Brief Research Report: In-depth Immunophenotyping Reveals Stability of CD19 CAR T-cells Over Time

Ivan Odak¹ Lâle May Bayir^{1, 2} Lennart Riemann^{1, 3} Ruth Sikora^{1, 2} Jessica Schneider^{1, 2} Yankai Xiao¹ Nora Möhn⁴ Thomas Skripuletz⁴ Gernot Beutel² Matthias Eder² Arnold Ganser² Reinhold Forster¹ Christian R. Schultze-Florey^{1, 2*} Christian Koenecke^{1, 2*}

• ¹Institute of Immunology, Hannover Medical School, Germany
• ²Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Germany
• ³Department of Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, Germany
• ⁴Department of Neurology, Hannover Medical School, Germany

Variability or stability might have an impact on treatment success and toxicity of CD19 CAR T-cells. We conducted a prospective observational study of 12 patients treated with Tisagenlecleucel for CD19 + B-cell malignancies. Using a 31-color spectral flow cytometry panel, we analyzed differentiation stages and exhaustion markers of CAR T-cell subsets prior to CAR T-cell infusion and longitudinally during 6 months of follow-up. The majority of activation markers on CAR T-cells showed stable expression patterns over time and were not associated with response to therapy or toxicity. Unsupervised cluster analysis revealed an immune signature of CAR T-cell products associated with the development of immune cellassociated neurotoxicity syndrome. Warranting validation in an independent patient cohort, in-depth phenotyping of CAR T-cell products as well as longitudinal monitoring post cell transfer might become a valuable tool to increase efficacy and safety of CAR T-cell therapy.