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  • 101. Orlova, Anna
    et al.
    Nilsson, Fredrik Y.
    Wikman, Maria
    Widstrom, Charles
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Carlsson, Jorgen
    Tolmachev, Vladimir
    Comparative in vivo evaluation of technetium and iodine labels on an anti-HER2 Affibody for single-photon imaging of HER2 expression in tumors2006Inngår i: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 47, nr 3, s. 512-519Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In vivo diagnosis with cancer-specific targeting agents that have optimal characteristics for imaging is an important development in treatment planning for cancer patients. Overexpression of the HER2 antigen is high in several types of carcinomas and has predictive and prognostic value, especially for breast cancer. A new type of targeting agent, the Affibody molecule, was described recently. An Affibody dimer, HiS(6)-(ZHER(2:4))(2) (15.4 kDa), binds to HER2 with an affinity of 3 nmol/L and might be used for the imaging of HER2 expression. The use of Tc-99m might improve the availability of the labeled conjugate, and Tc(l)-carbonyl chemistry enables the site-specific labeling of the histidine tag on the Affibody molecule. The goals of the present study were to prepare Tc-99m-labeled Hi0S(6)-(Z(HER2:4))(2) and to evaluate its targeting properties compared with the targeting properties of I-125 -4-iodobenzoate-HiS(6)-(Z(HER2:4))(2) [I-125-HiS(6)-(Z(HER2:4))(2)]- Methods: The labeling of HiS6-(Z(HER2:4))2 with Tc-99m was performed with an IsoLink kit. The specificity of Tc-99m-HiS(6)-(Z(HER2:4))(2) binding to HER2 was evaluated in vitro with SK-OV-3 ovarian carcinoma cells. The comparative biodistributions of Tc-99m-HiS(6)-(Z(HER2,4))(2) and I-125-HiS(6)-(Z(HER2:4))(2) in tumor-bearing BALB/c nulnu mice were determined. Results: The labeling yield for Tc-99m-HIS6(Z(HER2:4))(2) was similar to 60% (50 degrees C), and the radiochernical purity was greater than 97%. The conjugate was stable during storage and under histicline and cysteine challenges and demonstrated receptor-specific binding. The biodistribution study demonstrated tumor-specific uptake levels (percentage injected activity per gram of tissue [%]A/gj) of 2.6 %IA/g for Tc-99m-HiS(6)-(Z(HER2:4))(2) and 2.3 % IA/g for I-125-HiS6-(Z(HER2:4))(2) at 4 h after injection. Both conjugates provided clear imaging of SK-OV-3 xenografts at 6 h after injection. The tumor-to-nontumor ratios were much more favorable for the radioiodinated Affibody. Conclusion: The use of Tc(l)-carbonyl chemistry enabled us to prepare a stable, site-specifically labeled 99mTc-HiS(6)-(Z(HER2:4))(2) conjugate that was able to bind to HER2-expressing cells in vitro and in vivo. The indirectly radioiodinated conjugate provided better tumor-to-liver ratios. The labeling of Affibody molecules with Tc-99m should be investigated further.

  • 102. Pinitkiatisakul, S.
    et al.
    Mattsson, J.G.
    Wikman, Maria
    KTH, Skolan för bioteknologi (BIO).
    Friedman, Mikaela
    KTH, Skolan för bioteknologi (BIO).
    Bengtsson, K.L.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO).
    Lundén, Anne
    Immunisation of mice against neosporosis with recombinant NcSRS2 iscoms2005Inngår i: Veterinary parasitology, ISSN 0304-4017, E-ISSN 1873-2550, Vol. 129, nr 1-2, s. 25-34Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 103. Pinitkiatisakul, Sunan
    et al.
    Friedman, Mikaela
    Wikman, Maria
    Mattsson, Jens G.
    Lovgren-Bengtsson, Karin
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Lunden, Anna
    Immunogenicity and protective effect against murine cerebral neosporosis of recombinant NcSRS2 in different iscom formulations2007Inngår i: Vaccine, ISSN 0264-410X, E-ISSN 1873-2518, Vol. 25, nr 18, s. 3658-3668Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Recombinant NcSRS2, a major immunodominant surface antigen of the intracellular protozoan parasite Neospora caninum, was used as a model antigen to compare the immunogenicity of iscoms prepared according to three different methods. Two NcSRS2 fusion proteins were used, one that was biotinylated upon expression in Escherichia coli and linked to Ni2+-loaded iscom matrix (iscom without any protein) via a hexabistidyl (HiS(6)-tagged streptavidin fusion protein, and another that contained both a HiS(6)-tag and streptavidin (HiS(6)-SA-SRS2') and was coupled to either Ni2+-loaded or biotinylated matrix. While all three iscom preparations induced N. caninum specific antibodies at similar levels, HiS(6)-SA-SRS2' coupled to biotinylated matrix generated the strongest cellular responses measured as in vitro proliferation and production of interferon-gamma and interleukin-4 after antigen stimulation of spleen cells. However, the relationship between the levels of these cytokines as well as between IgG1 and IgG2a titres in serum induced by the three iscom preparations were similar, indicating that the balance between Th1 and Th2 responses did not differ. After challenge infection, mice immunised with His(6)-SA-SRS2' coupled to biotinylated matrix had significantly lower amounts of parasite DNA in their brains compared to the other immunised groups. Possible reasons for the performance of the different iscom formulations are discussed.

