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Originally published as JCO Early Release 10.1200/JCO.2005.08.375 on August 1 2005 © 2005 American Society of Clinical Oncology. Tumoral and Immunologic Response After Vaccination of Melanoma Patients With an ALVAC Virus Encoding MAGE Antigens Recognized by T Cells
From the Ludwig Institute for Cancer Research, Brussels Branch; Génétique Cellulaire, Université de Louvain; Centre du Cancer, Cliniques Universitaires Saint-Luc, Brussels, Belgium; Aventis Pasteur, Lyon; Hôtel-Dieu, Centre Hospitalier Universitaire de Nantes; Institut Curie, Paris; Institut Gustave-Roussy, Villejuif; Centre Léon Bérard, Lyon; Institut Paoli-Calmettes, Marseille, France; Ludwig Institute for Cancer Research, Lausanne Branch, Lausanne; Hôpital Cantonal Universitaire, Genève, Switzerland; Leiden University Medical Center, Leiden, the Netherlands; Johannes Gutenberg University, Mainz, Germany; Ludwig Institute for Cancer Research, New York Branch, New York, NY; and Aventis Pasteur, Toronto, Ontario, Canada Address reprint requests to N. van Baren, Ludwig Institute for Cancer Research, 74 avenue Hippocrate, UCL7459, B-1200 Brussels, Belgium; e-mail: nicolas.vanbaren{at}bru.licr.org.
PURPOSE: To evaluate the toxicity, antitumoral effectiveness, and immunogenicity of repeated vaccinations with ALVAC miniMAGE-1/3, a recombinant canarypox virus containing a minigene encoding antigenic peptides MAGE-3168-176 and MAGE-1161-169, which are presented by HLA-A1 and B35 on tumor cells and can be recognized by cytolytic T lymphocytes (CTLs). MATERIALS AND METHODS: The vaccination schedule comprised four sequential injections of the recombinant virus, followed by three booster vaccinations with the MAGE-3168-176 and MAGE-1161-169 peptides. The vaccines were administered, both intradermally and subcutaneously, at 3-week intervals. RESULTS: Forty patients with advanced cancer were treated, including 37 melanoma patients. The vaccines were generally well tolerated with moderate adverse events, consisting mainly of transient inflammatory reactions at the virus injection sites. Among the 30 melanoma patients assessable for tumor response, a partial response was observed in one patient, and disease stabilization in two others. The remaining patients had progressive disease. Among the patients with stable or progressive disease, five showed evidence of tumor regression. A CTL response against the MAGE-3 vaccine antigen was detected in three of four patients with tumor regression, and in only one of 11 patients without regression. CONCLUSION: Repeated vaccination with ALVAC miniMAGE-1/3 is associated with tumor regression and with a detectable CTL response in a minority of melanoma patients. There is a significant correlation between tumor regression and CTL response. The contribution of vaccine-induced CTL in the tumor regression process is discussed in view of the immunologic events that could be analyzed in detail in one patient.
Tumor cells carry antigenic peptides bound to HLA class I molecules that can be recognized by autologous cytolytic T lymphocytes (CTLs). Some of these antigens are absent from normal tissues, and thus constitute safe targets for T cell-mediated immunotherapy of cancer.1 An important category of tumor specific antigens include those encoded by cancer-germline genes such as members of the MAGE, BAGE, GAGE and LAGE-1/NY-ESO-1 gene families. These antigens are expressed by many melanomas, transitional bladder cancers, head and neck squamous cell carcinomas, non–small-cell lung cancers, esophageal cancers, and multiple myelomas.2 Tumor vaccine candidates containing the MAGE-3.A1 antigen, nonapeptide MAGE-3168-176 presented by HLA-A1, have been investigated in small-scale clinical trials. In a pilot study, the synthetic MAGE-3.A1 peptide was administered to 45 HLA-A1 patients with MAGE-3 expressing melanoma, by subcutaneous (SC) and intradermal (ID) injections of 100 or 300 µg of peptide on three occasions at monthly intervals. No significant toxicity was reported. Of the 25 melanoma patients with measurable disease who received all three immunizations, seven displayed tumor regressions. We observed three complete responses, one partial response, and three mixed responses.3 In a phase I/II trial, the recombinant MAGE-3 protein was tested as a vaccine formulation in patients with MAGE-3 expressing cancer, mainly melanoma. The patients received either 30, 100, or 300 µg of the protein, with or without the immunological adjuvants MPL and QS21, repeatedly by intramuscular