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Adoptive T cell therapy has shown significant clinical success for patients with advanced melanoma and other tumors. Further development of T cell therapy requires improved strategies to select effective, yet nonself-reactive, TCRs. In this study, we isolated 10 TCR sequences against four MAGE-C2 (MC2) epitopes from melanoma patients who showed clinical responses following vaccination that were accompanied by significant frequencies of anti-MC2 CD8 T cells in blood and tumor without apparent side effects. We introduced these TCRs into T cells, pretreated tumor cells of different histological origins with the epigenetic drugs azacytidine and valproate, and tested tumor and self-reactivities of these TCRs. Pretreatment of tumor cells upregulated MC2 gene expression and enhanced recognition by T cells. In contrast, a panel of normal cell types did not express MC2 mRNA, and similar pretreatment did not result in recognition by MC2-directed T cells. Interestingly, the expression levels of MC2, but not those of CD80, CD86, or programmed death-ligand 1 or 2, correlated with T cell responsiveness. One of the tested TCRs consistently recognized pretreated MC2(+) cell lines from melanoma, head and neck, bladder, and triple-negative breast cancers but showed no response to MHC-eluted peptides or peptides highly similar to MC2. We conclude that targeting MC2 Ag, combined with epigenetic drug-enhanced antigenicity, allows for significant and tumor-selective T cell responses., (Copyright © 2016 by The American Association of Immunologists, Inc.)

In this Timeline, we describe the characteristics of tumour antigens that are recognized by spontaneous T cell responses in cancer patients and the paths that led to their identification. We explain on what genetic basis most, but not all, of these antigens are tumour specific: that is, present on tumour cells but not on normal cells. We also discuss how strategies that target these tumour-specific antigens can lead either to tumour-specific or to crossreactive T cell responses, which is an issue that has important safety implications in immunotherapy. These safety issues are even more of a concern for strategies targeting antigens that are not known to induce spontaneous T cell responses in patients.

Cancer vaccines have recently been shown to induce some clinical benefits. The relationship between clinical activity and anti-vaccine T cell responses is somewhat controversial. Indeed, in many trials it has been documented that the induction of vaccine-specific T cells exceeds the clinical responses observed. Here, we evaluate immunological and clinical responses in 23 MAGE-A3(+) melanoma patients treated with autologous lymphocytes genetically engineered to express the tumor antigen MAGE-A3 and the viral gene product thymidine kinase of the herpes simplex virus (HSV-TK). HSV-TK was used as safety system in case of adverse events and as tracer antigen to monitor the immune competence of treated patients. The increase of anti-TK and anti-MAGE-A3 T-cells after vaccination was observed in 90 and 27% of patients, respectively. Among 19 patients with measurable disease, we observed a disease control rate of 26.3%, with one objective clinical response, and four durable, stable diseases. Three patients out of five with no evidence of disease (NED) at the time of vaccination remained NED after 73+, 70+ and 50+ months. Notably, we report that only patients experiencing MAGE-A3-specific immune responses showed a clinical benefit. Additionally, we report that responder and non-responder patients activate and expand T cells against the tracer antigen TK in a similar way, suggesting that local rather than systemic immune suppression might be involved in limiting clinically relevant antitumor immune responses., (Copyright © 2012 UICC.)

The ability of T cells that have been genetically engineered to express T-cell receptors (TCRs) directed against tumor antigens to mediate tumor regression has been demonstrated in several clinical trials. These TCRs have primarily targeted HLA-A*0201-restricted TCRs, as approximately 50% of whites, who represent the predominant population of patients who develop melanomas, expresses this HLA class I allele. These therapies could be extended to additional patients through the use of TCRs that target epitopes that are presented by additional class I alleles that are prevalent in this population such as HLA-C*07 and HLA-A*01, which are expressed by approximately 50% and 30% of the patient population respectively. Therefore, 2 TCRs that recognize an epitope of MAGE-A12 in the context of HLA-C*07 and 2 TCRs that recognize an epitope of MAGE-A3 in the context of HLA-A*01 were isolated from tumor-reactive T-cell clones and cloned in a recombinant retroviral expression vector. Comparative studies indicated that one of the 2 MAGE-A3-reactive TCRs and one of the 2 MAGE-A12-reactive TCRs were superior to the additional TCRs in conferring transduced peripheral blood mononuclear cells with the capacity to recognize a broad array of antigen and MHC-positive target cells. These results provide support for the use of these TCRs in cancer adoptive immunotherapy trials.

