels were unchanged, but the subfraction of large HDL-C was higher (p <= 0.0001) after n-3 PUFAs than after placebo, and intermediate HDL-C and small HDL-C were reduced after n-3 PUFAs compared to placebo (p = 0.02 and p <= 0.001, respectively). No changes were found in apolipoproteins and HDL-C function. N-3 PUFAs supplements reduced atherogenic lipoproteins in FH subjects, leaving HDL-C function unaffected.
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| 15. |
- Hjelm, Linnea C., 1993-, et al.
(författare)
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Affibody Molecules Intended for Receptor-Mediated Transcytosis via the Transferrin Receptor
- 2023
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Ingår i: Pharmaceuticals. - : MDPI. - 1424-8247. ; 16:7
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Tidskriftsartikel (refereegranskat)abstract
- The development of biologics for diseases affecting the central nervous system has been less successful compared to other disease areas, in part due to the challenge of delivering drugs to the brain. The most well-investigated and successful strategy for increasing brain uptake of biological drugs is using receptor-mediated transcytosis over the blood-brain barrier and, in particular, targeting the transferrin receptor-1 (TfR). Here, affibody molecules are selected for TfR using phage display technology. The two most interesting candidates demonstrated binding to human TfR, cross-reactivity to the murine orthologue, non-competitive binding with human transferrin, and binding to TfR-expressing brain endothelial cell lines. Single amino acid mutagenesis of the affibody molecules revealed the binding contribution of individual residues and was used to develop second-generation variants with improved properties. The second-generation variants were further analyzed and showed an ability for transcytosis in an in vitro transwell assay. The new TfR-specific affibody molecules have the potential for the development of small brain shuttles for increasing the uptake of various compounds to the central nervous system and thus warrant further investigations.
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| 16. |
- Hjelm, Linnea C., et al.
(författare)
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Construction and Validation of a New Naive Sequestrin Library for Directed Evolution of Binders against Aggregation-Prone Peptides
- 2023
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 24:1
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules are small affinity proteins that have excellent properties for many different applications, ranging from biotechnology to diagnostics and therapy. The relatively flat binding surface is typically resulting in high affinity and specificity when developing binding reagents for globular target proteins. For smaller unstructured peptides, the paratope of affibody molecules makes it more challenging to achieve a sufficiently large binding surface for high-affinity interactions. Here, we describe the development of a new type of protein scaffold based on a dimeric form of affibodies with a secondary structure content and mode of binding that is distinct from conventional affibody molecules. The interaction is characterized by encapsulation of the target peptide in a tunnel-like cavity upon binding. The new scaffold was used for construction of a high-complexity phage-displayed library and selections from the library against the amyloid beta peptide resulted in identification of high-affinity binders that effectively inhibited amyloid aggregation.
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| 17. |
- Hjelm, Linnea C., 1993-
(författare)
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Development of new affinity proteins for neurodegenerative disorders
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Neurodegenerative disorders include a full spectrum of diagnoses, including dementias and other neuronal diseases, characterised by degradation of neurons in the brain occurring along with disease progression. Amongst the dementias, the most prevalent are Alzheimer’s (AD) and Parkinson’s disease (PD) that affect millions of people worldwide. During the last years, advancements in potential treatments have been made where the first two clinical antibodies have been approved by the US Food and Drug Administration (FDA) for a disease modifying effect on Alzheimer’s disease.As alternatives to antibodies, other types of affinity reagents that are based on non-immunoglobulin protein scaffolds are also investigated. Such alternative scaffolds often demonstrate distinct and complementary properties compared to antibodies. In this thesis, the development of a new type of affinity protein scaffold called sequestrin is described. Sequestrins are derived from the affibody molecule and comprise two heterogenic subunits with truncated N-terminals fused as a head-to-tail construct. Sequestrins undergo a structural rearrangement upon target binding and forms a stabile complex. The scaffold is designed for interactions with disease-related amyloidogenic peptides e.g. amyloid beta and alpha-synuclein involved in AD and PD, respectively. In the first paper, a sequestrin library was developed and its compatibility with phage display was investigated. Successful panning against the amyloid beta peptide resulted in binders with high affinity. Further on in paper II, the alpha-synuclein peptide was targeted and sequestrins with low nanomolar affinities were obtained. All sequestrins displayed structural rearrangement upon target engagement, which stabilized the interaction to the target peptides and further inhibited toxic aggregation, opening up for future studies of disease modifying effects in vivo.When targeting the brain, passage through the blood–brain barrier (BBB) is an obstacle that needs to be addressed to reach sufficiently high therapeutic concentrations. To overcome this barrier, brain shuttles have been developed with the capability to transport a cargo over the BBB. One such mechanism of transportation is by receptor-mediated transcytosis, which is utilized by e.g. the transferrin receptor (TfR). In paper III, a TfR-targeting shuttle was investigated for BBB passage when fused to a sequestrin targeting the amyloid beta peptide, resulting in a higher penetration through the BBB, and maintained functionality of the sequestrin.High-throughput in vitro methods would facilitate development of novel brain shuttles. Thus, in paper IV, a transwell system based on nanofibrillar silkmembranes with murine brain endothelial cells was developed. Evaluation of the method using a TfR-specific antibody demonstrated higher transfer over the barrier compared to an isotype control and the method has potential to facilitate screening of transcytosis capability of brain shuttles.In paper V, TfR-specific affibody-based brain shuttles were developed and investigated for transcytosis capability using the in vitro transcytosis assay. A panel of affibody molecules were evaluated, demonstrating both cross-species reactivity to murine and human TfR and active receptor-mediated transcytosis. These candidates could thus potentially be used in further development of CNS-targeting therapeutics.In conclusion, a new sequestrin scaffold was developed that can be utilised for targeting amyloidogenic peptides found in neurodegenerative disorders. An affibody-based brain shuttle was also developed, which showed transcytosis capability. In the future, the new brain shuttle might be combined with sequestrins to create multifunctional fusion proteins for facilitated delivery over the BBB, which hopefully can result in therapeutic concentrations in the brain even when administered with a lower dosage.
