EXTH-25. ENHANCING GLIOBLASTOMA CELL STRESS RESPONSE TO IMPROVE GAMMA DELTA T-CELL IMMUNOTHERAPY

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi168-vi168
Author(s):  
Amber Jones ◽  
Kate Rochlin ◽  
Lawrence Lamb ◽  
Anita Hjelmeland
Keyword(s):  
T Cells ◽  
T Cell ◽  
Stress Response ◽  
Tumor Cells ◽  
Stress Responses ◽  
Cell Stress ◽  
Immune Recognition ◽  
Gamma Delta ◽  
Gamma Delta T Cell

Abstract Glioblastoma (GBM) is an aggressive cancer that has been largely intractable to novel therapies, however, enhancing the efficacy of immunotherapy could potentially overcome immunosuppression and potentially improve patient outcomes. The cellular stress induced by Temozolomide (TMZ) increases innate immune ligands, which could be exploited to promote immune recognition. TMZ-induced DNA damage can activate the stress response pathway, increasing the expression of NKG2D ligands (NKG2DL) on tumor cells. This leads to an increase in NKG2DL recognition by NKG2D receptors on both natural killer and cytotoxic T-cells to elicit a cytotoxic effect. The lymphodepleting effect of TMZ, however, can limit the ability of these cells to recognize and kill tumor cells. TMZ was shown to induce NKG2DL in gliomas both in vitro and in vivo, providing the basis for clinical trials of TMZ in combination with genetically engineered TMZ-resistant gamma delta T-cells (NCT04165941). To further promote immune recognition, we sought to augment the TMZ-induced stress response by exploring the combination of DNA alkylation with either PARP (Niraparib) or ATM Kinase inhibition (AZD1390). Combinatorial therapy significantly, but heterogeneously, increased differential subsets of NKG2DL genes in comparison to TMZ alone in GBM cells isolated from patient derived xenografts (PDX): 1) MICA and MICB were increased at least 10-fold in D456 (proneural) cells; 2) ULBP1 and ULBP2 were increased at least 2-fold in JX39 (classical) cells; and 3) minimal increases in NKG2DLs were observed in JX22 (mesenchymal) cells. Repression of NKG2DLs by hypoxia/low glucose was also heterogeneous, being observed in two of three GBM models tested. We are currently determining whether these combinatorial treatments improve gamma delta T-cell cytotoxicity against GBM cells and in vivo tumor models. Taken together, our data suggest that enhancing cell stress responses induced by chemotherapies may permit novel immunotherapy therapeutic interventions for brain tumor patients.

scholarly journals 688 In vivo expansion of gamma delta T cells by a CD19-targeted butyrophilin heterodimer leads to elimination of peripheral B cells

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A727-A727
Author(s):  
Suresh De Silva ◽  
George Fromm ◽  
Louis Gonzalez ◽  
Arpita Patel ◽  
Kyung Yoon ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Mechanism Of Action ◽  
Peripheral Blood ◽  
Fusion Proteins ◽  
Gamma Delta T Cells ◽  
Gamma Delta ◽  
Gamma Delta T Cell

BackgroundA primary mechanism of cancer immunotherapy resistance involves downregulation of specific antigens or major histocompatibility complex based antigen presentation, which renders tumor cells invisible to alpha-beta T cells, but not gamma-delta T cells. Recently, a two-step model of gamma-delta T cell activation has emerged, wherein one butyrophilin (BTN, ie. BTN2A1) directly binds the gamma-delta TCR but is only activated if certain molecular patterns (eg. phosphoantigens) facilitate recruitment of a second BTN (ie. BTN3A1) into a complex to form a BTN2A1/3A1 heterodimer. The BTN2A1/3A1 complex specifically activates the predominant gamma-delta T cell population in the peripheral blood, comprising the Vg9d2 T cell receptor (TCR), but does not activate the primary gamma-delta T cell population in mucosal tissues, comprising the Vg4 TCR. The unique mechanism of action and specificity of gamma-delta TCR/BTN interactions suggests that therapeutic proteins comprising specific BTN heterodimers could be used to target specific gamma-delta T cell populations, with a lower risk of off-target activation common with CD3-directed T cell engagers.MethodsHuman BTN2A1/3A1-Fc-CD19scFv and mouse BTNL1/6-Fc-CD19scFv heterodimeric fusion proteins were purified and binding to CD19 or the respective gamma-delta TCRs was assessed by ELISA, Octet and flow cytometry using gd T-cells isolated from human peripheral blood and mouse intestinal tissue. The functionality of the constructs to activate gamma-delta T cells and mediate killing of tumor cells was assessed using live cell imaging in vitro as well as a murine B-cell lymphoma model in vivo.ResultsThe CD19-targeting scFv domains of the BTN heterodimer fusion proteins bound to human and mouse CD19 with low nanomolar affinity. The BTN2A1/3A1-Fc-CD19scFv compound specifically bound to the Vg9d2 TCR on human gd T cells while the mouse BTNL1/6-Fc-CD19scFv bound to Vg7d4 TCR on mouse gd T cells. Both compounds were able to activate gd T cells in a co-culture assay resulting in degranulation and increased surface expression of CD107a and also increased apoptosis of CD19+ tumor cells. Intraperitoneal administration of the mouse BTNL1/6-Fc-CD19scFv led to anti-tumor effects in A20 tumor bearing BALB/c mice. Phenotyping from BTNL1/6-Fc-CD19scFv treated mice revealed profound and rapid expansion of the endogenous gamma-delta T cells in the circulation and tumor, with concomitant depletion of peripheral CD19+ B-cells, confirming the mechanism of action of the heterodimer as a gamma-delta T cell specific engager.ConclusionsThese results provide proof of mechanism for in vivo manipulation of gamma-delta T cells using antigen-targeted butyrophilin heterodimeric fusion proteins for the treatment of cancer.

