Intravenous delivery of a multi-mechanistic cancer-targeted oncolytic poxvirus in humans

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The efficacy and safety of biological molecules in cancer therapy, such as peptides and small interfering RNAs (siRNAs), could be markedly increased if high concentrations could be achieved and amplified selectively in tumour tissues versus normal tissues after intravenous administration. This has not been achievable so far in humans. We hypothesized that a poxvirus, which evolved for blood-borne systemic spread in mammals, could be engineered for cancer-selective replication and used as a vehicle for the intravenous delivery and expression of transgenes in tumours. JX-594 is an oncolytic poxvirus engineered for replication, transgene expression and amplification in cancer cells harbouring activation of the epidermal growth factor receptor (EGFR)/Ras pathway, followed by cell lysis and anticancer immunity1. Here we show in a clinical trial that JX-594 selectively infects, replicates and expresses transgene products in cancer tissue after intravenous infusion, in a dose-related fashion. Normal tissues were not affected clinically. This platform technology opens up the possibility of multifunctional products that selectively express high concentrations of several complementary therapeutic and imaging molecules in metastatic solid tumours in humans.

At a glance


  1. Ex vivo infection of explants of tumour and normal tissue from patients reveals tumour-selective JX-594 gene expression.
    Figure 1: Ex vivo infection of explants of tumour and normal tissue from patients reveals tumour-selective JX-594 gene expression.

    JX-594 expressing green fluorescent protein (GFP) (JX-594-GFP+/β-gal) was used to infect primary live-tissue specimens from cancer patients undergoing surgical resection. Matched tumour and adjacent normal tissues were infected overnight to assess the selectivity of transgene expression and replication, or were treated with PBS as a negative control. GFP expression from JX-594-GFP+/β-gal-infected cells was assessed using a fluorescence microscope. N = normal tissue, C = cancer tissue.

  2. JX-594 is selectively delivered to, and amplified within, tumours after intravenous infusion.
    Figure 2: JX-594 is selectively delivered to, and amplified within, tumours after intravenous infusion.

    a, Acute pharmacokinetics of JX-594 genomes (qPCR) after a single intravenous infusion, plotted by dose cohort. Error bars are s.e.m. b, Dose-dependent delivery of JX-594, as demonstrated by PCR and/or immunohistochemical (IHC) analysis of tumour biopsies collected 8–10days after treatment. c, Dose-dependent induction of antibodies to β-galactosidase in patients evaluable for this endpoint. ρ = Spearman’s rank correlation coefficient. The number of patients evaluable for each group (n) is indicated.

  3. Immunohistochemical staining reveals JX-594 infection and [bgr]-galactosidase expression in tumours.
    Figure 3: Immunohistochemical staining reveals JX-594 infection and β-galactosidase expression in tumours.

    a, Immunohistochemistry for vaccinia (patient 20, 10days after treatment). Scale bar, 200μm. b, Immunohistochemistry, no primary antibody. Scale bar, 200μm. c, Immunohistochemistry for vaccinia in pre-treatment biopsy. Scale bar, 50μm. df, As in ac for patient 18, biopsy at 8days after treatment. Scale bars, 50μm. g, h, Three-dimensional reconstruction of vaccinia (green) throughout tumour in patient 20. Scale bars 200μm. i, Immunohistochemistry for vaccinia at low magnification (patient 20). Scale bar, 500μm. Black arrows indicate tumour; red arrows indicate normal tissue. j, Immunohistochemistry for β-galactosidase (patient 20). Scale bar, 50μm. k, Immunohistochemistry for vaccinia. Scale bar, 50μm. l, Negative control. Scale bar, 50μm. Linear adjustment to brightness and contrast was applied to jl.


