Yonsei Med J.  2011 Nov;52(6):990-998. 10.3349/ymj.2011.52.6.990.

Immunotherapy of Malignant Melanoma with Tumor Lysate-Pulsed Autologous Monocyte-Derived Dendritic Cells

• Kim DS
• Kim DH
• Goo B
• Cho YH
• Park JM
• Lee TH
• Kim HO
• Kim HS
• Lee H
• Lee JD
• Byamba D
• Je JH
• Lee MG

• KMID: 1058821
• DOI: http://doi.org/10.3349/ymj.2011.52.6.990

Abstract

PURPOSE
Dendritic cell (DC) vaccination for melanoma was introduced because melanoma carries distinct tumor-associated antigens. The purpose of this study was to investigate the efficacy and safety of DC vaccination for melanoma in Korea.
MATERIALS AND METHODS
Five patients with stage IV and one with stage II were enrolled. Autologous monocyte-derived DCs (MoDCs) were cultured and pulsed with tumor-lysate, keyhole limpet hemocyanin, and cytokine cocktail for mature antigen-loaded DC. DC vaccination was repeated four times at 2-week intervals and 2-4x107 DC were injected each time.
RESULTS
Reduced tumor volume was observed by PET-CT in three patients after DC vaccination. Delayed type hypersensitivity responses against tumor antigen were induced in five patients. Tumor antigen-specific IFN-gamma-producing peripheral blood mononuclear cells were detected with enzyme-linked immunosorbent spot in two patients. However, the overall clinical outcome showed disease progression in all patients.
CONCLUSION
In this study, DC vaccination using tumor antigen-loaded, mature MoDCs led to tumor regression in individual melanoma patients. Further standardization of DC vaccination protocol is required to determine which parameters lead to better anti-tumor responses and clinical outcomes.

Keyword

Dendritic cell; immunotherapy; malignant melanoma

MeSH Terms

Dendritic Cells/*cytology
Enzyme-Linked Immunosorbent Assay
Flow Cytometry
Humans
Immunotherapy/*methods
Melanoma/*therapy
Monocytes/*cytology
Treatment Outcome

Figure

• 

  Fig. 1 Patients had multiple cutaneous metastases and recurrent melanoma in the tonsil. Because the whole tumor-lysate was used for antigen pulsing, it was important to have sufficient tumor volume.

• 

  Fig. 2 The immunophenotypic profiles of enriched monocytes (A); immature DC on day 6 of culture with GM-CSF and IL-4 (B); and mature DC cultured after an additional 2 days with tumor lysate, KLH and cytokine cocktail (TNF-a, IL-1b, IL-6, and PGE2) (C). Each of the cells was stained with monoclonal antibodies specific to CD1a, CD14, CD45, CD80, CD83, CD86, HLA-ABC and HLA-DR. Mature DCs have a higher expression of HLA-DR, CD86, and CD80 than immature DCs. Only mature DCs express CD83. The data shown are from one representative experiment. DC, dendritic cell; KLH, keyhole limpet hemocyanin.

• 

  Fig. 3 Tumor volume reduction in PET-CT. In the case of patient 3, reduction of tumor volume was noted in PET-CT after the 4th vaccination. The lymph nodes at the Lt. supraclavicular fossa, bilateral retroperitoneal lymph node chain, Rt. inguinal area and upper part of right axillary lymph nodes were pointed out as sites of improvement.

• 

  Fig. 4 Patient 3 showing positive reactions to the DTH test. DTH was examined before and then 2 weeks after the 4th vaccination. Patients received i.d. injections of 100 µL of normal saline, 100 µg tumor lysate, 50 µg KLH, 100 µg tumor lysate, plus 50 µg KLH, at separate sites on the forearm. Forty-eight hours later, DTH was scored positive if the areas of erythema and induration were greater than 5 mm. DTH, delayed type hypersensitivity; KLH, keyhole limpet hemocyanin.

• 

  Fig. 5 ELISPOT for the detection of Ag-specific IFN-γ producing PBMCs in patient 2, 3. Both patients showed positive results. ELISPOT, enzyme-linked immunosorbent spot.

Reference

1. Hall HI, Miller DR, Rogers JD, Bewerse B. Update on the incidence and mortality from melanoma in the United States. J Am Acad Dermatol. 1999. 40:35–42.

2. Houghton AN, Gold JS, Blachere NE. Immunity against cancer: lessons learned from melanoma. Curr Opin Immunol. 2001. 13:134–140.