scholarly journals Genome-wide profiling of druggable active tumor defense mechanisms to enhance cancer immunotherapy

2019 ◽  
Author(s):  
Rigel J. Kishton ◽  
Shashank J. Patel ◽  
Suman K. Vodnala ◽  
Amy E. Decker ◽  
Yogin Patel ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Defense Mechanisms ◽  
Transcriptional Analysis ◽  
Immune Recognition ◽  
Loss Of Function ◽  
Chemokine Production ◽  
Tumor Destruction

SummaryAll current highly effective anti-tumor immunotherapeutics depend on the activity of T cells, but tumor cells can escape immune recognition by several mechanisms including loss of function in antigen presentation and inflammatory response genes, expression of immunomodulatory proteins and an immunosuppressive tumor microenvironment. In contrast, the comprehensive identification of strategies that sensitize tumor cells to immunotherapy in vivo has remained challenging. Here, we combine a two-cell type (2CT) whole-genome CRISPR-Cas9 screen with dynamic transcriptional analysis (DTA) of tumor upon T cell encounter to identify a set of genes that tumor cells express as an active defense against T cell-mediated killing. We then employed small molecule and biologic screens designed to antagonize gene products employed by tumor cells to actively defend against T cell-mediated tumor destruction and found that the inhibition of BIRC2, ITGAV or DNPEP enhanced tumor cell destruction by T cells. Mechanistically, we found that BIRC2 promoted immunotherapy resistance through inhibiting non-canonical NF-κB signaling and limiting inflammatory chemokine production. These findings show the path forward to improving T cell-mediated tumor destruction in the clinic.

scholarly journals Chimeric Antigen Receptor T-Cells for the Treatment of Gamma-Delta T-Cell Malignancies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3338-3338
Author(s):  
Paul Maciocia ◽  
Patrycja Wawrzyniecka ◽  
Leo Kassimatis ◽  
Martin Pule
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Gamma Delta ◽  
Car T Cells ◽  
Car T ◽  
Gamma Delta T Cell

