Advertisement

Lack of Fucose on Human IgG1 N-Linked Oligosaccharide Improves Binding to Human FcγRIII and Antibody-dependent Cellular Toxicity*

      Lec13 cells, a variant Chinese hamster ovary cell line, were used to produce human IgG1 that were deficient in fucose attached to the Asn297-linked carbohydrate but were otherwise similar to that found in IgG1 produced in normal Chinese hamster ovary cell lines and from human serum. Lack of fucose on the IgG1 had no effect on binding to human FcγRI, C1q, or the neonatal Fc receptor. Although no change in affinity was found for the His131 polymorphic form of human FcγRIIA, a slight improvement in binding was evident for FcγRIIB and the Arg131 FcγRIIA polymorphic form. In contrast, binding of the fucose-deficient IgG1 to human FcγRIIIA was improved up to 50-fold. Antibody-dependent cellular cytotoxicity assays using purified peripheral blood monocytes or natural killer cells from several donors showed enhanced cytotoxicity, especially evident at lower antibody concentrations. When combined with an IgG1 Fc protein variant that exhibited enhanced antibody-dependent cellular cytotoxicity, the lack of fucose was synergistic.
      FcRn
      neonatal Fc receptor
      ADCC
      antibody-dependent cellular cytotoxicity
      CHO
      Chinese hamster ovary
      DHFR
      dihydrofolate reductase
      ELISA
      enzyme-linked immunosorbent assay
      mAb
      monoclonal antibody
      NK
      natural killer cell
      PBMC
      peripheral blood monocytic cell
      The nature and importance of the conserved, Asn297-linked carbohydrate in influencing immunoglobulin G effector functions has long been recognized (
      • Nose M.
      • Wigzell H.
      ,
      • Rudd M.P.
      • Ellliot T.
      • Cresswell P.
      • Wilson I.A.
      • Dwek R.A.
      ,
      • Jefferis R.
      • Lund J.
      • Pound J.D.
      ,
      • Wright A.
      • Morrison S.L.
      ). Variations in composition of the carbohydrate have been shown to affect the affinity of IgG for the three classes of FcγR (
      • Jefferis R.
      • Lund J.
      • Pound J.D.
      ,
      • Wright A.
      • Morrison S.L.
      ) (FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16)) that link IgG antibody-mediated immune responses with cellular effector functions (
      • Ravetch J.V.
      • Bolland S.
      ,
      • Gessner J.E.
      • Heiken H.
      • Tamm A.
      • Schmidt R.E.
      ,
      • Gavin A.
      • Hulett M.
      • Hogarth P.M.
      ). Carbohydrate composition also influences the activity of IgG in the classical pathway of complement activation, which is initiated by IgG1 binding to C1q (
      • Jefferis R.
      • Lund J.
      • Pound J.D.
      ,
      • Wright A.
      • Morrison S.L.
      ,
      • Makrides S.C.
      ) and to mannose-binding protein, which structurally resembles C1q (
      • Weis W.I.
      • Taylor M.E.
      • Drickamer K.
      ,
      • Malhotra R.
      • Wormald M.R.
      • Rudd P.M.
      • Fischer P.B.
      • Dwek R.A.
      • Sim R.B.
      ,
      • Wright A.
      • Morrison S.L.
      ). The role of carbohydrate in the clearance rate of IgG remains unclear. Binding of aglycosyl IgG to the neonatal Fc receptor (FcRn)1 appears unperturbed (
      • Shields R.L.
      • Namenuk A.K.
      • Hong K.
      • Meng Y.G.
      • Rae J.
      • Briggs J.
      • Xie D.
      • Lai J.
      • Stadlen A., Li, B.
      • Fox J.A.
      • Presta L.G.
      ,
      • Hobbs S.M.
      • Jackson L.E.
      • Hoadley J.
      ,
      • Kim J.-K.
      • Tsen M.-F.
      • Ghetie V.
      • Ward E.S.
      ); however, in some studies, glycosylated and aglycosyl-matched mAb show no appreciable difference in clearance rate in mice (
      • Hobbs S.M.
      • Jackson L.E.
      • Hoadley J.
      ,
      • Tao M.H.
      • Morrison S.L.
      ,
      • Horan Hand P.H.
      • Calvo B.
      • Milenic D.
      • Yokota T.
      • Finch M.
      • Snoy P.
      • Garmestani K.
      • Gansow O.
      • Schlom J.
      • Kashmiri S.V.S.
      ), whereas in other studies differences have been detected (
      • Tao M.H.
      • Morrison S.L.
      ,
      • Horan Hand P.H.
      • Calvo B.
      • Milenic D.
      • Yokota T.
      • Finch M.
      • Snoy P.
      • Garmestani K.
      • Gansow O.
      • Schlom J.
      • Kashmiri S.V.S.
      ,
      • Wawrzynczak E.J.
      • Cumber A.J.
      • Parnell G.D.
      • Jones P.T.
      • Winter G.
      ). Clearance of mAb may also be affected by variation in galactosylation through binding of agalactosyl IgG to mannose-binding protein or mannose receptors (
      • Malhotra R.
      • Wormald M.R.
      • Rudd P.M.
      • Fischer P.B.
      • Dwek R.A.
      • Sim R.B.
      ,
      • Wright A.
      • Morrison S.L.
      ).
      For IgG from either serum or produced ex vivo in hybridomas or engineered cells, the IgG are heterogeneous with respect to the Asn297-linked carbohydrate (
      • Jefferis R.
      • Lund J.
      • Pound J.D.
      ,
      • Wright A.
      • Morrison S.L.
      ,
      • Jefferis R.
      • Lund J.
      • Mizutani H.
      • Nakagawa H.
      • Kawazoe Y.
      • Arata Y.
      • Takahashi N.
      ,
      • Raju S.
      • Briggs J.B.
      • Borge S.M.
      • Jones A.J.S.
      ). For human IgG the core oligosaccharide normally consists of Asn297-Nδ2- GlcNAc(fucose)-GlcNAc-mannose-(mannose-GlcNAc)2(Fig. 1). Variation among individual IgG includes attachment of galactose and/or galactose-sialic acid at one or both of the terminal GlcNAc and/or attachment of a third GlcNAc arm (bisecting GlcNAc). The presence or absence of terminal galatose or sialic acid residues affects IgG function (
      • Wright A.
      • Morrison S.L.
      ,
      • Jassal R.
      • Jenkins N.
      • Charlwood J.
      • Camilleri P.
      • Jefferis R.
      • Lund J.
      ,
      • Groenink J.
      • Spijker J.
      • van den Herik-Oudijk I.E.
      • Boeije L.
      • Rook G.
      • Aarden L.
      • van de Winkel J.G.J.
      • van den Broek M.F.
      ,
      • Boyd P.N.
      • Lines A.C.
      • Patel A.K.
      ,
      • Kumpel B.M.
      • Rademacher T.W.
      • Rook G.A.W.
      • Williams P.J.
      • Wilson I.R.H.
      ,
      • Tsuchiya N .
      • Endo T.
      • Matsuta K.
      • Yoshinoya S.
      • Aikawa T.
      • Kosuge E.
      • Takeuchi F.
      • Miyamoto T.
      • Kobata A.
      ), and attachment of a third arm to the carbohydrate via bisecting GlcNAc has been reported to improve antibody-dependent cellular cytotoxicity (ADCC) (
      • Umaña P.
      • Jean-Mairet J.
      • Moudry R.
      • Amstutz H.
      • Bailey J.E.
      ,
      • Davies J.
      • Jiang L.
      • Pan L.-Y.
      • LaBarre M.J.
      • Anderson D.
      • Reff M.
      ). A connection between IgG glycosylation and some human diseases has been shown (e.g. the level of galactosylation appears correlated with rheumatoid arthritis) (
      • Parekh 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.
      ).
      Figure thumbnail gr1
      Figure 1Schematic of IgG glycoforms. The core structure (Gal0 + Fucose) normally is composed of Asn297-Nδ2-GlcNAc(fucose)-GlcNAc-mannose-(mannose-GlcNAc)2, where GlcNAc is N-acetylglucosamine. Variation among individual glycoforms includes attachment of one (Gal1 +Fucose) or two (Gal2 + Fucose) terminal galactose units, attachment of sialic acid to the terminal galactose, and/or attachment of a third GlcNAc arm (bisecting GlcNAc).
      Several recombinant monoclonal antibodies (mAbs) are being used as human therapeutics (
      • Glennie M.J.
      • Johnson P.W.M.
      ). Some of these (e.g. mAbs that bind to a receptor or soluble ligand and thereby block ligand-receptor interaction) may function without utilizing antibody effector mechanisms. Abrogating immune system recruitment for these mAbs can be achieved by altering IgG residues in the lower hinge region (
      • Armour K.L.
      • Clark M.R.
      • Hadley A.G.
      • Williamson L.M.
      ,
      • Duncan A.R.
      • Woof J.M.
      • Partridge L.J.
      • Burton D.R.
      • Winter G.
      ), using IgG2 or IgG4 subclasses, which are relatively less efficient in effector function, or using F(ab) or F(ab′)2 fragments (although these may have undesirable clearance rates). Other mAbs may need to recruit the immune system to kill the target cell (
      • Sampson J.H.
      • Crotty L.E.
      • Lee S.
      • Archer G.E.
      • Ashley D.M.
      • Wikstrand C.J.
      • Hale L.P.
      • Small C.
      • Dranoff G.
      • Friedman A.H.
      • Friedman H.S.
      • Bigner D.D.
      ,
      • Clynes R.A.
      • Towers T.L.
      • Presta L.G.
      • Ravetch J.V.
      ,
      • Clynes R.
      • Takechi Y.
      • Moroi Y.
      • Houghton A.
      • Ravetch J.V.
      ,
      • Anderson D.R.
      • Grillo-Lopez A.
      • Varns C.
      • Chambers K.S.
      • Hanna N.
      ). In those circumstances where recruitment of immune effector cells is desirable for therapeutic mAb efficacy, engineering the IgG Fc portion to improve effector function (via improved binding to IgG receptors and/or complement) could be a valuable enhancement to antibody therapeutics. Currently, methods that improve the immune system recruitment include bispecific antibodies in which one arm of the antibody binds to an IgG receptor (
      • Segal D.M.
      • Weiner G.J.
      • Weiner L.M.
      ), IgG-cytokine fusion proteins (
      • Penichet M.L.
      • Morrison S.L.
      ), alteration of the IgG Fc sequence for improved binding to FcγR (
      • Shields R.L.
      • Namenuk A.K.
      • Hong K.
      • Meng Y.G.
      • Rae J.
      • Briggs J.
      • Xie D.
      • Lai J.
      • Stadlen A., Li, B.
      • Fox J.A.
      • Presta L.G.
      ), and optimization of the Asn297-linked carbohydrate (
      • Umaña P.
      • Jean-Mairet J.
      • Moudry R.
      • Amstutz H.
      • Bailey J.E.
      ,
      • Davies J.
      • Jiang L.
      • Pan L.-Y.
      • LaBarre M.J.
      • Anderson D.
      • Reff M.
      ,
      • Lifely M.R.
      • Hale C.
      • Royce S.
      • Keen M.J.
      • Phillips J.
      ).
      To evaluate the role of fucosylated oligosaccharide in IgG function, the Lec13 cell line (
      • Ripka J.
      • Adamany A.
      • Stanley P.
      ) was utilized to express human IgG1. This Chinese hamster ovary (CHO) cell line is deficient in its ability to add fucose but provided IgG with oligosaccharide that was otherwise similar to that found in normal CHO cell lines and from human serum (
      • Jefferis R.
      • Lund J.
      • Mizutani H.
      • Nakagawa H.
      • Kawazoe Y.
      • Arata Y.
      • Takahashi N.
      ,
      • Raju S.
      • Briggs J.B.
      • Borge S.M.
      • Jones A.J.S.
      ,
      • Routier F.H.
      • Davies M.J.
      • Bergemann K.
      • Hounsell E.F.
      ). The resultant IgGs were used to evaluate the effect of fucosylated carbohydrate on antibody effector functions, including binding to human FcγR, human C1q, human FcRn, and ADCC using human effector cells.