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| 18. |
- Leitao, Charles Dahlsson, 1992-, et al.
(författare)
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Bacterial Cell Display for Selection of Affibody Molecules
- 2023
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Ingår i: Genotype Phenotype Coupling. - : Springer Nature. ; , s. 99-112
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- This review describes the principles for generation of affibody molecules using bacterial display on the Gram-negative Escherichia coli and the Gram-positive Staphylococcus carnosus, respectively. Affibody molecules are small and robust alternative scaffold proteins that have been explored for therapeutic, diagnostic, and biotechnological applications. They typically exhibit high-stability, affinity, and specificity with high modularity of functional domains. Due to the small size of the scaffold, affibody molecules are rapidly excreted through renal filtration and can efficiently extravasate from blood and penetrate tissues. Preclinical and clinical studies have demonstrated that affibody molecules are promising and safe complements to antibodies for in vivo diagnostic imaging and therapy. Sorting of affibody libraries displayed on bacteria using fluorescence-activated cell sorting is an effective and straightforward methodology and has been used successfully to generate novel affibody molecules with high affinity for a diverse range of molecular targets.
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| 19. |
- Leitao, Charles Dahlsson, 1992-, et al.
(författare)
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Conditionally activated affibody-based prodrug targeting EGFR demonstrates improved tumour selectivity
- 2023
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Ingår i: Journal of Controlled Release. - : Elsevier BV. - 0168-3659 .- 1873-4995. ; 357, s. 185-195
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Tidskriftsartikel (refereegranskat)abstract
- Safety and efficacy of cancer-targeting treatments can be improved by conditional activation enabled by the distinct milieu of the tumour microenvironment. Proteases are intricately involved in tumourigenesis and commonly dysregulated with elevated expression and activity. Design of prodrug molecules with protease -dependent activation has the potential to increase tumour-selective targeting while decreasing exposure to healthy tissues, thus improving the safety profile for patients. Higher selectivity could also allow for adminis-tration of higher doses or use of more aggressive treatment options, leading to higher therapeutic efficacy. We have previously developed an affibody-based prodrug with conditional targeting of EGFR conferred by an anti-idiotypic affibody masking domain (ZB05). We could show that binding to endogenous EGFR on cancer cells in vitro was restored following proteolytic removal of ZB05. In this study we evaluate a novel affibody-based pro -drug design, which incorporates a protease substrate sequence recognized by cancer-associated proteases and demonstrate the potential of this approach for selective tumour-targeting and shielded uptake in healthy tissues in vivo using tumour-bearing mice. This may widen the therapeutic index of cytotoxic EGFR-targeted thera-peutics by decreasing side effects, improving selectivity of drug delivery, and enabling the use of more potent cytotoxic drugs.
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| 20. |
- Leitao, Charles Dahlsson, 1992-, et al.