It is generally thought that dendritic cells (DCs) loaded with full-length tumor antigen could improve immunotherapy by stimulating broad T-cell responses and by allowing treatment irrespective of the patient's human leukocyte antigen (HLA) type. To investigate this, we determined the specificity of T cells from melanoma patients treated with DCs loaded with mRNA encoding a full-length tumor antigen fused to a signal peptide and an HLA class II sorting signal, allowing presentation in HLA class I and II. In delayed-type hypersensitive (DTH)-biopsies and blood, we found functional CD8(+) and CD4(+) T cells recognizing novel treatment-antigen-derived epitopes, presented by several HLA types. Additionally, we identified a CD8(+) response specific for the signal peptide incorporated to elicit presentation by HLA class II and a CD4(+) response specific for the fusion region of the signal peptide and one of the antigens. This demonstrates that the fusion proteins contain newly created immunogenic sequences and provides evidence that ex vivo-generated mRNA-modified DCs can induce effector CD8(+) and CD4(+) T cells from the naive T-cell repertoire of melanoma patients. Thus, this work provides definitive proof that DCs presenting the full antigenic spectrum of tumor antigens can induce T cells specific for novel epitopes and can be administered to patients irrespective of their HLA type.

We have pursued our analysis of a melanoma patient who showed almost complete tumor regression following vaccination with MAGE-A1 and MAGE-A3 antigens. We previously described high frequencies of tumor-specific CTL precursors in blood samples collected after but also before vaccination. A set of CTL clones were derived that recognized antigens different from those of the vaccine. Two of these antigens were peptides encoded by another MAGE gene, MAGE-C2. Here we describe the antigen recognized by another tumor-specific CTL clone. It proved to be a third antigenic peptide encoded by gene MAGE-C2, ASSTLYLVF. It is presented by HLA-B57 molecules and proteasome-dependent. Tumor cells exposed to interferon-gamma (IFN-γ) were better recognized by the anti-MAGE-C2(42-50) CTL clone. This mainly resulted from a better processing of the peptide by the immunoproteasome as compared to the standard proteasome. Mass spectrometric analyses showed that the latter destroyed the antigenic peptide by cleaving between two internal hydrophobic residues. Despite its higher "chymotryptic-like" (posthydrophobic) activity, the immunoproteasome did not cleave at this position, in line with the suggestion that hydrophobic residues immediately downstream from a cleavage site impair cleavage by the immunoproteasome. We previously reported that one of the other MAGE-C2 peptides recognized by CTL from this patient was also better processed by the immunoproteasome. Together, these results support the notion that the tumor regression of this patient was mediated by an antitumor response shaped by IFN-γ and dominated by CTL directed against peptides that are better produced by the immunoproteasome, such as the MAGE-C2 peptides., (Copyright © 2011 UICC.)

T cell-based cancer immunotherapy relies on advancements made over the last 20 years on the molecular mechanisms underlying the antigenicity of tumors. This review focuses on human tumor antigens recognized by T lymphocytes, particularly the reasons why some are tumor-specific but others are not, and on the immunologic adjuvants used in clinical trials on therapeutic vaccination with defined tumor antigens.

