"Cancer-germline" genes such as those of the MAGE family are expressed in many tumors and in male germline cells, but are silent in normal tissues. They encode shared tumor-specific antigens, which have been used in therapeutic vaccination trials of cancer patients. MAGE-6 is expressed in more than 70% of metastatic melanomas and more than 50% of carcinomas of the lung, esophagus, bladder, and head and neck. We report here the identification of a new MAGE-6 antigenic peptide, which is recognized by a tumor-specific cytolytic T lymphocyte clone isolated from a melanoma patient. The peptide, ISGGPRISY, corresponds to positions 293 to 301 of the MAGE-6 protein sequence and is presented by HLA-Cw1601 molecules.
This article was published in Cancer Immunity, a Cancer Research Institute journal that ceased publication in 2013 and is now provided online in association with Cancer Immunology Research.
Patient MZ2 was diagnosed with a metastatic melanoma in 1982. A cell line, MZ2-MEL, was established from an adrenal metastasis. After ineffective cytostatic chemotherapy, multiple lymph nodes and visceral metastases were resected in 1982 and 1983. From 1983 onwards, the patient received multiple injections of mutagenized and lethally irradiated autologous melanoma cell clones (1). She has been free of detectable melanoma for eighteen years.
A panel of stable cytolytic T lymphocyte (CTL) clones directed against the autologous MZ2-MEL cells has been obtained by mixed lymphocyte-tumor cell cultures (1, 2). These CTL clones showed specificity for the tumor cells, insofar as they did not lyse autologous fibroblasts, autologous Epstein Barr virus-transformed B cells, or the natural killer target cells K562 (3). They were used to select variants of MZ2-MEL cells that resisted lysis by some of the CTL clones. We concluded from the analysis of these antigen-loss variants that several distinct antigens were recognized on MZ2-MEL cells by autologous CTLs (3). Gene transfection approaches led to the identification of seven of these antigens: MAGE-1 peptides presented by HLA-A1 or Cw16, a MAGE-3 peptide presented by A1, a BAGE-1 peptide presented by Cw16, GAGE peptides presented by A29 or Cw6, and a tyrosinase peptide presented by B44 (4, 5, 6, 7, 8, 9, 10). We report here the identification of an eighth antigen recognized on MZ2-MEL cells by autologous CTLs.
Isolation of an anti-tumor CTL restricted by HLA-Cw*1601
Blood lymphocytes from melanoma patient MZ2 were stimulated with the autologous tumor cell line MZ2-MEL.43 and anti-tumor CTL clones were obtained by limiting dilution of the responder T cell population. Several clones failed to lyse the MZ2-MEL.3.1 subline, which had been passaged in vitro for more than 150 generations (Figures 1 and 2). These CTL clones, including CTL 82/21, were labeled anti-MZ2-B, under the assumption that they were directed against a single antigen lost by MZ2-MEL.3.1 (3). However subsequent analysis indicated that subline MZ2-MEL.3.1 had lost the genes coding for HLA-A29, B4403, and Cw1601 (5) (Figure 1). CTL 82/21 recognized MZ2-MEL.3.1 cells when they were transfected with the autologous HLA-Cw*1601 cDNA (data not shown). This suggested that the antigenic peptide recognized by this CTL clone was presented by Cw1601 molecules.
Identification of the gene encoding the antigenic peptide
A cDNA library prepared with RNA extracted from MZ2-MEL.43, and containing approximately 66,000 inserts, was divided into pools of 200-400 bacteria (7). Plasmid DNA from each pool was transfected together with the Cw*1601 construct into duplicate microcultures of COS-7 cells. The transfected cells were screened for expression of the antigen by adding CTL 82/21 and measuring TNF production. Two positive cDNA pools were identified. One of them was cloned to obtain cDNA GEP3/317/2B7, which transferred the expression of the antigen (Figure 3C). The sequence of this cDNA corresponded to that of MAGE-6.
Identification of the MAGE-6 antigenic peptide
To localize the peptide-coding sequence, we prepared several truncated MAGE-6 cDNA sequences that were cloned into expression plasmids and transfected into COS-7 cells together with the Cw*1601 construct (Figure 4). Transfected cells were tested for their ability to stimulate CTL 82/21. We concluded that the antigenic peptide was encoded by the last 86 nucleotides of the MAGE-6 open reading frame. Peptides encoded in this region were synthesized and tested for recognition by the CTLs (Figures 4 and 5). Nonapeptide ISGGPRISY was the shortest peptide recognized, with half-maximal lysis obtained at 20 nM. Nonapeptide ISGGPRISY corresponds to positions 336 to 344 of the MAGE-6 protein sequence. The homologous MAGE-3 peptide, which differs from the MAGE-6 peptide only at position 6, was not recognized by CTL 82/21 (Figure 5).
The anti-tumor T cell response of patient MZ2 is almost exclusively directed against antigens encoded by "cancer-germline genes". The MAGE, BAGE, GAGE antigenic peptides are presented by HLA-A, B, and C molecules. HLA-C molecules are considered to be expressed at the cell surface at about 10% of the levels of the HLA-A and B molecules and their importance for antigen presentation has been questioned (11). Of note, out of the eight peptides that have been identified with anti-tumor CTLs from patient MZ2, three are presented by HLA-Cw*1601 molecules and a fourth by HLA-Cw*0601. The relevance of HLA-C for antigen presentation should perhaps be reconsidered.