injection. No severe toxicity was reported. Among 33 assessable melanoma patients, four experienced regressions of metastatic lesions, two partial and two mixed responses. A partial response was also observed in a patient with metastatic bladder cancer.4 The same MAGE-3 protein was administered ID and SC without immunologic adjuvant to 26 patients with metastatic, nonvisceral melanoma. Five regressions, one partial response and four mixed responses, were reported (W.H.J. Kruit et al, unpublished observations). In another clinical study, patients with advanced metastatic melanoma were vaccinated with autologous dendritic cells pulsed with the MAGE-3.A1 peptide administered subcutaneously and intravenously. Six of eleven patients immunized with this vaccine showed tumor regressions, all being mixed responses.5 In our initial vaccination study with the MAGE-3.A1 peptide, we did not observe anti-vaccine CTL responses even in those patients who showed tumor regressions. This indicated the absence of strong CTL responses (ie, responses involving CTL frequency above 10–4 of CD8 T cells, our detection threshold at that time). More recently, a new approach with an improved sensitivity of approximately 8 x 10–7, involving lymphocyte-peptide culture and the use of HLA/peptide tetramers, was used to document a significant increase in cytolytic T lymphocyte precursor (CTLp) frequency in a patient who showed tumor regression following vaccination with the MAGE-3.A1 peptide. This method also showed that the CTL response was monoclonal.6 It was extended to 19 other patients who received this peptide without adjuvant. None had a detectable CTL response, indicating that this vaccine is weakly immunogenic.7 In patients vaccinated with the MAGE-3 protein, sensitive monitoring of T lymphocyte responses with HLA/peptide tetramers was not possible because of the great diversity of antigenic peptides that a protein vaccine can generate. Among other potential vaccine candidates aimed at inducing CTL responses to tumor antigens, viral vector–based approaches offer potential advantages. More specifically, viral antigens are often very immunogenic, as indicated by the strong cellular immune responses that can be observed during human viral diseases. One of the possible reasons for this strong immunogenicity is the fact that viruses are potent activators of innate immunity, which in turn can boost specific immune responses. Cells infected by viruses can express foreign genes that have been inserted into the viral genome. Like endogenous proteins, the foreign gene products can be processed into peptides that are displayed at the cell surface by HLA molecules, allowing primary cellular immune responses to such antigens to be induced by infecting professional antigen-presenting cells with recombinant viruses.8 Among possible vector candidates, avian poxviruses deserve particular attention. They have the ability to infect a wide variety of cell types in various hosts, including mammals, but their replication is restricted to avian cells, which prevents them from causing viral disease in humans. ALVAC is an attenuated canarypox virus that has been extensively tested in animal models.9 Its excellent safety profile and its capacity to induce immune responses in humans have been established in a series of clinical trials.10-15 We report here clinical observations made on 40 patients with advanced cancer who received vaccinations with ALVAC miniMAGE-1/3, a recombinant ALVAC virus that contains a minigene coding for a MAGE-3 and a MAGE-1 antigen. These priming immunizations were followed by booster vaccinations with the two corresponding peptides. We also provide a synthesis of the analysis of the CTL responses of the patients, carried out with a sensitive detection approach.
CTL Recognition of Cells Infected With ALVAC miniMAGE-1/3 Virus Dendritic cells were derived from monocytes isolated from the blood of an HLA-A1 hemochromatosis patient, as described previously.16 They were distributed in microwells at 10,000 cells per well and were infected at increasing multiplicities of infection for 2 hours with either ALVAC miniMAGE-1/3 or a control ALVAC virus expressing ß-galactosidase. Infected cells were then washed and incubated for 20 hours with 3,000 cells of either CTL clone 82/30 or CTL clone 20/38, which recognize the MAGE-1.A1 and MAGE-3.A1 antigens, respectively.17,18 The concentration of interferon (IFN) -  , which is released by activated CTL, was measured in the supernatant by enzyme-linked immunosorbent assay (ELISA).
