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  • 1. Altai, M.
    et al.
    Ding, Haozhong
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Rinne, S.
    Dept Med Chem, Uppsala, Sweden..
    Vorobyeva, A.
    Gräslund, Torbjörn
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Tolmachev, V.
    Orlova, A.
    Dept Med Chem, Uppsala, Sweden..
    Evaluation Of Several Newly Designed Affibody-based Drug Conjugates Using Radionuclide-based Techniques: A Powerful Tool For Drug Development2019Ingår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 46, nr SUPPL 1, s. S715-S716Artikel i tidskrift (Övrigt vetenskapligt)
  • 2.
    Altai, M.
    et al.
    Immunology, Genetics and Pathology, Uppsala, SWEDEN, .
    Vorobyeva, A.
    Immunology, Genetics and Pathology, Uppsala, SWEDEN, .
    Westerlund, Kristina
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Mitran, B.
    Div Mol Imaging, Uppsala, Sweden..
    Orlova, A.
    Div Mol Imaging, Uppsala, Sweden..
    Eriksson Karlström, Amelie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Tolmachev, V.
    Immunology, Genetics and Pathology, Uppsala, SWEDEN, .
    A novel method for conjugation of PNA to antibodies for radionuclide based pretargeting: proof of principal2018Ingår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 45, s. S648-S648Artikel i tidskrift (Övrigt vetenskapligt)
  • 3. Altai, Mohamed
    et al.
    Liu, Hao
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Ding, Haozhong
    Mitran, Bogdan
    Edqvist, Per-Henrik
    Tolmachev, Vladimir
    Orlova, Anna
    Gräslund, Torbjorn
    Affibody-derived Drug Conjugates: Potent Cytotoxic Drugs ForTreatment Of HER2 Over-Expressing TumorsManuskript (preprint) (Övrigt vetenskapligt)
  • 4.
    Cavallaro, Sara
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Fotonik.
    Horak, Josef
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Haag, Petra
    Karolinska Inst, Karolinska Univ Hosp, Dept Oncol Pathol, Theme Canc,Patient Area,Pelvis, Akad Straket 1, S-17164 Stockholm, Sweden..
    Gupta, Dhanu
    Karolinska Inst, Clin Res Ctr, Dept Lab Med, S-17177 Stockholm, Sweden.;Evox Therapeut Ltd, Oxford OX4 4HG, England..
    Stiller, Christiane
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Sahu, Siddharth S.
    Uppsala Univ, Angstrom Lab, Dept Solid State Elect, Box 534, SE-75121 Uppsala, Sweden..
    Gorgens, Andre
    Karolinska Inst, Clin Res Ctr, Dept Lab Med, S-17177 Stockholm, Sweden.;Evox Therapeut Ltd, Oxford OX4 4HG, England.;Univ Duisburg Essen, Univ Hosp Essen, Inst Transfus Med, D-45122 Essen, Germany..
    Gatty, Hithesh K.
    Uppsala Univ, Angstrom Lab, Dept Solid State Elect, Box 534, SE-75121 Uppsala, Sweden..
    Viktorsson, Kristina
    Karolinska Inst, Dept Oncol Pathol, Karolinska Univ Hosp, Theme Canc,Patient Area,Head & Neck Lung & Skin, Akad Straket 1, S-17164 Solna, Sweden..
    El Andaloussi, Samir
    Karolinska Inst, Clin Res Ctr, Dept Lab Med, S-17177 Stockholm, Sweden.;Evox Therapeut Ltd, Oxford OX4 4HG, England..
    Lewensohn, Rolf
    Karolinska Inst, Dept Oncol Pathol, Karolinska Univ Hosp, Theme Canc,Patient Area,Head & Neck Lung & Skin, Akad Straket 1, S-17164 Solna, Sweden..
    Eriksson Karlström, Amelie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering. KTH Royal Inst Technol, Sch Engn Sci Chem Biotechnol & Hlth, Dept Prot Sci, AlbalNova Univ Ctr, S-10691 Stockholm, Sweden..
    Linnros, Jan
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Fotonik. KTH Royal Inst Technol, Sch Engn Sci, Dept Appl Phys, S-16440 Kista, Sweden..
    Dev, Apurba
    Uppsala Univ, Angstrom Lab, Dept Solid State Elect, Box 534, SE-75121 Uppsala, Sweden..
    Label-Free Surface Protein Profiling of Extracellular Vesicles by an Electrokinetic Sensor2019Ingår i: ACS SENSORS, ISSN 2379-3694, Vol. 4, nr 5, s. 1399-1408Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Small extracellular vesicles (sEVs) generated from the endolysosomal system, often referred to as exosomes, have attracted interest as a suitable biomarker for cancer diagnostics, as they carry valuable biological information and reflect their cells of origin. Herein, we propose a simple and inexpensive electrical method for label-free detection and profiling of sEVs in the size range of exosomes. The detection method is based on the electrokinetic principle, where the change in the streaming current is monitored as the surface markers of the sEVs interact with the affinity reagents immobilized on the inner surface of a silica microcapillary. As a proof-of-concept, we detected sEVs derived from the non-small-cell lung cancer (NSCLC) cell line H1975 for a set of representative surface markers, such as epidermal growth factor receptor (EGFR), CD9, and CD63. The detection sensitivity was estimated to be similar to 175000 sEVs, which represents a sensor surface coverage of only 0.04%. We further validated the ability of the sensor to measure the expression level of a membrane protein by using sEVs displaying artificially altered expressions of EGFR and CD63, which were derived from NSCLC and human embryonic kidney (HEK) 293T cells, respectively. The analysis revealed that the changes in EGFR and CD63 expressions in sEVs can be detected with a sensitivity in the order of 10% and 3%, respectively, of their parental cell expressions. The method can be easily parallelized and combined with existing microfluidic-based EV isolation technologies, allowing for rapid detection and monitoring of sEVs for cancer diagnosis.

