Calibrated CAR activation potential directs alternative T cell fates and therapeutic potency
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE121226 | GEO | 2018/12/17
REPOSITORIES: GEO
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Project description:The adoptive transfer of chimeric antigen receptor- (CAR) modified T cells is revolutionizing the treatment of B cell malignancies and has the potential to be applied to other diseases. CARs redirect T cell specificity by linking an antigen recognition domain to T cell signaling modules comprised of CD3z to provide signal 1, and CD28 or 4-1BB to provide costimulation. CD28/CD3z and 4-1BB/CD3z CARs confer differences in effector function and cell fate that affect clinical efficacy and toxicity. These differences may result from activation of divergent transcriptional programs. To gain this insight, we analyzed changes in gene expression in stimulated and resting CD28/CD3z or 4-1BB/CD3z CAR T cells. CD28/CD3z CAR stimulation initiated more marked early transcriptional changes with greater fold increases in the expression of effector molecules including GZMB, IFNG, IL2, TNF, and IL6. Direct comparison of CD28/CD3z and 4-1BB/CD3z samples stimulated for 6 hours identified 1,673 differentially expressed genes. Of these, the memory T cell-associated genes KLF2, IL7R, and FAM65B were expressed at lower levels in CD28/CD3z CAR T cells. KLF2 and IL7R are FOXO transcription factor family targets and we found that FOXO4 expression was similarly reduced in CD28/CD3z CAR T cells. CD28/CD3z CAR stimulation induces an effector T cell-like transcriptional profile that may underlie the decreased persistence and increased risks of toxicities observed with CD28/CD3z CAR T cells in early clinical trials.
2018-08-19 | GSE109161 | GEO
Project description:Chimeric antigen receptors (CARs) are synthetic proteins that redirect T cell specificity by linking an extracellular ligand binding domain to intracellular T cell signaling domains. CAR-expressing T (CAR-T) cells have demonstrated significant efficacy for the treatment of refractory B cell malignancies and are being evaluated as immunotherapeutic reagents for many other cancers. CAR designs are based on the fundamental principles of TCR recognition and most CARs employ the T cell-activating CD3z endodomain alongside a costimulatory domain from CD28 or 4-1BB. However, emerging data suggest that CD28/CD3z and 4-1BB/CD3z signaling modules promote divergent metabolic pathways, gene expression programs, and cell fates. To determine how CAR phosphoprotein signaling drives these disparate cell fates, we analyzed CAR ligation-induced signaling networks in primary human T cells using shotgun mass spectrometry. We isolated CD8+CD62L+ T cells from healthy donors and introduced a CD28/CD3z or 4-1BB/CD3z CAR by lentiviral transduction. Transduced T cells were purified by FACS and expanded once in vitro. When the cells returned to a resting state, CD28/CD3z or 4-1BB/CD3z CAR-T cells were stimulated for 10 or 45 minutes with magnetic microbeads coated with a monoclonal antibody specific for a 9 amino acid tag in the CAR extracellular sequence. CAR-T cells were also left unstimulated for 10 or 45 minutes to serve as controls. Altogether, 8 unique conditions were tested in an experiment and three independent experiments were performed.
2018-08-13 | PXD007921 | Pride
Project description:Adoptive transfer of anti-CD19 chimeric antigen receptor (CAR)-engineered T cells has shown impressive clinical responses in patients with refractory B-cell malignancies. However, therapeutic effects of CAR-T cells targeting other hematologic malignancies and solid tumors are not yet satisfactory. Although inefficient tumor trafficking and multiple immunosuppressive molecules impede CAR-T cell effector responses, signals delivered by the current CAR constructs may still be insufficient to fully activate antitumor T cell functions. Optimal T cell activation and proliferation requires multiple signals including T cell receptor (TCR) engagement (signal 1), costimulation (signal 2), and cytokine engagement (signal 3). CAR genes developed to date contain a CD3z domain and costimulatory domain(s), but not a domain to transmit signal 3. In this study, we have developed a novel CAR construct capable of inducing cytokine signaling in an antigen-dependent manner. The new generation CD19 CAR encodes a cytoplasmic domain of IL-2RB and STAT3-binding YXXQ motif together with CD3z and CD28 domains (28-IL2RB-z (YXXQ)). The 28-IL2RB-z (YXXQ) CAR-T cells showed antigen-dependent JAK-STAT, especially the STAT3-mediated pathway activation, which promoted their proliferation and prevented terminal differentiation. The 28-IL2RB-z (YXXQ) CAR-T cells demonstrated superior in vivo persistence and antileukemia effects compared with the currently used CARs in multiple tumor models.