Vaccine Production Peptides MAGE-1.A1 (amino acid sequence EADPTGHSY) and MAGE-3.A1 (EVDPIGHLY) were synthesized by Multiple Peptide Systems (Sunnyvale, CA) and were provided by Aventis Pasteur in accordance with GMP (batches D01164 and D01165, respectively). They were formulated in solution in phosphate buffered saline, pH 7.4, at a concentration of 600 µg/ml. Vials were kept frozen at –80°C and were thawed just before injection.
Patient Eligibility Criteria
Study Design Patients with a favorable course of the disease were offered the possibility to receive additional vaccinations with the two peptides at decreasing frequency, on a compassionate basis. In addition, a few of these patients received booster vaccinations with peptides and ALVAC for the purpose of analyzing their impact on the anti-vaccine CTL response. These complementary research activities were compliant to national regulations and received prior IRB approval and written informed consent from the patients.
Clinical Evaluation of the Patients Tumor response was defined according to the WHO criteria.21,22 Evaluation took place 4 weeks after the seventh vaccination or at time of study removal when appropriate. Objective responses and disease stabilizations were confirmed at least 4 weeks thereafter. For cutaneous melanoma, mixed responses (ie, regression of some target lesions while others remain stable, progress, or appear simultaneously), although formally classified as stable or progressive disease in the WHO classification, were documented as well. A long-term follow-up of all included patients was realized at regular intervals until death.
Analysis of the Immune Response to the Vaccine Antibody responses to the ALVAC virus and to the MAGE-1 and MAGE-3 proteins were assessed in the serum by ELISA, as described previously.26
A pilot clinical study was performed to evaluate the effect of priming with an ALVAC-MAGE vaccine candidate in patients with advanced malignancy. The ALVAC-MAGE construct coded for two antigenic peptides, MAGE-3168-176 and MAGE-1161-169, which are recognized by T cells on both HLA-A1 and HLA-B35. The MAGE expression cassette is represented schematically in Figure 1. Expression of the MAGE epitopes in infected cells was verified by the fact that ALVAC miniMAGE-1/3 virus rendered infected dendritic cells from an HLA-A1 individual capable of presenting the HLA-A1-restricted MAGE-3168-176 and MAGE-1161-169 epitopes to specific CTL clones (Fig 2). Similar results were obtained with HLA-B35 dendritic cells.27,28
The complete immunization of patients involved four vaccinations with this ALVAC miniMAGE-1/3 virus, followed by three boosting vaccinations with the synthetic peptides MAGE-3168-176 and MAGE-1161-169 without adjuvant, all administered intradermally and subcutaneously at 3-week intervals (Fig 3). These routes of administration were chosen because the skin contains a high density of antigen-presenting cells and lymphatic vessels. The choice of four ALVAC administrations was based on previous experience with an ALVAC-HIV construct, which showed that the rate of anti-HIV CTL responses increased further after the third and fourth vaccination, but remained stable after the fifth.29
Patient Characteristics From September 1999 to December 2001, 40 patients were enrolled on the study. They comprised 18 men and 22 women with age ranging from 28 to 86 years, and with a mean age of 54 years. Thirty-seven patients had stage III or IV cutaneous melanoma, two had stage IV NSCLC and one had stage IV head and neck carcinoma. The main features of the patients are displayed in Table 1. Nine patients were removed from the study before they received the fourth ALVAC vaccination, due to the early death of two individuals, and to rapidly progressing disease for the others. Among the 31 patients who received four ALVAC vaccinations, 12 also completed the three peptide vaccinations.
