Abstract LB068: A novel CD20/CD3 T cell engager designed from Rituximab targeting CD20+ cancer with multiple mechanisms
Authors: Wenyan Cai, Jianbo Dong, Sachith Gallolu Kankanamalage, Allison Titong, Jiadong Shi, Zhejun Jia, Bo Wang, Cai Huang, Jing Zhang, Jun Lin, Steven Z. Kan, Joe Zhou, Yue Liu
Journal: Cancer Research (2021)

Abstract Background Bispecific T cell engaging antibodies (TEAs) with one arm targeting a cancer antigen and another arm binding to CD3 have demonstrated impressive efficacy in multiple clinical studies. However, establishing a balance of safety versus efficacy remains challenging. For instance, some TEAs have severe safety issues such as causing cytokine release symptom. Additionally, due to the heterogeneity of cancer cells, not all patients or all cancer cells of one patient respond equally to TEA treatments. It has been shown that cancer cells with PD-L1 overexpression respond to TEA therapy worse than other cancer cells. To develop next generation bispecific TEAs with a better balance of safety, efficacy and expanded mechanism of actions, we replaced heavy chain complementarity-determining regions (HCDRs) in one arm of Rituximab with HCDRs from a CD3 antibody to make a novel CD20/CD3 bispecific antibody, Rituximab/CD3 (GB261). After several rounds of computer aided sequence optimization, the final lead molecule demonstrated a superior balance of safety and efficacy in both in vitro and animal studies. Methods The affinities of antibodies to antigens were determined using FACS and SPR. T cell activation was determined by measuring CD69+/CD2+ cell percentage using FACS and cytokine release. Cancer cell killing assays were performed by measuring target cell depletion after incubating with PBMCs, ADCC assays were performed by measuring target cell depletion after incubating with NK92-CD16 cells and CDC was performed by measuring target cell depletion after incubating with complements-enriched human serum. In vivo cancer cell killing was performed using human PBMC engrafted NDG mouse models. Pre-tox and PK study were performed with cynomolgus monkey.Results GB261 exhibited high affinity to CD20 and an extremely low affinity to CD3. It showed similar T cell activation and cancer cell killing in Rituximab resistant Raji cells (RRCL) while reducing cytokine secretion compared to a benchmark CD20/CD3 (BM). GB261-induced ADCC and CDC only killed CD20+ cells but not CD3+ cells. When RRCL and PBMC cells (1:1) were co-injected with antibodies, it exhibited cancer killing effect comparable to the BM in a PBMC engrafted mouse model. However, if RRCL cells were injected first, then PBMC cells and antibodies were co-injected 4 days later, it showed superior cancer cell killing. Moreover, it delayed cancer relapse more effectively than Rutuximab in mice. Pre-tox and PK data in a cyno model also suggested that GB261 has good safety and PK. Finally, GB261 also demonstrated excellent manufacturability based on expression level and bio-analytic data.Conclusions GB261 exhibits enhanced efficacy and safety, and great manufacturability, presenting several advantages over the BM antibody. Thus, GB261 is a promising bispecific therapeutic antibody against CD20+ cancers. Citation Format: Wenyan Cai, Jianbo Dong, Sachith Gallolu Kankanamalage, Allison Titong, Jiadong Shi, Zhejun Jia, Bo Wang, Cai Huang, Jing Zhang, Jun Lin, Steven Z. Kan, Joe Zhou, Yue Liu. A novel CD20/CD3 T cell engager designed from Rituximab targeting CD20+ cancer with multiple mechanisms [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 LB068.