Recombinant poxviruses expressing immunomodulatory molecules together with specific antigens represent powerful vaccines for cancer immunotherapy. Recently, we and others have demonstrated, in vitro and in vivo, that coexpression of CD80 and CD86 costimulatory molecules enhances the immunogenic capacity of a recombinant vaccinia virus (rVV) encoding different tumor-associated antigens. To further investigate the capacity of these vectors to provide ligands for different costimulatory pathways relevant in the generation of T cell responses, we constructed a recombinant virus (rVV) expressing CD40 ligand or CD154 (CD154rVV). Upon binding the CD40 receptor expressed on antigen presenting cells (APC), this molecule, physiologically expressed on activated CD4+ T cells, increases their antigen presentation and immunostimulatory capacities. Therefore, we evaluated the effects of CD154rVV infection on APC activation and its consequences on T cell stimulation. CD154rVV infection of autologous fibroblasts, monocytes, or iDC promoted the expression of a number of cytokines, including GM-CSF, TNF-alpha, and IL-15 in iDC. Most importantly, IL-12 p40 gene expression and protein secretion were induced by CD154rVV but not by wild-type VV (WT VV) in either CD14+ cells or iDC, and these effects could be blocked by anti-CD40 monoclonal antibodies. Furthermore, phenotypic characterization of CD154rVV infected iDC revealed enhanced expression of CD83 and CD86 surface markers as compared with wild-type vaccinia virus infection. As expected, VV infection triggered cytokines gene expression in cultures including APC and T cells from VV immune donors. However, cytokine genes typically expressed by T cell receptor triggered T cells such as those encoding IL-2 and IFN-gamma, or T cell proliferation, were detectable to a significantly higher extent in CD154rVV infected cultures, as compared with WT VV. Activation of specific CD8+ T cells was then investigated using MART-1/Melan-A(27-35) epitope as the model of tumor-associated antigen (TAA). In the presence of CD154rVV activated APCs, significantly higher numbers of specific cytotoxic CD8+ T cells were detected, as compared with cultures performed in the presence of WT VV or in the absence of virus. Taken together, these data indicate that functional CD154 expression from rVV infected cells promotes APC activation, thereby enhancing antigen-specific T cell generation. Such a recombinant vector might help bypass the requirement for activated helper cells during CTL priming, thus qualifying as a potentially relevant vector in the generation of CD8+ T cell responses in cancer immunotherapy.

The characterization of the expression pattern of different families of cancer/testis (C/T) antigens in different tumors, at the protein level, might be of relevance in the development of multiantigen vaccine preparations for active specific immunotherapy. We have used tissue microarray (TMA) technology to explore in large numbers of tumor specimens the expression of NY‐ESO‐1/LAGE‐1 C/T antigens and its correlation with MAGE‐A expression by using D8.38 and 57B monoclonal antibodies (MAb). The epitopes recognized by these reagents in C/T antigens were identified by molecular mapping by using a bacterial expression system. Out of 2,052 samples, 119 (5.8%) scored positive upon staining with D8.38 NY‐ESO‐1/LAGE‐1‐specific MAb. Expression in >10% of cases was detectable in melanoma and basalioma (31.6 and 18.2%, respectively), large cell carcinomas and adenocarcinomas of the lung (17.8 and 10.5%, respectively), stomach adenocarcinomas of the intestinal type (13.2%), pT2‐4 bladder TCC (18.2%), nonseminomatous carcinomas of the testis (10.4%) and liposarcomas (15.4%). Simultaneous expression of NY‐ESO‐1/LAGE‐1 and MAGE‐A C/T antigens was then addressed in a TMA where 101/845 and 73/845 samples (12 and 8.6%, respectively) showed evidence of MAGE‐A or NY‐ESO‐1/LAGE‐1 specific staining, respectively. In 35/845 specimens (4.1%) concomitant expression of MAGE‐A and NY‐ESO‐1/LAGE‐1 was observed ( p = 0.0002). Discrepancies in the expression of NY‐ESO‐1/LAGE‐1 and MAGE‐A were conspicuously detectable in squamous cell carcinomas of the skin (MAGE‐A positive but NY‐ESO‐1/LAGE‐1 negative) and in liposarcomas (NY‐ESO‐1/LAGE‐1 positive, but MAGE‐A negative). Taken together, these data suggest novel areas of application of C/T antigens targeted active specific immunotherapy possibly based on multiantigen vaccine preparations. © 2005 Wiley‐Liss, Inc.

