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  • 1.
    Andersson, Christin
    et al.
    KTH, Superseded Departments, Biotechnology.
    Wikman, Maria
    KTH, Superseded Departments, Biotechnology.
    Lövgren-Bengtsson, Karin
    Lundén, Anne
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    In vivo and in vitro lipidation of recombinant immunogens for direct iscom incorporation2001In: Journal of Immunological Methods, ISSN 0022-1759, Vol. 255, no 1-2, p. 135-148Article in journal (Refereed)
    Abstract [en]

    We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic tags to improve their capacity to be incorporated into an adjuvant formulation (J. Immunol. Methods 222 (1999) 171; 238 (2000) 181). Here, we have explored the possibility to use in vivo or in vitro lipidation of recombinant immunogens as means to achieve iscom incorporation through hydrophobic interaction. For the in vivo lipidation strategy, a general expression vector was constructed encoding a composite tag consisting of a sequence (lpp) of the major lipoprotein of E. coli, fused to a dual affinity fusion tag to allow efficient recovery by affinity chromatography. Upon expression in E. coli, fatty acids would be linked to the produced gene products. To achieve in vitro lipidation, the target immunogen would be expressed in frame with an N-terminal His6-ABP affinity tag, in which the hexahistidyl tag was utilized to obtain lipidation via a Cu2+-chelating lipid. A 238 amino acid segment ΔSAG1, from the central region of the major surface antigen SAG1 of Toxoplasma gondii, served as model immunogen in this study. The two generated fusion proteins, lpp-His6-ABP-ΔSAG1 and His6-ABP-ΔSAG1, both expressed at high levels (approximately 5 and 100 mg/l, respectively), could be recovered to high purity by ABP-mediated affinity chromatography, and were evaluated in iscom-incorporation experiments. The His6-ABP-ΔSAG1 fusion protein was associated to iscom matrix with pre-incorporated chelating lipid. Both fusion proteins were found in the iscom fractions after analytical ultracentrifugation in a sucrose gradient, indicating successful iscom incorporation/association. Iscom formation was further supported by electron microscopy analysis. In addition, these iscom preparations were demonstrated to induce high-titer antigen-specific antibody responses upon immunization of mice. For this particular target immunogen, ΔSAG1, the induced antibodies demonstrated poor reactivity to the native antigen, although slightly better for the preparation employing the in vitro lipidation strategy, indicating that ΔSAG1 was suboptimally folded or presented. Nevertheless, we believe that the presented strategies offer convenient alternative ways to achieve efficient adjuvant incorporation for recombinant immunogens.

  • 2. Lindholm, L
    et al.
    Andersson, K
    Boulanger, P
    Frykholm, K
    Henning, P
    Hoeben, R
    Hong, S S
    Johannisson, J
    Magnusson, M
    Myhre, S
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Pettersson, E
    Stahl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Vellinga, J
    Wikman, Maria
    KTH, Superseded Departments, Biotechnology.
    Genetic re-targeting of adenovirus using a hyperstable scFv domain and an affibody (R) molecule against Her2/neu2004In: Molecular Therapy, ISSN 1525-0016, E-ISSN 1525-0024, Vol. 9, p. S250-S250Article in journal (Refereed)
    Abstract [en]

    One important goal in gene therapy is to develop adenovirus (Ad) vectors that are genetically de-targeted as well as re-targeted. Genetic re-targeting of Ad using complex cell-binding ligands has previously not been possible. We have previously demonstrated that ligands for genetic re-targeting of adenoviruses must be able to fold correctly in the cytoplasm of virus producing cells, a milieu that is not conducive to the formation of disulphide bonds.

    Here, we describe functional Ad5 viruses with fibers and pIX capsid proteins genetically modified to contain two types of complex ligands. One is affibody® molecules, corresponding to small (6 kDa) binding proteins developed by combinatorial protein engineering using a single three-helix bundle staphylococcal protein A domain. The other type is hyperstable antibody scFv domains.

    The affibody molecule used here (ZH2N) is directed against Her2/neu. Recombinant viruses were constructed with ZH2N in three different positions: (i) at the C-terminus of shaft repeat 7 of de-knobbed fibers; (ii) at the C-terminus of pIX; and (iii) in the HI-loop of the fiber knob. Each of the viruses exhibited a characteristic phenotype regarding fiber content, growth and ability to infect Her2/neu expressing cells.

