scholarly journals Deletion of Cbl-b inhibits CD8+ T-cell exhaustion and promotes CAR T-cell function

2021 ◽  
Vol 9 (1) ◽  
pp. e001688
Author(s):  
Jitendra Kumar ◽  
Ritesh Kumar ◽  
Amir Kumar Singh ◽  
Elviche L Tsakem ◽  
Mahesh Kathania ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
T Cell Exhaustion ◽  
Sequencing Data ◽  
T Cell Therapy ◽  
Cell Exhaustion ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundChimeric antigen receptor (CAR) T-cell therapy is an emerging option for cancer treatment, but its efficacy is limited, especially in solid tumors. This is partly because the CAR T cells become dysfunctional and exhausted in the tumor microenvironment. However, the key pathways responsible for impaired function of exhausted cells remain unclear, which is essential to overcome CAR T-cell exhaustion.MethodsAnalysis of RNA-sequencing data from CD8+ tumor-infiltrating lymphocytes (TILs) led to identification of Cbl-b as a potential target. The sequencing data were validated using a syngeneic MC38 colon cancer model. To analyze the in vivo role of Cbl-b in T-cell exhaustion, tumor growth, % PD1+Tim3+ cells, and expression of effector cytokines were analyzed in cbl-b+/+ and cbl-b–/– mice. To evaluate the therapeutic potential of Cbl-b depletion, we generated a new CAR construct, hCEAscFv-CD28-CD3ζ.GFP, that recognizes human carcinoembryonic antigen (CEA). cbl-b+/+ and cbl-b–/– CEA-CAR T cells were generated by retroviral transduction. Rag–/– mice bearing MC38-CEA cells were injected with cbl-b+/+ and cbl-b–/–; CEA-CAR T cells, tumor growth, % PD1+Tim3+ cells and expression of effector cytokines were analyzed.ResultsOur results show that the E3 ubiquitin ligase Cbl-b is upregulated in exhausted (PD1+Tim3+) CD8+ TILs. CRISPR-Cas9-mediated inhibition of Cbl-b restores the effector function of exhausted CD8+ TILs. Importantly, the reduced growth of syngeneic MC38 tumors in cbl-b–/– mice was associated with a marked reduction of PD1+Tim3+ CD8+ TILs. Depletion of Cbl-b inhibited CAR T-cell exhaustion, resulting in reduced MC38-CEA tumor growth, reduced PD1+Tim3+ cells and increased expression of interferon gamma, tumor necrosis factor alpha, and increased tumor cell killing.ConclusionOur studies demonstrate that deficiency of Cbl-b overcomes endogenous CD8+ T-cell exhaustion, and deletion of Cbl-b in CAR T cells renders them resistant to exhaustion. Our results could facilitate the development of efficient CAR T-cell therapy for solid tumors by targeting Cbl-b.

scholarly journals Chimeric Antigen Receptor T Cell Exhaustion during Treatment for Hematological Malignancies

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Chunyi Shen ◽  
Zhen Zhang ◽  
Yi Zhang
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Hematological Malignancies ◽  
Antigen Receptor ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Immunotherapy, especially based on chimeric antigen receptor (CAR) T cells, has achieved prominent success in the treatment of hematological malignancies. However, approximately 30-50% of patients will have disease relapse following remission after receiving CD19-targeting CAR-T cells, with failure of maintaining a long-term effect. Mechanisms underlying CAR-T therapy inefficiency consist of loss or modulation of target antigen and CAR-T cell poor persistence which mostly results from T cell exhaustion. The unique features and restoration strategies of exhausted T cells (Tex) have been well described in solid tumors. However, the overview associated with CAR-T cell exhaustion is relatively rare in hematological malignancies. In this review, we summarize the characteristics, cellular, and molecular mechanisms of Tex cells as well as approaches to reverse CAR-T cell exhaustion in hematological malignancies, providing novel strategies for immunotherapies.

