Two mechanisms of the enhanced antibody-dependent cellular cytotoxicity (ADCC) efficacy of non-fucosylated therapeutic antibodies in human blood
© Iida et al; licensee BioMed Central Ltd. 2009
Received: 11 December 2007
Accepted: 18 February 2009
Published: 18 February 2009
Antibody-dependent cellular cytotoxicity (ADCC) has recently been identified as one of the critical mechanisms underlying the clinical efficacy of therapeutic antibodies, especially anticancer antibodies. Therapeutic antibodies fully lacking the core fucose of the Fc oligosaccharides have been found to exhibit much higher ADCC in humans than their fucosylated counterparts. However, data which show how fully non-fucosylated antibodies achieve such a high ADCC in human whole blood have not yet been disclosed. The precise mechanisms responsible for the high ADCC mediated by fully non-fucosylated therapeutic antibodies, even in the presence of human plasma, should be explained based on direct evidence of non-fucosylated antibody action in human blood.
Using a human ex vivo B-cell depletion assay with non-fucosylated and fucosylated anti-CD20 IgG1s rituximab, we monitored the binding of the therapeutic agents both to antigens on target cells (target side interaction) and to leukocyte receptors (FcγR) on effector cells (effector side interaction), comparing the intensities of ADCC in human blood.
In the target side interaction, down-modulation of CD20 on B cells mediated by anti-CD20 was not observed. Simple competition for binding to the antigens on target B cells between fucosylated and non-fucosylated anti-CD20s was detected in human blood to cause inhibition of the enhanced ADCC of non-fucosylated anti-CD20 by fucosylated anti-CD20. In the effector side interaction, non-fucosylated anti-CD20 showed sufficiently high FcγRIIIa binding activity to overcome competition from plasma IgG for binding to FcγRIIIa on natural killer (NK) cells, whereas the binding of fucosylated anti-CD20 to FcγRIIIa was almost abolished in the presence of human plasma and failed to recruit NK cells effectively. The core fucosylation levels of individual serum IgG1 from healthy donors was found to be so slightly different that it did not affect the inhibitory effect on the ADCC of fucosylated anti-CD20.
Our results demonstrate that removal of fucosylated antibody ingredients from antibody therapeutics elicits high ADCC in human blood by two mechanisms: namely, by evading the inhibitory effects both of plasma IgG on FcγRIIIa binding (effector side interaction) and of fucosylated antibodies on antigen binding (target side interaction).
The clinical successes of therapeutic antibodies have been demonstrated by improvement of overall survival and time to disease progression in various types of malignant diseases, such as breast, colon, and hematological cancer [1–5]. As molecular-targeted therapeutics, therapeutic antibodies that bind to specific cell-surface antigens on target cells can induce cytotoxicity via the effector functions of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) through the constant region of the antibody (Fc), and apoptosis of target cells directly [6–9]. Recently, some clinical evidence based on genetic analysis of leukocyte receptor (FcγR) polymorphisms of cancer patients treated with anti-CD20 IgG1 rituximab and anti-HER2 IgG1 trastuzumab therapies has revealed that ADCC is one of the critical mechanisms responsible for the clinical efficacy of these therapeutic antibodies [10–14]. A significant correlation has also been reported between the clinical responses to the therapies and the low-affinity FcγR polymorphisms in rituximab-treated patients of systemic lupus erythematosus (SLE) and Waldenstrom's macroglobulinemia, in Crohn's disease patients treated with anti-TNF-α IgG1 infliximab, and in pregnant women with fetal hemolytic disease treated with anti-RhD [12, 15–17]. Hence, ADCC enhancement technology is expected to play a key role in the development of therapeutic antibodies with improved clinical efficacy. Indeed, a number of preclinical and clinical trials using such non-fucosylated antibody therapeutics are now underway.
