91 Directly link T cell phenotype and function to genotype with the opto™ cell therapy development 1.0 workflow

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A102-A102
Author(s):  
Yelena Bronevetsky
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Tumor Cell ◽  
Target Cell ◽  
Cell Killing ◽  
Cytokine Secretion ◽  
Tumor Cell Death ◽  
Car T ◽  
Therapy Development

BackgroundThe key challenges to developing T cell-based therapies center on the fact that T cell-mediated tumor death relies on complicated cell-cell interactions and several complex mechanisms. These therapies have also been associated with significant side effects related to cytokine release syndrome (CRS) and neurotoxicity, placing importance on understanding T cell anti-tumor functions like cytokine release and killing kinetics. Ideally, T cell therapies would be tailored to mediate the rapid destruction of multiple tumor cells while reducing these side effects.MethodsThe Berkeley Lights Opto™ Cell Therapy Development Workflow is a collection of software capabilities, reagents, and protocols that allow scientists to selectively measure cytokine secretion, visualize killing behavior, and sequence TCRs from individual cells in parallel. Here, we demonstrate its use for CAR-T cell phenotypic and functional screening as well as the discovery of TCRs associated with specific T cell behaviors.ResultsThe cumulative percentage of pens with tumor cell caspase-3 activity increased over time in pens loaded with CD19+ tumors, peaking at 50% tumor cell death after 16 hours of incubation. This is in contrast to only 10% of pens displaying tumor cell death in control pens loaded with CD19- tumor cells; control pens also exhibited slower killing kinetics. The single-cell resolution of the OptoSelect™ microfluidic chip enabled us to analyze each significant T cell-tumor cell interaction. We were able to directly compare differences in killing kinetics of individual T cells and link this tumor killing behavior to IFNγ secretion. We identified fast-killing and slow-killing CAR-T cells in a single-day experiment, which could then be exported for genomic analysis. We highlight an example where TCR alpha and beta sequences are recovered from single T cells after export.ConclusionsThe Opto™ Cell Therapy Development Workflow on Berkeley Lights systems enables researchers to correlate cytokine secretion to target cell killing behavior in CAR-mediated antigen recognition, discriminate CAR-T cell subsets based on kinetics of target cell killing, and link cytokine secretion and target cell killing behavior to TCR sequence in TCR-mediated antigen recognition.

scholarly journals O2 Directly linking single T cell phenotype and function to genotype

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A4.1-A4
Author(s):  
Y Bronevetsky
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
Cell Killing ◽  
Cell Phenotype ◽  
Functional Assay ◽  
Functional Screening ◽  
Tumor Cell Death ◽  
And Function ◽  
Tumor Cell Killing

T cell therapies for cancer treatment are challenging to develop because of the complex mechanisms and cell interactions that underly T cell-mediated tumor killing. Current technologies rely on correlating phenotype, function, and gene expression based on experiments performed on different populations of T cells because no one platform is able to assess cell surface marker expression, cytokine secretion, and tumor cell killing activity of the same T cell and recover this cell for downstream genomic analysis. Here we share two use cases - CAR-T cell functional screening and TCR sequence recovery following functional assay - that demonstrate how the T Cell Analysis Suite on the LightningTM optofluidic platform can be used to directly link T cell phenotype and function (IFNγ secretion and tumor cell killing) to genotype (TCR sequence recovery) at a single-cell level and on the same T cell, enabling deeper and more thorough characterization of how T cells mediate tumor cell death and potentially the development of more efficacious therapies.Disclosure InformationY. Bronevetsky: A. Employment (full or part-time); Significant; Berkeley Lights Inc.

scholarly journals A Protease-Mediated Regulatory Chimeric Antigen Receptor (CAR): "Double Arm" CAR System Improves Tumor-Cell-Specificity of CAR-T Cell Therapy

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 41-41
Author(s):  
Satoru Aoyama ◽  
Shunichiro Yasuda ◽  
Daisuke Watanabe ◽  
Hiroki Akiyama ◽  
Yoshihiro Umezawa ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Target Cell ◽  
Target Cells ◽  
T Cell Therapy ◽  
Car T Cell ◽  
Cell Specificity ◽  
Car T Cell Therapy ◽  
Car T

