Over the last decade, at least 20 HLA class I-restricted peptide epitopes have been defined from multiple melanoma differentiation and cancer-testis antigens. They are reviewed in ref. 15. Peptide vaccine trials have been conducted with individual or multiple peptides in patients with stage IV disease in aqueous solution or using different adjuvants, with different cytokines, or via DNA plasmid delivery. Some trials have already been conducted in high-risk resected patients, which would appear to be an optimal population for conducting cancer vaccine trials. Significant controversy exists, however, over the optimal surrogate immune end point to measure in peptide vaccine trials, appropriate dose, the duration of treatment, and scheduling.
The first tumor antigen recognized by T cells that was cloned was MAGE-1, a member of a multigene family dubbed a "cancer-testis" antigen because it was found to be encoded normally in the germline but expressed only in tumor tissue, testis, and placenta (16,17). Since the testis was regarded as an immune sanctuary due to lack of class I expression on its parenchymal and germ cells, the MAGE genes appeared to constitute an optimal group of tumor antigens for vaccine strategies because of their preferential expression by tumors. Clinical trials of an HLA-A1 peptide encoded by MAGE-3, present on 70-80% of metastatic melanomas, resulted in evidence of tumor regression in 6 of 25 patients that received a monthly regimen of peptide in aqueous solution. Surprisingly, no evidence of antigen-specific T cells could be detected when standard techniques such as limiting dilution analysis, ELISPOT, and tetramer assays were used (18). Only after multiple restimulations ex vivo could reactivity to MAGE-3 be detected in patients' peripheral blood. In one study by Coulie, MAGE-3 tetramers were used to detect 1:40,000 circulating CD8+ T cells with a monoclonal T-cell receptor in a patient that received repetitive vaccinations with the MAGE-3 peptide alone (19). In another trial of the same MAGE-3 epitope administered with incomplete Freund's adjuvant (IFA) to patients with resected melanoma, modest levels of immunity were seen in 5 of 18 patients, with functional lytic T cells detected in circulating peripheral blood mononuclear cells (PBMCs) (20).
The definition of a number of differentiation antigens expressed by both melanocytes and melanomas such as MART-1, tyrosinase, gp100, TRP-1, and TRP-2 followed the cloning of the MAGE genes (21-28). Surprisingly, endogenous T-cell reactivity to MART-1 could be detected in a significant minority of patients with melanoma, but not normals (29,30). Clinical trials of an immunodominant MART-1 epitope peptide comprising amino acids 27-35, or its amino acid-substituted counterpart amino acids 26-35 (27L) have been conducted with peptide in aqueous solution or with adjuvants like QS-21 or Montanide ISA 51, equivalent to the IFA used in mice. The Rosenberg group demonstrated that the immunodominant epitope with IFA-induced immune responses by a restimulation assay in the majority of patients, but without tumor regression (31,32). The substituted epitope was clearly more potent as an immunogen in vivo (32). Romero and colleagues have conducted extensive in vitro and in vivo testing of MART-1 peptides and have demonstrated that immune responses by ELISPOT and tetramer could be detected in the majority of patients in peripheral blood; that draining lymph nodes harbored large numbers of MART-1 antigen-specific cells that were antigen-experienced classic memory-effector cells; that T-cell receptor populations after vaccination were not focused but were quite diverse; and that occasional tumor regression resulted from vaccination with MART-1 peptides in aqueous solution or with adjuvants (33-37). Weber and colleagues have shown that in patients with resected stages III/IV melanoma at high risk for relapse, the MART-1 immunodominant epitope with IFA-induced immune responses in the majority of patients that correlated with time to relapse, suggesting that clinical benefit might be associated with MART-1 vaccination (38).