Although various therapeutic antibodies confer great benefits to patients, they also present serious issues of cost-effective performance; as it has been pointed out, some of the currently approved therapeutic antibodies are unable to induce more than remission of cancer despite the fairly high cost of the therapy. Recently, the inhibition of therapeutic-antibody ADCC by plasma IgG, probably through competition for binding of the therapeutics to FcγR on effector cells, is found to be at least partly responsible for the large dose requirement in antibody therapies [18, 19]. On the other hand, fully non-fucosylated anti-CD20 rituximab is shown to exhibit strong ADCC at lower concentrations (10 to 100 ng/mL) with much higher efficacy than that of its fucosylated counterparts, even in the presence of plasma IgG [20, 21]. However, the basic data elucidating how fully non-fucosylated antibodies show such a high ADCC in human whole blood have not yet been disclosed. The precise mechanisms responsible for the high ADCC mediated by fully non-fucosylated therapeutic antibodies, even in the presence of human plasma, should be explained based on direct evidence of non-fucosylated antibody action in human blood. Understanding the behavioral differences between non-fucosylated and fucosylated therapeutic antibodies in human blood can provide a new clue for improving antibody therapies.
In this study, we explored the mechanisms by which non-fucosylated therapeutic antibodies induce much higher ADCC in human blood than do fucosylated therapeutics. As a model of human in vivo, we employed a human B-cell depletion assay with anti-CD20 IgG1 rituximab, and focused on the actual binding to the two key cellular components of effector and target cells in human blood: namely, the binding through FcγRIIIa to natural killer (NK) cells (effector side interaction) and the binding through the antigen to target cells (target side interaction). This ex vivo model was considered to be suitable for analyzing the functions of therapeutic antibodies in human blood because it has been shown to reflect well the in vivo intravascular environment [18, 20, 22, 23]. Here, we discuss two ingenious mechanisms underlying the enhanced ADCC efficacy of fully non-fucosylated therapeutic antibodies in human blood, based on data reflecting the actions of antibody molecules in human blood.
Blood donors were randomly selected from healthy volunteers registered at Tokyo Research Laboratories, Kyowa Hakko, Co., Ltd. All donors gave written informed consent before the analyses. Human serum samples collected from 12 individuals (5 women and 7 men, aged 20 to 57 years) with written informed consent were purchased from ProMedDx (Norton, MA).
Non-fucosylated and fucosylated anti-CD20 IgG1s, and a triple amino acid-substituted anti-CD20 IgG1 rituximab mutant (S239D/S298A/I332E) were prepared as described previously [20, 24]. Anti-CD20 IgG1s were biotinylated using EZ-Link Sulfo-NHS-LC-biotin (Pierce, Rock-ford, IL) according to the manufacturer's instructions. The labeled anti-CD20s were purified by dialysis with Spectra/Pro® 2 Dialysis Membrane (Spectrum Laboratories, Inc., Rancho Dominguez, CA) in phosphate-buffered saline (PBS). The antigen-binding activity of the labeled anti-CD20s was measured by CD20-binding ELISA , and confirmed to be equivalent to that of the non-labeled anti-CD20 and rituximab (Rituxan) purchased from Genentech, Inc. (South San Francisco, CA) prior to use.
Analyses of human endogenous IgG1
Human endogenous IgG was fractionated from human serum samples of 20 healthy donors using MabSelect (Amersham Biosciences, Piscataway, NJ). The bound fraction contained IgG1, 2 and 4, whereas the bulk of IgG3 passed through the column. The pool of IgG1, 2, and 4, eluted from the MabSelect, was stored in 10 mM citrate buffer pH 6.0 with 0.15 M NaCl. The concentration of endogenous IgG1 in the pool was measured by human IgG1-specific ELISA using peroxidase-conjugated mouse anti-human IgG1 monoclonal antibodies (clone HP6069; Zymed Laboratories, South San Francisco, CA) as previously described . The Fc of the IgG1 was prepared from the pool by a common procedure including papain cleavage, purification with MabSelect, and Superose12 (Amersham Biosciences) gel filtration chromatography to analyze the Fc oligosaccharide structure.
Analyses of antibody-derived N-linked oligosaccharides
N-linked oligosaccharides were released by digestion of the purified anti-CD20 IgG1 and the Fc of human endogenous IgG1 with N-glycosidase F (Takara, Shiga, Japan). The released carbohydrates were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with a positive-ion mode as described previously . Monosaccharide composition of IgG1 was characterized by modified high-performance anion exchange chromatography (HPAEC) analysis as previously described .
