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Träfflista för sökning "AMNE:(NATURAL SCIENCES Biological Sciences Biochemistry and Molecular Biology) ;pers:(Ståhl Stefan)"

Sökning: AMNE:(NATURAL SCIENCES Biological Sciences Biochemistry and Molecular Biology) > Ståhl Stefan

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1.
  • Lindberg, Hanna, et al. (författare)
  • A truncated and dimeric format of an Affibody library on bacteria enables FACS-mediated isolation of amyloid-beta aggregation inhibitors with subnanomolar affinity
  • 2015
  • Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 10:11, s. 1707-1718
  • Tidskriftsartikel (refereegranskat)abstract
    • The amyloid hypothesis suggests that accumulation of amyloid β (Aβ) peptides in the brain is involved in development of Alzheimer's disease. We previously generated a small dimeric affinity protein that inhibited Aβ aggregation by sequestering the aggregation prone parts of the peptide. The affinity protein is originally based on the Affibody scaffold, but is evolved to a distinct interaction mechanism involving complex structural rearrangement in both the Aβ peptide and the affinity proteins upon binding. The aim of this study was to decrease the size of the dimeric affinity protein and significantly improve its affinity for the Aβ peptide to increase its potential as a future therapeutic agent. We combined a rational design approach with combinatorial protein engineering to generate two different affinity maturation libraries. The libraries were displayed on staphylococcal cells and high-affinity Aβ-binding molecules were isolated using flow-cytometric sorting. The best performing candidate binds Aβ with a KD value of around 300 pM, corresponding to a 50-fold improvement in affinity relative to the first-generation binder. The new dimeric Affibody molecule was shown to capture Aβ1-42 peptides from spiked E. coli lysate. Altogether, our results demonstrate successful engineering of this complex binder for increased affinity to the Aβ peptide.
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2.
  • Luheshi, Leila M., et al. (författare)
  • Sequestration of the A beta Peptide Prevents Toxicity and Promotes Degradation In Vivo
  • 2010
  • Ingår i: PLoS biology. - : Public Library of Science (PLoS). - 1544-9173 .- 1545-7885. ; 8:3, s. e1000334-
  • Tidskriftsartikel (refereegranskat)abstract
    • Protein aggregation, arising from the failure of the cell to regulate the synthesis or degradation of aggregation-prone proteins, underlies many neurodegenerative disorders. However, the balance between the synthesis, clearance, and assembly of misfolded proteins into neurotoxic aggregates remains poorly understood. Here we study the effects of modulating this balance for the amyloid-beta (A beta) peptide by using a small engineered binding protein (Z(A beta 3)) that binds with nanomolar affinity to A beta, completely sequestering the aggregation-prone regions of the peptide and preventing its aggregation. Co-expression of Z(A beta 3) in the brains of Drosophila melanogaster expressing either A beta(42) or the aggressive familial Alzheimer's disease (AD) associated E22G variant of A beta(42) abolishes their neurotoxic effects. Biochemical analysis indicates that monomer A beta binding results in degradation of the peptide in vivo. Complementary biophysical studies emphasize the dynamic nature of A beta aggregation and reveal that Z(A beta 3) not only inhibits the initial association of A beta monomers into oligomers or fibrils, but also dissociates pre-formed oligomeric aggregates and, although very slowly, amyloid fibrils. Toxic effects of peptide aggregation in vivo can therefore be eliminated by sequestration of hydrophobic regions in monomeric peptides, even when these are extremely aggregation prone. Our studies also underline how a combination of in vivo and in vitro experiments provide mechanistic insight with regard to the relationship between protein aggregation and clearance and show that engineered binding proteins may provide powerful tools with which to address the physiological and pathological consequences of protein aggregation.
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3.