  1. Kirn, D. H. & Thorne, S. H. Targeted and armed oncolytic poxviruses: a novel multi-mechanistic therapeutic class for cancer. Nature Rev. Cancer 9, 6471 (2009)
  2. Parato, K. A., Senger, D., Forsyth, P. A. & Bell, J. C. Recent progress in the battle between oncolytic viruses and tumours. Nature Rev. Cancer 5, 965976 (2005)
  3. Liu, T. C., Galanis, E. & Kirn, D. Clinical trial results with oncolytic virotherapy: a century of promise, a decade of progress. Nat. Clin. Pract. Oncol. 4, 101117 (2007)
  4. Vanderplasschen, A., Hollinshead, M. & Smith, G. L. Antibodies against vaccinia virus do not neutralize extracellular enveloped virus but prevent virus release from infected cells and comet formation. J. Gen. Virol. 78, 20412048 (1997)
  5. Vanderplasschen, A., Mathew, E., Hollinshead, M., Sim, R. B. & Smith, G. L. Extracellular enveloped vaccinia virus is resistant to complement because of incorporation of host complement control proteins into its envelope. Proc. Natl Acad. Sci. USA 95, 75447549 (1998)
  6. Wein, L. M., Wu, J. T. & Kirn, D. H. Validation and analysis of a mathematical model of a replication-competent oncolytic virus for cancer treatment: implications for virus design and delivery. Cancer Res. 63, 13171324 (2003)
  7. Smith, G. L., Murphy, B. J. & Law, M. Vaccinia virus motility. Annu. Rev. Microbiol. 57, 323342 (2003)
  8. Katsafanas, G. C. & Moss, B. Vaccinia virus intermediate stage transcription is complemented by Ras-GTPase-activating protein SH3 domain-binding protein (G3BP) and cytoplasmic activation/proliferation-associated protein (p137) individually or as a heterodimer. J. Biol. Chem. 279, 5221052217 (2004)
  9. Yang, H. et al. Antiviral chemotherapy facilitates control of poxvirus infections through inhibition of cellular signal transduction. J. Clin. Invest. 115, 379387 (2005)
  10. Hanahan, D. & Weinberg, R. A. The hallmarks of cancer. Cell 100, 5770 (2000)
  11. Mastrangelo, M. J. et al. Intratumoral recombinant GM-CSF-encoding virus as gene therapy in patients with cutaneous melanoma. Cancer Gene Ther. 6, 409422 (1999)
  12. Kim, J. H. et al. Systemic armed oncolytic and immunologic therapy for cancer with JX-594, a targeted poxvirus expressing GM-CSF. Mol. Ther. 14, 361370 (2006)
  13. Buller, R. M., Smith, G. L., Cremer, K., Notkins, A. L. & Moss, B. Decreased virulence of recombinant vaccinia virus expression vectors is associated with a thymidine kinase-negative phenotype. Nature 317, 813815 (1985)
  14. Park, B. H. et al. Use of a targeted oncolytic poxvirus, JX-594, in patients with refractory primary or metastatic liver cancer: a phase I trial. Lancet Oncol. 9, 533542 (2008)
  15. Hovgaard, D., Mortensen, B. T., Schifter, S. & Nissen, N. I. Clinical pharmacokinetic studies of a human haemopoietic growth factor, GM-CSF. Eur. J. Clin. Invest. 22, 4549 (1992)
  16. Choi, H. et al. Correlation of computed tomography and positron emission tomography in patients with metastatic gastrointestinal stromal tumor treated at a single institution with imatinib mesylate: proposal of new computed tomography response criteria. J. Clin. Oncol. 25, 17531759 (2007)
  17. Byrne, M. J. & Nowak, A. K. Modified RECIST criteria for assessment of response in malignant pleural mesothelioma. Ann. Oncol. 15, 257260 (2004)
  18. Moss, B. Vaccinia virus: a tool for research and vaccine development. Science 252, 16621667 (1991)
  19. Davis, M. E. et al. Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature 464, 10671070 (2010)
  20. Peng, K. W. et al. Intraperitoneal therapy of ovarian cancer using an engineered measles virus. Cancer Res. 62, 46564662 (2002)
  21. McCart, J. A. et al. Oncolytic vaccinia virus expressing the human somatostatin receptor SSTR2: molecular imaging after systemic delivery using 111In-pentetreotide. Mol. Ther. 10, 553561 (2004)
  22. Msaouel, P. et al. Noninvasive imaging and radiovirotherapy of prostate cancer using an oncolytic measles virus expressing the sodium iodide symporter. Mol. Ther. 17, 20412048 (2009)
  23. McCart, J. A. et al. Systemic cancer therapy with a tumor-selective vaccinia virus mutant lacking thymidine kinase and vaccinia growth factor genes. Cancer Res. 61, 87518757 (2001)
  24. Thorne, S. H. et al. Rational strain selection and engineering creates a broad-spectrum, systemically effective oncolytic poxvirus, JX-963. J. Clin. Invest. 117, 33503358 (2007)
  25. Thorne, S. H. et al. Targeting localized immune suppression within the tumor through repeat cycles of immune cell-oncolytic virus combination therapy. Mol. Ther. 18, 16981705 (2010)
  26. Rozen, S. & Skaletsky, H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol. 132, 365386 (2000)
  27. Kulesh, D. A. et al. Smallpox and pan-orthopox virus detection by real-time 3′-minor groove binder TaqMan assays on the Roche LightCycler and the Cepheid Smart Cycler platforms. J. Clin. Microbiol. 42, 601609 (2004)
  28. Therasse, P. et al. New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J. Natl. Cancer Inst. 92, 205216 (2000)
  29. Choi, H. et al. CT evaluation of the response of gastrointestinal stromal tumors after imatinib mesylate treatment: a quantitative analysis correlated with FDG PET findings. AJR Am. J. Roentgenol. 183, 16191628 (2004)
  30. Myers, J. L. W. & Arnold, D. Research Design and Statistical Analysis. 2nd edn, 505512 (Laurence Erlbaum, 2003)

