Reversible ON- and OFF-switch chimeric antigen receptors controlled by lenalidomide

Cell-based therapies are emerging as effective agents against cancer and other diseases. As autonomous “living drugs,” these therapies lack precise control. Chimeric antigen receptor (CAR) T cells effectively target hematologic malignancies but can proliferate rapidly and cause toxicity. We developed ON and OFF switches for CAR T cells using the clinically approved drug lenalidomide, which mediates the proteasomal degradation of several target proteins by inducing interactions between the CRL4CRBN E3 ubiquitin ligase and a C2H2 zinc finger degron motif. We performed a systematic screen to identify “super-degron” tags with enhanced sensitivity to lenalidomide-induced degradation and used these degradable tags to generate OFF-switch degradable CARs. To create an ON switch, we engineered a lenalidomide-inducible dimerization system and developed split CARs that required both lenalidomide and target antigen for activation. Subtherapeutic lenalidomide concentrations controlled the effector functions of ON- and OFF-switch CAR T cells. In vivo, ON-switch split CARs demonstrated lenalidomide-dependent antitumor activity, and OFF-switch degradable CARs were depleted by drug treatment to limit inflammatory cytokine production while retaining antitumor efficacy. Together, the data showed that these lenalidomide-gated switches are rapid, reversible, and clinically suitable systems to control transgene function in diverse gene- and cell-based therapies.

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Engineering better chimeric antigen receptor T cells

Experimental Hematology and Oncology ◽

10.1186/s40164-020-00190-2 ◽

2020 ◽

Vol 9 (1) ◽

Author(s):

Hao Zhang ◽

Pu Zhao ◽

He Huang

Keyword(s):

T Cells ◽

Intracellular Signaling ◽

Influence Factors ◽

Target Antigen ◽

Car T Cells ◽

Car T ◽

Underlying Mechanisms ◽

Almost All

AbstractCD19-targeted CAR T cells therapy has shown remarkable efficacy in treatment of B cell malignancies. However, relapse of primary disease remains a major obstacle after CAR T cells therapy, and the majority of relapses present a tumor phenotype with retention of target antigen (antigen-positive relapse), which highly correlate with poor CAR T cells persistence. Therefore, study on factors and mechanisms that limit the in vivo persistence of CAR T cells is crucial for developing strategies to overcome these limitations. In this review, we summarize the rapidly developing knowledge regarding the factors that influence CAR T cells in vivo persistence and the underlying mechanisms. The factors involve the CAR constructs (extracellular structures, transmembrane and intracellular signaling domains, as well as the accessory structures), activation signaling (CAR signaling and TCR engagement), methods for in vitro culture (T cells collection, purification, activation, gene transduction and cells expansion), epigenetic regulations, tumor environment, CD4/CD8 subsets, CAR T cells differentiation and exhaustion. Of note, among these influence factors, CAR T cells differentiation and exhaustion are identified as the central part due to the fact that almost all factors eventually alter the state of cells differentiation and exhaustion. Moreover, we review the potential coping strategies aiming at these limitations throughout this study.

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CAR T-cell therapy for glioblastoma: recent clinical advances and future challenges

Neuro-Oncology ◽

10.1093/neuonc/noy032 ◽

2018 ◽

Vol 20 (11) ◽

pp. 1429-1438 ◽

Cited By ~ 58

Author(s):

Stephen J Bagley ◽

Arati S Desai ◽

Gerald P Linette ◽

Carl H June ◽

Donald M O’Rourke

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

Hematologic Malignancies ◽

Antigen Receptors ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell ◽

Car T Cell Therapy ◽

Car T

Abstract In patients with certain hematologic malignancies, the use of autologous T cells genetically modified to express chimeric antigen receptors (CARs) has led to unprecedented clinical responses. Although progress in solid tumors has been elusive, recent clinical studies have demonstrated the feasibility and safety of CAR T-cell therapy for glioblastoma. In addition, despite formidable barriers to T-cell localization and effector function in glioblastoma, signs of efficacy have been observed in select patients. In this review, we begin with a discussion of established obstacles to systemic therapy in glioblastoma and how these may be overcome by CAR T cells. We continue with a summary of previously published CAR T-cell trials in GBM, and end by outlining the key therapeutic challenges associated with the use of CAR T cells in this disease.

