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  • 1. Altai, M.
    et al.
    Liu, Hao
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Ding, Haozhong
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Mitran, B.
    Edqvist, P. -H
    Tolmachev, V.
    Orlova, A.
    Gräslund, Torbjörn
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Affibody-derived drug conjugates: Potent cytotoxic molecules for treatment of HER2 over-expressing tumors2018In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 288, p. 84-95Article in journal (Refereed)
    Abstract [en]

    Patients with HER2-positive tumors often suffer resistance to therapy, warranting development of novel treatment modalities. Affibody molecules are small affinity proteins which can be engineered to bind to desired targets. They have in recent years been found to allow precise targeting of cancer specific molecular signatures such as the HER2 receptor. In this study, we have investigated the potential of an affibody molecule targeting HER2, ZHER2:2891, conjugated with the cytotoxic maytansine derivate MC-DM1, for targeted cancer therapy. ZHER2:2891 was expressed as a monomer (ZHER2:2891), dimer ((ZHER2:2891)2) and dimer with an albumin binding domain (ABD) for half-life extension ((ZHER2:2891)2-ABD). All proteins had a unique C-terminal cysteine that could be used for efficient and site-specific conjugation with MC-DM1. The resulting affibody drug conjugates were potent cytotoxic molecules for human cells over-expressing HER2, with sub-nanomolar IC50-values similar to trastuzumab emtansine, and did not affect cells with low HER2 expression. A biodistribution study of a radiolabeled version of (ZHER2:2891)2-ABD-MC-DM1, showed that it was taken up by the tumor. The major site of off-target uptake was the kidneys and to some extent the liver. (ZHER2:2891)2-ABD-MC-DM1 was found to have a half-life in circulation of 14 h. The compound was tolerated well by mice at 8.5 mg/kg and was shown to extend survival of mice bearing HER2 over-expressing tumors. The findings in this study show that affibody molecules are a promising class of engineered affinity proteins to specifically deliver small molecular drugs to cancer cells and that such conjugates are potential candidates for clinical evaluation on HER2-overexpressing cancers. 

  • 2. Altai, Mohamed
    et al.
    Liu, Hao
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Ding, Haozhong
    Mitran, Bogdan
    Edqvist, Per-Henrik
    Tolmachev, Vladimir
    Orlova, Anna
    Gräslund, Torbjorn
    Affibody-derived Drug Conjugates: Potent Cytotoxic Drugs ForTreatment Of HER2 Over-Expressing TumorsManuscript (preprint) (Other academic)
  • 3.
    Annelies, Nonneman
    et al.
    KU Leuven Univ Leuven, Dept Neurosci, Lab Neurobiol & Expt Neurol, Herestr 49, B-3000 Leuven, Belgium.;LBI, Herestr 49, B-3000 Leuven, Belgium.;Ctr Brain & Dis Res, VIB, Herestr 49, B-3000 Leuven, Belgium..
    Nathan, Criem
    Ctr Brain & Dis Res, VIB, Herestr 49, B-3000 Leuven, Belgium.;KU Leuven Univ Leuven, Dept Cardiovasc Sci, Ctr Mol & Vasc Biol, Herestr 49, B-3000 Leuven, Belgium.;KU Leuven Univ Leuven, Dept Human Genet, Herestr 49, B-3000 Leuven, Belgium..
    Lewandowski, Sebastian
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Inst, Dept Clin Neurosci, S-17177 Stockholm, Sweden..
    Rik, Nuyts
    KU Leuven Univ Leuven, Dept Neurosci, Lab Neurobiol & Expt Neurol, Herestr 49, B-3000 Leuven, Belgium.;LBI, Herestr 49, B-3000 Leuven, Belgium.;Ctr Brain & Dis Res, VIB, Herestr 49, B-3000 Leuven, Belgium..
    Dietmar, Thal R.
    KU Leuven Univ Leuven, Dept Neurosci, Lab Neuropathol, Herestr 49, B-3000 Leuven, Belgium.;Univ Hosp Leuven, Dept Neurol, Herestr 49, B-3000 Leuven, Belgium..
    Frank, Pfrieger W.
    Univ Strasbourg, CNRS UPR 3212, Inst Cellular & Integrat Neurosci, F-67084 Strasbourg, France..
    John, Ravits
    Univ Calif San Diego, Dept Neurosci, 9500 Gilman Dr, San Diego, CA 92093 USA..
    Philip, Van Damme
    KU Leuven Univ Leuven, Dept Neurosci, Lab Neurobiol & Expt Neurol, Herestr 49, B-3000 Leuven, Belgium.;LBI, Herestr 49, B-3000 Leuven, Belgium.;Ctr Brain & Dis Res, VIB, Herestr 49, B-3000 Leuven, Belgium.;Univ Hosp Leuven, Dept Neurol, Herestr 49, B-3000 Leuven, Belgium..
    An, Zwijsen
    Ctr Brain & Dis Res, VIB, Herestr 49, B-3000 Leuven, Belgium.;KU Leuven Univ Leuven, Dept Cardiovasc Sci, Ctr Mol & Vasc Biol, Herestr 49, B-3000 Leuven, Belgium.;KU Leuven Univ Leuven, Dept Human Genet, Herestr 49, B-3000 Leuven, Belgium..
    Ludo, Van Den Bosch
    KU Leuven Univ Leuven, Dept Neurosci, Lab Neurobiol & Expt Neurol, Herestr 49, B-3000 Leuven, Belgium.;LBI, Herestr 49, B-3000 Leuven, Belgium.;Ctr Brain & Dis Res, VIB, Herestr 49, B-3000 Leuven, Belgium..
    Wim, Robberecht
    KU Leuven Univ Leuven, Dept Neurosci, Lab Neurobiol & Expt Neurol, Herestr 49, B-3000 Leuven, Belgium.;LBI, Herestr 49, B-3000 Leuven, Belgium.;Ctr Brain & Dis Res, VIB, Herestr 49, B-3000 Leuven, Belgium.;Univ Hosp Leuven, Dept Neurol, Herestr 49, B-3000 Leuven, Belgium..
    Astrocyte-derived Jagged-1 mitigates deleterious Notch signaling in amyotrophic lateral sclerosis2018In: Neurobiology of Disease, ISSN 0969-9961, E-ISSN 1095-953X, Vol. 119, p. 26-40Article in journal (Refereed)
    Abstract [en]

    Amyotrophic lateral sclerosis (ALS) is a late-onset devastating degenerative disease mainly affecting motor neurons. Motor neuron degeneration is accompanied and aggravated by oligodendroglial pathology and the presence of reactive astrocytes and microglia. We studied the role of the Notch signaling pathway in ALS, as it is implicated in several processes that may contribute to this disease, including axonal retraction, microgliosis, astrocytosis, oligodendrocyte precursor cell proliferation and differentiation, and cell death. We observed abnormal activation of the Notch signaling pathway in the spinal cord of SOD1(G93A) mice, a well-established model for ALS, as well as in the spinal cord of patients with sporadic ALS (sALS). This increased activation was particularly evident in reactive GFAP-positive astrocytes. In addition, one of the main Notch ligands, Jagged-1, was ectopically expressed in reactive astrocytes in spinal cord from ALS mice and patients, but absent in resting astrocytes. Astrocyte-specific inactivation of Jagged-1 in presymptomatic SOD1(G93A) mice further exacerbated the activation of the Notch signaling pathway and aggravated the course of the disease in these animals without affecting disease onset. These data suggest that aberrant Notch signaling activation contributes to the pathogenesis of ALS, both in sALS patients and SOD1(G93A) mice, and that it is mitigated in part by the upregulation of astrocytic Jagged-1.

