The Carbohydrate at FcγRIIIa Asn-162

Abstract

FcγRIIIa plays a prominent role in the elimination of tumor cells by antibody-based cancer therapies. Non-fucosylated bisected IgGs bind this receptor with increased affinity and trigger FcγRIII-mediated effector functions more efficiently than native, fucosylated antibodies. In this study the contribution of the carbohydrates of both binding partners to the strength of the complex was analyzed. Glycoengineering of the antibody increased affinity for two polymorphic forms of soluble human FcγRIIIa (by up to 50-fold) but did not affect binding to the inhibitory FcγRIIb receptor. While the absence of carbohydrate at FcγRIIIa’s Asn-162 increased affinity for native IgG, presumably due to the removal of steric hindrance caused by the bulky sugars, it unexpectedly reduced affinity for glycoengineered (GE) antibodies by over one order of magnitude, bringing the affinity down to the same level as for native IgG. We conclude that the high affinity between GE antibodies and FcγRIII is mediated by productive interactions formed between the receptor carbohydrate attached at Asn-162 and regions of the Fc that are only accessible when it is nonfucosylated. As FcγRIIIa and FcγRIIIb are the only human Fcγ receptors glycosylated at this position, the proposed interactions explain the observed selective affinity increase of GE antibodies for only these receptors. Furthermore, we predict from our structural model that only one of the two Fc-fucose residues needs to be absent for increased binding affinity toward FcγRIII. This information can be exploited for the design of new antibodies with altered Fc receptor binding affinity and enhanced therapeutic potential. FcγRIIIa plays a prominent role in the elimination of tumor cells by antibody-based cancer therapies. Non-fucosylated bisected IgGs bind this receptor with increased affinity and trigger FcγRIII-mediated effector functions more efficiently than native, fucosylated antibodies. In this study the contribution of the carbohydrates of both binding partners to the strength of the complex was analyzed. Glycoengineering of the antibody increased affinity for two polymorphic forms of soluble human FcγRIIIa (by up to 50-fold) but did not affect binding to the inhibitory FcγRIIb receptor. While the absence of carbohydrate at FcγRIIIa’s Asn-162 increased affinity for native IgG, presumably due to the removal of steric hindrance caused by the bulky sugars, it unexpectedly reduced affinity for glycoengineered (GE) antibodies by over one order of magnitude, bringing the affinity down to the same level as for native IgG. We conclude that the high affinity between GE antibodies and FcγRIII is mediated by productive interactions formed between the receptor carbohydrate attached at Asn-162 and regions of the Fc that are only accessible when it is nonfucosylated. As FcγRIIIa and FcγRIIIb are the only human Fcγ receptors glycosylated at this position, the proposed interactions explain the observed selective affinity increase of GE antibodies for only these receptors. Furthermore, we predict from our structural model that only one of the two Fc-fucose residues needs to be absent for increased binding affinity toward FcγRIII. This information can be exploited for the design of new antibodies with altered Fc receptor binding affinity and enhanced therapeutic potential. Antibodies provide a link between the humoral and the cellular immune system with IgG 2The abbreviations used are: IgG, immunoglobulin G; GE, glycoengineered; Fuc, fucose; GnT-III, β1,4-N-acetylglucosaminyltransferase III; FcγR, Fcγ receptor; mAb, monoclonal antibody; SPR, surface plasmon resonance; h, human; s, soluble; m, mouse. 