  • 5. Chouhan, Dimple
    et al.
    Thatikonda, Naresh
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Nilebäck, Linnea
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Widhe, Mona
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Hedhammar, My
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Mandal, Biman B.
    Recombinant Spider Silk Functionalized Silkworm Silk Matrices as Potential Bioactive Wound Dressings and Skin Grafts2018Ingår i: ACS Applied Materials and Interfaces, ISSN 1944-8244, E-ISSN 1944-8252, Vol. 10, nr 28, s. 23560-23572Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Silk is considered to be a potential biomaterial for a wide number of biomedical applications. Silk fibroin (SF) can be retrieved in sufficient quantities from the cocoons produced by silkworms. While it is easy to formulate into scaffolds with favorable mechanical properties, the natural SF does not contain bioactive functions. Spider silk proteins, on the contrary, can be produced in fusion with bioactive protein domains, but the recombinant procedures are expensive, and large-scale production is challenging. We combine the two types of silk to fabricate affordable, functional tissue-engineered constructs for wound-healing applications. Nanofibrous mats and microporous scaffolds made of natural silkworm SF are used as a bulk material that are top-coated with the recombinant spider silk protein (4RepCT) in fusion with a cell-binding motif, antimicrobial peptides, and a growth factor. For this, the inherent silk properties are utilized to form interactions between the two silk types by self-assembly. The intended function, that is, improved cell adhesion, antimicrobial activity, and growth factor stimulation, could be demonstrated for the obtained functionalized silk mats. As a skin prototype, SF scaffolds coated with functionalized silk are cocultured with multiple cell types to demonstrate formation of a bilayered tissue construct with a keratinized epidermal layer under in vitro conditions. The encouraging results support this strategy of fabrication of an affordable bioactive SF-spider silk-based biomaterial for wound dressings and skin substitutes.

  • 6.
    Ding, Haozhong
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Altai, Mohamed
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden..
    Rinne, Sara S.
    Uppsala Univ, Dept Med Chem, S-75123 Uppsala, Sweden..
    Vorobyeva, Anzhelika
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden..
    Tolmachev, Vladimir
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden..
    Gräslund, Torbjörn
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Orlova, Anna
    Uppsala Univ, Dept Med Chem, S-75123 Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, S-75123 Uppsala, Sweden..
    Incorporation of a Hydrophilic Spacer Reduces Hepatic Uptake of HER2-Targeting Affibody-DM1 Drug Conjugates2019Ingår i: Cancers, ISSN 2072-6694, Vol. 11, nr 8, artikel-id 1168Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Affibody molecules are small affinity-engineered scaffold proteins which can be engineered to bind to desired targets. The therapeutic potential of using an affibody molecule targeting HER2, fused to an albumin-binding domain (ABD) and conjugated with the cytotoxic maytansine derivate MC-DM1 (AffiDC), has been validated. Biodistribution studies in mice revealed an elevated hepatic uptake of the AffiDC, but histopathological examination of livers showed no major signs of toxicity. However, previous clinical experience with antibody drug conjugates have revealed a moderateto high-grade hepatotoxicity in treated patients, which merits efforts to also minimize hepatic uptake of the AffiDCs. In this study, the aim was to reduce the hepatic uptake of AffiDCs and optimize their in vivo targeting properties. We have investigated if incorporation of hydrophilic glutamate-based spacers adjacent to MC-DM1 in the AffiDC, (Z(HER2:2891))(2) -ABD-MC-DM1, would counteract the hydrophobic nature of MC-DM1 and, hence, reduce hepatic uptake. Two new AffiDCs including either a triglutamate-spacer-, (Z(HER2:2891))(2)-ABD-E-3-MC-DM1, or a hexaglutamate-spacer-, (Z(HER2:2891))(2)-ABD-E-6-MC-DM1 next to the site of MC-DM1 conjugation were designed. We radiolabeled the hydrophilized AffiDCs and compared them, both in vitro and in vivo, with the previously investigated (Z(HER2:2891))(2)-ABD-MC-DM1 drug conjugate containing no glutamate spacer. All three AffiDCs demonstrated specific binding to HER2 and comparable in vitro cytotoxicity. A comparative biodistribution study of the three radiolabeled AffiDCs showed that the addition of glutamates reduced drug accumulation in the liver while preserving the tumor uptake. These results confirmed the relation between DM1 hydrophobicity and liver accumulation. We believe that the drug development approach described here may also be useful for other affinity protein-based drug conjugates to further improve their in vivo properties and facilitate their clinical translatability.

  • 7.
    Garousi, J.
    et al.
    Uppsala Univ, Uppsala, Sweden..
    Lindbo, Sarah
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Mitran, B.
    Uppsala Univ, Uppsala, Sweden..
    Vorobyeva, A.
    Uppsala Univ, Uppsala, Sweden..
    Oroujeni, M.
    Uppsala Univ, Uppsala, Sweden..
    Orlova, A.
    Uppsala Univ, Uppsala, Sweden..
    Hober, Sophia
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Tolmachev, V.
    Uppsala Univ, Uppsala, Sweden..
    Selection of the most optimal ADAPT6-based probe for imaging of HER2 using PET and SPECT2018Ingår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 45, s. S77-S78Artikel i tidskrift (Övrigt vetenskapligt)
  • 8.
    Garousi, Javad
    et al.
    Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden..
    Huizing, Fokko J.
    Radboud Univ Nijmegen, Dept Radiat Oncol, Med Ctr, Nijmegen, Netherlands..
    Vorobyeva, Anzhelika
    Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden..
    Mitran, Bogdan
    Uppsala Univ, Dept Med Chem, Uppsala, Sweden..
    Andersson, Ken G.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Leitao, Charles Dahlsson
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Frejd, Fredrik Y.
    Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden..
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Bussink, Johan
    Radboud Univ Nijmegen, Dept Radiat Oncol, Med Ctr, Nijmegen, Netherlands..
    Orlova, Anna
    Uppsala Univ, Dept Med Chem, Uppsala, Sweden..
    Heskamp, Sandra
    Radboud Univ Nijmegen, Dept Radiol & Nucl Med, Med Ctr, Nijmegen, Netherlands..
    Tolmachev, Vladimir
    Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden..
    Comparative evaluation of affibody- and antibody fragments-based CAIX imaging probes in mice bearing renal cell carcinoma xenografts2019Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, artikel-id 14907Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Carbonic anhydrase IX (CAIX) is a cancer-associated molecular target for several classes of therapeutics. CAIX is overexpressed in a large fraction of renal cell carcinomas (RCC). Radionuclide molecular imaging of CAIX-expression might offer a non-invasive methodology for stratification of patients with disseminated RCC for CAIX-targeting therapeutics. Radiolabeled monoclonal antibodies and their fragments are actively investigated for imaging of CAIX expression. Promising alternatives are small non-immunoglobulin scaffold proteins, such as affibody molecules. A CAIX-targeting affibody ZCAIX:2 was re-designed with the aim to decrease off-target interactions and increase imaging contrast. The new tracer, DOTA-HE3-ZCAIX:2, was labeled with In-111 and characterized in vitro. Tumor-targeting properties of [In-111]In-DOTA-HE3-ZCAIX:2 were compared head-to-head with properties of the parental variant, [(99)mTc]Tc(CO)(3)-HE3-ZCAIX:2, and the most promising antibody fragment-based tracer, [In-111]In-DTPA-G250(Fab')(2), in the same batch of nude mice bearing CAIX-expressing RCC xenografts. Compared to the (99)mTc-labeled parental variant, [In-111]In-DOTA-HE3-ZCAIX:2 provides significantly higher tumor-to-lung, tumor-to-bone and tumor-to-liver ratios, which is essential for imaging of CAIX expression in the major metastatic sites of RCC. [In-111]In-DOTA-HE3-ZCAIX:2 offers significantly higher tumor-to-organ ratios compared with [In-111]In-G250(Fab']2. In conclusion, [In-111]In-DOTA-HE3-ZCAIX:2 can be considered as a highly promising tracer for imaging of CAIX expression in RCC metastases based on our results and literature data.