      EXPERIMENTAL PROCEDURES

       cDNA Constructs for Stable Cell Lines

      The heavy and light chains of Hu4D5 (
      • Carter P.
      • Presta L.
      • Gorman C.M.
      • Ridgway J.B.B.
      • Henner D.
      • Wong W.L.T.
      • Rowland A.M.
      • Kotts C.
      • Carver M.E.
      • Shepard M.H.
      ) and HuE27 (
      • Presta L.G.
      • Lahr S.J.
      • Shields R.L.
      • Porter J.P.
      • Gorman C.M.
      • Jardieu P.M.
      ) were subcloned into a previously described mammalian cell expression vector (
      • Lucas B..
      • Giere L.M.
      • DeMarco R.A.
      • Shen A.
      • Chisholm V.
      • Crowley C.W.
      ) with the modification of a puromycin site fused 5′ to the dihydrofolate reductase (DHFR) site to form a fusion gene. Puromycin is used as a selective marker in DHFR(+) cells such as the Lec13 cells and the DHFR site was retained for methotrexate amplification of the stable cell line.

       Transfection and Culturing of Lec13 and Wild-type CHO Cells

      The CHO cell line Pro-Lec13.6a (Lec13), was obtained from Professor Pamela Stanley of Albert Einstein College of Medicine of Yeshiva University. Parental lines were Pro (proline auxotroph) and GAT (glycine, adenosine, thymidine auxotroph). The CHO-DP12 cell line used for wild-type antibodies is a derivative of the CHO-K1 cell line (ATCC number CCL-61) which is DHFR-deficient and has a reduced requirement for insulin. Cell lines were transfected with cDNA using the Superfect method (Qiagen, Valencia, CA). Selection of stably transfected Lec13 cells expressing mAbs was performed using puromycin dihydrochloride (Calbiochem) at 10 μg/ml in growth medium containing minimal essential medium-α medium with l-glutamine, ribonucleosides, and deoxyribonucleosides (Invitrogen), supplemented with 10% inactivated fetal bovine serum (Invitrogen), 10 mm HEPES, and 1× penicillin/streptomycin (Invitrogen). Selection of wild-type CHO-DP12 cells was performed in growth medium containing Ham's F-12 without GHT (glycine, hypoxanthine, thymidine), low glucose Dulbecco's modified Eagle's medium without glycine with NaHCO3, supplemented with 5% dialyzed fetal bovine serum (Invitrogen), 10 mm HEPES, 2 mml-glutamine, and penicillin/streptomycin. Colonies formed within 2–3 weeks and were pooled for expansion. The pools were seeded initially at 3 × 106 cells/10-cm plate for small batch protein expression. The cells were converted to serum-free medium once they grew to 90–95% confluence, and after 3–5 days cell supernatants were collected and quantified for mAb expression using both an anti-IgG-Fc and an anti-intact IgG ELISA.

       Protein Expression and Analysis

      For each batch of mAb, Lec13 and wild-type CHO cells were seeded at ∼8 × 106 cells/15-cm plate 1 day prior to converting to PSO4 (

      Mather, J. P., and Tsao, M. C. (June 16, 1992) U. S. Patent 5,122,469

      ) production medium, supplemented with 10 mg/liter recombinant human insulin and 1 mg/liter trace elements (

      Mather, J. P., and Tsao, M. C. (June 16, 1992) U. S. Patent 5,122,469

      ). Cells remained in serum-free production medium for 3–5 days. Supernatants were collected and clarified by centrifugation in 150-ml conical tubes to remove cells and debris. The protease inhibitors phenylmethylsulfonyl fluoride and aprotinin (Sigma) were added, and the supernatants were concentrated 5-fold on stirred cells using MWCO30 filters (Amicon, Beverly, MA) prior to immediate purification using protein A chromatography (Amersham Biosciences). All proteins were buffer-exchanged into phosphate-buffered saline using Centriprep-30 concentrators (Amicon) and analyzed by SDS-polyacrylamide gel electrophoresis. Protein concentrations were determined using A280 and verified using amino acid composition analysis. On average, the Lec13 cells generated 10 μg of mAb per 15-cm plate; expression in control CHO cells for all antibodies was 4–5 times higher than in the Lec13 cells. Antibodies generated from CHO-DP12 grown on plates will be denoted as CHO-P.
      CHO-DP12 cells were also grown in spinner flasks. Cells were seeded at 6 × 105 cells/ml and grown at 37 °C for 2 days. On the third day, the temperature was shifted to 33 °C, and the cells were allowed to grow until viability dropped to 70% due to the pH dropping to ∼6.5. Antibodies derived from CHO-DP12 cells grown in spinner flasks will be denoted as CHO-S.
      Matrix-assisted laser desorption/ionization time-of-flight mass spectral analysis was used to characterize the Asn297-linked oligosaccharides on the mAbs following a previously described protocol (
      • Shields R.L.
      • Namenuk A.K.
      • Hong K.
      • Meng Y.G.
      • Rae J.
      • Briggs J.
      • Xie D.
      • Lai J.
      • Stadlen A., Li, B.
      • Fox J.A.
      • Presta L.G.
      ,
      • Papac D.I.
      • Briggs J.B.
      • Chin E.T.
      • Jones A.J.S.
      ).

