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BACKGROUND. Chimeric antigen receptor (CAR) T cell immunotherapy has resulted in complete remission (CR) and durable response in highly refractory patients. However, logistical complexity and high costs of manufacturing autologous viral products limit CAR T cell availability. METHODS. We report the early results of a phase I/II trial in B cell acute lymphoblastic leukemia (B-ALL) patients relapsed after allogeneic hematopoietic stem cell transplantation (HSCT) using donor-derived CD19 CAR T cells generated with the Sleeping Beauty (SB) transposon and differentiated into cytokine-induced killer (CIK) cells. RESULTS. The cellular product was produced successfully for all patients from the donor peripheral blood (PB) and consisted mostly of [CD3.sup.+] lymphocytes with 43% CAR expression. Four pediatric and 9 adult patients were infused with a single dose of CAR T cells. Toxicities reported were 2 grade I and 1 grade II cytokine-release syndrome (CRS) cases at the highest dose in the absence of graft-versus-host disease (GVHD), neurotoxicity, or dose-limiting toxicities. Six out of 7 patients receiving the highest doses achieved CR and CR with incomplete blood count recovery (CRi) at day 28. Five out of 6 patients in CR were also minimal residual disease negative (MRD). Robust expansion was achieved in the majority of the patients. CAR T cells were measurable by transgene copy PCR up to 10 months. Integration site analysis showed a positive safety profile and highly polyclonal repertoire in vitro and at early time points after infusion. CONCLUSION. SB-engineered CAR T cells expand and persist in pediatric and adult B-ALL patients relapsed after HSCT. Antileukemic activity was achieved without severe toxicities. TRIAL REGISTRATION. ClinicalTrials.gov NCT03389035. FUNDING. This study was supported by grants from the Fondazione AIRC per la Ricerca sul Cancro (AIRC); Cancer Research UK (CRUK); the Fundacion Cientifica de la Asociacion Espanola Contra el Cancer (FC AECC); Ministero Della Salute; Fondazione Regionale per la Ricerca Biomedica (FRRB)., Introduction Substantial efforts over the past few years have led chimeric antigen receptor (CAR) T cell therapy to success in relapsed and refractory (r/r) B cell malignancies. The early concept [...]

CD19‐targeted chimeric antigen receptor T (CAR‐T) cell therapy has shown unprecedented results in patients with B cell relapsed/refractory acute lymphoblastic leukemia (R/R‐ALL) and B cell non‐Hodgkin lymphomas where no other curative options are available. In vivo monitoring of CAR‐T cell kinetics is fundamental to understand the correlation between CAR‐T cells expansion and persistence with treatment response and toxicity development. The aim of this study was to define a robust, sensitive, and universal method for CAR‐T cell detection using flow cytometry. We set up and compared with each other three assays for CD19 CAR‐T cell detection, all based on commercially available reagents. All methods used a recombinant human CD19 protein fragment recognized by the single‐chain variable fragment of the CAR construct. The two indirect staining assays (CD19his + APC‐conjugated antihistidine antibody and CD19bio + APC‐conjugated antibiotin antibody) showed better sensitivity and specificity compared with the direct staining with CD19‐FITC, and CD19his had a better cost‐effective profile. We validated CAR detection with CD19his with parallel quantitative real‐time polymerase chain reaction data and we could demonstrate a strong positive correlation. We also showed that CD19his staining can be easily included in a multicolor flow cytometry panel to achieve additional information about the cell phenotype of CAR‐T cell positive subpopulations. Finally, this method can be used for different anti‐CD19 CAR‐T cell products and for different sample sources. These data demonstrate that detection of CAR‐T cells by CD19his flow cytometry staining is a reliable, robust, and broadly applicable tool for in vivo monitoring of CAR‐T cells. [ABSTRACT FROM AUTHOR]