  • 104. Qazi, K. R.
    et al.
    Wikman, M.
    Vasconcelos, N. M.
    Berzins, K.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Fernandez, C.
    Enhancement of DNA vaccine potency by linkage of Plasmodium falciparum malarial antigen gene fused with a fragment of HSP70 gene2005Inngår i: Vaccine, ISSN 0264-410X, E-ISSN 1873-2518, Vol. 23, nr 9, s. 1114-1125Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Finding an appropriate adjuvant for human vaccination is crucial. HSPs have been shown to act as adjuvants when coadministered with peptide antigens or given as fusion proteins. However, there is a potential risk of autoimmunity when using the complete molecules because HSPs are evolutionary conserved. To overcome this. we first evaluated the adjuvant effect of a less conserved fragment of Plasmodium falciparum HSP70 (Pf70C) as compared it to that of the whole HSP70 molecule from Trypanosoma cruzi (TcHSP70). We found that Pf70C exhibited similar adjuvant properties as the whole molecule. We then evaluated the adjuvant potential of Pf70C for the malarial antigen EB200 in a chimeric DNA construct. No appreciable levels of EB200 specific antibodies were detected in mice immunized with the DNA constructs only. However, the DNA immunization efficiently primed the immune system, as indicated by the strong Th-1 antibody response elicited by a subsequent boosting with the corresponding recombinant fusion proteins. In contrast, while no such priming effect was observed for ex vivo IFN-gamma production, stimulation with the HSP chimeric fusion protein induced an enhanced secretion of IFN-gamma in vitro as compared to other proteins used. Our results emphasize the potential of HSPs as adjuvants in subunit vaccines.

  • 105.
    Rinne, Sara S.
    et al.
    Uppsala Univ, Dept Med Chem, Uppsala, Sweden..
    Leitao, Charles Dahlsson
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Mitran, Bogdan
    Uppsala Univ, Dept Med Chem, Uppsala, Sweden..
    Bass, Tarek
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Andersson, Ken G.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Tolmachev, Vladimir
    Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden..
    Ståhl, Stefan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Orlova, Anna
    Uppsala Univ, Dept Med Chem, Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, Uppsala, Sweden..
    Optimization of HER3 expression imaging using affibody molecules: Influence of chelator for labeling with indium-1112019Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, artikkel-id 655Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Radionuclide molecular imaging of human epidermal growth factor receptor 3 (HER3) expression using affibody molecules could be used for patient stratification for HER3-targeted cancer therapeutics. We hypothesized that the properties of HER3-targeting affibody molecules might be improved through modification of the radiometal-chelator complex. Macrocyclic chelators NOTA (1,4,7-triazacyclononane-N,N',N ''-triacetic acid), NODAGA (1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-triazacyclononane), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid), and DOTAGA (1,4,7,10-tetraazacyclododececane, 1-(glutaric acid)-4,7,10-triacetic acid) were conjugated to the C-terminus of anti-HER3 affibody molecule Z(08698) and conjugates were labeled with indium-111. All conjugates bound specifically and with picomolar affinity to HER3 in vitro. In mice bearing HER3-expressing xenografts, no significant difference in tumor uptake between the conjugates was observed. Presence of the negatively charged In-111-DOTAGA-complex resulted in the lowest hepatic uptake and the highest tumor-to-liver ratio. In conclusion, the choice of chelator influences the biodistribution of indium-111 labeled anti-HER3 affibody molecules. Hepatic uptake of anti-HER3 affibody molecules could be reduced by the increase of negative charge of the radiometal-chelator complex on the C-terminus without significantly influencing the tumor uptake.

  • 106.
    Rockberg, Johan
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Hjelm, Barbara
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Epitope mapping of antibodies using bacterial surface display2008Inngår i: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 5, nr 12, s. 1039-1045Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We describe a method for mapping the epitopes recognized by antibodies, based on bacterial surface expression of antigen protein fragments followed by antibody-based flow-cytometric sorting. We analyzed the binding sites of both monoclonal and polyclonal antibodies directed to three human protein targets: (i) the human epidermal growth factor receptor 2 (HER2), (ii) ephrin-B3 and (iii) the transcription factor SATB2. All monoclonal antibodies bound a single epitope, whereas the polyclonal antibodies showed, in each case, a binding pattern with one to five separate epitopes. A comparison of polyclonal and monoclonal antibodies raised to the same antigen showed overlapping binding epitopes. We also demonstrated that bacterial cells with displayed protein fragments can be used as affinity ligands to generate epitope-specific antibodies. Our approach shows a path forward for systematic validation of antibodies for epitope specificity and cross-reactivity on a whole-proteome level.

  • 107.
    Rockberg, Johan
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Hjelm, Barbara
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Epitope mapping using gram-positive surface display2010Inngår i: Current Protocols in Immunology, ISSN 1934-3671, nr SUPPL. 90, s. 9.9.1-9.9.17Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Antibodies have proven to be invaluable tools for a vast number of applications during the last decades, including protein purification and characterization, medical diagnosis and imaging, and treatment using therapeutic antibiotics. No matter what the aims of the application are, the antibodys binding characteristics will still be the main features determining the assays reliability. Here, we describe a protocol for determination of antibody-binding epitopes using an antigen-focused, library-based approach where library members are generated by fragmentation of antigen DNA and presented as cloned peptides on the cell surface of the Gram-positive bacterium Staphylococcus carnosus. The rigid cell structure of this organism allows for multivalent expression and permits rapid library analysis and sorting of antibody-binding cells using flow-sorting devices. Epitopes are determined by DNA sequencing of the sorted cells and alignment back to the antigen sequence. The protocol described here has been shown useful for mapping of both monoclonal and polyclonal binders with varying epitope lengths.

