Background: Tumors are heterogeneous in nature with highly suppressive microenvironment. The heterogeneity is distinguished by the presence of distinct cell populations, where many tumors are partial suppressed, thus lead to tumor relapse and develop drug-resistance after initial therapy. Most importantly, the heterogeneity in tumors raises considerable challenges to design effective treatment strategy with predictable outcomes. Paracellular permeability at tight junctions is largely determined by Claudins. Under aberrant condition, Claudins disrupt tight junction functions and promote tumor-initiation. Claudin 18.2 is significantly expressed in gastric and pancreatic cancers, while normal tissue expression is limited to the epithelium of the stomach, making it a promising target for cancer therapy. Although great efficacy has been observed for T-cell engagers, balance of safety-efficacy remains a major concern. Bispecific antibody (BsAb) with lower CD3-binding arm could have a huge potential to minimize unspecific T-cell activation while maintaining better-efficacy. Here, we designed Claudin 18.2/CD3 BsAb with weaker CD3-binding arm and maintain Fc-effector functions to broaden mechanisms of action including antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Methods: Claudin 18.2 expressing and T-cells were used for Claudin 18.2 and CD3 binding analysis, respectively. T-cell activation was performed by co-culturing T-cells with Claudin 18.2+ and Chaudin 18.2- cells. Pancreatic cancer cell was co-cultured with human PBMCs for ADCC, T-cell activation and T-cell cytotoxicity studies. Co-culture of Jurkat and CD16+ NK cells were used for Fc-mediated T-cell activation study. Claudin 18.2+ and T-cell cells were used for CDC studies in presence of human complement serum. Results: While comparable Claudin 18.2 binding affinity was maintained, a significant reduction in CD3 binding was observed by Claudin 18.2/CD3 BsAb compared to a benchmark antibody. Although weaker CD3 binding, BsAb induced equivalent Claudin 18.2 specific T-cell activation in presence of Jurkat and/or human PBMCs compared to the benchmark. Moreover, it also induced ADCC of pancreatic cancer cell and T-cell activation in presence of PBMCs in dose-dependent manner. Claudin 18.2/CD3 BsAb induced CDC of Claudin 18.2+ cells but not T-cell cells in presence of human complement serum. While Claudin 18.2/CD3 BsAb maintained the Fc-mediated ADCC and CDC functions, it had no significant effect on Fc-mediated T-cell activation compared to the benchmark antibody in presence of CD16+ NK cells. Conclusion: We designed Claudin 18.2/CD3 BsAb with weaker CD3 binding to minimize unspecific T-cell activation while maintaining Fc-mediated-effector functions. These data suggest the development of Claudin 18.2/CD3 BsAb as a drug target for patients with gastric and pancreatic cancers.

Citation Format: Amit K. Chaudhary, Mohd S. Zaman, Cai Huang, Jianbo Dong, Yue Liu. Development of Claudin 18.2/CD3 bispecific antibody maintaining effector functions for better cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB070.