Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Adenovirus 5 vector genetically re-targeted by an Affibody molecule with specificity for tumor antigen HER2/neu

Cancer Gene Therapy volume 14, pages 468–479 (2007)Cite this article

Abstract

In order to use adenovirus (Ad) type 5 (Ad5) for cancer gene therapy, Ad needs to be de-targeted from its native receptors and re-targeted to a tumor antigen. A limiting factor for this has been to find a ligand that (i) binds a relevant target, (ii) is able to fold correctly in the reducing environment of the cytoplasm and (iii) when incorporated at an optimal position on the virion results in a virus with a low physical particle to plaque-forming units ratio to diminish the viral load to be administered to a future patient. Here, we present a solution to these problems by producing a genetically re-targeted Ad with a tandem repeat of the HER2/neu reactive Affibody molecule (ZH) in the HI-loop of a Coxsackie B virus and Ad receptor (CAR) binding ablated fiber genetically modified to contain sequences for flexible linkers between the ZH and the knob sequences. ZH is an Affibody molecule specific for the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) that is overexpressed in inter alia breast and ovarian carcinomas. The virus presented here exhibits near wild-type growth characteristics, infects cells via HER2/neu instead of CAR and represents an important step toward the development of genetically re-targeted adenoviruses with clinical relevance.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9

Similar content being viewed by others

References

  1. Russell WC . Update on adenovirus and its vectors. J Gen Virol 2000; 81: 2573–2604.

    Article  CAS  PubMed  Google Scholar 

  2. Bergelson JM, Cunningham JA, Droguett G, Kurt-Jones EA, Krithivas A, Hong JS et al. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science 1997; 275: 1320–1323.

    Article  CAS  PubMed  Google Scholar 

  3. Dechecchi MC, Melotti P, Bonizzato A, Santacatterina M, Chilosi M, Cabrini G . Heparan sulfate glycosaminoglycans are receptors sufficient to mediate the initial binding of adenovirus types 2 and 5. J Virol 2001; 75: 8772–8780.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Wickham TJ, Mathias P, Cheresh DA, Nemerow GR . Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not virus attachment. Cell 1993; 73: 309–319.

    Article  CAS  PubMed  Google Scholar 

  5. Akiyama M, Thorne S, Kirn D, Roelvink PW, Einfeld DA, King CR et al. Ablating CAR and integrin binding in adenovirus vectors reduces nontarget organ transduction and permits sustained bloodstream persistence following intraperitoneal administration. Mol Ther 2004; 9: 218–230.

    Article  CAS  PubMed  Google Scholar 

  6. Roelvink PW, Mi Lee G, Einfeld DA, Kovesdi I, Wickham TJ . Identification of a conserved receptor-binding site on the fiber proteins of CAR-recognizing adenoviridae. Science 1999; 286: 1568–1571.

    Article  CAS  PubMed  Google Scholar 

  7. Kirby I, Davison E, Beavil AJ, Soh CP, Wickham TJ, Roelvink PW et al. Mutations in the DG loop of adenovirus type 5 fiber knob protein abolish high-affinity binding to its cellular receptor CAR. J Virol 1999; 73: 9508–9514.

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Bewley MC, Springer K, Zhang YB, Freimuth P, Flanagan JM . Structural analysis of the mechanism of adenovirus binding to its human cellular receptor, CAR. Science 1999; 286: 1579–1583.

    Article  CAS  PubMed  Google Scholar 

  9. Kirby I, Davison E, Beavil AJ, Soh CP, Wickham TJ, Roelvink PW et al. Identification of contact residues and definition of the CAR-binding site of adenovirus type 5 fiber protein. J Virol Mar 2000; 74: 2804–2813.

    Article  CAS  Google Scholar 

  10. Magnusson MK, Hong SS, Boulanger P, Lindholm L . Genetic retargeting of adenovirus: novel strategy employing ‘de-knobbing’ of the fiber. J Virol 2001; 75: 7280–7289.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Magnusson MK, Hong SS, Henning P, Boulanger P, Lindholm L . Genetic retargeting of adenovirus vectors: functionality of targeting ligands and their influence on virus viability. J Gene Med 2002; 4: 356–370.

    Article  CAS  PubMed  Google Scholar 

  12. Henning P, Magnusson MK, Gunneriusson E, Hong SS, Boulanger P, Nygren PA et al. Genetic modification of adenovirus 5 tropism by a novel class of ligands based on a three-helix bundle scaffold derived from Staphylococcal protein A. Hum Gene Ther 2002; 13: 1427–1439.

