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
2003; Elsevier BV; Volume: 278; Issue: 5 Linguagem: Inglês
10.1074/jbc.m210665200
ISSN1083-351X
AutoresToyohide Shinkawa, Kazuyasu Nakamura, Naoko Yamane, Emi Shoji-Hosaka, Yutaka Kanda, Mikiko Sakurada, Kazuhisa Uchida, Hideharu Anazawa, Mitsuo Satoh, Motoo Yamasaki, Nobuo Hanai, Kenya Shitara,
Tópico(s)T-cell and B-cell Immunology
ResumoAn anti-human interleukin 5 receptor (hIL-5R) humanized immunoglobulin G1 (IgG1) and an anti-CD20 chimeric IgG1 produced by rat hybridoma YB2/0 cell lines showed more than 50-fold higher antibody-dependent cellular cytotoxicity (ADCC) using purified human peripheral blood mononuclear cells as effector than those produced by Chinese hamster ovary (CHO) cell lines. Monosaccharide composition and oligosaccharide profiling analysis showed that low fucose (Fuc) content of complex-type oligosaccharides was characteristic in YB2/0-produced IgG1s compared with high Fuc content of CHO-produced IgG1s. YB2/0-produced anti-hIL-5R IgG1 was subjected to Lens culinaris aggulutin affinity column and fractionated based on the contents of Fuc. The lower Fuc IgG1 had higher ADCC than the IgG1 before separation. In contrast, the content of bisecting GlcNAc of the IgG1 affected ADCC much less than that of Fuc. In addition, the correlation between Gal and ADCC was not observed. When the combined effect of Fuc and bisecting GlcNAc was examined in anti-CD20 IgG1, only a severalfold increase of ADCC was observed by the addition of GlcNAc to highly fucosylated IgG1. Quantitative PCR analysis indicated that YB2/0 cells had lower expression level of FUT8 mRNA, which codes α1,6-fucosyltransferase, than CHO cells. Overexpression of FUT8 mRNA in YB2/0 cells led to an increase of fucosylated oligosaccharides and decrease of ADCC of the IgG1. These results indicate that the lack of fucosylation of IgG1 has the most critical role in enhancement of ADCC, although several reports have suggested the importance of Gal or bisecting GlcNAc and provide important information to produce the effective therapeutic antibody. An anti-human interleukin 5 receptor (hIL-5R) humanized immunoglobulin G1 (IgG1) and an anti-CD20 chimeric IgG1 produced by rat hybridoma YB2/0 cell lines showed more than 50-fold higher antibody-dependent cellular cytotoxicity (ADCC) using purified human peripheral blood mononuclear cells as effector than those produced by Chinese hamster ovary (CHO) cell lines. Monosaccharide composition and oligosaccharide profiling analysis showed that low fucose (Fuc) content of complex-type oligosaccharides was characteristic in YB2/0-produced IgG1s compared with high Fuc content of CHO-produced IgG1s. YB2/0-produced anti-hIL-5R IgG1 was subjected to Lens culinaris aggulutin affinity column and fractionated based on the contents of Fuc. The lower Fuc IgG1 had higher ADCC than the IgG1 before separation. In contrast, the content of bisecting GlcNAc of the IgG1 affected ADCC much less than that of Fuc. In addition, the correlation between Gal and ADCC was not observed. When the combined effect of Fuc and bisecting GlcNAc was examined in anti-CD20 IgG1, only a severalfold increase of ADCC was observed by the addition of GlcNAc to highly fucosylated IgG1. Quantitative PCR analysis indicated that YB2/0 cells had lower expression level of FUT8 mRNA, which codes α1,6-fucosyltransferase, than CHO cells. Overexpression of FUT8 mRNA in YB2/0 cells led to an increase of fucosylated oligosaccharides and decrease of ADCC of the IgG1. These results indicate that the lack of fucosylation of IgG1 has the most critical role in enhancement of ADCC, although several reports have suggested the importance of Gal or bisecting GlcNAc and provide important information to produce