scholarly journals Sensitive manipulation of CAR T cell activity using a chimeric endocytosing receptor

2020 ◽  
Vol 8 (2) ◽  
pp. e000756
Author(s):  
Boning Zhang ◽  
John Victor Napoleon ◽  
Xin Liu ◽  
Qian Luo ◽  
Madduri Srinivasarao ◽  
...  
Keyword(s):  
T Cell ◽  
Ligand Binding ◽  
Folate Receptor ◽  
Cell Activity ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Car T Cell ◽  
Car T ◽  
Fusion Receptor

BackgroundMost adoptive cell therapies (ACTs) suffer from an inability to control the therapeutic cell’s behavior following its transplantation into a patient. Thus, efforts to inhibit, activate, differentiate or terminate an ACT after patient reinfusion can be futile, because the required drug adversely affects other cells in the patient.MethodsWe describe here a two domain fusion receptor composed of a ligand-binding domain linked to a recycling domain that allows constitutive internalization and trafficking of the fusion receptor back to the cell surface. Because the ligand-binding domain is designed to bind a ligand not normally present in humans, any drug conjugated to this ligand will bind and endocytose selectively into the ACT.ResultsIn two embodiments of our strategy, we fuse the chronically endocytosing domain of human folate receptor alpha to either a murine scFv that binds fluorescein or human FK506 binding protein that binds FK506, thereby creating a fusion receptor composed of largely human components. We then create the ligand-targeted drug by conjugating any desired drug to either fluorescein or FK506, thereby generating a ligand-drug conjugate with ~10-9 M affinity for its fusion receptor. Using these tools, we demonstrate that CAR T cell activities can be sensitively tuned down or turned off in vitro as well as tightly controlled following their reinfusion into tumor-bearing mice.ConclusionsWe suggest this ‘chimeric endocytosing receptor’ can be exploited to manipulate not only CAR T cells but other ACTs following their reinfusion into patients. With efforts to develop ACTs to treat diseases including diabetes, heart failure, osteoarthritis, cancer and sickle cell anemia accelerating, we argue an ability to manipulate ACT activities postinfusion will be important.

scholarly journals Bone marrow MSC from pediatric patients with B-ALL highly immunosuppress T-cell responses but do not compromise CD19-CAR T-cell activity

2020 ◽  
Vol 8 (2) ◽  
pp. e001419
Author(s):  
Samanta Romina Zanetti ◽  
Paola Alejandra Romecin ◽  
Meritxell Vinyoles ◽  
Manel Juan ◽  
José Luis Fuster ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Activity ◽  
Cell Responses ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Severe Colitis

BackgroundAlthough adoptive transfer of CD19-directed chimeric antigen receptor (CAR) T-cells (CD19-CAR T-cells) achieves high rates of complete response in patients with B-cell acute lymphoblastic leukemia (B-ALL), relapse is common. Bone marrow (BM) mesenchymal stem/stromal cells (BM-MSC) are key components of the hematopoietic niche and are implicated in B-ALL pathogenesis and therapy resistance. MSC exert an immunosuppressive effect on T-cells; however, their impact on CD19-CAR T-cell activity is understudied.MethodsWe performed a detailed characterization of BM-MSC from pediatric patients with B-ALL (B-ALL BM-MSC), evaluated their immunomodulatory properties and their impact on CD19-CAR T-cell activity in vitro using microscopy, qRT-PCR, ELISA, flow cytometry analysis and in vivo using a preclinical model of severe colitis and a B-ALL xenograft model.ResultsWhile B-ALL BM-MSC were less proliferative than those from age-matched healthy donors (HD), the morphology, immunophenotype, differentiation potential and chemoprotection was very similar. Likewise, both BM-MSC populations were equally immunosuppressive in vitro and anti-inflammatory in an in vivo model of severe colitis. Interestingly, BM-MSC failed to impair CD19-CAR T-cell cytotoxicity or cytokine production in vitro using B-ALL cell lines and primary B-ALL cells. Finally, the growth of NALM6 cells was controlled in vivo by CD19-CAR T-cells irrespective of the absence/presence of BM-MSC.ConclusionsCollectively, our data demonstrate that pediatric B-ALL and HD BM-MSC equally immunosuppress T-cell responses but do not compromise CD19-CAR T-cell activity.

scholarly journals Ligand-Induced Degradation of a CAR Permits Reversible Remote Control of CAR T Cell Activity In Vitro and In Vivo

2020 ◽  
Vol 28 (8) ◽  
pp. 1932
Author(s):  
Sarah A. Richman ◽  
Liang-Chuan Wang ◽  
Edmund K. Moon ◽  
Uday R. Khire ◽  
Steven M. Albelda ◽  
...  
Keyword(s):  
Remote Control ◽  
T Cell ◽  
Cell Activity ◽  
Car T Cell ◽  
Car T

scholarly journals Ligand-Induced Degradation of a CAR Permits Reversible Remote Control of CAR T Cell Activity In Vitro and In Vivo

