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

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.