PURPOSE: Cancer vaccines targeting tumor-associated antigens are being investigated for the therapy of tumors. Numerous strategies, including the direct intratumoral (i.t.) vaccination route, have been examined. For tumors expressing carcinoembryonic antigen (CEA) as a model tumor-associated antigen, we previously designed poxviral vectors that contain the transgenes for CEA and a triad of T-cell costimulatory molecules, B7-1, intercellular adhesion molecule-1, (ICAM-1), and leukocyte function associated antigen-3 (LFA-3) (CEA/TRICOM). Two types of poxvirus vectors were developed: replication-competent recombinant vaccinia and replication-defective recombinant fowlpox. We have shown previously that a vaccine regimen composed of priming mice s.c. with recombinant vaccinia-CEA/TRICOM and boosting i.t. with recombinant fowlpox-CEA/TRICOM was superior to priming and boosting vaccinations using the conventional s.c. route in inducing T-cell responses specific for CEA. These studies also showed that CEA was needed to be present both in the vaccine and in the tumor for therapeutic effects. EXPERIMENTAL DESIGN: To determine specific immune responses associated with vaccination-mediated tumor regression, CEA-transgenic mice bearing CEA(+) tumors were vaccinated with the CEA/TRICOM s.c./i.t. regimen, and T-cell immune responses were assessed. RESULTS: In CEA(+) tumor-bearing mice vaccinated with the CEA/TRICOM s.c./i.t. regimen, T-cell responses could be detected not only to CEA encoded in vaccine vectors but also to other antigens expressed on the tumor itself: wild-type p53 and an endogenous retroviral epitope of gp70. Moreover, the magnitude of CD8(+) T-cell immune responses to gp70 was far greater than that induced to CEA or p53. Finally, the predominant T-cell population infiltrating the regressing CEA(+) tumor after therapy was specific for gp70. CONCLUSION: These studies show that the breadth and magnitude of antitumor immune cascades to multiple antigens could be critical in the therapy of established tumors.