  • 4.
    Antypas, H.
    et al.
    Karolinska Inst, Dept Neurosci, Swedish Med Nanosci Ctr, Stockholm, Sweden..
    Veses-Garcia, M.
    Karolinska Inst, Dept Neurosci, Swedish Med Nanosci Ctr, Stockholm, Sweden..
    Weibull, Emelie
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Svahn Andersson, Helene
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Richter-Dahlfors, A.
    Karolinska Inst, Dept Neurosci, Swedish Med Nanosci Ctr, Stockholm, Sweden..
    A universal platform for selection and high-resolution phenotypic screening of bacterial mutants using the nanowell slide2018In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 18, no 12, p. 1767-1777Article in journal (Refereed)
    Abstract [en]

    The Petri dish and microtiter plate are the golden standard for selection and screening of bacteria in microbiological research. To improve on the limited resolution and throughput of these methods, we developed a universal, user-friendly platform for selection and high-resolution phenotypic screening based on the nanowell slide. This miniaturized platform has an optimal ratio between throughput and assay complexity, holding 672 nanowells of 500 nl each. As monoclonality is essential in bacterial genetics, we used FACS to inoculate each nanowell with a single bacterium in 15 min. We further extended the protocol to select and sort only bacteria of interest from a mixed culture. We demonstrated this by isolating single transposon mutants generated by a custom-made transposon with dual selection for GFP fluorescence and kanamycin resistance. Optical compatibility of the nanowell slide enabled phenotypic screening of sorted mutants by spectrophotometric recording during incubation. By processing the absorbance data with our custom algorithm, a phenotypic screen for growth-associated mutations was performed. Alternatively, by processing fluorescence data, we detected metabolism-associated mutations, exemplified by a screen for -galactosidase activity. Besides spectrophotometry, optical compatibility enabled us to perform microscopic analysis directly in the nanowells to screen for mutants with altered morphologies. Despite the miniaturized format, easy transition from nano- to macroscale cultures allowed retrieval of bacterial mutants for downstream genetic analysis, demonstrated here by a cloning-free single-primer PCR protocol. Taken together, our FACS-linked nanowell slide replaces manual selection of mutants on agar plates, and enables combined selection and phenotypic screening in a one-step process. The versatility of the nanowell slide, and the modular workflow built on mainstream technologies, makes our universal platform widely applicable in microbiological research.

  • 5.
    Ayoglu, Burcu
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nilsson, Peter
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Multiplexed antigen bead arrays for the assessment of antibody selectivity and epitope mapping2018In: Epitope Mapping Protocols, Humana Press Inc. , 2018, p. 239-248Chapter in book (Refereed)
    Abstract [en]

    With the increasing number of binding reagents for affinity-based investigations of the human proteome, high-throughput tools for the characterization of the used reagents become essential. For the analysis of binding selectivity, bead-based antigen arrays offer a miniaturized and parallelized assay platform to meet such needs, as they enable two-dimensional multiplexing to analyze up to 384 samples against up to 500 analytes in a single round of analysis. In this chapter, we describe our protocols for the generation of multiplex bead arrays built on immobilized protein fragments, as well as biotinylated peptides. Combined together, these two versions of antigen arrays offer a versatile approach for multiplexed characterization of antibody binding selectivity, off-target interactions, as well as mapping for the amino acids of epitopes involved in antibody binding.

  • 6.
    Banerjee, Indradumna
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Russom, Aman
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Lab-on-DVD: Optical Disk Drive-Based Platforms for Point-of-Care Diagnostics2018In: Frugal Innovation in Bioengineering for the Detection of Infectious Diseases / [ed] AK Chavali, R Ramji, Switzerland: Springer, 2018, 2, p. 23-38Chapter in book (Refereed)
    Abstract [en]

    There is a growing demand for simple, affordable, reliable and quality-assured point-of-care (POC) diagnostics for use in resource-limited settings. Among the top ten leading causes of death worldwide, three are infectious diseases, namely, respiratory infections, HIV/AIDS and diarrheal diseases (World Health Organization 2012). Although high-quality diagnostic tests are available, these are often not available to patients in developing countries. While recent development in microfluidics and “lab-on-a-chip” devices has the potential to spur the development of protocols and affordable instruments for diagnosis of infectious disease at POC, integration of complex sample preparation and detection into automated molecular and cellular systems remain a bottleneck for implementation of these systems at resource-limited settings. Towards this, we describe here how low-cost optical drives can, with minor modifications, be turned into POC diagnostic platforms. A DVD drive is essentially a highly advanced and low-cost optical laser-scanning microscope, with the capability to deliver high-resolution images for biological applications. Furthermore, the inherent centrifugal force on rotational discs is elegantly used for sample preparation and integration. Hence, the merging of low-cost optical disc drives with centrifugal microfluidics is feasible concept for POC diagnostics, specifically designed to meet the needs at resource-limited settings.

  • 7. Berger, Ashton C
    et al.
    Korkut, Anil
    Kanchi, Rupa S
    Hegde, Apurva M
    Lenoir, Walter
    Liu, Wenbin
    Liu, Yuexin
    Fan, Huihui
    Shen, Hui
    Ravikumar, Visweswaran
    Rao, Arvind
    Schultz, Andre
    Li, Xubin
    Sumazin, Pavel
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Cellular and Clinical Proteomics.
    Mestdagh, Pieter
    Gunaratne, Preethi H
    Yau, Christina
    Bowlby, Reanne
    Robertson, A Gordon
    Tiezzi, Daniel G
    Wang, Chen
    Cherniack, Andrew D
    Godwin, Andrew K
    Kuderer, Nicole M
    Rader, Janet S
    Zuna, Rosemary E
    Sood, Anil K
    Lazar, Alexander J
    Ojesina, Akinyemi I
    Adebamowo, Clement
    Adebamowo, Sally N
    Baggerly, Keith A
    Chen, Ting-Wen
    Chiu, Hua-Sheng
    Lefever, Steve
    Liu, Liang
    MacKenzie, Karen
    Orsulic, Sandra
    Roszik, Jason
    Shelley, Carl Simon
    Song, Qianqian
    Vellano, Christopher P
    Wentzensen, Nicolas
    Weinstein, John N
    Mills, Gordon B
    Levine, Douglas A
    Akbani, Rehan
    A Comprehensive Pan-Cancer Molecular Study of Gynecologic and Breast Cancers.2018In: Cancer Cell, ISSN 1535-6108, E-ISSN 1878-3686, Vol. 33, no 4, p. 690-705.e9, article id S1535-6108(18)30119-3Article in journal (Refereed)
    Abstract [en]

    We analyzed molecular data on 2,579 tumors from The Cancer Genome Atlas (TCGA) of four gynecological types plus breast. Our aims were to identify shared and unique molecular features, clinically significant subtypes, and potential therapeutic targets. We found 61 somatic copy-number alterations (SCNAs) and 46 significantly mutated genes (SMGs). Eleven SCNAs and 11 SMGs had not been identified in previous TCGA studies of the individual tumor types. We found functionally significant estrogen receptor-regulated long non-coding RNAs (lncRNAs) and gene/lncRNA interaction networks. Pathway analysis identified subtypes with high leukocyte infiltration, raising potential implications for immunotherapy. Using 16 key molecular features, we identified five prognostic subtypes and developed a decision tree that classified patients into the subtypes based on just six features that are assessable in clinical laboratories.

  • 8.
    Bremer, Hanna D.
    et al.
    Swedish Univ Agr Sci, Dept Clin Sci, SE-75007 Uppsala, Sweden..
    Landegren, Nils
    Karolinska Inst, Karolinska Univ Hosp, Dept Med Solna, CMM, L8 01, SE-17176 Stockholm, Sweden.;Uppsala Univ, Dept Med Sci, Sci Life Lab, Uppsala, Sweden..
    Sjoberg, Ronald
    KTH Royal Inst Technol, Sch Biotechnol, Affin Prote, SciLifeLab, SE-17121 Solna, Sweden..
    Hallgren, Asa
    Karolinska Inst, Karolinska Univ Hosp, Dept Med Solna, CMM, L8 01, SE-17176 Stockholm, Sweden..
    Renneker, Stefanie
    Euroimmun AG, D-23560 Lubeck, Germany..
    Lattwein, Erik
    Euroimmun AG, D-23560 Lubeck, Germany..
    Leonard, Dag
    Uppsala Univ, Rheumatol & Sci Life Lab, Dept Med Sci, SE-75185 Uppsala, Sweden..
    Eloranta, Maija-Leena
    Uppsala Univ, Rheumatol & Sci Life Lab, Dept Med Sci, SE-75185 Uppsala, Sweden..
    Ronnblom, Lars
    Uppsala Univ, Rheumatol & Sci Life Lab, Dept Med Sci, SE-75185 Uppsala, Sweden..
    Nordmark, Gunnel
    Uppsala Univ, Rheumatol & Sci Life Lab, Dept Med Sci, SE-75185 Uppsala, Sweden..
    Nilsson, Peter
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics.
    Andersson, Goran
    Swedish Univ Agr Sci, Dept Anim Breeding & Genet, SE-75007 Uppsala, Sweden..
    Lilliehook, Inger
    Swedish Univ Agr Sci, Dept Clin Sci, SE-75007 Uppsala, Sweden..
    Lindblad-Toh, Kerstin
    Broad Inst Harvard & MIT, Cambridge, MA 02142 USA.;Uppsala Univ, Sci Life Lab, IMBIM, SE-75123 Uppsala, Sweden..
    Kampe, Olle
    Karolinska Inst, Karolinska Univ Hosp, Dept Med Solna, CMM, L8 01, SE-17176 Stockholm, Sweden.;Uppsala Univ, Dept Med Sci, Sci Life Lab, Uppsala, Sweden.;Univ Bergen, Dept Clin Sci, N-5021 Bergen, Norway.;Univ Bergen, KG Jebsen Ctr Autoimmune Disorders, N-5021 Bergen, Norway.;Haukeland Hosp, Dept Med, N-5021 Bergen, Norway..
    Hansson-Hamlin, Helene
    Swedish Univ Agr Sci, Dept Clin Sci, SE-75007 Uppsala, Sweden..
    ILF2 and ILF3 are autoantigens in canine systemic autoimmune disease2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 4852Article in journal (Refereed)
    Abstract [en]