       Binding of Antibodies to FcγR, FcRn, and C1q: Antibody-dependent Cell Cytotoxicity Assays

      Assays for measuring binding of monomeric IgG1 to FcγRI and FcRn (
      • Shields R.L.
      • Namenuk A.K.
      • Hong K.
      • Meng Y.G.
      • Rae J.
      • Briggs J.
      • Xie D.
      • Lai J.
      • Stadlen A., Li, B.
      • Fox J.A.
      • Presta L.G.
      ), binding of trimeric IgG1 to FcγRII and FcγRIII (
      • Shields R.L.
      • Namenuk A.K.
      • Hong K.
      • Meng Y.G.
      • Rae J.
      • Briggs J.
      • Xie D.
      • Lai J.
      • Stadlen A., Li, B.
      • Fox J.A.
      • Presta L.G.
      ), and binding of dimeric IgG1 to FcγR (
      • Huizinga T.W.J.
      • Kerst M.
      • Nuyens J.H.
      • Vlug A.
      • von dem Borne A.E.G.K.
      • Roos D.
      • Tetteroo P.A.T.
      ) and ADCC assays with natural killer (NK) cells and peripheral blood monocytes (PBMC) of FcγRIIIA genotyped donors have been described previously (
      • Shields R.L.
      • Namenuk A.K.
      • Hong K.
      • Meng Y.G.
      • Rae J.
      • Briggs J.
      • Xie D.
      • Lai J.
      • Stadlen A., Li, B.
      • Fox J.A.
      • Presta L.G.
      ). The assay used to measure binding to human C1q has also been previously described (
      • Idusogie E.E.
      • Presta L.G.
      • Gazzano-Santoro H.
      • Totpal K.
      • Wong P.Y.
      • Ultsch M.
      • Meng Y.G.
      • Mulkerrin M.G.
      ).
      Immunofluorescence staining of purified NK cells from an FcγRIIIA(Phe158/Phe158) donor was performed using Hu4D5 variants. NK cells were purified by negative selection and gated with phycoerythrin-conjugated anti-CD56 and fluorescein isothiocyanate-conjugated anti-CD16 (Pharmingen, San Diego, CA). Cells were adjusted to 2 × 106/ml and incubated with 2 μg/ml Hu4D5 variants for 30 min in ice-cold phosphate-buffered saline containing 0.1% bovine serum albumin, 0.01% sodium azide. Cells were then washed, and antibody binding was detected by incubating with fluorescein isothiocyanate-conjugated F(ab′)2 goat anti-human IgG for 30 min.