2The abbreviations used are: IgG, immunoglobulin G; GE, glycoengineered; Fuc, fucose; GnT-III, β1,4-N-acetylglucosaminyltransferase III; FcγR, Fcγ receptor; mAb, monoclonal antibody; SPR, surface plasmon resonance; h, human; s, soluble; m, mouse. being the most abundant serum immunoglobulin. While the Fab regions of the antibody recognize antigens, the Fc part interacts with membrane-bound Fcγ receptors (FcγRs) that are differentially expressed by all immune competent cells. Receptor crosslinking by a multivalent antigen-antibody complex triggers degranulation, cytolysis or phagocytosis of the target cell, and transcriptional activation of cytokine-encoding genes (1Deo Y.M. Graziano R.F. Repp R. van de Winkel J.G. Immunol. Today. 1997; 18: 127-135Abstract Full Text PDF PubMed Scopus (219) Google Scholar). Recently, the importance of the activating receptor FcγRIIIa for the in vivo elimination of tumor cells in humans has been demonstrated. In follicular non-Hodgkin’s lymphoma patients, a relationship was discovered between the FcγRIIIa genotype and clinical and molecular responses to rituximab, an anti-CD20 chimeric antibody used against hematological malignancies (2Cartron G. Dacheux L. Salles G. Solal-Celigny P. Bardos P. Colombat P. Watier H. Blood. 2002; 99: 754-758Crossref PubMed Scopus (1603) Google Scholar). The authors demonstrated that the efficacy of rituximab was higher in patients homozygous for the “high affinity” FcγRIIIa, characterized by a valine at position 158 (FcγRIIIa[Val-158]), than in patients heterozygous or homozygous for the “low affinity” FcγRIIIa, which has a phenylalanine residue at this position (FcγRIIIa[Phe-158]) and has lower affinity for IgG (3Koene H.R. Kleijer M. Algra J. Roos D. von dem Borne A.E. de Haas M. Blood. 1997; 90: 1109-1114Crossref PubMed Google Scholar). Increased survival of lymphoma patients that mount an anti-tumor humoral response after anti-idiotypic vaccination has also been correlated with homozygocity for FcγRIIIa[Val-158] (4Weng W.K. Czerwinski D. Timmerman J. Hsu F.J. Levy R. J. Clin. Oncol. 2004; 22: 4717-4724Crossref PubMed Google Scholar). The above observations imply a crucial role for FcγRIIIa in the elimination of tumor cells and support the idea that therapeutic monoclonal antibodies (mAbs) with increased affinity for FcγRIIIa will have improved biological activity. One route to increase the affinity of monoclonal antibodies toward FcγRIIIa and consequently to enhance their effector functions is manipulation of their carbohydrate moiety (5Umaña P. Jean-Mairet J. Moudry R. Amstutz H. Bailey J.E. Nat. Biotechnol. 1999; 17: 176-180Crossref PubMed Scopus (620) Google Scholar, 6Shields 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: 26733-26740Abstract Full Text Full Text PDF PubMed Scopus (1266) Google Scholar, 7Ferrara C. Brünker P. Suter T. Moser S. Püntener U. Umaña P. Biotechnol. Bioeng. 2006; (in press)PubMed Google Scholar). The N-glycosylation of the Fc fragment at Asn-297 in both Cγ2 domains is crucial to the affinity for all FcγRs (8Tao M.H. Morrison S.L. J. Immunol. 1989; 143: 2595-2601PubMed Google Scholar, 9Mimura Y. Sondermann P. Ghirlando R. Lund J. Young S.P. Goodall M. Jefferis R. J. Biol. Chem. 2001; 276: 45539-45547Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar) and is required to elicit proper effector functions (10Wright A. Morrison S.L. J. Exp. Med. 1994; 180: 1087-1096Crossref PubMed Scopus (158) Google Scholar, 11Sarmay G. Lund J. Rozsnyay Z. Gergely J. Jefferis R. Mol. Immunol. 1992; 29: 633-639Crossref PubMed Scopus (100) Google Scholar). It is comprised of a conserved pentasaccharide structure with variable addition of fucose and outer arm sugars (12Jefferis R. Lund J. Pound J.D. Immunol. Rev. 1998; 163: 59-76Crossref PubMed Scopus (283) Google Scholar). The N-glycosylation pattern of mAbs can be manipulated by engineering the glycosylation pathway of the production cell line using enzyme activities that lead to naturally occurring carbohydrates. Umaña and co-workers (5Umaña P. Jean-Mairet J. Moudry R. Amstutz H. Bailey J.E. Nat. Biotechnol. 