We performed a phase I/II clinical trial in metastatic melanoma patients with an ultraviolet (UV)-inactivated nonreplicating recombinant vaccinia virus enabling the expression, from a single construct, of endoplasmic reticulum-targeted HLA-A0201-restricted Melan-A/MART-1(27-35), gp100(280-288), and tyrosinase(1-9) epitopes, together with CD80 and CD86 costimulatory proteins. Corresponding soluble peptides were used to boost responses and granulocyte-macrophage colony-stimulating factor was used as systemic adjuvant. Safety and immunogenicity, as monitored with in vitro-restimulated peripheral blood mononuclear cells by cytotoxic T lymphocyte precursor (CTLp) frequency analysis and tetramer staining, were specifically addressed. Of 20 patients entering the protocol, 2 had to withdraw because of rapidly progressing disease. Immune responses were evaluated in 18 patients (stage III, n = 5; stage IV, n = 13) and increases in specific CTLp frequencies were observed in 15. In 16 patients responsiveness against all 3 antigens could be analyzed: 7 (43%), including all stage III cases, showed evidence of induction of CTLs specific for the three epitopes, and 2 (12%) and 4 (25%), respectively, showed reactivity against two or one tumor-associated antigen. In three stage IV patients no specific CTL reactivity could be induced. Increases in CTLp frequency were detected mostly after viral vaccine injections. However, in a majority of patients final CTLp levels were comparable to initial levels. Tetramer characterization of Melan-A/MART-1(27-35)-specific CTLs during the protocol also suggested preferential expansion after recombinant virus administration. Vector-specific humoral responses, frequently undetectable in stage IV patients, did not appear to prevent tumor-associated antigen-specific CTL induction. Aside from a single occurrence of transient grade 3 leukopenia, no major clinical toxicity was reported. Seventeen of 18 patients completed the 3-month trial (one patient died before the last delayed-type hypersensitivity test). Three displayed regression of individual metastases, seven had stable disease, and progressive disease was observed in seven patients. This is the first report on the administration of a UV-inactivated recombinant vaccinia virus coexpressing five transgenes in cancer patients. The results described here, in terms of safety and immunogenicity, support the use of this reagent in active specific immunotherapy.

We investigated the expression of tumor-associated antigens (TAA) of the cancer/testis (C/T) gene family in cervical squamous cell carcinomas. First, we focused on the HeLa cervical cancer derived cell line, and we found that it expresses MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A12, GAGE-3/6, LAGE-1, and PRAME genes, encoding defined C/T TAA. In contrast, no expression of MAGE-A10, BAGE, GAGE-1/2, or NY-ESO-1 genes was observed. Corresponding gene products could also be detected by immunoblotting and immunocytochemistry, taking advantage of monoclonal antibodies recognizing discrete TAA. Capitalizing on these data, a monoclonal antibody predominantly recognizing MAGE-A4 TAA in paraffin-embedded sections (57B) was used to investigate the C/T gene expression in clinical tumor samples. A group of 60 patients was studied, and 57B positivity was detectable to different extents in 33% of the cases (20/60). In 13 of them (21%), staining of over 50% of the tumor cells was evident, whereas healthy cells always scored negative. Remarkably, MAGE-A4 expression was significantly (p < 0.05) more frequently detectable in poorly differentiated tumors (8/13) than in well-differentiated or moderately differentiated cancers (3/15 and 9/32, respectively) and in stage FIGO II as compared with stage FIGO Ib tumors (12/23 and 5/24, respectively, p = 0.04). Interestingly, staining was mostly nuclear in well-differentiated tumors, but involved both nuclei and cytoplasm in less differentiated cancers. Positivities of comparable frequency were also detectable in a smaller series of specimens upon staining with MAGE-A1- or NY-ESO-1/LAGE-1-specific reagents. Considering the high tumor specificity of C/T TAA, our data provide the rationale for the design of immunotherapy procedures targeting these antigens in cervical cancers.

Cancer/testis tumour-associated antigens (C/T TAA) were the first human tumour-associated antigens to be characterised at the molecular level. Specific genes are expressed in the testis and in tumours of varying histological origin. The tissue expression pattern supports the notion that these antigens could be targets for active specific immunotherapy. Specific serological reagents have been developed and have helped to clarify biochemical characteristics of C/T TAA and to assess their distribution within clinical tumour samples. We review immunohistochemical evidence of the expression of C/T TAA known to be recognised by specific cytotoxic T lymphocytes. The emerging picture is consistent with a mostly heterogeneous expression in human cancers. These findings support the concept of multiantigenic tumour vaccine preparations. Moreover, the wide range of tumours in which C/T TAA have been detected urges further efforts to develop effective specific immunotherapeutic procedures.

TAAs of the MAGE family are mostly studied as targets of specific immune responses. Their potential relevance as tumor markers has also been underlined. We used a MAb, 57B, recognizing MAGE‐A4 protein in paraffin‐embedded sections, to evaluate its expression in bladder cancers by employing TMA including 2,317 samples from 1,849 patients. In 2,090/2,317 cases (90.2%), immunostaining yielded interpretable results. Since for some patients more than 1 sample was available, only interpretable first biopsies (n = 1,628) were considered. MAGE‐A4 protein was expressed at significantly (p < 0.001) higher frequency in squamous (25/55, 45.5%) than in adeno (4/15, 26.7%), sarcomatoid (4/14, 28.6%), small cell (5/20, 25%) or transitional cell (281/1,522, 18.5%) carcinomas. In TCCs, overall MAGE‐A4 positivity was significantly correlated with invasive phenotype (p < 0.001) and high tumor grade (p < 0.0001). Clinical data from 908 TCC patients were retrospectively evaluated, revealing that strong 57B staining was highly significantly associated with decreased tumor‐specific survival (p < 0.0001). These data suggest that evaluation of MAGE‐A4 protein expression is useful in the identification of groups of TCCs characterized by severe prognosis, thus possibly providing indications for early MAGE TAA‐targeted immunotherapy. © 2002 Wiley‐Liss, Inc.

NY-ESO-1 gene encodes a novel member of the cancer/testis (CT) family of human tumour-associated antigens (TAA). Specific monoclonal antibodies (mAb) have identified the corresponding gene product in lysates of tumour cell lines as a 22 kDa protein but no data are available concerning its intracellular location or distribution within neoplastic tissues. We have generated NY-ESO-1 specific mAbs recognizing the target molecule in cytospin preparations and in sections from clinical tumour specimens. These reagents identify NY-ESO-1 TAA in melanoma cell lines expressing the specific gene as a cytoplasmic protein, sharing the intracellular location of most MAGE TAA. In a series of 12 melanoma specimens, specific staining, limited to neoplastic cells, was detectable in the five cases where NY-ESO-1 gene expression was observed. In two of them over 90% of tumour cells showed evidence of positive staining. Lower percentages of positive neoplastic cells ranging between single cells and 50% were observed in the remaining tumours. These data suggest that active specific immunotherapies targeting NY-ESO-1, alone or in combination with other TAA could be of high clinical relevance in sizeable subgroups of melanoma patients. © 2000 Cancer Research Campaign

