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Background Development of T cell engagers (TCEs) for solid tumors is hampered by limited availability of specific tumor-associated antigens (TAAs), resulting in reduced TCE selectivity and increased risk of on-target/off-tumor toxicity due to antigen-expression in healthy tissues. Particularly immunotherapy in ovarian cancer (OC) has had limited success due to the absence of ‘clean’ TAAs, as well as the presence of dysfunctional T cells and an immunosuppressive tumor microenvironment. OC remains the leading cause of cancer mortality in women, with a 5-year overall survival rate of <50% due to late diagnosis and a high relapse rate. We developed a MSLN and EpCAM-targeted multi-specific, logic-gated CD3 TCE with a CD2 co-stimulatory effector function. The TAA-dependent AND-gate function of this conditional Switch-DARPin molecule ensures that the CD3-engaging moiety can be released independent of proteases or pH, and activates T cells only when both TAAs are co-expressed on target cells. This design allows potent and sustained antitumor efficacy, while reducing systemic toxicity and on-target/off-tumor effects on single TAA-expressing healthy cells.Methods The MSLNxEpCAM-targeted conditional CD2xCD3 Switch-DARPin was generated using our designed ankyrin repeat protein (DARPin) platform. It contains a masked CD3 binder that is released only upon simultaneous binding to MSLN and EpCAM. Safety and antitumor activity of our Switch-DARPin were assessed in vitro and ex vivo using reporter cells, primary T cells from healthy donors and OC patient ascites, whole blood cytokine release assays, and in vivo using an OVCAR-3 ovarian cancer xenograft model in PBMC-humanized NXG mice.Results In vitro, the MSLNxEpCAM Switch-DARPin demonstrated selective T cell cytotoxicity against OC tumor cells co-expressing both targets, with attenuated activity against cells expressing only MSLN or EpCAM. No inhibition of activity in the presence of clinically relevant concentrations of soluble MSLN was observed, suggesting cytotoxicity is maintained independent of MSLN shedding. In addition, CD2 co-engagement induced sustained T cell proliferation and activity, preventing T cell dysfunction. In vivo, the Switch-DARPin induced significant tumor regression in a MSLN+EpCAM+ xenograft tumor model without causing systemic release of cytokines. In human whole blood, low cytokine release was observed despite the additional CD2 co-engagement.Conclusions Our logic-gated MSLNxEpCAM Switch-DARPin demonstrates selective antitumor activity against dual MSLN+EpCAM+ tumors (e.g. OC) without signs of peripheral T cell activation. The conditional Switch further allows additional CD2 co-stimulation to enhance T cell activity. This approach provides an opportunity for novel cancer treatments through logic-gated tumor-directed immune activation with increased efficacy and safety.