Anti-EMMPRIN monoclonal antibody as a novel agent for therapy of head and neck cancer

Abstract

Purpose: Extracellular matrix metalloprotease inducer (EMMPRIN) is a tumor surface protein that promotes growth and is overexpressed in head and neck cancer. These features make it a potential therapeutic target for monoclonal antibody (mAb)-based therapy. Because molecular therapy is considered more effective when delivered with conventional cytotoxic agents, anti-EMMPRIN therapy was assessed alone and in combination with external beam radiation.

Experimental design: Using a murine flank model, loss of EMMPRIN function was achieved by transfection with a small interfering RNA against EMMPRIN or treatment with a chimeric anti-EMMPRIN blocking mAb. Cytokine expression was assessed for xenografts, tumor cells, fibroblasts, and endothelial cells.

Results: Animals treated with anti-EMMPRIN mAb had delayed tumor growth compared with untreated controls, whereas treatment with combination radiation and anti-EMMPRIN mAb showed the greatest reduction in tumor growth (P = 0.001). Radiation-treated EMMPRIN knockdown xenografts showed a reduction in tumor growth compared with untreated knockdown controls (P = 0.01), whereas radiation-treated EMMPRIN-expressing xenografts did not show a delay in tumor growth. Immunohistochemical evaluation for Ki67 and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) resulted in a reduction in proliferation (P = 0.007) and increased apoptosis in anti-EMMPRIN mAb-treated xenografts compared with untreated controls (P = 0.087). In addition, we provide evidence that EMMPRIN suppression results in decreased interleukin 1beta (IL-1beta), IL-6, and IL-8 cytokine production, in vitro and in vivo.

Conclusions: These data suggest that anti-EMMPRIN antibody inhibits tumor cell proliferation in vivo and may represent a novel targeted treatment option in head and neck squamous cell carcinoma.

Fig. 1

A, CNTO3899 binds to immobilized human EMMPRIN extracellular domain. The EC₅₀ for mAb binding in the ELISA format is ~ 10 ng/mL. B, CNTO3899 inhibits soluble EMMPRIN – induced MMP-1generation. The expression of MMP-1 by EMMPRIN extracellular domain stimulated normal human lung fibroblast cells is inhibited in a dose-dependent manner by CNTO3899. The IC₅₀ for CNTO3899 in this assay format is between 5 and 10 mg/mL. C, CNTO3899 inhibits MMP-2 expression induced by cocultured normal dermal fibroblast/G361 cells. MMP-2 levels are elevated above background when normal dermal fibroblast and G361cells are cocultured, as indicated in the intensity of the band observed by zymogram. Intensity decreases in response to increasing concentrations of CNTO3899 mAb, showing bioactivity without the addition of exogenously added soluble EMMPRIN. No change in intensity is observed with the isotype control.

Fig. 2

A, SCC-1and normal dermal fibroblast cells were incubated with increasing concentrations of CNTO3899 alone and in combination. After 72 h, anti-EMMPRIN mAb – treated SCC-1cells were 15% (50 μg/mL), 30% (100 μg/mL), and 57% (200 μg/mL) less than control, with a significant reduction in tumor cell viability obtained with100 μg/mL (P = 0.02) and 200 μg/mL of CNTO3899 (P < 0.001). No reduction in tumor cell viability was observed in normal dermal fibroblast cells treated with CNTO3899. SCC-1cells cocultured with normal dermal fibroblasts showed a significant reduction in tumor growth with 100 and 200 μg/mL of CNTO3899 (P < 0.001). B, FaDu and normal dermal fibroblast cells were incubated with CNTO3899. A reduction in cell proliferation was seen with 100 μg/mL (P = 0.01) and 200 μg/mL (P = 0.001) of CNTO3899. C, FaDu and FaDu/siE cells were plated on type I collagen with normal dermal fibroblasts. A significant decrease in collagenolysis was observed in EMMPRIN knockdown cells compared with FaDu controls (P = 0.01). D, FaDu and normal dermal fibroblast cells (1 × 10⁵ of each) were plated on type I collagen and treated with 100 μg/mL of CNTO3899. A significant inhibition in collagenolysis was observed (P = 0.005).

Fig. 3

CNTO3899 inhibits head and neck squamous cell carcinoma xenograft growth. Monoclonal anti-EMMPRIN antibody (200 μg i.p. twice weekly for 3 wk) was administered to severe combined immunodeficient mice bearing SCC-1tumors (n = 7 per group). A, xenografts treated with CNTO3899 showed a significant reduction in tumor growth (tumor doubling time, 34.3 ± 0.9 d) when compared with untreated controls (21.1 ± 1.0 d; P = 0.004). B, lysates from treated and untreated xenografts were analyzed by ELISA. CNTO3899 treatment decreased expression of IL-1β (P = 0.0079), IL-6 (P = 0.077), IL-8 (P = 0.1481), and VEGF (P = 0.0538) when compared with animals receiving no treatment. C, analysis for Ki67 (cell proliferation) revealed that treated tumors had fewer proliferating cells compared with the untreated group (P = 0.007). TUNEL analysis of xenografts treated with CNTO3899 showed a higher percentage of positively stained cells compared with control tumors, although the difference was not significant (P = 0.087). Bars, SEM. *, raw values multiplied by 100 for scale. Original magnification, × 200.