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"IF-Better" Gating: Combinatorial Targeting and Synergistic Signaling for Enhanced CAR T Cell Efficacy

Blood ◽

10.1182/blood-2021-149263 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2774-2774

Author(s):

Sascha Haubner ◽

Jorge Mansilla-Soto ◽

Sarah Nataraj ◽

Xingyue He ◽

Jae H Park ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Target Antigen ◽

Normal Cells ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Provision Of Services ◽

Xenograft Models

Abstract CAR T cell therapy provides a potent therapeutic option in various B cell-related hematologic malignancies. One of the major efficacy challenges is escape of tumor cells with low antigen density, which has been clinically observed in several malignancies treated with CAR therapy. Novel concepts of CAR design are needed to address phenotypic heterogeneity including clonal variability of target antigen expression. In the study presented here, we focused on AML and selected ADGRE2 as CAR target due to its high rate of positivity on AML bulk and leukemic stem cells (LSC) in a molecularly heterogeneous AML patient population. We chose an ADGRE2-CAR with optimized scFv affinity and fine-tuned CD3zeta signaling to achieve an ideal killing threshold that would allow for sparing of ADGRE2-low normal cells. We hypothesized that co-targeting of a second AML-related antigen may mitigate potential CAR target antigen-low AML escape and we identified CLEC12A as preferential co-target due to its non-overlapping expression profiles in normal hematopoiesis and other vital tissues. We developed ADCLEC.syn1, a novel combinatorial CAR construct consisting of an ADGRE2-targeting 28z1XX-CAR and a CLEC12A-targeting chimeric costimulatory receptor (CCR). ADCLEC.syn1 operates based on what we describe as "IF-BETTER" gate: High CAR target expression alone triggers killing, whereas low CAR target expression does not, unless a CCR target is present. Additional CCR interaction lowers the threshold for CAR-mediated killing through increased avidity and costimulation, allowing for higher CAR sensitivity that is purposefully limited to target cells expressing both antigens. In the context of ADCLEC.syn1, ADGRE2-high/CLEC12A-negative AML cells can trigger cell lysis while ADGRE2-low/CLEC12A-negative normal cells are spared. Importantly, ADGRE2-low/CLEC12A-high AML cells are also potently eliminated, preventing ADGRE2-low AML escape. Using NSG in-vivo xenograft models of engineered MOLM13 AML cell line variants with low levels of ADGRE2 to model antigen escape, we found that ADCLEC.syn1 outperforms a single-ADGRE2-CAR lacking assistance via CLEC12A-CCR. Importantly, ADCLEC.syn1 also outperformed an otherwise identical alternative dual-CAR version (OR-gated ADGRE2-CAR+CLEC12A-CAR) in the setting of both ADGRE2-high and ADGRE2-low MOLM13, further underlining the importance of fine-tuned overall signaling. We confirmed high in-vivo potency against diverse AML cell lines with a wide range of ADGRE2 and CLEC12A levels reflecting population-wide AML heterogeneity. At clinically relevant CAR T cell doses, ADCLEC.syn1 induced complete and durable remissions in xenograft models of MOLM13 (ADGRE2-high/CLEC12A-low) and U937 (ADGRE2-low/CLEC12A-high). ADCLEC.syn1 CAR T cells were found to be functionally persistent for >70 days, with a single CAR T cell dose potently averting relapse modeled via AML re-challenges. In summary, we provide pre-clinical evidence that an "IF-BETTER"-gated CAR+CCR T cell (ADCLEC.syn1) can outperform a single-CAR T cell (ADGRE2-CAR) and a dual-CAR T cell (ADGRE2-CAR+CLEC12A-CAR). ADCLEC.syn1 enhances antileukemic efficacy and prevents antigen-low AML escape via detection of a rationally selected combinatorial target antigen signature that is commonly found in AML but limited in vital normal cells. Using phenotypically representative AML xenograft models and clinically relevant T cell doses, we demonstrate high therapeutic potential of ADCLEC.syn1 CAR T cells, further supporting clinical translation of an "IF-BETTER"-gated CAR concept into a phase 1 trial. Disclosures Haubner: Takeda Pharmaceuticals Company Ltd.: Patents & Royalties: Co-inventor of IP that MSK licensed to Takeda, Research Funding. Mansilla-Soto: Takeda Pharmaceuticals Company Ltd.: Patents & Royalties; Atara Biotherapeutics: Patents & Royalties; Fate Therapeutics: Patents & Royalties; Mnemo Therapeutics: Patents & Royalties. He: Takeda Pharmaceuticals Company Ltd.: Ended employment in the past 24 months, Patents & Royalties. Park: Curocel: Consultancy; BMS: Consultancy; Innate Pharma: Consultancy; Autolus: Consultancy; Servier: Consultancy; Kite Pharma: Consultancy; Affyimmune: Consultancy; Intellia: Consultancy; Minerva: Consultancy; PrecisionBio: Consultancy; Amgen: Consultancy; Kura Oncology: Consultancy; Artiva: Consultancy; Novartis: Consultancy. Rivière: Juno Therapeutics: Patents & Royalties; Fate Therapeutics: Other: Provision of Services, Patents & Royalties; Centre for Commercialization of Cancer Immunotherapy: Other: Provision of Services; The Georgia Tech Research Corporation (GTRC): Other: Provision of Services (uncompensated); FloDesign Sonics: Other: Provision of Services. Sadelain: NHLBI Gene Therapy Resource Program: Other: Provision of Services (uncompensated); St. Jude Children's Research Hospital: Other: Provision of Services; Minerva Biotechnologies: Patents & Royalties; Mnemo Therapeutics: Patents & Royalties; Juno Therapeutics: Patents & Royalties; Fate Therapeutics: Other: Provision of Services (uncompensated), Patents & Royalties; Ceramedix: Patents & Royalties; Takeda Pharmaceuticals: Other: Provision of Services, Patents & Royalties; Atara Biotherapeutics: Patents & Royalties.