(författare)
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Display of a naïve affibody library on staphylococci for selection of binders by means of flow cytometry sorting
- 2023
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 655, s. 75-81
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Tidskriftsartikel (refereegranskat)abstract
- Within the field of combinatorial protein engineering there is a great demand for robust high-throughput selection platforms that allow for unbiased protein library display, affinity-based screening, and amplification of selected clones. We have previously described the development of a staphylococcal display system used for displaying both alternative-scaffolds and antibody-derived pro-teins. In this study, the objective was to generate an improved expression vector for displaying and screening a high-complexity naive affibody library, and to facilitate downstream validation of isolated clones. A high-affinity normalization tag, consisting of two ABD-moieties, was introduced to simplify off-rate screening procedures. In addition, the vector was furnished with a TEV protease substrate recog-nition sequence upstream of the protein library which enables proteolytic processing of the displayed construct for improved binding signal. In the library design, 13 of the 58 surface-exposed amino acid positions were selected for full randomization (except proline and cysteine) using trinucleotide tech-nology. The genetic library was successfully transformed to Staphylococcus carnosus cells, generating a protein library exceeding 109 members. De novo selections against three target proteins (CD14, MAPK9 and the affibody ZEGFR:2377) were successfully performed using magnetic bead-based capture followed by flow-cytometric sorting, yielding affibody molecules binding their respective target with nanomolar affinity. Taken together, the results demonstrate the feasibility of the staphylococcal display system and the proposed selection procedure to generate new affibody molecules with high affinity.
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| 21. |
- Leitao, Charles Dahlsson, et al.
(författare)
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Evaluating the Therapeutic Efficacy of Mono- and Bivalent Affibody-Based Fusion Proteins Targeting HER3 in a Pancreatic Cancer Xenograft Model
- 2020
-
Ingår i: Pharmaceutics. - : MDPI. - 1999-4923. ; 12:6
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Tidskriftsartikel (refereegranskat)abstract
- Human epidermal growth factor receptor 3 (HER3) has been increasingly scrutinized as a potential drug target since the elucidation of its role in mediating tumor growth and acquired therapy resistance. Affibody molecules are so-called scaffold proteins with favorable biophysical properties, such as a small size for improved tissue penetration and extravasation, thermal and chemical stability, and a high tolerance to modifications. Additionally, affibody molecules are efficiently produced in prokaryotic hosts or by chemical peptide synthesis. We have previously evaluated the biodistribution profiles of five mono- and bivalent anti-HER3 affibody molecules (designated as 3) fused to an albumin-binding domain (designated as A), 3A, 33A, 3A3, A33, and A3, that inhibit ligand-dependent phosphorylation. In the present study, we examined the therapeutic efficacy of the three most promising variants, 3A, 33A, and 3A3, in a direct comparison with the HER3-targeting monoclonal antibody seribantumab (MM-121) in a preclinical BxPC-3 pancreatic cancer model. Xenografted mice were treated with either an affibody construct or MM-121 and the tumor growth was compared to a vehicle group. Receptor occupancy was estimated by positron emission tomography/computed tomography (PET/CT) imaging using a HER3-targeting affibody imaging agent [Ga-68]Ga-(HE)(3)-Z(08698)-NODAGA. The affibody molecules could inhibit ligand-dependent phosphorylation and cell proliferation in vitro and demonstrated tumor growth inhibition in vivo comparable to that of MM-121. PET/CT imaging showed full receptor occupancy for all tested drug candidates. Treatment with 3A and 3A3 affibody constructs was more efficient than with 33A and similar to the anti-HER3 antibody seribantumab, showing that the molecular design of affibody-based therapeutics targeting HER3 in terms of the relative position of functional domains and valency has an impact on therapeutic effect.
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| 22. |
- Meister, Sebastian, et al.
(författare)
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An Affibody Molecule Is Actively Transported into the Cerebrospinal Fluid via Binding to the Transferrin Receptor
- 2020
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 21:8, s. 2999-
-
Tidskriftsartikel (refereegranskat)abstract
- Theuseofbiotherapeuticsforthetreatmentofdiseasesofthecentralnervoussystem(CNS) is typically impeded by insufficient transport across the blood–brain barrier. Here, we investigate a strategy to potentially increase the uptake into the CNS of an affibody molecule (ZSYM73) via binding to the transferrin receptor (TfR). ZSYM73 binds monomeric amyloid beta, a peptide involved in Alzheimer’s disease pathogenesis, with subnanomolar affinity. We generated a tri-specific fusion protein by genetically linking a single-chain variable fragment of the TfR-binding antibody 8D3 and an albumin-binding domain to the affibody molecule ZSYM73. Simultaneous tri-specific target engagementwasconfirmedinabiosensorexperimentandtheaffinityformurineTfRwasdetermined to 5 nM. Blockable binding to TfR on endothelial cells was demonstrated using flow cytometry and in a preclinical study we observed increased uptake of the tri-specific fusion protein into the cerebrospinal fluid 24 h after injection.
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| 23. |
- Meister, Sebastian, et al.
