scholarly journals 8 Multiparameter characterization of CAR T cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A8-A8
Author(s):  
Xueting Wang ◽  
Christina Pitzka ◽  
Daniela Rheindorf ◽  
Nadine Mockel-Tenbrinck ◽  
Tatjana Holzer ◽  
...  
Keyword(s):  
T Cells ◽  
Effector Function ◽  
Adoptive Cell Transfer ◽  
Intrinsic Variability ◽  
Freezing And Thawing ◽  
Therapeutic Success ◽  
Car T Cells ◽  
Number Of Patients ◽  
Car T

BackgroundAdoptive cell transfer of chimeric antigen receptor (CAR) modified T cells has demonstrated great therapeutic success against certain hematological malignancies. However, a substantial number of patients experienced relapse at some point after treatment with the underlying mechanisms not fully understood. Emerging data suggest that the undesired clinical outcome is related to different aspects, which include: the tumor heterogeneity, the tumor microenvironment, as well as intrinsic characteristics of the CAR T cells. In this work, we aimed to understand the diversity of CAR T cells generated from different donors, using multiparameter in vitro characterization.MethodsLeukapheresis from healthy donors were collected to generate CAR T cells using the GMP-compliant CliniMACS Prodigy® platform, enabling an automated and closed engineering of CAR T cells in a highly reproducible manner. We performed an in-depth characterization of the resulting CAR T cells by exploring differences in the immunophenotype, cell fitness and effector function of the freshly prepared as compared to frozen CAR T cell samples. Specifically, we designed several flow cytometry panels for the extensive characterization of immunophenotypes of interest such as: proliferative capacity, differentiation, activation and exhaustion. Cell fitness status was determined by the rate at which cells undergo apoptosis following stress. Finally, effector function was determined by the ability of the activated CAR T cells to secrete proinflammatory cytokines including IFN-g, TNF-a and IL-2. The associations between these different parameters were analyzed using comprehensive statistical approaches.ResultsWith our established workflow, over 20 healthy-donor derived CAR T cells were generated and characterized. We have observed donor-dependent variations and responses for most of the explored parameters. In general, the freezing and thawing process negatively affected cell fitness and effector function of the CAR T cells and resulted in altered immunophenotypes. Additionally, correlations between certain immunophenotypes and cell fitness/effector function were identified.ConclusionsCollectively, we established a workflow for multiparameter characterization of CAR T cells and assessed the intrinsic variability of CAR T cells for both research and clinical application.

A Novel High Throughput Process for Generation and Characterization of Chimeric Antigen Receptor (CAR) T Cells

2017 ◽  
Vol 17 ◽  
pp. S381-S382
Author(s):  
Sabarinath Venniyil Radhakrishnan ◽  
Adam Miles ◽  
Djordje Atanackovic ◽  
Tim Luetkens
Keyword(s):  
T Cells ◽  
High Throughput ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Car T

scholarly journals Early Investigations and Recent Advances in Intraperitoneal Immunotherapy for Peritoneal Metastasis

Vaccines ◽  
2018 ◽  
Vol 6 (3) ◽  
pp. 54 ◽  
Author(s):  
Anusha Thadi ◽  
Marian Khalili ◽  
William Morano ◽  
Scott Richard ◽  
Steven Katz ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Peritoneal Metastasis ◽  
Checkpoint Inhibitors ◽  
Folate Receptor ◽  
Malignant Ascites ◽  
T Cell Response ◽  
Adoptive Cell Transfer ◽  
Car T Cells ◽  
Car T

Peritoneal metastasis (PM) is an advanced stage malignancy largely refractory to modern therapy. Intraperitoneal (IP) immunotherapy offers a novel approach for the control of regional disease of the peritoneal cavity by breaking immune tolerance. These strategies include heightening T-cell response and vaccine induction of anti-cancer memory against tumor-associated antigens. Early investigations with chimeric antigen receptor T cells (CAR-T cells), vaccine-based therapies, dendritic cells (DCs) in combination with pro-inflammatory cytokines and natural killer cells (NKs), adoptive cell transfer, and immune checkpoint inhibitors represent significant advances in the treatment of PM. IP delivery of CAR-T cells has shown demonstrable suppression of tumors expressing carcinoembryonic antigen. This response was enhanced when IP injected CAR-T cells were combined with anti-PD-L1 or anti-Gr1. Similarly, CAR-T cells against folate receptor α expressing tumors improved T-cell tumor localization and survival when combined with CD137 co-stimulatory signaling. Moreover, IP immunotherapy with catumaxomab, a trifunctional antibody approved in Europe, targets epithelial cell adhesion molecule (EpCAM) and has shown considerable promise with control of malignant ascites. Herein, we discuss immunologic approaches under investigation for treatment of PM.

