| 1. |
- Deyev, S., et al.
(författare)
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Comparative Evaluation of Two DARPin Variants : Effect of Affinity, Size, and Label on Tumor Targeting Properties
- 2019
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Ingår i: Molecular Pharmaceutics. - : American Chemical Society (ACS). - 1543-8384 .- 1543-8392. ; 16:3, s. 995-1008
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Tidskriftsartikel (refereegranskat)abstract
- Designed ankyrin repeat proteins (DARPins) are small engineered scaffold proteins that can be selected for binding to desirable molecular targets. High affinity and small size of DARPins render them promising probes for radionuclide molecular imaging. However, detailed knowledge on many factors influencing their imaging properties is still lacking. We have evaluated two human epidermal growth factor 2 (HER2)-specific DARPins with different size and binding properties. DARPins 9-29-H 6 and G3-H 6 were radiolabeled with iodine-125 and tricarbonyl technetium-99m and evaluated in vitro. A side-by-side comparison of biodistribution and tumor targeting was performed. HER2-specific tumor accumulation of G3-H 6 was demonstrated. A combination of smaller size and higher affinity resulted in a higher tumor uptake of G3-H 6 in comparison to 9-29-H 6 . Technetium-99m labeled G3-H 6 demonstrated a better biodistribution profile than 9-29-H 6 , with several-fold lower uptake in liver. Radioiodinated G3-H 6 showed the best tumor-to-organ ratios. The combined effect of affinity, molecular weight, scaffold composition, and nonresidualizing properties of iodine label provided radioiodinated G3-H 6 with high clinical potential for imaging of HER2.
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| 2. |
- Fleetwood, Filippa, et al.
(författare)
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Novel affinity binders for neutralization of vascular endothelial growth factor (VEGF) signaling
- 2016
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Ingår i: Cellular and Molecular Life Sciences (CMLS). - : Birkhauser Verlag. - 1420-682X .- 1420-9071. ; 73:8, s. 1671-1683
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Tidskriftsartikel (refereegranskat)abstract
- Angiogenesis denotes the formation of new blood vessels from pre-existing vasculature. Progression of diseases such as cancer and several ophthalmological disorders may be promoted by excess angiogenesis. Novel therapeutics to inhibit angiogenesis and diagnostic tools for monitoring angiogenesis during therapy, hold great potential for improving treatment of such diseases. We have previously generated so-called biparatopic Affibody constructs with high affinity for the vascular endothelial growth factor receptor-2 (VEGFR2), which recognize two non-overlapping epitopes in the ligand-binding site on the receptor. Affibody molecules have previously been demonstrated suitable for imaging purposes. Their small size also makes them attractive for applications where an alternative route of administration is beneficial, such as topical delivery using eye drops. In this study, we show that decreasing linker length between the two Affibody domains resulted in even slower dissociation from the receptor. The new variants of the biparatopic Affibody bound to VEGFR2-expressing cells, blocked VEGFA binding, and inhibited VEGFA-induced signaling of VEGFR2 over expressing cells. Moreover, the biparatopic Affibody inhibited sprout formation of endothelial cells in an in vitro angiogenesis assay with similar potency as the bivalent monoclonal antibody ramucirumab. This study demonstrates that the biparatopic Affibody constructs show promise for future therapeutic as well as in vivo imaging applications.
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| 3. |
- Gast, Veronica, 1992, et al.
(författare)
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Engineering Saccharomyces cerevisiae for the production and secretion of Affibody molecules
- 2022
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 21:1
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Affibody molecules are synthetic peptides with a variety of therapeutic and diagnostic applications. To date, Affibody molecules have mainly been produced by the bacterial production host Escherichia coli. There is an interest in exploring alternative production hosts to identify potential improvements in terms of yield, ease of production and purification advantages. In this study, we evaluated the feasibility of Saccharomyces cerevisiae as a production chassis for this group of proteins. RESULTS: We examined the production of three different Affibody molecules in S. cerevisiae and found that these Affibody molecules were partially degraded. An albumin-binding domain, which may be attached to the Affibody molecules to increase their half-life, was identified to be a substrate for several S. cerevisiae proteases. We tested the removal of three vacuolar proteases, proteinase A, proteinase B and carboxypeptidase Y. Removal of one of these, proteinase A, resulted in intact secretion of one of the targeted Affibody molecules. Removal of either or both of the two additional proteases, carboxypeptidase Y and proteinase B, resulted in intact secretion of the two remaining Affibody molecules. The produced Affibody molecules were verified to bind their target, human HER3, as potently as the corresponding molecules produced in E. coli in an in vitro surface-plasmon resonance binding assay. Finally, we performed a fed-batch fermentation with one of the engineered protease-deficient S. cerevisiae strains and achieved a protein titer of 530 mg Affibody molecule/L. CONCLUSION: This study shows that engineered S. cerevisiae has a great potential as a production host for recombinant Affibody molecules, reaching a high titer, and for proteins where endotoxin removal could be challenging, the use of S. cerevisiae obviates the need for endotoxin removal from protein produced in E. coli.