Rosenberg and colleagues have shown that an immunodominant gp100 epitope 209217 modified by the substitution of an amino acid at the second anchor position for class I was more effective at generation of an antigen-specific T-cell response in vitro and in vivo (39). The substituted epitope peptide 209-217 (210M) with Montanide ISA 51 was immunogenic in virtually all HLA A *0201 patients, and in vitro and in vivo was superior to the native peptide at the generation of immune responses. When the substituted epitope peptide was administered with interleukin-2 (IL-2), regression of tumor in a high proportion of patients was observed (40). The T-cell repertoire after gp100 immunization was found to be diverse and oligoclonal, and the kinetics of gp100-specific T-cell responses indicated that increases were seen after each vaccination, with a plateau only after multiple vaccinations and an increase in T-cell receptor avidity over time after multiple vaccinations (41-44). Significant levels of gp100-specific circulating T cells were achieved, although there did not appear to be a correlation between detection of functional T cells and tumor regression (45). Surprisingly, only 1 of 11 patients treated with gp100 peptide/IFA with IL-2 had detectable tetramer-positive antigen-specific T cells, compared to six of six with the adjuvant IFA alone (46). The gp100-specific T cells over time were found to be antigen-experienced, functional, effector-memory T cells that were
CCR7-, CD45RA+, CD44+, and CD27+ (47,48). After gp100 vaccination, there was evidence of epitope spreading to the cancer-testis antigen MAGE-12, indicating that indirect mechanisms for tumor regression might occur. Large numbers of antigen-specific T cells were found to infiltrate tumors after peptide vaccination (49), even though the levels of circulating cells varied enormously, suggesting that an assessment of the tumor milieu was probably important for any evaluation of an antigen peptide vaccine strategy. That same group found that the substituted gp100 209-217 (210M) and 280-288 (288V) peptides were potent immunogens, and that tumor-infiltrating lymphocytes that were therapeutically effective recognized those gp100 epitopes. That group isolated tetramer-positive antigen-specific T cells and found that gp100-specific effector cells from the fresh blood were able to produce interferon (IFN)-y after exposure to antigen in the form of peptide- or antigen-expressing tumor cells, and that there was a correlation between intensity of tetramer staining and production of IFN-y by cytokine flow cytometry or quantitative RT-PCR assay (43). In patients with metastatic melanoma who had a good immune response to the gp100 peptides and survived 2 yr after being vaccinated, antigen-specific effector cells could still be found in the bloodstream (44). In another trial, effector-memory T cells that were detected by functional ELISPOT assay were found to be highly conserved in patients with resected stages III or IV melanoma over a period of 1.5 and 3 yr after finishing a 6-mo vaccination regimen. Their phenotype was CCR7 negative, CD45RA positive, CD44 positive indicating that they were activated effector-memory T cells (Chiong et al., manuscript in preparation). Similar data have been observed by the Rosenberg group for patients with metastatic melanoma, although in their assays, there was no correlation between increased frequency of vaccine-induced gp100-specific T cells by tetramer assay and regression of tumor, although tetramer-positive cells tended to also be y IFN producers (42).
The same substituted gp100 peptide has been used in combination with a tyrosinase peptide, and with the MART-1 immunodominant 26-35 (27L) epitope with different cytokines in patients with resected melanoma who received a prolonged series of vaccinations. Weber and colleagues demonstrated that IL-12 administered with a multipeptide vaccine was an effective immune booster (50), as was granulocyte-macrophage colony-stimulating factor (GM-CSF). Approximately 80-90% of patients had augmented antigen-specific immunity by tetramer and cytokine release assays, and the time to plateauing of the immune response was quite prolonged, with six to eight vaccinations required for optimal immunity, in agreement with findings by the Rosenberg group. In the trial of gp100/tyrosinase peptides with IFA and GM-CSF, epitope spreading against MART-1 was observed (51), confirming the prior finding that an expanded T-cell receptor repertoire may be important for clinical benefit.
