Webster and Xiong et al. placed an anti-CD19 CAR (CAR19) construct under the control of AP1-NF-kB or STAT3 promoters to generate a self-driving anti-CD19 CAR system. AP1-NF-kB-CAR19 maintained a low level of CAR19 expression on the T cell surface, which rapidly increased in the presence of the CD19 antigen on target cells. AP1-NF-kB-CAR19 showed comparable tumor cytotoxicity, superior expansion, and lower exhaustion compared to constitutively expressed CAR19 in vitro and in vivo. Dominant-negative TGFβ receptor II (TGFβRIIdn) also expressed under the AP1-NF-kB promoter effectively countered TGFβ-mediated suppression.
Contributed by Shishir Pant
ABSTRACT: Chimeric antigen receptor (CAR) T cells typically use a strong constitutive promoter to ensure maximal long-term CAR expression. However, recent evidence suggests that restricting the timing and magnitude of CAR expression is functionally beneficial, whereas constitutive CAR activation may lead to exhaustion and loss of function. We created a self-driving CD19-targeting CAR, which regulates its own function based on the presence of a CD19 antigen engaged by the CAR itself, by placing self-driving CAR19 constructs under transcriptional control of synthetic activator protein 1 (AP1)-nuclear factor κB (NF-κB) or signal transducer and activator of transcription (STAT)5 promoters. CD19 antigen-regulated expression was observed for self-driving AP1-NFκB-CAR19, with CAR19 upregulation within 18 h after exposure to target CD19, and corresponded to the level of tumor burden. Self-driving CAR-T cells showed enhanced tumor-dependent activation, expansion, and low exhaustion in vitro as compared to constitutively expressed EF1α and murine stem cell virus (MSCV) CARs and mediated tumor regression and survival in Raji-bearing NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Long-term CAR function correlated with upregulated CAR expression within 24 h of exposure to tumor antigen. The self-driving AP1-NFκB-CAR19 circuit was also used to inducibly express dominant-negative transforming growth factor β receptor II (TGFBRIIdn), which effectively countered the negative effects of TGF-β on CAR-T activation. Thus, a self-driving CAR approach may offer a new modality to express CAR and auxiliary proteins by enhancing CAR-T functional activity and limiting exhaustion.
Author Info: (1) Translational Research and Development, Lentigen, a Miltenyi Biotec Company, 1201 Clopper Road, 20878 Gaithersburg, MD, USA, United States; Research and Development, Miltenyi B
Author Info: (1) Translational Research and Development, Lentigen, a Miltenyi Biotec Company, 1201 Clopper Road, 20878 Gaithersburg, MD, USA, United States; Research and Development, Miltenyi Biotec, Friedrich-Ebert-Stra§e 68, 51429 Bergisch Gladbach, Germany. Electronic address: brianwe@miltenyibiotec.de. (2) Translational Research and Development, Lentigen, a Miltenyi Biotec Company, 1201 Clopper Road, 20878 Gaithersburg, MD, USA, United States. (3) Translational Research and Development, Lentigen, a Miltenyi Biotec Company, 1201 Clopper Road, 20878 Gaithersburg, MD, USA, United States. (4) Translational Research and Development, Lentigen, a Miltenyi Biotec Company, 1201 Clopper Road, 20878 Gaithersburg, MD, USA, United States. (5) Translational Research and Development, Lentigen, a Miltenyi Biotec Company, 1201 Clopper Road, 20878 Gaithersburg, MD, USA, United States. (6) Translational Research and Development, Lentigen, a Miltenyi Biotec Company, 1201 Clopper Road, 20878 Gaithersburg, MD, USA, United States; Seattle Children's Research Institute and Department of Pediatrics, University of Washington School of Medicine, 4800 Sand Point Way NE, 98105 Seattle, WA, United States. (7) Translational Research and Development, Lentigen, a Miltenyi Biotec Company, 1201 Clopper Road, 20878 Gaithersburg, MD, USA, United States. Electronic address: brianwe@miltenyibiotec.de. (8) Translational Research and Development, Lentigen, a Miltenyi Biotec Company, 1201 Clopper Road, 20878 Gaithersburg, MD, USA, United States. Electronic address: brianwe@miltenyibiotec.de.