A core challenge in cancer immunotherapy is to understand the basis for efficacious vaccine responses in human patients. In previous work we identified a melanoma patient who displayed a low-level antivaccine cytolytic T-cell (CTL) response in blood with tumor regression after vaccination with melanoma antigens (MAGE). Using a genetic approach including T-cell receptor β (TCRβ) cDNA libraries, we found very few antivaccine CTLs in regressing metastases. However, a far greater number of TCRβ sequences were found with several of these corresponding to CTL clones specific for nonvaccine tumor antigens, suggesting that antigen spreading was occurring in regressing metastases. In this study, we found another TCR belonging to tumor-specific CTL enriched in regressing metastases and detectable in blood only after vaccination. We used the TCRβ sequence to detect and clone the desired T cells from tumor-infiltrating lymphocytes isolated from the patient. This CD8 clone specifically lysed autologous melanoma cells and displayed HLA-A2 restriction. Its target antigen was identified as the mitochondrial enzyme caseinolytic protease. The target antigen gene was mutated in the tumor, resulting in production of a neoantigen. Melanoma cell lysis by the CTL was increased by IFN-γ treatment due to preferential processing of the antigenic peptide by the immunoproteasome. These results argue that tumor rejection effectors in the patient were indeed CTL responding to nonvaccine tumor-specific antigens, further supporting our hypothesis. Among such antigens, the mutated antigen we found is the only antigen against which no T cells could be detected before vaccination. We propose that antigen spreading of an antitumor T-cell response to truly tumor-specific antigens contributes decisively to tumor regression., (©2011 AACR.)

Dendritic cell (DC) targeting in vivo has recently been shown to confer strong and protective cytotoxic T lymphocyte (CTL)-based immunity in tumor murine models. Our group has recently demonstrated in preclinical models that the infusion of genetically modified lymphocytes (GMLs) expressing the self/tumor antigen TRP-2 is able to elicit functional TRP-2-specific effectors with antitumor activity by targeting DCs in vivo. Here we have analyzed vaccine- and tumor-specific immune responses of 10 melanoma patients treated with autologous GMLs expressing the cancer germline gene MAGE-A3. Three of 10 patients treated with MAGE-A3-GML showed an increase of circulating anti-MAGE-A3 T cells, and developed skin delayed-type hypersensitivity to MAGE-A3. Interestingly, in 2 of these patients, with progressive and measurable tumors at study entry, anti-MAGE-A3 T cells were detected not only in the blood but also within tumors resected after vaccination. These results demonstrate that the infusion of MAGE-A3-GML elicits antitumor T cells, which are capable of trafficking to inflamed tissues and of infiltrating tumors. Clinical studies on a larger group of patients are needed to evaluate the clinical efficacy of such a strategy.

We identified an antigen recognized on a human non-small-cell lung carcinoma by a cytotoxic T lymphocyte clone derived from autologous tumor-infiltrating lymphocytes. The antigenic peptide is presented by HLA-A2 and is encoded by the CALCA gene, which codes for calcitonin and for the alpha-calcitonin gene-related peptide. The peptide is derived from the carboxy-terminal region of the preprocalcitonin signal peptide and is processed independently of proteasomes and the transporter associated with antigen processing. Processing occurs within the endoplasmic reticulum of all tumoral and normal cells tested, including dendritic cells, and it involves signal peptidase and the aspartic protease, signal peptide peptidase. The CALCA gene is overexpressed in medullary thyroid carcinomas and in several lung carcinomas compared with normal tissues, leading to recognition by the T cell clone. This new epitope is, therefore, a promising candidate for cancer immunotherapy.

Tumor regressions have been observed in a small proportion of melanoma patients vaccinated with a MAGE-A3 peptide presented by HLA-A1, administered as peptide, ALVAC canarypox virus containing a MAGE-A3 minigene, or peptide-pulsed dendritic cells (DC). There was a correlation between tumor regression and the detection of anti-MAGE-3.A1 CTL responses. These responses were monoclonal and often of a very low magnitude after vaccination with peptide or ALVAC, and usually polyclonal and of a higher magnitude after DC vaccination. These results suggested that, at least in some patients, surprisingly few anti-MAGE-3.A1 T-cells could initiate a tumor regression process. To understand the role of these T cells, we carried out a functional analysis of anti-MAGE-3.A1 CTL clones derived from vaccinated patients who displayed tumor regression. The functional avidities of these CTL clones, evaluated in lysis assays, were surprisingly low, suggesting that high avidity was not part of the putative capability of these CTL to trigger tumor rejection. Most anti-MAGE-3.A1 CTL clones obtained after DC vaccination, but not after peptide or ALVAC vaccination, produced interleukin 10. Transcript profiling confirmed these results and indicated that approximately 20 genes, including CD40L, prostaglandin D2 synthase, granzyme K, and granzyme H, were highly differentially expressed between the anti-MAGE-3.A1 CTL clones derived from patients vaccinated with either peptide-ALVAC or peptide-pulsed DC. These results indicate that the modality of vaccination with a tumor-specific antigen influences the differentiation pathway of the antivaccine CD8 T-cells, which may have an effect on their capacity to trigger a tumor rejection response.

