scholarly journals Interleukin-6-knockdown of chimeric antigen receptor-modified T cells significantly reduces IL-6 release from monocytes

2020 ◽  
Vol 9 (1) ◽  
Author(s):  
Liqing Kang ◽  
Xiaowen Tang ◽  
Jian Zhang ◽  
Minghao Li ◽  
Nan Xu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Interleukin 6 ◽  
Lymphoma Cells ◽  
B Lymphoma ◽  
Car T Cell ◽  
B Lymphoma Cells ◽  
Car T

Abstract Background T cells expressing a chimeric antigen receptor (CAR) engineered to target CD19 can treat leukemia effectively but also increase the risk of complications such as cytokine release syndrome (CRS) and CAR T cell related encephalopathy (CRES) driven by interleukin-6 (IL-6). Here, we investigated whether IL-6 knockdown in CART-19 cells can reduce IL-6 secretion from monocytes, which may reduce the risk of adverse events. Methods Supernatants from cocultures of regular CART-19 cells and B lymphoma cells were added to monocytes in vitro, and the IL-6 levels in monocyte supernatants were measured 24 h later. IL-6 expression was knocked down in regular CART-19 cells by adding a short hairpin RNA (shRNA) (termed ssCART-19) expression cassette specific for IL-6 to the conventional CAR vector. Transduction efficiency and cell proliferation were measured by flow cytometry, and cytotoxicity was measured by evaluating the release of lactate dehydrogenase into the medium. Gene expression was assessed by qRT-PCR and RNA sequencing. A xenograft leukemia mouse model was established by injecting NOD/SCID/γc-/- mice with luciferase-expressing B lymphoma cells, and then the animals were treated with regular CART-19 cells or ssCART-19. Tumor growth was assessed by bioluminescence imaging. Results Both recombinant IL-6 and CART-19 derived IL-6 significantly triggered IL-6 release by monocytes. IL-6 knockdown in ssCART-19 cells dramatically reduced IL-6 release from monocytes in vitro stduy. In vivo study further demonstrated that the mice bearing Raji cells treated with ssCART-19 cells showed significant lower IL-6 levels in serum than those treated with regular CART-19 cells, but comparable anti-tumor efficacy between the animal groups. Conclusion CAR T-derived IL-6 is one of the most important initiators to amplify release of IL-6 from monocytes that further drive sCRS development. IL-6 knockdown in ssCART-19 cells by shRNA technology provide a promising strategy to improve the safety of CAR T cell therapy.

scholarly journals Interleukin-6-knockdown of chimeric antigen receptor-modified T cells significantly reduces IL-6 release from monocytes

2020 ◽  
Author(s):  
Liqing Kang ◽  
Xiaowen Tang ◽  
Jian Zhang ◽  
Minghao Li ◽  
Nan Xu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Chimeric Antigen Receptor ◽  
Interleukin 6 ◽  
Lymphoma Cells ◽  
B Lymphoma ◽  
B Lymphoma Cells ◽  
Car T

Abstract Background T cells expressing a chimeric antigen receptor (CAR) engineered to target CD19 can treat leukemia effectively but also increase the risk of complications such as cytokine release syndrome (CRS) and CAR T cell related encephalopathy (CRES) driven by interleukin-6 (IL-6). Here, we investigated whether IL-6 knockdown in CART-19 cells can reduce IL-6 secretion from monocytes, which may reduce the risk of adverse events. Methods Supernatants from cocultures of regular CART-19 cells and B lymphoma cells were added to monocytes in vitro, and the IL-6 levels in monocyte supernatants were measured 24 h later. IL-6 expression was knocked down in regular CART-19 cells by adding a short hairpin RNA (shRNA) (termed ssCART-19) expression cassette specific for IL-6 to the conventional CAR vector. Transduction efficiency and cell proliferation were measured by flow cytometry, and cytotoxicity was measured by evaluating the release of lactate dehydrogenase into the medium. Gene expression was assessed by qRT-PCR and RNA sequencing. A xenograft leukemia mouse model was established by injecting NOD/SCID/γc-/- mice with luciferase-expressing B lymphoma cells, and then the animals were treated with regular CART-19 cells or ssCART-19. Tumor growth was assessed by bioluminescence imaging. Results Both recombinant IL-6 and activated regular CART-19 cells expressing IL-6 triggered IL-6 release by monocytes. IL-6 knockdown in ssCART-19 cells dramatically reduced IL-6 release from monocytes without reducing cytotoxic activity. Mice treated with ssCART-19 cells showed lower IL-6 levels in the serum than mice treated with regular CART-19 cells, but tumor growth and survival were similar between the animal groups. Conclusion IL-6 released from activated CAR T cells may be one of the main initiators of the release of IL-6 from monocytes that can drive CRS. IL-6 knockdown in ssCART-19 cells reduces monocyte release of IL-6 both in vitro and in vivo without affecting antitumor efficacy. The IL-6 knockdown strategy may provide a useful and promising way to improve the safety of CAR T cell therapy.