scholarly journals Mutation of the CD28 Costimulatory Domain Confers Decreased CAR T Cell Exhaustion

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 966-966 ◽  
Author(s):  
Justin C. Boucher ◽  
Gongbo Li ◽  
Bishwas Shrestha ◽  
Maria Cabral ◽  
Dylan Morrissey ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract The therapeutic promise of chimeric antigen receptor (CAR) T cells was realized when complete remission rates of 90% were reported after treating B cell acute lymphoblastic leukemia (B-ALL) with CD19-targeted CAR T cells. However, a major obstacle with continued clinical development of CAR T cells is the limited understanding of CAR T cell biology and its mechanisms of immunity. We and others have shown that CARs with a CD28 co-stimulatory domain drive high levels of T cell activation causing acute toxicities, but also lead to T cell exhaustion and shortened persistence. The CD28 domain includes 3 intracellular subdomains (YMNM, PRRP, and PYAP) that regulate signaling pathways post TCR-stimulation, but it is unknown how they modulate activation and/or exhaustion of CAR T cells. A detailed understanding of the mechanism of CD28-dependent exhaustion in CAR T cells will allow the design of a CAR less prone to exhaustion and reduce relapse rates. We hypothesized that by incorporating null mutations of the CD28 subdomains (YMNM, PRRP, or PYAP) we could optimize CAR T cell signaling and reduce exhaustion. In vitro, we found mutated CAR T cells with only a functional PYAP (mut06) subdomain secrete significantly less IFNγ (Fig1A), IL6, and TNFα after 24hr stimulation compared to non-mutated CD28 CAR T cells, but greater than the 1st generation m19z CAR. Also, cytoxicity was enhanced with the PYAP only CAR T cells compared to non-mutated CARs (Fig1B). When we examined the PYAP (mut06) only mutant in an immune competent mouse model we found similar B cell aplasia and CAR T cell persistence compared to non-mutated CD28 CAR T cells. Additionally, PYAP only CAR T cells injected into mice had decreased (82% to 62%) expression of PD1 in the BM. Using a pre-clinical immunocompetent mouse tumor model we found the PYAP only CAR T cell treated mice had a significant survival advantage compared to non-mutated CD28 CAR T cells, with 100% survival of mice given PAYP only CAR T cells compared to 50% survival of mice given non-mutated CAR T cells (Fig1C). We next sought to determine what role CAR T cell exhaustion was playing using a Rag knockout mouse system. CAR T cells were given to Rag-/- mice and 1 week later mice were challenged with tumor. Studies in Rag-/- mice also showed PYAP only CAR T cells were increased 35% in the BM and 92% in the spleen compared to non-mutated CD28 CAR T cells. We also found PYAP only CAR T cells had significantly less expression of PD1 compared to non-mutated CAR T cells (Fig1D). We then co-cultured CAR T cells with target cells expressing CD19 and PDL1 and found PYAP only CAR T cells had increased IFNγ (42%), TNFα (62%) and IL2 (73%) secretion compared to exhausted non-mutated CD28 CAR T cells. This shows that PYAP only CAR T cells are more resistant to exhaustion. To find a mechanistic explanation for this observation we examined CAR T cell signaling. Using Nur77, pAkt, and pmTOR to measure CAR signaling we found PYAP only CAR T cells had significantly reduced levels of Nur77 while still having higher expression then first generation CAR T cells. We then examined what affect the PYAP only CAR had on transcription factors. We found similar AP1 and NF-kB expression between PYAP only and non-mutated CD28 CAR T cells but a significant reduction of NFAT in the PYAP only mutants compared to non-mutated CD28 CAR T cells. This suggests reduced NFAT expression contributes to the PYAP only CAR's resistance to exhaustion. Finally, we made human CAR constructs of the PYAP only mutant. We found PYAP only human CAR T cells had increased cytoxicity and decreased exhaustion in vitro compared to non-mutated human CD28 CAR T cells. NFAT levels in human PYAP only CAR T cells were significantly reduced compared to non-mutated CAR T cells supporting our findings in mice. Our results demonstrate that CAR T cells with only a PYAP CD28 subdomain have better cytoxicity and decreased exhaustion compared to non-mutated CD28 CAR T cells. Our results suggest this is the result of decreased CAR and NFAT signaling. Additionally, we were able to validate these findings using human CAR constructs. This work allows for development of an enhanced 2nd and 3rd generation CAR T cell therapies for B cell malignancies by optimizing CAR T cell activation and persistence which may reduce relapse rates and severe toxicities. Figure 1 Figure 1. Disclosures Davila: Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Chimeric antigen receptor T cells targeting PD-L1 suppress tumor growth

