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An ex vivo loop system models the toxicity and efficacy of PEGylated and unmodified adenovirus serotype 5 in whole human blood

Gene Therapy volume 17, pages 752–762 (2010)Cite this article

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Abstract

Polyethylene glycol coating (PEGylation) of adenovirus serotype 5 (Ad5) has been shown to effectively reduce immunogenicity and increase circulation time of intravenously administered virus in mouse models. Herein, we monitored clot formation, complement activation, cytokine release and blood cell association upon addition of uncoated or PEGylated Ad5 to human whole blood. We used a novel blood loop model where human blood from healthy donors was mixed with virus and incubated in heparin-coated PVC tubing while rotating at 37 °C for up to 8 h. Production of the complement components C3a and C5a and the cytokines IL-8, RANTES and MCP-1 was significantly lower with 20K-PEGylated Ad5 than with uncoated Ad5. PEGylation prevented clotting and reduced Ad5 binding to blood cells in blood with low ability to neutralize Ad5. The effect was particularly pronounced in monocytes, granulocytes, B-cells and T-cells, but could also be observed in erythrocytes and platelets. In conclusion, PEGylation of Ad5 can reduce the immune response mounted in human blood, although the protective effects are rather modest in contrast to published mouse data. Our findings underline the importance of developing reliable models and we propose the use of human whole blood models in pre-clinical screening of gene therapy vectors.

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References

  1. Rein DT, Breidenbach M, Curiel DT . Current developments in adenovirus-based cancer gene therapy. Future Oncol 2006; 2: 137–143.

    Article  CAS  PubMed  Google Scholar 

  2. Danielsson A, Dzojic H, Nilsson B, Essand M . Increased therapeutic efficacy of the prostate-specific oncolytic adenovirus Ad[I/PPT-E1A] by reduction of the insulator size and introduction of the full-length E3 region. Cancer Gene Ther 2008; 15: 203–213.

    Article  CAS  PubMed  Google Scholar 

  3. Vaha-Koskela MJ, Heikkila JE, Hinkkanen AE . Oncolytic viruses in cancer therapy. Cancer Lett 2007; 254: 178–216.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Nemunaitis J, Senzer N, Sarmiento S, Zhang YA, Arzaga R, Sands B et al. A phase I trial of intravenous infusion of ONYX-015 and enbrel in solid tumor patients. Cancer Gene Ther 2007; 14: 885–893.

    Article  CAS  PubMed  Google Scholar 

  5. Vecil GG, Lang FF . Clinical trials of adenoviruses in brain tumors: a review of Ad-p53 and oncolytic adenoviruses. J Neurooncol 2003; 65: 237–246.

    Article  PubMed  Google Scholar 

  6. Small EJ, Carducci MA, Burke JM, Rodriguez R, Fong L, van Ummersen L et al. A phase I trial of intravenous CG7870, a replication-selective, prostate-specific antigen-targeted oncolytic adenovirus, for the treatment of hormone-refractory, metastatic prostate cancer. Mol Ther 2006; 14: 107–117.

    Article  CAS  PubMed  Google Scholar 

  7. Freytag SO, Stricker H, Peabody J, Pegg J, Paielli D, Movsas B et al. Five-year follow-up of trial of replication-competent adenovirus-mediated suicide gene therapy for treatment of prostate cancer. Mol Ther 2007; 15: 636–642.

    Article  CAS  PubMed  Google Scholar 

  8. Yu W, Fang H . Clinical trials with oncolytic adenovirus in China. Curr Cancer Drug Targets 2007; 7: 141–148.

    Article  PubMed  Google Scholar 

  9. Kim S, Peng Z, Kaneda Y . Current status of gene therapy in Asia. Mol Ther 2008; 16: 237–243.

    Article  CAS  PubMed  Google Scholar 

  10. Peng Z . Current status of gendicine in China: recombinant human Ad-p53 agent for treatment of cancers. Hum Gene Ther 2005; 16: 1016–1027.

