Figure 1.
Insertion of ASS and OTC enzymes enhances CAR-T cell proliferation in vitro. Biochrom amino acid analysis of (A) AML (n = 10) and (B) neuroblastoma (n = 11) plasma samples revealing low concentrations of arginine, cysteine, methionine, a-amino-n-butyric, aspartic acid, citrulline, and taurine at diagnosis. (C) Plasma arginine levels at diagnosis are significantly lower than healthy controls in AML and solid pediatric cancer patients. (D) Schematic of CAR-T constructs containing the basic anti-XX-CAR scFv-CD8 hinge-41BB-CD3ζ in concert with ASS or OTC or ASS+OTC enzyme. A truncated CD34 is expressed for CAR-T identification and purification. (E) Western blot of ASS and OTC expression, with β-actin control, in control and modified anti-GD2-CAR-T cells posttransduction and expansion. (Representative of n = 7.) (F) ASS enzyme activity, measured by citrulline catabolism, is increased in ASS expressing CAR-T cells compared with controls. (G) OTC enzyme activity, measured by citrulline production, is increased in OTC expressing CAR-T cells compared with controls. (H) Highest proliferation of 2.5 × 106 modified anti-GD2-Jurkat CAR-T cell line following engraftment into NOG-SCID mice treated with recombinant human arginase (BCT-100, daily 50 mg/kg IV). (I) Proliferation of modified human CAR-T cells is significantly increased in low arginine media conditions, in vitro, as measured by flow cytometry after 72 hours. (J) Proliferation of modified anti-GD2 or anti-CD33-CAR-T cells is significantly increased in neuroblastoma (LAN-1 neuroblastoma or K562 AML, 72-hour conditioned supernatants) conditions, in vitro as measured by flow cytometry after 72 hours. (K) Expansion of modified CAR-T cells is enhanced in low arginine vs excess arginine (RPMI10%) conditions as measured by flow cytometry after 72 hours. (L) Liquid chromatography mass spectometry analysis of modified CAR-T cells after proliferation in low arginine conditions (72 hours) compared with each modified version. Heatmap demonstrating metabolomic changes in arginine and proline metabolism, pyrimidine metabolism, and purine metabolism pathways.

Insertion of ASS and OTC enzymes enhances CAR-T cell proliferation in vitro. Biochrom amino acid analysis of (A) AML (n = 10) and (B) neuroblastoma (n = 11) plasma samples revealing low concentrations of arginine, cysteine, methionine, a-amino-n-butyric, aspartic acid, citrulline, and taurine at diagnosis. (C) Plasma arginine levels at diagnosis are significantly lower than healthy controls in AML and solid pediatric cancer patients. (D) Schematic of CAR-T constructs containing the basic anti-XX-CAR scFv-CD8 hinge-41BB-CD3ζ in concert with ASS or OTC or ASS+OTC enzyme. A truncated CD34 is expressed for CAR-T identification and purification. (E) Western blot of ASS and OTC expression, with β-actin control, in control and modified anti-GD2-CAR-T cells posttransduction and expansion. (Representative of n = 7.) (F) ASS enzyme activity, measured by citrulline catabolism, is increased in ASS expressing CAR-T cells compared with controls. (G) OTC enzyme activity, measured by citrulline production, is increased in OTC expressing CAR-T cells compared with controls. (H) Highest proliferation of 2.5 × 106 modified anti-GD2-Jurkat CAR-T cell line following engraftment into NOG-SCID mice treated with recombinant human arginase (BCT-100, daily 50 mg/kg IV). (I) Proliferation of modified human CAR-T cells is significantly increased in low arginine media conditions, in vitro, as measured by flow cytometry after 72 hours. (J) Proliferation of modified anti-GD2 or anti-CD33-CAR-T cells is significantly increased in neuroblastoma (LAN-1 neuroblastoma or K562 AML, 72-hour conditioned supernatants) conditions, in vitro as measured by flow cytometry after 72 hours. (K) Expansion of modified CAR-T cells is enhanced in low arginine vs excess arginine (RPMI10%) conditions as measured by flow cytometry after 72 hours. (L) Liquid chromatography mass spectometry analysis of modified CAR-T cells after proliferation in low arginine conditions (72 hours) compared with each modified version. Heatmap demonstrating metabolomic changes in arginine and proline metabolism, pyrimidine metabolism, and purine metabolism pathways.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal