T cells possess several valuable properties for tumor therapy. With up to 80% different T cell subsets account for the majority of the lymphocyte population within the peripheral blood, which proliferate strongly and are highly cytotoxic after activation. One opportunity to use T cells for tumor therapy is the development of T cell recruiting bi-specific antibodies. However, the therapeutic application of those antibodies is often accompanied by severe adverse events that mainly depend on cytokine release by T cells as well as the non-selective expression of targeted tumor antigen. Most tumor antigens in clinical use are characterized by upregulated expression on tumor cells but show broad expression on normal healthy tissue as well. To reduce effects on healthy cells antigens that are exclusively expressed on tumor cells are of highest interest. One such promising tumor antigen is TA-MUC1. During this study different T cell recruiting bi-specific antibodies that target TA-MUC1 were produced and characterized to analyze if TA-MUC1 is a suitable antigen for T cell recruitment. Four different anti-CD3 antibodies were analyzed regarding T cell binding and activation to choose the most promising candidates for expression as bi-specific antibodies. As a first step three constructs with three different anti-CD3 scFv’s fused to the Cκ domain of an IgG1 were compared in functional assays. Despite comparable CD3 binding the scFv’s showed clear differences in T cell activation and cytokine release. The antibody construct containing scFvOkt3.2 induced less target cell independent cytokine release and T cell activation compared to the other constructs. Following the analysis of the different scFv’s two constructs, scFvOkt3.2 fused to the Cκ or CH3 domain, were characterized in more detail to analyze a potential influence of the chosen construct format. The subsequent functional tests showed best results for the CH3 fusion. This construct mediated improved CD3 binding leading to enhanced T cell activation and proliferation as well as to increased cytotoxicity against TA-MUC1 positive tumor cells. Surprisingly stronger target cell independent activation and costimulation was mediated by the Cκ construct. In summary this study shows that TA-MUC1 is a valuable tumor antigen for T cell recruiting bi-specific antibodies. The analyzed PM-CD3 constructs are able to induce in vitro T cell activation and proliferation as well as T cell dependent cytotoxicity. However, for clinical application additional assays regarding the safety profile are necessary.