Treatment of chondrosarcomas is limited to resection because these tumors are unresponsive to standard adjuvant treatments, such as chemotherapy and radiation. We have previously shown that high-grade chondrosarcomas express unspecified members of the Melanoma Antigen (MAGE) gene family. We show here that FS human chondrosarcoma (FS) cells express MAGE-A3 gene and HLA-A1 molecules. In vitro assays show that a cytolytic T-lymphocyte clone (CTL) specific for a MAGE-A3 peptide presented by HLA-A1 specifically lysed FS chondrosarcoma cells. Addition of antigenic peptide did not increase the susceptibility of FS cells to CTL mediated lysis, suggesting that HLA-A1 expression by the chondrosarcoma cells limited their susceptibility to lysis by the anti-MAGE-A3 CTL clone. Incubation of FS cells with 50 U/mL interferon-gamma increased surface expression of HLA class-I molecules, increased their susceptibility to lysis, and had no effect on MAGE-A3 gene expression. These results suggest that immunotherapy targeted against chondrosarcoma cells is possible., ((c) 2007 Orthopaedic Research Society.)

Gene MAGE-A3 encodes tumor-specific antigenic peptides recognized by T cells on many tumors. MAGE-A3 peptides presented by HLA class I molecules have been identified using CD8 lymphocytes stimulated with cells that either expressed gene MAGE-A3 or were pulsed with candidate peptides. One antigen identified with the latter method is peptide MAGE-A3(195-203) IMPKAGLLI, presented by HLA-A24 molecules. It has been used to vaccinate advanced cancer patients. Here, we have used HLA/peptide tetramers to detect T cells recognizing this peptide. Their frequency was estimated to be 2 x 10(-8) of the blood CD8 cells in non-cancerous HLA-A24(+) individuals, which is tenfold lower than the reported frequencies of T cells against other MAGE peptides. In the blood of a patient vaccinated with MAGE-A3, the estimated frequency was 5 x 10(-7). Anti-MAGE-3.A24 cytolytic T cell clones were derived, that lysed peptide-pulsed cells with half-maximal effect at the low concentration of 500 pM. However, these CTL did not recognize a panel of HLA-A24(+) tumor cells that expressed MAGE-A3 at levels similar to those found in HLA-A1(+) tumor cells recognized by anti-MAGE-3.A1 CTLs. Furthermore, 293-EBNA cells transfected with MAGE-A3 and HLA-A24 constructs were hardly recognized by the anti-MAGE-3.A24 CTL clones. These results suggest that peptide MAGE-A3(195-203) is poorly processed and is not an appropriate target for cancer immunotherapy.

Cancer-germline genes (CGGs) code for immunogenic antigens that are present on various human tumors but not on normal tissues. The importance of CGGs in cancer immunotherapy has led to detailed studies of their expression in a range of human tumors. We measured the levels of expression of 12 CGGs in various pediatric solid tumors to identify targets for therapeutic cancer vaccines. Quantitative real-time PCR (qPCR) was used to measure the expression of 8 MAGE genes and of genes LAGE-2/NY-ESO-1 and GAGE-1, 2, 8 in 9 osteosarcomas, 10 neuroblastomas, 12 rhabdomyosarcomas and 18 Ewing's sarcomas. Nine tumors were also examined by immunohistochemistry with monoclonal antibodies specific for the MAGE-A1, MAGE-A4 and NY-ESO-1 proteins. All osteosarcoma and 80% of neuroblastoma samples expressed several CGGs at high levels. Six of 12 rhabdomyosarcomas and 11 of 18 Ewing's sarcomas expressed at least one CGG. Immunohistochemistry data correlated well with qPCR results and showed a homogeneous protein distribution pattern in most positive tumors. No correlation was found between the levels of CGG expression in the tumors and clinicopathological parameters of the patients. Pediatric solid tumors express several CGGs, which encode antigens that could be targeted in therapeutic vaccination trials. Several CGGs of the MAGE, GAGE and LAGE families are coexpressed in a large proportion of osteosarcoma and neuroblastoma samples. Some rhabdomyosarcomas express several of these genes at high levels. Ewing's sarcomas have an overall low CGG expression.