    Dogs can spontaneously develop complex systemic autoimmune disorders, with similarities to human autoimmune disease. Autoantibodies directed at self-antigens are a key feature of these autoimmune diseases. Here we report the identification of interleukin enhancer-binding factors 2 and 3 (ILF2 and ILF3) as autoantigens in canine immune-mediated rheumatic disease. The ILF2 autoantibodies were discovered in a small, selected canine cohort through the use of human protein arrays; a method not previously described in dogs. Subsequently, ILF3 autoantibodies were also identified in the same cohort. The results were validated with an independent method in a larger cohort of dogs. ILF2 and ILF3 autoantibodies were found exclusively, and at a high frequency, in dogs that showed a speckled pattern of antinuclear antibodies on immunofluorescence. ILF2 and ILF3 autoantibodies were also found at low frequency in human patients with SLE and Sjogren's syndrome. These autoantibodies have the potential to be used as diagnostic biomarkers for canine, and possibly also human, autoimmune disease.

  • 9. Chen, Ziqing
    et al.
    Dodig-Crnkovic, Tea
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Tao, Sheng-ce
    Current applications of antibody microarrays2018In: Clinical Proteomics, ISSN 1542-6416, E-ISSN 1559-0275, Vol. 15, article id 7Article, review/survey (Refereed)
    Abstract [en]

    The concept of antibody microarrays is one of the most versatile approaches within multiplexed immunoassay technologies. These types of arrays have increasingly become an attractive tool for the exploratory detection and study of protein abundance, function, pathways, and potential drug targets. Due to the properties of the antibody microarrays and their potential use in basic research and clinical analytics, various types of antibody microarrays have already been developed. In spite of the growing number of studies utilizing this technique, few reviews about antibody microarray technology have been presented to reflect the quality and future uses of the generated data. In this review, we provide a summary of the recent applications of antibody microarray techniques in basic biology and clinical studies, providing insights into the current trends and future of protein analysis.

  • 10. 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.

  • 11.
    Costeira-Paulo, Joana
    et al.
    Uppsala Univ, Dept Chem BMC, Box 576, S-75123 Uppsala, Sweden..
    Gault, Joseph
    Univ Oxford, Dept Chem, Phys & Theoret Chem Lab, South Parks Rd, Oxford OX1 3QZ, England..
    Popova, Gergana
    Karolinska Inst, Dept Microbiol Tumour & Cell Biol, Nobels Vag 16, S-17177 Stockholm, Sweden..
    Ladds, Marcus J. G. W.
    Karolinska Inst, Dept Microbiol Tumour & Cell Biol, Nobels Vag 16, S-17177 Stockholm, Sweden..
    van Leeuwen, Ingeborg M. M.
    Karolinska Inst, Dept Microbiol Tumour & Cell Biol, Nobels Vag 16, S-17177 Stockholm, Sweden..
    Sarr, Medoune
    Karolinska Inst, Ctr Alzheimer Res, Dept Neurobiol Care Sci & Soc NVS, Div Neurogeriatr,Dept Neurobiol, S-14157 Huddinge, Sweden..
    Olsson, Anders
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Lane, David P.
    Karolinska Inst, Dept Microbiol Tumour & Cell Biol, Nobels Vag 16, S-17177 Stockholm, Sweden.;Karolinska Inst, Sci Life Lab, Dept Microbiol Tumour & Cell Biol, Tomtebodavagen 23A, S-17165 Stockholm, Sweden..
    Lain, Sonia
    Karolinska Inst, Dept Microbiol Tumour & Cell Biol, Nobels Vag 16, S-17177 Stockholm, Sweden.;Karolinska Inst, Sci Life Lab, Dept Microbiol Tumour & Cell Biol, Tomtebodavagen 23A, S-17165 Stockholm, Sweden..
    Marklund, Erik G.
    Uppsala Univ, Dept Chem BMC, Box 576, S-75123 Uppsala, Sweden..
    Landreh, Michael
    Karolinska Inst, Dept Microbiol Tumour & Cell Biol, Nobels Vag 16, S-17177 Stockholm, Sweden.;Karolinska Inst, Sci Life Lab, Dept Microbiol Tumour & Cell Biol, Tomtebodavagen 23A, S-17165 Stockholm, Sweden..
    Lipids Shape the Electron Acceptor-Binding Site of the Peripheral Membrane Protein Dihydroorotate Dehydrogenase2018In: Cell Chemical Biology, ISSN 2451-9456, E-ISSN 2451-9448, Vol. 25, no 3, p. 309-+Article in journal (Refereed)
    Abstract [en]

    The interactions between proteins and biological membranes are important for drug development, but remain notoriously refractory to structural investigation. We combine non-denaturing mass spectrometry (MS) with molecular dynamics (MD) simulations to unravel the connections among cofactor, lipid, and inhibitor binding in the peripheral membrane protein dihydroorotate dehydrogenase (DHODH), a key anticancer target. Interrogation of intact DHODH complexes by MS reveals that phospholipids bind via their charged head groups at a limited number of sites, while binding of the inhibitor brequinar involves simultaneous association with detergent molecules. MD simulations show that lipids support flexible segments in the membrane-binding domain and position the inhibitor and electron acceptor-binding site away from the membrane surface, similar to the electron acceptor-binding site in respiratory chain complex I. By complementing MS with MD simulations, we demonstrate how a peripheral membrane protein uses lipids to modulate its structure in a similar manner as integral membrane proteins.

  • 12.
    Danielsson, Frida
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Cellular and Clinical Proteomics. Royal Inst Technol, Sci Life Lab, S-17165 Stockholm, Sweden..
    Peterson, McKenzie Kirsten
    Univ Utah, Sch Med, Dept Pathol, Salt Lake City, UT 84112 USA..
    Araujo, Helena Caldeira
    Univ Madeira, Ctr Quim, P-9020105 Funchal, Portugal..
    Lautenschlaeger, Franziska
    Saarland Univ, Leibniz Inst Neue Mat gGmbH INM & Expt Phys, NT Fac, Campus D2 2,E 2 6, D-66123 Saarbrucken, Germany..
    Britt Gad, Annica Karin
    Univ Madeira, Ctr Quim, P-9020105 Funchal, Portugal.;Uppsala Univ, Dept Med Biochem & Microbiol, S-75237 Uppsala, Sweden..
    Vimentin Diversity in Health and Disease2018In: CELLS, ISSN 2073-4409, Vol. 7, no 10, article id 147Article, review/survey (Refereed)
    Abstract [en]

    Vimentin is a protein that has been linked to a large variety of pathophysiological conditions, including cataracts, Crohn's disease, rheumatoid arthritis, HIV and cancer. Vimentin has also been shown to regulate a wide spectrum of basic cellular functions. In cells, vimentin assembles into a network of filaments that spans the cytoplasm. It can also be found in smaller, non-filamentous forms that can localise both within cells and within the extracellular microenvironment. The vimentin structure can be altered by subunit exchange, cleavage into different sizes, re-annealing, post-translational modifications and interacting proteins. Together with the observation that different domains of vimentin might have evolved under different selection pressures that defined distinct biological functions for different parts of the protein, the many diverse variants of vimentin might be the cause of its functional diversity. A number of review articles have focussed on the biology and medical aspects of intermediate filament proteins without particular commitment to vimentin, and other reviews have focussed on intermediate filaments in an in vitro context. In contrast, the present review focusses almost exclusively on vimentin, and covers both ex vivo and in vivo data from tissue culture and from living organisms, including a summary of the many phenotypes of vimentin knockout animals. Our aim is to provide a comprehensive overview of the current understanding of the many diverse aspects of vimentin, from biochemical, mechanical, cellular, systems biology and medical perspectives.