1999; 17: 176-180Crossref PubMed Scopus (620) Google Scholar, 7Ferrara C. Brünker P. Suter T. Moser S. Püntener U. Umaña P. Biotechnol. Bioeng. 2006; (in press)PubMed Google Scholar) reported the production of glycoengineered (GE) antibodies, which feature high proportions of bisected, non-fucosylated oligosaccharides, improved affinity for FcγRIIIa and enhanced antibody-dependent cellular cytotoxicity. Antibodies with increased binding to FcγRIIIa have also been obtained using a cell line which is unable to add fucose residues to N-linked oligosaccharides (6Shields 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: 26733-26740Abstract Full Text Full Text PDF PubMed Scopus (1266) Google Scholar, 13Shinkawa T. Nakamura K. Yamane N. Shoji-Hosaka E. Kanda Y. Sakurada M. Uchida K. Anazawa H. Satoh M. Yamasaki M. Hanai N. Shitara K. J. Biol. Chem. 2003; 278: 3466-3473Abstract Full Text Full Text PDF PubMed Scopus (1044) Google Scholar). Little information is available on the influence of FcγRIIIa carbohydrates on the affinity for IgG. The crystal structure of unglycosylated FcγRIII in complex with the Fc fragment of human (h) IgG1 indicates that a carbohydrate moiety attached at Asn-162 of FcγRIII would point into the central cavity within the Fc fragment (14Sondermann P. Huber R. Oosthuizen V. Jacob U. Nature. 2000; 406: 267-273Crossref PubMed Scopus (576) Google Scholar), where the rigid core glycans attached to IgG-Asn-297 are also located (15Huber R. Deisenhofer J. Colman P.M. Matsushima M. Palm W. Nature. 1976; 264: 415-420Crossref PubMed Scopus (303) Google Scholar). In the present study, binding of glycosylated soluble (s) hFcγRIIIa variants to distinct antibody glycovariants was evaluated by surface plasmon resonance (SPR) and in a cellular system to dissect the interaction between IgG1 and glycosylated FcγRIIIa on a molecular level. Cell Lines, Expression Vectors, and Antibodies—HEK293-EBNA cells were a kind gift from Rene Fischer (Laboratory of Organic Chemistry, Zürich, Switzerland). Additional cell lines used in this study were Jurkat cells (human lymphoblastic T cell, ATCC number TIB-152) and FcγRIIIa[Val-158]- and FcγRIIIa[Val-158/Gln-162]-expressing Jurkat cell lines, generated as described previously (7Ferrara C. Brünker P. Suter T. Moser S. Püntener U. Umaña P. Biotechnol. Bioeng. 2006; (in press)PubMed Google Scholar). The cells were cultivated according to the instructions of the supplier. DNA encoding the shFcγRIIIa[Val-158] and shFcγRIIIa[Phe-158] variants were fused after residue 191 to a hexahistidine tag (NH2-MRTEDL... GYQG(H6)-COOH, numbering is based on the mature protein) using PCR as described (16Shields 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: 6591-6604Abstract Full Text Full Text PDF PubMed Scopus (898) Google Scholar). Asn-162 of shFcγRIIIa[Val-158] was exchanged for Gln by PCR. All expression vectors contained the replication origin oriP from the Epstein-Barr virus for expression in HEK293-EBNA cells. GE and native anti-CD20 antibodies were produced in HEK-293 EBNA cells and characterized by standard methods. Neutral oligosaccharide profiles for the antibodies were analyzed by mass spectrometry (Autoflex, Bruker Daltonics GmbH, Faellanden, Switzerland) in positive ion mode (17Papac D.I. Briggs J.B. Chin E.T. Jones A.J. Glycobiology. 