scholarly journals 720 CUE-102 selectively activates and expands WT1-specific T cells for the treatment of patients with WT1+ malignancies

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A749-A749
Author(s):  
Christie Zhang ◽  
Natasha Girgis ◽  
Zohra Merazga ◽  
Steven Hatfield ◽  
Alex Histed ◽  
...  
Keyword(s):  
T Cells ◽  
Transgenic Mice ◽  
Cd8 T Cells ◽  
Wilms Tumor ◽  
Interleukin 2 ◽  
Target Cells ◽  
Tolerability Profile ◽  
Activation Markers

BackgroundWilms' Tumor 1 (WT1) was ranked as the highest priority antigen for therapeutic targeting in an effort by the National Cancer Institute. Development of novel modalities targeting WT1 provide a significant opportunity to address high unmet medical need in WT1-positive malignancies, including AML, ovarian, endometrial, breast, lung, colorectal and pancreatic cancer. Leveraging the Immuno-STAT platform of targeted IL-2 therapies, and the ongoing development of CUE-101, CUE-102 is being developed as a novel therapeutic fusion protein to selectively activate tumor antigen-specific T cells to treat WT1-expressing cancers. CUE-102 consists of two human leukocyte antigen (HLA) molecules presenting a WT1 peptide, four affinity-attenuated human interleukin-2 (IL-2) molecules, and an effector attenuated human immunoglobulin G (IgG1) Fc domain.MethodsHuman PBMCs were tested to demonstrate cellular activity and specificity of CUE-102, while in vivo activity of CUE-102 was assessed in HLA-A2 transgenic mice. HLA-A2/WT1-specific TCRs were validated and expressed in primary human CD8 T cells. Tetramer staining and flow cytometry identified cell populations and activation markers.ResultsMultiple in vitro assessments demonstrate that CUE-102 selectively activates and expands WT1-specific CD8+ T cells from PBMC of healthy and cancer bearing donors. These CUE-102-expanded CD8+ T cells exhibit polyfunctional and cytotoxic responses upon challenge with WT1-presenting target cells. In addition, significant functional attenuation of the IL-2 components of CUE-102 was shown, similar to preclinical results obtained with CUE-101. In vivo studies in HLA-A2 transgenic mice confirm that CUE-102 elicits and expands polyfunctional WT1-specific CD8+ T cells from naïve and previously immunized mice without significantly altering the frequencies of other immune lineages. The WT1-specific CD8+ T cells expanded in vivo exhibit polyfunctionality in response to peptide-loaded target cells, and selectively kill WT1-presenting target cells in vivo.ConclusionsCUE-102 elicits selective expansion of a WT1-specific population of cytotoxic CD8+ T cells both in vitro and in vivo. These results, together with its similarity to CUE-101, support its anticipated tolerability profile and potential for clinical efficacy in a Phase 1 trial planned to initiate in 2022.Ethics ApprovalAll animal studies followed guidance from the SmartLabs Institutional Animal Care and Use Committee protocol MIL-100 and were performed in compliance with federal guidelines.

scholarly journals Resting Memory CD8+ T Cells are Hyperreactive to Antigenic Challenge In Vitro

1996 ◽  
Vol 184 (6) ◽  
pp. 2141-2152 ◽  
Author(s):  
Maria Pihlgren ◽  
Patrice M. Dubois ◽  
Martine Tomkowiak ◽  
Tove Sjögren ◽  
Jacqueline Marvel
Keyword(s):  
T Cells ◽  
T Cell ◽  
Transgenic Mice ◽  
Cd8 T Cells ◽  
Cell Receptor ◽  
Relative Role ◽  
Activation Markers ◽  
Homogeneous Population

The characteristics of CD8+ T cells responsible for memory responses are still largely unknown. Particularly, it has not been determined whether different activation thresholds distinguish naive from memory CD8+ T cell populations. In most experimental systems, heterogeneous populations of primed CD8+ T cells can be identified in vivo after immunization. These cells differ in terms of cell cycle status, surface phenotype, and/or effector function. This heterogeneity has made it difficult to assess the activation threshold and the relative role of these subpopulations in memory responses. In this study we have used F5 T cell receptor transgenic mice to generate a homogeneous population of primed CD8+ T cells. In the F5 transgenic mice, peptide injection in vivo leads to activation of most peripheral CD8+ T cells. In vivo BrdU labeling has been used to follow primed T cells over time periods spanning several weeks after peptide immunization. Our results show that the majority of primed CD8+ T cells generated in this system are not cycling and express increased levels of CD44 and Ly6C. These cells remain responsive to secondary peptide challenge in vivo as evidenced by short term upregulation of activation markers such as CD69 and CD44. The activation thresholds of naive and primed CD8+ T cells were compared in vitro. We found that CD8+ T cells from primed mice are activated by peptide concentrations 10–50-fold lower than naive mice. In addition, the kinetics of interleukin 2Rα chain upregulation by primed CD8+ T cells differ from naive CD8+ T cells. These primed hyperresponsive CD8+ T cells might play an important role in the memory response.