2020 ◽  
Author(s):  
Le Qin ◽  
Ruocong Zhao ◽  
Dongmei Chen ◽  
Xinru Wei ◽  
Qiting Wu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Solid Tumors ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Car T Cells ◽  
Car T

Abstract Background: Chimeric antigen receptor T cells (CAR-T cells) therapy has been well recognized for treating B cell-derived malignancy. However, the efficacy of CAR-T cells against solid tumors remains dissatisfactory, partially due to the heterogeneity of solid tumors and T cell exhaustion in tumor microenvironment. PD-L1 is up-regulated in multiple solid tumors, resulting in T cell exhaustion upon binding to its receptor PD-1. Methods: Here, we designed a dominant-negative form of PD-1 , dPD1z, a vector containing the extracellular and transmembrane regions of human PD-1, and a CAR vector against PD-L1, CARPD-L1z, a vector employs a high-affinity single-chain variable fragment (scFv) against human PD-L1. These two vectors shared the same intracellular structure, including 4-1BB and TLR2 co-stimulatory domains, and the CD3ζ signaling domain. Results: dPD1z T and CARPD-L1z T cells efficiently lysed PD-L1 + tumor cells and had enhanced cytokine secretion in vitro and suppressed the growth of non-small cell lung cancer (NSCLC), gastric cancer and hepatoma carcinoma in patient-derived xenograft (PDX). However, the combination of anti-mesothelin CAR-T cells (CARMSLNz T) with dPD1z T or CARPD-L1z T cells did not repress tumor growth synergistically in PDX, as CARMSLNz T cells upregulated PD-L1 expression upon activation and were subsequently attacked by dPD1z T or CARPD-L1z T cells. Conclusions: In conclusion, we demonstrate CAR-T cells targeting PD-L1 were effective for suppressing the growth of multiple types of solid tumors in PDX models though their safety needs to be carefully examined.

scholarly journals Dynamic chromatin regulatory landscape of human CAR T cell exhaustion

2021 ◽  
Vol 118 (30) ◽  
pp. e2104758118
Author(s):  
David G. Gennert ◽  
Rachel C. Lynn ◽  
Jeff M. Granja ◽  
Evan W. Weber ◽  
Maxwell R. Mumbach ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cancer Immunotherapy ◽  
Target Genes ◽  
Ex Vivo ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Dysfunction in T cells limits the efficacy of cancer immunotherapy. We profiled the epigenome, transcriptome, and enhancer connectome of exhaustion-prone GD2-targeting HA-28z chimeric antigen receptor (CAR) T cells and control CD19-targeting CAR T cells, which present less exhaustion-inducing tonic signaling, at multiple points during their ex vivo expansion. We found widespread, dynamic changes in chromatin accessibility and three-dimensional (3D) chromosome conformation preceding changes in gene expression, notably at loci proximal to exhaustion-associated genes such as PDCD1, CTLA4, and HAVCR2, and increased DNA motif access for AP-1 family transcription factors, which are known to promote exhaustion. Although T cell exhaustion has been studied in detail in mice, we find that the regulatory networks of T cell exhaustion differ between species and involve distinct loci of accessible chromatin and cis-regulated target genes in human CAR T cell exhaustion. Deletion of exhaustion-specific candidate enhancers of PDCD1 suppress the expression of PD-1 in an in vitro model of T cell dysfunction and in HA-28z CAR T cells, suggesting enhancer editing as a path forward in improving cancer immunotherapy.

