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  • 1.
    Abouzayed, A.
    et al.
    Uppsala Univ, Uppsala, Sweden..
    Rinne, S. S.
    Uppsala Univ, Uppsala, Sweden..
    Wadeea, F.
    Uppsala Univ, Uppsala, Sweden..
    Tano, Hanna
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Nagy, Abel
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Eriksson Karlström, Amelie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Tolmachev, V.
    Uppsala Univ, Uppsala, Sweden..
    Orlova, A.
    Uppsala Univ, Uppsala, Sweden..
    Conjugation of GRPR-targeting antagonist RM26 to albumin-binding domain extends antagonist's blood circulation and residence in tumours2020Inngår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 47, nr SUPPL 1, s. S652-S652Artikkel i tidsskrift (Annet vitenskapelig)
  • 2.
    Abouzayed, Ayman
    et al.
    Uppsala Univ, Dept Med Chem, S-75183 Uppsala, Sweden..
    Tano, Hanna
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Nagy, Abel
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Rinne, Sara S.
    Uppsala Univ, Dept Med Chem, S-75183 Uppsala, Sweden..
    Wadeea, Fadya
    Uppsala Univ, Dept Med Chem, S-75183 Uppsala, Sweden..
    Kumar, Sharmishtaa
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Westerlund, Kristina
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Tolmachev, Vladimir
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 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. KTH Royal Inst Technol, AlbaNova Univ Ctr, Dept Prot Sci, Sch Engn Sci Chem Biotechnol & Hlth, S-10691 Stockholm, Sweden..
    Orlova, Anna
    Uppsala Univ, Dept Med Chem, S-75183 Uppsala, Sweden.;Tomsk Polytech Univ, Res Sch Chem & Appl Biomed Sci, Res Centrum Oncotheranost, Tomsk 634050, Russia.;Uppsala Univ, Sci Life Lab, S-75105 Uppsala, Sweden..
    Preclinical Evaluation of the GRPR-Targeting Antagonist RM26 Conjugated to the Albumin-Binding Domain for GRPR-Targeting Therapy of Cancer2020Inngår i: Pharmaceutics, E-ISSN 1999-4923, Vol. 12, nr 10, artikkel-id 977Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The targeting of gastrin-releasing peptide receptors (GRPR) was recently proposed for targeted therapy, e.g., radiotherapy. Multiple and frequent injections of peptide-based therapeutic agents would be required due to rapid blood clearance. By conjugation of the GRPR antagonist RM26 (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) to an ABD (albumin-binding domain), we aimed to extend the blood circulation of peptides. The synthesized conjugate DOTA-ABD-RM26 was labelled with indium-111 and evaluated in vitro and in vivo. The labelled conjugate was stable in PBS and retained specificity and its antagonistic function against GRPR. The half-maximal inhibitory concentration (IC50) of In-nat-DOTA-ABD-RM26 in the presence of human serum albumin was 49 +/- 5 nM. [In-111]In-DOTA-ABD-RM26 had a significantly longer residence time in blood and in tumors (without a significant decrease of up to 144 h pi) than the parental RM26 peptide. We conclude that the ABD-RM26 conjugate can be used for GRPR-targeted therapy and delivery of cytotoxic drugs. However, the undesirable elevated activity uptake in kidneys abolishes its use for radionuclide therapy. This proof-of-principle study justified further optimization of the molecular design of the ABD-RM26 conjugate.

  • 3. Altai, M.
    et al.
    Ding, Haozhong
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Rinne, S.
    Dept Med Chem, Uppsala, Sweden..
    Vorobyeva, A.
    Gräslund, Torbjörn
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    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 Development2019Inngår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 46, nr SUPPL 1, s. S715-S716Artikkel i tidsskrift (Annet vitenskapelig)
  • 4.
    Altai, M.
    et al.
    Dept Clin Sci, Div Oncol & Pathol, Lund, Sweden..
    Vorobyeva, A.
    Myrhammar, Anders
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Westerlund, Kristina
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Yoneoka, S.
    Lab Adv Nucl Energy, Tokyo, Japan..
    Tsukahara, T.
    Lab Adv Nucl Energy, Tokyo, Japan..
    Eriksson Karlström, Amelie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Orlova, A.
    Dept Med Chem, Uppsala, Sweden..
    Tolmachev, V.
    Design and evaluation oflactosaminated cetuximabas a clearing agent for antibody-based PNA-mediated pretargeting2020Inngår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 47, nr SUPPL 1, s. S343-S344Artikkel i tidsskrift (Annet vitenskapelig)
  • 5.
    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, Proteinvetenskap.
    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, Proteinvetenskap.
    Tolmachev, V.
    Immunology, Genetics and Pathology, Uppsala, SWEDEN, .
    A novel method for conjugation of PNA to antibodies for radionuclide based pretargeting: proof of principal2018Inngår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 45, s. S648-S648Artikkel i tidsskrift (Annet vitenskapelig)
  • 6. Altai, Mohamed
    et al.
    Liu, Hao
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    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) (Annet vitenskapelig)
  • 7. Altai, Mohamed
    et al.
    Vorobyeva, Anzhelika
    Tolmachev, Vladimir
    Eriksson Karlström, Amelie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Westerlund, Kristina
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Preparation of Conjugates for Affibody-Based PNA-Mediated Pretargeting2020Inngår i: Methods in Molecular Biology, Humana Press Inc. , 2020, s. 283-304Kapittel i bok, del av antologi (Fagfellevurdert)
    Abstract [en]

    Affibody molecules are small engineered scaffold proteins suitable for in vivo tumor targeting. Radionuclide molecular imaging using directly radiolabelled affibody molecules provides excellent imaging. However, affibody molecules have a high renal reabsorption, which complicates their use for radionuclide therapy. The high renal reabsorption is a common problem for the use of engineered scaffold proteins for radionuclide therapy. Affibody-based PNA-mediated pretargeting reduces dramatically the absorbed dose to the kidneys and makes affibody-based radionuclide therapy possible. This methodology might, hopefully, solve the problem of high renal reabsorption for radionuclide therapy mediated by other engineered scaffold proteins. 

  • 8.
    Banijamali, Mahsan
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Höjer, Pontus
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Nagy, Abel
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Haag, Petra
    Karolinska Inst, Dept Oncol Pathol, Solna, Sweden..
    Paz Gomero, Elizabeth
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Stiller, Christiane
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Kaminskyy, Vitaliy O.
    Karolinska Inst, Dept Oncol Pathol, Solna, Sweden.;Karolinska Inst, Dept Physiol & Pharmacol, Stockholm, Sweden..
    Ekman, Simon
    Karolinska Inst, Dept Oncol Pathol, Solna, Sweden.;Karolinska Univ Hosp, Theme Canc, Med Unit Head & Neck Lung & Skin Tumors, Thorac Oncol Ctr, Solna, Sweden..
    Lewensohn, Rolf
    Karolinska Inst, Dept Oncol Pathol, Solna, Sweden.;Karolinska Univ Hosp, Theme Canc, Med Unit Head & Neck Lung & Skin Tumors, Thorac Oncol Ctr, Solna, 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, Proteinvetenskap.
    Viktorsson, Kristina
    Karolinska Inst, Dept Oncol Pathol, Solna, Sweden..
    Ahmadian, Afshin
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Characterizing single extracellular vesicles by droplet barcode sequencing for protein analysis2022Inngår i: Journal of Extracellular Vesicles, E-ISSN 2001-3078, Vol. 11, nr 11, artikkel-id 12277Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Small extracellular vesicles (sEVs) have in recent years evolved as a source of biomarkers for disease diagnosis and therapeutic follow up. sEV samples derived from multicellular organisms exhibit a high heterogeneous repertoire of vesicles which current methods based on ensemble measurements cannot capture. In this work we present droplet barcode sequencing for protein analysis (DBS-Pro) to profile surface proteins on individual sEVs, facilitating identification of sEV-subtypes within and between samples. The method allows for analysis of multiple proteins through use of DNA barcoded affinity reagents and sequencing as readout. High throughput single vesicle profiling is enabled through compartmentalization of individual sEVs in emulsion droplets followed by droplet barcoding through PCR. In this proof-of-concept study we demonstrate that DBS-Pro allows for analysis of single sEVs, with a mixing rate below 2%. A total of over 120,000 individual sEVs obtained from a NSCLC cell line and from malignant pleural effusion (MPE) fluid of NSCLC patients have been analyzed based on their surface proteins. We also show that the method enables single vesicle surface protein profiling and by extension characterization of sEV-subtypes, which is essential to identify the cellular origin of vesicles in heterogenous samples.

  • 9.
    Björk, Sara
    et al.
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Schappert, Martin
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Nanobioteknologi.
    Jönsson, Håkan
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Droplet microfluidic microcolony sorting by fluorescence area for high throughput, yield-based screening of triacyl glycerides in S. Cerevisiae2020Inngår i: MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Chemical and Biological Microsystems Society , 2020, s. 1015-1016Konferansepaper (Fagfellevurdert)
    Abstract [en]

    Here we present a droplet microfluidics workflow for cell factory screening by yield of an intracellular product from isogenic microcolonies, i.e. minimal cell populations, encapsulated in picoliter droplets. This allows us to utilize all the benefits of droplet microfluidic screening in terms of throughput, but based on the signal from a population average, rather than the noisy single cell signal. We demonstrate microcolony sorting by integrated droplet fluorescence area of encapsulated E. coli, optimize triglyceride (TG) microcolony assay in droplets and apply the microcolony screening concept to analyze triglyceride (TG) production in S. cerevisiae.

