Dynamic chromatin regulatory landscape of human CAR T cell exhaustion

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.

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Dynamic chromatin regulatory landscape of human CAR T cell exhaustion

10.1101/2021.04.02.438240 ◽

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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Chimeric Antigen Receptor T Cell Exhaustion during Treatment for Hematological Malignancies

BioMed Research International ◽

10.1155/2020/8765028 ◽

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.

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

Blood ◽

10.1182/blood-2018-99-110645 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 966-966 ◽

Cited By ~ 2

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.

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

Neuro-Oncology ◽

10.1093/neuonc/noab196.116 ◽

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.

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

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-001688 ◽

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.

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Enhancing the Efficacy of CAR T Cells

Blood ◽

10.1182/blood.v130.suppl_1.sci-15.sci-15 ◽

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.

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

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0142 ◽

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.

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Presence of Endogenous TCR Antigen in Vivo Attenuates Efficacy of Anti-CD19 Targeted CAR T Cell Therapy

Blood ◽

10.1182/blood.v124.21.3721.3721 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3721-3721

Author(s):

Yinmeng Yang ◽

Christopher Daniel Chien ◽

Elad Jacoby ◽

Haiying Qin ◽

Waleed Haso ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Ex Vivo ◽

Ifn Gamma ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell ◽

Car T Cell Therapy ◽

Car T

Abstract Adoptive therapy using T cells genetically engineered to express chimeric antigen receptors (CAR) has proven extremely effective against acute lymphoblastic leukemia (ALL) in clinical trials with the use of anti-CD19 CAR T cells. Most CAR T cell protocols use autologous T cells, which are then activated, transduced with the anti-CD19 CAR, and expanded ex-vivo before infusion back into the patient. This approach minimizes the risk of graft-versus-host disease (GVHD) even in allogeneic transplant recipients, due to tolerization of the donor T cell repertoire in the recipient. However, many patients have heavy disease burden and lymphopenia due to previous treatments, which makes the isolation of healthy T cells difficult. Thus, centers are exploring the potential of allogeneic T cell donors and the possibility of universal T cell donors for CAR-based therapy including the use of virus-specific T cells. In these cases, in addition to the chimeric receptor specificity, the transduced T cell population will also have reactivity against target antigens through the endogenous TCR. However, little is known about the impact of signaling of the endogenous TCR on CAR T cell activity, particularly in vivo. To test this, we used a syngeneic transplantable ALL murine model, E2aPBx, in which CD19 CAR T cells can effectively eradicate ALL. CD4 (Marilyn) and CD8 (Matahari) T cells from syngeneic HY-TCR transgenic donors specific for the minor histocompatibility male antigen, HY, were used as CAR T cell donors to control for endogenous TCR reactivity. Splenic T cells isolated from Matahari, Marilyn, or B6 mice were activated ex-vivo using anti-CD3/anti-CD28 beads, with the addition of IL2 and IL7. T cells were transduced with a retroviral vector expressing a murine CAR composed of anti-CD19 scfv/CD28/CD3ζ on days two and three. CAR T cells are evaluated in vitro by CD107a degranulation assay and INF gamma ELISA. In response to HY peptide alone or HY+CD19- line M39M, transduced CD8 HY (Matahari) cells produced IFN gamma and expressed CD107a whereas transduced CD4 HY (Marilyn) cells only produced IFN gamma. Interestingly, in response to CD19+HY- ALL, both Matahari and Marilyn expressed CD107a and produced IFN gamma indicating that CD4 T cells can acquire CD8-like lytic activity when stimulated through a CAR receptor. When CD19 CAR transduced Marilyns and Mataharis were stimulated in the presence of HY and CD19, CD8 Mataharis had an attenuated effect against CD19, suggesting that the presence of antigen activated TCR adversely affects the potency of the CAR receptor. Efficacy of the HY and polyclonal CAR T cells were next tested in-vivo in male and female B6 mice. Mice were given 1E6 E2aPBx ALL leukemia cells on day 1, and received 500 rads sub-lethal total body irradiation on day 4 as a lymphodepleting regimen. On day 5, mice were given a low (1E5) or high (5E6) dose of CAR T cells. There was a statistically significant (p=0.0177) improvement in the survival of female versus male mice after treatment with the CD4+ HY specific anti-CD19 CAR T cells, and female mice that received HY anti-CD19 CAR T cells survived longer than untreated control females (p=0.01). Remarkably, the survival of male mice that received HY anti-CD19 CAR T cells was statistically worse than untreated control males (p=0.008). This suggests that the presence of TCR antigen negatively impacts the function of CAR T cells. Furthermore, in a separate experiment using an equally mixed population of Marilyn (CD4+) and Matahari (CD8+) HY specific T cells, males has a statistically significantly (p=0.0116) worse survival compared to females after receiving 5E5 HY specific T cells. In conclusion, simultaneous stimulation through both CAR and TCR results in attenuated cytokine production and degranulation by CD8 T cells. In vivo, in the presence of the endogenous TCR antigen, both CD4 and CD8 CAR T cells are less potent at eradicating leukemia. These have implications for the development of universal donors for CAR T cell therapy. Disclosures No relevant conflicts of interest to declare.

