Abstract 677: Development of an in vitro exhaustion assay for functional screening of CAR T cell enhancements.

Abstract Chimeric antigen receptor (CAR) T cells have emerged as a powerful tool in cancer immunotherapy, particularly in hematologic malignancies such as B-cell leukemia and lymphoma. CAR T cell therapy involves genetically engineering patient-derived T cells to express synthetic receptors that target tumor-associated antigens, enabling precise and potent immune responses against cancer cells. Despite remarkable clinical successes, especially with CD19-targeted CAR T cells, the therapy has limitations that hinder broader application and long-term efficacy. A significant challenge is T cell exhaustion, a dysfunctional state involving reduced proliferation, diminished cytokine production, and impaired cytotoxicity. Exhaustion often arises in the tumor microenvironment, where persistent antigen stimulation, immunosuppressive signals, and metabolic stress collectively impair CAR T cell function. This limits durability and contributes to relapse in many patients. Overcoming CAR T cell exhaustion is essential to improving efficacy and durability of cellular therapies. Strategies under investigation include optimizing CAR architecture by using alternative co-stimulatory domains, transiently modulating inhibitory pathways, and refining manufacturing protocols to preserve T cell fitness. An in vitro assay that reliably mimics exhaustion is essential for identifying and validating improvements. In this study, we present several approaches to inducing exhaustion-like phenotypes in CAR T cells. Chronic, antigen-agnostic stimulation and repeated co-culture with antigen-expressing target cells are used to model persistent activation, and to compare various functional readouts. We discuss the advantages and limitations of different assay setups to help identify the most suitable approach depending on the specific experimental needs. A robust and reproducible assay system is key to reliably screening novel CAR T cell designs and therapeutic interventions aimed at mitigating T cell exhaustion. We compare T cell functionality using multiple readout technologies, including impedance-based measurements using xCelligence for adherent and selected suspension cells and luminescence assays with luciferase-expressing target cells as well as flow cytometry. This allows us to highlight the strengths and constraints of each method in capturing functional decline and exhaustion phenotypes. Our findings support the use of standardized in vitro exhaustion models as valuable tools for the preclinical evaluation of next-generation CAR T cell therapies and combination strategies. Furthermore, our antigen-agnostic model enables evaluation of the impact of any T cell modulation approach on exhaustion dynamics. These models enable more predictive and efficient development of strategies to enhance therapeutic performance. Citation Format: Carla N. Castro, Veronica Bergo, Marina Zintchenko, Susana Minguet, Philipp Metzger, Cynthia Obodozie, Holger Weber. Development of an in vitro exhaustion assay for functional screening of CAR T cell enhancements abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 677.