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The colon adenocarcinoma C26, carrying two endogenous tumor-associated antigens (TAA) recognized by CTL, has been transduced with the gene coding for the human folate receptor alpha (FR alpha) as an additional antigen in order to study the efficacy of vaccination against a tumor expressing multiple antigens. A dicistronic vector was used to transduce the IL-12 genes to create C26/IL-12/FR alpha that has been used as a cellular vaccine to treat mice bearing lung metastases of C26/FR alpha. After vaccination mice were partially splenectomized and splenic lymphocytes frozen and used retrospectively to study in vitro CD8 T cell response related to the treatment outcome. Vaccination cured 50% of mice and the effect was CD8 T cell dependent. Mice either cured (responders) or not cured (nonresponders) by vaccination developed tumor-specific CTL. However, analysis of CTL specificity and pCTL frequencies revealed that responders had a predominant CTL activity against endogenous C26-related tumor antigens, whereas nonresponders had CTL that recognized preferentially the FR alpha antigen. CD8 from responder mice were characterized to release high levels of granulocyte-macrophage (GM)-CSF upon antigen stimulation. Tumors obtained from mice that died despite vaccination lost expression of the FR alpha transgene but maintained expression of endogenous C26 antigens. Immunoselection against FR alpha antigen was not observed in tumors from non-vaccinated controls and from CD8-depleted vaccinated mice. Down-regulation of FR alpha antigen expression was due, at least in part, to methylation of retroviral vector long terminal repeat promoter since FR alpha expression was partially restored, ex vivo, by treatment with 5-aza-2'-deoxy-cytidine (aza). These results indicate that CD8 T cell-mediated immunoselection and production of GM-CSF are determining factors for the efficacy of tumor vaccines.

We have compared the therapeutic activity and characterized the antitumor response induced by IL-12 and IL-2 gene-transduced tumor cell vaccines. Mice bearing lung metastases of the BALB/c colon carcinoma C51 were treated with syngenic, histologically related, and antigenically cross-reacting irradiated IL-12 (C26/IL12) or IL-2 (C26/IL2) gene-transduced C26 tumor cells given s.c. Vaccination with C26/IL12 cells cured 40% of mice, while vaccination with C26/IL2 cells reduced the number of metastatic nodules without affecting survival. Despite this difference, similar antitumor CTL activation was shown in mice treated with C26/IL12 or C26/IL2 cells. The lytic pattern of CTL was shown to be directed to tumor-associated Ags (TAA) shared between the colon carcinomas C51, C26, and CC36 as well as with other syngenic tumors. Both treatments induced anti-TAA Abs, but only sera from mice treated with C26/IL12 contained Ab that lysed tumor cells in a C-dependent cytotoxicity assay. Early infiltration of activated T cells was found in the lungs of mice vaccinated with C26/IL12. CD4+ lymphocytes purified from the lymph nodes draining the vaccination site or from the spleen showed a higher production of IFN-gamma in response to anti-CD3 mAb in C26/IL12 vaccinated mice, while a higher production of IL-4 was shown in mice vaccinated with C26/IL2 cells. These results indicate that the better therapeutic efficacy of vaccination with C26/IL12 is associated with the production of C-binding Ab, an early infiltration of the metastatic lungs by activated T lymphocytes and a predominant systemic activation of Th1 more than Th2 cells.