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Synthetic Chimeric Antigen Receptors (CARs) Rapidly Induce Exhaustion and Augmented Glycolytic Metabolism In Human T Cells and Implicate Persistent CD28 Signaling As a Driver Of Exhaustion In Human T Cells

Blood ◽

10.1182/blood.v122.21.192.192 ◽

2013 ◽

Vol 122 (21) ◽

pp. 192-192

Author(s):

Adrienne H. Long ◽

Rimas J. Orentas ◽

Crystal L. Mackall

Keyword(s):

T Cells ◽

Antitumor Efficacy ◽

Antigen Receptors ◽

Antitumor Effects ◽

Car T Cells ◽

Human T Cells ◽

Car T ◽

In Vitro Expansion

Abstract Introduction Chimeric antigen receptors (CARs) provide a promising new approach for the adoptive immunotherapy of cancer. Though impressive antitumor activity has been observed with some CAR T cells, other CAR T cells demonstrate poor antitumor efficacy in vivo despite high cytolytic capacity in vitro due to poor expansion and persistence. Whether exhaustion of CAR T cells mirrors exhaustion that occurs naturally in chronically stimulated human T cells has not yet been studied. Here, we report that expression of select CD28 containing CARs in normal human T cells rapidly induces an exhausted state characterized by high PD-1 expression, poor persistence and poor antitumor efficacy, whereas other CARs do not induce this phenotype. Results Human T cells were expanded with anti-CD3/CD28 beads, and then transduced with a second-generation (CD28-CD3ζ) disialoganglioside 2 (GD2) specific CAR or a second-generation (CD28-CD3ζ) CD19 specific CAR. By day 7 of in vitro expansion, GD2 CAR T cells developed a metabolism more highly dependent on glycolysis compared to CD19 CAR T cells or untransduced controls. Neither CAR population was exposed to antigen during this expansion period. Using a Seahorse Extracellular Flux Analyzer, the ratio of glycolysis to oxidative phosphorylation rates (ECAR:OCR ratio) of GD2 CAR T cells was found to be double that of CD19 CAR T cells or controls on day 7. The highly glycolytic metabolism of GD2 CAR T cells was associated with an exhausted phenotype. GD2 CAR T cells expressed higher levels of PD-1, TIM-3 and LAG-3, and transcription repressor BLIMP-1, compared to CD19 CAR T cells or untransduced controls. Additionally, GD2 CAR T cells were poor cytokine producers, generating <10x lower levels of IL2, TNFα and IFNγ than CD19 CAR T cells upon in vitro co-incubation with a GD2+CD19+ osteosarcoma line (143B-CD19), despite maintaining comparable in vitro cytolytic ability. GD2 CAR T cells showed poor in vitro expansion and increased rates of apoptosis compared to controls. GD2 CAR T cells also did not persist and did not mediate antitumor effects against GD2+CD19+ tumors in a murine xenograft model in vivo, whereas CD19 CAR T cells completely eradicated CD19+ tumors and persisted in both the spleen and tumor compartments. To rule out the possibility that diminished cytokine production and in vivo efficacy was related to antigen specific effects, T cells were co-transduced with both the GD2 and CD19 CARs. Though single-transduced CD19 CAR T cells show no signs of an altered metabolism or exhaustion and have strong antitumor efficacy, CD19 CAR