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CD4(+) Th cells play an important role in the induction and maintenance of adequate CD8(+) T cell-mediated antitumor responses. Therefore, identification of MHC class II-restricted tumor antigenic epitopes is of major importance for the development of effective immunotherapies with synthetic peptides. CAMEL and NY-ESO-ORF2 are tumor Ags translated in an alternative open reading frame from the highly homologous LAGE-1 and NY-ESO-1 genes, respectively. In this study, we investigated whether CD4(+) T cell responses could be induced in vitro by autologous, mature dendritic cells pulsed with recombinant CAMEL protein. The data show efficient induction of CAMEL-specific CD4(+) T cells with mixed Th1/Th2 phenotype in two healthy donors. Isolation of CD4(+) T cell clones from the T cell cultures of both donors led to the identification of four naturally processed HLA-DR-binding CAMEL epitopes: CAMEL(1-20), CAMEL(14-33), CAMEL(46-65), and CAMEL(81-102). Two peptides (CAMEL(1-20) and CAMEL(14-33)) also contain previously identified HLA class I-binding CD8(+) T cell epitopes shared by CAMEL and NY-ESO-ORF2 and are therefore interesting tools to explore for immunotherapy. Furthermore, two CD4(+) T cell clones that recognized the CAMEL(14-33) peptide with similar affinities were shown to differ in recognition of tumor cells. These CD4(+) T cell clones recognized the same minimal epitope and expressed similar levels of adhesion, costimulatory, and inhibitory molecules. TCR analysis demonstrated that these clones expressed identical TCR beta-chains, but different complementarity-determining region 3 loops of the TCR alpha-chains. Introduction of the TCRs into proper recipient cells should reveal whether the different complementarity-determining region 3 alpha loops are important for tumor cell recognition.

Delivery of the full-length tumor antigen might be more successful in immunotherapy than single peptides and has the advantage that patients no longer need to be selected for their HLA type. In this study, we tested the in vitro induction of CAMEL/NY-ESO-ORF2-specific T cells by dendritic cells infected with an adenovirus (Ad) type 5 vector containing the fiber shaft and knob of human serotype Ad35 (Ad5F35 vector). Our data show induction of CD8(+) T cells specific for the known HLA-A(*)0201-binding CAMEL/NY-ESO-ORF2(1-11) epitope by DC infected with Ad5F35-CAMEL, but not by DC pulsed with the recombinant CAMEL protein. In one healthy donor, even CD8(+) T cells specific for a new HLA-B7-binding CAMEL/NY-ESO-ORF2(46-54) epitope were raised. In conclusion, the in vitro induction of CAMEL/NY-ESO-ORF2-specific CD8(+) T cells in healthy donors by DC infected with Ad5F35-CAMEL strongly supports further investigation of the Ad5F35 vector as a vehicle for gene transfer into DC for the generation of tumor antigen-specific CD8(+) T cell responses in vivo.

Tumor Ag NY-ESO-1 is an attractive target for immunotherapy of cancer, since both CD8(+) CTL and CD4(+) Th cells against NY-ESO-1 have been described. Moreover, NY-ESO-1 as well as the highly homologous tumor Ag LAGE-1 are broadly expressed in various tumor types. Interestingly, the NY-ESO-1 and LAGE-1 genes also encode for proteins translated in an alternative open reading frame. These alternatively translated NY-ESO-ORF2 and CAMEL proteins, derived from the NY-ESO-1 and LAGE-1 genes, respectively, have been demonstrated to be immunogenic, since CTL specific for these proteins have been isolated from melanoma patients. In this study a panel of advanced melanoma patients was screened for the presence of Th cells specific for the alternatively translated tumor Ags NY-ESO-ORF2 and CAMEL. PBMC of melanoma patients were stimulated for 4 days with mixes of overlapping peptides covering the entire NY-ESO-ORF2 and CAMEL protein sequences and were tested for the release of type 1 (IFN-gamma) and type 2 (IL-13) cytokines in ELISPOT assays. In three of 15 patients, T cells specific for two CAMEL peptides (CAMEL(71-92) and CAMEL(81-102)) could be detected. From one of these patients, CD4(+) T cell clones specific for CAMEL(81-102) could be generated. These clones recognized a naturally processed epitope presented in both HLA-DR11 and HLA-DR12 and produced high levels of IL-4, IL-5, and IL-13. In conclusion, this study shows the presence of Th cells specific for the alternatively translated tumor Ag CAMEL in melanoma patients and is the first report that describes the isolation of tumor Ag-specific CD4(+) Th 2 clones.