Biology subjects

Fleming, R. A.

Publications and source records attributed to Fleming, R. A..

3 recordsLinked to original sources

bioRxiv2025

PTPN2-KO CAR-T Cells Demonstrate Enhanced Effector Function, CNS Infiltration, and Toxicity in a Non-Human Primate CAR-T Model

B-cell targeting CAR-T cell therapies achieve high remission rates, yet durable responses occur in fewer than 40% of patients. Deletion of negative T-cell regulators, such as PTPN2, a key inhibitor of TCR and cytokine signaling, represents a promising strategy to enhance the efficacy of CAR-T cells. While transfer of PTPN2 knockout (KO) T cells has demonstrated antitumor benefits in murine models, its impact on human-derived CAR-T cells and, importantly, the associated in vivo efficacy and toxicity remain unclear. Here, we demonstrate that PTPN2-KO human CD19 CAR-T cells exhibit enhanced cytokine production, cytotoxicity, TCR and CAR affinity and signaling, leading to superior in vitro elimination of leukemic cells with low CD19 expression. To assess in vivo efficacy and toxicity, we performed a dose-escalation study using a non-human primate (NHP) model of B-cell-targeting CD20 CAR-T cell therapy. We demonstrated that PTPN2-KO CD20 CAR-T cells exhibited superior in vivo expansion and B-cell depletion compared to WT CAR-T cells, in a dose-dependent manner. At the highest dose level, CAR-T expansion was associated with increased toxicities, particularly ICANS, compared to PTPN2 WT CD20 CAR-T cells driven by enhanced CNS-infiltration. Transcriptional profiling revealed a dominant effector and proliferative signature, with cytotoxic CNS-infiltrating CD8+ PTPN2-KO CAR-T cells implicated in ICANS pathogenesis. This study details the comprehensive evaluation of PTPN2-KO CAR-T cells in an immunocompetent model, demonstrating their enhanced on-target functionality, while highlighting increased toxicity risks, underscoring the need for rigorous preclinical assessment of potent genetic modifications in CAR-T therapy. Key pointsO_LIPTPN2-KO CAR-T cells exhibit enhanced effector function C_LIO_LIIn a dose escalation study in rhesus macaques, PTPN2-KO mediated enhanced proliferation and CNS infiltration was associated with increased ICANS C_LI

immunology↗

bioRxiv2023

B cell directed CAR-T cell therapy results in activation of CD8+ cytotoxic CAR-negative bystander T cells in both non-human primates and patients

There is growing appreciation for the emergence of CARneg bystander T cells after CAR-T cell infusion. However, their phenotypic and transcriptomic hallmarks and mechanisms of activation remain uncertain. We performed single-cell RNA-Seq (scRNA-Seq) on non-human primate (NHP) and patient-derived T cells to interrogate CARneg T cells following B cell targeted CAR-T cell therapy. In a NHP model, we observed a distinct population of activated CD8+ CARneg T cells emerging during CAR-T cell expansion. These bystander CD8+ CARneg T cells exhibited a unique transcriptional signature with upregulation of NK-cell markers (KIR3DL2, CD160, KLRD1), chemokines and chemokine receptors (CCL5, XCL1, CCR9), and downregulation of naive T cell-associated genes (SELL, CD28). A transcriptionally similar population was identified in patients following Tisangelecleucel infusion. Mechanistic studies revealed that IL-2 and IL-15 exposure induced bystander-like CD8+ T cells. These T cells efficiently killed leukemic cells through a TCR-independent mechanism. Together, these data identify bystander CD8+ T cells as a novel mechanism by which CAR-T cell infusion can induce further anti-leukemic activity, measurable in both NHP and in patients. Statement of SignificanceWe have deeply interrogated CARneg bystander CD8+ T cells during CAR-T cell expansion in non{-}human primates and patients receiving Tisangelecleucel to identify the unique transcriptomic signature defining these cells, and to determine that IL-2-and IL-15-induced cytotoxic bystander T cells are capable of killing in a TCR-independent manner. These data highlight the potential of bystander T cells for leukemia control and provide a critical foundation for their future analysis.

immunology↗

bioRxiv2021

Identification and tracking of alloreactive T cell clones in Rhesus Macaques through the RM-scTCR-Seq platform.

T cell receptor clonotype tracking is a powerful tool for interrogating T cell mediated immune processes. New methods to pair a single cells transcriptional program with its T cell receptor (TCR) identity allow monitoring of T cell clonotype-specific transcriptional dynamics. While these technologies have been available for human and mouse T cells studies, they have not been developed for Rhesus Macaques, a critical translational organism for autoimmune diseases, vaccine development and transplantation. We describe a new pipeline, RM-scTCR-Seq, which, for the first time, enables RM specific single cell TCR amplification, reconstitution and pairing of RM TCRs with their transcriptional profiles. We apply this method to a RM model of GVHD, and identify and track in vitro detected alloreactive clonotypes in GVHD target organs and explore their GVHD driven cytotoxic T cell signature. This novel, state-of-the-art platform fundamentally advances the utility of RM to study protective and pathogenic T cell responses.

immunology↗