the effective therapeutic antibody. antibody-dependent cellular cytotoxicity human interleukin 5 receptor fucose mannose Chinese hamster ovary the constant region of the antibody peripheral blood mononuclear cell L. culinaris aggulutin P. vulgaris E4 high performance liquid chromatography long terminal repeat N-acetylglucosaminyltransferase III Antibody-dependent cellular cytotoxicity (ADCC),1 a lytic attack on antibody-targeted cells, is triggered upon binding of lymphocyte receptors (FcγRs) to the constant region (Fc) of the antibodies. ADCC is considered to be a major function of some of the therapeutic antibodies, although antibodies have multiple therapeutic functions (e.g. antigen binding, induction of apoptosis, and complement-dependent cellular cytotoxicity) (1Lewis G.D. Figari I. Fendly B. Wong W.L. Carter P. Gorman C. Shepard H.M. Cancer Immunol. Immunother. 1993; 37: 255-263Google Scholar, 2Clynes R.A. Towers T.L. Presta L.G. Ravetch J.V. Nat. Med. 2000; 6: 443-446Google Scholar). One IgG molecule contains two N-linked oligosaccharide sites in its Fc region (3Rademacher T.W. Homans S.W. Perekh R.B. Dwek R.A. Biochem. Soc. Symp. 1986; 51: 131-148Google Scholar). The general structure of N-linked oligosaccharide on IgG is complex-type, characterized by a mannosyl-chitobiose core (Man3GlcNAc2-Asn) with or without bisecting GlcNAc/l-fucose (Fuc) and other chain variants including the presence or absence of Gal and sialic acid. In addition, oligosaccharides may contain zero (G0), one (G1), or two (G2) Gal. Recent studies have shown that engineering the oligosaccharides of IgGs may yield optimized ADCC. ADCC requires the presence of oligosaccharides covalently attached at the conserved Asn297 in the Fc region and is sensitive to change in the oligosaccharide structure. In the oligosaccharide, sialic acid of IgG has no effect on ADCC (4Boyd P.N. Lines A.C. Patel A.K. Mol. Immunol. 1995; 32: 1311-1318Google Scholar). The relationship between the Gal residue and ADCC is controversial. Boyd et al. (4Boyd P.N. Lines A.C. Patel A.K. Mol. Immunol. 1995; 32: 1311-1318Google Scholar) have shown that obvious change was not found in ADCC after removal of the majority of the Gal residues. However, several reports have shown that Gal residues enhance ADCC (5Kumpel B.M. Rademacher T.W. Rook G.A. Williams P.J. Wilson I.B. Hum. Antib. Hybrid. 1994; 5: 143-151Google Scholar, 6Kumpel B.M. Wang Y. Griffiths H.L. Hadley A.G. Rook G.A. Hum. Antib. Hybrid. 1995; 6: 82-88Google Scholar). Several groups have focused on bisecting GlcNAc, which is a β1,4-GlcNAc residue transferred to a core β-mannose (Man) residue, and it has been implicated in biological activity of therapeutic antibodies (7Lifely M.R. Hale C. Boyce S. Keen M.J. Phillips J. Glycobiology. 1995; 5: 813-822Google Scholar). N-Acetylglucosaminyltransferase III (GnTIII), which catalyzes the addition of the bisecting GlcNAc residue to the N-linked oligosaccharide (8Narisimhan S. J. Biol. Chem. 1982; 257: 10235-10242Google Scholar), has been expressed in a Chinese hamster ovary (CHO) cell line with an anti-neuroblastoma IgG1 and resulted in greater ADCC (9Umana P. Jean-Mairet J. Moudry R. Amstutz H. Bailey J.E. Nat. Biotechnol. 1999; 17: 176-180Google Scholar). Moreover, expression of GnTIII in a recombinant CHO cell line has led to the increase in ADCC of the anti-CD20 antibody (10Davies J. Jiang L. Pan L.Z. LaBarre M.J. Anderson D. Reff M. Biotechnol. Bioeng. 