Coordinated Activation of Toll-Like Receptor8 (TLR8) and NLRP3 by the TLR8 Agonist, VTX-2337, Ignites Tumoricidal Natural Killer Cell Activity

PLoS One. 2016 Feb 29;11(2):e0148764. doi: 10.1371/journal.pone.0148764. eCollection 2016.

Abstract

VTX-2337 (USAN: motolimod) is a selective toll-like receptor 8 (TLR8) agonist, which is in clinical development as an immunotherapy for multiple oncology indications, including squamous cell carcinoma of the head and neck (SCCHN). Activation of TLR8 enhances natural killer cell activation, increases antibody-dependent cell-mediated cytotoxicity, and induces Th1 polarizing cytokines. Here, we show that VTX-2337 stimulates the release of mature IL-1β and IL-18 from monocytic cells through coordinated actions on both TLR8 and the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome complex. In vitro, VTX-2337 primed monocytic cells to produce pro-IL-1β, pro-IL-18, and caspase-1, and also activated the NLRP3 inflammasome, thereby mediating the release of mature IL-1β family cytokines. Inhibition of caspase-1 blocked VTX-2337-mediated NLRP3 inflammasome activation, but had little impact on production of other TLR8-induced mediators such as TNFα. IL-18 activated natural killer cells and complemented other stimulatory pathways, including FcγRIII and NKG2D, resulting in IFNγ production and expression of CD107a. NLRP3 activation in vivo was confirmed by a dose-related increase in plasma IL-1β and IL-18 levels in cynomolgus monkeys administered VTX-2337. These results are highly relevant to clinical studies of combination VTX-2337/cetuximab treatment. Cetuximab, a clinically approved, epidermal growth factor receptor-specific monoclonal antibody, activates NK cells through interactions with FcγRIII and facilitates ADCC of tumor cells. Our preliminary findings from a Phase I open-label, dose-escalation, trial that enrolled 13 patients with recurrent or metastatic SCCHN show that patient NK cells become more responsive to stimulation by NKG2D or FcγRIII following VTX-2337 treatment. Together, these results indicate that TLR8 stimulation and inflammasome activation by VTX-2337 can complement FcγRIII engagement and may augment clinical responses in SCCHN patients treated with cetuximab.

Trial registration: ClinicalTrials.gov NCT01334177.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Benzazepines / pharmacology*
  • Benzazepines / therapeutic use
  • Carrier Proteins / agonists*
  • Caspase 1 / metabolism
  • Cell Degranulation / drug effects
  • Cell Degranulation / immunology
  • Cell Line, Tumor
  • Cytotoxicity, Immunologic / immunology
  • Disease Models, Animal
  • Female
  • Humans
  • Inflammasomes / metabolism
  • Interleukin-18 / biosynthesis
  • Interleukin-1beta / biosynthesis
  • K562 Cells
  • Killer Cells, Natural / drug effects*
  • Killer Cells, Natural / immunology*
  • Killer Cells, Natural / metabolism
  • Leukocytes, Mononuclear / drug effects
  • Leukocytes, Mononuclear / immunology
  • Leukocytes, Mononuclear / metabolism
  • Lymphocyte Activation / drug effects
  • Lymphocyte Activation / immunology
  • Macaca fascicularis
  • Male
  • NLR Family, Pyrin Domain-Containing 3 Protein
  • Neoplasms / genetics
  • Neoplasms / immunology*
  • Neoplasms / metabolism*
  • Receptors, IgG / metabolism
  • Toll-Like Receptor 8 / agonists*

Substances

  • Benzazepines
  • Carrier Proteins
  • Inflammasomes
  • Interleukin-18
  • Interleukin-1beta
  • NLR Family, Pyrin Domain-Containing 3 Protein
  • NLRP3 protein, human
  • Receptors, IgG
  • Toll-Like Receptor 8
  • VTX-2337
  • Caspase 1

Associated data

  • ClinicalTrials.gov/NCT01334177

Grant support

Funding for both basic research and the Phase 1b Clinical trial of VTX-2337 in squamous cell carcinoma of the head and neck (SCCHN) described in this manuscript was provided by VentiRx Pharmaceuticals. The company website is www.ventirx.com. The funder provided support in the form of salaries for multiple authors (HL, YY, MD). LC and CM are employed by the University of Washington, which conducted the clinical study sponsored by VentiRx Pharmaceuticals. Role of funders: VentiRx Pharmaceuticals employees Gregory N. Dietsch and Robert M. Hershberg played an active role in the study designs described in this manuscript, were involved in data collection and analysis of study data, the decision to publish, and the preparation of this manuscript. The specific roles of these authors (GD, RH) are articulated in the “author contributions” section.