Session: 711. Cell Collection and Manufacturing of HSPCs, CAR-T Cells, and Other Cellular Therapy Products: Poster III
Hematology Disease Topics & Pathways:
Research, Translational Research, Assays, Immunology, Biological Processes, Technology and Procedures
Methods: Eight CAR T-cell clones were constructed via genetically modifying peripheral blood mononuclear cells from eight healthy donors (21–69 years old) using lentivirus to express an anti-CD19 CAR (FMC63-41BBz), as previously described by Kochenderfer et al. (J Immunol, 2009). The cytotoxic activity of these clones was then evaluated in vitro. Each clone was co-cultured with K562-CD19 cells that stably expressed the reporter gene encoding firefly luciferase (FLuc) for 15 h to assess the killing activity using a luciferase-mediated bioluminescence imaging assay. These clones were then stimulated with a fluorescently labeled CD19 protein for an hour. The change in CAR localization was determined via analyzing the intensity, area, and distribution (spot counts) of CAR expression in detail using a MI-FCM. The antitumor activity of some clones of CAR T-cells in vivo using a xenotransplantation model. NOG mice were injected intravenously with the CD19 positive human B-cell precursor leukemia cell line (NALM6) transfected with the FLuc reporter gene was also determined. Each CAR T-cell clone (5×106) cells was injected intravenously seven days after NALM6 injection. To assess antitumor activity, tumor volume was quantified using the IVIS imaging system to detect luminescent intensity.
Results: We found CAR molecules on cell surface aggregates in the immune response and detected the concentration as a decrease in CAR expression area. The percentage of CAR T-cells with single-spot CAR concentration was significantly correlated with cytotoxic activity (Pearson R = 0.83). We also confirmed that the clone with a higher percentage could demonstrate antitumor response and prolong the survival in xenotransplantation mouse models compared with non-treated mice, whereas the clone with the lowest percentage did not show these effects. We also confirmed that this method could be applied to CAR T-cell therapy products.
Conclusions: Quantifying the superficial CAR T-cell concentration using MI-FCM could be useful for evaluating their immune function.
Disclosures: Yasui: Sysmex Corporation: Research Funding; TokioTHERA Holdings: Current equity holder in private company; Kyowa Kirin Co., Ltd.: Research Funding; Shionogi & Co., Ltd.: Research Funding; DAIICHI SANKYO, INC.: Research Funding. Hirano: Sysmex Corporation: Current Employment, Current equity holder in publicly-traded company. Takahashi: Sysmex Corporation: Current Employment, Current equity holder in publicly-traded company. Artemenko: Sysmex Corporation: Current Employment, Current equity holder in publicly-traded company. Noda: Sysmex Corporation: Current Employment, Current equity holder in publicly-traded company. Arai: Sanofi K.K.: Honoraria; Asahi Kasei Pharma Corporation: Research Funding; Shionogi & Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Novartis Pharma K.K.: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Honoraria, Research Funding; Ono Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Kyowa Kirin Co., Ltd.: Honoraria, Research Funding; Eisai Co., Ltd.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Bristol-Myers Squibb K.K.: Honoraria; AbbVie GK: Honoraria; Janssen Pharmaceutical K.K: Honoraria. Yanagida: Sysmex Corporation: Current Employment, Current equity holder in publicly-traded company.