  • 9.
    Güler, Rezan
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Thatikonda, Naresh
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Ghani, Hawraa Ali
    Hedhammar, My
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Artificial VEGFR2-Specific Growth Factors Demonstrate Agonistic Effects in Both Soluble Form and When Immobilized Via Spider SilkManuskript (preprint) (Övrigt vetenskapligt)
  • 10.
    Horak, Josef
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Jansson, Ronnie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Dev, Apurba
    Uppsala Univ, Ångström Lab, Solid State Elect, Uppsala Box 534, SE-75121 Uppsala, Sweden..
    Nilebäck, Linnea
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Behnam, Kiarash
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Linnros, Jan
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Fotonik.
    Hedhammar, My
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Eriksson Karlström, Amelie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Recombinant Spider Silk as Mediator for One-Step, Chemical-Free Surface Biofunctionalization2018Ingår i: Advanced Functional Materials, ISSN 1616-301X, E-ISSN 1616-3028, Vol. 28, nr 21, artikel-id 1800206Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A unique strategy for effective, versatile, and facile surface biofunctionalization employing a recombinant spider silk protein genetically functionalized with the antibody-binding Z domain (Z-4RepCT) is reported. It is demonstrated that Z-silk can be applied to a variety of materials and platform designs as a truly one-step and chemical-free surface modification that site specifically captures antibodies while simultaneously reducing nonspecific adsorption. As a model surface, SiO2 is used to optimize and characterize Z-silk performance compared to the Z domain immobilized by a standard silanization method. First, Z-silk adsorption is investigated and verified its biofunctionality in a long-term stability experiment. To assess the binding capacity and protein-protein interaction stability of Z-silk, the coating is used to capture human antibodies in various assay formats. An eightfold higher binding capacity and 40-fold lower detection limit are obtained in the immunofluorescence assay, and the complex stability of captured antibodies is shown to be improved by a factor of 20. Applicability of Z-silk to functionalize microfluidic devices is demonstrated by antibody detection in an electrokinetic microcapillary biosensor. To test Z-silk for biomarker applications, real-time detection and quantification of human immunoglobulin G are performed in a plasma sample and C1q capture from human serum using an anti-C1q antibody.

  • 11.
    Kanje, Sara
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Venskutonytė, Raminta
    Lund University.
    Scheffel, Julia
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Nilvebrant, Johan
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Lindkvist-Petersson, Karin
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Centra, Centrum för Bioprocessteknik, CBioPT. KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Protein engineering allows for mild affinity-based elution of therapeutic antibodies2018Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 430, nr 18, s. 3427-3438Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Presented here is an engineered protein domain, based on Protein A, that displays a calcium-dependent binding to antibodies. This protein, ZCa, is shown to efficiently function as an affinity ligand for mild purification of antibodies through elution with ethylenediaminetetraacetic acid. Antibodies are commonly used tools in the area of biological sciences and as therapeutics, and the most commonly used approach for antibody purification is based on Protein A using acidic elution. Although this affinity-based method is robust and efficient, the requirement for low pH elution can be detrimental to the protein being purified. By introducing a calcium-binding loop in the Protein A-derived Z domain, it has been re-engineered to provide efficient antibody purification under mild conditions. Through comprehensive analyses of the domain as well as the ZCa–Fc complex, the features of this domain are well understood. This novel protein domain provides a very valuable tool for effective and gentle antibody and Fc-fusion protein purification

  • 12.
    Liu, Hao
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Lindbo, Sarah
    Ding, Haozhong
    Hober, Sophia
    Gräslund, Torbjorn
    Potent and specific fusion toxins consisting of a HER2-binding ABD-derived affinity protein (ADAPT), fused to truncated versions of Pseudomonas exotoxin AManuskript (preprint) (Övrigt vetenskapligt)
  • 13.
    Lundqvist, Magnus
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Thalén, Niklas
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Volk, Anna-Luisa
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Hansen, Henning Gram
    Tech Univ Denmark, Novo Nordisk Fdn Ctr Biosustainabil, Lyngby, Denmark..
    von Otter, Eric
    Nanyang Technol Univ, Sch Biol Sci, Singapore 637551, Singapore..
    Nygren, Per-Åke
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap. The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark.
    Rockberg, Johan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Chromophore pre-maturation for improved speed and sensitivity of split-GFP monitoring of protein secretion2019Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, artikel-id 310Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Complementation-dependent fluorescence is a powerful way to study co-localization or interactions between biomolecules. A split-GFP variant, involving the self-associating GFP 1-10 and GFP 11, has previously provided a convenient approach to measure recombinant protein titers in cell supernatants. A limitation of this approach is the slow chromophore formation after complementation. Here, we alleviate this lag in signal generation by allowing the GFP 1-10 chromophore to mature on a solid support containing GFP 11 before applying GFP 1-10 in analyses. The pre-maturated GFP 1-10 provided up to 150-fold faster signal generation compared to the non-maturated version. Moreover, pre-maturated GFP 1-10 significantly improved the ability of discriminating between Chinese hamster ovary (CHO) cell lines secreting GFP 11-tagged erythropoietin protein at varying rates. Its improved kinetics make the pre-maturated GFP 1-10 a suitable reporter molecule for cell biology research in general, especially for ranking individual cell lines based on secretion rates of recombinant proteins.