Toxicity The 40 patients enrolled in the trial received at least one vaccination with ALVAC and were evaluated for safety and toxicity after each vaccination according to the NCIC CTG scale. A total of 146 vaccinations with ALVAC and 46 vaccinations with the two peptides were performed. They were generally well tolerated. No patient was removed from the trial as a result of toxicity. No adverse reactions above grade 3 were reported. The frequency or severity of adverse reactions did not increase with the number of injections. Adverse reactions at the ALVAC injection sites occurred very frequently. They were immediate and usually mild to moderate in intensity. They consisted mainly of extended redness, but edema, induration, and pain were also observed. No instance of skin necrosis was observed. There were no major differences between reactions at intradermal and subcutaneous injection sites, except that intradermal reactions usually appeared earlier, lasted for longer and were slightly less severe. Local inflammation was already noticeable after the first ALVAC injection, and did not vary strongly with subsequent administrations. These observations, coupled to the appearance of occasional flu-like symptoms and a frequent increase in plasma levels of C-reactive protein (data not shown), are consistent with the activation of innate immunity mechanisms. Local adverse reactions at peptide injection sites were rare and mild. All local reactions resolved within days. Grade 1 and 2 systemic reactions reported after ALVAC vaccination occurred frequently, whereas grade 3 reactions were rare (Table 1). These systemic reactions consisted of asthenia (reported after 33% of ALVAC injections), grade 2 or 3 fever (22%), headache (19%), myalgia (16%), arthralgia (14%), nausea (10%), and pain (6%). Systemic reactions after peptide vaccinations were less frequent and less severe. They consisted of asthenia (after 15% of peptide injections), grade 2 fever (15%), pain (10%), headache (7%), myalgia (7%), and arthralgia (7%). This apparent difference between the ALVAC and peptides vaccines might be biased by the early removal of patients with the poorest clinical condition, who presumably have a higher probability of experiencing adverse events. Nineteen serious adverse events (SAEs) were reported in 18 patients (Table 1). Fifteen of these SAEs were considered clearly as unrelated to the study drugs. The four remaining SAEs were considered as possibly or probably related to the ALVAC vaccinations. Patient IGR38 had a prolonged hospitalization following appearance of grade 2 fever 9 hours after his second ALVAC vaccination. The temperature normalized after 24 hours. Patient CP71 had a prolonged hospitalization after dyspnea and fever appeared 2 hours after her first ALVAC vaccination. The symptoms disappeared after 24 hours. The patient received the next three ALVAC injections without subsequent fever. Patient MA38 had his first vaccination delayed because of a sepsis. The fever resolved after two days of antibiotherapy, which was maintained for two additional days. He was vaccinated the next day. Later that day, he experienced tachycardia and anxiety. The next days he developed dyspnea and fever, and died as a result of cardiac failure 4 days after the start of the treatment. No autopsy was performed and the precise cause of death was not determined. The most probable cause was sepsis. Patient LE1 was hospitalized for abdominal pain occurring 10 days after the third peptide vaccination. The pain was attributed to necrosis of abdominal metastatic lesions.
Tumor Responses
Even though they have no evident clinical benefit, mixed responses (ie, the regression of a subset of the metastases in patients with stable or progressive disease) deserve to be reported and described in detail because they may be important to understanding the relevance of the observed immune responses. A mixed response was observed in patients NAP34, CP67, LAU147, LAU624, and NAP33. Figure 5 displays the clinical evolution of the six melanoma patients who showed evidence of tumor regression. One notices that these regressions started several weeks after the onset of treatment and were often slow to proceed. Often, regressing lesions did not shrink simultaneously. In the case of skin metastases, no local inflammation was noticed. These features are similar to those described in previous trials with MAGE peptides or protein.3,4 A brief description of these six patients follows.
Patient EB81, who achieved a partial response, had many cutaneous in-transit metastases on her right leg at the onset of treatment that were progressing in size and number. Approximately 18 lesions were nodular, including some that were ulcerated, whereas the others were completely flat. All were pigmented (Fig 6). By the third ALVAC vaccination, the ulcerated nodules had dried out and flattened. Some large nodules increased in size, but became scabby. No new lesions had appeared. By the second peptide vaccination, flattening of almost all the nodular lesions was noted. A right inguinal adenopathy appeared, but no melanoma cells were observed in a fine-needle aspirate. The size of the adenopathy remained unchanged during 3 months, then increased and it was removed surgically. This lymph node was invaded by a large metastatic nodule, surrounded by a fibrotic shell infiltrated by lymphocytes and melanophages.30 A right inguinal lymph node clearance was performed thereafter. None of the 10 removed lymph nodes was invaded by the tumor. The patient received additional vaccinations with the two peptides at increasing intervals. Some remaining pigmented patches were excised and were found to contain melanophages, but no tumor cells. The melanin tattooing disappeared very slowly. Patient EB81 had no evidence of disease 1 year after treatment onset and has remained disease-free for more than 4 years. During that period, she has received several vaccinations with either the MAGE-3.A1 peptide or the ALVAC virus for the purpose of analyzing their impact on the frequency of anti-vaccine CTL.23
NAP34, one of the two patients who achieved disease stabilization, showed regression of one in-transit metastasis of the leg and stabilization of 11 others following vaccination. This was followed by slow disease progression, then by further regression of most in-transit metastases. A mixed response was seen in four patients with disease progression. In patient CP67, vaccination was associated with regression of regional subcutaneous and lymph node metastases. Eventually, all four skin nodules disappeared completely, but the adenopathy increased in size and new metastases appeared. Patient LAU147 had only one regional subcutaneous metastasis, documented by cytologic examination, present at study entry. This nodule regressed completely during vaccination, but two new metastases appeared, one in the breast and one in the brain, and were removed surgically. New metastases appeared thereafter, and were treated initially by surgery, then by a combination of chemotherapy with temozolomide and vaccination with the MAGE-3.A1 and MAGE-3.A24 peptides associated with the immunologic adjuvant Montanide ISA 51(Seppic, Paris, France). A complete response was obtained with the latter treatments, and has been maintained for more than a year. Patient LAU624 experienced regression of four distant subcutaneous nodules following vaccination, but two distant lymph node and one spleen metastases showed progression. Patient NAP33 had a slow regression of three SC in-transit nodules. Two of them disappeared completely, while the third remained detectable only by ultrasound for many months. Then it increased in size again, and new in-transit nodules appeared.