2020 ◽  
Vol 28 (7) ◽  
pp. 1600-1613 ◽  
Author(s):  
Sarah A. Richman ◽  
Liang-Chuan Wang ◽  
Edmund K. Moon ◽  
Uday R. Khire ◽  
Steven M. Albelda ◽  
...  
Keyword(s):  
Remote Control ◽  
T Cell ◽  
Cell Activity ◽  
Car T Cell ◽  
Car T

scholarly journals Identification of CD318, TSPAN8 and CD66c as target candidates for CAR T cell based immunotherapy of pancreatic adenocarcinoma

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Daniel Schäfer ◽  
Stefan Tomiuk ◽  
Laura N. Küster ◽  
Wa’el Al Rawashdeh ◽  
Janina Henze ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Activity ◽  
Ductal Adenocarcinoma ◽  
Cellular Immunotherapy ◽  
Car T Cell ◽  
Car T

AbstractA major roadblock prohibiting effective cellular immunotherapy of pancreatic ductal adenocarcinoma (PDAC) is the lack of suitable tumor-specific antigens. To address this challenge, here we combine flow cytometry screenings, bioinformatic expression analyses and a cyclic immunofluorescence platform. We identify CLA, CD66c, CD318 and TSPAN8 as target candidates among 371 antigens and generate 32 CARs specific for these molecules. CAR T cell activity is evaluated in vitro based on target cell lysis, T cell activation and cytokine release. Promising constructs are evaluated in vivo. CAR T cells specific for CD66c, CD318 and TSPAN8 demonstrate efficacies ranging from stabilized disease to complete tumor eradication with CD318 followed by TSPAN8 being the most promising candidates for clinical translation based on functionality and predicted safety profiles. This study reveals potential target candidates for CAR T cell based immunotherapy of PDAC together with a functional set of CAR constructs specific for these molecules.

scholarly journals Engineering clinically-approved drug gated CAR circuits

2020 ◽  
Author(s):  
Hui-Shan Li ◽  
Nicole M. Wong ◽  
Elliot Tague ◽  
John T. Ngo ◽  
Ahmad S. Khalil ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Therapy ◽  
Logic Gate ◽  
Cell Activity ◽  
T Cell Therapy ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

SUMMARYChimeric antigen receptor (CAR) T cell immunotherapy has the potential to revolutionize cancer medicine. However, excessive CAR activation, lack of tumor-specific surface markers, and antigen escape have limited the safety and efficacy of CAR T cell therapy. A multi-antigen targeting CAR system that is regulated by safe, clinically-approved pharmaceutical agents is urgently needed, yet only a few simple systems have been developed, and even fewer have been evaluated for efficacy in vivo. Here, we present NASCAR (NS3 ASsociated CAR), a collection of induc-ible ON and OFF switch CAR circuits engineered with a NS3 protease domain deriving from the Hepatitis C Virus (HCV). We establish their ability to regulate CAR activity using multiple FDA-approved antiviral protease inhibitors, including grazoprevir (GZV), both in vitro and in a xenograft tumor model. In addition, we have engineered several dual-gated NASCAR circuits, consisting of an AND logic gate CAR, universal ON-OFF CAR, and a switchboard CAR. These engineered receptors enhance control over T cell activity and tumor-targeting specificity. Together, our com-prehensive set of multiplex drug-gated CAR circuits represent a dynamic, tunable, and clinically-ready set of modules for enhancing the safety of CAR T cell therapy.

scholarly journals Immunotherapy using IgE or CAR T cells for cancers expressing the tumor antigen SLC3A2

2021 ◽  
Vol 9 (6) ◽  
pp. e002140
Author(s):  
Giulia Pellizzari ◽  
Olivier Martinez ◽  
Silvia Crescioli ◽  
Robert Page ◽  
Ashley Di Meo ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
T Cells ◽  
T Cell ◽  
Type I ◽  
Cell Therapies ◽  
Car T Cells ◽  
Car T Cell ◽  
Tumor Associated Antigen ◽  
Car T