HLA-Cw16 molecules cannot be detected by serology and there is limited information about their population distribution. They seem to be expressed by 7-10% of Caucasians (5, 12, 13). Six alleles have been described, and Cw*1601 represents 90% of the expression. No peptide-binding motif has been proposed yet and only three Cw16-binding peptides have been described: the MAGE-1 peptide SAYGEPRKL, the BAGE-1 peptide AARAVFLAL, and this MAGE-6 peptide ISGGPRISY. The Cw16 binding motif may therefore comprise a hydrophobic residue, L or Y, at the C-terminus, as is the case for many other HLA class I molecules.
MAGE-6 is expressed in 72% of metastatic melanomas, 68% of esophageal carcinomas and more than 50% of carcinomas of the head and neck, lung and bladder (14). Two MAGE-6 peptides presented by HLA class I molecules have been described previously: REPVTKAEML (MAGE-6127-136), which was recognized on B3701 by anti-tumor CTLs isolated from a melanoma patient, and VKISGGPR (MAGE-6290-298), which was recognized on A34 by CTLs infiltrating a spontaneously regressing human primary melanoma (15, 16). These results indicate that some MAGE-6 antigenic peptides are immunogenic in cancer patients and can therefore be used as cancer vaccine targets.
Materials and methods
Cell lines and T cells
Patient MZ2 was typed HLA-A1, A29, B37, B*4403, Cw*0601, Cw*1601. Melanoma cell line MZ2-MEL was derived from an abdominal metastasis of patient MZ2 and a number of sublines were obtained. Subclone MZ2-MEL.3.0 was obtained by limiting dilution (Figure 1). Subline MZ2-MEL.3.1 was obtained by extending the culture of subclone MZ2-MEL.3.0 for more than 150 generations. Subline MZ2-MEL.43 was derived by limiting dilution from MZ2-MEL.3.0 cells that had survived a mutagen treatment (2, 3). Melanoma cell lines were grown as previously described (3, 17). Blood lymphocytes collected in 1984 from patient MZ2 were stimulated with MZ2-MEL.43 cells in standard mixed lymphocyte-tumor cell culture conditions and MZ2-CTL clone 82/21 was obtained by limiting dilution (3). It was grown in conditions similar to those described previously (17). CTL clone 82/21 expresses a TCRBV5-8*01/J2-7*01 rearrangement. Lymphoblastoid cell line MZ2-EBV was derived from patient MZ2 by standard techniques.
The construction of the cDNA library has already been described in Boël et al. (7). Poly-A+ RNA was extracted from MZ2-MEL.43 cells using the FastTrack® mRNA extraction kit (Invitrogen Corp., Oxon, UK). mRNA was converted to cDNA using random primers, ligated to adaptors as described in the SuperScript plasmid system kit (Life Technologies, Gaithersburg, MD, USA), and inserted into the EcoRI site of expression vector pCD-SRalpha (18). Recombinant plasmids were electroporated into E. coli JM101 and selected with ampicillin (50 µg/ml). The library contained 66,000 inserts and was divided into 87 pools of 400 bacteria and 297 pools of 200 bacteria. Each of these pools comprised approximately 280 or 140 different cDNAs respectively, as about 70% of the plasmids carried an insert. Each pool of bacteria was amplified to saturation and plasmid DNA was extracted by the alkaline lysis method.
Transfection of COS-7 cells
Transfection experiments were performed by the DEAE-dextran-chloroquine method (18, 19, 20). Briefly, 1.5x104 COS-7 cells were transfected with 50 ng of plasmid pCD-SRalpha containing an HLA-Cw*1601 cDNA insert, and 50 ng of a pool of the cDNA library or 50 ng of a cDNA clone. The HLA-Cw*1601 cDNA was isolated from a cDNA library prepared with RNA extracted from subline MZ2-MEL.43 (5).
CTL stimulation assay
Transfectants were tested for their ability to stimulate the production of Tumor Necrosis Factor (TNF) by CTLs 24-48 h after transfection (17). Briefly, 1,500 CTLs were added in microwells containing target cells in 100 µl of Iscove medium (Life Technologies) containing 10% human serum and 20 U/ml r-hu-IL2. After 24 h, the supernatant was collected and its TNF content was determined by testing its cytotoxic effect on WEHI-164 clone 13 cells in an MTT colorimetric assay (17, 21, 22).
To identify the region of the MAGE-6 sequence that codes for the antigenic peptide presented by HLA-Cw*1601, we constructed several truncated cDNA clones that were obtained by PCR. The PCR products were inserted in vector pCR3 (Invitrogen, Paisley, UK).
Antigenic peptides and CTL assay
Lyophilized peptides were dissolved first in 1 volume DMSO, and 9 volumes of 10 mM acetic acid were then added. Diluted peptides were stored at -20°C. Lysis of target cells by CTLs was tested by chromium release as previously described (23). In the peptide sensitization assay, target cells were labeled with 51Cr for one hour at 37°C and washed extensively. One thousand target cells were then incubated in 96-well microplates in the presence of various concentrations of peptide for 15 min at room temperature. The CTLs were then added and chromium release was measured after 4 h at 37°C.
We thank Sébastien Loeuille for expert technical assistance, and Mrs. Nathalie Krack for editorial assistance. Valérie Vantomme was supported by the TELEVIE fund (Belgium).
- Received October 17, 2003.
- Accepted November 14, 2003.
- Copyright © 2003 by Pierre van der Bruggen