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.