2001; 74: 288-294Google Scholar). Recently, Shields et al. have revealed the effect of fucosylated oligosaccharide on antibody effector functions, including binding to human FcγR, human C1q, human FcRn, and ADCC (11Shields R.L. Lai J. Keck R. O'Connell L.Y. Hong K. Meng Y.G. Weikert S.H. Presta L.G. J. Biol. Chem. 2002; 277: 26733-26740Google Scholar). The Fuc-deficient IgG1s have shown 50-fold increased binding to FcγRIIIa and enhanced ADCC. Nevertheless, there are no data on comparison of the effect of Fuc, Gal, and GlcNAc or the combined effect of Fuc and bisecting GlcNAc. Here, we describe the correlation between glycosylation of human IgG1 and ADCC and demonstrate that Fuc showed the critical role for enhancing ADCC out of several sugar residues reported previously. We unexpectedly found that human IgG1 produced by rat hybridoma YB2/0 cells showed extremely high ADCC at more than 50-fold lower concentration of those produced by CHO cells. YB2/0-produced IgG1 had lower Fuc content than CHO-produced IgG1. IgG1 containing lower fucosylated oligosaccharides, which was fractionated by Lens culinaris aggulutin (LCA) lectin affinity chromatography, showed higher ADCC before separation. In contrast, the addition of bisecting GlcNAc to IgG1 enhanced ADCC much less effectively than defucosylation. The effect of bisecting GlcNAc was only observed in highly fucosylated IgG1. YB2/0 cells expressed a lower level of FUT8 (α1,6-fucosyltransferase gene) mRNA than CHO cells, and overexpression of FUT8 in YB2/0 led the increase of fucosylation of IgG1 and the decrease of ADCC. Rat hybridoma YB2/0 cells were purchased from the American Type Culture Collection (ATCC; CRL-1662). CHO cell line DG44 (12Urlaub G. Mitchell P.J. Kas E. Chasin L.A. Funanage V.L. Myoda T.T. Hamlin J. Somatic Cell Mol. Genet. 1986; 12: 555-566Google Scholar), for wild type IgG1 production, was kindly provided by Dr. Lawrence Chasin (Columbia University). LEC10, a variant CHO cell line overexpressing GnTIII (13Campbell C. Stanley P. J. Biol. Chem. 1984; 259: 13370-13378Google Scholar), was kindly provided by Dr. Pamela Stanley (Albert Einstein College of Medicine). For the generation of human IgG1 of humanized anti-human interleukin-5 receptor (hIL-5R) α-chain and chimeric anti-CD20, the appropriate humanized or murine VL and VH cDNAs were subcloned into the previously described pKANTEX93 vector (14Nakamura K. Tanaka Y. Fujino I. Hirayama N. Shitara K. Hanai N. Mol. Immunol. 2000; 37: 1035-1046Google Scholar). The cDNA coding for the VL and VH region of each antibody was constructed by the PCR-based method (14Nakamura K. Tanaka Y. Fujino I. Hirayama N. Shitara K. Hanai N. Mol. Immunol. 2000; 37: 1035-1046Google Scholar). In the case of chimeric anti-CD20 antibody, the cDNA sequence of each V region was designed as the same with that of RituxanTM (VL: GenBankTM accession number AR015962; VH: GenBankTM accession number AR000013). Establishment of anti-hIL-5R humanized IgG1 will be described elsewhere. 2M. Koike, E. Shoji-Hosaka, K. Nakamura, A. Furuya, K. Shitara, and N. Hanai, manuscript in preparation. Antibody expression vectors were introduced into YB2/0 cells or DG44 cells via electroporation and selected for gene amplification in methotrexate-containing medium (14Nakamura K. Tanaka Y. Fujino I. Hirayama N. Shitara K. Hanai N. Mol. Immunol. 2000; 37: 1035-1046Google Scholar). The anti-hIL-5R IgG1-producing YB2/0 cell line was suspended in GIT medium (Wako, Osaka, Japan) containing 0.5 mg/ml G418 and 200 nm methotrexate to give a density of 3 × 105 cells/ml and dispensed in suspension culture flasks (Greiner, Frickenhausen, Germany). The anti-hIL-5R IgG1-producing CHO cell line was suspended in the EX-CELL302 medium (JRH, Kansas City, MO) containing 3 mml-Gln, 0.5% chemically defined lipid concentrate (Invitrogen), and 0.3% PLURONIC F-68 (Invitrogen) to give a density of 3 × 105 cells/ml and cultured using spinner flasks (Asahi Techno Glass, Tokyo, Japan) under agitating at a rate of 100 rpm. The anti-CD20 IgG1-producing YB2/0 cell line was suspended in the