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  • 1. Chouhan, Dimple
    et al.
    Thatikonda, Naresh
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Nilebäck, Linnea
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Widhe, Mona
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Hedhammar, My
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Mandal, Biman B.
    Recombinant Spider Silk Functionalized Silkworm Silk Matrices as Potential Bioactive Wound Dressings and Skin Grafts2018In: ACS Applied Materials and Interfaces, ISSN 1944-8244, E-ISSN 1944-8252, Vol. 10, no 28, p. 23560-23572Article in journal (Refereed)
    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.

  • 2.
    Garousi, J.
    et al.
    Uppsala Univ, Uppsala, Sweden..
    Lindbo, Sarah
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    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, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Tolmachev, V.
    Uppsala Univ, Uppsala, Sweden..
    Selection of the most optimal ADAPT6-based probe for imaging of HER2 using PET and SPECT2018In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 45, p. S77-S78Article in journal (Other academic)
  • 3.
    Garousi, Javad
    et al.
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden..
    Lindbo, Sarah
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Borin, Jesper
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    von Witting, Emma
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Vorobyeva, Anzhelika
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden..
    Oroujeni, Maryam
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden..
    Mitran, Bogdan
    Uppsala Univ, Dept Med Chem, Uppsala, Sweden..
    Orlova, Anna
    Uppsala Univ, Dept Med Chem, Uppsala, Sweden..
    Buijs, Jos
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden..
    Tolmachev, Vladimir
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden..
    Hober, Sophia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Comparative evaluation of dimeric and monomeric forms of ADAPT scaffold protein for targeting of HER2-expressing tumours2019In: European journal of pharmaceutics and biopharmaceutics, ISSN 0939-6411, E-ISSN 1873-3441, Vol. 134, p. 37-48Article in journal (Refereed)
    Abstract [en]

    ADAPTs are small engineered non-immunoglobulin scaffold proteins, which have demonstrated very promising features as vectors for radionuclide tumour targeting. Radionuclide imaging of human epidermal growth factor 2 (HER2) expression in vivo might be used for stratification of patients for HER2-targeting therapies. ADAPT6, which specifically binds to HER2, has earlier been shown to have very promising features for in vivo targeting of HER2 expressing tumours. In this study we tested the hypothesis that dimerization of ADAPT6 would increase the apparent affinity to HER2 and accordingly improve tumour targeting. To find an optimal molecular design of dimers, a series of ADAPT dimers with different linkers, -SSSG- (DiADAPT6L1), -(SSSG)(2)- (DiADAPT6L2), and -(SSSG)(3)- (DiADAPT6L3) was evaluated. Dimers in combination with optimal linker lengths demonstrated increased apparent affinity to HER2. The best variants, DiADAPT6L2 and DiADAPT6L3 were site-specifically labelled with In-111 and I-125, and compared with a monomeric ADAPT6 in mice bearing HER2-expressing tumours. Despite higher affinity, both dimers had lower tumour uptake and lower tumour-to-organ ratios compared to the monomer. We conclude that improved affinity of a dimeric form of ADAPT does not compensate the disadvantage of increased size. Therefore, increase of affinity should be obtained by affinity maturation and not by dimerization.

  • 4.
    Gomez-Cid, L.
    et al.
    Hosp GU Gregorio Maranon, Serv Cardiol, CIBERCV, Madrid, Spain..
    Fuentes, L.
    Hosp GU Gregorio Maranon, Serv Cardiol, CIBERCV, Madrid, Spain..
    Fernandez-Santos, M. E.
    Hosp GU Gregorio Maranon, Serv Cardiol, CIBERCV, Madrid, Spain..
    Suarez-Sancho, S.
    Hosp GU Gregorio Maranon, Serv Cardiol, CIBERCV, Madrid, Spain..
    Plasencia, V.
    Hosp GU Gregorio Maranon, Serv Cardiol, CIBERCV, Madrid, Spain..
    Climent, A. M.
    Hosp GU Gregorio Maranon, Serv Cardiol, CIBERCV, Madrid, Spain..
    Sanz-Ruiz, R.
    Hosp GU Gregorio Maranon, Serv Cardiol, CIBERCV, Madrid, Spain..
    Hedhammar, My
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Atienza, F.
    Hosp GU Gregorio Maranon, Serv Cardiol, CIBERCV, Madrid, Spain..
    Aviles, F. F.
    Hosp GU Gregorio Maranon, Serv Cardiol, CIBERCV, Madrid, Spain..
    Effect of spider silk matrix on cardiac tissue regeneration of mesenchymal stem cells2018In: European Journal of Clinical Investigation, ISSN 0014-2972, E-ISSN 1365-2362, Vol. 48, p. 150-150Article in journal (Other academic)
  • 5.
    Guo, Weijin
    et al.
    KTH, School of Electrical Engineering and Computer Science (EECS), Micro and Nanosystems.
    Gustafsson, Linnea
    KTH, School of Electrical Engineering and Computer Science (EECS), Micro and Nanosystems.
    Jansson, Ronnie
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Hedhammar, My
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology. KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    van der Wijngaart, Wouter
    KTH, School of Electrical Engineering and Computer Science (EECS), Micro and Nanosystems.
    Formation of a thin-walled Spider Silk Tube on a Micromachined Scaffold2018In: Proceeding of 2018 IEEE 31st International Conference on Micro Electro Mechanical Systems (MEMS), Institute of Electrical and Electronics Engineers (IEEE), 2018, Vol. 2018, p. 83-85Conference paper (Refereed)
    Abstract [en]

    This paper reports on the first formation of a thin bio-functionalized spider silk tube, supported by an internal micromachined scaffold, in which both the inside and outside of the tube wall are freely accessible. The silk tube could potentially be used as an artificial blood vessel in an in vitro tissue scaffold, where endothelial cells and tissue cells can grow on both sides of the silk tube.

