Generación de una plataforma para la producción de células CAR-T alogénicasestudios de eficacia y seguridad

Abstract

Autologous T cells expressing chimeric antigen receptors (CARs) have been approved as advanced therapy medicinal products (ATMPs) against several type B malignancies. However, a main drawback comes from their autologous nature which limits the amount of patients that can be treated and precludes its standardization. Allogeneic CAR-T cells are an alternative to simplify this complex and time-consuming process. In this Doctoral Thesis, we have studied the feasibility (efficacy and safety) of TCR knockdown (TCRKO) to generate universal allogeneic CAR-T cells. In addition, we have investigated metabolic intervention with small molecules as a way to improve CAR-T cell products, including TCRKO CAR-T cells. Specifically, the work carried out has been divided into two main objectives: Determination of genome editing efficacy and safety to generate Universal CAR-T cells. For this, we had the collaboration of Dr Manel Juan who donated the CAR ARI-0001, a second generation αCD19 CAR approved by the Spanish Agency for Medicines and Medical Devices (AEMPS) within the framework of Hospital Exemption for the treatment of patients older than 25 years with Relapsed/Refractory acute B cell lymphoblastic leukemia (R/R B-ALL). First, the efficacy and potential safety concerns that arise after disruption of the TRAC locus using the CRISPR/Cas9 system were analyzed. We show that the use of CRISPR/Cas9 delivered as ribonucleoparticles (RNPs) allows highly efficient TCR depletion (over 80%) without significant alterations in T cell phenotype. However, we have also found that efficient TCRKO can lead to on-target large deletions in the genome, indicating a potential risk of this procedure. As expected, TCR elimination of ARI-CAR-T cells efficiently prevented allogeneic responses. Importantly, the procedure did not significantly alter ARI-CAR-T cell phenotype, while maintaining a similar antitumor activity ex vivo and in vivo compared to unedited ARI-CAR-T cells (WT ARI-CAR-T cells). These results demonstrate that, although some risks of genotoxicity still exist, TRAC disruption is a feasible strategy for the generation of functional universal ARI-CAR-T cells. In this sense, our next step will be to validate the process under escalation and good manufacturing practices (GMP) to propose a clinical trial and/or hospital exemption for the treatment of patients with type B lymphomas or leukemias who have no other options and who can not access commercial CAR-T therapy. Metabolic intervention with small molecules to favour CAR-T products enriched in memory T cells. On the other hand, in this thesis we have also tried to address one of the problems that current CAR-T therapies encounter: their exhaustion/lack of persistence before eliminating all the tumor cells in patients, especially important in allogeneic CAR-T cell therapies. In this direction, there is strong evidence that a greater presence of memory T cells, with a high capacity for self-renewal and pluripotency (T stem cell memory, TSCM), is directly correlated with a better therapeutic response. Based on these studies, we entered in collaboration with Prof. Pedro Romero, who had developed various procedures to reprogram T cell phenotype through metabolic intervention. In this direction, we studied whether metabolic intervention during ex vivo expansion could be a method to improve the antitumor activity of allogeneic TCRKO CAR-T cells. Specifically, we evaluated two inhibitors, one for the mitochondrial enzyme isocitrate dehydrogenase 2 (IDH2) and another for the mitochondrial pyruvate transporter (MPC). These studies revealed an increase in TSCM phenotype in WT and TCRKO ARI-CAR-T cells treated with the inhibitor for MPC (MPCi), although not with the inhibitor for IDH2 (IDHi). MPC inhibition did not compromise CAR-T cell expansion or cytokine production. ARI-CAR-T cells pre-treated with MPCi showed a higher antitumor effector function which varies depending on the model used. This variability in increasing efficacy needs to be investigated in animal models and, based on the results, we will propose its future application in protocols for the generation of allogeneic CAR-T cells.