      RESULTS

       Binding of mAbs to Human FcR

      Anti-HER2 Hu4D5 mAb (
      • Carter P.
      • Presta L.
      • Gorman C.M.
      • Ridgway J.B.B.
      • Henner D.
      • Wong W.L.T.
      • Rowland A.M.
      • Kotts C.
      • Carver M.E.
      • Shepard M.H.
      ) expressed in stably transfected Lec13 cells consistently had about 10% fucosylated carbohydrate (TableI). In contrast, Hu4D5 mAbs from stably transfected CHO-DP12 cells cultured in spinner flasks (CHO-S) or plates (CHO-P) showed about 98% fucosylated carbohydrate. The variation in galactosylation for the Lec13 mAbs was similar to that found for CHO-S mAbs (as opposed to CHO-P) (Table I); hence, CHO-S mAbs were used as reference in the binding assays.
      Table ICarbohydrate analysis of antibodies
      Variant
      Hu4D5, humanized anti-p185HER2 IgG1 (40); HuE27, humanized anti-IgE IgG1 (41); CHO-S, IgG expressed by CHO cells in spinner flasks; CHO-P, IgG expressed by CHO cells on 15-cm plates; Lec, IgG expressed by Lec13 cells on 15-cm plates; HEK, IgG expressed by human embryonic kidney 293 cells on 15-cm plates; AAA, S298A/E333A/K334A IgG1 variant (12). For Lec-X and Lec-AAA-X, X represents independently expressed lots of IgG.
      Without fucoseWith fucose
      Man
      Values are percentage of total oligosaccharide. Glycoforms were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectral analysis following a previously described protocol (12, 44). Man, incompletely processed, hypermannosylated oligosaccharides containing 5–8 mannose units (19). Gal0, Gal1, Gal2 are oligosaccharides with no (agalactosyl), one (monogalactosyl), or two (digalactosyl) galactose residues covalently linked to the terminal GlcNAc residues (see Fig. 1). Mean ± S.D. is provided for six independently expressed lots of Hu4D5-Lec13 (A–E), three independently expressed lots of Hu4D5 Lec13-AAA (A–C), and three independently expressed lots of HuE27-HEK293. None of the mAbs contained measurable triantennary oligosaccharide (bisecting GlcNAc), and sialylated oligosaccharide was less than 6% of total oligosaccharide.
      Gal0Gal1Gal2TotalGal0Gal1Gal2Total
      Hu4D5
       CHO-S120035042698
       CHO-P020027025398
       Lec-A048385912428
       Lec-B0513549044210
       Lec-C049395932417
       Lec-D1463658847213
       Lec-E3503138766113
       Lec-F1513148756213
       Lec-Avg1 ± 149 ± 235 ± 34 ± 189 ± 34 ± 25 ± 12 ± 111 ± 3
       HEK-AAA162202047181580
       Lec-AAA-A073212962114
       Lec-AAA-B071183924408
       Lec-AAA-C565183914329
       Lec-AAA-Avg2 ± 370 ± 419 ± 23 ± 193 ± 33 ± 13 ± 21 ± 17 ± 3
      HuE27
       HEK-A102211549251084
       HEK-B18111215019978
       HEK-C110201343301588
       HEK-Avg13 ± 41 ± 11 ± 11 ± 116 ± 447 ± 425 ± 611 ± 383 ± 5
       CHO-P020027026298
       Lec1363657878621
       HEK-AAA171101951171381
      1-a Hu4D5, humanized anti-p185HER2 IgG1 (
      • Carter P.
      • Presta L.
      • Gorman C.M.
      • Ridgway J.B.B.
      • Henner D.
      • Wong W.L.T.
      • Rowland A.M.
      • Kotts C.
      • Carver M.E.
      • Shepard M.H.
      ); HuE27, humanized anti-IgE IgG1 (
      • Presta L.G.
      • Lahr S.J.
      • Shields R.L.
      • Porter J.P.
      • Gorman C.M.
      • Jardieu P.M.
      ); CHO-S, IgG expressed by CHO cells in spinner flasks; CHO-P, IgG expressed by CHO cells on 15-cm plates; Lec, IgG expressed by Lec13 cells on 15-cm plates; HEK, IgG expressed by human embryonic kidney 293 cells on 15-cm plates; AAA, S298A/E333A/K334A IgG1 variant (
      • Shields R.L.
      • Namenuk A.K.
      • Hong K.
      • Meng Y.G.
      • Rae J.
      • Briggs J.
      • Xie D.
      • Lai J.
      • Stadlen A., Li, B.
      • Fox J.A.
      • Presta L.G.
      ). For Lec-X and Lec-AAA-X, X represents independently expressed lots of IgG.
      1-b Values are percentage of total oligosaccharide. Glycoforms were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectral analysis following a previously described protocol (
      • Shields R.L.
      • Namenuk A.K.
      • Hong K.
      • Meng Y.G.
      • Rae J.
      • Briggs J.
      • Xie D.
      • Lai J.
      • Stadlen A., Li, B.
      • Fox J.A.
      • Presta L.G.
      ,
      • Papac D.I.
      • Briggs J.B.
      • Chin E.T.
      • Jones A.J.S.
      ). Man, incompletely processed, hypermannosylated oligosaccharides containing 5–8 mannose units (
      • Raju S.
      • Briggs J.B.
      • Borge S.M.
      • Jones A.J.S.
      ). Gal0, Gal1, Gal2 are oligosaccharides with no (agalactosyl), one (monogalactosyl), or two (digalactosyl) galactose residues covalently linked to the terminal GlcNAc residues (see Fig. 1). Mean ± S.D. is provided for six independently expressed lots of Hu4D5-Lec13 (A–E), three independently expressed lots of Hu4D5 Lec13-AAA (A–C), and three independently expressed lots of HuE27-HEK293. None of the mAbs contained measurable triantennary oligosaccharide (bisecting GlcNAc), and sialylated oligosaccharide was less than 6% of total oligosaccharide.
      Monomeric Hu4D5-Lec13 IgG1 bound to human FcγRI equivalent to binding of Hu4D5-CHO-S (Table II). The EC50 values (∼0.1 μg/ml) correspond to an affinity constant of 0.7 nm, at the lower end of the range of previously determined values (
      • Gessner J.E.
      • Heiken H.
      • Tamm A.
      • Schmidt R.E.
      ,
      • Gavin A.
      • Hulett M.
      • Hogarth P.M.
      ). In contrast to FcγRI, IgG binding to the low affinity FcγR (FcγRII and FcγRIII) required the formation of dimers (Hu4D5 and HuE27) or trimers (HuE27) (
      • Shields R.L.
      • Namenuk A.K.
      • Hong K.
      • Meng Y.G.
      • Rae J.
      • Briggs J.
      • Xie D.
      • Lai J.
      • Stadlen A., Li, B.
      • Fox J.A.
      • Presta L.G.
      ,
      • Liu J.
      • Lester P.
      • Builder S.
      • Shire S.J.
      ) to detect binding. For FcγRIIA there are naturally occurring allotypes at position 131 that result in different binding avidities for IgG2 (
      • Clark M.R.
      • Clarkson S.B.
      • Ory P.A.
      • Stollman N.
      • Goldstein I.M.
      ,
      • Warmerdam P.A.M.
      • van de Winkel J.G.J.
      • Vlug A.
      • Westerdaal N.A.C.
      • Capel P.J.A.
      ). Binding of Hu4D5-Lec13 dimers to the FcγRIIA(Arg131) polymorphic form and to FcγRIIB exhibited only a slight improvement compared with Hu4D5-CHO-S (Fig.2, A and B). Lack of fucose did not affect binding to the FcγRIIA(His131) polymorphic form (Fig. 2C). Note that in contrast to FcγRI and FcγRIIIA, binding of the antibodies to FcγRIIA and FcγRIIB did not reach a plateau at the highest concentration used (10 μg/ml) (Fig. 2, A and B); hence, EC50 values could not be calculated. At concentrations above 10 μg/ml, aggregation of mAb was problematic.
      Table IIBinding of antibodies to human FcγRI and FcγRIIIA
      Variant
      Hu4D5, humanized anti-p185HER2 IgG1 (40); HuE27, humanized anti-IgE IgG1 (41); CHO-S, IgG expressed by CHO cells in spinner flasks; CHO-P, IgG expressed by CHO cells on 15-cm plates; Lec13, IgG expressed by Lec13 cells on 15-cm plates; HEK293, IgG expressed by human embryonic kidney 293 cells on 15-cm plates; AAA, S298A/E333A/K334A IgG1 variant (12).
      EC50n
      MeanS.D.
      μg/ml
      FcγRI/Hu4D5 monomers
      Two independent lots of Hu4D5-Lec13 (A and B) used with four assays for lot A and five assays for lot B.
       CHO-S0.090.045
       CHO-P0.110.075
       Lec13-Avg0.090.059
      FcγRIIIA (Phe158)/Hu4D5 dimers
      Four independent lots of Hu4D5-Lec13 (A, B, C, and D) used with three assays per lot; three independent lots of Hu4D5-Lec13-AAA (A, B, and C) used with 1–3 assays per lot.
       CHO-S>106
       CHO-P>103
       Lec13-Avg0.240.0416
       HEK293-AAA0.280.032
       Lec13-AAA-Avg0.160.016
      FcγRIIIA (Phe158)/HuE27 dimers
       HEK293∼31
       CHO-P>100.013
       Lec130.190.043
      FcγRIIIA (Phe158)/HuE27 trimers
       HEK2930.200.033
       CHO-P0.630.343
       Lec130.050.023
       HEK293-AAA0.070.013
      FcγRIIIA (Val158)/Hu4D5 dimers
      Four independent lots of Hu4D5-Lec13 (A, B, C, and D) used with three assays per lot; three independent lots of Hu4D5-Lec13-AAA (A, B, and C) used with 1–3 assays per lot.
       CHO-S1.300.606
       CHO-P1.660.182
       Lec13-Avg0.070.0214
       HEK293-AAA0.110.012
       Lec13-AAA-Avg0.040.016
      FcγRIIIA (Val158)/HuE27 dimers
       HEK2930.501
       CHO-P2.500.072
       Lec130.050.013
      FcγRIIIA (Val158)/HuE27 trimers
       HEK2930.060.033
       CHO-P0.060.023
       Lec130.040.023
       HEK293-AAA0.040.013
      VariantA490(Var)/A490(CHO-S)n
