The MAGE‐encoded antigens that are recognized by cytolytic T lymphocytes (CTL) are shared by many tumors and are 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. We have used a method to identify CTL epitopes, which selects naturally processed peptides. CD8+ T cells, obtained from individuals without cancer, were stimulated with autologous dendritic cells infected with a recombinant adenovirus containing the MAGE‐A4 coding sequence. Responder cell microcultures that specifically lysed autologous EBV‐transformed B cells infected with vaccinia‐MAGE‐A4 were cloned using autologous stimulator cells infected with a Yersinia enterocolitica carrying the MAGE‐A4 sequence. An anti‐MAGE‐A4 CTL clone was obtained and the epitope was found to be decapeptide GVYDGREHTV (amino acids 230 – 239) presented by HLA‐A2 molecules. The CTL clone lysed HLA‐A2 tumor cells expressing MAGE‐A4. This is the first reported antigenic peptide encoded by MAGE‐A4. It may be valuable for cancer immunotherapy because MAGE‐A4 is expressed in 51 % of lung carcinomas and 63 % of esophageal carcinomas, whereas about 50 % of Caucasians and Asians express HLA‐A2.

The murine mastocytoma cell line P815 was used as a model to evaluate the effect on its tumorigenic capacity following interleukin-2 (IL-2) gene transfer into the tumor cells using a replication-defective adenovirus vector. The data show that P815 cells infected in vitro with this recombinant adenovirus secreted significant amounts of functional IL-2 as tested on CTL-L2 cells. Furthermore, when injected into syngeneic DBA/2 mice, the tumorigenic phenotype is lost in up to 80% of the animals. The rejection of the infected cells was host dependent, because co-injection at the same site or concomitant injection at the opposite side of the animal with a tumorigenic dose of noninfected P815 cells did not lead to tumor development in 50-70% of the mice. Moreover, protected animals developed a long-lasting state of immunization against the P815 tumor cells and their splenocytes were able to transfer the immunity to syngeneic naive recipients.

Regression of P815 tumors established on naive syngeneic mice can be obtained by the intratumoral injection of a single dose of an adenoviral vector expressing the IL-2 gene (Ad.IL2). Injection triggers local IL-2 production for at least 10 days. We measured a number of immunologic parameters in situ following intratumoral Ad.IL2 treatment. We also analyzed the situation of regression obtained upon challenge with P815 cells of mice previously immunized against the tumor and compared both systems. While IFN-gamma messenger RNA expression was found to be elevated in both situations of tumor regression, the level of infiltration by tumor-specific CTL was different. A small amount of tumor-specific CD8+ T cells were present in growing, untreated tumors. Such cells are found in much larger numbers in tumors rejected upon challenge, consistent with a CTL-mediated rejection. In contrast, they were found not to proliferate following Ad.IL2 injection. The latter caused an increased infiltration of a polyclonal, presumably nonspecific, T cell population. These results suggest that the initial regression of established P815 tumors following Ad.IL2 treatment in vivo is mostly due to nonspecific effectors.

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