  • 108. Ronnmark, J.
    et al.
    Hansson, M.
    Nguyen, T.
    Uhlén, Mathias
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Robert, A.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Construction and characterization of affibody-Fc chimeras produced in Escherichia coli2002Inngår i: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 261, nr 02-jan, s. 199-211Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Affibody-Fc chimeras were constructed by genetic fusion between different affibody affinity proteins with prescribed specificities and an Fc fragment derived from human I-G. Using affibody ligands previously selected for binding to respiratory syncytial virus (RSV) surface protein G and Thermus aquaticus (Taq) DNA polymerase, respectively, affibody-Fc fusion proteins showing spontaneous Fc fragment-mediated homodimerization via disulfide bridges were produced in Escherichia coli and affinity purified on protein A Sepharose from bacterial periplasms at yields ranging between 1 and 6 mg/l culture, Further characterization of the chimeras using biosensor technology showed that the affibody moieties have retained high selectivities for their respective targets after fusion to the Fc fragment. Avidity effects in the target binding were observed for the affibody-Fc chimeras compared to monovalent affibody fusion proteins, indicating that both affibody moieties in the chimeras were accessible and contributed in the binding. Fusion of a head-to-tail dimeric affibody moiety to the Fc fragment resulted in tetravalent affibody constructs which showed even more pronounced avidity effects. In addition, the Fc moiety of the chimeras was demonstrated to be specifically recognized by anti-human IgG antibody enzyme conjugates. One application for this class of artificial antibodies was demonstrated in a western blotting experiment in which one of the anti-RSV surface protein G affibody-Fc chimeras was demonstrated to be useful for specific detection of the target protein in a complex background consisting of a total E. coli lysate. The results show that through the replacement of the Fab portion of an antibody for an alternative binding domain based on a less complicated structure, chimeric proteins compatible with bacterial production routes containing both antigen recognition domains and Fc domains can be constructed. Such artificial antibodies should be interesting alternatives to, for example, whole antibodies or scFv-Fc fusions as detection devices and in diagnostic or therapeutic applications.

  • 109. Rosestedt, M.
    et al.
    Andersson, Ken G.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Mitran, B.
    Rinne, S. S.
    Tolmachev, V.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Orlova, A.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Evaluation of a radiocobalt-labelled affibody molecule for imaging of human epidermal growth factor receptor 3 expression2017Inngår i: International Journal of Oncology, ISSN 1019-6439, Vol. 51, nr 6, s. 1765-1774Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The human epidermal growth factor receptor 3 (HER3) is involved in the development of cancer resistance towards tyrosine kinase-Targeted therapies. Several HER3-Targeting therapeutics are currently under clinical evaluation. Non-invasive imaging of HER3 expression could improve patient management. Affibody molecules are small engineered scaffold proteins demonstrating superior properties as targeting probes for molecular imaging compared with monoclonal antibodies. Feasibility of in vivo HER3 imaging using affibody molecules has been previously demonstrated. Preclinical studies have shown that the contrast when imaging using anti-HER3 affibody molecules can be improved over time. We aim to develop an agent for PET imaging of HER3 expression using the long-lived positron-emitting radionuclide cobalt-55 (55Co) (T1/2=17.5 h). A long-lived cobalt isotope 57Co was used as a surrogate for 55Co in this study. The anti-HER3 affibody molecule HEHEHE-ZHER3-NOTA was labelled with radiocobalt with high yield, purity and stability. Biodistribution of 57Co-HEHEHE-ZHER3-NOTA was measured in mice bearing DU145 (prostate carcinoma) and LS174T (colorectal carcinoma) xenografts at 3 and 24 h post injection (p.i.). Tumour-To-blood ratios significantly increased between 3 and 24 h p.i. (p<0.05). At 24 h p.i., tumour-To-blood ratios were 6 for DU145 and 8 for LS174T xenografts, respectively. HER3-expressing xenografts were clearly visualized in a preclinical imaging setting already 3 h p.i., and contrast further improved at 24 h p.i. In conclusion, the radiocobalt-labelled anti-HER3 affibody molecule, HEHEHE-ZHER3-NOTA, is a promising tracer for imaging of HER3 expression in tumours.

  • 110. Rosestedt, M.
    et al.
    Andersson, Ken
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Mitran, B.
    Tolmachev, V.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Orlova, A.
    PET Imaging of HER3-Expression in Tumours Using a 68Ga-Labeled Affibody Molecule2014Inngår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 41, s. S310-S310Artikkel i tidsskrift (Annet vitenskapelig)
  • 111. Rosestedt, M.
    et al.
    Andersson, Ken
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Mitran, B.
    Tolmachev, V.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Orlova, A.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Affibody-mediated PET imaging of HER3 expression in malignant tumours2015Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Human epidermal growth factor receptor 3(HER3) is involved in the progression of various cancers and in resistance to therapies targeting the HER family. In vivo imaging of HER3 expression would enable patient stratification for anti-HER3 immunotherapy. Key challenges with HER3-targeting are the relatively low expression in HER3-positive tumours and HER3 expression in normal tissues. The use of positron-emission tomography (PET) provides advantages of high resolution, sensitivity and quantification accuracy compared to SPECT. Affibody molecules, imaging probes based on a non-immunoglobulin scaffold, provide high imaging contrast shortly after injection. The aim of this study was to evaluate feasibility of PET imaging of HER3 expression using 68Ga-labeled affibody molecules. The anti-HER3 affibody molecule HEHEHE-Z08698-NOTA was successfully labelled with 68Ga with high yield, purity and stability. The agent bound specifically to HER3-expressing cancer cells in vitro and in vivo. At 3 h pi, uptake of 68Ga-HEHEHE-Z08698-NOTA was significantly higher in xenografts with high HER3 expression (BT474, BxPC-3) than in xenografts with low HER3 expression (A431). In xenografts with high expression, tumour-to-blood ratios were >20, tumour-to-muscle >15, and tumour-to-bone >7. HER3-positive xenografts were visualised using microPET 3 h pi. In conclusion, PET imaging of HER3 expression is feasible using 68Ga-HEHEHE-Z08698-NOTA shortly after administration.

