Safety and immunologic correlates of Melanoma GVAX, a GM-CSF secreting allogeneic melanoma cell vaccine administered in the adjuvant setting

Abstract

Background: Limited adjuvant treatment options exist for patients with high-risk surgically resected melanoma. This first-in-human study investigated the safety, tolerability and immunologic correlates of Melanoma GVAX, a lethally irradiated granulocyte-macrophage colony stimulating factor (GM-CSF)-secreting allogeneic whole-cell melanoma vaccine, administered in the adjuvant setting.

Methods: Patients with stage IIB-IV melanoma were enrolled following complete surgical resection. Melanoma GVAX was administered intradermally once every 28 days for four cycles, at 5E7 cells/cycle (n = 3), 2E8 cells/cycle (n = 9), or 2E8 cells/cycle preceded by cyclophosphamide 200 mg/m(2) to deplete T regulatory cells (Tregs; n = 8). Blood was collected before each vaccination and at 4 and 6 months after treatment initiation for immunologic studies. Vaccine injection site biopsies and additional blood samples were obtained 2 days after the 1st and 4th vaccines.

Results: Among 20 treated patients, 18 completed 4 vaccinations. Minimal treatment-related toxicity was observed. One patient developed vitiligo and patches of white hair during the treatment and follow-up period. Vaccine site biopsies demonstrated complex inflammatory infiltrates, including significant increases in eosinophils and PD-1+ lymphocytes from cycle 1 to cycle 4 (P < 0.05). Serum GM-CSF concentrations increased significantly in a dose-dependent manner 48 h after vaccination (P = 0.0086), accompanied by increased numbers of activated circulating monocytes (P < 0.0001) and decreased percentages of myeloid-derived suppressor cells among monocytes (CD14+ , CD11b+ , HLA-DR low or negative; P = 0.002). Cyclophosphamide did not affect numbers of circulating Tregs. No significant changes in anti-melanoma immunity were observed in peripheral T cells by interferon-gamma ELIPSOT, or immunoglobulins by serum Western blotting.

Conclusion: Melanoma GVAX was safe and tolerable in the adjuvant setting. Pharmacodynamic testing revealed complex vaccine site immune infiltrates and an immune-reactive profile in circulating monocytic cell subsets. These findings support the optimization of Melanoma GVAX with additional monocyte and dendritic cell activators, and the potential development of combinatorial treatment regimens with synergistic agents.

Trial registration: NCT01435499.

Figures

Figure 1

Transient melanoma progression in a patient treated in Cohort A, accompanied by vitiligo. a This patient developed the onset of vitiligo and greying hair after receiving four cycles of Melanoma GVAX. Vitiligo was initially confined to a prior lymphadenectomy incision site, but progressed to other skin sites and hair over the next few months. b Pelvic CT scan at the 6-month evaluation interval showed enlarging left pelvic lymph nodes (yellow and red arrows), biopsy-proven to be recurrent melanoma. At 8 months, these lesions had regressed without further intervention. Two months later, they enlarged again and the patient went on to receive other therapies.

Figure 2

Vaccine site biopsy reveals a mixed inflammatory infiltrate. Representative photomicrograph of a skin punch biopsy obtained from a patient in Cohort A, 2 days after receiving the first dose of Melanoma GVAX. H&E staining reveals a dense, mixed inflammatory infiltrate centered primarily in the superficial subcutaneous adipose tissue (blue inset box), including lymphocytes, histiocytes, eosinophils, and irradiated melanoma vaccine cells. Left image, ×10 original magnification; right image, ×200 original magnification. Black arrowhead, dermal/subcutaneous junction; black arrows, Melanoma GVAX cells; green arrows, eosinophils. Immunohistochemical characterization is shown in Additional file 3: Figure S2.

Figure 3

Vaccine site immune cell changes during treatment. Significant increases in the mean scores of dermal eosinophils and lymphocyte PD-1 expression from C1 to C4 were observed in Melanoma GVAX vaccine site biopsies. Paired analyses performed using the Wilcoxon signed-rank test; asterisks indicate p < 0.05. Scoring criteria are described in “Methods”. Vertical bars depict SEM. C treatment cycle.

Figure 4

Melanoma GVAX administered intradermally increases systemic GM-CSF levels in a dose-dependent manner. a Serum GM-CSF concentrations measured 2 days after the first administration of Melanoma GVAX were significantly higher in patients receiving a dose of 2E8 cells (Cohorts B and C, mean 19.8 ± 2.72) compared to 5E7 cells (Cohort A, mean 4.8 ± 1.5). There was no significant difference between patients receiving high-dose vaccine in Cohorts B (no CPM) versus C (with CPM) (not shown). All patients had detectable serum GM-CSF after the first vaccination (detection limit 1 pg/ml). b Serum GM-CSF concentrations were significantly lower 2 days after the fourth vaccine (6.8 ± 1.5) compared to the first vaccine (17.5 ± 2.6). Bars depict the mean ± SEM; p values from 2-sided Mann–Whitney U test (a) or paired Wilcoxon signed-rank test (b). C treatment cycle, D treatment day. Pre-vaccine sera from Cohort C were collected on D0 prior to CPM administration, and from Cohorts A and B, on D1 prior to vaccine administration.

Figure 5

Melanoma GVAX coordinately increases numbers of activated circulating monocytes and decreases circulating MDSCs. Flow cytometric analysis revealed significantly increased numbers (a) and activation (b) of circulating monocytes (CD14+ , CD11b+) 2 days following the first and fourth vaccinations. Monocyte activation was quantified as mean fluorescence intensity (MFI) of HLA-DR expression. Decreased percentages of myeloid-derived suppressor cells (CD14+ , CD11b+ , HLA-DR low or negative) among monocytes were observed at the same time intervals (c). Bars depict the mean ± SEM; p values from 2-sided Wilcoxon signed-rank test. MDSC myeloid derived suppressor cells, C treatment cycle, D treatment day.

Figure 6

Changes in monocyte numbers and activation state 2 days following the first dose of Melanoma GVAX correlate with serum GM-CSF concentrations at C1D3. Changes (Δ) were calculated by subtracting baseline values (C1D1 for cohorts A and B, or C1D0 for Cohort C) from values obtained at cycle 1, day 3. Data from 19 patients are shown. Monocyte activation was quantified by measuring mean fluorescence intensity (MFI) of HLA-DR expression on CD14+ , CD11b+ events by flow cytometry. r values are from a 2-sided Spearman Correlation Analysis.

Figure 7

Increased melanoma peptide-specific reactivity generated in one patient over time on treatment with Melanoma GVAX. This patient in Cohort A was HLA-A2+ . IFN-g ELISPOT was used to measure the specific reactivity of short-term in vitro stimulated T cell cultures against a pool of melanoma peptides restricted by HLA-A2. Stimulation index = [IFN-g spot forming colonies/1E6 cells stimulated with melanoma peptide pool]/[IFN-g spot forming colonies/1E6 cells stimulated with HBV control peptide]. Trends were similar in two separate assays, showing peak reactivity after two cycles of treatment. C treatment cycle; D treatment day.