Monoclonal antibodies against recombinant-MAGE-1 protein identify a cross-reacting 72-kDa antigen which is co-expressed with MAGE-1 protein in melanoma cells

Abstract

The MAGE-I gene codes for tumor-associated peptides recognized by cytolytic T lymphocytes in association with MHCclass-I molecules such as HLA-AI and HLA-Cw16. In the course of a study aiming at the immunohistochemical detection of the MACE-I gene product in tumor samples, 2 mouse monoclonal antibodies (MAbs) directed against a full-length recombinant MAGE-I fusion protein were found to react strongly not only with the 46-kDa MAGE-I protein, but also with a 72-kDa product in immunoblots of lysates obtained from several MAGE-I -mRNA-positive melanoma cell lines. Pre-incubation of the antibodies with the recombinant MAGE-I fusion protein abolished their reactivity both with MAGE-I protein and with the 72-kDa product, thus confirming the occurrence of antigenic determinant@) shared by the 2 proteins. The 72-kDa protein is not an alternative product of MACE-I, since it was still detected in lysates of a MACE-I loss variant derived from a MAGE-Ipositive melanoma cell line. Moreover, the 72-kDa protein does not appear to be a product of the other members of the MACE gene family known to be expressed in tumors (such as MACE-I, -3, -4 and -12). Interestingly, expression of the 72-kDa protein was found to be correlated with that of MAGE-I protein. Thus, in 30 tumor cell lines analyzed by immunoblotting and RT-PCR, the 72-kDa protein was never detected in MAGE-I-mRNAnegative cell lines, while it was co-expressed with MAGE-I protein in I 2 out of I 5 cell lines expressing MAGE-I. Furthermore, the 72-kDa protein was detected in lysates of human testis, the only normal tissue known to express MAGE-I. Finally, treatment of MAGE-I -mRNA-negative cell lines with 5-Aza-2'-deoxycytidine, a hypomethylating agent known to induce MAGE-I expression, resulted in the expression of the 72-kDa protein. Taken collectively, these findings suggest that expression of the gene encoding the 72-kDa protein identified in this study through antigenic determinant(s) shared with MAGE-I protein is regulated in a way similar to that of MAGE-I.8 1996 Wiley-Liss, Inc.The human MACE-1 gene directs the expression of a peptide antigen originally identified on the human melanoma cell line MZ2-MEL 3.0 by autologous CD8+ cytolytic T lymphocytes (CTL) (Van der Bruggen et al., 1991). The restriction element for this antigen, referred to as MZ2-E, is HLA-A1. The MZ2-E antigen can be mimicked by a synthetic nonapeptide corresponding to a sequence encoded by the third exon of the MAGE-1 gene (Traversari et al., 1992). A second antigenic peptide encoded by MAGE-1 and presented by HLA-Cw16 has been described (Van der Bruggen et al., 1994). It has further been shown that MAGE-1 belongs to a family of 12 closely related genes that are located on chromosome X (De Plaen et al., 1994).The MAGE genes encode relatively large proteins, and it is likely that a number of peptides derived from these proteins might combine to various HLA molecules and constitute distinct tumor-rejection antigens. The function of theMAGE-1 gene product and of the other members of the MAGE famil...

Expression of MAGE-1 and -3 genes and gene products in human hepatocellular carcinoma

“…This discrepancy might be caused by a lower antigenisity of the proteins in paraffinembedded samples. With respect to the 72-kDa protein which cross-reacts with the mAb 77B and is frequently co-expressed with the MAGE-1 protein in melanomas (Carrel et al, 1996;Gudat et al, 1996). In the present study, only two of 15 HCCs expressed this protein and these two HCCs also expressed MAGE-1 protein.…”

“…The samples were also immediately frozen after resection, and stored at -80°C until RNA and protein extraction. The erythroleukemia cell line K562 was used as a positive control of MAGE-1 and -3 gene expression (Serrano et al, 1995), while human testicular tissues were also used as a positive control for the immunostaining and the Western blot analysis (Carrel et al, 1996).…”

“…To generate these monoclonal antibodies BALB/c mice were immunized with 20 µg of the respective recombinant protein. Hybridoma supernatants were screened by ELISA for binding to the each protein used for immunization (Kocher et al, 1995;Carrel et al, 1996). After overnight incubation with the respective undiluted mAb at 4°C, the membrane was washed with PBS/Tween-20, incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse second-step antibody (ECL Detection System, Amersham Japan, Tokyo, Japan) for 1 h. Chemiluminescence detection system was used to reveal specific binding (ECL Detection System, Amersham Japan, Tokyo, Japan), and protein bands were visualized on autoradiogrphy film.…”

“…48-kDa (Chen et al, 1994;Schultz-Thater et al, 1994;Kocker et al, 1995;Carrel et al, 1996;Gudat et al, 1996;Gunther et al, 1997). Immunohistochemical studies in malignant melanomas have demonstrated that MAGE-1 gene product is a cytoplasmic protein clustered in paranuclear organelle-like structures (Schultz-Thater et al, 1994).…”

Efficient Simultaneous Presentation of NY-ESO-1/LAGE-1 Primary and Nonprimary Open Reading Frame-Derived CTL Epitopes in Melanoma

“…Western blotting was performed as previously described (19). The anti-NY-ESO-1 mouse mAbs used were ES121 (20) …”