One of the major limitations to the immunotherapy of ovarian carcinoma based on the use of anti-CD3/antitumor bispecific monoclonal antibodies (bi-mAb) is the need for preactivation of effector cells ex vivo, because cross-linking of the T cell receptor-CD3 complex per se may lead to T-cell unresponsiveness or even apoptosis. The bi-mAb OC/TR, which recognizes the folate-binding protein (FBP) overexpressed in 90% of ovarian carcinomas and the CD3 molecule on T cells, has demonstrated efficacy in a clinical setting. Here we investigated the possibility of delivering accessory signals to OC/TR-retargeted peripheral blood mononuclear cells (PBMCs) via an anti-CD28 mAb or an anti-FBP/anti-CD28 bi-mAb. Coculture of resting PBMCs from healthy donors with OC/TR, anti-FBP/anti-CD28 bi-mAb, and FBP+ tumor cell lines resulted in a highly activated phenotype of effector cells and in a dramatic in vitro growth inhibition of the target cells without an increase in OC/TR-redirected lysis. Whereas both the CD4 and CD8 T cell subsets were involved in the growth inhibition, only the CD8 subpopulation accounted for the cytotoxic activity. The in vitro tumor growth inhibition was mediated mainly by soluble factors, which were active on both FBP+ and FBP- ("bystander effect") cell lines. Activation and antitumor activity were also observed, albeit to a lesser extent, using OC/TR and monospecific bivalent anti-CD28 mAb. In vitro analysis demonstrated that cross-linking between tumor and effector cells for at least 24 h was needed to achieve T-cell activation and development of antitumor activities. Thus, ex vivo CD3-CD28 costimulation on resting PBMCs might be of therapeutic utility for local treatment of minimal residual disease.