(författare)
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Engineering of TEV protease variants with redesigned substrate specificity
- 2023
-
Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 18:11
-
Tidskriftsartikel (refereegranskat)abstract
- Due to their ability to catalytically cleave proteins and peptides, proteases present unique opportunities for the use in industrial, biotechnological, and therapeutic applications. Engineered proteases with redesigned substrate specificities have the potential to expand the scope of practical applications of this enzyme class. We here apply a combinatorial protease engineering-based screening method that links proteolytic activity to the solubility and correct folding of a fluorescent reporter protein to redesign the substrate specificity of tobacco etch virus (TEV) protease. The target substrate EKLVFQA differs at three out of seven positions from the TEV consensus substrate sequence. Flow cytometric sorting of a semi-rational TEV protease library, consisting of focused mutations of the substrate binding pockets as well as random mutations throughout the enzyme, led to the enrichment of a set of protease variants that recognize and cleave the novel target substrate.
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| 24. |
- Meister, Sebastian W., 1988-
(författare)
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Amyloid Beta – Biotherapy Target and Biotechnological Tool
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Alzheimer’s disease is a progressive neurodegenerative disease characterized primarily by memory impairment and cognitive decline. As of 2020, an estimated 50 million people suffer from Alzheimer’s disease or related dementia and no disease-modifying treatment options are yet approved for clinical practice. A major pathological feature of Alzheimer’s disease is the presence of cerebral senile plaques with aggregated amyloid beta (Aβ) peptides as the main constituent. In this thesis, Aβ is used in five separate studies either as a target for the development of potential biotherapeutical interventions or as a tool in biotechnological research.In the first study, a high-throughput screening method was developed that enables functiondriven selection of protein-based aggregation inhibitors from combinatorial libraries. The method employs a reporter protein consisting of Aβ42 fused to the N-terminus of green fluorescent protein (GFP). The reporter protein misfolds due to the aggregating nature of Aβ42. Following protein expression in Escherichia coli a low whole-cell GFP fluorescence signal was detected using flow cytometry. However, when co-expressed with an affibodybased aggregation inhibitor, the reporter protein was rescued from aggregation and an increased whole-cell GFP fluorescence signal was detected in flow cytometry. By combining the screening method with flow cytometric cell sorting, the aggregation-inhibiting affibody molecule could successfully be enriched from a large background of non-inhibiting affibody molecules. The results thus demonstrated that the developed method enables highthroughput screening and sorting of combinatorial protein libraries based on the Aβ aggregation inhibiting ability.The second study explored a strategy to increase the uptake of a biotherapeutical candidate protein into the central nervous system (CNS) via receptor-mediated transcytosis across the blood-brain barrier (BBB). The affibody-based candidate ZSYM73 binds monomeric Aβ and inhibits Aβ aggregation. Here, ZSYM73 was fused to the C-terminus of a single-chain variable fragment (scFv8D3) binding the transferrin receptor (TfR); a receptor expressed on the BBB. An engineered albumin-binding domain (ABD) was fused to ZSYM73 to extend the circulatory half-life of the fusion protein. In a mouse study, the tri-specific fusion protein scFv8D3-ZSYM73-ABD exhibited increased cerebrospinal fluid (CSF) bioavailability compared to the control protein ZSYM73-ABD, indicating an active transport mechanism into the CNS.In the third study, a novel method for combinatorial protease engineering was developed and applied to generate highly proteolytic active variants of the coxsackievirus 3C protease. The method is based on the findings form the first study and employs a reporter protein consisting of Aβ42 fused to the N-terminus of GFP via a peptide linker containing a protease substrate sequence. The reporter protein misfolds upon expression in E. coli, which resulted in a low whole-cell GFP fluorescence signal detected in flow cytometry. Co-expression of a protease with activity on the substrate sequence led to proteolytic separation of the aggregation-prone Aβ42 peptide from GFP and restored whole-cell fluorescence. This method was used in combination with flow cytometric cell sorting to isolate highly proteolytic active variants from a randomly mutated 3C protease library. The aim of the fourth study was to evaluate the potential of the newly developed method from the third study to engineer the substrate specificity of proteases. A semi-rational tobacco etch virus (TEV) protease library was screened for variants with proteolytic activity on a novel target substrate. The target substrate differed substantially from the wild-type TEV consensus substrate and exhibited high sequence similarity to the aggregation-inducing hydrophobic core region of Aβ. After three rounds of flow cytometric cell sorting, a set of TEV protease variants was enriched that exhibited proteolytic activity on the novel substrate.In the fifth study, a methodology employing flow cytometric sorting of multiprotein aggregates was developed to investigate the protein interactome related to Aβ plaques. It was demonstrated that in human serum or human CSF, Aβ aggregates bound to a fluorescent probe can be detected and isolated using flow cytometry. Quantitative mass spectrometry analysis was performed on Aβ aggregates isolated from human CSF. The abundances and functional features of proteins found in the isolated aggregates were investigated, and a hypothetical model of the layered architecture of Aβ aggregates was proposed. In conclusion, this thesis describes the development of new concepts and methods that will hopefully contribute to increasing the understanding and improving the therapy of Alzheimer’s disease and other diseases associated with protein aggregation.