【 Introduction 】 CAR-T cell therapy has shown excellent therapeutic effects against some malignancies including refractory B cell lymphoma or leukemia. This adoptive T cell transfer therapy is an attractive methodology; however, there are several disadvantages to be overcome. Current CAR-T cell therapy targets single cell-surface molecule, which leads to damage of normal cells expressing the target protein. Such "On-target / off-tumor" effect is one of the major adverse effects. Improvement of target-cell-specificity is needed to avoid this serious adverse effect and to expand target diseases of this therapy. Here, we show the protease-mediated "Double-Arm" CAR-T cell system, which improved the specificity of CAR-T cell therapy by recognizing two distinct cell-surface proteins. We designed two types of CARs: "Effector CAR" and "Scissors CAR". The "Effector CAR" is constituted of a single chain Fv fragment (scFv) targeting a cell-surface protein (protein X) on tumor cells, Human Immunodeficiency Virus protease (HIVPR) recognition polypeptide sequence, and a functional domain of CD3-zeta. The "scissors CAR" is constituted of a recognition portion targeting another protein (protein Y) and HIVPR. The HIVPR induces cleavage of the recognition polypeptide sequence in the effector CAR leading to inactivation of the effector CAR when the CAR-T cells contact with cells expressing both proteins X and Y. 【 Material and Methods 】 For proof of principle, we first constructed "anti-CD19 mCherry CAR" harboring mCherry fluorescence protein in the cytoplasmic region under the HIVPR recognition polypeptide sequence. Also, we constructed "anti-CD19 scissors CAR", low affinity "anti-HER2 (4D5-3) scissors CAR" and high affinity "anti-HER2 (4D5-8) scissors CAR". To analyze the target-cell-dependent cleavage of mCherry CAR, 293T cells expressing these CARs were co-cultured with target cells, including K562 (CD19-, HER2-), Raji (CD19+, HER2+), or SK-BR-3 (CD19-, HER2+). To obtain target cells expressing both CD19 and HER2, Raji and SK-BR-3 cells were molecularly manipulated. (1) To evaluate efficiency of this system, after co-cultivation of CAR-transduced 293T and the target cells (K562, Raji, SK-BR-3), the localization of mCherry was examined under the microscopy and Western blotting. (2) To assess the T cell activation, we constructed "anti-CD19 effector CAR" and established Jurkat cells expressing both the "effector CAR" and the "anti-HER2 scissors CAR". These cells were co-cultured with wild type or engineered Raji or SK-BR-3 cells. T cell activation was analyzed with flowcytometric analysis. (3) To assess the CAR activity of this system, we transduced these CARs to primary T cells from healthy donors. After co-cultivation with the target cells, we measured target-specific cytotoxic activity. 【 Results 】 (1) Transduced "anti-CD19 mCherry CAR" was detected as a membrane-bound protein in 293T cells. Co-cultivation of 293T cells expressing both the "anti-CD19 mCherry CAR" and "anti-HER2 scissors CAR" with engineered Raji cells expressing both CD19 and HER2 induced cleavage of the recognition site and translocation of the mCherry from the membrane to the cytoplasm. These results suggested that this system would regulate activities of CAR-T cell through HIVPR-mediated cleavage of the "effector CAR" in vitro. (2) Jurkat cells expressing "anti-CD19 effector CAR" were activated through a target-cell-dependent manner. Furthermore, "anti-HER2 scissors CAR" attenuated T cell activation driven by "anti-CD19 effector CAR" when Jurkat cells expressing both the "anti-CD19 effector CAR" and the "anti-HER2 scissors CAR" contacted with target-cells expressing both CD19 and HER2. In addition, high affinity 4D5-8 scissors CAR showed more potent attenuation than the low affinity 4D5-3 scissors CAR. (3) Primary human T cells expressing "anti-CD19 effector CAR" showed CD19-expressing target-cell-specific cytotoxic activity. We also demonstrated that "Double Arm" primary human CAR-T cells showed tumor-cell-specificity as seen in cell-line models described above. 【Discussion】 Our "Double-Arm" CAR-T cell system has improved tumor-cell-specificity even in primary human T-cells as we expected. This system would attenuate the adverse effects of clinical CAR-T cell therapies. Disclosures No relevant conflicts of interest to declare.

scholarly journals 653 Dasatinib as a rapid pharmacological ON/OFF switch for T cell bispecific antibody-induced T cell activation and cytokine release

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A690-A690
Author(s):  
Gabrielle Leclercq ◽  
Helene Haegel ◽  
Anneliese Schneider ◽  
Estelle Marrer Berger ◽  
Antje Walz ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Target Cell ◽  
Cell Activation ◽  
Bispecific Antibody ◽  
Tumor Antigens ◽  
Cell Killing ◽  
Cytokine Release ◽  
Car T