  • 112. Rosestedt, M.
    et al.
    Mitran, B.
    Andersson, K. G.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Löfblom, J.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Ståhl, S.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Tolmachev, V.
    Orlova, A.
    Development and Evaluation of Radiocobalt-labelled Affibody Molecule for Next Day PET Imaging of HER3 Expression2016Inngår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 43, s. S37-S38Artikkel i tidsskrift (Fagfellevurdert)
  • 113.
    Samuelson, Patrik
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Gunneriusson, E.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Display of proteins on bacteria2002Inngår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 96, nr 2, s. 129-154Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Display of heterologous proteins on the surface of microorganisms, enabled by means of recombinant DNA technology, has become an increasingly used strategy in various applications in microbiology, biotechnology and vaccinology. Gram-negative, Gram-positive bacteria, viruses and phages are all being investigated in such applications. This review will focus on the bacterial display systems and applications. Live bacterial vaccine delivery vehicles are being developed through the surface display of foreign antigens on the bacterial surfaces. In this field, 'second generation' vaccine delivery vehicles are at present being generated by the addition of mucosal targeting signals, through co-display of adhesins, in order to achieve targeting of the live bacteria to immunoreactive sites to thereby increase immune responses. Engineered bacteria are further being evaluated as novel microbial biocatalysts with heterologous enzymes immobilized as surface exposed on the bacterial cell surface. A discussion has started whether bacteria can find use as new types of whole-cell diagnostic devices since single-chain antibodies and other type of tailor-made binding proteins can be displayed on bacteria. Bacteria with increased binding capacity for certain metal ions can be created and potential environmental or biosensor applications for such recombinant bacteria as biosorbents are being discussed. Certain bacteria have also been employed for display of various poly-peptide libraries for use as devices in in vitro selection applications. Through various selection principles, individual clones with desired properties can be selected from such libraries. This article explains the basic principles of the different bacterial display systems, and disc-asses current uses and possible future trends of these emerging technologies.

  • 114.
    Samuelson, Patrik
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Wernérus, Henrik
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Svedberg, M.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Staphylococcal surface display of metal-binding polyhistidyl peptides2000Inngår i: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 66, nr 3, s. 1243-1248Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Recombinant Staphylococcus xylosus and Staphylococcus carnosus strains were generated with surface-exposed chimeric proteins containing polyhistidyl peptides designed for binding to divalent metal ions. Surface accessibility of the chimeric surface proteins was demonstrated and the chimeric surface proteins were found to be functional in terms of metal binding, since the recombinant staphylococcal cells were shown to have gained Ni2+- and Cd2+-binding capacity, suggesting that such bacteria could find use in bioremediation of heavy metals. This is, to our knowledge, the first time that recombinant, surface-exposed metal-binding peptides have been expressed on gram-positive bacteria. Potential environmental or biosensor applications for such recombinant staphylococci as biosorbents are discussed.

  • 115. Steffen, A.C
    et al.
    Orlova, A
    Wikman, Maria
    Nilsson, F.Y
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Adams, G.P
    Tolmachev, V
    Carlsson, J
    Affibody mediated tumor targeting of HER-2 expressing xenografts in mice2006Inngår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 33, nr 6, s. 631-638Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 116. Steffen, Ann Charlott
    et al.
    Wikman, Maria
    Tolmachev, Vladimir
    Adams, G.P.
    Nilsson, F.Y.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO).
    Carlsson, J.
    In vitro characterization of a bivalent anti-HER-2 affibody with potential for radionuclide-based diagnostics2005Inngår i: Cancer Biotherapy and Radiopharmaceuticals, ISSN 1084-9785, E-ISSN 1557-8852, Vol. 20, nr 3, s. 239-248Artikkel i tidsskrift (Fagfellevurdert)
    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).

  • 117.
    Ståhl, Stefan
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Friedman, Mikaela
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Carlsson, Jörgen
    Rudbeck Lab, Uppsala University.
    Tolmachev, Vladimir
    Rudbeck Lab, Uppsala University.
    Frejd, Fredrik
    Affibody AB.
    Affibody Molecules for Targeted Radionuclide Therapy2011Inngår i: Targeted Radionuclide Therapy: Immunology and Targeted Constructs / [ed] Tod W. Speer, Philadelphia, USA: Lippincott Williams & Wilkins, 2011, s. 49-58Kapittel i bok, del av antologi (Fagfellevurdert)
  • 118.
    Ståhl, Stefan
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Gräslund, Torbjörn
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Eriksson Karlström, Amelie
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Frejd, Fredrik Y.
    Nygren, Per-Åke
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Affibody Molecules in Biotechnological and Medical Applications2017Inngår i: Trends in Biotechnology, ISSN 0167-7799, E-ISSN 1879-3096, Vol. 35, nr 8, s. 691-712Artikkel, forskningsoversikt (Fagfellevurdert)
    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.

