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Clinical Cancer Research 13, 1875-1882, March 15, 2007. doi: 10.1158/1078-0432.CCR-06-1335
© 2007 American Association for Cancer Research
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Cancer Therapy: Preclinical

A Nonfucosylated Anti-HER2 Antibody Augments Antibody-Dependent Cellular Cytotoxicity in Breast Cancer Patients

Eiji Suzuki1, Rinpei Niwa2, Shigehira Saji1, Mariko Muta1, Makiko Hirose1, Shigeru Iida2, Yukimasa Shiotsu2, Mitsuo Satoh2, Kenya Shitara2, Masahide Kondo3 and Masakazu Toi1

Authors' Affiliations: 1 Breast Group, Komagome Hospital, Tokyo Metropolitan Cancer and Infectious Diseases Center; 2 Pharmaceutical Research Center, Kyowa Hakko Kogyo Co. Ltd.; and 3 Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tokyo, Japan

Requests for reprints: Masakazu Toi, Breast Group, Komagome Hospital, Tokyo Metropolitan Cancer and Infectious Diseases Center, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8677, Japan. Phone: 81-3-3823-2101; Fax: 81-3-3824-1552; E-mail: maktoi77{at}wa2.so-net.ne.jp.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Removal of fucose residues from the oligosaccharides of human antibody is a powerful approach to enhance antibody-dependent cellular cytotoxicity (ADCC), a potential important antitumor mechanism of therapeutic antibodies. To provide clinically relevant evidence of this mechanism, we investigated ADCC of a fucose-negative version of trastuzumab [anti–human epidermal growth factor receptor 2 (HER2) humanized antibody] using peripheral blood mononuclear cells (PBMC) from breast cancer patients as effector cells.

Experimental Design: Thirty volunteers, including 20 breast cancer patients and 10 normal healthy control donors, were recruited randomly, and aliquots of peripheral blood were collected. ADCC of commercial trastuzumab (fucosylated) and its fucose-negative version were measured using PBMCs drawn from the volunteers as effector cells and two breast cancer cell lines with different HER2 expression levels as target cells. Relationships between cytotoxicity and characteristics of the patients, such as content of natural killer cells in PBMCs, type of therapy, FCGR3A genotypes, etc. were also analyzed.

Results: ADCC was significantly enhanced with the fucose-negative antibody compared with the fucose-positive antibody using PBMCs from either normal donors or breast cancer patients. Enhancement of ADCC was observed irrespective of the various clinical backgrounds of the patients, even in the chemotherapy cohort that presented with a reduced number of natural killer cells and weaker ADCC.

Conclusions: This preliminary study suggests that the use of fucose-negative antibodies may improve the therapeutic effects of anti-HER2 therapy for patients independent of clinical backgrounds.

Human epidermal growth factor receptor 2 (HER2) is a key contributor to normal cell growth and differentiation (1). However, when overexpressed, it is associated with neoplastic transformation of cells. Approximately 15% to 20% of breast cancers show HER2 overexpression and/or HER2 gene amplification. Clinical benefit has been shown either as monotherapy with trastuzumab (Herceptin), a humanized anti-HER2 monoclonal antibody of human IgG1/{kappa} isotype, or in combination with chemotherapy (2–8). Trastuzumab is known to show efficacy only in breast cancer patients with tumors strongly overexpressing HER2, graded 3+ by immunohistochemistry, or graded 2+ with HER2 gene amplification as determined by fluorescence in situ hybridization (9); however, even in those HER2 overexpressors, some of the patients do not achieve clinical benefits with trastuzumab. Therefore, search for the prediction marker, which can select patients who would have better clinical benefits by trastuzumab, is important. On the other hand, to achieve better clinical outcome on HER2 overexpressors but with less efficacy, improvement of potential efficacy of conventional trastuzumab could be one of the important options.