  • 10.
    Bronge, M.
    et al.
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden..
    Asplund Hogelin, K.
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden..
    Thomas, O. G.
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden..
    Ruhrmann, S.
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden..
    Carvalho-Querioz, C.
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden..
    Nilsson, O.
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden..
    Kaiser, A.
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden..
    Holmgren, E.
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden..
    Linnerbauer, M.
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden..
    Adzemovic, M. Z.
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden..
    Zeitelhofer, M.
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden..
    Hellström, Cecilia
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik.
    Jelcic, I.
    Univ Hosp Zurich, Neuroimmunol & MS Res, Zurich, Switzerland..
    Liu, Hailong
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Fiber- och polymerteknologi, Polymerteknologi.
    Nilsson, Peter
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik.
    Hillert, J.
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden..
    Brundin, L.
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden..
    Fink, K.
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden..
    Martin, R.
    Univ Hosp Zurich, Neuroimmunol & MS Res, Zurich, Switzerland..
    Tegel, Hanna
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Gräslund, Torbjörn
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Al Nimer, F.
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden..
    Guerreiro-Cacais, A. O.
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden..
    Khademi, M.
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden..
    Gafvelin, G.
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden..
    Olsson, T.
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden..
    Gronlund, H.
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden..
    T cell reactivity screening reveals four novel CNS autoantigens in multiple sclerosis2021Inngår i: Multiple Sclerosis Journal, ISSN 1352-4585, E-ISSN 1477-0970, Vol. 27, nr 2_SUPPL, s. 344-345Artikkel i tidsskrift (Annet vitenskapelig)
  • 11.
    Bronge, Mattias
    et al.
    Karolinska Inst, Ctr Mol Med, Dept Clin Neurosci, Therapeut Immune Design, S-17176 Stockholm, Sweden..
    Hogelin, Klara Asplund
    Karolinska Inst, Ctr Mol Med, Dept Clin Neurosci, Neuroimmunol Unit, S-17176 Stockholm, Sweden..
    Thomas, Olivia G.
    Karolinska Inst, Ctr Mol Med, Dept Clin Neurosci, Therapeut Immune Design, S-17176 Stockholm, Sweden..
    Ruhrmann, Sabrina
    Karolinska Inst, Ctr Mol Med, Dept Clin Neurosci, Therapeut Immune Design, S-17176 Stockholm, Sweden..
    Carvalho-Queiroz, Claudia
    Karolinska Inst, Ctr Mol Med, Dept Clin Neurosci, Therapeut Immune Design, S-17176 Stockholm, Sweden..
    Nilsson, Ola B.
    Karolinska Inst, Ctr Mol Med, Dept Clin Neurosci, Therapeut Immune Design, S-17176 Stockholm, Sweden..
    Kaiser, Andreas
    Karolinska Inst, Ctr Mol Med, Dept Clin Neurosci, Therapeut Immune Design, S-17176 Stockholm, Sweden..
    Zeitelhofer, Manuel
    Karolinska Inst, Dept Med Biochem & Biophys, Div Vasc Biol, S-17177 Stockholm, Sweden..
    Holmgren, Erik
    Karolinska Inst, Ctr Mol Med, Dept Clin Neurosci, Therapeut Immune Design, S-17176 Stockholm, Sweden..
    Linnerbauer, Mathias
    Karolinska Inst, Ctr Mol Med, Dept Clin Neurosci, Neuroimmunol Unit, S-17176 Stockholm, Sweden..
    Adzemovic, Milena Z.
    Karolinska Inst, Ctr Mol Med, Dept Clin Neurosci, Neuroimmunol Unit, S-17176 Stockholm, Sweden..
    Hellström, Cecilia
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik.
    Jelcic, Ivan
    Univ Zurich, Univ Hosp Zurich, Neuroimmunol & MS Res Sect NIMS, Neurol Clin, CH-8091 Zurich, Switzerland..
    Liu, Hao
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Nilsson, Peter
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik.
    Hillert, Jan
    Karolinska Inst, Karolinska Univ Hosp, Dept Clin Neurosci, Div Neurol, S-17176 Stockholm, Sweden..
    Brundin, Lou
    Karolinska Inst, Karolinska Univ Hosp, Dept Clin Neurosci, Div Neurol, S-17176 Stockholm, Sweden..
    Fink, Katharina
    Karolinska Inst, Karolinska Univ Hosp, Dept Clin Neurosci, Div Neurol, S-17176 Stockholm, Sweden..
    Kockum, Ingrid
    Karolinska Inst, Ctr Mol Med, Dept Clin Neurosci, Neuroimmunol Unit, S-17176 Stockholm, Sweden..
    Tengvall, Katarina
    Karolinska Inst, Ctr Mol Med, Dept Clin Neurosci, Neuroimmunol Unit, S-17176 Stockholm, Sweden.;Uppsala Univ, Dept Med Biochem & Microbiol, Sci Life Lab, S-75237 Uppsala, Sweden..
    Martin, Roland
    Univ Zurich, Univ Hosp Zurich, Neuroimmunol & MS Res Sect NIMS, Neurol Clin, CH-8091 Zurich, Switzerland..
    Tegel, Hanna
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Gräslund, Torbjörn
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Al Nimer, Faiez
    Karolinska Inst, Ctr Mol Med, Dept Clin Neurosci, Neuroimmunol Unit, S-17176 Stockholm, Sweden..
    Guerreiro-Cacais, Andre Ortlieb
    Karolinska Inst, Ctr Mol Med, Dept Clin Neurosci, Neuroimmunol Unit, S-17176 Stockholm, Sweden..
    Khademi, Mohsen
    Karolinska Inst, Ctr Mol Med, Dept Clin Neurosci, Neuroimmunol Unit, S-17176 Stockholm, Sweden..
    Gafvelin, Guro
    Karolinska Inst, Ctr Mol Med, Dept Clin Neurosci, Therapeut Immune Design, S-17176 Stockholm, Sweden..
    Olsson, Tomas
    Karolinska Inst, Ctr Mol Med, Dept Clin Neurosci, Neuroimmunol Unit, S-17176 Stockholm, Sweden..
    Gronlund, Hans
    Karolinska Inst, Ctr Mol Med, Dept Clin Neurosci, Therapeut Immune Design, S-17176 Stockholm, Sweden..
    Identification of four novel T cell autoantigens and personal autoreactive profiles in multiple sclerosis2022Inngår i: Science Advances, E-ISSN 2375-2548, Vol. 8, nr 17, artikkel-id eabn1823Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS), in which pathological T cells, likely autoimmune, play a key role. Despite its central importance, the autoantigen repertoire remains largely uncharacterized. Using a novel in vitro antigen delivery method combined with the Human Protein Atlas library, we screened for T cell autoreactivity against 63 CNS-expressed proteins. We identified four previously unreported autoantigens in MS: fatty acid-binding protein 7, prokineticin-2, reticulon-3, and synaptosomal-associated protein 91, which were verified to induce interferon-gamma responses in MS in two cohorts. Autoreactive profiles were heterogeneous, and reactivity to several autoantigens was MS-selective. Autoreactive T cells were predominantly CD4(+) and human leukocyte antigen-DR restricted. Mouse immunization induced antigen-specific responses and CNS leukocyte infiltration. This represents one of the largest systematic efforts to date in the search for MS autoantigens, demonstrates the heterogeneity of autoreactive profiles, and highlights promising targets for future diagnostic tools and immunomodulatory therapies in MS.

  • 12. Caers, Jo
    et al.
    Duray, Elodie
    Dumoulin, Mireille
    D'Huyvetter, Matthias
    Eriksson Karlström, Amelie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Anti-Cd38 Single-Domain Antibodies in Disease Monitoring and Treatment2023Patent (Annet (populærvitenskap, debatt, mm))
    Abstract [en]

    NOVELTY - A pre-targeting system comprising an anti-cluster of differentiation (CD)38 single-domain antibody (sdAb) and a second agent capable of specifically binding to the anti-CD38 sdAb and comprising a second molecule is new, where the antibody comprises an amino acid sequence that comprises 3 complementary determining regions (CDR1-CDR3). The CDR1 is chosen from (a) an amino acid sequence of SEQ ID NO: 1, (b) polypeptides that have at least 80% amino acid sequence identity with SEQ ID NO: 1, and (c) polypeptides that have 3, 2 or 1 amino acid difference with SEQ ID NO: 1. The CDR2 is chosen from (a) an amino acid sequence of SEQ ID NO: 2, (b) polypeptides that have at least 80% amino acid sequence identity with SEQ ID NO: 2, (c) polypeptides that have 3, 2 or 1 amino acid difference with SEQ ID NO: 2.

    USE - The pre-targeting system is useful in kit of parts or medicine or diagnostics for diagnosing, monitoring and treating neoplastic disease in subject, and evaluating or monitoring presence, location and/or amount of CD38-expressing cells in subject. The neoplastic disease is a solid tumor. The neoplastic disease is hepatocellular carcinoma, lung cancer, melanoma, breast cancer or glioma, preferably hematological malignancy. The neoplastic disease is multiple myeloma, non-Hodgkin lymphoma (NHL) or chronic lymphoid leukemia (CLL), preferably multiple myeloma (all claimed).

    ADVANTAGE - The system exhibits excellent cytotoxic effect on CD38-expressing neoplastic cells.

    DETAILED DESCRIPTION - A pre-targeting system comprising an anti-cluster of differentiation (CD)38 single-domain antibody (sdAb) and a second agent capable of specifically binding to the anti-CD38 sdAb and comprising a second molecule is new, where the antibody comprises an amino acid sequence that comprises 3 complementary determining regions (CDR1-CDR3). The CDR1 is chosen from (a) an amino acid sequence of SEQ ID NO: 1, (b) polypeptides that have at least 80% amino acid sequence identity with SEQ ID NO: 1, and (c) polypeptides that have 3, 2 or 1 amino acid difference with SEQ ID NO: 1. The CDR2 is chosen from (a) an amino acid sequence of SEQ ID NO: 2, (b) polypeptides that have at least 80% amino acid sequence identity with SEQ ID NO: 2, (c) polypeptides that have 3, 2 or 1 amino acid difference with SEQ ID NO: 2. The CDR3 is chosen from (a) a 18 amino acid sequence (SEQ ID NO: 3) fully defined in the specification, (b) polypeptides that have at least 80% amino acid sequence identity with SEQ ID NO: 3, and (c) polypeptides that have 3, 2 or 1 amino acid difference with SEQ ID NO: 3. Tyr-Thr-Asp-Ser-Asp-Tyr-Ile (SEQ ID NO: 1), and Thr-Ile-Tyr-Ile-Gly-Gly-Thr-Tyr-Ile-His (SEQ ID NO: 2).

    INDEPENDENT CLAIMS are included for the following:

    • kit of parts comprising the pre-targeting system;
    • use of pre-targeting system or kit of parts in medicine or diagnostics for diagnosing, monitoring and treating a neoplastic disease in a subject; and
    • imaging method for evaluating or monitoring presence, location and/or amount of CD38-expressing cells in a subject involves (i) detecting, in a subject to whom a detectable quantity of the pre-targeting system, and (ii) generating an image representative of the location and/or quantity or intensity of the signal, where the second agent comprises a signal-emitting molecule, has been administered, signal emitted by said signal-emitting molecule.
  • 13.
    Caers, Jo
    et al.
    Univ Liege, Lab Hematol, GIGA I3, Liege, Belgium.;CHU Liege, Dept Hematol, Liege, Belgium..
    Duray, Elodie
    Univ Liege, Lab Hematol, GIGA I3, Liege, Belgium.;Univ Liege, Ctr Prot Engn, Inbios, Liege, Belgium..
    Vrancken, Louise
    Univ Liege, Lab Hematol, GIGA I3, Liege, Belgium.;CHU Liege, Dept Hematol, Liege, Belgium..
    Marcion, Guillaume
    Univ Liege, Lab Hematol, GIGA I3, Liege, Belgium..
    Bocuzzi, Valentina
    Univ Liege, Lab Hematol, GIGA I3, Liege, Belgium..
    De Veirman, Kim
    Vrije Univ Brussel, Dept Hematol & Immunol, Brussels, Belgium..
    Krasniqi, Ahmet
    Vrije Univ Brussel, Lab In Vivo Cellular & Mol Imaging Lab ICMI, Brussels, Belgium..
    Lejeune, Margaux
    Univ Liege, Lab Hematol, GIGA I3, Liege, Belgium..
    Withofs, Nadia
    CHU Liege, Dept Nucl Med, Liege, Belgium..
    Devoogdt, Nick
    Vrije Univ Brussel, Lab In Vivo Cellular & Mol Imaging Lab ICMI, Brussels, Belgium..
    Dumoulin, Mireille
    Univ Liege, Ctr Prot Engn, Inbios, Liege, Belgium..
    Eriksson Karlström, Amelie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    D'Huyvetter, Matthias
    Vrije Univ Brussel, Lab In Vivo Cellular & Mol Imaging Lab ICMI, Brussels, Belgium..
    Radiotheranostic Agents in Hematological Malignancies2022Inngår i: Frontiers in Immunology, E-ISSN 1664-3224, Vol. 13, artikkel-id 911080Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Radioimmunotherapy (RIT) is a cancer treatment that combines radiation therapy with tumor-directed monoclonal antibodies (Abs). Although RIT had been introduced for the treatment of CD20 positive non-Hodgkin lymphoma decades ago, it never found a broad clinical application. In recent years, researchers have developed theranostic agents based on Ab fragments or small Ab mimetics such as peptides, affibodies or single-chain Abs with improved tumor-targeting capacities. Theranostics combine diagnostic and therapeutic capabilities into a single pharmaceutical agent; this dual application can be easily achieved after conjugation to radionuclides. The past decade has seen a trend to increased specificity, fastened pharmacokinetics, and personalized medicine. In this review, we discuss the different strategies introduced for the noninvasive detection and treatment of hematological malignancies by radiopharmaceuticals. We also discuss the future applications of these radiotheranostic agents.

