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

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.

Conflict of interest statement

Competing Interests: Gregory N. Dietsch and Robert M. Hershberg are both employees and have ownership (shares) in VentiRx Pharmaceuticals, Seattle, WA, United States of America. Gregory N. Dietsch and Robert M. Hershberg have Patent applications (pending and actual) to which the authors are affiliated and from which the authors may benefit. Robert M. Hershberg is a Board Member of VentiRx Pharmaceuticals. Mary L. Disis is a paid consultant for VentiRx Pharmaceuticals, Seattle, WA, United States of America. The association of Gregory N. Dietsch, Robert M. Hershberg and Mary L. Disis with VentiRx Pharmaceuticals does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1. CONSORT Flow Diagram for Phase 1 clinical study in adult patients with advanced recurrent squamous cell carcinomas of the head and neck (SCCHN).

Study subjects received VTX-2337 administered by the SC route on days 1, 8 and 15 of a 28-day treatment cycle. The first cohort received a 2.5 mg/m2 dose of VTX-2337 following cetuximab administration; this dose was escalated in subsequent cohorts using a 3+3 design to 3.0 mg/m2 and finally 3.5 mg/m2.

Fig 2. VTX-2337 induces mature IL-1β and IL-18 via NLRP3 inflammasome activation in THP-1 cells.

(A-B) VTX-2337 induced pro-IL-1β and pro-IL-18 mRNA expression in THP-1 cells. Cells were cultured overnight in medium or VTX-2337 (3 or 10 μM), and expression of IL-1β and IL-18 mRNA, normalized to β-actin levels, were measured by real-time RT-PCR. (C) Western blot analysis of IL-1β and caspase-1 protein levels in THP-1 culture supernatants, and pro-IL-1β protein levels in cell lysates, following overnight activation with medium (Ø), or VTX-2337 (3 or 10 μM). (D) Pretreatment with caspase-1 inhibitor z-VAD blocks VTX-2337-induced secretion of mature IL-1β. Triplicate THP-1 cultures (10 μg/ml) were treated with VTX-2337. Levels of IL-1β in the presence or absence of z-VAD-FMK were assessed using HEK-Blue™ IL-1β cells (mean±sem). (E and F) Levels of secreted IL-1β and IL-18 following overnight VTX-2337 stimulation (0.03 to 25 μM in 1:3 serial dilutions) in wild type THP-1 (●) and NLRP3 deficient THP-1 (THP-1/NLRP3def) cells (○). Levels of IL-1β and IL-18 (mean±sem) in culture supernatant from triplicate wells were measured by ELISA. All results shown are representative of three independent experiments. **, p<0.01, ***, p<0.001 by unpaired Student t-test.

Fig 3. VTX-2337 induces IL-1β and IL-18 in human PBMC.

(A-B) IL-1β and IL-18 secretion from VTX-2337 activated PBMC. Dose responses are shown for one representative donor, where data point represents the mean for duplicate culture wells at the indicated concentration of VTX-2337 after 24 h. (C-D) Summary graphs of VTX-2337-induced IL-1β and IL-18 in whole blood from 11 donors, as analyzed by TruCulture. Whole blood was incubated with medium (Ø) or VTX-2337 (0.3 or 1 μM) for 24 h, levels of secreted IL-1β and IL-18 were analyzed in a multiplexed assay. Each symbol represents data from an individual donor. The horizontal bar represents group average. **, p<0.01; ***, p<0.001 by Wilcoxon signed-rank test.

Fig 4. Caspase-1 activation and IL-18 induction by VTX-2337 leads to NK cell activation.

(A-D) The effects of caspase-1 inhibition on IL-1β, IL-18, IFNγ, and TNFα production by PBMC. Duplicate PBMC cultures from four healthy donors (indicated as D1, D2, D3 and D4) were treated overnight with medium (Ø), VTX-2337 (1 μM), or VTX-2337 plus z-VAD-FMK (10 μg/ml). Levels of IL-1β, IL-18, IFNγ, and TNFα in culture supernatant were measured by ELISA. (E-F) The effects of IL-18 blockade on VTX-2337-induced IFNγ and CD107a expression in NK cells. Summary graphs show the percentage of IFNγ+ and CD107a+ NK cells from PBMC cultured with medium (Ø), VTX-2337 (0.5 μM), or VTX-2337 and anti-IL18 neutralizing antibody (20 μg/ml) for 9 donors where each symbol represents one individual donor. The horizontal bar represents group the mean for the treatment. Data shown are the summary of four independent experiments from the 9 donors, *, p<0.05 by Wilcoxon signed-rank test.

Fig 5. VTX-2337 enhances NK activation in response to stimulation by K562 cells and anti-CD16 in vitro.

(A) FACS plots showing IFNγ and CD107a expression by NK cells following overnight cultured in medium or VTX-2337 (0.5 μM), and then left unstimulated, stimulated with either K562 cells, or plate-bound anti-CD16 mAb. (B-C) Summary graphs of FACS analyses of NK cell activation in PBMC from 11 donors. Each data point represents an individual donor. The horizontal bar represents group average. Each donor was analyzed in an independent experiment. *, p<0.05, **, p<0.01, ***, p<0.001 by Wilcoxon signed-rank test.

Fig 6. VTX-2337 administration to cynomolgus monkeys induces IL-1β and IL-18.

Male cynomolgus monkeys (n = 6 per group) received a subcutaneously injection of VTX-2337 (1 or 10 mg/kg). Plasma collected predose, and 6, 12, 24 and 96 h following dosing was assessed for levels of IL-1β, IL-18 and IL-6. Cytokine levels in pg/mL (mean±sem) at each time point for the two VTX-2337 dose levels are shown. At each VTX-2337 dose level, plasma cytokine levels at 6, 12, 24 and 96 h were compared to predose levels *, p<0.05, **, p<0.01, by t-test.

Fig 7. Administration of VTX-2337 to SCCHN cancer patients enhances NK cell degranulation.

Representative FACS plots from two patients illustrate NK cell activation measured by increased CD107a expression 24 h following the administration of VTX-2337 (3.0 mg/m2) compared to pre-treatment. Freshly isolated PBMC from each patient were unstimulated (negative control), or were stimulated with K562-15mb-41BBL cells or plate-bound anti-CD16 mAb.