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  • 1.
    Friedman, Mikaela
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Affibody molecules targeting the epidermal growth factor receptor for tumor imaging applications2008Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    Tumor targeting and molecular imaging of protein markers specific for or overexpressed in tumors can add useful information in deciding upon treatment and assessing the response to treatment for a cancer patient. The epidermal growth factor receptor (EGFR) is one such tumor-associated receptor, which expression is abnormal or upregulated in various cancers and associated with a poor patient prognosis. It is therefore considered a good target for imaging and therapy. Monoclonal antibodies and recently also antibody fragments have been investigated for in vivo medical applications, like therapy and imaging. In molecular imaging a small sized targeting agent is favorable to give high contrast and therefore, antibody fragments and lately also small affinity proteins based on a scaffold structure constitute promising alternatives to monoclonal antibodies. Affbody molecules are such affinity proteins that are developed by combinatorial protein engineering of the 58 amino acid residue Z-domain scaffold, derived from protein A.

    In this thesis, novel Affibody molecules specific for the EGFR have been selected from a combinatorial library using phage display technology. Affibody molecules with moderate high affinity demonstrated specific binding to native EGFR on the EGFR-expressing epithelial carcinoma A431 cell line. Further cellular assays showed that the EGFR-binding Affibody molecules could be labeled with radiohalogens or radiometals with preserved specific binding to EGFR-expressing cells. In vitro, the Affibody molecule demonstrated a high uptake and good retention to EGFR-expressing cells and was found to internalize. Furthermore, successful imaging of tumors in tumor-bearing mice was demonstrated. Low nanomolar or subnanomolar affinities are considered to be desired for successful molecular imaging and a directed evolution to increase the affinity was thus performed. This resulted in an approximately 30-fold improvement in affinity, yielding EGFR-binding Affibody molecules with KD´s in the 5-10 nM range, and successful targeting of A431 tumors in tumor-bearing mice. To find a suitable format and labeling, monomeric and dimeric forms of one affinity matured binder were labeled with 125I and 111In. The radiometal-labeled monomeric construct, 111In-labeled-ZEGFR:1907, was found to provide the best tumor-to-organ ratio due to good tumor localization and tumor retention. The tumor-to-blood ratio, which is often used as a measure of contrast, was 31±8 at 24 h post injection and the tumor was clearly visualized by gamma-camera imaging.

    Altogether, the EGFR-binding Affibody molecule is considered a promising candidate for further development of tumor imaging tracers for EGFR-expressing tumors and metastases. This could simplify the stratification of patients for treatment and the assessment of the response of treatment in patients.

  • 2.
    Friedman, Mikaela
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Lindström, Sara
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Engineering and characterization of a bispecific HER2 × EGFR-binding affibody molecule2009In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 54, p. 121-131Article in journal (Refereed)
    Abstract [en]

    HER2 (human epidermal-growth-factor receptor-2; ErbB2) and EGFR (epidermal-growth-factor receptor) are overexpressed in various forms of cancer, and the co-expression of both HER2 and EGFR has been reported in a number of studies. The simultaneous targeting of HER2 and EGFR has been discussed as a strategy with which to potentially increase efficiency and selectivity in molecular imaging and therapy of certain cancers. In an effort to generate a molecule capable of bispecifically targeting HER2 and EGFR, a gene fragment encoding a bivalent HER2-binding affibody molecule was genetically fused in-frame with a bivalent EGFR-binding affibody molecule via a (G(4)S)(3) [(Gly(4)-Ser)(3)]-encoding gene fragment. The encoded 30 kDa affibody construct (Z(HER2))(2)-(G(4)S)(3)-(Z(EGFR))(2), with potential for bs (bispecific) binding to HER2 and EGFR, was expressed in Escherichia coli and characterized in terms of its binding capabilities. The retained ability to bind HER2 and EGFR separately was demonstrated using both biosensor technology and flow-cytometric analysis, the latter using HER2- and EGFR-overexpressing cells. Furthermore, simultaneous binding to HER2 and EGFR was demonstrated in: (i) a sandwich format employing real-time biospecific interaction analysis where the bs affibody molecule bound immobilized EGFR and soluble HER2; (ii) immunofluorescence microscopy, where the bs affibody molecule bound EGFR-overexpressing cells and soluble HER2; and (iii) a cell-cell interaction analysis where the bs affibody molecule bound HER2-overexpressing SKBR-3 cells and EGFR-overexpressing A-431 cells. This is, to our knowledge, the first reported bs affinity protein with potential ability for the simultaneous targeting of HER2 and EGFR. The potential future use of this and similar constructs, capable of bs targeting of receptors to increase the efficacy and selectivity in imaging and therapy, is discussed.

