Cancer Immunology, Immunotherapy

, 60:1309

Tumor immunotherapy using adenovirus vaccines in combination with intratumoral doses of CpG ODN

  • S. M. Geary
  • C. D. Lemke
  • D. M. Lubaroff
  • A. K. Salem
Original article


The combination of viral vaccination with intratumoral (IT) administration of CpG ODNs is yet to be investigated as an immunotherapeutic treatment for solid tumors. Here, we show that such a treatment regime can benefit survival of tumor-challenged mice. C57BL/6 mice bearing ovalbumin (OVA)-expressing EG.7 thymoma tumors were therapeutically vaccinated with adenovirus type 5 encoding OVA (Ad5-OVA), and the tumors subsequently injected with the immunostimulatory TLR9 agonist, CpG-B ODN 1826 (CpG), 4, 7, 10, and 13 days later. This therapeutic combination resulted in enhanced mean survival times that were more than 3.5× longer than naïve mice, and greater than 40% of mice were cured and capable of resisting subsequent tumor challenge. This suggests that an adaptive immune response was generated. Both Ad5-OVA and Ad5-OVA + CpG IT treatments led to significantly increased levels of H-2 Kb-OVA-specific CD8+ lymphocytes in the peripheral blood and intratumorally. Lymphocyte depletion studies performed in vivo implicated both NK cells and CD8+ lymphocytes as co-contributors to the therapeutic effect. Analysis of tumor infiltrating lymphocytes (TILs) on day 12 post-tumor challenge revealed that mice treated with Ad5-OVA + CpG IT possessed a significantly reduced percentage of regulatory T lymphocytes (Tregs) within the CD4+ lymphocyte population, compared with TILs isolated from mice treated with Ad5-OVA only. In addition, the proportion of CD8+ TILs that were OVA-specific was reproducibly higher in the mice treated with Ad5-OVA + CpG IT compared with other treatment groups. These findings highlight the therapeutic potential of combining intratumoral CpG and vaccination with virus encoding tumor antigen.


Toll-like receptor Adenovirus Intratumoral therapy Immunotherapy Tumor immunology CpG ODN 

Supplementary material

262_2011_1038_MOESM1_ESM.pdf (63 kb)
Supplementary material 1 (PDF 34 kb)