  • 14.
    Orlova, A.
    et al.
    Uppsala Univ, Uppsala, Sweden..
    Rosestedt, M.
    Uppsala Univ, Uppsala, Sweden..
    Rinne, S. S.
    Uppsala Univ, Uppsala, Sweden..
    Mitran, B.
    Uppsala Univ, Uppsala, Sweden..
    Vorobyeva, A.
    Uppsala Univ, Uppsala, Sweden..
    Andersson, Ken G.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Tolmachev, V.
    Uppsala Univ, Uppsala, Sweden..
    Imaging contrast of HER3 expression using monomeric affibody-based imaging probe can be improved by co-injection of affibody trimer2018Ingår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 45, s. S673-S673Artikel i tidskrift (Övrigt vetenskapligt)
  • 15.
    Oroujeni, M.
    et al.
    Uppsala Univ, Uppsala, Sweden..
    Garousi, J.
    Uppsala Univ, Uppsala, Sweden..
    Andersson, Ken G.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Mitran, B.
    Uppsala Univ, Uppsala, Sweden..
    Orlova, A.
    Uppsala Univ, Uppsala, Sweden..
    Tolmachev, V.
    Uppsala Univ, Uppsala, Sweden..
    Comparative evaluation of anti-EFGR affibody molecules labelled with gallium-68 and zirconium-89 using desferrioxamine B as a chelator2018Ingår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 45, s. S674-S675Artikel i tidskrift (Övrigt vetenskapligt)
  • 16.
    Oroujeni, Maryam
    et al.
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75181 Uppsala, Sweden..
    Andersson, Ken G.
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75181 Uppsala, Sweden..
    Steinhardt, Xenia
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Altai, Mohamed
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75181 Uppsala, Sweden..
    Orlova, Anna
    Uppsala Univ, Dept Med Chem, Uppsala, Sweden..
    Mitran, Bogdan
    Uppsala Univ, Dept Med Chem, Uppsala, Sweden..
    Vorobyeva, Anzhelika
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75181 Uppsala, Sweden..
    Garousi, Javad
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75181 Uppsala, Sweden..
    Tolmachev, Vladimir
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75181 Uppsala, Sweden..
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Influence of composition of cysteine-containing peptide-based chelators on biodistribution of Tc-99m-labeled anti-EGFR affibody molecules2018Ingår i: Amino Acids, ISSN 0939-4451, E-ISSN 1438-2199, Vol. 50, nr 8, s. 981-994Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Epidermal growth factor receptor (EGFR) is overexpressed in a number of cancers and is the molecular target for several anti-cancer therapeutics. Radionuclide molecular imaging of EGFR expression should enable personalization of anti-cancer treatment. Affibody molecule is a promising type of high-affinity imaging probes based on a non-immunoglobulin scaffold. A series of derivatives of the anti-EGFR affibody molecule ZEGFR:2377, having peptide-based cysteine-containing chelators for conjugation of Tc-99m, was designed and evaluated. It was found that glutamate-containing chelators Gly-Gly-Glu-Cys (GGEC), Gly-Glu-Glu-Cys (GEEC) and Glu-Glu-Glu-Cys (EEEC) provide the best labeling stability. The glutamate containing conjugates bound to EGFR-expressing cells specifically and with high affinity. Specific targeting of EGFR-expressing xenografts in mice was demonstrated. The number of glutamate residues in the chelator had strong influence on biodistribution of radiolabeled affibody molecules. Increase of glutamate content was associated with lower uptake in normal tissues. The Tc-99m-labeled variant containing the EEEC chelator provided the highest tumor-to-organ ratios. In conclusion, optimizing the composition of peptide-based chelators enhances contrast of imaging of EGFR-expression using affibody molecules.

  • 17.
    Petrou, Georgia
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Jansson, Ronnie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Hogqvist, Mark
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH).
    Erlandsson, Johan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Fiber- och polymerteknologi.
    Wågberg, Lars
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Fiber- och polymerteknologi.
    Hedhammar, My
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Crouzier, Thomas
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Glykovetenskap.
    Genetically Engineered Mucoadhesive Spider Silk2018Ingår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 19, nr 8, s. 3268-3279Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Mucoadhesion is defined as the adhesion of a material to the mucus gel covering the mucous membranes. The mechanisms controlling mucoadhesion include nonspecific electrostatic interactions and specific interactions between the materials and the mucins, the heavily glycosylated proteins that form the mucus gel. Mucoadhesive materials can be used to develop mucosal wound dressings and noninvasive transmucosal drug delivery systems. Spider silk, which is strong, biocompatible, biodegradable, nontoxic, and lightweight would serve as an excellent base for the development of such materials. Here, we investigated two variants of the partial spider silk protein 4RepCT genetically engineered in order to functionalize them with mucoadhesive properties. The pLys-4RepCT variant was functionalized with six cationically charged lysines, aiming to provide nonspecific adhesion from electrostatic interactions with the anionically charged mucins, while the hGal3-4RepCT variant was genetically fused with the Human Galectin-3 Carbohydrate Recognition Domain which specifically binds the mucin glycans Gal beta 1-3GlcNAc and Gal beta 1-4GlcNAc. First, we demonstrated that coatings, fibers, meshes, and foams can be readily made from both silk variants. Measured by the adsorption of both bovine submaxillary mucin and pig gastric mucin, the newly produced silk materials showed enhanced mucin binding properties compared with materials of wild-type (4RepCT) silk. Moreover, we showed that pLys-4RepCT silk coatings bind mucins through electrostatic interactions, while hGal3-4RepCT silk coatings bind mucins through specific glycan-protein interactions. We envision that the two new mucoadhesive silk variants pLys-4RepCT and hGal3-4RepCT, alone or combined with other biofunctional silk proteins, constitute useful new building blocks for a range of silk protein-based materials for mucosal treatments.

