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Host Responses to Melanoma
Implications for Immunotherapy
Julian A. Kim and Ernest Borden
C
ONTENTSINTRODUCTION
OBSERVATIONS OF NATURAL IMMUNE RESPONSES TO MELANOMA
IMMUNOTHERAPY OF MELANOMA
CONCLUSIONS AND PERSPECTIVES
REFERENCES
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Summary
Development of immunotherapeutic strategies for the treatment of patients with melanoma have far exceeded that of any other solid malignancy, with the possible exception of renal cell carcinoma. The rationale for use of melanoma as a model for development of immunotherapies is derived from the fol- lowing observations:
1. Melanoma metastases and primary cutaneous melanomas are among the most common solid tumors to exhibit spontaneous regression.
2. Local intratumoral injections of bacillus-camillus-guerin can lead to regression of not only the injected lesions but also distant lesions, suggesting the successful induction of a systemic immune response.
3. The ability to culture tumor-infiltrating lymphocytes, which recognize melanoma cell lines that share specific major histocompatibility complex alleles could be used to identify shared melanoma-associ- ated rejection antigens.
Key Words: Melanoma; regression; interferon; interleukin; tumor necrosis factor.
INTRODUCTION
The purpose of this chapter will be to highlight observations that demonstrate a natural host immune response to melanoma, which make melanoma particularly suitable to attempts at immunotherapeutic control. Major strategies of immunotherapy will be pre- sented, along with how these strategies have been tested in human clinical trials. The basic categories that will be discussed include “active” forms of immunotherapy, which require a host immune response to exert their activity; and “passive” forms of immuno- therapy, which directly target the tumor and can theoretically generate an antitumor
From: From Melanocytes to Melanoma: The Progression to Malignancy Edited by: V. J. Hearing and S. P. L. Leong © Humana Press Inc., Totowa, NJ
effect without a host immune response. Active nonspecific immunotherapies, in the form of systemic administration of cytokines, and active specific immunotherapies, such as cancer vaccines, will be discussed and compared with passive immunotherapies, which consist primarily of monoclonal antibodies and adoptive transfer of T-cells into the tumor-bearing host. Emphasis will be placed on the proposed mechanism of action, antigen(s) that are being targeted, and results of phase II and phase III human clinical trials. Although a detailed analysis of promising therapies that are in preclinical and early clinical development is beyond the scope of this chapter, particularly novel strategies for next-generation immunotherapies will be presented to provide a sense of the future direction of the field.
OBSERVATIONS OF NATURAL IMMUNE RESPONSES TO MELANOMA Spontaneous Regression of Melanoma
Melanoma is arguably one of the most unpredictable solid tumors in terms of biologi- cal behavior, in which there is a documented incidence of spontaneous regression of both the primary cutaneous tumor as well as distant metastases. The percentage of patients who present with partial or complete histological regression of the primary tumor is estimated to be as high as 25 to 40%, although the reports of sustained long-term partial regression are rare (1–3). Interestingly, up to 15% of patients with stage IV metastatic melanoma will live 5 yr, independent of whether they are receiving therapy (4). Many investigators have attributed the regression of primary and metastatic melanomas to the adaptive immune response, although others have shown evidence of alternative mecha- nisms of tumor growth control, such as apoptosis (5,6).
Several lines of evidence support a central role of immune mediators in spontaneous melanoma regression. Biopsies of regressing lesions have demonstrated overexpression of T-helper (Th)-1 cytokines, such as interferon (IFN)-J, interleukin (IL)-2, and tumor- necrosis factor-D, when compared with nonregressing lesions (7). In addition, although the reports in the literature are sparse, isolated reports of spontaneous regression of metastatic melanoma have been associated with evidence of increased immune param- eters, such as delayed-type hypersensitivity response to skin testing (8). Indirect evi- dence supporting immune surveillance of primary cutaneous melanoma has been reported in renal transplant patients who not only have a higher risk because of immu- nosuppression, but also develop cutaneous melanoma from precursor dysplastic nevi, which demonstrated a marked absence of lymphocytic and macrophage infiltrates (9,10).
Finally, the presence of a brisk lymphocytic infiltrate in primary cutaneous melanomas has been correlated with improved survival (11). A retrospective analysis of 259 patients with localized primary cutaneous melanoma with median follow-up of 12.3 yr con- firmed that the degree of tumor infiltration by lymphocytes was an independent predictor of melanoma-specific mortality (12).