  • Wållberg, Helena, et al. (författare)
  • Affinity recovery of eight HER2-binding affibody variants using an anti-idiotypic affibody molecule as capture ligand
  • 2011
  • Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 76:1, s. 127-135
  • Tidskriftsartikel (refereegranskat)abstract
    • Affibody molecules generated by combinatorial protein engineering to bind the human epidermal growth factor receptor 2 (HER2) have in earlier studies proven to be promising tracers for HER2-mediated molecular imaging of cancer. Amino acid extensions either at the N- or C-terminus of these Z(HER2) affibody molecules, have been successfully employed for site-specific radiolabeling of the tracer candidates. Hexahistidyls or other tags, which would be convenient for recovery purposes, should be avoided since they could negatively influence the tumor targeting efficacy and biodistribution properties of the tracer. Using a new beta-lactamase-based protein fragment complementation assay (PCA), an affibody molecule was isolated which bound a Z(HER2) affibody molecule with sub-micromolar affinity, but not unrelated affibody molecules. This suggests that the interacting area include the HER2-binding surface of Z(HER2). This novel anti-idiotypic affibody molecule Z(E01) was produced in Escherichia coli, purified, and chemically coupled to a chromatography resin in order to generate an affibody-based affinity column, suitable for recovery of different variants of Z(HER2) affibody molecules, having a common binding surface for HER2. Eight such Z(HER2) affibody molecules, designed for future radioimaging investigations, having different C-terminal peptide extensions aimed for radioisotope (Tc-99m)-chelation, were successfully produced and recovered in a single step to high purity using the anti-idiotypic affibody ligand for the affinity purification. These results clearly suggest a potential for the development of anti-idiotypic affibody-based resins for efficient recovery of related variants of a target protein that might have altered biochemical properties, thus avoiding the cumbersome design of specific recovery schemes for each variant of a target protein.
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4.
  • Altai, Mohamed, et al. (författare)
  • Influence of Molecular Design on the Targeting Properties of ABD-Fused Mono- and Bi-Valent Anti-HER3 Affibody Therapeutic Constructs
  • 2018
  • Ingår i: Cells. - : MDPI AG. - 2073-4409. ; 7:10
  • Tidskriftsartikel (refereegranskat)abstract
    • Overexpression of human epidermal growth factor receptor type 3 (HER3) is associated with tumour cell resistance to HER-targeted therapies. Monoclonal antibodies (mAbs) targeting HER3 are currently being investigated for treatment of various types of cancers. Cumulative evidence suggests that affibody molecules may be appropriate alternatives to mAbs. We previously reported a fusion construct (3A3) containing two HER3-targeting affibody molecules flanking an engineered albumin-binding domain (ABD 035) included for the extension of half-life in circulation. The 3A3 fusion protein (19.7 kDa) was shown to delay tumour growth in mice bearing HER3-expressing xenografts and was equipotent to the mAb seribantumab. Here, we have designed and explored a series of novel formats of anti-HER3 affibody molecules fused to the ABD in different orientations. All constructs inhibited heregulin-induced phosphorylation in HER3-expressing BxPC-3 and DU-145 cell lines. Biodistribution studies demonstrated extended the half-life of all ABD-fused constructs, although at different levels. The capacity of our ABD-fused proteins to accumulate in HER3-expressing tumours was demonstrated in nude mice bearing BxPC-3 xenografts. Formats where the ABD was located on the C-terminus of affibody binding domains (3A, 33A, and 3A3) provided the best tumour targeting properties in vivo. Further development of these promising candidates for treatment of HER3-overexpressing tumours is therefore justified.
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5.
  • Dahlsson Leitao, Charles, et al. (författare)
  • Molecular Design of HER3-Targeting Affibody Molecules : Influence of Chelator and Presence of HEHEHE-Tag on Biodistribution of 68Ga-Labeled Tracers
  • 2019
  • Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 20:5
  • Tidskriftsartikel (refereegranskat)abstract
    • Affibody-based imaging of HER3 is a promising approach for patient stratification. We investigated the influence of a hydrophilic HEHEHE-tag ((HE)3-tag) and two different gallium-68/chelator-complexes on the biodistribution of Z08698 with the aim to improve the tracer for PET imaging. Affibody molecules (HE)3-Z08698-X and Z08698-X (X = NOTA, NODAGA) were produced and labeled with gallium-68. Binding specificity and cellular processing were studied in HER3-expressing human cancer cell lines BxPC-3 and DU145. Biodistribution was studied 3 h p.i. in Balb/c nu/nu mice bearing BxPC-3 xenografts. Mice were imaged 3 h p.i. using microPET/CT. Conjugates were stably labeled with gallium-68 and bound specifically to HER3 in vitro and in vivo. Association to cells was rapid but internalization was slow. Uptake in tissues, including tumors, was lower for (HE)3-Z08698-X than for non-tagged variants. The neutral [68Ga]Ga-NODAGA complex reduced the hepatic uptake of Z08698 compared to positively charged [68Ga]Ga-NOTA-conjugated variants. The influence of the chelator was more pronounced in variants without (HE)3-tag. In conclusion, hydrophilic (HE)3-tag and neutral charge of the [68Ga]Ga-NODAGA complex promoted blood clearance and lowered hepatic uptake of Z08698. [68Ga]Ga-(HE)3-Z08698-NODAGA was considered most promising, providing the lowest blood and hepatic uptake and the best imaging contrast among the tested variants.
