Mejora de la Inmunoterapia CAR frente a neoplasias tipo B refractarias mediante edición genómica

Abstract

Adoptive Cell Therapy (ACT) with genetically modified T cells expressing Chimeric Antigen Receptor (CAR) has emerged as a promising option for patients with refractory leukemia or lymphoma. Despite its success in B-cell malignancies, CAR-T cell therapy still faces challenges such as toxicity, inactivation by the tumour microenvironment (TME), and low cell persistence in patients. In this context, the PD-1/PD-L1 axis plays a crucial role in various tumour types. The main aim of this Doctoral Thesis is to develop a gene editing (GE) strategy to generate CAR-T cells capable of overcoming inhibitory signals induced by PD-L1 expression in tumor cells. The initial approach involved PD-1 deletion in CAR-T cells, with a detailed characterization of their phenotypic and metabolic features. The CAR utilized in this Thesis, CAR ARI-0001, is a second-generation CAR specific for CD19, developed in collaboration with Dr. Manel Juan. PD-1 elimination was achieved through CRISPR/Cas9 ribonucleoprotein (RNP) delivery targeting exon 1 of PDCD1 gene. The study demonstrates that CRISPR/Cas9 delivered as RNP enables highly efficient PD-1 depletion (80%) without significant alterations in T cell phenotype or generating large deletions. Subsequent cellular and metabolic characterizations of CAR-T PD- 1 KO cells revealed a reduced proliferative capacity and lower available respiratory capacity (SRC), indicating a decreased mitochondrial oxidative potential. However, PD-1 editing induced T cells toward memory populations. Additionally, CAR-T PD-1 KO cells exhibited enhanced persistence and cytotoxicity capacity when were co-cultured with PD-L1-expressing target tumor cells compared to CAR-T WT cells, although expression of exhaustion markers remained consistent. These findings suggest that while PD-1 editing improves CAR-T cell persistence in the presence of PD-L1, it simultaneously induces a fitness reduction (proliferative capacity and metabolic impairment) that needs to be improved. Based on these results, it was proposed that, in addition to PD-1 elimination, controlled expression of some soluble factors enhancing fitness is needed like an additional step in the GE strategy, focusing on the cytokine IL-15. IL-15 plays a crucial role in the cytotoxic response against tumor cells, including improving T cell proliferation and metabolism. Due to its high biological activity across multiple targets, controlling its expression is essential. Therefore, a CRISPR/Cas9 – Homology-Directed Repair (HDR) platform was aimed at expressing a gene of interest (IL-15, other cytokines, antibodies, etc.) at the PD-1 locus. This platform could allow not only PD-1 abrogation but also the expression of genes further enhancing CAR-T cell activity. Additionally, these enhancing genes would be expressed following the PD-1 gene pattern, ensuring a controlled expression and avoiding the negative effects from constitutive expression. To achieve this objective, various DNA donor delivery strategies (IDLVs, AAVs, PCR product DNA, and ADNcs) were evaluated, and optimizations of IDLVs were performed using an eGFP-expressing donor through a strong promoter directed to a locus (TRAC). The results showed that AAV6 was the most efficient vector for delivering DNA donors to primary T cells, with efficiencies around 60% for specific integrations in the generated pdTRUCKIL-15 T cell product. Consequently, the platform for generating these 4th generation CAR-T PD-1KO would use RNPs for CRISPR/Cas9 delivery and AAV6 for IL-15 ADNc delivery to PDCD1 locus to generate pdTRUCKIL-15 cells. Once pdTRUCKIL-15 cells were generated, it was determined that IL-15 expression followed the PD-1 expression profile. Furthermore, the impact of PD-1 deletion and controlled IL-15 expression on the phenotype, proliferation, respiratory capacity, anti-apoptotic proteins, and lytic activity of pdTRUCKIL-15 cells was evaluated. Results demonstrated that IL-15 expression completely reversed the proliferative deficiency of CAR-T PD-1 KO cells, with a decrease in BIM (pro-apoptotic protein) expression and an increase in Bcl-xL (anti-apoptotic protein) expression. Moreover, pdTRUCKIL-15 cells exhibited an increase in memory populations together with and improved metabolic characteristics in terms of SRC rates (a crucial feature for longevity, persistence, and effective lytic function) compared to CAR-T PD-1 KO cells. Additionally, pdTRUCKIL-15 cells showed higher lytic capacity than CAR-T PD-1 KO and CAR-T WT cells, although the difference was significant only against CAR-T WT cells. In summary, this Thesis demonstrates that PD-1 deletion in CAR-T cells enhances their activity against PD-L1+ tumour cells but may also reduce their metabolic potential and proliferative activity. Furthermore, controlled expression of IL-15 reverses these two deficiencies, endowing pdTRUCKIL-15 cells with a greater ability to withstand the challenging conditions of the tumor microenvironment, ensuring a prolonged survival and persistence. Ultimately, the presented strategy as a genomic editing platform to generate PD-1 KO TRUCKS represents a significant advance in CAR-T cell engineering, paving the way for more effective and durable therapies in the treatment of leukemia, lymphoma, and other refractory hematologic malignancies.