Temporal control of CAR expression enables thymic generation of autoreactive T cells targeting tumor-associated antigens

Abstract

Abstract Chimeric antigen receptor (CAR) T cell therapy represents one of several cancer immunotherapies leveraging the immune system to elicit potent antitumor responses. CAR T cell therapy has demonstrated remarkable clinical efficacy by enabling MHC-independent, tumor-specific targeting, illustrated by the success of CD19 CAR T cell therapy for the treatment of relapsed or refractory B cell malignancies. Nevertheless, long-term durable disease control remains a significant hurdle, with both antigen-positive and antigen-negative relapses contributing to treatment failure. Currently approved conventional CAR T cell products, largely composed of ex vivo expanded effector T cells, often exhibit limited persistence. To overcome these limitations, strategies such as cytokine modulation during manufacturing and transcriptional or epigenetic reprogramming are being explored to improve CAR T cell fitness and durability. Building upon our previous findings that intrathymic administration of hematopoietic stem and progenitor cells (HSPCs) can give rise to tumor antigen-specific T cells that persist long-term in murine models, we utilized the thymus as an in vivo bioreactor for the sustained production of CAR T cells from intrathymically injected CD19 CAR engineered HSPCs. This strategy is particularly advantageous given the superior antitumor efficacy associated with less differentiated T cell subsets, such as naïve T cells. Our investigation focused on CAR T cells targeting CD19, a well-characterized B cell antigen conserved across both human and murine systems. However, due to the expression of CD19 on normal B cells, CARs targeting this antigen exhibit intrinsic autoreactivity. Analogous to endogenous autoreactive T cell receptors, CD19-specific CAR expression during thymic T cell development is expected to trigger negative selection via clonal deletion, a central mechanism of immune tolerance. To circumvent this barrier, we evaluated several approaches to enhance thymic development and facilitate the successful selection of CAR T cells targeting autoreactive tumor-associated antigens. We demonstrate via single-cell transcriptomics, surface protein profiling and serial transplantation studies that the thymic microenvironment supports the engraftment of short-term, but not long-term, hematopoietic stem cells, while initiating a transcriptional program that drives T cell lineage commitment and suppresses myeloid commitment of multipotent HSPCs. Furthermore, we conducted in vivo studies systematically comparing the fate of intrathymically injected HSPCs that were engineered with different CD19 CAR constructs designed to increase the likelihood of CD19 CAR expressing developing thymocytes to survive thymic selection checkpoints. Constructs that were tested included CARs with decreased costimulation, CARs under the control of a distal LCK promoter (distal LCK activity is restricted to the later stages of thymic T cell development), and a Cre-dependent tamoxifen-inducible CAR. We used autologous HSPCs – either wild type or Pmel-1 TCR transgenic – for CD19 HSPC manufacturing. Our data indicate that modifying CAR constructs to reduce costimulatory signaling, as well as altering the endogenous TCR repertoire, improves the survival of developing thymocytes expressing an autoreactive CAR. Importantly, we show that temporal control of CAR gene expression via an inducible CAR expression system enables sustained thymic generation of autoreactive CD19-specific CAR T cells, which effectively deplete CD19-expressing tumor cells. Our findings offer new insights into the molecular and developmental pathways governing the fate of CAR-engineered T cell precursors within the thymus and establish proof-of-concept for thymus-derived CAR T cell therapy. We demonstrate that CD19 CAR expression can be successfully induced months after intrathymic delivery of Cre-dependent, CAR-transduced HSPCs; however, as anticipated, thymic negative selection eliminates a subset of CAR-expressing single positive thymocytes. Future directions will focus on developing clinically translatable strategies for thymic CAR T cell generation, including the use of tetracycline-inducible expression systems or the design of CARs specific for truly tumor-restricted, non-self-antigens, thereby circumventing central tolerance barriers imposed by autoreactivity.