Impaired Antibody-Dependent Cellular Cytotoxicity Mediated by Herceptin in Patients with Gastric Cancer1

[CANCER RESEARCH 62, 5813–5817, October 15, 2002] Impaired Antibody-dependent Cellular Cytotoxicity Mediated by Herceptin in Patients with Gastric Cancer1

Koji Kono,2 Akihiro Takahashi, Fumiko Ichihara, Hidemitsu Sugai, Hideki Fujii, and Yoshirou Matsumoto First Department of Surgery, Yamanashi Medical University, Yamanashi 409-3898, Japan

ABSTRACT progression of gastric cancer (10, 11). Abundant examples from experimental models and clinical trials suggest that HER-2/neu can be The humanized monoclonal antibody Herceptin, which specifically tar- immunogenic and generate antibody, CTL-, and helper T-cell-specific gets HER-2/neu, exhibits growth inhibitory activity against HER-2/neu- responses in individuals with HER-2/neu-overexpressing tumors (12, overexpressing tumors and is approved for therapeutic use with proved survival benefit in patients with HER-2/neu-positive breast cancer. In the 13). On the basis of the above reports, anti-HER-2/neu immune present study, we investigated whether Herceptin could affect the HER- targeting may be used as an attractive approach to treat gastric cancer. 3 2/neu-overexpressing gastric cancer cells based on antibody-dependent The humanized mAb Herceptin, which specifically targets HER- cell-mediated cytotoxicity (ADCC) and compared immune effector cells 2/neu, exhibits potent growth inhibitory activity against HER-2/neu- from gastric cancer patients with normal individuals on ADCC. HER-2/ overexpressing tumors (14). The underlying mechanisms of the action neu-expressing gastric cancer cells could be killed by Herceptin-mediated of Herceptin include the inhibition of tumor cell growth such as the ADCC and the Herceptin-induced ADCC correlated with the degree of down-regulation of the HER-2/neu receptor and the enhancement of HER-2/neu expression on the gastric cancer cells. However, the Hercep- the immune system such as ADCC (14). Herceptin has boosted the tin-mediated ADCC was significantly impaired in peripheral blood mono- interest of clinicians in immunotherapy based on this molecule, be- compared with that (10 ؍ nuclear cells from advanced disease patients (n cause it represents the first mAb approved for therapeutic use with ,10 ؍ or healthy individuals (n (0.04 ؍ P ;12 ؍ in early disease (n Moreover, natural killer (NK) cells purified from patients with proven survival benefit in patients with HER-2/neu-positive breast .(0.02 ؍ P advanced disease indicated less Herceptin-mediated ADCC in comparison cancer with metastasis (15, 16). Thus, it is desirable to apply the .whereas monocytes purified treatment with Herceptin to other HER-2/neu-expressing tumors ,(0.04 ؍ with that from healthy donors (P from the patients showed an almost equal amount of Herceptin-mediated However, there has been no previous report describing the possibility ADCC in comparison with that from healthy individuals, indicating that of applying Herceptin to gastric cancer patients. NK cell dysfunction contributed to the impaired Herceptin-mediated In the present study, we: (a) investigated the biological activity of ADCC in gastric cancer patients. Furthermore, the NK-cell dysfunction Herceptin against HER-2/neu-overexpressing gastric cancer cells with on Herceptin-mediated ADCC correlated with the down-regulation of respect to ADCC; and (b) compared the degree of ADCC associated ␨ CD16 expression in the patients, and interleukin 2 ex vivo treatment of with immune effector cells obtained from gastric cancer patients to NK cells could restore the impairment of Herceptin-mediated ADCC, those from normal individuals. concomitant to the normalization of the expression of CD16␨ molecules. Thus, some modalities such as interleukin 2 treatment aimed at reversing NK dysfunction may be necessary for successful