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4851 Novel Method to Evaluate the Quantitative Immune Functionality of CAR T-Cells to Analyze the Localization Change of CAR Molecules

Program: Oral and Poster Abstracts
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
Monday, December 9, 2024, 6:00 PM-8:00 PM

Hiroshi Yasui, MD, PhD1,2, Keiji Hirano3*, Asako Kobayashi1*, Lihua Li, PhD1*, Takuya Takahashi3*, Margarita Artemenko, MD, PhD3*, Naotaka Noda, PhD3*, Daichi Sadato, PhD4*, Kyoko Haraguchi, MD, PhD5*, Ayako Arai, MD, PhD2, Kohzoh Imai, MD, PhD6*, Noriko Doki7, Masatoshi Yanagida, PhD3* and Tomohiro Ishigaki, MD, PhD8*

1The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan
2Department of Hematology and Oncology, St. Marianna University School of Medicine, Kawasaki, Kanagawa, Japan
3Central Research Laboratories, Sysmex Corporation, Kobe, Japan
4Clinical Research and Trials Center, Tokyo Metropolitan Cancer and Infectious Diseases Center, Komagome Hospital, Tokyo, Japan
5Division of Transfusion and Cell Therapy, Tokyo Metropolitan Cancer and Infectious Diseases Center, Komagome Hospital, Bunkyo-ku, Tokyo, Japan
6Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan
7Hematology Division, Tokyo Metropolitan Cancer and Infectious Diseases Center, Komagome Hospital, Tokyo, Japan
8Department of Laboratory Medicine, IMSUT Hospital, The Institute of Medical Science, The University of Tokyo, Minato-Ku, Tokyo, JPN

Background: Chimeric antigen receptor (CAR) T cells, genetically synthesized to reprogram T cells to recognize tumor antigen inducing intracellular signaling to destruct tumor cells, is a ground approach to hematological malignancies. As either loss of immune function or exhaustion in CAR T cells is a recurrence mechanism in patients with hematological malignancies who receive CAR T-cell therapy, evaluating CAR T-cell immune functions is a crucial clinical need. In this study, we used molecular imaging flow cytometry (MI-FCM) to develop a new evaluation method to quantitatively determine the immune functionality of CAR T-cells to analyze the change in CAR molecule localization.

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.

*signifies non-member of ASH