Immune-Cell Infiltrate of Melanoma
Elegant studies directed toward characterizing the immune-cell infiltrate of primary and metastatic malignant melanoma lesions have provided ample evidence of host–
tumor immune interaction. Immunohistochemical analysis of immune-cell infiltration have confirmed a preponderance of T-cells, which can range from memory CD4 cells in early (<0.75 mm) superficial spreading melanomas to CD8 cells of cutaneous origin that express HECA 452 (13). Dermal dendritic cells that express human leukocyte anti-
gen-D related (HLA-DR) also appear to infiltrate both vertical growth-phase and radial growth-phase melanomas but not benign compound nevi, and the expression of HLA-DR correlates with the degree of T-cell infiltrate, consistent with the idea that melanoma- associated antigens are being presented to infiltrating T-cells (14). Isolation of tumor- infiltrating lymphocytes (TILs) and long-term culture have resulted in the generation of T-cell clones that recognize melanoma-associated antigens in the context of major his- tocompatibility complex (MHC) (15).
A variety of cytokines has been identified within the host–melanoma microenviron- ment that may play a role in either recruitment of T-cells, generation of T-cell effector responses, or both. As has been identified in other solid malignancies, melanoma cells are known to secrete proinflammatory cytokines, such as granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-1 and IL-6, and growth factors, such as vascular endothelial growth factor and basic fibroblast growth factor, which may serve not only as autocrine growth factors but also regulate local cell-mediated immune responses (16–18). Interestingly, melanoma cells have been shown to express a func- tional IL-4 receptor, which, after ligation, results in decreased cell proliferation and increased MHC II molecule expression (19). Finally, gene-profiling studies of fine- needle aspirations from primary and metastatic subcutaneous melanoma define a sub- group of approx 30 genes, such as chemokines and immune response transcription factors, which were predictive of clinical response to immunotherapy (20). Approxi- mately one-half of the 30 genes with predictive value had function related to T-cell regulation, suggesting that the molecular events which underlie melanoma regression confirm T-cell-mediated mechanisms (21,22).
Tumor-Induced Mechanism of Immune Suppression
Immune-mediated tumor regression can be influenced not only by the efficiency of the host immune response but also counterbalanced by tumor-induced mechanisms of immune evasion. Several observations support the concept that melanoma can either directly or indirectly induce mechanisms of immune evasion. Plasmacytoid dendritic cells that express the tryptophan-degrading enzyme, indoleamine 2,3-dioxygenase, have been found to induce T-cell anergy and direct toward Th2 responses. These cells have been identified in melanoma draining regional lymph nodes in higher numbers as compared with control lymph nodes, suggesting the induction of immunoregulatory antigen-presenting cells (APC) as a mechanism of immune evasion (23). CD4 T-cell clones derived from human melanoma metastases have been shown to decrease cyto- toxic T-cell activity in vitro in an antigen-dependent fashion, suggesting the possibility of a subset of T-cells with a suppressor phenotype (24). Melanoma secretion of immu- nosuppressive cytokines, such as transforming growth factor (TGF)-E and IL-10, and secretion of soluble Fas ligand may also contribute to suppression of local immune responses and deletion of effector T-cells (25–27). Gene-profiling studies have identi- fied suppression of interferon-stimulated genes within primary and metastatic melano- mas, and downregulation of MHC class I expression by melanoma cells in vivo has also been observed and is postulated as a mechanism of evasion from antigen-specific CD8 cells (28,29).
Two observations that underscore the importance of melanoma-induced immune evasion are the presence of T-cells in melanoma patients with defective signal transduc- tion pathways and the apparent lack of correlation of the presence of T-cell clonal expansion and melanoma regression. Several studies in patients with both renal cell
carcinoma and melanoma have demonstrated T-cells with defective T-cell receptor tyrosine kinase activity, which involves the Src family kinases, lck/fyn, and the ]-chain of the T-cell receptor (30–33). The putative mechanism of tumor-induced T-cell dys- function appears to be via soluble factors, and the signaling defects are reversible by administration of certain cytokines, such as IL-2. Melanoma-induced T-cell dysfunction may also explain why recent studies have failed to show a correlation between T-cell clonal expansion within TIL populations and tumor regression (34). Thus, although natural immune responses to melanoma-associated antigens do appear to exist, limita- tions of the innate and adaptive immune responses may be caused by the inability to overcome melanoma-induced immunosuppressive mechanisms within the tumor–host microenvironment.