  • 3.
    Friedman, Mikaela
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Nordberg, Erika
    Uppsala Univ, Dept Oncol Radiol & Clin Immunol, Rudbeck Lab.
    Höidén-Guthenberg, Ingmarie
    Affibody AB, Bromma.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Adams, Gregory P.
    Fox Chase Canc Ctr, Dept Med Oncol, Philadelphia.
    Nilsson, Fredrik Y.
    Uppsala Univ, Dept Oncol Radiol & Clin Immunol, Rudbeck Lab.
    Carlsson, Jörgen
    Uppsala Univ, Dept Oncol Radiol & Clin Immunol, Rudbeck Lab.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Phage display selection of Affibody molecules with specific binding to the extracellular domain of the epidermal growth factor receptor2007In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 20, no 4, p. 189-199Article in journal (Refereed)
    Abstract [en]

    Affibody molecules specific for the epidermal growth factor receptor (EGFR) have been selected by Phage display technology from a combinatorial protein library based on the 58-residue, protein A-derived Z domain. EGFR is overexpressed in various malignancies and is frequently associated with poor patient prognosis, and the information provided by targeting this receptor could facilitate both patient diagnostics and treatment. Three selected Affibody variants were shown to selectively bind to the extracellular domain of EGFR (EGFR-ECD). Kinetic biosensor analysis revealed that the three monomeric Affibody molecules bound with similar affinity, ranging from 130 to 185 nM. Head-to-tail dimers of the Affibody molecules were compared for their binding to recombinant EGFR-ECD in biosensor analysis and in human epithelial cancer A431 cells. Although the dimeric Affibody variants were found to bind in a range of 2550 nM affinities in biosensor analysis, they were found to be low nanomolar binders in the cellular assays. Competition assays using radiolabeled Affibody dimers confirmed specific EGFR-binding and demonstrated that the three Affibody molecules competed for the same epitope. Immunofluorescence microscopy demonstrated that the selected Affibody dimers were initially binding to EGFR at the cell surface of A431, and confocal microscopy analysis showed that the Affibody dimers could thereafter be internalized. The potential use of the described Affibody molecules as targeting agents; for radionuclide based imaging applications in various carcinomas ils discussed.

  • 4.
    Friedman, Mikaela
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Orlova, Anna
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
    Johansson, Eva
    Affibody AB, Bromma.
    Eriksson, Tove L. J.
    Affibody AB, Bromma.
    Höidén-Guthenberg, Ingmarie
    Affibody AB, Bromma.
    Tolmachev, Vladimir
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
    Nilsson, Fredrik Y.
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding Affibody molecule2008In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 376, no 5, p. 1388-1402Article in journal (Refereed)
    Abstract [en]

    The epidermal growth factor receptor 1 (EGFR) is overexpressed in various malignancies and is associated with a poor patient prognosis. A small, receptor-specific, high-affinity imaging agent would be a useful tool in diagnosing malignant tumors and in deciding upon treatment and assessing the response to treatment. We describe here the affinity maturation procedure for the generation of Affibody molecules binding with high affinity and specificity to EGFR. A library for affinity maturation was constructed by rerandomization of selected positions after the alignment of first-generation binding variants. New binders were selected with phage display technology, using a single oligonucleotide in a single-library effort, and the best second-generation binders had an approximately 30-fold improvement in affinity (K-d = 5-10 nM) for the soluble extracellular domain of EGFR in biospecific interaction analysis using Biacore. The dissociation equilibrium constant, Kd, was also determined for the Affibody with highest affinity using EGFR-expressing A431 cells in flow cytometric analysis (K-d = 2.8 nM). A retained high specificity for EGFR was verified by a dot blot assay showing staining only of EGFR proteins among a panel of serum proteins and other EGFR family member proteins (HER2, HER3, and HER4). The EGFR-binding Affibody molecules were radiolabeled with indium-111, showing specific binding to EGFR-expressing A431 cells and successful targeting of the A431 tumor xenografts with 4-6% injected activity per gram accumulated in the tumor 4 h postinjection.