3. de Braud F, Khayat D, Kroon BB, Valdagni R, Bruzzi P, Cascinelli N. Malignant melanoma. Crit Rev Oncol Hematol. 2003. 47:35–63.

4. Hsu FJ, Benike C, Fagnoni F, Liles TM, Czerwinski D, Taidi B, et al. Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells. Nat Med. 1996. 2:52–58.

5. Schuler G, Schuler-Thurner B, Steinman RM. The use of dendritic cells in cancer immunotherapy. Curr Opin Immunol. 2003. 15:138–147.

6. Kim S, Kim HO, Baek EJ, Choi Y, Kim HS, Lee MG. Monocyte enrichment from leukapheresis products by using the Elutra cell separator. Transfusion. 2007. 47:2290–2296.

7. Eubel J, Enk AH. Dendritic cell vaccination as a treatment modality for melanoma. Expert Rev Anticancer Ther. 2009. 9:1631–1642.

8. Nakai N, Asai J, Ueda E, Takenaka H, Katoh N, Kishimoto S. Vaccination of Japanese patients with advanced melanoma with peptide, tumor lysate or both peptide and tumor lysate-pulsed mature, monocyte-derived dendritic cells. J Dermatol. 2006. 33:462–472.

9. López MN, Pereda C, Segal G, Muñoz L, Aguilera R, González FE, et al. Prolonged survival of dendritic cell-vaccinated melanoma patients correlates with tumor-specific delayed type IV hypersensitivity response and reduction of tumor growth factor beta-expressing T cells. J Clin Oncol. 2009. 27:945–952.

10. Jonuleit H, Schmitt E, Stassen M, Tuettenberg A, Knop J, Enk AH. Identification and functional characterization of human CD4(+)CD25(+) T cells with regulatory properties isolated from peripheral blood. J Exp Med. 2001. 193:1285–1294.

11. Liyanage UK, Moore TT, Joo HG, Tanaka Y, Herrmann V, Doherty G, et al. Prevalence of regulatory T cells is increased in peripheral blood and tumor microenvironment of patients with pancreas or breast adenocarcinoma. J Immunol. 2002. 169:2756–2761.

12. Curiel TJ, Coukos G, Zou L, Alvarez X, Cheng P, Mottram P, et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med. 2004. 10:942–949.

13. Mahnke K, Schmitt E, Bonifaz L, Enk AH, Jonuleit H. Immature, but not inactive: the tolerogenic function of immature dendritic cells. Immunol Cell Biol. 2002. 80:477–483.

14. Dannull J, Su Z, Rizzieri D, Yang BK, Coleman D, Yancey D, et al. Enhancement of vaccine-mediated antitumor immunity in cancer patients after depletion of regulatory T cells. J Clin Invest. 2005. 115:3623–3633.

15. Mahnke K, Schönfeld K, Fondel S, Ring S, Karakhanova S, Wiedemeyer K, et al. Depletion of CD4+CD25+ human regulatory T cells in vivo: kinetics of Treg depletion and alterations in immune functions in vivo and in vitro. Int J Cancer. 2007. 120:2723–2733.

16. Rasku MA, Clem AL, Telang S, Taft B, Gettings K, Gragg H, et al. Transient T cell depletion causes regression of melanoma metastases. J Transl Med. 2008. 6:12.

17. Attia P, Maker AV, Haworth LR, Rogers-Freezer L, Rosenberg SA. Inability of a fusion protein of IL-2 and diphtheria toxin (Denileukin Diftitox, DAB389IL-2, ONTAK) to eliminate regulatory T lymphocytes in patients with melanoma. J Immunother. 2005. 28:582–592.

18. Lee TH, Cho HK, Cho YH, Lee MG. Development of an effective method for dendritic cell immunotherapy of mouse melanoma. Scand J Immunol. 2009. 70:85–92.

19. Jonuleit H, Giesecke-Tuettenberg A, Tuting T, Thurner-Schuler B, Stuge TB, Paragnik L, et al. A comparison of two types of dendritic cell as adjuvants for the induction of melanoma-specific T-cell responses in humans following intranodal injection. Int J Cancer. 2001. 93:243–251.

20. Tuettenberg A, Becker C, Huter E, Knop J, Enk AH, Jonuleit H. Induction of strong and persistent MelanA/MART-1-specific immune responses by adjuvant dendritic cell-based vaccination of stage II melanoma patients. Int J Cancer. 2006. 118:2617–2627.

21. Whiteside TL. The tumor microenvironment and its role in promoting tumor growth. Oncogene. 2008. 27:5904–5912.