T cells co-transduced with the GD2 CAR demonstrate an exhausted phenotype and diminished antitumor efficacy similar to that of single-transduced GD2 CAR T cells. Thus, expression of the GD2 CAR confers a dominant exhausted phenotype in T cells, and prevents otherwise efficacious CARs from mediating strong antitumor effects. We hypothesized that chronic signaling of CD3ζ and CD28 via the GD2 CAR results in exhaustion. Interestingly, however, we did not identify GD2 expression in the culture system. Point mutations in the CAR antigen-binding site, though abrogating GD2 binding, did not prevent the development of exhaustion. Thus, we postulate that constitutive receptor signaling may occur via interactions between the framework regions of the CAR receptors. Importantly however, substitution of 4-1BB for the CD28 domain in the GD2 CAR substantially diminished PD-1 expression, one of the hallmark features of exhausted T cells. Conclusions We report that expression of a CD28 containing GD2 CAR induces both an altered metabolism and an exhausted state in human T cells, resulting in poor in vivo persistence and antitumor efficacy. We hypothesize that tonic signaling through the GD2 CAR induces this phenotype and have identified the CD28 domain as an important component contributing to this phenotype. Rapid induction of exhaustion mediated via a synthetic receptor provides a novel model system to identify mechanistic factors required for this phenotype in human T cells. Work is currently underway to molecularly define the basis for the exhaustion of GD2 CAR T cells and to probe a potential role for altered T cell metabolism as a contributor to T cell exhaustion in human T cells. Disclosures: No relevant conflicts of interest to declare.

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Modular Chimeric Antigen Receptor Systems for Universal CAR T Cell Retargeting

International Journal of Molecular Sciences ◽

10.3390/ijms21197222 ◽

2020 ◽

Vol 21 (19) ◽

pp. 7222

Author(s):

Ashley R. Sutherland ◽

Madeline N. Owens ◽

C. Ronald Geyer

Keyword(s):

T Cells ◽

T Cell ◽

Cell Activity ◽

Cellular Immunotherapy ◽

Antigen Receptors ◽

Antigen Specificity ◽

Precise Control ◽

Car T Cells ◽

Multiple Antigen ◽

Car T

The engineering of T cells through expression of chimeric antigen receptors (CARs) against tumor-associated antigens (TAAs) has shown significant potential for use as an anti-cancer therapeutic. The development of strategies for flexible and modular CAR T systems is accelerating, allowing for multiple antigen targeting, precise programming, and adaptable solutions in the field of cellular immunotherapy. Moving beyond the fixed antigen specificity of traditional CAR T systems, the modular CAR T technology splits the T cell signaling domains and the targeting elements through use of a switch molecule. The activity of CAR T cells depends on the presence of the switch, offering dose-titratable response and precise control over CAR T cells. In this review, we summarize developments in universal or modular CAR T strategies that expand on current CAR T systems and open the door for more customizable T cell activity.