Antigen binding on target cells
Human peripheral blood mononuclear cells (PBMC) were prepared from healthy donors, and washed and resuspended in stain buffer (PBS containing 1% bovine serum albumin (BSA)). B cells were purified from PBMC by B Cell Isolation Kit II (Miltenyi Biotec, Bergisch-Gladbach, Germany) as negative fractions passed through a magnetic separation column (autoMACS™; Miltenyi Biotec). The purity of isolated B cells was more than 95%, as assessed by flow cytometer FACSCalibur (BD Biosciences, San Jose, CA) using anti-CD19-FITC (clone J4.119; Beckman Coulter, Coulter, Miami, FL). Purified B cells were adjusted to 1 × 106 cells/mL and incubated with serial dilutions (0.001 to 10 μg/mL) of anti-CD20 s for 30 min to 4 h at 37°C. The cells were then washed, and incubated with phycoerythrin (PE)-conjugated mouse anti-human IgG (BD Biosciences Pharmingen, San Diego, CA) for 30 min at 4°C to detect binding of anti-CD20s on B cells using FACSCalibur.
Ex vivoB-cell depletion assay
Heparinized peripheral blood samples from healthy donors were incubated with serial dilutions (0.0001 to 10 μg/mL) of anti-CD20s for 4 h at 37°C, and CD19+CD2- B cells were analyzed by FACSCalibur as described previously . The cytotoxicity against CD19+CD2- B cells was calculated according to the following formula:
Cytotoxicity (%) = (1 - (%CD19+CD2- B cells treated with anti-CD20 IgG1)/(%CD19+CD2- B cells treated with PBS)) × 100.
Antigen-binding analysis in human blood
The antigen-binding activity of anti-CD20s on human B cells in human whole blood was analyzed by competitive inhibition assay with biotin-labeled anti-CD20 using flow cytometry. Heparinized peripheral blood from healthy donors was incubated simultaneously with 3.3 μg/mL of the biotinylated anti-CD20 (fucosylated or non-fucosylated) and serial dilutions (1 to 100 μg/mL) of non-labeled competitor anti-CD20s on ice for 30 min. Aliquots of samples were washed once with the stain buffer, and incubated with PE-conjugated streptavidin (Beckman Coulter) and anti-CD19-FITC on ice for 30 min. Stained cells were suspended in FACS Lysing Solution (BD Biosciences) to lyse erythrocytes and washed twice with the stain buffer. The binding intensity of biotinylated anti-CD20 to B cells detected with CD19+ was analyzed by FACSCalibur.
FcγRIIIa-binding analysis in human blood
The FcγRIIIa-binding activity of anti-CD20s on human NK cells in human whole blood was analyzed by flow cytometry using anti-idiotype monoclonal antibodies against rituximab . Heparinized peripheral blood samples from healthy donors or samples, depleted of plasma by washing and reconstituted with stain buffer, were incubated with serial dilutions (1.1 to 30 μg/mL) of anti-CD20s on ice for 30 min. After washing with the stain buffer, the cells were stained with anti-rituximab-FITC (clone MB2 A4, Abcam, Cambridge, UK) to detect anti-CD20s bound to FcγR, and simultaneously stained with PE-Cyanin 5.1 (PC5)-labeled anti-CD3 antibodies PC5 (clone UCHT1, Beckman Coulter) and PE-labeled anti-CD56 antibodies (clone N901, Beckman Coulter) to detect CD3-CD56+ NK cells. Following incubation on ice for 30 min, the intensity of anti-rituximab-FITC bound to NK cells detected with CD3-CD56+ was analyzed by FACSCalibur. To analyze whether or not the binding of anti-CD20 to NK cells was through FcγRIIIa, samples were incubated simultaneously with 100 μg/mL of non-labeled anti-CD16 antibodies (clone 3G8, Ancell, Bayport, MN) and 10 μg/mL of non-fucosylated anti-CD20 on ice for 30 min, and the anti-CD20 bound to NK cells was analyzed by FACSCalibur. Mouse IgG1 (clone 15H6s, Beckman Coulter) was used as an isotype control.
In vitroADCC assay
Human PBMC, including effector NK cells and target B cells, was prepared from healthy donors and suspended in RPMI 1640 medium supplemented with 5% fetal bovine serum. Then, the PBMC suspension (240 μL including 0.6 × 106 cells), auto plasma or commercial human serum (300 μL), and serial dilutions (0.001 to 10 μg/mL) of anti-CD20s (60 μL in PBS) were dispensed into each well of 24-well culture plates and incubated at 37°C for 4 h. The cytotoxicity against CD19+CD2- B cells was estimated as described above in an ex vivo B-cell depletion assay. The ADCC inhibitory activity of human serum was calculated according to the following formula:
ADCC inhibition (%) = (1 - (cytotoxicity (%) with human serum)/(cytotoxicity (%) without human serum)) × 100.