International Journal of CancerVolume 86, Issue 5 p. 749-751 LETTER TO THE EDITORFree Access Anti-MAGE-3 antibody 57b and anti-MAGE-1 antibody 6C1 can be used to study different proteins of the MAGE-A family Donata Rimoldi, Corresponding Author Donata Rimoldi donata.rimoldi@isrec.unil.ch Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, SwitzerlandLudwig Institute for Cancer Research, Ch. des Boveresses 155, 1066 Epalinges, Switzerland. Fax: +41-21-653 4474Search for more papers by this authorSuzanne Salvi, Suzanne Salvi Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, SwitzerlandSearch for more papers by this authorElke Schultz-Thater, Elke Schultz-Thater Departments of Surgery and Research, University of Basel, SwitzerlandSearch for more papers by this authorGiulio C. Spagnoli, Giulio C. Spagnoli Departments of Surgery and Research, University of Basel, SwitzerlandSearch for more papers by this authorJean-Charles Cerottini, Jean-Charles Cerottini Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, SwitzerlandSearch for more papers by this author Donata Rimoldi, Corresponding Author Donata Rimoldi donata.rimoldi@isrec.unil.ch Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, SwitzerlandLudwig Institute for Cancer Research, Ch. des Boveresses 155, 1066 Epalinges, Switzerland. Fax: +41-21-653 4474Search for more papers by this authorSuzanne Salvi, Suzanne Salvi Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, SwitzerlandSearch for more papers by this authorElke Schultz-Thater, Elke Schultz-Thater Departments of Surgery and Research, University of Basel, SwitzerlandSearch for more papers by this authorGiulio C. Spagnoli, Giulio C. Spagnoli Departments of Surgery and Research, University of Basel, SwitzerlandSearch for more papers by this authorJean-Charles Cerottini, Jean-Charles Cerottini Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, SwitzerlandSearch for more papers by this author First published: 27 April 2000 https://doi.org/10.1002/(SICI)1097-0215(20000601)86:5<749::AID-IJC24>3.0.CO;2-OCitations: 64AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Dear Sir, 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. Figure 1Open in figure viewerPowerPoint 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. Table I. CROSS-Reactivities of mAb and 6C1 Mage-a Proteins MAGE-A product Anti-MAGE-3 57B Anti-MAGE-1 6Cl Westerna ICCb Western ICC MAGE-1 + + + + MAGE-2 + + + + MAGE-3 + + + + MAGE-4 + + + + MAGE-6 + + + + MAGE-10 − − + + MAGE-12 + + + + a Reactivities were assessed as in Fig. 1. b Immunocytochemistry (CC) was performed on transiently transfected 293T and NA8-MEL cells using a fluorescence-labeled secondary antibody. 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. Figure 2Open in figure viewerPowerPoint 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, REFERENCES Carrel, S., Schreyer, M., Spagnoli, G., Cerottini, J.C. and Rimoldi, D., Monoclonal antibodies against r-MAGE-1 protein identify a cross-reacting 72-kDa antigen which is co-expressed with MAGE-1 protein in melanoma cells. Int. J. Cancer, 67, 417- 422 (1996). Chaux, P., Vantomme, V., Stroobant, V., Thielemans, K., Corthals, J., Luiten, R., Eggermont, A. M. M., Boon, T. and van der Bruggen,P., Identification of MAGE-3 epitopes presented by HLA-DR molecules to CD4(+) T lymphocytes. J. exp. Med., 189, 767- 778 (1999). Chen, Y.-T., Stockert, E., Chen, Y., Garin-Chesa, P., Rettig, W. J., Van der Bruggen, P., Boon, T. and Old, L. J., Identification of the MAGE-1 gene product by monoclonal and polyclonal antibodies. Proc. nat. Acad. Sci. (Wash.), 91, 1004- 1008 (1994). De Plaen, E. and 14 others, Structure, chromosomal localization, and expression of 12 genes of the MAGE family. Immunogenetics, 40, 360- 369 (1994). Hofbauer, G. F., Schaefer, C., Noppen, C., Boni, R., Kamarashev, J., Nestle, F. O., Spagnoli, G. and Dummer, R., MAGE-3 immunoreactivity in formalin-fixed, paraffin-embedded primary and metastatic melanoma: frequency and distribution. Amer. J. Pathol., 151, 1549- 1553 (1997). Huang, L-Q., Brasseur, F., Serrano, A., De Plaen, E., Van der Bruggen, P., Boon, T. and Van Pel, A., Cytolytic T lymphocytes recognize an antigen encoded by MAGE-A10 on a human melanoma. J. Immunol., 162, 6849- 6854 (1999). Jurk, M., Kremmer, E., SchwarZ, U., Forster, R. and Winnacker, E.L., MAGE-11 protein is highly conserved in higher organisms and located predominantly in the nucleus. Int. J. Cancer, 75, 762- 766 (1998). Kocher, T., Schultz-Thater, E., Gudat, F., Schaefer, C., Casorati, G., Juretic, A., Willimann, T., Harder, F., Heberer, M. and Spagnoli, G.C. Identification and intracellular location of MAGE-3 gene product. Cancer Res., 55, 2236- 2239 (1995). Manici, S., Sturniolo, T., Imro, M. A., Hammer, J., Sinigaglia, F., Noppen, C., Spagnoli, G., Mazzi, B., Bellone, M., Dellabona, P. and Protti, M. P., Melanoma cells present a MAGE-3 epitope to CD4(+) cytotoxic T cells in association with histocompatibility leukocyte antigen DR11. J. Exp. Med., 189, 871- 876 (1999). Marchand, M. and 24 others, Tumor regressions observed in patients with metastatic melanoma treated with an antigenic peptide encoded by gene MAGE-3 and presented by HLA-A1. Int. J. Cancer, 80, 219- 230 (1999). Nestle, F. O., Alijagic, S., Gilliet, Y., Sun, S., Grabbe, S., Dummer, R., Burg, G. and Schadendorf, D., Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nature (Med.), 4, 328- 332 (1998). Rimoldi, D., Salvi, D., Reed, D., Coulie, P., Jongeneel, V.C., De Plaen, E., Brasseur, F., Rodriguez, A-M., Boon, T. and Cerottini,J.-C., cDNA and protein characterization of human MAGE-10. Int. J. Cancer, 32, 901- 907 (1999). Shichijo, S., Tsunosue, R., Kubo, K., Kuramoto, T., Tanaka, Y., Hayashi, A. and Itoh, K., Establishment of an enzyme-linked immunosorbent assay (ELISA) for measuring cellular MAGE-4 protein on human cancers. J. immunol. Methods, 186, 137- 149 (1995). Van den Eynde, B. J. and van der Bruggen, P. T cell defined tumor antigens. Curr. Opin. Immunol., 9, 684- 693 (1997). Zorn, E. and Hercend, T., A MAGE-6 encoded peptide is recognized by expanded lymphocytes infiltrating a spontaneously regressing human primary melanoma lesion. Eur. J. Immunol., 29, 602- 607 (1999). Donata Rimoldi donata.rimoldi@isrec.unil.ch*, Suzanne Salvi*, Elke Schultz-Thater , Giulio C. Spagnoli , Jean-Charles Cerottini* Citing Literature Volume86, Issue51 June 2000Pages 749-751 FiguresReferencesRelatedInformation

