The development of effective means for immunizing patients against their growing cancer is a major goal of studies in human tumor immunology. A variety of shared tumor-associated antigens present on melanomas represent possible candidates for such immunization1,2
. The gp100 antigen was selected for evaluation because of its recognition by TILs, whose adoptive transfer was highly associated with tumor regression in patients with melanoma8
. In prior phase I studies, patients were immunized with escalating doses of one of the immunodominant gp100 peptides (g154–162, g209–217, g280–288). The immunologic reactivity of patients before and after immunization was evaluated by in vitro
sensitization of PBMCs to the peptide pulsed onto antigen-presenting cells10–14
. Patients receiving the g209–217 and g280–288 peptides, but not the g154–162 peptide, developed specific immune precursors, although multiple in vitro
sensitizations were necessary to elicit reproducible immune responses14
. In in vitro
studies, a synthetic modification of the g209–217 peptide (called 209-2M), which exhibited increased binding to HLA-A2 molecules, had an increased ability to generate melanoma-reactive CTLs after multiple stimulation of the PBMCs of HLA-A2+
. Because of these findings, we conducted the current study in which patients were immunized with this modified peptide.
The present study makes two major points. It represents the first time that a self-peptide (in this case a synthetic modification designed to increase MHC binding) has provided a consistent and powerful means of immunizing patients to generate lymphocyte precursors against growing tumor. In addition, immunization with this peptide plus IL-2 appears to provide significantly higher cancer regression rates than those seen with either agent alone.
In the present study, 2 of 8 patients that received the native g209–217 peptide, compared with 10 of 11 patients immunized with the modified g209-2M peptide, developed highly reproducible reactivity against the native g209–217 peptide and against melanoma cells (P
= 0.006). Of importance was the ability to detect this reactivity after a single exposure of PBMCs to peptide without the need for any restimulation in vitro
. Thus, in contrast to our prior studies with unmodified peptides18–19
and the reported studies mentioned above, the degree of immunization was substantially higher using the g209-2M peptide than had previously been seen. Significant reactivity to the native g209–217 peptide was seen within 4 days after exposure to peptide in vitro
and continued for at least 18 days in culture (data not shown).
Further evidence for the strong immunization against the native g209–217 peptide came from studies of the precursor frequencies present in circulating PBMCs. In our prior studies and those of others, it has rarely been possible to measure precursor frequencies against melanoma antigens because of their very low frequency30–32
. In the present series, precursor frequencies against the native peptide were less than 1 in 30,000 (the lower limit of detection in this assay) PBMCs before immunization compared with frequencies between 1 in 2800 and 1 in 5900 after immunization. In these patients, therefore, precursor frequencies were of the same magnitude often seen against viral or allogeneic antigens33,34
The mechanism by which immunization with the g209-2M modified peptide in IFA resulted in high levels of circulating cellular immunity against the native peptide, as well as against melanoma cells, is unclear. The emulsification of peptide in adjuvant is thought to provide prolonged exposure to antigen and to activate nonspecific inflammatory cells and possible recruitment of antigen-presenting cells to the site of immunization. Nonspecific recruitment of helper cells at the sites of vaccination or at regional draining sites may provide the necessary help to stimulate immune reactions. It is known, however, that specifically reactive CD8+
cells can be generated following immunization with MHC class I-restricted peptides in the absence of CD4+
. The decrease in circulating precursor cells when IL-2 was administered with peptide may be due to activation of these cells and traffic to the tumor site, and attempts to reisolate these cells from the tumor are in progress.
Despite the induction of high levels of these tumor-reactive cells following immunization with the g209-2M peptide in IFA alone, none of the 11 patients in this study experienced an objective tumor response, although several patients had mixed responses with shrinkage of some lesions. Several possibilities exist to explain this paradox, although it is likely that tumor cells do not contain the appropriate costimulatory or adhesion molecules required to activate the resting precursor cells that circulate in peripheral blood as a result of immunization. Peripheral anergy may thus result from contact with antigen in the absence of costimulation. An additional source of helper function may be required. In experimental systems, provision of helper epitopes, as well as cytokines such as IL-2 or IL-12, have been shown to increase the immunizing capacity of MHC class I-reactive antigens20,21,23,36–38
Although the 42% response rate seen in the present study using the peptide vaccine plus IL-2 appears higher than the 17% response rate seen in prior studies using IL-2 alone29
, randomized trials to evaluate the efficacy of g209-2M peptide immunization plus IL-2 are required. Several modifications to attempt to improve upon these results are in progress. We have recently initiated a clinical trial in which patients with melanoma are immunized simultaneously with four separate peptides (g209-2M, g280-9V, MART-1:27–35 and tyrosinase:369–377) from three different melanoma antigens (gp100, MART-1 and tyrosinase), all recognized by TIL cells associated with tumor rejection. In another trial, PBMCs from patients after immunization with g209-2M in IFA are being activated by antigen and IL-2 in vitro
and used for adoptive transfer to the autologous tumor-bearing patient.
This study demonstrates that it is possible, in patients with metastatic cancer, to increase significantly the number of lymphocyte precursors reactive with normal nonmutated differentiation proteins such as gp100 by immunization with synthetic high binding peptides in IFA and to mediate tumor regression. Many cancers such as those that occur in the breast, prostate and ovary express proteins unique to, or overexpressed by, the tissue of origin of the tumor, and these differentiation proteins may also be suitable targets for immunotherapy.