Helper T-cell responses and clinical activity of a melanoma vaccine with multiple peptides from MAGE and melanocytic differentiation antigens

Abstract

Purpose: A phase I/II trial was performed to evaluate the safety and immunogenicity of a novel melanoma vaccine comprising six melanoma-associated peptides defined as antigenic targets for melanoma-reactive helper T cells. Source proteins for these peptides include MAGE proteins, MART-1/MelanA, gp100, and tyrosinase.

Patients and methods: Thirty-nine patients with stage IIIB to IV melanoma were vaccinated with this six-peptide mixture weekly at three dose levels, with a preceding phase I dose escalation and subsequent random assignment among the dose levels. Helper T-lymphocyte responses were assessed by in vitro proliferation assay and delayed-type hypersensitivity skin testing. Patients with measurable disease were evaluated for objective clinical response by Response Evaluation Criteria in Solid Tumors.

Results: Vaccination with the helper peptide vaccine was well tolerated. Proliferation assays revealed induction of T-cell responses to the melanoma helper peptides in 81% of patients. Among 17 patients with measurable disease, objective clinical responses were observed in two patients (12%), with response durations of 1 and 3.9+ years. Durable stable disease was observed in two additional patients for periods of 1.8 and 4.6+ years.

Conclusion: Results of this study support the safety and immunogenicity of a vaccine comprised of six melanoma helper peptides. There is also early evidence of clinical activity.

Fig 1.

Flow diagram for Mel41 clinical trial. For each vaccine, the peptides were administered with granulocyte-macrophage colony-stimulating factor 110 μg in a stable emulsion with Montanide ISA-51 adjuvant 1 mL on days 1, 8, 15, 29, 36, and 43 (weeks 0, 1, 2, 4, 5, and 6). The first three vaccinations were divided between two injection sites (primary and replicate), and the last three were delivered to the primary injection site only. At each injection site, half of the vaccination was administered subcutaneously, and half was administered intradermally. DTH, delayed-type hypersensitivity.

Fig 2.

Cellular proliferative responses to six melanoma helper peptides (6MHP) in peripheral-blood mononuclear cells (PBMCs) and the sentinel immunized node (SIN). (A) Proliferation data are shown for one patient VMM699 at 5 days after stimulation with the 6MHP pool, individual peptides, or an irrelevant tetanus helper peptide. Calculated stimulation index (SI) data are shown; SIs above the red line (SI > 4) are considered positive. The patient is HLA-DR4 and HLA-DR12 positive; described HLA-DR restrictions for peptides with responses (TSY, WNR, FLL, and AQN) are DR11, DR4 and DR1, DR15, and DR4, respectively. W, weeks; T, vaccine treatment; SIN W = 3, sentinel immunized node harvested at week 3. (B) SIs reported for the pool of 6MHP correlated with the sum of maximum SIs for each peptide at all time points evaluated in the PBMC or SIN. (C) Time course for proliferation assay SI data over time for all evaluated patients, with box plots (boxes = 25th to 75th percentiles; vertical lines = minimum to maximum). Numbers at the top of each bar are the number of assessable patients at the time shown. W, weeks from study initiation; M, months from study initiation; T, treatment/vaccine number; SIN, day of SIN biopsy; NA, not available. These are PBMC data only. Blood was not drawn at weeks 2, 4, and 6 and was not evaluated at month 12. (D) Box plot of SIs for immune responders, through week 12, in the PBMC and SIN for each study group (groups A, B, and C = 200, 400, and 800 μg dose, respectively).

Fig 3.

Identification and characterization of the cells responding to the six melanoma helper peptides (6MHP). (A) Two patients were evaluated for proliferative response to the 6MHP pool after labeling with carboxyfluorescein diacetate succinimidyl ester (CFSE). The proliferative response was observed among the CD4 T-cell population in both patients. (B) The proportion of CD4⁺ FoxP3-positive cells among circulating peripheral-blood mononuclear cells (PBMCs) or lymphocytes from the sentinel immunized node (SIN) was evaluated for five patients on the Mel41 trial. Flow cytometry plots are shown for one patient (VMM681); summary data (n = 5) are shown in a box plot, in which the box represents the 25th to 75th percentiles, the vertical lines represent minimum and maximum, and plus signs represent medians. PBMC data are for patients VMM625, VMM677, VMM681, VMM683, and VMM699; SIN data are for four of those patients, plus patient VMM727 instead of patient VMM677. Postvaccine data are inclusive of PBMC data analyzed at weeks 3, 5, 7, and 26. PreVax, before vaccine.

Fig 4.

Delayed-type hypersensitivity (DTH) assay data. DTH reactivity was identified in seven of 24 assessable patients. (A) The diameter of induration is shown at 24 and 48 hours for those seven patients, both before vaccine and at 12 weeks (post). (B) The change, from before vaccine to week 12, in induration diameter (maximum of 24 hours and 48 hours, subtracting maximum prevaccine value, averaged for all seven patients) is shown for reactions to the six melanoma helper peptides (6MHP) and to the standard recall antigens. (C) The association between DTH reactivity and proliferative responses is shown in peripheral-blood mononuclear cell (PBMC) and sentinel immunized node (SIN). (D) Associations between DTH reactivity and study group, disease status, and disease stage are presented based on the proportions of patients in each subgroup with DTH reactivity. The denominators for subgroups are as follows: overall (n = 24), group A (n = 10), group B (n = 8), group C (n = 6), measurable disease (Dz; n = 8), nonmeasurable disease (n = 16), stage IV (n = 14), and stage III (n = 10).