    Article  CAS  PubMed  Google Scholar 

  11. Xia ZJ, Chang JH, Zhang L, Jiang WQ, Guan ZZ, Liu JW et al. [Phase III randomized clinical trial of intratumoral injection of E1B gene-deleted adenovirus (H101) combined with cisplatin-based chemotherapy in treating squamous cell cancer of head and neck or esophagus]. Ai Zheng 2004; 23: 1666–1670.

    PubMed  Google Scholar 

  12. Nemunaitis J, Khuri F, Ganly I, Arseneau J, Posner M, Vokes E et al. Phase II trial of intratumoral administration of ONYX-015, a replication-selective adenovirus, in patients with refractory head and neck cancer. J Clin Oncol 2001; 19: 289–298.

    Article  CAS  PubMed  Google Scholar 

  13. Garnett CT, Erdman D, Xu W, Gooding LR . Prevalence and quantitation of species C adenovirus DNA in human mucosal lymphocytes. J Virol 2002; 76: 10608–10616.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Sumida SM, Truitt DM, Lemckert AA, Vogels R, Custers JH, Addo MM et al. Neutralizing antibodies to adenovirus serotype 5 vaccine vectors are directed primarily against the adenovirus hexon protein. J Immunol 2005; 174: 7179–7185.

    Article  CAS  PubMed  Google Scholar 

  15. Nwanegbo E, Vardas E, Gao W, Whittle H, Sun H, Rowe D et al. Prevalence of neutralizing antibodies to adenoviral serotypes 5 and 35 in the adult populations of The Gambia, South Africa, and the United States. Clin Diagn Lab Immunol 2004; 11: 351–357.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Reid T, Galanis E, Abbruzzese J, Sze D, Wein LM, Andrews J et al. Hepatic arterial infusion of a replication-selective oncolytic adenovirus (dl1520): phase II viral, immunologic, and clinical endpoints. Cancer Res 2002; 62: 6070–6079.

    CAS  PubMed  Google Scholar 

  17. Lyons M, Onion D, Green NK, Aslan K, Rajaratnam R, Bazan-Peregrino M et al. Adenovirus type 5 interactions with human blood cells may compromise systemic delivery. Mol Ther 2006; 14: 118–128.

    Article  CAS  PubMed  Google Scholar 

  18. Muruve DA . The innate immune response to adenovirus vectors. Hum Gene Ther 2004; 15: 1157–1166.

    Article  CAS  PubMed  Google Scholar 

  19. Jiang H, Wang Z, Serra D, Frank MM, Amalfitano A . Recombinant adenovirus vectors activate the alternative complement pathway, leading to the binding of human complement protein C3 independent of anti-ad antibodies. Mol Ther 2004; 10: 1140–1142.

    Article  CAS  PubMed  Google Scholar 

  20. Kiang A, Hartman ZC, Everett RS, Serra D, Jiang H, Frank MM et al. Multiple innate inflammatory responses induced after systemic adenovirus vector delivery depend on a functional complement system. Mol Ther 2006; 14: 588–598.

    Article  CAS  PubMed  Google Scholar 

  21. Delgado C, Francis GE, Fisher D . The uses and properties of PEG-linked proteins. Crit Rev Ther Drug Carrier Syst 1992; 9: 249–304.

    CAS  PubMed  Google Scholar 

  22. Croyle MA, Yu QC, Wilson JM . Development of a rapid method for the PEGylation of adenoviruses with enhanced transduction and improved stability under harsh storage conditions. Hum Gene Ther 2000; 11: 1713–1722.

    Article  CAS  PubMed  Google Scholar 

  23. Hofherr SE, Mok H, Gushiken FC, Lopez JA, Barry MA . Polyethylene glycol modification of adenovirus reduces platelet activation, endothelial cell activation, and thrombocytopenia. Hum Gene Ther 2007; 18: 837–848.