BackgroundT cell bispecific antibodies (TCBs) are extremely potent T cell engagers, harboring a 2+1 format with one binder to the CD3ε chain and two binders to specific tumor antigens. Crosslinking of CD3 with tumor antigens triggers T cell activation, proliferation and cytokine release, leading to tumor cell killing.1 2 TCB treatment is sometimes associated with safety liabilities due to on-target on-tumor, on-target off-tumor cytotoxic activity and cytokine release. Patients treated with TCBs may experience a Cytokine Release Syndrome (CRS), characterized by fever, hypotension and respiratory deficiency and associated with the release of pro-inflammatory cytokines such as IL-6, TNF-α, IFN-γ, and IL-1β.3 Off-tumor toxicity may occur if target antigens are expressed in healthy cells, which may potentially result in tissue damages and compromise the patient‘s safety. Rapid pharmacological blockade of T cell activation and proliferation is a promising approach to mitigate these life-threatening toxicities. Tyrosine kinases such as SRC, LCK or ZAP70 are involved in downstream signaling pathways after engagement of the T cell receptor and blocking these kinases might serve to abrogate T cell activation when required. Dasatinib was identified as a potent candidate that switches off CAR T cell functionality.4 5MethodsUsing an in vitro model of target cell killing by human peripheral blood mononuclear cells, we assessed the reversible effects of dasatinib combined with CEA-TCB or HLA-A2-WT1-TCB on T cell activation and proliferation, target cell killing and cytokine release. At assay endpoints, T cell phenotype and target cell killing were measured by flow cytometry and supernatants were analyzed by Luminex to assess cytokine release. To determine the effective dose of dasatinib, the Incucyte system was used to follow kinetics of target cells killing by TCB in the presence of a dose response of dasatinib concentrations.Results100 nM dasatinib prevented TCB-mediated target cell killing when added in the system upon restimulation of activated T cells (figure 1). Dasatinib concentrations above 50 nM fully switched off target cell killing (figure 2) which was restored upon removal of dasatinib. These data confirm that dasatinib act as a potent and reversible on/off switch for activated T cells at pharmacologically relevant doses as they are applied in patients according to the label.6ConclusionsTaken together, we provide evidence for the use of dasatinib as a pharmacological on/off switch to mitigate off-tumor toxicities or CRS by T cell engaging therapies. These data are being validated in vivo.ReferencesBacac M, Fauti T, Sam J, Colombetti S, Weinzierl T, Ouaret D, et al. A novel carcinoembryonic antigen T-Cell Bispecific Antibody [CEA TCB] for the treatment of solid tumors. Clin Cancer Res 2016;22(13):3286–97.Bacac M, Klein C, Umana P. CEA TCB: A novel head-to-tail 2:1 T cell bispecific antibody for treatment of CEA-positive solid tumors. Oncoimmunology 2016;5(8):e1203498.Shimabukuro-Vornhagen A, Gödel P, Subklewe M, Stemmler HJ, Schlößer HA, Schlaak M, et al. Cytokine release syndrome. J Immunother Cancer 2018;6(1):56.Weber EW, Lynn RC, Sotillo E, Lattin J, Xu P, Mackall CL. Pharmacologic control of CAR-T cell function using dasatinib. Blood Advances 2019;3(5):711–7.Mestermann K, Giavridis T, Weber J, Rydzek J, Frenz S, Nerreter T, et al. The tyrosine kinase inhibitor dasatinib acts as a pharmacologic on/off switch for CAR T cells. Science Translational Medicine 2019;11(499):eaau5907.Wang X, Roy A, Hochhaus A, Kantarjian HM, Chen TT, Shah NP. Differential effects of dosing regimen on the safety and efficacy of dasatinib: retrospective exposure-response analysis of a Phase III study. Clinical pharmacology : advances and applications 2013;5:85–97.Abstract 653 Figure 1Representative flow cytometry experiment reporting SKM-1 target cell viability upon first stimulation with 10 nM HLA-A2 WT-1-TCB in the absence of dasatinib (left pannel) and upon second stimulation with 10 nM HLA-A2 WT-1-TCB in the presence of 100 nM dasatinib (right pannel)Abstract 653 Figure 2Real time killing (Incucyte) of red fluorescent A375 cells loaded with RMF peptides by 10 nM HLA-A2 WT-1-TCB (left pannel) and of red fluorescent MKN45 cells by 1 nM CEA-TCB (right pannel) in the presence of different dasatinib concentrations ranging from 100 nM to 0 nM. Mean of technical duplicates + SEM

scholarly journals Evaluating on-Target Toxicity of Hematopoietic-Targeting Cars Demonstrates Target-Nonspecific Suppression of Marrow Progenitors

