BACKGROUND: Ovarian cancer remains a formidable challenge in oncology, necessitating innovative therapeutic approaches. Claudin-6 (CLDN6), a member of the tight junction molecule CLDN family, exhibits negligible expression in healthy tissues but displays aberrant upregulation in various malignancies, including ovarian cancer. Although several therapeutic modalities targeting CLDN6 are currently under investigation, there is still a need for more potent therapeutic options. While T-cell engagers (TCEs) hold substantial promise as potent immunotherapeutic agents, their current efficacy and safety in terms of target antigen selection and T-cell exhaustion due to only CD3 stimulation without co-stimulation must be improved, particularly against solid tumors. To provide an efficacious treatment option for ovarian cancer, we generated SAIL66, a tri-specific antibody against CLDN6/CD3/CD137. METHODS: Using our proprietary next-generation TCE technology (Dual-Ig), SAIL66 was designed to bind to CLDN6 with one Fab and CD3/CD137 with the other, thereby activating T cells through CD3 activation and CD137 co-stimulation. The preclinical characterization of SAIL66 was performed in a series of in vitro and in vivo studies which included comparisons to a conventional TCE targeting CLDN6 and CD3. RESULTS: Despite the high similarity between CLDN6 and other CLDN family members, SAIL66 demonstrated high specificity for CLDN6, reducing the risk of off-target toxicity. In an in vitro co-culture assay with CLDN6-positive cancer cells, we confirmed that SAIL66 strongly activated the CD137 signal in the Jurkat reporter system, and preferentially induced activation of both CD4 and CD8 T cells isolated from human peripheral blood mononuclear cells compared to conventional TCEs. In vivo studies demonstrated that SAIL66 led to a more pronounced increase in intratumor T-cell infiltration and a decrease in exhausted T cells compared with conventional CLDN6 TCE by contribution of CD137 co-stimulation, resulting in better antitumor efficacy in tumor-bearing mouse models. CONCLUSION: Our data demonstrate that SAIL66, designed to engage CLDN6, CD3, and CD137, has the potential to enhance antitumor activity and provide a potent therapeutic option for patients with ovarian and other solid tumors expressing CLDN6. Clinical trials are currently underway to evaluate the safety and efficacy of SAIL66.