1998; 8: 445-454Crossref PubMed Scopus (135) Google Scholar). Production and Purification of Recombinant shFcγRIIIa Receptors— The shFcγRIIIa variants were produced by transient expression in HEK-293-EBNA cells (18Jordan M. Schallhorn A. Wurm F.M. Nucleic Acids Res. 1996; 24: 596-601Crossref PubMed Scopus (704) Google Scholar) and purified using a HiTrap Chelating HP column (Amersham Biosciences, Otelfingen, Switzerland) and a size exclusion chromatography step with HBS-EP buffer (0.01 m HEPES, pH 7.4, 0.15 m NaCl, 3 mm EDTA, 0.005% Surfactant P20). Human sFcγRIIb and mouse (m) sFcγRIIb were produced and purified as described (19Sondermann P. Jacob U. Biol. Chem. 1999; 380: 717-721Crossref PubMed Scopus (32) Google Scholar). The concentration of proteins was determined as described (20Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (4969) Google Scholar). SPR—SPR experiments were performed on a Biacore3000 with HBS-EP as running buffer (Biacore, Freiburg, Germany). Direct coupling of around 1,000 resonance units of human IgG glycovariants was performed on a CM5 chip using the standard amine coupling kit (Biacore). Different concentrations of soluble FcγRs were passed with a flow rate of 10 μl/min through the flow cells. Increasing the flow rate did not influence the binding curves. Bulk refractive index differences were corrected for by subtracting the response obtained on flowing sample over a bovine serum albumin-coupled surface. The steady state response was used to obtain the dissociation constant KD by non-linear curve fitting of the Langmuir binding isotherm. Kinetic constants were obtained using the BIAevaluation program curve-fitting facility (v3.0, Biacore), to fit rate equations for 1:1 Langmuir binding by numerical integration. Binding of IgG to FcγRIIIa-expressing Cells—The experiment was conducted as described previously (7Ferrara C. Brünker P. Suter T. Moser S. Püntener U. Umaña P. Biotechnol. Bioeng. 2006; (in press)PubMed Google Scholar). Briefly, hFcγRIIIa-expressing Jurkat cells were incubated with IgG variants in phosphate-buffered saline, 0.1% bovine serum albumin. After two washes with phosphate-buffered saline, 0.1% bovine serum albumin, antibody binding was detected by incubating with 1:200 fluorescein isothiocyanate-conjugated goat anti-human F(ab′)2, F(ab′)2 fragments (Jackson ImmunoResearch, West Grove, PA) (16Shields 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: 6591-6604Abstract Full Text Full Text PDF PubMed Scopus (898) Google Scholar). The fluorescence intensity of the bound antibody variants was determined on a FACS Calibur (BD Biosciences, Allschwil, Switzerland). Modeling—We visualized the interaction of the Fc fragment derived from native IgG and the FcγRIII glycans after creating a carbohydrate in silico, attached at the position Asn-162 of the receptor. The glycan unit was modeled on to the crystal structure of FcγRIII in complex with Fc-IgG (Protein Data Bank code 1e4k). The interaction between FcγRIII and IgG was modeled by directing the Fc-linked pentasaccharide core to the fucose residue of oligosaccharide linked to the Fc-Asn-297. The model was not energy minimized and only created to visualize the proposed binding mode. Biochemical Characterization of Soluble hFcγRIIIa Receptors and Antibody Glycovariants—ShFcγRIIIa[Val-158], shFcγRIIIa[Phe-158], and shFcγRIIIa[Val-158/Gln-162] were expressed in HEK293-EBNA cells and purified to homogeneity. The purified shFcγRIIIa[Val-158] and [Phe-158] migrate as broad bands in the apparent molecular weight range of 40–50 kDa when subjected to reducing SDS-PAGE. The apparent molecular weight is slightly lower for the mutant shFcγRIIIa[Val-158/Gln-162] (data not shown). This can be explained by the elimination of the carbohydrates linked to Asn-162. Upon enzymatic N-deglycosylation all three receptor variants migrate identically in the apparent molecular weight range of 25–30 kDa and feature three bands as observed previously for the membrane form of N-deglycosylated hFcγRIII (21Edberg J.C. Kimberly J. Immunol. 1997; Scholar, B. J. Exp. Med. 1989; PubMed Scopus Google Scholar). This pattern from the of carbohydrates. The native antibody glycosylation pattern is characterized by fucosylated complex oligosaccharides and with to antibodies were produced in a cell line β1,4-N-acetylglucosaminyltransferase an enzyme the addition of a to the of the GE antibody variants were was produced by of and by of and 7Ferrara C. Brünker P. Suter T. Moser S. Püntener U. Umaña P. Biotechnol. Bioeng. 