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6.
  • Rinne, Sara S., et al. (författare)
  • 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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7.
  • Rinne, Sara S., et al. (författare)
  • 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
  • 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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8.
  • Löfblom, John, et al. (författare)
  • Evaluation of staphylococcal cell surface display and flow cytometry for postselectional characterization of affinity proteins in combinatorial protein engineering applications
  • 2007
  • Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 73:21, s. 6714-6721
  • Tidskriftsartikel (refereegranskat)abstract
    • For efficient generation of high-affinity protein-based binding molecules, fast and reliable downstream characterization platforms are needed. In this work, we have explored the use of staphylococcal cell surface display together with How cytometry for affinity characterization of candidate affibody molecules directly on the cell surface. A model system comprising three closely related affibody molecules with different affinities for immunoglobulin G and an albumin binding domain with affinity for human serum albumin was used to investigate advantages and differences compared to biosensor technology in a side-by-side manner. Equilibrium dissociation constant (K-D) determinations as well as dissociation rate analysis were performed using both methods, and the results show that the on-cell determinations give both KD and dissociation rate values in a very fast and reproducible manner and that the relative affinities are very similar to the biosensor results. Interestingly, the results also show that there are differences between the absolute affinities determined with the two different technologies, and possible explanations for this are discussed. This work demonstrates the advantages of cell surface display for directed evolution of affinity proteins in terms of fast postselectional, on-cell characterization of candidate clones without the need for subcloning and subsequent protein expression and purification but also demonstrates that it is important to be aware that absolute affinities determined using different methods often vary substantially and that such comparisons therefore could be difficult.
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9.
  • Löfblom, John, et al. (författare)
  • Fine affinity discrimination by normalized fluorescence activated cell sorting in staphylococcal surface display
  • 2005
  • Ingår i: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 0378-1097 .- 1574-6968. ; 248:2, s. 189-198
  • Tidskriftsartikel (refereegranskat)abstract
    • We have investigated a staphylococcal surface display system for its potential future use as a protein library display system ill combinatorial biochemistry. Efficient affinity-based selections require a system capable of fine affinity discrimination of closely related binders to minimize the loss of potentially improved variants. In this Study, a significant breakthrough was achieved to avoid biases due to potential cell-to-cell variations in surface expression levels, since it was found that a generic protein tag, present within the displayed recombinant surface proteins on the cells, could be successfully employed to obtain normalization of the target-binding signal. Four mutated variants of a staphylococcal protein A domain with different affinity to human IgG were successfully expressed on the surface of recombinant Staphylococcus carnosus cells. The system was evaluated for affinity-based cell sorting experiments, where cell-displayed protein A domains with an 8-fold difference in target affinity were mixed at a ratio of 1: 1000 and sorted using FACS. Enrichment factors around 140-fold were obtained from a single round of sorting under normal library sorting conditions when the top 0.1% fraction having the highest antigen binding to Surface expression level ratio was sorted. The results demonstrate that the system would have a potential as a selection system in protein library display applications, and the normalization strategy should indeed make it possible to achieve fine affinity discriminations in future library selections. (c) 2005 Federation of European Microbiological Societies.
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10.
  • Rockberg, Johan, et al. (författare)
  • Epitope mapping of antibodies using bacterial surface display
  • 2008
  • Ingår i: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 5:12, s. 1039-1045
  • Tidskriftsartikel (refereegranskat)abstract
    • We describe a method for mapping the epitopes recognized by antibodies, based on bacterial surface expression of antigen protein fragments followed by antibody-based flow-cytometric sorting. We analyzed the binding sites of both monoclonal and polyclonal antibodies directed to three human protein targets: (i) the human epidermal growth factor receptor 2 (HER2), (ii) ephrin-B3 and (iii) the transcription factor SATB2. All monoclonal antibodies bound a single epitope, whereas the polyclonal antibodies showed, in each case, a binding pattern with one to five separate epitopes. A comparison of polyclonal and monoclonal antibodies raised to the same antigen showed overlapping binding epitopes. We also demonstrated that bacterial cells with displayed protein fragments can be used as affinity ligands to generate epitope-specific antibodies. Our approach shows a path forward for systematic validation of antibodies for epitope specificity and cross-reactivity on a whole-proteome level.
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