High Resolution Mapping of the Binding Site on Human IgG1 for Fcgamma RI, Fcgamma RII, Fcgamma RIII, and FcRn and Design of IgG1 Variants with Improved Binding to the Fcgamma R*

Robert L. ShieldsDagger , Angela K. NamenukDagger , Kyu Hong§, Y. Gloria Meng§, Julie RaeDagger , John Briggs, Dong Xie||, Jadine LaiDagger , Andrew StadlenDagger , Betty LiDagger , Judith A. Fox||, and Leonard G. PrestaDagger **

From the Dagger  Department of Immunology, the § Department of BioAnalytical Technology, the  Department of Analytical Chemistry, and the || Department of Pharmacokinetics and Metabolism, Genentech, Inc., South San Francisco, California 94080

Received for publication, October 17, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Immunoglobulin G (IgG) Fc receptors play a critical role in linking IgG antibody-mediated immune responses with cellular effector functions. A high resolution map of the binding site on human IgG1 for human Fcgamma RI, Fcgamma RIIA, Fcgamma RIIB, Fcgamma RIIIA, and FcRn receptors has been determined. A common set of IgG1 residues is involved in binding to all Fcgamma R; Fcgamma RII and Fcgamma RIII also utilize residues outside this common set. In addition to residues which, when altered, abrogated binding to one or more of the receptors, several residues were found that improved binding only to specific receptors or simultaneously improved binding to one type of receptor and reduced binding to another type. Select IgG1 variants with improved binding to Fcgamma RIIIA exhibited up to 100% enhancement in antibody-dependent cell cytotoxicity using human effector cells; these variants included changes at residues not found at the binding interface in the IgG/Fcgamma RIIIA co-crystal structure (Sondermann, P., Huber, R., Oosthuizen, V., and Jacob, U. (2000) Nature 406, 267-273). These engineered antibodies may have important implications for improving antibody therapeutic efficacy.



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ABSTRACT
INTRODUCTION
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Monoclonal antibodies (mAbs)1 are increasingly being used as therapeutics in human disease (1-3). Although some of these, e.g. mAbs that bind to a receptor or ligand and thereby block ligand-receptor interaction, may function without utilizing antibody effector mechanisms, other mAbs may need to recruit the immune system to kill the target cell (4-6). If immune system recruitment is desirable for a therapeutic mAb, 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, immune system recruitment can be abrogated by altering IgG residues in the lower hinge region (7, 8), using human IgG2 or IgG4 subclasses that are comparatively inefficient in effector function or using antibody F(ab) or F(ab')2 fragments (although these may have undesirable rapid clearance rates). There are few methods that improve immune system recruitment; these include bispecific antibodies, in which one arm of the antibody binds to an IgG receptor (9), cytokine-IgG fusion proteins (10), and optimization of the Asn297-linked carbohydrate (11, 12). Alteration of clearance rate is also being investigated (13).

IgG Fc receptors play a critical role in linking IgG antibody-mediated immune responses with cellular effector functions. The latter include release of inflammatory mediators, endocytosis of immune complexes, phagocytosis of microorganisms, antibody-dependent cellular cytotoxicity (ADCC), and regulation of immune system cell activation (14-17). One group of IgG Fc receptors, Fcgamma R, are expressed on leukocytes and are composed of three distinct classes as follows: Fcgamma RI (CD64), Fcgamma RII (CD32), and Fcgamma RIII (CD16). In humans, the latter two classes can be further divided into Fcgamma RIIA and Fcgamma RIIB, Fcgamma RIIIA and Fcgamma RIIIB. Structurally, the Fcgamma R are all members of the immunoglobulin superfamily, having an IgG-binding alpha -chain with an extracellular portion composed of either two (Fcgamma RII and Fcgamma RIII) or three (Fcgamma RI) Ig-like domains. In addition, Fcgamma RI and Fcgamma RIII have accessory protein chains (gamma  and zeta ) associated with the alpha -chain that function in signal transduction. The receptors are also distinguished by their affinity for IgG. Fcgamma RI exhibits a high affinity for IgG, Ka = 108-109 M-1 (14), and can bind monomeric IgG. In contrast, Fcgamma RII and Fcgamma RIII show a weaker affinity for monomeric IgG, Ka <=  107 M-1 (14), and hence can only interact effectively with multimeric immune complexes.

Given the interest in and increasing use of antibody therapeutics, a comprehensive mapping of the binding site on human IgG for the different Fcgamma R could provide for alternative methods of either abrogating or enhancing immune recruitment via Fcgamma R. Previous studies mapped the binding site on human and murine IgG for Fcgamma R primarily to the lower hinge region composed of IgG residues 233-239 (Eu numbering, see Ref. 18) (8, 14-17, 19-22). Other studies proposed additional broad segments, e.g. Gly316-Lys338 for human Fcgamma RI (21), Lys274-Arg301 and Tyr407-Arg416 for human Fcgamma RIII (23, 24), or found few specific residues outside the lower hinge, e.g. Asn297 and Glu318 for murine IgG2b interacting with murine Fcgamma RII (25). The very recent report of the 3.2-Å crystal structure of the human IgG1 Fc fragment with human Fcgamma RIIIA delineated IgG1 residues Leu234-Ser239, Asp265-Glu269, Asn297-Thr299, and Ala327-Ile332 as involved in binding to Fcgamma RIIIA (26).

The current study provides a complete, high resolution mapping of human IgG1 for human Fcgamma R receptors (Fcgamma RI, Fcgamma RIIA, Fcgamma RIIB, and Fcgamma RIIIA) as well as for human FcRn, an Fc receptor belonging to the major histocompatability complex structural class, which is involved in IgG transport and clearance (27, 28). The binding site on human IgG1 for the various receptors was determined by individually changing all solvent-exposed amino acids in human IgG1 CH2 and CH3 domains, based on the crystal structure of human IgG1 Fc (30), to Ala. A common set of IgG1 residues is involved in binding to all Fcgamma R; Fcgamma RII and Fcgamma RIII also utilize distinct residues in addition to this common set. As well as residues that abrogated binding to one or more Fc receptors when changed to Ala, several positions were found that improved binding only to specific receptors or simultaneously improved binding to one type of Fcgamma R and reduced binding to another type. Notably, for both Fcgamma RIIIA and FcRn, which have crystal structures of complexes with IgG available (26, 29), several IgG residues not found at the IgG:receptor interface had a profound effect on binding and biological activity. Select IgG1 variants with improved binding to Fcgamma RIIIA showed an enhancement in ADCC when either peripheral blood monocyte cells (PBMC) or natural killer cells (NK) were used. These variants may have important implications for using Fc-engineered antibodies for improved therapeutic efficacy.


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cDNA Constructs and Soluble Receptor Expression-- The cDNAs encoding extracellular and transmembrane domains of human Fcgamma RIIA (CD32A; His131 allotype), Fcgamma RIIB (CD32B), and Fcgamma RIIIA (CD16A; Val158 allotype) were provided by Dr. J. Ravetch (Rockefeller University, New York). Fcgamma RIIA-Arg131 allotype and Fcgamma RIIIA-Phe158 allotype were generated by site-directed mutagenesis (31). The cDNA for Fcgamma RI (CD64) was isolated by reverse transcriptase-PCR (GeneAmp, PerkinElmer Life Sciences) of oligo(dT)-primed RNA from U937 cells using primers that generated a fragment encoding the alpha -chain extracellular domain. The cDNAs encoding human neonatal Fc receptor (FcRn) alpha -chain, beta 2-microglobulin subunit, and human Fcgamma R gamma -chain were obtained from the I.M.A.G.E. Consortium (32). The coding regions of all receptors were subcloned into previously described pRK mammalian cell expression vectors (33).

For all Fcgamma R and the FcRn alpha -chain pRK plasmids, the transmembrane and intracellular domains were replaced by DNA encoding a Gly-His6 tag and human glutathione S-transferase (GST). The 234-amino acid GST sequence was obtained by PCR from the pGEX-4T2 plasmid (Amersham Pharmacia Biotech) with NheI and XbaI restriction sites at the 5' and 3' ends, respectively. Thus, the expressed proteins contained the extracellular domains of the alpha -chain fused at their carboxyl termini to Gly/His6/GST at amino acid positions as follows: Fcgamma RI, His292; Fcgamma RIIA, Met216; Fcgamma RIIB, Met195; Fcgamma RIIIA, Gln191; FcRn, Ser297 (residue numbers include signal peptides).