  • 18.
    Rinne, S.
    et al.
    Uppsala Univ, Uppsala, Sweden..
    Leitao, Charles Dahlsson
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Mitran, B.
    Uppsala Univ, Uppsala, Sweden..
    Tolmachev, V.
    Uppsala Univ, Uppsala, Sweden..
    Ståhl, Stefan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Orlova, A.
    Uppsala Univ, Uppsala, Sweden..
    Optimizing affibody-mediated PET imaging of HER3 expression using long-lived radiocobalt for the next day PET image2019Ingår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 46, nr SUPPL 1, s. S436-S436Artikel i tidskrift (Övrigt vetenskapligt)
  • 19.
    Rinne, S.
    et al.
    Uppsala Univ, Uppsala, Sweden..
    Mitran, B.
    Uppsala Univ, Uppsala, Sweden..
    Vorobyeva, A.
    Uppsala Univ, Uppsala, Sweden..
    Leitao, Charles Dahlsson
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Andersson, K.
    KTH.
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Tolmachev, V.
    Uppsala Univ, Uppsala, Sweden..
    Orlova, A.
    Uppsala Univ, Uppsala, Sweden..
    Optimization of molecular design of Ga-68-labeled affibody molecule for PET imaging of HER3 expression2018Ingår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 45, s. S109-S109Artikel i tidskrift (Övrigt vetenskapligt)
  • 20.
    Rinne, Sara
    et al.
    Uppsala Univ, Uppsala, Sweden..
    Mitran, Bogdan
    Uppsala Univ, Uppsala, Sweden..
    Gentry, Joshua
    Uppsala Univ, Uppsala, Sweden..
    Vorobyeva, Anzhelika
    Uppsala Univ, Uppsala, Sweden..
    Leitao, Charles Dahlsson
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap. KTH Royal Inst Technol, Stockholm, Sweden..
    Andersson, Ken G.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap. KTH Royal Inst Technol, Stockholm, Sweden..
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Tolmachev, Vladimir
    Uppsala Univ, Uppsala, Sweden..
    Orlova, Anna
    Uppsala Univ, Uppsala, Sweden..
    Optimizing the molecular design of Ga-68-labeled affibody molecules for in vivo PET imaging of HER3 expression2019Ingår i: Journal of labelled compounds & radiopharmaceuticals, ISSN 0362-4803, E-ISSN 1099-1344, Vol. 62, s. S468-S470Artikel i tidskrift (Övrigt vetenskapligt)
  • 21.
    Stiller, Christiane
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Aghelpasand, Hooman
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Frick, Tobias
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Westerlund, Kristina
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Ahmadian, Afshin
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Eriksson Karlström, Amelie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Fast and Efficient Fc-Specific Photoaffinity Labeling To Produce Antibody-DNA Conjugates2019Ingår i: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 30, nr 11, s. 2790-2798Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Antibody DNA conjugates are powerful tools for DNA-assisted protein analysis. Growing usage of these methods demands efficient production of high-quality conjugates. We developed an easy and fast synthesis route yielding covalent antibody-DNA conjugates with a defined conjugation site and low batch-to-batch variability. We utilize the Z domain from protein A, containing the unnatural amino acid 4-benzoylphenylalanine (BPA) for photoaffinity labeling of the antibodies' Fc region. Z(xBPA) domains are C-terminally modified with triple-glycine (G(3))-modified DNA-oligonucleotides enzymatic Sortase A coupling. We reliable modification of the most commonly used IgG's. To prove our conjugates' functionality, we detected antibody-antigen binding events in an assay called Droplet Barcode Sequencing for Protein analysis (DBS-Pro). It confirms not only retained functionality for both conjugate parts but also the potential of using DBS-Pro for quantifying protein abundances. As intermediates are easily storable and our approach is modular, it offers a convenient strategy for screening various antibody-DNA conjugates using the same starting material.

  • 22.
    Vorobyeva, A.
    et al.
    Uppsala Univ, Uppsala, Sweden..
    Schulga, A.
    Shemyakin & Ovchinnikov Inst Bioorgan Chem, Moscow, Russia..
    Abouzayed, A.
    Uppsala Univ, Uppsala, Sweden..
    Konovalova, E.
    Uppsala Univ, Uppsala, Sweden..
    Gunther, T.
    Uppsala Univ, Uppsala, Sweden..
    Ding, Haozhong
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Gräslund, Torbjörn
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Orlova, A.
    Uppsala Univ, Uppsala, Sweden..
    Deyev, S. M.
    Shemyakin & Ovchinnikov Inst Bioorgan Chem, Moscow, Russia.;Natl Res Nucl Univ MEPhI, Moscow, Russia..
    Tolmachev, V.
    Uppsala Univ, Uppsala, Sweden..
    Imaging of EpCAM expression in pancreatic cancer using radiolabelled DARPin Ec12019Ingår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 46, nr SUPPL 1, s. S749-S750Artikel i tidskrift (Övrigt vetenskapligt)
  • 23.
    Vorobyeva, A.
    et al.
    Uppsala Univ, Uppsala, Sweden..
    Schulga, A.
    Shemyakin & Ovchinnikov Inst Bioorgan Chem, Moscow, Russia..
    Konovalova, E.
    Shemyakin & Ovchinnikov Inst Bioorgan Chem, Moscow, Russia..
    Güler, Rezan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Sandstrom, M.
    Uppsala Univ, Uppsala, Sweden..
    Garousi, J.
    Uppsala Univ, Uppsala, Sweden..
    Chernov, V.
    Russian Acad Sci, Canc Res Inst, Tomsk Natl Res Med Ctr, Tomsk, Russia..
    Bragina, O.
    Russian Acad Sci, Canc Res Inst, Tomsk Natl Res Med Ctr, Tomsk, Russia..
    Orlova, A.
    Uppsala Univ, Uppsala, Sweden..
    Tolmachev, V.
    Uppsala Univ, Uppsala, Sweden..
    Deyev, S. M.
    Shemyakin & Ovchinnikov Inst Bioorgan Chem, Moscow, Russia.;Natl Res Nucl Univ MEPhI, Moscow, Russia..
    N-terminal position of histidine-glutamate-containing tag improves biodistribution of [Tc-99m]Tc-labeled DARPin G32019Ingår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 46, nr SUPPL 1, s. S749-S749Artikel i tidskrift (Övrigt vetenskapligt)
  • 24.
    Vorobyeva, A.
    et al.
    Uppsala Univ, Uppsala, Sweden..
    Westerlund, Kristina
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Mitran, B.
    Uppsala Univ, Uppsala, Sweden..
    Altai, M.
    Uppsala Univ, Uppsala, Sweden..
    Rinne, S.
    Uppsala Univ, Uppsala, Sweden..
    Sorensen, J.
    Uppsala Univ, Uppsala, Sweden..
    Orlova, A.
    Uppsala Univ, Uppsala, Sweden..
    Eriksson Karlström, Amelie
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Tolmachev, V.
    Uppsala Univ, Uppsala, Sweden..
    Development of a PET Imaging Approach for Selection of Patients for Affibody-Based PNA-Mediated Pretargeted Radionuclide Therapy2018Ingår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 45, s. S104-S104Artikel i tidskrift (Övrigt vetenskapligt)
  • 25. Vorobyeva, A.
    et al.
    Westerlund, Kristina
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Mitran, B.
    Altai, M.
    Rinne, S.
    Sörensen, J.
    Orlova, A.
    Tolmachev, V.
    Eriksson Karlström, Amelie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Development of an optimal imaging strategy for selection of patients for affibody-based PNA-mediated radionuclide therapy2018Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, nr 1, artikel-id 9643Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Affibody molecules are engineered scaffold proteins, which demonstrated excellent binding to selected tumor-associated molecular abnormalities in vivo and highly sensitive and specific radionuclide imaging of Her2-expressing tumors in clinics. Recently, we have shown that peptide nucleic acid (PNA)-mediated affibody-based pretargeted radionuclide therapy using beta-emitting radionuclide 177Lu extended significantly survival of mice bearing human Her2-expressing tumor xenografts. In this study, we evaluated two approaches to use positron emission tomography (PET) for stratification of patients for affibody-based pretargeting therapy. The primary targeting probe ZHER2:342-SR-HP1 and the secondary probe HP2 (both conjugated with DOTA chelator) were labeled with the positron-emitting radionuclide 68Ga. Biodistribution of both probes was measured in BALB/C nu/nu mice bearing either SKOV-3 xenografts with high Her2 expression or DU-145 xenografts with low Her2 expression. 68Ga-HP2 was evaluated in the pretargeting setting. Tumor uptake of both probes was compared with the uptake of pretargeted 177Lu-HP2. The uptake of both 68Ga-ZHER2:342-SR-HP1 and 68Ga-HP2 depended on Her2-expression level providing clear discrimination of between tumors with high and low Her2 expression. Tumor uptake of 68Ga-HP2 correlated better with the uptake of 177Lu-HP2 than the uptake of 68Ga-ZHER2:342-SR-HP1. The use of 68Ga-HP2 as a theranostics counterpart would be preferable approach for clinical translation. 

