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Overexpression of the epidermal growth factor receptor (EGFR) is a hallmark of squamous cell carcinoma of the head and neck (SCCHN). Monoclonal antibodies (mAbs) against EGFR are currently used for therapy of recurrent or metastatic disease; however, their mode of action is not completely understood. To investigate the immunological effects of anti-EGFR mAb, we generated a three-dimensional spheroid model of EGFR-expressing SCCHN and used this model to study the effect of anti-EGFR mAb on leukocyte migration toward tumors. Pretreatment with the blocking anti-EGFR mAb EMD 72000, its F(ab')2 fragments or an EGFR tyrosine kinase inhibitor led to substantially increased leukocyte infiltration into EGFR overexpressing tumor spheroids, but not into those with low EGFR expression. Nonblocking anti-EGFR mAb or fibroblast-specific mAb did not affect leukocyte infiltration, suggesting that the observed increase in leukocyte infiltration depends on interference with EGFR activation. Using a human cytokine macroarray, we demonstrated that the blockade of EGFR by anti-EGFR mAb in EGFR-overexpressing SCCHN cells leads to differential expression of several cytokines and chemokines, including the chemokine MCP-1/CCL-2. The significant upregulation of MCP-1/CCL2 on exposure to anti-EGFR mAb was confirmed by quantitative PCR and enzyme-linked immunospot analyses. Moreover, blocking anti-MCP-1 antibody inhibited leukocyte migration toward tumor cells induced by anti-EGFR mAb, pointing to an important role of MCP-1/CCL2 in anti-EGFR mAb-induced leukocyte migration. Our findings demonstrate that anti-EGFR mAb induces leukocyte infiltration to tumor spheroids by upregulating chemokine expression. This novel mechanism for anti-EGFR mAb action may contribute to the antitumor effects of anti-EGFR mAb in vivo.

In this open uncontrolled phase I study, nine patients with stage III and IV squamous cell carcinoma of the head and neck (SCCHN) were treated with five administrations of the humanized antiepidermal growth factor receptor monoclonal antibody EMD 72000 in three consecutive ascending dose groups. Loading doses of 100 mg (group I), 200 mg (group II), and 400 mg (group III) were followed by four weekly maintenance doses of half the loading doses, i.e. 50, 100, and 200 mg, respectively. Two EMD 72000 administrations were scheduled before and three after surgery. The objectives of this trial were (a) to investigate the safety and toxicity of multiple EMD 72000 doses, (b) to determine the cumulative maximum tolerated dose of EMD 72000 at dosages between 300 mg and 1,200 mg, and (c) to determine the serum pharmacokinetics of EMD 72000. In total, 102 adverse events (AEs) were reported: five of toxicity grade 3, 18 of toxicity grade 2, 66 of toxicity grade 1, and 38 of toxicity grade 0. All AEs of toxicity grade 3 were considered to be not or remotely related to EMD 72000. The most frequent study drug-related AEs were fever and a transient elevation of liver enzymes. In all patients, the time to reach peak serum concentrations (tmax) was within 1-3 h of the start of each EMD 72000 infusion. Average peak serum concentrations (Cmax) after correction for dosage appeared to be dose-independent, whereas the half-life (t1/2) showed dose dependency. In conclusion, EMD 72000 was very well tolerated in patients with advanced stage SCCHN. The pharmacokinetic data from this trial suggest the feasibility of conducting future studies with weekly doses of 200 mg EMD 72000.

