Abstract 6333: A high-parameter mass cytometry panel for the functional characterization of CAR T cells
Authors: Mahamed Deeqa , Awong Geneve , Selvanatham Thiru 
Published: March 2024

Abstract
High-parameter flow cytometry provides a powerful tool for in-depth analysis of CAR T cell therapy, from characterizing CAR T cell populations to understanding their interactions within the tumor microenvironment. This information is crucial for advancing CAR T cell therapies and improving their effectiveness in treating cancer.
To address the need for deep immunoprofiling at all stages of CAR T cell therapy, we created a robust and comprehensive mass cytometry panel of antibodies against 44 cell surface and intracellular markers. This assay allows for comprehensive characterization of CAR T cell features including surface marker expression, activation state, cytokine production, and differentiation status. These data will provide researchers with a better understanding of the phenotype of the CAR T cells used in therapy and can guide optimization efforts to enhance expansion, persistence, and self-renewal. For example, newer-generation CAR T cells (T cells redirected for antigen-unrestricted cytokine-initiated killing, or TRUCKs) that are engineered to produce cytokines such as IL-2 to enhance their own survival can be evaluated in vitro to select CAR constructs with the most potent anti-tumor activity.
The lyophilized and validated 30-marker Maxpar® Direct™ Immune Profiling Assay™ enables comprehensive characterization of immune cell populations in both whole blood and PBMC. The 7-marker Maxpar Direct T Cell Expansion Panel 3 (OX40, TIGIT, CD69, PD-1, Tim-3, ICOS, and 4-1BB) and the Maxpar Direct Basic Activation Expansion Panel (CD107a, IL-2, TNFα, IFNγ, perforin, and granzyme B) are add-on modules that can further characterize CAR T cell exhaustion, degranulation, cytokine production, and cytotoxicity against target cells. We also compared CAR T detection options for sensitivity and specificity using a CD19 CAR-transduced cell line spiked into healthy donor PBMC. Indirect staining with Miltenyi Biotec biotinylated CD19 CAR Detection Reagent followed by anti-biotin- or streptavidin-conjugated metal tags was compared against a directly conjugated or biotinylated anti-G4S linker antibody. We included sample multiplexing using 6 metal-tagged CD45 antibodies to address the need to minimize batch effects, crucial to multi-site and longitudinal studies. Samples were acquired on a CyTOF® XT™ instrument, and preliminary data analysis was performed using Maxpar Pathsetter™ for automated enumeration of immune cells.
By combining the lyophilized single-tube Maxpar Immune Profiling Assay and Expansion Panels with specific detection of CAR T cells, this assay enables researchers to analyze multiple key parameters simultaneously at the single-cell level at all stages of CAR T therapy development.
For Research Use Only. Not for use in diagnostic procedures.
Citation Format: Deeqa Mahamed, Geneve Awong, Thiru Selvanatham. A high-parameter mass cytometry panel for the functional characterization of CAR T cells [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(6_Suppl):Abstract nr 6333.