    Article  CAS  PubMed  Google Scholar 

  24. O’Riordan CR, Lachapelle A, Delgado C, Parkes V, Wadsworth SC, Smith AE et al. PEGylation of adenovirus with retention of infectivity and protection from neutralizing antibody in vitro and in vivo. Hum Gene Ther 1999; 10: 1349–1358.

    Article  PubMed  Google Scholar 

  25. Gao JQ, Eto Y, Yoshioka Y, Sekiguchi F, Kurachi S, Morishige T et al. Effective tumor targeted gene transfer using PEGylated adenovirus vector via systemic administration. J Control Release 2007; 122: 102–110.

    Article  CAS  PubMed  Google Scholar 

  26. Mok H, Palmer DJ, Ng P, Barry MA . Evaluation of polyethylene glycol modification of first-generation and helper-dependent adenoviral vectors to reduce innate immune responses. Mol Ther 2005; 11: 66–79.

    Article  CAS  PubMed  Google Scholar 

  27. Kreppel F, Kochanek S . Modification of adenovirus gene transfer vectors with synthetic polymers: a scientific review and technical guide. Mol Ther 2008; 16: 16–29.

    Article  CAS  PubMed  Google Scholar 

  28. Wortmann A, Vohringer S, Engler T, Corjon S, Schirmbeck R, Reimann J et al. Fully detargeted polyethylene glycol-coated adenovirus vectors are potent genetic vaccines and escape from pre-existing anti-adenovirus antibodies. Mol Ther 2008; 16: 154–162.

    Article  CAS  PubMed  Google Scholar 

  29. Hofherr SE, Shashkova EV, Weaver EA, Khare R, Barry MA . Modification of adenoviral vectors with polyethylene glycol modulates in vivo tissue tropism and gene expression. Mol Ther 2008; 16: 1276–1282.

    Article  CAS  PubMed  Google Scholar 

  30. Wisse E, Jacobs F, Topal B, Frederik P, De Geest B . The size of endothelial fenestrae in human liver sinusoids: implications for hepatocyte-directed gene transfer. Gene Therapy 2008; 15: 1193–1199.

    Article  CAS  PubMed  Google Scholar 

  31. Carlisle RC, Di Y, Cerny AM, Sonnen AF, Sim RB, Green NK et al. Human erythrocytes bind and inactivate type 5 adenovirus by presenting coxsackievirus-adenovirus receptor and complement receptor 1. Blood 2009; 113: 1909–1918.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Repik A, Pincus SE, Ghiran I, Nicholson-Weller A, Asher DR, Cerny AM et al. A transgenic mouse model for studying the clearance of blood-borne pathogens via human complement receptor 1 (CR1). Clin Exp Immunol 2005; 140: 230–240.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Asher DR, Cerny AM, Finberg RW . The erythrocyte viral trap: transgenic expression of viral receptor on erythrocytes attenuates coxsackievirus B infection. Proc Natl Acad Sci USA 2005; 102: 12897–12902.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Alemany R, Suzuki K, Curiel DT . Blood clearance rates of adenovirus type 5 in mice. J Gen Virol 2000; 81: 2605–2609.

    Article  CAS  PubMed  Google Scholar 

  35. Dong JF, Moake JL, Nolasco L, Bernardo A, Arceneaux W, Shrimpton CN et al. ADAMTS-13 rapidly cleaves newly secreted ultralarge von Willebrand factor multimers on the endothelial surface under flowing conditions. Blood 2002; 100: 4033–4039.

    Article  CAS  PubMed  Google Scholar 

  36. Tian J, Xu Z, Smith JS, Hofherr SE, Barry MA, Byrnes AP . Adenovirus activates complement by distinctly different mechanisms in vitro and in vivo: indirect complement activation by virions in vivo. J Virol 2009; 83: 5648–5658.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Appledorn DM, McBride A, Seregin S, Scott JM, Schuldt N, Kiang A et al. Complex interactions with several arms of the complement system dictate innate and humoral immunity to adenoviral vectors. Gene Therapy 2008; 15: 1606–1617.