  • 119.
    Ståhl, Stefan
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Kronqvist, N.
    Jonsson, Andreas
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Affinity proteins and their generation2013Inngår i: Journal of chemical technology and biotechnology (1986), ISSN 0268-2575, E-ISSN 1097-4660, Vol. 88, nr 1, s. 25-38Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Engineered affinity proteins have, together with antibodies and antibody derivatives, become indispensable tools in many areas of life science and with an increasing number of applications. The need for high-throughput methods for generation of these different affinity proteins is evident. Today, combinatorial protein engineering is the most successful strategy to generate novel affinity proteins of non-immunoglobulin origin. In this approach, high-complexity combinatorial libraries are constructed from which affinity proteins are isolated using appropriate selection methods, thus circumventing the need for detailed knowledge of the protein structure and the binding mechanism that is necessary in more rational approaches. Since the introduction of the phage display technology, several alternative selection systems have been developed for this purpose. This review presents briefly some of the more commonly used affinity proteins, and gives an overview of the different methods and challenges related to the generation of library diversity and the selection methods available for the isolation of affinity proteins with desired properties.

  • 120.
    Tolmachev, Vladimir
    et al.
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
    Friedman, Mikaela
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    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, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    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 Chemistry2009Inngår i: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 50, nr 2, s. 274-283Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 121. Wahlberg, Elisabet
    et al.
    Rahman, Mahafuzur
    Lindberg, Hanna
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Gunneriusson, Elin
    Schmuck, Benjamin
    Lendel, Christofer
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Härd, Torleif
    Selection of binding proteins that specifically recognize protofibrillar aggregates of amyloid-βManuskript (preprint) (Annet vitenskapelig)
  • 122.
    Wernérus, Henrik
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Lehtio, J.
    Teeri, Tuula T.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Generation of metal-binding staphylococci through surface display of combinatorially engineered cellulose-binding domains2001Inngår i: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 67, nr 10, s. 4678-4684Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Ni2+-binding staphylococci were generated through surface display of combinatorially engineered variants of a fungal cellulose-binding domain (CBD) from Trichoderma reesei cellulase CeI7A. Novel CBD variants were generated by combinatorial protein engineering through the randomization of 11 amino acid positions, and eight potentially Ni2+-binding CBDs were selected by phage display technology. These new variants were subsequently genetically introduced into chimeric surface proteins for surface display on Staphylococcus carnosus cells. The expressed chimeric proteins were shown to be properly targeted to the cell wall of S. carnosus cells, since full-length proteins could be extracted and affinity purified. Surface accessibility for the chimeric proteins was demonstrated, and furthermore, the engineered CBDs, now devoid of cellulose-binding capacity, were shown to be functional with regard to metal binding, since the recombinant staphylococci had gained Ni2+-binding capacity. Potential environmental applications for such tailor-made metal-binding bacteria as bioadsorbents in biofilters or biosensors are discussed.

  • 123.
    Wernérus, Henrik
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Lehtiö, Janne
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Samuelson, Patrik
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Engineering of staphylococcal surfaces for biotechnological applications2002Inngår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 96, nr 1, s. 67-78Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Novel surface proteins can be introduced onto bacterial cell surfaces by recombinant means. Here, we describe various applications of two such display systems for the food-grade bacteria Staphylococcus carnosus and Staphylococcus xylosus, respectively. The achievements in the use of such staphylococci as live bacterial vaccine delivery vehicles will be described. Co-display of proteins and peptides with adhesive properties to enable targeting of the bacteria, have significantly improved the vaccine delivery potential. Recently, protective immunity to respiratory syncytial virus (RSV) could be evoked in mice by intranasal immunization using such 'second generation' vaccine delivery systems. Furthermore, antibody fragments and other 'affinity proteins' with capacity to specifically bind a certain protein, e.g. Staphylococcus aureus protein A-based affibodies, have been surface-displayed on staphylococci as initial efforts to create whole-cell diagnostic devices. Surface display of metal-binding peptides, or protein domains into which metal binding properties has been engineered by combinatorial protein engineering, have been exploited to create staphylococcal bioadsorbents for potential environmental or biosensor applications. The use of these staphylococcal surface display systems as alternatives for display of large protein libraries and subsequent affinity selection of relevant binding proteins by fluorescence-activated cell sorting (FACS) will be discussed.

  • 124.
    Wernérus, Henrik
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Samuelson, Patrik
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Fluorescence-activated cell sorting of specific affibody-displaying staphylococci2003Inngår i: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 69, nr 9, s. 5328-5335Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Efficient enrichment of staphylococcal cells displaying specific heterologous affinity ligands on their cell surfaces was demonstrated by using fluorescence-activated cell sorting. Using bacterial surface display of peptide or protein libraries for the purpose of combinatorial protein engineering has previously been investigated by using gram-negative bacteria. Here, the potential for using a gram-positive bacterium was evaluated by employing the well-established surface expression system for Staphylococcus carnosus. Staphylococcus aureus protein A domains with binding specificity to immunoglobulin G or engineered specificity for the G protein of human respiratory syncytial virus were expressed as surface display on S. carnosus cells. The surface accessibility and retained binding specificity of expressed proteins were demonstrated in whole-cell enzyme and flow cytometry assays. Also, affibody-expressing target cells could be sorted essentially quantitatively from a moderate excess of background cells in a single step by using a high-stringency sorting mode. Furthermore, in a simulated library selection experiment, a more-than-25,000-fold enrichment of target cells could be achieved through only two rounds of cell sorting and regrowth. The results obtained indicate that staphylococcal surface display of affibody libraries combined with fluoresence-activated cell sorting might indeed constitute an attractive alternative to existing technology platforms for affinity-based selections.