Hintergrund: Bei Melanomen können Tumor-assoziierte Antigene (TAA) Ziele einer spezifischen zytotoxischen T-Zellantwort sein. Das NY-ESO-1-Gen kodiert ein neues TAA sogenanntes „tumorspezifisches Antigen“. Bislang wurde die Antigenexpression nur auf der Transkriptionsebene nachgewiesen, und es war nicht bekannt, ob das entsprechende Protein in Tumorzellen produziert wird. Wir haben deshalb NY-ESO-1-spezifische monoklonale Antikörper (mAk) hergestellt, die den Nachweis des NY-ESO-1-TAA auf Proteinebene ermöglichen.

The human MAGE-3 gene encodes a melanoma antigenic epitope recognized by specific cytotoxic T lymphocytes, but its gene product has not been identified thus far. We produced a recombinant MAGE-3 gene product by expression cloning of the entire reading frame in the context of a fusion protein characterized by a 10-histidine tail, allowing purification by metal chelation on a nickel Sepharose column. The semipurified product was used to generate MAGE-3-specific monoclonal antibodies. One reagent could identify by immunoblotting the native MAGE-3 gene product as a M(r) 48,000 protein in lysates of cell lines showing evidence of MAGE-3 gene expression. No apparent cross-reactivity with recombinant or native MAGE-1 gene product was observed. Immunohistochemistry shows that, closely resembling the MAGE-1 gene product, MAGE-3 is a cytoplasmic protein.