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132 HLA-independent T cell receptors effectively target low abundance antigens

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.132 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A142-A142

Author(s):

Jorge Mansilla-Soto ◽

Justin Eyquem ◽

Sascha Haubner ◽

Mohamad Hamieh ◽

Judith Feucht ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

High Sensitivity ◽

Target Antigen ◽

Dependent Manner ◽

Cell Surface Antigens ◽

Car T Cells ◽

Car T ◽

Increased Sensitivity

BackgroundChimeric antigen receptors (CARs) engage antigen independently of HLA and enable sustained T cell proliferation when they are endowed with both activating and costimulatory functions. While remission rates have been noticeably elevated in numerous clinical trials targeting CD19, CD22 or BCMA, relapses are common. One of the several underlying relapse mechanisms is antigen escape, which refers to a relapsing tumor that is either negative for the targeted antigen or expresses the latter at a low level. Failure to eliminate antigen-low tumors raises questions about the sensitivity of CARs and the minimum antigen density that is required for effective tumor eradication. Unlike CARs, TCRs engage antigen in an HLA-dependent manner, and they do so with high sensitivity. We hypothesized that a TCR/CD3 complex containing the same heavy and light immunoglobulin chains as a CAR will display increased sensitivity to the target antigen.MethodsWe edited the TRAC locus in human primary T cells to establish a novel antigen receptor structure, termed HLA-independent TCR or HIT receptor, by incorporating into the TCR/CD3 complex the same heavy and light chains as those of a corresponding CAR. We assessed their antigen sensitivity against a panel of cell lines expressing different antigen levels, analyzing their cytotoxicity, cytokine secretion, signaling response and degranulation activity. HIT and CAR T cells were further evaluated for their anti-tumor response using established ALL and AML mouse models.ResultsCD19-TRAC-HIT and CD19-TRAC-CAR T cells lysed wild-type NALM6 (~27,000 CD19 molecules) and NALM6 variants with 100-fold less CD19. As CD19 levels decreased further, CAR T cells no longer killed their target, in contrast to HIT T cells. HIT T cells showed increased expression of IFN-gamma, IL-2 and TNF-alpha upon exposure to NALM6 cells expressing ~20 CD19 molecules per cell, compared to CAR T cells. This increased sensitivity of HIT receptors correlated to their greater signaling response, upon exposure to the low-antigen-density NALM6. Phospho-proteomic analyses further confirmed this increased response of HIT T cells to low antigen levels. Altogether, these results confirm that HIT receptors endow T cells with greater antigen sensitivity than canonical CARs. We further showed that HIT T cells have higher in vivo anti-tumor activity compared to CAR T cells in mice bearing low-antigen-density ALL or AML.ConclusionsHIT receptors consistently afford high antigen sensitivity and mediate tumor recognition beyond what current CARs can provide. HIT receptors open new prospects for targeting cell surface antigens of low abundance.Ethics ApprovalEight- to 12-week-old NOD/SCID/IL-2Rgamma-null (NSG) male mice (Jackson Laboratory) were used under a protocol approved by the MSKCC Institutional Animal Care and Use Committee.

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Novel full-human CD22-CAR therapy overcomes resistance to previous CD19/22-CAR regimens in ALL

10.21203/rs.3.rs-147858/v1 ◽

2021 ◽

Author(s):

Yue Tan ◽

Haodong Cai ◽

Chuo Li ◽

Biping Deng ◽

Weiliang Song ◽

...

Keyword(s):