hybridoma-SFM medium (Invitrogen) containing 5% Daigo's GF21 (Wako) and 200 nm methotrexate to give a density of 1 × 105 cells/ml and dispensed in suspension culture flasks. The flasks were incubated under conditions of 37 °C in humid air containing 5% CO2. After 8 or 10 days of incubation, the culture supernatants were recovered. The culture supernatants containing anti-hIL-5R IgG1 from YB2/0 cells and CHO cells and anti-CD20 IgG1 from YB2/0 cells were clarified by centrifugation and passed through a 0.2-μm filter. The IgG1 bound to a PROSEP-A (Millipore) column was eluted with 0.1 m citrate buffer (pH 3.5). Then the antibody was subjected to a Sephacryl S-300 (Amersham Biosciences) column. The buffer composition of the YB2/0-produced anti-CD20 IgG1 was changed to that for RituxanTM (9.0 mg/ml sodium chloride, 7.35 mg/ml sodium citrate dihydrate, 0.7 mg/ml polysorbate 80). The purity of IgG1 was confirmed by SDS-PAGE. YB2/0- and CHO-produced humanized anti-hIL-5R IgG1 were designated as KM8399 and KM8404, respectively. YB2/0-produced chimeric anti-CD20 IgG1 was designated as KM3065. RituxanTM (chimeric mouse/human anti-CD20 monoclonal antibody derived from the CHO cell line) was purchased from Genentech (South San Francisco, CA)/IDEC Pharmaceutical (San Diego, CA). An ADCC assay was performed by 51Cr release assay as reported previously (15Ohta S. Honda A. Tokutake Y. Yoshida H. Hanai N. Cancer Immunol. Immunother. 1993; 36: 260-266Google Scholar). Briefly, target cells (1 × 106), a murine T cell line CTLL-2 (h5R) expressing hIL-5R α-chain and β-chain (16Takaki S. Murata Y. Kitamura T. Miyajima A. Tominaga A. Takatsu K. J. Exp. Med. 1993; 177: 1523-1529Google Scholar), were labeled with 3.7 MBq of Na251CrO4 at 37 °C for 1.5 h. Human effector cells were peripheral blood mononuclear cells (PBMC) purified from healthy donors using Polymorphprep (Nycomed Pharma AS, Roskilde, Norway). Aliquots of the 51Cr-labeled target cells were dispensed into 96-well U-bottomed plates (1 × 104/50 μl) and incubated with serial dilutions of antibodies (50 μl) in the presence of human effector cells (100 μl) at an E/T ratio of 90/1. After 4 h of incubation at 37 °C, the plates were centrifuged, and the radioactivity in the supernatants was measured using a γ counter. The percentage of specific cytolysis was calculated from the counts of samples according to the formula, %specific lysis=100×(E−S)/(M−S)Equation 1 where E represents the experimental release (cpm in the supernatant from target cells incubated with antibody and effector cells), S is the spontaneous release (cpm in the supernatant from target cells incubated with medium alone), and M is the maximum release (cpm released from target cells lysed with 1 mol/liter HCl). An ADCC assay was performed by a lactate dehydrogenase release assay. Aliquots of target cells, a human B lymphoma cell line Raji (number 9012, purchased from JCRB, Tokyo, Japan), were distributed into 96-well U-bottomed plates (1 × 104/50 μl) and incubated with serial dilutions of antibodies (50 μl) in the presence of human effector cells (100 μl) at an E/T ratio of 25:1 or 20:1. Human effector cells were PBMC purified from healthy donors using Lymphoprep (Axis Shield, Dundee, UK). After a 4-h incubation at 37 °C, the plate was centrifuged, and the lactate dehydrogenase activity in the supernatants was measured using a nonradioactive cytotoxicity assay kit (Promega, Madison, WI). The percentage of specific cytolysis was calculated from the activities of samples according to the formula, %specific lysis=100×(E−SE−ST)/(M−ST)Equation 2 where E represents the experimental release (activity in the supernatant from target cells incubated with antibody and effector cells), S E is the spontaneous release in the presence of effector cells (activity in the supernatant from effector cells), S T is the spontaneous release of target cells (activity in the supernatant from target cells incubated with medium alone), and M is the maximum release of target cells (activity released from target cells lysed with 9% Triton X-100). Oligosaccharides were prepared from 100 μg of IgG1 by hydrazinolysis and N-acetylation by the methods reported previously (17Hase S. Ibuki T. Ikenaka T. J. Biochem. (Tokyo). 