We will explain why major histocompatibility complex (MHC) molecules presenting peptides derived from tumour-associated antigens can be recognized not only by T cell receptors (TCR), but also by soluble proteins endowed with TCR-like reactivity. To understand how an antibody can display high affinity and specificity for a particular MHC:peptide complex, we have employed X-ray crystallography to determine the structure of a recombinant antibody, Hyb3, bound to human HLA-A1 molecules presenting the peptide EADPTGHSY that is derived from the tumour-associated antigen MAGE-Al. The results indicate that although Hyb3 recgonizes its target in a TCR-like diagonal binding mode, important differences between the two types of proteins exist that are probably due to the fact that TCR are part of a molecular assembly on the surface of effector cells, while antibodies such as Hyb3 have to carry out their function as individual molecules.

We have identified an antigen recognized by cytolytic T lymphocytes (CTL) on the autologous tumor cells of a nonsmall cell lung cancer patient. The antigenic peptide, presented by HLA-B*5201 molecules, was encoded by a mutated sequence in the gene coding for the C subunit of transcription factor NF-Y. The mutation was present in the tumor sample from which the cell line was derived, and appeared to be unique to the tumor of this patient. In a lymph node draining the tumor, precursors of CTL recognizing the autologous tumor cells were detected at a frequency of about 1/30,000 of the CD8 cells, and 85% of them recognized the mutated NF-YC peptide, suggesting that the patient mounted a T cell response against this antigen. These results strengthened the notion that unique tumor-specific antigens are highly represented not only in melanoma but also in other types of tumors, like nonsmall cell lung cancer., (Copyright (c) 2005 Wiley-Liss, Inc.)

We describe an HLA-A1 melanoma patient who has mounted a spontaneous cytolytic T cell (CTL) response against an antigenic peptide encoded by gene MAGE-A3 and presented by HLA-A1. The frequency of anti-MAGE-3.A1 CTLp was 5 x 10(-7) of the blood CD8 cells, with a dominant clonotype which was present in six out of seven independent anti-MAGE-3.A1 CTL clones. After vaccination with a recombinant poxvirus coding for the MAGE-3.A1 antigen, the blood frequency of anti-MAGE-3.A1 CTLp increased tenfold. Twenty-two independent CTL clones were derived. Surprisingly, only one of them corresponded to the dominant clonotype present before vaccination. Two new clonotypes were repeated 12 and 7 times, respectively. Our interpretation of these results is that the spontaneous anti-MAGE-3.A1 CTL response pre-existing to vaccination was polyclonal, and that the vaccine restimulated only some of these clones. To estimate the incidence of spontaneous anti-MAGE-3.A1 CTL responses in melanoma patients with a tumor expressing gene MAGE-A3, we measured the blood frequency of anti-MAGE-3.A1 T cells in 45 patients, and found only two clear responses.

In recent years, several clinical trials have involved the vaccination of cancer patients with tumor-specific antigens that are recognized by T lymphocytes. Anti-vaccine T-cell responses in these patients have been monitored on the assumption that their magnitude would correlate with clinical efficacy. Although analysis of these data show that such a correlation is emerging, detailed analyses of the few patients who benefit clinically from the vaccinations suggest that the function of the anti-vaccine T cells might be more important than their number. Recent studies show that in cancer patients numerous tumor-specific T cells appear to be quiescent in the presence of the tumor. Understanding how an efficient vaccine interferes with this coexistence is one of the current challenges of cancer immunotherapy.