Generation of non-fucosylated and fucosylated anti-CD20s
Ex vivoADCC activity of anti-CD20s
Inhibitory effect of human plasma on binding of anti-CD20s to FcγRIIIa on NK cells
Inhibitory effect of fucosylated anti-CD20 on binding of non-fucosylated anti-CD20 to the target antigen on B cells
Inhibitory effect of individual human plasma on in vitroADCC induced by anti-CD20s
Currently, the application of human IgG1 fully lacking core fucose in the Fc oligosaccharides is expected to be a promising approach for next-generation therapeutic antibodies with improved efficacy, even when administrated at low doses in humans in vivo. In order to develop fully non-fucosylated therapeutic antibodies as new medicine, the pharmacodynamic characteristics of the antibodies should be elucidated: how fully non-fucosylated antibodies effectively mediate two key cellular component interactions of effector and target cells to exercise high ADCC and what the differences are between non-fucosylated and fucosylated antibodies in these actions. In this study, we addressed non-fucosylated antibody actions in human blood.
Firstly, we were surprised that the binding of fucosylated antibodies to NK cells was not detected, even at the high concentration of 30 μg/mL, in the presence of human plasma (Fig. 5). A common feature of the antibody therapies against cancers is that their anti-tumor efficacies typically require weekly administration of a high dose (2–8 mg/kg) over several months to maintain effective serum concentrations exceeding 10 μg/mL [32–34]. Thus, it is realized that currently-licensed therapeutic antibodies mostly do not bind to NK cells in human blood after administration. On the other hand, the binding of antibodies to target cells was detected at a concentration of 3.3 μg/mL, irrespective of the presence of human plasma (Fig. 1, 6). The antigen binding of therapeutic antibodies to target cells appears to be simply determined by their specificity and affinity. In current antibody therapies, administered therapeutic antibodies seem to have sufficient capability to concentrate in the target cells accessible from the bloodstream, but not to capture effector cells in the target sites effectively.
We confirmed the inhibitory effects of human serum IgG on the ADCC of therapeutic antibodies, consistent with previous reports [18–21, 35]. ADCC of fucosylated antibodies was significantly affected by human plasma IgG (Fig. 7a). In contrast, non-fucosylated antibodies showed much stronger binding to FcγRIIIa on NK cells, even in the presence of human plasma IgG, resulting in much higher potency and efficacy of non-fucosylated compared to fucosylated antibodies (Fig. 3a). In our experiments, the concentrations of therapeutic antibodies required for inducing ex vivo ADCC and binding to FcγRIIIa on NK cells were not necessarily consistent. This discrepancy seemed to reflect the fact that human serum IgG bound to FcγRIIIa on NK cells might be more effectively displaced by therapeutic antibodies bound to the surface of target cells than unbound forms, possibly due to an avidity effect via formation of a polyvalent FcγRIIIa-binding matrix, and consequently promoting recruitment of NK cells. Interestingly, a triple amino acid-substituted mutant (S239D/S298A/I332E) showed almost identical ex vivo ADCC and NK cell binding to that of the non-fucosylated wild type (Fig. 3a, 5), although the mutant was shown to have higher affinity than the non-fucosylated wild type for recombinant FcγRIIIa in vitro . The binding affinity estimated by solid-phase assay with recombinant protein forms might not necessarily be the same as the affinity to the native structure forms on the cell surface; thus, the equivalency in anti-CD20 efficacy was observed.