  1. 1.
    Farkas AM, Finn OJ (2010) Vaccines based on abnormal self-antigens as tumor-associated antigens: immune regulation. Semin ImmunolGoogle Scholar
  2. 2.
    Gilboa E (2004) The promise of cancer vaccines. Nat Rev Cancer 4:401–411PubMedCrossRefGoogle Scholar
  3. 3.
    Finn OJ (2006) Human tumor antigens, immunosurveillance, and cancer vaccines. Immunol Res 36:73–82PubMedCrossRefGoogle Scholar
  4. 4.
    Waldmann TA (2003) Immunotherapy: past, present and future. Nat Med 9:269–277PubMedCrossRefGoogle Scholar
  5. 5.
    Rosenberg SA, Yang JC, Restifo NP (2004) Cancer immunotherapy: moving beyond current vaccines. Nat Med 10:909–915PubMedCrossRefGoogle Scholar
  6. 6.
    Liniger M, Zuniga A, Naim HY (2007) Use of viral vectors for the development of vaccines. Expert Rev Vaccines 6:255–266PubMedCrossRefGoogle Scholar
  7. 7.
    Cheng C, Gall JG, Kong WP, Sheets RL, Gomez PL et al (2007) Mechanism of ad5 vaccine immunity and toxicity: fiber shaft targeting of dendritic cells. PLoS Pathog 3:e25PubMedCrossRefGoogle Scholar
  8. 8.
    Miller G, Lahrs S, Pillarisetty VG, Shah AB, DeMatteo RP (2002) Adenovirus infection enhances dendritic cell immunostimulatory properties and induces natural killer and T-cell-mediated tumor protection. Cancer Res 62:5260–5266PubMedGoogle Scholar
  9. 9.
    Amato RJ (2010) 5T4-modified vaccinia Ankara: progress in tumor-associated antigen-based immunotherapy. Expert Opin Biol Ther 10:281–287PubMedCrossRefGoogle Scholar
  10. 10.
    McCluskie MJ, Weeratna RD (2001) Novel adjuvant systems. Curr Drug Targets Infect Disord 1:263–271PubMedCrossRefGoogle Scholar
  11. 11.
    Krieg AM (2007) Development of TLR9 agonists for cancer therapy. J Clin Invest 117:1184–1194PubMedCrossRefGoogle Scholar
  12. 12.
    Salucci V, Mennuni C, Calvaruso F, Cerino R, Neuner P et al (2006) CD8+ T-cell tolerance can be broken by an adenoviral vaccine while CD4+ T-cell tolerance is broken by additional co-administration of a Toll-like receptor ligand. Scand J Immunol 63:35–41PubMedCrossRefGoogle Scholar
  13. 13.
    Karan D, Krieg AM, Lubaroff DM (2007) Paradoxical enhancement of CD8 T cell-dependent anti-tumor protection despite reduced CD8 T cell responses with addition of a TLR9 agonist to a tumor vaccine. Int J Cancer 121:1520–1528PubMedCrossRefGoogle Scholar
  14. 14.
    Moore MW, Carbone FR, Bevan MJ (1988) Introduction of soluble protein into the class I pathway of antigen processing and presentation. Cell 54:777–785PubMedCrossRefGoogle Scholar
  15. 15.
    Giermasz AS, Urban JA, Nakamura Y, Watchmaker P, Cumberland RL et al (2009) Type-1 polarized dendritic cells primed for high IL-12 production show enhanced activity as cancer vaccines. Cancer Immunol Immunother 58:1329–1336PubMedCrossRefGoogle Scholar
  16. 16.
    Zhou F, Rouse BT, Huang L (1992) Prolonged survival of thymoma-bearing mice after vaccination with a soluble protein antigen entrapped in liposomes: a model study. Cancer Res 52:6287–6291PubMedGoogle Scholar
  17. 17.
    Kim TS, Chung SW, Kim SH, Kang SN, Kang BY (2000) Therapeutic anti-tumor response induced with epitope-pulsed fibroblasts genetically engineered for B7.1 expression and IFN-gamma secretion. Int J Cancer 87:427–433PubMedCrossRefGoogle Scholar
  18. 18.
    Hariharan K, Braslawsky G, Black A, Raychaudhuri S, Hanna N (1995) The induction of cytotoxic T cells and tumor regression by soluble antigen formulation. Cancer Res 55:3486–3489PubMedGoogle Scholar
  19. 19.
    Chung SW, Cohen EP, Kim TS (2004) Generation of tumor-specific cytotoxic T lymphocyte and prolongation of the survival of tumor-bearing mice using interleukin-18-secreting fibroblasts loaded with an epitope peptide. Vaccine 22:2547–2557PubMedCrossRefGoogle Scholar
  20. 20.
    Song W, Levy R (2005) Therapeutic vaccination against murine lymphoma by intratumoral injection of naive dendritic cells. Cancer Res 65:5958–5964PubMedCrossRefGoogle Scholar
  21. 21.
    Lonsdorf AS, Kuekrek H, Stern BV, Boehm BO, Lehmann PV et al (2003) Intratumor CpG-oligodeoxynucleotide injection induces protective antitumor T cell immunity. J Immunol 171:3941–3946PubMedGoogle Scholar
  22. 22.
    Elzey BD, Siemens DR, Ratliff TL, Lubaroff DM (2001) Immunization with type 5 adenovirus recombinant for a tumor antigen in combination with recombinant canarypox virus (ALVAC) cytokine gene delivery induces destruction of established prostate tumors. Int J Cancer 94:842–849PubMedCrossRefGoogle Scholar
  23. 23.
    Banchereau J, Briere F, Caux C, Davoust J, Lebecque S et al (2000) Immunobiology of dendritic cells. Annu Rev Immunol 18:767–811PubMedCrossRefGoogle Scholar