    In order to test the potentials of scFv liganded Ad vectors, a hyperstable antibody scFv against b-galactosidase was genetically incorporated into knobless fibers, in tandem with a mutated protein A domain reactive with IgG1 Fc that targeted the virus to Fc-expressing 293 cells. These fibers could be rescued into viable virions that retained the original antigen binding specificity of the scFv, demonstrating the basic potential of hyperstable scFvs for genetic re-targeting of Ad. Quite unexpectedly, the fiber content of Ad with knobless, scFv containing fibers was close to normal in contrast to other Ad with knobless fibers that generally has a much reduced fiber content. The hyperstable scFv was further fused to the C-terminus of the capsid protein pIX. The recombinant molecules could be rescued into viable viruses with wt fibers. The scFv retained its binding-specificity on the recombinant virions.

    The results demonstrate that, contrary to current beliefs, it is possible to construct Ad that genetically incorporates functional scFvs and other complex ligands into the virus fiber and pIX. The feasibility is demonstrated by the creation of different viruses that are re-targeted to Her2/neu. These viruses are currently in pre-clinical development.

  • 3. 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.

  • 4. Steffen, A.C
    et al.
    Orlova, A
    Wikman, Maria
    Nilsson, F.Y
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Adams, G.P
    Tolmachev, V
    Carlsson, J
    Affibody mediated tumor targeting of HER-2 expressing xenografts in mice2006In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 33, no 6, p. 631-638Article in journal (Refereed)
    Abstract [en]

    PURPOSE:

    Targeted delivery of radionuclides for diagnostic and therapeutic applications has until recently largely been limited to receptor ligands, antibodies and antibody-derived molecules. Here, we present a new type of molecule, a 15-kDa bivalent affibody called (Z(HER2:4))(2), with potential for such applications. The (Z(HER2:4))(2) affibody showed high apparent affinity (K (D)=3 nM) towards the oncogene product HER-2 (also called p185/neu or c-erbB-2), which is often overexpressed in breast and ovarian cancers. The purpose of this study was to investigate the in vivo properties of the new targeting agent.

    METHODS:

    The biodistribution and tumour uptake of the radioiodinated (Z(HER2:4))(2) affibody was studied in nude mice carrying tumours from xenografted HER-2 overexpressing SKOV-3 cells.

    RESULTS:

    The radioiodinated (Z(HER2:4))(2) affibody was primarily excreted through the kidneys, and significant amounts of radioactivity were specifically targeted to the tumours. The blood-borne radioactivity was, at all times, mainly in the macromolecular fraction. A tumour-to-blood ratio of about 10:1 was obtained 8 h post injection, and the tumours could be easily visualised with a gamma camera at this time point.

    CONCLUSION:

    The results indicate that the (Z(HER2:4))(2) affibody is an interesting candidate for applications in nuclear medicine, such as radionuclide-based tumour imaging and therapy.

  • 5. Steffen, Ann Charlott
    et al.
    Wikman, Maria
    Tolmachev, Vladimir
    Adams, G.P.
    Nilsson, F.Y.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO).
    Carlsson, J.
    In vitro characterization of a bivalent anti-HER-2 affibody with potential for radionuclide-based diagnostics2005In: Cancer Biotherapy and Radiopharmaceuticals, ISSN 1084-9785, E-ISSN 1557-8852, Vol. 20, no 3, p. 239-248Article in journal (Refereed)
    Abstract [en]

    The 185 kDa transmembrane glycoprotein human epidermal growth factor receptor 2 (HER-2) (p185/neu, c-ErbB-2) is overexpressed in breast and ovarian cancers. Overexpression in breast cancer correlates with poor patient prognosis, and visualization of HER-2 expression might provide valuable diagnostic information influencing patient management. We have previously described the generation of a new type of affinity ligand, a 58-amino-acid affibody (Z HER2:4) with specific binding to HER-2. In order to benefit from avidity effects, we have created a bivalent form of the affibody ligand, (Z HER2:4)2. The monovalent and bivalent ligands were compared in various assays. The new bivalent affibody has a molecular weight of 15.6 kDa and an apparent affinity (KD) against HER-2 of 3 nM. After radioiodination, using the linker molecule N-succinimidyl p-(trimethylstannyl) benzoate (SPMB), in vitro binding assays showed specific binding to HER-2 overexpressing cells. Internalization of 125I was shown after delivery with both the monovalent and the bivalent affibody. The cellular retention of 125I was longer after delivery with the bivalent affibody when compared to delivery with the monovalent affibody. With approximately the same affinity as the monoclonal antibody trastuzumab (Herceptin™) but only one tenth of the size, this new bivalent molecule is a promising candidate for radionuclide-based detection of HER-2 expression in tumors. 125I was used in this study as a surrogate marker for the diagnostically relevant radioisotopes 123I for single photon emission computed tomography (SPECT)/gamma-camera imaging and 124I for positron emission tomography (PET).