IMMUNOTHERAPY OF MELANOMA Systemic Cytokine Therapy
Immunotherapeutic strategies using systemic administration of recombinant human cytokines represent the most widely studied approach to the treatment of patients with melanoma. The concept of enhancing pre-existing natural immune responses to mela- noma-associated antigens by the systemic administration of cytokines has been tested both in the settings of patients with refractory metastatic disease and as adjuvant therapy in patients with high-risk surgically resected disease. Although the efficacy of systemic cytokine administration in generating specific immune responses may be limited by dose-limiting toxicity and a narrow therapeutic window, systemic cytokine administra- tion remains the only Food and Drug Administration (FDA)-approved biological therapy for patients with either metastatic (IL-2) or surgically resected, high-risk (IFN-D-2b) melanoma.
INTERFEROND-2B
Human IFNs are pleiotropic cytokines that exert a broad range of biological effects, including antiviral, antiproliferative, antiangiogenic, apoptosis induction, and immune cell modulation. Preclinical studies in more than 40 freshly derived melanoma tumor cells have confirmed that IFNs exert direct antiproliferative effects and induce apoptosis in vitro via the IFN-stimulated gene, Apo2L ligand (TRAIL) (35). IFNs appear to exert their effects in part via phosphorylation of the intracellular-signaling protein, STAT-1, because the antitumor effects of interferon are abrogated when administered to STAT- 1-deficient mice (36). Interestingly, the antitumor efficacy of IFN is preserved in vivo against STAT-1-deficient melanoma tumors growing in syngeneic immunocompetent mice, which demonstrates that stimulation of the host immune response remains an important mechanism of activity (37). Indeed, IFNs have been known to be central regulators of cell-mediated immune responses that involve Th and cytolytic T-cells, as well as professional APCs, such as dendritic cells (38). Finally, IFNs mediate significant antiangiogenic effects in preclinical tumor models via downmodulation of vascular endothelial growth factor gene expression (39,40). These preclinical data provided the rationale for human clinical trials of IFN-D-2b in patients with advanced melanoma.
Human Clinical Trials Using IFN-D-2b. Recombinant IFN-D-2b administered in patients with metastatic disease resulted in an approx 20% clinical response rate. Of note, was that nearly one-third of the responders exhibited complete radiographic responses, with duration of response extending from 1 to 3 yr, suggesting that IFN had significant
activity as a single agent (41). Initial early phase studies of combinations of IFN-D-2b with dacarbazine demonstrated clinical response rates approaching 40 to 50%. However, a randomized phase III study comparing combinations of dacarbazine with IFN-D-2b and tamoxifen showed no improvement in time to treatment failure and survival over dacarbazine alone (42). However, because of the significant activity of IFN-D-2b as a single agent, phase III studies in patients with melanoma at high-risk for relapse soon followed.
Numerous clinical trials of adjuvant IFN-D-2b have been conducted in patients with primary cutaneous melanoma greater than 4 mm in thickness or in patients with stage III disease after confirmation of regional lymph node metastasis. The first Eastern Coop- erative Oncology Group (ECOG) 1684 study consisted of 287 patients with deep pri- mary (>4 mm thick) or regional lymph node metastatic (N1) disease, which were completely resected and patients were subsequently randomized to treatment with either 20 million U/m2/d of IFN-D-2b administered intravenously for 4 wk, followed by 10 million U/m2 of IFN-D-2b three times weekly administered subcutaneously for 48 wk, vs observation. The study demonstrated a significant improvement in relapse-free sur- vival (p < 0.002) and overall survival (p < 0.02) with a median follow-up of 6.9 yr, and based on this data, IFN-D-2b was later approved by the FDA for adjuvant treatment of patients with resected high-risk melanoma (43). In an attempt to determine whether a low-dose regimen of IFN could retain the same antitumor activity while reducing treat- ment-related toxicity, an Intergroup study E1690 compared the previously tested high- dose IFN (HDI) regimen for 1 yr with a low-dose regimen for a total of 2 yr (44). At 52 mo median follow-up, the relapse free-survival for the HDI was significantly improved as compared with the low-dose regimen and control observation groups (p < 0.03, two- sided) in both node-positive and node-negative patients. Laboratory correlative studies demonstrated increases in tumor cell MHC class II expression and adhesion molecule expression that were dose-dependent but did not predict prolonged disease-free survival (45). Importantly, no improvement in overall survival was demonstrated between the three groups, although this was confounded by the fact that the overall survival in the observation group in E1684 was significantly higher than the same group of patients in E1690 (6 yr vs 2.8 yr). Despite several attempts to reconcile the discrepancy between overall survival in the observation arms of E1690 and E1684, a third study E1694 was initiated that compared 1 yr of adjuvant therapy with either HDI or a ganglioside vaccine, GMK, consisting of the GM2 ganglioside and the adjuvant QS-21 (46). Patients with resected stage IIb or III were randomized to either one of the two treatment arms, because there was no observation control group. The 880 patients were randomized and the trial was closed prematurely, after interim analysis, when it was determined that there was a statistically significant improvement in relapse-free and overall survival in the HDI group as compared with the GMK group (p < 0.0015 and p < 0.009, respectively).
