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. 2012 Sep;152(3):431-40.
doi: 10.1016/j.surg.2012.05.035. Epub 2012 Jul 6.

Cetuximab therapy in head and neck cancer: immune modulation with interleukin-12 and other natural killer cell-activating cytokines

Eric Luedke  1 , Alena Cristina Jaime-Ramirez, Neela Bhave, Julie Roda, Moaz Maqbool Choudhary, Bhavna Kumar, Theodoros N Teknos, William E Carson 3rd
Affiliations

Cetuximab therapy in head and neck cancer: immune modulation with interleukin-12 and other natural killer cell-activating cytokines

Eric Luedke et al. Surgery. 2012 Sep.

Abstract

Background: Squamous cell carcinoma of the head and neck (SCCHN) is the sixth most common cancer worldwide. Greater than 90% of SCCHN of the oropharynx overexpress the epidermal growth factor receptor (EGFR or HER1). Cetuximab (Erbitux-TM) is a humanized anti-HER1 monoclonal antibody (mAb) that binds to HER1 overexpressing tumor cells. Cetuximab has a direct effect on HER1-positive cancer cells, but it also can activate immune cells that bear receptors for the Fc (constant portion) of IgG such as natural killer (NK) cells. NK cells have an activating Fc receptor for IgG (FcγRIIIa), which mediates Ab dependent cellular cytotoxicity (ADCC) and enhances production of interferon-γ (IFN-γ) in response to Ab-coated targets. Interleukin-12 (IL-12) is a cytokine produced by antigen-presenting cells that stimulates IFN-γ production from NK cells. We hypothesized that IL-12 would enhance the anti-tumor activity of cetuximab by activating the FcR effector mechanisms of NK cells.

Methods: Expression of HER1 was measured on human papilloma virus (HPV)-positive (UD-SCC2, UM-SCC47) and HPV-negative (Cal27, UM-SCC74B) SCCHN cell lines by immunoblot analysis and flow cytometry. NK cells from normal donors were treated overnight with IL-2 (100 U), IL-12, IL-15, or IL-21 (all 10 ng/mL) and tested for ADCC versus cetuximab-coated cancer cells in a 4 hr (51)Cr assay. Release of cytokines by NK cells in response to cetuximab-coated cells was measured by ELISA. Phosphorylation of the ERK transcription factor in NK cells was measured by flow cytometry. The efficacy of combination therapy with cetuximab plus IL-12 was evaluated in a murine tumor model of head and neck cancer.

Results: All cell lines showed >99% expression of HER1 by flow cytometry and immunoblot analysis except UM-SCC74B (73%). Normal NK cells mediated 49.4% lysis of cetuximab-coated SCCHN cell lines as compared to 7.6% lysis of cells treated with control IgG (P = .0002). NK cell lysis of cetuximab-coated SCCHN cells was markedly enhanced by 12 hr pre-treatment of NK cells with IL-12 (71.6% lysis, P = .005 vs cetuximab alone). As a control, IL-12-activated NK cells were tested against IgG-treated cells. ADCC under these conditions was just 21.7%. Similar levels of lysis were noted for both HPV-positive and HPV-negative and cell lines. Other NK cell activating factors such as IL-2, IL-15, and IL-21 were also able to enhance NK cell ADCC. The stimulus of IL-12 and cetuximab-coated tumor cells induced the synergistic production of nanogram levels of IFN-γ (>6-fold increase over controls) (P < .001). A similar effect was seen for NK cell production of the chemokines RANTES, MIP-1α, and IL-8. Phosphorylation of ERK (which is critical for FcR-mediated ADCC and cytokine production) was enhanced in NK cells exposed to IL-12 and IgG as compared to control conditions. The combination of cetuximab plus IL-12 resulted in a reduction in tumor burden when compared to either agent alone in a murine xenograft model of SCCHN.

Conclusion: Cytokine stimulation of NK cells in the presence of cetuximab-coated head and neck cancer cells leads to enhanced NK cell mediated ADCC and cytokine secretion independent of tumor cell HPV-status. Cytokine administration could be a useful adjuvant in the cetuximab treatment of HER1-positive head and neck cancer.