Characterization of HLH-Like Manifestations as a CRS Variant in Patients Receiving CD22 CAR T-Cells

Blood ◽  
2021 ◽  
Author(s):  
Daniel A Lichtenstein ◽  
Fiorella Schischlik ◽  
Lipei Shao ◽  
Seth M Steinberg ◽  
Bonnie Yates ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Expansion ◽  
Nk Cell ◽  
Severe Neutropenia ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
T Cell Expansion

CAR T-cell toxicities resembling hemophagocytic lymphohistiocytosis (HLH) occur in a subset of patients with cytokine release syndrome (CRS). As a variant of conventional CRS, a comprehensive characterization of CAR T-cell associated HLH (carHLH) and investigations into associated risk factors are lacking. In the context of 59 patients infused with CD22 CAR T-cells where a substantial proportion developed carHLH, we comprehensively describe the manifestations and timing of carHLH as a CRS variant and explore factors associated with this clinical profile. Amongst 52 subjects with CRS, 21 (40.4%) developed carHLH. Clinical features of carHLH included hyperferritinemia, hypertriglyceridemia, hypofibrinogenemia, coagulopathy, hepatic transaminitis, hyperbilirubinemia, severe neutropenia, elevated lactate dehydrogenase and occasionally hemophagocytosis. Development of carHLH was associated with pre-infusion NK-cell lymphopenia and higher bone marrow T/NK-cell ratio, which was further amplified with CAR T-cell expansion. Following CRS, more robust CAR T-cell and CD8 T-cell expansion in concert with pronounced NK-cell lymphopenia amplified pre-infusion differences in those with carHLH without evidence for defects in NK-cell mediated cytotoxicity. CarHLH was further characterized by persistent elevation of HLH-associated inflammatory cytokines, which contrasted with declining levels in those without carHLH. In the setting of CAR T-cell mediated expansion, clinical manifestations and immunophenotypic profiling in those with carHLH overlap with features of secondary HLH, prompting consideration of an alternative framework for identification and management of this toxicity profile to optimize outcomes following CAR T-cell infusion.

Abstract 1204: Design and development of anti-linker antibodies for the detection and characterization of CAR T cells

2019 ◽  
Author(s):  
Stuart A. Sievers ◽  
Keith A. Kelley ◽  
Stephanie H. Astrow ◽  
Adrian Bot ◽  
Jed J. Wiltzius
Keyword(s):  
T Cells ◽  
Design And Development ◽  
Car T Cells ◽  
Car T

scholarly journals 198. CD4+ IL13Rα2-Specific CAR T Cells Exhibit Potent Effector Function Against Glioblastoma

2016 ◽  
Vol 24 ◽  
pp. S77
Author(s):  
Dongrui Wang ◽  
Renate Starr ◽  
Brenda Aguilar ◽  
Alfonso Brito ◽  
Brenda Chang ◽  
...  
Keyword(s):  
T Cells ◽  
Effector Function ◽  
Car T Cells ◽  
Car T ◽  
Potent Effector

scholarly journals Production and Application of CAR T Cells: Current and Future Role of Europe

2021 ◽  
Vol 8 ◽  
Author(s):  
Vladan Vucinic ◽  
Andrea Quaiser ◽  
Philipp Lückemeier ◽  
Stephan Fricke ◽  
Uwe Platzbecker ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Automated Manufacturing ◽  
Car T Cells ◽  
Car T Cell ◽  
Number Of Patients ◽  
Car T ◽  
Digitally Controlled ◽  
The One ◽  
Processing Techniques

Rapid developments in the field of CAR T cells offer important new opportunities while at the same time increasing numbers of patients pose major challenges. This review is summarizing on the one hand the state of the art in CAR T cell trials with a unique perspective on the role that Europe is playing. On the other hand, an overview of reproducible processing techniques is presented, from manual or semi-automated up to fully automated manufacturing of clinical-grade CAR T cells. Besides regulatory requirements, an outlook is given in the direction of digitally controlled automated manufacturing in order to lower cost and complexity and to address CAR T cell products for a greater number of patients and a variety of malignant diseases.