Electroporation of Dicer-Substrate siRNA Duplexes Targeting Endogenous TCR Enhance Tumor Killing Activity of Wilms' Tumor 1 (WT1)-Specific TCR-Redirected Cytotoxic T Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 813-813 ◽  
Author(s):  
Diana Campillo-Davo ◽  
Fumihiro Fujiki ◽  
Johan M.J. Van den Bergh ◽  
Evelien L. Smits ◽  
Haruo Sugiyama ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Wilms Tumor ◽  
Target Cells ◽  
Wild Type ◽  
Activation Markers ◽  
Killing Activity ◽  
Ifn Γ ◽  
Mrna Electroporation

Abstract In adoptive cellular immunotherapy, T cells can be genetically engineered to express a novel T-cell receptor (TCR) that recognizes a tumor-associated antigen. However, mispairing between transgene and endogenous TCR chains may result in a reduction of transgene TCR expression and potentially harmful off-target reactivities. Here, we sought to develop a novel clinically safe strategy to promote transgene expression of a Wilms' tumor 1 (WT1)-specific TCR by Dicer-substrate small interfering RNA (DsiRNA)-mediated silencing of the endogenous TCR, using a double electroporation protocol. First, we isolated and cloned an HLA-A*0201-restricted WT1 peptide-specific TCR derived from a leukemia patient who demonstrated clinical benefit after receiving a WT1-targeted DC vaccine. Next, we produced a codon-optimized TCR sequence from the wild-type TCR construct and both TCR mRNAs were generated by in vitro transcription. TCR expression levels were validated by electroporation of TCR-deficient Jurkat J76.7 cells stably transduced with CD8 and an NFAT-driven GFP reporter gene. TCR functionality was confirmed by high expression levels of GFP (70% GFP+cells) upon TCR signaling after co-culture with WT1 peptide-pulsed T2 cells. In order to suppress the translation of endogenous TCR mRNA in CD8+ T cells, DsiRNA duplexes were designed to specifically target the constant regions of wild-type TCR α- and β-chains, but not the codon-optimized TCR. We further developed a double electroporation protocol combining DsiRNA and TCR mRNA transfection in which DsiRNA electroporation was performed 24 hours prior to TCR mRNA electroporation. Our results show more than 2-fold increase in WT1-specific TCR expression by HLA-A2/WT1 tetramer staining after DsiRNA treatment as compared to TCR mRNA electroporation only. This specific TCR expression was maintained at least 5 days after TCR mRNA electroporation in resting peripheral blood CD8+ lymphocytes from healthy donors. The enhanced TCR expression in DsiRNA-transfected CD8+T cells was also correlated with an increase of epitope recognition as shown by interferon (IFN)-γ ELISpot. To determine the killing capacity of DsiRNA/TCR mRNA-transfected CD8+ T cells against epitope-bearing target cells, we performed a flow cytometry-based cytotoxicity assay using WT1 peptide-pulsed T2 cells. Specific cytotoxicity, which was already present in WT1 TCR-transfected cells, was significantly enhanced in TCR mRNA-electroporated T cells following suppression of the endogenous TCR expression by DsiRNA treatment. Accordingly, DsiRNA-treated TCR mRNA transfected CD8+T cells presented higher levels of CD137 and CD69 activation markers and secretion of cytokines (IFN-γ and tumor necrosis factor-α), granzyme B, and perforin upon TCR triggering as compared to the non-DsiRNA treated T cells. In summary, we show a marked enhancement of transgene WT1-specific TCR expression upon silencing of the endogenous TCR using DsiRNA electroporation prior to TCR mRNA electroporation. Importantly, this enhancement in TCR expression was correlated with a significant increase in WT1-specific CD8+ T-cell killing activity, expression of CD69 and CD137 activation markers and cytokine secretion after recognition of WT1 peptide-bearing target cells. These results pave the way for developing a clinically safer strategy for T cell-based adoptive immunotherapy of patients with WT1-expressing malignancies. Disclosures No relevant conflicts of interest to declare.

scholarly journals T cell-mediated eradication of murine metastatic melanoma induced by targeted interleukin 2 therapy.

