Creation of next generation CAR-T by Repurposing endogenous immune pathways

M Cortijo-Gutiérrez(1) N Maldonado-Pérez(1) M Tristán-Manzano(1) K Pavlovic-Pavlovic(1,2) P Justicia-Lirio(1,4) C Blanco(1,4) M Juan(5) M Castella(5) I C Herrera(2,3) F Martin(1) K Benabdellah(1)

1:GENyO; 2:IMIBIC; 3:Hematology Unit, Reina Sofía Hospital; 4:Lentistem Biotech; 5:Clinic Hospital

Adoptive Cell therapy (ACT) is an emerging field that shows promise for refractory leukemia or lymphoma patients. Among these, genetically engineered T cells expressing Chimeric Antigen Receptors (CARs), have highlighted as a successful therapy for type B malignancies. Although CAR-T cell therapies hold great promise, still face multiple challenges, including toxicity, inactivation by the tumour microenvironment and low persistence in patients. In the present study, we explore the application of genome editing (GE) approach based on CRISPR/Cas9 to repurpose TCR and PD-1, two major players of the T cell activation pathway. Our final aim is to engineer the TRAC locus to avoid Graft versus host disease (GVHD) (generating universal CAR-T cells) and the PD-1 locus expressing IL-15 (generating T cells that respond to PD-L1 increasing their activity). For proof-of-principle, we first designed a donor DNA to insert IL-15-2A-eGFP into PD-1 locus. We used PCR products for donor delivery and RNP nucleofection with CRISPR/Cas9. Our first results show the appearance of CD3- T cells due to the TCR Knock-out, and GFP+PD-1- T cells indicating a specific integration of the donor in the target locus. We observed a high proportion of death cells (from 50% to 12% alive cells) as consequence of GE, probably due to the use of PCR product as donors. The same donors will be delivered as AAV6 in next experiments as an alternative to reduce cytotoxicity of the approach.

This strategy allows generating CAR-T cells with exhaustion/activation dependent IL-15 secretion, which should lead to improved therapeutic efficacy.