Before starting these experiments, we speculated that the concentration of human plasma IgG1 is so huge that its fucosylation level might affect the inhibitory activity of ADCC induced by therapeutic antibodies, even though the proportion of non-fucosylated IgG1 is very low. Indeed, human plasma contains 5 mg/mL of endogenous IgG1 , which is approximately 100-fold higher than the serum trough concentration of rituximab . However, contrary to our speculation, we could not detect any significant difference caused by variations in serum IgG1 fucosylation levels in healthy individuals (Fig. 7) although individual human serum IgG1 from 12 healthy donors showed slight variations in the concentration (5.3 to 7.5 mg/mL) and the content of non-fucosylated oligosaccharide structures (1.2 to 6.5%). Factors other than endogenous IgG1 concentration and fucosylation levels, such as variations of other endogenous IgG subclasses, might also be involved in the ADCC inhibitory activity of human serum IgG from healthy individuals. Some leukemia patients are reported to increase their relative levels of non-fucosylated Fc oligosaccharides . It is still thought to be interesting to explore whether individual patient variations in endogenous IgG1 fucosylation level might affect the efficacy of antibody therapies.
We also confirmed that enhanced ex vivo ADCC of non-fucosylated therapeutic antibodies is inhibited by their fucosylated counterparts, consistent with previous reports [20, 21, 27]. In our experiments, down-modulation of CD20 antigen on B cells mediated by anti-CD20 was not observed (Fig. 1), and simple competition for binding to the antigens on target B cells between fucosylated and non-fucosylated anti-CD20s was detected in human blood (Fig. 6), resulting in inhibition of the enhanced ADCC of non-fucosylated anti-CD20 by fucosylated anti-CD20. Thus, the mechanism by which ADCC induced by non-fucosylated antibodies is inhibited by fucosylated forms is mainly through competition of the two forms for the antigens on target cells. In other words, the density of the non-fucosylated form on the target cells is reduced by fucosylated form occupation, which causes an effect to shed the target antigens from capture by the therapeutic agent having high ADCC activity even in plasma. To overcome interference from human plasma IgG and recruit NK cells to the target cells effectively, the key is to coat the target cells with therapeutic agents having higher binding affinity to FcγRIIIa than plasma IgG, such as fully non-fucosylated IgG1. The non-fucosylated form of IgG1 is a natural component in human serum IgG, and is also observed as a trace ingredient in currently-licensed therapeutic antibodies [37–40]. It will be great benefits for patients if the proportion of the non-fucosylated form is increased in antibody therapeutics.
Our findings demonstrate that removal of fucosylated antibody ingredients from antibody therapeutics elicits high ADCC, even in human blood, via two mechanisms: namely, by evading the inhibitory effects both of plasma IgG on the binding of FcγRIIIa on NK cells and of fucosylated counterparts on the binding of the antigen on target cells. Thus, antibody therapy consisting of fully non-fucosylated IgG1, not including the fucosylated form, is worth to apply as next-generation antibody therapy with improved efficacy at a reduced dose for patients, which will have a great impact especially on human cancer therapy based on the ADCC mechanism.
The authors would like to thank Dr. George Spitalny for helpful suggestions and critical reading of the manuscript. We are also very grateful for the technical support provided by Miss Masako Wakitani and Ms. Ai Sato-Kubota.
