Developing exhaustion-resistant CAR-T cells for the treatment of solid tumors

[eng] Adoptive cell therapy using Chimeric Antigen Receptor (CAR)-T cells has demonstrated remarkable results in patients with certain types of cancer. CARs are synthetic receptors designed to redirect T cells against tumors by including an extracellular antigen-binding domain that recognizes a tumor antigen, and an intracellular domain that induces T cell activation after antigen ligation. The tremendous success of CAR-T cells in the treatment of hematological malignancies has prompt efforts to extend this technology to the more challenging setting of solid tumors. Although clinical responses in solid tumors have generally been limited, recent reports have highlighted measurable antitumor activity of CAR-T cells in patients with advanced-stage cancer. Nevertheless, several obstacles remain to be addressed to broaden the application of CAR-T therapy for solid tumors. One of the primary causes of CAR-T resistance in solid tumors is the inadequate function of CAR-T cells against cancer cells, which hinders effective disease control. The principal aim of this thesis was to elucidate the T cell intrinsic mechanisms that drive to CAR-T cell dysfunction and therapy failure in solid tumors. To address this issue, we optimized an in vivo model of xenograft solid tumors in which infused CAR-T cells induce tumor regression but eventually become dysfunctional due to chronic antigen exposure that leads to tumor escape. Tumor infiltrating CAR-T cells (CAR-TILs) were isolated to study phenotype, function and transcriptome by RNA-sequencing at early and late timepoints after treatment. Based on the obtained transcriptomic data on CAR-TILs, we performed a CRISPR/Cas9 screen to identify potential mediators of CAR-T cells dysfunction in vivo. Our findings indicate that dysfunction in CAR-T cells is a primary cause of tumor escape in mouse models of solid tumors, although other factors may contribute to therapy failure. Dysfunction induced by chronic antigen exposure in our model is characterized by an irreversible loss of T cell effector functions, including cytotoxicity and cytokine production. Interestingly, transcriptional signatures of T cell exhaustion were early enriched in CAR-T cells after treatment, despite observing antitumor activity in vivo at this point. Furthermore, the performance of CAR-T cells in a mouse model of xenograft solid tumors can be enhanced by deleting specific candidate genes.