  • 26.
    Vorobyeva, Anzhelika
    et al.
    Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden..
    Schulga, Alexey
    Russian Acad Sci, Shemyakin & Ovchinnikov Inst Bioorgan Chem, Mol Immunol Lab, Moscow, Russia..
    Konovalova, Elena
    Russian Acad Sci, Shemyakin & Ovchinnikov Inst Bioorgan Chem, Mol Immunol Lab, Moscow, Russia..
    Güler, Rezan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Löfblom, John
    KTH, Skolan för teknikvetenskap (SCI). KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Sandstrom, Mattias
    Uppsala Univ, Dept Surg Sci, Nucl Med & PET, Uppsala, Sweden..
    Garousi, Javad
    Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden..
    Chernov, Vladimir
    Russian Acad Sci, Tomsk Natl Res Med Ctr, Canc Res Inst, Nucl Med Dept, Tomsk, Russia..
    Bragina, Olga
    Russian Acad Sci, Tomsk Natl Res Med Ctr, Canc Res Inst, Nucl Med Dept, Tomsk, Russia..
    Orlova, Anna
    Uppsala Univ, Dept Med Chem, Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, Uppsala, Sweden..
    Tolmachev, Vladimir
    Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden..
    Deyev, Sergey M.
    Russian Acad Sci, Shemyakin & Ovchinnikov Inst Bioorgan Chem, Mol Immunol Lab, Moscow, Russia.;Natl Res Tomsk Polytech Univ, Tomsk, Russia.;Natl Res Nucl Univ MEPhI, Inst Engn Phys Biomed PhysBio, Bionanophoton Lab, Moscow, Russia..
    Optimal composition and position of histidine-containing tags improves biodistribution of Tc-99m-labeled DARP in G32019Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, artikel-id 9405Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Radionuclide molecular imaging of HER2 expression in disseminated cancer enables stratification of patients for HER2-targeted therapies. DARP in G3, a small (14 kDa) engineered scaffold protein, is a promising probe for imaging of HER2. We hypothesized that position (C- or N-terminus) and composition (hexahistidine or (HE)(3)) of histidine-containing tags would influence the biodistribution of [Tc-99m]Tc(CO)(3)-labeled DARP in G3. To test the hypothesis, G3 variants containing tags at N-terminus (H-6-G3 and (HE)(3)-G3) or at C-terminus (G3-H-6 and G3-(HE)(3)) were labeled with [Tc-99m]Tc(CO)(3). Labeling yield, label stability, specificity and affinity of the binding to HER2, biodistribution and tumor targeting properties of these variants were compared side-by-side. There was no substantial influence of position and composition of the tags on binding of [Tc-99m]Tc(CO)(3)-labeled variants to HER2. The specificity of HER2 targeting in vivo was confirmed. The tumor uptake in BALB/c nu/nu mice bearing SKOV3 xenografts was similar for all variants. On the opposite, there was a strong influence of the tags on uptake in normal tissues. The tumor-to-liver ratio for [Tc-99m]Tc(CO)(3)-(HE)(3)-G3 was three-fold higher compared to the hexahistidine-tag containing variants. Overall, [Tc-99m]Tc(CO)(3)-(HE)(3)-G3 variant provided the highest tumor-to-lung, tumor-to-liver, tumor-to-bone and tumor-to-muscle ratios, which should improve sensitivity of HER2 imaging in these common metastatic sites.