  • 6.
    Güler, Rezan
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Thatikonda, Naresh
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Ghani, Hawraa Ali
    Hedhammar, My
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Löfblom, John
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Artificial VEGFR2-Specific Growth Factors Demonstrate Agonistic Effects in Both Soluble Form and When Immobilized Via Spider SilkManuscript (preprint) (Other academic)
  • 7.
    Hendrikse, Natalie
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Swedish Orphan Biovitrum AB, Stockholm, Sweden.
    Charpentier, Gwenaelle
    KTH, Centres, Science for Life Laboratory, SciLifeLab. ESCOM, 1 Allee Reseau Jean Marie Buckmaster, F-60200 Compiegne, France..
    Nordling, Erik
    Swedish Orphan Biovitrum AB, Stockholm, Sweden..
    Syrén, Per-Olof
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Swedish Orphan Biovitrum AB, Stockholm, Sweden.
    Ancestral diterpene cyclases show increased thermostability and substrate acceptance2018In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 285, no 24, p. 4660-4673Article in journal (Refereed)
    Abstract [en]

    Bacterial diterpene cyclases are receiving increasing attention in biocatalysis and synthetic biology for the sustainable generation of complex multicyclic building blocks. Herein, we explore the potential of ancestral sequence reconstruction (ASR) to generate remodeled cyclases with enhanced stability, activity, and promiscuity. Putative ancestors of spiroviolene synthase, a bacterial class I diterpene cyclase, display an increased yield of soluble protein of up to fourfold upon expression in the model organism Escherichia coli. Two of the resurrected enzymes, with an estimated age of approximately 1.7 million years, display an upward shift in thermostability of 7-13 degrees C. Ancestral spiroviolene synthases catalyze cyclization of the natural C-20-substrate geranylgeranyl diphosphate (GGPP) and also accept C-15 farnesyl diphosphate (FPP), which is not converted by the extant enzyme. In contrast, the consensus sequence generated from the corresponding multiple sequence alignment was found to be inactive toward both substrates. Mutation of a nonconserved position within the aspartate-rich motif of the reconstructed ancestral cyclases was associated with modest effects on activity and relative substrate specificity (i.e., k(cat)/K-M for GGPP over k(cat)/K-M for FPP). Kinetic analyses performed at different temperatures reveal a loss of substrate saturation, when going from the ancestor with highest thermostability to the modern enzyme. The kinetics data also illustrate how an increase in temperature optimum of biocatalysis is reflected in altered entropy and enthalpy of activation. Our findings further highlight the potential and limitations of applying ASR to biosynthetic machineries in secondary metabolism.

  • 8.
    Hober, Sophia
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Lindbo, Sarah
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Nilvebrant, Johan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Bispecific applications of non-immunoglobulin scaffold binders2019In: Methods, ISSN 1046-2023, E-ISSN 1095-9130, Vol. 154, p. 143-152Article in journal (Refereed)
    Abstract [en]

    Non-immunoglobulin scaffolds represent a proven group of small affinity proteins that can be engineered in vitro to similar affinity and potency as monoclonal antibodies. Several novel candidate biotherapeutics that exploit the potential advantages scaffold proteins hold over larger and more complex antibodies have been developed over the past decade. The ease of using small and robust binding proteins as flexible and modular building blocks has led to the development of a wide range of innovative approaches to combine them in various bi- and multispecific formats. This progress is expected to aid the ongoing challenge of identifying niche applications where clear differentiation from antibody-based molecules will be key to success. Given the many engineering options that are available for non-immunoglobulin scaffold proteins, they have potential to not only complement but probably also surpass antibodies in certain applications.

  • 9. Hu, Francis Jingxin
    et al.
    Lundqvist, Magnus
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Uhlén, Mathias
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Rockberg, Johan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    SAMURAI (Solid-phase Assisted Mutagenesis by Uracil Restricted Ablation In vitro) for Antibody Affinity Maturation and Paratope MappingManuscript (preprint) (Other academic)
    Abstract [en]

    Mutagenesis libraries are essential for combinatorial protein engineering. Despite improve- ments in gene synthesis and directed mutagenesis, current methodologies still have limitations regarding the synthesis of intact antibody scFv genes and simultaneous diversification of all six CDRs. Here, we de- scribe the generation of mutagenesis libraries for antibody affinity maturation, using a cell-free solid-phase technique for annealing of single-strand mutagenic oligonucleotides. This procedure consists of PCR-based incorporation of uracil into a wild-type template, bead-based capture, and elution of single-strand DNA, and in vitro uracil excision enzyme based degradation of the template DNA. Our approach enabled rapid (8 hours) mutagenesis and automated cloning of 50 position specific alanine mutants for mapping of a scFv antibody paratope. We further exemplify our method by generating affinity maturation libraries with di- versity introduced in critical, nonessential, or all CDR positions randomly. Assessment with Illumina deep sequencing showed >99% functional diversity in two libraries and the ability to diversify all CDR positions simultaneously. Selections of the libraries with bacterial display and deep sequencing evaluation of the selection output showed that diversity introduced in non-essential positions allowed quicker enrichment of improved binders compared to the other two diversification strategies.

  • 10.
    Hu, Francis Jingxin
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Lundqvist, Magnus
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Uhlén, Mathias
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab. Tech Univ Denmark, Novo Nord Fdn Ctr Biosustainabil, DK-2970 Horsholm, Denmark..
    Rockberg, Johan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    SAMURAI (Solid-phase Assisted Mutagenesis by Uracil Restriction for Accurate Integration) for antibody affinity maturation and paratope mapping2019In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 47, no 6, article id e34Article in journal (Refereed)
    Abstract [en]

    Mutagenesis libraries are essential for combinatorial protein engineering. Despite improvements in gene synthesis and directed mutagenesis, current methodologies still have limitations regarding the synthesis of complete antibody single-chain variable fragment (scFv) genes and simultaneous diversification of all six CDRs. Here, we describe the generation of mutagenesis libraries for antibody affinity maturation using a cell-free solid-phase technique for annealing of single-strand mutagenic oligonucleotides. The procedure consists of PCR-based incorporation of uracil into a wild-type template, bead-based capture, elution of single-strand DNA, and in vitro uracil excision enzyme based degradation of the template DNA. Our approach enabled rapid (8 hours) mutagenesis and automated cloning of 50 position-specific alanine mutants for mapping of a scFv antibody paratope. We further exemplify our method by generating affinity maturation libraries with diversity introduced in critical, nonessential, or all CDR positions randomly. Assessment with Illumina deep sequencing showed less than 1% wild-type in two libraries and the ability to diversify all CDR positions simultaneously. Selections of the libraries with bacterial display and deep sequencing evaluation of the selection output showed that diversity introduced in non-essential positions allowed for a more effective enrichment of improved binders compared to the other two diversification strategies.