After vaccination of melanoma patients with MAGE antigens, we observed that even in the few patients showing tumor regression, the frequency of anti-vaccine T cells in the blood was often either undetectable or <10(-5) of CD8 T cells. This frequency being arguably too low for these cells to be sole effectors of rejection, we reexamined the contribution of T cells recognizing other tumor antigens. The presence of such antitumor T cells in melanoma patients has been widely reported. To begin assessing their contribution to vaccine-induced rejection, we evaluated their blood frequency in five vaccinated patients. The antitumor cytotoxic T lymphocyte (CTL) precursors ranged from 10(-4) to 3 x 10(-3), which is 10-10,000 times higher than the anti-vaccine CTL in the same patient. High frequencies were also observed before vaccination. In a patient showing nearly complete regression after vaccination with a MAGE-3 antigen, we observed a remarkably focused antitumoral response. A majority of CTL precursors (CTLp's) recognized antigens encoded by MAGE-C2, another cancer-germline gene. Others recognized gp100 antigens. CTLp's recognizing MAGE-C2 and gp100 antigens were already present before vaccination, but new clonotypes appeared afterwards. These results suggest that a spontaneous antitumor T cell response, which has become ineffective, can be reawakened by vaccination and contribute to tumor rejection. This notion is reinforced by the frequencies of anti-vaccine and antitumor CTLs observed inside metastases, as presented by Lurquin et al. (Lurquin, C., B. Lethe, V. Corbiere, I. Theate, N. van Baren, P.G. Coulie, and T. Boon. 2004. J. Exp. Med. 201:249-257).

Melanoma patients have high frequencies of T cells directed against antigens of their tumor. The frequency of these antitumor T cells in the blood is usually well above that of the anti-vaccine T cells observed after vaccination with tumor antigens. In a patient vaccinated with a MAGE-3 antigen presented by HLA-A1, we measured the frequencies of anti-vaccine and antitumor T cells in several metastases to evaluate their respective potential contribution to tumor rejection. The frequency of anti-MAGE-3.A1 T cells was 1.5 x 10(-5) of CD8 T cells in an invaded lymph node, sixfold higher than in the blood. An antitumor cytotoxic T lymphocyte (CTL) recognizing a MAGE-C2 antigen showed a much higher enrichment with a frequency of approximately 10%, 1,000 times higher than its blood frequency. Several other antitumor T clonotypes had frequencies >1%. Similar findings were made on a regressing cutaneous metastasis. Thus, antitumor T cells were approximately 10,000 times more frequent than anti-vaccine T cells inside metastases, representing the majority of T cells present there. This suggests that the anti-vaccine CTLs are not the effectors that kill the bulk of the tumor cells, but that their interaction with the tumor generates conditions enabling the stimulation of large numbers of antitumor CTLs that proceed to destroy the tumor cells. Naive T cells appear to be stimulated in the course of this process as new antitumor clonotypes arise after vaccination.

We have identified 2 antigens recognized by several melanoma-specific cytolytic T lymphocyte clones isolated from a melanoma patient with a clinical history of tumor regression after immunotherapy. Both antigens are presented by HLA-A2 and encoded by gene MAGE-C2, a cancer-germline gene shown previously to be silent in normal somatic tissues and expressed in 40% of melanomas and in other tumor types. One antigen corresponds to peptide ALKDVEERV(336-344), whereas the other corresponds to peptide LLFGLALIEV(191-200). The CTL clones recognizing these 2 peptides also recognized allogeneic tumor cell lines expressing MAGE-C2 and HLA-A2. These 2 new peptides are the first known MAGE-C antigens and represent promising targets for cancer immunotherapy., (Copyright 2004 Wiley-Liss, Inc.)