Immunologic Responses to the Vaccine Antigens The CTL response against the MAGE-3.A1 and MAGE-1.A1 vaccine antigens was evaluated with a sensitive approach which measures CTLp frequencies as low as 8 x 10–7. This approach involves the stimulation of blood lymphocytes repeatedly with the antigen during 3 weeks in microwell plates under limiting dilution conditions, followed by the staining of responder cells with an A1/MAGE-3 or A1/MAGE-1 tetramer, and detection of the positive microcultures by flow cytometry. The CTLp frequency is deduced from the proportion of positive wells. Importantly, individual tetramer-stained CTL are cloned and the lytic activity of the CTL clones is verified to be specific for the MAGE antigens. The T cell receptor genes are sequenced so as to distinguish different clonotypes recognizing the same antigen. A detailed account of this MLPC/tetramer/cloning approach and a description of the MAGE-3.A1 CTL responses in 17 patients from the present trial, selected according to the availability and quality of frozen PBMCs, and including four patients with evidence of tumor regression, have been reported previously.7,23 A synthesis of these results extended to the MAGE-1.A1 antigen is shown in Table 2. Two patients, VUB39 and NAP37, were found to have a pre-existing CTL response against the MAGE-3.A1 antigen. Since this precluded the demonstration of a response triggered by the vaccine candidate, they were excluded from the analysis. In the remaining 15 patients, we observed a significant correlation between anti-MAGE-3.A1 CTL response and the occurrence of tumor regressions: 3 of 4 patients with tumor regression and only 1 of 11 patients without regression mounted a detectable CTL response (Fisher's exact test, P = .033). These responses were detectable in the blood collected after ALVAC vaccination and before peptide vaccination in all positive patients. No anti-MAGE-1.A1 CTLp could be detected in the postimmune blood of 12 HLA-A1 patients, including four with tumor regression.
Antigenic peptides MAGE-3168-176 and MAGE-1161-169 are also presented by HLA-B*3501. Three patients carrying the HLA-B  3501 allele, NAP34, NAP35 and LB2196, were evaluated for their CTL response. No CTL response was detected after four ALVAC vaccinations (data not shown). However, further vaccination of patient NAP34 with the two peptides and additional booster vaccinations with ALVAC were associated with the appearance of an anti-MAGE-1.B35 CTL clone in her blood (data not shown). This weak CTL response might be relevant, because this patient experienced tumor regressions that started late after the onset of the vaccinations and proceeded quite slowly. Patients with B35 alleles other than B3501 were not analyzed, because the appropriate HLA/peptide tetramers were not available.
CTL responses against vaccine-encoded tumor antigens were also assessed in 27 evaluable patients by IFN- ELISA was used to monitor vaccine induced antibody responses against the wild-type ALVAC virus. Consistent with previous reports involving vaccination with ALVAC, an antibody response against the virus was detected in the serum of all patients (data not shown). No antibody response was found against the MAGE-357-219 and MAGE-157-219 polypeptides, which contain the epitopes expressed by the ALVAC-based vaccine candidate (data not shown).