Plasmids were transfected into the adenovirus-transformed human embryonic kidney cell line 293 by calcium phosphate precipitation (34). Supernatants were collected 72 h after conversion to serum-free PSO4 medium supplemented with 10 mg/liter recombinant bovine insulin, 1 mg/liter human transferrin, and trace elements. Proteins were purified by nickel-nitrilotriacetic acid chromatography (Qiagen, Valencia, CA) and buffer exchanged into phosphate-buffered saline (PBS) using Centriprep-30 concentrators (Millipore, Bedford, MA). Proteins were analyzed on 4-20% SDS-polyacrylamide gels (NOVEX, San Diego, CA), transferred to polyvinylidene difluoride membranes (NOVEX), and their amino termini sequenced to ensure proper signal sequence cleavage. Receptor conformation was evaluated by ELISA using murine monoclonals 32.2 (anti-Fcgamma RI), IV.3 (anti-Fcgamma RII), 3G8 (anti-Fcgamma RIII) (Medarex, Annandale, NJ), and B1G6 (anti-beta 2-microglobulin) (Beckman Coulter, Palo Alto, CA). Receptor concentrations were determined by amino acid analysis.

Human IgG1 Mutagenesis-- The humanized IgG1 anti-IgE E27, an affinity-matured variant of anti-IgE E25, binds to the Fcepsilon 3 domain of human IgE (35). When mixed with human IgE in a 1:1 molar ratio, the IgE and anti-IgE form a hexameric complex composed of three IgE and three anti-IgE (36). Site-directed mutagenesis (31) on E27 IgG1 was used to generate IgG1 variants in which all solvent-exposed residues in the CH2 and CH3 domains were individually altered to Ala; selection of solvent-exposed residues was based on the crystal structure of human IgG1 Fc (30). Human IgG2, IgG3, and IgG4 isotypes of E27 were constructed by subcloning the appropriate heavy chain Fc cDNAs from a human spleen cDNA library into a pRK vector containing the E27 variable heavy domain. All IgG isotypes and variants were expressed using the same E27 kappa  light chain.

Following cotransfection of heavy and light chain plasmids into 293 cells, IgG1, IgG2, IgG4, and variants were purified by protein A chromatography (Amersham Pharmacia Biotech). IgG3 isotype was purified using protein G chromatography (Amersham Pharmacia Biotech). All proteins were analyzed by SDS-polyacrylamide gel electrophoresis. Protein concentrations were determined using A280 and verified using amino acid composition analysis and a human IgG Fc ELISA. IgGs were also tested for their binding to human IgE in an ELISA format to ensure that they bound IgE as well as native E27. Structural integrity of the variants was also evident by their ability to be purified using protein A (which binds at the CH2:CH3 domain interface (30)) as well as all variants, except P329A, binding similar to native IgG1 to at least one of the five receptors.

IgG Immune Complexes and IgG Binding to Fcgamma R-- Fcgamma RIIA, Fcgamma RIIB, and Fcgamma RIIIA fusion proteins at 1 µg/ml in PBS, pH 7.4, were coated onto ELISA plates (Nalge-Nunc, Naperville, IL) for 48 h at 4 °C. Plates were blocked with Tris-buffered saline, 0.5% bovine serum albumin, 0.05% polysorbate-20, 2 mM EDTA, pH 7.45 (assay buffer), at 25 °C for 1 h. E27-IgE hexameric complexes were prepared in assay buffer by mixing equimolar amounts of E27 and human myeloma IgE (37) at 25 °C for 1 h. Serial 3-fold dilutions of native E27 standard or variant complexes (10.0-0.0045 µg/ml) were added to plates and incubated for 2 h. After washing plates with assay buffer, bound complexes to Fcgamma RIIA and Fcgamma RIIB were detected with peroxidase-conjugated F(ab')2 fragment of goat anti-human F(ab')2-specific IgG (Jackson ImmunoResearch, West Grove, PA). Binding of complexes to Fcgamma RIIIA was detected with peroxidase-conjugated protein G (Bio-Rad). The substrate used was o-phenylenediamine dihydrochloride (Sigma). Absorbance at 490 nm was read using a Vmax plate reader (Molecular Devices, Mountain View, CA). Any contribution to binding via interaction of the IgE in the E27-IgE complexes with the human Fcgamma RII and Fcgamma RIIIA was not apparent based on the lack of binding of several Ala variants (Class 1, Table I).

For the high affinity Fcgamma RI, the receptor fusion protein at 1.5 µg/ml in PBS, pH 7.4, was coated onto ELISA plates (Nunc) for 18 h at 4 °C. Plates were blocked with assay buffer at 25 °C for 1 h. Serial 3-fold dilutions of monomeric E27 and variants (10.0-0.0045 µg/ml) were added to plates and incubated for 2 h. After washing plates with assay buffer, IgG bound to Fcgamma RI was detected with peroxidase-conjugated F(ab')2 fragment of goat anti-human F(ab')2-specific IgG (Jackson ImmunoResearch) or with peroxidase-conjugated protein G (Bio-Rad). The substrate used was o-phenylenediamine dihydrochloride (Sigma). Absorbance at 490 nm was read using a Vmax plate reader (Molecular Devices).

For all Fcgamma R, binding values reported are the binding of each E27 variant relative to native E27, taken as (A490 nm(variant)/A490 nm(native IgG1)) at 0.33 or 1 µg/ml for Fcgamma RII and Fcgamma RIIIA and 2 µg/ml for Fcgamma RI. A value greater than 1 denotes binding of the variant was improved compared with native IgG1, whereas a ratio less than 1 denotes reduced binding compared with native IgG1. Reduced binding to any given receptor was defined as a reduction of >= 40% compared with native IgG; better binding was defined as an improvement of >= 25% compared with native IgG1. The latter was chosen based on the observation that variants with >= 25% improved binding in the ELISA format assay, such as E333A, K334A, and S298A, also showed improved efficacy in the cell-based binding and ADCC assays.

IgG Binding to FcRn-- ELISA plates (Nunc) were coated with 2 µg/ml NeutrAvidin (Pierce) or streptavidin (Zymed Laboratories Inc., South San Francisco, CA) in 50 mM carbonate buffer, pH 9.6, at 4 °C overnight (the same results were obtained with either molecule). Plates were blocked with PBS, 0.5% BSA, 10 ppm Proclin 300 (Supelco, Bellefonte, PA), pH 7.2, at 25 °C for 1 h. FcRn-Gly-His6-GST was biotinylated using a standard protocol with biotin-X-NHS (Research Organics, Cleveland, OH) and bound to NeutrAvidin-coated plates at 2 µg/ml in PBS, 0.5% BSA, 0.05% polysorbate-20 (sample buffer), pH 7.2, at 25 °C for 1 h. Plates were then rinsed with sample buffer, pH 6.0. Eight serial 2-fold dilutions of E27 standard or variants (1.6-200 ng/ml) in sample buffer at pH 6.0 were incubated for 2 h. Plates were rinsed with sample buffer, pH 6.0, and bound IgG was detected with peroxidase-conjugated goat F(ab')2 anti-human IgG F(ab')2 (Jackson ImmunoResearch) in pH 6.0 sample buffer using 3,3',5,5'-tetramethylbenzidine (Kirkegaard & Perry Laboratories, Gaithersburg, MD) as substrate. Absorbance at 450 nm was read on a Vmax plate reader (Molecular Devices). Titration curves were analyzed by a four-parameter nonlinear regression fit (KaleidaGraph, Synergy Software, Reading, PA). For each ELISA plate assay, a full titration curve of E27 standard was done. The absorbance at the midpoint of the titration curve (mid-OD) and its corresponding E27 concentration were determined. Then the concentration of each variant at this mid-OD was determined, and the concentration of E27 was divided by the concentration of each variant. Hence, the values are a ratio of the binding of each variant relative to native IgG1 standard. A control human IgG1 was run on each ELISA plate as a control and had a ratio of 1.12 ± 0.07 (n = 92).

A second format was also evaluated in which IgE was coated on plates instead of the FcRn. MaxiSorp 96-well microwell plates (Nunc) were coated with 1 µg/ml IgE in 50 mM carbonate buffer, pH 9.6, at 4 °C overnight. Plates were blocked with PBS, 0.5% bovine serum albumin, 10 ppm Proclin 300, pH 7.2, at 25 °C for 1 h. 2-fold serial dilutions of anti-IgE antibodies (1.6-200 ng/ml) in PBS, 0.5% BSA, 0.05% polysorbate 20, pH 6.0 (sample buffer), were added to the plates, and plates were incubated for 2 h at 25 °C. Biotinylated FcRn at 3.6 µg/ml in sample buffer was added to the plates. After a 1-h incubation, bound FcRn was detected by adding streptavidin-labeled peroxidase (Amdex, Copenhagen, Denmark) in sample buffer, incubating the plates for 1 h and adding 3,3',5,5'-tetramethylbenzidine as the substrate. Plates were washed between steps with PBS, 0.5% BSA, 0.05% polysorbate 20, pH 6.0. Absorbance was read at 450 nm on a Vmax plate reader, and titration curves were fit as described above.

