Cancer Immunology, Immunotherapy

, Volume 62, Issue 1, pp 171–182 | Cite as

Low-dose cyclophosphamide administered as daily or single dose enhances the antitumor effects of a therapeutic HPV vaccine

  • Shiwen Peng
  • Sofia Lyford-Pike
  • Belinda Akpeng
  • Annie Wu
  • Chien-Fu Hung
  • Drew Hannaman
  • John R. Saunders
  • T.-C. Wu
  • Sara I. PaiEmail author
Original Article


Although therapeutic HPV vaccines are able to elicit systemic HPV-specific immunity, clinical responses have not always correlated with levels of vaccine-induced CD8+ T cells in human clinical trials. This observed discrepancy may be attributable to an immunosuppressive tumor microenvironment in which the CD8+ T cells are recruited. Regulatory T cells (Tregs) are cells that can dampen cytotoxic CD8+ T-cell function. Cyclophosphamide (CTX) is a systemic chemotherapeutic agent, which can eradicate immune cells, including inhibitory Tregs. The optimal dose and schedule of CTX administration in combination with immunotherapy to eliminate the Treg population without adversely affecting vaccine-induced T-cell responses is unknown. Therefore, we investigated various dosing and administration schedules of CTX in combination with a therapeutic HPV vaccine in a preclinical tumor model. HPV tumor-bearing mice received either a single preconditioning dose or a daily dose of CTX in combination with the pNGVL4a-CRT/E7(detox) DNA vaccine. Both single and daily dosing of CTX in combination with vaccine had a synergistic antitumor effect as compared to monotherapy alone. The potent antitumor responses were attributed to the reduction in Treg frequency and increased infiltration of HPV16 E7-specific CD8+ T cells, which led to higher ratios of CD8+/Treg and CD8+/CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs). There was an observed trend toward decreased vaccine-induced CD8+ T-cell frequency with daily dosing of CTX. We recommend a single, preconditioning dose of CTX prior to vaccination due to its efficacy, ease of administration, and reduced cumulative adverse effect on vaccine-induced T cells.


Cyclophosphamide Human papillomavirus Head and neck cancer Regulatory T cells Vaccine Immunomodulatory agents 



This work was supported by the National Institutes of Health (NIH)/National Institute of Dental and Craniofacial Research (NIDCR) Head and Neck SPORE Program P50 DE019032 grant and the Milton J. Dance Jr. Head and Neck Center at Greater Baltimore Medical Center, Baltimore, MD.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