2006; (in press)PubMed Google and and feature high proportions of bisected, non-fucosylated oligosaccharides and We have previously that both forms in affinity for FcγRIIIa and increased antibody-dependent cellular to native but in their in (7Ferrara C. Brünker P. Suter T. Moser S. Püntener U. Umaña P. Biotechnol. Bioeng. 2006; (in press)PubMed Google Scholar). IgG to Antibodies with Increased for interaction of antibody glycovariants with shFcγRIIIa variants and and was analyzed by Binding of shFcγRIIIa[Val-158] to the GE antibodies was up to than to the native antibody The affinity polymorphic form of the shFcγRIIIa[Phe-158], also bound to the GE antibodies with higher affinity than to the native antibody of affinity constants determined by and Data are the of two not for for for for for for for for for for for for for for for for for for for for for for for for Kinetic for in a new the dissociation of both receptor variants from native IgG was to a of constants for these the that a of the antibodies is dissociation from the receptors dissociation from native IgG dissociation were using rate constants and with the (data not shown). that the increase in affinity be for by dissociation rate constant The rate constants of the two polymorphic forms of shFcγRIIIa for GE antibodies were but the dissociation rate of was and for the lower affinity of this receptor The affinity of the antibodies for human and FcγRIIb was also GE and native IgGs bound the human inhibitory receptor with affinity the of this receptor the affinity for human IgG1 was also by antibody but was that of the human FcγRIIb receptor The dissociation constant for the interaction of the native antibody with only be determined by steady state the was for a FcγRIIIa Binding to Antibody mutant form of hFcγRIIIa that is not glycosylated at Asn-162 was used to the influence of the carbohydrate on complex with IgG. removal of N-glycosylation at native IgG a increase in affinity for the GE antibodies an over in affinity binding to GE antibodies, removal of the receptor glycosylation in an increase in but an over increase in state and determined KD by for binding of shFcγRIIIa[Val-158/Gln-162] to the antibodies. This most from a high in fitting the dissociation The were using Jurkat cells membrane bound FcγRIIIa, which a for FcγRIIIa expression E. van de Winkel J.G. van M. R.L. Cell Immunol. 1992; 143: PubMed Scopus Google Scholar). We used the which not between FcγRIIIa[Val-158] and B. T. 2003; PubMed Scopus Google Scholar), to FcγRIII expression in these cell In this experiment GE antibodies bound FcγRIIIa[Val-158] than the native antibody Binding to reduced for all IgG native IgG The dissociation rate constants in the experiment for binding of to all three IgG variants explain the lower binding in the cellular Kinetic of the our KD for the interaction of IgG1 with glycosylated FcγRIIIa with previously by A. Shoji-Hosaka E. Nakamura K. M. Uchida K. S. K. Shitara K. J. Mol. Biol. 2004; PubMed Scopus Google Scholar). authors that the affinity increase of the non-fucosylated (GE) antibody is caused by an increase in In we not and for binding to native IgG due to the high of the of the binding for native and GE antibodies dissociation of the receptor variants from native IgG We conclude that antibody new interactions between the binding partners are formed or the present are we that glycoengineered antibodies bind with higher affinity to the more affinity of FcγRIIIa than native antibodies to the high affinity of the receptor. This the of antibody for with this The of FcγRIIIa at Asn-162 Binding to of origin is a glycosylated with N-linked glycosylation the crystal structure of in complex with unglycosylated FcγRIII (14Sondermann P. Huber R. Oosthuizen V. Jacob U. Nature. 