Heterogeneous T-cell response to MAGE-A10(254-262): high avidity-specific cytolytic T lymphocytes show superior antitumor activity.

MAGE-encoded antigens, which are expressed by tumors of many histological types but not in normal tissues, are suitable candidates for vaccine-based immunotherapy of cancers. Thus far, however, T-cell responses to MAGE antigens have been detected only occasionally in cancer patients. In contrast, by using HLA/peptide fluorescent tetramers, we have observed recently that CD8(+) T cells specific for peptide MAGE-A10(254-262) can be detected frequently in peptide-stimulated peripheral blood mononuclear cells from HLA-A2-expressing melanoma patients and healthy donors. On the basis of these results, antitumoral vaccination trials using peptide MAGE-A10(254-262) have been implemented recently. In the present study, we have characterized MAGE-A10(254-262)-specific CD8(+) T cells in polyclonal cultures and at the clonal level. The results indicate that the repertoire of MAGE-A10(254-262)-specific CD8(+) T cells is diverse both in terms of clonal composition, efficiency of peptide recognition, and tumor-specific lytic activity. Importantly, only CD8(+) T cells able to recognize the antigenic peptide with high efficiency are able to lyse MAGE-A10-expressing tumor cells. Under defined experimental conditions, the tetramer staining intensity exhibited by MAGE-A10(254-262)-specific CD8(+) T cells correlates with efficiency of peptide recognition so that "high" and "low" avidity cells can be separated by FACS. Altogether, the data reported here provide evidence for functional diversity of MAGE-A10(254-262)-specific T cells and will be instrumental for the monitoring of peptide MAGE-A10(254-262)-based clinical trials.

Correlation of tumor-associated antigens MAGE, NY-ESO-1 and P53 expression with clinical and pathological relationships of patients with oral squamous cell carcinoma

Despite advances in the diagnosisand treatment of head and neck cancer,survival rates have not improvedover recent years. New therapeuticstrategies, including immunotherapy,are the subject of extensive research.In several types of tumors, the presenceof tumor infiltrating lymphocytes(TILs), notably CD8+ T cellsand dendritic cells, has been correlatedwith improved prognosis. Moreover,some T cells among TILs havebeen shown to kill tumor cells in vitroupon recognition of tumor-associatedantigens. Tumor associated antigensare expressed in a significant proportionof squamous cell carcinoma ofthe head and neck and apparently mayplay a role in the regulation of cancercell growth notably by inhibition ofp53 protein function in some cancers.The MAGE family CT antigens couldtherefore potentially be used as definedtargets for immunotherapy andtheir study bring new insight in tumorgrowth regulation mechanisms. Between1995 - 2005 54 patients weretreated surgically in our institution forsquamous cell carcinoma of the oralcavity. Patient and clinical data wasobtained from patient files and collectedinto a computerized database.For each patient, paraffin embeddedtumor specimens were retrieved andexpression of MAGE CT antigens,p53, NY-OESO-1 were analyzed byimmunohistochemistry. Results werethen correlated with histopathologicalparameter such as tumor depth,front invasion according to Bryne andboth, local control and disease freesurvival. MAGE-A was expressed in52% of patients. NY-ESO-1 and p53expression was found in 7% and 52%cases respectively. A higher tumordepth was significantly correlatedwith expression of MAGE-Aproteins(p = 0.03). No significant correlationcould be made between the expressionof both p53 andNY-OESO-1 andhistopathological parameters. Expressionof tumor-associated antigendid not seem to impact significantlyon patient prognosis. As does thedemonstration of p53 function inhibitionby CT antigens of MAGE family,our results suggest, that tumor associatedantigens may be implicated in tumorprogression mechanisms. Thishypothesis need further investigationto clarify the relationship betweenhost immune response and local tumorbiology.

Frequent MAGE mutations in human melanoma.