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Manufacturing an Enhanced CAR T Cell Product By Inhibition of the PI3K/Akt Pathway During T Cell Expansion Results in Improved In Vivo Efficacy of Anti-BCMA CAR T Cells

Blood ◽

10.1182/blood.v126.23.1893.1893 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1893-1893 ◽

Cited By ~ 6

Author(s):

Molly R. Perkins ◽

Shannon Grande ◽

Amanda Hamel ◽

Holly M. Horton ◽

Tracy E. Garrett ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Ex Vivo ◽

Employment Equity ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Equity Ownership ◽

Cells Cultured

Abstract Patients treated with chimeric antigen receptor (CAR) T cells targeting CD19 for B cell malignancies have experienced rapid and durable tumor regressions. Manufacture of CAR T cells is challenged by the necessity to produce a unique drug product for each patient. Each treatment requires ex vivo culture of patient T cells to facilitate CAR gene transfer and to achieve therapeutic amounts of T cells. Paradoxically, ex vivo culture with IL-2 also decreases CAR T cell activity. Some investigators have proposed isolating central memory T cells (thought to be enriched for therapeutic T cells), yet isolation techniques are cumbersome and costly to scale commercially. Culture of T cells in IL-7 and IL-15 has also been shown by several investigators to improve therapeutic activity. Here we explored the potential for culture modifications to improve the therapeutic potential of CAR T cells without adding complexity to manufacturing. We tested this hypothesis using CAR T cells specific to B cell maturation antigen (BCMA) manufactured using standard IL-2 culture with an inhibitor of PI3K added to the media, or with IL-7 and IL-15 in place of IL-2. The in vivo activity was studied in NSG mouse models of human Burkitt's lymphoma (Daudi), and multiple myeloma (RPMI-8226), both of which express BCMA. In the lymphoma model, NSG mice were injected intravenously (IV) with 2 x 106 Daudi cells and allowed to accumulate a large tumor burden before being treated with 4 x 106 CAR+ T cells on day 18 post-tumor injection. At this late time point post implantation, mice had highly disseminated Daudi tumor (our goal was to model late stage disease observed in relapsed and refractory lymphoma). In this model of advanced disease, IL-2 cultured anti-BCMA CAR T cells had no effect on tumor growth (p = 0.22) and all mice succumbed to the tumors within two weeks after treatment. Anti-BCMA CAR T cells grown in IL-7 and IL-15 also failed to control tumor growth (p = 0.23). In sharp contrast, all animals treated with anti-BCMA CAR T cells cultured with the PI3K inhibitor survived and experienced complete long-term tumor regression (p=0.003). The same anti-BCMA CAR T cells were used in a model of multiple myeloma. NSG mice were injected subcutaneously (SC) with 107 RPMI-8226 MM cells, and at 22 days post-implantation mice received a single IV administration of anti-BCMA CAR T cells (4 x 105 CAR+ T cells/mouse) cultured under various conditions. In this model, all treatment groups demonstrated tumor regression, regardless of the in vitro culture conditions. To evaluate CAR T cell durability, two weeks after initial tumor clearance, surviving animals were then re-challenged with RPMI-8226 cells on the opposite flank to model tumor relapse. We found that only animals that had been treated with anti-BCMA CAR T cells cultured with PI3K inhibition were immune to subsequent tumor challenge (p=0.005). Given the superior in vivo efficacy of anti-BCMA CAR T cells cultured with PI3K inhibition, we sought to identify phenotypic characteristics associated with the improved therapeutic activity. Anti-BCMA CAR T cells cultured with PI3K inhibition contained an increased frequency of CD62L+ CD8 T cells in the final product (p < 0.001) suggesting improved expansion of a distinct CD8 T cell subset. These data suggest that inhibition of PI3K during ex vivo expansion with IL-2 may generate a superior anti-BCMA CAR T cell product for clinical use. Furthermore, this approach could potentially be used in the manufacture of other T cell therapies. Disclosures Perkins: bluebird bio: Employment, Equity Ownership. Grande:bluebird bio: Employment, Equity Ownership. Hamel:bluebird bio: Employment, Equity Ownership. Horton:bluebird bio: Employment, Equity Ownership. Garrett:bluebird bio: Employment, Equity Ownership. Miller:bluebird bio: Employment, Equity Ownership. Latimer:bluebird bio: Employment, Equity Ownership. Horvath:bluebird bio: Employment, Equity Ownership. Kuczewski:bluebird bio: Employment, Equity Ownership. Friedman:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership.