Trastuzumab has been shown to have multiple mechanisms of action based on in vitro studies: antibody-dependent cellular cytotoxicity (ADCC) and direct growth inhibition of tumor cells (10–15). ADCC, a lytic attack on antibody-targeted cells, is triggered following binding of the Fc region of an antibody to the Fc{gamma} receptor IIIa expressed on natural killer (NK) cells. The clinical importance of ADCC was first shown with rituximab (Rituxan), an anti-CD20 chimeric antibody approved for non-Hodgkin's lymphoma treatment in 1998 (16–18). These studies have focused on the relationships between the clinical response and Fc{gamma} receptor IIIa gene (FCGR3A) functional polymorphism that generates either phenylalanine (F) or valine (V) at amino acid position 158, with significantly better clinical responses for patients having FCGR3A-158V allele associated with strong IgG binding to the receptor and ADCC activation (19, 20). More recently, ADCC involvement in the clinical response was also suggested for trastuzumab therapy with methods seemingly more direct than FCGR3A genotyping. Gennari et al. (21) showed a significant correlation between clinical responses and ADCC-mediated killing by patients' peripheral blood mononuclear cells (PBMC). Furthermore, Arnould et al. (22) showed an increased infiltration of NK cells into tumor tissue of trastuzumab-responding patients. These reports support an in vivo role of ADCC in trastuzumab therapy and imply that ADCC enhancement could be a potential approach to improve its efficacy.

We and others reported that removal of fucose from antibody oligosaccharides attached to Asn297 of the heavy chain (defucosylation) significantly enhanced ADCC compared with that of conventional antibody (23–27). Thus, this modulation of antibody could be one of the most powerful approaches to improve efficacy in antibody therapy. One possible problem is that all these studies used PBMCs from normal healthy donors as effector cells. It is unclear whether this activity is also functioning for PBMCs from cancer patients whose immune system could be impaired either by the therapeutic agent used or by the immunosuppressing activity of tumor cells.

In this study, using PBMCs from breast cancer patients as effector cells, we evaluated ADCC of a defucosylated trastuzumab compared with commercial trastuzumab, which contained highly fucosylated oligosaccharides. In addition, relationships between ADCC and various clinical backgrounds of the patients were also analyzed.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
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Blood samples. From April to October 2005, blood samples were randomly collected from both breast cancer patients (n = 20) who underwent various types of treatment, including surgery, chemotherapy, radiotherapy, hormonal therapy, and antibody treatment trastuzumab, and healthy volunteers (n = 10) registered at Breast Group, Komagome Hospital, Tokyo Metropolitan Cancer and Infectious Diseases Center (Tokyo, Japan). The numbers of patients composing each classification are shown in Table 1 . The protocol of this study was approved by Institutional Review Board. All patients and healthy volunteers signed written informed consent statements before samples were taken and analyzed.


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  		Table 1. Patients' characteristics

 
Cell lines. Human breast adenocarcinoma cell lines MCF-7 and SK-BR-3 were purchased from the American Type Culture Collection (Manassas, VA).

Anti-HER2 humanized antibodies. Trastuzumab (Herceptin) IgG1/{kappa}-type anti-HER2 humanized antibody was purchased from Chugai Pharmaceutical Co. Ltd. (Tokyo, Japan). For the generation of the nonfucosylated version of trastuzumab, the cDNA sequences of the VL and VH region were designed as the same with that of trastuzumab (2) and constructed by PCR-based method. The expression plasmid was constructed using humanized IgG1 expression plasmid pKANTEX93 (28) by joining the VL and VH cDNAs with human {kappa} and {gamma}1 constant region cDNAs, respectively. The expression vector was then introduced into {alpha}-1,6 fucosyltransferase gene knockout Chinese hamster ovary cells (29) via electroporation, and transfectant clones were selected in medium lacking hypoxanthine and thymidine as described previously (29). High-producing clones were selected by comparing IgG amounts in culture supernatants using an IgG-detecting ELISA method (28). Antibody was then purified from supernatant of confluent transfectant clone cultured in Excell 301 Medium (JRH Biosciences, Lenexa, KS) using protein A-Sepharose (Millipore, Billerica, MS).

Oligosaccharide analysis of anti-HER2 humanized antibodies. N-linked oligosaccharides were released by digestion of the antibodies with N-glycosidase F (Takara, Shiga, Japan). The released carbohydrates were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with positive ion mode as described previously (30).

Analysis of HER2 expression in breast tumor cell lines. Expression of cell surface HER2 molecules was determined by flow cytometry. Tumor cells (2 x 106) were stained with 10 µg/mL trastuzumab for 1 h on ice in the presence of 250 µg/mL human IgG (Welfide, Osaka, Japan) as blocking reagent. FITC-conjugated anti-human IgG (H+L; R&D Systems, Minneapolis, MN) was used as the secondary reagent. The stained cells were analyzed using an EPICS XL-MCL flow cytometer (Beckman Coulter, Tokyo, Japan).