scholarly journals P08.06 Combining RIG-I-targeted immune activation with CAR T cell therapy induces efficient tumor control in murine pancreatic cancer models

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A27.1-A27
Author(s):  
AM Senz ◽  
SL Formisano ◽  
B Cadilha ◽  
T Lorenzini ◽  
S Endres ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Tumor Growth ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

BackgroundThe efficacy of chimeric antigen receptor (CAR) T cells against solid tumors remains unsatisfactory due to impaired trafficking of the CAR T cells into the tumor microenvironment (TME) and the presence of immunosuppressive factors and cells. 5’- triphosphate double-stranded RNA (3p-RNA) is recognized by the intracellular pattern recognition receptor retinoic acid-induced gene I (RIG-I). RIG-I activates a downstream signaling cascade, triggering the expression of type I interferons (IFN), proinflammatory cytokines and chemokines enhancing immune surveillance in the TME. We hypothesized that priming the TME with RIG-I ligands increases the efficacy of CAR T cell therapy.Materials and MethodsT110299 pancreatic tumor cells (derived from a genetically-engineered Kras and p53 mutant murine PDAC model) were engineered to express murine epithelial cell adhesion molecule (EpCAM) and used to induce subcutaneous or orthotopic tumors in C57BL/6J female mice. Mice bearing T110299 EpCAM+ tumors were treated with intratumoral or i.v. injections of 3p-RNA followed by i.v. injection of syngeneic murine T cells that were retrovirally tranduced to express anti-EpCAM CARs. Three days after CAR T cell injection, immune cell composition and CAR T cell infiltration in the TME were assessed by flow cytometry. Additionally, tumor growth and survival were monitored.ResultsIntratumoral injections of 3p-RNA reshaped the myeloid immune compartment in the TME by significantly reducing suppressive polymorphonuclear-MDSC and macrophages while increasing Ly6Chigh inflammatory monocytes. Moreover, antigen-presenting cells, such as dendritic cells and macrophages, were activated as evidenced by increased MHC-I expression levels. This was paralleled by a significant increase in the infiltration of CAR T cells into the TME in the combination therapy group. Interestingly, anti-EpCAM CAR T cells alone failed to control the tumor growth of T110299 EpCAM+ tumors, while monotherapy with 3p-RNA slightly delayed tumor growth in the subcutaneous model. Combination of 3p-RNA with anti-EpCAM CAR T cells induced a significant clinical benefit with tumor regression in 50% of the treated mice in the subcutaneous tumor model and prolonged survival in an orthotopic model.ConclusionsRemodeling the immunosuppressive TME using RIG-I ligands is a promising strategy for overcoming therapeutic resistance of CAR T cells in solid tumors, such as pancreatic cancer.FundingThe project was supported by the international doctoral program ‘i-Target: Immunotargeting of cancer’ funded by the Elite Network of Bavaria and the Stiftungen zu Gunsten der Medizinischen Fakultät.Disclosure InformationA.M. Senz: None. S.L. Formisano: None. B. Cadilha: None. T. Lorenzini: None. S. Endres: None. S. Kobold: None. M. Schnurr: None. L.M. König: None.

CBIO-16. DOSE INTENSIFIED TEMOZOLOMIDE IMPROVES INFILTRATION OF CD70 CAR T CELLS INTO THE GBM TUMOR MICROENVIRONMENT

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi30-vi30
Author(s):  
Aida Karachi ◽  
Jianping Huang ◽  
Haipeng Tao ◽  
Farhad Dastmalchi ◽  
Linchun Jin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
T Cell Exhaustion ◽  
Systemic Blood ◽  
Cell Exhaustion ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
And Function