Interestingly, in those patients treated with GMK, antibody titers to GM2 in treated patients’ serum were detected and did appear to correlate with improved relapse-free and overall survival. In addition, although patients treated with HDI demonstrated improved survival overall, the largest increase in survival appeared to be in those patients with thick primary tumors that were node negative.
Despite data from these three randomized studies that demonstrated significant improvements in relapse-free and, in two instances, overall survival in patients treated with adjuvant HDI, treatment-related toxicity, such as depression, fatigue, and hepato- toxicity emerged as substantial concerns. A quality-of-life adjusted survival analysis
was performed in patients from E1684 that suggested that patients who received HDI experienced severe treatment-related toxicity for an average of 5.8 mo while gaining a mean of 8.9 mo without relapse and 7.0 mo of overall survival. When stratified for tumor burden, it was determined that the greatest quality-of-life adjusted benefit was in the patients who were node positive (47). A recent pooled analysis of the data from the three ECOG and Intergroup trials evaluating over 2000 randomized patients with long-term follow-up (April 2001) confirmed that patients with high-risk, resected melanoma stage IIb or III demonstrated improved relapse-free but not overall survival as compared with observation (48). Although the meta-analysis confirms that IFN-D-2b has significant activity as a single agent in the adjuvant therapy of these patients, there is still consid- erable need to improve IFN-based adjuvant therapy regarding the overall survival ben- efit and quality of life (49).
INTERLEUKIN-2
IL-2, first described as T-cell growth factor, was initially isolated from the superna- tants of human peripheral blood mononuclear cells after stimulation with lectins, such as phytohemagglutinin (50,51). In vitro studies of recombinant human IL-2 have dem- onstrated that, after binding of the high affinity IL-2 receptor on the surface of T-cells, activation of signaling molecules within the Janus kinase/signal transducers of and activators of transcription pathways leads to increased transcriptional activity that favors cell proliferation. Because T-cell activation and proliferation is a central component of most strategies of immunotherapy, IL-2 has been one of the most widely studied cyto- kines in terms of its ability to modulate a therapeutic antitumor immune response.
Early preclinical animal models demonstrated that IL-2 was effective in maintaining long-term T-cell lines and tumor-reactive T-cell clones in culture and represented a significant improvement over lectins and phorbol esters (52). Culture of human periph- eral blood lymphocytes with high concentrations of IL-2 (1000–6000 U/mL) resulted in morphological changes that included blast formation and cytoplasmic granules (53). The ability of the resulting cells to lyse a variety of tumor cell targets in vitro led to their classification as lymphokine-activated killer cells (LAK). The hallmark of the LAK phenomenon was that the effector cells lysed a number of tumor cell targets with no apparent MHC restriction but did not lyse normal autologous peripheral blood cells. The theory that emerged from these observations was that tumors of different histological origins shared common or shared antigens that were recognized by LAK cells. The fact that LAK cells did not lyse normal cells provided a significant therapeutic window and the rationale for translation into human clinical trials.
Human Clinical Trials of Systemic IL-2 in Patients With Metastatic Melanoma.
Based on the observation of MHC-unrestricted lysis of a variety of tumor cells by lymphocytes in cultures with high concentrations of IL-2, systemic administration of recombinant human IL-2 was initiated in patients with metastatic disease of various histologies (54). Rosenberg et al. reported the experience of the Surgery Branch of the National Cancer Institute, in which over 1039 courses of high-dose IL-2 (720,000 IU/
kg of IL-2, every 8 h for 5-d cycles) were administered in 652 patients, 596 of whom had metastatic cancer. Patients treated with either IL-2 alone (n = 155) or IL-2 in combination with ex vivo-generated autologous LAK cells (n = 214) demonstrated a 20 to 35%
objective response rate in the setting of metastatic melanoma, renal carcinoma, colorectal cancer, and non-Hodgkin’s lymphoma. Although the majority of the objective responses
were partial regression, of the 18 patients who demonstrated complete radiographic response, 10 demonstrated a duration of complete response from 18 to 52 mo.
