Abstract 5590: BCG024: A novel DLL3×CD3×4-1BB trispecific T cell engager enhances T cell persistence and functionality in preclinical models

Abstract Background: CD3-based T cell engagers (TCEs) as powerful therapeutic agents have demonstrated clinical success in both hematologic malignancies and solid tumor indications. One major obstacle to TCE therapies in treating solid tumors is T-cell exhaustion due to conventional TCEs lacking co-stimulatory signaling, limiting their therapeutic durability. To address this challenge, integrating a co-stimulatory signal such as 4-1BB into TCEs could be an attractive approach to enhance T cell function and durability. Methods: We developed a trispecific antibody (TriAb) targeting DLL3, CD3, and 4-1BB using a VHH 4-1BB agonist derived from RenNano® mice to achieve tumor antigen-dependent co-stimulation. In vitro assays included repeated stimulation assay and cytotoxicity against DLL3-positive small cell lung cancer (SCLC) cell line. In vivo efficacy and T cell proliferation were evaluated in xenograft mouse models bearing established SCLC tumors. The toxicity study was evaluated in CD3ε×4-1BB humanized mice. Results: The DLL3 TriAb demonstrated significantly enhanced T cell proliferation while maintaining high cell viability and cytotoxicity following repeated stimulation. However, the benchmark and bsAb counterparts showed marked reductions in cell viability and activity. In vivo, mice treated with benchmark caused dose-dependent reductions in serum T cell number, whereas those treated with DLL3 TriAb maintained stable T cell populations. That indicated TCE integrated 4-1BB nanobody co-stimulation indeed enhances T cell durability and functionality. In SCLC xenograft models, DLL3 TriAb exhibited robust antitumor efficacy in a monovalent antigen-binding format rather than a bivalent antigen-binding format. Notably, contrasting with non-specific TriAb controls, DLL3 TriAb displayed favorable safety profiles in CD3ε×4-1BB humanized mice even at high doses, with no significant body weight loss or systemic cytokine elevation observed. Conclusions: Our DLL3×CD3×4-1BB TriAb demonstrates tumor-dependent co-stimulation, enhanced T cell persistence and functionality, further exhibiting superior anti-tumor efficacy in preclinical SCLC model and good safety profiles at high dose. These findings highlight the DLL3 TriAb as a next-generation immunotherapy, offering enhanced T cell functionality and clinical translatability. Citation Format: Guan Wang, Baihong Liu, Liu Yang, Xue Li, Shuangshi Liu, Yu Qi, Jinhua Zhao, Yang Chen, Frank An, Yi Yang. BCG024: A novel DLL3×CD3×4-1BB trispecific T cell engager enhances T cell persistence and functionality in preclinical models abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5590.