1984; 95: 197-203Google Scholar). Oligosaccharides were pyridylaminated (17Hase S. Ibuki T. Ikenaka T. J. Biochem. (Tokyo). 1984; 95: 197-203Google Scholar) and analyzed by reverse-phase HPLC using a Shim-pack CLC-ODS column (60 × 150 mm; Shimadzu, Kyoto, Japan) (18Takahashi N. Hotta T. Ishihara H. Mori M. Tejima S. Bligny R. Akazawa T. Biochemistry. 1986; 25: 388-395Google Scholar) with slight modifications. Elution was performed at a flow rate of 1.0 ml/min at 55 °C using two solvents, A and B. Solvent A was 10 mmsodium phosphate buffer (pH 3.8), and solvent B was 10 mmsodium phosphate buffer (pH 3.8) containing 0.5% 1-butanol (Sigma). The column was equilibrated with solvent A. After injection of sample, the ratio of solvent B to A was increased with a linear gradient to 60:40 in 80 min. The elution profile was monitored by fluorescence detection with excitation at 320 nm and emission at 400 nm. The oligosaccharide peak assignments were made according to retention time comparison with PA-labeled oligosaccharide standards (TaKaRa Bio, Otsu, Japan). The peak area was used to calculate the percentage of each oligosaccharide, since the relative fluorescence was the same on a molar basis for each component (17Hase S. Ibuki T. Ikenaka T. J. Biochem. (Tokyo). 1984; 95: 197-203Google Scholar). To identify the structure, each oligosaccharide fraction that separated as a peak was collected and evaporated to dryness under vacuum prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Monosaccharides were released from an aliquoted quantity of IgG1 by heating with 4m trifluoroacetic acid at 100 °C for 2 h. Monosaccharides were dried under vacuum and reconstituted in water prior to high performance anion exchange chromatography analysis. Monosaccharides were analyzed using a waveform and DX500 system (DIONEX, Sunnyvale, CA) described previously (19McGuire J.M. Douglas M. Smith K.D. Carbohydr. Res. 1996; 292: 1-9Google Scholar). A CarboPac PA-1 column (DIONEX) was used to resolve monosaccharides with a flow rate of 0.8 ml/min at 35 °C. After injection of samples, the monosaccharides were resolved with 18 mm NaOH for 30 min, and the column was regenerated by elution with 500 mm NaOH for 10 min. The column was held 18 mm NaOH for 30 min prior to the next injection. LCA or Phaseolus vulgaris E4 (PHA-E4) were used as ligands of lectin affinity HPLC for separation of IgG1 based on the content of Fuc or bisecting GlcNAc, respectively. An LA-LCA (4.6 × 150 mm; HONEN, Tokyo, Japan) column was installed in the LC-6A HPLC system (Shimadzu). Purified antibodies dissolved in 10 mmKH2PO4 were applied to the column previously equilibrated with 50 mm Tris-H2SO4(pH 7.3). The column was eluted with a linear gradient of the buffer containing 0.2 mα-methyl-d-mannoside (Nacalai Tesque, Kyoto, Japan) in 60 min. An LA-PHA-E4 (4.6 × 150 mm; HONEN) column was installed in the LC-6A HPLC system (Shimadzu). Purified antibodies were applied to the column, previously equilibrated with 50 mm Tris-H2SO4 (pH 8.0) (A buffer). The column was eluted with the buffer containing 0.1 mK2B4O7 (B buffer). Elution was followed by linear gradients from 0 to 58% B buffer in 35 min and then 100% B buffer for 5 min. The column was equilibrated with 100% A buffer for 20 min before the