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  • 51.
    Qin, Haiyan
    et al.
    KTH, School of Biotechnology (BIO), Theoretical Chemistry.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Quantum dot-affibody fluorescence probes for single molecule tracking in living cell membranesManuscript (preprint) (Other academic)
  • 52.
    Seijsing, Johan
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Johansson, P.
    Eklund, P.
    Höidén-Guthenberg, I.
    Frejd, F.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Protein Technology.
    Reduction of immunoglobulin G in mice by an affibody molecule blocking the interaction between immunoglobulin G and the neonatal Fc receptorManuscript (preprint) (Other academic)
  • 53.
    Seijsing, Johan
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Lindborg, Malin
    Höidén-Guthenberg, I.
    Bönisch, H.
    Gunneriusson, E.
    Frejd, F.
    Abramsén, L.
    Ekblad, C.
    Löfblom, J.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Protein Technology.
    An engineered affibody molecule with pHdependent binding to FcRn mediates extended circulatory half-lifeof a fusion proteinManuscript (preprint) (Other academic)
  • 54.
    Seijsing, Johan
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Lindborg, Malin
    Höidén-Guthenberg, Ingmarie
    Bönisch, Heiko
    Guneriusson, Elin
    Frejd, Fredrik Y.
    Abrahmsén, Lars
    Ekblad, Caroline
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Protein Technology.
    An engineered affibody molecule with pH-dependent binding to FcRn mediates extended circulatory half-life of a fusion protein2014In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 111, no 48, p. 17110-17115Article in journal (Refereed)
    Abstract [en]

    Proteins endocytosed from serum are degraded in the lysosomes. However, serum albumin (SA) and IgG, through its Fc part, bind to the neonatal Fc receptor (FcRn) at low pH in the endosome after endocytosis, and are transported back to the cellular surface, where they are released into the bloodstream, resulting in an extended serum circulation time. Association with Fc or SA has been used to prolong the in vivo half-life of biopharmaceuticals, using the interaction with FcRn to improve treatment regimens. This has been achieved either directly, by fusion or conjugation to Fc or SA, or indirectly, using SA-binding proteins. The present work takes this principle one step further, presenting small affinity proteins that bind directly to FcRn, mediating extension of the serum half-life of fused biomolecules. Phage display technology was used to select affibody molecules that can bind to FcRn in the pH-dependent manner required for rescue by FcRn. The biophysical and binding properties were characterized in vitro, and the affibody molecules were found to bind to FcRn more strongly at low pH than at neutral pH. Attachment of the affibody molecules to a recombinant protein, already engineered for increased halflife, resulted in a nearly threefold longer half-life in mice. These tags should have general use as fusion partners to biopharmaceuticals to extend their half-lives in vivo.

  • 55.
    Seijsing, Johan
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Lindborg, Malin
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Protein Technology.
    Robust Expression of the Human Neonatal Fc Receptor in a Truncated Soluble Form and as a Full-Length Membrane-Bound Protein in Fusion with eGFP2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 11, p. e81350-Article in journal (Refereed)
    Abstract [en]

    Studies on the neonatal Fc receptor (FcRn) have revealed a multitude of important functions in mammals, including protection of IgG and serum albumin (SA) from lysosomal degradation. The pharmacokinetic behavior of therapeutic antibodies, IgG-Fc- and SA-containing drugs is therefore influenced by their interaction with FcRn. Pre-clinical development of such drugs is facilitated if their interaction with FcRn can be studied in vitro. For this reason we have developed a robust system for production of the soluble extracellular domain of human FcRn as well as the full-length receptor as fusion to green fluorescent protein, taking advantage of a lentivirus-based gene delivery system where stable over-expressing cells are easily and rapidly generated. Production of the extracellular domain in multiple-layered culture flasks, followed by affinity purification using immobilized IgG, resulted in capture of milligram amounts of soluble receptor per liter cell culture with retained IgG binding. The receptor was further characterized by SDS-PAGE, western blotting, circular dichroism spectroscopy, ELISA, surface plasmon resonance and a temperature stability assay showing a functional and stable protein of high purity. The full-length receptor was found to be successfully over-expressed in a membrane-bound form with retained pH-dependent IgG- and SA-binding.