262_2012_1322_MOESM1_ESM.tif (130 kb)
Supplemental Figure 1 High frequency of regulatory T cells (Treg) are present in HPV-related head and neck cancers. The frequency of Tregs was evaluated in the peripheral blood, contralateral normal tonsil tissue, and tumors of HPV-related head and neck cancer patients. Single-cell suspensions were prepared from tonsil tumor or contralateral normal tissue. PBMC was prepared by Ficoll density centrifugation. Cells were stained for surface CD4 and CD25, permeabilized, fixed, and stained for intracellular Foxp3 expression. a. Top panel: Representative flow cytometry data (based on gating of total CD4+ population). Bottom panel: Summary of the flow cytometry data demonstrating increased frequency of Tregs within the tumor microenvironment as compared to the contralateral normal tonsil and circulating peripheral blood (p = 0.01) of HPV-related head and neck cancer patients. b. A higher frequency of Tregs was found in the tumor microenvironment of HPV-related head and neck cancer patients (n = 5) compared to that of benign tonsil controls (n = 7) (p = 0.03). (TIFF 130 kb)
262_2012_1322_MOESM2_ESM.tif (68 kb)
Supplemental Figure 2 Daily administration of CTX can inhibit vaccine induced immunologic responses at a threshold dose. 1 × 105 TC-1 tumor cells per mouse were injected subcutaneously (s.c.) in the right flank of 5- to 8-wk-old C57BL/6 mice (five mice per group) and were treated with either pNGVL4a-CRT/E7(detox) DNA vaccine alone, daily dose of 10 mg/kg of CTX alone, daily dose of 20 mg/kg of CTX alone, daily dose of 10 mg/kg of CTX in combination with pNGVL4a-CRT/E7(detox) DNA vaccine, daily dose of 20 mg/kg of CTX in combination with pNGVL4a-CRT/E7(detox) DNA vaccine, or no treatment. 7 days after the last vaccination, single cells were prepared from spleen and tumors. To detect regulatory T cells, the lymphocytes were surface stained with anti-mouse CD4 followed by intracellular Foxp3 staining according to the manufacturer’s protocol. HPV-16 E7aa49-57 peptide-specific CD8+ T cells were evaluated with tetramer staining. Specifically, 1 × 105 tumor-infiltrating lymphocytes were stained with purified anti-mouse CD16/32, and then stained with FITC-conjugated anti-mouse CD8a, PE-conjugated HPV-16 E7aa49-57 peptide loaded H-2Db tetramer (provided by National Institute of Allergy and Infectious Disease MHC Tetramer Core Facility), and APC-conjugated anti-mouse CD3. a Summary of the flow cytometry data of regulatory T cells from tumor-infiltrating lymphocytes. b Summary of the flow cytometry data of CD3+CD8+E7 tetramer+ T cells from tumor-infiltrating lymphocytes. (TIFF 67 kb)
262_2012_1322_MOESM3_ESM.tif (64 kb)
Supplemental Figure 3 Administration of CTX on TC-1 tumor growth in T cell deficient athymic nude mice has no anti-tumor effect. a Schedule of CTX treatment of TC-1 tumor-bearing athymic nude mice. b Tumor growth curve from TC-1 tumor-bearing athymic nude mice treated with CTX either daily or single dose. Briefly, 1 × 105 TC-1 tumor cells per mouse were injected into 5- to 8-week-old athymic nude mice (five mice per group) s.c. in the right flank. After 8 days, the mice were treated as indicated in the schematic diagram of the CTX treatment schedule (A). Tumor volume was calculated as described in the material and method section. (TIFF 64 kb)