Despite the limitations of this reported series of patients with mixed histologies and treatment regimens, two major themes emerged. The first was that systemic administra- tion of a biological agent that had no direct antiproliferative or cytotoxic effect against tumor cells could result in tumor regression in a minority of patients. Thus, in contrast to the interferons, which have been shown to exert some direct effects on tumor cells in vitro, IL-2 therapy demonstrated “proof of principal” that immune mechanisms alone could mediate tumor regression. The second important observation from this study was that subsets of patients with melanoma and renal carcinoma appeared to have the highest chance of tumor regression, which was an important finding because effective conven- tional cytotoxic chemotherapy was lacking in those particular tumor types. Thus, the initial experience with systemic IL-2 administration led to further randomized studies to define the therapeutic efficacy and mechanism of action in patients with these histologies.
Subsequent studies included a phase II single-armed protocol of high-dose IL-2 therapy in patients with metastatic melanoma and renal cell carcinoma (55). Between September 1985 and December 1992, 283 consecutive patients were treated, and a total of 447 courses were administered. Objective response rates were achieved in 17% of patients with melanoma (7% complete response [CR], 10% partial response [PR]) and 20% of patients with renal carcinoma (7% CR, 13% PR). Based on these and subsequent studies, recombinant human IL-2 was approved for the treatment of patients with meta- static melanoma in 1998. A comparison study of LAK plus IL-2 vs IL-2 alone in 157 patients demonstrated a higher objective response rate in the patients treated with cells plus IL-2, but the difference in patients achieving complete response as well as the development of activated T-cells with MHC-restricted tumor antigen reactivity lead to a gradual decline in the study of LAK adoptive immunotherapy (56).
FUTURE DIRECTIONS
Despite the inherent toxicities of high-dose cytokine therapy, IFN-D-2B and IL-2 remain the only FDA-approved therapies for patients either in the high-risk adjuvant or metastatic melanoma groups. Studies that examined regimens combining IFNs and IL-2 with each other or cytotoxic chemotherapy have yielded only modest increases in objective response rate, but are generally limited by poor tolerability. Methods of deliv- ering cytokines by chemical modification (pegylation of interferon) or the use of carrier proteins, such as monoclonal antibody fragments, has resulted in diminished treatment- related toxicity with apparent maintenance of therapeutic efficacy (57). Preclinical stud- ies identifying novel IFN-stimulated gene pathways to enhance therapeutic efficacy, as well as decrease resistance to IFN therapy, represent an active area of ongoing investi- gation that may result in targeted therapies for patients with melanoma (35,58,59).
Active Specific Immunotherapy Using Vaccines
Development of therapeutic cancer vaccines for melanoma has been an area of intense investigation in both preclinical animal models and human clinical trials. Although the component structure of vaccines may vary from whole tumor cell preparations, tumor cell lysates, and subcellular fractions, whole proteins and defined peptides administered alone or with professional APCs or gene-modified tumor cells or APCs, the goal of the vaccination is the same. Vaccines are classified as active specific immunotherapy
because they require active participation of the host immune system to develop T-cell responses to tumor-associated antigens. Although some vaccines have been demon- strated to result in generation of Th responses that result in antibody production that specifically recognizes and binds the immunogen, most vaccine approaches have focused on the generation of antigen-specific CD8+ cytolytic T-cells. A comprehensive review of the many vaccine strategies is beyond the scope of this chapter. However, examples of several types of vaccines that differ in their composition, as well as resultant immune response, will be highlighted.