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Preservation of T-Cell Stemness with a Novel Expansionless CAR-T Manufacturing Process, Which Reduces Manufacturing Time to Less Than Two Days, Drives Enhanced CAR-T Cell Efficacy

Blood ◽

10.1182/blood-2021-146246 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2848-2848

Author(s):

Boris Engels ◽

Xu Zhu ◽

Jennifer Yang ◽

Andrew Price ◽

Akash Sohoni ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Ex Vivo ◽

Antitumor Efficacy ◽

Publicly Traded ◽

Car T Cells ◽

Car T Cell ◽

Car T

Abstract Background: Extended T-cell culture periods in vitro deplete the CAR-T final product of naive and stem cell memory T-cell (T scm) subpopulations that are associated with improved antitumor efficacy. YTB323 is an autologous CD19-directed CAR-T cell therapy with dramatically simplified manufacturing, which eliminates complexities such as long culture periods. This improved T-Charge™ process preserves T-cell stemness, an important characteristic closely tied to therapeutic potential, which leads to enhanced expansion ability and greater antitumor activity of CAR-T cells. Methods: The new T-Charge TM manufacturing platform, which reduces ex vivo culture time to about 24 hours and takes <2 days to manufacture the final product, was evaluated in a preclinical setting. T cells were enriched from healthy donor leukapheresis, followed by activation and transduction with a lentiviral vector encoding for the same CAR used for tisagenlecleucel. After ≈24 hours of culture, cells were harvested, washed, and formulated (YTB323). In parallel, CAR-T cells (CTL*019) were generated using a traditional ex vivo expansion CAR-T manufacturing protocol (TM process) from the same healthy donor T cells and identical lentiviral vector. Post manufacturing, CAR-T products were assessed in T-cell functional assays in vitro and in vivo, in immunodeficient NSG mice (NOD-scid IL2Rg-null) inoculated with a pre-B-ALL cell line (NALM6) or a DLBCL cell line (TMD-8) to evaluate antitumor activity and CAR-T expansion. Initial data from the dose escalation portion of the Phase 1 study will be reported separately. Results: YTB323 CAR-T products, generated via this novel expansionless manufacturing process, retained the immunophenotype of the input leukapheresis; specifically, naive/T scm cells (CD45RO -/CCR7 +) were retained as shown by flow cytometry. In contrast, the TM process with ex vivo expansion generated a final product consisting mainly of central memory T cells (T cm) (CD45RO +/CCR7 +) (Fig A). Further evidence to support the preservation of the initial phenotype is illustrated by bulk and single-cell RNA sequencing experiments, comparing leukapheresis and final products from CAR-Ts generated using the T-Charge™ and TM protocols. YTB323 CAR-T cell potency was assessed in vitro using a cytokine secretion assay and a tumor repeat stimulation assay, designed to test the persistence and exhaustion of the cell product. YTB323 T cells exhibited 10- to 17-fold higher