Abstract 2982: TriTCE CPI, next generation trispecific T cell engagers with integrated checkpoint inhibition (CPI) for the treatment of solid tumors
Authors: Poffenberger Maya C. , Verstraete Meghan M. , Von Rossum Anna , Zwierzchowski Patricia , Zago Matteo , Luu Veronica , Arrafi Sifa , Cao Siran , Pratap Harsh , Piscitelli Chayne L. , Weisser Nina E. , von Kreudenstein Thomas Spreter 
Published: April 2023

Abstract
CD3-bispecific T cell engager (TCE) therapies have exhibited clinical utility against hematological malignancies, but successes in solid tumor indications have been limited. Compared to heme malignancies, treatment of solid tumors is hindered by immunosuppressed microenvironments that can be refractory to traditional CD3-bispecific TCEs. Immunosuppression in the tumor microenvironment limits treatment responses in part due to the expression of inhibitory immune checkpoints, such as PD-1 on exhausted T cells and PD-L1 on tumor cells. To improve T cell responses and anti-tumor activity in immunosuppressed solid tumors, we generated trispecific TCE antibodies (Abs) that target a tumor associated antigen (TAA), CD3 and PD-L1 (via a PD1 moiety) to stimulate tumor-directed T cell killing and checkpoint blockade at the tumor site.
In this engineering approach we harnessed the flexibility of our AzymetricTM technology to screen multiple antibody formats, geometries, paratopes, and PD-1 domain affinities in parallel. We screened the TriTCE CPI antibodies, targeting different TAAs, for tumor-directed T cell cytotoxicity and CPI activity on TAA+PD-L1+ and TAA-PD-L1+ tumor cells. We identified multiple TriTCE CPI Abs that induced potent TAA-dependent T cell killing of TAA+PD-L1+, but not TAA-PD-L1+, tumor cells. Evaluation of CPI using a PD-1/PD-L1 checkpoint reporter gene assay identified antibody formats that stimulated simultaneous TAA dependent T cell engagement and enhanced checkpoint inhibition superior to bispecific Ab plus anti-PD-L1 Ab combination treatments. Additionally, in a human PBMC-engrafted xenograft model, TriTCE CPI Abs showed increased anti-tumor activity compared to a bispecific Ab control +/- anti-PD-L1 Ab treatment. Furthermore, the benefits of increased anti-tumor activity and CPI was observed across multiple TriTCE CPI Abs targeting different TAAs.
We generated multiple TriTCE CPI Abs that combine tumor-dependent T cell cytotoxicity with checkpoint blockade, which may translate to improved T cell responses in immunosuppressed solid tumors. The evaluation of multiple Ab formats, geometries and paratope affinities allowed for optimization of TAA-dependent cytotoxicity and CPI to identify Abs with enhanced anti-tumor activity and superior site-specific CPI, key factors that may contribute to a wide therapeutic index and improved clinical outcomes.
Citation Format: Maya C. Poffenberger, Meghan M. Verstraete, Anna Von Rossum, Patricia Zwierzchowski, Matteo Zago, Veronica Luu, Sifa Arrafi, Siran Cao, Harsh Pratap, Chayne L. Piscitelli, Nina E. Weisser, Thomas Spreter von Kreudenstein. TriTCE CPI, next generation trispecific T cell engagers with integrated checkpoint inhibition (CPI) for the treatment of solid tumors [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 2982.