  • 5.
    Friedman, Mikaela
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Engineered affinity proteins for tumour-targeting applications2009In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 53, p. 1-29Article, review/survey (Refereed)
    Abstract [en]

    Targeting of tumour-associated antigens is an expanding treatment modality in clinical oncology as an alternative to, or in combination with, conventional treatments, such as chemotherapy, external-radiation therapy and surgery. Targeting of antigens that are unique or more highly expressed in tumours than in normal tissues can be used to increase the specificity and reduce the cytotoxic effect on normal tissues. Several targeting agents have been studied for clinical use, where monoclonal antibodies have been the ones most widely used. More than 20 monoclonal antibodies are approved for therapy today and the largest field is oncology. Advances in genetic engineering and in vitro selection technology has enabled the feasible high-throughput generation of monoclonal antibodies, antibody derivatives [e.g. scFvs, Fab molecules, dAbs (single-domain antibodies), diabodies and minibodies] and more recently also non-immunoglobulin scaffold proteins. Several of these affinity proteins have been investigated for both in vivo diagnostics and therapy. Affinity proteins in tumour-targeted therapy can affect tumour progression by altering signal transduction or by delivering a payload of toxin, drug or radionuclide. The ErbB receptor family has been extensively studied as biomarkers in tumour targeting, primarily for therapy using monoclonal antibodies. Two receptors in the ErbB family, EGFR (epidermal growth factor receptor) and HER2 (epidermal growth factor receptor 2), are over-expressed in various malignancies and associated with poor patient prognosis and are therefore interesting targets for solid turnours. In the present review, strategies are described for tumour targeting of solid turnours using affinity proteins to deliver radionuclides, either for molecular imaging or radiotherapy. Antibodies, antibody derivatives and non-immunoglobulin scaffold proteins are discussed with a certain focus on the affibody (Affibody (R)) molecule.

  • 6.
    Myhre, Susanna
    et al.
    Univ Gothenburg, Inst Biomed, Gothenburg, Sweden..
    Magnusson, Maria
    Univ Gothenburg, Inst Biomed, Gothenburg, Sweden..
    Friedman, Mikaela
    KTH.
    Henning, Petra
    Univ Gothenburg, Inst Biomed, Gothenburg, Sweden..
    Ståhl, Stefan
    KTH.
    Lindholm, Leif
    Univ Gothenburg, Inst Biomed, Gothenburg, Sweden..
    A re-targeted adenovirus with dual specificity: Evaluation of binding specificities for affibody molecules at different positions in the HI-loop of the fiber knob2007In: Human Gene Therapy, ISSN 1043-0342, E-ISSN 1557-7422, Vol. 18, no 10, p. 1002-1002Article in journal (Other academic)
  • 7.
    Nordberg, Erika
    et al.
    Rudbeck Laboratory, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences, Uppsala University.
    Friedman, Mikaela
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Göstring, Lovisa
    Rudbeck Laboratory, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences, Uppsala University.
    Adams, Gregory P.
    Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Nilsson, Fredrik Y.
    Rudbeck Laboratory, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences, Uppsala University.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Glimelius, Bengt
    Rudbeck Laboratory, Oncology, Radiology and Clinical Immunology, Uppsala University.
    Carlsson, Jörgen
    Rudbeck Laboratory, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences, Uppsala University.
    Cellular studies of binding, internalization and retention of a radiolabeled EGFR-binding affibody molecule2007In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 34, no 6, p. 609-618Article in journal (Refereed)
    Abstract [en]

    Introduction: The cellular binding and processing of an epidermal growth factor receptor (EGFR) targeting affibody molecule, (Z(EGFR:955))(2), was studied. This new and small molecule is aimed for applications in nuclear medicine. The natural ligand epidermal growth factor (EGF) and the antibody cetuximab were studied for comparison.

    Methods: All experiments were made with cultured A431 squamous carcinoma cells. Receptor specificity, binding time patterns, retention and preliminary receptor binding site localization studies were all made after (125) I, labeling. Internalization was studied using Oregon Green 488, Alexa Fluor 488 and CypHer5E markers.