next injection. Each chromatography was performed at room temperature and a flow rate of 0.5 ml/min. A mammalian expression vector, pAGE249, which was derived by excision of a 2.7-kbSphI-SphI fragment containing the dihydrofolate reductase gene expression cassette from pAGE249 (20Sasaki K. Kurata K. Funayama K. Nagata M. Watanabe E. Ohta S. Hanai N. Nishi T. J. Biol. Chem. 1994; 269: 14730-14737Google Scholar), was employed. This plasmid contained a hygromycin-resistant gene driven by the herpes simplex virus thymidine kinase gene promoter. The murine FUT8 cDNA (1728 bp) (21Hayashi H. Yoneda A. Asada M. Ikekita M. Imamura T. DNA Seq. 2000; 11: 91-96Google Scholar) was inserted into pAGE248 under the control of the Moloney murine leukemia virus 3′-LTR promoter to form a plasmid designated as pAGEmfFUT8. Two FspI restriction sites located within the plasmid backbone enabled linearization of the expression vector construct, prior to transfection of cells. FUT8 expression vector, pAGEmfFUT8, was introduced into anti-CD20-IgG1-producing YB2/0 cells via electroporation, and FUT8-overexpressing YB2/0 cells, 3065ft8–72, were selected in 0.5 mg/ml hygromycin-B (Sigma)-containing medium. Total RNA was isolated from 1.0 × 107 YB2/0 cells, FUT8-overexpressing YB2/0 cells, or CHO/DG44 cells using the RNeasy minikit (Qiagen, Tokyo, Japan) and incubated for 1 h at 37 °C with 20 units of RNase-free DNase (RQ1; Promega) to degrade genomic DNA. After DNA digestion, the total RNA was purified again using the RNeasy minikit. The single-strand cDNA was synthesized from 3 μg of each total RNA using the Superscript first strand synthesis system for RT-PCR (Invitrogen). The 50-fold diluted reaction mixture was used as a template for the following competitive RT-PCR. Quantification of FUT8 transcripts was carried out using competitive RT-PCR in which a 979-bp partial fragment of rat FUT8 cDNA was used as a standard DNA and a 772-bp fragment deleting a 207-bp ScaI (blunt)-HindIII (blunt) fragment from standard DNA was used as a competitor. Standard DNA was amplified from single-stranded cDNAs of YB2/0 cells by PCR using primers 5′-ACTCATCTTGGAATCTCAGAATTGG-3′ and 5′-CTTGACCGTTTCTATCTTCTCTCG-3′. PCRs for detection of FUT8 were carried out by heating at 94 °C for 3 min and subsequent 32 cycles of 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min in 20 μl of reaction mixture containing 5 μl of the 50-fold diluted single-stranded cDNA, 10 fg of linearized competitors, 10 pmol of FUT8-specific primers, 4 nmol of dNTP mixture, 5% dimethyl sulfoxide, and ExTaq polymerase (Takara Bio). The sense primer 5′-GTCCATGGTGATCCTGCAGTGTGG-3′ and antisense primer 5′-CACCAATGATATCTCCAGGTTCC-3′ were employed to amplify an FUT8 fragment. Aliquots of PCR products (7 μl) were subjected to electrophoresis in 1.75% agarose gel and stained with SYBR Green I nucleic gel stain (Molecular Probes, Inc., Eugene, OR). The amount of products was quantified by measuring luminescence intensity using a FluoroImager SI (Amersham Biosciences), calculated from standard curves, and converted into molar numbers. To normalize the synthesis efficiency of first-strand cDNAs, the amount of β-actin transcripts was also quantified by competitive RT-PCR in which a 1128-bp partial fragment of rat β-actin cDNA was used as a standard DNA and a 948-bp fragment deleting a 180-bpDraIII (blunt)-DraIII (blunt) fragment from standard DNA as a competitor were employed. Standard DNA was amplified from single-stranded cDNAs of YB2/0 cells by PCR using primers 5′-ATTTAAGGTACCGAAGCATTTGCGGTGCACGATGGAGGGG-3′ and 5′-AAGTATAAGCTTACATGGATGACGATATCGCTGCGCTCGT-3′. PCRs for detection of β-actin were carried out by heating at 94 °C for 3 min and subsequent 17 cycles of 94 °C for 30 s, 65 °C for 1 