  • 56.
    Seijsing, Johan
    et al.
    KTH, School of Biotechnology (BIO). Stockholm Univ, Wenner Gren Inst, Dept Mol Biosci, Stockholm, Sweden..
    Yu, Shengze
    KTH, School of Biotechnology (BIO).
    Frejd, Fredrik Y.
    Affibody AB, Gunnar Asplunds Alle 24, S-17163 Solna, Sweden..
    Hoiden-Guthenberg, Ingmarie
    Affibody AB, Gunnar Asplunds Alle 24, S-17163 Solna, Sweden..
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO).
    In vivo depletion of serum IgG by an affibody molecule binding the neonatal Fc receptor2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 5141Article in journal (Refereed)
    Abstract [en]

    Lowering the total level of Immunoglobulin G (IgG) in circulation is a promising general treatment option for many autoimmune diseases driven by pathogenic autoantibodies. The half-life of IgG in circulation is unusually long as a consequence of its interaction with the neonatal Fc receptor (FcRn), which protects it from lysosomal degradation by cells in contact with blood. Blocking the IgG/FcRn interaction prevents FcRn-mediated rescue, which may lead to increased catabolism and a lowering of the total IgG level. Here, we find that an engineered alternative scaffold protein, an affibody molecule, interacting specifically with FcRn, is able to block the IgG/FcRn interaction in vitro. The affibody molecule (Z(FcRn)) was expressed alone or as a fusion to an albumin binding domain (ABD), to extend its half-life in circulation, in both cases with retained affinity and blocking potential. Repeated i.v. injections in mice of Z(FcRn) and Z(FcRn)-ABD were found to result in an up to 40% reduction of the IgG serum-level after 5 days. Potential applications of Z(FcRn) as a general treatment modality for autoimmune diseases are discussed.

  • 57. Sekiguchi, M.
    et al.
    Iwasaki, T.
    Shibasaki, S.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Protein Technology.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Protein Technology.
    Sano, H.
    Affibody Molecules Inhibiting The Interaction Between Ras And Raf Suppress The Proliferation And The Production Of Inflammatory Mediators By Synovial Cells2014In: Annals of the Rheumatic Diseases, ISSN 0003-4967, E-ISSN 1468-2060, Vol. 73, p. 848-848Article in journal (Other academic)
  • 58. Shibasaki, Seiji
    et al.
    Karasaki, Miki
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Protein Technology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Protein Technology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Sano, Hajime
    Iwasaki, Tsuyoshi
    Inhibitory effects of H-Ras/Raf-1–binding affibody molecules on synovial cell function2014In: AMB Express, ISSN 2191-0855, E-ISSN 2191-0855, Vol. 4, no 82Article in journal (Refereed)
    Abstract [en]

    Affibody molecules specific for H-Ras and Raf-1 were evaluated for their ability to inhibit synovial cell function. Affibody molecules targeting H-Ras (Zras122, Zras220, and Zras521) or Raf-1 (Zraf322) were introduced into the MH7A synovial cell line using two delivery methods: transfection with plasmids encoding the affibody molecules or direct introduction of affibody protein using a cell-penetrating peptide reagent. Interleukin-6 (IL-6) and prostaglandin E2 (PGE2) production by MH7A cells were analyzed by enzyme-linked immunosorbent assay after stimulation with tumor necrosis factor-alpha (TNF-α). Cell proliferation was also analyzed. Phosphorylation of extracellular signal-regulated kinase (ERK) was analyzed by western blot. All affibody molecules could inhibit IL-6 and PGE2 production in TNF-α-stimulated MH7A cells. The inhibitory effect was stronger when affibody molecules were delivered as proteins via a cell-penetrating peptide reagent than when plasmid-DNA encoding the affibody moelcules was transfected into the cells. Plasmid-expressed Zras220 inhibited phosphorylation of ERK in TNF-α-stimulated MH7A cells. Protein-introduced Zraf322 inhibited the production of IL-6 and PGE2 and inhibited cell proliferation in MH7A cells. These findings suggest that affibody molecules specific for H-Ras and Raf-1 can affect intracellular signal transduction through the MAP kinase pathway to inhibit cell proliferation and production of inflammatory mediators by synovial cells.