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| 25. |
- Mestre Borras, Anna, 1994-, et al.
(författare)
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Generation of an anti-idiotypic affibody-based masking domain for conditional activation of EGFR-targeting
- 2023
-
Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 73, s. 9-18
-
Tidskriftsartikel (refereegranskat)abstract
- Conditional activation of engineered affinity proteins by proteolytic processing is an interesting approach for a wide range of applications. We have generated an anti-idiotypic masking domain with specificity for the binding surface of an EGFR-targeting affibody molecule using an in-house developed staphylococcal display method. The masking domain could specifically abrogate EGFR-binding on cancer cells when fused to the EGFR-targeting affibody molecule via a linker comprising a protease cleavage site. EGFR-binding was restored by proteolytic cleavage of the linker region resulting in release of the masking domain. A saturation mutagenesis study provided detailed information on the interaction between the EGFR-targeting affibody molecule and the masking domain. Introducing an anti-idiotypic masking affibody domain is a viable approach for blocking EGFR-binding and al-lows for conditional activation by proteolytic processing. The results warrant further studies evaluating the therapeutic and diagnostic applicability both in vitro and in vivo.
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| 26. |
- Moberg, Christina, et al.
(författare)
-
De unga gör helt rätt när de stämmer staten
- 2022
-
Ingår i: Aftonbladet. - 1103-9000.
-
Tidskriftsartikel (populärvet., debatt m.m.)abstract
- 1 620 forskare och lärare i forskarvärlden: Vi ställer oss bakom Auroras klimatkrav
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| 27. |
- Mähl, Stefan, 1972-, et al.
(författare)
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Edition of Johannes Hildebrandi's texts from Paris
- 2021
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Ingår i: Swedish Students at the University of Paris in the Middle Ages I. - Stockholm : Sällskapet Runica et Mediaevalia. - 9789188568793 ; , s. 845-873
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Bokkapitel (refereegranskat)
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| 28. |
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| 29. |
- Parks, Luke
(författare)
-
Development of directed-evolution methods utilizing combinatorial protein libraries in Escherichia coli
- 2024
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Directed evolution using combinatorial protein libraries is a tremendously powerful technique for the generation of proteins with new or improved properties. A key aspect in such techniques is the link between individual protein variants and their corresponding genetic information. To provide this link, the most successful combinatorial protein engineering methods employ microorganisms, such as bacteriophages, bacteria or yeast for the production and display of libraries. This thesis focuses on the development and application of new directed evolution methods utilizing the bacterium Escherichia coli (E. coli), for the engineering of affinity proteins and proteases.The first study aimed to engineer the substrate specificity of a protease from tobacco etch virus (TEV). For this purpose, a novel method was devised based on expression of intracellular protease libraries, and employed a reporter fusion protein consisting of amyloid β peptide fused to the N-terminus of enhanced green fluorescent protein (EGFP). Variants were screened for proteolytic activity on co-expressed target substrate by means of fluorescence-activated cell sorting (FACS). After three rounds of FACS, a set of TEV protease variants were enriched that exhibited improved proteolytic activity on the novel substrate.Studies two to four describe the development of an E. coli surface expression system that was explored for directed evolution applications. The method is based on display of recombinant proteins on the outer membrane via fusion to a bacterial autotransporter, adhesin involved in diffuse adherence I (AIDA-I). The second study focused on the optimization of the surface display system and its application to directed evolution. In this effort, several affinity protein classes were evaluated for surface display via AIDA-I in a panel of E. coli strains. Results showed that smaller and less complicated affibody molecules were displayed at high levels, while more complex proteins, such as antibody fragments, varied in their performance and functioned best in certain engineered strains. A mock affibody library was used to develop a high-throughput magnetic-assisted cell sorting (MACS) protocol for enrichment of binders from very large libraries.In the third and fourth study, the new E. coli display method combined with the MACS protocol was evaluated for generation of new affibody molecules.In the third study, a large naïve affibody library (>1.5×1011 members) was constructed, displayed on E. coli and characterized. The performance of the method and library was evaluated by selection of binders against two cancer-associated targets, tumor-associated calcium signal transducer 2 (TROP-2) and lymphocyte-activation gene 3 (LAG-3). MACS and FACS were performed, with flow cytometry assessment between rounds to monitor enrichment. Both selections produced high affinity binders to their respective targets.In the fourth study, a maturation library was constructed for improving the properties of an affibody molecule toward the renal cell carcinoma biomarker carbonic anhydrase IX (CAIX). Selections included stringent off-rate procedures and yielded variants with improved affinities and folding stability compared to previously reported binders.In summary, the work in this thesis demonstrate the potential of E. coli-based directed evolution methods for selection of new proteins with altered or improved properties.
