705 Anti-tumor activity of CB-668, a potent, selective and orally bioavailable small-molecule inhibitor of the immuno-suppressive enzyme Interleukin 4 (IL-4)-Induced Gene 1 (IL4I1)

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A747-A747
Author(s):  
Andrew MacKinnon ◽  
Deepthi Bhupathi ◽  
Jason Chen ◽  
Tony Huang ◽  
Weiqun Li ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Tumor Microenvironment ◽  
Immune Cell ◽  
Interleukin 4 ◽  
Mouse Tumor ◽  
Syngeneic Mouse ◽  
Molecule Inhibitor ◽  
Anti Tumor Activity ◽  
Mouse Tumor Models

BackgroundTumors evade destruction by the immune system through multiple mechanisms including altering metabolism in the tumor microenvironment. Metabolic control of immune responses occurs through depletion of essential nutrients or accumulation of toxic metabolites that impair immune cell function and promote tumor growth. The secreted enzyme interleukin 4 (IL-4)-induced gene 1 (IL4I1) is an L-phenylalanine oxidase that catabolizes phenylalanine and produces phenyl-pyruvate and hydrogen peroxide. IL4I1 regulates several aspects of adaptive immunity in mice, including inhibition of cytotoxic T cells through its production of hydrogen peroxide (reviewed in1). In human tumors, IL4I1 expression is significantly elevated relative to normal tissues and is notably high in ovarian tumors and B cell lymphomas. Motivated by the hypothesis that IL4I1 is an immuno-metabolic enzyme that suppresses anti-tumor immunity, we discovered CB-668, the first known small-molecule inhibitor of IL4I1.MethodsIL4I1 enzymatic activity was measured using an HRP-coupled enzyme assay. RNA in-situ hybridization was carried out on the RNAScope platform. Syngeneic mouse tumor models were used to evaluate the anti-tumor activity of CB-668. The level of phenyl-pyruvate in tumor homogenates was measured by LC/MS.ResultsOur clinical candidate, CB-668 is a potent and selective non-competitive inhibitor of IL4I1 (IC50 = 15 nM). CB-668 has favorable in vitro ADME properties and showed low clearance and high oral bioavailability in rodents. Twice-daily oral administration of CB-668 was well-tolerated in mice and resulted in single-agent anti-tumor activity in the syngeneic mouse tumor models B16-F10, A20, and EG7. Oral CB-668 administration reduced the levels of phenyl-pyruvate in the tumor, consistent with inhibition of IL4I1 enzymatic activity. Anti-tumor activity of CB-668 was immune cell-mediated since efficacy was abrogated in CD8-depleted mice, and CB-668 treatment caused increased expression of pro-inflammatory immune genes in the tumor. Moreover, CB-668 had no direct anti-proliferative activity on tumor cells grown in vitro (IC50 > 50 µM). CB-668 also favorably combined with anti-PD-L1 therapy to reduce tumor growth in the B16-F10 tumor model.ConclusionsThese data support an immune-mediated anti-tumor effect of IL4I1 inhibition by CB-668, and suggest inhibition of IL4I1 represents a novel strategy for cancer immuno-therapy.ReferencesMolinier-Frenkel V, Prévost-Blondel A, and Castellano F. The IL4I1 Enzyme: A New Player in the Immunosuppressive Tumor Microenvironment. Cells 2019;8:1–9.

scholarly journals 859 Tuning the tumor microenvironment by reprogramming TREM1+ myeloid cells to unleash anti-tumor immunity in solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A900-A900
Author(s):  
Nadine Jahchan ◽  
Hanna Ramoth ◽  
Vladi Juric ◽  
Erin Mayes ◽  
Shilpa Mankikar ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Tumor Microenvironment ◽  
Tumor Immunity ◽  
Single Agent ◽  
Myeloid Cells ◽  
Mouse Tumor ◽  
Syngeneic Mouse ◽  
Tumor Tissues ◽  
Mouse Tumor Models

BackgroundThe tumor microenvironment (TME) often contains high levels of suppressive myeloid cells that contribute to innate checkpoint inhibitor (CPI) resistance. Pionyr’s Myeloid Tuning approach involves altering the composition and/or the function of myeloid cells in the TME. Myeloid reprogramming alters the function of immunosuppressive myeloid cells to acquire an immunostimulatory phenotype. Triggering receptor expressed on myeloid cells-1 (TREM1) is an immunoglobulin superfamily cell surface receptor enriched on tumor-associated myeloid cells. To investigate the potential of TREM1 modulation as an anti-cancer therapeutic strategy, Pionyr developed an afucosylated humanized anti-TREM1 monoclonal antibody termed PY159 and characterized it in pre-clinical and translational biomarker assays described below.MethodsPY159 responses in human whole blood and dissociated primary tumor cells in vitro were evaluated by flow cytometry and measurement of secreted cytokines and chemokines by MSD. TREM1 expression in human tumors was validated by scRNAseq, flow cytometry, and immunohistochemistry (IHC). In vivo efficacy and pharmacodynamic studies of a surrogate anti-mouse TREM1 antibody, termed PY159m, were evaluated using syngeneic mouse tumor models, either as a single-agent or in combination with anti-PD-1. To select tumor types and patients most likely to benefit from PY159 therapy, Pionyr developed qualitative and quantitative monoplex and multiplex IHC assays that detect TREM1 expression levels in human tumor tissues.ResultsPY159 treatment in vitro induced signaling, upregulated monocyte activation markers, and induced proinflammatory cytokines. In human tumors, TREM1 was detected on tumor-associated neutrophils, tumor-associated macrophages, and monocytic myeloid-derived suppressive cells. The surrogate PY159m anti-mouse TREM1 antibody exhibited anti-tumor efficacy in several syngeneic mouse tumor models, both as single-agent and in combination with anti-PD-1. Screening for TREM1 expression in tumor tissues demonstrated that TREM1+ tumor associated myeloid cells were highly enriched in the TME of multiple solid tumor indications. The monoplex and multiplex IHC assays offered insights into the localization of TREM1+ myeloid cells and their spatial relationship with other immune cells present in the TME to determine what immune composition will be more favorable for response to PY159 therapy.ConclusionsCollectively, the available nonclinical data support PY159 as a TREM1 agonist that reprograms myeloid cells and unleashes anti-tumor immunity. PY159 safety and efficacy are currently being evaluated in first-in-human clinical trial (NCT04682431) involving select advanced solid tumors patients resistant and refractory to standard of care therapies alone and in combination with a CPI. The TREM1 IHC assay is successfully being used on FFPE archival tumor tissues from enrolled patients to determine TREM1 expression levels.

scholarly journals 778 Modulating tumor microenvironment with arginase-1 specific T cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A813-A813
Author(s):  
Evelina Martinenaite ◽  
Mia Aaboe Jørgensen ◽  
Rasmus Erik Johansson Mortensen ◽  
Shamaila Munir Ahmad ◽  
Stine Emilie Weis-Banke ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Memory T Cells ◽  
Ethics Committee ◽  
Mouse Tumor ◽  
Immune Modulators ◽  
Arginase 1 ◽  
Mouse Tumor Models