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3357-3357 ◽  
Author(s):  
Christopher Tor Sauter ◽  
Christopher Daniel Chien ◽  
Feng Shen ◽  
Sarah K Tasian ◽  
Terry J Fry
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Cytokine Secretion ◽  
Target Antigen ◽  
T Cell Therapy ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Abstract Chimeric Antigen Receptor (CAR)-modified T cells are a class of immunotherapy, most known for producing durable remissions in B-cell malignancies. To date, On-Target/Off-Tumor effects, systemic cytokine syndromes, and neurotoxicity are some types of toxicities encountered in early CAR T cell therapy trials. Initially, we sought to investigate potential toxicity by a novel, FLT3-targeting CAR T cell (FLT3 CAR). As a tumor-associated antigen, FLT3 is expressed on AML, ALL and MLL, as well as non-malignant hematopoietic subsets, warranting evaluation for On-Target toxicity. To examine this, human peripheral mobilized CD34+ stem cells were engrafted into either NSG or NSGSÑan NSG-derived knock-in expressing human interleukin-3, GM-CSF, and stem cell factorÑimmunodeficient murine stains. After establishing hematopoietic xenografts and allowing for reconstitution of circulating human myeloid cells, we treated mice with either FLT3 CARs or mock-transduced T cells derived from a marrow-autologous donor [Figure 1A]. In the presence of FLT3 CARs, we observed loss of circulating mature monocytes compared to mock T cell treated controls. To elucidate if myeloid loss originated from an On-Target toxicity towards a FLT3 expressing progenitor or by some other mechanism, we treated marrow-humanized mice with either FLT3 CAR, a lymphoid restricted CD22-targeting CAR, a mature myeloid restricted CD33-targeting CAR or mock transduced T cells. Two weeks following treatment, we analyzed progenitor subsets including human hematopoietic stem cells (HSC), multipotent progenitors (MPP), common myeloid progenitors (CMP), granulocyte-macrophage progenitors (GMP), maturing marrow granulocytes, common lymphoid progenitors (CLP), and megakaryocyte erythroid progenitors (MEP). Surprisingly, we observed significant marrow loss of CMPs, GMPs, MEPs and CLPs across all mice receiving CARs compared to mice treated with mock transduced T cells alone [Figure 1B]. Absolute numbers of HSCs, MPPs, and total humans cells were equivalent across treatment groups. As the CD19-targeting CAR therapy is most well characterized, we repeated this experiment using CD19 CARs with consistent outcomes [Figure 1C]. Flow cytometric characterization of these progenitor populations confirms that loss still occurs in the absence of target-antigen expression. Taken together, these results suggest that CAR T cell therapy may exert a deleterious effect on specific marrow progenitors through a target antigen-independent mechanism. Furthermore, while a FLT3-CAR On-Target toxicity towards FLT3 expressing progenitors (CLP, GMP, and CMP) can not be fully excluded, an exacerbated progenitor loss compared to target antigen-null subset toxicity in the CD33 CAR and CD22 CAR treated groups was not observed. Interestingly, prolonged cytopenias are anecdotally observed in Phase I CAR trials, often attributed to heavy pretreatment of enrollees. Local inflammatory cytokine milieu, particularly IFNg, is implicated in marrow suppression in other settingsÑchronic viral infections, donor lymphocyte infusion and disseminated non-tuberculous mycobacterial infections. We hypothesize that prolonged CAR T cell cytokine secretion may exert similar marrow suppression. This could be due to either continuous CAR-engagement with reconstituting antigen-positive progenitors or an intrinsic supraphysiologic basal level of cytokine secretion by CAR T cells. We are currently analyzing cytokine profiles in our humanized murine model to further evaluate this hypothesis. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Anti-Mesothelin CAR T cell therapy for malignant mesothelioma

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Laura Castelletti ◽  
Dannel Yeo ◽  
Nico van Zandwijk ◽  
John E. J. Rasko
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Malignant Mesothelioma ◽  
Oncolytic Viruses ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