Abstract Introduction Cancers derived from the malignant transformation of gamma delta T-cells are rare but carry very poor prognosis. Hepatosplenic T-cell lymphoma is a highly aggressive condition characterised by hepatosplenic and bone marrow involvement. It has among the worst outcomes of all lymphoma subtypes, with a median survival of only 6-8 months. 95% of cases express the gamma delta T-cell receptor (GDTCR), which is also expressed on a proportion of cases of T-ALL. Treatment for these cancers is based on cytotoxic chemotherapy, with no tumour-specific therapies including immunotherapy available. We have developed a novel chimeric antigen receptor targeting GDTCR and here demonstrate specific in vitro and in vivo efficacy against gamma delta T-cell malignancies. Results We cloned anti-GDTCR antibody as a single chain variable fragment (ScFv), and confirmed specific binding to GDTCR-positive T-cell cell lines and primary GD cells. Next, we cloned anti-GDTCR ScFv into a 2ndgeneration chimeric antigen receptor (CAR) format, including a spacer derived from CD8-stalk, CD28 transmembrane domain and 41BB-zeta endodomain. This construct was stably introduced to primary alpha-beta T-cells by retroviral transduction and surface expression was confirmed by flow cytometry. We established 48-hour co-cultures of anti-GDTCR CAR T-cells or control anti-CD19 CAR T-cells with T-cell lines positive (Loucy, BE13, MOLT-13) or negative for surface GDTCR (Jurkat, SupT1-CD19). While control anti-CD19 CAR killed only SupT1-CD19 cells, specific cytotoxicity was seen by anti-GDTCR CAR T-cells only against GDTCR-positive cell lines. In addition, anti-GDTCR CAR T-cells demonstrated specific secretion of cytokines including interferon gamma and IL-2, and robust antigen-specific proliferation only in co-culture with GDTCR-positive cells. Expression of exhaustion, activation and differentiation markers in long term co-cultures with target cells was similar to that seen with control anti-CD19 CAR. To assess the in vivo potency of anti-GDTCR CAR T-cells, we established a murine model of disseminated GDTCR-positive leukaemia. NSG mice were intravenously injected with 4x10^6 Loucy cells, engineered to stably express Firefly luciferase. Tumour engraftment in bone marrow was confirmed at D7 following injection, and mice were treated with 0.8x10^6 anti-GDTCR or control anti-CD19 CAR T-cells. Disease burden was monitored by bioluminescence imaging. Mice receiving anti-GDTCR CAR demonstrated substantial reduction of tumour burden, increased expansion of CAR T-cells and prolonged survival compared to control-CAR treated animals. Conclusions We have developed a novel chimeric antigen receptor T-cell treatment for gamma-delta TCR-positive malignancies, including hepatosplenic T-cell lymphoma and some cases of T-ALL. Our approach is, to our knowledge, the first immunotherapeutic strategy proposed for these conditions. Given the restricted expression of GD-TCR on a small subset (0.5-5%) of peripheral T-cells and the absence of a clear human phenotype associated with GD T-cell deficiency, we suggest that this therapy may be well tolerated. Given the very poor prognosis and lack of effective therapies for GD-TCR-positive malignancies, as well as the considerable efficacy of CAR T-cell therapy in analogous B-cell disorders, our approach could bring much needed benefit to patients suffering these conditions. Disclosures Maciocia: Autolus: Equity Ownership, Patents & Royalties: UCLB. Pule:UCLB: Patents & Royalties; Autolus: Employment, Equity Ownership.

scholarly journals Structure and specificity of T cell receptor gamma/delta on major histocompatibility complex antigen-specific CD3+, CD4-, CD8- T lymphocytes.

1988 ◽  
Vol 168 (5) ◽  
pp. 1899-1916 ◽  
Author(s):  
J A Bluestone ◽  
R Q Cron ◽  
M Cotterman ◽  
B A Houlden ◽  
L A Matis
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Gene Segment ◽  
Cell Receptor ◽  
Gamma Delta ◽  
Human T Cells ◽  
T Cell Lines ◽  
Gamma Delta T Cell ◽  
Delta Cells

Analyses of TCR-bearing murine and human T cells have defined a unique subpopulation of T cells that express the TCR-gamma/delta proteins. The specificity of TCR-gamma/delta T cells and their role in the immune response have not yet been elucidated. Here we examine alloreactive TCR-gamma/delta T cell lines and clones that recognize MHC-encoded antigens. A BALB/c nu/nu (H-2d)-derived H-2k specific T cell line and derived clones were both cytolytic and released lymphokines after recognition of a non-classical H-2 antigen encoded in the TL region of the MHC. These cells expressed the V gamma 2/C gamma 1 protein in association with a TCR-delta gene product encoded by a Va gene segment rearranged to two D delta and one J delta variable elements. A second MHC-specific B10 nu/nu (H-2b) TCR-gamma/delta T cell line appeared to recognize a classical H-2D-encoded MHC molecule and expressed a distinct V gamma/C gamma 4-encoded protein. These data suggest that many TCR-gamma/delta-expressing T cells may recognize MHC-linked antigens encoded within distinct subregions of the MHC. The role of MHC-specific TCR-gamma/delta cells in immune responses and their immunological significance are discussed.

scholarly journals 121 ICAM-1-specific affinity tuned CAR T cells expressing SSTR2 for real-time imaging

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A130-A130
Author(s):  
Jingmei Hsu ◽  
Eric von Hofe ◽  
Michael Hsu ◽  
Koen Van Besien ◽  
Thomas Fahey ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Phase I ◽  
Tumor Cells ◽  
Somatostatin Receptor ◽  
Laboratory Animals ◽  
Therapeutic Window ◽  
Car T Cells ◽  
Car T