IgG Binding to Cell-bound Fcgamma R-- The expression of Fcgamma RI on THP-1 cells (38) was confirmed using the anti-Fcgamma RI antibody 32.2 (Medarex). Binding of IgG to Fcgamma RI on THP-1 cells was performed by incubating monomeric IgG in staining buffer (PBS, 0.1% BSA, 0.01% sodium azide) with 5 × 105 cells/well in 96-well round-bottom tissue culture plates (Costar, Cambridge, MA) at 4 °C for 30 min. Cells were washed three times with staining buffer, and IgG binding was detected by addition of 1:200 fluorescein isothiocyanate-F(ab')2 fragment of goat anti-human F(ab')2 specific for human IgG (Jackson ImmunoResearch). Immunofluorescence staining was analyzed on a FACScan flow cytometer using Cellquest software (Becton Dickinson, San Jose, CA). Dead cells were excluded from analysis by the addition of 1 µg/ml propidium iodide.

The CHO stable cell lines expressing Fcgamma RIIIA-Phe158 and Fcgamma RIIIA-Val158 with human gamma -chain were generated by subcloning the alpha -chain and gamma -chains into a previously described vector that includes DNA encoding a green fluorescent protein (39). CHO cell transfection was carried out using Superfect (Qiagen) according to the manufacturer's instructions. Fluorescence-activated cell sorting was done based on green fluorescent protein expression as described previously (40). Receptor expression levels were determined by staining with anti-Fcgamma RIII monoclonal antibody 3G8 (Medarex). Binding of IgG1 variants was performed by adding monomeric IgG in staining buffer to 5 × 105 cells and incubating in 96-well round-bottom tissue culture plates (Costar, Cambridge, MA) at 4 °C for 30 min. Cells were washed three times with staining buffer, and IgG binding was detected by addition of 1:200 PE-F(ab')2 fragment of goat anti-human IgG and incubation for 30 min at 4 °C. After washing, immunofluorescence staining was analyzed on a FACScan flow cytometer using Cellquest software (Becton Dickinson). Dead cells were excluded from analysis by addition of 1 µg/ml propidium iodide.

MALDI Analysis of Released N-Linked Oligosaccharides-- To determine whether differences in binding among variants was related to variation in the oligosaccharide at the conserved Asn297-linked glycosylation site, oligosaccharides of various IgG variants were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) as described previously (41). Following immobilization of ~50 µg of IgG to polyvinylidene difluoride membranes in 96-well MultiScreen IP plates (Millipore), proteins were reduced with 50 µl of 0.1 M dithiothreitol in 8 M urea, 360 mM Tris, 3.2 mM EDTA, pH 8.6 (RCM buffer). Resultant free sulfhydryl groups were subsequently carboxymethylated by incubation with 0.1 M iodoacetic acid in RCM buffer at 25 °C for 30 min in the dark. Prior to enzymatic release of glycoproteins, membrane-bound proteins were incubated in 1% aqueous polyvinylpyrrolidone 360 (Sigma) solution at 25 °C for 1 h. Oligosaccharides were released by incubating protein with 32 units of peptide:N-glycosidase F (New England Biolabs, Beverly, MA) in 25 µl of Tris acetate buffer, pH 8.4, at 37 °C for 3 h, followed by acidification by addition of 2.5 µl of 1.5 M acetic acid and then incubated for 25 °C for 3 h. Samples were then purified by cation exchange chromatography using hydrogen form, 100-200 mesh AG50W-X8 resin (Bio-Rad). Released oligosaccharides were analyzed by MALDI-TOF-MS in both positive and negative modes using matrices containing 2,5-dihydroxybenzoic acid and 2,4,6-trihydroxyacetophenone, respectively (42). Analysis was performed on a Voyager DE mass spectrometer (Perspective Biosystems, Foster City, CA) by transferring 0.5 µl of sample to a stainless steel target containing 0.4 µl of the appropriate matrix. Following vacuum dessication, the samples were ionized by irradiation with an N2 laser (337 nm wavelength), and ions were accelerated with a 20-kV voltage. Ion mass assignment was made using oligosaccharide standards (Oxford Glycosciences, Rosedale, NY) in a two-point external calibration. Final spectra were the result of the summation of the individual spectral data from 240 laser ignitions.

ADCC Assays Using PBMC as Effector Cells-- SK-BR-3 (ATCC HTB-30) breast tumor cell line expressing p185HER2 (43) as target cell was purchased from the American Type Culture Collection. Effector cells were PBMC purified from four healthy donors using Lymphocyte Separation Medium (LSM, Organon Technika, Durham, NC) on the day of the experiment. ADCC was conducted by measuring the lactate dehydrogenase (LDH) activity released from the dead or plasma membrane damaged cells. SK-BR-3 (target) cells were detached from plates using Versene (Life Technologies, Inc.), washed three times with RPMI 1640 medium (Life Technologies, Inc.), and incubated with humanized anti-p185HER2 IgG1 HERCEPTIN® (44) at 1 µg/ml (maximum ADCC) or 2 ng/ml for 30 min at 25 °C. HERCEPTIN® IgG variants were evaluated only at 2 ng/ml. Purified PBMC effector cells were washed three times with medium and placed in 96-well U-bottom Falcon plates (Becton Dickinson) using 2-fold serial dilution from 3 × 105 cells/well (100:1 effector/target ratio) to 600 cells/well (0.2:1). Opsonized SK-BR-3 cells were added to each well at 3 × 103 cells/well. AICC was measured by adding effector and target cells without antibody. Spontaneous release (SR, negative control) was measured by adding only target or effector cells; maximum release (MR, positive control) was measured by adding 2% Triton X-100 to target cells. After 4 h of incubation at 37 °C in 5% CO2, assay plates were centrifuged. The supernatant was transferred to a 96-well flat-bottom Falcon plates and incubated with LDH reaction mixture (LDH Detection Kit, Roche Molecular Biochemicals) for 30 min at 25 °C. The reaction was stopped by adding 50 µl of 1 N HCl. The samples were measured at 490 nm with reference wavelength of 650 nm. The percent cytotoxicity was calculated as ((LDH releasesample - SReffector - SRtarget)/(MRtarget - SRtarget)) × 100. For each assay and antibody, the percent cytotoxicity versus log(effector/target ratio) was plotted and the area under the curve (AUC) calculated.

ADCC Assays Using NK as Effector Cells-- Fcgamma RIIIA allotype of human donors was determined using a previously reported two-step PCR of genomic DNA (45). Following LSM (Cappel-ICN, Aurora, OH) purification of whole blood PBMCs, NK cells were purified by negative selection using a magnetic bead NK cell isolation kit (Miltenyi Biotech, Auburn, CA) and suspended in 50:50 Ham's F-12/Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 1% heat-inactivated fetal bovine serum (HyClone, Logan, UT), 1% penicillin/streptomycin (Life Technologies, Inc.), 2 mM L-glutamine, 0.01 M HEPES buffer. Cell purity was assessed by staining with PE-conjugated anti-CD56 and PE-conjugated anti-CD16 (PharMingen, San Diego, CA). SK-BR-3 cells were opsonized with 1 ng/ml of either native or variant humanized anti-p185HER2 IgG1 HERCEPTIN® for 30 min at 25 °C in 50:50 Ham's F-12/Dulbecco's modified Eagle's medium in sterile 96-well round-bottom plates. Serial 2-fold dilutions of NK cells were added to provide a final effector/target ratio ranging from 10:1 to 0.156:1. The plates were incubated for 4 h at 37 °C in a humidified 5% CO2 incubator. Cytotoxicity of targets was measured by LDH release using the colorimetric LDH Detection Kit (Roche Molecular Biochemicals). Spontaneous release (SR) was measured in wells with target and effector cells only; maximal release (MR) was measured by addition of 1% Triton X-100 detergent to control wells. Percent cytotoxicity was expressed as (LDH releasesample/MR) × 100.


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Binding of IgG Subclasses to Fcgamma R-- IgG1 binding to Fcgamma RI is of sufficient affinity to allow detection of monomeric IgG1. In contrast, monomeric IgG1 does not bind well to Fcgamma RII and Fcgamma RIII, and these receptors require a multimeric complex for assay. Previous studies investigating Fcgamma RII and Fcgamma RIII have utilized rosetting assays (46, 47), heat-denatured (aggregated) IgG1 binding (48, 49), and dimeric IgG (46, 47, 50). In this study, a stable hexameric immune complex composed of three IgE and three anti-IgE molecules (36) was used to evaluate IgG1 binding to Fcgamma RII and Fcgamma RIII. Fig. 1 shows that these complexes bind to Fcgamma RII and Fcgamma RIII in a concentration-dependent, saturable manner.