AbstractMalignant mesothelioma (MM) is a treatment-resistant tumor originating in the mesothelial lining of the pleura or the abdominal cavity with very limited treatment options. More effective therapeutic approaches are urgently needed to improve the poor prognosis of MM patients. Chimeric Antigen Receptor (CAR) T cell therapy has emerged as a novel potential treatment for this incurable solid tumor. The tumor-associated antigen mesothelin (MSLN) is an attractive target for cell therapy in MM, as this antigen is expressed at high levels in the diseased pleura or peritoneum in the majority of MM patients and not (or very modestly) present in healthy tissues. Clinical trials using anti-MSLN CAR T cells in MM have shown that this potential therapeutic is relatively safe. However, efficacy remains modest, likely due to the MM tumor microenvironment (TME), which creates strong immunosuppressive conditions and thus reduces anti-MSLN CAR T cell tumor infiltration, efficacy and persistence. Various approaches to overcome these challenges are reviewed here. They include local (intratumoral) delivery of anti-MSLN CAR T cells, improved CAR design and co-stimulation, and measures to avoid T cell exhaustion. Combination therapies with checkpoint inhibitors as well as oncolytic viruses are also discussed. Preclinical studies have confirmed that increased efficacy of anti-MSLN CAR T cells is within reach and offer hope that this form of cellular immunotherapy may soon improve the prognosis of MM patients.

scholarly journals Metabolic and Mitochondrial Functioning in Chimeric Antigen Receptor (CAR)—T Cells

Cancers ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1229
Author(s):  
Ali Hosseini Rad S. M. ◽  
Joshua Colin Halpin ◽  
Mojtaba Mollaei ◽  
Samuel W. J. Smith Bell ◽  
Nattiya Hirankarn ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Chimeric Antigen Receptor ◽  
Metabolic State ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized adoptive cell therapy with impressive therapeutic outcomes of >80% complete remission (CR) rates in some haematological malignancies. Despite this, CAR T cell therapy for the treatment of solid tumours has invariably been unsuccessful in the clinic. Immunosuppressive factors and metabolic stresses in the tumour microenvironment (TME) result in the dysfunction and exhaustion of CAR T cells. A growing body of evidence demonstrates the importance of the mitochondrial and metabolic state of CAR T cells prior to infusion into patients. The different T cell subtypes utilise distinct metabolic pathways to fulfil their energy demands associated with their function. The reprogramming of CAR T cell metabolism is a viable approach to manufacture CAR T cells with superior antitumour functions and increased longevity, whilst also facilitating their adaptation to the nutrient restricted TME. This review discusses the mitochondrial and metabolic state of T cells, and describes the potential of the latest metabolic interventions to maximise CAR T cell efficacy for solid tumours.

IMMU-45. COMBINATION OF EGFRVIII CAR T-CELL THERAPY AND PARACRINE GAM MODULATION FOR THE TREATMENT OF GBM

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi102-vi103
Author(s):  
Tomás A Martins ◽  
Marie-Françoise Ritz ◽  
Tala Shekarian ◽  
Philip Schmassmann ◽  
Deniz Kaymak ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Differential Expression Analysis ◽  
Dependent Manner ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Abstract The GBM immune tumor microenvironment mainly consists of protumoral glioma-associated microglia and macrophages (GAMs). We have previously shown that blockade of CD47, a ‘don't eat me’-signal overexpressed by GBM cells, rescued GAMs' phagocytic function in mice. However, monotherapy with CD47 blockade has been ineffective in treating human solid tumors to date. Thus, we propose a combinatorial approach of local CAR T cell therapy with paracrine GAM modulation for a synergistic elimination of GBM. We generated humanized EGFRvIII CAR T-cells by lentiviral transduction of healthy donor human T-cells and engineered them to constitutively release a soluble SIRPγ-related protein (SGRP) with high affinity towards CD47. Tumor viability and CAR T-cell proliferation were assessed by timelapse imaging analysis in co-cultures with endogenous EGFRvIII-expressing BS153 cells. Tumor-induced CAR T-cell activation and degranulation were confirmed by flow cytometry. CAR T-cell secretomes were analyzed by liquid chromatography-mass spectrometry. Immunocompromised mice were orthotopically implanted with EGFRvIII+ BS153 cells and treated intratumorally with a single CAR T-cell injection. EGFRvIII and EGFRvIII-SGRP CAR T-cells killed tumor cells in a dose-dependent manner (72h-timepoint; complete cytotoxicity at effector-target ratio 1:1) compared to CD19 controls. CAR T-cells proliferated and specifically co-expressed CD25 and CD107a in the presence of tumor antigen (24h-timepoint; EGFRvIII: 59.3±3.00%, EGFRvIII-SGRP: 52.6±1.42%, CD19: 0.1±0.07%). Differential expression analysis of CAR T-cell secretomes identified SGRP from EGFRvIII-SGRP CAR T-cell supernatants (-Log10qValue/Log2fold-change= 3.84/6.15). Consistent with studies of systemic EGFRvIII CAR T-cell therapy, our data suggest that intratumoral EGFRvIII CAR T-cells were insufficient to eliminate BS153 tumors with homogeneous EGFRvIII expression in mice (Overall survival; EGFRvIII-treated: 20%, CD19-treated: 0%, n= 5 per group). Our current work focuses on the functional characterization of SGRP binding, SGRP-mediated phagocytosis, and on the development of a translational preclinical model of heterogeneous EGFRvIII expression to investigate an additive effect of CAR T-cell therapy and GAM modulation.

