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

Abstract BackgroundAdoptive cell therapy (ACT) with genetically engineered T cells expressing chimeric antigen receptors (CARs) has emerged as a promising treatment option for patients with refractory leukaemia or lymphoma. Despite its success in type B malignancies, CAR-T cell therapy still faces some challenges such as toxicity, functional suppression by the tumour microenvironment (TME), and poor persistence in treated patients.MethodsThis study employed a second-generation CD19-targeting CAR construct to generate engineered CAR-T cells with enhanced functionality through precise genome editing. Using CRISPR/Cas9 technology, the PDCD1 gene was to mitigate T cell exhaustion, and in a parallel knock-in strategy, an IL-15 transgene was inserted at the PDCD1 locus. Gene editing was performed via electroporation of RNP complexes, with AAV6 vectors used for homology-directed IL-15 integration. Editing efficiency and off-target activity were assessed by flow cytometry, Sanger sequencing, ICE, and CAST-Seq. Functional characterization included bulk RNA sequencing, metabolic profiling using Seahorse technology, and cytotoxicity assays against CD19⁺ target cells.ResultsWe initially demonstrated that αCD19 CAR-T cells lacking PD-1 expression (PD-1 KO) exhibited reduced expansion capacity and overall fitness compared to control CAR-T cells but showed a superior cytotoxicity against PDL1⁺ target cells. To address the impaired fitness of PD-1 KO CAR-T cells, we generated PD-1KIL-15 CAR-T cells, which combine PD-1 KO with the expression of 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 displayed improved phenotype, viability, and metabolism. More importantly, they also demonstrated enhanced cytolytic capacity of PDL1⁺ CD19 + target cells, which correlated with increased resistance to apoptosis and improved cell fitness.ConclusionsIn summary, we present a next 4th generation CAR-T cells platform (TRUCKs) that integrates PD-1 deletion with the inducible expression of IL-15 upon T cell activation and/or exhaustion. This strategy 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 locus.