  • 13. Djureinovic, D.
    et al.
    Dodig-Crnkovic, Tea
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hellström, Cecilia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics.
    Holgersson, G.
    Bergqvist, M.
    Mattsson, J. S. M.
    Pontén, F.
    Ståhle, E.
    Schwenk, Jochen M.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics.
    Micke, P.
    Detection of autoantibodies against cancer-testis antigens in non-small cell lung cancer2018In: Lung Cancer, ISSN 0169-5002, E-ISSN 1872-8332, Vol. 125, p. 157-163Article in journal (Refereed)
    Abstract [en]

    Objectives: Cancer-testis antigens (CTAs) are defined as proteins that are specifically expressed in testis or placenta and their expression is frequently activated in cancer. Due to their ability to induce an immune response, CTAs may serve as suitable targets for immunotherapy. The aim of this study was to evaluate if there is reactivity against CTAs in the plasma of non-small cell lung cancer (NSCLC) patients through the detection of circulating antibodies. Materials and methods: To comprehensively analyze autoantibodies against CTAs the multiplexing capacities of suspension bead array technology was used. Bead arrays were created with 120 protein fragments, representing 112 CTAs. Reactivity profiles were measured in plasma samples from 133 NSCLC patients and 57 cases with benign lung diseases. Results: Altogether reactivity against 69 antigens, representing 81 CTAs, was demonstrated in at least one of the analyzed samples. Twenty-nine of the antigens (45 CTAs) demonstrated exclusive reactivity in NSCLC samples. Reactivity against cancer-testis antigen family 47; member A (CT47A) genes, P antigen family member 3 (PAGE3), variable charge X-linked (VCX), melanoma antigen family B1 (MAGEB1), lin-28 homolog B (LIN28B) and chromosome 12 open reading frame 54 (C12orf54) were only found in NSCLC patients at a frequency of 1%–4%. The presence of autoantibodies towards these six antigens was confirmed in an independent group of 34 NSCLC patients. Conclusion: We identified autoantibodies against CTAs in the plasma of lung cancer patients. The reactivity pattern of autoantibodies was higher in cancer patients compared to the benign group, stable over time, but low in frequency of occurrence. The findings suggest that some CTAs are immunogenic and that these properties can be utilized as immune targets. 

  • 14.
    Drobin, Kimi
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Antibody-based bead arrays for high-throughput protein profiling in human plasma and serum2018Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    Affinity-based proteomics utilizes affinity binders to detect target proteins in a large-scale manner. This thesis describes a high-throughput method, which enables the search for biomarker candidates in human plasma and serum. A highly multiplexed antibody-based suspension bead array is created by coupling antibodies generated in the Human Protein Atlas project to color-coded beads. The beads are combined for parallel analysis of up to 384 analytes in patient and control samples. This provides data to compare protein levels from the different groups.

    In paper I osteoporosis patients are compared to healthy individuals to find disease-linked proteins. An untargeted discovery screening was conducted using 4608 antibodies in 16 cases and 6 controls. This revealed 72 unique proteins, which appeared differentially abundant. A validation screening of 91 cases and 89 controls confirmed that the protein autocrine motility factor receptor (AMFR) is decreased in the osteoporosis patients.

    Paper II investigates the risk proteome of inflammatory bowel disease (IBD). Antibodies targeting 209 proteins corresponding to 163 IBD genetic risk loci were selected. To find proteins related to IBD or its subgroups, sera from 49 patients with Crohn’s disease, 51 with ulcerative colitis and 50 matched controls were analyzed. From these targeted assays, the known inflammation-related marker serum amyloid protein A (SAA) was shown to be elevated in the IBD cases. In addition, the protein laccase (multi-copper oxidoreductase) domain containing 1 (LACC1) was found to be decreased in the IBD subjects.

    In conclusion, assays using affinity-based bead arrays were developed and applied to screen human plasma and serum samples in two disease contexts. Untargeted and targeted screening strategies were applied to discover disease-associated proteins. Upon further validation, these potential biomarker candidates could be valuable in future disease studies.

  • 15.
    Drobin, Kimi
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics.
    Assadi, Ghazaleh
    Hong, Mun-Gwan
    Andersson, Eni
    Fredolini, Claudia
    Forsström, Björn
    Reznichenko, Anna
    Akhter, Tahmina
    Ek, Weronica
    Bonfiglio, Ferdinando
    Berner Hansen, Mark
    Sandberg, Kristian
    Greco, Dario
    Repsilber, Dirk
    Schwenk, Jochen
    D'Amato, Mauro
    Halfvarson, Jonas
    Targeted analysis of serum proteins encoded at known inflammatory bowel disease risk lociManuscript (preprint) (Other academic)
  • 16.
    Edfors, Fredrik
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hober, Andreas
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Linderbäck, Klas
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Maddalo, Gianluca
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Azimi, Alireza
    Karolinska Inst, Karolinska Univ Hosp, Dept Oncol Pathol, SE-17177 Stockholm, Sweden..
    Sivertsson, Åsa
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Tegel, Hanna
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hober, Sophia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Al-Khalili Szigyarto, Cristina
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fagerberg, Linn
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    von Feilitzen, Kalle
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Oksvold, Per
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lindskog, Cecilia
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden..
    Forsström, Björn
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab. Biosustainabil, DK-2970 Horsholm, Denmark..
    Enhanced validation of antibodies for research applications2018In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, article id 4130Article in journal (Refereed)
    Abstract [en]

    There is a need for standardized validation methods for antibody specificity and selectivity. Recently, five alternative validation pillars were proposed to explore the specificity of research antibodies using methods with no need for prior knowledge about the protein target. Here, we show that these principles can be used in a streamlined manner for enhanced validation of research antibodies in Western blot applications. More than 6,000 antibodies were validated with at least one of these strategies involving orthogonal methods, genetic knockdown, recombinant expression, independent antibodies, and capture mass spectrometry analysis. The results show a path forward for efforts to validate antibodies in an application-specific manner suitable for both providers and users.

  • 17.
    Faridi, Muhammad Asim
    et al.
    KTH.
    Shahzad, Adnan Faqui
    KTH.
    Russom, Aman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Milliliter scale acoustophoresis based bioparticle processing platform2018In: ASME 2018 16th International Conference on Nanochannels, Microchannels, and Minichannels, ICNMM 2018, ASME Press, 2018Conference paper (Refereed)
    Abstract [en]

    Bioparticles such as mammalian cells and bacteria can be manipulated directly or indirectly for multiple applications such as sample preparation for diagnostic applications mainly up-concentration, enrichment & separation as well as immunoassay development. There are various active and passive microfluidic particle manipulation techniques where Acoustophoresis is a powerful technique showing high cell viability. The use of disposable glass capillaries for acoustophoresis, instead of cleanroom fabricated glass-silicon chip can potentially bring down the cost factor substantially, aiding the realization of this technique for real-world diagnostic devices. Unlike available chips and capillary-based microfluidic devices, we report milliliter-scale platform able to accommodate 1ml of a sample for acoustophoresis based processing on a market available glass capillary. Although it is presented as a generic platform but as a demonstration we have shown that polystyrene suspending medium sample can be processed with trapping efficiency of 87% and the up-concentration factor of 10 times in a flow through manner i.e., at 35µl/min. For stationary volume accommodation, this platform practically offers 50 times more sample handling capacity than most of the microfluidic setups. Furthermore, we have also shown that with diluted blood (0.6%) in a flow-through manner, 82% of the white blood cells (WBCs) per ml could be kept trapped. This milliliter platform could potentially be utilized for assisting in sample preparation, plasma separation as well as a flow-through immunoassay assay development for clinical diagnostic applications.