Fig. 4

EMMPRIN knockdown alters in vitro cytokine expression. To determine the role of EMMPRIN in autocrine and paracrine cytokine production. A, FaDu and FaDu/siE tumor cells were cultured in DMEM with 0.1% bovine serum albumin, serum-free media was collected after 18 h, and analyzed by ELISA for IL-1β, IL-6, IL-8, and VEGF cytokine levels. IL-1β (P = 0.0047) and VEGF (P = 0.0031) production by FaDu cells were significantly higher than from FaDu/siE cells. IL-8 production was greater in FaDu cells versus FaDu/siE, but the difference was not statistically significant (P = 0.1635), whereas IL-6 production was lower in FaDu versus FaDu/siE cells (P = 0.004). Normal dermal fibroblasts (B) and human umbilical vascular endothelial cells (C) were stimulated with cell membrane preparations from FaDu and FaDu/siE cells; serum-free media was collected after 18 h and analyzed for IL-1β, IL-6, IL-8, and VEGF cytokine levels. The differences between normal dermal fibroblast stimulation with FaDu and FaDu/siE were significant for IL-1β (B, P = 0.0009), IL-6 (P = 0.0003), and VEGF (P = 0.0164), but not IL-8 (P = 0.1074). C, the increases in cytokine expression after stimulation of human umbilical vascular endothelial cells with EMMPRIN-positive cell membranes were significant for IL-6 (P = 0.0001) and IL-8 (P = 0.0001), but not for IL-1β (P = 0.2969) or VEGF (P = 0.4300). Bars, SEM. *, raw values multiplied by 100 for scale.

Fig. 5

Anti-EMMPRIN mAb provides greater radiation response in vitro. SCC-1 and FaDu cells were plated with and without normal dermal fibroblast cells in the presence of CNTO3899 (100 μg/mL) and exposed to 4 Gy of ⁶⁰Co radiation. A, after 72 h, SCC-1cells exposed to radiation were 68% less than control (P = 0.0003), whereas those that received anti-EMMPRIN and radiation were 76% less than control (P = 0.0002). A significant difference was observed between radiated groups treated with and without CNTO3899 (P = 0.006). When SCC-1 cells were combined with normal dermal fibroblasts, a greater reduction in tumor cell proliferation was seen with combination treatment than with radiation alone (P < 0.0001). B, FaDu cells exposed to radiation were 62% less than control (P = 0.0003), whereas cells treated with radiation and CNTO3899 were 76% less than control (P = 0.0002). Tumor reduction was greater for cells treated with radiation and CNTO3899 than radiation alone (P = 0.009). FaDu/normal dermal fibroblast tumor cell proliferation was not significantly reduced with combination therapy when compared with radiation alone (P = 0.16).

Fig. 6

Enhanced radiation response in knockdown or anti-EMMPRIN antibody treated xenografts. A, FaDuand FaDu/siE tumor cells (2 × 10⁶)were injected in to the flanks of severe combined immunodeficient mice, and animals were divided into radiation and control groups (n = 8 per group). Mice in treatment groups received 12 Gy of ⁶⁰Co radiation divided into six 2 Gy fractions. No significant difference was observed between FaDu control (tumor doubling time, 29 ± 1.9 d) and FaDu radiation groups (29 ± 1.8 d; P = 0.61), whereas irradiated FaDu/siE xenografts showed significant growth delay (42 ± 2.1d) in comparison with nonirradiated FaDu/siE controls (33 ± 1.9 d; P = 0.012). B, severe combined immunodeficient mice bearing SCC-1xenografts were divided into control, CNTO3899, radiation, and combined CNTO3899 and radiation groups (n = 7 per group). Treatment groups received 1.2 mg of antibody and/or 12 Gy of⁶⁰ Co radiation. Combination therapy with CNTO3899 and radiation significantly inhibited tumor growth (tumor doubling time, 40 ± 8.3 d) compared with control (8 ±0.8 d; P = 0.001). CNTO3899 treated xenografts showed inhibition of tumor growth (tumor tripling time, 27 ±4.6 d) when compared with untreated xenografts (14 ± 1.2; P = 0.019), whereas radiation alone did not produce a significant reduction in tumor growth (22 ± 7.1; P = 0.510).