Abstract 7292: HY05350: A trispecific T-cell engager with immune checkpoint inhibitor targeting mesothelin for solid cancer

Abstract Objective: Limited efficacy is the major road block when developing T cell engager (TCE) for solid cancers, which is partly due to the suppressive tumor microenvironment (TME) created by the expression of inhibitory immune checkpoints, such as PD-L1. To enhance anti-tumor activity of T cells, we engineered a trispecific T cell engager (HY05350), targeting CD3 and tumor-associated antigens Mesothelin (MSLN) and PD-L1 for solid cancers. Methods: CD3 antibody obtained by optimizing affinity of SP34. Alpaca was immunized with MSLN and PD-L1. VHH antibodies were obtained and humanized. Various trispecific formats evaluated by T cell activation, T cell-dependent cytotoxicity (TDCC), cytokine release and PD-L1 checkpoint blockade assays. Anti-tumor efficacy was studied in CDX mouse models, MoA studied by single cell NGS in the CD3xPD1xPDL1 transgenic mice, PK/TK and GLP toxicity evaluated in cynolmogus. Results: HY05350 in the format of 2(MSLN)+1(PD-L1)+1(CD3) was selected. It could induce strong target- & dose-dependent cytotoxicity in MSLN+ cancer cells and effectively block binding of PD-L1 to T cell PD1. It demonstrated a strong anti-tumor activity in a variety of the mouse CDX models. Cynomolgus GLP toxicity study demonstrated HY05350 was well-tolerated with a toxicity profile comparable to typical CD3 based bispecific antibodies and had an acceptable PK/TK profile with a T1/2 of 12-35 hours. We explored MoA of HY05350 in vitro and in vivo. Our in vitro experiments showed (1) HY05350 could induce a stronger 2nd signal than the bispecific antibody (CD3xMSLN), consistent with the observation by Liu et al (2019) that the CD3xPD-L1 arm of HY05350 could target the PD-L1+ APC cells to activate T cells and induce more effective antitumor immunity than directly bridging T cells to cancer cells; (2) As expected, activation of T cells induced PD-L1 expression in most tested cancer cells. Furthermore, HY05350 showed more potent TDCC activity against MSLN and PDL1 double-positive than single-positive tumor cells, suggesting PD-L1, together with MSLN, could enhance stronger TDCC activity, presumably via a feedforward mechanism, than the bispecific antibody CD3xMSLN; (3) HY05350 induced strong cytotoxicity against PD-L1+ DMSC, suggesting remodeling TME potential. Single cell sequencing analysis of ∼10K immune cells of MC38 tumors revealed that in pre- and post-dosed CDX mice, HY05350 induced a wide range of immune responses. In particularly, it significantly increased proportions of three major anti-tumor groups of lymphocytes: T cells, NK cells and interestingly gamma-delta T cells. Conclusion: We engineered a trispecific T cell engager HY05350 targeting CD3, MSLN and PD-L1 to treat solid cancers. Our in vitro and in vivo studies demonstrated that HY05350 was well-tolerated, induced strong T cell activation, cytotoxicity and potent antitumor efficacy, supporting for further clinical investigation. Citation Format: Yan Hu, Sanyou Guo, Yujing Zhang, Mengyun Zhang, Bo An, Dan Sun, Ao Fu, Xiaoqiang Chai, Jiangshan Dai, Qing Xiong, Tao Wei. HY05350: A trispecific T-cell engager with immune checkpoint inhibitor targeting mesothelin for solid cancer [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 7292.