  1. 1.
    Gillison ML, Koch WM, Capone RB et al (2000) Evidence for a causal association between human papillomavirus and a subset of head and neck cancers. J Natl Cancer Inst 92:709–720PubMedCrossRefGoogle Scholar
  2. 2.
    Chung CH, Gillison ML (2009) Human papillomavirus in head and neck cancer: its role in pathogenesis and clinical implications. Clin Cancer Res 15(22):6758–6762PubMedCrossRefGoogle Scholar
  3. 3.
    Wu AA, Niparko KJ, Pai SI (2008) Immunotherapy for head and neck cancer. J Biomed Sci 15:275–289PubMedCrossRefGoogle Scholar
  4. 4.
    Trimble CL, Peng S, Kos F et al (2009) A phase I trial of a human papillomavirus DNA vaccine for HPV16 + cervical intraepithelial neoplasia 2/3. Clin Cancer Res 15:361–367PubMedCrossRefGoogle Scholar
  5. 5.
    Kenter GG, Welters MJ, Valentijn AR et al (2009) Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia. N Engl J Med 361:1838–1847PubMedCrossRefGoogle Scholar
  6. 6.
    Radulovic S, Brankovic-Magic M, Malisic E et al (2009) Therapeutic cancer vaccines in cervical cancer: phase I study of Lovaxin-C. J BUON 14(Suppl 1):S165–S168PubMedGoogle Scholar
  7. 7.
    Emens LA, Reilly RT, Jaffee EM (2005) Cancer vaccines in combination with multimodality therapy. Cancer Treat Res 123:227–245PubMedCrossRefGoogle Scholar
  8. 8.
    Andersen MH, Sørensen RB, Schrama D et al (2008) Cancer treatment: the combination of vaccination with other therapies. Cancer Immunol Immunother 57(11):1735–1743PubMedCrossRefGoogle Scholar
  9. 9.
    Machiels JP, Reilly RT, Emens LA et al (2001) Cyclophosphamide, doxorubicin, and paclitaxel enhance the antitumor immune response of granulocyte/macrophage-colony stimulating factor-secreting whole-cell vaccines in HER-2/neu tolerized mice. Cancer Res 61:3689–3697PubMedGoogle Scholar
  10. 10.
    Emens LA, Armstrong D, Biedrzycki B et al (2004) A phase I vaccine safety and chemotherapy dose-finding trial of an allogeneic GM-CSF-secreting breast cancer vaccine given in a specifically timed sequence with immunomodulatory doses of cyclophosphamide and doxorubicin. Hum Gene Ther 15:313–337PubMedCrossRefGoogle Scholar
  11. 11.
    Bass KK, Mastrangelo MJ (1998) Immunopotentiation with low-dose cyclophosphamide in the active specific immunotherapy of cancer. Cancer Immunol Immunother 47:1–12PubMedCrossRefGoogle Scholar
  12. 12.
    Mastrangelo MJ, Berd D, Maguire H Jr (1986) The immunoaugmenting effects of cancer chemotherapeutic agents. Semin Oncol 13:186–194PubMedGoogle Scholar
  13. 13.
    Wada S, Yoshimura K, Hipkiss EL et al (2009) Cyclophosphamide augments antitumor immunity: studies in an autochthonous prostate cancer model. Cancer Res 69:4309–4318PubMedCrossRefGoogle Scholar
  14. 14.
    Ghiringhelli F, Larmonier N, Schmitt E et al (2004) CD4 + CD25 + regulatory T cells suppress tumor immunity but are sensitive to cyclophosphamide which allows immunotherapy of established tumors to be curative. Eur J Immunol 34:336–344PubMedCrossRefGoogle Scholar
  15. 15.
    Lutsiak ME, Semnani RT, De Pascalis R, Kashmiri SV, Schlom J, Sabzevari H (2005) Inhibition of CD4(+)25 + T regulatory cell function implicated in enhanced immune response by low-dose cyclophosphamide. Blood 105:2862–2868PubMedCrossRefGoogle Scholar
  16. 16.
    Josefowicz SZ, Lu LF, Rudensky AY (2012) Regulatory T cells: mechanisms of differentiation and function. Annu Rev Immunol 30:531–564PubMedCrossRefGoogle Scholar
  17. 17.
    Albers AE, Ferris RL, Kim GG, Chikamatsu K, DeLeo AB, Whiteside TL (2005) Immune responses to p53 in patients with cancer: enrichment in tetramer + p53 peptide-specific T cells and regulatory T cells at tumor sites. Cancer Immunol Immunother 54:1072–8118PubMedCrossRefGoogle Scholar
  18. 18.
    Lin KY, Guarnieri FG, Staveley-O’Carroll KF et al (1996) Treatment of established tumors with a novel vaccine that enhances major histocompatibility class II presentation of tumor antigen. Cancer Res 56:21–26PubMedGoogle Scholar