scholarly journals Single-cell antigen-specific activation landscape of CAR T infusion product identifies determinants of CD19 positive relapse in patients with ALL

2021 ◽  
Author(s):  
Zhiliang Bai ◽  
Steven Woodhouse ◽  
Dongjoo Kim ◽  
Stefan Lundh ◽  
Hongxing Sun ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Molecular Mechanisms ◽  
T Cell Subsets ◽  
Durable Response ◽  
Car T Cells ◽  
Number Of Patients ◽  
Car T

Chimeric antigen receptor modified (CAR) T cells targeting CD19 have mediated dramatic responses in relapsed or refractory acute lymphoblastic leukemia (ALL), yet a notable number of patients have CD19-positive relapse within one year of treatment. It remains unclear if the long-term response is associated with the characteristics of CAR T cells in infusion products, hindering the identification of biomarkers to predict therapeutic outcomes prior to treatment. Herein we present 101,326 single cell transcriptomes and surface protein landscape from the CAR T infusion products of 12 pediatric ALL patients upon CAR antigen-specific stimulation in comparison with TCR mediated activation and controls. We observed substantial heterogeneity in the antigen-specific activation states, among which a deficiency of Th2 function was associated with CD19 positive relapsed patients (median remission 9.6 months) compared with very durable responders (remission over 54 months). Proteomic profiles also revealed that the frequency of early memory T cell subsets, rather than activation or co-inhibitory signatures could distinguish CD19-positive relapse. Additionally, a deficit of type 1 helper and cytotoxic effector function and an enrichment for terminally differentiated CD8+ T cells exhibiting low cytokine polyfunctionality was associated with initial non-responders. By contrast, the single-cell transcriptomic data of unstimulated or TCR-activated CAR T cells failed to predict clinical responses. In aggregate, our results dissect the landscape of CAR-specific activation states in infusion products that can identify patients who do not develop a durable response to the therapy, and unveil the molecular mechanisms that may inform strategies to boost specific T cell function to maintain long term remission.

scholarly journals The S enantiomer of 2-hydroxyglutarate increases central memory CD8 populations and improves CAR-T therapy outcome

2020 ◽  
Vol 4 (18) ◽  
pp. 4483-4493
Author(s):  
Iosifina P. Foskolou ◽  
Laura Barbieri ◽  
Aude Vernet ◽  
David Bargiela ◽  
Pedro P. Cunha ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antitumor Activity ◽  
Cd8 T Cells ◽  
Ex Vivo ◽  
Phase 1 ◽  
Therapeutic Success ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T

Abstract Cancer immunotherapy is advancing rapidly and gene-modified T cells expressing chimeric antigen receptors (CARs) show particular promise. A challenge of CAR-T cell therapy is that the ex vivo–generated CAR-T cells become exhausted during expansion in culture, and do not persist when transferred back to patients. It has become clear that naive and memory CD8 T cells perform better than the total CD8 T-cell populations in CAR-T immunotherapy because of better expansion, antitumor activity, and persistence, which are necessary features for therapeutic success and prevention of disease relapse. However, memory CAR-T cells are rarely used in the clinic due to generation challenges. We previously reported that mouse CD8 T cells cultured with the S enantiomer of the immunometabolite 2-hydroxyglutarate (S-2HG) exhibit enhanced antitumor activity. Here, we show that clinical-grade human donor CAR-T cells can be generated from naive precursors after culture with S-2HG. S-2HG–treated CAR-T cells establish long-term memory cells in vivo and show superior antitumor responses when compared with CAR-T cells generated with standard clinical protocols. This study provides the basis for a phase 1 clinical trial evaluating the activity of S-2HG–treated CD19-CAR-T cells in patients with B-cell malignancies.

Abstract 2188: Generation and characterization of human placental-derived CD19 CAR-T cells using viral vectors

2020 ◽  
Author(s):  
Kathy Karasiewicz ◽  
Shuyang He ◽  
Kristina Tess ◽  
Weifang Ling ◽  
Kevin Jhun ◽  
...  
Keyword(s):  
T Cells ◽  
Viral Vectors ◽  
Car T Cells ◽  
Car T
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