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Fig. 1.   Binding of anti-IgE E27 IgG1 to human Fcgamma R. A, binding of E27 monomers (solid circles), dimeric (solid squares), and hexameric (open squares) complexes to Fcgamma RIIA-Arg131. B, binding of E27 monomers (solid circles), dimeric (solid squares), and hexameric (open squares) complexes to Fcgamma RIIIA-Phe158. Dimers were formed by mixing E27 IgG1 and a F(ab')2 fragment of goat anti-human kappa  light chain at 1:0.5 molar ratio at 25 °C for 1 h (50). Hexameric complexes (i.e. trimeric in E27 IgG1) were formed by mixing E27 IgG1 with human IgE in a 1:1 molar ratio at 25 °C for 1 h (36).

To ensure that the results of binding of the IgE-anti-IgE hexamer complexes were not an idiosyncratic feature of the anti-IgE IgG1, complexes composed of VEGF and anti-VEGF IgG1 were also assayed for selected variants. These complexes were formed by mixing human VEGF and humanized anti-VEGF (51) in a 1:1 molar ratio. These complexes have not been fully characterized as to their size, but sedimentation ultracentrifugation experiments show that they form octamers (four VEGF:four anti-VEGF) and larger.2 The pattern of binding for the variants was the same regardless of whether the IgE-anti-IgE or VEGF-anti-VEGF complexes were used (data not shown).

The relative affinities of human IgG subclasses IgG1, IgG2, IgG3, and IgG4 and of murine subclasses IgG1, IgG2a, and IgG2b to the Fcgamma R have been previously determined (14-16). The pattern of binding of human and murine subclasses to human Fcgamma RI, Fcgamma RIIA, and Fcgamma RIIIA was verified using the ELISA format assays (data not shown). Human Fcgamma RIIA has two known, naturally occurring allotypes that are determined by the amino acid at position 131 (52). In this study, the Fcgamma RIIA-Arg131 allotype was primarily used, but some IgG variants were tested against the Fcgamma RIIA-His131 form. Human Fcgamma RIIIA has naturally occurring allotypes at position 48 (Leu, His, or Arg) and at position 158 (Val or Phe). For the Fcgamma RIIIA assays in this study, the Fcgamma RIIIA-Arg48/Phe158 allotype was used. Binding to the Fcgamma RIIIA-Arg48/Val158 allotype was also evaluated for selected variants.

To ensure that the results from the ELISA format assays reflected binding to Fcgamma R on cells, selected variants were tested in cell-based assays. The binding of variants to THP-1 cells, which express Fcgamma RI, was the same as for the ELISA-based assays; indeed, for Fcgamma RI the cell-based results were included in the mean values cited in Table I. For Fcgamma RIIIA, a select panel of variants was assayed on a stable-transfected CHO cell line expressing human FcgRIIIA-Phe158 (alpha - and gamma -chains) and the pattern of binding reflected that found in the ELISA-based format (data not shown).


                              
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Table I
Binding of human IgG1 variants to human FcRn and Fcgamma R

Segregation of Individual Variants into Classes-- The IgG1 variants can be separated into distinct classes based on their effects on binding to the various receptors. Class 1 consists of variants that showed reduced binding to all Fcgamma R (Table I) and are clustered near the region of the CH2 domain where the hinge joins CH2 (Fig. 2A). In the E233P/L234V/L235A/G236deleted variant, part of the so-called lower hinge region (residues 233-239) of human IgG1 was exchanged with that of human IgG2. The reduction in binding to all Fcgamma R is in agreement with previous studies (8, 19, 21, 22, 25, 53, 54); this variant also showed impaired binding to FcRn. Two other residues in the lower hinge region were individually changed to Ala; P238A (Class 1) had a more pronounced effect than S239A (Class 8). If the P238A effect is due to a conformation change, this change was beneficial for binding to FcRn. In contrast, P329A showed a relatively modest reduction in binding to FcRn compared with the significant reduction in binding to the Fcgamma R. In the IgG1 Fc:Fcgamma RIIIA crystal structure (26), Pro329 intimately interacts with two Trp side chains of the receptor, and the loss of these interactions by P329A may account for the severe reduction in binding. Pro329 is also involved in binding of human IgG1 to human C1q (55).



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Fig. 2.   Binding sites of human IgG1 for Fcgamma R. A, IgG1 residues comprising the binding site for Fcgamma RI and Fcgamma RII. The two Fc heavy chains are in light and medium gray; carbohydrate is in dark gray. Residues that affected binding to all Fcgamma R are in red; the Fcgamma RI binding site is composed only of red residues. Residues that showed improved binding to Fcgamma RII and Fcgamma RIIIA are in magenta. Residues that showed reduced binding only to Fcgamma RII are in yellow. Residues that showed >=  50% improved binding only to Fcgamma RII are in green. B, IgG1 residues comprising the binding site for Fcgamma RI and Fcgamma RIIIA. The two Fc heavy chains are in light and medium gray; carbohydrate is in dark gray. Residues that affected binding to all Fcgamma R are in red; the Fcgamma RI binding site is composed only of red residues. Residues that showed improved binding to Fcgamma RII and Fcgamma RIIIA are in magenta. Residues that showed reduced binding only to Fcgamma RIIIA are in yellow. Residues that showed >=  25% improved binding only to Fcgamma RIIIA are in green. Glu430, involved in a salt-bridge with Lys338, is shown in blue.

Removal of the conserved Asn-linked glycosylation site in the CH2 domain, N297A, abolished binding, in agreement with earlier studies (56, 57). Another residue that interacts with carbohydrate, Asp265, has also been found previously to be important in human IgG3 binding to human Fcgamma RI (56, 57). In human IgG1, changing Asp265 to Ala, Asn, or Glu nullified binding (Tables I and II), suggesting that both the charge and size are important. The results of the A327Q (Class 1) and A327S (Class 2) variants imply that the region around Ala327 involved in binding to the Fcgamma R may require a close fit between receptor and IgG1, as enlarging this side chain diminished binding. This position is an Asp in mouse IgG2a and IgG2b, and therefore changing the human IgG1 Ala to a larger side chain is unlikely to have affected the conformation. The A327G (Class 8) variant reduced binding only to Fcgamma RIIIA suggesting that this receptor requires the presence of a small amino acid side chain at this position, whereas the other receptors do not.

Class 1 variants (in addition to the hinge residues, which were not investigated in this study) compose the entire binding site on IgG1 for Fcgamma RI. Residues in the F(ab) portions of the IgG1 do not contribute to Fcgamma RI binding as evidenced by both a CD4-immunoadhesin (58) and an Fc fragment binding to Fcgamma RI as effectively as did intact IgG1 (data not shown). Notably, no variants were found that reduced binding only to Fcgamma RI.

Class 2 consists of three variants with reduced binding to Fcgamma RII and Fcgamma RIII but not Fcgamma RI. Like the residues in Class 1, Asp270 and Gln295 are located near the hinge (Fig. 2A). In crystal structures of human IgG1 Fc (30, 59), Gln295 is completely solvent-exposed, whereas Asp270, although exposed, forms hydrogen bonds from its side chain O-delta atom to the backbone nitrogens of Lys326 and Ala327 and to the side chain N-delta of Asn325. Disruption of these interactions by D270A could cause a local conformational perturbation that affected the severe reduction in binding to Fcgamma RII and Fcgamma RIIIA. However, D270A did not affect binding to Fcgamma RI or FcRn. Furthermore, D270N, which could maintain the aforementioned hydrogen bonds, also abolished binding to Fcgamma RII and Fcgamma RIIIA, and D270E bound to Fcgamma RIIIA as effectively as did native IgG1 (Table II). Taken together, these data suggest that the side chain charge of Asp270 is important for interaction with Fcgamma RII and Fcgamma RIIIA.


                              
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Table II
Binding of human IgG1 non-Ala variants to human Fcgamma RII and Fcgamma RIIIA

Class 3 consists of two variants with improved binding to Fcgamma RIIA, Fcgamma RIIB, and Fcgamma RIIIA. Thr256 and Lys290 are located near one another in the CH2 domain (Fig. 2). T256A also exhibited improved binding to FcRn; indeed altering Thr256 in murine IgG1 to other residues improved binding to murine FcRn (13).

Class 4 variants were characterized by improved binding only to Fcgamma RIIA and Fcgamma RIIB. Those that improved binding to Fcgamma RII the most, R255A, E258A, S267A, E272A, and D280A, are distant from one another in the CH2 domain (Fig. 2A). Of these, only Ser267 was cited as an interacting residue in the Fc:Fcgamma RIIA crystal structure (26). S267A improved binding only to Fcgamma RII, S267G abolished binding only to Fcgamma RIIIA, and S267T reduced binding to Fcgamma RII and Fcgamma RIIIA (Tables I and II). D280N (Table II), like D280A (Table I), improved binding only to Fcgamma RII. Three of the Class 4 residues also exhibited improved binding to FcRn (E272A, T307A, and A378Q), whereas R255A exhibited reduced binding. Ala378 interacts with CH2 domain loop AB, which contains residues that interact directly with FcRn, and hence may influence binding to FcRn indirectly.