BackgroundIO112 is an immune modulatory cancer therapy under preclinical development to target arginase-1-expressing tumor cells and immune inhibitory myeloid cells, such as myeloid derived suppressor cells (MDSCs), and tumor associated macrophages (TAMs). Arginase-1 acts as a metabolic immune regulator at the tumor site by reducing availability of L-arginine to the infiltrating immune cells thus reducing T cell functionality and proliferation. Previously, we demonstrated that IO112 triggers activation of spontaneous CD4+ and CD8+ T-cell responses against arginase-1, found in both cancer patients and healthy individuals.1 These T cells are present in the memory T cell compartment, and are activated in arginase-1 inducing conditions, such as presence of TH2 cytokines IL-4 or IL-13 in vitro.2 3 In this study we aimed to explore the role of arginase-1-specific T cells as immune modulators in immune homeostasis and tumor microenvironment for the development of IO112 immunomodulatory therapy.MethodsHuman arginase-1-specific T cells were isolated and expanded for functional characterization of reactivity against arginase-1 expressing target cells as well as subsequent phenotyping of the targeted arginase-1 positive cells. Syngeneic C57BL/6 mouse tumor models were used to assess the therapeutic efficacy of IO112.ResultsWe show that arginase-1-specific memory T cells specifically recognize arginase-1 expressing cells, such as mRNA transfected autologous dendritic cells (DCs) and B cells as well as M2 polarized macrophages in vitro. In addition, activated arginase-1-specific T cells produce pro-inflammatory cytokines IFNγ and TNFα. Secretion of TH1 cytokines by these T cells suggests that they may act as potent immune modulators in the tumor microenvironment, since many arginase-1 expressing myeloid cells are not terminally differentiated and they can be re-polarized to an immunostimulatory, M1-like phenotype. We also observed that targeting of M2-polarized arginase-1 expressing monocytic leukemia cell line THP-1 with arginase-1-specific CD4+ T cells induces upregulation of PD-L1 on the THP-1 cells. Furthermore, we demonstrate anti-tumor activity of IO112 in syngeneic mouse tumor models (B16 and MC38), both as monotherapy and in combination with anti-PD-1 treatment. The therapeutic effect was associated with increased immune infiltration in the IO112-treated mice compared to the control.ConclusionsWe demonstrate that arginase-1 specific T cells can influence the polarization of arginase-1-expressing immune cells. Our study provides evidence that IO112 immune therapy against arginase-1 is an attractive way of modulating the immune suppressive tumor microenvironment for therapeutic benefit. With this rationale, we are currently undertaking Investigational New Drug (IND) application enabling studies to explore this approach in a clinical setting.ReferencesMartinenaite E, Mortensen REJ, Hansen M, Holmström MO, Ahmad SM, Jørgensen NGD, Met Ö, Donia M, Svane IM, Andersen MH. Frequent adaptive immune responses against arginase-1. Oncoimmunology 2018;7(3):e1404215.Martinenaite E, Ahmad SM, Svane IM, Andersen MH. Peripheral memory T cells specific for Arginase-1. Cell Mol Immunol 2019;16(8):718–719.Martinenaite E, Ahmad SM, Bendtsen SK, Jørgensen MA, Weis-Banke SE, Svane IM, Andersen MH. Arginase-1-based vaccination against the tumor microenvironment: the identification of an optimal T-cell epitope. Cancer Immunol Immunother 2019;68(11):1901–1907.Ethics ApprovalThis study was approved by the Scientific Ethics Committee for The Capital Region of Denmark and Danish Ethics Committee on experimental animal welfare.

scholarly journals 737 Inhibition of P21-activated kinase 4 (PAK4) reverts immune exclusion and restores anti-tumor immunity in the tumor microenvironment

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A768-A768
Author(s):  
Yu 'Jerry' Zhou ◽  
Gina Chu ◽  
Eleanore Hendrickson ◽  
Johnni Gullo-Brown ◽  
Brandy Chavez ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Microenvironment ◽  
Tumor Cells ◽  
Immune Cell ◽  
Mouse Tumor ◽  
Type I ◽  
List Type ◽  
P21 Activated Kinase ◽  
Immune Exclusion

