Adoptively transferred CAIX-CAR T cells show limited peripheral persistence and induce both humoral and cellular immunity. (A) Kinetics of absolute numbers of circulating CAIX-CAR T cells (Å) and CAIX-CAR DNA copies (■) in blood samples of patients 4 and 5 obtained before, during, and after immunogene therapy with CAIX-CAR⁺ T cells. Treatment periods are indicated at the top of each panel. Individual CAIX-CAR infusions are represented by dots; IL-2 treatment by lines; (B) Kinetics of HACA (○; expressed as ng/μL at left y-axis) blood levels and capacity of patient plasma to inhibit CAIX-CAR mediated cytolysis (▴; expressed as percent inhibition compared with pretreatment serum at right y-axis). Data are shown for 2 representative patients (patient 4 and 5), showing, respectively, 1 development of HACA and serum inhibitory capacity (patient 4 representative for patients 1-4, 6, and 7), and no development of HACA and inhibitory activity (patient 5 representative for patients 5, 8-11). Negative tests were repeated 2-3× for confirmation. Inhibition of CAIX-CAR–mediated cytolysis was expressed relative to cytolysis in autologous pretreatment serum. Treatment intervals are indicated by horizontal lines in the top of each panel. (C) Kinetics of cellular anti– CAIX-CAR immune reactivity in patients 4 and 5. PBMC obtained before, during, and after treatment were cocultured for 5 weeks on autologous CAIX-CAR T cells as described (see Figure 1) and subsequently assayed for anti–CAIX-CAR T-cell reactivity in a cytotoxicity assay using ⁵¹Cr-labeled autologous CAIX-CAR T cells as “positive control” target cells (•) and autologous NTD cultured T cells (○) as “negative control” target cells. Percent cytolysis of target cells is expressed as weighted mean of specific cytolysis at effector cell to target cell ratio of 20:1 (WMSL_{[ET 20:1]}). Representative experiments are shown.