  • 125.
    Wernérus, Henrik
    et al.
    KTH, Tidigare Institutioner, Bioteknologi.
    Ståhl, Stefan
    KTH, Tidigare Institutioner, Bioteknologi.
    Biotechnological applications for surface-engineered bacteria2004Inngår i: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 40, s. 209-228Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Display of heterologous proteins on the surface of micro-organisms, enabled by means of recombinant DNA technology, has become an increasingly popular strategy in microbiology, biotechnology and vaccinology. Both Gram-negative and Gram-positive bacteria have been investigated for potential applications. The present review will describe the most commonly used systems for bacterial display, with a focus on the biotechnology applications. Live bacterial vaccine-delivery vehicles have long been investigated through the surface display of foreign antigens and, recently, 'second-generation' vaccine-delivery vehicles have been generated by the addition of mucosal targeting signals, as a means to increase immune responses. Engineered bacteria have also the potential to act as novel microbial biocatalysts with heterologous enzymes immobilized as surface exposed on the bacterial cell surface. They provide the potential for new types of whole-cell diagnostic devices, since single-chain antibodies and other type of tailor-made binding proteins can be displayed on bacteria. Bacteria with increased binding capacity for certain metal ions can be created, and potential environmental or biosensor applications for such recombinant bacteria as biosorbents are being explored. Certain bacteria have also been employed to display various polypeptide libraries for use as devices in in vitro selection applications. Part of the present review has been devoted to a more in-depth description of a promising Gram-positive display system, i.e. Staphylococcus carnosus, and its applications. The review describes the basic principles of the different bacterial display systems and discusses current uses and possible future trends of these emerging technologies.

  • 126.
    Wernérus, Henrik
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Vector engineering to improve a staphylococcal surface display system2002Inngår i: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 212, nr 1, s. 47-54Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A previously developed expression system for surface display of heterologous proteins on the surface of Staphylococcus carnosus employs the secretion signals from a Staphylococcus hyicus lipase and the cell wall anchoring part of Staphylococcus aureus protein A (SpA) to achieve surface display of expressed recombinant proteins. The system has been successfully used in various applications but the vector has not been considered genetically stable enough to allow protein library display applications, which would be of obvious interest. A new set of vectors, differing in size and devoid of a phage f1 origin of replication, were constructed and evaluated in terms of bacterial growth characteristics and vector stability. Furthermore, surface expression of a model surface protein was monitored by an enzymatic whole-cell assay and flow cytometry. The engineered expression vectors demonstrated dramatically improved stability and growth properties and two of the novel vectors demonstrated retained high surface density of the displayed model protein. The flow cytometry was found to be a powerful toot for observing the surface density of displayed heterologous proteins, and would thus be a rational strategy for monitoring the optimisation of any surface display system. The implications of these improved display vectors for future protein library applications are discussed.

  • 127.
    Wikman, Maria
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Friedman, Mikaela
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Pinitkiatisakul, S.
    Andersson, Christin
    KTH, Skolan för bioteknologi (BIO).
    Hemphill, A.
    Lovgren-Bengtsson, K.
    Lunden, A.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    General strategies for efficient adjuvant incorporation of recombinant subunit immunogens2005Inngår i: Vaccine, ISSN 0264-410X, E-ISSN 1873-2518, Vol. 23, nr 17-18, s. 2331-2335Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 128.
    Wikman, Maria
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Friedman, Mikaela
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Pinitkiatisakul, S.
    Hemphill, A.
    Lövgren-Bengtsson, K.
    Lunden, A.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Applying biotin-streptavidin binding for iscom (immunostimulating complex) association of recombinant immunogens2005Inngår i: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 41, s. 163-174Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 129. Wikman, Maria
    et al.
    Friedman, Mikaela
    Pinitkiatisakul, Sunan
    Andersson, Christin
    Lovgren-Bengtsson, Karin
    Lunden, Anna
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Achieving directed immunostimulating complex incorporation2006Inngår i: Expert Review of Vaccines, ISSN 1476-0584, E-ISSN 1744-8395, Vol. 5, nr 3, s. 395-403Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    In recent years, several studies have been reported with the common aim of generating general expression systems for straightforward production and subsequent coupling of expressed antigens to an adjuvant system. Here, we describe a series of such efforts with a common theme of using gene fusion technology for association of recombinant antigens to immunostimulating complexes (iscoms). In the early stages of vaccine development, uniform antigen preparations are crucial to allow the comparison of immune responses to different antigens, or even subdomains thereof, and we believe that the described systems constitute an important development in this context.

  • 130. Wikman, Maria
    et al.
    Rowcliffe, Eric
    Friedman, Mikaela
    Henning, Petra
    Lindholm, Leif
    Olofsson, Sigvard
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Selection and characterization of an HIV-1 gp120-binding affibody ligand2006Inngår i: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 45, s. 93-105Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    To evaluate the possibility of generating novel proteins binding to highly glycosylated viral proteins, affibody ligands were selected by bactericiphage display technology to the HIV-1 envelope glycoprotein gp120 (glycoprotein 120), from a combinatorial protein library based on the 58-amino-acid-residue staphylococcal Protein A domain. The predominant variant from the bacteriophage selection was produced in Escherichia coli and characterized by biosenscir analyses. Both univalent and bivalent affibody molecules were shown to bind selectively to the gp120 target molecule in a biosensor analysis. The dissociation equilibrium constants (K.) were determined to be approx. 100 nM for the univalent affibody and 10 nM for the bivalent affibody, confirming the stronger gp120 binding of the bivalent affibody ligand. The affibody constructs were further introduced into the AdS (adenovirus type 5) fibre gene, and the recombinant fibres were shown to bind selectively to gp 120 in a biosensor analysis and to gp160 transiently expressed in African-green-monkey (Cercopithecus aethiops) kidney cells. Neither the affibody ligand nor the AdS fibres showed any virus neutralization activity, suggesting that the affibody bound to a non-neutralizing site on gp 120. To investigate the binding site for the affibody ligand on gp120, CD4 (cluster of differentiation 4) and a panel of mAbs (monoclonal antibodies) known to bind to gp 120 were allowed to compete with the affibody ligand in a biosensor study. Two mAbs, 670-30D and 697-30D, were found to compete with gp120 for overlapping binding sites. Although neutralization effects were not achieved in this initial investigation, the successful selection of a gp120-binding affibody ligand indicates that future affibody-based strategies might evolve to complement antibody-based efforts for HIV-I therapy. Strategies for directed selection of affibody ligands binding to neutralizing epitopes and the potential of using adenovirus for gene-therapy-mediated efforts are discussed.

