Abstract 2125: The trispecific T cell engager platform enhances T cell function and therapeutic efficacy via integrated 4-1BB nanobody co-stimulation

Abstract CD3 T cell engagers (TCEs) have gained significant clinical interest, with 9 approvals in the past decade. Advances in solid tumor-targeting TCEs, such as DLL3xCD3, GP100xCD3, and STEAP1xCD3, indicate the potential to overcome the challenges posed by solid tumor barriers. However, issues like cytokine release syndrome (CRS), low T cell infiltration, treatment resistance, and limited patient response continue to pose hurdles. To optimize efficacy and minimize adverse effects, CD3 TCEs need further development. The inclusion of the co-stimulatory molecule 4-1BB, which promotes T cell proliferation and upregulates T cell survival genes expression and activation, in the design of trispecific TCE presents an attractive approach. CAR-T cell therapies suggest that combining 4-1BB signaling achieves more robust and durable anti-tumor responses. Therefore, we developed a tumor antigen -dependent CD3×4-1BB trispecific antibody (TriAb) platform by integrating 4-1BB nanobody generated from RenNanoTM mice to maximize T cell activation and sustain the T cell response. Compared with the canonical CD3 TCE, Our TriAb platform features a weaker binding activity to the CD3 receptor through steric hindrance, reducing safety risk relative to canonical CD3 TCEs. We validated the TriAb platform with three targets: Claudin18.2, DLL3 and CDH17. In vitro, TriAbs demonstrated robust antigen-dependent cytotoxic activity against tumor cell lines, surpassing bispecifc antibodies and benchmark counterparts in T cell activation and cytokine production. Importantly, TriAb did not induce cytotoxicity in tumor antigen-negative cells or non-specific cytokine release. In xenograft model with established tumors, TriAbs showed significant tumor growth inhibition. Additionally, TriAbs exhibited an antibody-like PK profile in CD3ε×4-1BB humanized mice, supporting intermittent dosing in human. Taken together, these data indicate our TriAb platform holds promise as a next-generation therapeutic option for clinical development. Citation Format: Kaiqing Zhang, Baihong Liu, Liu Yang, Saina Ma, Jiawei Wu, Jinhua Zhao, Yang Chen, Xueyuan Jiang, Peiyan Pan, Jing Zhang, Yi Yang. The trispecific T cell engager platform enhances T cell function and therapeutic efficacy via integrated 4-1BB nanobody co-stimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2125.