    Article  CAS  PubMed  Google Scholar 

  38. Seregin SS, Aldhamen YA, Appledorn DM, Schuldt NJ, McBride AJ, Bujold M et al. CR1/2 is an important suppressor of Adenovirus-induced innate immune responses and is required for induction of neutralizing antibodies. Gene Therapy 2009; 16: 1245–1259.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Wakimoto H, Ikeda K, Abe T, Ichikawa T, Hochberg FH, Ezekowitz RA et al. The complement response against an oncolytic virus is species-specific in its activation pathways. Mol Ther 2002; 5: 275–282.

    Article  CAS  PubMed  Google Scholar 

  40. Xu Z, Tian J, Smith JS, Byrnes AP . Clearance of adenovirus by Kupffer cells is mediated by scavenger receptors, natural antibodies, and complement. J Virol 2008; 82: 11705–11713.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Sahu A, Kay BK, Lambris JD . Inhibition of human complement by a C3-binding peptide isolated from a phage-displayed random peptide library. J Immunol 1996; 157: 884–891.

    CAS  PubMed  Google Scholar 

  42. Janssen BJ, Halff EF, Lambris JD, Gros P . Structure of compstatin in complex with complement component C3c reveals a new mechanism of complement inhibition. J Biol Chem 2007; 282: 29241–29247.

    Article  CAS  PubMed  Google Scholar 

  43. Cichon G, Boeckh-Herwig S, Schmidt HH, Wehnes E, Muller T, Pring-Akerblom P et al. Complement activation by recombinant adenoviruses. Gene Therapy 2001; 8: 1794–1800.

    Article  CAS  PubMed  Google Scholar 

  44. Kreppel F, Biermann V, Kochanek S, Schiedner G . A DNA-based method to assay total and infectious particle contents and helper virus contamination in high-capacity adenoviral vector preparations. Hum Gene Ther 2002; 13: 1151–1156.

    Article  CAS  PubMed  Google Scholar 

  45. Kreppel F, Gackowski J, Schmidt E, Kochanek S . Combined genetic and chemical capsid modifications enable flexible and efficient de- and retargeting of adenovirus vectors. Mol Ther 2005; 12: 107–117.

    Article  CAS  PubMed  Google Scholar 

  46. Katragadda M, Magotti P, Sfyroera G, Lambris JD . Hydrophobic effect and hydrogen bonds account for the improved activity of a complement inhibitor, compstatin. J Med Chem 2006; 49: 4616–4622.

    Article  CAS  PubMed  Google Scholar 

  47. Fairbanks VF, Tefferi A . Normal ranges for packed cell volume and hemoglobin concentration in adults: relevance to ‘apparent polycythemia’. Eur J Haematol 2000; 65: 285–296.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Jaan Hong (Div. Clinical Immunology, Uppsala University, Uppsala, Sweden) for drawing blood samples used in the experiments. This work was supported by funding from The Swedish Cancer Society (Grant CAN 2007/885 and CAN 2009/55), the Swedish Research Council (Grant K2008-68X-15270-04-3), Gunnar Nilsson's Cancer Foundation (Grant E50/08) and the European Community on behalf of GIANT (Grant LSHB-CT-2004-512087). M Essand is a recipient of the Swedish Cancer Society Senior Investigator Award.

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Authors and Affiliations

  1. Division of Clinical Immunology, Uppsala University, Uppsala, Sweden

    A Danielsson, G Elgue, B M Nilsson, B Nilsson, T H Tötterman & M Essand

  2. Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA

    J D Lambris

  3. Division of Gene Therapy, University of Ulm, Ulm, Germany

    S Kochanek & F Kreppel

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  1. A Danielsson
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Danielsson, A., Elgue, G., Nilsson, B. et al. An ex vivo loop system models the toxicity and efficacy of PEGylated and unmodified adenovirus serotype 5 in whole human blood. Gene Ther 17, 752–762 (2010). https://doi.org/10.1038/gt.2010.18

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