Investigators have shown that several normal self antigens that are overexpressed by human carcinomas can be used as targets for active immunotherapy. We have been interested in studying the feasibility of mounting an immunological response to a carcinoma antigen which also serves as a tissue-specific normal, self antigen. One such antigen is human carcinoembryonic antigen (CEA). Studies have been undertaken to investigate the immunological parameters involved in generating host immunity to CEA in a murine experimental model in which CEA is expressed as a transgene (CEA.Tg mice). The tisse distribution for human CEA in the CEA.Tg mice is similar to that found in humans (i.e., stomach, colon). Other traditionally CEA-negative tissues, such as the trachea and uterus, were found to also constitutively express CEA. Initial studies established that the CEA.Tg mice were tolerant to CEA as determined by the (i) absence of any host immunity to CEA in naive CEA.Tg mice and (ii) inability to generate either a CEA-specific humoral or cellular response following the administration of soluble CEA protein in adjuvant to the CEA.Tg mice. Subsequent studies demonstrated that tolerance to CEA could be broken following the administration of a recombinant vaccinia virus expressing CEA (rV-CEA) to the CEA.Tg mice. Immunization of naive CEA.Tg with rV-CEA induced both anti-CEA antibody and cell-mediated responses. Upon further analyses, the anti-CEA IgM and IgG titers were found to be strong and indicative of Ig class switching. CEA.Tg mice immunized with rV-CEA also developed CEA-specific lymphoproliferative responses and CEA-peptide specific cell-mediated cytotoxicity. The ability to generate CEA-specific host immunity following rV-CEA immunization correlated with the ability to protect those CEA.Tg mice from subsequent tumor challenge. The protection from tumor was accomplished with no overt toxicity directed at CEA-positive normal tissues. The experimental findings demonstrate the ability to mount an effective antitumor immune response to a self tumor antigen by immunizing with a recombinant vaccinia virus. Even with the delivery of a self tumor antigen, such as CEA, in a recombinant vaccinia virus, it was apparent that the resultant host immune response was relatively weak. Therefore, additional studies were undertaken to (i) identify cytokines which can augment antitumor host immunity and (ii) develop potentially novel delivery systems for those cytokines. In an experimental model in which CEA serves as a foreign antigen, granulocyte-macrophage colony-stimulating factor (GM-CSF) was found to increase CEA-specific T cell growth and cell-mediated cytotoxicity when administered in conjunction with rV-CEA immunization. Moreover, when GM-CSF is administered concurrently with rV-CEA immunization and subsequently for three additional days at the immunization site, both protection from tumor challenge and tumor therapy were augmented in B6 mice. The cDNA of GM-CSF was isolated by RT-PCR from mouse spleen cells and ligated into a vaccinia virus transfer vector and the recombinant vaccinia-GM-CSF virus (rV-GM-CSF) was generated by homologous recombination. Infection of cells with rV-GM-CSF produced high quantities of biologically active GM-CSF and, in a previous study, infection of syngeneic tumor cells suppressed their growth as primary tumors in immunocompetent mice. Studies were undertaken to compare the effects of rV-GM-CSF with GM-CSF in an antigen-specific experimental model murine model. A single rV-GM-CSF administration produced biologically active GM-CSF in vivo for seven days as evidenced by the changes of cellular class II expression in draining regional lymph nodes. Combining rV-CEA and rV-GM-CSF immunization was equally effective as the combination of rV-CEA with GM-CSF in protecting mice from tumor challenge. Thus, murine GM-CSF can be delivered effectively as a recombinant virus and a single rV-GM-CSF administration was as effective as daily (x4) treatments of GM-CSF. Moreover, the results argue that rV-GM-CSF is compatible when used in combination with another replicating, recombinant virus (rV-CEA) and functions as an effective vaccine adjuvant by faciltating the development of antigen-specific host immunity. The CEA.Tg model will be an excellent experimental setting in which to identify cytokines, costimulatory models and other factors that can augment host immunity directed to a self tumor antigen.