  • 27.
    Vorobyeva, Anzhelika
    et al.
    Uppsala Univ, Dept Immunol Genet & Pathol, Dag Hammarskjolds V 20, SE-75185 Uppsala, Sweden. hulga, Alexey; Konovalova, Elena; Deyev, Sergey.
    Schulga, Alexey
    Konovalova, Elena
    Güler, Rezan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Mitran, Bogdan
    Garousi, Javad
    Rinne, Sara
    Löfblom, John
    KTH, Skolan för teknikvetenskap (SCI).
    Orlova, Anna
    Deyev, Sergey
    Tolmachev, Vladimir
    Comparison of tumor-targeting properties of directly and indirectly radioiodinated designed ankyrin repeat protein (DARPin) G3 variants for molecular imaging of HER22019Ingår i: International Journal of Oncology, ISSN 1019-6439, Vol. 54, nr 4, s. 1209-1220Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Evaluation of human epidermal growth factor receptor 2 (HER2) expression levels in breast and gastroesophageal cancer is used for the stratification of patients for HER2-targeting therapies. The use of radionuclide molecular imaging may facilitate such evaluation in a non-invasive way. Designed ankyrin repeat proteins (DARPins) are engineered scaffold proteins with high potential as probes for radionuclide molecular imaging. DARPin G3 binds with high affinity to HER2 and may be used to visualize this important therapeutic target. Studies on other engineered scaffold proteins have demonstrated that selection of the optimal labeling approach improves the sensitivity and specificity of radionuclide imaging. The present study compared two methods of labeling G3, direct and indirect radioiodination, to select an approach providing the best imaging contrast. G3-H6 was labeled with iodine-124, iodine-125 and iodine-131 using a direct method. A novel construct bearing a C-terminal cysteine, G3-GGGC, was site-specifically labeled using [125I]I-iodo-[(4-hydroxyphenyl)ethyl]maleimide (HPEM). The two radiolabeled G3 variants preserved binding specificity and high affinity to HER2-expressing cells. The specificity of tumor targeting in vivo was demonstrated. Biodistribution comparison of [131I]I-G3-H6 and [125I]I-HPEM-G3-GGGC in mice, bearing HER2-expressing SKOV3 xenografts, demonstrated an appreciable contribution of hepatobiliary excretion to the clearance of [125I]I-HPEM-G3-GGGC and a decreased tumor uptake compared to [131I]I-G3-H6. The direct label provided higher tumor-to-blood and tumor-to-organ ratios compared with the indirect label at 4 h post-injection. The feasibility of high contrast PET/CT imaging of HER2 expression in SKOV3 xenografts in mice using [124I]I-G3-H6 was demonstrated. In conclusion, direct radioiodination is the preferable approach for labeling DARPin G3 with iodine-123 and iodine-124 for clinical single photon emission computed tomography and positron emission tomography imaging.

  • 28.
    Westerlund, Kristina
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Altai, Mohamed
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75181 Uppsala, Sweden..
    Mitran, Bogdan
    Uppsala Univ, Div Mol Imaging, Dept Med Chem, Uppsala, Sweden..
    Konijnenberg, Mark
    Erasmus MC, Dept Radiol & Nucl Med, Rotterdam, Netherlands..
    Oroujeni, Maryam
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75181 Uppsala, Sweden..
    Atterby, Christina
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75181 Uppsala, Sweden..
    de Jong, Marion
    Erasmus MC, Dept Radiol & Nucl Med, Rotterdam, Netherlands..
    Orlova, Anna
    Uppsala Univ, Div Mol Imaging, Dept Med Chem, Uppsala, Sweden..
    Mattsson, Johanna
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75181 Uppsala, Sweden..
    Micke, Patrick
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75181 Uppsala, Sweden..
    Eriksson Karlström, Amelie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Tolmachev, Vladimir
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75181 Uppsala, Sweden..
    Radionuclide Therapy of HER2-Expressing Human Xenografts Using Affibody-Based Peptide Nucleic Acid-Mediated Pretargeting: In Vivo Proof of Principle2018Ingår i: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 59, nr 7, s. 1092-1098Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Affibody molecules are small proteins engineered using a nonanti-body scaffold. Radiolabeled Affibody molecules are excellent imaging probes, but their application to radionuclide therapy has been prevented by high renal reabsorption. The aim of this study was to test the hypothesis that Affibody-based peptide nucleic acid (PNA)-mediated pretargeted therapy of human epidermal growth factor receptor 2 (HER2)-expressing cancer extends survival without accompanying renal toxicity. Methods: A HER2-targeting Affibody molecule ligated with an AGTCGTGATGTAGTC PNA hybridization probe (Z(HER2:342)-SR-HP1) was used as the primary pretargeting agent. A complementary AGTCGTGATGTAGTC PNA conjugated to the chelator DOTA and labeled with the radionuclide Lu-177 (Lu-177-HP2) was used as the secondary agent. The influence of different factors on pretargeting was investigated. Experimental radionuclide therapy in mice bearing SKOV-3 xenografts was performed in 6 cycles separated by 7 d. Results: Optimal tumor targeting was achieved when 16 MBq/3.5 mu g (0.65 nmol) of Lu-177-HP2 was injected 16 h after injection of 100 mu g (7.7 nmol) of Z(HER2:342)-SR-HP1. The calculated absorbed dose to tumors was 1,075 mGy/MBq, whereas the absorbed dose to kidneys was 206 mGy/MBq and the absorbed dose to blood (surrogate of bone marrow) was 4 mGy/MBq. Survival of mice was significantly longer (P < 0.05) in the treatment group (66 d) than in the control groups treated with the same amount of Z(HER2:342)-SR-HP1 only (37 d), the same amount and activity of Lu-177-HP2 only (32 d), or phosphate-buffered saline (37 d). Conclusion: The studied pretargeting system can deliver an absorbed dose to tumors appreciably exceeding absorbed doses to critical organs, making Affibody-based PNA-mediated pretargeted radionuclide therapy highly attractive.

