University of Newcastle, NSW
Treatment of melanoma by immunotherapy remains a topic of much interest. Initial studies were based on stimulation of immune responses by immunisation with whole cells or lysates of whole cells. Some of these trials are still in progress but results with these or purified melanoma antigen vaccines have not been particularly effective. Recent approaches have been directed to overcoming inhibitory homeostatic mechanisms by use of antibodies which block the CTLA-4 receptor involved in downregulation of cytotoxic T cells and the activity of regulatory T cells. Adoptive immunotherapy after immunodepletion of recipients is also showing promise. Future trials that combine immunotherapy with agents that overcome resistance to apoptosis of melanoma cells may provide the breakthrough being sought in treatment of melanoma.
Treatment of melanoma by immunotherapy has been of much interest to clinicians and scientists for at least the past three decades. Such endeavour has been stimulated by evidence suggesting that immune responses against the tumour have an important role in evolution of the disease. This includes partial or complete regression of cutaneous melanoma usually associated with lymphoid infiltration into the primary tumour and more rarely spontaneous regression of advanced metastatic disease. In vitro studies have also shown abundant evidence of antibody and T cell responses against autologous tumours and more recently studies have shown that adoptive transfer of tumour infiltrating lymphocytes (TILs) may be associated with regression of metastatic melanoma.1
The source of antigens in vaccines include whole melanoma cells, melanoma lysates, whole protein antigens, peptide epitopes and RNA or DNA coding for the antigen. In addition, various vaccine “platforms” have been utilised in the studies, such as administration with adjuvants, dendritic cells or direct injection of antigen into skin. The antigen sources and types of vaccines are summarised in Figure 1.2
The hypotheses being tested in vaccine studies have evolved over the years as follows.
Hypothesis 1 – “The immune response against melanoma is too weak to control melanoma growth. Immunisation with vaccines will increase strength of response and control tumour growth.”
This hypothesis has now been tested in several relatively large randomised trials. The first of these was by Wallack et al, who used vaccinia viral lysates from three melanoma cells. The result showed a non-significant trend in favour of the vaccine. The second and so far largest of the randomised trials was that conducted by Hersey et al in Australia.3 The vaccine was a vaccinia viral lysate of one melanoma cell and vaccines were given over a three year period to patients with stage IIB or III melanoma in the old AJCC staging system.
The results summarised in Table 1 indicated that there were 20 more deaths in the control group and that the hazard of dying was reduced to 0.81 compared to control untreated patients. Nevertheless, the confidence intervals were wide (.64 – 1.02) and the P value of 0.068 was just outside the accepted .05 value. All patients in the study survived very well with an overall median survival of 100 months (see Figure 2).
The third trial was conducted by the South West Oncology Group (SWOG) in the US in 689 patients with AJCC stage II disease. The vaccine was a sonicated lysate of two melanoma cells given in an adjuvant called Detox. The results for all patients showed a small effect on disease free survival (DFS) but no effect on overall survival. However, when the patients were stratified according to their HLA-A2 and/or C3 status it was found that the vaccine induced a significant increase in survival in patients who were HLA-A2 and/or C3 positive.4 A follow-up study with the vaccine has not yet been commenced.
The fourth trial used a vaccine made from three whole viable irradiated melanoma cells (Canvaxin) given initially with BCG as an adjuvant. Trials in patients with resected stage IV disease were discontinued in April 2005 by the Data Monitoring Committee because of no detectable effect on survival. A total of 496 patients had been accrued after entry of 1160 patients. A larger trial in patients with resected stage III disease was closed to accrual in September 2004 and results are awaited.
Hypothesis 2 – “Vaccines made from peptide epitopes or purified proteins will be more effective than use of whole cells or whole cell lysates.”
As information about the specific melanoma antigens recognised by the immune system became available, it was possible to produce vaccines containing only well-defined antigens, such as peptide epitopes recognised by T cells. Such vaccines were relatively cheap to produce and were shown to be effective in inducing T cell responses against the antigen concerned. In practice however, such vaccines have proved relatively ineffective in inducing clinical responses in patients with melanoma. Our experience is shown in Table 2. In studies on 36 patients the best response was stabilisation of disease in six patients defined as no progression over a 12 week period.5 Similar experiences have been reported by other investigators6 so that there is now little enthusiasm for purified peptide vaccines in treatment of melanoma.
