Tumour rejection Antigen P815 (35-43)

A DNA immunization approach was used to induce an immune response against the tumor-specific antigen P815A in DBA/2 mice. The P1A gene, which encodes the P815A antigen, was modified by the addition of a short sequence coding for a tag epitope recognized by the monoclonal antibody AU1, and cloned into the eukaryotic expression vector pBKCMV, resulting in plasmid pBKCMV-P1A. L1210 cells stably transfected with pBKCMV-P1A expressed P1A mRNA and were lysed by the syngeneic P815A-specific cytotoxic clone CTL-P1:5, thus confirming that the tag-modified P1A protein underwent correct processing and presentation. A single intramuscular injection of 100 microg of pBKCMV-P1A induced the expression of P1A mRNA for at least 4 months. Eighty percent of DBA/2 mice injected three times with 100 microg of pBKCMV-P1A generated cytotoxic T lymphocytes (CTL) that lysed P815 tumor cells, whereas mock-inoculated animals failed to show any cytotoxicity. Moreover, experiments designed to evaluate the protection of pBKCMV-P1A-immunized mice against a lethal challenge with P815 tumor cells showed that 6 of 10 immunized mice rejected the tumor, and 2 mice showed prolonged survival compared to control animals.

The weakly immunogenic murine P1A Ag is a useful experimental model for the development of new vaccination strategies that could potentially be used against human tumors. An i.m. DNA-based immunization procedure, consisting of three inoculations with the P1A-coding pBKCMV-P1A plasmid at 10-day intervals, resulted in CTL generation in all treated BALB/c mice. Surprisingly, gene gun skin bombardment with the pBKCMV-P1A vector did not induce CTL, nor was it protective against a lethal challenge with the syngeneic P1A-positive J558 tumor cell line. To speed up the immunization procedure, we pretreated the tibialis anterior muscles with cardiotoxin, which induces degeneration of myocytes while sparing immature satellite cells. The high muscle-regenerative activity observable after cardiotoxin inoculation was associated with infiltration of inflammatory cells and expression of proinflammatory cytokines. A single pBKCMV-P1A plasmid inoculation in cardiotoxin-treated BALB/c mice allowed for sustained expansion of P1A-specific CTL and the induction of strong lytic activity in <2 wk. Cardiotoxin adjuvanticity could not be replaced by another muscle-degenerating substance, such as bupivacaine, or by MF59, a Th1 response-promoting adjuvant. Although this vaccination schedule failed to induce tumor rejection in all immunized mice, the analysis of CD8 T cell responses at an individual mouse level disclosed that the cytotoxic activity of P1A-specific CTL was correlated to the antitumor efficacy. These results highlight the critical need to identify reliable, specific immunological parameters that may predict success or failure of an immune response against cancer.

In order to examine the use of DNA immunization to block tumor growth, we have developed a model system in which a defined 9-amino-acid epitope from the nucleoprotein of influenza virus is used as a surrogate tumor-associated antigen. A mastocytoma cell line of DBA/2 origin (P815) was transfected with a plasmid encoding the minimal H-2Kd-restricted NP(147-155) cytotoxic T lymphocyte (CTL) epitope, pCMV/NPep, to generate the cell line designated P815-NPep. Mice primed and boosted once with a plasmid encoding the full-length NP gene, pCMV/NP, but not with the minigene pCMV/NPep, developed a strong NP(147-155)-specific CTL response within 2 weeks after the boost. When challenged with 10(4) P815-NPep cells, pCMV/NP-immunized DBA/2 mice were protected from tumor challenge, whereas control mice immunized with the vector backbone rapidly developed lethal tumor. Importantly, the P815-NPep-immune mice were also protected from a subsequent challenge with the untransfected parental tumor P815. By depleting the NP-immune mice of either CD4+ or CD8+ T cells and then challenging with 10(4) P815-NPep tumor cells, it was determined that the CD8-depleted mice rapidly developed tumors, whereas the CD4-depleted or non-treated mice were protected. These data clearly indicate that intramuscular (i.m.) plasmid DNA immunization can be used to mobilize an effective CD8+ CTL-mediated antitumor response.