Herceptin treatment of MATERIALS AND METHODS gastric cancer. Patients. PBMCs were isolated from gastric cancer patients with early disease corresponding to stage I (n ϭ 12, 65 Ϯ 14 years old), with advanced INTRODUCTION disease (n ϭ 10, 68 Ϯ 16 years old), corresponding to stage II (n ϭ 2), III (n ϭ 2), IV (n ϭ 6), and healthy individuals (n ϭ 10, 63 Ϯ 18 years old). The Gastric cancer is one of the most common cancers in Japan today. stage of the disease was defined according to the Unio Internationale Contra Despite the efforts to introduce new treatment modalities such as Cancrum classification. All of the patients were treated at Yamanashi Medical surgery combined with chemotherapy (1), hyperthermia (2), or che- University Hospital and pathologically diagnosed with gastric cancer. None of moradiotherapy (3), control of gastric cancer at the advanced stage the patients received radiotherapy, chemotherapy, or other medical interven- remains difficult. Therefore, the utilization of antitumor T cells or tions during this study. This study was approved by the ethical committee of antibody against tumor antigens as immunoadjuvant therapy for gas- Yamanashi Medical University, and informed consent for blood donations was tric cancer is extremely appealing. We and others have reported that obtained for all of the individuals. MHC class I-restricted CTLs from gastric-cancer patients can react Preparation of Cells. PBMCs were isolated from heparinized peripheral specifically against autologous tumor cells (4–7). In fact, we have blood by Ficoll-Paque (Pharmacia, Uppsala, Sweden) density gradient centrifugation. reported recently that adoptive transfer of ex vivo cultured T cells To prepare monocytes with negative selection, monocytes were isolated derived from tumor-associated lymphocytes resulted in a survival from PBMCs by Ficoll-Paque (Pharmacia) density gradient centrifugation after effect in a randomized control study in patients with gastric cancer (8). being incubated with RosetteSep antibody mixture for monocytes (StemCell The HER-2/neu proto-oncogene encodes a Mr 185,000 transmem- Technologies Inc., Vancouver, British Columbia, Canada). RosetteSep anti- brane glycoprotein that contains an extracellular domain and intracel- body was bound in bispecific antibody complexes, which are directed against lular domain with tyrosine-specific kinase activity (9). We reported cell surface antigens on human hematopoietic cells (CD2, CD3, CD8, CD19, that amplification and overexpression of the HER-2/neu proto-onco- CD56, and C66b) and glycophorin A on RBCs. Unwanted cells, which adhered gene were seen in ϳ20% of gastric cancers and correlated with stage to RBCs, and desired cells were separated on a Ficoll-Paque (Pharmacia) density gradient. Monocytes resulted in Ͼ87% purity confirmed by flow cytometric analysis using anti-CD14 mAb (Becton Dickinson, Mountain Received 4/8/02; accepted 8/9/02. The costs of publication of this article were defrayed in part by the payment of page View, CA). charges. This article must therefore be hereby marked advertisement in accordance with To prepare NK cells with negative selection, NK cells were isolated by 18 U.S.C. Section 1734 solely to indicate this fact. Ficoll-Paque density gradient after being incubated with RosetteSep antibody 1 Supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and from Haraguchi Memorial Cancer Research Fund. 2 To whom requests for reprints should be addressed, at First Department of Surgery, 3 The abbreviations used are: mAb, monoclonal antibody; ADCC, antibody-dependent Yamanashi Medical University, 1110 Tamaho, Yamanashi 409-3898, Japan. Phone: cell-mediated cytotoxicity; PBMC, peripheral blood mononuclear cell; NK, natural killer; 81-552-73-7390; Fax 81-552-73-6751; E-mail: [email protected]. IL, interleukin; MFI, mean fluorescence intensity; PE, phycoerythrin. 5813