scholarly journals HIV-1-Specific Chimeric Antigen Receptor T Cells Fail To Recognize and Eliminate the Follicular Dendritic Cell HIV Reservoir In Vitro

2020 ◽  
Vol 94 (10) ◽  
Author(s):  
Matthew T. Ollerton ◽  
Edward A. Berger ◽  
Elizabeth Connick ◽  
Gregory F. Burton
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antiretroviral Therapy ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Follicular Dendritic

ABSTRACT The major obstacle to a cure for HIV infection is the persistence of replication-competent viral reservoirs during antiretroviral therapy. HIV-specific chimeric antigen receptor (CAR) T cells have been developed to target latently infected CD4+ T cells that express virus either spontaneously or after intentional latency reversal. Whether HIV-specific CAR-T cells can recognize and eliminate the follicular dendritic cell (FDC) reservoir of HIV-bound immune complexes (ICs) is unknown. We created HIV-specific CAR-T cells using human peripheral blood mononuclear cells (PBMCs) and a CAR construct that enables the expression of CD4 (domains 1 and 2) and the carbohydrate recognition domain of mannose binding lectin (MBL) to target native HIV Env (CD4-MBL CAR). We assessed CAR-T cell cytotoxicity using a carboxyfluorescein succinimidyl ester (CFSE) release assay and evaluated CAR-T cell activation through interferon gamma (IFN-γ) production and CD107a membrane accumulation by flow cytometry. CD4-MBL CAR-T cells displayed potent lytic and functional responses to Env-expressing cell lines and HIV-infected CD4+ T cells but were ineffective at targeting FDC bearing HIV-ICs. CD4-MBL CAR-T cells were unresponsive to cell-free HIV or concentrated, immobilized HIV-ICs in cell-free experiments. Blocking intercellular adhesion molecule-1 (ICAM-1) inhibited the cytolytic response of CD4-MBL CAR-T cells to Env-expressing cell lines and HIV-infected CD4+ T cells, suggesting that factors such as adhesion molecules are necessary for the stabilization of the CAR-Env interaction to elicit a cytotoxic response. Thus, CD4-MBL CAR-T cells are unable to eliminate the FDC-associated HIV reservoir, and alternative strategies to eradicate this reservoir must be sought. IMPORTANCE Efforts to cure HIV infection have focused primarily on the elimination of latently infected CD4+ T cells. Few studies have addressed the unique reservoir of infectious HIV that exists on follicular dendritic cells (FDCs), persists in vivo during antiretroviral therapy, and likely contributes to viral rebound upon cessation of antiretroviral therapy. We assessed the efficacy of a novel HIV-specific chimeric antigen receptor (CAR) T cell to target both HIV-infected CD4+ T cells and the FDC reservoir in vitro. Although CAR-T cells eliminated CD4+ T cells that express HIV, they did not respond to or eliminate FDC bound to HIV. These findings reveal a fundamental limitation to CAR-T cell therapy to eradicate HIV.

Effect of Rituximab On the Activity of T Cells Expressing CD20-Specific Chimeric Antigen Receptors

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4222-4222
Author(s):  
Gregory A. Rufener ◽  
Michael C. Jensen ◽  
Shaunda Brouns ◽  
Lihua E. Budde ◽  
David G. Maloney ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Cell Function ◽  
Cytokine Secretion ◽  
Lymphoma Cells ◽  
Car T Cells ◽  
Car T ◽  
Anti Cd20

