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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Appending appropriate esters to anti-inflammatory compounds leads to monocyte/macrophage selectivity. This has been demonstrated in p38 alpha MAP kinase in which we observe differential inhibition of phosphorylation of the substrate MAPKAPK2 in stimulated cells. This selectivity is due to differential expression of carboxylesterases within the cells, which cause the active form of the compounds to accumulate with the cells.

The therapeutic and toxic effects of drugs are often generated through effects on distinct cell types in the body. Selective delivery of drugs to specific cells or cell lineages would, therefore, have major advantages, in particular, the potential to significantly improve the therapeutic window of an agent. Cells of the monocyte-macrophage lineage represent an important target for many therapeutic agents because of their central involvement in a wide range of diseases including inflammation, cancer, atherosclerosis, and diabetes. We have developed a versatile chemistry platform that is designed to enhance the potency and delivery of small-molecule drugs to intracellular molecular targets. One facet of the technology involves the selective delivery of drugs to cells of the monocyte-macrophage lineage, using the intracellular carboxylesterase, human carboxylesterase-1 (hCE-1), which is expressed predominantly in these cells. Here, we demonstrate selective delivery of many types of intracellularly targeted small molecules to monocytes and macrophages by attaching a small esterase-sensitive chemical motif (ESM) that is selectively hydrolyzed within these cells to a charged, pharmacologically active drug. ESM versions of histone deacetylase (HDAC) inhibitors, for example, are extremely potent anticytokine and antiarthritic agents with a wider therapeutic window than conventional HDAC inhibitors. In human blood, effects on monocytes (hCE-1-positive) are seen at concentrations 1000-fold lower than those that affect other cell types (hCE-1-negative). Chemical conjugates of this type, by limiting effects on other cells, could find widespread applicability in the treatment of human diseases where monocyte-macrophages play a key role in disease pathology.

Inhibition of histone deacetylase activity represents a promising new modality in the treatment of a number of cancers. A novel HDAC series demonstrating inhibitory activity in cell proliferation assays is described. Optimisation based on the introduction of basic amine linkers to effect good drug distribution to tumour led to the identification of a compound with oral activity in a human colon cancer xenograft study associated with increased histone H3 acetylation in tumour tissue.

A novel series of HDAC inhibitors demonstrating class I subtype selectivity and good oral bioavailability is described. The compounds are potent enzyme inhibitors (IC50 values less than 100 nM), and improved activity in cell proliferation assays was achieved by modulation of polar surface area (PSA) through the introduction of novel linking groups. Employing oral pharmacokinetic studies in mice, comparing drug levels in spleen to plasma, we selected compounds that were tested for efficacy in human tumor xenograft studies based on their potential to distribute into tumor. One compound, 21r (CHR-3996), showed good oral activity in these models, including dose-related activity in a LoVo xenograft. In addition 21r showed good activity in combination with other anticancer agents in in vitro studies. On the basis of these results, 21r was nominated for clinical development.