Abstract CD70 CAR T cells developed by our group have anti-tumor efficacy in syngeneic murine GBM model and are currently being developed for first-in-human testing. The objective of this study was to evaluate the impact of temozolomide (TMZ) in various dosing strategies on the expansion, persistence and function of the CD70 CAR T cells after infusion. C57BL/6 mice underwent intracranial implantation of KR-158 overexpressing CD70 tumor cells. Tumor bearing animals were treated with standard dose (SD) (50 mg/kg x 5 days), metronomic dose (MD) (25mg/kg x 10 days), or dose intensified (DI) (75mg/kg x 5 days) of TMZ, followed by 10×106 CAR T cells infusion. Peripheral blood was collected to monitor the persistence of CAR T cells in the systemic blood circulation. Six weeks post treatments, spleens and tumors were collected and CAR T cells abundance and function were measured. TMZ preconditioning resulted in the expected lymphopenia in animals by generation of dose dependent lymphopenia. Circulating CD70 CAR T cells peaked in the systemic blood 2 weeks after infusion (3-fold, p< 0.0001). Markers of T cell exhaustion including PD-1 and TOX expression on CAR T cells were not different between the three TMZ treatment groups compared to control. Six weeks post treatment, CD70 CAR T cells were found to be highly infiltrated within the tumor microenvironment in the DI TMZ group compared to the other groups (4-fold,p < 0.0001). Tumor infiltrating CD70 CAR T cells in the DI TMZ group did not have an increase in PD-1 and TOX expression which was seen in the MD and SD TMZ groups. DI TMZ preconditioning results in greater CD70 CAR T cell trafficking to the tumor without T cell exhaustion compared to lower doses of TMZ. The affects of TMZ on the immune microenvironment to enhance CAR T cell regimens warrants further study.

scholarly journals Enhancing the Efficacy of CAR T Cells

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. SCI-15-SCI-15
Author(s):  
Crystal L. Mackall
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Board Of Directors ◽  
T Cell Exhaustion ◽  
Advisory Committees ◽  
Cell Exhaustion ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

The impressive response rates of chimeric antigen receptor (CAR) modified T cells in B-ALL and high grade B cell lymphomas illustrate their remarkable capacity to kill chemoresistant cancer. However, much work remains to be done if CAR T cells are to cure a high fraction of patients with B cell malignancies and if this potency is to be extended to other chemoresistant cancers. Experience in B cell malignancies has identified primary mechanisms of resistance to CAR T cells as antigen escape and T cell failure, which are generally mutually exclusive, such that antigen escape occurs in cases where CAR T cells persist whereas antigen positive recurrence is associated with disappearance of CAR T cells. A significant factor driving antigen escape is a requirement for high antigen density for CAR T cell function. Unlike natural T cells which are activated by low antigen density (e.g. ~10 peptides/cell), CAR T cells require high antigen density for activation, estimated to be ~10,000 antigens/cell. This is most well illustrated by experience with the CD22-CARs for B-ALL. CD22-CARs induce high response rates in both CD19+ and CD19neg/lo B-ALL, however this is followed by a significant rate of relapse associated with expression of CD22lo B-ALL. The basis for the CAR T cell requirement for high antigen density remains incompletely understood and efforts are underway to lower the antigen density required for CAR T cell activation. But this property could be leveraged to provide a therapeutic window for targeting antigens with low level expression on normal tissues, which may be useful for extending the efficacy of CAR T cells to solid tumors. Furthermore, acknowledgement of the CAR requirement for high antigen density as well as the general property of tumor heterogeneity leads to the prediction that monospecific CAR T cells are unlikely to eradicate most cancers. We have therefore focused efforts on optimizing approaches to develop bispecific CAR T cells and are currently testing bivalent, bispecific CD19/22-CAR T cells capable of activating in response to CD19 and/or CD22 expression. We hypothesize that simultaneous targeting of two antigens will lower the risk of antigen loss escape in B cell malignancies. The second major cause of resistance following CAR T cell therapy is T cell failure, most commonly as a result of T cell exhaustion. Current understanding of the biology of human T cell exhaustion remains incomplete. My laboratory has developed a model of "T cell exhaustion in a dish", which uses a synthetic biology model of tonically signaling CAR T cells to induce excessive TCR zeta phosphorylation and drive T cell exhaustion. We have observed that temporary cessation of TCR zeta phosphorylation using a small molecule based system to regulate CAR protein expression levels leads to remarkable rejuvenation of exhausted T cells. The rejuvenation induced via temporary cessation of zeta phosphorylation is significantly more potent than that induced by PD-1 blockade. These results suggest that regulatable CAR expression systems could prevent and/or reverse T cell exhaustion by attacking the "root cause", namely excessive or prolonged zeta phosphorylation. Disclosures Mackall: Vor Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Adapt immune: Membership on an entity's Board of Directors or advisory committees; Glaxo-Smith-Kline: Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Patents & Royalties: CD22-CAR; Bluebird Bio: Research Funding.