  • 14.
    Cavallaro, Sara
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Fotonik.
    Horak, Josef
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    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, Proteinvetenskap.
    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, Proteinvetenskap. 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 Sensor2019Inngår i: ACS Sensors, E-ISSN 2379-3694, Vol. 4, nr 5, s. 1399-1408Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 15. Chaudhary, Himanshu
    et al.
    Meister, Sebastian
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Zetterberg, Henrik
    Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, the Sahlgrenska Academy at the University of Gothenburg, Mölndal, Sweden..
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Lendel, Christofer
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi, Tillämpad fysikalisk kemi.
    Dissecting the structural organization of multiprotein amyloid aggregates using a bottom-up approach2020Inngår i: ACS Chemical Neuroscience, E-ISSN 1948-7193, Vol. 11, nr 10, s. 1447-1457Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Deposition of fibrillar amyloid β (Aβ) in senile plaques is a pathological signature of Alzheimer's disease. However, senile plaques also contain many other components, including a range of different proteins. Although the composition of the plaques can be analyzed in post mortem tissue, knowledge of the molecular details of these multiprotein inclusions and their assembly processes is limited, which impedes the progress in deciphering the biochemical mechanisms associated with Aβ pathology. We here describe a bottom-up approach to monitor how proteins from human cerebrospinal fluid associate with Aβ amyloid fibrils to form plaque particles. The method combines flow cytometry and mass spectrometry proteomics and allowed us to identify and quantify 128 components of the captured multiprotein aggregates. The results provide insights in the functional characteristics of the sequestered proteins and reveal distinct interactome responses for the two investigated Aβ variants, Aβ(1-40) and Aβ(1-42). Furthermore, the quantitative data is used to build models of the structural organization of the multiprotein aggregates, which suggests that Aβ is not the primary binding target for all the proteins; secondary interactions account for the majority of the assembled components. The study elucidates how different proteins are recruited into senile plaques and establishes a new model system for exploring the pathological mechanisms of Alzheimer's disease from a molecular perspective.

  • 16.
    Cheung, Pierre
    et al.
    Science for Life Laboratory, Department of Medicinal Chemistry, Uppsala University, Uppsala, Sweden.
    Thorngren, Julia
    Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden.
    Zhang, Bo
    Science for Life Laboratory, Department of Medicinal Chemistry, Uppsala University, Uppsala, Sweden.
    Vasylovska, Svitlana
    Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden.
    Lechi, Francesco
    Science for Life Laboratory, Department of Medicinal Chemistry, Uppsala University, Uppsala, Sweden.
    Persson, Jonas
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Ståhl, Stefan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Korsgren, Olle
    Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
    Eriksson, Jonas
    Science for Life Laboratory, Department of Medicinal Chemistry, Uppsala University, Uppsala, Sweden.
    Lau, Joey
    Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden.
    Eriksson, Olof
    Science for Life Laboratory, Department of Medicinal Chemistry, Uppsala University, Uppsala, Sweden.
    Preclinical evaluation of Affibody molecule for PET imaging of human pancreatic islets derived from stem cells2023Inngår i: EJNMMI Research, E-ISSN 2191-219X, Vol. 13, nr 1, artikkel-id 107Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background: Beta-cell replacement methods such as transplantation of isolated donor islets have been proposed as a curative treatment of type 1 diabetes, but widespread application is challenging due to shortages of donor tissue and the need for continuous immunosuppressive treatments. Stem-cell-derived islets have been suggested as an alternative source of beta cells, but face transplantation protocols optimization difficulties, mainly due to a lack of available methods and markers to directly monitor grafts survival, as well as their localization and function. Molecular imaging techniques and particularly positron emission tomography has been suggested as a tool for monitoring the fate of islets after clinical transplantation. The integral membrane protein DGCR2 has been demonstrated to be a potential pancreatic islet biomarker, with specific expression on insulin-positive human embryonic stem-cell-derived pancreatic progenitor cells. The candidate Affibody molecule ZDGCR2:AM106 was radiolabeled with fluorine-18 using a novel click chemistry-based approach. The resulting positron emission tomography tracer [18F]ZDGCR2:AM106 was evaluated for binding to recombinant human DGCR2 and cryosections of stem-cell-derived islets, as well as in vivo using an immune-deficient mouse model transplanted with stem-cell-derived islets. Biodistribution of the [18F]ZDGCR2:AM106 was also assessed in healthy rats and pigs. Results: [18F]ZDGCR2:AM106 was successfully synthesized with high radiochemical purity and yield via a pretargeting approach. [18F]ZDGCR2:AM106 retained binding to recombinant human DCGR2 as well as to cryosectioned stem-cell-derived islets, but in vivo binding to native pancreatic tissue in both rat and pig was low. However, in vivo uptake of [18F]ZDGCR2:AM106 in stem-cell-derived islets transplanted in the immunodeficient mice was observed, albeit only within the early imaging frames after injection of the radiotracer. Conclusion: Targeting of DGCR2 is a promising approach for in vivo detection of stem-cell-derived islets grafts by molecular imaging. The synthesis of [18F]ZDGCR2:AM106 was successfully performed via a pretargeting method to label a site-specific covalently bonded fluorine-18 to the Affibody molecule. However, the rapid washout of [18F]ZDGCR2:AM106 from the stem-cell-derived islets graft indicates that dissociation kinetics can be improved. Further studies using alternative binders of similar classes with improved binding potential are warranted.

  • 17. 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, Proteinvetenskap.
    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 Grafts2018Inngår i: ACS Applied Materials and Interfaces, ISSN 1944-8244, E-ISSN 1944-8252, Vol. 10, nr 28, s. 23560-23572Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 18.
    Clinton, Jacob
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Westerlund, Kristina
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Eriksson Karlström, Amelie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Gräslund, Torbjörn
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Affibody-mediated peptide nucleic acid pretargeting for delivery of cytotoxic payloads to HER2 positive carcinoma2024Inngår i: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 30Artikkel i tidsskrift (Annet vitenskapelig)
  • 19.
    Damiati, Safa A.
    et al.
    King Abdulaziz Univ KAU, Dept Pharmaceut, Fac Pharm, Jeddah 21589, Saudi Arabia..
    Rossi, Damiano
    Blacktrace Holdings Ltd, Dolomite Microfluid, Royston SG8 5TW, England..
    Jönsson, Håkan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap. Novo Nordisk Foundation Center for Biosustainability at KTH, Stockholm. Sweden.
    Damiati, Samar
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap. King Abdulaziz Univ KAU, Fac Sci, Dept Biochem, Jeddah, Saudi Arabia..
    Artificial intelligence application for rapid fabrication of size-tunable PLGA microparticles in microfluidics2020Inngår i: Scientific Reports, E-ISSN 2045-2322, Vol. 10, nr 1, artikkel-id 19517Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In this study, synthetic polymeric particles were effectively fabricated by combining modern technologies of artificial intelligence (AI) and microfluidics. Because size uniformity is a key factor that significantly influences the stability of polymeric particles, therefore, this work aimed to establish a new AI application using machine learning technology for prediction of the size of poly(d,l-lactide-co-glycolide) (PLGA) microparticles produced by diverse microfluidic systems either in the form of single or multiple particles. Experimentally, the most effective factors for tuning droplet/particle sizes are PLGA concentrations and the flow rates of dispersed and aqueous phases in microfluidics. These factors were utilized to develop five different and simple in structure artificial neural network (ANN) models that are capable of predicting PLGA particle sizes produced by different microfluidic systems either individually or jointly merged. The systematic development of ANN models allowed ultimate construction of a single in silico model which consists of data for three different microfluidic systems. This ANN model eventually allowed rapid prediction of particle sizes produced using various microfluidic systems. This AI application offers a new platform for further rapid and economical exploration of polymer particles production in defined sizes for various applications including biomimetic studies, biomedicine, and pharmaceutics.

  • 20.
    Delaney, Samantha
    et al.
    Department of Chemistry, Hunter College, City University of New York, New York, NY, USA; Ph.D. Program in Biochemistry, The Graduate Center of the City University of New York, New York, NY, USA; Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
    Nagy, Abel
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Eriksson Karlström, Amelie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Zeglis, Brian M.
    Department of Chemistry, Hunter College, City University of New York, New York, NY, USA; Ph.D. Program in Biochemistry, The Graduate Center of the City University of New York, New York, NY, USA; Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY, USA; Ph.D. Program in Chemistry, The Graduate Center of the City University of New York, New York, NY, USA; Department of Radiology, Weill Cornell Medical College, New York, NY, USA.
    Site-Specific Photoaffinity Bioconjugation for the Creation of <sup>89</sup>Zr-Labeled Radioimmunoconjugates2023Inngår i: Molecular Imaging and Biology, ISSN 1536-1632, E-ISSN 1860-2002, Vol. 25, nr 6, s. 1104-1114Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Purpose: Site-specific approaches to bioconjugation produce well-defined and homogeneous immunoconjugates with potential for superior in vivo behavior compared to analogs synthesized using traditional, stochastic methods. The possibility of incorporating photoaffinity chemistry into a site-specific bioconjugation strategy is particularly enticing, as it could simplify and accelerate the preparation of homogeneous immunoconjugates for the clinic. In this investigation, we report the synthesis, in vitro characterization, and in vivo evaluation of a site-specifically modified, 89Zr-labeled radioimmunoconjugate created via the reaction between an mAb and an Fc-binding protein bearing a photoactivatable 4-benzoylphenylalanine residue. Procedures: A variant of the Fc-binding Z domain of protein A containing a photoactivatable, 4-benzoylphenylalanine residue — Z(35BPA) — was modified with desferrioxamine (DFO), combined with the A33 antigen-targeting mAb huA33, and irradiated with UV light. The resulting immunoconjugate — DFOZ(35BPA)-huA33 — was purified and characterized via SDS-PAGE, MALDI-ToF mass spectrometry, surface plasmon resonance, and flow cytometry. The radiolabeling of DFOZ(35BPA)-huA33 was optimized to produce [89Zr]Zr-DFOZ(35BPA)-huA33, and the immunoreactivity of the radioimmunoconjugate was determined with SW1222 human colorectal cancer cells. Finally, the in vivo performance of [89Zr]Zr-DFOZ(35BPA)-huA33 in mice bearing subcutaneous SW1222 xenografts was interrogated via PET imaging and biodistribution experiments and compared to that of a stochastically labeled control radioimmunoconjugate, [89Zr]Zr-DFO-huA33. Results: HuA33 was site-specifically modified with Z(35BPA)-DFO, producing an immunoconjugate with on average 1 DFO/mAb, high in vitro stability, and high affinity for its target. [89Zr]Zr-DFOZ(35BPA)-huA33 was synthesized in 95% radiochemical yield and exhibited a specific activity of 2 mCi/mg and an immunoreactive fraction of ~ 0.85. PET imaging and biodistribution experiments revealed that high concentrations of the radioimmunoconjugate accumulated in tumor tissue (i.e., ~ 40%ID/g at 120 h p.i.) but also that the Z(35BPA)-bearing immunoPET probe produced higher uptake in the liver, spleen, and kidneys than its stochastically modified cousin, [89Zr]Zr-DFO-huA33. Conclusions: Photoaffinity chemistry and an Fc-binding variant of the Z domain were successfully leveraged to create a novel site-specific strategy for the synthesis of radioimmunoconjugates. The probe synthesized using this method — DFOZ(35BPA)-huA33 — was well-defined and homogeneous, and the resulting radioimmunoconjugate ([89Zr]Zr-DFOZ(35BPA)-huA33) boasted high specific activity, stability, and immunoreactivity. While the site-specifically modified radioimmunoconjugate produced high activity concentrations in tumor tissue, it also yielded higher uptake in healthy organs than a stochastically modified analog, suggesting that optimization of this system is necessary prior to clinical translation.