Hypothesis 3 – “Melanoma antigens given on dendritic cell vaccines will be more effective than direct cutaneous injections of the vaccine.”
This approach was based largely on the view that dendritic cell (DC) antigen presenting cells (APCs) were non-functional or present in low cell numbers around tumours. Therefore if DCs were produced in vitro and injected into patients after incubation with melanoma antigens the immune responses to melanoma would be stronger and result in regression of melanoma. Again, the results from a number of trials have not lived up to expectations. The results of our first phase II trial on 33 patients shown in Table 3 resulted in three partial responses, one mixed response and nine with stabilisation of disease.7 Similar results were reported by Smithers et al but studies by O’Rourke et al8 were more impressive, resulting in 3 CR and 3 PR (Table 4).
Hypothesis 4 – “Immune responses against melanoma are inhibited by physiological down-regulatory mechanisms in the immune system.” “Taking off the brake approach.”
The immune system has a number of homeostatic mechanisms which return activated lymphocytes to a resting state. One such mechanism is the presence of signal pathways that inhibit the activation signals resulting from contact with antigen and co-stimulatory receptors. The inhibitory pathways are activated by ligands on APC that interact with receptors on T cells. One of the most important of these is the cytotoxic T lymphocyte antigen 4 (CTLA-4) receptor which interacts with CD80 (B7.1) and CD86 (B7.2) on APC. Studies by Allison and colleagues9 have shown that antibodies against CTLA-4 allow the activated T cells to persist and proliferate, resulting in a more prolonged vigorous T cell response. Blockade of the CTLA-4 receptor may also inhibit a subpopulation of regulatory T cells that act to inhibit immune responses by down-regulation of stimulatory signals from APC. Regulatory T cells constitutively express the CD25 (IL-2Ra) IL-2 receptor and CTLA-4 and MAb against CTLA-4 may limit their production of inhibitory cytokines such as IL-10 and/or TGF-b.
These studies have been commercially exploited by Medarex/Bristol Myers Squibb, which is testing the MDX-010 monoclonal antibody (MAb), and by Pfizer, who are testing another MAb, CP-675206. The phase I dose finding studies by Pfizer (shown in Table 5) were very promising in that durable CR and PR were seen at the higher dose levels in patients who had failed other treatments. Overall the response rate was 20%. A large phase II study at the 15mg/kg dose level each three months is now planned.
MDX-010 has been tested in combination with peptide vaccines by the NCI group10 and by Weber et al.11 Again, impressive durable CR and PR were seen (Table 5). A large international randomised three arm study in previously treated patients is now in progress. Treatment with MDX-010 has been complicated by severe side effects, particularly diarrhea which can require admission to hospital and fluid replacement. Skin rashes are common and rarely hypophysitis may occur.
Hypothesis 5 – “Lymphocyte depletion will allow selective expansion of tumour specific lymphocytes produced by vaccines or adoptively transferred after expansion in vitro.”
Homeostatic mechanisms control lymphocyte numbers at surprisingly constant levels and are believed to limit the proliferation of lymphocytes activated against tumours. Rosenberg and colleagues have depleted lymphocytes from patients with cyclophosphamide and fludarabine, and then adoptively transferred T.I.L.s that have been expanded in vitro. The results (Table 6) have been most impressive, with durable CR and PR seen in 50% of a series of 35 patients so treated.12 The in vitro cultivation of T.I.L. requires considerable laboratory resources and other groups (Urba, Fox, et al) are testing whether immunisation with vaccines following lymphocyte depletion may achieve the same outcome.13,14
Hypothesis 6 – “Immune therapy alone is limited by resistance of melanoma cells to apoptosis. Combination with agents which reduce resistance to apoptosis is required for optimal results.”