The proto-oncogene products erbB-l (EGF-Receptor) and erbB-2 (HER-2/neu), distinct members of the epidermal growth factor receptor family, are frequently overexpressed in squamous cell carcinoma of the head and neck (SCCHN). The accumulation of these transmembrane proteins may lead to significant amounts of the respective extracellular receptor domains (ECD) that are shed from the tumour cell surface and enter blood circulation, thus representing potential serum tumour markers. For erbB-l and erbB-2, we determined the ECD serum levels with enzyme-linked immunosorbent assays and evaluated the protein expression in tumour tissue by immunohistochemistry. The present study included 49 patients (37 untreated, 12 recurrences) and the same number of age- and sex-matched healthy controls. In 24 patients ECD serum levels were determined before and 6 weeks after surgery. Mean ECD serum levels for erbB-1 and erbB-2 were 54.8+/-1.6 and 153.7+/-6.1 fmol/ml in cancer patients, and 54+/-1.5 and 147.9+/-4.5 fmol/ml in healthy controls, respectively. There was no significant difference between untreated and recurrent disease. Serum ECD follow-ups 6 weeks after surgery revealed a significant 12.3% decline of erbB-1 but no change of erbB-2 values. Immunohistochemistry showed strong staining for erbB-1 in 78% and erbB-2 in 47% of the SCCHN specimens. No correlation was detectable between receptor ECD serum levels and receptor tissue expression, tumour stage, and tumour differentiation. Hence, ECD serum levels of erbB-1 and erbB-2 are not considered to be valuable tumour markers in SCCHN.

Squamous cell carcinomas of the head and neck (SCCHN) frequently display high levels of the epidermal growth factor receptor (EGFR). Since EGFR is expressed on the cell surface it may form a suitable target for anticancer therapy with anti-receptor monoclonal antibodies (mAb). Besides the interference with receptor/ligand interactions, binding of mAb to EGFR leads to immunoglobulin-coated tumour cells that may induce or enhance non-specific immune effector mechanisms like antibody-dependent cell-mediated cytotoxicity (ADCC). In established cell lines of SCCHN (UM-SCC 11B, 14C, 22B, and 8029 NA) we investigated the antitumour activity of allogeneic peripheral blood mononuclear cells (PBMC) in combination with rat (ICR 62), mouse (EMD 55900), and humanized (EMD 72000) anti-EGFR mAb. In addition, autologous PBMC were available for tumour line UD-SCC 4. The EGFR protein content of the tumour cell lines ranged between 170 fmol/mg protein and 8100 fmol/mg protein, and MCF-7 cells served as receptor-negative controls. PBMC activity against SCCHN targets was determined in 96-well microtitre-plate monolayer cultures by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after coincubation for 4 h, 24 h and 72 h at effector target ratios of 1:1, 5:1, 10:1 and 20:1. PBMC subpopulations were obtained by macrophage depletion (plastic adherence) or natural killer (NK) cell enrichment (magnetic bead negative selection). Prolonged time of exposure and increased effector:target ratios revealed marked antitumour activity of PBMC alone. This non-specific immune destruction was enhanced considerably by humanized and rat, but not mouse anti-EGFR mAb. Increased EGFR protein in tumour cells partly correlated with an intensification of ADCC but was accompanied by decreased primary PBMC cytotoxicity. The utilization of PBMC subpopulations suggested a mainly NK-cell-mediated ADCC, which appeared to benefit directly or indirectly, e.g. via the secretion of cytokines, from other PBMC components. In conclusion, humanized (EMD 72000) and rat (ICR 62) anti-EGFR mAb were able to generate strong antitumour ADCC in target monolayers of SCCHN.

Head and neck squamous cell carcinomas (HNSCC) frequently display increased levels of epidermal growth factor receptor (EGFR) and since the receptor is located on the cell surface, anti-EGFR antibodies appear to be suitable agents for antitumor therapy. We investigated the effect of murine EMD 55900 and rat ICR 62 monoclonal antibodies (MAb) directed against EGFR both as single agents and in combination with cisplatin. ELISA detection showed the amount of EGFR protein in HNSCC lines UM-SCC-10A, -10B, -11B, -14A, -14B, 14C, -22B and HLac 79, 8029NA, 8029DDP to range between 20 and 8100 fmol/mg protein. Compared to A431 cells, seven HNSCC lines were high and three low receptor expressors. Only low levels of TGF alpha were found in the supernatants of some untreated HNSCC lines, probably due to the consumption of TGF alpha by EGFR. Consequently, occupation of EGFR by MAb led to marked accumulation of TGF alpha in cell supernatants. Colorimetric MTT assay showed both MAbs (0.3-30nM) to have comparable dose-dependent growth inhibition which correlated with the EGFR content of the respective cell lines (p < 0.05). Using 30nM MAb, seven high receptor expressing HNSCC lines were growth inhibited by at least 20% to a maximum of 61% (mean = 38%). Combined treatment with MAb and cisplatin led to a significant decrease in cisplatin IC50 values in 5 cell lines expressing more than 1200 fmol EGFR/mg (dose modification by factor 2.1-4.1). In conclusion, anti-EGFR MAb exert direct antiproliferative activity in HNSCC lines and show additive effects in combination with cisplatin.