ADCC assay. Cytotoxicity was determined by the lactate dehydrogenase release assay as described previously (29), using human PBMCs as effector cells and either MCF-7 cells or SK-BR-3 cells as target cells at an E:T ratio of 15:1. Briefly, target cells (1 x 104) and effector cells (1.5 x 105) were distributed into 96-well U-bottomed plates and incubated with serial dilutions of antibodies for 4 h at 37°C. PBMCs were purified from peripheral blood using Lymphoprep (Axis-Shield, Dundee, United Kingdom). The supernatant lactate dehydrogenase activity was measured using a nonradioactive cytotoxicity assay kit (Promega, Madison, WI). Percentage cytotoxicity was calculated according to the formula: cytotoxicity (%) = 100 x (E – SE – ST) / (M – SE), where E is the experimental release, SE is the spontaneous release of effector cells, ST is the spontaneous release of target cells, and M is the maximum release of target cells lysed with 9% Triton X-100. ADCC was calculated according to the formula: ADCC (%) = cytotoxicity (%) – antigen-independent cellular cytotoxicity (AICC; %), where AICC is the nonspecific cytotoxicity in the absence of antibody. In some experiments, ADCC was normalized to the number of NK cells (104 cells) by the following equation: ADCC/104 NK (%) = ADCC (%) x 104 / [E:T ratio x target cell number in an experimental well (104) x NK percentage in PBMC].

Fc{gamma} receptor IIIa-158F/V genotyping. Genotyping of the Fc{gamma} receptor IIIa (FCGR3A)-158V/F polymorphism was done by PCR-based allele-specific restriction analysis assay using genomic DNA prepared from aliquots of peripheral blood as described previously (19).

Quantitation of effector cell proportions in PBMC. Quantitation of the NK cells in PBMCs were determined by flow cytometric analysis as follows: PBMCs (1 x 106) were incubated on ice for 30 min with both FITC-labeled anti-CD3 antibody and PE-labeled anti-CD56 antibody (Beckman Coulter) in the presence of 3.8 mg/mL human IgG as blocking reagent. After incubation, cells were washed twice with PBS and analyzed using FACSCalibur flow cytometer (Beckton Dickinson, Mountain View, CA). The CD56+CD3– cells were defined as NK cells.

Statistical analysis. Data comparing differences between two groups and three groups were assessed using unpaired Student's t test and two-way ANOVA, respectively. Multivariate analysis of patient clinicopathologic factors affecting the ADCC activity was done using a stepwise linear regression model. Differences were considered significant when P < 0.05. Statistical analysis was conducted using the StatView 5.0 for Windows program.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Generation and characterization of humanized anti-HER2 antibodies. A fucose-negative variant of trastuzumab was produced with {alpha}-1,6 fucosyltransferase gene (FUT8)-knockout Chinese hamster ovary cells (FUT8–/– Chinese hamster ovary cells) transfected with the expression plasmid encoding heavy and light chain sequences identical to those of trastuzumab (2). The antibody purified from the culture supernatant, designated dFu-{alpha}HER2, has an identical amino acid sequence to commercially available trastuzumab. Consequently, the two antibodies showed similar binding activities to the surface of HER2+ tumor cell lines confirmed by flow cytometry (data not shown).

Oligosaccharide analysis revealed that Asn297-linked oligosaccharides of dFu-{alpha}HER2 were of a complex-biantennary type and completely defucosylated (100% nonfucosylated), whereas a large fraction of oligosaccharides of trastuzumab were fucosylated (90.0% fucosylated).

FCGR3A genotyping of the patients. It has been known that FCGR3A gene allelic polymorphism correlates with ADCC intensity, with higher activity for individuals having FCGR3A-158V allele via strong binding of this variant on NK cells to the antibody Fc region (19, 20, 27). Hence, FCGR3A-158F/158V genotyping was conducted for the 20 patients recruited in this study. The number of patients with F/F genotype was 11, 7 patients had F/V genotype, and 2 patients had V/V genotype. The allelic frequency calculated was 0.725 for FCGR3A-158F allele and 0.275 for FCGR3A-158V allele. The distribution observed was similar to that in previous reports of the Japanese population (allelic frequency of FCGR3A-158F was 0.72-0.74; refs. 31, 32). The distribution of FCGR3A genotype among various subgroups based on clinical backgrounds is shown in Fig. 1 . The chemotherapy cohort (n = 14) included 12 patients with recurrent disease and their profile was composed of six F/F, four F/V, and two V/V genotypes of FCGR3A gene. The remaining two patients, without recurrent tumor, underwent neoadjuvant chemotherapy and were shown to possess the F/V genotype. The nonchemotherapy cohort (n = 6) included four patients that had received hormonal therapy all having F/F genotype and two patients (one F/F and one F/V) that had undergone trastuzumab monotherapy.