1996 ◽  
Vol 183 (5) ◽  
pp. 2361-2366 ◽  
Author(s):  
J C Becker ◽  
J D Pancook ◽  
S D Gillies ◽  
K Furukawa ◽  
R A Reisfeld
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Interleukin 2 ◽  
Tumor Model ◽  
Melanoma Metastases ◽  
Tumor Eradication

Induction of a T-cell mediated antitumor response is the ultimate goal for tumor immunotherapy. We demonstrate here that antibody-targeted IL2 therapy is effective against established pulmonary and hepatic melanoma metastases in a syngeneic murine tumor model. The effector mechanisms involved in this tumor eradication are not dependent on NK cells, since the therapeutic effect of antibody-IL2 fusion protein was not altered in NK cell-deficient mice. In contrast, T cells are essential for the observed antitumor effect, since therapy with antibody IL2 fusion proteins is unable to induce tumor eradication in T cell-deficient SCID mice. In vivo depletion studies characterized the essential effector cell population further as CD8 + T cells. Such CD8 + T cells, isolated from tumor bearing mice after antibody-directed IL2 therapy, exerted a MHC class I-restricted cytotoxicity against the same tumor in vitro. These data demonstrate the ability of antibody-targeted IL2 delivery to induce a T cell-dependent host immune response that is capable of eradicating established melanoma metastases in clinically relevant organs.

scholarly journals Characterization of common variable immunodeficiency: identification of a subset of patients with distinctive immunophenotypic and clinical features [see comments]

Blood ◽  
1990 ◽  
Vol 76 (10) ◽  
pp. 2046-2051 ◽  
Author(s):  
JJ Wright ◽  
DK Wagner ◽  
RM Blaese ◽  
C Hagengruber ◽  
TA Waldmann ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Clinical Features ◽  
Cd8 T Cells ◽  
Common Variable Immunodeficiency ◽  
Interleukin 2 ◽  
Delayed Type Hypersensitivity ◽  
Activation Markers ◽  
Common Variable

The peripheral blood lymphocyte surface markers and clinical features of 38 patients with common variable immunodeficiency (CVID) were assessed. These studies identified a subset of CVID consisting of 14 of the 38 patients with a distinctive T-cell immunophenotype and clinical findings. The phenotypic changes were characterized by an abnormally low CD4/CD8 ratio (less than or equal to 0.9) due primarily to a significant increase in CD8 T cells. In addition, there was an expansion in CD8 T cells coexpressing CD57 and increased expression of the activation markers HLA-DR and interleukin-2 receptor (IL-2R) by these cells. This group of immunophenotypically abnormal CVID patients also had characteristic clinical features, including splenomegaly (P less than .02) and in vivo T-cell dysfunction based on the evaluation of delayed-type hypersensitivity (P less than .05). Approximately 71% of these patients had splenomegaly and 42% were anergic in contrast to the remaining group of CVID patients, in which 29% had splenomegaly and 7% were anergic. These findings define a subgroup of CVID patients that have specific immunophenotypic features and functional T-cell abnormalities.

scholarly journals 5-Azacytidine Promotes an Inhibitory T-Cell Phenotype and Impairs Immune Mediated Antileukemic Activity

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Thomas Stübig ◽  
Anita Badbaran ◽  
Tim Luetkens ◽  
York Hildebrandt ◽  
Djordje Atanackovic ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Phenotype ◽  
Target Cells ◽  
T Helper 1 ◽  
Cell Immunity ◽  
The Impact

