In prior work, we found that melanoma vaccines using mixtures of synthetic MHC-associated peptides are immunogenic, and that 10 of the 12 melanoma peptides in MELITAC 12.1 were individually immunogenic.11
The present study enrolled 119 eligible patients who were evaluable for immune responses, and each of the 12 peptides was immunogenic, when assayed directly, without ex vivo
stimulation. However, some were weakly immunogenic, and those patterns match our prior studies. Overall, 54% of patients developed immune responses detectable by direct ELIspot assay, which compares favorably to a 37% immune response rate measured in a multicenter clinical trial using three HLA-A2 peptides, using a very similar assay system.18
For the 61 patients vaccinated with peptides in Montanide ISA-51 without GM-CSF, immune responses to the 12 melanoma peptides were detected in 73% of patients (), which exceeds response rates in prior studies using direct ELIspot.
Also, in prior work, we used an ELIspot assay on lymphocytes stimulated ex vivo
, and immune responses detected using this assay did correlate with disease-free survival.11
A direct ELIspot assay is widely considered a more biologically relevant assay; however, that remains to be determined. A stimulated ELIspot assay may be relevant in measuring, in part, proliferative capacity of T cells, which may have some connection with memory responses. In a subset of patients on this trial (n = 45), we performed stimulated ELIspot assays also, which were associated with immune response rates on the order of 90% for the study overall, and 100% for patients vaccinated without GM-CSF (). In those assays, even for individual peptides, immune response rates were 100% for 4 peptides and close to 90% for two others (, Supplemental Table 3
). These findings highlight the immunogenicity of peptide vaccines administered in an incomplete Freund’s adjuvant.
One question addressed by the present study is whether vaccination in two extremity sites leads to better immune responses than vaccination in just one site. There was a trend to greater CD8+
T cell response with 2 vaccine sites, and a weak trend to a greater CD4+
T cell response with one vaccine site, neither of which was statistically significant (). Thus, we conclude that there is insufficient evidence in the present data to claim that response rates are increased by vaccinating at two sites. However, other data strongly suggest that trafficking of T cells to tumor may depend on where they are primed by a vaccine 19
; thus, there is still more work to do to answer the question of where to vaccinate, depending on the location of melanoma metastases.
The main question that was addressed in the present study was whether GM-CSF administered locally in the vaccine emulsion would increase immunogenicity. Prior murine studies found that this strategy increased immune responses to a peptide vaccine 2
, and supported the value of GM-CSF delivered in whole-cell vaccines.1
However, recent studies are conflicting on the role of GM-CSF as an immune potentiator or therapeutic agent.20
GM-CSF administered with a heat shock protein vaccine has been implicated in the induction of myeloid-derived suppressor cells in melanoma patients 21
, and GM-CSF at high dose, in murine models, may increase myeloid-derived suppressor cells (MDSC).22
However, daily subcutaneous administration of GM-CSF (125 mcg/m2/d) increased circulating mature dendritic cells (DC), but did not increase MDSC in melanoma patients.23
Prior studies of GM-CSF as a vaccine adjuvant were primarily small non-randomized studies, or did not include defined antigens. We designed the present study to address the role of GM-CSF administered locally as a vaccine adjuvant, in a randomized multicenter design, with adequate power to detect a significant immunologic effect. The resulting data show that the circulating T cell response to the multipeptide vaccine is significantly lower in patients whose vaccines included GM-CSF. This was true both for CD8+
T cell responses to 12 melanoma peptides and for CD4+
T cell responses to a tetanus helper peptide. Reasons for this remain to be defined, but the findings cast doubt on the benefit of GM-CSF protein as local adjuvant. This finding is consistent with a prior small study with local administration of GM-CSF using a different delivery strategy 9
. It has been suggested that negative immunologic effects of GM-CSF may be associated primarily with high doses of GM-CSF, in the range of 100-500 mcg/day 20
. The dose used in this trial was 110 mcg administered only once a week. Since it was given in a slow-release emulsion, the effective daily dose is arguably less than 20 mcg/day. Thus, even low doses of GM-CSF administered with a multipeptide vaccine may have negative immunologic effects. A possible mechanism by which GM-CSF may interfere with immune function is through milk fat globule EGF-8 (MFG-E8). 24;25
GM-CSF induces expression of MFG-E8 by macrophages and DC; MFG-E8 can maintain regulatory T cells and can downregulate Th1 responses.24;25
This mechanism deserves further investigation as a possible explanation for some negative effects of GM-CSF as an adjuvant. Additional studies are also needed to determine if GM-CSF alters the quality of function of vaccine-induced T cells and whether inclusion of GM-CSF with the vaccine may have an impact on clinical outcome.
In addition to questions about whether GM-CSF may have value as an immune adjuvant, it has been suggested that GM-CSF may have therapeutic value when administered subcutaneously as a single-agent therapy for melanoma 26
, and that finding is being tested in a randomized phase III trial of systemic subcutaneous GM-CSF for the adjuvant therapy of high-risk melanoma, alone or combined with a multipeptide vaccine (ECOG trial E4697). That study has been closed to accrual, and final analysis is pending. In a recent non-randomized study, clinical outcome of 39 patients with stage III-IV melanoma treated with subcutaneous GM-CSF has been presented as additional support for the therapeutic potential of single agent GM-CSF.23
However, the results of non-randomized clinical studies of small size has been notoriously subject to selection bias, and the clinical outcome in the present study with and without GM-CSF compares favorably to what was reported in that study 23
. With longer follow-up, we will be able to address whether clinical outcome mirrors immunologic outcomes.
In summary, we have found that immunization with the MELITAC 12.1 multipeptide melanoma vaccine is immunogenic in a majority of patients with high-risk melanoma, that vaccination in two extremities does not increase T cell response, and that addition of GM-CSF to the vaccine decreases circulating CD4+ and CD8+ T cell responses to the vaccine. Caution should be taken in future use of GM-CSF as a local vaccine adjuvant.