| 1. |
- Lindholm, L, et al.
(author)
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Genetic re-targeting of adenovirus using a hyperstable scFv domain and an affibody (R) molecule against Her2/neu
- 2004
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In: Molecular Therapy. - : Elsevier BV. - 1525-0016 .- 1525-0024. ; 9, s. S250-S250
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Journal article (peer-reviewed)abstract
- 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.
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| 2. |
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| 3. |
- Frachon, A., et al.
(author)
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Sensitivity of numerical simulation to input data
- 2000
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In: Proceedings of PM2000 Powder metallurgy world congress and exhibition. - Tokyo : Japan Society of Powder and Powder Metallurgy. - 9784990021481
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Conference paper (peer-reviewed)
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| 4. |
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| 5. |
- Nicholls, Ian A., et al.
(author)
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Molecularly imprinted polymers: unique possibilities for environmental monitoring
- 2002
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In: Proceedings of Kalmar Eco-Tech'01 : conference on leachate and waste water treatment with high-tech and natural systems : the 3rd International Conference on the Establishment of Cooperation Between Companies/Institutions in the Nordic Countries and the Countries in the Baltic Sea Region : November 26 to 28, 2001 Kalmar, Sweden. - : Högskolan i Kalmar. ; , s. 285-288, s. 285-288
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Conference paper (other academic/artistic)
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| 6. |
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| 7. |
- Wikman, Maria, et al.
(author)
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Selection and characterization of HER2/neu-binding affibody ligands
- 2004
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In: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 17:5, s. 455-462
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Journal article (peer-reviewed)abstract
- 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 (approximately 50 nM) to the HER2-ECD molecule at a different site than the monoclonal antibody trastuzumab. Furthermore, radiolabeled His6-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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