      MeanS.D.
      FcγRIIIA (Phe158)-transfected CHO cells/Hu4D5 monomers
      [mAb] = 2.2 μg/ml. Three independent lots of Hu4D5-Lec13 (D, E, and F) used with three or four assays per lot; two independent lots of Hu4D5-Lec13-AAA (B and C) used with three assays per lot.
       CHO-S1.004
       Lec13-Avg16.12.510
       HEK293-AAA10.71.43
       Lec13-AAA-Avg26.45.66
      2-a Hu4D5, humanized anti-p185HER2 IgG1 (
      • Carter P.
      • Presta L.
      • Gorman C.M.
      • Ridgway J.B.B.
      • Henner D.
      • Wong W.L.T.
      • Rowland A.M.
      • Kotts C.
      • Carver M.E.
      • Shepard M.H.
      ); HuE27, humanized anti-IgE IgG1 (
      • Presta L.G.
      • Lahr S.J.
      • Shields R.L.
      • Porter J.P.
      • Gorman C.M.
      • Jardieu P.M.
      ); CHO-S, IgG expressed by CHO cells in spinner flasks; CHO-P, IgG expressed by CHO cells on 15-cm plates; Lec13, IgG expressed by Lec13 cells on 15-cm plates; HEK293, IgG expressed by human embryonic kidney 293 cells on 15-cm plates; AAA, S298A/E333A/K334A IgG1 variant (
      • Shields R.L.
      • Namenuk A.K.
      • Hong K.
      • Meng Y.G.
      • Rae J.
      • Briggs J.
      • Xie D.
      • Lai J.
      • Stadlen A., Li, B.
      • Fox J.A.
      • Presta L.G.
      ).
      2-b Two independent lots of Hu4D5-Lec13 (A and B) used with four assays for lot A and five assays for lot B.
      2-c Four independent lots of Hu4D5-Lec13 (A, B, C, and D) used with three assays per lot; three independent lots of Hu4D5-Lec13-AAA (A, B, and C) used with 1–3 assays per lot.
      2-d [mAb] = 2.2 μg/ml. Three independent lots of Hu4D5-Lec13 (D, E, and F) used with three or four assays per lot; two independent lots of Hu4D5-Lec13-AAA (B and C) used with three assays per lot.
      Figure thumbnail gr2
      Figure 2Binding of anti-HER2 Hu4D5 dimers to human FcγRII in ELISA format assays (representative plot for one of three assays). A, FcγRIIB;B, FcγRIIA(Arg131); C, FcγRIIA(His131). Open circles, Hu4D5 CHO-S; filled circles, Hu4D5 Lec13-A;filled squares, Hu4D5 Lec13-C; filled diamonds, Hu4D5 Lec13-D. Hu4D5 Lec13-A, -C, and -D are three different lots of mAb.
      Human FcγRIIIA has naturally occurring allotypes at position 48 (Leu, His, or Arg) and at position 158 (Val or Phe); the FcγRIIIA(Val158) allotype interacts with human IgG better than the FcγRIIIA(Phe158) allotype (
      • Shields R.L.
      • Namenuk A.K.
      • Hong K.
      • Meng Y.G.
      • Rae J.
      • Briggs J.
      • Xie D.
      • Lai J.
      • Stadlen A., Li, B.
      • Fox J.A.
      • Presta L.G.
      ,
      • Koene H.R.
      • Kleijer M.
      • Algra J.
      • Roos D.
      • von dem Borne E.G.K.
      • de Hass M.
      ,
      • Wu J.
      • Edberg J.C.
      • Redecha P.B.
      • Bansal V.
      • Guyre P.M.
      • Coleman K.
      • Salmon J.E.
      • Kimberly R.P.
      ). Both the Phe158 and Val158 polymorphic forms of FcγRIIIA exhibited significantly improved binding to IgG1 that lacked fucose. Binding of dimeric Hu4D5-Lec13 to FcγRIIIA(Phe158) showed at least a 42-fold improvement (Fig. 3A; Table II), and binding to FcγRIIIA(Val158) showed a 19-fold improvement over Hu4D5-CHO-S (Fig. 3B; Table II). HuE27-Lec13 dimers also exhibited a 50-fold enhancement in binding to both polymorphic forms of FcγRIIIA compared with HuE27 CHO-P (Fig.4A; Table II). The binding of HuE27 expressed in HEK293 cells was intermediate to HuE27-Lec13 and CHO-P (Fig. 4A; Table II) and correlates to the percentage of HuE27 mAb without fucose: CHO-P, 2%; HEK293, 16%; Lec13, 78% (Table I). The EC50 values for Hu4D5-Lec13 and HuE27-Lec13 (as dimers) were 0.24 and 0.19 μg/ml, respectively, for FcγRIIIA(Phe158) and 0.07 and 0.05 μg/ml, respectively, for FcγRIIIA(Val158) (Table II), showing that the binding of the Lec13 antibodies was independent of antibody specificity.
      Figure thumbnail gr3
      Figure 3Binding of anti-HER2 Hu4D5 dimers to human FcγRIIIA in ELISA format assays (representative plot for one assay). A, FcγRIIIA(Phe158);B, FcγRIIIA(Val158). Open circles, Hu4D5 CHO-S; open squares, Hu4D5 Lec13; filled circles, Hu4D5 HEK293;filled squares, Hu4D5 HEK293-AAA;filled diamonds, Hu4D5 Lec13-AAA. The plot for Hu4D5 Lec13 is the average of three different lots of mAb witherror bars shown; the plot for Hu4D5 Lec13-AAA is the average of two different lots of mAb; error bars were small enough to be contained within the diamonds.
      Figure thumbnail gr4
      Figure 4Binding of anti-IgE HuE27 to human FcγRIIIA in ELISA format assays (representative plot for one assay). A, FcγRIIIA(Phe158); HuE27 dimers. B, FcγRIIIA(Phe158); HuE27 trimers. C, FcγRIIIA(Val158); HuE27 trimers.Open circles, HuE27 CHO-P; filled circles, HuE27-Lec13-B; filled squares, HuE27 HEK293; filled diamonds, HuE27 HEK293-AAA.
      In a previous study of the effect of protein sequence variants of human IgG1 on binding to human FcγR, an S298A/E333A/K334A-IgG1 variant showed improved binding to FcγRIIIA as well as more potent cytotoxicity in ADCC assays (
      • Shields R.L.
      • Namenuk A.K.
      • Hong K.
      • Meng Y.G.
      • Rae J.
      • Briggs J.
      • Xie D.
      • Lai J.
      • Stadlen A., Li, B.
      • Fox J.A.
      • Presta L.G.
      ). Expressing Hu4D5 S298A/E333A/K334A-IgG1 in Lec13 cells added fucose deficiency to this protein-sequence variant (Table I). When assayed as dimers, the Hu4D5 S298A/E333A/K334A-IgG1 with fucose exhibited 36- and 12-fold enhancement in binding to FcγRIIIA(Phe158) and FcγRIIIA(Val158), respectively. The same variant without fucose showed even more improvement in binding to FcγRIIIA(Phe158) and FcγRIIIA(Val158) of 63- and 33-fold, respectively (Fig. 3, A and B; Table II), suggesting that the protein and carbohydrate alterations are synergistic.
      In addition to dimers, complexes consisting of three anti-IgE HuE27 and three IgE were used to test binding to FcγRIIIA; these complexes are trimeric in anti-IgE HuE27 (
      • Shields R.L.
      • Namenuk A.K.
      • Hong K.
      • Meng Y.G.
      • Rae J.
      • Briggs J.
      • Xie D.
      • Lai J.
      • Stadlen A., Li, B.
      • Fox J.A.
      • Presta L.G.
      ). Binding to FcγRIIIA by the trimeric complexes was stronger than for the corresponding dimeric complexes (Fig. 4, A and B; Table II), suggesting an appreciable avidity component. The avidity resulted in a smaller improvement in binding to FcγRIIIA(Phe158) for trimeric HuE27-Lec13, only 13-fold, compared with 53-fold for dimeric HuE27. In a previous study, the improvement in binding of trimeric HuE27 S298A/E333A/K334A-IgG1 variant to FcγRIIIA(Phe158) and FcγRIIIA(Val158) was 2- and 1.1-fold, respectively; whereas the improvement in binding was small, the effect on ADCC was significant (
      • Shields R.L.
      • Namenuk A.K.
      • Hong K.
      • Meng Y.G.
      • Rae J.
      • Briggs J.
      • Xie D.
      • Lai J.
      • Stadlen A., Li, B.
      • Fox J.A.
      • Presta L.G.
      ). In the current study, the trimeric complex of HuE27 S298A/E333A/K334A-IgG1 showed improved binding to both FcγRIIIA polymorphic forms in line with the values from the previous study (Fig.4, B and C; Table II).
      The improved binding of fucose-deficient IgG1 to FcγRIIIA was confirmed with FcγRIIIA(Phe158) full-length α-chain co-expressed with γ-chain on CHO cells (Fig.5; Table II). As for the α-chain fusion protein alone in ELISA format, fucose deficiency was synergistic with the S298A/E333A/K334A-IgG1 variant.
      Figure thumbnail gr5
      Figure 5Binding of anti-HER2 Hu4D5 monomers to CHO cell line stably transfected with human FcγRIIIA α-chain and γ-chain (representative plot for one assay). Open circles, Hu4D5 CHO-S;open squares, Hu4D5 Lec13-D; open diamonds, Hu4D5 Lec13-E; open triangles, Hu4D5 Lec13-F; filled circles, Hu4D5 HEK293-AAA; filled squares, Hu4D5 Lec13-AAA-B; filled diamonds, Hu4D5 Lec13-AAA-C.
      Binding of the native and fucose-deficient IgG1 to murine FcγRII and FcγRIII was also evaluated. As trimeric complexes, the fucose-deficient HuE27 showed slightly reduced binding to both receptors compared with fucosylated HuE27 (data not shown). The major histocompatibility complex class I-like neonatal Fc receptor (FcRn) is structurally unrelated to the FcγR (
      • Raghavan M.
      • Bjorkman P.J.
      ) and has been proposed to be involved in a number of biological processes including clearance rate of IgG (
      • Ghetie V.
      • Ward E.S.
      ). Binding of nonfucosylated and fucosylated IgG1 to FcRn was equivalent (ratio of 0.98 at 5 μg/ml) (data not shown).