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2002 American Association for Cancer Research. HERCEPTIN IN GASTRIC CANCER mixture (CD3, CD4, CD36, CD19, and CD66b) for NK cells as described FITC-CD16 (Becton Dickinson). The stained cells were assessed by flow above. NK cells resulted in Ͼ88% purity confirmed by flow cytometric cytometric analysis. analysis using anti-CD16 mAb (Becton Dickinson). Biochemical Analysis for ␨ Chain. Negatively selected NK cells (2 ϫ 105/ Cell Lines. MKN-7 (a well-differentiated gastric adenocarcinoma) and ml) were lysed in NP40 lysis buffer [1% NP40, 150 nM NaCl, 1 mM EDTA, KATOIII (a signet ring cell gastric carcinoma) cell lines were obtained from 1mM phenylmethylsulfonyl fluoride, and 50 mM Tris (pH 8.0)]. Cellular the Immuno-Biological Laboratories cell bank (Gunma, Japan). MKN-28 lysates were electrophoresed in 10% SDS gels, transferred to nitrocellulose (a well-differentiated gastric adenocarcinoma) and MKN-45 (a poorly differ- membrane, and blotted with anti-␨ mAb (TIA-2) or isotype-matched control entiated gastric adenocarcinoma) cell lines were provided by Chugai (Tokyo, mAb. The membrane was incubated with antimouse immunoglobulin-horse- Japan). AME-1 gastric cancer cell line (a poorly differentiated gastric adeno- radish peroxidase (Amersham, Arlington Heights, IL) followed by the carcinoma) was established by our laboratory. All of the gastric cancer cell enhanced chemiluminescence reagent (enhanced chemiluminescence; lines were maintained in RPMI 1640 (Life Technologies, Inc., Grand Island, Amersham). NY) supplemented with 7.5% of FCS (Life Technologies, Inc.), penicillin IL-2 Treatment of NK Cells. Negatively selected NK cells (1 ϫ 106 (100 units/ml; Sigma Chemical Co., St. Louis, MO), and streptomycin cell/ml) from gastric cancer patients were incubated with AIM-V medium (100 ␮g/l; Sigma). (Life Technologies, Inc.) in 48-well culture plates in the presence or absence ADCC Assay. After the target cells were labeled with 100 ␮Ci 51Cr for 60 of IL-2 (500 IU/ml; Shionogi, Tokyo, Japan) for 5 days. min, target cells (5 ϫ 103/well) and effector cells at various E:T ratios were Antibodies. Clinically marketed anti-HER-2/neu mAb Herceptin and anti- coincubated in 200 ␮l of RPMI 1640 in a 96-well U-bottomed plate in CD20 mAb Rituxan, as an isotype-matched control mAb for Herceptin, pur- triplicate for6hat37°C with Herceptin (2 ␮g/well; Roche) or control chased from Roche were used in the ADCC experiment. For the evaluation of antibody, Rituxan (2 ␮g/well; Roche). Then, the radioactivity of the superna- HER-2/neu expression on the tumor cells, a PE-labeled anti-HER-2/neu mAb tant (100 ␮l) was measured with a gamma counter. The percentage of specific (Becton Dickinson, San Jose, CA) and PE-labeled mouse IgG1 mAb (Beck- lysis was calculated according to the formula: % specific lysis ϭ 100 ϫ (ex- man-Coulter, Miami, FL) as a negative control were used for immunostaining perimental cpm Ϫ spontaneous cpm)/(maximum cpm Ϫ spontaneous cpm). by flow cytometric analysis. Controls included the incubation of target cells alone with Herceptin. Rituxan Statistics. To determine statistical differences between groups, nonpaired was used as a control mAb in some experiments. Student’s t test was used. Statistical significance was determined when Ps were Flow Cytometric Analysis. For CD16 ␨ expression on NK cells, intracel- Ͻ0.05. lular staining was performed as described previously by us (17). Briefly, the isolated PBMCs were fixed with 0.5% formaldehyde in PBS for 20 min on ice RESULTS after permeabilization by digitonin (10 ␮g/ml; Sigma) for 15 min on ice. The intracellular component of ␨ molecules in the CD16 complex was stained by Herceptin Mediates ADCC against HER-2/neu-overexpressing anti-␨ mAb conjugated with PE (TIA-2, IgG1; Coulter) or by IgG1 isotype Gastric Cancers. To test whether Herceptin induces ADCC against control mAb at a saturating concentration, followed by counterstaining with gastric cancer cells, we performed an ADCC assay by PBMCs from