  • 18.
    Fasterius, Erik
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Analysis of public RNA-sequencing data reveals biological consequences of genetic heterogeneity in cell line populations2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 11226Article in journal (Refereed)
    Abstract [en]

    Meta-analysis of datasets available in public repositories are used to gather and summarise experiments performed across laboratories, as well as to explore consistency of scientific findings. As data quality and biological equivalency across samples may obscure such analyses and consequently their conclusions, we investigated the comparability of 85 public RNA-seq cell line datasets. Thousands of pairwise comparisons of single nucleotide variants in 139 samples revealed variable genetic heterogeneity of the eight cell line populations analysed as well as variable data quality. The H9 and HCT116 cell lines were found to be remarkably stable across laboratories (with median concordances of 99.2% and 98.5%, respectively), in contrast to the highly variable HeLa cells (89.3%). We show that the genetic heterogeneity encountered greatly affects gene expression between same-cell comparisons, highlighting the importance of interrogating the biological equivalency of samples when comparing experimental datasets. Both the number of differentially expressed genes and the expression levels negatively correlate with the genetic heterogeneity. Finally, we demonstrate how comparing genetically heterogeneous datasets affect gene expression analyses and that high dissimilarity between same-cell datasets alters the expression of more than 300 cancer-related genes, which are often the focus of studies using cell lines.

  • 19.
    Fasterius, Erik
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    seqCAT: a Bioconductor R-package for variant analysis of high throughput sequencing dataManuscript (preprint) (Other academic)
  • 20.
    Fasterius, Erik
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Uhlén, Mathias
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Al-Khalili Szigyarto, Cristina
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Single cell RNA-seq variant analysis for exploration of inter- and intra-tumour genetic heterogeneityManuscript (preprint) (Other academic)
  • 21. Frostegård, J.
    et al.
    Hellström, Cecilia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Nilsson, Peter
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Frostegård, A. G.
    Ajeganova, S.
    Autoantibody profiling reveals four protein candidate autoantigens associated with systemic lupus erythematosus2018In: Lupus, ISSN 0961-2033, E-ISSN 1477-0962, Vol. 27, no 10, p. 1670-1678Article in journal (Refereed)
    Abstract [en]

    Objectives In systemic lupus erythematosus (SLE) there are typically many autoantibodies. The disease heterogeneity could be better understood with discovery of phenotype-specific antigens targeted by autoantibodies. We here aimed to identify novel autoantigens potentially related to SLE disease and a major complication, atherosclerosis. Methods Antigen microarrays were used to profile IgG autoantibody reactivity against 77 protein fragments (20-140 amino acids (aa) long, median 89 aa) produced within the Human Protein Atlas project, in serum samples from SLE patients (n=107) and age- and sex-matched population-based controls (n=107). Common carotid intima-media thickness, plaque occurrence and echogenicity were determined by B-mode ultrasound. Results We determined significant differences between patients and controls in IgG reactivity against four proteins. In patients compared to controls, there was an increase of IgG reactivity against zinc finger protein 688 (ZNF688), early B cell factor 2 (EBF2), crystallin, alpha B (CRYAB) and tumor necrosis factor receptor superfamily member 13C (TNFRSF13C). Of these four antigens, only anti-ZNF688 was associated with carotid atherosclerosis (plaque occurrence) and vulnerable plaques in SLE. There was a weak association between anti-EBF2 and SLE disease activity but no significant associations were determined for other measured IgG reactivity. Conclusions In this discovery screening we here demonstrate new candidate autoantigens with differential reactivity (reflecting autoantibody levels) in SLE patients and in controls and in relation to atherosclerosis in SLE.

  • 22. Glimelius, Bengt
    et al.
    Melin, Beatrice
    Enblad, Gunilla
    Alafuzoff, Irina
    Beskow, Anna
    Ahlström, Håkan
    Bill-Axelson, Anna
    Birgisson, Helgi
    Björ, Ove
    Edqvist, Per-Henrik
    Hansson, Tony
    Helleday, Thomas
    Hellman, Per
    Henriksson, Kerstin
    Hesselager, Göran
    Hultdin, Magnus
    Häggman, Michael
    Höglund, Martin
    Jonsson, Håkan
    Larsson, Chatarina
    Lindman, Henrik
    Ljuslinder, Ingrid
    Mindus, Stephanie
    Nygren, Peter
    Pontén, Fredrik
    Riklund, Katrine
    Rosenquist, Richard
    Sandin, Fredrik
    Schwenk, Jochen M.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Stenling, Roger
    Stålberg, Karin
    Stålberg, Peter
    Sundström, Christer
    Karlsson, Camilla Thellenberg
    Westermark, Bengt
    Bergh, Anders
    Claesson-Welsh, Lena
    Palmqvist, Richard
    Sjöblom, Tobias
    U-CAN: a prospective longitudinal collection of biomaterials and clinical information from adult cancer patients in Sweden2018In: Acta Oncologica, ISSN 0284-186X, E-ISSN 1651-226X, Vol. 57, no 2, p. 187-194Article in journal (Refereed)
    Abstract [en]

    Background: Progress in cancer biomarker discovery is dependent on access to high-quality biological materials and high-resolution clinical data from the same cases. To overcome current limitations, a systematic prospective longitudinal sampling of multidisciplinary clinical data, blood and tissue from cancer patients was therefore initiated in 2010 by Uppsala and Umea Universities and involving their corresponding University Hospitals, which are referral centers for one third of the Swedish population. Material and Methods: Patients with cancer of selected types who are treated at one of the participating hospitals are eligible for inclusion. The healthcare-integrated sampling scheme encompasses clinical data, questionnaires, blood, fresh frozen and formalin-fixed paraffin-embedded tissue specimens, diagnostic slides and radiology bioimaging data. Results: In this ongoing effort, 12,265 patients with brain tumors, breast cancers, colorectal cancers, gynecological cancers, hematological malignancies, lung cancers, neuroendocrine tumors or prostate cancers have been included until the end of 2016. From the 6914 patients included during the first five years, 98% were sampled for blood at diagnosis, 83% had paraffin-embedded and 58% had fresh frozen tissues collected. For Uppsala County, 55% of all cancer patients were included in the cohort. Conclusions: Close collaboration between participating hospitals and universities enabled prospective, longitudinal biobanking of blood and tissues and collection of multidisciplinary clinical data from cancer patients in the U-CAN cohort. Here, we summarize the first five years of operations, present U-CAN as a highly valuable cohort that will contribute to enhanced cancer research and describe the procedures to access samples and data.

  • 23.
    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)
  • 24.
    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.

  • 25.
    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)
  • 26.
    Horak, Josef
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Jansson, Ronnie
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Dev, Apurba
    Uppsala Univ, Ångström Lab, Solid State Elect, Uppsala Box 534, SE-75121 Uppsala, Sweden..
    Nilebäck, Linnea
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Behnam, Kiarash
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Linnros, Jan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Photonics.
    Hedhammar, My
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Eriksson Karlström, Amelie
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Recombinant Spider Silk as Mediator for One-Step, Chemical-Free Surface Biofunctionalization2018In: Advanced Functional Materials, ISSN 1616-301X, E-ISSN 1616-3028, Vol. 28, no 21, article id 1800206Article in journal (Refereed)
    Abstract [en]

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

  • 27. 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.

  • 28.
    Jahn, Michael
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab. K.
    Vialas, Vital
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Karlsen, Jan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Maddalo, Gianluca
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Edfors, Fredrik
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Forsström, Björn
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Käll, Lukas
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hudson, Elton P.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Growth of Cyanobacteria Is Constrained by the Abundance of Light and Carbon Assimilation Proteins2018In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 25, no 2, p. 478-+Article in journal (Refereed)
    Abstract [en]

    Cyanobacteria must balance separate demands for energy generation, carbon assimilation, and biomass synthesis. We used shotgun proteomics to investigate proteome allocation strategies in the model cyanobacterium Synechocystis sp. PCC 6803 as it adapted to light and inorganic carbon (C-i) limitation. When partitioning the proteome into seven functional sectors, we find that sector sizes change linearly with growth rate. The sector encompassing ribosomes is significantly smaller than in E. coli, which may explain the lower maximum growth rate in Synechocystis. Limitation of light dramatically affects multiple proteome sectors, whereas the effect of C-i limitation is weak. Carbon assimilation proteins respond more strongly to changes in light intensity than to C-i. A coarse-grained cell economy model generally explains proteome trends. However, deviations from model predictions suggest that the large proteome sectors for carbon and light assimilation are not optimally utilized under some growth conditions and may constrain the proteome space available to ribosomes.