BackgroundP21-activated kinase 4 (PAK4) is a serine/threonine protein kinase that is mostly expressed in tumor and stroma cells. PAK4 activates tumor WNT/β-catenin pathway and regulates cellular morphology, motility, EMT, cell proliferation and survival. Recent studies also showed that PAK4 can actively exclude T cells from tumors, suggesting that therapeutic inhibition of PAK4 can increase T cell infiltration in tumor microenvironment and overcome resistance to checkpoint inhibitor immunotherapy.1MethodsWe generated PAK4 knockout (KO) clones in human and mouse tumor cells to validate its biology in vitro and in vivo. We also performed pharmacological evaluation of PAK4 inhibition using Pfizer compounds (referred to as 'PAK4i compounds' below) for their potential tumor-intrinsic and immune-regulatory roles.ResultsNanostring, qPCR and RNASeq analysis showed that PAK4 depletion led to increase of cytokine expression in tumor, including conventional dendritic cell (cDC)- recruiting chemokine CCL4, and type I IFN / ISG pathway genes that are associated with MHC upregulation such as CXCL10. In addition, PAK4 KO sensitizes B16F10 tumors to anti-PD-1 treatment and increases infiltration of cDC and T cells in the tumor microenvironment.We also showed that small molecule PAK4i compounds induced more potent cancer cell growth inhibition over treated normal PBMCs. PAK4i compounds also increased immune-activating and decreased immune exclusion genes in B16F10 cells and tumor explants in vitro. Although PAK4 target engagement is demonstrated by CETSA assay, the compound potency on modulating PAK4 downstream Wnt/ β-catenin pathway is low, suggesting that the aforementioned phenotypic changes induced by PAK4i compounds may be partially attributed to other off-target effects.ConclusionsCollectively, our data suggests that genetic depletion or pharmacological inhibition of PAK4 may induce immune-activating cytokine production in tumor cells, revert immune cell exclusion in tumor microenvironment, and synergize with checkpoint blockade therapies. However, further optimization on these PAK4i compounds is needed to improve its specificity on modulating PAK4 enzyme activities.ReferenceAbril-Rodriguez G, Torrejon DY, Liu W, Zaretsky JM, Nowicki TS, Tsoi J, Puig-Saus C, Baselga-Carretero I, Medina E, Quist MJ, Garcia AJ, Senapedis W, Baloglu E, Kalbasi A, Cheung-Lau G, Berent-Maoz B, Comin-Anduix B, Hu-Lieskovan S, CWang CY, Grasso CS & Ribas A. PAK4 inhibition improves PD-1 blockade immunotherapy. Nat Cancer 2020;1:46–58.Ethics ApprovalAll animal studies were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of Pfizer. Approved protocol # LAJ-2019-01347

591 Comparison of two oHSV Vectors for the treatment of glioblastoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A626-A626
Author(s):  
Joseph Jackson ◽  
Bonnie Hall ◽  
Lisa Bailey ◽  
E Chiocca ◽  
Justus Cohen
Keyword(s):  
Tumor Microenvironment ◽  
Immune Cell ◽  
Polymorphonuclear Cells ◽  
List Type ◽  
Cell Composition ◽  
Animal Survival ◽  
Strain Derivative ◽  
Anti Tumor Activity

