Identification of a promiscuous T-cell epitope encoded by multiple members of the MAGE family.

One of the major limitations of tumor-specific vaccination is the generation of antigen-loss variants that are able to escape the immune response elicited by a monoantigenic peptide epitope. Here, we report the identification of a new HLA-B*3701-restricted epitope shared by four different members of the MAGE family. Peripheral blood lymphocytes isolated from a melanoma patient were stimulated in vitro with the autologous HLA-negative melanoma line transfected with autologous HLA B*3701 molecule. This protocol led to the induction of tumor-specific, B*3701-restricted CTLs specific for a peptide epitope encoded by codons 127-136 of the gene MAGE-1. The same epitope is also encoded by the homologous region of three other members of the MAGE family, MAGE-2, -3, and -6. Consistent with the notion that the peptide encoded by MAGE-1 codons 127-136 is, indeed, processed from the proteins encoded by all four MAGE family members, the CTLs were able to specifically recognize Cos-7 cells cotransfected with HLA-B*3701 and any of these MAGE genes. Moreover, the CTLs also recognized a MAGE-6-positive melanoma line transfected with the B*3701 molecule. These findings allow the inclusion of a new set of tumor patients into clinical cancer vaccination trials. Furthermore, they suggest that some promiscuous peptide epitopes shared by different members of the MAGE family might be less prone to escape the immune response by generation of MAGE antigen loss variants.

Generation of tumour-specific cytotoxic T-cell clones from histocompatibility leucocyte antigen-identical siblings of patients with melanoma.

Lymphodepletion and infusion of autologous expanded tumour-infiltrating lymphocytes is effective therapy for patients with malignant melanoma. Antitumour responses are likely to be mediated by HLA class I- and II-restricted immune responses directed at tumour antigens. We assessed whether the peripheral blood of normal HLA-matched siblings of patients with melanoma could be used to generate lymphocytes with antimelanoma activity for adoptive immunotherapy after allogeneic blood or marrow transplantation. Melanoma cell lines were derived from two donors and were used to stimulate the mononuclear cells of three HLA-identical siblings. CD4(+) clones dominated cultures. Of these, approximately half were directly cytotoxic towards recipient melanoma cells and secreted interferon-gamma in response to tumour stimulation. More than half of the noncytotoxic clones also secreted interferon-gamma after melanoma stimulation. No CD4(+) clones responded to stimulation with recipient haemopoietic cells. The majority of CD8(+) clones directly lysed recipient melanoma, but did not persist in long-term culture in vitro. No crossreactivity with recipient haemopoietic cells was observed. The antigenic target of one CD4(+) clone was determined to be an HLA-DR11-restricted MAGE-3 epitope. Antigenic targets of the remaining clones were not elucidated, but appeared to be restricted through a non-HLA-DR class II molecule. We conclude that the blood of allogeneic HLA-matched sibling donors contains melanoma-reactive lymphocyte precursors directed at tumour-associated antigens. Adoptive immunotherapy with unselected or ex vivo-stimulated donor lymphocytes after allogeneic stem cell transplantation has a rational basis for the treatment of malignant melanoma.

Multiple HLA-A alleles can present an immunodominant peptide of the human melanoma antigen Melan-A/MART-1 to a peptide-specific HLA-A*0201+ cytotoxic T cell line.

The majority of HLA-A*0201-restricted tumor-infiltrating lymphocytes from melanoma patients recognize a peptide, MT(27-35), derived from the Melan-A/MART-1 Ag. This study reports that six variants of HLA-A2 and the HLA-A28 subtype A*6901 can present peptide MT(27-35). A CTL line specific for peptide MT(27-35) was generated by in vitro stimulation of PBL of an HLA-A*0201+, healthy donor with peptide-pulsed, activated autologous B lymphoblasts. This CTL line was shown to recognize peptide MT(27-35) after endogenous processing on Melan-A/MART-1+/HLA-A2+ tumor cells. Moreover, a panel of B lymphoblastoid cell lines (BLCLs) expressing A*0202, A*0204, A*0205, A*0206, A*0209, and with lower efficiency A*6901, could be sensitized to lysis upon incubation with the relevant peptide. As demonstrated by the levels of ED50 and CD8 dependency of lysis, HLA-A*0204 and HLA-A*0205 presented the peptide as efficiently as HLA-A*0201, while the other four alleles were less efficient. Peptide-binding studies suggest that TCR- rather than peptide-binding affinity determines the T cell recognition levels of peptide-pulsed EBV-BLCLs expressing A*0201, A*0204, A*0206, and A*0209. Peptide-pulsed BLCLs expressing HLA-A*0207 or two additional subtypes of HLA-A28 were not recognized. MT(27-35)-specific CTL could also be raised from donors expressing HLA-A*0205. These findings have implications on the applicability of peptide vaccination with peptide MT(27-35) on melanoma patients.

Functional heterogeneity of HLA-A*02 subtypes revealed by presentation of a MAGE-3-encoded peptide to cytotoxic T cell clones.