scholarly journals First-in-Human Study of ET019003, a Next Generation Anti-CD19 T-Cell Therapy, in Patients with Relapsed/Refractory Diffuse Large B Cell Lymphoma (r/r DLBCL)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2870-2870 ◽  
Author(s):  
Pengcheng He ◽  
Hong Liu ◽  
Haibo Liu ◽  
Mina Luo ◽  
Hui Feng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Cell Expansion ◽  
T Cell Therapy ◽  
Car T ◽  
Equity Ownership ◽  
T Cell Expansion ◽  
The Absolute

Background : CD19-targeted CAR-T therapies have shown promising efficacy in treating B-cell malignancies. However, treatment-related toxicities, such as cytokine-release syndrome (CRS) and CAR T-cell-related encephalopathy syndrome (CRES), have been one of the major obstacles limiting the use of CAR-T therapies. How to minimize occurrence and severity of toxicity while maintaining efficacy is a major focus for T-cell therapies in development. ET019003 is a next generation CD19-targeted T-cell therapy developed by Eureka Therapeutics, built on the proprietary ARTEMISTM T-cell platform. The ET019003 construct is optimized with the co-expression of an ET190L1 Antibody-TCR (Xu et al, 2018) and novel co-stimulation molecule. We are conducting a First-in-human (FIH) study of ET019003 T cells in CD19+ r/r DLBCL patients. Methods: This FIH study aims to evaluate the safety and efficacy of ET019003 T-cell therapy in CD19+ patients with r/r DLBCL. As of July 2019, six subjects were administered ET019003 T cells. These subjects were pathologically confirmed with DLBCL that is CD19+ (by immunohistochemistry), whose disease have progressed or relapsed after 2-5 lines of prior therapies. All were high-risk patients with rapid tumor progression and heavy tumor burden. Each subject had a Ki67 proliferative index over 60%, 2/6 of the subjects had a Ki67 proliferative index over 90%. Moreover, 5/6 of the subjects had extra-nodal involvement. Following a 3-day preconditioning treatment with Fludarabine (25mg/m2/day)/ Cyclophosphamide (250mg/m2/day), patients received i.v. infusions of ET019003 T cells at an initial dose of 2-3×106 cells/kg. Additional doses at 3×106 cells/kg were administered at 14 to 30-day intervals. Adverse events were monitored and assessed based on CTCAE 5.0. Clinical responses were assessed based on Lugano 2014 criteria. Results: As of July 2019, six subjects have received at least one ET019003 T-cell infusion, and four subjects have received two or more ET019003 T-cell infusions. No Grade 2 or higher CRS was observed in the six subjects. One subject developed convulsions and cognitive disturbance. This subject had lymphoma invasion in the central nervous system before ET019003 T-cell therapy. The subject was treated with glucocorticoid and the symptoms resolved within 24 hours. Other adverse events included fever (6/6, 100%), fatigue (3/6, 50%), thrombocytopenia (3/6, 50%), diarrhea (2/6, 33%), and herpes zoster (1/6, 17%). ET019003 T-cell expansion in vivo (monitored by flow cytometry and qPCR) was observed in all six subjects after first infusion. The absolute peak value of detected ET019003 T cells ranged between 26,000 - 348,240 (median 235,500) per ml of peripheral blood. Tmax (time to reach the absolute peak value) was 6 - 14 days (median 7.5 days). For the four subjects who received multiple ET019003 T-cell infusions, the absolute peak values of detected ET019003 T cells after the second infusion were significantly lower than the absolute peak values achieved after the first infusion. For the two subjects who received three or more infusions of ET019003 T cells, no significant ET019003 T-cell expansion in vivo was observed after the third infusion. All six subjects completed the evaluation of clinical responses at 1 month after ET019003 T-cell therapy. All subjects responded to ET019003 T cells and achieved either a partial remission (PR) or complete response (CR). Conclusions: Preliminary results from six CD19+ r/r DLBCL patients in a FIH study show that ET019003 T-cell therapy is safe with robust in vivo T-cell expansion. The clinical study is on-going and we are monitoring safety as well as duration of response in longer follow-up. Reference: Xu et al. Nature Cell Discovery, 2018 Disclosures Liu: Eureka Therapeutics: Employment, Equity Ownership. Chang:Eureka Therapeutics: Equity Ownership. Liu:Eureka Therapeutics: Employment, Equity Ownership.