Class 5 variants exhibited improved binding to Fcgamma RIIA and Fcgamma RIIB but, in contrast to Class 4, also showed reduced binding to Fcgamma RIIIA. Of these, Lys322 has also been implicated in human C1q binding (55). The aliphatic portion of the Arg301 side chain is buried and interacts with the Tyr296 side chain, at least in some crystal structures, whereas the Arg301 guanidinium group may interact with the Asn297-linked carbohydrate (30, 59, 60). The R301A variant effected a modest improvement in binding to Fcgamma RIIB and a pronounced reduction in binding to Fcgamma RIIIA (Table I); the R301M variant, which may maintain the aliphatic interaction of the Arg301 side chain, showed improved binding to Fcgamma RIIA and Fcgamma RIIB and a less pronounced reduction of binding to Fcgamma RIIIA compared with R301A (Table II).

Class 6 residues show diminished binding to Fcgamma RII only. R292A is located in the CH2 domain distant from the hinge. Lys414 is at the "bottom" of the IgG1, spatially removed from all other residues having an effect on Fcgamma RII binding, suggesting that it may play only a minor role in binding (discussed below).

Class 7 is composed of S298A that reduced binding to Fcgamma RII but improved binding to Fcgamma RIIIA. Situated among the Class 1 residues near the hinge (Fig. 2), Ser298 is also part of the Asn-linked glycosylation sequence Asn297-Ser298-Thr299. S298T followed the pattern of S298A, whereas S298N abolished binding to Fcgamma RIIIA as well as Fcgamma RII (Table II).

Reduced binding only to Fcgamma RIIIA characterizes Class 8 and includes five residues in the CH2 domain and two in the CH3 domain (Ala327 is in Class 1). Ser239 has been previously identified as playing a minor role in murine IgG2b binding to murine Fcgamma RII (25), and in the IgG1 Fc:Fcgamma RIIIA crystal structure (26), the Ser239 in one of the two heavy chains forms a hydrogen bond to the Lys117 side chain of Fcgamma RIIIA. In contrast, E293A (Table I) and E293D (Table II) reduced binding as much as did S239A even though Glu293 is not located near the Fc:Fcgamma RIIIA interface in the crystal structure. In some crystal structures (30, 59, 60), the Tyr296 side chain interacts intimately with the aliphatic portion of Arg301 (Class 5); altering either of these reduced binding to Fcgamma RIIIA. Note, however, that Tyr296 was changed to Phe (not Ala) and the 50% reduction in binding to Fcgamma RIIIA was due only to removal of the side chain hydroxyl group.

At position Lys338, altering the side chain to Ala or Met affected reduction in binding to Fcgamma RIIIA, suggesting that both the side chain charge and aliphatic portions are required. The Lys338 side chain forms part of the interface between the CH2 and CH3 domains and participates in a salt bridge with Glu430 in several crystal structures (30, 60, 61) (Fig. 2B). Although it is possible that altering Lys338 may disrupt the CH2:CH3 interface and thereby influence binding, K338A (Table I) and K338M (Table II) did not disrupt binding to Fcgamma RI, Fcgamma RII, or FcRn. Since it is known that binding of IgG1 to FcRn involves residues in both CH2 and CH3 (27-29), this suggests that any conformational effect of K338A must be local and minimal. Note also that while K338A and K338M reduced binding to Fcgamma RIIIA, E430A (Class 4) improved binding, suggesting that the Lys338:Glu430 salt bridge is not essential in maintaining binding. Another CH3 residue affecting Fcgamma RIIIA is Asp376 that interacts with the CH2 domain.

Class 9 is characterized by improved binding only to Fcgamma RIIIA and includes E333A, K334A, and A339T. A previous study found that A339T improved binding to Fcgamma RI (62); in this study the A339T variant bound better than native IgG1 to Fcgamma RIIIA but not Fcgamma RI (Table I). Several non-Ala variants were tested at Glu333 and Lys334. E333D also improved binding to Fcgamma RIIIA, whereas E333N reduced binding to Fcgamma RII as well as Fcgamma RIIIA (Table II). At position 334, changing Lys to Gln, Glu, or Val maintained the improved binding to Fcgamma RIIIA (Table II). Surprisingly, the K334R variant reversed the receptor preference, i.e. this variant bound better to Fcgamma RIIB and not Fcgamma RIIIA as for the K334A variant. Taken together these data suggest that Fcgamma RIIIA interacts with Lys334 even though this residue is not among the IgG1 residues found to interact with Fcgamma RIIIA in the co-crystal structure (26).

Class 10 residues influenced binding only to FcRn. Note that residues in other classes may also have affected binding to FcRn but were classified according to their effect on Fcgamma R. Positions that effectively abrogated binding to FcRn when changed to alanine include Ile253, Ser254, His435, and Tyr436. Other positions showed a less pronounced reduction in binding as follows: Glu233-Gly236 (Class 1), Arg255 (Class 4), Lys288, Ser415, and His433. Several amino acid positions exhibited an improvement in FcRn binding when changed to alanine; notable among these are Pro238 (Class 1), Thr256 (Class 3), Thr307 (Class 4), Gln311, Asp312, Glu380, Glu382, and Asn434. The pattern of binding was the same when a second assay format was used, e.g. with IgE-coated plates rather than FcRn-coated plates.

Binding of Combination Variants-- A number of combination variants were tested in which two or more residues were simultaneously altered to Ala. Some of these combinations showed additive effects. An example is the E258A/S267A variant that exhibited binding to Fcgamma RIIA, and Fcgamma RIIB that was better than the E258A (Class 4) and S267A (Class 4) variants (Tables I and III). A similar outcome was found for the S298A/E333A and S298A/K334A variants in which the binding to Fcgamma RIIIA improved over the parental variants (Table III). In other combinations, one residue dominated the other, e.g. the T256A/S298A variant showed reduced binding to Fcgamma RIIA and Fcgamma RIIB similar to the S298A variant even though the T256A change effected better binding to both these receptors (Class 3).


                              
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Table III
Binding of human IgG1 combination variants to human FcRn and Fcgamma R

The most pronounced additivity was found for combination variants with improved binding to FcRn. At pH 6.0, the E380A/N343A variant showed over 8-fold better binding to FcRn, relative to native IgG1, compared with 2-fold for E380A and 3.5-fold for N434A (Tables I and III). Adding T307A to this effected a 13-fold improvement in binding relative to native IgG1. Likewise, combining E380A and L309A, the latter being deleterious to FcRn binding, resulted in a variant that was intermediate between the two parental variants (Table III). As with the Fcgamma R, some combinations showed dominance of one residue over the other; for the K288A/N434A variant, the better binding due to N434A clearly overcame the reduction in binding from K288A (Table III). As expected from previous studies (28), at pH 7.2 none of variants bound well (data not shown).

Role of IgG Residues Affecting Carbohydrate-- Previously it was noted that replacing human IgG3 residues that contact the oligosaccharide, e.g. Asp265, Tyr296, and Arg301, with Ala resulted in increased galactosylation and sialylation relative to native IgG3 and in reduced binding to both Fcgamma R and C1q (56). To determine if the effect seen for specific Ala substitutions (either deleterious or advantageous) was due to differences in glycosylation, oligosaccharide analysis was performed for selected variants (Table IV). The D265A, R301A, and R301M variants showed increased galactosylation, a relatively small amount of sialylation, and a small percentage of triantennary carbohydrate, in agreement with Lund et al. (56). The R301A and R301M variants also showed an increase in fucose and a decrease in mannose not seen previously. For the Y296F variant, there were no differences from native IgG1, in contrast to the decrease in galactose and fucose and increase in mannose reported by Lund et al. (56). These differences may be due to the different mammalian cells used to express the antibodies (human kidney 293 cells in this study and Chinese hamster ovary cells in the previous study) or may reflect that Tyr296 was changed to Phe296 in this study, whereas it was changed to Ala296 in the Lund et al. (56) study.


                              
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Table IV
Percent of total oligosaccharide area by glycan type

The Lys334 side chain is near the carbohydrate in IgG crystal structures but does not interact with it as intimately as do Asp265, Tyr296, and Arg301. The K334A variant exhibited a small increase in mannose and small decrease in fucose compared with native IgG1 (Table IV). Ser298 interacts with the carbohydrate only through its O-gamma atom, which forms a hydrogen bond to the Asn297 O-delta , and no difference in carbohydrate for the S298A was evident compared with native IgG1. Neither the Glu258 or Val303 side chains interact with the carbohydrate, and indeed both are located on the opposite face of the CH2 domain from the carbohydrate. However, the E258A variant showed an increase in galactosylation and a small amount of sialic acid, whereas the V303A variant only showed a small amount of sialic acid. Hence, variation in galactosylation and sialic acid for a given variant (compared with native IgG1) may occur regardless of whether the amino acid side chain interacts with the carbohydrate.

