1. Identification of five MAGE-A1 epitopes recognized by cytolytic T lymphocytes obtained by in vitro stimulation with dendritic cells transduced with MAGE-A1
P Chaux, R Luiten, N Demotte, V Vantomme, V Stroobant, C Traversari, V Russo, E Schultz, G R Cornelis, T Boon, P van der Bruggen J Immunol. 1999 Sep 1;163(5):2928-36.
MAGE genes are expressed by many human tumors of different histological types but not by normal cells, except for male germline cells. The Ags encoded by MAGE genes and recognized by T cells are therefore strictly tumor-specific. Clinical trials involving therapeutic vaccination of cancer patients with MAGE antigenic peptides or proteins are in progress. To increase the range of patients eligible for therapy with peptides, it is important to identify additional MAGE epitopes recognized by CTL. Candidate peptides known to bind to a given HLA have been used to stimulate T lymphocytes in vitro. In some instances, CTL clones directed against these synthetic peptides have been obtained, but these clones often failed to recognize tumor cells expressing the relevant gene. Therefore, we designed a method to identify CTL epitopes that selects naturally processed peptides. Monocyte-derived dendritic cells infected with a recombinant canarypoxvirus (ALVAC) containing the entire MAGE-A1 gene were used to stimulate CD8+ T lymphocytes from the blood of individuals without cancer. Responder cell microcultures that specifically lysed autologous cells expressing MAGE-A1 were cloned using autologous stimulator cells either transduced with a retrovirus coding for MAGE-A1 or infected with recombinant Yersinia-MAGE-A1 bacteria. The CTL clones were tested for their ability to lyse autologous cells loaded with each of a set of overlapping MAGE-A1 peptides. This strategy led to the identification of five new MAGE-A1 epitopes recognized by CTL clones on HLA-A3, -A28, -B53, -Cw2, and -Cw3 molecules. All of these CTL clones recognized target cells expressing gene MAGE-A1.
2. Identification of a MAGE-2-encoded human leukocyte antigen-A24-binding synthetic peptide that induces specific antitumor cytotoxic T lymphocytes
K Tahara, K Takesako, A Sette, E Celis, S Kitano, T Akiyoshi Clin Cancer Res. 1999 Aug;5(8):2236-41.
Because MAGE-2 gene is expressed in a wide variety of malignant tumors and HLA-A24 is the most common allele in the Japanese population and is also frequently present in Caucasians, the identification of MAGE-2-encoded peptide presented by HLA-A24 is, therefore, considered to be important in order to develop specific immunotherapy for malignant tumors using peptides as a vaccine. By using a MHC-binding assay, eight peptides derived from MAGE-2 were found to bind with sufficient affinity to the HLA-A24 molecule. When the induction of specific cytotoxic T lymphocytes (CTLs) was examined using a simplified method, the highest human lymphocyte antigen (HLA) binder (EYLQLVFGI) in these peptides was able to elicit CTLs from unseparated peripheral blood mononuclear cells in HLA-A24 healthy donors by stimulation with freshly isolated, peptide-pulsed peripheral blood mononuclear cells as antigen-presenting cells and also by using interleukin 7 and keyhole-limpet hemocyanin in a primary culture. The induced CTL could, thus, lyse HLA-A24 tumor cells expressing MAGE-2, as well as the peptide-pulsed target cells, with antigen specificity in a HLA class I-restricted manner. The identification of this peptide may, thus, be of therapeutic value in peptide-based vaccines for the treatment of several types of malignant tumors expressing MAGE-2.
3. Analysis of MAGE-3-specific cytolytic T lymphocytes in human leukocyte antigen-A2 melanoma patients
D Valmori, D Liénard, G Waanders, D Rimoldi, J C Cerottini, P Romero Cancer Res. 1997 Feb 15;57(4):735-41.
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.