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Figures

Figure 1
Figure 1
Expression of HER1 in SCCHN cell lines. (A) Lysates were prepared from human head and neck cancer cell lines and subjected to immunoblot analysis with antibodies directed against HER1. β-actin was the loading control. HeLa and SKBR3 served as positive and negative controls. (B) Representative histograms for human head and neck cancer cell lines stained with anti-HER1-PE mAb and analyzed by flow cytometry.
Figure 1
Figure 1
Expression of HER1 in SCCHN cell lines. (A) Lysates were prepared from human head and neck cancer cell lines and subjected to immunoblot analysis with antibodies directed against HER1. β-actin was the loading control. HeLa and SKBR3 served as positive and negative controls. (B) Representative histograms for human head and neck cancer cell lines stained with anti-HER1-PE mAb and analyzed by flow cytometry.
Figure 2
Figure 2
Expression of HPV-16 E7 in SCCHN cell lines. Purified DNA from the indicated cell lines was analyzed for the expression of HPV-16 E7 (224 bp) by PCR using HPV-16 E7A (forward) and HPV-16 E7B (reverse) primers. HeLa and Raji tumor cell lines served as positive and negative controls, respectively.
Figure 3
Figure 3
IL-12 enhances ADCC of NK cells against SCCHN cell lines. Purified NK cells were incubated overnight with medium ± IL-12 (10 ng/mL). The lytic activity of NK cells against cetuximab-coated HPV-positive (A) and HPV-negative (B) head and neck cancer cell lines was assessed via a standard 4 hr Cr-51 release assay. Each graph depicts results from one representative donor. At least 3 normal donors were tested per cell line. p<0.02 for combination treatment vs. cytokine activated NK cells (*).
Figure 3
Figure 3
IL-12 enhances ADCC of NK cells against SCCHN cell lines. Purified NK cells were incubated overnight with medium ± IL-12 (10 ng/mL). The lytic activity of NK cells against cetuximab-coated HPV-positive (A) and HPV-negative (B) head and neck cancer cell lines was assessed via a standard 4 hr Cr-51 release assay. Each graph depicts results from one representative donor. At least 3 normal donors were tested per cell line. p<0.02 for combination treatment vs. cytokine activated NK cells (*).
Figure 4
Figure 4
ADCC of NK cells against SCCHN cell lines is enhanced by cytokines. Purified NK cells were incubated overnight with medium ± IL-2 (100 IU), IL-15 (10 ng/mL), or IL-21 (10 ng/mL). The lytic activity of NK cells against cetuximab-coated HPV-negative (A) and HPV-positive (B) cell lines was assessed via a 4-hr Cr-51 release assay. Each graph depicts results from one donor. At least 3 normal donors were tested per cell line. p<0.03 for combination treatment vs. control conditions (*).
Figure 4
Figure 4
ADCC of NK cells against SCCHN cell lines is enhanced by cytokines. Purified NK cells were incubated overnight with medium ± IL-2 (100 IU), IL-15 (10 ng/mL), or IL-21 (10 ng/mL). The lytic activity of NK cells against cetuximab-coated HPV-negative (A) and HPV-positive (B) cell lines was assessed via a 4-hr Cr-51 release assay. Each graph depicts results from one donor. At least 3 normal donors were tested per cell line. p<0.03 for combination treatment vs. control conditions (*).
Figure 5
Figure 5
Human NK cell cytokine secretion. Cetuximab-coated SCCHN cells were cultured with purified human NK cells in media supplemented with various NK cell-activating cytokines in an in vitro co-culture assay. Control conditions consisted of PBS, cytokine alone, or cetuximab-coated tumor cells alone. Culture supernatants were harvested at 48 hrs and analyzed by ELISA for IFN-γ, MIP-1α, RANTES, and IL-8 production in both HPV-negative (A and C) and HPV-positive (B and D) cell lines. Each graph depicts results from one representative donor. A minimum of 3 normal donors was tested per cell line. *= (p<0.001).
Figure 5
Figure 5
Human NK cell cytokine secretion. Cetuximab-coated SCCHN cells were cultured with purified human NK cells in media supplemented with various NK cell-activating cytokines in an in vitro co-culture assay. Control conditions consisted of PBS, cytokine alone, or cetuximab-coated tumor cells alone. Culture supernatants were harvested at 48 hrs and analyzed by ELISA for IFN-γ, MIP-1α, RANTES, and IL-8 production in both HPV-negative (A and C) and HPV-positive (B and D) cell lines. Each graph depicts results from one representative donor. A minimum of 3 normal donors was tested per cell line. *= (p<0.001).
Figure 5
Figure 5
Human NK cell cytokine secretion. Cetuximab-coated SCCHN cells were cultured with purified human NK cells in media supplemented with various NK cell-activating cytokines in an in vitro co-culture assay. Control conditions consisted of PBS, cytokine alone, or cetuximab-coated tumor cells alone. Culture supernatants were harvested at 48 hrs and analyzed by ELISA for IFN-γ, MIP-1α, RANTES, and IL-8 production in both HPV-negative (A and C) and HPV-positive (B and D) cell lines. Each graph depicts results from one representative donor. A minimum of 3 normal donors was tested per cell line. *= (p<0.001).
Figure 5
Figure 5
Human NK cell cytokine secretion. Cetuximab-coated SCCHN cells were cultured with purified human NK cells in media supplemented with various NK cell-activating cytokines in an in vitro co-culture assay. Control conditions consisted of PBS, cytokine alone, or cetuximab-coated tumor cells alone. Culture supernatants were harvested at 48 hrs and analyzed by ELISA for IFN-γ, MIP-1α, RANTES, and IL-8 production in both HPV-negative (A and C) and HPV-positive (B and D) cell lines. Each graph depicts results from one representative donor. A minimum of 3 normal donors was tested per cell line. *= (p<0.001).
Figure 6
Figure 6
Supernatants from NK cells co-stimulated with immobilized antibody plus cytokine induce chemotaxis of activated T cells. Activated T cells were assayed for chemotaxis in response to culture supernatants derived from NK cells costimulated with cetuximab-coated tumor cells plus IL-2, IL-12, IL-15, or IL-21. Supernatants from control-stimulated NK cells were used as a comparison.
Figure 7
Figure 7
ERK activation. Purified human NK cells were co cultured with cetuximab coated head and neck tumor cells in the presence of IL-12 for 2 hrs at 37°C. Control conditions consisted of media alone, IL-12 alone, and cetuximab-coated tumor cells alone. Following incubation, cells were collected and underwent dual flow staining using anti-CD56 and anti-phospho-ERK1/2 Abs. The percentage of cells positive for p-ERK is depicted in the figure by the addition of cells in the upper right and lower right quadrants. The flow data depicts results from one representative donor, however, the percentages represent the average from n=3 donors.
Figure 8
Figure 8
The combination of IL-12 and cetuximab results in reduced tumor burden in a mouse xenograft model. The effects of thrice weekly i.p. treatments of PBS, IL-12 (50 ng), cetuximab (0.5 mg/kg) or the combination on tumor volume were assessed in athymic mice bearing Cal-27 HER1+ tumor xenografts. n=3 mice/group, bars=SE.
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