Anti-MAGE-3 antibody 57b and anti-MAGE-1 antibody 6C1 can be used to study different proteins of the MAGE-A family

MAGE-A genes are expressed by a variety of cancers but not by normal adult tissues, except for testis and placenta. They encode 12 highly homologous proteins of 309-369 aminoacids (De Plaen et al., 1994). Several peptide epitopes from MAGE-A proteins, including MAGE-1, 3, 6 and 10, recognized by specific cytolytic T lymphocytes derived from tumors or peripheral blood have been described (Van den Eynde and van der Bruggen, 1997; Huang et al., 1999; Zorn and Hercend, 1999). In addition, HLA class II restricted epitopes have been described for MAGE-3 (Chaux et al., 1999; Manici et al., 1999). MAGE-A genes are therefore regarded as attractive candidates for specific immunotherapy and clinical trials of cancer patients involving immunization with MAGE-A1 and 3 peptides are in progress (Marchand et al., 1999; Nestle et al., 1998). Monoclonal antibodies (MAbs) against MAGE-1, 3, 4 and 11 proteins have been developed using the respective recombinant E. coli proteins as immunogen (Chen et al., 1994; Kocher et al., 1995; Shichijo et al., 1995; Carrel et al., 1996; Jurk et al., 1998). The anti-MAGE-3 MAb 57B appeared to be particularly interesting as it was shown to be suitable for immunocytochemistry for both frozen and paraffin-embedded tumor samples (Hofbauer et al., 1997). However, the possibility that cross-reactivity of this antibody with other members of the MAGE-A family may exist, due to the high degree of homology of these proteins (MAGE-3 and 6, in particular, differ only for 11 aminoacids), has not been formally ruled out. To this regard, we have demonstrated that a MAb raised against recombinant MAGE-1, named 6C1, strongly cross-reacts with MAGE-10 (Carrel et al., 1996; Rimoldi et al., 1999). To resolve this issue, we have investigated in detail the cross-reactivities of anti-MAGE-3 MAb 57B and anti-MAGE-1 MAb 6C1 using a transient transfection approach where human embryonic kidney 293T cells were used as recipient. Plasmids containing cDNAs encoding the more commonly expressed MAGE genes (MAGE-1, 2, 3, 4, 6, 10 and 12) were transfected individually and cell lysates analyzed by Western blotting. Figure 1a shows that both MAbs were able to recognize all the different MAGE proteins, except for MAGE-10 that was not recognized by MAb 57B. These results demonstrate the high degree of cross-reactivity of both MAbs and also show that all MAGE-A proteins have an apparent m.w. of 45–50 kDa, except for MAGE-10, which displays an unexpected MW of 72 kDa (Rimoldi et al., 1999). The extent of cross-reactivity towards the different proteins appeared to be variable. However, quantitative interpretations cannot be drawn from these experiments due to possible variations in transfection efficiencies. To confirm that cross-reactivity by the MAbs can take place when MAGE genes are expressed at a normal physiological level, such as found in tumors, a series of cell lines expressing different combinations of MAGE genes, as determined by RT/PCR, was analyzed. Figure 1b confirms the ability of the 2 MAbs to recognize multiple members of the MAGE family. In particular, MAb 57B detected a band of approximately 50 kDa (similar to the size of MAGE-3, Kocher et al., 1995) in melanoma Me242 and Me244 cells (expressing only MAGE-2/6 and 1/2/6/10, respectively) and fibrosarcoma LB23-SAR cells (expressing only MAGE-4). In contrast, the 45 kDa/MAGE-A1 band was not detected by MAb 57B under these conditions. Interestingly, the pattern of proteins detected by MAb 6C1 closely resembled that of MAb 57B, with the addition of the 72 kDa/MAGE-10 and the 45 kDa/MAGE-1 bands. The ability of the 2 MAbs to detect multiple MAGE proteins was further assessed by immunocytochemistry on transiently transfected 293T and MAGE negative melanoma cells. The results paralleled those described above for the Western blot analyses (not shown). A summary of the cross-reactivities of MAbs 57B and 6C1 is given in Table I. Western blot analyses of human cells with anti-MAGE-1 MAb 6C1 and anti-MAGE-3 MAb 57B. (a) 293T cells transiently transfected by the calcium/phosphate technique with plasmids containing the indicated MAGE cDNAs. Western blotting was performed as described (Carrel et al., 1996); (b) A series of cell lines derived from melanoma metastases and a fibrosarcoma cell line (LB23-SAR) were analyzed for expression of MAGE genes by RT/PCR as described (Carrel et al., 1996) and cell lysates analyzed by Western blotting. Equal amounts of proteins (50 μg) were loaded in each lane. Finally, to corroborate our findings, we sought to characterize the epitopes recognized by the MAbs. By screening several peptide sequences from MAGE-1 and 3 proteins in Elisa tests, we have identified 2 overlapping peptides from MAGE-1 (amino-acid 250-267 and 257-273) and 1 peptide from MAGE-3 (amino-acid 14-31) strongly recognized by MAb 6C1 and 57B, respectively, and capable of blocking the MAb activity against the respective recombinant protein (not shown). Figure 2 shows the MAGE-1 and 3 sequences containing the MAb epitopes aligned with the other MAGE-A sequences. In the case of MAb 6C1, the sequence recognized is identical for MAGE-1 and 10, confirming the high degree of cross-reactivity previously observed (Rimoldi et al., 1999), while the homologous MAGE-2, 3, 4, 6 and 12 sequences differ for one amino-acid. Elisa tests with recombinant MAGE-1 and 3 proteins indicated that the single aminoacid mismatch resulted in approximately 100-fold difference in affinity of 6C1 towards these proteins (not shown). For MAb 57B, the sequence recognized is identical in MAGE-3, 6 and 2, and thus these proteins are expeps;7cted to be similarly recognized. In contrast, a low degree of homology is observed for the corresponding MAGE-10 sequence, confirming the lack of reactivity of mAb 57B with this protein. The different localization of the epitopes, amino-terminal for mAb 57B and carboxy-terminal for 6C1, may explain the Western blot results on MAGE-4 transfected proteins, where anti-MAGE-3 57B, but not MAb 6C1, detected additional lower m.w. products. Similar probable degradation products have been observed by Shichijo et al. (1995) with an anti-MAGE-4 MAb. Comparison of MAGE-3 and MAGE-1 sequences containing the epitopes recognized by MAb 57B and 6C1 with the corresponding homologous sequences from other MAGE-A proteins. In conclusion, we have described that 2 antibodies raised against specific recombinant MAGE proteins can in fact recognize most of the MAGE-A family members. Thus, other MAGE proteins not yet characterized because of lack of specific reagents can now be studied, provided that they are used individually. While MAb 57B remains a valid immunohistochemical tool, studies with this Ab should, however, be interpreted considering the cross-reactivities described here. Yours sincerely, Donata Rimoldi donata.rimoldi@isrec.unil.ch*, Suzanne Salvi*, Elke Schultz-Thater , Giulio C.