This clinical trial establishes a good safety profile for vaccination with a recombinant ALVAC virus expressing a MAGE-1/3 minigene. The reported adverse reactions were in line with previous clinical reports on the toxicity of various ALVAC recombinants.10-14 Moreover, ALVAC was well tolerated after ID injection, which had not been investigated before in humans. Our study also shows that this vaccine has a weak antitumor effectiveness. Among the 30 melanoma patients who received four ALVAC vaccinations, only one partial response and two stabilized diseases were recorded. The clinical efficacy of the experimental vaccine may be underestimated due to the rather short observation period, because two patients, NAP34 and NAP33, experienced regression of most tumor lesions after their tumor response had been evaluated as stable disease and progressive disease, respectively. In both cases, these regressions persisted for more than two years, in the absence of any other treatment. Our clinical data do not allow to conclude that the ALVAC vaccine candidate is either superior or inferior to other vaccine modalities in terms of anti-tumor effectiveness, as similarly low rates of tumor response were observed with the MAGE-3.A1 peptide and the MAGE-3 protein (W.H.J. Kruit, unpublished observations).3,4 Other vaccine modalities with different tumor antigens have met with similarly low clinical success.31,32 We have considered three principal potential causes for the failure of a cancer vaccine to induce tumor regression. The vaccine might fail to induce a CTL response. The activated CTL might fail to reach the tumor sites. Finally, tumor resistance or local immunosuppression might prevent the anti-vaccine T cells from attacking the tumor cells. As a first step in evaluating these causes for failure, we have engaged in a detailed analysis of the T cell responses in vaccinated patients. We first investigated whether our vaccine candidate had induced anti-vaccine CTL responses, ie, CTL directed at the MAGE-3 and MAGE-1 antigens. Preliminary work had shown that these CTL responses would usually be too low to be evaluated by the usual CTL monitoring techniques such as the commonly performed ELISPOT tests or the direct tetramer and intracellular cytokine assays. Accordingly, the pre- and postimmunization CTL precursor frequencies were measured with a sensitive monitoring approach, involving limiting dilution in vitro restimulation, tetramer analysis, and cloning.23 Using this approach, a CTL response was detected in four of 17 evaluated HLA-A1 patients. With one exception, these CTL responses provided a frequency of CTL precursors in the blood that was lower than 10–5 of CD8 T cells. We conclude that the ALVAC vaccine has a weak capacity to trigger anti-MAGE CTL responses. It is noteworthy that all the observed CTL responses were directed against the MAGE-3.A1 antigen. Our in vitro analysis of the immunogenicity of the viral vaccine candidate showed that both the MAGE-3.A1 and the MAGE-1.A1 antigens were appropriately processed by infected dendritic cells. However, the presenting cells were better recognized by the anti-MAGE-3.A1 than by the anti-MAGE-1.A1 CTL clone, raising the possibility that MAGE-3.A1 peptide was better processed. This may explain the preferential CTL response observed in this trial. Notwithstanding their weakness, the observed CTL responses appear to be significant, because they are correlated with clinical evidence of tumor regression. Three of four evaluated patients who showed evidence of tumor regression had a CTL response, as opposed to only one of 11 patients without regression. For this type of analysis, aimed at generating hypotheses regarding the process of tumoral regression following vaccination as opposed to assessing therapeutic efficacy, we strongly feel that it is important to take into account all instances of observed tumor regression, whether or not they qualify as objective responses. It is noteworthy that there was no correlation between the magnitude of the observed vaccine-induced CTL response and that of the clinical response. NAP33 had an almost undetectable CTL response, but has remained free of active disease for more than 2 years, whereas LAU147 demonstrated major disease progression despite a strong CTL response. For the four detectable CTL responses, analysis of T-cell receptor usage indicated that these responses were monoclonal.7,23 The CTL response that had been observed in a patient vaccinated with the MAGE-3.A1 peptide without adjuvant was also monoclonal.6 On the other hand, in a small series of patients vaccinated with autologous dendritic cells pulsed with the MAGE-3.A1 peptide, polyclonal CTL responses were observed.33 To try to understand the paradox of observing tumor regression in patients with a low level of antivaccine T cells in the blood, we examined in patient EB81 the frequency of these T cells inside various metastases. We felt that a high enrichment of these T cells relative to other T cells might solve the paradox. But little enrichment was observed.30 Accordingly, we considered it unlikely that antivaccine T cells could be the sole specific