  • 11.
    Johansson, Ulrika
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology. Linnæus Center of Biomaterials Chemistry, Linnæus University, Kalmar, Sweden Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
    Widhe, Mona
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Shalaly, Nancy Dekki
    Arregui, Irene Linares
    Nilebäck, Linnea
    Tasiopoulos, Christos Panagiotis
    Åstrand, Carolina
    Berggren, Per-Olof
    Gasser, Christian
    Hedhammar, My
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Assembly of functionalized silk together with cells to obtain proliferative 3D cultures integrated in a network of ECM-like microfibers.2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, no 1, article id 6291Article in journal (Refereed)
    Abstract [en]

    Tissues are built of cells integrated in an extracellular matrix (ECM) which provides a three-dimensional (3D) microfiber network with specific sites for cell anchorage. By genetic engineering, motifs from the ECM can be functionally fused to recombinant silk proteins. Such a silk protein, FN-silk, which harbours a motif from fibronectin, has the ability to self-assemble into networks of microfibers under physiological-like conditions. Herein we describe a method by which mammalian cells are added to the silk solution before assembly, and thereby get uniformly integrated between the formed microfibers. In the resulting 3D scaffold, the cells are highly proliferative and spread out more efficiently than when encapsulated in a hydrogel. Elongated cells containing filamentous actin and defined focal adhesion points confirm proper cell attachment to the FN-silk. The cells remain viable in culture for at least 90 days. The method is also scalable to macro-sized 3D cultures. Silk microfibers formed in a bundle with integrated cells are both strong and extendable, with mechanical properties similar to that of artery walls. The described method enables differentiation of stem cells in 3D as well as facile co-culture of several different cell types. We show that inclusion of endothelial cells leads to the formation of vessel-like structures throughout the tissue constructs. Hence, silk-assembly in presence of cells constitutes a viable option for 3D culture of cells integrated in a ECM-like network, with potential as base for engineering of functional tissue.

  • 12.
    Kanje, Sara
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Venskutonytė, Raminta
    Lund University.
    Scheffel, Julia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Nilvebrant, Johan
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Lindkvist-Petersson, Karin
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT. KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Protein engineering allows for mild affinity-based elution of therapeutic antibodies2018In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 430, no 18, p. 3427-3438Article in journal (Refereed)
    Abstract [en]

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

  • 13.
    Lindbo, Sarah
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Generation and engineering of ABD-derived affinity proteins for clinical applications2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Proteins that specifically recognize and bind to other molecules or structures are important tools in industrial and medical applications. Binding proteins engineered from small stable scaffold proteins have been utilized for several purposes due to their favorable biophysical properties, tolerance to mutagenesis, efficient tissue penetration and ease of production. The 46 amino acid long albumin-binding domain (ABD) derived from the bacterial receptor Protein G is a promising scaffold that has been explored in this thesis. The scaffold was subjected to combinatorial protein engineering for generation of ABD-derived binding proteins with novel specificities. Furthermore, the medical potential of engineered ABD- derived affinity proteins (ADAPTs) was evaluated in a series of pre-clinical studies.

    In the first studies, ADAPTs suitability as tracers for radionuclide molecular imaging was evaluated. Factors influencing biodistribution and tumor targeting properties were assessed in mice models bearing HER2 positive xenografts. All tested ADAPT constructs demonstrated high and specific targeting of HER2-expressing tumor cells as well as fast clearance from circulation. The results also showed that the size and character of the N- terminus affected the biodistribution profile of ADAPTs. Moreover, the targeting properties of ADAPTs proved to be highly influenced by the residualizing properties of the attached radionuclide label. Taken together, the results provided the first evidence that tumor imaging can be performed using ADAPTs and the favorable pharmacokinetic profiles in the studied mice models suggest that the scaffold is a promising candidate for clinical applications.

    In the last study, a platform for generation of stable ABD-derived affinity proteins with novel binding specificities was established using a multi-step approach combining directed evolution and rational protein design. A broad combinatorial protein library with 20 randomized positions in ABD was designed and binders against three distinct targets were selected using phage display. Characterization of the selected binders provided information regarding optimal positions to randomize in a final library. In addition, the isolated binders were subjected to mutagenesis in certain surface exposed positions and mutations that provided increased stability were introduced into the original scaffold. Finally, a more focused combinatorial protein library consisting of 11 randomized positions was designed and constructed. The library was validated by selections against the same set of targets as for the first, broad library. The isolation of highly stable affinity ligands confirms that the library can be used for generation of diverse and stable affinity molecules.

  • 14.
    Lindbo, Sarah
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Garousi, Javad
    Uppsala university.
    Mitran, Bogdan
    Uppsala university.
    Vorobyeva, Anzhelika
    Uppsala university.
    Oroujeni, Maryam
    Uppsala university.
    Orlova, Anna
    Uppsala university.
    Hober, Sophia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH). KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT. KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Tolmachev, Vladimir
    Uppsala university.
    Optimized molecular design of ADAPT-based HER2-imaging probes labelled with 111In and 68Ga2018In: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 15, no 7, p. 2674-2683Article in journal (Refereed)
    Abstract [en]