T Cells ◽

Lymphoblastic Leukemia ◽

Considerable Proportion ◽

Target Antigen ◽

Single Chain ◽

T Cell Therapy ◽

Car T Cells ◽

Car T

Abstract BackgroundCD19- and/or CD22-targeted chimeric antigen receptor (CAR) T cells efficiently induced remission in patients with B acute lymphoblastic leukemia (B-ALL), but a considerable proportion of patients relapsed after both CD19- and CD22-CAR therapies associated with the loss or downregulation of target antigen. Re-infusions of the prior used CAR T cells were usually ineffective. In contrast to the frequent loss of CD19, low level of CD22 is usually present on leukemia cells post CAR therapy, suggesting that newly designed CD22-CAR therapies may be effective in these patients.MethodsA yeast full-human single-chain variable fragment (scFv) library and a high-throughput NFAT reporter assay were utilized to screen several full-human CD22-CAR candidates; CD107 assay and in vitro cytotoxicity assay was used to evaluate the effector function of CAR T cells; membrane proteome assay was conducted to determine the specificity of the CAR toward the target antigen; a leukemia animal models was used to test the in vivo efficacy of CAR T cells. A phase I trial (ChiCTR2000028793) was conducted to assess the safety and effectiveness of CD22-CARFH80 therapy in 8 children with B-ALL resistant to or relapsed after prior CD19- and CD22-CAR treatment.ResultsWe identified a full-human CD22-CAR construct termed CD22CARFH80 which could mediate superior anti-leukemia activity in vitro and in a leukemia animal model and had good specificity to the target antigen. Data from the trial showed that with CD22-CARFH80 T-cell therapy, 6/8 (75%) patients including 2 who had CD22low blasts achieved complete remission; 1 patient had a partial response. CAR T cells efficiently expanded in vivo, while the toxic effect is low in most patients. At a median follow-up of 5 months, 4/6 (57%) patients remained in remission.ConclusionsTherapy with a newly invented CD22-CARFH80 overcomes the resistance to prior versions of CD19- and CD22-CAR formats and elicits potent anti-leukemia responses with an acceptable safety profile, representing a promising salvage regimen for B-ALL that fails in prior CD19- and CD22-CAR treatments.Trial registrationClinicalTrials.gov: ChiCTR2000028793; registered 4 January, 2020. http://www.chictr.org.cn/showproj.aspx?proj=47857

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Locally secreted BiTEs complement CAR T cells by enhancing solid tumor killing

10.1101/2021.06.08.447602 ◽

2021 ◽

Author(s):

Yibo Yin ◽

Jesse Rodriguez ◽

Nannan Li ◽

Radhika Thokala ◽

MacLean P Nasrallah ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Solid Tumor ◽

Tumor Therapy ◽

Target Antigen ◽

Car T Cells ◽

Car T ◽

Anti Tumor Activity

Bispecific T-cell engagers (BiTEs) are bispecific antibodies that redirect T cells to target antigen-expressing tumors. BiTEs can be secreted by T cells through genetic engineering and perform anti-tumor activity. We hypothesized that BiTE-secreting T cells could be a valuable T cell-directed therapy in solid tumors, with distinct properties in mono- or multi-valent strategies incorporating chimeric antigen receptor (CAR) T cells. Glioblastomas represent a good model for solid tumor heterogeneity and represent a significant therapeutic challenge. We detected expression of tumor-associated epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), and interleukin-13 receptor alpha 2 (IL13Rα2) on glioma tissues and glioma cancer stem cells. These antigens formed the basis of a multivalent approach, using a conformation-specific tumor-related EGFR targeting antibody (806) and Hu08, an IL13Rα2-targeting antibody, as the scFvs to generate new BiTE molecules. Compared with 806CAR T cells and Hu08CAR T cells, BiTE T cells demonstrated prominent activation, cytokine production, and cytotoxicity in response to target-positive gliomas. Superior response activity was also demonstrated in BiTE secreting bivalent targeting T cells compared with bivalent targeting CAR T cells, which significantly delayed tumor growth in a glioma mouse model. In summary, BiTEs secreted by mono- or multi- valent targeting T cells have potent anti-tumor activity in vitro and in vivo with significant sensitivity and specificity, demonstrating a promising strategy in solid tumor therapy.