BackgroundThe use of CAR T cells for solid tumors has a number of challenges, such as lack of tumor-specific targets, CAR T cell exhaustion, and the immunosuppressive tumor microenvironment. To address these challenges, AffyImmune has developed technologies to affinity tune and track CAR T cells in patients. The targeting moiety is affinity tuned to preferentially bind to tumor cells overexpressing the target while leaving normal cells with low basal levels untouched, thereby increasing the therapeutic window and allowing for more physiological T cell killing. The CAR T cells are designed to express SSTR2 (somatostatin receptor 2), which allows for the tracking of CAR T cells in vivo via PET/CT scan using FDA-approved DOTATATE.MethodsAIC100 was generated by affinity tuning the I-domain of LFA-1, the physiological ligand to ICAM-1. Various mutants with 106-fold difference in affinity were evaluated for affinity. This allowed structure activity relationships to be conducted using CAR T cells expressing the various affinity mutants against targets with varying antigen densities. The variant with micromolar affinity was clearly the most effective in non-clinical animal models. AIC100 is currently being evaluated to assess safety, CAR T expansion, tumor localization, and preliminary activity in patients with advanced thyroid cancer in a phase I study (NCT04420754). Our study uses a modified toxicity probability interval design with three dosage groups of 10 x 106, 100 x 106, and 500 x 106 cells.ResultsPreclinical studies demonstrated greater in vivo anti-tumor activity and safety with lower affinity CAR T cells. A single dose of AIC100 resulted in tumor elimination and significantly improved survival of animals. AIC100 activity was confirmed in other high ICAM-1 tumor models including breast, gastric, and multiple myeloma. In a Phase I patient given 10-million CAR T cells, near synchronous imaging of FDG and DOTATATE revealed preliminary evidence of transient CAR T expansion and tumor reduction at multiple tumor lesions, with the peak of CAR T density coinciding with the spike in CAR T numbers in blood.ConclusionsWe have developed affinity tuned CAR T cells designed to selectively target ICAM-1 overexpressing tumor cells and to spatiotemporally image CAR T cells. Near-synchronous FDG and DOTATATE scans will enhance patient safety by early detection of off-tumor CAR T activity and validation of tumor response. We anticipate that our ‘tune and track’ technology will be widely applicable to developing potent yet safe CAR T cells against hard-to-treat solid cancers.Trial RegistrationNCT04420754Ethics ApprovalIRB number19-12021154IACUC (animal welfare): All animal experiments were performed in accordance with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals. Animal handling protocols were approved by the Institutional Laboratory Animal Use and Care Committee of Weill Cornell Medicine (Permit Number: 2012–0063).

scholarly journals A delta T-cell receptor deleting element transgenic reporter construct is rearranged in alpha beta but not gamma delta T-cell lineages.

1995 ◽  
Vol 15 (12) ◽  
pp. 7022-7031 ◽  
Author(s):  
J Shutter ◽  
J A Cain ◽  
S Ledbetter ◽  
M D Rogers ◽  
R D Hockett
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Receptor ◽  
Chain Gene ◽  
Gamma Delta ◽  
Alpha Chain ◽  
Chain Locus ◽  
Discrete Population ◽  
Alpha Beta ◽  
Gamma Delta T Cell

T cells can be divided into two groups on the basis of the expression of either alpha beta or gamma delta T-cell receptors (TCRs). Because the TCR delta chain locus lies within the larger TCR alpha chain locus, control of the utilization of these two receptors is important in T-cell development, specifically for determination of T-cell type: rearrangement of the alpha locus results in deletion of the delta coding segments and commitment to the alpha beta lineage. In the developing thymus, a relative site-specific recombination occurs by which the TCR delta chain gene segments are deleted. This deletion removes all D delta, J delta, and C delta genes and occurs on both alleles. This delta deletional mechanism is evolutionarily conserved between mice and humans. Transgenic mice which contain the human delta deleting elements and as much internal TCR delta chain coding sequence as possible without allowing the formation of a complete delta chain gene were developed. Several transgenic lines showing recombinations between deleting elements within the transgene were developed. These lines demonstrate that utilization of the delta deleting elements occurs in alpha beta T cells of the spleen and thymus. These recombinations are rare in the gamma delta population, indicating that the machinery for utilization of delta deleting elements is functional in alpha beta T cells but absent in gamma delta T cells. Furthermore, a discrete population of early thymocytes containing delta deleting element recombinations but not V alpha-to-J alpha rearrangements has been identified. These data are consistent with a model in which delta deletion contributes to the implementation of a signal by which the TCR alpha chain locus is rearranged and expressed and thus becomes an alpha beta T cell.

scholarly journals T cell-intrinsic role of IL-6 signaling in primary and memory responses