- de Bono JS, Rowinsky EK: The ErbB receptor family: a therapeutic target for cancer. Trends Mol Med. 2002, 8 (Suppl 4): S19-26. 10.1016/S1471-4914(02)02306-7.View ArticlePubMedGoogle Scholar
- Forero A, Lobuglio AF: History of antibody therapy for non-Hodgkin's lymphoma. Semin Oncol. 2003, 30: 1-5. 10.1053/j.seminoncol.2003.10.002.View ArticlePubMedGoogle Scholar
- Grillo-Lopez AJ: Rituximab (Rituxan/Mab Thera): the first decade (1993–2003). Expert Rev Anticancer Ther. 2003, 3: 767-769. 10.1586/1473718.104.22.1687.View ArticlePubMedGoogle Scholar
- Vogel CL, Franco SX: Clinical experience with trastuzumab (Herceptin). Breast J. 2003, 9: 452-462. 10.1046/j.1524-4741.2003.09602.x.View ArticlePubMedGoogle Scholar
- Reichert JM, Rosensweig CJ, Faden LB, Dewitz MC: Monoclonal antibody successes in the clinic. Nat Biotechnol. 2005, 23: 1073-1078. 10.1038/nbt0905-1073.View ArticlePubMedGoogle Scholar
- Shan D, Ledbetter JA, Press OW: Signaling events involved in anti-CD20-induced apoptosis of malignant human B cells. Cancer Immunol Immunother. 2000, 48: 673-683. 10.1007/s002620050016.View ArticlePubMedGoogle Scholar
- Carter P: Improving the efficacy of antibody-based cancer therapies. Nat Rev Cancer. 2001, 1: 118-129. 10.1038/35101072.View ArticlePubMedGoogle Scholar
- Glennie MJ, Winkel van de JG: Renaissance of cancer therapeutic antibodies. Drug Discov Today. 2003, 8: 503-510. 10.1016/S1359-6446(03)02714-4.View ArticlePubMedGoogle Scholar
- Smith MR: Rituximab (monoclonal anti-CD20 antibody): mechanisms of action and resistance. Oncogene. 2003, 22: 7359-7368. 10.1038/sj.onc.1206939.View ArticlePubMedGoogle Scholar
- Cartron G, Dacheux L, Salles G, Solal-Celigny P, Bardos P, Colombat P, Watier H: Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor FcγRIIIa gene. Blood. 2002, 99: 754-758. 10.1182/blood.V99.3.754.View ArticlePubMedGoogle Scholar
- Anolik JH, Campbell D, Felgar RE, Young F, Sanz I, Rosenblatt J, Looney RJ: The relationship of FcγRIIIa genotype to degree of B cell depletion by rituximab in the treatment of systemic lupus erythematosus. Arthritis Rheum. 2003, 48: 455-459. 10.1002/art.10764.View ArticlePubMedGoogle Scholar
- Weng WK, Levy R: Two immunoglobulin G fragment C receptor polymorphisms independently predict response to rituximab in patients with follicular lymphoma. J Clin Oncol. 2003, 21: 3940-3947. 10.1200/JCO.2003.05.013.View ArticlePubMedGoogle Scholar
- Dall'Ozzo S, Tartas S, Paintaud G, Cartron G, Colombat P, Bardos P, Watier H, Thibault G: Rituximab-dependent cytotoxicity by natural killer cells: influence of FCGR3A polymorphism on the concentration-effect relationship. Cancer Res. 2004, 64: 4664-4669. 10.1158/0008-5472.CAN-03-2862.View ArticlePubMedGoogle Scholar
- Gennari R, Menard S, Fagnoni F, Ponchio L, Scelsi M, Tagliabue E, Castiglioni F, Villani L, Magalotti C, Gibelli N, Oliviero B, Ballardini B, Da Prada G, Zambelli A, Costa A: Pilot study of the mechanism of action of preoperative trastuzumab in patients with primary operable breast tumors overexpressing HER2. Clin Cancer Res. 2004, 10: 5650-5655. 10.1158/1078-0432.CCR-04-0225.View ArticlePubMedGoogle Scholar
- Louis E, El Ghoul Z, Vermeire S, Dall'Ozzo S, Rutgeerts P, Paintaud G, Belaiche J, De Vos M, Van Gossum A, Colombel JF, Watier H: Association between polymorphism in IgG Fc receptor IIIa coding gene and biological response to infliximab in Crohn's disease. Aliment Pharmacol Ther. 2004, 19: 511-519. 10.1111/j.1365-2036.2004.01871.x.View ArticlePubMedGoogle Scholar