  • 59.
    Ståhl, Stefan
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Protein Technology.
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Protein Technology.
    Frejd, Fredrik Y.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Protein Technology.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Affibody Molecules in Biotechnological and Medical Applications2017In: Trends in Biotechnology, ISSN 0167-7799, E-ISSN 1879-3096, Vol. 35, no 8, p. 691-712Article, review/survey (Refereed)
    Abstract [en]

    Affibody molecules are small (6.5-kDa) affinity proteins based on a three-helix bundle domain framework. Since their introduction 20 years ago as an alternative to antibodies for biotechnological applications, the first therapeutic affibody molecules have now entered clinical development and more than 400 studies have been published in which affibody molecules have been developed and used in a variety of contexts. In this review, we focus primarily on efforts over the past 5 years to explore the potential of affibody molecules for medical applications in oncology, neurodegenerative, and inflammation disorders, including molecular imaging, receptor signal blocking, and delivery of toxic payloads. In addition, we describe recent examples of biotechnological applications, in which affibody molecules have been exploited as modular affinity fusion partners.

  • 60. Tolmachev, V.
    et al.
    Hofström, Camilla
    KTH, School of Biotechnology (BIO), Protein Technology.
    Altai, M.
    Honarvar, H.
    Strand, J.
    Malmberg, J.
    Hosseinimehr, S. J.
    Orlova, A.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Protein Technology.
    HAHAHA-, HIHIHI-. HKHKHK. HHHHHH- and HEHEHE- tags: Influence of position and composition of histidine-containing tags on biodistribution of [99mTc(CO) 3]+- labeled affibody molecules2013In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 40, p. S185-S185Article in journal (Other academic)
  • 61. Tolmachev, V.
    et al.
    Hofström, Camilla
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Strand, J.
    Varasteh, Z.
    Sandström, M.
    Orlova, A.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    99mTc(CO)3-HEHEHE-ZIGF1R:4551 affibody molecule, a poteintial imaging agent for visualization of insulin-like growth factor-1 receptor in malignant tumors.2012In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 39, p. S300-S300Article in journal (Other academic)
  • 62. Tolmachev, Vladimir
    et al.
    Hofström, Camilla
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Malmberg, Jennie
    Ahlgren, Sara
    Hosseinimehr, Seyed Jalal
    Sandström, Mattias
    Abrahmsen, Lars
    Orlova, Anna
    Gräslund, Torbjorn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    HEHEHE-Tagged Affibody Molecule May Be Purified by IMAC, Is Conveniently Labeled with [Tc-99m(CO)(3)](+), and Shows Improved Biodistribution with Reduced Hepatic Radioactivity Accumulation2010In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 21, no 11, p. 2013-2022Article in journal (Refereed)
    Abstract [en]

    Affibody molecules are a class of small (ca. 7 kDa) robust scaffold proteins suitable for radionuclide molecular imaging of therapeutic targets in vivo. A hexahistidine tag at the N-terminus streamlines development of new imaging probes by enabling facile purification using immobilized metal ion affinity chromatography (IMAC), as well as convenient [Tc-99m(CO)(3)](+)-labeling. However, previous studies in mice have demonstrated that Affibody molecules labeled by this method yield higher liver accumulation of radioactivity, compared to the same tracer lacking the hexahistidine tag and labeled by an alternative method. Two variants of the HER2-binding Affibody molecule Z(HER2:342) were made in an attempt to create a tagged tracer that could be purified by immobilized metal affinity chromatography, yet would not result in anomalous hepatic radioactivity accumulation following labeling with [Tc-99m(CO)(3)](+). In one construct, the hexahistidine tag was moved to the C-terminus. In the other construct, every second histidine residue in the hexahistidine tag was replaced by the more hydrophilic glutamate, resulting in a HEHEHE-tag. Both variants, denoted Z(HER2:342)-H-6 and (HE)(3)-Z(HER2:342), respectively, could be efficiently purified using IMAC and stably labeled with [Tc-99m(CO)(3)](+) and were subsequently compared with the parental H-6-Z(HER2:342) having an N-terminal hexahistidine tag. All three variants were demonstrated to specifically bind to HER2-expressing cells in vitro. The hepatic accumulation of radioactivity in a murine model was 2-fold lower with [Tc-99m(CO)(3)](+)-Z(HER2:342)-H-6 compared to [Tc-99m(CO)(3)](+)-H-6-Z(HER2:342), and more than 10-fold lower with [Tc-99m(CO)(3)](+)-(HE)(3)-Z(HER2:342). These differences translated into appreciably superior tumor-to-liver ratio for [Tc-99m(CO)(3)](+)-(HE)(3)-Z(HER2:342) compared to the alternative conjugates. This information might be useful for development of other scaffold-based molecular imaging probes.

