Abstract 5555: A bispecific antibody targeting CLDN18.2 and 4-1BB induces potent anti-tumor efficacy with an extraordinary safety profile
Authors: Chen Junying , Huang Weifeng , Liang Zhiquan , Miao Xiaoniu , Peng Shaogang , Wang Chao , Dong Tiantian , Tsun Andy , Luo Yi 
Published: June 2022

Abstract
Background: Gastric cancer is one of the most common cancers worldwide, which is estimated to have 32.1 and 13.2 cases per 100,000 individuals in males and females, respectively. Claudin-18 isoform 2 (CLDN18.2), a membrane-bound protein involved in the formation of tight junctions, has been identified to be highly expressed in various types of cancers such as gastric and pancreatic cancer. Targeting CLDN18.2 with the monoclonal antibody (mAb) Zolbetuximab has achieved moderate clinical benefit with controllable toxicity. 4-1BB is a potent stimulator of T cells and NK cells, and when activated, can improve effector and/or memory responses. However, inherent on-target related toxic effects in the liver was observed during the clinical development of the monoclonal agonist antibody Urelumab. Here, we report the discovery of a humanized bispecific antibody (biAb) anti-CLDN18.2 x 4-1BB, which activates immune cells such as T cells via CLDN18.2-mediated crosslinking of 4-1BB. This bispecific antibody induces potent anti-tumor efficacy with limited toxicity.
Methods: The anti-CLDN18.2 x 4-1BB biAb was generated by the fusion of an anti-4-1BB VHH targeting the CRD4 domain for 4-1BB, to the C-terminus of an anti-CLDN18.2 mAb via a G4S linker. Binding affinity and specificity testing of each arm were studied by flow cytometry and on the Retrogenix Cell Microarray Technology platform, a human membrane/secreted protein binding array. The immunomodulatory functions were evaluated using luciferase reporter cell assays in vitro and human 4-1BB KI mouse models in vivo.
Results: The CLDN18.2 antibody interacted specifically to CLDN18.2 on the Retrogenix Cell Microarray platform among over 5,500 proteins. Next, in vitro studies showed how the molecule specifically induced T cell activation and cytokine release against CLDN18.2-positive cells. More importantly, such activation was correlated to the expression level of CLDN18.2, which indicates that the molecule may preferentially recognize CLDN18.2-high-expressing cancer cells. In mouse models, the anti-CLDN18.2 x 4-1BB biAb induced superior anti-tumor efficacy compared to anti-CLDN18.2 and anti-4-1BB mAbs alone with acceptable PK attributes. Furthermore, the anti-CLDN18.2 x 4-1BB biAb induced strong immunological memory where tumors could not grow in re-challenged tumor-free mice. Finally, the CLDN18.2 x 4-1BB biAb showed a safe profile in a GLP NHP study of which no obvious signs of toxicity were observed at 50 mg/kg, 100 mg/kg or 150 mg/kg doses.
Conclusion: In summary, a bispecific antibody targeting CLDN18.2 and 4-1BB was developed, which displayed potent anti-tumor efficacy with strong immunological memory and has shown good safety in NHPs. Moreover, the molecule has high selectivity to CLDN18.2. The molecule shall enter clinical trials by early 2022.
Citation Format: Junying Chen, Weifeng Huang, Zhiquan Liang, Xiaoniu Miao, Shaogang Peng, Chao Wang, Tiantian Dong, Andy Tsun, Yi Luo. A bispecific antibody targeting CLDN18.2 and 4-1BB induces potent anti-tumor efficacy with an extraordinary safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5555.