The gp100 280-288 peptide has also been shown to be immunogenic. Slingluff and colleagues have immunized 22 patients who had resected stage III or IV melanoma with that peptide with or without a tetanus epitope peptide. Of 22 patients, 79% had T-helper responses to tetanus, but only 14% to the gp100 280-288 epitope (52). That same group has also demonstrated that high levels of gp100-specific T cells may be isolated from the lymph nodes draining vaccine sites, which might be a surrogate marker for the success of a peptide vaccine (53). A variety of factors may affect the ability to mount an immune reaction directed against class I-restricted peptide vaccines. Class I HLA antigen A2 is the most common allelotype in the United States, comprising 46% of the population. The locus is quite polymorphic, with at least 12 A2 alleles, of which A*0201 is the most common in the United States. Marincola and colleagues found that different HLA-A2-restricted melanoma peptides bound with differing affinities to A2 alleles, so that the immunodominant gp100 and MART-1 epitope peptides were recognized by PBMCs from patients expressing the A*0201 allele but not A*0204 or A*0207, for example (54). A vaccine that might work effectively in a Northern European population that was predominantly A*0201 might not work well in Southern Europe where other alleles may be common.
Many of the defined melanoma epitope peptides are derived from differentiation antigens like MART-1, tyrosinase, or gp100, which are both heterogeneously expressed on many tumors or simply downregulated in many metastatic lesions. The lack of expression of those antigens on more advanced tumors suggests that earlier-stage tumors may be more effectively targeted by peptide immunization strategies, and also that the use of multiple epitopes in a vaccine would diminish the chance of immune selection resulting in tumor variants that could escape recognition. There are contradictory data on this issue. In one study, the majority of relapsing patients continued to express the antigen (gp100) against which they were vaccinated (51). In several other studies, relapsing patients' tumors were shown to lose expression of melanoma antigens with which they were immunized (55,56). In one study, MART-1-specific T cells were shown to correlate with spontaneous regression of primary melanomas, and when tumors progressed after regression of the primary, loss of MART-1 expression was observed, consistent with an immune selection hypothesis and the idea that MART-1 is a tumor regression antigen (57). Melanomas have also been shown to downregulate or completely delete expression of class I MHC or P-2 microglobulin, which could also result in the phenomenon of immune escape (58).
Further evidence for the idea that MART-1 and gp100 are true "tumor regression" antigens comes from the demonstration that tumor-infiltrating lymphocytes (TILs) often recognize those antigens. In one study, 19 of 59 TIL cultures recognized known immunodominant MART-1 or gp100 peptides (59). The group of Jager and Knuth has conducted several multipeptide vaccine trials in patients with metastatic melanoma, and have demonstrated that tumor regression in a small trial with GM-CSF administered as an adjuvant cytokine correlated with antigen-specific immune responses (60). The same group has also intensively studied T-cell responses to MART-1 and has shown that development of a clonal T-cell response to that antigen correlated with tumor regression, strengthening the idea that a successful peptide vaccination conferred clinical benefit (61).
Immunization with melanoma epitope peptides might result in a "bystander" reaction in which the phenomenon of epitope spreading occurs. Reactivity to epitopes not included among those in the vaccine have been demonstrated for MART-1 (51) after vaccination with gp100 and tyrosinase epitopes, and for MAGE-12 after a gp100 vaccination (47). This might expand the number and type of antimelanoma T cells, and provide clinical benefit.
Tyrosinase encodes a number of HLA-A2-restricted epitopes, and several have been shown to be immunogenic in patients. The tyrosinase 1-9 and 368-376 (370D) peptides have been used to vaccinated patients with stage IV melanoma, and modest levels of reactivity were observed by ELISPOT assays. The 368-376 (370D) peptide is unique in that it results from a post-translational modification to the 370 amino acid, and is actually expressed on tumor cells. Lewis and colleagues immunized nine patients with the modified tyrosinase 368-376 peptide with adjuvant QS-21, and found that only two of nine had an augmented frequency of cytolytic T cells (CTLs), without evidence of clinical benefit (62). Scheibenbogen immunized 16 patients with the tyrosinase 1-9 epitope, and an additional 2 patients who had no evidence of disease (NED) (63). The peptides were administered in aqueous solution with GM-CSF. Only five patients were able to receive six injections over 14 wk. One mixed response and two patients with stable disease were seen. There was evidence of augmented tyrosinase-specific T-cell immunity by ELISPOT assay in four patients, including the one patient with a mixed response, one patient with stable disease, and the two patients who were NED.
