Insertion of arginase enzymes enhances CAR T-cell activity in vitro and in vivo. (A) Schematic of CAR T-cell constructs containing the basic anti-CD33-CAR scFv-CD8 hinge-41BB-CD3ζ or the basic anti-GD2-CAR scFv with CH2CH3 spacer-CD8 hinge-41BB-CD3ζ, in conjunction with Arg1 or Arg2. A truncated CD34 is expressed for CAR T identification and purification. (B) Representative expression of Arg1 or Arg2 by anti-GD2 CAR T cells as demonstrated by western blot, with actin loading control. Representative of N = 3 individual donors. (C) Arginase enzyme activity, measured by catabolism of arginine into ornithine and urea, is increased in Arg1- or Arg2–modified anti-GD2 or anti-CD33 CAR T cells compared with unmodified CAR T cells. Data from n = 20 human CAR T-cell donors. (D) Arginase enzyme insertions lead to no detrimental effect on CAR T-cell–specific cytotoxicity against anti-GD2⁺ tumor cell targets, as measured by chromium release. (E) Heatmap of the differential gene expression analysis comparing Arg1- or Arg2–modified anti-GD2 CAR T-cells with unmodified anti-GD2 CAR T-cells from n = 3 human donors. Top 100 genes shown. (F) Arginase–modified anti-GD2 CAR T-cells have enhanced proliferation in the presence of anti-GD2 tumor target cells, compared with unmodified CAR T cells, in vitro. CAR T-cell proliferation was measured by flow cytometry after 72 hours. (G) Arginase–modified anti-GD2 CAR T cells have enhanced proliferation under low-arginine (75% free) or tumor-conditioned media culture conditions, compared with unmodified CAR T cells, in vitro. CAR T-cell proliferation was measured by ³H-thymidine uptake after 96 hours. (H) Arginase–modified anti-GD2 CAR T cells have enhanced proliferation when culture conditions are supplemented with 100μM arginine, compared with unmodified CAR T cells, in vitro. CAR T-cell proliferation was measured by ³H-thymidine uptake after 96 hours. (I) Diagram illustrating subcutaneous engraftment of GD2⁺ KELLY cell line in nude mice, before the administration of CAR T cells on Day +10. (J) Proliferation of unmodified control or Arg1- or Arg2–modified anti-GD2 CAR T cells in nude mice with established engraftment of GD2⁺ tumor cells (KELLY). Data from the day of euthanization. CAR T-cell copies determined by qPCR. (K) Arg1- or Arg2–modified anti-GD2 CAR T cells reduce tumor volume, compared with those treated with unmodified CAR T cells. qPCR, quantitative polymerase chain reaction.