Abstract 6704: Development and characterization of a tri-specific selective CD8 T cell engager (CD8xCD3xCD19) for treatment of B cell lymphoma

Abstract The recent development of CD3 bi-specific antibodies (BsAbs) targeting CD20, BCMA, GPRC5D and CD19 to treat B cell malignancies have resulted in significant clinical benefit to patients. However, long-term remission rates and survival remain to be demonstrated. Current CD3 BsAbs preferentially bind to and activate CD4 T cells. However, CD8 cytotoxic T cells also play the key role in anti-tumor response and T cell exhaustion in cancer immunity. To potentially enhance anti-tumor efficacy by augmenting CD8 T-cell response, we developed a novel tri-specific T cell engager (TriTE) IM-8319, which simultaneously binds human CD3, CD8 and CD19 epitopes with KD at 14. 2 nM, 3. 3 nM and 0. 3 nM, respectively. The binding affinity to human CD8 T cells and CD4 T cells was measured with EC50 at 0. 02 nM and 1. 2 nM, respectively. In human PBMC-killing assays, IM-8319 was found to preferentially activate CD8 T cells with EC50 of 6. 0 pM, compared to an EC50 of 94 pM for activating CD4 T cells. Unique cytokine release responses were seen with IM-8319 treatment. Compared to bi-specific TCE controls without CD8 binding domain, IM-8319 in human PBMC assays increased INFγ and TNFα in CD8 T cells and blunted IL-2 release in CD4 T cells. We observed robust cytotoxic killing of CD19+ B cells with IM-8319. Compared the bi-specific TCE controls lacking CD8 binding domain, in vitro CD19+ B-cell depletion was increased 6-fold with IM-8319. In addition, we observed notable structure activity relationships, with 100-fold increases in TriTE cytotoxicity with a weak CD3 binding domain. T-cell fratricide has hindered the development of antibodies containing 2+ T-cell epitopes. We observed correctable, dose-dependent CD8 T cell depletion at high concentrations of CD8xCD3xCD19 TriTEs in vitro and xenograft mouse models. This unwanted biological effect was not a surprise when the TriTE binds to CD8 T cells in trans. However, by structure engineering IM-8319 to allow for cis binding of CD3 and CD8, we effectively eliminated CD8 T cell fratricide at therapeutic doses. In a Raji-inoculated murine xenograft that was reconstituted with human PBMC, IM-8319 significantly inhibited tumor growth without obvious CD8 T cell depletion. In summary, we report a novel TriTE (IM-8319) that selectively binds to and activates CD8 T cells. IM-8319 results in robust on-target cytotoxicty with specific activation of CD8 T cells, suggesting potential to reduce systemic toxicities such as cytokine release syndrome (CRS) while augmenting direct antitumor efficacy. Further preclinical evaluation of IM-8319 is warranted in patients with B-cell leukemias and lymphomas. Citation Format: Zheng Yang, Wuzhong Shen, Guiyun Tu, Shenjie Xue, Ying Tan, Bingbin Xie, Jason Xu, Hanyang Chen, Shui Liu, Xiaoqiang Yan. Development and characterization of a tri-specific selective CD8 T cell engager (CD8xCD3xCD19) for treatment of B cell lymphoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 6704.