The peptide-binding and presentation characteristics of seven naturally occurring HLA-A2 subtypes were studied using M3(271), a peptide derived from the tumor-specific Ag encoded by gene MAGE-3, which has been shown to be processed and presented by A*0201+ melanoma lines. Three independent M3(271)-specific CTL clones were obtained from two unrelated A*0201+ donors. B lymphoblastoid cell lines (BLCLs) expressing A*0201, A*0207, or A*0209 could be sensitized to lysis by all three clones upon incubation with the relevant peptide. Furthermore, the same BLCLs were able to present endogenous M3(271) in IFN-gamma release assays. These findings demonstrate, for the first time, the existence of a functional overlap between A*0207 and other A*02 subtypes. One of the CTL clones also lysed M3(271)-pulsed BLCLs expressing A*0204 and A*0206, while the other two clones recognized M3(271) only in the context of either of these two subtypes. Peptide-pulsed BLCLs expressing A*0202 or A*0205 were not lysed, although A*0205 and, with lower affinity, A*0202 molecules were shown to bind peptide M3(271). These findings have implications for the selection of cancer patients for specific immunotherapy with peptide M3(271).

Dendritic cells acquire the MAGE-3 human tumor antigen from apoptotic cells and induce a class I-restricted T cell response.

In an attempt to transduce monocyte-derived dendritic cells (DCs) with a retroviral vector coding for an intracytoplasmic tumor antigen (TAA), we were confronted by the evident dissociation between the ability of the treated DCs to induce a TAA-specific response, and the presence of integrated vector proviral DNA. The TAA, i.e., MAGE-3, was acquired by DCs and presented to immune effectors, thanks to the property of DCs to uptake the apoptotic bodies released by the irradiated vector-producing cells. Indeed, we observed that upon irradiation vector-producing cells underwent apoptotic cell death, monitored by annexin V and propidium iodide staining, and were phagocytosed by DCs. Lymphocytes obtained from a patient affected by a MAGE-3(+) melanoma, were stimulated in vitro with autologous DCs previously exposed to irradiated MAGE-3-expressing cells. This procedure led to the induction of MAGE-3-specific cytotoxic effectors, directed against a yet unknown MAGE-3 epitope presented by HLA-A*B5201 molecules. These data demonstrate that DCs can present engulfed human TAAs, thus providing strategies for cancer vaccination.

Rexpression of HLA class I antigens and restoration of antigen-specific CTL response in melanoma cells following 5-aza-2'-deoxycytidine treatment.

Cell surface expression of HLA class I/peptide complexes on tumor cells is a key step in the generation of T-cell-based immune responses. Several genetic defects underlying the lack of HLA class I expression have been characterized. Here we describe another molecular mechanism that accounts for the complete absence of HLA class I molecule expression in a tumor line (MSR3-mel) derived from a melanoma patient. Hypermethylation of the MSR3-mel DNA, specifically of HLA-A and -B genes, was identified, which resulted in loss of HLA class I heavy chain transcription. Treatment of MSR3-mel cells with the demethylating agent 5'-aza-2'-deoxycytidine (DAC) allowed HLA-A and -B transcription, restoring cell surface expression of HLA class I antigens and tumor cell recognition by MAGE-specific cytotoxic T lymphocytes. The MSR3-mel line was obtained from a metastatic lesion of a nonresponding patient undergoing MAGE-3.A1 T-cell-based peptide immunotherapy. It is tempting to speculate that the hypermethylation-induced lack of HLA class I expression is the cause of the impaired response to vaccination. This study provides the first evidence that DNA hypermethylation is used by human neoplastic cells to switch off HLA class I genes, thus providing a new route of escape from immune recognition.

Characterization of antigenic peptides presented by HLA-B44 molecules on tumor cells expressing the gene MAGE-3.

The amino acid sequence of the protein encoded by the gene MAGE-3 was screened for peptides containing the binding motif for HLA-B44. Nine peptides were synthesized, and their binding affinity for HLA-B*4402 and -B*4403 was analyzed in an HLA class I alpha-chain refolding assay. Four peptides with binding affinity for HLA-B*4403 were chosen for in vitro cytotoxic T-lymphocyte induction assays using as antigen-presenting cells peptide-pulsed, autologous activated B lymphoblasts from a healthy, B*4403+ donor. Peptide-specific effectors could be raised only against one peptide, M3-167. Cytotoxic T lymphocytes specific for this peptide were also able to recognize melanoma cell lines expressing HLA-B44 and the gene MAGE-3, strongly suggesting that M3-167 is a naturally processed MAGE-3-encoded epitope presented by HLA-B44. M3-167 is a I amino acid N-terminal extension of M3-168, a naturally processed epitope MAGE-3-encoded epitope presented by HLA-A1 that has been previously described. TAP binding studies of these 2 peptides revealed that the TAP affinity of M3-167 is about 9-fold higher than that of M3-168. M3-167 or a longer precursor could be transported into the endoplasmatic reticulum, where it could be trimmed for presentation by HLA-A1 or -B44 molecules. Taken together, our data suggest that M3-167 could be an immunodominant peptide encoded by the gene MAGE-3.