min, and 72 °C for 2 min in 20 μl of reaction mixture containing 5 μl of the 50-fold single-stranded cDNAs, 1 pg of linearized competitors, 10 pmol of β-actin-specific primers, 4 nmol of dNTP mixture, 5% dimethyl sulfoxide, and ExTaq polymerase (TakaRa Bio). The sense primer 5′-GATATCTGCTGCGCTCGTCGTCGAC-3′ and antisense primer 5′-CAGGAAGGAAGGCTGGAAGAGAGC-3′ were designed to amplify an β-actin fragment. Aliquots of PCR products (7 μl) were subjected to electrophoresis in 1.75% agarose gel for analysis. The purified humanized anti-hIL-5R IgG1 antibodies, KM8399 (YB2/0-produced) and KM8404 (CHO-produced), were compared for their ability to induce ADCC against a murine T cell line CTLL-2 (h5R) expressing hIL-5R α-chain and β-chain. Human peripheral blood mononuclear cells were used as effector cells for ADCC. Both humanized KM8399 and KM8404 showed high affinity to the soluble hIL-5R α-chain antigen and had no differences in antigen binding in enzyme-linked immunosorbent assay (data not shown). In contrast, the ADCC of KM8399 was ∼50-fold higher than that of KM8404 at the concentration of antibody of 25% cytotoxicity that was the maximum activity of KM8404 (Fig. 1 A), indicating that YB2/0-produced IgG1 promoted killing of IL-5R-positive cells at an ∼50-fold lower concentration than the CHO-produced IgG1. To confirm the reproducibility of this result, we assessed the ADCC of another antibody, chimeric anti-CD20 IgG1. RituxanTM was CHO-produced chimeric anti-CD20 IgG1 approved as a therapeutic agent in non-Hodgkin's lymphoma. We originally established YB2/0-produced chimeric anti-CD20 IgG1, KM3065, that had the same V region amino acid sequences as RituxanTM. Both chimeric RituxanTMand KM3065 exhibited the same antigen binding activity in flow cytometric analysis using CD20-positive cell lines (data not shown). On the other hand, the ADCC of KM3065 was at least 100-fold higher than that of RituxanTM at the concentration of antibody of 20% cytotoxicity that was the maximum activity of RituxanTM (Fig. 1 B). Moreover, the maximum cytotoxicity of YB2/0-produced KM8399 and KM3065 was 2–3-fold higher than that of CHO-produced KM8404 and RituxanTM, respectively (Fig. 1, A and B). To elucidate the molecular basis of the difference of ADCC between YB2/0- and CHO-produced IgG1, we analyzed the protein portion and oligosaccharide portion of KM8399 and KM8404. There was no significant difference in SDS-PAGE, peptide mapping, and CD (data not shown), suggesting the importance of oligosaccharides for controlling ADCC. Hydrazinolysis-derived oligosaccharides were labeled with 2-aminopyrizine and separated by HPLC (Fig. 2, A and B). As shown in Fig. 2, A and B, peak patterns and content of oligosaccharide between YB2/0-produced KM8399 and CHO-produced KM8404 were quite different. KM8399 contained nine major oligosaccharides (peaks a, b, c, e, f, g, h, k, and l); in contrast, KM8404 contains five major oligosaccharides (peaks a, e, f, g, and h). The oligosaccharide structure of each peak is shown in Fig. 2 C, and all structures have been found in human IgGN-linked oligosaccharides as natural structures (22Takahashi N. Ishii I. Ishihara H. Mori M. Tejima S. Jefferis R. Endo S. Arata Y. Biochemistry. 1987; 26: 1137-1144Google Scholar,23Parekh R.B. Dwek R.A. Sutton B.J. Fernandes D.L. Leung A. Stanworth D. Rademacher T.W. Mizuochi T. Taniguchi T. Matsuda K. Takeuchi F. Nagano Y. Miyamoto T. Kobata A. Nature. 