Abstract Abstract 4222 BACKGROUND: Adoptive cellular therapy using autologous T cells that have been genetically modified to express a chimeric antigen receptor (CAR) has emerged as a promising therapy for lymphoma. Clinical trials for lymphoid malignancies to date have primarily targeted either the CD19 or CD20 antigens. While CD20 has a more established track record as an immunotherapy target, one potential drawback of targeting CD20 with CAR+ T cells is the theoretical possibility that residual levels of circulating anti-CD20 antibodies (Ab) from prior chemoimmunotherapy regimens could partially or completely block CAR-antigen interactions. This could negatively impact the efficacy of CD20-targeted CAR+ T cells. However, previous data from our group and others indicate that CD20 CAR+T cell function is only partially blocked by anti-CD20 Ab, and T cell function in the setting of anti-CD3 × anti-CD20 bispecific Ab is not blocked by rituximab (R) levels of up to 100 μg/ml. Collectively, these data suggest that a very low number of available CD20 binding sites may be sufficient to trigger CAR signaling and T cell activation. METHODS: We tested the effect of different levels of R on in vitro function of polyclonal T cells from healthy donors negatively selected by MACS, activated with anti-CD3/CD28 beads, and transduced with epHIV7 lentiviral vectors encoding 1st or 3rdgeneration (αCD20-ζ or αCD20-CD28–41BB-ζ) anti-CD20 CARs. T cells were re-stimulated 1 week after initial activation by co-culture with antigen presenting cells (APCs) that had been pre-incubated for 30 minutes with varying concentrations of R (ranging from 0 to 800 μg/ml). APCs were K562 cells transduced to express CD80 with or without CD20 (denoted “K80” and “K80-20”), or Ramos lymphoma cells. Proliferation, cytokine secretion, and cytotoxicity were then assessed as discussed below. RESULTS: We first used flow cytometry to test whether varying concentrations of R blocked binding of the Leu16 Ab and, as expected, found a dose-dependent blockade of CD20 on each cell line, with 50 μg/ml and 200 μg/ml causing near-complete blockade of K80-20 and Ramos cells, respectively. However, despite this apparent blockade, proliferation was largely unimpaired in CFSE-labeled 1st or 3rd generation CAR+ T cells cultured with K80-20 or Ramos cells pre-incubated with R concentrations of up to 400 μg/ml. We concurrently measured cytokine secretion of these T cells using Luminex assays and found that IL-2 and IFN-γ secretion decreased with increasing R levels, but 50–85% of baseline levels were still achieved at R concentrations of up to 100 μg/ml. Cytotoxicity against K80-20 and Ramos target cells in standard 51Cr-release assays by 1st and 3rd generation CAR+ T cells was largely preserved at low R concentrations, and 50–75% of cytolytic activity was retained at 100 μg/ml. Nonspecific proliferation, cytokine secretion, and cytotoxicity were excluded in these experiments by using CAR+ T cells incubated with K80 cells lacking CD20 expression, or T cells transduced with an empty vector as negative controls. Mouse xenograft experiments are currently ongoing to test the effect of serum R levels on the in vivo anti-tumor efficacy of CD20-CAR T cells. CONCLUSIONS: These in vitro results suggest that despite apparent blockade of the CD20 antigen, CAR+ T cells targeting CD20 retain significant activity in the presence of R concentrations of up to 100 ug/ml. Patients receiving 2–3 cycles of R-chemotherapy have serum R trough levels in the range of 30–70 μg/ml. We therefore predict that residual serum R levels will not present a significant impediment to CD20-targeted adoptive T cell therapy given after salvage R-chemotherapy. Disclosures: Jensen: ZetaRx: Equity Ownership, Patents & Royalties. Maloney:Roche: Consultancy; Genentech: Consultancy. Off Label Use: Lentiviral vector encoding a CD20-specific chimeric antigen receptor, used to re-direct autologous T cells to recognize B cell lymphoma cells.

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.

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.

CD4+ T cells kill Id+ B-lymphoma cells: FasLigand-Fas interaction is dominant in vitro but is redundant in vivo

2004 ◽  
Vol 53 (12) ◽  
pp. 1135-1145 ◽  
Author(s):  
Katrin U. Lundin ◽  
Valentina Screpanti ◽  
Hilde Omholt ◽  
Peter O. Hofgaard ◽  
Hideo Yagita ◽  
...  
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Lymphoma Cells ◽  
B Lymphoma ◽  
B Lymphoma Cells

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).

scholarly journals 221 CRISPR screen identifies loss of IFNγR signaling and downstream adhesion as a resistance mechanism to CAR T-cell cytotoxicity in solid but not liquid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A234-A234
Author(s):  
Rebecca Larson ◽  
Michael Kann ◽  
Stefanie Bailey ◽  
Nicholas Haradhvala ◽  
Kai Stewart ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Solid Tumors ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