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| 30. |
- Parks, Luke, et al.
(författare)
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Investigation of an AIDA-I based expression system for display of various affinity proteins on Escherichia coli
- 2024
-
Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 696
-
Tidskriftsartikel (refereegranskat)abstract
- Autotransporters constitute a large family of natural proteins that are essential for delivering many types of proteins and peptides across the outer membrane in Gram-negative bacteria. In biotechnology, autotransporters have been explored for display of recombinant proteins and peptides on the surface of Escherichia coli and have potential as tools for directed evolution of affinity proteins. Here, we investigate conditions for AIDA-I autotransporter-mediated display of recombinant proteins. A new expression vector was designed and engineered for this purpose, and a panel of proteins from different affinity-protein classes were subcloned to the vector, followed by evaluation of expression, surface display and functionality. Surface expression was explored in ten different E. coli strains together with assessment of transformation efficiencies. Furthermore, the most promising strain and expression vector combination was used in mock library selections for evaluation of magnetic-assisted cell sortings (MACS). The results demonstrated dramatically different performances depending on the type of affinity protein and choice of E. coli strain. The optimized MACS protocol showed efficient enrichment, and thus potential for the new AIDA-I display system to be used in methods for directed evolution of affinity proteins.
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| 31. |
- Persson, Jonas, et al.
(författare)
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Discovery, optimization and biodistribution of an Affibody molecule for imaging of CD69
- 2021
-
Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 11:1
-
Tidskriftsartikel (refereegranskat)abstract
- Due to the wide scale of inflammatory processes in different types of disease, more sensitive and specific biomarkers are required to improve prevention and treatment. Cluster of differentiation 69 (CD69) is one of the earliest cell surface proteins expressed by activated leukocytes. Here we characterize and optimize potential new imaging probes, Affibody molecules targeting CD69 for imaging of activated immune cells. Analysis of candidates isolated in a previously performed selection from a Z variant E. coli library to the recombinant extracellular domain of human CD69, identified one cross-reactive Z variant with affinity to murine and human CD69. Affinity maturation was performed by randomization of the primary Z variant, followed by selections from the library. The resulting Z variants were evaluated for affinity towards human and murine CD69 and thermal stability. The in vivo biodistribution was assessed by SPECT/CT in rats following conjugation of the Z variants by a DOTA chelator and radiolabeling with Indium-111. A primary Z variant with a K-d of approximately 50 nM affinity to human and murine CD69 was identified. Affinity maturation generated 5 additional Z variants with improved or similar affinity. All clones exhibited suitable stability. Radiolabeling and in vivo biodistribution in rat demonstrated rapid renal clearance for all variants, while the background uptake and washout varied. The variant Z(CD69:4) had the highest affinity for human and murine CD69 (34 nM) as well as the lowest in vivo background binding. In summary, we describe the discovery, optimization and evaluation of novel Affibody molecules with affinity for CD69. Affibody molecule Z(CD69:4) is suitable for further development for imaging of activated immune cells.
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| 32. |
- Persson, Rebecca, et al.
(författare)
-
Analysis of Peak Currents Reported by the Swedish Lightning Location System During 2015-2020
- 2022
-
Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The purpose of this work is to process lightning data from the Swedish Meteorological and Hydrological Institutes (SMHI) lightning location system to analyze which currents occur during lightning strikes in Sweden. The result can then be compared with the values currently used in dimensioning lightning protection for the transmission network, to evaluate whether the design of the protection is relevant with regard to the currents registered by the lightning location system. The results based on the years 2015-2020 show that the current distribution fits a lognormal and generalized extreme value distribution, the latter yielding a slightly better representation. The national analysis shows a significantly lower median peak current in Sweden compared to the reference median peak current recommended by CIGRE.
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| 33. |
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| 34. |
- Persson, Rebecca, et al.
(författare)
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Distribution of the current from lightning in Sweden
- 2022
-
Ingår i: Proceedings of the 2022 International Symposium on Electromagnetic Compatibility (EMC Europe 2022). - : IEEE. ; , s. 285-289
-
Konferensbidrag (refereegranskat)abstract
- Data from the lightning detection system owned by SMHI mainly from 2015–2020 was used to model the peak current. The log-normal distribution and the generalised extreme value distribution were tried and the later was slightly better. The median of the peak current was found to be lower than the value recommended by Cigre, in particular for the first stroke in a flash. In part, this can be due that the lightning detection system is calibrated for the subsequent negative strokes. The duration and the interval of the strokes were as expected. No correlation between current and rise time was found, whereas there were some differences between the regions in Sweden.
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| 35. |
- Persson, Rebecca, et al.
