ASS and OTC enzymes increases CAR-T proliferation in vivo and activity against solid or hematological cancer. Specificity of modified CAR-T cell killing is unchanged by the addition of ASS or OTC enzymes, against (A) neuroblastoma or (B) AML target cells. Chromium (51Cr) release assays demonstrating antigen-specific killing of tumor cell targets by CAR-T cells. Minimal killing is seen against tumor cells that do not express the corresponding antigen. (C) Increased CAR-T proliferation correlates with a decreased AML burden in xenograft bone marrow. Linear regression line shown. (D) Increased proliferation of ASS-modified anti-CD33 CAR-T cells in NOG-SCID mice with established engraftment of AML blasts (HL60). (E) Decrease in established AML (HL60) engraftment in murine cell line xenografts treated with anti-CD33-ASS CAR-T cells. Data from day of euthanization. Dotted red line showing mean AML engraftment (43%) in the bone marrow of untreated mice. (F) Kaplan-Meier curves of GD2+ tumor murine xenografts, showing increased survival following treatment with CAR-T constructs. All remaining live mice were euthanized on day 57 for analysis. P value compared with control. (G) Increased proliferation of ASS, OTC, or ASS+OTC modified anti-GD2 CAR-T cells in nude mice with established s/c GD2+ tumors. Data from day of euthanization. (H) Anti-GD2-OTC CAR-T cells significantly decrease tumor growth in nude mice. (I) Kaplan-Meier curves of GD2+ tumor murine xenograft survival, which is increased with OTC-modified CAR-T constructs. All remaining live mice were euthanized on day 57 for analysis. P value compared with control. (J) Linear regression demonstrating the magnitude of anti-GD2-CAR-T cell proliferation correlates with GD2+ tumor xenograft survival. (K) Serum in tumor-bearing xenografts is deplete in arginine. Arginine concentrations are restored toward normal in response to CAR-T cell antitumor activity.