WHOLE TUMOR CELL VACCINES
Morton and colleagues pioneered the modern era of the use of irradiated tumor cells as the source of antigen for active specific immunotherapy of melanoma. Initial attempts were focused on the use of autologous melanoma cells irradiated and admixed with bacillus-camillus-guerin (BCG) administered intradermally. However, technical issues surrounding the isolation and expansion of suitable numbers of melanoma cells made the use of autologous tumor cells problematic. Culture of hundreds of melanoma explants resulted in the outgrowth of several melanoma cell lines that could be maintained in vitro and stored using cryopreservation methods. An allogeneic polyvalent melanoma tumor cell vaccine was constructed by combining several cell lines that are now known to express several known melanoma antigens, such as gangliosides, tyrosinase, gp100, and the melanoma antigen recognized by T-cells/melanoma antigen gene proteins. The ini- tial results of a clinical trial using the polyvalent melanoma cell vaccine (MCV) in 136 stage IIIA and IV patients demonstrated significantly increased survival in both groups of patients as compared with historical controls, with no change in the natural history of melanoma in a database of over 1400 patients during that time period (60). Importantly, improved survival correlated significantly with the presence of a delayed-type hypersen- sitivity reaction and antibody responses to the MCV, suggesting that both cell-mediated and humoral immune responses might be contributing to the survival benefit. Although some of the patients with stage IV disease underwent surgical resection before admin- istration of the vaccine, 9 of 40 patients (23%) who had measurable disease demonstrated evidence of tumor regression, 3 of which were complete responses. These initial obser- vations were followed by three separate reports of prolonged survival as compared with historical controls in patients undergoing vaccination with an allogeneic whole mela- noma cell vaccine in patients with stage II, III, and IV disease after complete surgical resection (61–63). These data are the basis for ongoing randomized phase III studies comparing disease-free and overall survival in patients undergoing treatment with either BCG alone or BCG in combination with an allogeneic MCV (Canvaxin™) in patients with either stage III or stage IV melanoma after complete surgical resection. The accrual goal to the trial in stage III patients was met in Fall, 2004, at which time the study was closed to further accrual. The results of the randomized trial in stage III patients may provide an alternative adjuvant therapy for patients who do not wish to be treated with adjuvant IFN-D-2b, although without data comparing Canvaxin and IFN-D-2b there will be continued debate regarding the standard adjuvant therapy for this patient population.
The correlative laboratory studies that accompanied the MCV trials have provided several intriguing results, which may provide insight into potential contributing factors to the mechanism of action. For example, HLA typing of a subset of 69 patients who underwent therapy with MCV demonstrated a significant correlation between the overall
survival of the patient and the degree of HLA class I phenotype match with the MCV component cell lines (64). Specific HLA subtypes were also correlated with a better (HLA-A25) or poorer (HLA-B35) outcome. Interestingly, a subset analysis performed in stage III patients treated with an allogeneic melanoma cell-lysate vaccine, as part of a randomized Intergroup study, demonstrated significant correlations with improved overall survival and patients with HLA-A2 or HLA-C3 phenotype (65). Finally, patients treated with Canvaxin that develop immune complexes against the tumor-associated glycoprotein, TA90, have prolonged survival compared with patients that had the TA90 immune complexes before initiation of therapy (66).
TUMOR LYSATE VACCINES
An alternative method to the use of whole tumor cells is the development of tumor lysate vaccines. These preparations have the theoretical advantage over whole tumor cell vaccines of eliminating immunosuppressive factors released by tumor cells (such as IL-10 and TGF-E) but retaining tumor-associated antigens that can be processed and presented by professional APCs. Two examples of melanoma lysate vaccines that have been tested in randomized clinical trials are the Vaccinia Melanoma Oncolysate (VMO) vaccine and Melacine. Interestingly, both vaccines were shown to be ineffective in improving survival in the intent-to-treat analysis, but post hoc subset analyses identified subgroups that had improved survival.
The VMO was developed using a live vaccinia virus-augmented allogeneic mela- noma lysate. A phase Ib study conducted in patients with American Joint Committee on Cancer (AJCC) stage III melanoma demonstrated a statistically significant increase in disease-free survival as compared with a matched historical control cohort (67). Labo- ratory studies confirmed a positive correlation between serum titers of antimelanoma immunoglobulin (Ig)G antibodies and disease-free survival. Based on the data, which suggested that VMO may have a protective effect against recurrence in high-risk, sur- gically resected melanoma patients, a phase III randomized double-blind study was conducted in patients with AJCC stage III, node-positive melanoma. Patients were ran- domized to receive either adjuvant VMO or vaccinia and followed for disease-free and overall survival. The interim analysis of 217 patients demonstrated no benefit in disease- free survival in patients treated with either agent (68). However, a post-hoc subset analysis suggested that male patients between the ages of 44 and 57 yr with one to five positive nodes appeared to have a survival benefit from VMO therapy. Two subsequent follow-up reports confirmed this observation, with 18.9, 26.8, and 21.3% improvements in survival at 2, 3, and 5 yr, respectively, in the specific male subset who were treated with VMO as compared with vaccinia alone (69,70). In an attempt to determine whether VMO demonstrated therapeutic efficacy as compared with other adjuvant therapy trials in patients with AJCC stage III melanoma, a statistical analysis was performed compar- ing patients treated with VMO with the survival of patients in the treatment arms of several cooperative group trials of adjuvant IFN-D-2a (71). The results suggested that the survival of patients treated with VMO was comparable to patients treated with adjuvant IFN-D-2a in the ECOG EST 1684, which the authors propose as evidence of therapeutic effect related to treatment with VMO.