Demethylating agent, 5-Azacytidine (5-Aza), has been shown to be active in treatment of myeloid malignancies. 5-Aza enhances anticancer immunity, by increasing expression of tumor-associated antigens. However, the impact of 5-Aza immune responses remains poorly understood. Here, T-cell mediated tumor immunity effects of 5-Aza, are investigatedin vitroandin vivo. T-cells from healthy donors were treated with 5-Aza and analyzed by qRT-PCR and flow cytometry for changes in gene expression and phenotype. Functionality was assessed by a tumor lysis assay. Peripheral blood from patients treated with 5-Aza after alloSCT was monitored for changes in T-cell subpopulations. 5-Aza treatment resulted in a decrease in CD8+ T-cells, whereas CD4+ T-cells increased. Furthermore, numbers of IFN-γ+ T-helper 1 cells (Th1) were reduced, while Treg-cells showed substantial increase. Additionally, CD8+ T-cells exhibited limited killing capacity against leukemic target cells.In vivodata confirm the increase of Treg compartment, while CD8+ T-effector cell numbers were reduced. 5-Aza treatment results in a shift from cytotoxic to regulatory T-cells with a functional phenotype and a major reduction in proinflammatory Th1-cells, indicating a strong inhibition of tumor-specific T-cell immunity by 5-Aza.

scholarly journals Characterization of common variable immunodeficiency: identification of a subset of patients with distinctive immunophenotypic and clinical features [see comments]

Blood ◽  
1990 ◽  
Vol 76 (10) ◽  
pp. 2046-2051 ◽  
Author(s):  
JJ Wright ◽  
DK Wagner ◽  
RM Blaese ◽  
C Hagengruber ◽  
TA Waldmann ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Clinical Features ◽  
Cd8 T Cells ◽  
Common Variable Immunodeficiency ◽  
Interleukin 2 ◽  
Delayed Type Hypersensitivity ◽  
Activation Markers ◽  
Common Variable

Abstract The peripheral blood lymphocyte surface markers and clinical features of 38 patients with common variable immunodeficiency (CVID) were assessed. These studies identified a subset of CVID consisting of 14 of the 38 patients with a distinctive T-cell immunophenotype and clinical findings. The phenotypic changes were characterized by an abnormally low CD4/CD8 ratio (less than or equal to 0.9) due primarily to a significant increase in CD8 T cells. In addition, there was an expansion in CD8 T cells coexpressing CD57 and increased expression of the activation markers HLA-DR and interleukin-2 receptor (IL-2R) by these cells. This group of immunophenotypically abnormal CVID patients also had characteristic clinical features, including splenomegaly (P less than .02) and in vivo T-cell dysfunction based on the evaluation of delayed-type hypersensitivity (P less than .05). Approximately 71% of these patients had splenomegaly and 42% were anergic in contrast to the remaining group of CVID patients, in which 29% had splenomegaly and 7% were anergic. These findings define a subgroup of CVID patients that have specific immunophenotypic features and functional T-cell abnormalities.

scholarly journals Strong priming of T cells adoptively transferred into scid mice.

1991 ◽  
Vol 174 (6) ◽  
pp. 1653-1656 ◽  
Author(s):  
G H Sunshine ◽  
B L Jimmo ◽  
C Ianelli ◽  
L Jarvis
Keyword(s):  
T Cells ◽  
B Cells ◽  
Lymph Nodes ◽  
Cd8 T Cells ◽  
Scid Mouse ◽  
Interleukin 2 ◽  
Keyhole Limpet Hemocyanin ◽  
Scid Mice

We have examined the requirements for activating unprimed T cells in vivo by transferring T cells into scid mice, which lack mature B and T cells. Purified adult thymocytes and a protein antigen, keyhole limpet hemocyanin (KLH), were injected into scid mice. scid mice injected with T cells and KLH developed cellular lymph nodes containing CD4+ and CD8+ T cells. Cells recovered from the lymph nodes of injected scid mice proliferated and secreted interleukin 2 in response to KLH in vitro. The results indicate that T cells can be primed to KLH in the scid mouse in the absence of B cells.

scholarly journals A bispecific antibody agonist of the IL-2 heterodimeric receptor preferentially promotes in vivo expansion of CD8 and NK cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Katherine E. Harris ◽  
Kyle J. Lorentsen ◽  
Harbani K. Malik-Chaudhry ◽  
Kaitlyn Loughlin ◽  
Harish Medlari Basappa ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Bispecific Antibody ◽  
T Regulatory Cells ◽  
Tumor Regression ◽  
Interleukin 2 ◽  
In Vivo Studies ◽  
Preferential Binding