levels of IL-2 and IFN-γ secretion upon CD19-specific activation compared with CTL*019. Moreover, YTB323 cells were able to control the tumor at a 30-fold lower Effector:Tumor cell ratio and for a minimum of 7 more stimulations in the repeat stimulation assay. Both assays clearly demonstrated enhanced potency of the YTB323 CAR-T cells in vitro. The ultimate preclinical assessment of the YTB323 cell potency was through comparison with CTL*019 regarding in vivo expansion and antitumor efficacy against B-cell tumors in immunodeficient NSG mouse models at multiple doses. Expansion of CD3+/CAR+ T-cells in blood was analyzed weekly by flow cytometry for up to 4 weeks postinfusion. Dose-dependent expansion (C max and AUC 0-21d) was observed for both YTB323 and CTL*019. C max was ≈40-times higher and AUC 0-21d was ≈33-times higher for YTB323 compared with CTL*019 across multiple doses. Delayed peak expansion (T max) of YTB323 by at least 1 week compared with CTL*019 was observed, supporting that increased expansion was driven by the less differentiated T-cell phenotype of YTB323. YTB323 controlled NALM6 B-ALL tumor growth at a lower dose of 0.1×10 6 CAR+ cells compared to 0.5×10 6 CAR+ cells required for CTL*019 (Fig B). In the DLBCL model TMD-8, only YTB323 was able to control the tumors while CTL*019 led to tumor progression at the respective dose groups. This ability of YTB323 cells to control the tumor at lower doses confirms their robustness and potency. Conclusions: The novel manufacturing platform T-Charge™ used for YTB323 is simplified, shortened, and expansionless. It thereby preserves T-cell stemness, associated with improved in vivo CAR-T expansion and antitumor efficacy. Compared to approved CAR-T therapies, YTB323 has the potential to achieve higher clinical efficacy at its respective lower doses. T-Charge™ is aiming to substantially revolutionize CAR-T manufacturing, with concomitant higher likelihood of long-term deep responses. Figure 1 Figure 1. Disclosures Engels: Novartis: Current Employment, Current equity holder in publicly-traded company. Zhu: Novartis: Current Employment, Current equity holder in publicly-traded company. Yang: Novartis: Current Employment, Patents & Royalties. Price: Novartis: Current Employment. Sohoni: Novartis: Current Employment. Stein: Novartis: Current Employment. Parent: Novartis: Ended employment in the past 24 months; iVexSol, Inc: Current Employment. Greene: iVexSol, Inc: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Niederst: Novartis: Current Employment, Current equity holder in publicly-traded company. Whalen: Novartis: Current Employment. Orlando: Novartis: Current Employment. Treanor: Novartis: Current Employment, Current holder of individual stocks in a privately-held company, Divested equity in a private or publicly-traded company in the past 24 months, Patents & Royalties: no royalties as company-held patents. Brogdon: Novartis Institutes for Biomedical Research: Current Employment.

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