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Simone A Nish ◽  
Dominik Schenten ◽  
F Thomas Wunderlich ◽  
Scott D Pope ◽  
Yan Gao ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Suppressive Effect ◽  
Immune Recognition ◽  
Th17 Response ◽  
Cell Responses ◽  
Underlying Mechanisms ◽  
Specific Deletion

Innate immune recognition is critical for the induction of adaptive immune responses; however the underlying mechanisms remain incompletely understood. In this study, we demonstrate that T cell-specific deletion of the IL-6 receptor α chain (IL-6Rα) results in impaired Th1 and Th17 T cell responses in vivo, and a defect in Tfh function. Depletion of Tregs in these mice rescued the Th1 but not the Th17 response. Our data suggest that IL-6 signaling in effector T cells is required to overcome Treg-mediated suppression in vivo. We show that IL-6 cooperates with IL-1β to block the suppressive effect of Tregs on CD4+ T cells, at least in part by controlling their responsiveness to IL-2. In addition, although IL-6Rα-deficient T cells mount normal primary Th1 responses in the absence of Tregs, they fail to mature into functional memory cells, demonstrating a key role for IL-6 in CD4+ T cell memory formation.

The Plant-Derived Agent Silvestrol Has B-Cell Selective Activity In Vitro in Chronic Lymphocytic Leukemia Patient Cells and In Vivo in the Tcl-1 Mouse Model of CLL.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3123-3123 ◽  
Author(s):  
David M. Lucas ◽  
Ryan B. Edwards ◽  
Michael D. De Lay ◽  
Derek A. West ◽  
Gerard Lozanski ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Tumor Cells ◽  
Lymphocytic Leukemia ◽  
Pathological Examination ◽  
Cell Percentage ◽  
Leukocyte Counts

Abstract Chronic Lymphocytic Leukemia (CLL) is an incurable disease with limited therapeutic options, especially for high-risk populations such as the del(17p13) patient subset. Currently available therapies for CLL, even if effective, can have significant detrimental effects on remaining T cells, leaving patients at risk of potentially lethal opportunistic infections. New agents with unique mechanisms of action, independence of key resistance pathways, and selectivity for tumor cells are crucial to make an impact on patient survival. Silvestrol, a structurally unique compound isolated from the plant genus Aglaia, exhibited potent activity against several tumor cell lines and moderate in vivo activity in the P388 mouse leukemia model (J. Org. Chem. 2004, 69:3350; ibid. 69:6156). Based on these results, we tested silvestrol against tumor cells obtained from CLL patients. The LC50 (concentration lethal to 50% of cells relative to untreated control) of silvestrol was 6.5 nM at 72 hours by MTT assay. We performed assays to determine CLL patient cell viability at 72 hours with or without drug washout at various times. In these studies, silvestrol showed up to 50% killing at 72 hours with only a four hour exposure, and reached maximum efficacy with a 24 hour exposure. Silvestrol was similarly effective against cells from CLL patients with or without del(17p13). Furthermore, there was no significant difference in silvestrol-mediated cytotoxicity between lymphoblastic cells with a ten-fold overexpression of Bcl-2 relative to control cells. In MTT assays using isolated CD3+ or CD19+ cells, and in whole blood from healthy volunteers and CLL patients, silvestrol demonstrated substantially more cytotoxicity toward B cells than T cells. We then tested silvestrol using Tcl-1 transgenic mice, which are initially normal but develop a slow-progressing B cell leukemia very similar to human CLL. Lymphocytes obtained from spleens of Tcl-1 mice with leukemia were incubated ex vivo with 80 nM silvestrol and analyzed by flow cytometry. Silvestrol produced an 88% reduction in the B cell percentage after 24 hours with no negative effect on the T cell percentage (8% increase), in contrast to 1 μM fludarabine, which affected both B cell (22% reduction) and T cell (14% reduction) subsets. Non-leukemic mice of the Tcl-1 background strain were treated with 1.0, 1.5 and 2.5 mg/kg/day silvestrol for 5 days to determine a tolerable dose. Three of five mice treated with 2.5 mg/kg/day died at the beginning of the second week of treatment. However, none of the animals treated at 1.0 or 1.5 mg/kg showed signs of toxicity or weight loss even after two full weeks of treatment and were normal at pathological examination. Tcl-1 mice with evidence of leukemia as determined by elevated leukocyte counts and enlarged spleens were then treated with silvestrol at 1.5 mg/kg/day × 5 days for two weeks. Treated mice experienced decreased overall leukocyte counts relative to vehicle controls. Furthermore, CD19+ cell numbers and percentages diminished substantially while the T cells were only mildly affected. Additional leukemic Tcl-1 mice are currently being treated and studies are underway examining the mechanism of action of silvestrol in CLL cells.

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