Cetuximab induce antibody‐dependent cellular cytotoxicity against EGFR‐expressing esophageal squamous cell carcinoma

To evaluate the possibility of treatment with antiepidermal growth factor receptor (EGFR) mAb, Cetuximab against esophageal squamous cell carcinoma (SCC), we performed detail analysis of the antibody‐dependent cellular cytotoxicity (ADCC) mediated by Cetuximab against esophageal SCC. Esophageal SCC cell lines with various levels of EGFR (n = 8) were evaluated for their Cetuximab‐mediated ADCC by 51Cr‐release assay. As a result, Cetuximab was able to induce ADCC against EGFR‐expressing esophageal SCC and the activities reflected the degree of EGFR expression on the esophageal SCC. The activities of Cetuximab‐mediated ADCC by patients' PBMC were impaired in comparison with those by healthy donors' PBMC. Moreover, the inhibition of transforming growth factor (TGF)‐β could enhance Cetuximab‐mediated ADCC against TGF‐β‐producing SCC. In conclusion, Cetuximab was able to induce ADCC against EGFR‐expressing esophageal SCC. Some modalities aiming at enhancing the Cetuximab‐mediated ADCC may be necessary for successful Cetuximab treatment of patients with esophageal SCC. © 2006 Wiley‐Liss, Inc.

Esophageal cancer is the sixth most frequent cause of cancer death worldwide. 1 Most patients with esophageal cancer in Japan have squamous cell carcinoma (SCC), while most of those in Western countries have adenocarcinoma. Despite improvements in surgical techniques and perioperative management, 2,3 and surgery combined with chemotherapy 4 and radiotherapy, 5 the prognosis remains poor. Therefore, for esophageal SCC patients, novel therapies such as molecular targeted therapy including small molecule inhibitors of tyrosine kinases or humanized monoclonal antibodies are very much needed. Since it has been reported that epidermal growth factor receptor (EGFR), 6 cyclin D1, 7 int-2/hst-1, 8 MDM 9 or E-cadherin 10 are related to the progression of esophageal SCC, these molecules are candidates for molecular targeted therapy.
The EGFR is a member of a family of 4 closely related receptors: EGFR (HER-1), HER-2, HER-3 and HER-4. EGFR is a 170kilodalton glycoprotein, which is composed of 3 domains: an internal domain with tyrosine kinase activity, an external ligandbinding domain and a transmembrane domain. 11 When the ligands such as EGF bind to EGFR, EGFR is dimerized, which leads to the subsequent activation of EGFR tyrosine kinase, resulting in it playing a key role in the regulation of cell proliferation, survival and differentiation. Overexpression of EGFR has been found in a variety of human cancers such as gastrointestinal tract cancers, 12 breast cancers 13 and bladder cancers, 14 and is correlated in a wide variety of tumors with progression. Furthermore, we and others have recently reported that the overexpression of esophageal SCC, partially accounted for by gene amplification, is found in 50-70% of cases, 15,16 and may be indicative of a poor prognosis. 17,18 EGFR has recently attracted much attention due to the development of clinical therapies targeted with EGFR. Cetuximab (Erbitux 2 ), a human-mouse chimerized IgG 1 antibody with a high affinity for the EGFR, has now been approved for use in patients with colorectal cancers. This antibody blocks binding of the ligands, and is able to induce the internalization and downregulation of EGFR. 19,20 Many mechanisms are thought to contribute to the antitumor activity of Cetuximab, including the direct inhibition of EGFR tyrosine kinase activity, 21 the inhibition of cell cycle progression, 22,23 increased levels and activities of proapoptotic molecules 22,24 and enhanced cytotoxicity of chemotherapy and radiotherapy. 25 In addition, it has been shown to inhibit angiogenesis, invasion and metastasis. 26 However, there is no previous report describing the application of Cetuximab for esophageal SCC with a detailed analysis of antibody-dependent cellular cytotoxicity (ADCC) mediated by Cetuximab.
In the present study, we performed detail analysis of Cetuximab-mediated ADCC activity against esophageal SCC cell lines with various levels of EGFR, and furthermore, the synergic effect of Cetuximab and anti-TGF-b 2 (TGF, transforming growth factor) blocking mAb in a SCC cell line producing TGF-b 2 .

Cell lines
Esophageal SCC cell lines TE1, TE2, TE3, TE4 and TE5 were a kind gift from Dr. Nishihara (Institute of Development, Aging and Cancer, University of Tohoku, Sendai, Japan). Esophageal SCC cell lines KYSE30, KYSE50 and KYSE110 were purchased from the Health Science Research Resources Bank (Osaka, Japan). Epidermoid carcinoma A431 was obtained from the American Type Culture Collection (Rockville, MD). All cells were cultured in RPMI 1640 medium with 5% fetal bovine serum, 100 U/ml penicillin, 100 lg/ml streptomycin and 2 mmol/l L-glutamine.