BackgroundGlioblastoma multiforme (GBM) is the most common human brain cancer. Despite a well-established standard of care, the 5-year mortality rate of GBM patients is 95%, highlighting the need for innovative therapeutic interventions. A variety of oncolytic viruses, including those derived from herpes simplex virus (oHSV), have been designed for GBM therapy, but early-phase clinical trials have reported few complete responses and no evidence of durable anti-tumor immunity. Potential reasons for the lack of efficacy are limited vector potency (i.e., virulence) and the presence of a highly immunosuppressive tumor microenvironment (TME) comprised of few activated lymphocytes, large numbers of immunosuppressive myeloid cells (macrophages, myeloid derived suppressor cells [MDSCs], microglia), and an agglomerate of immunosuppressive cytokines (IL-10, VEGF, MIF, etc.).1 Herein we explore these obstacles by comparing the anti-tumor activity two different oHSV designs, an HSV-1 KOS strain derivative designated KG4:T124, and an F strain derivative designated rQNestin34.5v.1 (a similar oHSV, rQNestin34.5v.2, is currently in a phase I clinical trial for GBM).2MethodsUsing the murine syngeneic GBM models, GL261N4 and CT2A, we compared the anti-tumor activity of KG4:T124 and rQNestin34.5v1. In vitro, we evaluated the viral entry, replication capacity, and cytotoxicity of both oHSVs. In vivo, we measured the impact of both vectors on tumor progression, TME immune cell composition, and animal survival.ResultsVirus entry into cancer cells of KG4:T124 or rQNestin34.5v1 was relatively similar, but rQNestin34.5v.1 replicated more effectively and generally induced greater viral mediated cytotoxicity. In syngeneic mice, rQNestin34.5v.1 reduced orthotopic GL261N4 tumor burden and enhanced animal survival compared to KG4:T124. However, preliminary data indicate that multiple injections of KG4:T124 but not rQNestin34.5 enhance GL261N4 survival outcome. Neither oHSV impacted survival outcomes in the more pernicious CT2A model. Analysis, of either the GL261N4 or CT2A TME two days post virus administration revealed that both viruses had reduced microglia cell frequency, induced the influx of tumor associated macrophages and polymorphonuclear cells, but did not alter the frequency of monocytic MDSCs, natural killer cells, CD8+ or CD4+ T-cells.ConclusionsrQNestin34.5 had greater oncolytic activity In vivo and in vitro, but did not benefit from multiple oHSV injections. Both viruses induced similar changes in the TME immune cell composition. However, the presence of vital adaptive immune cell types within the TME was not observed at 2 days post oHSV treatment.AcknowledgementsResearch reported in this abstract was supported by the National Institutes of Health grants P01CA163205 (EAC & JCG) and 5T32CA082084-19 (JWJ).ReferencesMuller S, Kohanbash G, Liu SJ, Alvarado B, Carrera D, Bhaduri A, Watchmaker PB, Yagnik G, Di Lullo E, Malatesta M, Amankulor NM, Kriegstein AR, Lim DA, Aghi M, Okada H, Diaz A. Single-cell profiling of human gliomas reveals macrophage ontogeny as a basis for regional differences in macrophage activation in the tumor microenvironment. Genome Biol. 2017;18(1):234. Epub 2017/12/22. doi: 10.1186/s13059-017-1362-4. PubMed PMID: 29262845; PMCID: PMC5738907.Chiocca EA, Nakashima H, Kasai K, Fernandez SA, Oglesbee M. Preclinical Toxicology of rQNestin34.5v.2: An Oncolytic Herpes Virus with Transcriptional Regulation of the ICP34.5 Neurovirulence Gene. Mol Ther Methods Clin Dev 2020;17:871–93. Epub 2020/05/07. doi: 10.1016/j.omtm.2020.03.028. PubMed PMID: 32373649; PMCID: PMC7195500.

scholarly journals In Vitro Evaluation of CD276-CAR NK-92 Functionality, Migration and Invasion Potential in the Presence of Immune Inhibitory Factors of the Tumor Microenvironment

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1020
Author(s):  
Stefan Grote ◽  
Guillermo Ureña-Bailén ◽  
Kenneth Chun-Ho Chan ◽  
Caroline Baden ◽  
Markus Mezger ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Solid Tumors ◽  
Immune Cells ◽  
Immune Cell ◽  
Nk Cell ◽  
Migration And Invasion ◽  
Cellular Immunotherapy ◽  
Knock Out ◽  
Immunosuppressive Tumor Microenvironment