  • 29.
    Just, David
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Månberg, Anna
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Carlström, Eva Lindholm
    Uppsala Univ, Uppsala, Sweden..
    Cunningham, Janet
    Uppsala Univ, Uppsala, Sweden..
    Nilsson, Peter
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Towards Molecular Insights Into Psychiatric Disorders Using Affinity Proteomics2018In: Schizophrenia Bulletin, ISSN 0586-7614, E-ISSN 1745-1701, Vol. 44, p. S223-S223Article in journal (Other academic)
  • 30.
    Kanje, Sara
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Venskutonyte, Raminta
    Lund Univ, Expt Med Sci, Med Struct Biol, BMC C13, SE-22184 Lund, Sweden..
    Scheffel, Julia
    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, Protein Engineering.
    Lindkvist-Petersson, Karin
    Lund Univ, Expt Med Sci, Med Struct Biol, BMC C13, SE-22184 Lund, Sweden..
    Hobert, Sophia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
    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, Z(ca), 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 Z(ca)-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. 

  • 31.
    Karlsen, Jan
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Asplund-Samuelsson, Johannes
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Thomas, Quentin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH). KTH, Centres, Science for Life Laboratory, SciLifeLab. Univ Copenhagen, Copenhagen Plant Sci Ctr, Dept Plant & Environm Sci, Frederiksberg, Denmark..
    Jahn, Michael
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hudson, Elton Paul
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ribosome Profiling of Synechocystis Reveals Altered Ribosome Allocation at Carbon Starvation2018In: MSYSTEMS, ISSN 2379-5077, Vol. 3, no 5, article id e00126-18Article in journal (Refereed)
    Abstract [en]

    Cyanobacteria experience both rapid and periodic fluctuations in light and inorganic carbon (C-i) and have evolved regulatory mechanisms to respond to these, including extensive posttranscriptional gene regulation. We report the first genome-wide ribosome profiling data set for cyanobacteria, where ribosome occupancy on mRNA is quantified with codon-level precision. We measured the transcriptome and translatome of Synechocystis during autotrophic growth before (high carbon [HC] condition) and 24 h after removing CO2 from the feedgas (low carbon [LC] condition). Ribosome occupancy patterns in the 5' untranslated region suggest that ribosomes can assemble there and slide to the Shine-Dalgarno site, where they pause. At LC, total translation was reduced by 80% and ribosome pausing was increased at stop and start codons and in untranslated regions, which may be a sequestration mechanism to inactivate ribosomes in response to rapid C-i depletion. Several stress response genes, such as thioredoxin M (sll1057), a putative endonuclease (slr0915), protease HtrA (slr1204), and heat shock protein HspA (sll1514) showed marked increases in translational efficiency at LC, indicating translational control in response to Ci depletion. Ribosome pause scores within open reading frames were mostly constant, though several ribosomal proteins had significantly altered pause score distributions at LC, which might indicate translational regulation of ribosome biosynthesis in response to Ci depletion. We show that ribosome profiling is a powerful tool to decipher dynamic gene regulation strategies in cyanobacteria. IMPORTANCE Ribosome profiling accesses the translational step of gene expression via deep sequencing of ribosome-protected mRNA footprints. Pairing of ribosome profiling and transcriptomics data provides a translational efficiency for each gene. Here, the translatome and transcriptome of the model cyanobacterium Synechocystis were compared under carbon-replete and carbon starvation conditions. The latter may be experienced when cyanobacteria are cultivated in poorly mixed bioreactors or engineered to be product-secreting cell factories. A small fraction of genes (<200), including stress response genes, showed changes in translational efficiency during carbon starvation, indicating condition-dependent translation-level regulation. We observed ribosome occupancy in untranslated regions, possibly due to an alternative translation initiation mechanism in Synechocystis. The higher proportion of ribosomes residing in untranslated regions during carbon starvation may be a mechanism to quickly inactivate superfluous ribosomes. This work provides the first ribosome profiling data for cyanobacteria and reveals new regulation strategies for coping with nutrient limitation.

  • 32. Karnevi, E.
    et al.
    Dror, L. B.
    Mardinoglu, Adil
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Elebro, J.
    Heby, M.
    Olofsson, S. -E
    Nodin, B.
    Eberhard, J.
    Gallagher, W.
    Uhlén, Mathias
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Jirström, K.
    Translational study reveals a two-faced role of RBM3 in pancreatic cancer and suggests its potential value as a biomarker for improved patient stratification2018In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 9, no 5, p. 6188-6200Article in journal (Refereed)
    Abstract [en]

    Periampullary adenocarcinoma, including pancreatic cancer, is a heterogeneous group of tumors with dismal prognosis, partially due to lack of reliable targetable and predictive biomarkers. RNA-binding motif protein 3 (RBM3) has previously been shown to be an independent prognostic and predictive biomarker in several types of cancer. Herein, we examined the prognostic value of RBM3 in periampullary adenocarcinoma, as well as the effects following RBM3 suppression in pancreatic cancer cells in vitro. RBM3 mRNA levels were examined in 176 pancreatic cancer patients from The Cancer Genome Atlas. Immunohistochemical expression of RBM3 was analyzed in tissue microarrays with primary tumors and paired lymph node metastases from 175 consecutive patients with resected periampullary adenocarcinoma. Pancreatic cancer cells were transfected with anti-RBM3 siRNA in vitro and the influence on cell viability following chemotherapy, transwell migration and invasion was assessed. The results demonstrated that high mRNA-levels of RBM3 were significantly associated with a reduced overall survival (p = 0.026). RBM3 protein expression was significantly higher in lymph node metastases than in primary tumors (p = 0.005). High RBM3 protein expression was an independent predictive factor for the effect of adjuvant chemotherapy and an independent negative prognostic factor in untreated patients (p for interaction = 0.003). After siRNA suppression of RBM3 in vitro, pancreatic cancer cells displayed reduced migration and invasion compared to control, as well as a significantly increased resistance to chemotherapy. In conclusion, the strong indication of a positive response predictive effect of RBM3 expression in pancreatic cancer may be highly relevant in the clinical setting and merits further validation.

  • 33. Kennedy, S
    et al.
    Fasterius, Erik
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Al-Khalili Szigyarto, Cristina
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Kolch, W
    et al.,
    Adaptive rewiring of protein-protein interactions and signal flow in the EGFR signaling network by mutant RASManuscript (preprint) (Other academic)
  • 34.
    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.

  • 35.
    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.

  • 36.
    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.

  • 37.
    Liu, Hao
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Lindbo, Sarah
    Ding, Haozhong
    Hober, Sophia
    Gräslund, Torbjorn
    Potent and specific fusion toxins consisting of a HER2-binding ABD-derived affinity protein (ADAPT), fused to truncated versions of Pseudomonas exotoxin AManuscript (preprint) (Other academic)
  • 38.
    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.

  • 39.
    Mikus, Maria
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Array-based identification of disease-associated proteins2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    To increase our understanding of the human body in both health and disease, proteins can be studied in samples such as plasma and serum to provide a molecular profile of the physiological status. In the work presented in this thesis, array-based methods were used to study associations of protein and autoantibody profiles with disease. The methods included antibody suspension bead arrays for protein profiling and planar antigen arrays or antigen suspension bead arrays for autoantibody profiling.

    In Paper I, we studied protein levels in the context of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). We identified three proteins, NEFM, RGS18 and SLC25A20, to be significantly elevated in patients with ALS. We also evaluated the diagnostic potential of these proteins, reaching areas under the curves (AUCs) between 0.78 and 0.86 for each of the three proteins individually.

    In Paper II, drug-induced liver injury (DILI) cases and controls were studied in four independent cohorts of longitudinal and cross-sectional design and covering a range of drugs. The protein FABP1 was elevated in DILI cases upon initiation of treatment whereas CDH5 were elevated before treatment. Furthermore, we compared FABP1 with the clinically measured alanine aminotransferase (ALT), and identified some aspects in which FABP1 was superior: tissue distribution – FABP1 was not found in skeletal and heart muscle tissue, injuries in which can cause elevations of ALT; kinetics – FABP1 is smaller and has a lower half-life compared to ALT. Both of these circumstances mean that FABP1 as a biomarker has the potential to more accurately reflect ongoing injury.

    In Paper III, asthma of different severities, chronic obstructive pulmonary disease and healthy controls from two independent cohorts were studied. The levels of ten proteins were verified to be significantly elevated in severe asthma compared to both mild-to-moderate asthma and healthy controls in both cohorts. We also clustered asthma patients based on their protein profiles and identified six subgroups that could help to guide the appropriate treatment.

    In Paper IV, atopic dermatitis (AD) of different severities and healthy controls were studied. Increased autoantibody reactivity to four antigens, KRTAP17-1, HSPA4, S100A12 and S100Z, were observed in AD patients or in any of the two severity disease subgroups compared to controls.