Flow cytometry
For the analysis of EGFR expression by flow cytometry, mouse anti-human EGFR mAb (DakoCytomation) as the primary mAb and a FITC-conjugated polyclonal rabbit anti-mouse mAb as the secondary mAb (DakoCytomation) were used. As a negative control for the primary mAb, mouse immunoglobulin G1 mAb (Beckman-Coulter, Miami, FL) was used. Each step of the incubation with mAb was performed at 4°C for 30 min. After cells were washed twice in PBS, the stained cells were analyzed on a flow cytometer.

Antibody-dependent cell-mediated cytotoxicity assay
Peripheral blood mononuclear cells (PBMC) were separated from peripheral blood obtained from healthy donors and esophageal SCC patients prior to treatment by centrifugation with Ficoll-Paque (Pharmacia, Uppsala, Sweden). After the target cells were labeled with 50 lCi of 51 Cr for 60 min, target cells (5 3 10 3 /well) and PBMC from healthy donors or esophageal cancer patients as effector cells were coincubated at various effector/target ratios in 200 ll of X-VIVO medium in a 96-well U-bottomed plate in triplicate with indicated doses of Cetuximab or a control antibody, Rituxan. After 6 hr of incubation, the radioactivity of the supernatant (100 ll) was measured with a g-counter. The percentage of specific lysis 5 100 3 (experimental cpm 2 spontaneous cpm)/ (maximum cpm 2 spontaneous cpm).

Apoptosis
Each cell line (2 3 10 5 cells) was incubated in 2 ml of X-VIVO with Cetuximab (0.5 lg/ml), Gefitinib (0.1 lM), control mAb or Cetuximab (0.5 lg/ml) plus Gefitinib (0.1 lM) at 37°C in a 6-well plate. After incubation for 24 hr, apoptosis in each cell line was measured by staining with FITC-conjugated Annexin-V and Propidium Iodide (PI) using a MEBCYTO Apoptosis kit (MBL, Nagoya, Japan) following the manufacturer's recommendations. The populations of early apoptotic cells (annexin-V positive/PI negative) and late apoptotic cells (PI positive) as a percent of total cells were evaluated.

MTT cell proliferation assay
Each cell line (2,500 cells) was incubated in 200 ll of X-VIVO with Cetuximab (0.5 lg/ml), Gefitinib (0.1 lM), control mAb or Cetuximab (0.5 lg/ml) plus Gefitinib (0.1 lM) in a 96-well flatbottomed plate in triplicate. After incubation for 96 hr at 37°C, 50 ll of MTT (3-(4,5-dimenthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 2 mg/ml, Sigma, St. Louis, MO) were added to each well and incubation was carried out for 4 hr. Then, the supernatant was discarded and the crystal products were eluted with DMSO (50 ll/ well, Sigma). The colorimetric evaluation was tested using a spectrophotometer at 570 nm. The inhibition of proliferation was shown as percentage of cell growth induced by Cetuximab or Gefitinib in comparison with that induced by control mAb.

Quantitative determination of EGF, TGF-b 2 or TGF-a
Each cell line (2 3 10 5 cells) was cultured in 2 ml of X-VIVO at 37°C for 24 hr in a 6-well plate. After incubation, the culture supernatants in each cell line were collected and tested for their contents using a Quantikine ELISA kit for Human EGF, TGF-a or TGF-b 2 (R&D Systems) following the recommendations of the manufacture.

Treatment with anti-TGF-b 2 neutralizing mAb of Cetuximab-mediated ADCC
After the target cells were labeled with 50 lCi of 51 Cr for 60 min, target cells (5 3 10 3 /well) and PBMC were coincubated at various ratios in 200 ll of X-VIVO medium in a 96-well U-bottomed plate in triplicate with or without anti-TGF-b 2 mAb (5 ng/ ml, R&D Systems) in the presence of Cetuximab (0.5 lg/ml). After incubation for 6 hr at 37°C, the radioactivity of the supernatant (100 ll) was measured with a g-counter.
IL-2 treatment of PBMC from the patients PBMC (1 3 10 6 cell/ml) from esophageal cancer patients were incubated with X-VIVO medium in 48-well culture plates in the presence or absence of IL-2 (500 IU/ml, Shionogi, Japan) for 5 days.

Statistics
To evaluate statistical differences between groups, Student's t test was performed. Significant differences were considered at p < 0.05.