    Radionuclide molecular imaging is a promising tool for visualization of cancer associated molecular abnormalities in vivo and stratification of patients for specific therapies. ADAPT is a new type of small engineered proteins based on the scaffold of an albumin binding domain of protein G. ADAPTs have been utilized to select and develop high affinity binders to different proteinaceous targets. ADAPT6 binds to human epidermal growth factor 2 (HER2) with low nanomolar affinity and can be used for its in vivo visualization. Molecular design of 111In-labeled anti-HER2 ADAPT has been optimized in several earlier studies. In this study, we made a direct comparison of two of the most promising variants, having either a DEAVDANS or a (HE)3DANS sequence at the N-terminus, conjugated with a maleimido derivative of DOTA to a GSSC amino acids sequence at the C-terminus. The variants (designated DOTA-C59-DEAVDANS-ADAPT6-GSSC and DOTA-C61-(HE)3DANS-ADAPT6-GSSC) were stably labeled with 111In for SPECT and 68Ga for PET. Biodistribution of labeled ADAPT variants was evaluated in nude mice bearing human tumor xenografts with different levels of HER2 expression. Both variants enabled clear discrimination between tumors with high and low levels of HER2 expression. 111In-labeled ADAPT6 derivatives provided higher tumor-to-organ ratios compared to 68Ga-labeled counterparts. The best performing variant was DOTA-C61-(HE)3DANS-ADAPT6-GSSC, which provided tumor-to-blood ratios of 208 ± 36 and 109 ± 17 at 3 h for 111In and 68Ga labels, respectively.

  • 15.
    Lindbo, Sarah
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Gunneriusson, Elin
    Affibody AB.
    Ekblad, Caroline
    Affibody AB.
    Fant, Gunilla
    Affibody AB.
    Hober, Sophia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Design, construction and characterization of an ABD-based library with improved stabilityManuscript (preprint) (Other academic)
    Abstract [en]

    Recombinant affinity proteins binding specifically to other molecules are important tools for many clinical and industrial applications. Small robust protein scaffolds have proven to be well suited as frameworks for generation of novel affinity binders due to their stability. Here we used the albumin-binding domain (ABD) of protein G from Streptococcus G148 as scaffold to design a new combinatorial library capable of generating stable binders to various target proteins with high affinity and specificity. To create a robust framework able to generate highly stable binders, mutations in the non-binding region were evaluated and residues providing increased stability were introduced into the scaffold. By combining rational design with combinatorial protein engineering we also evaluated the surface exposed amino acids at the albumin-binding interface and identified 11 residues suitable for randomization. The potency of the novel scaffold library was assessed by screening for binders using phage display against three distinct targets; complement factor 4, (C4), insulin and interleukin-6 (IL-6). Binders in the nanomolar range with melting temperature above 57°C were selected for all three targets. Notably, the identified IL-6 binders were characterized by extreme thermal stability with variants demonstrating organized structures even at 90°C. This demonstrates that stable binders with distinct specificities can be generated and thus proves the high potential of the library.

  • 16.
    Liu, Hao
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Tumor targeted delivery of cytotoxic payloads using affibody molecules and ABD-derived affinity proteins2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Cancer treatment cost billions of dollars every year, but the mortality rate is still high. An ideal treatment is the so-called “magic bullets” that recognize and kill tumor cells while leaving normal cells untouched. In recent years, some nonimmunoglobulin alternative scaffold affinity proteins, such as affibody molecules and ADAPTs, have emerged and been used to specifically recognize different tumor antigens. In this thesis, I studied the properties and anti-tumor activities of affibody and ADAPT fusion toxins and affibody drug conjugates. In the first two papers, I studied a panel of recombinant affitoxins (affibody toxin fusion proteins) consisting of an anti-HER2 affibody molecule (ZHER2), an albumin binding domain (ABD) and a truncated version of Pseudomonas Exotoxin A(PE38X8). The affitoxins demonstrated specific anti-tumor activity on HER2-overexpressing tumor cells in vitro. A biodistribution experiment showed that addition of an ABD increased the blood retention by 28-fold and a (HE)3 N-terminal purification tag decreased hepatic uptake of the affitoxin compared with a His6 tag. In paper III, I studied immunotoxins consisting of an anti-HER2 ABD-derived affinity protein (ADAPT), an ABD and a minimized and deimmunized version of Pseudomonas exotoxin A (PE25). These immunotoxins demonstrated potent and specific cytotoxicity toward HER2 overexpressing tumor cells in vitro similar to affitoxins. In paper IV, I produced a panel of affibody drug conjugates consisting of ZHER2, ABD and malemidocaproylmertansine (mc-DM1). The conjugates had selective toxic activity on HER2-overexpressing tumor cells in vitro comparable with the approved drug trastuzumab emtansine. The conjugate, ZHER2-ZHER2-ABD-mc-DM1 was found to prolong the life span of tumor bearing mice and delayed the growth ofxenografted SKOV-3 tumors. In conclusion, affibody molecules and ADAPTs are promising alternatives to antibodies for targeted tumor therapy.

  • 17.
    Lundqvist, Magnus
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Methods for cell line and protein engineering2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Therapeutic proteins are becoming increasingly important. They are desirable, as they typically possess low adverse effects and higher specificity compared to the traditional, small molecule drugs. But they are also more complex and involve different intricate and expensive development and production processes. Through new technologies in protein and cell line development, more efficient and safer drugs can be readily available and at a lower cost. This thesis gives an overview of how protein therapeutics are developed and produced. It explores strategies to improve the efficacy and safety of protein drugs and how to improve production yields. In the present investigation, two papers present new methods for high-throughput cloning and site-directed mutagenesis using solid-phase immobilization of DNA fragments. These methods were designed to generate new drug candidates with swiftness and ease. Three papers show the development of a new cell line screening system that combines droplet microfluidics and the split-GFP reporter system. This combination allows for relative quantification of secreted recombinant proteins between individual cells and provides a tool for the selection of the best-producing clones for final production from a heterologous cell pool. The final paper explores the possibility to produce proteins at a higher cell density by examining how the metabolome and proteome of a perfusion bioreactor evolve as the cell density reaches exceptionally high levels. The consistent goal of all of these studies is to expedite the development and improve the production of therapeutic proteins, to assist the discovery of new drugs and to bring down production and development costs. Engineered proteins can be used to cure previously incurable diseases or give current medications a higher efficacy. Lower production and development costs can make the treatments available to more people.