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Author information

  1. These authors contributed equally to this work.

    • John C. Bell &
    • David H. Kirn


  1. Jennerex Inc., 450 Sansome Street, 16th floor, San Francisco, California 94111, USA

    • Caroline J. Breitbach,
    • Anne Moon,
    • Adina Pelusio,
    • Terri Robertson &
    • David H. Kirn
  2. Department of Hematology/Oncology, 801 North 29th Street, Billings Clinic, Billings, Montana 59101, USA

    • James Burke &
    • Jorge Nieva
  3. Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, Ontario K1H 8L6, Canada

    • Derek Jonker,
    • Laura Q. M. Chow,
    • Fabrice Le Boeuf,
    • Joe Burns,
    • Laura Evgin,
    • Naomi De Silva,
    • Sara Cvancic,
    • Kelley Parato,
    • Jean-Simon Diallo,
    • Manijeh Daneshmand &
    • John C. Bell
  4. University of Ottawa, 75 Laurier Avenue East, Ottawa, Ontario K1N 6N5, Canada

    • Derek Jonker,
    • Laura Q. M. Chow,
    • Joe Burns,
    • Laura Evgin,
    • Naomi De Silva,
    • Sara Cvancic,
    • Manijeh Daneshmand &
    • John C. Bell
  5. Cancer Centers of the Carolinas, 3 Butternut Drive, Greenville, South Carolina 29605, USA

    • Joe Stephenson
  6. University of Pennsylvania Medical Center, 3400 Spruce Street, Philadelphia, Pennsylvania 19104, USA

    • Andrew R. Haas
  7. Pusan National University, Jangjeon-dong GeumJeong-gu, Busan 609-735, South Korea

    • Tae-Ho Hwang,
    • Ji-Eun Je &
    • Yeon-Sook Lee
  8. RadMD, 712 Hyde Park, Doylestown, Pennsylvania 18901, USA

    • Richard Patt
  9. Robarts Research Institute, 100 Perth Drive, P.O. Box 5015, London, Ontario N6A 5K8, Canada

    • Aaron Fenster
  10. Present address: Jennerex Inc., 450 Sansome Street, 16th floor, San Francisco, California 94111, USA.

    • James Burke


Study design: D.H.K. and J.C.B. Data analysis and study write-up: C.J.B., D.H.K., T.-H.H., A.M., R.P., A.P., T.R., J.C.B. and A.F. Enrolment and management of patients: J.B., D.J., J.S., A.R.H., L.Q.M.C. and J.N. Laboratory work: F.L.B., J.B., N.D.S., S.C., J.-E.J., L.E., Y.-S.L., K.P., J.S.D., M.D. and J.-S.D. C.J.B. and D.H.K. had access to all the data in the trial. C.J.B. and D.H.K. took the final decision to submit for publication.

Competing financial interests

C.J.B., J.B., A.M., A.P., T.R. and D.H.K. are employees of Jennerex Inc. and hold stock options in Jennerex Inc. T.-H.H. and J.C.B. consult for and hold stock options in Jennerex Inc. R.P., Y.-S.L. and M.D. consult for Jennerex Inc.

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Supplementary information

PDF files

  1. Supplementary Information (636K)

    The file contains Supplementary Figures 1-4 with legends, a Supplementary Discussion and Supplementary References.


  1. Supplementary Movie 1 (1.5M)

    This movie shows the 360° view of a colorectal carcinoma tumour in which vaccinia (JX-594) antigens were detected by immunohistochemistry. Green staining represents areas of tumour staining for vaccinia (JX-594).


  1. Report this comment #31286

    Miriam Gothe said:

    On behalf of Tibor Bakacs

    Oncolytic NDV therapy effective for years

    There is no need to speculate whether intravenous pharmacological dosing of JX-594 oncolytic poxvirus is able to transiently saturate native mechanisms of viral clearance such that repeated intravenous delivery will be feasible (1). More than a decade ago Csatary et al. (2) had demonstrated in 30 cancer patients receiving an attenuated Newcastle disease virus (NDV) vaccine for up to 8 years that repeated intravenous delivery of virus is not only feasible but necessary for clinical efficacy in the presence of neutralizing antibodies.

