P014
CRISPR-mediated targeting of IL15 to the PD1 locus to improve the phenotype of CD19 CAR T cells.
M Cortijo-Gutiérrez(1) N Maldonado-Pérez(1) M Tristán-Manzano(1,4) K Pavlovic-Pavlovic(1,2) P Justicia-Lirio(1,4) M Juan(6) M Castella(6) I C Herrera(2) R O Bak(7) K Benabdellah(1) F Martin(1,5)
1:1. Gene and Cell Therapy Group. Genomic medicine department, GENYO, Granada, 14016, Spain.; 2:2. GC1 4Cell Therapy, IMIBIC. University of Cordoba, Reina Sofia University Hospital, Cordoba, 14004, Spain.; 3:3. Hematology Unit, Reina Sofia Hospital, Cordoba, Cordoba, 14004, Spain.; 4:4. Lentistem Granada, 14016, Spain.; 5:5. Departmento de Bioquímica y Biología Molecular III e Immunology, Facultad de Medicina, Universidad de Granada, Av. de la Investigación 11, 18007 Granada, Spain; 6:6. Hospital Clinic, Barcelona, 08036, Spain.; 7:7. Department of Biomedicine - Forskning og uddannelse, Skou-bygningen, 8000 Aarhus C, Denmark.
Adoptive cell therapy (ACT) with genetically engineered T cells expressing chimeric antigen receptors (CARs) has emerged as a promising treatment option for refractory leukemia or lymphoma patients. Despite its success in type B malignancies, CAR-T cell therapy still faces challenges such as toxicity, inactivation by the tumor microenvironment, and low cell persistence in patients. This study aims to investigate the potential use of genome editing using the CRISPR/Cas9 system to repurpose the PD-1 locus, which is a crucial component of the T cell activation-suppression pathway.
The goal is to enhance the effectiveness and longevity of CAR-T cells by inserting IL-15 into the PD-1 locus using AAV6 as Donor delivery tool achieving this way a controlled expression of IL-15 under this endogenous promoter. These pdTRUCKIL-15 CAR-T cells will be able to express IL-15 in a controlled manner and resistant to PD-L1 blockade.
In this study we evaluated the impact of PD-1 KO and the IL-15 controlled expression on the phenotype, proliferation, respiratory capacity, anti-apoptotic proteins, and lytic activity of newly generated CAR-T cells.
CAR-T cells lacking PD-1 exhibited decreased expansion and fitness. However, when IL-15 was Knocked-In in this locus, it resulted in CAR-T cells PD-1KO with an improved phenotype, viability, and overall cell wellness together with an improved lytic capacity showing more persistence on killing assays and an improved specific lysis.
Furthermore, pdTRUCKIL-15 T cells exhibited a significantly decreased expression of pro-apoptotic proteins like BIM and a significant increased anti-apoptotic proteins expression like Bcl-2 and Bcl-xL showing an increased resistance to apoptosis.