       C1q Binding

      Binding of C1q to antibodies is the first step in the classical pathway of complement activation (
      • Makrides S.C.
      ). The nature of the carbohydrate on the IgG has been shown to influence its interaction with C1q (
      • Jefferis R.
      • Lund J.
      • Pound J.D.
      ,
      • Wright A.
      • Morrison S.L.
      ,
      • Boyd P.N.
      • Lines A.C.
      • Patel A.K.
      ,
      • Tsuchiya N .
      • Endo T.
      • Matsuta K.
      • Yoshinoya S.
      • Aikawa T.
      • Kosuge E.
      • Takeuchi F.
      • Miyamoto T.
      • Kobata A.
      ). However, the lack of fucose did not affect the ability of Hu4D5 to interact with human C1q (data not shown).

       ADCC

      The effect of fucosylated carbohydrate on ADCC was evaluated using Hu4D5-Lec13 IgG1 on the human breast cancer cell line SK-BR-3 (
      • Hudziak R.M.
      • Lewis G.D.
      • Winget M.
      • Fendly B.M.
      • Shepard H.M.
      • Ullrich A.
      ). SK-BR-3 cells were opsonized with varying Hu4D5 concentrations, and PBMCs from three FcγRIIIA(Val158/Phe158) donors and three FcγRIIIA(Phe158/Phe158) donors were used as effector cells at a 30:1 effector/target ratio; representative ADCC assays are shown in Fig. 6, Aand B. For all donors studied, the fucose-deficient IgG1 exhibited significant improvement in ADCC compared with IgG1 with fucose, especially at the lower antibody concentrations (Fig. 6).
      Figure thumbnail gr6
      Figure 6ADCC assays using anti-HER2 Hu4D5 mAbs, SK-BR-3 cells as target, and PBMCs as effector cells. The effector/target ratio was held constant at 30:1, and [mAb] varied.A, representative plot for one of three FcγRIIIA(Val158/Phe158) donors. B, representative plot for one of three FcγRIIIA(Phe158/Phe158) donors.Open squares, Hu4D5 Lec13-A; filled circles, Hu4D5 CHO-S; open circles, spontaneous lysis. Each assay was performed in duplicate witherror bars shown.
      To verify that the combination of fucose deficiency and protein sequence variant exhibited a synergistic effect, as seen in the ELISA and cell binding assays (Figs. Figure 3, Figure 4, Figure 5), immunofluorescence staining and ADCC assays were performed comparing native Hu4D5 and S298A/E333A/K334A-IgG1 variant with and without fucose against NK cells from two FcγRIIIA(Phe158/Phe158) donors. The intensity of staining showed the pattern: Hu4D5 S298A/E333A/K334A-IgG1 Lec13 > Hu4D5 Lec13 > Hu4D5 CHO-S (Fig.7A). For the ADCC assays, SK-BR-3 cells were opsonized with 1 ng/ml mAb, and NK cells were used as effector cells. Both donors showed similar results; the fucose-deficient S298A/E333A/K334A-IgG1 was more potent than either fucose-deficient Hu4D5 or fucosylated S298A/E333A/K334A-IgG1 (Fig. 7B) and correlated with the immunofluorescence staining.
      Figure thumbnail gr7
      Figure 7A, immunofluorescence staining of purified NK cells from an FcγRIIIA(Phe158/Phe158) donor by Hu4D5 variants. NK cells were purified by negative selection. Cells were adjusted to 2 × 106/ml and incubated with 2 μg/ml Hu4D5 variants for 30 min in ice-cold phosphate-buffered saline containing 0.1% bovine serum albumin, 0.01% sodium azide. Cells were then washed, and antibody binding was detected by incubating with fluorescein isothiocyanate-conjugated F(ab′)2 goat anti-human IgG and phycoerythrin-conjugated anti-CD56 for 30 min. The plot shows the fluorescence intensity on gated CD56+ cells for the secondary detecting antibody (shaded histogram) (1), Hu4D5-CHO-S (2), Hu4D5 Lec13-A (3), and Hu4D5 Lec13-AAA (4) (relative mean fluorescence intensities of 4.82, 15.8, 190, and 372, respectively).B, ADCC assay using anti-HER2 Hu4D5 mAbs, SK-BR-3 cells as target, and NK cells from an FcγRIIIA(Phe158/Phe158) donor as effector cells. SK-BR-3 cells were opsonized with 1 ng/ml mAb, and the effector/target ratio was varied. Closed circles, Hu4D5 CHO-S; filled squares, Hu4D5 Lec13-A;filled diamonds, Hu4D5 HEK293-AAA;filled triangles, Hu4D5 Lec13-AAA-C;open circles, spontaneous lysis. The assay was performed in duplicate with error barsshown.