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| 4. |
- 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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| 5. |
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| 6. |
- Güler, Rezan, et al.
(författare)
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Estimating the Deformability of Elastic Materials using Optical Flow and Position-based Dynamics
- 2015
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Ingår i: Humanoid Robots (Humanoids), 2015 IEEE-RAS 15th International Conference on. - : IEEE conference proceedings. ; , s. 965-971
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Konferensbidrag (refereegranskat)abstract
- Knowledge of the physical properties of objects is essential in a wide range of robotic manipulation scenarios. A robot may not always be aware of such properties prior to interaction. If an object is incorrectly assumed to be rigid, it may exhibit unpredictable behavior when grasped. In this paper, we use vision based observation of the behavior of an object a robot is interacting with and use it as the basis for estimation of its elastic deformability. This is estimated in a local region around the interaction point using a physics simulator. We use optical flow to estimate the parameters of a position-based dynamics simulation using meshless shape matching (MSM). MSM has been widely used in computer graphics due to its computational efficiency, which is also important for closed-loop control in robotics. In a controlled experiment we demonstrate that our method can qualitatively estimate the physical properties of objects with different degrees of deformability.
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| 7. |
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| 8. |
- Güler, Rezan, et al.
(författare)
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Increasing thermal stability and improving biodistribution of VEGFR2-binding affibody molecules by a combination of in silico and directed evolution approaches
- 2020
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Ingår i: Scientific Reports. - : Nature Research. - 2045-2322. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- The family of vascular endothelial growth factor (VEGF) ligands and their interactions with VEGF receptors (VEGFRs) play important roles in both pathological and physiological angiogenesis. Hence, agonistic and antagonistic ligands targeting this signaling pathway have potential for both studies on fundamental biology and for development of therapies and diagnostics. Here, we engineer VEGFR2-binding affibody molecules for increased thermostability, refolding and improved biodistribution. We designed libraries based on the original monomeric binders with the intention of reducing hydrophobicity, while retaining high affinity for VEGFR2. Libraries were displayed on bacteria and binders were isolated by fluorescence-activated cell sorting (FACS). In parallel, we used an automated sequence- and structure-based in silico algorithm to identify potentially stabilizing mutations. Monomeric variants isolated from the screening and the in silico approach, respectively, were characterized by circular dichroism spectroscopy and biosensor assays. The most promising mutations were combined into new monomeric constructs which were finally fused into a dimeric construct, resulting in a 15 degrees C increase in melting temperature, complete refolding capability after heat-induced denaturation, retained low picomolar affinity and improved biodistribution profile in an in vivo mouse model. These VEGFR2-binding affibody molecules show promise as candidates for further in vivo studies to assess their suitability as molecular imaging and therapeutic agents.
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| 9. |
- Güler, Rezan, et al.
(författare)
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VEGFR2-Specific Ligands Based on Affibody Molecules Demonstrate Agonistic Effects when Tetrameric in the Soluble Form or Immobilized via Spider Silk
- 2019
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Ingår i: ACS Biomaterials Science & Engineering. - : American Chemical Society (ACS). - 2373-9878. ; 5:12, s. 6474-6484
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Tidskriftsartikel (refereegranskat)abstract
- Strategies to promote vascularization are being developed in order to improve long-term survival of artificial tissue constructs. Vascular endothelial growth factor A (VEGFA) has an important role in both pathological and physiological angiogenesis, mediated by binding to VEGF receptors (VEGFRs). In nature, signaling can be modulated by presentation of growth factors in either soluble form or bound to the extracellular matrix. Herein, a previously reported VEGFR2-binding antagonistic affibody heterodimer (di-Z(VEGFR2)) was formatted into a tetrameric construct (tetra-Z(VEGFR2)) with the intention of generating artificial agonistic ligands for VEGFR2 signaling. In vitro cell assays demonstrated that tetra-Z(VEGFR2) induced VEGFR2 phosphorylation and increased cell proliferation, in contrast to di-Z(VEGFR2). In order to simulate matrix-bound factors, both constructs were fused at the genetic level to a partial spider silk protein, 4RepCT. Assembly of the silk fusion proteins onto a solid surface was verified by quartz crystal microbalance with dissipation analysis. Moreover, surface plasmon resonance studies demonstrated retained VEGFR2 binding ability of di-Z(VEGFR2) silk and tetra-Z(VEGFR2)-silk after silk-mediated immobilization. Cell culture studies demonstrated that VEGFR2-overexpressing cells adhered to di-Z(VEGFR2) -silk and tetra-Z(VEGFR2) -silk and had activated VEGFR2 signaling. Altogether, we demonstrate the potential of especially tetra-Z(VEGFR2)-silk to promote angiogenesis in tissue-engineering applications. The results from the study also show that molecules can obtain completely new functions when presented on materials, and verifying the biological effects after functionalizing materials is thus always recommended.
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| 10. |
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