  • 63. Tolmachev, Vladimir
    et al.
    Malmberg, Jennie
    Hofström, Camilla
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Abrahmsén, Lars
    Bergman, Thomas
    Sjöberg, Anna
    Sandström, Mattias
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Orlova, Anna
    Imaging of Insulinlike Growth Factor Type 1 Receptor in Prostate Cancer Xenografts Using the Affibody Molecule (111)In-DOTA-Z(IGF1R:4551)2012In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 53, no 1, p. 90-97Article in journal (Refereed)
    Abstract [en]

    One of the pathways leading to androgen independence in prostate cancer involves upregulation of insulinlike growth factor type 1 receptor (IGF-1R). Radionuclide imaging of IGF-1R in tumors might be used for selection of patients who would most likely benefit from IGF-1R-targeted therapy. The goal of this study was to evaluate the feasibility of in vivo radionuclide imaging of IGF-1R expression in prostate cancer xenografts using a small nonimmunoglobulin-derived binding protein called an Affibody molecule. Methods: The IGF-1R-binding Z(IGF1R:4551) Affibody molecule was site-specifically conjugated with a maleimido derivative of DOTA and labeled with (111)In. The binding of radiolabeled Z(IGF1R:4551) to IGF-1R-expressing cells was evaluated in vitro and in vivo. Results: DOTA-Z(IGF1R:4551) can be stably labeled with (111)In with preserved specific binding to IGF-1R-expressing cells in vitro. In mice, (111)In-DOTAZ(IGF1R):(4551) accumulated in IGF-1R-expressing organs (pancreas, stomach, lung, and salivary gland). Receptor saturation experiments demonstrated that targeting of DU-145 prostate cancer xenografts in NMRI nu/nu mice was IGF-1R-specific. The tumor uptake was 1.1 +/- 0.3 percentage injected dose per gram, and the tumor-to-blood ratio was 3.2 +/- 0.2 at 8 h after injection. Conclusion: This study demonstrates the feasibility of in vivo targeting of IGF-1R-expressing prostate cancer xenografts using an Affibody molecule. Further development of radiolabeled Affibody molecules might provide a useful clinical tool for stratification of patients with prostate cancer for IGF-1R-targeting therapy.

  • 64.
    Vernet, Erik
    et al.
    KTH, School of Biotechnology (BIO).
    Konrad, Anna
    KTH, School of Biotechnology (BIO).
    Lundberg, Emma
    KTH, School of Biotechnology (BIO).
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO).
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO).
    Affinity-based entrapment of the HER2 receptor in the endoplasmic reticulum using an affibody molecule2008In: Journal of immunological methods, ISSN 0022-1759, Vol. 338, p. 1-6Article in journal (Refereed)
    Abstract [en]

    Interference with the export of cell surface receptors can be performed through co-expression of specific affinity molecules designed for entrapment in the endoplasmic reticulum during the export process. We describe the investigation of a small (6 kDa) non-immunoglobulin-based HER2 receptor binding affibody molecule (ZHER2:00477), for use in affinity mediated entrapment of the HER2 receptor in the ER. Constructs encoding ZHER2:00477 or a control affibody protein, with or without ER-retention peptide extensions (KDEL), were expressed in the HER2 over-expressing cell line SKOV-3. Intracellular expression of the full-length affibody constructs could be confirmed by probing cell extracts by Western blotting. Confocal immunofluorescence microscopy experiments showed extensive co-localization of the HER2 receptor and ZHER2:00477-KDEL in the ER, whereas the use of a KDEL-extended control affibody molecule resulted in distinct and separate signals from cell surface-localized HER2 receptor and ER-localized affibody protein. This indicated a capability of the ZHER2:00477-KDEL fusion protein to functionally interfere with the export process of HER2 receptor in a specific manner. Using flow cytometry and cell proliferation analyses, it could be shown that expression of the ZHER2:00477-KDEL fusion construct in the SKOV-3 cell line resulted both in a marked reduction in cell surface level of HER2 receptors and that the cell population doubling time was significantly increased. Expression of the ZHER2:00477-KDEL fusion protein in additional cell lines of different origin and with different expression levels of endogenous HER2 receptor compared to SKOV-3, also resulted in depletion of the cell surface levels of HER2 receptor. This indicated upon a general ability of the ZHER2:00477-KDEL fusion protein to functionally interfere with the export process of HER2.