A similar sequence of events surrounded a phase III randomized study comparing an allogeneic melanoma lysate (Melacine) to observation in patients with surgically resected, AJCC stage III melanoma (65). Despite a lack of therapeutic efficacy in the
intent-to-treat analysis, a subset analysis of patient survival based on HLA type demon- strated that patients who were either HLA-A2 or HLA-C3 had an improved survival when treated with Melacine as compared with observation, with 5-yr, relapse-free sur- vival of 77 vs 64% (p = 0.004). These particular HLA serotypes were associated with response to Melacine therapy in patients with stage IV melanoma, suggesting that the allogeneic lysate may contain processed tumor antigens that are presented by profes- sional APCs in the context of these HLA (72). Although the therapeutic efficacy of Melacine was not established by the prospective randomized trial, the subset analysis supports the idea that tumor antigens processed and presented as peptides may represent a valid strategy for vaccine development.
VACCINES USING DEFINED ANTIGENS
The discovery of melanoma-associated antigens, such as those in melanoma antigen gene and melanoma antigen recognized by T-cells families, as well as gp100 and tyro- sinase, ushered a new era in active specific immunotherapy using defined peptides that would bind particular MHC motifs (73,74). Preclinical as well as clinical trials of immu- notherapy of melanoma over the past 5 yr have been dominated by vaccine strategies using defined antigens. Whole protein antigens or peptides that bind to specific MHC molecules have been used alone, in combination with adjuvants, or, most commonly, after pulsing with autologous dendritic cells (75). Route of administration of antigen- pulsed dendritic cells has ranged from intradermal, subcutaneous, intravenous, and even intranodal.
Although no phase III randomized studies have been reported using defined-antigen melanoma vaccines, a recent review of vaccine trials for patients with melanoma sum- marizes the current state of the field (76). A review of cancer vaccine clinical trials performed at the Surgery Branch of the National Cancer Institute in 440 patients with metastatic melanoma yielded an objective response rate of only 2.6%. Unfortunately, this response rate was similar to other trials performed in patients with metastatic disease using defined melanoma antigen vaccines. Interestingly, in several of these patients, there was measurable evidence of immunization against the defined antigen, as mea- sured by an increase in the frequency of antigen-specific T-cells in the peripheral blood after vaccine administration (77,78). One hypothesis that the authors present for the presence of antigen-specific T-cells in the face of a lack of melanoma regression is that the cells are not becoming activated at the tumor site to perform their effector function.
Evidence of immune selection within melanomas after antigen-specific vaccine therapy has been reported in 532 melanoma lesions in 204 patients in which the tumor expression of the target antigen was reduced, particularly when the vaccine was administered with systemic IL-2 (79). Thus, although there appears to be clear evidence that antigen- specific T-cells can be generated with defined antigen vaccines, with a subsequent elimination of antigen-expressing melanoma cells, clinical tumor regression has been difficult to achieve.
FUTURE DIRECTIONS
Active specific immunotherapy for melanoma appears to be at a crossroads with respect to the management of patients with melanoma. Whole cell vaccination with allogeneic melanoma cell lines that express a variety of melanoma antigens are currently being tested in the adjuvant setting in patients with surgically resected stage III mela-
noma, the results of which may significantly impact treatment options for patients with melanoma. Newer generations of whole cell vaccines using melanoma cells fused to autologous dendritic cells may provide some improvement over allogeneic vaccines because the melanoma antigens will be presented in the proper MHC context for optimal host T-cell response (80,81). Based on studies that have demonstrated clinical responses in patients with metastatic melanoma after administration of intralesional granulocyte macrophage colony-stimulating factor (GM-CSF) or in combination with a melanoma vaccine (82,83), gene-modified whole cell melanoma vaccines that secrete cytokines, such as GM-CSF or IL-4, are currently under investigation in human clinical trials (84,85). Defined melanoma vaccines have demonstrated the ability to stimulate antigen- specific T-cells in patients with melanoma. However, the observation that the immune response may be too restricted, leading to immune selection and outgrowth of low antigen-expressing melanoma cells, suggests that multiepitope vaccines may provide a better potential for success in future trials.