Fig. 1. Herceptin mediates ADCC against HER-2/neu-overexpressing gastric cancers. PBMCs from healthy donors were tested for ADCC in the presence of Herceptin against several different gastric cancer cell lines with various levels of HER-2/neu. HER-2/neu expression on the tumor cells was evaluated by flow cytometric analysis (A). Representative data of ADCC against MKN-7 with HER-2/neu-overexpression (B), MKN-45 with moderate levels of HER-2/neu (C), or AME-1 with low levels of HER-2/neu (D) are shown. 5814

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2002 American Association for Cancer Research. HERCEPTIN IN GASTRIC CANCER healthy donors against several different gastric cancer cell lines with various levels of HER-2/neu. Representative data showed that Her- ceptin induced much higher cytotoxicity against MKN-7 with HER- 2/neu overexpression in comparison to that against MKN-45 with moderate levels of HER-2/neu or AME-1 with low levels of HER-2/ neu (Fig. 1). There were no direct cytotoxicities for the three targets by Herceptin (Fig. 1). Summarized data from healthy individuals (n ϭ 10) showed that Herceptin significantly mediated the lysis of HER-2/neu-overexpressing MKN-7 and the Herceptin-induced cytotoxicity positively correlated with the degree of HER-2/neu expression on the gastric cancer cells (Fig. 2). There was no significant ADCC by control antibody Rituxan against any targets (Fig. 2). Herceptin-mediated ADCC Is Impaired in PBMCs from the Patients with Gastric Cancer. To evaluate Herceptin-mediated ADCC in gastric cancer patients, we compared the ADCC in PBMCs from patients with that from healthy individuals. The Herceptin- mediated cytotoxicity against HER-2/neu- overexpressing MKN-7 was significantly impaired in advanced disease patients in comparison with that in early disease (P ϭ 0.04) or healthy individuals (P ϭ 0.02) as indicated in Fig. 3. These results indicated that Herceptin-mediated ADCC was impaired in PBMCs from the patients with advanced gastric cancer.

Fig. 4. Impaired Herceptin-mediated ADCC was dependent on NK cell dysfunction. NK cells (A) and monocytes (B) were purified from PBMCs with negative selection from the patients and healthy individuals, and were tested for ADCC in the presence of Herceptin or control antibody (Rituxan) against HER-2/neu-overexpressing MKN-7 gas- .P Ͻ 0.05; bars, ϮSDء .tric cancer cell line

Impaired Herceptin-mediated ADCC Was Dependent on NK Fig. 2. Herceptin-induced ADCC correlated with the degree of HER-2/neu expression Cell Dysfunction. Monocytes and NK cells are known to be impor- on the gastric cancer cells. PBMCs from healthy donors were tested for ADCC in the tant effectors in the role of ADCC (18). To additionally investigate the presence of Herceptin or control antibody (Rituxan) against several different gastric impaired ADCC by Herceptin in the patients, monocytes and NK cells cancer cell lines with various levels of HER-2/neu. HER-2/neu expression on the tumor cells was expressed as MFI by flow cytometric analysis; bars, ϮSD. were purified from PBMCs with negative selection from the patients and healthy individuals. NK cells purified from the patients with advanced disease (n ϭ 8) indicated less Herceptin-mediated ADCC against HER-2/neu overexpressing MKN-7 in comparison with that from healthy donors (n ϭ 10; P ϭ 0.04), as shown in Fig. 4A.Onthe other hand, monocytes purified from the patients showed an almost equal amount of Herceptin-mediated ADCC against HER-2/neu-overexpressing MKN-7 in comparison with that from healthy individuals (Fig. 4B). These results indicated that impaired Herceptin-mediated ADCC in PBMCs from the patients contributes to NK cell- dysfunction. Because Herceptin exhibits ADCC depending on FC␥RIII (CD16) molecules (19, 20) and the phosphorylation of CD16 ␨ molecules is one of the major signal transductions in NK cell-mediated ADCC through CD16 molecules (21, 22), we additionally studied the expression of signal-transducing CD16␨ molecules on NK cells using an intracellular staining method and Western blotting. As expected, the expression of signal-transducing CD16␨ molecules on NK cells from Fig. 3. Herceptin-mediated ADCC is impaired in PBMCs from the patients with the gastric cancer patients was significantly down-regulated compared advanced gastric cancer. PBMCs from healthy donors, gastric cancer patients with early with that from healthy donors (Fig. 5, A–C; P ϭ 0.03). Thus, NK cell disease, or gastric cancer patients with advanced disease were tested for ADCC in the presence of Herceptin or control antibody (Rituxan) against HER-2/neu-overexpressing dysfunction on ADCC mediated by Herceptin in gastric cancer pa- .P Ͻ 0.05; bars, ϮSD. tients correlated to the down-regulation of CD16␨ moleculesء .MKN-7 gastric cancer cell line 5815

