title: Cetuximab therapy in head and neck cancer: immune modulation with interleukin-12 and other natural killer cell-activating cytokines.
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.
  
  
  CONCLUSIONS
  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.
authors: Department of Surgery, The Ohio State University, Columbus, OH 43210, USA.
authors: Eric
authors: E
authors: Luedke
authors: Alena Cristina
authors: AC
authors: Jaime-Ramirez
authors: Neela
authors: N
authors: Bhave
authors: Julie
authors: J
authors: Roda
authors: Moaz Maqbool
authors: MM
authors: Choudhary
authors: Bhavna
authors: B
authors: Kumar
authors: Theodoros N
authors: TN
authors: Teknos
authors: William E
authors: WE
authors: Carson
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage*; D061067:Antibodies, Monoclonal, Humanized; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy* / Q000276:immunology* / Q000378:metabolism; D045744:Cell Line, Tumor; D000068818:Cetuximab; D018920:Coculture Techniques; D016207:Cytokines / Q000008:administration & dosage / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D005434:Flow Cytometry; D006258:Head and Neck Neoplasms / Q000188:drug therapy* / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007155:Immunologic Factors / Q000008:administration & dosage*; D018664:Interleukin-12 / Q000008:administration & dosage*; D007694:Killer Cells, Natural / Q000187:drug effects* / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D000077195:Squamous Cell Carcinoma of Head and Neck
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D016207:Cytokines; D007155:Immunologic Factors; D018664:Interleukin-12; C512478:EGFR protein, human; D066246:ErbB Receptors; D000068818:Cetuximab