  • 21.
    Deyev, Sergey M.
    et al.
    Molecular Immunology Laboratory, Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia; Research Centrum for Oncotheranostics, Research School of Chemistry and Applied Biomedical Sciences, Tomsk Polytechnic University, 634050 Tomsk, Russia.
    Oroujeni, Maryam
    Department of Immunology, Genetics and Pathology, Uppsala University, 751 85 Uppsala, Sweden;; Affibody AB, 171 65 Solna, Sweden.
    Garousi, Javad
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap. Department of Immunology, Genetics and Pathology, Uppsala University, 751 85 Uppsala, Sweden.
    Gräslund, Torbjörn
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Li, Ruonan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Rosly, Alia Hani Binti
    Department of Immunology, Genetics and Pathology, Uppsala University, 751 85 Uppsala, Sweden;.
    Orlova, Anna
    Department of Medicinal Chemistry, Uppsala University, 751 83 Uppsala, Sweden;.
    Konovalova, Elena
    Molecular Immunology Laboratory, Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia.
    Schulga, Alexey
    Molecular Immunology Laboratory, Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia; Research Centrum for Oncotheranostics, Research School of Chemistry and Applied Biomedical Sciences, Tomsk Polytechnic University, 634050 Tomsk, Russia.
    Vorobyeva, Anzhelika
    Department of Immunology, Genetics and Pathology, Uppsala University, 751 85 Uppsala, Sweden;.
    Tolmachev, Vladimir
    Department of Immunology, Genetics and Pathology, Uppsala University, 751 85 Uppsala, Sweden;.
    Preclinical Evaluation of HER2-Targeting DARPin G3: Impact of Albumin-Binding Domain (ABD) Fusion2024Inngår i: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 25, nr 8, artikkel-id 4246Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Designed ankyrin repeat protein (DARPin) G3 is an engineered scaffold protein. This small (14.5 kDa) targeting protein binds with high affinity to human epidermal growth factor receptor 2 (HER2). HER2 is overexpressed in several cancers. The use of the DARPin G3 for radionuclide therapy is complicated by its high renal reabsorption after clearance via the glomeruli. We tested the hypothesis that a fusion of the DARPin G3 with an albumin-binding domain (ABD) would prevent rapid renal excretion and high renal reabsorption resulting in better tumour targeting. Two fusion proteins were produced, one with the ABD at the C-terminus (G3-ABD) and another at the N-terminus (ABD-G3). Both variants were labelled with 177Lu. The binding properties of the novel constructs were evaluated in vitro and their biodistribution was compared in mice with implanted human HER2-expressing tumours. Fusion with the ABD increased the retention time of both constructs in blood compared with the non-ABD-fused control. The effect of fusion with the ABD depended strongly on the order of the domains in the constructs, resulting in appreciably better targeting properties of [177Lu]Lu-G3-ABD. Our data suggest that the order of domains is critical for the design of targeting constructs based on scaffold proteins.

  • 22.
    Deyev, Sergey M.
    et al.
    Russian Acad Sci, Mol Immunol Lab, Shemyakin & Ovchinnikov Inst Bioorgan Chem, Moscow, Russia.;Res Tomsk Polytech Univ, Res Sch Chem & Appl Biomed Sci, Tomsk, Russia.;Sechenov Univ, Ctr Biomed Engn, Moscow, Russia..
    Vorobyeva, Anzhelika
    Res Tomsk Polytech Univ, Res Sch Chem & Appl Biomed Sci, Tomsk, Russia.;Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden..
    Schulga, Alexey
    Russian Acad Sci, Mol Immunol Lab, Shemyakin & Ovchinnikov Inst Bioorgan Chem, Moscow, Russia.;Res Tomsk Polytech Univ, Res Sch Chem & Appl Biomed Sci, Tomsk, Russia..
    Abouzayed, Ayman
    Uppsala Univ, Dept Med Chem, Uppsala, Sweden..
    Gunther, Tyran
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden..
    Garousi, Javad
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden..
    Konovalova, Elena
    Russian Acad Sci, Mol Immunol Lab, Shemyakin & Ovchinnikov Inst Bioorgan Chem, Moscow, Russia..
    Ding, Haozhong
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Gräslund, Torbjörn
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Orlova, Anna
    Res Tomsk Polytech Univ, Res Sch Chem & Appl Biomed Sci, Tomsk, Russia.;Uppsala Univ, Dept Med Chem, Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, Uppsala, Sweden..
    Tolmachev, Vladimir
    Res Tomsk Polytech Univ, Res Sch Chem & Appl Biomed Sci, Tomsk, Russia.;Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden..
    Effect of a radiolabel biochemical nature on tumor-targeting properties of EpCAM-binding engineered scaffold protein DARPin Ec12020Inngår i: International Journal of Biological Macromolecules, ISSN 0141-8130, E-ISSN 1879-0003, Vol. 145, s. 216-225Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Radionuclide-based imaging of molecular therapeutic targets might facilitate stratifying patients for specific biotherapeutics. New type of imaging probes, based on designed ankyrin repeat proteins (DARPins), have demonstrated excellent contrast of imaging of human epidermal growth factor type 2 (HER2) expression in preclinical models. We hypothesized that labeling approaches, which result in lipophilic radiometabolites (non-residualizing labels), would provide the best imaging contrast for DARPins that internalize slowly after binding to cancer cells. The hypothesis was tested using DARPin Ec1 that binds to epithelial cell adhesion molecule (EpCAM). EpCAM is a promising therapeutic target. Ec1 was labeled with I-125 using two methods to obtain the non-residualizing labels, while residualizing labels were obtained by labeling it with Tc-99m. All labeled Ec1 variants preserved target specificity and picomolar binding affinity to EpCAM-expressing pancreatic adenocarcinoma BxPC-3 cells. In murine models, all the variants provided similar tumor uptake. However, I-125-PIB-H-6-Ec1 had noticeably lower retention in normal tissues, which provided appreciably higher tumor-to-organ ratios. Furthermore, I-125-PIB-H-6-Ec1 demonstrated the highest imaging contrast in preclinical models than any other EpCAM-imaging agent tested so far. In conclusion, DARPin Ec1 in combination with a non-residualizing label is a promising probe for imaging EpCAM expression a few hours after injection.

  • 23.
    Deyev, Sergey M.
    et al.
    Tomsk Polytech Univ, Res Ctr Oncotheranost, Res Sch Chem & Appl Biomed Sci, Tomsk 634050, Russia.;Russian Acad Sci, Shemyakin & Ovchinnikov Inst Bioorgan Chem, Mol Immunol Lab, Moscow 117997, Russia.;Natl Res Nucl Univ MEPhI, Inst Engn Phys Biomed PhysBio, Bionanophoton Lab, Moscow 115409, Russia..
    Xu, Tianqi
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden..
    Liu, Yongsheng
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden..
    Schulga, Alexey
    Tomsk Polytech Univ, Res Ctr Oncotheranost, Res Sch Chem & Appl Biomed Sci, Tomsk 634050, Russia.;Russian Acad Sci, Shemyakin & Ovchinnikov Inst Bioorgan Chem, Mol Immunol Lab, Moscow 117997, Russia..
    Konovalova, Elena
    Russian Acad Sci, Shemyakin & Ovchinnikov Inst Bioorgan Chem, Mol Immunol Lab, Moscow 117997, Russia..
    Garousi, Javad
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap. Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden..
    Rinne, Sara S.
    Uppsala Univ, Dept Med Chem, S-75123 Uppsala, Sweden..
    Larkina, Maria
    Tomsk Polytech Univ, Res Ctr Oncotheranost, Res Sch Chem & Appl Biomed Sci, Tomsk 634050, Russia.;Siberian State Med Univ SSMU, Dept Pharmaceut Anal, 2 Moscow Trakt, Tomsk 634050, Russia..
    Ding, Haozhong
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap. KTH Royal Inst Technol, Dept Prot Sci, Sch Engn Sci Chem Biotechnol & Hlth, S-11417 Stockholm, Sweden..
    Gräslund, Torbjörn
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap. KTH Royal Inst Technol, Dept Prot Sci, Sch Engn Sci Chem Biotechnol & Hlth, S-11417 Stockholm, Sweden..
    Orlova, Anna
    Tomsk Polytech Univ, Res Ctr Oncotheranost, Res Sch Chem & Appl Biomed Sci, Tomsk 634050, Russia.;Uppsala Univ, Dept Med Chem, S-75123 Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, S-75123 Uppsala, Sweden..
    Tolmachev, Vladimir
    Tomsk Polytech Univ, Res Ctr Oncotheranost, Res Sch Chem & Appl Biomed Sci, Tomsk 634050, Russia.;Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden..
    Vorobyeva, Anzhelika
    Tomsk Polytech Univ, Res Ctr Oncotheranost, Res Sch Chem & Appl Biomed Sci, Tomsk 634050, Russia.;Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden..
    Influence of the Position and Composition of Radiometals and Radioiodine Labels on Imaging of Epcam Expression in Prostate Cancer Model Using the DARPin Ec12021Inngår i: Cancers, ISSN 2072-6694, Vol. 13, nr 14, artikkel-id 3589Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Simple Summary Metastasis-targeting therapy might improve outcomes in oligometastatic prostate cancer. Epithelial cell adhesion molecule (EpCAM) is overexpressed in 40-60% of prostate cancer cases and might be used as a target for specific delivery of toxins and drugs. Radionuclide molecular imaging could enable non-invasive detection of EpCAM and stratification of patients for targeted therapy. Designed ankyrin repeat proteins (DARPins) are scaffold proteins, which can be selected for specific binding to different targets. The DARPin Ec1 binds strongly to EpCAM. To determine an optimal design of Ec1-based probes, we labeled Ec1 at two different positions with four different nuclides (Ga-68, In-111, Co-57 and I-125) and investigated the impact on Ec1 biodistribution. We found that the C-terminus is the best position for labeling and that In-111 and I-125 provide the best imaging contrast. This study might be helpful for scientists developing imaging probes based on scaffold proteins. The epithelial cell adhesion molecule (EpCAM) is intensively overexpressed in 40-60% of prostate cancer (PCa) cases and can be used as a target for the delivery of drugs and toxins. The designed ankyrin repeat protein (DARPin) Ec1 has a high affinity to EpCAM (68 pM) and a small size (18 kDa). Radiolabeled Ec1 might be used as a companion diagnostic for the selection of PCa patients for therapy. The study aimed to investigate the influence of radiolabel position (N- or C-terminal) and composition on the targeting and imaging properties of Ec1. Two variants, having an N- or C-terminal cysteine, were produced, site-specifically conjugated to a DOTA chelator and labeled with cobalt-57, gallium-68 or indium-111. Site-specific radioiodination was performed using ((4-hydroxyphenyl)-ethyl)maleimide (HPEM). Biodistribution of eight radiolabeled Ec1-probes was measured in nude mice bearing PCa DU145 xenografts. In all cases, positioning of a label at the C-terminus provided the best tumor-to-organ ratios. The non-residualizing [I-125]I-HPEM label provided the highest tumor-to-muscle and tumor-to-bone ratios and is more suitable for EpCAM imaging in early-stage PCa. Among the radiometals, indium-111 provided the highest tumor-to-blood, tumor-to-lung and tumor-to-liver ratios and could be used at late-stage PCa. In conclusion, label position and composition are important for the DARPin Ec1.