(författare)
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Distribution of the current from lightning in Sweden
- 2022
-
Ingår i: 2022 International Symposium on Electromagnetic Compatibility (EMC Europe 2022). - : Institute of Electrical and Electronics Engineers (IEEE). - 9781665407885 - 9781665407892 ; , s. 285-289
-
Konferensbidrag (refereegranskat)abstract
- Data from the lightning detection system owned by SMHI mainly from 2015-2020 was used to model the peak current. The log-normal distribution and the generalised extreme value distribution were tried and the later was slightly better. The median of the peak current was found to be lower than the value recommended by Cigre, in particular for the first stroke in a flash. In part, this can be due that the lightning detection system is calibrated for the subsequent negative strokes. The duration and the interval of the strokes were as expected. No correlation between current and rise time was found, whereas there were some differences between the regions in Sweden.
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| 36. |
- Puuvuori, E., et al.
(författare)
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PET-CT imaging of CD69 in rheumatoid arthritis model
- 2022
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Ingår i: European Journal of Nuclear Medicine and Molecular Imaging. - : SPRINGER. - 1619-7070 .- 1619-7089. ; 49:SUPPL 1, s. S279-S280
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 37. |
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| 38. |
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| 39. |
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| 40. |
- Rinne, Sara S., et al.
(författare)
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Benefit of Later-Time-Point PET Imaging of HER3 Expression Using Optimized Radiocobalt-Labeled Affibody Molecules
- 2020
-
Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 21:6
-
Tidskriftsartikel (refereegranskat)abstract
- HER3-binding affibody molecules are a promising format for visualization of HER3 expression. Cobalt-55, a positron-emitting isotope, with a half-life of 17.5 h, allows for next-day imaging. We investigated the influence of the charge of the radiocobalt-chelator complex on the biodistribution of anti-HER3 affibody molecule (HE)(3)-Z(HER3) and compared the best radiocobalt-labeled variant with a recently optimized gallium-labeled variant. Affibody conjugates (HE)(3)-Z(HER3)-X (X = NOTA, NODAGA, DOTA, DOTAGA) were labeled with [Co-57]Co (surrogate for Co-55). Affinity measurements, binding specificity and cellular processing were studied in two HER3-expressing cancer cell lines. Biodistribution was studied 3 and 24 h post-injection (pi) in mice with HER3-expressing BxPC-3 xenografts and compared to [Ga-68]Ga-(HE)(3)-Z(HER3)-NODAGA. Micro-single-photon emission tomography/computed tomography (microSPECT/CT) and micro-positron emission tomography/computed tomography (microPET/CT) imaging was performed 3 and 24 h pi. Stably labeled conjugates bound to HER3 with subnanomolar affinity. [Co-57]Co-(HE)(3)-Z(HER3)-DOTA had the best tumor retention and a significantly lower concentration in blood than other conjugates, leading to superior tumor-to-blood and tumor-to-liver ratios 24 h pi. Compared to [Ga-68]Ga-(HE)(3)-Z(HER3)-NODAGA 3 h pi, [Co-57]Co-(HE)(3)-Z(HER3)-DOTA provided superior imaging contrast in liver 24 h pi. Concluding, the composition and charge of the [Co-57]Co-chelator complex influenced the uptake in tumors and normal tissue. [Co-57]Co-(HE)(3)-Z(HER3)-DOTA provided the best imaging properties among the cobalt-labeled conjugates. Delayed imaging of HER3 expression with [Co-57]Co-(HE)(3)-Z(HER3)-DOTA improved imaging contrast compared to early-time-point imaging with [Ga-68]Ga-(HE)(3)-Z(HER3)-NODAGA.
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| 41. |
- Rinne, Sara S., et al.
(författare)
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HER3 PET Imaging : 68Ga-Labeled Affibody Molecules Provide Superior HER3 Contrast to 89Zr-Labeled Antibody and Antibody-Fragment-Based Tracers
- 2021
-
Ingår i: Cancers. - : MDPI AG. - 2072-6694. ; 13:19, s. 4791-4791
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Tidskriftsartikel (refereegranskat)abstract
- HER3 (human epidermal growth factor receptor type 3) is a challenging target for diag- nostic radionuclide molecular imaging due to the relatively modest overexpression in tumors and substantial expression in healthy organs. In this study, we compared four HER3-targeting PET tracers based on different types of targeting molecules in a preclinical model: the 89Zr-labeled therapeutic antibody seribantumab, a seribantumab-derived F(ab)2-fragment labeled with 89Zr and 68Ga, and the 68Ga-labeled affibody molecule [68Ga]Ga-ZHER3. The novel conjugates were radiolabeled and characterized in vitro using HER3-expressing BxPC-3 and DU145 human cancer cells. Biodistribu- tion was studied using Balb/c nu/nu mice bearing BxPC-3 xenografts. HER3-negative RAMOS xenografts were used to demonstrate binding specificity in vivo. Autoradiography was conducted on the excised tumors. nanoPET/CT imaging was performed. New conjugates specifically bound to HER3 in vitro and in vivo. [68Ga]Ga-DFO-seribantumab-F(ab’)2 was considered unsuitable for imaging due to the low stability and high uptake in normal organs. The highest tumor-to-non-tumor contrast with [89Zr]Zr-DFO-seribantumab and [89Zr]Zr-DFO-seribantumab-F(ab’)2 was achieved at 96 h and 48 h pi, respectively. Despite lower tumor uptake, [68Ga]Ga-ZHER3 provided the best imaging contrast due to the fastest clearance from blood and normal organs. The results of our study suggest that affibody-based tracers are more suitable for PET imaging of HER3 expression than antibody- and antibody-fragment-based tracers.