    Article  CAS  PubMed  Google Scholar 

  13. van Beusechem VW, van Rijswijk AL, van Es HH, Haisma HJ, Pinedo HM, Gerritsen WR . Recombinant adenovirus vectors with knobless fibers for targeted gene transfer. Gene Therapy 2000; 7: 1940–1946.

    Article  CAS  PubMed  Google Scholar 

  14. Krasnykh V, Belousova N, Korokhov N, Mikheeva G, Curiel DT . Genetic targeting of an adenovirus vector via replacement of the fiber protein with the phage T4 fibritin. J Virol 2001; 75: 4176–4183.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Smith TA, Idamakanti N, Rollence ML, Marshall-Neff J, Kim J, Mulgrew K et al. Adenovirus serotype 5 fiber shaft influences in vivo gene transfer in mice. Hum Gene Ther 2003; 14: 777–787.

    Article  CAS  PubMed  Google Scholar 

  16. Karayan L, Hong SS, Gay B, Tournier J, d'Angeac AD, Boulanger P . Structural and functional determinants in adenovirus type 2 penton base recombinant protein. J Virol 1997; 71: 8678–8689.

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Curiel DT . Strategies to alter the tropism of adenoviral vectors via genetic capsid modifications. In: Curiel DT, Douglas JT (eds). Vector targeting for therapeutic gene delivery. Wiley-Liss Inc.: Hoboken, 2002, pp 171–200.

    Chapter  Google Scholar 

  18. Nilsson B, Moks T, Jansson B, Abrahmsen L, Elmblad A, Holmgren E et al. A synthetic IgG-binding domain based on Staphylococcal protein A. Protein Eng 1987; 1: 107–113.

    Article  CAS  PubMed  Google Scholar 

  19. Nord K, Gunneriusson E, Ringdahl J, Stahl S, Uhlen M, Nygren PA . Binding proteins selected from combinatorial libraries of an alpha-helical bacterial receptor domain. Nat Biotechnol 1997; 15: 772–777.

    Article  CAS  PubMed  Google Scholar 

  20. Legrand V, Spehner D, Schlesinger Y, Settelen N, Pavirani A, Mehtali M . Fiberless recombinant adenoviruses: virus maturation and infectivity in the absence of fiber. J Virol 1999; 73: 907–919.

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Miyazawa N, Leopold PL, Hackett NR, Ferris B, Worgall S, Falck-Pedersen E et al. Fiber swap between adenovirus subgroups B and C alters intracellular trafficking of adenovirus gene transfer vectors. J Virol 1999; 73: 6056–6065.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Miyazawa N, Crystal RG, Leopold PL . Adenovirus serotype 7 retention in a late endosomal compartment prior to cytosol escape is modulated by fiber protein. J Virol 2001; 75: 1387–1400.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Shayakhmetov DM, Li ZY, Ternovoi V, Gaggar A, Gharwan H, Lieber A . The interaction between the fiber knob domain and the cellular attachment receptor determines the intracellular trafficking route of adenoviruses. J Virol 2003; 77: 3712–3723.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Gaden F, Franqueville L, Magnusson MK, Hong SS, Merten MD, Lindholm L et al. Gene transduction and cell entry pathway of fiber-modified adenovirus type 5 vectors carrying novel endocytic peptide ligands selected on human tracheal glandular cells. J Virol 2004; 78: 7227–7247.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Miyazawa N, Crystal RG, Leopold PL . Adenovirus serotype 7 retention in a late endosomal compartment prior to cytosol escape is modulated by fiber protein. J Virol 2001; 75: 1387–1400.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Krasnykh V, Dmitriev I, Mikheeva G, Miller CR, Belousova N, Curiel DT . Characterization of an adenovirus vector containing a heterologous peptide epitope in the HI loop of the fiber knob. J Virol 1998; 72: 1844–1852.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Krasnykh VN, Douglas JT, van Beusechem VW . Genetic targeting of adenoviral vectors. Mol Ther 2000; 1 Part 1: 391–405.

    Article  CAS  PubMed  Google Scholar 

  28. Fontana L, Nuzzo M, Urbanelli L, Monaci P . General strategy for broadening adenovirus tropism. J Virol Oct 2003; 77: 11094–11104.

    Article  CAS  Google Scholar 

  29. Codony-Servat J, Albanell J, Lopez-Talavera JC, Arribas J, Baselga J . Cleavage of the HER2 ectodomain is a pervanadate-activable process that is inhibited by the tissue inhibitor of metalloproteases-1 in breast cancer cells. Cancer Res 1999; 59: 1196–1201.