1985; 316: 452-457Google Scholar). To clear the difference of oligosaccharides found in Fig. 2, quantitative monosaccharide compositions of IgG1s were determined (Table I). The contents of Fuc, Gal, and GlcNAc were different between YB2/0-produced IgG1s and CHO-produced IgG1s. KM8399 and KM3065 (YB2/0-produced) contained 0.8- and 0.09-fold lower content of Fuc than KM8404 and RituxanTM(CHO-produced), respectively. In contrast, the two YB2/0-produced IgG1s showed a higher content of GlcNAc than the two CHO-produced IgG1s. The difference in the content of Gal between YB2/0-produced IgG1s and CHO-produced IgG1s was not consistent. These results suggest the difference of ADCC between YB2/0-produced IgG1s and CHO-produced IgG1s is caused by that of oligosaccharide structure, especially Fuc- and/or GlcNAc-containing oligosaccharides.Table IMonosaccharide composition of IgGlsIgGlCell lineFucGalGlcNAcMan1-aMolar ratios calculatedversus 3 mannoses.KM8399YB2/00.721.014.513KM8404CHO0.910.273.803KM3065YB2/00.080.314.413Rituxan™CHO0.940.543.9831-a Molar ratios calculatedversus 3 mannoses. Open table in a new tab Oligosaccharide profiling analysis also showed that content of nonfucosylated oligosaccharides of KM8399 (34%, TableII) and KM3065 (91%, Table IV) were higher than those of KM8404 (9%, data not shown) and RituxanTM (6%, data not shown). Fuc composition of four IgG1s (KM8399, KM8404, KM3065, and RituxanTM), which was calculated from oligosaccharide profiling analysis (0.71, 0.91, 0.09, and 0.94, respectively) coincided well with the result of monosaccharide analysis (0.76, 0.91, 0.08, and 0.94, respectively) (Table I). Based on these results, we selected the oligosaccharide profiling analysis for further study.Table IIOligosaccharide composition of IgGlsIgGlabcdefghklTotalFuc(−)Bis(+)G0G1G2%%%%%%%%%%%%%%%%KM83992842ND3915533110034470273Fraction I751015NDNDNDNDNDNDND100100ND7525NDFraction II1311ND4921255310015867285Pyridylaminated oligosaccharides were analyzed for their compositions by reverse phase HPLC as described under "Experimental Procedures." The percentages of the total oligosaccharide are given on a molar basis. The structures of each oligosaccharide are represented in Fig. 2. ND, not detected. Fuc(−), total percentage of nonfucosylated oligosaccharides. Bis(+), total percentage of bisecting GlcNAc-binding oligosaccharides. G0, G1, and G2, total percentage of nongalactosylated, monogalactosylated, and digalactosylated oligosaccharides, respectively. Open table in a new tab Table IVOligosaccharide composition of PHA-E4-separated fractionsIgGlabcdefijklTotalFuc(−)Bis(+)G0G1G2%%%%%%%%%%%%%%%%KM30655411825112421100911973252Fraction V77611ND6NDNDNDNDND10094ND8317NDFraction VI6016725271NDND10093872262Fraction VII32121634ND29ND4ND100923369283Fraction VIII3010654ND162324100904552435ND, not detected. Fuc(−), total percentage of nonfucosylated oligosaccharides. Bis(+), total percentage of bisecting GlcNAc-binding oligosaccharides. G0, G1, and G2, total percentage of nongalactosylated, monogalactosylated, and digalactosylated oligosaccharides, respectively. Open table in a new tab Pyridylaminated oligosaccharides were analyzed for their compositions by reverse phase HPLC as described under "Experimental Procedures." The percentages of the total oligosaccharide are given on a molar basis. The structures of each oligosaccharide are represented in Fig. 2. ND, not detected. Fuc(−), total percentage of nonfucosylated oligosaccharides. Bis(+), total percentage of bisecting GlcNAc-binding oligosaccharides. G0, G1, and G2, total percentage of nongalactosylated, monogalactosylated, and digalactosylated oligosaccharides, respectively. ND, not detected. Fuc(−), total percentage of nonfucosylated oligosaccharides. Bis(+), total percentage of bisecting GlcNAc-binding oligosaccharides. G0, G1, and G2, total percentage of nongalactosylated, monogalactosylated, and digalactosylated oligosaccharides, respectively. To analyze the effect of Fuc-containing and bisecting GlcNAc-containing oligosaccharides on ADCC, YB2/0-produced anti-hIL-5R IgG1 KM
Referência(s)