  • 18.
    Lundqvist, Magnus
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Thalén, Niklas
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Volk, Anna-Luisa
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Hansen, Henning Gram
    Tech Univ Denmark, Novo Nordisk Fdn Ctr Biosustainabil, Lyngby, Denmark..
    von Otter, Eric
    Nanyang Technol Univ, Sch Biol Sci, Singapore 637551, Singapore..
    Nygren, Per-Åke
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Uhlén, Mathias
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark.
    Rockberg, Johan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Chromophore pre-maturation for improved speed and sensitivity of split-GFP monitoring of protein secretion2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 310Article in journal (Refereed)
    Abstract [en]

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

  • 19.
    Meister, Sebastian
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Hendrikse, Natalie
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology.
    Löfblom, John
    KTH, School of Engineering Sciences (SCI).
    Directed evolution of the 3C protease from coxsackievirus using a novel fluorescence-assisted intracellular method2019In: Biological chemistry (Print), ISSN 1431-6730, E-ISSN 1437-4315, Vol. 400, no 3, p. 405-415Article in journal (Refereed)
    Abstract [en]

    Proteases are crucial for regulating biological processes in organisms through hydrolysis of peptide bonds. Recombinant proteases have moreover become important tools in biotechnological, and biomedical research and as therapeutics. We have developed a label-free high-throughput method for quantitative assessment of proteolytic activity in Escherichia coli. The screening method is based on co-expression of a protease of interest and a reporter complex. This reporter consists of an aggregation-prone peptide fused to a fluorescent protein via a linker that contains the corresponding substrate sequence. Cleavage of the substrate rescues the fluorescent protein from aggregation, resulting in increased fluorescence that correlates to proteolytic activity, which can be monitored using flow cytometry. In one round of flow-cytometric cell sorting, we isolated an efficiently cleaved tobacco etch virus (TEV) substrate from a 1:100 000 background of non-cleavable sequences, with around 6000-fold enrichment. We then engineered the 3C protease from coxsackievirus B3 (CVB3 3C(pro)) towards improved proteolytic activity on the substrate LEVLFQ down arrow GP. We isolated highly proteolytic active variants from a randomly mutated CVB3 3C(pro) library with up to 4-fold increase in activity. The method enables simultaneous measurement of proteolytic activity and protease expression levels and can therefore be applied for protease substrate profiling, as well as directed evolution of proteases.

  • 20.
    Nilebäck, Linnea
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Expanded knowledge on silk assembly for development of bioactive silk coatings2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Silk is a fascinating natural material made from proteins that self-assemble through structural rearrangements into one of the toughest materials known. As silk is protein-based, durable and elastic, it has many features that makes it suitable as a scaffold material for tissue engineering. Natural silk proteins are complex and thus difficult to produce synthetically. Therefore, partial silk proteins have been designed for production in heterologous host cells such as expression strains of Escherichia coli. This thesis presents investigations of the properties of one such partial spider silk protein, 4RepCT, and its assembly process, and describes the development of bioactive silk coatings and their properties. The focus has been to develop coatings for implant surfaces to prevent infections and improve interactions with cells.

    In Paper I, the intrinsic properties and contribution to the self-assembly process of the two protein parts 4Rep and CT were investigated separately, in a mixture (4Rep+CT) and as a fusion protein (4RepCT). The results showed that assembly occurs both at the liquid-air and liquid-solid interfaces. CT reached the interface fast but did not refold to form β-sheets, characteristic for silk, on its own. 4Rep adsorbed rapidly, and extensive intermolecular interactions were formed, although unorganized. Covalent linkage between 4Rep and CT, as in 4RepCT, and thus close proximity between the two silk parts, was found to be crucial in order to obtain both conversion into β-sheet rich structures and a nanofibrillar topography of the adsorbed proteins.

    The finding that 4RepCT self-assembles into nanofibrillar coatings on solid surfaces could be useful for various applications, for example to improve implant surfaces. The coating process was thus further evaluated in Paper II, showing that the silk coatings were chemically resistant and could also be made from silk protein variants where additional peptide motifs had been fused to 4RepCT at the genetic level. Silk with a cell-binding motif (FN-silk) and an antimicrobial peptide (Mag-silk) could assemble onto titanium, stainless steel and hydroxyapatite, respectively, materials that are commonly used for implants. Fibroblasts and endothelial cells were successfully cultured on FN-silk coatings and proliferated well. Finally, coatings of Mag-silk were evaluated for their ability to prevent adhesion of Staphylococcus aureus.

    In Paper III, silk from silkworms were used to construct materials in three different formats suitable for wound healing applications. Microporous scaffolds, electrospun mats and thin coatings of silkworm silk could all be coated with 4RepCT. They thereby gained the functions added via 4RepCT fusion proteins with a cell-binding motif (FN-silk), an antibody binding domain (Z-silk) or an enzyme (Xyl-silk). This shows upon a versatile method for functionalization of materials in different formats with bioactive motifs and domains.

    In Paper IV, the aim was to develop dual-functional silk coatings to promote osseointegration and prevent bacterial adhesion to orthopedic and dental implants. Coatings of regular silk (4RepCT) and FN-silk were given additional functions by using the transpeptidase Sortase A to mediate conjugation with the biofilm dispersal enzyme Dispersin B, or the endolysins PlySs2 and SAL-1. The obtained coatings showed a reduced adhesion of S. aureus compared to regular silk and FN-silk. Moreover, osteosarcoma cells adhered and proliferated well on coatings of FN-silk also when conjugated with enzymes.

    Altogether, the work presented in this thesis suggests that 4RepCT silk coatings are valuable as a base for construction of bioactive surfaces. The coatings can be applied on many different surfaces, and the bioactive coatings developed herein show potential for wound healing applications and prevention of biomaterial-associated infections.