Restoration of Herceptin-mediated ADCC by ex Vivo IL-2 Treatment of NK Cells. We next investigated the effect of ex vivo IL-2 treatment of patient-derived NK cells. Negatively selected NK cells from gastric cancer patients (n ϭ 5) were incubated with AIM-V medium in the presence or absence of IL-2 (500 IU/ml) for 5 days. The amount of Herceptin-mediated ADCC by IL-2-treated NK cells was increased compared with that by nontreated NK cells (Fig. 6) and restored to the levels of Herceptin-mediated ADCC by healthy individuals. The expressions of signal-transducing CD16␨ molecules on IL-2-treated and untreated NK cells were 94.5 Ϯ 10.7 MFI and 61.5 Ϯ 12.1 MFI, respectively. Thus, the IL-2 treatment of patient- derived NK cells resulted in the restoration of Herceptin-mediated ADCC, concomitant to the recovery of the expression of CD16␨ molecules.

DISCUSSION Fig. 6. Restoration of Herceptin-mediated ADCC by ex vivo IL-2 treatment of NK cells. Negatively selected NK cells from gastric cancer patients were incubated with The present report contains several novel findings relevant to AIM-V medium in the presence or absence of IL-2 (500 IU/ml) for 5 days and were tested clinical therapeutic application of Herceptin against gastric cancer. for ADCC in the presence of Herceptin or control antibody (Rituxan) against MKN-7; Ϯ First, HER-2/neu-overexpressing gastric cancer cells could be killed bars, SD. by Herceptin-mediated ADCC; however, the Herceptin-mediated ADCC was impaired in PBMCs from the gastric cancer patients compared with that from healthy individuals, mainly because of the NK-cell dysfunction. Second, IL-2 ex vivo treatment of NK cells could restore the impairment of Herceptin-mediated ADCC in the patients with gastric cancer. The fully humanized anti-HER-2/neu mAb, Herceptin, has been designed to specifically antagonize the HER-2/neu function by direct- ing against the extracellular domain of the HER-2/neu protein (14). Herceptin was clinically proven to have survival effects for HER-2/ neu-overexpressing breast cancer (15, 16). Many mechanisms have been proposed to account for the therapeutic effect of Herceptin, including the blockade of signaling pathways (14), down-modulation of the HER-2/neu receptor (14), activation of apoptotic signals of the tumor cells (23), and the interaction with the immune system via its

immunoglobulin G1 Fc domain, such as ADCC (14). In the present study, we clearly showed that HER-2/neu-expressing gastric cancer cells, as well as breast cancer cells with HER-2/neu expression, could be killed by Herceptin-mediated ADCC. This result encourages us to apply the therapeutic approach with Herceptin to gastric cancer. In addition, we have indicated an important issue in the present study that this Herceptin-mediated ADCC was impaired in PBMCs from gastric cancer patients with advanced disease. NK cells, a principal cell type involved in ADCC, express the activation Fc␥ receptor, FC␥RIII (CD16), but do not express the inhibitory counterpart, FC␥RIIB, whereas monocytes express both FC␥RIII and FC␥RIIB (20). Clynes et al. (20) have reported that both NK cells and monocytes play a role in Herceptin-mediated ADCC by demonstrat- ing that mice deficient in the FC␥RIII receptor were unable to inhibit tumor growth by treatment of Herceptin, and Herceptin exhibit ADCC depending on FC␥RIII (CD16) molecules. In addition, it has also been reported that the inhibitory FC␥RIIB receptor is a potent regulator of ADCC mediated by Herceptin in vivo, modulating the activity of FC␥RIII on NK cells and monocytes (20). It was also shown in human PBMCs that Herceptin-mediated ADCC was CD16 molecule-dependent (19). In the present study, we showed that NK cells from gastric cancer patients were dysfunctional in Herceptin-mediated ADCC, whereas monocytes were not. These results indicated that NK cell- dysfunction contributed to the impaired Herceptin-mediated ADCC in gastric cancer patients. Similar to the present study, it has been shown Fig. 5. NK cell dysfunction coexisted with down-regulated CD16␨ expression in that there was impairment of ADCC activity in colon cancer patients gastric cancer patients. Representative data for CD16␨ molecules on NK cells are shown by intracellular staining (A) and Western blotting (B). Summarized data from an intra- in comparison with healthy donors (24). P Ͻ 0.05; bars, ϮSD. The signaling pathways leading to the activation of NK-cell killingء .cellular staining method are shown in C 5816

