m909 CAR T cells are reactive against cell surface FRβ on engineered C30-FRβ cell line. To first test the functionality of m909 CARs, the antigen-negative ovarian cancer cell line C30 was transduced to stably overexpress human FRβ cDNA. Cocultures were performed at an E:T ratio of 1:1 unless otherwise noted. Control MOV19-28Z CAR T cells are specific for FRα and do not express GFP. Control GFP T cells express only GFP. Error bars represent mean ± SEM. (A) FRβ expression on engineered C30-FRβ was detected by flow cytometry using biotinylated m909-IgG (black histogram). For comparison, the unmodified parental C30 cells were used as a control (gray histogram). (B) Antigen-specific IFN-γ (IFNg) production by m909 CAR T cells as detected by ELISA from 24-hour coculture supernatants. (C) m909 CAR⁺ T cells upregulate surface CD69 expression following 24-hour exposure to C30-FRβ. The m909 CAR⁺ cells are identified by GFP expression (y-axis). (D) m909-Z and m909-28Z CAR T cells proliferate in response to C30-FRβ. PKH26 dilution in labeled T cells was measured by flow cytometry after 5 days in coculture. Percentage of CD3⁺ cells proliferating (diluted PKH26 compared to day 0) is quantified. P values represent significant differences compared to MOV19-28Z CAR T cells. (E) m909-Z and m909-28Z exhibit specific lysis of C30-FRβ. Target cells were transduced to express fLuc and cocultured with CAR T cells at E:T ratios of 10:1, 3:1, and 1:1. Residual luciferase signal was determined after 18 hours. Percent lysis was determined by luminescence comparison with untreated target wells. (F) m909-Z and m909-28Z exhibit degranulation on coculture with C30-FRβ. CD107a/b surface expression was measured after 5 hours of coculture. CAR⁺ cells are identified by GFP expression (y-axis). Percentage of CAR⁺ cells with positive staining for CD107a/b is quantified to the right. P values represent significant increases compared to MOV19-28Z control T cells. *P < .05; **P < .01; ***P < .001.