Adoptive Immunotherapy Using Activated T-Cells
The passive transfer of immunity to the tumor-bearing host by virtue of the adminis- tration of activated immune cells is a method that was initially established in preclinical animal models. The translation of this technique into humans has required technological improvements in the culturing and expansion of cells ex vivo, and has evolved into a process that, although cumbersome, can result in the generation of billions of activated, antigen-specific T-cells. Despite the technical hurdles of cell processing, continued testing in human clinical trials is fueled by the occasional observation of striking regres- sion of metastatic melanoma at visceral sites.
TUMOR-INFILTRATING LYMPHOCYTES
The observation that melanoma lesions undergoing regression were infiltrated by immune cells stimulated interest in isolating these cells to determine their antitumor reactivity. Intense research in this area confirmed that long-term culture of TIL from melanoma lesions in both experimental murine models and humans resulted in the out- growth of T-cells with high avidity for melanoma-associated antigens (86–88). Genera- tion of high numbers of TIL in culture lead to clinical trials of adoptive transfer of these activated cells in combination with systemic IL-2 in patients with metastatic melanoma.
Occasional dramatic clinical responses with regression of bulky metastatic tumor provided proof-of-concept that passive transfer of immunity was achievable in select patients. However, the practical limitations of long-term T-cell culture appeared to outweigh the benefit to a small percentage of patients.
Recent studies have shed some insight into potential methods of improving adoptive immunotherapy using TIL. A phase I study of nonmyeloablative chemotherapy in com- bination with adoptive transfer of autologous melanoma-specific T-cells was devised to induce lymphodepletion before transfer of activated cells. The theoretical premise for this approach was to try to repopulate the tumor-bearing host immune system with the activated transferred cells as the dominant population, as well as to eradicate suppressor T-cells, which might limit the effectiveness of the transferred cells (89,90). Using this treatment strategy, 18 of 35 patients with metastatic melanoma demonstrated an objec- tive clinical response (>50% tumor reduction) (91). Interestingly, the activated trans- ferred cells had high avidity for melanoma antigens but were reactive against several
different antigens. This is in stark comparison to adoptive therapy using T-cell clones that were peptide-specific and derived from peripheral blood of patients who were undergoing defined-antigen vaccine therapy, which showed a marked lack of clinical effectiveness (92).
FUTURE DIRECTIONS: TUMOR-DRAINING LYMPH NODE CELLS
Extensive study in preclinical animal models as well as human clinical trials have demonstrated that lymph nodes draining a progressive subcutaneous tumor contain antigen-sensitized T-cells, which, after activation and expansion in culture with anti- CD3/IL-2, mediate effector function in vivo after adoptive transfer (93). One of the most interesting findings within this body of work was that the subpopulation of T-cells that downregulateL-selectin (CD62L) expression contain the subset of antigen-primed cells that mediate therapeutic antitumor effects after culture activation and adoptive transfer (94). Recent studies in mice suggest that restricted V E T-cell receptor usage within the cells that downregulate L-selectin may correspond to antigen-specific T-cells that are clonally derived (95). Human studies looking at lymph nodes draining a melanoma vaccine site demonstrate that T-cells with L-selectin downregulation are skewed toward a type 1 cytokine secretion pattern predictive of clinical response after adoptive transfer (96). These studies provide the rationale for the use of tumor-draining lymph node cells as a source of T-cells for adoptive immunotherapy protocols of the future.
CONCLUSIONS AND PERSPECTIVES
The observations of natural host immune responses to melanoma-associated antigens has resulted in intense research efforts to develop immunotherapy as a treatment option in these patients. The lessons learned from these studies appear to suggest that generation of broad immune responses, such as that seen with systemic cytokine administration, whole cell vaccine therapy, or adoptive transfer of T-cells with broad reactivity, may be beneficial, in that immune selectivity of antigen-expressing tumor cells is less likely.
Also, vaccine strategies can result in generation of T-cells with antigen-specificity, but the lack of activation of these T-cells at the tumor site may limit the efficacy in settings of bulky metastatic disease. Further understanding of the mechanism of action of immu- notherapy in vivo should provide investigators with a method of balancing the nature of the immune response generated by the treatment to the immune status and the bulk of metastatic disease of the patient. Lessons learned from human clinical trials will un- doubtedly extend the use of immunotherapy of patients with melanoma in the future.
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