MAGE-3 (168-176)

Vaccination of melanoma patients with tumor-specific antigens recognized by cytotoxic T lymphocytes (CTLs) may produce significant tumor regressions. Here, we suggest a novel type of tumor vaccines, with well-studied CTL epitopes presented on highly immunogenic virus-like particle (VLP) carriers. Cancer-germline gene MAGE-3 encodes for an antigenic nonapeptide (MAGE-3(168-176) peptide) that is recognized by CTLs on human leukocyte antigen (HLA)-A1 and HLA-B35 molecules. A set of recombinant genes encoding hepatitis B virus core protein carrying MAGE-3 epitope was constructed and expressed in Escherichia coli cells. Variants that led to formation of chimeric VLPs in vivo were purified and analyzed for their DNA binding properties in vitro. VLPs exhibiting the most pronounced nucleic acid binding affinity were selected and loaded either with single-stranded DNA oligodeoxynucleotides rich in nonmethylated CG motifs, or with longer double-stranded DNA fragments. Packaged DNA was protected, at least partially, against the action of bacterial DNase. Such highly purified chimeric VLPs with entrapped immunomodulatory sequences could possibly be used as antitumor vaccines.

The MAGE-3 gene is a member of a multigene family that is selectively expressed by subsets of different human tumor types, including malignant melanoma, but not by normal tissues except for testis and placenta. A cytolytic T lymphocyte (CTL)-defined MAGE-3 antigen, corresponding to the MAGE-3 peptide 271-279 associated with the human leukocyte antigen (HLA)-A2 molecule, has been recently identified using T lymphocytes from a normal individual stimulated in vitro with peptide-pulsed autologous antigen-presenting cells. Because MAGE-3 is expressed in 76% of metastatic melanomas, the HLA-A2-restricted MAGE-3 antigen should be expressed by approximately 37% of Caucasians bearing a metastatic melanoma tumor, thus representing an attractive candidate for the elicitation of specific CTL immune responses in vivo. In this study, we determined the proportion of HLA-A2+ melanoma patients displaying detectable MAGE-3 peptide 271-279-specific CTL precursors in peripheral blood. Peptide-specific CTL populations were obtained from at least 4 of 11 HLA-A2+ patients. Peptide-specific CTL lines derived from these populations readily lysed HLA-A2-positive target cells that were pulsed with MAGE-3 peptide 271-279 at nanomolar concentrations yet were unable to recognize (as assessed by cytolysis and cytokine production) MAGE-3-expressing autologous or allogeneic HLA-A2-positive melanoma lines. Similarly, the CTL lines failed to recognize MAGE-3-negative HLA-A2-positive tumor lines after transfection with the MAGE-3 gene, although they were able to recognize COS-7 cells transfected with MAGE-3. In contrast, HLA-A1-positive melanoma lines transfected with MAGE-3 were efficiently recognized by CTL lines directed against the MAGE-3 peptide 168-176, a known HLA-A1-restricted CTL epitope. These results suggest that the expression level of the MAGE-3 peptide 271-279, unlike that of MAGE-3 peptide 168-176, in melanomas may be too low to allow efficient recognition by specific CTLs. Thus, it appears that despite the presence of CTL precursors against MAGE-3 peptide 271-279 in some HLA-A2+ melanoma patients, the usefulness of this peptide for specific immunotherapy of melanoma may be limited.

MAGE-3 is the most commonly expressed cancer testis Ag and thus represents a prime target for cancer vaccines, despite infrequent natural occurrence of MAGE-3-specific immune responses in vivo. We report in this study the successful induction of Ab, CD8(+), and CD4(+) T cells in nonsmall cell lung cancer patients vaccinated with MAGE-3 recombinant protein. Two cohorts were analyzed: one receiving MAGE-3 protein alone, and one receiving MAGE-3 protein with adjuvant AS02B. Of nine patients in the first cohort, three developed marginal Ab titers and another one had a CD8(+) T cell response to HLA-A2-restricted peptide MAGE-3 271-279. In contrast, of eight patients from the second cohort vaccinated with MAGE-3 protein and adjuvant, seven developed high-titered Abs to MAGE-3, and four had a strong concomitant CD4(+) T cell response to HLA-DP4-restricted peptide 243-258. One patient simultaneously developed CD8(+) T cells to HLA-A1-restricted peptide 168-176. The novel monitoring methodology used in this MAGE-3 study establishes that protein vaccination induces clear CD4(+) T cell responses that correlate with Ab production. This development provides the framework for further evaluating integrated immune responses in vaccine settings and for optimizing these responses for clinical benefit.