Vaccination with mature, monocyte-derived dendritic cells (DC) pulsed with the MAGE-3(168-176) peptide, which is presented by HLA-A1, has been reported to induce tumor regressions and CTL in some advanced stage IV melanoma patients. We present here a precise evaluation of the level of some of these anti-MAGE-3.A1 CTL responses and an analysis of their clonal diversity. Blood lymphocytes were stimulated with the MAGE-3.A1 peptide under limiting dilution conditions and assayed with an A1/MAGE-3 tetramer. This was followed by the cloning of the tetramer-positive cells and by TCR sequence analysis of the CTL clones that lysed targets expressing MAGE-3.A1. We also used direct ex vivo tetramer staining of CD8 cells, sorting, and cloning of the positive cells. In three patients who showed regression of some of their metastases after vaccination, CTL responses were observed with frequencies ranging from 7 x 10(-6) to 9 x 10(-4) of CD8(+) blood T lymphocytes, representing an increase of 20- to 400-fold of the frequencies found before immunization. A fourth patient showed neither tumor regression nor an anti-MAGE-3.A1 CTL response. In each of the responses, several CTL clones were amplified. This polyclonality contrasts with the monoclonality of the CTL responses observed in patients vaccinated with MAGE-3.A1 peptide or with an ALVAC recombinant virus coding for this antigenic peptide.

We have analyzed the T cell responses of HLA-A1 metastatic melanoma patients with detectable disease, following vaccination with a recombinant ALVAC virus, which bears short MAGE-1 and MAGE-3 sequences coding for antigenic peptides presented by HLA-A1. To evaluate the anti-MAGE CTL responses, we resorted to antigenic stimulation of blood lymphocytes under limiting dilution conditions, followed by tetramer analysis and cloning of the tetramer-positive cells. The clones were tested for their specific lytic ability and their TCR sequences were obtained. Four patients who showed tumor regression were analyzed, and an anti-MAGE-3.A1 CTL response was observed in three of these patients. Postvaccination frequencies of anti-MAGE-3.A1 CTL were 3 x 10(-6), 3 x 10(-3), and 3 x 10(-7) of the blood CD8 T cells, respectively. These three responses were monoclonal. No anti-MAGE-1.A1 CTL response was observed. These results indicate that, like peptide immunization, ALVAC immunization produces monoclonal responses. They also suggest that low-level antivaccine CTL responses can initiate a tumor regression process. Taken together, our analysis of anti-MAGE-3.A1 T cell responses following peptide or ALVAC vaccination shows a degree of correlation between CTL response and tumor regression, but firm conclusions will require larger numbers.

'Cancer-germline' genes such as the MAGE gene family are expressed in many tumors and in male germline cells but not in normal tissues. They encode shared tumor-specific antigens, which have been used in therapeutic vaccination trials of metastatic melanoma patients. To establish whether there is a correlation between tumoral regressions and T-cell responses against the vaccine antigen, we evaluated the responses of patients vaccinated with a MAGE-3 antigenic peptide or a recombinant virus coding for the peptide. Blood lymphocytes were stimulated with antigenic peptide followed by detection with tetramer, T-cell cloning, and TCR analysis. In 4/9 regressor patients and in 1/14 progressors we found a low level, usually monoclonal cytolytic T lymphocyte response against the MAGE-3 peptide.

We have identified a gene encoding an antigen recognized by cytolytic T lymphocytes on the autologous tumor cells of a melanoma patient, AVL3. The gene shows homologies with members of the HERV-K family of human endogenous retroviruses, and it was provisionally named HERV-K-MEL. It contains many mutations that disrupt the open reading frames coding for all of the viral proteins. The HERV-K-MEL gene is not expressed in normal tissues with the exception of testis and some skin samples. It is expressed in most samples of cutaneous and ocular melanoma. It is also expressed in a majority of naevi and in a minority of carcinomas and sarcomas. The antigenic peptide, presented by HLA-A2 molecules, is encoded by a very short open reading frame present in the env region of a spliced HERV-K-MEL transcript. Anti-HERV.A2 CTLp could not be detected in the blood of three individuals without cancer but were present at a frequency of 3 x 10(-5) among blood CD8 T cells in patient AVL3 and 6 x 10(-7) in another HLA-A2 melanoma patient whose tumor expressed HERV-K-MEL. Anti-HERV.A2 CTL clones derived from each patient lysed melanoma cells. Analysis of T-cell receptor beta chain sequences indicated that the anti-HERV.A2 CTL population was oligoclonal in patient AVL3 and probably monoclonal in the other patient. These results suggest that HERV-K-MEL is a source of antigens that are targeted by CTLs in melanoma patients and could therefore be used for vaccination.