EGFR expression and production of EGF and TGF-a on esophageal SCC
We examined EGFR expression on esophageal SCC cell lines (n 5 8) and epidermoid carcinoma A431, which is a well known cell line with EGFR-overexpression 27 by flow cytometry. There were variable levels of EGFR expression on the esophageal SCC, and the levels of KYSE30 and TE3 were comparable to the EGFR-overexpressing A431 cell line (Table I).
The production of EGF and TGF-a, which are ligands for EGFR, were marginal in all tested cell lines (Table I).   The antiproliferative activity and apoptosis-inducing activity of Cetuximab against EGFR-expressing esophageal SCC To examine the antiproliferative activity and apoptosis-inducing activity of Cetuximab, an MTT assay and Annexin-PI assay were performed for high-EGFR expressing cell lines (KYSE30, TE3 and TE5) and low EGFR expressing cell lines (KYSE110, TE1 and TE4).
Significant inhibition of tumor cell growth induced by Cetuximab was found in cells with high-EGFR expression (TE3 and TE5) at comparable levels observed in A431, while inhibition in cells with low-EGFR expression was marginal (Fig. 1).
As representative data of Annexin-PI assay was shown in Figure 2, significant levels of apoptosis induced by Cetuximab were found in high-EGFR expressing cells (KYSE30, TE3 and TE5) as well as A431, while the remaining cells did not show apoptosis induced by Cetuximab (Table I). Taken together, the antiproliferative activity and apoptosis-inducing activity of Cetuximab was found in high-EGFR expressing SCC.
Since it has been shown that the combination of Cetuximab with Gefitinib could induce a complementary impact on apoptosis and proliferation, 28 the combination therapy of Cetuximab with Gefitinib for esophageal SCC was performed in MTT and apoptosis assay. As a result, some degrees of additional effect were seen in antiproliferative activity on only TE3 (Fig. 1). However, there was no complementary effect in apoptosis-inducing activity on tested cell lines (Table I).

Cetuximab-mediated ADCC for esophageal SCC in healthy donors
To investigate whether Cetuximab induces ADCC against esophageal SCC, Cetuximab-mediated ADCC for esophageal SCC with different levels of EGFR was determined by PBMC isolated from healthy donors (n 5 7). Representative data of ADCC against SCCs indicated that Cetuximab induced much higher cyto-toxicity against high-EGFR expressing KYSE30 in comparison to that against low-EGFR expressing TE4 (Fig. 3). Summarized data from healthy donors (n 5 7) showed that Cetuximab-induced ADCC reflected the degree of EGFR expression by flow cytometric analysis on the esophageal SCC cell lines (Fig. 4). There was no direct cytotoxicity for the targets using Cetuximab (Fig. 4). Furthermore, there was a dose-dependency of Cetuximab in Cetuximab-mediated ADCC against high-EGFR expressing TE5 (Fig. 5).
Since we previously reported that NK cell dysfunction contributed to impaired Trastuzumab-mediated ADCC in gastric cancer patients, 29 we next investigated if the impairment of Cetuximabmediated ADCC in the patients can be improved by the addition of exogenous IL-2 to activate the NK cells. Representative data showed that the amount of Cetuximab-mediated ADCC by IL-2treated PBMC was elevated in comparison to that by nontreated PBMC (Fig. 7). These results were confirmed in PBMC derived from 5 different patients with esophageal cancer.

Treatment with anti-TGF-b 2 neutralizing mAb of Cetuximab-mediated ADCC
Since we reported previously that the inhibition of TGF-b 2 enhanced an ADCC activity for cancer cells producing TGF-b 2 , 30 we next investigated if treatment with anti-TGF-b 2 neutralizing mAb enhanced Cetuximab-mediated ADCC. The amount of production of TGF-b 2 from TE1 (440.9 6 25.5 pg/ml) was much higher than TE5 (30.2 6 29.5 pg/ml) and KYSE30 (90.2 6 8.8 pg/ml). We found that the activities of Cetuximab-mediated ADCC for high TGF-b 2 producing TE1 was enhanced by the addition of anti-TGF-b 2 neutralizing mAb, while no significant effect for TE5 or KYSE30 was seen (Fig. 8). These observations were confirmed in the different E/T ratio settings (Fig. 8).
Furthermore, the inhibition of TGF-b 2 in Cetuximab-mediated ADCC was also confirmed by the exogenous addition of TGF-b 2 in the ADCC against non-TGF-b 2 producing TE5 cell, in which the Cetuximab-mediated ADCC activities for TE5 in the presence of TGF-b 2 (100 pg/ml) were significantly decreased than those in the absence of TGF-b 2 (60.3% 6 5.7% vs. 67.7% 6 7.9%, p < 0.05). Collectively, these results indicated that the inhibition of

