Abstract 1864: An improved trispecific antibody (TsAb) platform for optimally engaging T cells for the treatment of solid tumor malignancies
Authors: Nair Reshmi , Somasundaram Veena , Goswami Arvind , Bhatnagar Jaya , Paul Sourav , Dey Chaitali , Vaidyanathan Kalyanaraman , Paul Rajkumar , Periasamy Sankar , KG Mary Helen , NV Mahalakshmi , Sinha Abhishek , Salazar Rachel , Varshney Avanish K. 
Published: June 2023

Abstract
T-cell recruiting bispecific antibodies have been shown to be clinically effective in treating hematological malignancies. However, their application against solid tumors is limited by several challenges, including a narrow therapeutic window due to cytokine release syndrome (CRS) induced toxicity, limited number of functional T cells, and an immunosuppressive tumor microenvironment (TME). Engagement of CD28 co-stimulatory domain can promote T cell fitness and sustained activation to overcome T -cell anergy. Here, we describe the development of a novel tri-specific antibody (TsAb) platform which is designed to optimally engage T cells via co-stimulation of both CD3 and of CD28 to achieve more effective tumor cell killing without increased CRS induction. To that end, we identified three selected formats (TsAb-1, TsAb-2 and TsAb-3) based on improved properties of the antibody formats, such as safety, efficacy, stability and developability. These formats have been developed to target three different well-established solid tumor-associated antigens (TAAs) as proof of concept. The binding activity of TsAbs to TAA, CD3 and CD28 was evaluated by ELISA and flow cytometry. Tumor cell cytotoxicity was assessed and secretion of cytokines by immune cells using a tumor cell-PBMC co-culture assay. Effective engagement of CD3 and CD28 was evaluated by flow analysis of CD25 Bcl-xL expression, respectively. Additionally, flow immunophenotyping was performed to characterize the differentiation of memory T cell subsets. Our results show that while all three TsAb formats displayed comparable cytotoxicity, TsAb-3 triggered immune cell activation only upon TAA engagement and T cell synapse formation. TsAb-3 showed &gt; 90% purity, no CRS induction in PBMCs alone or in coculture with low TAA-expressing tumor cells. Additionally, tethered TsAb-3 format was unable to stimulate PBMCs in the absence of TAA. Upon further analysis of T cell phenotype differentiation, TsAb-3 showed enhanced CD25+ T cell activation, Bcl-xL induction and increased T-effector and memory cell phenotypes on day 3 and day 7. In conclusion, our data support further development of TsAb as a versatile and improved T cell engager therapeutics for the treatment of solid malignancies. These versatile formats can be utilized not only in T cell engager therapeutics, but also in rationale combinations with immune check point inhibitors.
Citation Format: Reshmi Nair, Veena Somasundaram, Arvind Goswami, Jaya Bhatnagar, Sourav Paul, Chaitali Dey, Kalyanaraman Vaidyanathan, Rajkumar Paul, Sankar Periasamy, Mary Helen KG, Mahalakshmi NV, Abhishek Sinha, Rachel Salazar, Avanish K. Varshney. An improved trispecific antibody (TsAb) platform for optimally engaging T cells for the treatment of solid tumor malignancies [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 1864.