- Miescher S, Spycher MO, Amstutz H, De Haas M, Kleijer M, Kalus UJ, Radtke H, Hubsch A, Andresen I, Martin RM, Bichler J: A single recombinant anti-RhD IgG prevents RhD immunization: association of RhD-positive red blood cell clearance rate with polymorphisms in the FcγRIIA and FcγIIIA genes. Blood. 2004, 103: 4028-4035. 10.1182/blood-2003-11-3929.View ArticlePubMedGoogle Scholar
- Treon SP, Hansen M, Branagan AR, Verselis S, Emmanouilides C, Kimby E, Frankel SR, Touroutoglou N, Turnbull B, Anderson KC, Maloney DG, Fox EA: Polymorphisms in FcγRIIIA (CD16) receptor expression are associated with clinical response to rituximab in Waldenström's macroglobulinemia. J Clin Oncol. 2005, 23: 474-481. 10.1200/JCO.2005.06.059.View ArticlePubMedGoogle Scholar
- Vugmeyster Y, Howell K: Rituximab-mediated depletion of cynomolgus monkey B cells in vitro in different matrices: possible inhibitory effect of IgG. Int Immunopharmacol. 2004, 4: 1117-1124. 10.1016/j.intimp.2004.04.015.View ArticlePubMedGoogle Scholar
- Preithner S, Elm S, Lippold S, Locher M, Wolf A, da Silva AJ, Baeuerle PA, Prang NS: High concentrations of therapeutic IgG1 antibodies are needed to compensate for inhibition of antibody-dependent cellular cytotoxicity by excess endogenous immunoglobulin G. Mol Immunol. 2006, 43: 1183-1193. 10.1016/j.molimm.2005.07.010.View ArticlePubMedGoogle Scholar
- Iida S, Misaka H, Inoue M, Shibata M, Nakano R, Yamane-Ohnuki N, Wakitani M, Yano K, Shitara K, Satoh M: Nonfucosylated therapeutic IgG1 antibody can evade the inhibitory effect of serum immunoglobulin G on antibody-dependent cellular cytotoxicity through its high binding to FcγRIIIa. Clin Cancer Res. 2006, 12: 2879-2887. 10.1158/1078-0432.CCR-05-2619.View ArticlePubMedGoogle Scholar
- Satoh M, Iida S, Shitara K: Non-fucosylated therapeutic antibodies as next-generation therapeutic antibodies. Expert Opin Biol Ther. 2006, 6: 1161-1173. 10.1517/14712522.214.171.1241.View ArticlePubMedGoogle Scholar
- Vugmeyster Y, Howell K, Bakshl A, Flores C, Canova-Davis E: Effect of anti-CD20 monoclonal antibody, Rituxan, on cynomolgus monkey and human B cells in a whole blood matrix. Cytometry A. 2003, 52: 101-109. 10.1002/cyto.a.10030.View ArticlePubMedGoogle Scholar
- Lazar GA, Dang W, Karki S, Vafa O, Peng JS, Hyun L, Chan C, Chung HS, Eivazi A, Yoder SC, Vielmetter J, Carmichael DF, Hayes RJ, Dahiyat BI: Engineered antibody Fc variants with enhanced effector function. Proc Natl Acad Sci USA. 2006, 103: 4005-4010. 10.1073/pnas.0508123103.View ArticlePubMedPubMed CentralGoogle Scholar
- Masuda K, Kubota T, Kaneko E, Iida S, Wakitani M, Kobayashi-Natsume Y, Kubota A, Shitara K, Nakamura K: Enhanced binding affinity for FcγRIIIa of fucose-negative antibody is sufficient to induce maximal antibody-dependent cellular cytotoxicity. Mol Immunol. 2007, 44: 3122-3131. 10.1016/j.molimm.2007.02.005.View ArticlePubMedGoogle Scholar
- Yamane-Ohnuki N, Kinoshita S, Inoue-Urakubo M, Kusunoki M, Iida S, Nakano R, Wakitani M, Niwa R, Sakurada M, Uchida K, Shitara K, Satoh M: Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity. Biotechnol Bioeng. 2004, 87: 614-622. 10.1002/bit.20151.View ArticlePubMedGoogle Scholar
- Jefferis R, Reimer CB, Skvaril F, de Lange GG, Goodall DM, Bentley TL, Phillips DJ, Vlug A, Harada S, Radl J, Claassen E, Boersma JA, Coolen J: Evaluation of monoclonal antibodies having specificity for human IgG subclasses: results of the 2nd IUIS/WHO collaborative study. Immunol Lett. 1992, 31: 143-168. 10.1016/0165-2478(92)90141-A.View ArticlePubMedGoogle Scholar
- Kanda Y, Yamada T, Mori K, Okazaki A, Inoue M, Kitajima-Miyama K, Kuni-Kamochi R, Nakano R, Yano K, Kakita S, Shitara K, Satoh M: Comparison of biological activity among nonfucosylated therapeutic IgG1 antibodies with three different N-linked Fc oligosaccharides: the high-mannose, hybrid, and complex types. Glycobiology. 2007, 17: 104-118. 10.1093/glycob/cwl057.View ArticlePubMedGoogle Scholar