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Project description:Adoptive transfer of anti-CD19 chimeric antigen receptor (CAR)-engineered T cells has shown impressive clinical responses in patients with refractory B-cell malignancies. However, therapeutic effects of CAR-T cells targeting other hematologic malignancies and solid tumors are not yet satisfactory. Although inefficient tumor trafficking and multiple immunosuppressive molecules impede CAR-T cell effector responses, signals delivered by the current CAR constructs may still be insufficient to fully activate antitumor T cell functions. Optimal T cell activation and proliferation requires multiple signals including T cell receptor (TCR) engagement (signal 1), costimulation (signal 2), and cytokine engagement (signal 3). CAR genes developed to date contain a CD3z domain and costimulatory domain(s), but not a domain to transmit signal 3. In this study, we have developed a novel CAR construct capable of inducing cytokine signaling in an antigen-dependent manner. The new generation CD19 CAR encodes a cytoplasmic domain of IL-2RB and STAT3-binding YXXQ motif together with CD3z and CD28 domains (28-DIL2RB-z (YXXQ)). The 28-DIL2RB-z (YXXQ) CAR-T cells showed antigen-dependent JAK-STAT, especially the STAT3-mediated pathway activation, which promoted their proliferation and prevented terminal differentiation. The 28-DIL2RB-z (YXXQ) CAR-T cells demonstrated superior in vivo persistence and antileukemia effects compared with the currently used CARs in multiple tumor models.
2018-01-21 | GSE103906 | GEO
Project description:In vivo persistence of chimeric antigen receptor (CAR) T cells correlates with therapeutic efficacy, yet CAR-specific factors that support persistence are not well resolved. Using a CAR containing a single chain variable fragment (scFv) specific for CD33 linked to a 4-1BB and CD3 zeta signaling domain that is currently in advanced clinical trials, we show that CAR-expression, in a ligand-independent manner, alters T cell differentiation during ex vivo expansion. CAR-transduced T cells displayed decreased naïve and stem memory populations and increased effector subsets relative to vector-transduced control cells, and this was associated with reduced in vivo persistence. Altered persistence was not due to antigen specificity or tumor presence, but was linked to tonic signaling through the CAR, most notably CD3 zeta ITAMs, prior to transfer. We identified the PI3K/AKT pathway in CD33 CAR T cells as responsible. Treatment with a PI3K inhibitor modulated the differentiation program of CAR T cells, preserved a less differentiated state without affecting T cell expansion, and improved in vivo persistence relative to control cells. These results help resolve mechanisms by which tonic signaling modulates CAR T cell fate, and identifies a novel pharmacologic approach to enhance the durability of CAR T cells for cell-based immunotherapy.
2018-05-17 | GSE93386 | GEO
Project description:CD19-targeted CAR therapies have successfully treated B cell leukemias, but many responders later relapse or experience toxicities. CAR ICDs are key to converting antigen recognition into anti-tumor effector functions. Despite the many possible immune signaling domain combinations that could be included in CARs, almost all CARs currently rely upon CD3, CD28, and/or 4-1BB signaling. To explore the signaling potential of CAR ICDs, we generated a library of 700,000 CD19 CAR molecules with diverse signaling domains and developed a high throughput screening platform to enable optimization of CAR signaling for anti-tumor functions. Our strategy identifies CARs with novel signaling domains that elicit distinct T cell behaviors from a clinically available CAR, including enhanced proliferation and persistence, lower exhaustion, potent cytotoxicity in an in vitro tumor rechallenge condition, and comparable tumor control in vivo. This approach is readily adaptable to numerous disease models, cell types, and selection conditions, making it a promising tool for rapidly improving adoptive cell therapies and expanding their utility to new disease indications.
2021-07-13 | GSE179767 | GEO
Project description:Poor CAR T persistence limits CAR T cell therapies for B cell malignancies and solid tumors1,2. The expression of memory-associated genes such as TCF7 (protein name TCF1) is linked to response and long-term persistence in patients3–7, thereby implicating memory programs in therapeutic efficacy. Here, we demonstrate that the pioneer transcription factor, FOXO1, is responsible for promoting memory programs and restraining exhaustion in human CAR T cells. Pharmacologic inhibition or gene editing of endogenous FOXO1 in human CAR T cells diminished the expression of memory-associated genes, promoted an exhaustion-like phenotype, and impaired antitumor activity in vitro and in vivo. FOXO1 overexpression induced a gene expression program consistent with T cell memory and increased chromatin accessibility at FOXO1 binding motifs. FOXO1-overexpressing cells retained function, memory potential, and metabolic fitness during settings of chronic stimulation and exhibited enhanced persistence and antitumor activity in vivo. In contrast, TCF1 overexpression failed to enforce canonical memory programs or enhance CAR T cell potency. Importantly, endogenous FOXO1 activity correlated with CAR T and TIL responses in patients, underscoring its clinical relevance in cancer immunotherapy. Our results demonstrate that memory reprogramming through FOXO1 can enhance the persistence and potency of human CAR T cells and highlights the utility of pioneer factors, which bind condensed chromatin and induce local epigenetic remodeling, for optimizing therapeutic T cell states.