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Blocking IFNγ in CAR-T Reduces Checkpoint Inhibitors and Cell-Mediated Toxicity without Compromising Therapeutic Efficacy in CD19 +malignancies

Blood ◽

10.1182/blood-2021-146042 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1723-1723

Author(s):

Stefanie R. Bailey ◽

Sonika Vatsa ◽

Rebecca Larson ◽

Amanda A Bouffard ◽

Irene Scarfò ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Immune Checkpoint ◽

Stock Options ◽

Macrophage Activation ◽

Hematologic Malignancies ◽

Car T Cells ◽

Car T

Abstract Background: Chimeric antigen receptor T cells (CAR-T) induce impressive responses in patients with hematologic malignancies but can also mediate a systemic inflammatory toxicity known as cytokine release syndrome (CRS), marked by elevated levels of pro-inflammatory cytokines and chemokines released from activated CAR-T and innate immune cells. Release of the pro-inflammatory cytokine interferon-gamma (IFNγ) in response to antigen is used as a potency assay for CAR-T cells, but elevated levels have been identified in patients suffering from CAR-T-associated toxicities such as CRS and neurotoxicity. Mutations in IFNγ receptor signaling have been identified as a mechanism of resistance in checkpoint blockade in melanoma and other solid tumors, and we have recently identified that IFNγ receptor signaling also confers resistance to CAR-T cell mediated cytotoxicity in solid tumors, but its biologic role in conferring responses in hematologic malignancies is not established. Methods: CD19-targeted CAR-T were generated using either 4-1BB or CD28 costimulatory domains. CAR-T effector functions in vitro and in vivo were assessed in the presence of absence of IFNγ-blocking antibody. Furthermore, we used CRISPR/Cas9 editing to knock out IFNγ in CD19-directed CAR-T cells. The effects of IFNγ inhibition in CAR-T by pharmacologic and genetic approaches on T cell function, immune checkpoint inhibitor expression, cancer cell lysis and macrophage activation/phenotype were assessed using ELISA, flow cytometry, in vitro/in vivo tumor models and Luminex/fluorescence microscopy/NanoString, respectively. Finally, serum from B cell lymphoma patients treated with the CAR-T products tisagenlecleucel or axicabtagene ciloleucel was collected 3 days post-CAR infusion and added to human macrophages in vitro in the presence of blocking antibodies to IFNγ versus the current clinical agents for managing CRS, including those targeting IL-1Rα and IL-6R. Macrophage phenotype and function was determined using NanoString, ELISA, and immunofluorescence microscopy. Results: We found that pharmacologic blockade or genetic knockout of IFNγ specifically reduces IFNγ signaling without compromising T cell phenotype or effector function, including production of GM-CSF, IL-2, Granzyme B and TNFα. We also observed reduced expression of the immune checkpoint proteins CTLA-4, PD-1, Lag3 and Tim3, which correlated with enhanced antigen-specific CAR-T proliferation. Cytotoxicity assays and NSG xenograft tumor-bearing mouse models revealed that blocking IFNγ has no effect on therapeutic efficacy of CAR T cells against CD19 + leukemias or lymphomas in vitro or in vivo. Furthermore, pharmacologic blockade or genetic knockout of IFNγ in CD19-directed CAR T cells abrogated macrophage activation in vitro and in hybrid in vitro/in vivo models of CRS, as shown by a reduction of activation markers (CD69, CD86) and pro-inflammatory proteins (IL-6, IP-10, MIP-1β and MCP-1). Further interrogation revealed that these findings were IFNγ-dependent but cell contact-independent. Finally, data herein reveals that blocking IFNγ in both healthy donor CAR-T cultures and lymphoma patient serum results in reduced macrophage activation/function to a similar, if not superior, extent as current clinical approaches targeting IL-1Rα and IL-6R. In addition to reduced macrophage function, NanoString analysis revealed a decreased expression of immune checkpoint inhibitor genes HAVCR2, VSIR and PDCD1LG2 and upregulation of co-stimulatory genes DPP4 and ICOSL. Conclusions: Collectively, these data show that IFNγ is dispensable for the efficacy of CAR-T against hematologic malignancies and blocking IFNγ could simultaneously mitigate cytokine-related toxicities while enhancing T cell proliferation and persistence via reduced expression of immune checkpoint proteins. Furthermore, direct comparison of IFNγ blockade or knockout in the CAR T cell product with current clinical strategies suggests that targeting IFNγ could mitigate major cytokine-related toxicities to a greater extent than existing approaches. Disclosures Frigault: Arcellx: Consultancy; Novartis: Consultancy, Research Funding; Kite: Consultancy, Research Funding; BMS: Consultancy; Iovance: Consultancy; Takeda: Consultancy; Editas: Consultancy. Maus: WindMIL: Consultancy; Torque: Consultancy, Current holder of stock options in a privately-held company; Tmunity: Consultancy; Novartis: Consultancy; Micromedicine: Consultancy, Current holder of stock options in a privately-held company; Kite Pharma: Consultancy, Research Funding; GSK: Consultancy; Intellia: Consultancy; In8bio (SAB): Consultancy; CRISPR therapeutics: Consultancy; Cabaletta Bio (SAB): Consultancy; BMS: Consultancy; Bayer: Consultancy; Atara: Consultancy; AstraZeneca: Consultancy; Astellas: Consultancy; Arcellx: Consultancy; Agenus: Consultancy; Adaptimmune: Consultancy; tcr2: Consultancy, Divested equity in a private or publicly-traded company in the past 24 months; century: Current equity holder in publicly-traded company; ichnos biosciences: Consultancy, Current holder of stock options in a privately-held company.