    In contrast to your editorial claim, (3) infection of tumours with oncolytic viruses is not a new type of treatment (4). Out of a total of 53 viruses tested as anticancer agents, 38 exerted antineoplastic effects in either animals or humans (5). Notwithstanding, developers of the JX-594 oncolytic poxvirus appear to be unmindful of r elevant experiences obtained in patients with NDV during the last 50 years. Therefore, it is perhaps useful to bring the recent past of oncolytic NDV virotherapy into sharper focus in order to avoid some risky strategies (e.g. immunosuppression by cyclophosphamide to potentiate viral replication and hence enhance tumour oncolysis (6)).

    Among the non-engineered oncolytic viruses NDV has a long history as a broad-spectrum oncolytic agent that can destroy tumour cells and stimulate the immune system. NDV is a single-stranded RNA virus, whose natural host is poultry. As early as 1965 NDV was reported to have interesting anti-neoplastic properties (7). Several NDV strains (MTH-68/H, NDV-PV701, NDV-Ulster, NDV-HUJ) have been the subject of systematic clinical studies in patients who had exhausted all conventional cancer treatments (8) (9) (10) (11) (12). NDV-infected patient-de rived tumor cell vaccines were also used to achieve long-lasting T cell mediated systemic anti-tumor immune memory (12). A randomized-controlled prospective study demonstrated that NDV helped to induce post-oncolytic anti-tumor immunity which improved 10-year survival of colon cancer patients operated from liver metastases (13). In addition, various studies from different institutions in Europe reported responsiveness of high-grade glioblastoma multiforme (GBM) to NDV treatment including tumour remissions and improved survival (14) (15) (16) (17).

    In contrast to native pox virus, native NDV shows tumor-selectivity a priori in its replication behavior in mammalian cells, including human (12) (18). NDV derived hemagglutinin-neuraminidase (HN) proteins were demonstrated to activate NKp46 receptors and tumor-killing activity in NK cells (19) and to stimula te a strong type I interferon response in monocytes, macrophages and plasmacytoid and myeloid dendritic cells (12) (18) (20). While such interferon response explains the very good tolerability of even high NDV doses, many tumor cells are incapable to prevent oncolytic NDV replication due to their deficient interferon response. Tumor targeting of NDV could be improved by bispecific antibodies (21). Delivery of additional therapeutic genes via recombinant NDV strains has also been reported (22).

    A tumor cell line which was entirely resistant to NDV (MTH 68/H) in vitro could nevertheless be affected in vivo after metastasis to the liver upon locoregional (but not upon intravenous) virus delivery (23). Lessons obtained from cancer treatment with oncolytic NDV in animals and cancer patients over decades in Germany demonstrated that oncolysis is not the only mechanism that matters in vivo. In sit u activation of host anti-tumor immune mechanisms including long-term T cell mediated tumor-specific memory may be equally if not more important for improvement of long-term overall survival with cancer.

    Why should neuroblastoma patients have to wait for matching tumour features with 131 different drugs that might help treat diseases for which they weren?t designed (24), when NDV has already proved to be a powerful weapon against the most malignant neuroectodermal tumour, GBM (15) (16) (17) (18) (25)?

    We have no competing financial interests.

    Tibor Bakacs (a)*, Volker Schirrmacher (b) and Ralph W. Moss (c)


    (a) Department of Probability, Alfred Renyi Institute of Mathematics, Hungarian Academy of Sciences, Realtanoda utca 13-15, H-1053 Budapest, Hungary; tel.: 361-483-8324;

    (b) German Cancer Resea rch Center, Division of Translational Immunology, 69120 Heidelberg, Germany and IOZK K?????? Germany ; tel.:0049-6221-29540;

    (c) Cancer Communications, Inc., PO Box 1076, Lemont, PA 16851; tel.: 814-238-3367;;