Figure 1
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  		Fig. 1. Diagram of distribution of FCGR3A genotypes among patients. Distributions of disease status, therapeutic type, and FCGR3A genotype among the 20 patients recruited in this study. These classifications correspond vertically with dashed lines; for example, 14 patients undergoing chemotherapy include 12 patients with recurrent disease composed of six F/F, four F/V, and two V/V genotypes of FCGR3A gene. Numerals indicate number of patients in each group. *, neoadjuvant chemotherapy; **, including combination with hormone therapy (four recurrent patients); and ***, trastuzumab monotherapy.

 
Enhanced cytotoxicity of fucose-negative variant of trastuzumab mediated by patient's PBMCs. We compared the cytotoxic activities mediated by either commercial trastuzumab or its fucose-negative counterpart, using PBMCs derived from the 20 cancer patients as effector cells (Fig. 1). Two breast cancer cell lines were chosen as the target cells: MCF-7 with low HER2 expression (~2 x 104 molecules per cell; ref. 33) and SK-BR-3 with high HER2 expression (~1 x 106 molecules per cell; Fig. 2A ; ref. 33). Cytotoxicity was measured by adding effector cells, target cells (E:T ratio, 15:1), and either anti-HER2 antibody at various concentrations or medium alone. AICC is mainly triggered by interaction of NK receptors expressed on NK cells and MHC-like ligands on tumor cells (34, 35). AICC is experimentally determined as percentage tumor lysis in the presence of tumor cells and PBMCs (without antibody). ADCC was calculated by subtracting AICC from cytotoxicity in the presence of antibody. Tumor lysis was calculated by a lactate dehydrogenase release method (29). As shown in Fig. 2B, augmentation of ADCC by fucose-negative antibody (dFu-{alpha}HER2) was detected in 18 patients for MCF-7 and in all patients for SK-BR-3 at the concentration of 1 ng/mL as representative data. Overall, the use of fucose-negative antibody showed significant enhancement of cytotoxicity in most of the cancer patients tested. This was seen previously using healthy donors (23–27). In both breast cancer group and healthy donors, augmentation of ADCC by dFu-{alpha}HER2 was more pronounced against MCF-7 target cells than against SK-BR-3 target cells. In this regard, the fucose-negative antibody always exhibited enhanced ADCC on MCF-7 at all concentrations; however, enhancement of ADCC with the fucose-negative antibody on SK-BR-3 was evident at the lower concentrations, but not at the higher levels of antibody tested (Fig. 2C).


Figure 2
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  		Fig. 2. ADCC of anti-HER2 antibodies against breast cancer cell lines mediated by PBMCs from patients. A, HER2 expression in MCF-7 cells (top) and SK-BR-3 cells (bottom) as shown by trastuzumab binding using flow cytometer. B, ADCC of trastuzumab and dFu-{alpha}HER2 (1 ng/mL each) in the presence of patient PBMCs as effector cells determined by 4-h lactate dehydrogenase release assay. Target cell lines used are shown above each panel. The plots of the same donor are connected with solid lines. C, mean ADCC of patients (n = 20; left) and that of healthy normal donors (n = 10; right). Target cell lines used (left). ***, P < 0.0001, statistically significant differences between ADCC of the two antibodies, as determined by two-tailed paired t test (B and C). {dagger}, P < 0.001; {dagger}{dagger}, P < 0.0001, the enhancement of cytotoxicity found by two-way ANOVA (C).

