UCLA

Chimeric antigen receptor (CAR) T cell therapy has demonstrated remarkable results in otherwise treatment-refractory diseases, most notably for B cell malignancies. However, there are several challenges to autologous CAR T therapy that limit its accessibility and widespread adoption. First, the de novo generation of each treatment can lead to CAR T cells that exhibit varying clinical responses, in part due to inherent variations in the T cell composition of each product. The variable outcomes are compounded by complex manufacturing processes and T cell expansion procedures that can produce a prematurely exhausted and less efficacious CAR T product. These challenges contribute to cancer relapse rates as high as 57-66% after treatment. Thus, there is a growing interest in generating functionally enhanced CAR T cells in vitro from a self-renewing source of induced pluripotent stem cells (iPSCs). iPSC-engineered T cell therapy would ensure immediate access to treatment that delivers a more durable and consistent clinical response.

Unlike peripheral blood T cells, the introduction or the perturbation of biologically active genes can pose serious challenges to the in vitro differentiation of iPSCs. Inappropriate signaling or developmental cues at critical stages of T cell development can result in a block in differentiation or diversion to an undesired lineage. We and others have identified two critical biologic barriers to the generation of iPSC-derived CAR T cells: (1) expression of the CAR in early, T cell precursors diverts differentiation towards the innate lineage, instead of the conventional T cell pathway, and (2) Removal of endogenous T cell receptor (TCR) to address the risk of alloreactivity halts T cell development due to the absence of positive selection. This dissertation introduces a novel CRISPR/Cas9 editing strategy that aim to address both CAR-mediated innate diversion and rescue of positive selection in the absence of endogenous TCR. We report the generation of iPSC-derived, TCR-null CAR T cells using the in vitro “Artificial Thymic Organoid” (ATO) system developed in our lab

Chapter 2 introduced the gene editing strategies used to circumvent CAR-mediated innate diversion and TCR-mediated alloreactivity. Our approach leverages the stage-specific expression profile of endogenous genes to regulate and drive the expression of the CAR transgene that is restricted to mature T cells, after commitment to the conventional T pathway. As a proof-of-concept, we showed that Granzyme A (GZMA) - regulated expression of a high tonic signaling CAR generated conventional and mature CD8+ CAR T cells with robust antigen-specific cytotoxicity. However, the removal of the endogenous TCR resulted in the halt in T cell development, which prompted the need to engineer an alternative source of positive selection signals.

Chapter 3 combined both stage-specific expression of the CAR and the introduction of the CAR cognate antigen in the ATO microenvironment to rescue development in the absence of endogenous TCR. We used the gene CD8B to drive and regulate CAR expression at the physiologically appropriate stage for positive selection. Using this approach, we showed the robust production of TCR-CD3- CD8+ CAR T cells that exhibit a uniform and homogenous “naïve-like” CD45RA+CD62L+CCR7+ phenotype. We further investigated the impact of ex vivo expansion on T cell function and alternative methods of DP activation on T cell phenotype. Single-cell RNA and ATAC sequencing comparing TCR- versus CAR-mediated development revealed possible key mediators that contribute to the production of TCR-null, naïve CAR T cells.