    In summary, the work included in this thesis highlights the applicability of protein array-based methods in various contexts and in studying various research questions. Disease-associated proteins were identified and further studies will determine their utility.

  • 40.
    Mikus, Maria
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Johansson, Catharina
    Acevedo, Nathalie
    Nilsson, Peter
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Scheynius, Annika
    The antimicrobial protein S100A12 identified as a potential autoantigen in a subgroup of atopic dermatitis patientsManuscript (preprint) (Other academic)
  • 41.
    Mikus, Maria
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kolmert, Johan
    James, Anna
    Andersson, Lars I
    Gomez, Cristina
    Ericsson, Magnus
    Thörngren, John-Olof
    Dahlén, Barbro
    Nilsson, Peter
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Dahlén, Sven-Erik
    Identification of proteins associated with asthma severityManuscript (preprint) (Other academic)
  • 42.
    Mitran, Bogdan
    et al.
    Uppsala Univ, Dept Med Chem, Dag Hammarskjoldsv 14C,3Tr, S-75183 Uppsala, Sweden..
    Güler, Rezan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Roche, Francis P.
    Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden..
    Lindstrom, Elin
    Uppsala Univ, Dept Med Chem, Dag Hammarskjoldsv 14C,3Tr, S-75183 Uppsala, Sweden..
    Selvaraju, Ram Kumar
    Uppsala Univ, Dept Med Chem, Dag Hammarskjoldsv 14C,3Tr, S-75183 Uppsala, Sweden.;Uppsala Univ, Dept Med Chem, Preclin PET MRI Platform, Uppsala, Sweden..
    Fleetwood, Filippa
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Rinne, Sara S.
    Uppsala Univ, Dept Med Chem, Dag Hammarskjoldsv 14C,3Tr, S-75183 Uppsala, Sweden..
    Claesson-Welsh, Lena
    Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, Uppsala, Sweden..
    Tolmachev, Vladimir
    Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden..
    Ståhl, Stefan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Orlova, Anna
    Uppsala Univ, Dept Med Chem, Dag Hammarskjoldsv 14C,3Tr, S-75183 Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, Uppsala, Sweden..
    Löfblom, John
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Radionuclide imaging of VEGFR2 in glioma vasculature using biparatopic affibody conjugate: proof-of-principle in a murine model2018In: Theranostics, ISSN 1838-7640, E-ISSN 1838-7640, Vol. 8, no 16, p. 4462-4476Article in journal (Refereed)
    Abstract [en]

    Vascular endothelial growth factor receptor-2 (VEGFR2) is a key mediator of angiogenesis and therefore a promising therapeutic target in malignancies including glioblastoma multiforme (GBM). Molecular imaging of VEGFR2 expression may enable patient stratification for antiangiogenic therapy. The goal of the current study was to evaluate the capacity of the novel anti-VEGFR2 biparatopic affibody conjugate (Z(VEGFR2)-Bp(2)) for in vivo visualization of VEGFR2 expression in GBM. Methods: Z(VEGFR2)-Bp(2) coupled to a NODAGA chelator was generated and radiolabeled with indium-111. The VEGFR2-expressing murine endothelial cell line MS1 was used to evaluate in vitro binding specificity and affinity, cellular processing and targeting specificity in mice. Further tumor targeting was studied in vivo in GL261 glioblastoma orthotopic tumors. Experimental imaging was performed. Results: [In-111]In-NODAGA-Z(VEGFR2)-Bp(2) bound specifically to VEGFR2 (K-D=33 +/- 18 pM). VEGFR2-mediated accumulation was observed in liver, spleen and lungs. The tumor-to-organ ratios 2 h post injection for mice bearing MS1 tumors were approximately 11 for blood, 15 for muscles and 78 for brain. Intracranial GL261 glioblastoma was visualized using SPECT/CT. The activity uptake in tumors was significantly higher than in normal brain tissue. The tumor-to-cerebellum ratios after injection of 4 mu g [In-111]In-NODAGA-Z(VEGFR2)-Bp(2) were significantly higher than the ratios observed for the 40 mu g injected dose and for the non-VEGFR2 binding size-matched conjugate, demonstrating target specificity. Microautoradiography of cryosectioned CNS tissue was in good agreement with the SPECT/CT images. Conclusion: The anti-VEGFR2 affibody conjugate [In-111]In-NODAGA-Z(VEGFR2)-Bp(2) specifically targeted VEGFR2 in vivo and visualized its expression in a murine GBM orthotopic model. Tumor-to-blood ratios for [In-111]In-NODAGA-Z(VEGFR2)-Bp(2) were higher compared to other VEGFR2 imaging probes. [In-111]In-NODAGA-Z(VEGFR2)-Bp(2) appears to be a promising probe for in vivo noninvasive visualization of tumor angiogenesis in glioblastoma.

  • 43. Mönnich, M.
    et al.
    Borgeskov, L.
    Breslin, L.
    Jakobsen, L.
    Rogowski, M.
    Doganli, C.
    Schrøder, J. M.
    Mogensen, J. B.
    Blinkenkjær, L.
    Harder, L. M.
    Lundberg, Emma
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Cellular and Clinical Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Geimer, S.
    Christensen, S. T.
    Andersen, J. S.
    Larsen, L. A.
    Pedersen, L. B.
    CEP128 Localizes to the Subdistal Appendages of the Mother Centriole and Regulates TGF-β/BMP Signaling at the Primary Cilium2018In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 22, no 10, p. 2601-2614Article in journal (Refereed)
    Abstract [en]

    The centrosome is the main microtubule-organizing center in animal cells and comprises a mother and daughter centriole surrounded by pericentriolar material. During formation of primary cilia, the mother centriole transforms into a basal body that templates the ciliary axoneme. Ciliogenesis depends on mother centriole-specific distal appendages, whereas the role of subdistal appendages in ciliary function is unclear. Here, we identify CEP128 as a centriole subdistal appendage protein required for regulating ciliary signaling. Loss of CEP128 did not grossly affect centrosomal or ciliary structure but caused impaired transforming growth factor-β/bone morphogenetic protein (TGF-β/BMP) signaling in zebrafish and at the primary cilium in cultured mammalian cells. This phenotype is likely the result of defective vesicle trafficking at the cilium as ciliary localization of RAB11 was impaired upon loss of CEP128, and quantitative phosphoproteomics revealed that CEP128 loss affects TGF-β1-induced phosphorylation of multiple proteins that regulate cilium-associated vesicle trafficking. Mönnich et al. show that CEP128 localizes to the subdistal appendages of the mother centriole and basal body of the primary cilium. CEP128 regulates vesicular trafficking and targeting of RAB11 to the primary cilium. CEP128 loss leads to impaired TGF-β/BMP signaling, which, in zebrafish, is associated with defective organ development.

  • 44.
    Nilebäck, Linnea
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Arola, Suvi
    Aalto Univ, Sch Chem Engn, Dept Bioprod & Biosyst, POB 16100, FI-00076 Aalto, Finland..
    Kvick, Mathias
    AlbaNova Univ Ctr, Spiber Technol AB, S-10691 Stockholm, Sweden..
    Paananen, Arja
    VTT Tech Res Ctr Finland Ltd, Tietotie 2, FI-02150 Espoo, Finland..
    Linder, Markus B.
    Aalto Univ, Sch Chem Engn, Dept Bioprod & Biosyst, POB 16100, FI-00076 Aalto, Finland..
    Hedhammar, My
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Interfacial Behavior of Recombinant Spider Silk Protein Parts Reveals Cues on the Silk Assembly Mechanism2018In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 34, no 39, p. 11795-11805Article in journal (Refereed)
    Abstract [en]

    The mechanism of silk assembly, and thus the cues for the extraordinary properties of silk, can be explored by studying the simplest protein parts needed for the formation of silk-like materials. The recombinant spider silk protein 4RepCT, consisting of four repeats of polyalanine and glycine-rich segments (4Rep) and a globular C-terminal domain (CT), has previously been shown to assemble into silk-like fibers at the liquid-air interface. Herein, we study the interfacial behavior of the two parts of 4RepCT, revealing new details on how each protein part is crucial for the silk assembly. Interfacial rheology and quartz crystal microbalance with dissipation show that 4Rep interacts readily at the interfaces. However, organized nanofibrillar structures are formed only when 4Rep is fused to CT. A strong interplay between the parts to direct the assembly is demonstrated. The presence of either a liquid-air or a liquid-solid interface had a surprisingly similar influence on the assembly.