 
Comparison of antibody-independent and antibody-dependent cytotoxicity according to various characteristics and backgrounds of the patients. Unlike healthy blood donors, the immune effector cells might be augmented or inhibited by the various types of therapy. Therefore, we reanalyzed the data obtained above to investigate whether various variables (normal or patient, type of therapy, NK percentage in PBMCs, recurrence or primary, HER2 index of the primary tumor tissues, FCGR3A genotype, and age) affect the ADCC of each cohort. The results of ADCC and AICC in each cohort are summarized in Table 2 . Values of ADCC at the concentration of 1 ng/mL of either fucose variant antibodies were chosen as representative data. Fourteen patients who had undergone chemotherapy (including 4 patients of combination with hormone therapy) showed lower AICC in MCF-7 as target cells and ADCC (for both two fucose variants) in SK-BR-3 as target cells than those in 6 patients who had received other types of therapy (4 for hormone therapy and 2 for trastuzumab monotherapy). These data suggested that the type of treatment might modulate cytotoxic activity, including AICC and ADCC. NK cells are thought to be principle mediators in ADCC; thus, activity or the number of NK cells and FCGR3A genotype on the cells possibly affect ADCC activity (27). Multivariate stepwise linear regression analyses showed that ADCC activity significantly depended on the NK percentage in PBMCs, which also significantly depended on the therapy type, such as hormonal treatment or chemotherapy (Table 3 ).


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  		Table 2. Percentage of NK cells and cytotoxicity separated by various clinical variables

 

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  		Table 3. Multivariate analysis revealed the factor affecting ADCC activity

 
To provide additional evidence of the effect of treatment type on ADCC, we further compared 10 patients who received chemotherapeutic agent alone and 4 patients who received hormonal treatment alone, each extracted from chemotherapy cohort and other therapy cohort, respectively. Consequently, ADCCs of chemotherapy alone cohort were significantly lower than those of hormone therapy cohort and normal cohort, indicating that the type of therapeutic agent could be an important factor that affects ADCC (Fig. 3 ). Interestingly, the degree of ADCC enhancement by fucose removal in this case seemed to compensate for the ADCC impairment by chemotherapy; percentage ADCC with dFu-{alpha}HER2 by PBMCs of chemotherapy alone was comparable with that with trastuzumab by PBMCs of hormone therapy for SK-BR-3 targets (35 ± 12% and 38 ± 16%, respectively) or even higher for MCF-7 targets (16 ± 11% and 6.8 ± 5.2%, respectively; Table 2).


Figure 3
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  		Fig. 3. Cytotoxicity according to the therapeutic types. Mean value of the ADCC of patients receiving hormone agent alone (Hormone), patients receiving chemotherapeutic agent alone (Chemo), and healthy normal donors (Normal) are separately plotted. Anti-HER2 antibodies and target cell lines used are shown above and left side of each panel, respectively. {dagger}, P < 0.01; {dagger}{dagger}, P < 0.001, statistically significant differences of ADCC versus chemotherapy cohort found by two-way ANOVA.

 
Inconsistent with earlier reports using healthy PBMC donors (23–27), no apparent difference on ADCC was found in any genotypes of FCGR3A in the current study. Heterogeneous background of the patients might mask the potential effect of FCGR3A genotype on ADCC; for example, 158Val carriers (F/V or V/V) associated with the strong ADCC induction were apparently biased toward chemotherapy cohort, which was shown to be associated with weak ADCC in this study, although it is not clear whether this deflection was related or not. Age of the patients and HER2 expression in primary tumor tissues determined by immunohistochemistry did not affect NK percentage, AICC, and ADCC.

Type of therapeutic agents might affect NK cell number in PBMCs and thus affects ADCC. The type of therapeutic agent seemed to have influence on NK cell number and ADCC. To understand whether the change in NK cell number was the major cause of modulation of cytotoxicity by therapeutic agents, we first analyzed the relationships between NK percentage in PBMC and AICC or ADCC for all patients (Fig. 4A ). Although the relationship between AICC and NK percentage was unclear with no significant correlation for MCF-7 target and weak correlation for SK-BR-3 target (P < 0.05), ADCC mediated by the two antibodies was strongly correlated with NK percentage for both targets, suggesting that the change in NK cell number by therapeutic agents might be one of factors in the modulation of ADCC.


Figure 4
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  		Fig. 4. Analyses on the effect of NK cell content on cytotoxicity. A, correlation between cytotoxicity and the percentage of NK cells in PBMCs. Cytotoxicity of all 20 patients recruited in the present study in the absence (AICC; left) or presence of antibodies (ADCC; right) and the regression lines. Concentrations of each antibody and target cell lines used are shown above and left side of each panel, respectively. *, P < 0.05; **, P < 0.01; ***, P < 0.001, statistically significant correlations, as determined by parametric correlation test. B, ADCC normalized to a fixed number (104) of NK cells. Mean values of ADCC of each cohort (left) were recalculated to normalize the individual heterogeneity of NK cell content (right). Differences of cytotoxicity found by two-way ANOVA (left) are diminished after the normalization (right).