MAGE-1 gene encodes a human melanoma antigen, recognized by syngeneic cytotoxic T lymphocytes (CTL). MAGE-1 transcripts are also detectable in breast cancers, in non-small-cell lung carcinomas and in central nervous system tumors. In order to identify, in cellular preparations, the protein encompassing the antigenic peptide, we generated a panel of monoclonal antibodies (MAbs) against the MAGE-1 gene product by using, as immunogen, a full-length recombinant preparation (rMAGE-1), obtained through expression cloning of the relevant gene in E. coli. Four reagents were obtained recognizing both rMAGE-1 and the 46-kDa native protein in cell lines expressing MAGE-1 mRNA. No positivity could be detected in MAGE-1-mRNA-negative melanoma lines. No surface labelling of MAGE-1-positive cell lines could be observed. In contrast, on permeabilization of MZ2 melanoma cells, all 4 MAbs induced efficient staining, as detected by cytofluorography. Fluorescence microscopy shows that MAGE-1 gene product is a cytoplasmic protein clustered in paranuclear organelle-like structures. Thus, MAGE-1 protein location closely resembles that of P91A and P198 murine-tumor antigens.

Cytokines are powerful modulators of immune responses, the local production of which could be relevant to the interaction between tumour and immune system. This study investigated the transcription of genes encoding interleukin (IL) 2, IL-4, IL-10 and interferon (IFN) gamma in lymphocyte-infiltrated renal cell carcinoma biopsies from ten patients using the reverse polymerase chain reaction technique. Autologous peripheral blood mononuclear cells and healthy renal parenchyma tissue were tested in parallel. The beta-actin gene, used as a positive control, was transcribed in all samples. In contrast, transcription of cytokine genes was confined to tumour biopsies: IL-2 gene transcripts were detectable in five biopsies and IL-10 transcripts in seven. IL-4 and IFN-gamma gene transcripts were detectable in one biopsy each. In two patients no cytokine gene transcription could be identified. These data underline that heterogeneous patterns of cytokine gene transcription can be observed in renal cell carcinoma biopsies. Although transcription of an immunostimulatory lymphokine such as IL-2 was observed in 50 per cent of biopsies, the most frequently transcribed cytokine gene coded for an inhibitory factor, IL-10.

Induction of cytokine gene transcription by recombinant human IL-2 (rhIL-2) in peripheral blood mononuclear cells (PBMC) from healthy donors was studied by qualitative polymerase chain reaction. In all donors tested, optimal doses of rhIL-2-induced transcription of genes encoding for IL-5, GM-CSF, IFN-γ and TNF-α whereas transcription of IL-1-α, IL-3, IL-4, and IL-6 genes could only be detected in about half of the donors. Moreover, we observed that different doses of rhIL-2 were needed to induce transcription of different cytokine genes. In contrast, transcription of IL-2 and IL-10 genes was only observed in a minority of donors, irrespective of the concentration of rhIL-2 used. Since IL-10 displays a well-characterized inhibitory activity on the synthesis of cytokines possibly involved in the generation of lymphokine-activated killer (LAK) cells, we asked whether the absence of IL- 10 gene transcription plays a role in the induction of LAK cells. Thus, we tested the effects of different doses of rhIL-10 on the rhIL-2-driven generation of LAK activity. Interestingly, rhIL-10 dose-dependently inhibited the production of IFN-γ and TNF-α induced by IL-2, but had no effects on PBMC proliferation and generation of LAK activity. Similarly, purified CD3-/CD16+ lymphocytes, the precursors of LAK effector cells, could be optimally induced by low doses of rhIL-2 to proliferate and generate MHC-unrestricted cytotoxic activity against NK-resistant targets in the presence of rhIL-10. Altogether, our results indicate that rhIL-2 induces transcription of a preferential pattern of cytokine genes, with the IL-10 gene being infrequently transcribed. On the other hand, rhIL-10 shows diverse effects on rhIL-2-triggered PBMC activation, in that it inhibits IFN-γ and TNF-α production but does not affect PBMC proliferation or generation of LAK activity.