  24. 24.
    Mahnke K, Johnson TS, Ring S, Enk AH (2007) Tolerogenic dendritic cells and regulatory T cells: a two-way relationship. J Dermatol Sci 46:159–167PubMedCrossRefGoogle Scholar
  25. 25.
    Warren TL, Bhatia SK, Acosta AM, Dahle CE, Ratliff TL et al (2000) APC stimulated by CpG oligodeoxynucleotide enhance activation of MHC class I-restricted T cells. J Immunol 165:6244–6251PubMedGoogle Scholar
  26. 26.
    Gray RC, Kuchtey J, Harding CV (2007) CpG-B ODNs potently induce low levels of IFN-alphabeta and induce IFN-alphabeta-dependent MHC-I cross-presentation in DCs as effectively as CpG-A and CpG-C ODNs. J Leukoc Biol 81:1075–1085PubMedCrossRefGoogle Scholar
  27. 27.
    Ballas ZK, Rasmussen WL, Krieg AM (1996) Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA. J Immunol 157:1840–1845PubMedGoogle Scholar
  28. 28.
    Melero I, Duarte M, Ruiz J, Sangro B, Galofre J et al (1999) Intratumoral injection of bone-marrow derived dendritic cells engineered to produce interleukin-12 induces complete regression of established murine transplantable colon adenocarcinomas. Gene Ther 6:1779–1784PubMedCrossRefGoogle Scholar
  29. 29.
    Nishioka Y, Hirao M, Robbins PD, Lotze MT, Tahara H (1999) Induction of systemic and therapeutic antitumor immunity using intratumoral injection of dendritic cells genetically modified to express interleukin 12. Cancer Res 59:4035–4041PubMedGoogle Scholar
  30. 30.
    Candido KA, Shimizu K, McLaughlin JC, Kunkel R, Fuller JA et al (2001) Local administration of dendritic cells inhibits established breast tumor growth: implications for apoptosis-inducing agents. Cancer Res 61:228–236PubMedGoogle Scholar
  31. 31.
    Li J, Song W, Czerwinski DK, Varghese B, Uematsu S et al (2007) Lymphoma immunotherapy with CpG oligodeoxynucleotides requires TLR9 either in the host or in the tumor itself. J Immunol 179:2493–2500PubMedGoogle Scholar
  32. 32.
    Gabrilovich DI, Nagaraj S (2009) Myeloid-derived suppressor cells as regulators of the immune system. Nat Rev Immunol 9:162–174PubMedCrossRefGoogle Scholar
  33. 33.
    Zoglmeier C, Bauer H, Noerenberg D, Wedekind G, Bittner P, et al (2011) CpG blocks immune suppression by myeloid-derived suppressor cells in tumor-bearing mice. Clin Cancer ResGoogle Scholar
  34. 34.
    Murad YM, Clay TM (2009) CpG oligodeoxynucleotides as TLR9 agonists: therapeutic applications in cancer. BioDrugs 23:361–375PubMedCrossRefGoogle Scholar
  35. 35.
    Hartman ZC, Osada T, Glass O, Yang XY, Lei GJ et al (2010) Ligand-independent toll-like receptor signals generated by ectopic overexpression of MyD88 generate local and systemic antitumor immunity. Cancer Res 70:7209–7220PubMedCrossRefGoogle Scholar
  36. 36.
    Carpentier A, Laigle-Donadey F, Zohar S, Capelle L, Behin A et al (2006) Phase 1 trial of a CpG oligodeoxynucleotide for patients with recurrent glioblastoma. Neuro Oncol 8:60–66PubMedCrossRefGoogle Scholar
  37. 37.
    Amato RJ, Shingler W, Goonewardena M, de Belin J, Naylor S et al (2009) Vaccination of renal cell cancer patients with modified vaccinia Ankara delivering the tumor antigen 5T4 (TroVax) alone or administered in combination with interferon-alpha (IFN-alpha): a phase 2 trial. J Immunother 32:765–772PubMedCrossRefGoogle Scholar
  38. 38.
    Krishnamachari Y, Geary SM, Lemke CD, Salem AK (2010) Nanoparticle Delivery systems in cancer vaccines. Pharm ResGoogle Scholar
  39. 39.
    Ramlau R, Quoix E, Rolski J, Pless M, Lena H et al (2008) A phase II study of Tg4010 (Mva-Muc1-Il2) in association with chemotherapy in patients with stage III/IV non-small cell lung cancer. J Thorac Oncol 3:735–744PubMedCrossRefGoogle Scholar
  40. 40.
    Lubaroff DM, Karan D (2009) CpG oligonucleotide as an adjuvant for the treatment of prostate cancer. Adv Drug Deliv Rev 61:268–274PubMedCrossRefGoogle Scholar
  41. 41.
    Shen Y, Nemunaitis J (2005) Fighting cancer with vaccinia virus: teaching new tricks to an old dog. Mol Ther 11:180–195PubMedCrossRefGoogle Scholar
  42. 42.
    Neri S, Mariani E, Meneghetti A, Cattini L, Facchini A (2001) Calcein-acetyoxymethyl cytotoxicity assay: standardization of a method allowing additional analyses on recovered effector cells and supernatants. Clin Diagn Lab Immunol 8:1131–1135PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • S. M. Geary
    • 1
  • C. D. Lemke
    • 1
  • D. M. Lubaroff
    • 2
  • A. K. Salem
    • 1
  1. 1.Division of Pharmaceutics, College of PharmacyUniversity of IowaIowa CityUSA
  2. 2.Departments of Urology & Microbiology, Holden Comprehensive Cancer CenterUniversity of IowaIowa CityUSA

Personalised recommendations