- Shinkawa T, Nakamura K, Yamane N, Shoji-Hosaka E, Kanda Y, Sakurada M, Uchida K, Anazawa H, Satoh M, Yamasaki M, Hanai N, Shitara K: 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: 3466-3473. 10.1074/jbc.M210665200.View ArticlePubMedGoogle Scholar
- Cragg MS, Bayne MB, Tutt AL, French RR, Beers S, Glennie MJ, Illidge TM: A new anti-idiotype antibody capable of binding rituximab on the surface of lymphoma cells. Blood. 2004, 104: 2540-2542. 10.1182/blood-2004-05-1733.View ArticlePubMedGoogle Scholar
- Schauer U, Stemberg F, Rieger CH, Borte M, Schubert S, Riedel F, Herz U, Renz H, Wick M, Carr-Smith HD, Bradwell AR, Herzog W: IgG subclass concentrations in certified reference material 470 and reference values for children and adults determined with the binding site reagents. Clin Chem. 2003, 49: 1924-1929. 10.1373/clinchem.2003.022350.View ArticlePubMedGoogle Scholar
- Kuroda Y, Nakata M, Makino A, Matsumoto A, Ohashi K, Itahashi K, Takeuchi F, Goto M, Kojima N, Mizuochi T: Structural studies on IgG oligosaccharides of patients with primary Sjogren's syndrome. Glycoconj J. 2002, 19: 23-31. 10.1023/A:1022528829799.View ArticlePubMedGoogle Scholar
- Berinstein NL, Grillo-López AJ, White CA, Bence-Bruckler I, Maloney D, Czuczman M, Green D, Rosenberg J, McLaughlin P, Shen D: Association of serum rituximab (IDEC-C2B8) concentration and anti-tumor response in the treatment of recurrent low-grade or follicular non-Hodgkin's lymphoma. Ann Oncol. 1998, 9: 995-1001. 10.1023/A:1008416911099.View ArticlePubMedGoogle Scholar
- Goldenberg MM: Trastuzumab, a recombinant DNA-derived humanized monoclonal antibody, a novel agent for the treatment of metastatic breast cancer. Clin Ther. 1999, 21: 309-318. 10.1016/S0149-2918(00)88288-0.View ArticlePubMedGoogle Scholar
- Baselga J, Albanell J: Mechanism of action of anti-HER2 monoclonal antibodies. Ann Oncol. 2001, 12 (Suppl 1): S35-41. 10.1023/A:1011163824080.View ArticlePubMedGoogle Scholar
- Nechansky A, Schuster M, Jost W, Siegl P, Wiederkum S, Gorr G, Kircheis R: Compensation of endogenous IgG mediated inhibition of antibody-dependent cellular cytotoxicity by glyco-engineering of therapeutic antibodies. Mol Immunol. 2007, 44: 1815-1817. 10.1016/j.molimm.2006.08.013.View ArticlePubMedGoogle Scholar
- Kondo A, Hosokawa Y, Kiso M, Hasegawa A, Kato I: Analysis of oligosaccharides of human IgG from serum of leukemia patients. Biochem Mol Biol Int. 1994, 32: 897-902.PubMedGoogle Scholar
- Mizuochi T, Taniguchi T, Shimizu A, Kobata A: Structural and numerical variations of the carbohydrate moiety of immunoglobulin G. J Immunol. 1982, 129: 2016-2020.PubMedGoogle Scholar
- Harada H, Kamei M, Tokumoto Y, Yui S, Koyama F, Kochibe N, Endo T, Kobata A: Systematic fractionation of oligosaccharides of human immunoglobulin G by serial affinity chromatography on immobilized lectin columns. Anal Biochem. 1987, 164: 374-381. 10.1016/0003-2697(87)90507-0.View ArticlePubMedGoogle Scholar
- Schenerman MA, Hope JN, Kletke C, Singh JK, Kimura R, Tsao EI, Folena-Wasserman G: Comparability testing of a humanized monoclonal antibody (Synagis) to support cell line stability, process validation, and scale-up for manufacturing. Biologicals. 1999, 27: 203-215. 10.1006/biol.1999.0179.View ArticlePubMedGoogle Scholar
- Kamoda S, Nomura C, Kinoshita M, Nishiura S, Ishikawa R, Kakehi K, Kawasaki N, Hayakawa T: Profiling analysis of oligosaccharides in antibody pharmaceuticals by capillary electrophoresis. J Chromatogr A. 2004, 1050: 211-216.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/9/58/prepub
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