The anti-tumor effects of cetuximab in combination with VTX-2337 are T cell dependent

Abstract

The Toll-like receptor 8 (TLR8) agonist VTX-2337 (motolimod) is an anti-cancer immunotherapeutic agent that is believed to augment natural killer (NK) and dendritic cell (DC) activity. The goal of this work is to examine the role of TLR8 expression/activity in head and neck squamous cell carcinoma (HNSCC) to facilitate the prediction of responders to VTX-2337-based therapy. The prognostic role of TLR8 expression in HNSCC patients was assessed by TCGA and tissue microarray analyses. The anti-tumor effect of VTX-2337 was determined in SCCVII/C3H, mEERL/C57Bl/6 and TUBO-human EGFR/BALB/c syngeneic mouse models. The effect of combined VTX-2337 and cetuximab treatment on tumor growth, survival and immune cell recruitment was assessed. TLR8 expression was associated with CD8+ T cell infiltration and favorable survival outcomes. VTX-2337 delayed tumor growth in all 3 syngeneic mouse models and significantly increased the survival of cetuximab-treated mice. The anti-tumor effects of VTX-2337+ cetuximab were accompanied by increased splenic lymphoid DCs and IFNγ+ CD4+ and tumor-specific CD8+ T cells. Depletion of CD4+ T cells, CD8+ T cells and NK cells were all able to abolish the anti-tumor effect of VTX-2337+ cetuximab. Altogether, VTX-2337 remains promising as an adjuvant for cetuximab-based therapy however patients with high TLR8 expression may be more likely to derive benefit from this drug combination compared to patients with low TLR8 expression.

Figure 1

TLR8 gene expression is associated with HNSCC patient survival. (A) Shown are Kaplan–Meier survival curves comparing overall survival of HNSCC patients (n = 522) from the TCGA database according to high (n = 91), medium (n = 316) or low (n = 115) TLR8 expression. (B, C): Dot-box plots illustrate the percentage of activated memory CD4⁺ (B) and CD8⁺ (C) tumor infiltrating T cells in HNSCC patients with high, medium and low TLR8 expression from (A). Error bars represent standard deviation of the mean. (D–F) Correlation analysis (Pearson’s correlation tests) between TLR8 and CD4 (D), CD8A (E) and ITGAX (F) of all HNSCC patients from (A). **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s.-non-significant (One-way ANOVA, Tukey’s multiple comparison test).

Figure 2

TLR8 protein expression is associated with HNSCC patient survival. (A) Representative examples of TLR8 immunostaining and expression scores in HNSCCs. (B) Kaplan–Meier estimates of overall survival according to TLR8 expression score(s). (C, D) Kaplan–Meier estimates of overall survival according to TLR8 expression score(s) in low EGFR-expressing (C) and high EGFR-expressing (D) HNSCCs. (E) Representative examples of CD8 immunostaining and expression scores in HNSCCs from (A). F: Percentage of tumors with high, moderate or low CD8 immunostaining based on TLR8 expression scores. G: Percentage of tumors with high, moderate or low CD8 immunostaining based on TLR8 + EGFR expression. IR: immunoreactivity. *p < 0.05, ****p < 0.0001, n.s.-non-significant (Fisher’s exact test).

Figure 3

A TLR8 agonist stimulates immune cell activity. (A–C) Peripheral blood mononuclear cells (PBMCs) were treated with serial dilutions of VTX-2337 for 24 h, then cell culture media harvested for analysis of IFN-γ (A), TNFα (B) and IL-1β (C) by ELISA. *p < 0.01 versus 0 µM. (D–I) SQ20B cells were co-cultured with PBMCs, treated with VTX and/or cetuximab (CTX) for 24 h, then activated CD4⁺ (D, G), CD8⁺ (E, H), and NK cells (F, I) analyzed by flow cytometry. Human IgG₁ was used as a control (CON). Bar graphs shown in (G–I) represent the mean of n = 3 experiments. Error bars represent standard deviation from the mean. *p < 0.05 versus CON, **p < 0.01 versus CON (One-way ANOVA, Tukey’s multiple comparison test).

Figure 4

VTX-2337 shows an anti-tumor effect in vivo. (A–C) C3H mice bearing SCCVII tumors (A), C57BL/6 mice bearing mEERL (B), and BALB/c mice bearing TUBO-hEGFR tumors (C) were treated with PBS (Control) or VTX-2337 (VTX) and tumor growth was measured. Arrow in (A) represents the time at which tumor growth in a control-treated mouse reached euthanasia criteria. N = 5–9 mice/treatment group. (D) VTX and cetuximab (CTX) treatment schema. (E–I) BALB/c mice (n = 10 mice/group, 5 male/5 female) bearing TUBO-hEGFR tumors were treated as in (D) with IgG + PBS used as controls. Separate tumor growth graphs are shown for male (F) and female (G) mice from E. Error bars represent standard error of the mean. (H–I) Shown are Kaplan Meier curves comparing overall and median survival of all mice (H) and CTX + VTX-treated mice from H separated into female and male (I). *p < 0.05. NS: non-significant (Linear regression).

Figure 5

VTX-2337 and Cetuximab increase immune cell infiltration. Blood, tumors and spleens were harvested from a subset of mice (n = 4–5) one day after drug treatment and analyzed for serum concentrations of TNFα (A), and tumor (B) and splenic (C) lymphoid DCs, CD69 + NK cells (D) and IFNγ + CD4 + T cells (E) using flow cytometry. (F) Images represent CD8 immunostaining in control (left image) and VTX + CTX (right image)-treated tumors. Bars = 100 µm (inset bars = 20 µm). (G–J) Female BALB/c mice (n = 9–10 mice/group) bearing TUBO-hEGFR tumors were treated with VTX in combination with CTX with or without anti-CD4, anti-CD8 or anti-asialo-GM1 (anti-NK) and tumor growth (G) was measured. Spleens were analyzed by flow cytometry for validation of CD4⁺ T cell (H), CD8⁺ T cell (I) and NK cell (J) depletion. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant (One-way ANOVA, Tukey’s multiple comparison test). Error bars represent standard deviation from the mean.