    Results: [(125) I](Z(EGFR:955))(2) and [(125) I]cetuximab gave a maximum cellular uptake of I-125 within 4 to 8 h of incubation, while [(125) I]EGF gave a maximum uptake already after 2 h. The retention studies showed that the cell-associated fraction of 125 1 after 48 It of incubation was similar to 20% when delivered as [(125) I](Z(FGFR:955))(2) and similar to 25% when delivered as [I-125] cetuximab. [(125) I]EGF-mediated delivery gave a faster (125) I release, where almost all cell-associated radioactivity had disappeared within 24 It. All three substances were internalized as demonstrated with confocal microscopy. Competitive binding studies showed that both EGF and cetuximab inhibited binding Of (Z(EGFR:955))(2) and indicated that the three substances competed for an overlapping binding site.

    Conclusion: The results gave information on cellular processing of radionuclides when delivered with (Z(EGFR:955))(2) in comparison to delivery with EGF and cetuximab. Competition assays suggested that [I-125](Z(EGFR:955))(2) bind to Domain III of EGFR. The affibody molecule (Z(EGFR:955))(2) can be a candidate for EGFR imaging applications in nuclear medicine.

  • 8.
    Nordberg, Erika
    et al.
    Uppsala Univ, Dept Oncol Radiol & Clin Immunol, Rudbeck Lab.
    Orlova, Anna
    Uppsala Univ, Dept Oncol Radiol & Clin Immunol, Rudbeck Lab.
    Friedman, Mikaela
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Tolmachev, Vladimir
    Uppsala Univ, Dept Oncol Radiol & Clin Immunol, Rudbeck Lab.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Nilsson, Fredrik Y.
    Uppsala Univ, Dept Oncol Radiol & Clin Immunol, Rudbeck Lab.
    Glimelius, Bengt
    Uppsala Univ, Akad Hosp, Rudbeck Lab.
    Carlsson, Jörgen
    Uppsala Univ, Dept Oncol Radiol & Clin Immunol, Rudbeck Lab.
    In vivo and in vitro uptake of 111In, delivered with the affibody molecule (ZEGFR:955)2, in EGFR expressing tumour cells2008In: Oncology Reports, ISSN 1021-335X, E-ISSN 1791-2431, Vol. 19, no 4, p. 853-857Article in journal (Refereed)
    Abstract [en]

    The epidermal growth factor receptor, EGFR, is overexpressed in many carcinomas. Targeting this receptor with radionuclides is important for imaging and therapy applications in nuclear medicine. We investigated the in vitro and in vivo properties of a new high affinity EGFR binding affibody molecule, (Z(EGFR:955))(2), when conjugated with CHXA"-DTPA and labelled with In-111. The binding time patterns and retention studies were performed using cultured squamous carcinoma A431 cells that overexpress EGFR. In the in vivo studies, female BALB/c nu/nu mice carrying tumours from xenografted A431 cells were used. The in vitro studies showed EGFR specific binding, high uptake and good retention of In-111 when delivered as [In-111](Z(EGFR:955))(2). The retention after 72 h of incubation was 38.0 +/- 1.15% of the initial level. The biodistribution study showed a tumour specific In-111 uptake of 3.8 +/- 1.4% of injected dose per gram turnout tissue 4 h post-injection. The tumour to blood ratio was 9.1 and the tumours could easily be visualized with a gamma camera at this time-point. In-111 delivered with [In-111](Z(EGFR:955))(2) gave an EGFR specific uptake and the results indicated that the (Z(EGFR:955))(2) affibody molecule is a candidate for radionuclide-based tumour imaging. Potential therapy applications are discussed.

  • 9.
    Orlova, A.
    et al.
    Uppsala Univ, Uppsala, Sweden..
    Friedman, M.
    KTH, School of Biotechnology (BIO).
    Sandstroem, M.
    Univ Uppsala Hosp, Uppsala, Sweden..
    Eriksson, T.
    Affibody AB, Bromma, Sweden..
    Ståhl, S.
    KTH, School of Biotechnology (BIO).
    Nilsson, F. Y.
    Affibody AB, Bromma, Sweden..
    Tolmachev, V.
    Affibody AB, Bromma, Sweden..
    Imaging of EGRF expression in xenografts using (111)InBz-DTPA-Z(1907) affibody molecule2007In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 34, p. S222-S222Article in journal (Other academic)
  • 10. Pinitkiatisakul, S.
    et al.
    Mattsson, J.G.
    Wikman, Maria
    KTH, School of Biotechnology (BIO).
    Friedman, Mikaela
    KTH, School of Biotechnology (BIO).
    Bengtsson, K.L.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO).
    Lundén, Anne
    Immunisation of mice against neosporosis with recombinant NcSRS2 iscoms2005In: Veterinary parasitology, ISSN 0304-4017, E-ISSN 1873-2550, Vol. 129, no 1-2, p. 25-34Article in journal (Refereed)
    Abstract [en]