For the Y296F, S298A, V303A, and K334A variants, the differences in glycosylation, compared with native IgG1, were minimal and most likely were not the cause of the differences in binding of these variants to the Fcgamma R. For the E258A, D265A, R301A, and R301M variants, it is difficult to discern whether reduction or improvement in Fcgamma R binding is due to the change in amino acid side chain or from differences in glycosylation.

Binding to Allotypic Forms of Fcgamma RIIA and Fcgamma RIIIA-- Selected variants were tested for binding to the Fcgamma RIIA-His131 and Fcgamma RIIIA-Val158 allotypic receptor forms based on their improved or reduced binding to the allotypic forms used for the assays (i.e. Fcgamma RIIA-Arg131 and Fcgamma RIIIA-Phe158). Table V shows that most of the variants bound equivalently to the Fcgamma RIIA-Arg131 and Fcgamma RIIA-His131 receptors. The exceptions were the S267A, H268A, and S267A/H268A variants that displayed binding to Fcgamma RIIA-His131 that was reduced compared with Fcgamma RIIA-Arg131 but still equivalent to native IgG1. The related S267G variant, however, showed a 50% reduction in binding to the Fcgamma RIIA-His131 receptor compared with native IgG1. In contrast to S267A and H268A, D270A reduced binding to Fcgamma RIIA-His131 by 50% but completely abrogated binding to Fcgamma RIIA-Arg131. This suggests that Ser267, His268, and Asp270 interact with Fcgamma RIIA in the vicinity of Fcgamma RIIA residue 131. 


                              
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Table V
Binding of human IgG1 variants to human Fcgamma RIIA-R131 and Fcgamma RIIA-H131 polymorphic receptors

For Fcgamma RIIIA, the selected variants were assayed in the ELISA format as well as on stable-transfected CHO cell lines expressing the alpha -chains (Fcgamma RIIIA-Phe158 or Fcgamma RIIIA-Val158) with the associated human gamma -chain. For Fcgamma RIIIA-Phe158, those variants that showed improved binding in the ELISA format exhibited even more improvement, compared with native IgG1, in the cell-based assay (Table VI). This could be due to the presence of the gamma -chain associated with the alpha -chain enhancing binding of the IgG to Fcgamma RIIIA (63). Alternatively, since the cell-based assay utilized monomeric IgG (in contrast to hexameric complexes used in the ELISA format assay), the cell-based assay may be less subject to an avidity component and thus more sensitive to changes in the binding interface. In contrast, none of the variants exhibited improved binding to the Fcgamma RIIIA-Val158 receptor in the ELISA format assay, although the S298A, K334A, and S298A/E333A/K334A variants did bind better than native IgG1 in the cell-based assay.


                              
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Table VI
Binding of human IgG1 variants to human Fcgamma RIIIA-Phe158 and Fcgamma RIIIA-Val158 polymorphic receptors

ADCC Assays-- ADCC assays were performed using a select set of variants to determine whether the improvement or reduction in binding seen in the ELISA format and cell-based assays were reiterated in a functional assay. For the ADCC assays, the IgG1 variants were generated in the Herceptin® (44) background since ADCC assays were not possible using the anti-IgE antibody. Chromium-57, calcein, and lactate dehydrogenase detection formats were used with either PBMCs or NKs, and the results were similar for all formats.

One set of ADCC assays with PBMCs compared the effect of D265A (Class 1), R292A (Class 6), and S298A (Class 7). The assay was repeated using four different donors. Fig. 3 shows that the ADCC pattern of the variants reiterated that seen in the ELISA binding assay for Fcgamma RIIIA; D265A prevented ADCC (p < 0.01; paired t test); R292A had no effect, and S298A statistically improved ADCC (p < 0.01; paired t test).



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Fig. 3.   Antibody-dependent cell cytotoxicity of anti-p185HER2 IgG1 variants. ADCC was performed using p185HER2-expressing SK-BR-3 cells as target. In four separate experiments, PBMCs isolated from four donors were used as effector cells. Cytotoxicity was detected by LDH release. SR of target cells and effector cells was measured using the respective cells alone; MR was measured by adding 2% Triton X-100 to target cells. AICC was measured using target and effector cells together (i.e. no antibody). Percent cytotoxicity was calculated as ((LDH releasesample - SReffector - SRtarget)/(MRtarget - SRtarget)) × 100. The log(effector/target ratio) was plotted versus percent cytotoxicity. The area below the curve (AUC) was calculated for each sample; AUC of AICC was subtracted from each sample, and the results were graphed in a bar plot for maximum cytotoxicity using 1 µg/ml anti-p185HER2 (wt-max), 2 ng/ml anti-p185HER2 (wt), 2 ng/ml S298A variant (Class 7), 2 ng/ml R292A (Class 6), and 2 ng/ml D265A (Class 1). p < 0.01 (paired t test) for S298A versus wt.

A second set of assays was performed in which the Fcgamma RIIIA allotype of the donors was determined. By using three Fcgamma RIIIA-Val158/Val158 and three Fcgamma RIIIA-Phe158/Phe158 donors, ADCC assays using only NK cells were repeated 3-4 times for each donor. Representative ADCC plots are shown in Fig. 4, A and B, and the summary of all assays is shown in Fig. 4C. The variants tested were as follows: S298A (Class 7), K334A (Class 9), S298A/K334A (Table III), and S298A/E333A/K334A (Table III). In agreement with the binding exhibited in the ELISA format assay (Tables I, III, and VI), the pattern of improved ADCC was S298A/E333A/K334A > S298A/K334A > S298A = K334A. This pattern was seen with both the Fcgamma RIIIA-Phe158/Phe158 and Fcgamma RIIIA-Val158/Val158 donors, although improvement in ADCC was less pronounced for the latter. Comparing the improvement in binding to receptor for these variants in the ELISA format assay (Tables I, III, and VI) with that in the cell-based (Table VI) and ADCC assays (Figs. 3 and 4) shows that the improvement in binding for any specific variant is enhanced when the receptor is expressed on cells.



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Fig. 4.   Antibody-dependent cell cytotoxicity of anti-p185HER2 IgG1 variants for Fcgamma RIIIA-Phe158 and Fcgamma RIIIA-Val158 allotypes. ADCC was performed using p185HER2-expressing SK-BR-3 cells as target, and NKs isolated from three Fcgamma RIIIA-Val158/Val158 donors or three Fcgamma RIIIA-Phe158/Phe158 donors were used as effector cells. Cytotoxicity was detected by LDH release. AICC was measured using target and effector cells together (i.e. no antibody). MR was measured by adding 1% Triton X-100 to target cells. Percent cytotoxicity was calculated as (LDH releasesample/MRtarget) × 100. The, effector/target ratio was plotted versus percent cytotoxicity. A, representative ADCC assay for IgG1 variants using NK cells from an Fcgamma RIIIA-Val158/Val158 donor. Native anti-p185HER2 IgG1 (solid circles), S298A (solid squares), K334A (solid triangles), S298A/K334A (open circles), S298A/E333A/K334A (open squares), AICC (open triangles). B, representative ADCC assay for IgG1 variants using NK cells from an Fcgamma RIIIA-Phe158/Phe158 donor. Native anti-p185HER2 IgG1 (solid circles), S298A (solid squares), K334A (solid triangles), S298A/K334A (open circles), S298A/E333A/K334A (open squares), AICC (open triangles). C, bar plot of mean percent increase in ADCC of variants compared with native anti-p185HER2 IgG1. Percent increase was calculated as (% cytotoxicityvariant - % cytotoxicitynative IgG1)/% cytotoxicitynative IgG1). For each variant, the mean and S.D. are for 13 data points using three different donors. For all variants the Fcgamma RIIIA-Phe158/Phe158 donors showed a significant increase in ADCC over the increase seen for Fcgamma RIIIA-Val158/Val158 donors (p < 0.0001 for all variants using paired t test).



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Set of Human IgG1 Residues Involved in Binding to All Human Fcgamma R-- The set of IgG1 residues involved in binding to all human Fcgamma R is represented by Class 1 (Table I). Indeed, this set comprises the entire binding site on IgG1 for Fcgamma RI. Class 1 residues are located in the CH2 domain proximal to the hinge and fall into two categories as follows: 1) positions that may interact directly with all Fcgamma R include Leu234-Pro238, Ala327, and Pro329 (and possibly Asp265); 2) positions that influence carbohydrate nature or position include Asp265 and Asn297.

Previous studies mapping the binding residues in mouse or human IgG have concentrated primarily on the lower hinge region, i.e. residues Leu234-Ser239, revealing Leu234 and Leu235 as the two most important for Fcgamma RI (8, 19) and Leu234 and Gly237 as the two most important for Fcgamma RII (25, 56). Of the two residues in the lower hinge investigated in this study, P238A affected binding to all Fcgamma R, whereas S239A affected binding only to Fcgamma RIIIA.