  • 6.
    Wikman, Maria
    KTH, School of Biotechnology (BIO).
    Rational and combinatorial protein engineering for vaccine delivery and drug targeting2005Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    This thesis describes recombinant proteins that have been generated by rational and combinatorial protein engineering strategies for use in subunit vaccine delivery and tumor targeting.

    In a first series of studies, recombinant methods for incorporating immunogens into an adjuvant formulation, e.g. immunostimulating complexes (iscoms), were evaluated. Protein immunogens, which are not typically immunogenic in themselves, are normally administered with an adjuvant to improve their immunogenicity. To accomplish iscom incorporation of a Toxoplasma gondii surface antigen through hydrophobic interaction, lipids were added either in vivo via E. coli expression, or in vitro via interaction of an introduced hexahistidyl (His6) peptide and a chelating lipid. The possibility of exploiting the strong interaction between biotin and streptavidin was also explored, in order to couple a Neospora caninum surface antigen to iscom matrix, i.e. iscom particles without any antigen. Subsequent analyses confirmed that the immunogens were successfully incorporated into iscoms by the investigated strategies. In addition, immunization of mice with the recombinant Neospora antigen NcSRS2, associated with iscoms through the biotin-streptavidin interaction, induced specific antibodies to native NcSRS2 and reduced clinical symptoms following challenge infection. The systems described in this thesis might offer convenient and efficient methods for incorporating recombinant immunogens into adjuvant formulations that might be considered for the generation of future recombinant subunit vaccines.

    In a second series of studies, Affibody® (affibody) ligands directed to the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu), which is known to be overexpressed in ∼ 20-30% of breast cancers, were isolated by phage display in vitro selection from a combinatorial protein library based on the 58 amino acid residue staphylococcal protein A-derived Z domain. Biosensor analyses demonstrated that one of the variants from the phage selection, denoted His6-ZHER2/neu:4, selectively bound with nanomolar affinity (KD ≈ 50 nM) to the extracellular domain of HER2/neu (HER2-ECD) at a different site than the monoclonal antibody trastuzumab. In order to exploit avidity effects, a bivalent affibody ligand was constructed by head-to-tail dimerization, resulting in a 15.6 kDa affibody ligand, termed His6-(ZHER2/neu:4)2, that was shown to have an improved apparent affinity to HER2-ECD (KD ≈ 3 nM) compared to the monovalent affibody. Moreover, radiolabeled monovalent and bivalent affibody ligands showed specific binding in vitro to native HER2/neu molecules expressed in human cancer cells. Biodistribution studies in mice carrying SKOV-3 xenografted tumors revealed that significant amounts of radioactivity were specifically targeted to the tumors in vivo, and the tumors could easily be visualized with a gamma camera. These results suggest that affibody ligands would be interesting candidates for specific tumor targeting in clinical applications, such as in vivo imaging and radiotherapy.

  • 7.
    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.

  • 8.
    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.

  • 9.
    Wikman, Maria
    et al.
    KTH, Superseded Departments, Biotechnology.
    Steffen, Ann Charlott
    Gunneriusson, Elin
    Tolmachev, Vladimir
    Adams, Gregg
    Carlsson, Jörgen
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Selection and characterization of HER2/neu-binding affibody ligands2004In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 17, no 5, p. 455-462Article in journal (Refereed)
    Abstract [en]

    Affibody® (affibody) ligands that are specific for the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) have been selected by phage display technology from a combinatorial protein library based on the 58 amino acid residue staphylococcal protein A-derived Z domain. The predominant variants from the phage selection were produced in Escherichia coli, purified by affinity chromatography, and characterized by biosensor analyses. Two affibody variants were shown to selectively bind to the extracellular domain of HER2/neu (HER2-ECD), but not to control proteins. One of the variants, denoted His6-ZHER2/neu:4, was demonstrated to bind with nanomolar affinity (∼50 nM) to the HER2-ECD molecule at a different site than the monoclonal antibody trastuzumab. Furthermore, radiolabeled His 6-ZHER2/neu:4 affibody showed specific binding to native HER2/neu, overexpressed on the SKBR-3 tumor cell line. Such affibody ligands might be considered in tumor targeting applications for radionuclide diagnostics and therapy of adenocarcinomas such as breast and ovarian cancers.

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