Self-driving armored CAR-T cells overcome a suppressive milieu and eradicate CD19+Raji lymphoma in preclinical models

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 kappa B (NF-kappa B) or signal transducer and activator of transcription (STAT)5 promoters. CD19 antigen-regulated expression was observed for self-driving AP1-NF kappa B-CAR19, with CAR19 up-regulation within 18 h after exposure to target CD19, and cor-responded 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 EF1 alpha and murine stem cell virus (MSCV) CARs and mediated tumor regression and survival in Raji-bearing NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (NSG) mice. Long-term CAR function correlated with upregulated CAR expression within 24 h of exposure to tumor antigen. The self-driving AP1-NF kappa B-CAR19 circuit was also used to inducibly express dominant-negative transforming growth factor beta receptor II (TGFBRIIdn), which effectively countered the negative effects of TGF-beta on CAR-T activation. Thus, a self-driving CAR approach may offer a new modality to express CAR and auxil-iary proteins by enhancing CAR-T functional activity and limiting exhaustion.

Automated summary

Restricting the timing and magnitude of CAR expression can be functionally beneficial, whereas constitutive CAR activation may lead to exhaustion. A self-driving CAR system has been created to regulate its own function based on the presence of a CD19 antigen engaged by the CAR itself.