      DISCUSSION

      Although the presence of carbohydrate is necessary for binding to FcγRI (
      • Shields R.L.
      • Namenuk A.K.
      • Hong K.
      • Meng Y.G.
      • Rae J.
      • Briggs J.
      • Xie D.
      • Lai J.
      • Stadlen A., Li, B.
      • Fox J.A.
      • Presta L.G.
      ,
      • Walker M.R.
      • Lund J.
      • Thompson K.M.
      • Jefferis R.
      ), the equivalent binding of IgG1 regardless of differences in fucose content (Lec13 versus CHO-S) or galactose content (CHO-P versus CHO-S) suggests that IgG1 binding to FcγRI is insensitive to the presence of these moieties on the carbohydrate. The effect of galactosylation on binding of IgG to human FcγRI has been previously studied (
      • Wright A.
      • Morrison S.L.
      ,
      • Boyd P.N.
      • Lines A.C.
      • Patel A.K.
      ,
      • Kumpel B.M.
      • Rademacher T.W.
      • Rook G.A.W.
      • Williams P.J.
      • Wilson I.R.H.
      ,
      • Tsuchiya N .
      • Endo T.
      • Matsuta K.
      • Yoshinoya S.
      • Aikawa T.
      • Kosuge E.
      • Takeuchi F.
      • Miyamoto T.
      • Kobata A.
      ), and review of the data suggests that if galactosylation affects binding to FcγRI, it is subtle and may be isotype-dependent (
      • Wright A.
      • Morrison S.L.
      ).
      The small improvement in binding of fucose-deficient IgG1 to both FcγRIIA(Arg131) and FcγRIIB, each having arginine at position 131, versus no effect on FcγRIIA(His131) suggests that the fucose may either directly interact with the FcγR residue at position 131 or alter the IgG1 conformation so as to effect a subtle, negative influence on binding when arginine is present at FcγR position 131. As with FcγRI, the IgG1 galactose content did not seem to affect binding to FcγRII.
      In contrast to the FcγRII class of receptors, lack of fucose effected a significant improvement in binding of IgG1 to FcγRIIIA. The absence of fucose not only increased binding of native IgG1 to FcγRIIIA but also augmented binding of the S298A/E333A/K334A-IgG1 variant. For both protein and carbohydrate variants of HuE27 (i.e.S298A/E333A/K334A-IgG1 and Lec13-derived), the improvement in binding of the trimeric complex was much smaller than that observed for the dimeric complex. For example, binding to FcγRIIIA(Phe158) by HuE27-Lec13 (21% fucosylated) showed a 53-fold improvement compared with HuE27-CHO-P (98% fucosylated) when in dimeric form but only a 13-fold improvement when in the trimeric form (Table II). This may result from the avidity effect evident when comparing binding of the dimeric and trimeric complexes (Fig. 4, A and B) and suggests that as the size of the immune complex is increased, the fucose effect diminishes.
      Comparison of the carbohydrate found on native IgG1 for the Hu4D5 Lec13-produced and CHO-S-produced antibodies showed no appreciable differences in the extent of galactosylation, and hence the difference in binding can be attributed solely to the presence/absence of fucose. However, for the S298A/E333A/K334A IgG1 variant, the Lec13-produced and HEK293-produced antibodies also showed variation in galactosylation. While this complicates the interpretation of the data, the combination of protein sequence variation and lack of fucose was synergistic in binding to recombinant FcγRIIIA, native FcγRIIIA expressed on transfected CHO cells or on purified NK cells, and in the ADCC activity of FcγRIIIA-expressing NK and PBMC cells.
      A previous study of protein sequence variants of human IgG1 found that alanine (and other) substitutions at some Fc positions could reduce or improve binding to FcγR as well as enhance ADCC (
      • Shields R.L.
      • Namenuk A.K.
      • Hong K.
      • Meng Y.G.
      • Rae J.
      • Briggs J.
      • Xie D.
      • Lai J.
      • Stadlen A., Li, B.
      • Fox J.A.
      • Presta L.G.
      ). Interestingly, some of these substitutions were not near the interaction interface found in crystal structures of human IgG Fc-human FcγRIIIA complex (
      • Shields R.L.
      • Namenuk A.K.
      • Hong K.
      • Meng Y.G.
      • Rae J.
      • Briggs J.
      • Xie D.
      • Lai J.
      • Stadlen A., Li, B.
      • Fox J.A.
      • Presta L.G.
      ,
      • Sondermann P.
      • Huber R.
      • Oosthuizen V.
      • Jacob U.
      ,
      • Radaev S.
      • Motyka S.
      • Fridman W.-H.
      • Sautes-Fridman C.
      • Sun P.D.
      ). For example, of the three alanine substitutions S298A/E333A/K334A used in this study, only Ser298 is at the Fc/FcγRIIIA interface in the crystal structures. Likewise, in the co-crystal structures, neither of the fucose residues on the two Fc heavy chains interact with FcγRIIIA. Inspection of crystal structures of human and rodent Fc or IgG shows that the fucose can adopt varying conformations and exhibits high B-factors (
      • Sondermann P.
      • Huber R.
      • Oosthuizen V.
      • Jacob U.
      ,
      • Radaev S.
      • Motyka S.
      • Fridman W.-H.
      • Sautes-Fridman C.
      • Sun P.D.
      ,
      • Deisenhofer J.
      ,
      • Harris L.J.
      • Skaletsky E.
      • McPherson A.
      ,
      • Harris L.J.
      • Larson S.B.
      • Hasel K.W.
      • McPherson A.
      ), suggesting a high degree of mobility. Although the presence or absence of fucose definitely affects the interaction of an IgG with FcγRIIIA (and, to a lesser extent, FcγRIIA(Arg131) and FcγRIIB), the current study cannot discern whether this is due to direct interaction of the fucose with receptor or to a conformational influence on the IgG itself.
      Improved binding to FcγRIIIA translated into improved ADCC in vitro. Notably, for all donors the enhancement in cytotoxicity was more apparent as the concentration of antibody was reduced. This may reflect the larger improvement in binding seen for dimers compared with that for trimers (i.e. fucose-deficient IgG1 may require fewer mAbs on the surface of the target cell in order to effect binding/activation of an effector cell). If this is the case, it suggests that for therapeutic antibodies that utilize ADCC, nonfucosylated antibody could conceivably be given at lower doses to effect an equivalent cell kill as higher doses of fucosylated antibody.
      In addition to the human FcγR, the influence of fucose on binding to C1q and FcRn were also investigated. Previous studies have shown that differences in IgG glycosylation can affect C1q binding, although the results of this study show that the presence/absence of fucose had no discernible effect. Likewise, the presence or absence of fucose did not affect IgG1 binding to FcRn. This is not surprising, since aglycosylated IgG1 binds this receptor similarly to glycosylated IgG1 (
      • Shields R.L.
      • Namenuk A.K.
      • Hong K.
      • Meng Y.G.
      • Rae J.
      • Briggs J.
      • Xie D.
      • Lai J.
      • Stadlen A., Li, B.
      • Fox J.A.
      • Presta L.G.
      ,
      • Hobbs S.M.
      • Jackson L.E.
      • Hoadley J.
      ,
      • Kim J.-K.
      • Tsen M.-F.
      • Ghetie V.
      • Ward E.S.
      ).
      Rothman et al. (
      • Rothman R.J.
      • Perussia B.
      • Herlyn D.
      • Warren L.
      ) tested the ADCC function of monoclonal murine IgG purified from hybridomas treated with glycosidase inhibitors that acted at different stages in the carbohydrate processing pathway. Treatment with castanospermine, which inhibits removal of glucose residues from the nascent oligosaccharide (
      • Kaushal G.P.
      • Elbein A.D.
      ), generated IgG that showed enhanced ADCC mediated by NK cells, which express only FcγRIIIA, but not by other types of effector cells such as monocytes. Lectin-binding analysis suggested that the castanospermine-treated IgG lacked fucose (
      • Rothman R.J.
      • Perussia B.
      • Herlyn D.
      • Warren L.
      ); however, the IgG resulting from such treatment probably had other carbohydrate structure, such as hypermannosylation as well as terminal glucose residues (
      • Wright A.
      • Morrison S.L.
      ,
      • Hashim O.H.
      • Cushley W.
      ,
      • Hashim O.H.
      • Cushley W.
      ), not routinely found on IgG secreted from nontreated cells or from human serum. In the current study, Lec13 cells were utilized to produce fucose-deficient IgG1. Other than the lack of fucose, the oligosaccharide on the IgG1 was similar to that found in normal CHO cell lines and from human serum (
      • Jefferis R.
      • Lund J.
      • Mizutani H.
      • Nakagawa H.
      • Kawazoe Y.
      • Arata Y.
      • Takahashi N.
      ,
      • Raju S.
      • Briggs J.B.
      • Borge S.M.
      • Jones A.J.S.
      ,
      • Routier F.H.
      • Davies M.J.
      • Bergemann K.
      • Hounsell E.F.
      ) and did not suffer from the hypermannosylation or hyperglucosylation resulting from the use of castanospermine. The improvement in FcγRIII binding and ADCC by the fucose-deficient IgG1 suggests that it is possible to generate monoclonal antibody with otherwise normal oligosaccharide that would have enhanced effector function.
      Normal CHO and HEK293 cells add fucose to IgG oligosaccharide to a high degree (80–98% in this study). IgGs from sera are also highly fucosylated (
      • Jefferis R.
      • Lund J.
      • Mizutani H.
      • Nakagawa H.
      • Kawazoe Y.
      • Arata Y.
      • Takahashi N.
      ,
      • Raju S.
      • Briggs J.B.
      • Borge S.M.
      • Jones A.J.S.
      ,
      • Parekh 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.
      ,
      • Routier F.H.
      • Davies M.J.
      • Bergemann K.
      • Hounsell E.F.
      ). Unfortunately, the Lec13 cell line is not robust enough to consider as a production cell line, because the expression levels of the anti-HER2 Hu4D5 and anti-IgE HuE27 antibodies in this cell line were low compared with the standard CHO cells used. Treatment of fucosylated IgG with fucosidases (
      • Okafo G.N.
      • Burrow L.M.
      • Neville W.
      • Truneh A.
      • Smith R.A.G.
      • Reff M.
      • Camilleri P.
      ) may be useful for generating enough afucosyl IgG to use for assays but might be impractical for production. Hence, a production cell line deficient in fucosylation of IgG remains a challenge.

