Bispecific antibodies (bsAbs) directed to tumor-associated antigens and to receptors mediating T-cell activation, such as the TCR/CD3 complex and the co-stimulatory CD28 molecule, are capable of activating T cells at the surface of tumor cells, resulting in tumor-cell killing. Here we report the pre-clinical characterization of bispecific-antibody fragments (bsFab2) directed to 2 different glioblastoma-associated antigens: the EGF receptor (EGFR) and a chondroitin-sulfate proteoglycan (CSPG). Using cultured glioblastoma cells expressing both target antigens, we found that the ability of anti-tumor x anti-CD28 bsFab2 to mediate "targeted T-cell co-stimulation" is superior for constructs targeting the CSPG molecule, correlating with an approximately 6-fold higher expression level of this antigen on the cell surface. In contrast, bsFab2 triggering CD3 are more effective if they contain EGFR-target specificity. This indicates that the activity of anti-tumor x anti-CD3 constructs critically depends on properties of the antigen other than its expression level on the cell surface, e.g., its mobility in the membrane. These findings prompted us to use EGFR-targeting bsFab2 in an ongoing clinical trial with glioma patients.