scholarly journals Chimeric antigen receptor T cells targeting PD-L1 suppress tumor growth

2020 ◽  
Author(s):  
Le Qin ◽  
Ruocong Zhao ◽  
Dongmei Chen ◽  
Xinru Wei ◽  
Qiting Wu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Solid Tumors ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Car T Cells ◽  
Car T

Abstract Background: Chimeric antigen receptor T cells (CAR-T cells) therapy has been well recognized for treating B cell-derived malignancy. However, the efficacy of CAR-T cells against solid tumors remains dissatisfactory, partially due to the heterogeneity of solid tumors and T cell exhaustion in tumor microenvironment. PD-L1 is up-regulated in multiple solid tumors, resulting in T cell exhaustion upon binding to its receptor PD-1. Methods: Here, we designed a dominant-negative form of PD-1 , dPD1z, a vector containing the extracellular and transmembrane regions of human PD-1, and a CAR vector against PD-L1, CARPD-L1z, a vector employs a high-affinity single-chain variable fragment (scFv) against human PD-L1. These two vectors shared the same intracellular structure, including 4-1BB and TLR2 co-stimulatory domains, and the CD3ζ signaling domain. Results: dPD1z T and CARPD-L1z T cells efficiently lysed PD-L1 + tumor cells and had enhanced cytokine secretion in vitro and suppressed the growth of non-small cell lung cancer (NSCLC), gastric cancer and hepatoma carcinoma in patient-derived xenograft (PDX). However, the combination of anti-mesothelin CAR-T cells (CARMSLNz T) with dPD1z T or CARPD-L1z T cells did not repress tumor growth synergistically in PDX, as CARMSLNz T cells upregulated PD-L1 expression upon activation and were subsequently attacked by dPD1z T or CARPD-L1z T cells. Conclusions: In conclusion, we demonstrate CAR-T cells targeting PD-L1 were effective for suppressing the growth of multiple types of solid tumors in PDX models though their safety needs to be carefully examined.

142 Contextual reprogramming of CAR T cells for the treatment of HER2-expressing cancers

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A156-A156
Author(s):  
Zhifen Yang ◽  
Lingyu Li ◽  
Ahu Turkoz ◽  
Pohan Chen ◽  
Hana Choi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Proliferation In Vitro