In the co-crystal structure of IgG1 Fc:Fcgamma RIIIA (26), Pro329 interacts with two Trp side chains from the receptor, and a similar interaction may occur with the other Fcgamma R. However, removal of the Pro side chain, as in P329A, might cause a localized conformational change that perturbs adjacent binding residues, supported by the report that P329A also affects C1q binding (55). A327Q could be causing steric hindrance to binding due to introduction of a large side chain at this position, although altering Ala327 to Ser did not affect binding to Fcgamma RI. Inspection of the IgG1 Fc:Fcgamma RIIIA crystal structure shows that Ala327 is near the IgG1:Fcgamma RIIIA interface and forms a van der Waals' interaction with the Trp87 side chain; however, it is not obvious why introduction of a larger side chain such as Ser or Gln should so severely reduce binding. For Asn297 and Asp265, earlier studies evaluated the requirement for carbohydrate attached at Asn297 as well as the influence of Asp265 on the nature of the carbohydrate (12, 56, 57); these will be discussed below.

Human IgG1 Binding to Fcgamma RI-- For Fcgamma RI the IgG segment Gly316-Ala339 has also been previously implicated based on sequence comparison and binding of IgG subclasses from different species (21, 64) and mutagenesis (19, 62). Within the segment Gly316-Ala339, however, only A327Q and P329A affected binding to Fcgamma RI (Class 1). All other exposed residues in the 316-339 segment had no effect. In contrast to a previous study in which changing residue 331 from Pro to Ser in human IgG3 reduced binding by 10-fold (19), in human IgG1 the P331A (Table I) and P331S (Table II) variants had no effect. Another previous report showed that an A339T substitution could improve binding to Fcgamma RI by 3-fold (62); in this study the A339T variant was only equivalent in binding to native IgG1 (Class 3).

It has been noted that the presence of the gamma -chain may augment the binding affinity of the Fcgamma RI alpha -chain (63). Since it is conceivable that some residues in the human IgG1 might interact directly with the gamma -chain, binding of the IgG1 variants to Fcgamma RI on THP-1 cells was tested as well to the Fcgamma RI alpha -chain coated on a plate. The results of the two assay formats were the same for the entire panel of variants, suggesting that the gamma -chain augments binding by the alpha -chain through a mechanism other than direct interaction with the IgG1.

Since Fcgamma RI binds monomeric IgG1 about 100-fold more strongly than do Fcgamma RII and Fcgamma RIII, one might expect that Fcgamma RI would utilize either different or additional IgG1 residues to effect the tighter binding. However, the set of IgG1 residues that control binding to Fcgamma RI are a subset of those effecting binding to Fcgamma RII and Fcgamma RIII (Class 1). This suggests that the comparatively strong binding of IgG1 to Fcgamma RI results from either 1) utilization of only two Ig-like domains of Fcgamma RI (analogous to the two Ig-like domains of Fcgamma RII and Fcgamma RIII) but with interaction of different amino acids on Fcgamma RI than are used by Fcgamma RII and Fcgamma RIII, and 2) utilization of the same amino acids on all three receptors but with additional direct interaction of amino acids in the third Fcgamma RI domain, or 3) the third domain of Fcgamma RI effects a conformational change in the other two Ig-like domains that result in more efficacious interaction of these domains with the common set of binding residues on IgG1. In both human and murine Fcgamma RI, removal of the third domain reduces affinity for monomeric IgG and alters specificity for IgG subclasses (65, 66). This would support, but does not discriminate between, possibilities 2 and 3.

Human IgG1 Binding to Human Fcgamma RIIA and Human Fcgamma RIIB-- In contrast to Fcgamma RI, Fcgamma RII requires the presence of two identical IgG heavy chains (67), suggesting that residues from both heavy chains may form the Fcgamma RII-binding site in IgG. The set of IgG1 residues, in addition to the common Class 1 residues, that affect binding to Fcgamma RII are as follows: (largest effect) Arg255, Thr256, Glu258, Ser267, Asp270, Glu272, Asp280, Arg292, Ser298, and (less effect) His268, Asn276, His285, Asn286, Lys290, Gln295, Arg301, Thr307, Leu309, Asn315, Lys322, Lys326, Pro331, Ser337, Ala339, Ala378, and Lys414.

A previous study elucidated the residues in murine IgG2b involved in binding to murine Fcgamma RII (25). Of the residues investigated in that study, only N297A and E318A showed a complete abrogation of binding. Several other murine IgG2b residues exhibited more modest reduction in binding to Fcgamma RII as follows: S239A, K248A, S267A, K322A, E333A, T335A, S337A, and K340A (25). Several of these residues also exhibited modest reduction in binding in the human system (e.g. S239A and T335A) or modest improvement in binding (e.g. K340A) but fell outside of the cut-off used in this study. Noteworthy differences between the two systems are as follows: D270A affecting only the human system, E318A affecting only the murine system, and K322A, S267A, and S337A exhibiting improved binding in the human system but slightly reduced binding in the murine.

In contrast to Fcgamma RI, several variants exhibited improved binding to Fcgamma RIIA and Fcgamma RIIB (Classes 3-5). Of special interest are Class 4 containing residues which, when changed to Ala, improved binding only to Fcgamma RII and Class 5 containing residues which, when changed to Ala, simultaneously improved binding to Fcgamma RII and reduced binding to Fcgamma RIIIA. These can be used to make IgG1 with improved specificity for Fcgamma RII over Fcgamma RIIIA.

Recently the crystal structures of human Fcgamma RIIA (68) and Fcgamma RIIB (69) have been solved. In the Fcgamma RIIA report, it was suggested that in addition to the lower hinge (Leu234-Gly237), residues in IgG CH2 domain loops FG (residues 326-330) and BC (residues 265-271) might play a role in binding, although it was noted that these had yet to be demonstrated by mutagenesis. Of the four exposed residues in loop FG, A330Q had no effect, A327Q, A327S, and P329A reduced binding, and K326A improved binding. Of the five exposed residues in loop BC, two reduced binding when altered to Ala (D265A and D270A) and two improved binding (S267A and H268A). Several of the residues found to influence binding to Fcgamma RII lie outside of the residues at the Fc:Fcgamma RIIIA interface in the co-crystal structure (26). One of these, Asp280, is not only distant from the Fc:Fcgamma RIIIA interface but is distant from other Fcgamma RII-influencing residues (Fig. 2A). However, both D280A and D280N improved binding to Fcgamma RII, suggesting that this residue does indeed interact with Fcgamma RII.

Human IgG1 Binding to Human Fcgamma RIIIA-- In addition to the Class 1 residues, positions that reduced binding to Fcgamma RIIIA by 40% or more (when changed to Ala) are as follows: Ser239, Ser267 (Gly only), His268, Glu293, Gln295, Tyr296, Arg301, Val303, Lys338, and Asp376. In the Fc crystal structure, these residues separate into two groups. Lys338 and Asp376 are at the CH2-CH3 interface and may affect the spatial relationship of these two domains, thereby affecting Fcgamma RIIIA binding; note that changing these two residues did not significantly reduce binding to Fcgamma RI, Fcgamma RII, or FcRn. The other eight positions are clustered together near the Class 1 residues at the hinge-proximal end of the CH2 domain; of these, only Ser239, Ser267, and His268 were cited as part of the binding site in the Fc:Fcgamma RIIIA crystal structure report (26). Of the remaining seven, a few might conceivably exert their effect by conformational change, e.g. Tyr296, Arg301, Val303, Lys338, and Asp376 (Fig. 2B). However, the Glu293 and Gln295 side chains are quite solvent-exposed, based on Fc crystal structures (30, 59), and are not involved in interactions which would hint at a conformational role. In addition to E293A reducing binding by 70% (Class 8), the more conservative change of E293D also showed a similar reduction (Table II) implying that Glu293 can indeed interact with Fcgamma RIIIA.

Variants that improved binding to Fcgamma RIIIA (Classes 3, 7, and 9) include T256A, K290A, S298A, E333A, K334A, and A339T. Of these, only Ser298 was cited as part of the binding site in the Fc:Fcgamma RIIIA crystal structure report (26). Although Glu333 and Lys334 do not interact with Fcgamma RIIIA in the co-crystal structure, their interaction with Fcgamma RIIIA is supported by four lines of evidence. First, murine IgG2b E333A exhibited a modest reduction in binding to murine Fcgamma RII (25). Although the same might not occur for murine Fcgamma RIII, this shows that residues distant from the hinge region can influence binding to Fcgamma R. Second, several non-Ala variants at Glu333 and Lys334 either improved or reduced binding to Fcgamma RIIIA (Table II). Third, binding of E333A and K334A to Fcgamma RIIIA-expressing CHO cells improved even more than seen in ELISA-based assays (Table VI). Finally, K334A exhibited a significant increase in ADCC. This increase in ADCC was additive when K334A was present with S298A and was further enhanced when E333A was present (Fig. 4).