 
To further confirm this hypothesis, ADCC values were recalculated by normalizing to a fixed number of NK cells (104 cells). No significant difference on ADCC between chemotherapy alone and hormonal therapy alone groups (especially for SK-BR-3 target) was found after the normalization (Fig. 4B). This may support the hypothesis that the major cause of the impaired ADCC in chemotherapy cohort was the reduced number of NK cells.


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The current study indicates that enhancement of ADCC by fucose removal from the structure of antibody molecules is shown using PBMCs from both healthy donors and cancer patients. Importantly, the defucosylated anti-HER2 antibody required roughly 10-fold less amount of antibody to achieve the comparable ADCC mediated by trastuzumab, as estimated from mean percentage cytotoxicity of all 20 patients (Fig. 2C). In particular, the difference in the efficacy was more pronounced for MCF-7 cells with less HER2 expression, with higher maximal (saturating) cytotoxicity for defucosylated antibody. Although the experimental in vitro data on augmentation of ADCC with fucose-negative trastuzumab may have no relevance in clinical setting, these data suggest that fucose-negative version of trastuzumab might show better clinical outcome in HER2-overexpressing breast cancer patients.

The second interesting finding is that the type of therapeutic agent might modulate ADCC. Patients who received chemotherapy alone (n = 10) showed reduced NK cell number and this might consequently have impaired AICC and ADCC. Fucose removal from trastuzumab could compensate for this ADCC impairment by elevating ADCC of chemotherapy cohort to the similar or higher level compared with that of hormonal therapy cohort. Enhancement of ADCC by fucose removal from the antibody structure may have some clinical importance because trastuzumab therapy is universally used in combination with chemotherapy and has been shown to exert clinical benefit (4–8).

Using two target cell lines, it was shown that ADCC of patients or healthy donors consistently showed higher level against the HER2-overexpressing SK-BR-3 cells than for MCF-7 cells, which express less HER2. Interestingly, ADCC activity in MCF-7 treated with dFu-{alpha}HER2 was comparable with that in SK-BR-3 treated with conventional trastuzumab, and this tendency was seen for any grouping of the patients as shown in Table 2. It is surprising because the difference in HER2 expression between both targets has been estimated to be two orders of magnitude (~2 x 104 and ~1 x 106 molecules per cell for MCF-7 and SK-BR-3, respectively; ref. 33). These data might suggest that dFu-{alpha}HER2 could overcome heterogeneous efficacy of antibody therapy for breast cancer depending on HER2 expression level, although it is unclear whether the antigen expression level is the sole factor that determines ADCC, and the use of target cell lines of different origins may not be an adequate system to investigate the quantitative relationships between antigen expression and ADCC (36). The reduction of antigen amount necessary for ADCC induction by fucose removal can be analyzed more quantitatively by using experimental target cell clones with different expression levels of exogenously transfected antigen gene (36).

As for effect of FCGR3A genotype on ADCC, in contrast to previous reports, in this study, variant of FCGR3A on PBMC did not affect ADCC activity. This could be explained by unexpected distribution of V carrier that is apparently biased toward chemotherapy that would decrease ADCC activity. Supporting this, after being normalized to NK cell number, ADCC showed a tendency of V carrier > F/F (data not shown) that is consistent with the fact that the presence of V allele is associated with higher ADCC (19, 20).

In conclusion, removal of a fucose from the antibody structure enhanced ADCC activity in vitro against two breast cancer cell lines with different HER2 expression levels as target cells. This may result in an improvement in the clinical effectiveness for therapeutic antibodies, although other effector functions of antibodies, such as complement-mediated cytotoxicity and apoptosis induction, should be taken into account to fully predict the clinical benefits. It is also suggested that nonfucosylated antibodies are more potent in ADCC compared with conventional fucosylated antibodies; therefore, the less amount of antibody compared with conventional trastuzumab may be required for treatment. Future clinical studies should be investigated to address these questions.


   Acknowledgments
 
We thank Dr. George Spitalny for helpful suggestions and critical reading of the manuscript.


   Footnotes
 
Grant support: A grant for optimization of new anticancer drugs from the Health and Labor Science Research Grants of Third Term Comprehensive Control Research for cancer from the Ministry of Health, Labor, and Welfare.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6/ 5/06; revised 12/26/06; accepted 1/ 3/07.


