Study of MAGE-3/Melan-A/gp 100/NA17 and rhIL-12 With/Out Low Dose IL-2 in Metastatic Melanoma

• The primary hypothesis is immunization of patients with 4 melanoma antigen peptides will induce augmented specific IFN-.-producing CD8+ T cells against all 4 antigens simultaneously, and to determine the clinical response rate. [ Time Frame: draft ] [ Designated as safety issue: No ]
  

Based on the above preclinical and Phase I results, a logical strategy for a second generation melanoma vaccine has emerged. A randomized Phase II study in metastatic melanoma patients will be undertaken. Patients first will be HLA-typed; HLA-A2-positive patients will be eligible for screening. When feasible, each patient will undergo a tumor biopsy to screen for expression of MAGE-3, Melan-A, gplOO, and NAI 7 using RT-PCR and immunohistochemistry, to determine whether T cells are present in the lesion, to measure cytokine gene expression by RT-PCR, and to perform gene array analysis. In addition, blood cells will be analyzed for certain parameters of T cell function.

Patients will be randomized to cohorts A (no IL-2) or B (with low-dose IL-2). For treatment, peripheral blood will be collected and fractionated by density centrifugation to isolate PBMC as a source of APC. The PBMC will be divided into four pools, each of which will be incubated with one of the following peptides: MAGE-3, Melan-A, gp 100, or Ni 7A. The peptide-loaded cells will then be washed and recombined into a single suspension in PBS, and lethally irradiated. Approximately 120 x 106 pulsed cells will be injected subcutaneously at a site near a lymph node not thought to be involved with tumor. The subcutaneous route has been selected for the reasons of safety, efficacy in the preclinical model, and the goal of targeting the vaccine to a draining lymph node. rhIL-12 (4 .tg straight dose) will then be given subcutaneously adjacent to the vaccine site days 1,3, and 5 of each cycle. This dose and schedule was found to be effective in our phase I study. In one-half of the patients (cohort B), IL-2 (I MU straight dose) will be administered subcutaneously daily, days 7-18. Re-immunization along with rhIL-12 followed by IL-2 (if assigned) will be performed at 3 week intervals as in cycle I.

On day 1 of each cycle, peripheral blood will be collected to measure peptide-specific IFN-y production. Before treatment and after every 3 cycles, PBMC will be collected to quantify peptide specific CD8 T cells by flow cytometric analysis with peptide/HLA-A2 tetramers, and evidence for a molecular response will be assessed by performing RT-PCR. for melanoma antigens on peripheral blood samples. In addition, prior to treatment, after the first 3 cycles, and at the time of going off- study, a tumor biopsy will be performed to assess the immune response in the tumor microenvironment, including gene array analysis. It is hoped that these studies will uncover the reason for lack of clinical response in patients with residual tumors. Clinical response will be assessed as a secondary outcome.