    CAS  PubMed  Google Scholar 

  30. Wikman M, Steffen AC, Gunneriusson E, Adams GP, Carlsson J, Stahl S . Selection and characterization of HER2/neu-binding affibody ligands. Protein Eng Des Sel 2004; 18: 18.

    Google Scholar 

  31. Menard S, Pupa SM, Campiglio M, Tagliabue E . Biologic and therapeutic role of HER2 in cancer. Oncogene 2003; 22: 6570–6578.

    Article  CAS  PubMed  Google Scholar 

  32. Steffen AC, Wikman M, Tolmachev V, Adams GP, Nilsson FY, Stahl S et al. In vitro characterization of a bivalent anti-HER-2 affibody with potential for radionuclide-based diagnostics. Cancer Biother Radiopharm 2005; 20: 239–248.

    Article  CAS  PubMed  Google Scholar 

  33. Wang SC, Hung MC . HER2 overexpression and cancer targeting. Semin Oncol 2001; 28: 115–124.

    Article  CAS  PubMed  Google Scholar 

  34. Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE et al. Studies of the HER-2/neu protooncogene in human breast and ovarian cancer. Science 1989; 244: 707–712.

    Article  CAS  PubMed  Google Scholar 

  35. Boyle P, Ferlay J . Cancer incidence and mortality in Europe, 2004. Ann Oncol 2005; 16: 481–488.

    Article  CAS  PubMed  Google Scholar 

  36. Hong JS, Engler JA . Domains required for assembly of adenovirus type 2 fiber trimers. J Virol 1996; 70: 7071–7078.

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Adams GP, Schier R, McCall AM, Simmons HH, Horak EM, Alpaugh RK et al. High affinity restricts the localization and tumor penetration of single-chain fv antibody molecules. Cancer Res 2001; 61: 4750–4755.

    CAS  PubMed  Google Scholar 

  38. Hansson M, Ringdahl J, Robert A, Power U, Goetsch L, Nguyen TN et al. An in vitro selected binding protein (affibody) shows conformation-dependent recognition of the respiratory syncytial virus (RSV) G protein. Immunotechnology 1999; 4: 237–252.

    Article  CAS  PubMed  Google Scholar 

  39. Hoppe HJ, Barlow PN, Reid KB . A parallel three stranded alpha-helical bundle at the nucleation site of collagen triple-helix formation. FEBS Lett 1994; 344: 191–195.

    Article  CAS  PubMed  Google Scholar 

  40. Henning P, Andersson KM, Frykholm K, Ali A, Magnusson MK, Nygren PA et al. Tumor cell targeted gene delivery by adenovirus 5 vectors carrying knobless fibers with antibody-binding domains. Gene Therapy 2005; 12: 211–224.

    Article  CAS  PubMed  Google Scholar 

  41. O'Reilly DR, Miller LK, Luckow VA . Virus methods. In: Press OU (ed). Baculovirus Expression Vectors. A Laboratory Manual. Oxford University Press Inc: Oxford, England, 1994, pp 124–138.

    Google Scholar 

  42. Mittereder N, March KL, Trapnell BC . Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy. J Virol 1996; 70: 7498–7509.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Gaden F, Franqueville L, Hong SS, Legrand V, Figarella C, Boulanger P . Mechanism of restriction of normal and cystic fibrosis transmembrane conductance regulator-deficient human tracheal gland cells to adenovirus infection and ad-mediated gene transfer. Am J Respir Cell Mol Biol 2002; 27: 628–640.

    Article  CAS  PubMed  Google Scholar 

  44. Brinkmann U, Buchner J, Pastan I . Independent domain folding of Pseudomonas exotoxin and single-chain immunotoxins: influence of interdomain connections. Proc Natl Acad Sci USA 1992; 89: 3075–3079.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Tang Y, Jiang N, Parakh C, Hilvert D . Selection of linkers for a catalytic single-chain antibody using phage display technology. J Biol Chem 1996; 271: 15682–15686.

    Article  CAS  PubMed  Google Scholar 

  46. Von Seggern DJ, Chiu CY, Fleck SK, Stewart PL, Nemerow GR . A helper-independent adenovirus vector with E1, E3, and fiber deleted: structure and infectivity of fiberless particles. J Virol 1999; 73: 1601–1608.

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Henning P, Lundgren E, Carlsson M, Frykholm K, Johannisson J, Magnusson MK et al. Adenovirus type 5 fiber knob domain has a critical role in fiber protein synthesis and encapsidation. J Gen Virol 2006; 87: 1351–1360.