BackgroundCombining checkpoint inhibition (CPI) to adoptive cell therapy (ACT) is a promising strategy to prevent chimeric antigen receptor (CAR)-engineered T cell exhaustion and improve outcomes. However, cumulative toxicities and costs limit this approach. Here, we apply a conditional, antigen-dependent non-editing CRISPR interference-(CRISPRi) modulation circuit that we originally described in yeast and eukaryotes1–3 (RB-340-1) to promote CAR T resilience to checkpoint suppression extending in vivo persistence and effectiveness.MethodsRB-340-1 is an CAR T cell product engineered via synthetic biology approaches to express a combination of molecules to prevent CAR T cell exhaustion and improve solid tumor treatment outcomes. The components include two constructs. The first construct encodes an anti-HER2 (4D5) CAR single chain variable fragment (scFv), with CD28 and CD3ζ co-stimulatory domains linked to a tobacco etch virus (TEV) protease and a programmed cell death protein 1 (PD1) promoter region-targeting single guide RNA (PD1sg). The second construct encodes a protein, linker for activation of T cells (LAT), complexed to nuclease-deactivated/dead Cas9 (dCas9)-Kruppel-associated box (Krab) via a TEV-cleavable linker. Activation of CAR brings CAR-TEV in close proximity to the LAT-dCas9-Krab complex releasing the enzyme for nuclear localization to the PD1 regulatory region to conditionally and reversibly suppress its expression. RB-340-1 was compared in vitro and in vivo against conventional and control (cRB-340-1, lacking PD1sg) HER2 CAR T cells in combination with CPI with Atezolizumab (5 mg/Kg administered intravenously twice a week).ResultsRB-340-1 consistently induced higher production of homeostatic cytokines such as IL-2 resulting in significantly enhanced proliferation in vitro (figure 1a). Our in vivo data showed significantly enhanced suppression of growth of HER2+ FADU oropharyngeal cancer xenografts upon intra-tumoral (figure 1b) and systemic administration (figure 1c) and prolonged persistence of CAR T cells in vivo.Abstract 142 Figure 1Rb-340-1 performance in vitro and in vivoRB-340-1 (orange) decreased PD-1 expression resulting in enhanced cytokine production and proliferation in vitro (figure 1a) and superior tumor suppression in vivo after intra-tumoral (figure 1b) or intravenous (figure 1c) administration compared to conventional CAR T cells (blue) or cRB-340-1 (green). Conventional CAR T cells or cRB-340-1 CAR T combination treatment with PDL1 blockade (Atezolizumab) is shown as dashed line. Colonization of tumors by human CD45+ cells is shown at the bottom of figure 1b & 1cConclusionsIntrinsic conditional regulation of checkpoint expression in CAR T cells provides a simplified approach to combination therapies that limits systemic toxicities and reduces costs. Since the expression of multiple genes can be simultaneously controlled by CRISPRi, broader applications can be envisioned in the future.Ethics ApprovalNot ApplicableConsentNot applicableReferencesGilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y, Whitehead EH, et al. Genome-Scale CRISPR-mediated control of gene repression and activation. Cell 2014;159(3):647-61.Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 2013;154(2):442–51.Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 2013;152(5):1173–83.

scholarly journals Dynamic chromatin regulatory landscape of human CAR T cell exhaustion

2021 ◽  
Author(s):  
David G Gennert ◽  
Rachel Christina Lynn ◽  
Jeffrey M Granja ◽  
Evan W Weber ◽  
Maxwell Robert Mumbach ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cancer Immunotherapy ◽  
Target Genes ◽  
Ex Vivo ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Dysfunction in T cells limits the efficacy of cancer immunotherapy. We profiled the epigenome, transcriptome, and enhancer connectome of exhaustion-prone GD2-targeting HA-28z chimeric antigen receptor (CAR) T cells and control CD19-targeting CAR T cells, which present less exhaustion-inducing tonic signaling, at multiple points during their ex vivo expansion. We found widespread, dynamic changes in chromatin accessibility and 3D chromosome conformation preceding changes in gene expression, notably at loci proximal to exhaustion-associated genes such as PDCD1, CTLA4, and HAVCR2, and increased DNA motif access for AP-1 family transcription factors, which are known to promote exhaustion. Although T cell exhaustion has been studied in detail in mouse, we find that the regulatory networks of T cell exhaustion differ between the species and involve distinct loci of accessible chromatin and cis-regulated target genes in human CAR T cell exhaustion. Deletion of exhaustion-specific candidate enhancers of PDCD1 suppress the expression of PD-1 in an in vitro model of T cell dysfunction and in HA-28z CAR T cells, suggesting enhancer editing as a path forward in improving cancer immunotherapy.

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