2000; 406: 267-273Crossref PubMed Scopus (576) Google Scholar), glycosylation at Asn-162 in FcγRIII has been to affinity for native IgG1 due to steric hindrance by the carbohydrate This has been with the glycosylation mutant of removal of carbohydrates at the N-glycosylation did not affect affinity for native IgG B. T. 2003; PubMed Scopus Google Scholar). the importance of glycosylation of IgG and FcγRIIIa for their a mutant of the high affinity receptor which is unglycosylated at position was As removal of the carbohydrate at Asn-162 of the receptor increased binding affinity for the native antibody the removal of the FcγRIIIa’s carbohydrate at Asn-162 unexpectedly to reduced binding affinity for GE antibodies by over an order of magnitude, bringing the affinity down to the level observed for the native The were in a cellular where GE antibodies bound to than to cells In two have to be for high affinity interaction between GE IgG and a carbohydrate has to be attached at and productive of this receptor carbohydrate with the can only be the is on these we a model in which the carbohydrate of FcγRIII a of the where a fucose residue is attached in native antibodies. This fucose residue from the surface of the Fc into and a of the Fc receptor carbohydrate productive interactions It be that a with the Fc is by a receptor carbohydrate with as three units Furthermore, the model that only one of the two Fc-fucose residues needs to be absent for increased binding affinity toward FcγRIII. In a study A. Shoji-Hosaka E. Nakamura K. M. Uchida K. S. K. Shitara K. J. Mol. Biol. 2004; PubMed Scopus Google Scholar) proposed that non-fucosylated antibodies bind FcγRIIIa with increased affinity as a of a formed between of the Fc and of the we that the increased affinity of non-fucosylated antibodies on glycosylation of the receptor which that an is to the affinity between GE antibodies and FcγRIIIa and FcγRIIIb forms are the only forms of the human FcγRs that N-glycosylation within the binding to IgG. We conclude that affinity for IgG will be by receptor glycosylation only for these two of the of FcγRIII from indicates that the N-glycosylation Asn-162 is by FcγRIII from and it is in the and mouse FcγRIII. mouse and number genes with high to the human FcγRIII and which proteins the Asn-162 glycosylation were 2002; PubMed Scopus Google Scholar), and expression of the was reported P. K. Full Text Full Text PDF PubMed Scopus Google Scholar). The of a glycosylation the immune system to the affinity toward FcγRIII by FcγRIII glycosylation (21Edberg J.C. Kimberly J. Immunol. 1997; Scholar) and by of the fucose of IgG. The between and has been proposed that an in the of activating to inhibitory enhance the efficacy of therapeutic antibodies Presta L.G. Nat. Med. 2000; PubMed Scopus Google Scholar). In the study, the inhibitory receptor was to have a affinity for native and GE antibodies The inhibitory receptors from mouse and human are not glycosylated at Asn-162. The of for GE antibodies by FcγRIIb is with glycosylation of activating FcγRIII at Asn-162 being for increased binding to non-fucosylated IgGs and that these GE antibodies enhanced therapeutic The that has higher affinity than human FcγRIIb for both native and GE be for the of in vivo experiments using mouse binding to the inhibitory receptor in a mouse model in a of the immune response than that observed in We demonstrated the importance of the carbohydrate of both FcγRIII and IgG for their provide into the complex and an interaction between the Asn-162 carbohydrate of FcγRIII and the Fc of non-fucosylated IgG This the design of new antibody variants that productive interactions with the carbohydrate of FcγRIIIa, which on with monoclonal antibodies.