    The coccidian parasite Neospora caninum is an intracellular protozoan, causing abortion in cattle in many countries around the world. In this study, the protective potential of the major N. caninum surface antigen NcSRS2, expressed in Escherichia coli and formulated into immunostimulating complexes (iscoms), was investigated in an experimental mouse model. The recombinant protein was specially designed for binding to iscoms via biotin-streptavidin interaction. Two groups of 10 BALB/c mice were immunised twice, on days 0 and 28 with iscoms containing either the recombinant NcSRS2 (NcSRS2 iscoms) or similar iscoms with NcSRS2 substituted by an unrelated recombinant malaria peptide (M5) as a control (M5 iscoms). A third group of 10 age-matched BALB/c mice served as an uninfected control group. Immunisation with recombinant NcSRS2 iscoms resulted in production of substantial antibody titres against N. caninum antigen, while the mice immunised with M5 iscoms produced only very low levels of antibodies reacting with N. caninum antigen. After challenge infection with N. caninum tachyzoites on day 69, mice immunised with NcSRS2 iscoms showed only mild and transient symptoms, whereas the group immunised with M5 iscoms showed clinical symptoms until the end of the experiment at 31 days post inoculation. A competitive PCR assay detecting Nc5-repeats was applied to evaluate the level of parasite DNA in the brain. The amount of Nc5-repeats in the group vaccinated with NcSRS2 iscoms was significantly lower than in the control group given M5 iscoms. In conclusion, it was found that the recombinant NcSRS2 iscoms induced specific antibodies to native NcSRS2 and immunity sufficient to reduce the proliferation of N. caninum in the brains of immunised mice.

  • 11.
    Ståhl, Stefan
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Friedman, Mikaela
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Carlsson, Jörgen
    Rudbeck Lab, Uppsala University.
    Tolmachev, Vladimir
    Rudbeck Lab, Uppsala University.
    Frejd, Fredrik
    Affibody AB.
    Affibody Molecules for Targeted Radionuclide Therapy2011In: Targeted Radionuclide Therapy: Immunology and Targeted Constructs / [ed] Tod W. Speer, Philadelphia, USA: Lippincott Williams & Wilkins, 2011, p. 49-58Chapter in book (Refereed)
  • 12.
    Tolmachev, Vladimir
    et al.
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
    Friedman, Mikaela
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Sandström, Mattias
    Section of Hospital Physics, Department of Oncology, Uppsala University Hospital.
    Eriksson, Tove L. J.
    Affibody AB, Bromma.
    Rosik, Daniel
    Affibody AB, Bromma.
    Hodik, Monika
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Frejd, Fredrik Y.
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
    Orlova, Anna
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
    Affibody Molecules for Epidermal Growth Factor Receptor Targeting In Vivo: Aspects of Dimerization and Labeling Chemistry2009In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 50, no 2, p. 274-283Article in journal (Refereed)
    Abstract [en]

    Noninvasive detection of epidermal growth factor receptor (EGFR) expression in malignant tumors by radionuclide molecular imaging may provide diagnostic information influencing patient management. The aim of this study was to evaluate a novel EGFR-targeting protein, the Z(EGFR:1907) Affibody molecule, for radionuclide imaging of EGFR expression, to determine a suitable tracer format (dimer or monomer) and optimal label. Methods: An EGFR-specific Affibody molecule, ZEGFR:1907, and its dimeric form, (Z(EGFR:1907))(2), were labeled with In-111 using benzyl-diethylenetriaminepentaacetic acid and with I-125 using p-iodobenzoate. Affinity and cellular retention of conjugates were evaluated in vitro. Biodistribution of radiolabeled Affibody molecules was compared in mice bearing EGFR-expressing A431 xenografts. Specificity of EGFR targeting was confirmed by comparison with biodistribution of non-EGFR-specific counterparts. Results: Head-to-tail dimerization of the Affibody molecule improved the dissociation rate. In vitro, dimeric forms demonstrated superior cellular retention of radioactivity. For both molecular set-ups, retention was better for the In-111-labeled tracer than for the radioiodinated counterpart. In vivo, all conjugates accumulated specifically in xenografts and in EGFRexpressing tissues. The retention of radioactivity in tumors was better in vivo for dimeric forms; however, the absolute uptake values were higher for monomeric tracers. The best tracer, In-111-labeled Z(EGFR:1907), provided a tumor-to-blood ratio of 100 (24 h after injection). Conclusion: The radiometal-labeled monomeric Aff ibody molecule Z(EGFR:1907) has a potential for radionuclide molecular imaging of EGFR expression in malignant tumors.