scholarly journals P08.05 Combined pharmacological targeting of adenosine 2a- and 2b-receptor enhances CAR T cell function

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A26.2-A27
Author(s):  
M Seifert ◽  
M Benmebarek ◽  
B Cadilha ◽  
J Jobst ◽  
J Dörr ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Combination Therapy ◽  
Cell Function ◽  
Cytokine Secretion ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Cell Effector

BackgroundDespite remarkable response rates mediated by anti-CD19 chimeric antigen receptor (CAR) T cells in selected B cell malignancies, CAR T cell therapy still lacks efficacy in the vast majority of tumors. A substantial limiting factor of CAR T cell function is the immunosuppressive tumor microenvironment. Among other mechanisms, the accumulation of adenosine within the tumor can contribute to disease progression by suppressing anti-tumor immune responses. Adenosine 2a- and 2b-receptor (A2A and A2B)-mediated cAMP build-up suppresses T cell effector functions. In the present study we hypothesize, that combination therapy with the selective A2A/A2B dual antagonist AB928 (etrumadenant) enhances CAR T cell efficacy.Materials and MethodsSecond generation murine (anti-EPCAM) and human (anti-MSLN) CAR constructs, containing intracellular CD28 and CD3ζ domains, were fused via overlap extension PCR cloning. Murine or human T cells were retrovirally transduced to stably express the CAR constructs. A2A/A2B signaling in CAR T cells was analyzed by phospho-specific flow cytometry of CREB (pS133)/ATF-1 (pS63). CAR T cell activation was quantified by flow cytometry and enzyme-linked immunosorbent assay (ELISA) of IFN-γ, IL-2 and TNF-α. CAR T cell proliferation was assessed by flow cytometry. CAR T cell cytotoxicity was assessed by impedance based real-time cell analysis.ResultsAB928 protected murine CAR T cells from cAMP response element-binding protein (CREB) phosphorylation in the presence of stable adenosine analogue 5′-N-ethylcarboxamidoadenosine (NECA). NECA inhibited antigen-dependent CAR T cell cytokine secretion in response to four murine tumor cell lines. CAR T cell-mediated tumor cell lysis as well as proliferation were decreased in the presence of NECA or adenosine. Importantly, AB928 fully restored CAR T cell cytotoxicity, proliferation, and cytokine secretion in a dose dependent manner. Further, AB928 also restored antigen dependent cytokine secretion of human CAR T cells in the presence of NECA.ConclusionsHere we used the A2A/A2B dual antagonist AB928 to overcome adenosine-mediated suppression of CAR T cells. We found that AB928 enhanced important CAR T cell effector functions in the presence of the adenosine analogue, suggesting that combination therapy with AB928 may improve CAR T cell efficacy. This study was limited to in vitro experiments. To confirm the relevance of our findings, this combination therapy must be further investigated in an in vivo setting.Disclosure InformationM. Seifert: None. M. Benmebarek : None. B. Cadilha : None. J. Jobst: None. J. Dörr: None. T. Lorenzini: None. D. Dhoqina: None. J. Zhang: None. J. Zhang: None. U. Schindler: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Amgen Inc., Arcus Biosciences. Other; Significant; Arcus Biosciences. S. Endres: None. S. Kobold: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Arcus Biosciences.

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