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Background CD3-based T cell engagers (TCEs) have demonstrated clinical success in solid tumors, including DLL3×CD3, GP100×CD3, and STEAP1×CD3 formats. However, conventional TCEs primarily deliver signal 1 (CD3-mediated activation) without co-stimulatory signaling, often leading to T cell exhaustion and diminished therapeutic efficacy. To address this limitation, integrating a co-stimulatory signal such as 4-1BB into TCEs may enhance T cell function and durability. Inspired by the success of second-generation CAR-T cells and the emerging clinical safety of 4-1BB bispecific antibodies, we developed a trispecific antibody (TriAb) targeting DLL3, CD3, and 4-1BB using a VHH antibody derived from RenNano ® mice to achieve tumor antigen-dependent co-stimulation.Methods We designed a DLL3×CD3×4-1BB TriAb with reduced CD3 affinity through structural steric hindrance to enhance safety. In vitro assays included T cell proliferation, cytokine release, and cytotoxicity against DLL3-positive small cell lung cancer (SCLC) cell lines. Cytokine release was assessed in co-cultures with DLL3+ or DLL3– tumor cells. In vivo efficacy was evaluated in xenograft mouse models bearing established DLL3+ tumors. T cell expansion, apoptosis, and anti-tumor activity were monitored in response to TriAb treatment compared with a benchmark CD3 TCE.Results The DLL3 TriAb induced stronger T cell proliferation and induced cytokines release only in the presence of DLL3+ tumor cells. No significant cytokine release was observed with DLL3– targets, indicating antigen-dependent activation. In cytotoxicity assays, TriAb elicited potent tumor cell killing even at low effector-to-target (E:T) ratios and promoted upregulation of anti-apoptotic markers in T cells. In xenograft models, TriAb significantly inhibited tumor growth, enhanced T cell proliferation in a dose-dependent manner, and avoided T cell apoptosis observed with benchmark TCEs.Conclusions Our DLL3×CD3×4-1BB trispecific antibody demonstrates tumor-dependent co-stimulation, enhanced T cell activation, and superior anti-tumor efficacy in preclinical SCLC models. These findings support its potential as a next-generation T cell engager for clinical development.