BackgroundChimeric Antigen Receptor (CAR) therapy has had a transformative impact on the treatment of hematologic malignancies1–6 but success in solid tumors remains elusive. We hypothesized solid tumors have cell-intrinsic resistance mechanisms to CAR T-cell cytotoxicity.MethodsTo systematically identify resistance pathways, we conducted a genome-wide CRISPR knockout screen in glioblastoma cells, a disease where CAR T-cells have had limited efficacy.7 8 We utilized the glioblastoma cell line U87 and targeted endogenously expressed EGFR with CAR T-cells generated from 6 normal donors for the screen. We validated findings in vitro and in vivo across a variety of human tumors and CAR T-cell antigens.ResultsLoss of genes in the interferon gamma receptor (IFNγR) signaling pathway (IFNγR1, JAK1, JAK2) rendered U87 cells resistant to CAR T-cell killing in vitro. IFNγR1 knockout tumors also showed resistance to CAR T cell treatment in vivo in a second glioblastoma line U251 in an orthotopic model. This phenomenon was irrespective of CAR target as we also observed resistance with IL13Ralpha2 CAR T-cells. In addition, resistance to CAR T-cell cytotoxicity through loss of IFNγR1 applied more broadly to solid tumors as pancreatic cell lines targeted with either Mesothelin or EGFR CAR T-cells also showed resistance. However, loss of IFNγR signaling did not impact sensitivity of liquid tumor lines (leukemia, lymphoma or multiple myeloma) to CAR T-cells in vitro or in an orthotopic model of leukemia treated with CD19 CAR. We isolated the effects of decreased cytotoxicity of IFNγR1 knockout glioblastoma tumors to be cancer-cell intrinsic because CAR T-cells had no observable differences in proliferation, activation (CD69 and LFA-1), or degranulation (CD107a) when exposed to wildtype versus knockout tumors. Using transcriptional profiling, we determined that glioblastoma cells lacking IFNγR1 had lower upregulation of cell adhesion pathways compared to wildtype glioblastoma cells after exposure to CAR T-cells. We found that loss of IFNγR1 reduced CAR T-cell binding avidity to glioblastoma.ConclusionsThe critical role of IFNγR signaling for susceptibility of solid tumors to CAR T-cells is surprising given that CAR T-cells do not require traditional antigen-presentation pathways. Instead, in glioblastoma tumors, IFNγR signaling was required for sufficient adhesion of CAR T-cells to mediate productive cytotoxicity. Our work demonstrates that liquid and solid tumors differ in their interactions with CAR T-cells and suggests that enhancing T-cell/tumor interactions may yield improved responses in solid tumors.AcknowledgementsRCL was supported by T32 GM007306, T32 AI007529, and the Richard N. Cross Fund. ML was supported by T32 2T32CA071345-21A1. SRB was supported by T32CA009216-38. NJH was supported by the Landry Cancer Biology Fellowship. JJ is supported by a NIH F31 fellowship (1F31-MH117886). GG was partially funded by the Paul C. Zamecnik Chair in Oncology at the Massachusetts General Hospital Cancer Center and NIH R01CA 252940. MVM and this work is supported by the Damon Runyon Cancer Research Foundation, Stand Up to Cancer, NIH R01CA 252940, R01CA238268, and R01CA249062.ReferencesMaude SL, et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med 2018;378:439–448.Neelapu SS, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med 2017;377:2531–2544.Locke FL, et al. Long-term safety and activity of axicabtagene ciloleucel in refractory large B-cell lymphoma (ZUMA-1): a single-arm, multicentre, phase 1–2 trial. The Lancet Oncology 2019;20:31–42.Schuster SJ, et al. Chimeric antigen receptor T cells in refractory B-cell lymphomas. N Engl J Med 2017;377:2545–2554.Wang M, et al. KTE-X19 CAR T-cell therapy in relapsed or refractory mantle-cell lymphoma. N Engl J Med 2020;382:1331–1342.Cohen AD, et al. B cell maturation antigen-specific CAR T cells are clinically active in multiple myeloma. J Clin Invest 2019;129:2210–2221.Bagley SJ, et al. CAR T-cell therapy for glioblastoma: recent clinical advances and future challenges. Neuro-oncology 2018;20:1429–1438.Choi BD, et al. Engineering chimeric antigen receptor T cells to treat glioblastoma. J Target Ther Cancer 2017;6:22–25.Ethics ApprovalAll human samples were obtained with informed consent and following institutional guidelines under protocols approved by the Institutional Review Boards (IRBs) at the Massachusetts General Hospital (2016P001219). Animal work was performed according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) (2015N000218 and 2020N000114).