  • 24.
    Ding, Haozhong
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Targeting HER2-expressing tumors with potent drug conjugates and fusion toxins based on scaffold proteins2022Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Targeted therapy is an emerging treatment for a variety of cancers. Small- sized scaffold proteins are an alternative to conventional antibody-based targeting molecules. Two small scaffold proteins—the 58-amino-acid protein class, the affibody molecules, and the 46-amino-acid protein class, the Albumin binding domain Derived Affinity Proteins (ADAPTs)—have previously been engineered to bind to a large variety of tumor-associated molecular targets with a high affinity. 

    The human epidermal growth factor receptor 2 (HER2) is a membrane-bound receptor for growth signal transmission. Expression of a high level of HER2 can cause cells to proliferate and may ultimately lead to cancer. It has earlier been shown that HER2 is involved in several different types of cancers, e.g., breast, ovarian, bladder, and gastric cancers. 

    HER2-targeted affibody and ADAPT molecules have previously been developed, such as ZHER2:2891 and ADAPT6 with strong affinity to HER2 with equilibrium dissociation constants of 76 pM and 2.5 nM, respectively. Their small size and high specificity have rendered these two scaffold proteins promising candidates for imaging of HER2-positive breast cancer tumors in clinical trials. 

    Delivery of cytotoxic agents to cancer cells, using a cell-targeting domain, may potentially precisely kill the cancer cells while having very low cytotoxic effects on normal cells. Many cancer-targeted antibody drug conjugates (ADCs) and toxic proteins (immunotoxins) have advanced the field of cancer treatment. Small-sized scaffold proteins hold promise as alternative targeting domains to build novel drug conjugates or fusion toxins for cancer treatment. 

    In this thesis, I first investigated an affibody-based drug conjugate (AffiDC) composed of an anti-HER2 affibody and an anti-mitotic maytansine-derived drug (DM1) for treatment of HER2-overexpressing cells. I studied a variety of targeting domain formats for efficacy optimization. All ZHER2:2891-based AffiDCs showed specific anti-tumor activity on HER2-overexpressing cancer cells in vitro as well as in mouse tumor xenografts. The hepatic uptake of the AffiDCs could be reduced by shielding the hydrophobic DM1 using a poly-glutamic-acid spacer, which might help to reduce potential liver toxicity allowing for administration of higher doses. In addition, tuning the valency of the affibody-targeting domain (ZHER2) from a divalent domain to a monovalent domain showed increased potency and reduced liver uptake. We also investigated the influence of the number of drug payloads on the pharmacokinetic profile of the AffiDCs. An AffiDC bearing three DM1s showed higher delivery of DM1 to the cancer cells in vivo, but fast blood clearance and an elevated liver retention was also observed. 

    With regards to fusion toxin design, we constructed a variety of recombinant toxins. The targeting domains were ZHER2:2891 and/or ADAPT6, which were genetically fused with truncated versions of the highly cytotoxic Pseudomonas Exotoxin A (PE). All fusion toxins we studied showed potent HER2-specific anti-tumor activity. The results suggested that both ZHER2:2891 and ADAPT6 could direct the PE-based cytotoxins specifically to HER2- overexpressing cancer cells. 

    In this work, we have demonstrated the potential of using ZHER2:2891 and ADAPT6 as targeting domains to carry the small molecule drug DM1, or cytotoxic PE-derived peptides to cancer cells. It can be concluded that careful molecular design of the targeting domain may considerably improve the potency and minimize the off-target uptake. 

    Fulltekst (pdf)
    HD_2022
  • 25.
    Ding, Haozhong
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    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, Proteinvetenskap.
    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 Conjugates2019Inngår i: Cancers, ISSN 2072-6694, Vol. 11, nr 8, artikkel-id 1168Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 26.
    Ding, Haozhong
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Altai, Mohamed
    Lund Univ, Dept Pathol & Oncol, Barngatan 4, S-22242 Lund, Sweden..
    Yin, Wen
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Lindbo, Sarah
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Liu, Hao
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Garousi, Javad
    Uppsala Univ, Dept Immunol Genet & Pathol, Dag Hammarskjolds Vag 20, S-75185 Uppsala, Sweden..
    Xu, Tianqi
    Uppsala Univ, Dept Immunol Genet & Pathol, Dag Hammarskjolds Vag 20, S-75185 Uppsala, Sweden..
    Orlova, Anna
    Uppsala Univ, Dept Med Chem, Dag Hammarskjolds Vag 14C, S-75123 Uppsala, Sweden.;Tomsk Polytech Univ, Res Sch Chem & Appl Biomed Sci, Res Ctr Oncotheranost, Tomsk 634050, Russia..
    Tolmachev, Vladimir
    Uppsala Univ, Dept Immunol Genet & Pathol, Dag Hammarskjolds Vag 20, S-75185 Uppsala, Sweden.;Tomsk Polytech Univ, Res Sch Chem & Appl Biomed Sci, Res Ctr Oncotheranost, Tomsk 634050, Russia..
    Hober, Sophia
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Gräslund, Torbjörn
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    HER2-Specific Pseudomonas Exotoxin A PE25 Based Fusions: Influence of Targeting Domain on Target Binding, Toxicity, and In Vivo Biodistribution2020Inngår i: Pharmaceutics, E-ISSN 1999-4923, Vol. 12, nr 4, artikkel-id 391Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The human epidermal growth factor receptor 2 (HER2) is a clinically validated target for cancer therapy, and targeted therapies are often used in regimens for patients with a high HER2 expression level. Despite the success of current drugs, a number of patients succumb to their disease, which motivates development of novel drugs with other modes of action. We have previously shown that an albumin binding domain-derived affinity protein with specific affinity for HER2, ADAPT(6), can be used to deliver the highly cytotoxic protein domain PE25, a derivative of Pseudomonas exotoxin A, to HER2 overexpressing malignant cells, leading to potent and specific cell killing. In this study we expanded the investigation for an optimal targeting domain and constructed two fusion toxins where a HER2-binding affibody molecule, Z(HER2:2891), or the dual-HER2-binding hybrid Z(HER2:2891)-ADAPT(6) were used for cancer cell targeting. We found that both targeting domains conferred strong binding to HER2; both to the purified extracellular domain and to the HER2 overexpressing cell line SKOV3. This resulted in fusion toxins with high cytotoxic potency toward cell lines with high expression levels of HER2, with EC50 values between 10 and 100 pM. For extension of the plasma half-life, an albumin binding domain was also included. Intravenous injection of the fusion toxins into mice showed a profound influence of the targeting domain on biodistribution. Compared to previous results, with ADAPT(6) as targeting domain, Z(HER2:2891) gave rise to further extension of the plasma half-life and also shifted the clearance route of the fusion toxin from the liver to the kidneys. Collectively, the results show that the targeting domain has a major impact on uptake of PE25-based fusion toxins in different organs. The results also show that PE25-based fusion toxins with high affinity to HER2 do not necessarily increase the cytotoxicity beyond a certain point in affinity. In conclusion, Z(HER2:2891) has the most favorable characteristics as targeting domain for PE25.

  • 27.
    Ding, Haozhong
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Xu, Tianqi
    Uppsala Univ, Dept Immunol Genet & Pathol, Dag Hammarskjolds Vag 20, S-75185 Uppsala, Sweden..
    Zhang, Jie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Tolmachev, Vladimir
    Uppsala Univ, Dept Immunol Genet & Pathol, Dag Hammarskjolds Vag 20, S-75185 Uppsala, Sweden.;Tomsk Polytech Univ, Res Sch Chem & Appl Biomed Sci, Res Ctr Oncotheranost, Tomsk 634050, Russia..
    Oroujeni, Maryam
    Uppsala Univ, Dept Immunol Genet & Pathol, Dag Hammarskjolds Vag 20, S-75185 Uppsala, Sweden..
    Orlova, Anna
    Tomsk Polytech Univ, Res Sch Chem & Appl Biomed Sci, Res Ctr Oncotheranost, Tomsk 634050, Russia.;Uppsala Univ, Dept Med Chem, Dag Hammarskjolds Vag 14C, S-75123 Uppsala, Sweden..
    Gräslund, Torbjörn
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Vorobyeva, Anzhelika
    Uppsala Univ, Dept Immunol Genet & Pathol, Dag Hammarskjolds Vag 20, S-75185 Uppsala, Sweden.;Tomsk Polytech Univ, Res Sch Chem & Appl Biomed Sci, Res Ctr Oncotheranost, Tomsk 634050, Russia..
    Affibody-Derived Drug Conjugates Targeting HER2: Effect of Drug Load on Cytotoxicity and Biodistribution2021Inngår i: Pharmaceutics, E-ISSN 1999-4923, Vol. 13, nr 3, artikkel-id 430Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Affibody molecules hold great promise as carriers of cytotoxic drugs for cancer therapy due to their typically high affinity, easy production, and inherent control of the drug molecules' loading and spatial arrangement. Here, the impact of increasing the drug load from one to three on the properties of an affibody drug conjugate targeting the human epidermal growth factor receptor 2 (HER2) was investigated. The affibody carrier was recombinantly expressed as a fusion to an albumin-binding domain (ABD) for plasma half-life extension. One or three cysteine amino acids were placed at the C-terminus to which cytotoxic mcDM1 molecules were conjugated. The resulting drug conjugates, Z(HER2)-ABD-mcDM1 and Z(HER2)-ABD-mcDM1(3), were characterized in vitro, and their biodistribution in mice carrying HER2-overexpressing SKOV3 xenografts was determined. Increasing the drug load from one to three led to a decrease in affinity for HER2, but a significantly more potent cytotoxic effect on SKOV3 cells with high HER2 expression. The difference in cytotoxic effect on other cell lines with high HER2 expression was not significant. In vivo, an increase in drug load led to a 1.45-fold higher amount of cytotoxic mcDM1 delivered to the tumors. The increase in drug load also led to more rapid hepatic clearance, warranting further optimization of the molecular design.