Discussion
The present study contains novel and important findings relevant to the Cetuximab-mediated ADCC against EGFR-expressing esophageal SCC. First, Cetuximab was able to induce ADCC against EGFR-expressing esophageal SCC and the activities reflected the degree of EGFR expression on the esophageal SCC. Second, the activities of Cetuximab-mediated ADCC by patients' PBMC were impaired in comparison with those by healthy donors' PBMC. Third, the inhibition of TGF-b could enhance Cetuximab-mediated ADCC against TGF-b-producing SCC. Since we have recently shown that EGFR-overexpression was observed in 50% of esophageal SCCs and was correlated with disease progression, 15 a therapeutic approach with Cetuximab is promising for the treatment of esophageal SCC.
Since EGFR is known to be overexpressed in variety of solid tumors with relatively high incidence and is correlated with the progression of disease, [12][13][14][15][16][17][18] anti-EGFR targeting therapy is a promising approach for cancer treatment. Recently, 2 anti-EGFR therapies have been in clinical development: small-molecule EGFR tyrosine kinase inhibitors such as Gefitinib, 31,32 and humanized antibodies against EGFR represented by Cetuximab. 33,34 Cetuximab (Erbitux), a human-mouse chimerized IgG 1 antibody with a high affinity to EGFR, is now approved by the FDA for use in patients with colorectal cancers. 35,36 This antibody blocks the binding of ligands, and is able to induce internalization of EGFR, and downregulate EGFR expression, 19,20 leading to the direct inhibition of EGFR tyrosine kinase activity, 21 the inhibition of cell cycle progression, 22,23 increased levels and activities of proapoptotic molecules 22,24 and enhanced cytotoxicity of chemotherapy and radiotherapy. 25 As a new finding in the present study, we clearly showed that EGFR-expressing esophageal SCC could be killed by Cetuximab-mediated ADCC. Moreover, the activities of Cetuximab-mediated ADCC reflected the degree of EGFR expression on esophageal SCC. However, the levels of EGFR expression on esophageal SCC lines was not only factor predicting the sensitivity to Cetuximab-induced ADCC, since SCC cells with almost same amount of Cetuximab-induced ADCC had a different level of EGFR expression. It has been shown that Cetuximabinduced antitumor activity did not correlate directly with the levels of EGFR expression in Human tumor xenograft models. 37 These observations suggest that molecular events other than EGFR levels are true determinants of in vivo responsiveness to Cetuximab treatment. Since the tumor xenograft models were insufficient to evaluate Cetuximab-mediated ADCC activities, to what extent ADCC are involved in antitumor effects of Cetuximab or if Cetuximabmediated ADCC activities correlate with EGFR expression levels in vivo remains to be studied.
In addition, we indicated an important issue in the present study that Cetuximab-mediated ADCC was impaired in the PBMC of patients with advanced disease in comparison to that of the healthy donors. It has been shown that both NK cells and monocytes play a role in the ADCC-mediated killing of cancer cells. 38 We previously reported that NK cell dysfunction contributed to impaired Trastuzumab-mediated ADCC in gastric cancer patients. 29 Thus, it is likely that the impaired ADCC mediated by Cetuximab in the present study might be due to the immunosuppressive state with NK cell dysfunction in esophageal SCC patients. It is well known that immunosuppressive cytokines such as IL-10 and TGF-b are produced within the tumor microenvironment and suppress the activity of NK cells, monocytes or T cells. [39][40][41] Therefore, to enhance the effect of Cetuximab-mediated ADCC, we next investigated the involvement of TGF-b in Cetuximab-mediated ADCC for esophageal SCC. As a result, Cetuximab-mediated ADCC against TGF-b-producing TE1 cells was enhanced by treatment with anti-TGF-b 2 neutralizing mAb. These observations indicated that anti-TGF-b blocking could enhance Cetuximab-mediated ADCC against esophageal SCC producing TGF-b. Considering these results, some modalities aiming at reversing NK dysfunction may be necessary for successful Cetuximab treatment of patients with esophageal SCC.
We showed in the present study that Cetuximab-mediated antiproliferative activity and apoptosis-inducing activity against esophageal SCC were relatively moderate. It has been shown that combining agents targeting distinct molecule domains of the EGFR such as the combination of Cetuximab with Gefitinib could induce a complementary impact on EGFR-downstream signaling, apoptosis and proliferation. 28 However, the present study failed to show the additional effect induced by the combination of Cetuximab with Gefitinib in antiproliferative activity and apoptosisinducing activity against esophageal SCC. Alternatively, other studies indicated that combinations of Cetuximab with radiation and chemotherapeutic agents produced synergistic growth inhibition in EGFR-expressing tumors. 42 Since esophageal SCC is well known to be sensitive to radiation and chemotherapy, anti-EGFR targeting with Cetuximab combined with chemoradiation may be a promising approach for esophageal SCC patients.
In conclusion, we reported that Cetuximab was able to induce ADCC against EGFR-expressing esophageal SCC and the activities reflected the degree of EGFR expression. These observations encourage the application of Cetuximab in the treatment of esophageal SCC. Some modalities aiming at enhancing the Cetuximabmediated antitumor effect may be necessary for the successful Cetuximab treatment of patients with esophageal SCC.