  • 29.
    Yu, Feifan
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Alesand, Veronica
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Nygren, Per-Åke
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Site-Specific Photoconjugation of Beta-Lactamase Fragments to Monoclonal Antibodies Enables Sensitive Analyte Detection via Split-Enzyme Complementation2018Ingår i: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 13, nr 7, artikel-id 1700688Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Protein fragment complementation assays (PCA) rely on a proximity-driven reconstitution of a split reporter protein activity, typically via interaction between bait and prey units separately fused to the reporter protein halves. The PCA principle can also be formatted for use in immunossays for analyte detection, e.g., via the use of small immunoglobulin binding proteins (IgBp) as fusion partners to split-reporter protein fragments for conversion of pairs of antibodies into split-protein half-probes. However, the non-covalent binding between IgBp and antibodies is not ideal for development of robust assays. Here, the authors describe how split-enzyme reporter halves can be both site-specifically and covalently photoconjugated at antibody Fc-parts for use in homogeneous dual-antibody in vitro immunoassays based on analyte-dependent split-enzyme fragment complementation. The half-probes consist of parts of a beta-lactamase split-protein reporter fused to an immunoglobulin Fc binding domain equipped with a unique cysteine residue at which a photoactivable maleimide benzophenone group (MBP) is attached. Using such antibody conjugates the authors obtain an analyte-driven complementation of the reporter enzyme fragments monitored via conversion of a chromogenic substrate. Results from detection of human interferon-gamma and the extracellular domain of HER2 is shown. The described principles for site-specific conjugation of proteins to antibodies should be broadly applicable.

  • 30.
    Yu, Shengze
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Exploitation of interactions with the neonatal Fc receptor to manipulate biological half-lives for therapeutic applications2019Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [sv]

    Proteinteknik tillhandahåller kraftfulla verktyg för att skapa användbara proteiner med önskade egenskaper. I denna avhandling har rationella designprinciper använts för utveckling av fusionsproteiner som kan interagera med neonatal Fc-receptorn (FcRn) för potentiella medicinska tillämpningar. 

    FcRn uttrycks allmänt i människokroppen. De naturliga liganderna aför FcRn är immunglobulin G (IgG) och serumalbumin (SA). FcRn kan binda till båda proteinerna på ett pH-beroende sätt och förse dem med en ovanligt lång halveringstid in vivo. Proteinbyggstenar som interagerar direkt eller indirekt med FcRn kan potentiellt användas för att antingen åka snålskjuts på FcRn-systemet för förlängning av halveringstiden in vivo eller för att mätta systemet för att minska halveringstiden in vivo för de naturliga liganderna. I den här avhandlingen har jag utforskat en FcRn-bindande affibody molekyl (ZFcRn) och en albuminbindande domän (ABD) för dessa ändamål. 

    I studie I och II befanns prolaktinreceptorn ofta uttryckt i glioblastoma multiforme tumörer från patienter såväl som i glioblastoma multiforme cellinjer. Vi undersökte en ny antagonist av prolaktinreceptorn in vitro och fann att den kunde blockera signalering genom receptorn såväl som cellulär invasivitet. En prolaktinreceptorantagonist kan således potentiellt bli ett läkemedel för behandling av glioblastoma multiforme. Men antagonisten kommer troligen att ha en kort halveringstid i blodet på grund av dess lilla storlek, vilket begränsar användbarheten. Därför uttrycktes det som en fusion till ABD, som interagerar indirekt med FcRn. Det producerade fusionsproteinet visade sig kunna blockera signalering genom prolaktinreceptorn in vitro och hade också en förlängd halveringstid i blodet in vivo

    Målet med studie III var att undersöka egenskaperna hos humant tillväxthormon (hGH) när det uttrycktes som en proteinfusion med ZFcRn som interagerar direkt med FcRn och/eller ABD. Fusionsproteinerna, ZFcRn-hGH, ABD-hGH och ZFcRn-ABD-hGH kunde uttryckas rekombinant och kunde framgångsrikt renas till homogenitet. De hade de förväntade bindningsförmågorna till FcRn, SA och hGH-receptorn. De visade sig alla kunna inducera signalering över plasmamembranet i en modellcellinje. 

    Patienter som lider av många autoimmuna sjukdomar producerar specifika IgG-molekyler som är ansvariga för sjukdomens symtom. En potentiell behandling kan vara att öka katabolismen av dessa IgG för att lindra sjukdomssymtomen. I studie IV undersöktes en FcRn-interagerande affibodymolekyl för IgG-minskning, detta genom att blockera IgG/FcRn-interaktionen. In vitro fann vi först att affibodymolekylerna delar ett gemensamt bindningsställe med IgG på FcRn, vilket indikerade att affibodymolekylen borde kunna blockera IgG från bindning till FcRn. In vivo injicerade vi stora mängder av affibodymolekylen i olika format i möss och fann upp till 39% reduktion av den totala mängden endogent IgG. I en klinisk kontext skulle reduktion av total IgG också minska nivån av de IgG som driver sjukdomen och potentiellt minska symtomen hos patienter som lider av IgG-drivna autoimmuna sjukdomar. 

    Sammantaget har jag i den här avhandlingen undersökt tillämpningar av FcRn-interagerande molekyler för förlängning av biologiska halveringstider av terapeutiskt relevanta proteiner och dessutom reduktion av totalt IgG genom FcRn-blockering. 

  • 31.
    Yu, Shengze
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Gräslund, Torbjörn
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Protein Engineering.
    Human growth hormone can transduce signals to phosphorylate STAT5 as a fusion with an affibody molecule binding the neonatal Fc receptor and an albumin binding domainManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Growth hormone replacement therapy has been used to treat children and adults with

    growth hormone deficiency for more than three decades. Growth hormone has a short

    biological half-life, requiring daily subcutaneous injections, and long acting versions are

    therefore desirable. In this study we have analyzed three fusion proteins, ZFcRn-hGH,

    ABD-hGH and ZFcRn-ABD-hGH, consisting of the human growth hormone (hGH) and an

    affibody molecule binding the neonatal Fc receptor (ZFcRn) and/or an albumin binding

    domain (ABD). Both ZFcRn and the ABD may have the ability to endow hGH with an

    extended plasma half-life. The fusion proteins could be recombinantly expressed in the

    periplasmic space of Escherichia coli and easily purified to homogeneity. All three fusion

    proteins appeared to have a strong interaction with the growth hormone receptor. ABDhGH

    and ZFcRn-ABD-hGH had a strong affinity for HSA (KD 0.006 and 0.02 nM,

    respectively). ZFcRn-hGH and ZFcRn-ABD-hGH had moderately strong affinity for mouse

    neonatal Fc receptor at pH 6.0 (KD 200 and 100 nM, respectively). The fusion proteins

    thus retained the expected binding abilities of the individual domains. Further

    characterization showed that the fusion proteins could induce phosphorylation of signal

    transducer and activator of transcription 5 (STAT5) in the model cell line U251-MG,

    further showing that the hGH-part of the fusion proteins was functional.

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