      REFERENCES

        • Nose M.
        • Wigzell H.
        Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6632-6636
        • Rudd M.P.
        • Ellliot T.
        • Cresswell P.
        • Wilson I.A.
        • Dwek R.A.
        Science. 2001; 291: 2370-2376
        • Jefferis R.
        • Lund J.
        • Pound J.D.
        Immunol. Rev. 1998; 163: 59-76
        • Wright A.
        • Morrison S.L.
        Trends Biotechnol. 1997; 15: 26-32
        • Ravetch J.V.
        • Bolland S.
        Annu. Rev. Immunol. 2001; 19: 275-290
        • Gessner J.E.
        • Heiken H.
        • Tamm A.
        • Schmidt R.E.
        Ann. Hematol. 1998; 76: 231-248
        • Gavin A.
        • Hulett M.
        • Hogarth P.M.
        van de Winkel J.G.J. Hogarth P.M. The Immunoglobulin Receptors and Their Physiological and Pathological Roles in Immunity. Kluwer Academic Publishers BV, Dordrecht, The Netherlands1998: 11-35
        • Makrides S.C.
        Pharmacol. Rev. 1998; 50: 59-87
        • Weis W.I.
        • Taylor M.E.
        • Drickamer K.
        Immunol. Rev. 1998; 163: 19-34
        • Malhotra R.
        • Wormald M.R.
        • Rudd P.M.
        • Fischer P.B.
        • Dwek R.A.
        • Sim R.B.
        Nat. Med. 1995; 1: 237-243
        • Wright A.
        • Morrison S.L.
        J. Immunol. 1998; 160: 3393-3402
        • Shields R.L.
        • Namenuk A.K.
        • Hong K.
        • Meng Y.G.
        • Rae J.
        • Briggs J.
        • Xie D.
        • Lai J.
        • Stadlen A., Li, B.
        • Fox J.A.
        • Presta L.G.
        J. Biol. Chem. 2001; 276: 6591-6604
        • Hobbs S.M.
        • Jackson L.E.
        • Hoadley J.
        Mol. Immunol. 1992; 29: 949-956
        • Kim J.-K.
        • Tsen M.-F.
        • Ghetie V.
        • Ward E.S.
        Eur. J. Immunol. 1994; 24: 542-548
        • Tao M.H.
        • Morrison S.L.
        J. Immunol. 1989; 143: 2595-2601
        • Horan Hand P.H.
        • Calvo B.
        • Milenic D.
        • Yokota T.
        • Finch M.
        • Snoy P.
        • Garmestani K.
        • Gansow O.
        • Schlom J.
        • Kashmiri S.V.S.
        Cancer Immunol. Immunother. 1992; 35: 165-174
        • Wawrzynczak E.J.
        • Cumber A.J.
        • Parnell G.D.
        • Jones P.T.
        • Winter G.
        Mol. Immunol. 1992; 29: 213-220
        • Jefferis R.
        • Lund J.
        • Mizutani H.
        • Nakagawa H.
        • Kawazoe Y.
        • Arata Y.
        • Takahashi N.
        Biochem. J. 1990; 268: 529-537
        • Raju S.
        • Briggs J.B.
        • Borge S.M.
        • Jones A.J.S.
        Glycobiology. 2000; 10: 477-486
        • Jassal R.
        • Jenkins N.
        • Charlwood J.
        • Camilleri P.
        • Jefferis R.
        • Lund J.
        Biochem. Biophys. Res. Commun. 2001; 286: 243-249
        • Groenink J.
        • Spijker J.
        • van den Herik-Oudijk I.E.
        • Boeije L.
        • Rook G.
        • Aarden L.
        • van de Winkel J.G.J.
        • van den Broek M.F.
        Eur. J. Immunol. 1996; 26: 1404-1407
        • Boyd P.N.
        • Lines A.C.
        • Patel A.K.
        Mol. Immunol. 1995; 32: 1311-1318
        • Kumpel B.M.
        • Rademacher T.W.
        • Rook G.A.W.
        • Williams P.J.
        • Wilson I.R.H.
        Hum. Antibod. Hybridomas. 1994; 5: 143-151
        • Tsuchiya N .
        • Endo T.
        • Matsuta K.
        • Yoshinoya S.
        • Aikawa T.
        • Kosuge E.
        • Takeuchi F.
        • Miyamoto T.
        • Kobata A.
        J. Rheumatol. 1989; 16: 285-290
        • Umaña P.
        • Jean-Mairet J.
        • Moudry R.
        • Amstutz H.
        • Bailey J.E.
        Nat. Biotechnol. 1999; 17: 176-180
        • Davies J.
        • Jiang L.
        • Pan L.-Y.
        • LaBarre M.J.
        • Anderson D.
        • Reff M.
        Biotechnol. Bioeng. 2001; 74: 288-294
        • Parekh 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: 452-457
        • Glennie M.J.
        • Johnson P.W.M.
        Immunol. Today. 2000; 21: 403-410
        • Armour K.L.
        • Clark M.R.
        • Hadley A.G.
        • Williamson L.M.
        Eur. J. Immunol. 1999; 29: 2613-2624
        • Duncan A.R.
        • Woof J.M.
        • Partridge L.J.
        • Burton D.R.
        • Winter G.
        Nature. 1988; 332: 563-564
        • Sampson J.H.
        • Crotty L.E.
        • Lee S.
        • Archer G.E.
        • Ashley D.M.
        • Wikstrand C.J.
        • Hale L.P.
        • Small C.
        • Dranoff G.
        • Friedman A.H.
        • Friedman H.S.
        • Bigner D.D.
        Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7503-7508
        • Clynes R.A.
        • Towers T.L.
        • Presta L.G.
        • Ravetch J.V.
        Nat. Med. 2000; 6: 443-446
        • Clynes R.
        • Takechi Y.
        • Moroi Y.
        • Houghton A.
        • Ravetch J.V.
        Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 652-656
        • Anderson D.R.
        • Grillo-Lopez A.
        • Varns C.
        • Chambers K.S.
        • Hanna N.
        Biochem. Soc. Trans. 1997; 25: 705-708
        • Segal D.M.
        • Weiner G.J.
        • Weiner L.M.
        J. Immunol. Methods. 2001; 248: 1-6
        • Penichet M.L.
        • Morrison S.L.
        J. Immunol. Methods. 2001; 248: 91-101
        • Lifely M.R.
        • Hale C.
        • Royce S.
        • Keen M.J.
        • Phillips J.
        Glycobiology. 1995; 5: 813-822
        • Ripka J.
        • Adamany A.
        • Stanley P.
        Arch. Biochem. Biophys. 1986; 249: 533-545
        • Routier F.H.
        • Davies M.J.
        • Bergemann K.
        • Hounsell E.F.
        Glycoconj. J. 1997; 14: 201-207
        • Carter P.
        • Presta L.
        • Gorman C.M.
        • Ridgway J.B.B.
        • Henner D.
        • Wong W.L.T.
        • Rowland A.M.
        • Kotts C.
        • Carver M.E.
        • Shepard M.H.
        Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4285-4291
        • Presta L.G.
        • Lahr S.J.
        • Shields R.L.
        • Porter J.P.
        • Gorman C.M.
        • Jardieu P.M.
        J. Immunol. 1993; 151: 2623-2632
        • Lucas B..
        • Giere L.M.
        • DeMarco R.A.
        • Shen A.
        • Chisholm V.
        • Crowley C.W.
        Nucleic Acids Res. 1996; 24: 1774-1779
      1. Mather, J. P., and Tsao, M. C. (June 16, 1992) U. S. Patent 5,122,469

        • Papac D.I.
        • Briggs J.B.
        • Chin E.T.
        • Jones A.J.S.
        Glycobiology. 1998; 8: 445-454
        • Huizinga T.W.J.
        • Kerst M.
        • Nuyens J.H.
        • Vlug A.
        • von dem Borne A.E.G.K.
        • Roos D.
        • Tetteroo P.A.T.
        J. Immunol. 1989; 142: 2359-2364
        • Idusogie E.E.
        • Presta L.G.
        • Gazzano-Santoro H.
        • Totpal K.
        • Wong P.Y.
        • Ultsch M.
        • Meng Y.G.
        • Mulkerrin M.G.
        J. Immunol. 2000; 164: 4178-4184
        • Liu J.
        • Lester P.
        • Builder S.
        • Shire S.J.
        Biochemistry. 1995; 34: 10474-10482
        • Clark M.R.
        • Clarkson S.B.
        • Ory P.A.
        • Stollman N.
        • Goldstein I.M.
        J. Immunol. 1989; 143: 1731-1734
        • Warmerdam P.A.M.
        • van de Winkel J.G.J.
        • Vlug A.
        • Westerdaal N.A.C.
        • Capel P.J.A.
        J. Immunol. 1991; 147: 1338-1343
        • Koene H.R.
        • Kleijer M.
        • Algra J.
        • Roos D.
        • von dem Borne E.G.K.
        • de Hass M.
        Blood. 1997; 90: 1109-1114
        • Wu J.
        • Edberg J.C.
        • Redecha P.B.
        • Bansal V.
        • Guyre P.M.
        • Coleman K.
        • Salmon J.E.
        • Kimberly R.P.
        J. Clin. Invest. 1997; 100: 1059-1070
        • Raghavan M.
        • Bjorkman P.J.
        Annu. Rev. Cell Dev. Biol. 1996; 12: 181-220
        • Ghetie V.
        • Ward E.S.
        Annu. Rev. Immunol. 2000; 18: 739-766
        • Hudziak R.M.
        • Lewis G.D.
        • Winget M.
        • Fendly B.M.
        • Shepard H.M.
        • Ullrich A.
        Mol. Cell. Biol. 1989; 9: 1165-1172
        • Walker M.R.
        • Lund J.
        • Thompson K.M.
        • Jefferis R.
        Biochem. J. 1989; 259: 347-353
        • Sondermann P.
        • Huber R.
        • Oosthuizen V.
        • Jacob U.
        Nature. 2000; 406: 267-273
        • Radaev S.
        • Motyka S.
        • Fridman W.-H.
        • Sautes-Fridman C.
        • Sun P.D.
        J. Biol. Chem. 2001; 276: 16469-16477
        • Deisenhofer J.
        Biochemistry. 1981; 20: 2361-2370
        • Harris L.J.
        • Skaletsky E.
        • McPherson A.
        J. Mol. Biol. 1998; 275: 861-872
        • Harris L.J.
        • Larson S.B.
        • Hasel K.W.
        • McPherson A.
        Biochemistry. 1997; 36: 1581-1597
        • Rothman R.J.
        • Perussia B.
        • Herlyn D.
        • Warren L.
        Mol. Immunol. 1989; 26: 1113-1123
        • Kaushal G.P.
        • Elbein A.D.
        Methods Enzymol. 1994; 230: 316-329
        • Hashim O.H.
        • Cushley W.
        Immunology. 1988; 63: 383-388
        • Hashim O.H.
        • Cushley W.
        Mol. Immunol. 1987; 24: 1087-1096
        • Okafo G.N.
        • Burrow L.M.
        • Neville W.
        • Truneh A.
        • Smith R.A.G.
        • Reff M.
        • Camilleri P.
        Anal. Biochem. 1996; 240: 68-74