Several trials have demonstrated that clinical benefit correlated with peptide-specific immunizations. Keilholz and colleagues reported that several patients who had a history of frequent relapses of stage IV melanoma received an extended series of immunizations with a tyrosinase peptide and adjuvant and experienced long-term freedom from recurrence while levels of circulating immune cells were detected against tyrosinase (63). Jager has shown that three patients in a small series immunized with multiple peptides derived from differentiation antigens with GM-CSF as an adjuvant had tumor regression, which correlated with immune response and DTH reactivity, as have other groups (60,61).
Another member of the "cancer-testis" family is NY-ESO-1, a protein that is highly expressed on a variety of epithelial carcinomas and melanomas. It is unique among the known melanoma antigens because it encodes antibody, T-helper, and cytolytic epitopes (65). In fact, it has been shown that CTL responses correlated with the detection of endogenous antibody responses in patients with NY-ESO-1 expressing melanomas, suggesting an interdependence of T-helper responses and CTL immunity (66). Three different class I HLA-A*0201-restricted epitope peptides have been tested in a small pilot trial in 12 patients with metastatic melanoma (67). Seven of 12 patients had no detectable preexisting NY-ESO-1-specific antibody response. Of those patients, four had increases in CTL reactivity, which correlated with disease stabilization or regression. Interestingly, that same group (68) immunized patients with the NY-ESO-1 157-167 epitope and noted that patients exhibited 159-167 specific response but that epitope was not naturally expressed by tumor cells. The 157-165 peptide is the "natural" epitope, yet was a minor component of the NY-ESO-1 reaction, indicating that patterns of peptide-specific reactivity were unpredictable.
Class II peptides have been defined from a number of melanoma antigens, were tested in transgenic mice and other in vitro systems, and found to be capable of generating immune responses in vitro (69-72). These peptides are restricted to common class II alleles such as DRB*04 01, or DP4, representing 25% and nearly 50% of the population, respectively. There is evidence that class II antigen-specific peptides might add to the ability to generate a class I-restricted CTL response in melanoma, and several trials are pursuing the idea of combining class I and class II peptides in a melanoma vaccine strategy (73).
The development of careful immunologic monitoring for melanoma peptide trials is critical for the success of the peptide vaccine approach. ELISPOT assays have been used by a number of investigators for cancer vaccine trials and for HIV vaccine studies. This assay is technically demanding and requires dedicated equipment, but provides a reliable functional and quantitative assay for the detection of circulating antigen-specific T cells
(74-76). Cytokine flow cytometry (CFC) is a technique that permits an assessment of function and permits enumeration of antigen-specific cells, but is not as well developed or as sensitive as the ELISPOT assay (77). The CFC assay has enormous flexibility to incorporate multiple markers of immune function, and will continue to be refined for future trials. The peptide-tetramer assay is a flow cytometry-based assay that provides great sensitivity, reliability, and quantitation, but is not a functional assay (78,79). In fact several groups have described a dichotomy between the enumeration of circulating cells by tetramer assay and the number of cells detected by the functional CFC or ELISPOT assays, which reinforces the notion that a quantitative and a functional assay are critical for proper interpretation of immune monitoring of a peptide vaccine trial (80).
The most extensive and convincing data for the generation of long-lasting melanoma-specific immunity exist for patients treated on peptide vaccine trials. There has been little toxicity to those approaches, but evidence is compelling for the detection of significant augmentation in levels of antigen-specific CTLs in the circulation, in lymph nodes draining vaccine sites, and even infiltrating tumors. Long-term immunity with the existence of effector-memory T cells lasting years after vaccination has been clearly shown. IL-12 and GM-CSF injected locally are likely to be potent adjuvants, and long duration of immunization is likely to be needed. There is no indication that a dose response above 1 mg exists for peptide vaccines. The antigen-specific CTLs induced after a peptide vaccination are a mixture of low- and high-affinity effectors, and reflect a diverse repertoire of T-cell clones. The factors that determine why very different levels of immunity with a range of1000-fold are generated in different patients remain to be determined, and are an obstacle to the further success of peptide vaccine strategies. The importance of class II peptides and their role in facilitating class I immunity remains to be defined.
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