The murine IgG2a monoclonal antibody (mAb) EMD 55900 was produced against the human epidermoid carcinoma cell line A431, with binding occurring to the polypeptide chain of the external domain of human epidermal growth factor receptor (EGF-R). In the present clinical study, 12 patients with advanced squamous cell carcinoma of the larynx or hypopharynx received a single dose of either 20, 100 or 400 mg EMD 55900 3 days prior to laryngectomy and neck dissection. Clinical signs and laboratory parameters of toxicity, development of human anti-mouse antibodies, and mAb plasma concentrations were monitored. In tumor specimens studied from primary tumors and lymph node metastases, expression of EGF-R, distribution of EMD 55900, and occupation of EGF-R by EMD 55900 (double staining) were determined by immunohistochemistry. Single-dose administration of EMD 55900 was very well tolerated in all patients, and good (100 mg) to excellent (400 mg) homogeneous binding of mAb to EGF-R was obtained in the advanced laryngeal and hypopharyngeal carcinomas studied.

Sixteen consecutive patients with stage III/IV laryngeal or hypopharyngeal cancer received 0, 20, 100 or 400 mg i.v. of the murine anti EGF-R IgG2a mab EMD 55900 three days before laryngectomy and neck dissection. Presence of macrophages, T-cells (CD3) and T-cell subpopulations (CD4/CD8), NK-cells, complement factors (iC3b, C1q, C4c), and expression of MHC class I, MHC class II, ICAM-1 and IL-2-R were determined in cryostat sections of the surgical specimens and compared to normal tissue of the same patient. The antibody showed good binding to EGF-R of malignant and normal tissue. However, no signs of strong inflammatory reactions were noticed. Infiltration with macrophages was directly correlated with the dose of the administered antibody, even in the basal layer of the normal mucosa. No morphological signs of direct destruction of normal and malignant tissue infiltrated by macrophages or other immunocytes were noticed. This finding was underlined by random infiltration with HLA-DR positive-, T- and NK-cells. Possible long-term effects of this antibody--e.g. non-specific stimulation or improved antigenic processing--must be evaluated in further studies.

A radioreceptor assay was used to determine the number and affinity of epidermal growth factor (EGF) receptors in a human squamous carcinoma cell line of the larynx (HLac 79) after exposure to cisplatin (DDP). Cells exposed to high concentrations of cisplatin showed about 30% fewer receptors compared to untreated and low-dose-treated cells. This reduction in the number of receptors might be responsible for the decreased growth when stimulated by EGF. Cisplatin appears to lower the concentration of EGF receptors on carcinoma cells, which might result in additional antiproliferative activity especially in carcinomas with high EGF receptor expression.

Representative tumor samples and mucosal samples were taken from three different groups of patients and were stained immunohistochemically for their expressions of epidermal growth factor receptors (EGFR). Patients in group 1 had oral squamous carcinoma, with specimens taken from the tumor as well as from the mucosa without tumor invasion. Patients in group 2 had no evidence of tumor but had heavy drinking and smoking habits. Tumor-free patients who do not drink or smoke served as the control group. The findings in the present study showed that the tumor and mucosal samples from groups 1 and 2 had increased EGFR expression while the control group showed significantly less EGFR. These results suggest that EGFR may play a role in the development of premalignant tissue changes, which are probably influenced by chronic toxic irritation.