Development of a Gene Editing Strategy to Enhance CAR-T Therapy Through Inducible IL-15 Expression at the PD-1 Locus

Background: Adoptive cell therapy (ACT) with genetically engineered T cells expressing chimeric antigen receptors (CARs) has emerged as a promising treatment option for refractory leukaemia or lymphoma patients. Despite its success in type B malignancies, CAR-T cell therapy still faces challenges such as toxicity, inactivation by the tumour microenvironment (TME), and low cell persistence in patients. Results: In this study, we developed an AAV6/Cas9-based knock-in platform to repurpose the PD-1 locus for inducible IL-15 expression. We initially demonstrated that anti-CD19 CAR-T cells lacking PD-1 expression exhibited reduced expansion capacity and overall fitness compared to control CAR-T cells. However, as expected, they showed an improved ability to lyse PDL1+ target cells compared to CAR-T WT cells. To enhance the fitness of PD-1 KO CAR-T cells, we generated PD-1KIL-15 CAR-T cells, which, in addition of been PD-1 KO, express IL-15 under the control of the PD-1 endogenous promoter. Compared to CAR T PD-1 KO cells, PD-1KIL-15 CAR-T cells I displayed improved phenotype, viability and metabolism... More importantly, they also demonstrated enhanced lytic capacity of PDL1+ CD19+ target cells, correlating with increased resistance to apoptosis and improved cell fitness. Conclusions: In summary, we present a new gene editing platform to generate 4th generation CAR-T cells (TRUCKs) that are PD-1 KO and express IL-15 upon T cell activation and/or exhaustion. This platform addresses the limitations associated with knocking-out PD-1 and those associated with sustained IL-15 cytokine expression. The same platform can be used to generate PD-1 KO TRUCKs targeting different antigens and expressing different cytokines under the control of the PD-1 gene promoter.