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| 42. |
- Rinne, Sara S., et al.
(författare)
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Influence of Residualizing Properties of the Radiolabel on Radionuclide Molecular Imaging of HER3 Using Affibody Molecules
- 2020
-
Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 21:4
-
Tidskriftsartikel (refereegranskat)abstract
- Human epidermal growth factor receptor type 3 (HER3) is an emerging therapeutic target in several malignancies. To select potential responders to HER3-targeted therapy, radionuclide molecular imaging of HER3 expression using affibody molecules could be performed. Due to physiological expression of HER3 in normal organs, high imaging contrast remains challenging. Due to slow internalization of affibody molecules by cancer cells, we hypothesized that labeling (HE)(3)-Z(HER3:08698)-DOTAGA affibody molecule with non-residualizing [I-125]-N-succinimidyl-4-iodobenzoate (PIB) label would improve the tumor-to-normal organs ratios compared to previously reported residualizing radiometal labels. The [I-125]I-PIB-(HE)(3)-Z(HER3:08698)-DOTAGA was compared side-by-side with [In-111]In-(HE)(3)-Z(HER3:08698)-DOTAGA. Both conjugates demonstrated specific high-affinity binding to HER3-expressing BxPC-3 and DU145 cancer cells. Biodistribution in mice bearing BxPC-3 xenografts at 4 and 24h pi showed faster clearance of the [I-125]I-PIB label compared to the indium-111 label from most tissues, except blood. This resulted in higher tumor-to-organ ratios in HER3-expressing organs for [I-125]I-PIB-(HE)(3)-Z(HER3:08698)-DOTAGA at 4 h, providing the tumor-to-liver ratio of 2.4 +/- 0.3. The tumor uptake of both conjugates was specific, however, it was lower for the [I-125]I-PIB label. In conclusion, the use of non-residualizing [I-125]I-PIB label for HER3-targeting affibody molecule provided higher tumor-to-liver ratio than the indium-111 label, however, further improvement in tumor uptake and retention is needed.
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| 43. |
- Rinne, Sara S., et al.
(författare)
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Targeting Tumor Cells Overexpressing the Human Epidermal Growth Factor Receptor 3 with Potent Drug Conjugates Based on Affibody Molecules
- 2022
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Ingår i: Biomedicines. - : MDPI AG. - 2227-9059. ; 10:6
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Tidskriftsartikel (refereegranskat)abstract
- Increasing evidence suggests that therapy targeting the human epidermal growth factor receptor 3 (HER3) could be a viable route for targeted cancer therapy. Here, we studied a novel drug conjugate, ZHER3-ABD-mcDM1, consisting of a HER3-targeting affibody molecule, coupled to the cytotoxic tubulin polymerization inhibitor DM1, and an albumin-binding domain for in vivo half-life extension. ZHER3-ABD-mcDM1 showed a strong affinity to the extracellular domain of HER3 (K-D 6 nM), and an even stronger affinity (KD 0.2 nM) to the HER3-overexpressing pancreatic carcinoma cell line, BxPC-3. The drug conjugate showed a potent cytotoxic effect on BxPC-3 cells with an IC50 value of 7 nM. Evaluation of a radiolabeled version, [99mTc]Tc-ZHER3-ABD-mcDM1, showed a relatively high rate of internalization, with a 27% internalized fraction after 8 h. Further in vivo evaluation showed that it could target BxPC-3 (pancreatic carcinoma) and DU145 (prostate carcinoma) xenografts in mice, with an uptake peaking at 6.3 +/- 0.4% IA/g at 6 h post-injection for the BxPC-3 xenografts. The general biodistribution showed uptake in the liver, lung, salivary gland, stomach, and small intestine, organs known to express murine ErbB3 naturally. The results from the study show that ZHER3-ABD-mcDM1 is a highly potent and selective drug conjugate with the ability to specifically target HER3 overexpressing cells. Further pre-clinical and clinical development is discussed.
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