Studies have shown that the delivery of tumor-associated antigens by recombinant pox-virus vaccines can induce T cell immunity against a variety of solid tumors. Those studies have also identified a number of limitations in the development of this approach for effective immunotherapy of solid tumors. One limitations is the relative weakness of the immune response to the tumor-associated antigens. The studies described here identifies novel cytokine-based approaches which augment the antitumor T cell response generated by poxvirus-based vaccines. Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates the maturation of professional antigen presenting cells which play a crucial role in the initiation of a primary or restimulation of a recall cellular immune response. A study was designed using experimental murine tumor models to determine whether GM-CSF administered either as a protein or delivered as a recombinant vaccinia viral construct, rV-GM- CSF, could augment an antitumro cellular immune response. rV-GM-CSF was generated by isolating the cDNA, by RT-PCR, the gene encoding GM-CSF from mouse spleen cells. It was ligated into a vaccinia virus transfer vector and the recombinat rV-GM-CSF was generated by homologous recombination. Murine colon tumor cells infected with rV-GM-CSF produced significant levels of the cytokine as measured in a bioassay. Moreover, rV-GM-CSF infected tumor cells failed to grow in syngeneic mice while when those same cells were infected with the wild-type vaccinia virus progressively growing tumors were established. In vivo T cell depletion prior to the implantation of rV-GM-CSF-infected murine tumor cells resulted in tumor growth, indicating that tumor suppression previously seen in untreated mice involved the presence of a cellular immune response. The presence of tumor-specific cellular immunity in mice immunized with rV-GM-CSF-infected tumor cells was further demonstrated by the protection of those mice from subsequent challenge with uninfected tumor cells. The effectiveness of rV-GM-CSF as well as GM-CSF to augment a cellular antitumor response was also analyzed in an experimental model using a defined tumor antigen - carcinoembryonic antigen (CEA). Mice immunized with a recombinant vaccinia-CEA construct (rV-CEA) were partially protected from either the growth of primary tumors or from challenge with CEA-expressing tumors. Subsequent mechanistic studies focused on the changes that occurred in regional lymph nodes following the administration of either GM-CSF or rV-GM-CSF. In both cases, significant proliferative responses were evident within the cellular component of the nodes and flow cytometric analyses further revealed a shift in the cellular phenotypic consistent with the emergence of higher nimbers of antigen presenting cells following treatment with either GM-CSF or rV-GM-CSF. Functional T cell assays revealed substantially higher CEA-specific proliferative response in those regional lymph nodes from mice treated with GM-CSF or rV-GM-CSF. Those changes correlated with enhanced protection of mice from challenge with CEA-expressing tumors as well as improved therapeutic effectiveness in mice treated with rV-CEA, GM-CSF in combination with cytoxan. The findings argue that the ability to better present a weak tumor antigen with the administration of GM-CSF protein or as a recombinant vaccinia construct can augment the antitumor cellular reponse to (i) an undefined tumor antigen in a gene therapy model as well as (ii) to CEA in a defined tumor model system.