  • 13.
    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.

  • 14.
    Wikman, Maria
    et al.
    KTH, School of Biotechnology (BIO).
    Friedman, Mikaela
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Pinitkiatisakul, S.
    Andersson, Christin
    KTH, School of Biotechnology (BIO).
    Hemphill, A.
    Lovgren-Bengtsson, K.
    Lunden, A.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    General strategies for efficient adjuvant incorporation of recombinant subunit immunogens2005In: Vaccine, ISSN 0264-410X, E-ISSN 1873-2518, Vol. 23, no 17-18, p. 2331-2335Article in journal (Refereed)
    Abstract [en]

    We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic peptides or lipid tags to improve their capacity to be incorporated into an adjuvant formulation, e.g., immunostimulating complexes (iscoms). Recently, we also explored the strong interaction between biotin and streptavidin to achieve iscom association of recombinant immunogens. Plasmodium falciparum, Toxoplasma gondii and Neospora caninum antigens have served as model immunogens in the different studies. Generated fusion proteins have been found to be successfully incorporated into iscoms and high-titer antigen-specific antibody responses have been obtained upon immunization of mice. We believe that the different concepts presented, utilizing either hydrophobic peptide or lipid tags, or the recently explored biotin-streptavidin principle, offer convenient methods to achieve efficient adjuvant incorporation of recombinant immunogens.

  • 15.
    Wikman, Maria
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Friedman, Mikaela
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Pinitkiatisakul, S.
    Hemphill, A.
    Lövgren-Bengtsson, K.
    Lunden, A.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Applying biotin-streptavidin binding for iscom (immunostimulating complex) association of recombinant immunogens2005In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 41, p. 163-174Article in journal (Refereed)
    Abstract [en]

    We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic peptide or lipid tags to improve their capacity to be incorporated into an adjuvant formulation. In the present study, we have explored the strong interaction between biotin and SA (streptavidin) (K-D approximate to 10(-15) M) to couple recombinant immunogens to iscoms (immunostimulating complexes). Two different concepts were evaluated. In the first concept, a His(6)-tagged SA fusion protein (His(6)-SA) was bound to Ni2+-loaded iscom matrix (iscom without associated protein), and biotinylated immunogens were thereafter associated with the SA-coated iscoms. The immunogens were either biotinylated in vivo on E. coli expression or double biotinylated in vivo and in vitro. In the second concept, the recombinant immunogens were expressed as SA fusion proteins, which were directly bound to a biotinylated iscom matrix. A 53-amino-acid malaria peptide (M), derived from the central repeat region of the Plasmodium faiciparum blood-stage antigen Pf155/RESA, and a 232-amino-acid segment (SRS2') from the central region (from Pro-97 to Lys-328) of the major surface antigen NcSRS2 of the protozoan parasite Neospora caninum, served as model immunogens in the present study. All fusion proteins generated were found to be efficiently expressed and could be recovered to high purity using affinity chromatography. The association between the different immunogen-containing fusion proteins and the corresponding iscom matrix was demonstrated by analytical ultracentrifugation in a sucrose density gradient. However, some fusion proteins were, to a certain extent, also found to associate unspecifically with a regular iscom matrix. Furthermore, selected iscom fractions were demonstrated to induce high-titre antigen-specific antibody responses on immunization of mice. For the particular target immunogen SRS2', the induced antibodies demonstrated reactivity to the native antigen NcSRS2. We believe that the presented concepts offer convenient methods to achieve efficient adjuvant association of recombinant immunogens, and the advantages and disadvantages of the two concepts are discussed.

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