Several residues that influenced binding, albeit modestly, to Fcgamma RIIIA, Fcgamma RIIA, and Fcgamma RIIB, are located at the "bottom" of the CH3 domain distant from the larger set of residues in the CH2 domain which exhibited a more pronounced effect on binding. Lys414 (Fig. 2A) showed a 40% reduction in binding for Fcgamma RIIA and Fcgamma RIIB (Class 6), Arg416 a 30% reduction for Fcgamma RIIA and Fcgamma RIIIA, Gln419 a 30% reduction to Fcgamma RIIA and a 40% reduction to Fcgamma RIIB, and Lys360 a 23% improvement to Fcgamma RIIIA (Class 10). Taken together, their effect on binding of IgG1 to Fcgamma RIIA, Fcgamma RIIB, and Fcgamma RIIIA suggests that the bottom of IgG1 may indeed be involved in the IgG1-Fcgamma R interaction, although it may play only a minor role.

Human IgG1 Binding to Human FcRn-- Previous studies have mapped the binding site of murine IgG for murine FcRn (13, 29, 70-74). These studies have implicated murine IgG residues Ile253, His310, Gln311, His433, Asn434, His435, and His436 as contacts for one FcRn molecule and Glu272 and His285 as contacts for a second FcRn molecule. In addition, the pH dependence of the IgG-FcRn interaction has been ascribed to His310 and His433 on IgG (as well as His250 and His251 on FcRn) (75).

In the current study of the human system, a larger number of residues were found that affected binding of IgG1 to human FcRn. Comparison of the human IgG1 sequence with the crystal structure of rat Fc bound to murine FcRn (29) shows that in the human Fc some of these residues could interact directly with human FcRn: Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435. Near the Fc:FcRn interface in the crystal structure but not interacting directly are Arg255, Thr256, Asp312, Glu380, Glu382, His433, and Tyr436. In the murine system it was found that altering Asn434 to Ala or Gln did not affect binding to murine FcRn (72, 74). However, in the human system N434A exhibited the largest improvement in binding seen for any single Ala substitution (Class 10) as well as showing additivity in combination variants (Table III). Note that whereas improvement in binding of the variants to FcRn occurred at pH 7.2 as well as at pH 6.0 (Table III), none of the variants bound well at pH 7.2. Hence, these single or combination variants may be useful in extending the half-life of human IgG1 in therapeutic antibodies, as previously found for murine IgG (13), and fulfill the requirement for binding at pH 6.0 and dissociating at pH 7.2.

Effect of Glycosylation-- The presence of carbohydrate linked at residue Asn297 is required for binding to Fcgamma R (25). In addition, the nature of the carbohydrate can influence binding (11, 12, 56, 57). In crystal structures of IgG (Fc and intact antibody), Asp265 interacts directly with the Asn297-linked carbohydrate via hydrogen bonds (30, 59-61). Previous studies found that an D265A change in human IgG3 altered the composition of the Asn297-linked carbohydrate and reduced binding to Fcgamma RI (56, 57). In human IgG1, D265A (Class 1) the carbohydrate also differed from that of native IgG1 (Table IV), and binding to Fcgamma RI was reduced. Variants at positions 258 and 301 also showed variation from native IgG1 and the other variants (Table IV). The two Arg301 variants exhibited an increase in binding to Fcgamma RIIB, a decrease in binding to Fcgamma RIIIA, and no effect on binding to Fcgamma RI or FcRn (Class 5). D265A (Class 1) showed decreased binding to all Fcgamma R, whereas E258A (Class 4) showed increased binding to Fcgamma RII only. Hence, although it is possible that the idiosyncratic carbohydrate on these variants influenced binding rather than the amino acid changes directly affecting interaction with the Fcgamma R, the data do not allow resolution of the two possibilities. For the Y296F, S298A, V303A, and K334A variants, there were no significant differences in carbohydrate from that of native IgG1 (Table IV). Hence the differences in binding to the various Fcgamma R exhibited by these variants are unlikely to be the result of glycosylation differences.

Human IgG1 Binding to Fcgamma RIIA and Fcgamma RIIIA Polymorphic Forms-- Human Fcgamma RIIA has two known, naturally occurring allotypes that are determined by the amino acid at position 131 (52). Among the human IgG1 variants tested against both Fcgamma RIIA-Arg131 and Fcgamma RIIA-His131, variants S267A, S267G, H268A, and D270 could discriminate between the polymorphic forms. This suggests that these IgG1 residues interact with Fcgamma RIIA in the vicinity of Fcgamma RIIA residue 131, and in the IgG1 Fc:Fcgamma RIIIA crystal structure (26), Ser267 is adjacent to His131.

Human Fcgamma RIIIA has naturally occurring allotypes at position 48 (Leu, His, or Arg) and at position 158 (Val or Phe). The Fcgamma RIIIA-Val158 allotype binds human IgG better than the Fcgamma RIIIA-Phe158 allotype (45, 76), and this difference is reiterated in the ELISA format, cell-based, and ADCC assays in this study. The IgG1 Fc:Fcgamma RIIIA crystal structure offers an explanation for this difference. In the crystal structure, Val158 interacts with the IgG1 lower hinge near Leu235-Gly236 and with the Fcgamma RIIIA Trp87 side chain (which in turn interacts with the important IgG1 Pro329); introduction of the larger Phe158 may alter either or both of these interactions and thereby reduce the binding.

Some of the IgG1 variants exhibited better binding to Fcgamma RIIIA-Phe158 (e.g. Classes 7 and 9) and could be further improved by combining individual variants (Table III). These same variants showed no improvement or minimal improvement in binding to Fcgamma RIIIA-Val158 in the ELISA format assay. However, when tested on cells expressing Fcgamma RIIIA-Val158 or in ADCC assays using Fcgamma RIIIA-Val158/Val158 donors, some of these variants did show superior interaction compared with native IgG1 (Table VI and Fig. 4). Comparing the ADCC results of select IgG1 variants with better binding to Fcgamma RIIIA, the variants exhibited a significant improvement in ADCC for both Fcgamma RIIIA-Phe158/Phe158 and Fcgamma RIIIA-Val158/Val158 donors (Fig. 4). Indeed, using the S298A/E333A/K334A variant, the Fcgamma RIIIA-Phe158/Phe158 donor ADCC could be increased over 100% (i.e. >2-fold) compared with native IgG1 (Fig. 4C).

Although the influence of Fcgamma RIIA polymorphic forms in various human diseases has been investigated for many years (reviewed in Ref. 77), the possible correlation between Fcgamma RIIIA polymorphic forms and human disease has only recently been investigated (76, 78, 79). Given the possible involvement of Fcgamma R in the mechanism of action of therapeutic antibodies (4-6), human IgG1 variants with improved binding capacity to human Fcgamma R, especially variants with better binding to Fcgamma RIIIA and simultaneous abrogation of binding to the inhibitory Fcgamma RIIB, could be used to provide more efficacious therapeutic antibodies. In addition, a recent report on the occurrence of polymorphic Fcgamma R forms in control populations showed that the Fcgamma RIIIA-Phe158 allele is more prevalent than the Fcgamma RIIIA-Val158 allele (77). Since the Fcgamma RIIIA-Phe158 receptor binds human IgG1 less well than the Fcgamma RIIIA-Val158 receptor, therapeutic antibodies with variant Fc portions that improve binding to Fcgamma RIIIA-Phe158 at least to the level seen for Fcgamma RIIIA-Val158 (if not more so) could provide increased therapeutic efficacy to the majority of the population.


    ACKNOWLEDGEMENTS

We thank Dr. Jeffrey V. Ravetch (Rockefeller University) for valuable discussion concerning this study and the role of Fcgamma R and Lori O'Connell for help in DNA sequencing and protein expression/purification.


    FOOTNOTES

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

** To whom correspondence should be addressed: Dept. of Immunology, MS 34, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080. E-mail: presta@gene.com.

Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M009483200

2 J. Liu and S. Shire, personal communication.


    ABBREVIATIONS

The abbreviations used are: mAb, monoclonal antibody; ADCC, antibody-dependent cell cytotoxicity; AICC, antibody-independent cell cytotoxicity; ELISA, enzyme-linked immunosorbent assay; Fcgamma R, IgG Fc gamma -receptor; FcRn, neonatal IgG Fc receptor; GST, human glutathione S-transferase; LDH, lactate dehydrogenase; MR, maximal response; NK, natural killer cells; PBMC, peripheral blood monocytes; PE, (R)-phycoerythrin; SR, spontaneous release; VEGF, vascular endothelial growth factor; PCR, polymerase chain reaction; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; BSA, bovine serum albumin.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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