  • 28. Duray, Elodie
    et al.
    Tano, Hanna
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Westerlund, Kristina
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Bocuzzi, Valentina
    Marcion, Guillaume
    Damicco, Silvestre
    Clinton, Jacob
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Caers, Jo
    Eriksson Karlström, Amelie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Development of an anti-CD38 single domain antibody fragment mediated PNA-based pretargeting strategyManuskript (preprint) (Annet vitenskapelig)
  • 29.
    Duru, Adil Doganay
    et al.
    Karolinska Inst, Dept Med Solna, Sci Life Lab, Stockholm, Sweden.;Karolinska Univ Hosp, Div Infect Dis, Stockholm, Sweden.;Nova Southeastern Univ, NSU Cell Therapy Inst, Ft Lauderdale, FL 33314 USA.;Nova Southeastern Univ, Kiran C Patel Coll Allopath Med, Ft Lauderdale, FL 33314 USA..
    Sun, Renhua
    Karolinska Inst, Dept Med Solna, Sci Life Lab, Stockholm, Sweden.;Karolinska Univ Hosp, Div Infect Dis, Stockholm, Sweden..
    Allerbring, Eva B.
    Karolinska Inst, Dept Med Solna, Sci Life Lab, Stockholm, Sweden.;Karolinska Univ Hosp, Div Infect Dis, Stockholm, Sweden..
    Chadderton, Jesseka
    Monash Univ, Biomed Discovery Inst, Dept Microbiol, Clayton, Vic, Australia..
    Kadri, Nadir
    Karolinska Inst, Dept Med Solna, Sci Life Lab, Stockholm, Sweden.;Karolinska Univ Hosp, Div Infect Dis, Stockholm, Sweden..
    Han, Xiao
    Karolinska Inst, Dept Med Solna, Sci Life Lab, Stockholm, Sweden.;Karolinska Univ Hosp, Div Infect Dis, Stockholm, Sweden..
    Peqini, Kaliroi
    Univ Milan, Sez Chim Gen & Organ, Dipartimento Sci Farmaceut, DISFARM, Milan, Italy..
    Uchtenhagen, Hannes
    Karolinska Inst, Dept Med Solna, Sci Life Lab, Stockholm, Sweden.;Karolinska Univ Hosp, Div Infect Dis, Stockholm, Sweden..
    Madhurantakam, Chaithanya
    Karolinska Inst, Dept Med Solna, Sci Life Lab, Stockholm, Sweden.;Karolinska Univ Hosp, Div Infect Dis, Stockholm, Sweden.;TERI, Dept Biotechnol, Struct & Mol Biol Lab, Sch Adv Studies, New Delhi, India..
    Pellegrino, Sara
    Univ Milan, Sez Chim Gen & Organ, Dipartimento Sci Farmaceut, DISFARM, Milan, Italy..
    Sandalova, Tatyana
    Karolinska Inst, Dept Med Solna, Sci Life Lab, Stockholm, Sweden.;Karolinska Univ Hosp, Div Infect Dis, Stockholm, Sweden..
    Nygren, Per-Åke
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Turner, Stephen J.
    Monash Univ, Biomed Discovery Inst, Dept Microbiol, Clayton, Vic, Australia..
    Achour, Adnane
    Karolinska Inst, Dept Med Solna, Sci Life Lab, Stockholm, Sweden.;Karolinska Univ Hosp, Div Infect Dis, Stockholm, Sweden..
    Tuning antiviral CD8 T-cell response via proline-altered peptide ligand vaccination2020Inngår i: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 16, nr 5, artikkel-id e1008244Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Viral escape from CD8(+) cytotoxic T lymphocyte responses correlates with disease progression and represents a significant challenge for vaccination. Here, we demonstrate that CD8(+) T cell recognition of the naturally occurring MHC-I-restricted LCMV-associated immune escape variant Y4F is restored following vaccination with a proline-altered peptide ligand (APL). The APL increases MHC/peptide (pMHC) complex stability, rigidifies the peptide and facilitates T cell receptor (TCR) recognition through reduced entropy costs. Structural analyses of pMHC complexes before and after TCR binding, combined with biophysical analyses, revealed that although the TCR binds similarly to all complexes, the p3P modification alters the conformations of a very limited amount of specific MHC and peptide residues, facilitating efficient TCR recognition. This approach can be easily introduced in peptides restricted to other MHC alleles, and can be combined with currently available and future vaccination protocols in order to prevent viral immune escape. Author summary Viral escape mutagenesis correlates often with disease progression and represents a major hurdle for vaccination-based therapies. Here, we have designed and developed a novel generation of altered epitopes that re-establish and enhance significantly CD8(+) T cell recognition of a naturally occurring viral immune escape variant. Biophysical and structural analyses provide a clear understanding of the molecular mechanisms underlying this reestablished recognition. We believe that this approach can be implemented to currently available or novel vaccination approaches to efficiently restore T cell recognition of virus escape variants to control disease progression.

  • 30.
    Falzarano, Maria Sofia
    et al.
    Univ Ferrara, UOL Unita Operat Logist Med Genet, Ferrara, Italy..
    Rossi, Rachele
    Univ Ferrara, UOL Unita Operat Logist Med Genet, Ferrara, Italy.;UCL Great Ormond St Inst Child Hlth, Dubowitz Neuromuscular Ctr, London, England..
    Grilli, Andrea
    Univ Modena & Reggio Emilia, Dept Life Sci, Modena, Italy..
    Fang, Mingyan
    Beijing Genom Inst BGI Shenzhen, Shenzhen, Peoples R China..
    Osman, Hana
    Univ Ferrara, UOL Unita Operat Logist Med Genet, Ferrara, Italy.;Univ Khartoum, Fac Med Lab Sci, Dept Med Microbiol, Khartoum, Sudan..
    Sabatelli, Patrizia
    CNR Inst Mol Genet Luigi Luca Cavalli Sforza, Unit Bologna, Bologna, Italy.;Ist Ortoped Rizzoli, Ist Ricovero & Cura Carattere Sci IRCCS, Bologna, Italy..
    Antoniel, Manuela
    CNR Inst Mol Genet Luigi Luca Cavalli Sforza, Unit Bologna, Bologna, Italy.;Ist Ortoped Rizzoli, Ist Ricovero & Cura Carattere Sci IRCCS, Bologna, Italy..
    Lu, Zhiyuan
    Beijing Genom Inst BGI Shenzhen, Shenzhen, Peoples R China..
    Li, Wenyan
    Beijing Genom Inst BGI Shenzhen, Shenzhen, Peoples R China..
    Selvatici, Rita
    Univ Ferrara, UOL Unita Operat Logist Med Genet, Ferrara, Italy..
    Al-Khalili Szigyarto, Cristina
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap. KTH Royal Inst Technol, Dept Prote, Stockholm, Sweden..
    Gualandi, Francesca
    Univ Ferrara, UOL Unita Operat Logist Med Genet, Ferrara, Italy..
    Bicciato, Silvio
    Univ Modena & Reggio Emilia, Dept Life Sci, Modena, Italy..
    Torelli, Silvia
    UCL Great Ormond St Inst Child Hlth, Dubowitz Neuromuscular Ctr, London, England.;UCL, Natl Inst Hlth Res, Great Ormond St Inst Child Hlth, Biomed Res Ctr, London, England..
    Ferlini, Alessandra
    Univ Ferrara, UOL Unita Operat Logist Med Genet, Ferrara, Italy.;UCL Great Ormond St Inst Child Hlth, Dubowitz Neuromuscular Ctr, London, England..
    Urine-Derived Stem Cells Express 571 Neuromuscular Disorders Causing Genes, Making Them a Potential in vitro Model for Rare Genetic Diseases2021Inngår i: Frontiers in Physiology, E-ISSN 1664-042X, Vol. 12, artikkel-id 716471Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background: Neuromuscular disorders (NMDs) are a heterogeneous group of genetic diseases, caused by mutations in genes involved in spinal cord, peripheral nerve, neuromuscular junction, and muscle functions. To advance the knowledge of the pathological mechanisms underlying NMDs and to eventually identify new potential drugs paving the way for personalized medicine, limitations regarding the availability of neuromuscular disease-related biological samples, rarely accessible from patients, are a major challenge.</p> & nbsp;</p> Aim: We characterized urinary stem cells (USCs) by in-depth transcriptome and protein profiling to evaluate whether this easily accessible source of patient-derived cells is suitable to study neuromuscular genetic diseases, focusing especially on those currently involved in clinical trials.</p> & nbsp;</p> Methods: The global transcriptomics of either native or MyoD transformed USCs obtained from control individuals was performed by RNA-seq. The expression of 610 genes belonging to 16 groups of disorders () whose mutations cause neuromuscular diseases, was investigated on the RNA-seq output. In addition, protein expression of 11 genes related to NMDs including COL6A, EMD, LMNA, SMN, UBA1, DYNC1H1, SOD1, C9orf72, DYSF, DAG1, and HTT was analyzed in native USCs by immunofluorescence and/or Western blot (WB).</p> & nbsp;</p> Results: RNA-seq profile of control USCs shows that 571 out of 610 genes known to be involved in NMDs, are expressed in USCs. Interestingly, the expression levels of the majority of NMD genes remain unmodified following USCs MyoD transformation. Most genes involved in the pathogenesis of all 16 groups of NMDs are well represented except for channelopathies and malignant hyperthermia related genes. All tested proteins showed high expression values, suggesting consistency between transcription and protein representation in USCs.</p> & nbsp;</p> Conclusion: Our data suggest that USCs are human cells, obtainable by non-invasive means, which might be used as a patient-specific cell model to study neuromuscular disease-causing genes and that they can be likely adopted for a variety of in vitro functional studies such as mutation characterization, pathway identification, and drug screening.</p>

  • 31. Faresjö, R.
    et al.
    Lindberg, Hanna
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Ståhl, Stefan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Syvänen, S.
    Sehlin, D.
    Transferrin Receptor Binding BBB-Shuttle Facilitates Brain Delivery of Anti-Aβ-Affibodies2022Inngår i: Pharmaceutical research, ISSN 0724-8741, E-ISSN 1573-904X, Vol. 39, nr 7, s. 1509-1521Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Affibodies targeting amyloid-beta (Aβ) could potentially be used as therapeutic and diagnostic agents in Alzheimer’s disease (AD). Affibodies display suitable characteristics for imaging applications such as high stability and a short biological half-life. The aim of this study was to explore brain delivery and retention of Aβ protofibril-targeted affibodies in wild-type (WT) and AD transgenic mice and to evaluate their potential as imaging agents. Two affibodies, Z5 and Z1, were fused with the blood–brain barrier (BBB) shuttle single-chain variable fragment scFv8D3. In vitro binding of 125I-labeled affibodies with and without scFv8D3 was evaluated by ELISA and autoradiography. Brain uptake and retention of the affibodies at 2 h and 24 h post injection was studied ex vivo in WT and transgenic (tg-Swe and tg-ArcSwe) mice. At 2 h post injection, [125I]I-Z5 and [125I]I-Z1 displayed brain concentrations of 0.37 ± 0.09% and 0.46 ± 0.08% ID/g brain, respectively. [125I]I-scFv8D3-Z5 and [125I]I-scFv8D3-Z1 showed increased brain concentrations of 0.53 ± 0.16% and 1.20 ± 0.35%ID/g brain. At 24 h post injection, brain retention of [125I]I-Z1 and [125I]I-Z5 was low, while [125I]I-scFv8D3-Z1 and [125I]I-scFv8D3-Z5 showed moderate brain retention, with a tendency towards higher retention of [125I]I-scFv8D3-Z5 in AD transgenic mice. Nuclear track emulsion autoradiography showed greater parenchymal distribution of [125I]I-scFv8D3-Z5 and [125I]I-scFv8D3-Z1 compared with the affibodies without scFv8D3, but could not confirm specific affibody accumulation around Aβ deposits. Affibody-scFv8D3 fusions displayed increased brain and parenchymal delivery compared with the non-fused affibodies. However, fast brain washout and a suboptimal balance between Aβ and mTfR1 affinity resulted in low intrabrain retention around Aβ deposits. 

  • 32.
    Frieberg, Paula
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Spatially resolved gene expression profiling of mouse brain tissue to study the impact of spaceflights2021Independent thesis Advanced level (degree of Master (Two Years)), 20 poäng / 30 hpOppgave
    Abstract [en]

    Since the first human spaceflight in 1961, hundreds of humans have been in space. Microgravity and high radiation are the main spaceflight hazards. The space environment is known to impact several aspects of human health, such as bone density and cognitive performance. However, the effects of long­duration spaceflights on a cellular and molecular level, utilizing biosamples and multiomic approaches, is poorly studied.

    In this project, the method Spatial Transcriptomics has been utilized to compare brain tissue from the hippocampus region of mice that have been in space with a control group of mice that have stayed on Earth. Spatial Transcriptomics allow for the quantification of gene expression, while maintaining the spatial information of the transcriptome. The results of this study suggest that spaceflights cause mitochondrial stress.   This thesis work is part of a more extensive study in collaboration with NASA, and more studies will be conducted to investigate the effects of spaceflights further. If these findings are confirmed, medicines used on Earth to treat patients with mitochondrial dysfunction could increase the well­being of astronauts in space.