effectors of the complete rejection of the skin metastases of this patient. We examined whether T cells could be found against other antigens borne by the tumor, and we found them at the remarkably high frequency around 10–3 of blood CD8 T cells (ie, approximately 1,000 times higher than that of the vaccine T cells).34 The same finding was made in four other melanoma patients. Remarkably, these T cells, which were labeled antitumoral as opposed to the antivaccine T cells, were already present at high frequency in all these patients before vaccination. In patient EB81, most of the antitumoral T cells were directed against various antigens encoded by another gene of the MAGE family, namely MAGE-C2. In patient EB81, the anti-tumor T cells showed enormous enrichment in the tumor, with some anti-MAGE-C2 CTL clones amounting to about 5% of the CD8 T cells present in the tumor.30 Similar observations have been made recently on a patient vaccinated with dendritic cells (A. Van Pel, unpublished observations). The presence of tumor-infiltrating lymphocytes (TILs) was reported many years ago by several groups.35-37 Moreover, some of these TILs were shown to be effective, after in vitro amplification, for adoptive transfer T-cell therapy in melanoma patients.38,39 TILs may slow tumor evolution in a number of patients, constituting a partially effective form of "immunosurveillance." Possibly, they may even eliminate some early tumors altogether. But this spontaneous response clearly becomes ineffective at one stage in the patients whose disease progresses. On the basis of our findings, we favor the following scenario for the elimination of the tumor that occasionally follows vaccination. Before vaccination, the tumor and the blood contain a high level of antitumor T cells. These cells have become ineffective, even though they can easily be reawakened by in vitro restimulation with tumor cells in the presence of IL-2. In some patients, vaccination produces CTL that reach the tumor and can resist the local immunosuppression long enough to attack some tumor cells. This results in a focal reversal of the immunosuppressive environment. This in turn enables the reawakening of old antitumor CTL clones or the generation of new antitumor CTL clones. These active antitumor CTLs expand in much larger numbers than the antivaccine CTLs and they are responsible for the elimination of the bulk of the tumor cells. In other terms, the antivaccine CTL serve only as a spark that reignites the bulk of the antitumor T cell response. Our results suggest that this spark generates new antitumor T cells, as new dominant T-cell receptor clonotypes appear after vaccination. Whether some previously present dormant T cells are reactivated remains an open question. The rejection scenario proposed in the preceding paragraphs has several implications. One is that tumor escape due to the selection of tumor variants that have lost the expression of the vaccine antigen may not be a limiting factor for the efficiency of antitumor vaccination, because such antigen-loss variants would still be sensitive to the many CTLs directed against other antigens of the tumor. Admittedly, loss of all HLA expression could have farther-reaching consequences, but these cells ought to be hypersensitive to natural killer cells.40 Another major implication is that vaccination might be combined usefully with treatments that alleviate the local immunosuppression of the tumor. One of many possibilities is to deplete regulatory T cells before vaccination.38 Selective usage of cytokines concomitant to vaccination might be effective also. It is possible that some T-cell clones have functional properties that render them more capable of serving as a spark by resisting the immunosuppressive environment of the tumor. Considering that many of our responses are monoclonal, it may be useful to vaccinate with a larger number of different antigens in order to increase the chances of obtaining a T-cell clone with the optimal functional properties. Finally, the frequency of antivaccine T cells may also be a limiting factor. Accordingly, we will try to vaccinate patients with higher doses of the ALVAC-MAGE vaccine candidate, because our in vitro studies indicated that injection of dendritic cells with higher doses of virus generated a higher CTL activation.
Although all authors completed the disclosure declaration, the following authors or their immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.
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Supported by Aventis Pasteur, Lyon, France Terms in blue are defined in the glossary, found at the end of this issue and online at www.jco.org. Authors' disclosures of potential conflicts of interest are found at the end of this article.
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ABSTRACT
INTRODUCTION
, interferon alfa; DTIC, dacarbazine; CDDP, cisplatin; tum +, tumor cells visible histologically; tum –, tumor cells not visible; MAGE-3 +, expression of gene MAGE-3; MAGE-3 –, no expression of gene MAGE-3; PR, partial response; SD, stable disease; PD, progressive disease; MxR, mixed response.
$100,000 (N/R) Not Required