  19. 19.
    Kim JW, Hung CF, Juang J, He L, Kim TW, Armstrong DK, Pai SI, Chen PJ, Lin CT, Boyd DA, Wu TC (2004) Comparison of HPV DNA vaccines employing intracellular targeting strategies. Gene Ther 11(12):1011–1018PubMedCrossRefGoogle Scholar
  20. 20.
    Münger K, Werness BA, Dyson N, Phelps WC, Harlow E, Howley PM (1989) Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product. EMBO J 8(13):4099–4105PubMedGoogle Scholar
  21. 21.
    Jurk M, Vollmer J (2007) Therapeutic applications of synthetic CpG oligodeoxynucleotides as TLR9 agonists for immune modulation. BioDrugs 21:387–401PubMedCrossRefGoogle Scholar
  22. 22.
    Luxembourg A, Hannaman D, Ellefsen B, Nakamura G, Bernard R (2006) Enhancement of immune responses to an HBV DNA vaccine by electroporation. Vaccine 24:4490–4493PubMedCrossRefGoogle Scholar
  23. 23.
    Mir O, Domont J, Cioffi A et al (2011) Feasibility of metronomic oral cyclophosphamide plus prednisolone in elderly patients with inoperable or metastatic soft tissue sarcoma. Eur J Cancer 47:515–519PubMedCrossRefGoogle Scholar
  24. 24.
    Lord R, Nair S, Schache A et al (2007) Low dose metronomic oral cyclophosphamide for hormone resistant prostate cancer: a phase II study. J Urol 177:2136–2140PubMedCrossRefGoogle Scholar
  25. 25.
    Sato E, Olson SH, Ahn J et al (2005) Intraepithelial CD8 + tumor-infiltrating lymphocytes and a high CD8 +/regulatory T cell ratio are associated with favorable prognosis in ovarian cancer. Proc Natl Acad Sci U S A 102(51):18538–18543PubMedCrossRefGoogle Scholar
  26. 26.
    Piersma SJ, Jordanova ES, van Poelgeest MI et al (2007) High number of intraepithelial CD8 + tumor-infiltrating lymphocytes is associated with the absence of lymph node metastases in patients with large early-stage cervical cancer. Cancer Res 67(1):354–361PubMedCrossRefGoogle Scholar
  27. 27.
    Angulo I, de las Heras FG, García-Bustos JF, Gargallo D, Muñoz-Fernández MA, Fresno M (2000) Nitric oxide-producing CD11b(+)Ly-6G(Gr-1)(+)CD31(ER-MP12)(+) cells in the spleen of cyclophosphamide-treated mice: implications for T-cell responses in immunosuppressed mice. Blood 95(1):212–220PubMedGoogle Scholar
  28. 28.
    Mikyšková R, Indrová M, Polláková V, Bieblová J, Símová J, Reiniš M (2012) Cyclophosphamide-induced myeloid-derived suppressor cell population is immunosuppressive but not identical to myeloid-derived suppressor cells induced by growing TC-1 tumors. J Immunother 35(5):374–384PubMedCrossRefGoogle Scholar
  29. 29.
    Leao IC, Ganesan P, Armstrong TD, Jaffee EM (2008) Effective depletion of regulatory T cells allows the recruitment of mesothelin-specific CD8 T cells to the antitumor immune response against a mesothelin-expressing mouse pancreatic adenocarcinoma. Clin Transl Sci 1(3):228–239PubMedCrossRefGoogle Scholar
  30. 30.
    Kerbel RS, Kamen BA (2004) The anti-angiogenic basis of metronomic chemotherapy. Nat Rev Cancer 4(6):423–436PubMedCrossRefGoogle Scholar
  31. 31.
    Bracci L, Moschella F, Sestili P et al (2007) Cyclophosphamide enhances the antitumor efficacy of adoptively transferred immune cells through the induction of cytokine expression, B-cell and T-cell homeostatic proliferation, and specific tumor infiltration. Clin Cancer Res 13:644–653PubMedCrossRefGoogle Scholar
  32. 32.
    Berraondo P, Nouze C, Preville X, Ladant D, Leclerc C (2007) Eradication of large tumors in mice by a tritherapy targeting the innate, adaptive, and regulatory components of the immune system. Cancer Res 67:8847–8855PubMedCrossRefGoogle Scholar
  33. 33.
    Damber JE, Vallbo C, Albertsson P, Lennernas B, Norrby K (2006) The anti-tumour effect of low-dose continuous chemotherapy may partly be mediated by thrombospondin. Cancer Chemother Pharmacol 58:354–360PubMedCrossRefGoogle Scholar
  34. 34.