    Fulltekst (pdf)
    fulltext
  • 33.
    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, Proteinvetenskap.
    Tolmachev, V.
    Uppsala Univ, Uppsala, Sweden..
    Selection of the most optimal ADAPT6-based probe for imaging of HER2 using PET and SPECT2018Inngår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 45, s. S77-S78Artikkel i tidsskrift (Annet vitenskapelig)
  • 34.
    Garousi, Javad
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap. Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden..
    Ding, Haozhong
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    von Witting, Emma
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Xu, Tianqi
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden..
    Vorobyeva, Anzhelika
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden.;Res Tomsk Polytech Univ, Res Ctr Oncotheranost, Res Sch Chem & Appl Biomed Sci, RU-634050 Tomsk, Russia..
    Oroujeni, Maryam
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden..
    Orlova, Anna
    Res Tomsk Polytech Univ, Res Ctr Oncotheranost, Res Sch Chem & Appl Biomed Sci, RU-634050 Tomsk, Russia.;Uppsala Univ, Dept Med Chem, SE-75123 Uppsala, Sweden..
    Hober, Sophia
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Gräslund, Torbjörn
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Tolmachev, Vladimir
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden..
    Targeting HER2 Expressing Tumors with a Potent Drug Conjugate Based on an Albumin Binding Domain-Derived Affinity Protein2021Inngår i: Pharmaceutics, E-ISSN 1999-4923, Vol. 13, nr 11, s. 1847-, artikkel-id 1847Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Albumin binding domain derived affinity proteins (ADAPTs) are a class of small and folded engineered scaffold proteins that holds great promise for targeting cancer tumors. Here, we have extended the in vivo half-life of an ADAPT, targeting the human epidermal growth factor receptor 2 (HER2) by fusion with an albumin binding domain (ABD), and armed it with the highly cytotoxic payload mertansine (DM1) for an investigation of its properties in vitro and in vivo. The resulting drug conjugate, ADAPT6-ABD-mcDM1, retained binding to its intended targets, namely HER2 and serum albumins. Further, it was able to specifically bind to cells with high HER2 expression, get internalized, and showed potent toxicity, with IC50 values ranging from 5 to 80 nM. Conversely, no toxic effect was found for cells with low HER2 expression. In vivo, ADAPT6-ABD-mcDM1, radiolabeled with Tc-99m, was characterized by low uptake in most normal organs, and the main excretion route was shown to be through the kidneys. The tumor uptake was 5.5% ID/g after 24 h, which was higher than the uptake in all normal organs at this time point except for the kidneys. The uptake in the tumors was blockable by pre-injection of an excess of the monoclonal antibody trastuzumab (having an overlapping epitope on the HER2 receptor). In conclusion, half-life extended drug conjugates based on the ADAPT platform of affinity proteins holds promise for further development towards targeted cancer therapy.

  • 35.
    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, Proteinvetenskap.
    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 xenografts2019Inngår i: Scientific Reports, E-ISSN 2045-2322, Vol. 9, artikkel-id 14907Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 36.
    Garousi, Javad
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Xu, Tianqi
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden..
    Liu, Yongsheng
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden..
    Vorontsova, Olga
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden..
    Hober, Sophia
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Orlova, Anna
    Uppsala Univ, Dept Med Chem, S-75123 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, Proteinvetenskap.
    Vorobyeva, Anzhelika
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden..
    Experimental HER2-Targeted Therapy Using ADAPT6-ABD-mcDM1 in Mice Bearing SKOV3 Ovarian Cancer Xenografts: Efficacy and Selection of Companion Imaging Counterpart2022Inngår i: Pharmaceutics, E-ISSN 1999-4923, Vol. 14, nr 8, artikkel-id 1612Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Overexpression of the human epidermal growth factor receptor 2 (HER2) in breast and gastric cancer is exploited for targeted therapy using monoclonal antibodies and antibody-drug conjugates. Small engineered scaffold proteins, such as the albumin binding domain (ABD) derived affinity proteins (ADAPTs), are a promising new format of targeting probes for development of drug conjugates with well-defined structure and tunable pharmacokinetics. Radiolabeled ADAPT6 has shown excellent tumor-targeting properties in clinical trials. Recently, we developed a drug conjugate based on the HER2-targeting ADAPT6 fused to an albumin binding domain (ABD) for increased bioavailability and conjugated to DM1 for cytotoxic action, designated as ADAPT6-ABD-mcDM1. In this study, we investigated the therapeutic efficacy of this conjugate in mice bearing HER2-expressing SKOV3 ovarian cancer xenografts. A secondary aim was to evaluate several formats of imaging probes for visualization of HER2 expression in tumors. Administration of ADAPT6-ABD-mcDM1 provided a significant delay of tumor growth and increased the median survival of the mice, in comparison with both a non-targeting homologous construct (ADAPT(Neg)-ABD-mcDM1) and the vehicle-treated groups, without inducing toxicity to liver or kidneys. Moreover, the evaluation of imaging probes showed that small scaffold proteins, such as Tc-99m(CO)(3)-ADAPT6 or the affibody molecule Tc-99m-Z(HER2:41071), are well suited as diagnostic companions for potential stratification of patients for ADAPT6-ABD-mcDM1-based therapy.

  • 37.
    Gestin, Maxime
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Westerlund, Kristina
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Tano, Hanna
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Clinton, Jacob
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Eriksson Karlström, Amelie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Evaluation of the impact of length of peptide nucleic acid probes for tumor pretargetingManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    Pretargeting is a strategy to improve the tumor-to-healthy tissue contrast in targeted nuclear imaging and therapy. The strategy relies on separating the tumor-targeting agent from the radioactive payload and combine the two in vivo. In the pretargeting approach previously studied by our group, the tumor targeting was mediated by an Affibody functionalized with a peptide nucleic acid (PNA) probe and the radionuclide was carried by a complementary PNA probe. Affibody-mediated PNA-based pretargeting was shown to increase the tumor-to-kidney ratio when evaluated in HER2-overexpressing tumor-bearing mice. The aim of the current study is to further optimize the design of the PNA probes to achieve better biodistribution properties and preconditions for a more cost-efficient production. The first important feature of the PNA pretargeting system is the tumor-to-kidney ratio, where the kidney retention is the dose-limiting factor for a clinical therapeutic application. The second aspect is the production of PNA, where the synthesis of PNA strands can be a challenge due to the steric repulsion between two PNA residues’ side chain and poor solubility in the synthesis solvent. In order to simplify the synthesis, we optimized the automation of the process using a microwave-assisted peptide synthesizer. Once the automated synthesis protocols were set up, we designed and synthesized a panel of new PNA probes, aimed at reducing the length of the PNA strands. The reduction in length was expected to simplify the synthesis workflow, but also to possibly decrease the kidney retention of the radioactive payload, as was shown in a previous study when reducing the length of the secondary PNA strand could improve the tumor-to-kidney ratio. The PNA duplexes were studied by CD and UV spectroscopy, and the binding kinetics of the interaction were studied by SPR to identify the limit in terms of number of base pairs needed to reach the high affinity expected to be required for an efficient pretargeting system. Our results showed that high affinity duplexes are formed between PNA probes having only 8 to 9 complementary bases, but that PNA probes with 6 or 7 complementary bases give rise to less stable duplexes having lower melting temperatures and faster dissociation rates.

  • 38.
    Giang, Kim Anh
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Boxaspen, Thorstein
    Department of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital, N-0424 Oslo, Norway; Precision Immunotherapy Alliance, Institute of Clinical Medicine, University of Oslo, N-0313 Oslo, Norway.
    Diao, Yumei
    Oncopeptides AB, S-171 48 Stockholm, Sweden.
    Nilvebrant, Johan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Kosugi-Kanaya, Mizuha
    Department of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital, N-0424 Oslo, Norway; Precision Immunotherapy Alliance, Institute of Clinical Medicine, University of Oslo, N-0313 Oslo, Norway.
    Kanaya, Minoru
    Department of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital, N-0424 Oslo, Norway; Precision Immunotherapy Alliance, Institute of Clinical Medicine, University of Oslo, N-0313 Oslo, Norway.
    Krokeide, Silje Zandstra
    Department of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital, N-0424 Oslo, Norway; Precision Immunotherapy Alliance, Institute of Clinical Medicine, University of Oslo, N-0313 Oslo, Norway.
    Lehmann, Fredrik
    Oncopeptides AB, S-171 48 Stockholm, Sweden.
    Svensson Gelius, Stefan
    Oncopeptides AB, S-171 48 Stockholm, Sweden.
    Malmberg, Karl Johan
    Department of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital, N-0424 Oslo, Norway; Precision Immunotherapy Alliance, Institute of Clinical Medicine, University of Oslo, N-0313 Oslo, Norway.
    Nygren, Per-Åke
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Affibody-based hBCMA x CD16 dual engagers for NK cell-mediated killing of multiple myeloma cells2023Inngår i: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 77, s. 139-148Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We describe the development and characterization of the (to date) smallest Natural Killer (NK) cell re-directing human B Cell Maturation Antigen (hBCMA) x CD16 dual engagers for potential treatment of multiple myeloma, based on combinations of small 58 amino acid, non-immunoglobulin, affibody affinity proteins. Affibody molecules to human CD16a were selected from a combinatorial library by phage display resulting in the identification of three unique binders with affinities (KD) for CD16a in the range of 100 nM–3 µM. The affibody exhibiting the highest affinity demonstrated insensitivity towards the CD16a allotype (158F/V) and did not interfere with IgG (Fc) binding to CD16a. For the construction of hBCMA x CD16 dual engagers, different CD16a binding arms, including bi-paratopic affibody combinations, were genetically fused to a high-affinity hBCMA-specific affibody. Such 15–23 kDa dual engager constructs showed simultaneous hBCMA and CD16a binding ability and could efficiently activate resting primary NK cells and trigger specific lysis of a panel of hBCMA-positive multiple myeloma cell lines. Hence, we report a novel class of uniquely small NK cell engagers with specific binding properties and potent functional profiles.

