ES-62, a glycoprotein secreted by the filarial nematode Acanthocheilonema viteae, exhibits anti-inflamma-tory properties by virtue of covalently attached phosphorylcholine moieties. Screening of a library of ES-62 phosphorylcholine-based small molecule analogues (SMAs) revealed that two compounds, termed 11a and 12b, mirrored the helminth product both in inhibiting mast cell degranulation and cytokine responses in vitro and in preventing ovalbumin-induced Th2-associated airway inflammation and eosin- ophil infiltration of the lungs in mice. Furthermore, the two SMAs inhibited neutrophil infiltration of the lungs when administered therapeutically. ES-62-SMAs 11a and 12b thus represent starting points for novel drug development for allergies such as asthma
Summary ES‐62 is a glycoprotein secreted by the filarial nematode Acanthocheilonema viteae that protects against ovalbumin (OVA)‐induced airway hyper‐responsiveness in mice by virtue of covalently attached anti‐inflammatory phosphorylcholine (PC) residues. We have recently generated a library of small molecule analogues (SMAs) of ES‐62 based around its active PC moiety as a starting point in novel drug development for asthma and identified two compounds – termed 11a and 12b – that mirror ES‐62's protective effects. In this study, we have moved away from OVA, a model allergen, to test the SMAs against two clinically relevant allergens – house dust mite (HDM) and cockroach allergen (CR) extract. We show that both SMAs offer some protection against development of lung allergic responses to CR, in particular reducing eosinophil infiltration, whereas only SMA 12b is effective in protecting against eosinophil‐dependent HDM‐induced allergy. These data therefore suggest that helminth molecule‐induced protection against model allergens may not necessarily translate to clinically relevant allergens. Nevertheless, in this study, we have managed to demonstrate that it is possible to produce synthetic drug‐like molecules based on a parasitic worm product that show therapeutic potential with respect to asthma resulting from known triggers in humans.
Tumor Associated Macrophages (TAMs), characteristic of an M2-like immune-suppressive phenotype, can induce proliferation and survival of tumor cells, facilitate angiogenesis, and suppress anti-tumor immune responses via expression of co-inhibitory molecules (e.g. PD-L1) and cytokines (e.g. IL-10, TGF-β). TAMs are therefore a highly attractive target of innovative cancer immunotherapies. Understanding the ability of pre-clinical candidate compounds to reverse TAM (M2-like)-mediated immune suppression and the potential for reprogramming of M2-like macrophages to a M1-like phenotype is key in the development of effective TAM-targeted cancer immunotherapies. Here we outline development of an assay to assess the capabilities of pre-clinical compounds to reverse M2 macrophage-mediated immune suppression. Monocytes were isolated from whole blood obtained from healthy volunteers and cultured under M2-polarising conditions. The resulting macrophages were phenotypically characterized and then used in co-culture with autologous PBMC, stimulated through T cell receptor ligation. Resulting cytokine production was assessed, alongside CD4+ and CD8+ T cell viability and cell cycle status (flow cytometry). M2-like macrophages polarized with M-CSF displayed an immune suppressive phenotype as shown by their production of IL-10 and their inhibition of IFN-γ production by, and cell cycle status of, T cells in co-culture assays. This suppressive activity was only partially reversed by PD-1-blockade. Modifications to the macrophage polarization protocol were seen to alter the resulting macrophage cell surface phenotype (e.g. expression levels of PD-L1, TIM-3 and CD200R) and their suppressive activity in the co-culture assay. These cell surface phenotypes broadly reflected those seen amongst tumor-derived macrophages from renal and ovarian carcinoma patients (CD14+ CD163+ with expression of TIM-3 and LAG-3). Moreover, compound-mediated changes in functionality seen with macrophages polarized from healthy PBMC monocytes could also be seen using patient-derived material. The assay outlined here therefore provides a M-o-A human in vitro system to test novel compound activity (either singly or in combination) upon TAM-like macrophage generation, phenotype and suppressive function. This allows selection of the most efficacious compounds for further investigation using patient-derived immune cells. Citation Format: Laura E. Gallagher, Andrew Hall, Lauren A. Patience, Lucia Janicova, Stephen Anderton. Interrogation and modulation of the immunosuppressive activity of human TAM-like macrophages using in vitro cultures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3808.
Compensatory release of immunosuppressive cytokines, such as IL-10, by macrophages present in the tumor microenvironment has been implicated as a mechanism for adaptive resistance to a number of immunotherapies. Our drug discovery effort utilises Esterase Motif TechnologyTM (ESMTM) which selectively targets myelomonocytic cells sparing the concomitant lymphocyte anti-tumor immune response. TLR agonists are known to be stimulators of the immune response, a key component of which is the production of myeloid cell IL-12p70. However, their therapeutic potential has been limited by their accompanying induction of IL-10 and other factors. MPL-5821 is an ESMTM p38 MAPK inhibitor which not only inhibits IL-10 but also enhances LPS stimulated IL-12p70 and in contrast to conventional p38 MAPK inhibitors provides enhancement of lymphocyte IFNγ production. The present studies contrast MPL-5821 with multiple non-targeted agents, including inhibitors of HDAC, JAK, PI3K, MEK and CSF-1R, in human PBMC assays. These demonstrated the benefit of ESMTM-targeting as applied to p38 MAP kinase inhibition to not only inhibit TLR agonist induced immunosuppression but also enhance IFNγ due to its sparing of the myeloid-lymphocyte axis. None of the other modalities were able to achieve the effects observed for MPL-5821. Having established the unique ability of MPL-5281 to inhibit LPS induced IL-10 production whilst still maintaining lymphocyte IFNγ production, we extended our studies to human cancer ex vivo models. We chose to evaluate MPL-5821 in combination with TLR agonists in ex vivo assays using tissue and ascites derived from ovarian and cervical cancer patients. A single cell suspension was prepared from cervical cancer tumor draining lymph nodes and incubated with MPL-5821 +/- the TLR 7/8 agonist R848 for 24 and 48 hours. MPL-5821 potently inhibited the R848 induced IL-10 production as measured by Cytometric Bead Array and in contrast to a conventional p38 MAP inhibitor LY2228820 also enhanced IFNγ production. We studied MPL-5821 in cell suspensions prepared from human ovarian tumor and ascites. For the tumor sample, a single cell homogenate was prepared by mechanical and enzymatic digestion and for the ascites the cells were isolated by centrifugation. The cell populations were then analysed by flow cytometry and the cell preparation cultured for 72 hours with anti-CD3 or TLR agonist in the presence of test compound. Cytokine production was measured after 72 hours by ELISA or Luminex bead array. MPL-5821 again showed potent inhibition of TLR agonist induced IL-10 with concomitant enhancement of IFNγ production. We conclude that application of ESMTM technology to macrophage selective delivery of p38 MAPK inhibitors has the potential to inhibit TLR agonist induction of IL-10, which is implicated in limiting the performance of TLR agonists in the clinic. Citation Format: David Moffat, Martin Perry, A. Marijne Heeren, Tanja D. de Gruijl, Justyna Rzepecka, Lucia Janicova, Anastasia Nika, Darryl Turner, Clare Doris, Claire Tebbutt, Kathryn Chapman, Gary Newton, Stephen Anderton. MPL-5821, a macrophage targeted ESMTM p38 MAPK inhibitor, inhibits the production of TLR agonist induced IL-10 whilst sparing T-cell functionality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3877.
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