ANALYSES OF RECOMBINANT VACCINIA AND FOWLPOX VACCINE VECTORS EXPRESSING TRANSGENES FOR TWO HUMAN TUMOR ANTIGENS AND THREE HUMAN COSTIMULATORY MOLECULES. The poor immunogenicity of tumor antigens and the antigenic heterogeneity of tumors call for vaccine strategies to enhance T-cell responses to multiple antigens. Two antigens expressed noncoordinately on most human carcinomas are carcinoembryonic antigen (CEA) and MUC-1. We have reported the construction and characterization of two viral vector vaccines to address these issues. The two viral vectors analyzed are the replication-competent recombinant vaccinia virus (rV-) and the avipox vector, fowlpox (rF-), which is replication incompetent in mammalian cells. Each vector encodes the transgenes for three human costimulatory molecules (B7-1, ICAM-1, and LFA-3, designated TRICOM) and the CEA and MUC-1 transgenes (which also contain agonist epitopes). The vectors are designated rV-CEA/MUC/TRICOM and rF-CEA/MUC/TRICOM. Each of the vectors is shown to be capable of faithfully expressing all five transgenes in human dendritic cells (DC). DCs infected with either vector are shown to activate both CEA- and MUC-1-specific T-cell lines to the same level as DCs infected with CEA-TRICOM or MUC-1-TRICOM vectors. Thus, no evidence of antigenic competition between CEA and MUC-1 was observed. Human DCs infected with rV-CEA/MUC/TRICOM or rF-CEA/MUC/ TRICOM are also shown to be capable of generating both MUC-1- and CEA-specific T-cell lines; these T-cell lines are in turn shown to be capable of lysing targets pulsed with MUC-1 or CEA peptides as well as human tumor cells endogenously expressing MUC-1 and/or CEA. These studies provided the rationale for the clinical evaluation of these multigene vectors in patients with a range of carcinomas expressing MUC-1 and/or CEA. INDUCTION OF HIGHER-AVIDITY HUMAN CTLS BY VECTOR-MEDIATED ENHANCED COSTIMULATION OF ANTIGEN-PRESENTING CELLS. The efficacy of antigen-specific CD8(+) CTLs depends not only on the quantity of CTLs generated but also perhaps, more importantly, on the avidity of the CTLs. To date, however, no strategy has been shown to preferentially induce higher-avidity human CTLs. In these studies, antigen-presenting cells (APC) generated from human peripheral blood mononuclear cells were infected with a recombinant avipox vector (rF-) containing the transgenes for a triad of costimulatory molecules (human B7.1, intercellular adhesion molecule-1, and LFA-3, designated as rF-TRICOM) and then used to elicit peptide-specific CTLs from autologous T cells. Compared with peptide-pulsed noninfected APCs or peptide-pulsed APCs infected with wild-type vector, peptide-pulsed APCs infected with rF-TRICOM induced not only more CTLs but also higher-avidity CTLs; this was shown by tetramer staining, tetramer dissociation, IFN-gamma production, and cytolytic assays. Peptide-pulsed rF-TRICOM-infected dendritic cells were also shown to induce CTLs with a >10-fold higher avidity than CTLs induced using CD40L-matured dendritic cells; the use of peptide-pulsed CD40L-matured dendritic cells infected with rF-TRICOM as APCs induced CTLs of even greater avidity. To our knowledge, these studies are the first to show a methodology to induce higher-avidity human CTLs and have implications for the development of more efficient vaccines for a range of human cancers. HUMAN B CELLS THAT HYPEREXPRESS A TRIAD OF COSTIMULATORY MOLECULES VIA AVIPOX-VECTOR INFECTION: AN ALTERNATIVE SOURCE OF EFFICIENT ANTIGEN-PRESENTING CELLS. Dendritic cells (DCs) are the most potent of the antigen-presenting cells (APCs). Preparation of sufficient numbers of mature DCs, however, is both costly and time-consuming. We have examined the possibility of using an alternative source of APCs that would be easier to obtain, would not require extensive culture, and thus would be more applicable to human immunotherapy protocols.
