Abstract 6338: Development of a novel MSLN × 4-1BB bispecific antibody

Abstract Background: 4-1BB (CD137) is not only expressed on the surface of activated T cells and NK cells, but also a marker for Treg cells. Mesothelin (MSLN) is a ~71 kDa cell surface glycoprotein that is rarely expressed in normal tissues but overexpressed in many types of tumors. The fastest progress of MSLN-targeted therapy is in clinical phase II currently, including antibodies, ADCs, immunotoxins, vaccines and CAR-T. Here, we developed a bispecific antibody (bsAb) targeting both MSLN and 4-1BB with an intact Fc fragment from human IgG1. It can simultaneously exert the cytotoxic effect of CD8+T cells and and NK cells on tumor cells expressing MSLN to achieve better antitumor efficacy. Methods: Firstly, two bsAbs with IgG-scFv format were constructed with IgG1 and IgG4, named as HK013-G1 and HK013-G4 respectively. Secondly, we evaluated the binding activities of two candidates to tumor cells with different MSLN expression levels and their effects on 4-1BB+ cell activation mediated by MSLN-crosslinking. Subsequently, the cell-killing abilities of NK and cytotoxic T cells induced by bsAbs were quantified. Finally, we compared the ability of bsAbs to inhibit tumor growth in vivo and their effects on intratumoral CD8+T cells and Tregs. The capacity of bsAb candidates to induce liver toxicity was investigated simultaneously in these immunocompetent mice. Moreover, to further confirm the safety of HK013-G1, the risk of cytokine release storm (CRS) and activation of 4-1BB signal mediated by Fc receptors (FcRs), which are responsible for the potential side effects of 4-1BB mAbs were also evaluated. Results: HK013-G1 is able to bind different MSLN-expressing cancer cells and bridge MSLN+ cells and 4-1BB+ cells. In luciferase reporter assay, the bsAb-induced 4-1BB activation is dependent on expression level of MSLN. While incubated with CD8+T cells, HK013-G1 increased IFN-γ production only in the presence of MSLN+ cells. Furthermore, HK013-G1 could exert its affects via both NK cell and activated PBMC. In MC38-hMSLN tumor models, HK013-G1 showed a more significant growth inhibition effect than HK013-G4, and did not show detectable liver damage. In addition, HK013-G1 could enhance the antitumor effect of anti-PD-1 in CT26-hMSLN tumor models. Also, HK013-G1 was shown no stronger ability to inducing CRS and activating 4-1BB signal in the presence of FcRs than HK013-G4 and parent anti-4-1BB mAb in vitro. Conclusions: HK013-G1, a MSLN × 4-1BB bsAb with human IgG1 Fc fragment prevents tumor development by killing tumor cells directly via effector functions mediated by NK and cytotoxic T cells. These results show that this bsAb has the potential to develop into a new clinical therapy for cancer types with high-level MSLN expression. Citation Format: Liangwei Li, Dayan Zhang, Guodong Shen, Wenting Liu, Xiaoli Zeng, Lingling Wu, Qun Zhao, Weiming Zhou, Yang Huang, Liansheng Cheng. Development of a novel MSLN × 4-1BB bispecific antibody. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6338.