    Article  Google Scholar 

  48. Wartlick H, Michaelis K, Balthasar S, Strebhardt K, Kreuter J, Langer K . Highly specific HER2-mediated cellular uptake of antibody-modified nanoparticles in tumour cells. J Drug Target 2004; 12: 461–471.

    Article  CAS  PubMed  Google Scholar 

  49. Germershaus O, Merdan T, Bakowsky U, Behe M, Kissel T . Trastuzumabpolyethylenimine-polyethylene glycol conjugates for targeting HER2-expressing tumors. Bioconjug Chem 2006; 17: 1190–1199.

    Article  CAS  PubMed  Google Scholar 

  50. Sarup JC, Johnson RM, King KL, Fendly BM, Lipari MT, Napier MA et al. Characterization of an anti-p185HER2 monoclonal antibody that stimulates receptor function and inhibits tumor cell growth. Growth Regul 1991; 1: 72–82.

    CAS  PubMed  Google Scholar 

  51. Nielsen UB, Kirpotin DB, Pickering EM, Hong K, Park JW, Refaat Shalaby M et al. Therapeutic efficacy of anti-ErbB2 immunoliposomes targeted by a phage antibody selected for cellular endocytosis. Biochim Biophys Acta 2002; 1591: 109–118.

    Article  CAS  PubMed  Google Scholar 

  52. Gill GN, Kawamoto T, Cochet C, Le A, Sato JD, Masui H et al. Monoclonal anti-epidermal growth factor receptor antibodies which are inhibitors of epidermal growth factor binding and antagonists of epidermal growth factor binding and antagonists of epidermal growth factor-stimulated tyrosine protein kinase activity. J Biol Chem 1984; 259: 7755–7760.

    CAS  PubMed  Google Scholar 

  53. Lesley J, Schulte R, Woods J . Modulation of transferrin receptor expression and function by anti-transferrin receptor antibodies and antibody fragments. Exp Cell Res 1989; 182: 215–233.

    Article  CAS  PubMed  Google Scholar 

  54. Nielsen UB, Marks JD . Internalizing antibodies and targeted cancer therapy: direct selection from phage display libraries. Pharm Sci Technol Today 2000; 3: 282–291.

    Article  CAS  PubMed  Google Scholar 

  55. Henry LJ, Xia D, Wilke ME, Deisenhofer J, Gerard RD . Characterization of the knob domain of the adenovirus type 5 fiber protein expressed in Escherichia coli. J Virol 1994; 68: 5239–5246.

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This study was supported by the European Community, Contract number: FP6-2003-LIFESCIHEALTH-I 512087, the GIANT and the JK Foundation, Gothenburg and Affibody AB. MF was supported by grant P25882-1 from the Swedish Agency of Innovation systems (VINNOVA).

Author information

Authors and Affiliations

  1. Got-a-Gene AB, Kullavik, Sweden

    M K Magnusson, P Henning, S Myhre, K M E Andersson & L Lindholm

  2. Department of Microbiology and Immunology, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden

    M K Magnusson, P Henning, S Myhre, K M E Andersson & L Lindholm

  3. Department of Biotechnology, Kungl Tekniska Högskolan, Albanova University Center, Stockholm, Sweden

    M Wikman, M Friedman & S Ståhl

  4. Department of Molecular Cell Biology, Leiden University Medical Centre, Leiden, The Netherlands

    T G Uil & R C Hoeben

  5. Laboratoire de Virologie et Pathogénèse Virale, Université Claude Bernard de Lyon, CNRS UMR-5537, Faculté de Médecine RTH Laennec, Lyon Cedex, France

    S S Hong & P Boulanger

  6. Department of Surgical Oncology and Technology, Faculty of Medicine, Imperial College, Hammersmith Hospital Campus, London, UK

    N A Habib

Authors
  1. M K Magnusson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. P Henning
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. S Myhre
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M Wikman
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. T G Uil
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. M Friedman
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. K M E Andersson
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. S S Hong
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. R C Hoeben
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. N A Habib
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. S Ståhl
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. P Boulanger
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. L Lindholm
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to L Lindholm.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Magnusson, M., Henning, P., Myhre, S. et al. Adenovirus 5 vector genetically re-targeted by an Affibody molecule with specificity for tumor antigen HER2/neu. Cancer Gene Ther 14, 468–479 (2007). https://doi.org/10.1038/sj.cgt.7701027

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.cgt.7701027

Keywords

This article is cited by

Search

Advanced search

Quick links