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767 Interferon gamma reduces CAR-T exhaustion and toxicity without compromising therapeutic efficacy in hematologic malignancies

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0767 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A815-A815

Author(s):

Stefanie Bailey ◽

Sonika Vatsa ◽

Amanda Bouffard ◽

Rebecca Larson ◽

Irene Scarfo ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Cancer Cells ◽

Tumor Cells ◽

Interferon Gamma ◽

Hematologic Malignancies ◽

Car T Cells ◽

Car T

BackgroundChimeric antigen receptor (CAR) T cell therapy has shown remarkable efficacy in hematologic malignancies, ultimately leading to its FDA approval for relapsed/refractory acute lymphoblastic leukemia and large cell lymphomas in 2017. Despite the success of CAR T cells in the clinic, toxicities such as cytokine release syndrome (CRS) can be severe. Attempts to mitigate these effects have primarily focused on the blockade of macrophage-derived cytokines, such as IL-6 and IL-1B. Herein, we show that the pharmaceutical blockade or genetic deletion of interferon gamma (IFNg, a CAR-T-derived cytokine that strongly correlates with CRS in the clinic, appears to be a viable target for the reduction of CAR-T-associated toxicities.MethodsPharmacologic (blocking antibody) and genetic (CRISPR/Cas9) approaches were used to block IFNg signaling and/or production by CAR T cells. In vitro CAR-T function and cytotoxicity was tested using ELISA, flow cytometry and short-/long-term killing assays prior to their assessment in vivo. NSG mice were injected with Nalm6 or JeKo-1 cancer cells prior to treatment with IFNg-modified CAR-T and tumor size and IFNg production were measured. To determine how the loss of IFNg might affect innate immune cells, CAR-T, macrophages and tumor cells were co-cultured and assessed by flow cytometry, immunofluorescence, Luminex and RNA sequencing.ResultsIFNg could be blocked using an anti-IFNg antibody or CRISPR/Cas9 editing of the CAR T cells without affecting T cell activation, proliferation or cytokine production (IL-2, TNFa, GM-CSF). Successful blockade of the IFNg signaling pathway was confirmed by reduced phosphorylation of JAK1, JAK2 and STAT1, even in the presence of exogenous IFNg. Loss of IFNg did not reduce the cytotoxic potential or persistence of CAR-T against hematologic malignancies in vitro or in vivo. When cultured with macrophages and cancer cells, IFNg knockout (IFNgKO) CAR-T yielded decreased levels of IL-1B, IL-6, IL-13, MCP1 and CXCL10, indicating a reduction in macrophage activation induced by CAR-T in the absence of IFNg. Serum from tumor-bearing mice treated with IFNgKO CAR-T elicited lower activation of macrophages in vitro compared to those treated with IFNg-producing CAR-T cells. Furthermore, IFNgKO CAR T cells co-cultured with tumor cells and macrophages demonstrated less exhaustion as shown by reduced expression of PD1, Tim3 and Lag3 and increased IFNgKO CAR-T expansion.ConclusionsCollectively, these data suggest that IFNg is not required for the efficacy of CAR-T in hematologic malignancies and can potentially be targeted to reduce toxicity and enhance CAR-T efficacy and persistence in the clinic.

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