   References
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 Abstract
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 Results
 Discussion
 References
 

  1. Hynes NE, Stern DF. The biology of erbB-2/neu/HER-2 and its role in cancer. Biochim Biophys Acta 1994;1198:165–84.[Medline]
  2. Carter P, Presta L, Gorman CM, et al. Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc Natl Acad Sci U S A 1992;89:4285–9.[Abstract/Free Full Text]
  3. Vogel CL, Cobleigh MA, Tripathy D, et al. Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. J Clin Oncol 2002;20:719–26.[Abstract/Free Full Text]
  4. Slamon DJ, Leyland-Jones B, Shak S, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 2001;344:783–92.[Abstract/Free Full Text]
  5. Marty M, Cognetti F, Maraninchi D, et al. Randomised phase II trial of the efficacy and safety of trastuzumab combined with docetaxel in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer administered as first-line treatment: the M77001 study group. J Clin Oncol 2005;23:4265–74.[Abstract/Free Full Text]
  6. Romond EH, Perez EA, Bryant J, et al. Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med 2005;353:1673–84.[Abstract/Free Full Text]
  7. Piccart-Gebhart MJ, Procter M, Leyland-Jones B, et al. Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med 2005;353:1659–72.[Abstract/Free Full Text]
  8. Buzdar AU, Ibrahim NK, Francis D, et al. Significantly higher pathologic complete remission rate after neoadjuvant therapy with trastuzumab, paclitaxel, and epirubicin chemotherapy: results of a randomized trial in human epidermal growth factor receptor 2-positive operable breast cancer. J Clin Oncol 2005;23:3676–85.[Abstract/Free Full Text]
  9. Pauletti G, Dandekar S, Rong H, et al. Assessment of methods for tissue-based detection of the HER-2/neu alteration in human breast cancer: a direct comparison of fluorescence in situ hybridization and immunohistochemistry. J Clin Oncol 2000;18:3651–64.[Abstract/Free Full Text]
  10. Molina MA, Codony-Servat J, Albanell J, Rojo F, Arribas J, Baselga J. Trastuzumab (Herceptin), a humanized anti-Her2 receptor monoclonal antibody, inhibits basal and activated Her2 ectodomain cleavage in breast cancer cells. Cancer Res 2001;61:4744–9.[Abstract/Free Full Text]
  11. Le XF, Claret FX, Lammayot A, et al. The role of cyclin-dependent kinase inhibitor p27Kip1 in anti-HER2 antibody-induced G1 cell cycle arrest and tumour growth inhibition. J Biol Chem 2003;278:23441–50.[Abstract/Free Full Text]
  12. Jackson JG, St Clair P, Sliwkowski MX, Brattain MG. Blockade of epidermal growth factor- or heregulin-dependent ErbB2 activation with the anti-ErbB2 monoclonal antibody 2C4 has divergent downstream signaling and growth effects. Cancer Res 2004;64:2601–9.[Abstract/Free Full Text]
  13. Yakes FM, Chinratanalab W, Ritter CA, King W, Seelig S, Arteaga CL. Herceptin-induced inhibition of phosphatidylinositol-3 kinase and Akt is required for antibody-mediated effects on p27, cyclin D1, and antitumour action. Cancer Res 2002;62:4132–41.[Abstract/Free Full Text]
  14. Mohsin SK, Weiss HL, Gutierrez MC, et al. Neoadjuvant trastuzumab induces apoptosis in primary breast cancers. J Clin Oncol 2005;23:2460–8.[Abstract/Free Full Text]
  15. Lewis GD, Figari I, Fendly B, et al. Differential responses of human tumor cell lines to anti-p185HER2 monoclonal antibodies. Cancer Immunol Immunother 1993;37:255–63.[CrossRef][Medline]
  16. Leget GA, Czuczman MS. Use of rituximab, the new FDA-approved antibody. Curr Opin Oncol 1998;10:548–51.[Medline]
  17. Gopal AK, Press OW. Clinical applications of anti-CD20 antibodies. J Lab Clin Med 1999;134:445–50.[CrossRef][Medline]
  18. Smith MR. Rituximab (monoclonal anti-CD20 antibody): mechanisms of action and resistance. Oncogene 2003;22:7359–68.[CrossRef][Medline]
