Abstract 3910: Cellular profiling of leukapheresis and infusion products of patients enrolled in CD19 CAR T cell therapy identifies biomarkers associated with clinical outcomes
Authors: Chour Tony , Hirayama Alexandre , Zheng Ye , Sheih Alyssa , Zhuang Summer , Wilson Ashley , Wu Vicky , Gottardo Raphael , Turtle Cameron , Gardner Rebecca , Newell Evan 
Published: June 2022

Abstract
CD19 CAR T cell therapy has emerged as a promising therapy in the treatment of B cell malignancies, such as B cell acute lymphoblastic leukemia (B-ALL). However, questions remain regarding the heterogeneity of the CAR T cell infusion products that lead to differential clinical outcomes and side effects, including cytokine release syndrome and neurotoxicity. The purpose of this study is to identify biomarkers and T cell subpopulations within 1) leukapheresis samples of patients receiving CD19 CAR T cell therapy and 2) pre-infusion manufactured CAR T cell products to identify correlates with clinical outcomes. We hypothesize that different manufacturing practices used to generate CAR T cells from the patient’s leukapheresis T cells drive heterogeneity within the infusion products that drive differential patient outcomes, and that cell intrinsic properties within the leukapheresis T cells drive phenotypic dispositions observed in the infusion products.
Leukapheresis samples and pre-infusion CAR T cells were obtained from pediatric and adult B-ALL patients enrolled in CD19 CAR T cell clinical trials. Briefly, leukapheresis T cells from pediatric patients were CD4/8 sorted and then stimulated with CD3/CD28 beads followed by CAR transduction. Pediatric CD4 CAR T cells were cultured with IL-7 + IL-15 while CD8 CAR T cells were cultured with IL-2 + IL-15. CAR T cells from adults were stimulated with LCLs expressing CD19. To identify T cell subpopulations and biomarkers leukapheresis T cells and CAR T cells from each patient were stained with two mass cytometry panels: 1) a phenotype panel of antibodies to characterize surface markers, transcription factors and chemokine receptors and 2) a functional panel of antibodies specific for cytokines to broadly characterize the secretion profile of leukapheresis and CAR T cells upon stimulation with PMA and Ionomycin. Additional multi-omic profiling was performed with CITE-seq and 10X Chromium on leukapheresis samples and CAR T cells of select adult patients to provide in depth analysis on transcriptomic drivers of phenotypes observed within CAR T cell infusion products.
We hierarchically clustered leukapheresis T cells and CAR T cells and correlated the abundance of T cell subclusters within each patient to their neurotoxicity grade (0-5). We determined that CD4 CAR T cells that secrete elevated levels of IL-3 upon stimulation correlate with an increased risk of severe neurotoxicity (designated as grades 3-5) within our pediatric cohort. Identification of IL-3 as a driver of neurotoxicity within the CD4 infusion products may inform strategies to improve manufacturing practices used to generate safer CAR T cell products.
Citation Format: Tony Chour, Alexandre Hirayama, Ye Zheng, Alyssa Sheih, Summer Zhuang, Ashley Wilson, Vicky Wu, Raphael Gottardo, Cameron Turtle, Rebecca Gardner, Evan Newell. Cellular profiling of leukapheresis and infusion products of patients enrolled in CD19 CAR T cell therapy identifies biomarkers associated with clinical outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3910.