  • 131.
    Wikman, Maria
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Steffen, Ann Charlott
    Gunneriusson, Elin
    Tolmachev, Vladimir
    Adams, Gregg
    Carlsson, Jörgen
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Selection and characterization of HER2/neu-binding affibody ligands2004Inngår i: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 17, nr 5, s. 455-462Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 132.
    Wållberg, Helena
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Grafström, Jonas
    Cheng, Qing
    Lu, Li
    Ahlzén, Hanna-Stina Martinsson
    Samén, Erik
    Thorell, Jan-Olov
    Johansson, Katarina
    Dunås, Finn
    Olofsson, Maria Hägg
    Stone-Elander, Sharon
    Arnér, Elias S. J.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    HER2-Positive Tumors Imaged Within 1 Hour Using a Site-Specifically C-11-Labeled Sel-Tagged Affibody Molecule2012Inngår i: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 53, nr 9, s. 1446-1453Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A rapid, reliable method for distinguishing tumors or metastases that overexpress human epidermal growth factor receptor 2 (HER2) from those that do not is highly desired for individualizing therapy and predicting prognoses. In vivo imaging methods are available but not yet in clinical practice; new methodologies improving speed, sensitivity, and specificity are required. Methods: A HER2-binding Affibody molecule, Z(HER2:342), was recombinantly fused with a C-terminal selenocysteine-containing tetrapeptide Sel-tag, allowing site-specific labeling with either C-11 or Ga-68, followed by biodistribution studies with small-animal PET. Dosimetry data for the 2 radiotracers were compared. Imaging of HER2-expressing human tumor xenografts was performed using the C-11-labeled Affibody molecule. Results: Both the C-11- and Ga-68-labeled tracers initially cleared rapidly from the blood, followed by a slower decrease to 4-5 percentage injected dose per gram of tissue at 1 h. Final retention in the kidneys was much lower (>5-fold) for the C-11-labeled protein, and its overall absorbed dose was considerably lower. C-11-Z(HER2:342) showed excellent tumor-targeting capability, with almost 10 percentage injected dose per gram of tissue in HER2-expressing tumors within 1 h. Specificity was demonstrated by preblocking binding sites with excess ligand, yielding significantly reduced radiotracer uptake (P = 0.002), comparable to uptake in tumors with low HER2 expression. Conclusion: To our knowledge, the Sel-tagging technique is the first that enables site-specific C-11-radiolabeling of proteins. Here we present the finding that, in a favorable combination between radionuclide half-life and in vivo pharmacokinetics of the Affibody molecules, C-11-labeled Set-tagged Z(HER2:342) can successfully be used for rapid and repeated PET studies of HER2 expression in tumors.

  • 133.
    Wållberg, Helena
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Grafström, Jonas
    Cheng, Qing
    Lu, Li
    Martinsson Ahlzén, Hanna-Stina
    Samén, Erik
    Thorell, Jan-Olov
    Johansson, Katarina
    Dunås, Finn
    Hägg Olofsson, Maria
    Stone-Elander, Sharon
    Arnér, Elias
    Shåhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Specific in vivo imaging of HER2-positive tumors within one hour using a site-specifically 11C-labeled Sel-tagged Affibody moleculeManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    A rapid, reliable method for distinguishing tumors or metastases that overexpress human epidermal growth factor receptor 2 (HER2) from those that do not is highly desired for improvement of cancer care. In v ivo imaging methods are available, but are not yet in clinical practice; new methodologies improving speed, sensitivity and specificity are required. Here we describe promising results with a HER2‐binding Affibody molecule, ZHER2:342, recombinantly fused with a C‐terminal selenocysteine‐containing tetrapeptide Sel‐tag and site‐specifically labeled with either 11C or 68Ga for molecular imaging applications with positron emissiontomography (PET).

    In mice, both the 11C‐ and 68Ga‐labeled tracers initially cleared rapidly from the blood, followed by a slower decrease to 4‐5 %ID/g at 1 h. Final uptake in kidneys was much lower (> 5‐fold) for the 11C‐labeled protein, leading to markedly reduced background radioactivity in the abdomen. Furthermore, 11C‐labeled Sel‐tagged ZHER2:342 showed excellent tumor targeting capability, with almost 10 %ID/g in HER2 expressing tumors within the first hour. High specificity was demonstrated by preblocking the binding sites with excess ligand, which yielded low radiotracer uptakes, comparable to those in tumors with low endogenous HER2 expression.

    To our knowledge the Sel‐tagging technique is the first that enables site‐specific 11C radiolabelingof proteins. Here we present that, in a favorable combination between radionuclide half‐life and in vivo pharmacokinetics of the Affibody molecules, 11C‐labeled Sel taggedZHER2:342 can successfully be used for rapid and repeated PET studies of HER2 expression in tumors.