  • 21.
    Nilebäck, Linnea
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Widhe, Mona
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Seijsing, Johan
    Bysell, Helena
    Sharma, Prashant K.
    Hedhammar, My
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Bioactive silk coatings reduce adhesion of Staphylococcus aureus while supporting growth of osteoblast-like cellsManuscript (preprint) (Other academic)
  • 22.
    Nilvebrant, Johan
    et al.
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Rockberg, Johan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    An introduction to epitope mapping2018In: Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029, Vol. 1785, p. 1-10Article in journal (Refereed)
    Abstract [en]

    Antibodies are protein molecules used routinely for therapeutic, diagnostic, and research purposes due to their exquisite ability to selectively recognize and bind a given antigen. The particular area of the antigen recognized by the antibody is called the epitope, and for proteinaceous antigens the epitope can be of complex nature. Information about the binding epitope of an antibody can provide important mechanistic insights and indicate for what applications an antibody might be useful. Therefore, a variety of epitope mapping techniques have been developed to localize such regions. Although the real picture is even more complex, epitopes in protein antigens are broadly grouped into linear or discontinuous epitopes depending on the positioning of the epitope residues in the antigen sequence and the requirement of structure. Specialized methods for mapping of the two different classes of epitopes, using high-throughput or high-resolution methods, have been developed. While different in their detail, all of the experimental methods rely on assessing the binding of the antibody to the antigen or a set of antigen mimics. Early approaches utilizing sets of truncated proteins, small numbers of synthesized peptides, and structural analyses of antibody-antigen complexes have been significantly refined. Current state-of-the-art methods involve combinations of mutational scanning, protein display, and high-throughput screening in conjunction with bioinformatic analyses of large datasets.

  • 23.
    Petrou, Georgia
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Jansson, Ronnie
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Hogqvist, Mark
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
    Erlandsson, Johan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Wågberg, Lars
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Hedhammar, My
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Crouzier, Thomas
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Genetically Engineered Mucoadhesive Spider Silk2018In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 19, no 8, p. 3268-3279Article in journal (Refereed)
    Abstract [en]

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

  • 24.
    Petrou, Georgia
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Jansson, Ronnie
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology. KTH Royal Inst Technol, Div Prot Technol, Stockholm, Sweden..
    Högqvist, Mark
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Hedhammar, My
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Crouzier, Thomas
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience. KTH Royal Inst Technol, Div Glycosci, Stockholm, Sweden..
    Engineering mucoadhesive silk2018In: Abstracts of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 255Article in journal (Other academic)
  • 25.
    Schwarz, Hubert
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Zhang, Ye
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Zhan, Caijuan
    KTH, School of Engineering Sciences (SCI). KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Malm, Magdalena
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Field, Raymond
    Biopharmaceutical Development, MedImmune, Cambridge, UK.
    Turner, Richard
    Biopharmaceutical Development, MedImmune, Cambridge, UK.
    Sellick, Christopher
    Biopharmaceutical Development, MedImmune, Cambridge, UK.
    Varley, Paul
    Biopharmaceutical Development, MedImmune, Cambridge, UK.
    Rockberg, Johan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Chotteau, Véronique
    KTH, School of Engineering Sciences (SCI).
    Small-scale bioreactor supports high density HEK293 cell perfusion culture for the production of recombinant ErythropoietinManuscript (preprint) (Other academic)
    Abstract [en]

    Process intensification in mammalian cell culture-based recombinant protein production has been achieved by high cell density perfusion exceeding 108 cells/mL in the recent years. As the majority of therapeutic proteins are produced in Chinese Hamster Ovary (CHO) cells, intensified perfusion processes have been mainly developed for this type of host cell line. However, the use of CHO cells can result in non-human posttranslational modifications of the protein of interest, which may be disadvantageous compared with human cell lines.

    In this study, we developed a high cell density perfusion process of Human Embryonic Kidney (HEK293) cells producing recombinant human Erythropoietin (rhEPO). Firstly, a small-scale perfusion system from commercial bench-top screening bioreactors was developed for <250 mL working volume. Then, after the first trial runs with CHO cells, the system was modified for HEK293 cells (more sensitive than CHO cells) to achieve a higher oxygen transfer under mild aeration and agitation conditions. Steady states for medium (20 x 106 cells/mL) and high cell densities (80 x 106 cells/mL), normal process temperature (37 °C) and mild hypothermia (33 °C) as well as different cell specific perfusion rates (CSPR) from 10 to 60 pL/cell/day were applied to study the performance of the culture. The volumetric productivity was maximized for the high cell density steady state but decreased when an extremely low CSPR of 10 pL/cell/day was applied. The shift from high to low CSPR strongly reduced the nutrient uptake rates. The results from our study show that human cell lines, such as HEK293 can be used for intensified perfusion processes. 

  • 26.
    Subramanian, Karthik
    et al.
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden..
    Neill, Daniel R.
    Univ Liverpool, Inst Infect & Global Hlth, Liverpool, Merseyside, England..
    Malak, Hesham A.
    Univ Liverpool, Inst Infect & Global Hlth, Liverpool, Merseyside, England..
    Spelmink, Laura
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden..
    Khandaker, Shadia
    Univ Liverpool, Inst Infect & Global Hlth, Liverpool, Merseyside, England..
    Marchiori, Giorgia Dalla Libera
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden..
    Dearing, Emma
    Univ Liverpool, Inst Infect & Global Hlth, Liverpool, Merseyside, England..
    Kirby, Alun
    Univ York, Ctr Immunol & Infect, Dept Biol, York, N Yorkshire, England.;Univ York, Hull York Med Sch, York, N Yorkshire, England..
    Yang, Marie
    Univ Liverpool, Inst Infect & Global Hlth, Liverpool, Merseyside, England..
    Achour, Adnane
    Karolinska Inst, Dept Med Solna, Sci Life Lab, Stockholm, Sweden.;Karolinska Univ Hosp Solna, Dept Infect Dis, Stockholm, Sweden..
    Nilvebrant, Johan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Nygren, Per-Åke
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Plant, Laura
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden..
    Kadioglu, Aras
    Univ Liverpool, Inst Infect & Global Hlth, Liverpool, Merseyside, England..
    Henriques-Normark, Birgitta
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden.;Karolinska Univ Hosp Solna, Clin Microbiol, Stockholm, Sweden.;Nanyang Technol Univ, Lee Kong Chian Sch Med LKC, Singapore, Singapore.;Nanyang Technol Univ, SCELSE, Singapore, Singapore..
    Pneumolysin binds to the mannose receptor C type 1 (MRC-1) leading to anti-inflammatory responses and enhanced pneumococcal survival2019In: Nature Microbiology, E-ISSN 2058-5276, Vol. 4, no 1, p. 62-70Article in journal (Refereed)
    Abstract [en]

