Fig. 1

A Lentiviral components of RB-340-1–RB-340-1 includes two lentiviral (LV) constructs. LV#1 (HER2-TEV) encodes an anti-HER2 (4D5 clone) scFv combined to the CD28 and CD3ζ co-stimulatory domains, the TEV protease and PD-1sg targeting the TSS of the endogenous PD-1 gene. LV#2 (LdCK) encodes LAT, fused to dCas9-KRAB via a TEV-cleavable sequence (TCS). RB-340-1 contains also two extracellular tags: Q8 part of LdCK and tNGFR part of HER2-TEV. In early experiments, GFP and mCherry were used for detection of LdCK or HER2-TEV respectively. B Figurative representation of RB-340-1–RB-340-1 conditionally suppresses expression of PD-1 upon activation of the CAR-T cells, while respective controls such as conventional HER2 CAR-T or RB-340-1 technical control (cRB-340-1) without targeting guide are not. C Mechanism of activation of HER2-TEV/LdCK CRISPRi platform—activation of HER2 CAR brings TEV in proximity of LdCK releasing dCas9-KRAB for nuclear translocation to the PD-1 TSS and conditionally and reversibly suppress PD-1 expression. The figure represents the CRISPRi logic at steady-state levels of LdCK expression. However, LdCK expression varies according to the physio-metabolic status of individual cells resulting in variable degree of transcriptional activity and consequently different substrate availability for TEV cleavage. This in turn contributes to conditionality as shown in Fig. 2A