Background: Melanoma is the most lethal of all skin-related cancers with incidences continuously rising. Novel therapeutic approaches are urgently needed, especially for the treatment of metastasizing or therapy-resistant melanoma. CAR-modified immune cells have shown excellent results in treating hematological malignancies and might represent a new treatment strategy for refractory melanoma. However, solid tumors pose some obstacles for cellular immunotherapy, including the identification of tumor-specific target antigens, insufficient homing and infiltration of immune cells as well as immune cell dysfunction in the immunosuppressive tumor microenvironment (TME). Methods: In order to investigate whether CAR NK cell-based immunotherapy can overcome the obstacles posed by the TME in melanoma, we generated CAR NK-92 cells targeting CD276 (B7-H3) which is abundantly expressed in solid tumors, including melanoma, and tested their effectivity in vitro in the presence of low pH, hypoxia and other known factors of the TME influencing anti-tumor responses. Moreover, the CRISPR/Cas9-induced disruption of the inhibitory receptor NKG2A was assessed for its potential enhancement of NK-92-mediated anti-tumor activity. Results: CD276-CAR NK-92 cells induced specific cytolysis of melanoma cell lines while being able to overcome a variety of the immunosuppressive effects normally exerted by the TME. NKG2A knock-out did not further improve CAR NK-92 cell-mediated cytotoxicity. Conclusions: The strong cytotoxic effect of a CD276-specific CAR in combination with an “off-the-shelf” NK-92 cell line not being impaired by some of the most prominent negative factors of the TME make CD276-CAR NK-92 cells a promising cellular product for the treatment of melanoma and beyond.

scholarly journals A Bioreactor Model of Mouse Tumor Progression

2007 ◽  
Vol 2007 ◽  
pp. 1-9 ◽  
Author(s):  
George A. Thouas ◽  
John Sheridan ◽  
Kerry Hourigan
Keyword(s):  
Tumor Progression ◽  
Cell Line ◽  
Interleukin 4 ◽  
Mouse Tumor ◽  
Mechanical Stimuli ◽  
Rotating Disc ◽  
Suitable Alternative ◽  
Stirred Bioreactor ◽  
Cell Biomass

The present study represents an investigation of a novel stirred bioreactor for culture of a transformed cell line under defined hydrodynamic conditions in vitro. Cell colonies of the EL-4 mouse lymphoma cell line grown for the first time in a rotating disc bioreactor (RDB), were observed to undergo changes in phenotype in comparison to standard, static flask cultures. RDB cultures, with or without agitation, promoted the formation of adherent EL-4 cell plaques that merged to form contiguous tumor-like masses in longer-term cultures, unlike the unattached spheroid aggregates of flask cultures. Plaques grown under agitated conditions were further altered in morphology and distribution in direct response to fluid mechanical stimuli. Plaque colonies growth in RDBs with or without agitation also exhibited significant increases in production of interleukin-4 (IL-4) and lactate, suggesting an inducible “Warburg effect.” Increases in cell biomass in RDB cultures were no different to flask cultures, though a trend toward a marginal increase was observed at specific rotational speeds. The RDB may therefore be a suitable alternative method to study mechanisms of tumor progression and invasiveness in vitro, under more complex physicochemical conditions that may approximate natural tissue environments.

scholarly journals The Immune Endocannabinoid System of the Tumor Microenvironment

2020 ◽  
Vol 21 (23) ◽  
pp. 8929
Author(s):  
Melanie Kienzl ◽  
Julia Kargl ◽  
Rudolf Schicho
Keyword(s):  
Tumor Microenvironment ◽  
Tumor Progression ◽  
Immune Cells ◽  
Cannabinoid Receptors ◽  
Immune Cell ◽  
Tumor Development ◽  
Endocannabinoid System ◽  
Cell Functions ◽  
In Vitro Effects

Leukocytes are part of the tumor microenvironment (TME) and are critical determinants of tumor progression. Because of the immunoregulatory properties of cannabinoids, the endocannabinoid system (ECS) may have an important role in shaping the TME. Members of the ECS, an entity that consists of cannabinoid receptors, endocannabinoids and their synthesizing/degrading enzymes, have been associated with both tumor growth and rejection. Immune cells express cannabinoid receptors and produce endocannabinoids, thereby forming an “immune endocannabinoid system”. Although in vitro effects of exogenous cannabinoids on immune cells are well described, the role of the ECS in the TME, and hence in tumor development and immunotherapy, is still elusive. This review/opinion discusses the possibility that the “immune endocannabinoid system” can fundamentally influence tumor progression. The widespread influence of cannabinoids on immune cell functions makes the members of the ECS an interesting target that could support immunotherapy.

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