Studies over the past few years have shown that lymphocytes kill tumour cells by induction of apoptosis (Table 7) and consequently mechanisms which reduce the sensitivity of melanoma to apoptosis limit the effectiveness of immunotherapy. The resistance mechanisms involved are the subject of intensive research but available evidence suggests that activation of MAP kinase extracellular receptor kinase (ERK1/2) and Akt signal pathways are responsible at least in part for resistance to apoptosis. We and others have shown that the ERK1/2 pathway is activated in advanced melanoma15and that inhibition of this pathway sensitises melanoma to killing by immune mediators of apoptosis such as TNF related apoptosis inducing ligand (TRAIL).16 Phase II studies using an inhibitor of this pathway produced by the Onyx/Bayer companies were associated with impressive responses (in previously treated patients) to chemotherapy. A randomised trial in Australia is now in progress to further test this combination. Inhibitors of the Akt pathway are also becoming available. One such inhibitor is the geldanamycin derivative (AAG) which inhibits the heat shock protein (HSP) 90, which chaperones several signal pathway proteins, including those in the Akt pathway.17 Phase II studies with this agent in stage IV melanoma patients are planned to commence soon in Australia.
The search for agents which sensitise melanoma cells to apoptosis is a fertile area of research and includes not only signal pathway inhibitors, but agents which target anti-apoptotic proteins in melanoma. The next few years will see many of these agents tested in clinical trials and may open the way for significant advances in treatment of this disease not seen over the past 30 years.
1. Hersey P, Morton DL, Eggermont A. Adjuvant therapy for high-risk primary melanoma and resected metastatic melanoma. In: Thompson JF, Morton DL, Kroon BBR, editors. Management of Melanoma. London: Martin Dunitz/Taylor and Francis; 2004. p. 573-585.
3. Hersey P, Coates AS, McCarthy WH, Thompson JF, Sillar RW, McLeod R, et al. Adjuvant immunotherapy of patients with high-risk melanoma using vaccinia viral lysates of melanoma: results of a randomised trial. J Clin Oncol. 2002;20(20):4181-90.
4. Sosman JA, Unger JM, Liu PY, Flaherty LE, Park MS, Kempf RA, et al. Adjuvant immunotherapy of resected, intermediate-thickness, node-negative melanoma with an allogeneic tumour vaccine: impact of HLA class I antigen expression on outcome. J Clin Oncol. 2002;20(8):2067-75.
5. Hersey P, Menzies SW, Coventry B, Nguyen T, Farrelly M, Collins S, et al. Phase I/II study of immunotherapy with T-cell peptide epitopes in patients with stage IV melanoma. Cancer Immunol Immunother. 2005;54:208-18.
7. Hersey P, Menzies SW, Halliday GM, Nugyen T, Farrelly ML, DeSilva C, Lett M. Phase I/II study of treatment with dendritic cell vaccines in patients with disseminated melanoma. Cancer Immunol Immunother. 2004;53:125-34.
8. O’Rourke MG, Johnson M, Lanagan C, See J, Yang J, Bell JR, et al. Durable complete clinical responses in a phase I/II trial using an autologous melanoma cell/dendritic cell vaccine. Cancer Immunol Immunother. 2003;52(6):387-95.
10. Phan GQ, Yang JC, Sherry RM, Hwu P, Topalian SL, Schwartzentruber DJ, et al. Cancer regression and autoimmunity induced by cytotoxic T lymphocyte-associated antigen 4 blockade in patients with metastatic melanoma. Proc Natl Acad Sci USA 2003;100(14):8372-7.
11. Sanderson K, Scotland R, Lee P, Liu D, Groshen S, Snively J, et al. Autoimmunity in a phase I trial of a fully human anti-cytotoxic T lymphocyte antigen-4 monoclonal antibody with multiple melanoma peptides and Montanide ISA 51 for patients with resected stages III and IV melanoma. J Clin Oncol. 2005;23(4):741-50.
12. Dudley ME, Wunderlich JR, Yang JC, Sherry RM, Topalian SL, Restifo NP, et al. Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma. J Clin Oncol. 2005;23(10):2346-57.
14. Asavaroengchai W, Kotera Y, Koike N, Pilon-Thomas S, Mule JJ. Augmentation of antitumour immune responses after adoptive transfer of bone marrow derived from donors immunized with tumour lysate-pulsed dendritic cells. Biol Blood Marrow Transplant. 2004;10(8):524-33.
16. Zhang XD, Borrow JM, Zhang XY, Nguyen T, Hersey P. Activation of ERK1/2 protects melanoma cells from TRAIL-induced apoptosis by inhibiting Smac/DIABLO release from mitochondria. Oncogene 2003;22:2869-2881.