Abstract: Background: Cancer/testis (CT) genes are expressed only in the germ line and certain tumors and are most frequently located on the X-chromosome (the CT-X genes). Amongst the best studied CT-X genes are those encoding several MAGE protein families. The function of MAGE proteins is not well understood, but several have been shown to potentially influence the tumorigenic phenotype. Methodology/Principal Findings: We undertook a mutational analysis of coding regions of four CT-X MAGE genes, MAGEA1, MAGEA4, MAGEC1, MAGEC2 and the ubiquitously expressed MAGEE1 in human melanoma samples. We first examined cell lines established from tumors and matching blood samples from 27 melanoma patients. We found that melanoma cell lines from 37% of patients contained at least one mutated MAGE gene. The frequency of mutations in the coding regions of individual MAGE genes varied from 3.7% for MAGEA1 and MAGEA4 to 14.8% for MAGEC2. We also examined 111 fresh melanoma samples collected from 86 patients. In this case, samples from 32% of the patients exhibited mutations in one or more MAGE genes with the frequency of mutations in individual MAGE genes ranging from 6% in MAGEA1 to 16% in MAGEC1. Significance: These results demonstrate for the first time that the MAGE gene family is frequently mutated in melanoma.

Amino acid identity and/or position determines the proteasomal cleavage of the HLA-A*0201-restricted peptide tumor antigen MAGE-3271-279.

The proteasome plays a crucial role in the proteolytic processing of antigens presented to T cells in the context of major histocompatibility complex class I molecules. However, the rules governing the specificity of cleavage sites are still largely unknown. We have previously shown that a cytolytic T lymphocyte-defined antigenic peptide derived from the MAGE-3 tumor-associated antigen (MAGE-3(271-279), FLWGPRALV in one-letter code) is not presented at the surface of melanoma cell lines expressing the MAGE-3 protein. By using purified proteasome and MAGE-3(271-279) peptides extended at the C terminus by 6 amino acids, we identified predominant cleavages after residues 278 and 280 but no detectable cleavage after residue Val(279), the C terminus of the antigenic peptide. In the present study, we have investigated the influence of Pro(275), Leu(278), and Glu(280) on the proteasomal digestion of MAGE-3(271-285) substituted at these positions. We show that positions 278 and 280 are major proteasomal cleavage sites because they tolerate most amino acid substitutions. In contrast, the peptide bond after Val(279) is a minor cleavage site, influenced by both distal and proximal amino acid residues.

Naturally acquired MAGE-A10- and SSX-2-specific CD8+ T cell responses in patients with hepatocellular carcinoma.

Immunotherapy is being proposed to treat patients with hepatocellular carcinoma (HCC). However, more detailed knowledge on tumor Ag expression and specific immune cells is required for the preparation of highly targeted vaccines. HCC express a variety of tumor-specific Ags, raising the question whether CTL specific for such Ags exist in HCC patients. Indeed, a recent study revealed CTLs specific for two cancer-testis (CT) Ags (MAGE-A1 and MAGE-A3) in tumor infiltrating lymphocytes of HCC patients. Here we assessed the presence of T cells specific for additional CT Ags: MAGE-A10, SSX-2, NY-ESO-1, and LAGE-1, which are naturally immunogenic as demonstrated in HLA-A2(+) melanoma patients. In two of six HLA-A2(+) HCC patients, we found that MAGE-A10- and/or SSX-2-specific CD8(+) T cells naturally responded to the disease, because they were enriched in tumor lesions but not in nontumoral liver. Isolated T cells specifically and strongly killed tumor cells in vitro, providing evidence that these CTL were selected in vivo for high avidity Ag recognition. Therefore, besides melanoma, HCC is the second solid human tumor with clear evidence for in vivo tumor recognition by T cells, providing the rational for specific immunotherapy, based on immunization with CT Ags such as MAGE-A10 and SSX-2.

MAGE-A3 and MAGE-A4 specific CD4(+) T cells in head and neck cancer patients: detection of naturally acquired responses and identification of new epitopes.