  • 134. Wållberg, Helena
    et al.
    Löfdahl, Per-Åke
    Tschapalda, Kirsten
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Tolmachev, Vladimir
    Nygren, Per-Åke
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Affinity recovery of eight HER2-binding affibody variants using an anti-idiotypic affibody molecule as capture ligand2011Inngår i: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 76, nr 1, s. 127-135Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Affibody molecules generated by combinatorial protein engineering to bind the human epidermal growth factor receptor 2 (HER2) have in earlier studies proven to be promising tracers for HER2-mediated molecular imaging of cancer. Amino acid extensions either at the N- or C-terminus of these Z(HER2) affibody molecules, have been successfully employed for site-specific radiolabeling of the tracer candidates. Hexahistidyls or other tags, which would be convenient for recovery purposes, should be avoided since they could negatively influence the tumor targeting efficacy and biodistribution properties of the tracer. Using a new beta-lactamase-based protein fragment complementation assay (PCA), an affibody molecule was isolated which bound a Z(HER2) affibody molecule with sub-micromolar affinity, but not unrelated affibody molecules. This suggests that the interacting area include the HER2-binding surface of Z(HER2). This novel anti-idiotypic affibody molecule Z(E01) was produced in Escherichia coli, purified, and chemically coupled to a chromatography resin in order to generate an affibody-based affinity column, suitable for recovery of different variants of Z(HER2) affibody molecules, having a common binding surface for HER2. Eight such Z(HER2) affibody molecules, designed for future radioimaging investigations, having different C-terminal peptide extensions aimed for radioisotope (Tc-99m)-chelation, were successfully produced and recovered in a single step to high purity using the anti-idiotypic affibody ligand for the affinity purification. These results clearly suggest a potential for the development of anti-idiotypic affibody-based resins for efficient recovery of related variants of a target protein that might have altered biochemical properties, thus avoiding the cumbersome design of specific recovery schemes for each variant of a target protein.

  • 135.
    Wållberg, Helena
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Orlova, Anna
    Altai, Mohammed
    Hosseinimehr, Seyed Jalal
    Widström, Charles
    Malmberg, Jennie
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Tolmachev, Vladimir
    Molecular Design and Optimization of Tc-99m-Labeled Recombinant Affibody Molecules Improves Their Biodistribution and Imaging Properties2011Inngår i: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 52, nr 3, s. 461-469Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Affibody molecules are a recently developed class of targeting proteins based on a nonimmunoglobulin scaffold. The small size (7 kDa) and subnanomolar affinity of Affibody molecules enables high-contrast imaging of tumor-associated molecular targets, particularly human epidermal growth factor receptor type 2 (HER2). Tc-99m as a label offers advantages in clinical practice, and earlier studies demonstrated that Tc-99m-labeled recombinant Affibody molecules with a C-terminal cysteine could be used for HER2 imaging. However, the renal retention of radioactivity exceeded tumor uptake, which might complicate imaging of metastases in the lumbar region. The aim of this study was to develop an agent with low renal uptake and preserved tumor targeting. Methods: A series of recombinant derivatives of the HER2-binding Z(HER2:342) Affibody molecule with a C-terminal chelating sequence, -GXXC (X denoting glycine, serine, lysine, or glutamate), was designed. The constructs were labeled with Tc-99m and evaluated in vitro and in vivo. Results: All variants were stably labeled with Tc-99m, with preserved capacity to bind specifically to HER2-expressing cells in vitro and in vivo. The composition of the chelating sequence had a clear influence on the cellular processing and biodistribution properties of the Affibody molecules. The best variant, Tc-99m-Z(HER2:V2), with the C-terminal chelating sequence -GGGC, provided the lowest radioactivity retention in all normal organs and tissues including the kidneys. Tc-99m-Z(HER2:V2) displayed high uptake of radioactivity in HER2-expressing xenografts, 22.6 +/- 4.0 and 7.7 +/- 1.5 percentage injected activity per gram of tissue at 4 h after injection in SKOV-3 (high HER2 expression) and DU-145 (low HER2 expression) tumors, respectively. In both models, the tumor uptake exceeded the renal uptake. Conclusion: These results demonstrate that the biodistribution properties of recombinant Tc-99m-labeled Affibody molecules can be optimized by modification of the C-terminal cysteine-containing chelating sequence. Tc-99m-Z(HER2:V2) is a promising candidate for further development as a diagnostic radiopharmaceutical for imaging of HER2-expressing tumors. These results may be useful for the development of imaging agents based on other Affibody molecules and, hopefully, other scaffolds.

  • 136.
    Wållberg, Helena
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Design and evaluation of radiolabeled tracers for tumor imaging2013Inngår i: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 60, nr 4, s. 365-383Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The growing understanding of tumor biology and the identification of tumor-specific genetic and molecular alterations, such as the overexpression of membrane receptors and other proteins, allows for personalization of patient management using targeted therapies. However, this puts stringent demands on the diagnostic tools used to identify patients who are likely to respond to a particular treatment. Radionuclide molecular imaging is a promising noninvasive method to visualize and characterize the expression of such targets. A number of different proteins, from full-length antibodies and their derivatives to small scaffold proteins and peptide receptor-ligands, have been applied to molecular imaging, each demonstrating strengths and weaknesses. Here, we discuss the concept of molecular targeting and, in particular, molecular imaging of cancer-associated targets. Additionally, we describe important biotechnological considerations and desired features when designing and developing tracers for radionuclide molecular imaging.

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