Figure 2. High affinity FRβ CAR T cells demonstrate greater in vitro reactivity against FRβ⁺ cell lines compared to low affinity FRβ CAR T cells.

(a) IFNγ secretion following overnight co-culture of LA-FRβ, HA-FRβ, or control CAR T cells with the indicated cell lines. Error bars represent mean ± SEM of n ≥5 independent experiments each performed in triplicate. (b) CD69 expression on CAR T cells following overnight co-culture with the indicated target cells. Live, CD3⁺ CAR⁺ flow cytometry gates were used to assess percent CD69⁺ CAR T cells. Error bars represent mean ± SEM of n=4 independent experiments each performed in triplicate. (Note: unstimulated CAR⁺ T-cells have ~20% CD69⁺ cells at baseline as revealed in the Media control). (c) High lytic activity from HA-FRβ CAR T cells against FRβ⁺ cell lines compared to LA-FRβ. T cells and fLuc⁺ target cells were co-cultured at the indicated E:T ratios. Percent lysis was assessed by residual target cell luminescence following overnight culture. Error bars represent mean ± SEM of n ≥5 independent experiments each performed in triplicate. Media indicates T cells cultured without target cells. GFP and/or CD19-28Z CAR T cells were used as controls. SEM – standard error, fLuc – firefly luciferase, LA – low affinity FRβ, HA – high affinity FRβ.