Frequent expression of cancer testis antigens (CTA) has been consistently observed in head and neck squamous cell carcinomas (HNSCC). For instance, in 52 HNSCC patients, MAGE-A3 and -A4 CTA were expressed in over 75% of tumors, regardless of the sites of primary tumors such as oral cavity or hypopharynx. Yet, T-cell responses against these CTA in tumor-bearing patients have not been investigated in detail. In this study, we assessed the naturally acquired T-cell response against MAGE-A3 and -A4 in nonvaccinated HNSCC patients. Autologous antigen-presenting cells pulsed with overlapping peptide pools were used to detect and isolate MAGE-A3 and MAGE-A4 specific CD4(+) T cells from healthy donors and seven head and neck cancer patients. CD4(+) T-cell clones were characterized by cytokine secretion. We could detect and isolate MAGE-A3 and MAGE-A4 specific CD4(+) T cells from 7/7 cancer patients analyzed. Moreover, we identified six previously described and three new epitopes for MAGE-A3. Among them, the MAGE-A3(111-125) and MAGE-A3(161-175) epitopes were shown to be naturally processed and presented by DC in association with HLA-DP and DR, respectively. All of the detected MAGE-A4 responses were specific for new helper epitopes. These data suggest that naturally acquired CD4(+) T-cell responses against CT antigens often occur in vivo in HNSCC cancer patients and provide a rationale for the development of active immunotherapeutic approaches in this type of tumor.

Correction: Frequent MAGE Mutations in Human Melanoma.

[This corrects the article on p. e12773 in vol. 5.].

Infrequent expression of the MAGE gene family in uveal melanomas.

It has previously been reported that MAGE-1, -2, -3 and -4 genes are expressed in human cancers including cutaneous melanoma. MAGE-1 and MAGE-3 represent targets for specific immunotherapy because they encode peptide antigens which are recognised by cytotoxic T lymphocytes (CTL) when presented by HLA class I molecules, and pilot clinical trials with these peptides are currently in progress. It is likely that other members of the MAGE gene family may also encode antigens recognised by CTL. Uveal melanomas, like cutaneous melanomas, arise from melanocytes that are derived from the neural crest. To determine if uveal melanoma patients would be suitable for MAGE-peptide immunotherapy, the expression of MAGE-1, -2, -3 and -4 genes was assessed by reverse transcription followed by polymerase chain reaction (RT-PCR) amplification and ethidium bromide staining. Expression of MAGE genes was not detected in any of 27 primary tumours. Either MAGE-1 or MAGE-4 was expressed in only 2 of 26 metastatic samples, but expression of MAGE-2 or -3 was not detected. Our data suggest that, unlike cutaneous melanomas, uveal melanomas may not be suitable candidates for MAGE-peptide immunotherapy.

A nonapeptide encoded by human gene MAGE-1 is recognized on HLA-A1 by cytolytic T lymphocytes directed against tumor antigen MZ2-E.

We have reported the identification of human gene MAGE-1, which directs the expression of an antigen recognized on a melanoma by autologous cytolytic T lymphocytes (CTL). We show here that CTL directed against this antigen, which was named MZ2-E, recognize a nonapeptide encoded by the third exon of gene MAGE-1. The CTL also recognize this peptide when it is presented by mouse cells transfected with an HLA-A1 gene, confirming the association of antigen MZ2-E with the HLA-A1 molecule. Other members of the MAGE gene family do not code for the same peptide, suggesting that only MAGE-1 produces the antigen recognized by the anti-MZ2-E CTL. Our results open the possibility of immunizing HLA-A1 patients whose tumor expresses MAGE-1 either with the antigenic peptide or with autologous antigen-presenting cells pulsed with the peptide.

Synthesis of a radioiodinated photoreactive MAGE-1 peptide derivative and photoaffinity labeling of cell-associated human leukocyte antigen-A1 molecules.

The synthesis of a photoreactive derivative of the human leukocyte antigen-A1 (HLA-A1)-restricted MAGE-1 peptide 161-169 (EADPTGHSY) is described. Using conventional automated solid-phase peptide synthesis, a photoreactive derivative of this peptide was synthesized by replacing histidine-167 with photo-reactive N-beta-4-azidosalicyloyl-L-2,3-diaminopropionic acid. The C-terminal tyrosine was incorporated as phosphotyrosine. This peptide derivative was radioiodinated in the presence of chloramine T. This iodination took place selectively at the photoreactive group, because the phosphate ester prevented tyrosine iodination. Following dephosphorylation with alkaline phosphatase and chromatographic purification, the radiolabeled peptide derivative was incubated with cells expressing HLA-A1 or other HLA molecules. Photoactivation resulted in efficient photoaffinity labeling of HLA-A1. Other HLA molecules or other cellular components were not detectably labeled. This labeling was inhibited by HLA-A1 but not by HLA-A2-binding peptides. This synthesis is generally applicable and can also be adapted to the synthesis of well-defined radiolabeled nonphotoreactive peptide derivatives.

