Project description:Allogeneic chimeric antigen receptor (CAR) T cell therapies offer a scalable, off-the-shelf option for cancer treatment, but their clinical use is limited by the risk of graft-versus-host disease (GvHD), mediated by the endogenous T cell receptor (TCR). Conventional strategies to eliminate TCR expression rely on genome editing tools such as CRISPR/Cas9 or base editing, which introduce permanent DNA changes and pose safety concerns. Here, we present an epigenetic editing approach that enables efficient, specific, and reversible silencing of the CD3ε gene, a critical component of the TCR complex, without altering the genome. Through systematic optimization of the epigenetic editor and guide RNA design, we achieved robust TCR silencing in primary T and CAR T cells while preserving CAR expression, activation, and effector function. Transcriptome analysis confirmed minimal off-target effects. In vivo observation suggest the epigenetically silenced T cells to prevent GvHD while persisting longer than TCR-knockout cells, supporting the notion that transient TCR suppression may help balance safety and long-term efficacy. Our findings establish epigenetic editing as a non-genotoxic alternative to genome editing, offering a flexible and safer route to generate next-generation allogeneic CAR T cells.
Project description:Chimeric antigen receptor–T (CAR-T) cell therapies can eliminate relapsed and refractory tumors, but the durability of antitumor activity requires in vivo persistence. Differential signaling through the CAR costimulatory domain can alter the T cell metabolism, memory differentiation, and influence long-term persistence. CAR-T cells costimulated with 4-1BB or ICOS persist in xenograft models but those constructed with CD28 exhibit rapid clearance. Here, we show that a single amino acid residue in CD28 drove T cell exhaustion and hindered the persistence of CD28-based CAR-T cells and changing this asparagine to phenylalanine (CD28-YMFM) promoted durable antitumor control. In addition, CD28-YMFM CAR-T cells exhibited reduced T cell differentiation and exhaustion as well as increased skewing toward Th17 cells. Reciprocal modification of ICOS-containing CAR-T cells abolished in vivo persistence and antitumor activity. This finding suggests modifications to the costimulatory domains of CAR-T cells can enable longer persistence and thereby improve antitumor response.
Project description:This is a single arm, open-label, uni-center, phase I-II study to evaluate the safety and effectiveness of CAR-T/TCR-T cell immunotherapy in treating with different malignancies patients.
Project description:Chimeric antigen receptor (CAR) T-cells induce responses in patients with relapsed/refractory leukemia; however, long-term efficacy is frequently limited by post-CAR relapses. The inability to target antigen-low cells is an intrinsic vulnerability of second-generation CAR T-cells and underlies the majority of relapses following CD22BBz CAR T-cell therapy. We interrogated CD22BBz CAR signaling in response to low antigen and found inefficient phosphorylation of LAT, limiting downstream signaling. To overcome this, we designed the Adjunctive LAT-Activating CAR T-cell (ALA-CART) platform, pairing a second-generation CAR with a LAT-CAR incorporating the intracellular domain of LAT. ALA-CART cells demonstrated reduced differentiation during manufacturing and increased LAT phosphorylation, MAPK signaling and AP-1 activity. Consequently, ALA-CART cells showed improved cytotoxicity, proliferation, persistence and efficacy against antigen-low leukemias that were refractory to clinically-active CD22BBz CAR T-cells. These data suggest restoration of LAT signaling through the ALA-CART platform represents a promising strategy for overcoming multiple mechanisms of CAR T-cell failure.
Project description:Chimeric antigen receptor (CAR) T cells are powerful tools against cancer and autoimmunity. CARs are typically introduced into T cells with endogenous T cell receptors (TCRs), enabling clonotype tracking. Also, CAR expression in T cells with virus-specific TCRs may enhance CAR-T efficacy. However, the functional impact of endogenous TCR activity on CAR-T behavior remains unclear. We here traced anti-CD19 CAR-T clonotypes in patients with B-cell malignancies using single-cell RNA-, TCR-, and CITE-seq. An IFNG-positive phenotype, but not short-term reactivity, predicted clinical CAR-T persistence. To test intrinsic TCR effects, we combined CAR transduction with orthotopic TCR replacement in human T cells. Inactive TCRs did not alter CAR-T function and may serve as molecular barcodes. In contrast, active TCRs modulated CAR signaling as agonists and CAR cytotoxicity as antagonists in an avidity-dependent manner, while CAR activity had no effect on TCR cytotoxicity. Therefore, spatial antigen separation alters TCR/CAR interplay with implications for therapeutic CAR-T design.