AbstractThe use of recombinant interleukin-2 (IL-2) as a therapeutic protein has been limited by significant toxicities despite its demonstrated ability to induce durable tumor-regression in cancer patients. The adverse events and limited efficacy of IL-2 treatment are due to the preferential binding of IL-2 to cells that express the high-affinity, trimeric receptor, IL-2Rαβγ such as endothelial cells and T-regulatory cells, respectively. Here, we describe a novel bispecific heavy-chain only antibody which binds to and activates signaling through the heterodimeric IL-2Rβγ receptor complex that is expressed on resting T-cells and NK cells. By avoiding binding to IL-2Rα, this molecule circumvents the preferential T-reg activation of native IL-2, while maintaining the robust stimulatory effects on T-cells and NK-cells in vitro. In vivo studies in both mice and cynomolgus monkeys confirm the molecule’s in vivo biological activity, extended pharmacodynamics due to the Fc portion of the molecule, and enhanced safety profile. Together, these results demonstrate that the bispecific antibody is a safe and effective IL-2R agonist that harnesses the benefits of the IL-2 signaling pathway as a potential anti-cancer therapy.

Role of NKG2D signaling in the cytotoxicity of activated and expanded CD8+ T cells

Blood ◽  
2004 ◽  
Vol 103 (8) ◽  
pp. 3065-3072 ◽  
Author(s):  
Michael R. Verneris ◽  
Mobin Karami ◽  
Jeanette Baker ◽  
Anishka Jayaswal ◽  
Robert S. Negrin
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Interleukin 2 ◽  
Cell Receptor ◽  
Malignant Cell ◽  
Leukemic Cells ◽  
Target Cells ◽  
High Dose ◽  
Effector Cells ◽  
Nkg2d Expression

Abstract Activating and expanding T cells using T-cell receptor (TCR) cross-linking antibodies and interleukin 2 (IL-2) results in potent cytotoxic effector cells capable of recognizing a broad range of malignant cell targets, including autologous leukemic cells. The mechanism of target cell recognition has previously been unknown. Recent studies show that ligation of NKG2D on natural killer (NK) cells directly induces cytotoxicity, whereas on T cells it costimulates TCR signaling. Here we demonstrate that NKG2D expression is up-regulated upon activation and expansion of human CD8+ T cells. Antibody blocking, redirected cytolysis, and small interfering RNA (siRNA) studies using purified CD8+ T cells demonstrate that cytotoxicity against malignant target cells occurs through NKG2D-mediated recognition and signaling and not through the TCR. Activated and expanded CD8+ T cells develop cytotoxicity after 10 to 14 days of culture, coincident with the expression of the adapter protein DAP10. T cells activated and expanded in low (30 U/mL) and high (300 U/mL) concentrations of IL-2 both up-regulated NKG2D expression equally, but only cells cultured in high-dose IL-2 expressed DAP10 and were cytotoxic. Collectively these results establish that NKG2D triggering accounts for the majority of major histocompatibility complex (MHC)–unrestricted cytotoxicity of activated and expanded CD8+ T cells, likely through DAP10-mediated signaling. (Blood. 2004;103: 3065-3072)

scholarly journals CD28 Costimulation Is Required for In Vivo Induction of Peripheral Tolerance in CD8 T Cells

2002 ◽  
Vol 197 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Melanie S. Vacchio ◽  
Richard J. Hodes
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Tolerance Induction ◽  
Peripheral Tolerance ◽  
Cd8 Cells ◽  
Costimulatory Signal ◽  
Cd28 Costimulation

Whereas ligation of CD28 is known to provide a critical costimulatory signal for activation of CD4 T cells, the requirement for CD28 as a costimulatory signal during activation of CD8 cells is less well defined. Even less is known about the involvement of CD28 signals during peripheral tolerance induction in CD8 T cells. In this study, comparison of T cell responses from CD28-deficient and CD28 wild-type H-Y–specific T cell receptor transgenic mice reveals that CD8 cells can proliferate, secrete cytokines, and generate cytotoxic T lymphocytes efficiently in the absence of CD28 costimulation in vitro. Surprisingly, using pregnancy as a model to study the H-Y–specific response of maternal T cells in the presence or absence of CD28 costimulation in vivo, it was found that peripheral tolerance does not occur in CD28KO pregnants in contrast to the partial clonal deletion and hyporesponsiveness of remaining T cells observed in CD28WT pregnants. These data demonstrate for the first time that CD28 is critical for tolerance induction of CD8 T cells, contrasting markedly with CD28 independence of in vitro activation, and suggest that the role of CD28/B7 interactions in peripheral tolerance of CD8 T cells may differ significantly from that of CD4 T cells.

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