Modulation of proteasomal activity required for the generation of a cytotoxic T lymphocyte-defined peptide derived from the tumor antigen MAGE-3.

We have analyzed the presentation of human histocompatability leukocyte antigen-A*0201-associated tumor peptide antigen MAGE-3271-279 by melanoma cells. We show that specific cytotoxic T lymphocyte (CTL)-recognizing cells transfected with a minigene encoding the preprocessed fragment MAGE-3271-279 failed to recognize cells expressing the full length MAGE-3 protein. Digestion of synthetic peptides extended at the NH2 or COOH terminus of MAGE-3271-279 with purified human proteasome revealed that the generation of the COOH terminus of the antigenic peptide was impaired. Surprisingly, addition of lactacystin to purified proteasome, though partially inhibitory, resulted in the generation of the antigenic peptide. Furthermore, treatment of melanoma cells expressing the MAGE-3 protein with lactacystin resulted in efficient lysis by MAGE-3271-279-specific CTL. We therefore postulate that the generation of antigenic peptides by the proteasome in cells can be modulated by the selective inhibition of certain of its enzymaticactivities.

Analysis of naturally acquired CD4+T-cell responses to MAGE-A3 and MAGE-A4 cancer/testis antigens in patients with resected head and neck squamous cell carcinoma

Despite advances in the medical and surgical treatment of Head and Neck (HN) squamous cell carcinoma (HNSCC), long term survival has remained unchanged in the last 20 years. The obvious limitations of traditional therapeutic options strongly urge the development of novel therapeutic approaches. The molecular cloning of tumor antigens recognized by T lymphocytes in recent years has provided targets for specific immunotherapy. In this regard, frequent expression of Cancer Testis Antigens (CTA) has been repeatedly observed among HN tumors. We analyzed CTA expression in 46 HNSCC patients and found that MAGE-A3 and/or -A4 CTA were positive in over 70% of samples, regardless of the anatomical site of primary tumors in the upper aerodigestive tract. Still, immune responses against these CTA in HNSCC patients have not yet been investigated in detail. In this study we assessed the responsiveness of HNSCC patient's lymphocytes against overlapping peptides spanning the entire MAGE-A3 and -A4 proteins. After depletion of CD4+CD25+ regulatory T cells, and following three rounds of in vitro stimulation with pools of overlapping peptides, peripheral blood mononuclear cells (PBMCs) of HNSCC patients were screened by IFN-g and TNF-a intracellular cytokine staining for reactivity against MAGE-A3 or -A4 derived peptides. Cytokine secreting CD4+ T cells, specific for several peptides, were detected in 7/7 patients. In contrast, only 2/5 PBMC from healthy donors showed weak T cell responses against 2 peptides. CD4+ T cells specific for one epitope MAGE-A3(281-295), previously described as an HLA-DR11 restricted epitope naturally processed and presented by dendritic cells and tumor cells, were detected in two patients. MAGE-A3(161-175) specific CD4+ T cells were found in one patient. Six MAGE-A3 and -A4 new epitopes are being characterized. Together, these data suggest that naturally acquired CD4+ T cell responses against CT antigens occur in vivo in HNSCC patients, providing a rational basis for the use of the identified peptides in vaccination protocols.

Frequent cytolytic T-cell responses to peptide MAGE-A10(254-262) in melanoma.

MAGE genes encode tumor-specific shared antigens that are among the most interesting candidates for cancer vaccines. Despite extensive studies, however, CD8+ T-cell responses to MAGE-derived epitopes have been detected only occasionally in cancer patients, even after vaccination. In contrast with these findings, we report here that HLA-A2 melanoma patients respond frequently to the recently identified peptide MAGE-A10(254-262). Indeed, as assessed by staining with fluorescent HLA-A2/peptide MAGE-A10(254-262) tetramers, CD8+ T cells directed against this peptide were readily detectable in a large proportion of HLA-A2+ melanoma patients. These results provide new insight into the immunogenicity of MAGE antigens and underline the potential usefulness of MAGE-A10 peptide-based cancer vaccines.