  • 45.
    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.

  • 46. O'Hagan, Steve
    et al.
    Muelas, Marina Wright
    Day, Philip J.
    Lundberg, Emma
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Cellular and Clinical Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kell, Douglas B.
    GeneGini: Assessment via the Gini Coefficient of Reference "Housekeeping'' Genes and Diverse Human Transporter Expression Profiles2018In: Cell Systems, ISSN 2405-4712, Vol. 6, no 2, p. 230-+Article in journal (Refereed)
    Abstract [en]

    The expression levels of SLC or ABC membrane transporter transcripts typically differ 100- to 10,000-fold between different tissues. The Gini coefficient characterizes such inequalities and here is used to describe the distribution of the expression of each transporter among different human tissues and cell lines. Many transporters exhibit extremely high Gini coefficients even for common substrates, indicating considerable specialization consistent with divergent evolution. The expression profiles of SLC transporters in different cell lines behave similarly, although Gini coefficients for ABC transporters tend to be larger in cell lines than in tissues, implying selection. Transporter genes are significantly more heterogeneously expressed than the members of most non-transporter gene classes. Transcripts with the stablest expression have a low Gini index and often differ significantly from the "housekeeping'' genes commonly used for normalization in transcriptomics/qPCR studies. PCBP1 has a low Gini coefficient, is reasonably expressed, and is an excellent novel reference gene. The approach, referred to as GeneGini, provides rapid and simple characterization of expression-profile distributions and improved normalization of genome-wide expression-profiling data.

  • 47.
    Olsson, T.
    et al.
    Karolinska Inst, Clin Neurosci, Stockholm, Sweden..
    Huang, J.
    Karolinska Inst, Clin Neurosci, Stockholm, Sweden..
    Hellström, Cecilia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. Royal Inst Technol, Sci Life Lab, Stockholm, Sweden..
    Tengvall, K.
    Karolinska Inst, Clin Neurosci, Stockholm, Sweden..
    Kammer, P.
    German Canc Res Ctr, Heidelberg, Germany..
    Bistrom, M.
    Umea Univ, Dept Pharmacol & Clin Neurosci, Umea, Sweden..
    Bomfim, I. Lima
    Karolinska Inst, Clin Neurosci, Stockholm, Sweden..
    Stridh, P.
    Karolinska Inst, Clin Neurosci, Stockholm, Sweden..
    Butt, J.
    German Canc Res Ctr, Heidellberg, Germany..
    Nicole, B.
    German Canc Res Ctr, Heidelberg, Germany..
    Michel, A.
    German Canc Res Ctr, Heidelberg, Germany..
    Ayoglu, Burcu
    KTH, Centres, Science for Life Laboratory, SciLifeLab. Royal Inst Technol, Sci Life Lab, Stockholm, Sweden..
    Lundberg, K.
    Karolinska Inst, Dept Med, Stockholm, Sweden..
    Lundberg, I.
    Karolinska Inst, Dept Med, Stockholm, Sweden..
    Olafsson, S.
    deCode Genet, Reykavik, Iceland..
    Jonsdottir, I.
    deCode Genet, Reykavik, Iceland..
    Stefansson, K.
    deCode Genet, Reykavik, Iceland..
    Dilthey, A.
    Welcome Trust Ctr Human Genet, Oxford, England..
    Hillert, J.
    Karolinska Inst, Clin Neurosci, Stockholm, Sweden..
    Alfredsson, L.
    Karolinska Inst, Inst Environm Med, Stockholm, Sweden..
    Sundstrom, P.
    Umea Univ, Pharmacol & Clin Neurosci, Umea, Sweden..
    Nilsson, Peter
    KTH, Centres, Science for Life Laboratory, SciLifeLab. Royal Inst Technol, Sci Life Lab, Stockholm, Sweden..
    Waterboer, T.
    German Canc Res Ctr, Heidelberg, Germany..
    Kockum, I.
    Karolinska Inst, Clin Neurosci, Stockholm, Sweden..
    Molecular mimicry between the autoantigen Anoctamin 2 and Epstein Barr virus nuclear antigen 1 associates with increased risk for multiple sclerosis2018In: Multiple Sclerosis, ISSN 1352-4585, E-ISSN 1477-0970, Vol. 24, p. 64-64Article in journal (Other academic)
  • 48.
    Orlova, Anna
    et al.
    Uppsala Univ, Dept Med Chem, SE-75283 Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, Uppsala, Sweden..
    Bass, Tarek
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Rinne, Sara S.
    Uppsala Univ, Dept Med Chem, SE-75283 Uppsala, Sweden..
    Leitao, Charles Dahlsson
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Rosestedt, Maria
    Uppsala Univ, Dept Med Chem, SE-75283 Uppsala, Sweden..
    Atterby, Christina
    Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden..
    Gudmundsdotter, Lindvi
    Affibody AB, Solna, Sweden..
    Frejd, Fredrik Y.
    Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden.;Affibody AB, Solna, Sweden..
    Löfblom, John
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Tolmachev, Vladimir
    Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden..
    Ståhl, Stefan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Evaluation of the Therapeutic Potential of a HER3-Binding Affibody Construct TAM-HER3 in Comparison with a Monoclonal Antibody, Seribantumab2018In: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 15, no 8, p. 3394-3403Article in journal (Refereed)
    Abstract [en]

    Human epidermal growth factor receptor type 3 (HER3) is recognized to be involved in resistance to HER targeting therapies. A number of HER3-targeting monoclonal antibodies are under clinical investigation as potential cancer therapeutics. Smaller high-affinity scaffold proteins are attractive non-Fc containing alternatives to antibodies. A previous study indicated that anti-HER3 affibody molecules could delay the growth of xenografted HER3-positive tumors. Here, we designed a second-generation HER3-targeting construct (TAM-HER3), containing two HER3-specific affibody molecules bridged by an albumin-binding domain (ABD) for extension of blood circulation. Receptor blocking activity was demonstrated in vitro. In mice bearing BxPC-3 xenografts, the therapeutic efficacy of TAM-HER3 was compared to the HER3-specific monoclonal antibody seribantumab (MM-121). TAM-HER3 inhibited heregulin-induced phosphorylation in a panel of HER3-expressing cancer cells and was found to be equally as potent as seribantumab in terms of therapeutic efficacy in vivo and with a similar safety profile. Median survival times were 60 days for TAM-HER3, 54 days for seribantumab, and 41 days for the control group. No pathological changes were observed in cytopathological examination. The multimeric HER3-binding affibody molecule in fusion to ABD seems promising for further evaluation as candidate therapeutics for treatment of HER3-overexpressing tumors.

  • 49. Pavankumar, A. R.
    et al.
    Zelenin, Sergey
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundin, A.
    Schulte, T.
    Rajarathinam, K.
    Rebellato, P.
    Ardabili, Sahar
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Salas, J.
    Achour, A.
    Russom, Aman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Bioanalytical advantages of a novel recombinant apyrase enzyme in ATP-based bioluminescence methods2018In: Analytica Chimica Acta, ISSN 0003-2670, E-ISSN 1873-4324, Vol. 1025, p. 118-123Article in journal (Refereed)
    Abstract [en]

    Ultrasensitive measurements of intracellular ATP (intATP) based on the firefly luciferase reactions are frequently used to enumerate bacterial or mammalian cells. During clinical applications, extracellular ATP (extATP) should be depleted in biological samples since it interferes with intATP and affects the quantification of bacteria. The extATP can be eliminated by ATP-degrading enzymes but complete hydrolysis of extATP remains a challenge for today's commercial enzymes. The catalytic efficiency of ATP-degrading enzymes depends on enzyme characteristics, sample composition and the ability to deplete diphosphates, triphosphates and their complexes generated during the reaction. This phenomenon restricts the usage of bioluminescence-based ATP methods in clinical diagnostics. In light of this, we have developed a recombinant Shigella flexneri apyrase (RSFA) enzyme and analysed its ATP depletion potential with five commercial biochemical sources including potato apyrase, acid phosphatase, alkaline phosphatase, hexokinase and glycerol kinase. The RSFA revealed superior activity by completely eliminating the extracellular ATP and ATP-complexes, even in biological samples like urine and serum. Therefore, our results can potentially unwrap the chemical and bio-analytical applications of ATP-based bioluminescence tests to develop highly sensitive point-of-care diagnostics.

  • 50.
    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.

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