Project description:Chimeric antigen receptor (CAR) T cells are powerful tools against cancer and autoimmunity. CARs are typically introduced into T cells with endogenous T cell receptors (TCRs), enabling clonotype tracking. Also, CAR expression in T cells with virus-specific TCRs may enhance CAR-T efficacy. However, the functional impact of endogenous TCR activity on CAR-T behavior remains unclear. We here traced anti-CD19 CAR-T clonotypes in patients with B-cell malignancies using single-cell RNA-, TCR-, and CITE-seq. An IFNG-positive phenotype, but not short-term reactivity, predicted clinical CAR-T persistence. To test intrinsic TCR effects, we combined CAR transduction with orthotopic TCR replacement in human T cells. Inactive TCRs did not alter CAR-T function and may serve as molecular barcodes. In contrast, active TCRs modulated CAR signaling as agonists and CAR cytotoxicity as antagonists in an avidity-dependent manner, while CAR activity had no effect on TCR cytotoxicity. Therefore, spatial antigen separation alters TCR/CAR interplay with implications for therapeutic CAR-T design.
Project description:transcriptional profiling was performed on Regnase-1 KO CAR and Regnase-1 TCF-1 DKO CAR T cells isolated 7days after co-transfer into tumor bearing mice. TCF-1 deficiency in Regnase-1 KO CAR T cells led to reduced long-term persistence and memory-like phenotype.
Project description:The underpinning mechanisms driving chimeric antigen receptor (CAR) T cell function and persistence after infusion, and their correlation with clinical outcome, remains poorly understood. Here, we applied single-cell multiomics, coupled with gene regulatory network analyses, to evaluate CAR-T cell transcriptional heterogeneity, dynamics and clonal evolution, in longitudinal bone marrow (BM) and peripheral blood (PB) paired samples, collected from MM patients treated with BCMA CAR T cells. We identify several regulons with increased activity, within CAR T cell infiltrating the BM, as key drivers of the more activated and exhausted phenotype observed in those cells compared to their PB counterparts. Interestingly, regulatory dysfunctions in the effector-to-memory axis promoted higher presence of terminally differentiated cells, leading to a lack of CAR T cell persistence. Moreover, we identified a hyperexpanded CAR T clone in the BM of a patient with partial response. Deeper characterization showed increased IL10 expression in that clone, that was associated to transcription factors related to exhausted CAR T cells, like PRDM1. Overall, our analyses provide insights into the regulatory mechanisms that could promote lack of CAR T cell persistence and dysfunction, representing potential targets to be modulated for the development of improved CAR T therapies for MM.
Project description:The production of autologous T cells expressing a chimaeric antigen receptor (CAR) is time-consuming, costly and occasionally unsuccessful. T cell-derived induced pluripotent stem cells are a promising source for the generation of ‘off-the-shelf’ CAR T cells, but their in vitro differentiation often yields T cells with suboptimal features. Here we show that premature expression of the T cell receptor (TCR) or a constitutively expressed CAR promote the acquisition of an innate phenotype, which can be averted by disabling the TCR and relying on the CAR to drive T cell differentiation. Delaying CAR expression and calibrating its signalling strength enabled the generation of human CD8αβ+ TCR– CAR+ T cells that perform overall similarly to peripheral blood CD8αβ+ CAR+ T cells in achieving effective tumour control upon systemic administration in a mouse model of leukaemia, without causing graft-versus-host disease. Driving T cell maturation in the absence of a TCR by taking advantage of a CAR may facilitate the large-scale development of potent allogeneic CD8αβ+ T cells for a broad range of immunotherapies. TRAC-1XX-iT cell generation: TiPS were differentiated to the DP T cell stage, and matured to CD8ab SP cells on 3T3-CD19-41BBL. CD8ab SP cells were purified by flow cytometry. Peripheral Blood Mononuclear cells were purified from healthy donor whole blood. CD4 and CD8 T cells were targeted with CD19-28z-1XX CAR into the TRAC locus, NK and gdT cells were retrovirally transduced to express the CD19-28z-1XX CAR. Cells were purified for CAR expression by flow cytometry.
Project description:Chimeric antigen receptor (CAR)-expressing T-cells induce durable remissions in patients with relapsed/refractory B-cell malignancies. CARs are artificial constructs introduced into mature T-cells conferring a second, non-MHC restricted specificity in addition to the endogenous T-cell receptor (TCR). The impact of TCR activation on CAR T-cell efficacy in vivo has important implications for clinical optimization of CAR T-cell therapy, but cannot be systematically evaluated in xenograft models. Using an immunocompetent, syngeneic murine model of CD19-targeted CAR T-cell therapy for pre-B cell ALL, we demonstrate loss of CD8 CAR T-cell mediated clearance of leukemia associated with T-cell exhaustion and apoptosis when TCR antigen is present. CD4 CAR T-cells demonstrate equivalent cytotoxicity, as compared to CD8 CAR T-cells, and in contrast, retain in vivo efficacy in the presence of TCR stimulation. Gene expression profiles confirm increased exhaustion and apoptosis of CAR8 upon dual receptor stimulation compared to CAR4, and indicate inherent differences in T-cell pathways. Chimeric antigen receptor (CAR) T cells express two activating receptors, the CAR and the endogenous T cell receptor (TCR). CAR T cells can be derived from either CD8 or CD4 T cells to generate CAR8 and CAR4 cells, respectively. In vivo, CAR8 and CAR4 cells respond differently when simultaneously stimulated through the CAR and TCR.