    Streptococcus pneumoniae (the pneumococcus) is a major cause of mortality and morbidity globally, and the leading cause of death in children under 5 years old. The pneumococcal cytolysin pneumolysin (PLY) is a major virulence determinant known to induce pore-dependent pro-inflammatory responses. These inflammatory responses are driven by PLY-host cell membrane cholesterol interactions, but binding to a host cell receptor has not been previously demonstrated. Here, we discovered a receptor for PLY, whereby pro-inflammatory cytokine responses and Toll-like receptor signalling are inhibited following PLY binding to the mannose receptor C type 1 (MRC-1) in human dendritic cells and mouse alveolar macrophages. The cytokine suppressor SOCS1 is also upregulated. Moreover, PLY-MRC-1 interactions mediate pneumococcal internalization into non-lysosomal compartments and polarize naive T cells into an interferon-gamma(low), interleukin-4(high) and FoxP3(+) immunoregulatory phenotype. In mice, PLY-expressing pneumococci colocalize with MRC-1 in alveolar macrophages, induce lower pro-inflammatory cytokine responses and reduce neutrophil infiltration compared with a PLY mutant. In vivo, reduced bacterial loads occur in the airways of MRC-1-deficient mice and in mice in which MRC-1 is inhibited using blocking antibodies. In conclusion, we show that pneumococci use PLY-MRC-1 interactions to downregulate inflammation and enhance bacterial survival in the airways. These findings have important implications for future vaccine design.

  • 27. Summer, D.
    et al.
    Garousi, J.
    Oroujeni, M.
    Mitran, B.
    Andersson, Ken G.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Vorobyeva, A.
    Löfblom, John
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Orlova, A.
    Tolmachev, V.
    Decristoforo, C.
    Cyclic versus Noncyclic Chelating Scaffold for 89Zr-Labeled ZEGFR:2377 Affibody Bioconjugates Targeting Epidermal Growth Factor Receptor Overexpression2018In: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 15, no 1, p. 175-185Article in journal (Refereed)
    Abstract [en]

    Zirconium-89 is an emerging radionuclide for positron emission tomography (PET) especially for biomolecules with slow pharmacokinetics as due to its longer half-life, in comparison to fluorine-18 and gallium-68, imaging at late time points is feasible. Desferrioxamine B (DFO), a linear bifunctional chelator (BFC) is mostly used for this radionuclide so far but shows limitations regarding stability. Our group recently reported on fusarinine C (FSC) with similar zirconium-89 complexing properties but potentially higher stability related to its cyclic structure. This study was designed to compare FSC and DFO head-to-head as bifunctional chelators for 89Zr-radiolabeled EGFR-targeting ZEGFR:2377 affibody bioconjugates. FSC-ZEGFR:2377 and DFO-ZEGFR:2377 were evaluated regarding radiolabeling, in vitro stability, specificity, cell uptake, receptor affinity, biodistribution, and microPET-CT imaging. Both conjugates were efficiently labeled with zirconium-89 at room temperature but radiochemical yields increased substantially at elevated temperature, 85 °C. Both 89Zr-FSC-ZEGFR:2377 and 89Zr-DFO-ZEGFR:2377 revealed remarkable specificity, affinity and slow cell-line dependent internalization. Radiolabeling at 85 °C showed comparable results in A431 tumor xenografted mice with minor differences regarding blood clearance, tumor and liver uptake. In comparison 89Zr-DFO-ZEGFR:2377, radiolabeled at room temperature, showed a significant difference regarding tumor-to-organ ratios. MicroPET-CT imaging studies of 89Zr-FSC-ZEGFR:2377 as well as 89Zr-DFO-ZEGFR:2377 confirmed these findings. In summary we were able to show that FSC is a suitable alternative to DFO for radiolabeling of biomolecules with zirconium-89. Furthermore, our findings indicate that 89Zr-radiolabeling of DFO conjugates at higher temperature reduces off-chelate binding leading to significantly improved tumor-to-organ ratios and therefore enhancing image contrast.

  • 28.
    Syrén, Per-Olof
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH Royal Inst Technol, Sch Engn Sci Chem Biotechnol & Hlth, Sci Life Lab, Dept Fibre & Polymer Technol, S-17165 Solna, Sweden.;KTH Royal Inst Technol, Dept Prot Sci, S-17165 Solna, Sweden..
    Enzymatic Hydrolysis of Tertiary Amide Bonds by anti Nucleophilic Attack and Protonation2018In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 83, no 21, p. 13543-13548Article in journal (Refereed)
    Abstract [en]

    The molecular mechanisms conferring high resistance of planar tertiary amide bonds to hydrolysis by most enzymes have remained elusive. To provide a chemical explanation to this unresolved puzzle, UB3LYP calculations were performed on an active site model of Xaa-Pro peptidases. The calculated reaction mechanism demonstrates that biocatalysts capable of tertiary amide bond hydrolysis capitalize on anti nucleophilic attack and protonation of the amide nitrogen, in contrast to the traditional syn displayed by amidases and proteases acting on secondary amide bonds.