scholarly journals 124 Functionalizing CAR T cells for selective proliferation and dual-targeting using the meditope technology

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A133-A133
Author(s):  
Cheng-Fu Kuo ◽  
Yi-Chiu Kuo ◽  
Miso Park ◽  
Zhen Tong ◽  
Brenda Aguilar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

BackgroundMeditope is a small cyclic peptide that was identified to bind to cetuximab within the Fab region. The meditope binding site can be grafted onto any Fab framework, creating a platform to uniquely and specifically target monoclonal antibodies. Here we demonstrate that the meditope binding site can be grafted onto chimeric antigen receptors (CARs) and utilized to regulate and extend CAR T cell function. We demonstrate that the platform can be used to overcome key barriers to CAR T cell therapy, including T cell exhaustion and antigen escape.MethodsMeditope-enabled CARs (meCARs) were generated by amino acid substitutions to create binding sites for meditope peptide (meP) within the Fab tumor targeting domain of the CAR. meCAR expression was validated by anti-Fc FITC or meP-Alexa 647 probes. In vitro and in vivo assays were performed and compared to standard scFv CAR T cells. For meCAR T cell proliferation and dual-targeting assays, the meditope peptide (meP) was conjugated to recombinant human IL15 fused to the CD215 sushi domain (meP-IL15:sushi) and anti-CD20 monoclonal antibody rituximab (meP-rituximab).ResultsWe generated meCAR T cells targeting HER2, CD19 and HER1/3 and demonstrate the selective specific binding of the meditope peptide along with potent meCAR T cell effector function. We next demonstrated the utility of a meP-IL15:sushi for enhancing meCAR T cell proliferation in vitro and in vivo. Proliferation and persistence of meCAR T cells was dose dependent, establishing the ability to regulate CAR T cell expansion using the meditope platform. We also demonstrate the ability to redirect meCAR T cells tumor killing using meP-antibody adaptors. As proof-of-concept, meHER2-CAR T cells were redirected to target CD20+ Raji tumors, establishing the potential of the meditope platform to alter the CAR specificity and overcome tumor heterogeneity.ConclusionsOur studies show the utility of the meCAR platform for overcoming key challenges for CAR T cell therapy by specifically regulating CAR T cell functionality. Specifically, the meP-IL15:sushi enhanced meCAR T cell persistence and proliferation following adoptive transfer in vivo and protects against T cell exhaustion. Further, meP-ritiuximab can redirect meCAR T cells to target CD20-tumors, showing the versatility of this platform to address the tumor antigen escape variants. Future studies are focused on conferring additional ‘add-on’ functionalities to meCAR T cells to potentiate the therapeutic effectiveness of CAR T cell therapy.

scholarly journals Evaluation of chimeric antigen receptor T cell therapy in non-human primates infected with SHIV or SIV

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0248973
Author(s):  
Nami Iwamoto ◽  
Bhavik Patel ◽  
Kaimei Song ◽  
Rosemarie Mason ◽  
Sara Bolivar-Wagers ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Chimeric Antigen Receptor ◽  
Viral Suppression ◽  
Antigen Receptor ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T

Achieving a functional cure is an important goal in the development of HIV therapy. Eliciting HIV-specific cellular immune responses has not been sufficient to achieve durable removal of HIV-infected cells due to the restriction on effective immune responses by mutation and establishment of latent reservoirs. Chimeric antigen receptor (CAR) T cells are an avenue to potentially develop more potent redirected cellular responses against infected T cells. We developed and tested a range of HIV- and SIV-specific chimeric antigen receptor (CAR) T cell reagents based on Env-binding proteins. In general, SHIV/SIV CAR T cells showed potent viral suppression in vitro, and adding additional CAR molecules in the same transduction resulted in more potent viral suppression than single CAR transduction. Importantly, the primary determinant of virus suppression potency by CAR was the accessibility to the Env epitope, and not the neutralization potency of the binding moiety. However, upon transduction of autologous T cells followed by infusion in vivo, none of these CAR T cells impacted either acquisition as a test of prevention, or viremia as a test of treatment. Our study illustrates limitations of the CAR T cells as possible antiviral therapeutics.

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