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2002 American Association for Cancer Research. HERCEPTIN IN GASTRIC CANCER have only been partly elucidated (22). Both the CD16 receptor com- peptide epitopes recognized by gastric cancer-specific CTLs. Int. J. Cancer, 78: plex and activatory counterpart of MHC-I inhibitory receptors or other 202–208, 1998. 8. Kono, K., Takahashi, A., Ichihara, F., Amemiya, H., Iizuka, H., Fujii, H., Sekikawa, NK triggering receptors are known to activate protein tyrosine kinase- T., and Matsumoto, Y. Prognostic significance of adoptive immunotherapy with dependent signaling pathways in NK cells (25). The protein tyrosine tumor-associated lymphocytes in patients with advanced gastric cancer: A random- kinase activity has been shown to constitute a mandatory step in the ized trial. Clin. Cancer Res., 8: 1762–1771, 2002. 9. Slamon, D. J., Godolphin, W., Jones, L. A., Holt, J. A., Wong, S. G., Keith, D. E., initiation of the CD16-mediated target cell killing (25). The ␣ subunit Levin, W. J., Stuart, S. G., Udove, J., and Ullrich, A. Studies of the HER-2/neu of CD16 associated noncovalently with the signal-transducing CD16␨ proto-oncogene in human breast and ovarian cancer. Science (Wash. DC), 244: and Fc⑀RI-␥ molecules (26). We and others showed previously that 707–712, 1989. 10. Kono, K., Takahashi, A., Amemiya, H., Ichihara, F., Sugai, H., Iizuka, H., Fujii, H., the expression of signal-transducing CD16␨ molecules on NK cells and Matsumoto, Y. Frequencies of HER-2/neu overexpression relating to HLA was down-regulated in cancer patients compared with that in healthy haplotype in patients with gastric cancer. Int. J. Cancer, 98: 216–220, 2002. 11. Takehana, T., Kunitomo, K., Kono, K., Kitahara, F., Iizuka, H., Matsumoto, Y., donors, and the alteration of signal transducing molecules was asso- Fujino, M., and Ooi, A. Status of c-erbB-2 in gastric adenocarcinoma: A comparative ciated with NK-cell dysfunction (17, 27, 28). Here, we emphasized study of immunohistochemistry, fluorescence in situ hybridization and enzyme-linked these observations again, in the case of Herceptin-induced ADCC for immuno-sorbent assay. Int. J. Cancer, 98: 833–837, 2002. 12. Disis, M. L., Grabstein, K. H., Sleath, P. R., and Cheever, M. A. Generation of the gastric cancer patients. The present study clearly showed that immunity to the HER-2/neu oncogenic protein in patients with breast and ovarian NK-cell dysfunction in Herceptin-mediated ADCC in the gastric cancer using a peptides-base vaccine. Clin. Cancer Res., 5:1289–1297, 1999. cancer patients was concomitant to down-regulated expression of 13. Disis, M. L., and Schiffman, K. Cancer vaccines targeting the HER-2/neu oncogenic ␨ ␨ protein. Semin. Oncol., 28: 12–20, 2001. CD16 molecules on NK cells, and the restoration of CD16 mole- 14. Sliwkowski, M. X., Lofgren, J. 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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2002 American Association for Cancer Research. Impaired Antibody-dependent Cellular Cytotoxicity Mediated by Herceptin in Patients with Gastric Cancer

Koji Kono, Akihiro Takahashi, Fumiko Ichihara, et al.

Cancer Res 2002;62:5813-5817.

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