BackgroundCancer immunotherapy with monoclonal antibodies and chimeric antigen receptor (CAR) T cell therapies can benefit from selection of new targets with high levels of tumor specificity and from early assessments of efficacy and safety to derisk potential therapies.MethodsEmploying mass spectrometry, bioinformatics, immuno-mass spectrometry and CRISPR/Cas9 we identified the target of the tumor-specific SF-25 antibody. We engineered IgE and CAR T cell immunotherapies derived from the SF-25 clone and evaluated potential for cancer therapy.ResultsWe identified the target of the SF-25 clone as the tumor-associated antigen SLC3A2, a cell surface protein with key roles in cancer metabolism. We generated IgE monoclonal antibody, and CAR T cell immunotherapies each recognizing SLC3A2. In concordance with preclinical and, more recently, clinical findings with the first-in-class IgE antibody MOv18 (recognizing the tumor-associated antigen Folate Receptor alpha), SF-25 IgE potentiated Fc-mediated effector functions against cancer cells in vitro and restricted human tumor xenograft growth in mice engrafted with human effector cells. The antibody did not trigger basophil activation in cancer patient blood ex vivo, suggesting failure to induce type I hypersensitivity, and supporting safe therapeutic administration. SLC3A2-specific CAR T cells demonstrated cytotoxicity against tumor cells, stimulated interferon-γ and interleukin-2 production in vitro. In vivo SLC3A2-specific CAR T cells significantly increased overall survival and reduced growth of subcutaneous PC3-LN3-luciferase xenografts. No weight loss, manifestations of cytokine release syndrome or graft-versus-host disease, were detected.ConclusionsThese findings identify efficacious and potentially safe tumor-targeting of SLC3A2 with novel immune-activating antibody and genetically modified cell therapies.

109 Dominant-negative TGFβ receptor 2 enhances GPC3-targeting CAR-T cell efficacy against hepatocellular carcinoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A121-A121
Author(s):  
Nina Chu ◽  
Michael Overstreet ◽  
Ryan Gilbreth ◽  
Lori Clarke ◽  
Christina Gesse ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Dominant Negative ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundChimeric antigen receptors (CARs) are engineered synthetic receptors that reprogram T cell specificity and function against a given antigen. Autologous CAR-T cell therapy has demonstrated potent efficacy against various hematological malignancies, but has yielded limited success against solid cancers. MEDI7028 is a CAR that targets oncofetal antigen glypican-3 (GPC3), which is expressed in 70–90% of hepatocellular carcinoma (HCC), but not in normal liver tissue. Transforming growth factor β (TGFβ) secretion is increased in advanced HCC, which creates an immunosuppressive milieu and facilitates cancer progression and poor prognosis. We tested whether the anti-tumor efficacy of a GPC3 CAR-T can be enhanced with the co-expression of dominant-negative TGFβRII (TGFβRIIDN).MethodsPrimary human T cells were lentivirally transduced to express GPC3 CAR both with and without TGFβRIIDN. Western blot and flow cytometry were performed on purified CAR-T cells to assess modulation of pathways and immune phenotypes driven by TGFβ in vitro. A xenograft model of human HCC cell line overexpressing TGFβ in immunodeficient mice was used to investigate the in vivo efficacy of TGFβRIIDN armored and unarmored CAR-T. Tumor infiltrating lymphocyte populations were analyzed by flow cytometry while serum cytokine levels were quantified with ELISA.ResultsArmoring GPC3 CAR-T with TGFβRIIDN nearly abolished phospho-SMAD2/3 expression upon exposure to recombinant human TGFβ in vitro, indicating that the TGFβ signaling axis was successfully blocked by expression of the dominant-negative receptor. Additionally, expression of TGFβRIIDN suppressed TGFβ-driven CD103 upregulation, further demonstrating attenuation of the pathway by this armoring strategy. In vivo, the TGFβRIIDN armored CAR-T achieved superior tumor regression and delayed tumor regrowth compared to the unarmored CAR-T. The armored CAR-T cells infiltrated HCC tumors more abundantly than their unarmored counterparts, and were phenotypically less exhausted and less differentiated. In line with these observations, we detected significantly more interferon gamma (IFNγ) at peak response and decreased alpha-fetoprotein in the serum of mice treated with armored cells compared to mice receiving unarmored CAR-T, demonstrating in vivo functional superiority of TGFβRIIDN armored CAR-T therapy.ConclusionsArmoring GPC3 CAR-T with TGFβRIIDN abrogates the signaling of TGFβ in vitro and enhances the anti-tumor efficacy of GPC3 CAR-T against TGFβ-expressing HCC tumors in vivo, proving TGFβRIIDN to be an effective armoring strategy against TGFβ-expressing solid malignancies in preclinical models.Ethics ApprovalThe study was approved by AstraZeneca’s Ethics Board and Institutional Animal Care and Use Committee (IACUC).

Abstract 60: Induction of T cell dysfunction and NK-like T cell differentiation in vitro and in patients after CAR T cell treatment

2021 ◽  
Author(s):  
Charly R. Good ◽  
Shunichiro Kuramitsu ◽  
Parisa Samareh ◽  
Greg Donahue ◽  
Kenichi Ishiyama ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
T Cell Differentiation ◽  
Cell Dysfunction ◽  
Car T Cell ◽  
Car T ◽  
T Cell Dysfunction
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