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Project description:Poor CAR T persistence limits CAR T cell therapies for B cell malignancies and solid tumors1,2. The expression of memory-associated genes such as TCF7 (protein name TCF1) is linked to response and long-term persistence in patients3–7, thereby implicating memory programs in therapeutic efficacy. Here, we demonstrate that the pioneer transcription factor, FOXO1, is responsible for promoting memory programs and restraining exhaustion in human CAR T cells. Pharmacologic inhibition or gene editing of endogenous FOXO1 in human CAR T cells diminished the expression of memory-associated genes, promoted an exhaustion-like phenotype, and impaired antitumor activity in vitro and in vivo. FOXO1 overexpression induced a gene expression program consistent with T cell memory and increased chromatin accessibility at FOXO1 binding motifs. FOXO1-overexpressing cells retained function, memory potential, and metabolic fitness during settings of chronic stimulation and exhibited enhanced persistence and antitumor activity in vivo. In contrast, TCF1 overexpression failed to enforce canonical memory programs or enhance CAR T cell potency. Importantly, endogenous FOXO1 activity correlated with CAR T and TIL responses in patients, underscoring its clinical relevance in cancer immunotherapy. Our results demonstrate that memory reprogramming through FOXO1 can enhance the persistence and potency of human CAR T cells and highlights the utility of pioneer factors, which bind condensed chromatin and induce local epigenetic remodeling, for optimizing therapeutic T cell states.
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Project description:Poor CAR T persistence limits CAR T cell therapies for B cell malignancies and solid tumors1,2. The expression of memory-associated genes such as TCF7 (protein name TCF1) is linked to response and long-term persistence in patients3–7, thereby implicating memory programs in therapeutic efficacy. Here, we demonstrate that the pioneer transcription factor, FOXO1, is responsible for promoting memory programs and restraining exhaustion in human CAR T cells. Pharmacologic inhibition or gene editing of endogenous FOXO1 in human CAR T cells diminished the expression of memory-associated genes, promoted an exhaustion-like phenotype, and impaired antitumor activity in vitro and in vivo. FOXO1 overexpression induced a gene expression program consistent with T cell memory and increased chromatin accessibility at FOXO1 binding motifs. FOXO1-overexpressing cells retained function, memory potential, and metabolic fitness during settings of chronic stimulation and exhibited enhanced persistence and antitumor activity in vivo. In contrast, TCF1 overexpression failed to enforce canonical memory programs or enhance CAR T cell potency. Importantly, endogenous FOXO1 activity correlated with CAR T and TIL responses in patients, underscoring its clinical relevance in cancer immunotherapy. Our results demonstrate that memory reprogramming through FOXO1 can enhance the persistence and potency of human CAR T cells and highlights the utility of pioneer factors, which bind condensed chromatin and induce local epigenetic remodeling, for optimizing therapeutic T cell states.
2024-02-29 | GSE255413 | GEO
Project description:Poor CAR T persistence limits CAR T cell therapies for B cell malignancies and solid tumors1,2. The expression of memory-associated genes such as TCF7 (protein name TCF1) is linked to response and long-term persistence in patients3–7, thereby implicating memory programs in therapeutic efficacy. Here, we demonstrate that the pioneer transcription factor, FOXO1, is responsible for promoting memory programs and restraining exhaustion in human CAR T cells. Pharmacologic inhibition or gene editing of endogenous FOXO1 in human CAR T cells diminished the expression of memory-associated genes, promoted an exhaustion-like phenotype, and impaired antitumor activity in vitro and in vivo. FOXO1 overexpression induced a gene expression program consistent with T cell memory and increased chromatin accessibility at FOXO1 binding motifs. FOXO1-overexpressing cells retained function, memory potential, and metabolic fitness during settings of chronic stimulation and exhibited enhanced persistence and antitumor activity in vivo. In contrast, TCF1 overexpression failed to enforce canonical memory programs or enhance CAR T cell potency. Importantly, endogenous FOXO1 activity correlated with CAR T and TIL responses in patients, underscoring its clinical relevance in cancer immunotherapy. Our results demonstrate that memory reprogramming through FOXO1 can enhance the persistence and potency of human CAR T cells and highlights the utility of pioneer factors, which bind condensed chromatin and induce local epigenetic remodeling, for optimizing therapeutic T cell states.
2024-02-29 | GSE255412 | GEO