MAGE-3 (243-258)

We previously reported results of a phase II trial in which recombinant MAGE-A3 protein was administered with or without adjuvant AS02B to 18 non-small-cell lung cancer (NSCLC) patients after tumor resection. We found that the presence of adjuvant was essential for the development of humoral and cellular responses against selected MAGE-A3 epitopes. In our current study, 14 patients that still had no evidence of disease up to 3 years after vaccination with MAGE-A3 protein with or without adjuvant received an additional four doses of MAGE-A3 protein with adjuvant AS02B. After just one boost injection, six of seven patients originally vaccinated with MAGE-A3 protein plus adjuvant reached again their peak antibody titers against MAGE-A3 attained during the first vaccination. All seven patients subsequently developed even stronger antibody responses. Furthermore, booster vaccination widened the spectrum of CD4(+) and CD8(+) T cells against various new and known MAGE-A3 epitopes. In contrast, only two of seven patients originally vaccinated with MAGE-A3 protein alone developed high-titer antibodies to MAGE-A3, and all these patients showed very limited CD4(+) and no CD8(+) T cell reactivity, despite now receiving antigen in the presence of adjuvant. Our results underscore the importance of appropriate antigen priming using an adjuvant for generating persistent B and T cell memory and allowing typical booster responses with reimmunization. In contrast, absence of adjuvant at priming compromises further immunization attempts. These data provide an immunological rationale for vaccine design in light of recently reported favorable clinical responses in NSCLC patients after vaccination with MAGE-A3 protein plus adjuvant AS02B.

During the last few years, HLA class I tetramers have been successfully used to demonstrate anti-vaccine CD8 CTL proliferation in cancer patients vaccinated with tumor antigens. Frequencies of CTL as low as 10(-6) among CD8 cells were observed even in patients showing tumor regression. Little is known about the role of tumor-antigen-specific CD4 T cells in the context of these anti-vaccine responses. Therefore, we developed a very sensitive approach using fluorescent class-II-peptide multimers to detect antigen-specific CD4 T cells in vaccinated cancer patients. We produced HLA-DP4 multimers loaded with the MAGE-3(243-258) peptide and used them to stain ex vivo PBL from melanoma patients injected with dendritic cells pulsed with several class I and class II tumor antigenic peptides, including the MAGE-3(243-258) peptide. The multimer(+) CD4 T cells were sorted and amplified in clonal conditions; specificity was assessed by their ability to secrete IFN-gamma upon contact with the MAGE-3 antigen. We detected frequencies of about 1x10(-6) anti-MAGE-3.DP4 cells among CD4 cells. A detailed analysis of one patient showed an anti-MAGE-3.DP4 CD4 T cell amplification of at least 3000-fold upon immunization. TCR analysis of the clones from this patient demonstrated a polyclonal response against the MAGE-3 peptide.

Vaccination with cancer/testis Ag MAGE-A3 in the form of recombinant protein often induces specific humoral and cellular immune responses. Although Ag-specific CD4+ T cells following vaccination are detectable by cytokine production after a single in vitro stimulation, their detection before vaccination is difficult because of low frequency. In this study, we have applied a sensitive method using CD154 (CD40L) staining to detect MAGE-A3-specific CD4+ T cells. MAGE-A3-specific T cell responses were analyzed in four healthy donors, two lung cancer patients with spontaneous serum Abs to MAGE-A3, and two baseline seronegative lung cancer patients throughout vaccination with MAGE-A3 protein. MAGE-A3-specific CD4+ T cells were detected in all individuals tested, at low frequency in healthy donors and seronegative cancer patients and higher frequency in patients seropositive for MAGE-A3. Polyclonal expansion of CD154-expressing CD4+ T cells after cell sorting generated a large number of MAGE-A3-specific CD4+ T cell lines from all individuals tested, enabling full characterization of peptide specificity, HLA-restriction, and avidity. Application of this method to cancer patients vaccinated with MAGE-A3 protein with or without adjuvant revealed that protein vaccination induced oligoclonal activation of MAGE-A3-specific CD4+ T cells. It appeared that MAGE-A3 protein vaccination in the presence of adjuvant selectively expanded high avidity CD4+ T cells, whereas high avidity T cells disappeared after multiple vaccinations with MAGE-A3 protein alone.