  • 65.
    Vernet, Erik
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Friedman, Mikaela
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Rigamonti, Nicolò
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Klausing, Sandra
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Affibody-mediated retention of the epidermal growth factor receptor in the secretory compartments leads to inhibition of phosphorylation in the kinase domain2009In: New biotechnology, ISSN 1871-6784, Vol. 25, no 6, p. 417-423Article in journal (Refereed)
    Abstract [en]

    Abnormal activity of the epidermal growth factor receptor (EGFR) is associated with various cancer-related processes and motivates the search for strategies that can selectively block EGFR signalling. In this study, functional knockdown of EGFR was achieved through expression of an affibody construct, (Z(EGFR:1907))(2)-KDEL, with high affinity for EGFR and extended with the amino acids KDEL to make it resident in the secretory compartments. Expression of (Z(EGFR:1907))(2)-KDEL resulted in 80% reduction of the cell surface level of EGFR, and fluorescent staining for EGFR and the (Z(EGFR:1907))(2)-KDEL construct showed overlapping intracellular localisation. Immunocapture of EGFR from cell lysates showed that an intracellular complex between EGFR and the affibody construct had been formed, further indicating a specific interaction between the affibody construct and EGFR. Surface depletion of EGFR led to a dramatic decrease in the amount of kinase domain phosphorylated EGFR, coincident with a significant decrease in the proliferation rate.

  • 66.
    Yu, Shengze
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Alkharusi, Amira
    Sultan Qaboos Univ, Coll Med & Hlth Sci, Muscat, Oman..
    Norstedt, Gunnar
    Sultan Qaboos Univ, Coll Med & Hlth Sci, Muscat, Oman.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden..
    Gräslund, Torbjörn
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    An in vivo half-life extended prolactin receptor antagonist can prevent STAT5 phosphorylation2019In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 14, no 5, article id e0215831Article in journal (Refereed)
    Abstract [en]

    Increasing evidence suggests that signaling through the prolactin/prolactin receptor axis is important for stimulation the growth of many cancers including glioblastoma multiforme, breast and ovarian carcinoma. Efficient inhibitors of signaling have previously been developed but their applicability as cancer drugs is limited by the short in vivo half-life. In this study, we show that a fusion protein, consisting of the prolactin receptor antagonist PrlRA and an albumin binding domain for half-life extension can be expressed as inclusion bodies in Escherichia coli and efficiently refolded and purified to homogeneity. The fusion protein was found to have strong affinity for the two intended targets: the prolactin receptor (K-D = 2.3 +/- 0.2 nM) and mouse serum albumin (K-D = 0.38 +/- 0.01 nM). Further investigation showed that it could efficiently prevent prolactin mediated phosphorylation of STAT5 at 100 nM concentration and above, similar to the PrlRA itself, suggesting a potential as drug for cancer therapy in the future. Complexion with HSA weakened the affinity for the receptor to 21 +/- 3 nM, however the ability to prevent phosphorylation of STAT5 was still prominent. Injection into rats showed a 100-fold higher concentration in blood after 24 h compared to PrlRA itself.

  • 67.
    Yu, Shengze
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Gräslund, Torbjörn
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Human growth hormone can transduce signals to phosphorylate STAT5 as a fusion with an affibody molecule binding the neonatal Fc receptor and an albumin binding domainManuscript (preprint) (Other academic)
    Abstract [en]

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

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

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

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

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

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

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

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

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

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

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

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

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

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

    characterization showed that the fusion proteins could induce phosphorylation of signal

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

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

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