Flow cytometric assays for CAR T cell manufacturing and patient immunomonitoring, comprising specific CAR detection reagents, stabilized pre-mixed cocktails, and automated data acquisition and analysis

Abstract

Background & Aim Unprecedented efficacy in targeting cancer has been demonstrated using adoptive cellular therapy with genetically engineered chimeric antigen receptor (CAR) T cells. However, the CAR T cell manufacturing process is highly complex and has extensive demands on personnel and infrastructure, which is still a major obstacle for their routine clinical use. Using a device for automated cell processing, the CliniMACS Prodigy, helps to overcome these hurdles, allowing generation of CAR T cells in a single automated and closed system. For assessment of CAR T cells during the manufacturing process and also for patient immunomonitoring, we aimed to establish a set of flow cytometric assays prepared from recombinant antibodies along with CAR detection reagents, e.g. for CD19, CD22, or BCMA. These assays were to be complemented by stabilized pre-mixed cocktails of those and additional tools for automated data acquisition and analysis. Methods, Results & Conclusion Our flow cytometric assays were developed for 1) in-process control, QC release testing, and CAR T cell phenotying during cell manufacturing, and 2) for determination of CAR T cell persistance and phenotyping during patient immunomonitoring. Currently already used in clinical trials, these assays allow to determine the general immune cell composition, the CAR transduction efficiency, and functional CAR T cell phenotyping like differentiation, activation, or exhaustion status. To further improve assay standardization, to simplify and speed up the IPC/QC process, and to reduce risks from manual handling, we developed stabilized pre-mixed antibody cocktails for these flow assays, featured in a single-tube format. In addition, so-called Express Modes were programmed that allow an automated sample acquisition and analysis on MACSQuant Analyzers. These Express Modes feature predefined experiment settings, automate the sample measurement, and apply a fully automated gating strategy with computational gate adjustment. In conclusion, elaborate flow assays specifically designed for CAR T cells, run with specific CAR detection reagents and high-quality recombinant antibodies in a stabilized pre-mixed format, along with fully automated flow analysis, provide a robust assessment for cell manufacturing and patient immunomonitoring. This will help with establishing complex individualized therapies and will enable us to understand in greater detail the phenotypic changes occuring throughout the life time of a CAR T cell.