    Man S, Bocci G, Francia G et al (2002) Antitumor effects in mice of low-dose (metronomic) cyclophosphamide administered continuously through the drinking water. Cancer Res 62:2731–2735PubMedGoogle Scholar
  35. 35.
    Burton JH, Mitchell L, Thamm DH, Dow SW, Biller BJ (2011) Low-dose cyclophosphamide selectively decreases regulatory T cells and inhibits angiogenesis in dogs with soft tissue sarcoma. J Vet Intern Med 25(4):920–926PubMedCrossRefGoogle Scholar
  36. 36.
    Khan OA, Blann AD, Payne MJ et al (2011) Continuous low-dose cyclophosphamide and methotrexate combined with celecoxib for patients with advanced cancer. Br J Cancer 104(12):1822–1827PubMedCrossRefGoogle Scholar
  37. 37.
    Hanahan D, Bergers G, Bergsland E (2000) Less is more, regularly: metronomic dosing of cytotoxic drugs can target tumor angiogenesis in mice. J Clin Invest 105:1045–1047PubMedCrossRefGoogle Scholar
  38. 38.
    Sanchez-Munoz A, Mendiola C, Perez-Ruiz E et al (2010) Bevacizumab plus low-dose metronomic oral cyclophosphamide in heavily pretreated patients with recurrent ovarian cancer. Oncology 79(1–2):98–104PubMedCrossRefGoogle Scholar
  39. 39.
    Colleoni M, Rocca A, Sandri MT et al (2002) Low-dose oral methotrexate and cyclophosphamide in metastatic breast cancer: antitumor activity and correlation with vascular endothelial growth factor levels. Ann Oncol 13:73–80PubMedCrossRefGoogle Scholar
  40. 40.
    Reinhardt J, Schott S, Mayer C, Sohn C, Eichbaum M (2011) Long-term remission of an advanced recurrent endometrial cancer in a heavily pretreated patient using a combined regimen with bevacizumab and metronomic cyclophosphamide. Anticancer Drugs 22:822–824PubMedCrossRefGoogle Scholar
  41. 41.
    Ladoire S, Eymard JC, Zanetta S et al (2010) Metronomic oral cyclophosphamide prednisolone chemotherapy is an effective treatment for metastatic hormone-refractory prostate cancer after docetaxel failure. Anticancer Res 30(10):4317–4323PubMedGoogle Scholar
  42. 42.
    Hermans IF, Chong TW, Palmowski MJ, Harris AL, Cerundolo V (2003) Synergistic effect of metronomic dosing of cyclophosphamide combined with specific antitumor immunotherapy in a murine melanoma model. Cancer Res 63:8408–8413PubMedGoogle Scholar
  43. 43.
    Ghiringhelli F, Menard C, Puig PE et al (2007) Metronomic cyclophosphamide regimen selectively depletes CD4 + CD25 + regulatory T cells and restores T and NK effector functions in end stage cancer patients. Cancer Immunol Immunother 56:641–648PubMedCrossRefGoogle Scholar
  44. 44.
    Emens LA, Asquith JM, Leatherman JM et al (2009) Timed sequential treatment with cyclophosphamide, doxorubicin, and an allogeneic granulocyte-macrophage colony-stimulating factor-secreting breast tumor vaccine: a chemotherapy dose-ranging factorial study of safety and immune activation. J Clin Oncol 27:5911–5918PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Shiwen Peng
    • 1
    • 2
  • Sofia Lyford-Pike
    • 1
  • Belinda Akpeng
    • 1
  • Annie Wu
    • 1
  • Chien-Fu Hung
    • 2
    • 3
  • Drew Hannaman
    • 4
  • John R. Saunders
    • 1
    • 5
  • T.-C. Wu
    • 2
    • 3
    • 6
    • 7
  • Sara I. Pai
    • 1
    • 3
    Email author
  1. 1.Department of Otolaryngology-Head and Neck SurgeryJohns Hopkins School of Medicine, Johns Hopkins Medical InstitutionsBaltimoreUSA
  2. 2.Department of PathologyJohns Hopkins School of MedicineBaltimoreUSA
  3. 3.Department of OncologyJohns Hopkins School of MedicineBaltimoreUSA
  4. 4.Ichor Medical Systems, Inc.San DiegoUSA
  5. 5.Milton J. Dance Jr. Head and Neck CenterGreater Baltimore Medical CenterBaltimoreUSA
  6. 6.Department of Obstetrics and GynecologyJohns Hopkins School of MedicineBaltimoreUSA
  7. 7.Department of Molecular Microbiology and ImmunologyJohns Hopkins School of MedicineBaltimoreUSA

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