  • 29. Uhlen, M.
    et al.
    Bjorling, E.
    Agaton, C.
    Szigyarto, C. A.
    Amini, B.
    Andersen, E.
    Andersson, A. C.
    Angelidou, P.
    Asplund, A.
    Asplund, C.
    Berglund, L.
    Bergstrom, K.
    Brumer, H.
    Cerjan, D.
    Ekstrom, M.
    Elobeid, A.
    Eriksson, C.
    Fagerberg, L.
    Falk, R.
    Fall, J.
    Forsberg, M.
    Bjorklund, M. G.
    Gumbel, K.
    Halimi, A.
    Hallin, I.
    Hamsten, C.
    Hansson, M.
    Hedhammar, M.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Hercules, G.
    Kampf, C.
    Larsson, K.
    Lindskog, M.
    Lodewyckx, W.
    Lund, J.
    Lundeberg, J.
    Magnusson, K.
    Malm, E.
    Nilsson, P.
    Odling, J.
    Oksvold, P.
    Olsson, I.
    Oster, E.
    Ottosson, J.
    Paavilainen, L.
    Persson, A.
    Rimini, R.
    Rockberg, J.
    Runeson, M.
    Sivertsson, A.
    Skollermo, A.
    Steen, J.
    Stenvall, M.
    Sterky, F.
    Stromberg, S.
    Sundberg, M.
    Tegel, H.
    Tourle, S.
    Wahlund, E.
    Walden, A.
    Wan, J.
    Wernerus, H.
    Westberg, J.
    Wester, K.
    Wrethagen, U.
    Xu, L. L.
    Hober, S.
    Ponten, F.
    A human protein atlas for normal and cancer tissues based on antibody proteomics2005In: Mol Cell Proteomics, Vol. 4, no 12, p. 1920-32Article in journal (Refereed)
    Abstract [en]

    Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, approximately 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.

  • 30.
    Volk, Anna-Luisa
    et al.
    KTH, School of Biotechnology (BIO).
    Rockberg, Johan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Discontinuous epitope mapping of antibodies using bacterial cell surface display of folded domains2018In: Epitope Mapping Protocols, Humana Press, 2018, p. 159-183Chapter in book (Refereed)
    Abstract [en]

    Knowledge of the exquisite-binding surface of an antibody on its target protein is of great value, in particular for therapeutic antibodies for understanding method of action and for stratification of patients carrying the necessary epitope for desired drug efficacy, but also for capture assays under native conditions. Several epitope mapping methodologies have been described for this purpose, with the laborious X-ray crystallography method being the ideal method for mapping of discontinuous epitopes in antibody-antigen crystal complexes and high-throughput peptide-based methods for mapping of linear epitopes. We here report on the usage of a bacterial surface display-based method for mapping of structural epitopes by display of folded domains on the surface of Gram positive bacteria, followed by domain-targeted mutagenesis and library analysis for the identification of key-residues by flow sorting and sequencing. Identified clones with reduced affinity are validated by single clone FACS and subsequent full-length expression in mammalian cells for validation.

  • 31.
    Wu, Yu-Tang
    et al.
    Univ Paris 11, Univ Paris Saclay, Inst Integrat Biol Cell, NanoBioPhoton Nanofret Com,CNRS,CEA, Orsay, France..
    Qiu, Xue
    Univ Paris 11, Univ Paris Saclay, Inst Integrat Biol Cell, NanoBioPhoton Nanofret Com,CNRS,CEA, Orsay, France..
    Lindbo, Sarah
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Susumu, Kimihiro
    US Naval Res Lab, Opt Sci Div, Code 5600, Washington, DC USA.;KeyW Corp, Hanover, MD 21076 USA..
    Medintz, Igor L.
    US Naval Res Lab, Ctr Bio Mol Sci & Engn, Code 6900, Washington, DC USA..
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT. KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Hildebrandt, Niko
    Univ Paris 11, Univ Paris Saclay, Inst Integrat Biol Cell, NanoBioPhoton Nanofret Com,CNRS,CEA, Orsay, France..
    Quantum Dot-Based FRET Immunoassay for HER2 Using Ultrasmall Affinity Proteins2018In: Small, ISSN 1613-6810, E-ISSN 1613-6829, Vol. 14, no 35, article id 1802266Article in journal (Refereed)
    Abstract [en]

    Engineered scaffold affinity proteins are used in many biological applications with the aim of replacing natural antibodies. Although their very small sizes are beneficial for multivalent nanoparticle conjugation and efficient Forster resonance energy transfer (FRET), the application of engineered affinity proteins in such nanobiosensing formats has been largely neglected. Here, it is shown that very small (approximate to 6.5 kDa) histidine-tagged albumin-binding domain-derived affinity proteins (ADAPTs) can efficiently self-assemble to zwitterionic ligand-coated quantum dots (QDs). These ADAPT-QD conjugates are significantly smaller than QD-conjugates based on IgG, Fab', or single-domain antibodies. Immediate applicability by the quantification of the human epidermal growth factor receptor 2 (HER2) in serum-containing samples using time-gated Tb-to-QD FRET detection on the clinical benchtop immunoassay analyzer KRYPTOR is demonstrated here. Limits of detection down to 40 x 10(-12)m (approximate to 8 ng mL(-1)) are in a relevant clinical concentration range and outperform previously tested assays with antibodies, antibody fragments, and nanobodies.

  • 32.
    Zhan, Caijuan
    et al.
    KTH, School of Engineering Sciences (SCI). KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Hubert, Schwarz
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Malm, Magdalena
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Sellick, Christopher
    Rockberg, Johan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Chotteau, Véronique
    KTH, School of Engineering Sciences (SCI). KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Hydrodynamic shear stress in hollow filter for perfusion culture of human cellsManuscript (preprint) (Other academic)
    Abstract [en]

    High cell density perfusion process is an economical way to produce biopharmaceuticals at high yield. To achieve high density of healthy cells, the cell culture conditions should be free from mechanically detriment. Human embryonic kidney (HEK) K293 cells, interesting for the production of therapeutic glycoproteins, are known as shear sensitive. In order to obtain the optimal hydrodynamics conditions with reduced mechanical damage, we investigated the fact of the shear stress compatible with HEK293 cells. We reviewed hollow filter based tangential flow filtration strategies, tangential flow filtration (TFF) and alternating tangential flow filtration (ATF). We studied shear stress introduced by these two flow filtration methods. By theoretical study, we obtained that lower shear stress introduced by alternating tangential flow filtration result in lower average shear stress comparing to tangential flow filtration with same flow rate.  In our experimental runs, we achieved different shear stress levels by applying different flow rates. 5-Days batch cultivations were performed to examine the influence of shear stress on cell growing and metabolic behaviour. We identified that the shear stress potentially reduce the growth rate and productivity of HEK293 cells and found the cell metabolism associated with shear stress levels. By documenting these cell responses to shear stress, we confirmed our theoretical results and could further optimize the hydrodynamic conditions for perfusion process of HEK 293 cells. 

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