  • 39.
    Giang, Kim Anh
    et al.
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Kanaya, Mizuha
    Oslo Univ Hosp, Inst Canc Res, Dept Canc Immunol, Oslo, Norway.;Univ Oslo, Inst Clin Med, Oslo, Norway..
    Boxaspen, Thorstein
    Oslo Univ Hosp, Inst Canc Res, Dept Canc Immunol, Oslo, Norway.;Univ Oslo, Inst Clin Med, Oslo, Norway.;Oslo Univ Hosp, Oslo Myeloma Ctr, Dept Hematol, Oslo, Norway..
    Kanaya, Minoru
    Oslo Univ Hosp, Inst Canc Res, Dept Canc Immunol, Oslo, Norway.;Univ Oslo, Inst Clin Med, Oslo, Norway..
    Krokeide, Silje Zandstra
    Oslo Univ Hosp, Inst Canc Res, Dept Canc Immunol, Oslo, Norway.;Univ Oslo, Inst Clin Med, Oslo, Norway..
    Diao, Yumei
    Oncopeptides AB, Stockholm, Sweden..
    Schjesvold, Fredrik
    Oslo Univ Hosp, Oslo Myeloma Ctr, Dept Hematol, Oslo, Norway.;Univ Oslo, KG Jebsen Ctr B Cell Malignancies, Oslo, Norway..
    Lehmann, Fredrik
    Oncopeptides AB, Stockholm, Sweden..
    Nilvebrant, Johan
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Gelius, Stefan Svensson
    Oncopeptides AB, Stockholm, Sweden..
    Malmberg, Karl-Johan
    Oslo Univ Hosp, Inst Canc Res, Dept Canc Immunol, Oslo, Norway.;Univ Oslo, Inst Clin Med, Oslo, Norway..
    Nygren, Per-Åke
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Affibody-Based BCMA x CD16 Dual Engagers for Activation of NK Cells Towards Multiple Myeloma2022Inngår i: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 140, s. 10699-10700Artikkel i tidsskrift (Annet vitenskapelig)
  • 40.
    Giang, Kim Anh
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Nygren, Per-Åke
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Nilvebrant, Johan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Selection of Affibody Affinity Proteins from Phagemid Libraries2023Inngår i: Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029, Vol. 2702, s. 373-392Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Herein, we describe a general protocol for the selection of target-binding affinity protein molecules from a phagemid-encoded library. The protocol is based on our experience with phage display selections of non-immunoglobulin affibody affinity proteins but can in principle be applied to perform biopanning experiments from any phage-displayed affinity protein library available in a similar phagemid vector. The procedure begins with an amplification of the library from frozen bacterial glycerol stocks via cultivation and helper phage superinfection, followed by a step-by-step instruction of target protein preparation, selection cycles, and post-selection analyses. The described procedures in this standard protocol are relatively conservative and rely on ordinary reagents and equipment available in most molecular biology laboratories.

  • 41.
    Giang, Kim Anh
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Sidhu, Sachdev S.
    School of Pharmacy, University of Waterloo, Kitchener, ON, Canada.
    Nilvebrant, Johan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Construction of Synthetic Antibody Phage Display Libraries2023Inngår i: Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029, Vol. 2702, s. 59-75Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Synthetic antibody libraries provide a vast resource of renewable antibody reagents that can rival natural antibodies and be rapidly isolated through controlled in vitro selections. Use of highly optimized human frameworks enables the incorporation of defined diversity at positions that are most likely to contribute to antigen recognition. This protocol describes the construction of synthetic antibody libraries based on a single engineered human autonomous variable heavy domain scaffold with diversity in all three complementarity-determining regions. The resulting libraries can be used to generate recombinant domain antibodies targeting a wide range of protein antigens using phage display. Furthermore, analogous methods can be used to construct antibody libraries based on larger antibody fragments or second-generation libraries aimed to fine-tune antibody characteristics including affinity, specificity, and manufacturability. The procedures rely on standard reagents and equipment available in most molecular biology laboratories.

  • 42.
    Greenberg, J. H.
    et al.
    NYU, Cognit Neurol, New York, NY USA..
    Lindberg, Hanna
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Orozco, J.
    Vama, B.
    Habbat, H.
    Löfblom, John
    KTH, Skolan för teknikvetenskap (SCI). KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Mejouate, O.
    Wisniewski, T.
    Boutajangout, A.
    The Role of Affibody in Aged Mouse Model of Alzheimer's Disease2020Inngår i: Journal of The American Geriatrics Society, ISSN 0002-8614, E-ISSN 1532-5415, Vol. 68, s. S341-S341Artikkel i tidsskrift (Annet vitenskapelig)
  • 43.
    Gräslund, Torbjörn
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Hedhammar, My
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Uhlén, Mathias
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Nygren, Per-Åke
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Hober, Sophia
    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.
    Integrated strategy for selective expanded bed ion-exchange adsorption and site-specific protein processing using gene fusion technology2002Inngår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 96, nr 1, s. 93-102Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The highly charged domain Z(basic) can be used as a fusion partner to enhance adsorption of target proteins to cation exchanging resins at high pH-values. In this paper, we describe a strategy for purification of target proteins fused to Z(basic) at a constant physiological pH using cation exchange chromatography in an expanded bed mode. We show that two proteins, Klenow DNA polymerase and the viral protease 3C, can be efficiently purified from unclarified Escherichia coli homogenates in a single step with a selectivity analogous to what is normally achieved by affinity chromatography. The strategy also includes an integrated site-specific removal of the Z(basic) purification handle to yield a free target protein.

  • 44.
    Gustafsson, Linnea
    et al.
    KTH, Skolan för elektroteknik och datavetenskap (EECS), Intelligenta system, Mikro- och nanosystemteknik.
    Dorka, Wilhelm Nicolai
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Åstrand, Carolina
    KTH.
    Ponsteen, Nienke
    KTH.
    Svanberg, Sara
    Hegrova, Veronica
    Jansson, Ronnie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Kvick, Mathias
    KTH.
    Horak, Josef
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Hedhammar, My
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    van der Wijngaart, Wouter
    KTH, Skolan för elektroteknik och datavetenskap (EECS), Intelligenta system, Mikro- och nanosystemteknik.
    Scalable Synthesis of Monodisperse Bioactive Spider Silk NanostrucutresManuskript (preprint) (Annet vitenskapelig)
  • 45.
    Gustafsson, Linnea
    et al.
    KTH, Skolan för elektroteknik och datavetenskap (EECS), Intelligenta system, Mikro- och nanosystemteknik. Spiber Technologies AB Roslagstullsbacken 15 Stockholm 114 21 Sweden.
    Kvick, Mathias
    Spiber Technologies AB Roslagstullsbacken 15 Stockholm 114 21 Sweden.
    Åstrand, Carolina
    Spiber Technologies AB Roslagstullsbacken 15 Stockholm 114 21 Sweden.
    Ponsteen, Nienke
    KTH, Skolan för elektroteknik och datavetenskap (EECS), Intelligenta system, Mikro- och nanosystemteknik.
    Dorka, Wilhelm Nicolai
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Hegrová, Veronika
    NenoVision s.r.o Purkyňova 127 Brno‐Medlánky 612 00 The Chezh Republic.
    Svanberg, Sara
    KTH, Skolan för elektroteknik och datavetenskap (EECS), Intelligenta system, Mikro- och nanosystemteknik.
    Horák, Josef
    NenoVision s.r.o Purkyňova 127 Brno‐Medlánky 612 00 The Chezh Republic.
    Jansson, Ronnie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Hedhammar, My
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    van der Wijngaart, Wouter
    KTH, Skolan för elektroteknik och datavetenskap (EECS), Intelligenta system, Mikro- och nanosystemteknik.
    Scalable Production of Monodisperse Bioactive Spider Silk Nanowires2023Inngår i: Macromolecular Bioscience, ISSN 1616-5187, E-ISSN 1616-5195, s. 2200450-2200450Artikkel i tidsskrift (Fagfellevurdert)
  • 46.
    Gustafsson, Linnea
    et al.
    KTH, Skolan för elektroteknik och datavetenskap (EECS), Intelligenta system, Mikro- och nanosystemteknik.
    Panagiotis Tasiopoulos, Christos
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Duursma, Thijs
    KTH, Skolan för elektroteknik och datavetenskap (EECS), Intelligenta system, Mikro- och nanosystemteknik.
    Jansson, Ronnie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Gasser, T. Christian
    KTH, Skolan för teknikvetenskap (SCI), Teknisk mekanik, Farkostteknik och Solidmekanik, Hållfasthetslära.
    Hedhammar, My
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    van der Wijngaart, Wouter
    KTH, Skolan för elektroteknik och datavetenskap (EECS), Intelligenta system, Mikro- och nanosystemteknik.
    Mechanical characterization of spider silk nanomembranes2020Inngår i: MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Chemical and Biological Microsystems Society , 2020, s. 418-419Konferansepaper (Fagfellevurdert)
    Abstract [en]

    In this work we present three different ways to characterize the mechanical properties of spider silk nanomembranes. The nanomembranes are formed by self-assembly at the liquid:air interface of a standing solution from which they can be lifted. The mechanical properties are evaluated by (1) manually dropping lead bullets onto the nanomembrane, (2) motorized lowering of a cylindrical indenter to record force-deformation characteristics, and (3) using a standard bulging experiments. Using these methods we show that the nanomembranes are both strong and flexible opening up for applications as pneumatic actuators in MEMS microvalves, or as cell layer actuators in organ-on-a-chip. 

  • 47.
    Gustafsson, Linnea
    et al.
    KTH, Skolan för elektroteknik och datavetenskap (EECS), Intelligenta system, Mikro- och nanosystemteknik.
    Panagiotis Tasiopoulos, Christos
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Jansson, Ronnie
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Kvick, Mathias
    Spiber technologies AB.
    Duursma, Thijs
    KTH, Skolan för elektroteknik och datavetenskap (EECS), Intelligenta system, Mikro- och nanosystemteknik.
    Gasser, T. Christian
    KTH, Skolan för teknikvetenskap (SCI), Teknisk mekanik, Farkostteknik och Solidmekanik, Hållfasthetslära.
    van der Wijngaart, Wouter
    KTH, Skolan för elektroteknik och datavetenskap (EECS), Intelligenta system, Mikro- och nanosystemteknik.
    Hedhammar, My
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Recombinant Spider Silk Forms Tough and Elastic Nanomembranes that are Protein‐Permeable and Support Cell Attachment and Growth2020Inngår i: Advanced Functional Materials, ISSN 1616-301X, E-ISSN 1616-3028, Vol. 30, nr 40, s. 2002982-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Biologically compatible membranes are of high interest for several biological and medical applications. Tissue engineering, for example, would greatly benefit from ultrathin, yet easy‐to‐handle, biodegradable membranes that are permeable to proteins and support cell growth. In this work, nanomembranes are formed by self‐assembly of a recombinant spider silk protein into a nanofibrillar network at the interface of a standing aqueous solution. The membranes are cm‐sized, free‐standing, bioactive and as thin as 250 nm. Despite their nanoscale thickness, the membranes feature an ultimate engineering strain of over 220% and a toughness of 5.2 MPa. Moreover, they are permeable to human blood plasma proteins and promote cell adherence and proliferation. Human keratinocytes seeded on either side of the membrane form a confluent monolayer within three days. The significance of these results lays in the unique combination of nanoscale thickness, elasticity, toughness, biodegradability, protein permeability and support for cell growth, as this may enable new applications in tissue engineering including bi‐layered in vitro tissue models and support for clinical transplantation of coherent cell layers.

    Fulltekst (pdf)
    fulltext
  • 48.
    Güler, Rezan
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Flemming Svedmark, Siri
    Abouzayed, Ayman
    Orlova, Anna
    Löfblom, John
    Increasing thermal stability and improving biodistribution of VEGFR2-binding affibody molecules by a combination of in silico and directed evolution approachesManuskript (preprint) (Annet vitenskapelig)
  • 49.
    Güler, Rezan
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Svedmark, Siri Flemming
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Abouzayed, Ayman
    Uppsala Univ, Dept Med Chem, Uppsala, Sweden..
    Orlova, Anna
    Uppsala Univ, Dept Med Chem, Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, Uppsala, Sweden.;Tomsk Polytech Univ, Res Ctr Oncotheranost, Res Sch Chem & Appl Biomed Sci, Tomsk, Russia..
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    Increasing thermal stability and improving biodistribution of VEGFR2-binding affibody molecules by a combination of in silico and directed evolution approaches2020Inngår i: Scientific Reports, E-ISSN 2045-2322, Vol. 10, nr 1, artikkel-id 18148Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 50.
    Güler, Rezan
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinvetenskap.
    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, Proteinvetenskap.
    Artificial VEGFR2-Specific Growth Factors Demonstrate Agonistic Effects in Both Soluble Form and When Immobilized Via Spider SilkManuskript (preprint) (Annet vitenskapelig)
1234 1 - 50 of 175
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