  19. Koene HR, Kleijer M, Algra J, von Roos D, dem Born AE, de Haas M. FcgRIIIa-158V/F polymorphism influences the binding of IgG by natural killer cell FcgRIIIa, independently of the FcgRIIIa-48L/R/H phenotype. Blood 1997;90:1109–14.[Abstract/Free Full Text]
  20. Wu J, Edberg JC, Redecha PB, et al. A novel polymorphism of FcgRIIIa (CD16) alters receptor function and predisposes to autoimmune disease. J Clin Invest 1997;100:1059–70.[Medline]
  21. Gennari R, Menard S, Fagnoni F, et al. Pilot study of the mechanism of action of preoperative trastuzumab in patients with primary operable breast tumours overexpressing HER2. Clin Cancer Res 2004;10:5650–5.[Abstract/Free Full Text]
  22. Arnould L, Gelly M, Penault-Liorca F, et al. Trastuzumab-based treatment of HER2-positive breast cancer: an antibody-dependent cellular cytotoxicity mechanism? Br J Cancer 2006;94:259–67.[CrossRef][Medline]
  23. Shields RL, Lai J, Keck R, et al. Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human FcgRIII and antibody-dependent cellular toxicity. J Biol Chem 2002;277:26733–40.[Abstract/Free Full Text]
  24. Shinkawa T, Nakamura K, Yamane N, et al. The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. J Biol Chem 2003;278:3466–73.[Abstract/Free Full Text]
  25. Niwa R, Shoji-Hosaka E, Sakurada M, et al. Defucosylated anti-CC chemokine receptor 4 IgG1 with enhanced antibody-dependent cellular cytotoxicity shows potent therapeutic activity to T cell leukemia and lymphoma. Cancer Res 2004;64:2127–33.[Abstract/Free Full Text]
  26. Okazaki A, Shoji-Hosaka E, Nakamura K, et al. Fucose depletion from human IgG1 oligosaccharide enhances binding enthalpy and association rate between IgG1 and FcgRIIIa. J Mol Biol 2004;336:1239–49.[CrossRef][Medline]
  27. Niwa R, Hatanaka S, Shoji-Hosaka E, et al. Enhancement of the antibody-dependent cellular cytotoxicity of low-fucose IgG1 is independent of FcgRIIIa functional polymorphism. Clin Cancer Res 2004;10:6248–55.[Abstract/Free Full Text]
  28. Nakamura K, Tanaka Y, Fujino I, Hirayama N, Shitara K, Hanai N. Dissection and optimization of immune effector functions of humanized anti-ganglioside GM2 monoclonal antibody. Mol Immunol 2000;37:1035–46.[CrossRef][Medline]
  29. Yamane-Ohnuki N, Kinoshita S, Inoue-Urakubo M, et al. Establishment of FUT8 knock-out CHO cells; an ideal host cell line for producing completely defucosylated antibodies with enhanced ADCC. Biotechnol Bioeng 2004;87:614–22.[CrossRef][Medline]
  30. Kanda Y, Yamane-Ohnuki N, Sakai N, et al. Comparison of cell lines for stable production of fucose-negative antibodies with enhanced ADCC. Biotechnol Bioeng 2006;94:680–8.[CrossRef][Medline]
  31. Leppers-van de Straat FGJ, van der Pol WL, Jansen MD, et al. A novel PCR-based method for direct Fcg receptor IIIa (CD16) allotyping. J Immunol Methods 2000;242:127–32.[CrossRef][Medline]
  32. Kobayashi T, Sugita N, van der Pol WL, et al. The Fc{gamma} receptor genotype as a risk factor for generalized early-onset periodontitis in Japanese patients. J Periodontol 2000;71:1425–32.[CrossRef][Medline]
  33. Prang N, Preithner S, Brischwein K, et al. Cellular and complement-dependent cytotoxicity of Ep-CAM-specific monoclonal antibody MT201 against breast cancer cell lines. Br J Cancer 2005;92:342–9.[Medline]
  34. Moretta A, Bottino C, Vitale M, et al. Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis. Annu Rev Immunol 2001;19:197–223.[CrossRef][Medline]
  35. Moretta A, Bottino C, Mingari MC, Biassoni R, Moretta L. What is a natural killer cell? Nat Immunol 2002;3:6–8.[CrossRef][Medline]
  36. Niwa R, Sakurada M, Kobayashi Y, et al. Enhanced natural killer cell binding and activation by low-fucose IgG1 antibody results in potent antibody-dependent cellular cytotoxicity induction at lower antigen density. Clin Cancer Res 2005;11:2327–36.[Abstract/Free Full Text]



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