    *Corresponding author: Tibor Bakacs

    Reference List

    1. Breitbach,C.J. et al. Intravenous delivery of a multi-mechanistic cancer-targeted oncolytic poxvirus in humans. Nature 477, 99-102 (2011). 2. Csatary,L.K. et al. Beneficial treatment of patients with advanced cancer using a Newcastle disease virus vaccine (MTH-68/H). Anticancer Res. 19, 635-638 (1999). 3. Galanis,E. Cancer: Tumour-fighting virus homes in. Nature 477, 40-41 (2011). 4. Csatary,L.K., Csatary,E., & Moss,R.W. Re: Scientific interest in newcastle disease virus is reviving. J Natl. Cancer Inst. 92, 493-494 (2000& #041;. 5. Webb,H.E. & Smith,C.E. Viruses in the treatment of cancer. Lancet 1, 1206-1208 (1970). 6. Prestwich,R.J. et al. The case of oncolytic viruses versus the immune system: waiting on the judgment of Solomon. Hum. Gene Ther. 20, 1119-1132 (2009). 7. Cassel,W.A. & Garrett,R.E. Newcastle disease virus as an antineoplastic agent. Cancer 18, 863-868 (1965). 8. Csatary,L.K. et al. Attenuated veterinary virus vaccine for the treatment of cancer. Cancer Detect. Prev. 17, 619-627 (1993). 9. Lorence,R.M. et al. Overview of phase I studies of intravenous administration of PV701, an oncolytic virus. Curr. Opin. Mol. Ther. 5, 618-624 (2003). 10. Pecora,A.L. et al. Phase I trial of intravenous administration of PV701, an oncolytic virus, in patients with advanced solid cancers. J. Clin. Oncol. 20, 2251-2266 (20 02). 11. Freeman,A.I. et al. Phase I/II trial of intravenous NDV-HUJ oncolytic virus in recurrent glioblastoma multiforme. Mol. Ther. 13, 221-228 (2006). 12. Schirrmacher,V. & Fournier,P. Newcastle disease virus: a promising vector for viral therapy, immune therapy, and gene therapy of cancer. Methods Mol. Biol. 542, 565-605 (2009). 13. Schulze,T. et al. Efficiency of adjuvant active specific immunization with Newcastle disease virus modified tumor cells in colorectal cancer patients following resection of liver metastases: results of a prospective randomized trial. Cancer Immunol. Immunother. 58, 61-69 (2009). 14. Steiner,H.H. et al. Antitumor vaccination of patients with glioblastoma multiforme: a pilot study to assess feasibility, safety, and clinical benefit. J Clin. Oncol. 22, 4272-4281 (2004). 15. Csatary,L.K. & Bakacs,T. Use of Newcastle disease virus vaccine (MTH-68/H) in a patient with high-grade glioblastoma. JAMA 281, 1588-1589 (1999). 16. Csatary,L.K. et al. MTH-68/H oncolytic viral treatment in human high-grade gliomas. J Neurooncol. 67, 83-93 (2004). 17. Nesselhut,J. et al. Improvement of dendritic cell therapy in glioblastoma multiforme WHO 4 by Newcastle disease virus. J.Clin.Oncol., ASCO Annual Meeting Proceedings (Post-Meeting Edition) 29[15_suppl]. 2011. Ref Type: Abstract 18. Fournier,P., Wilden,H., & Schirrmacher,V. Importance of retinoic acid-inducible gene I and of receptor for type I interferon for cellular resistance to infection by Newcastle disease virus. Int. J Oncol. 40, 287-298 (2012). 19. Jarahian,M. et al. Activation of natural killer cells by newcastle disease virus hemagglutinin-neuraminidase. J Virol. 83, 8108-8121 (2009). 09;20. Fournier,P., Arnold,A., & Schirrmacher,V. Polarization of human monocyte-derived dendritic cells to DC1 by in vitro stimulation with Newcastle Disease Virus. J BUON. 14 Suppl 1, S111-S122 (2009). 21. Bian,H., Wilden,H., Fournier,P., Peeters,B., & Schirrmacher,V. In vivo efficacy of systemic tumor targeting of a viral RNA vector with oncolytic properties using a bispecific adapter protein. Int. J Oncol. 29, 1359-1369 (2006). 22. Janke,M. et al. Recombinant Newcastle disease virus (NDV) with inserted gene coding for GM-CSF as a new vector for cancer immunogene therapy. Gene Ther. 14, 1639-1649 (2007). 23. Apostolidis,L., Schirrmacher,V., & Fournier,P. Host mediated anti-tumor effect of oncolytic Newcastle disease virus after locoregional application. Int. J Oncol. 31, 1009-1019 (2007). 24. Couzin-Frankel,J. Pushing the Envelope in N euroblastoma Therapy. Science 333, 1569-1571 (2011). 25. Reichard,K.W., Lorence,R.M., Katubig,B.B., Peeples,M.E., & Reyes,H.M. Retinoic acid enhances killing of neuroblastoma cells by Newcastle disease virus. J. Pediatr. Surg. 28, 1221-1225 (1993).

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