From the 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
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ABSTRACT |
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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 Fc 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, Fc Given the interest in and increasing use of antibody therapeutics, a
comprehensive mapping of the binding site on human IgG for the
different Fc The current study provides a complete, high resolution mapping of human
IgG1 for human Fc cDNA Constructs and Soluble Receptor Expression--
The
cDNAs encoding extracellular and transmembrane domains of human
Fc
For all Fc
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-Fc Human IgG1 Mutagenesis--
The humanized IgG1 anti-IgE E27, an
affinity-matured variant of anti-IgE E25, binds to the Fc
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 Fc
For the high affinity Fc
For all Fc 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 Fc
The CHO stable cell lines expressing Fc 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 ADCC Assays Using NK as Effector Cells--
Fc Binding of IgG Subclasses to Fc
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 Fc
To ensure that the results from the ELISA format assays reflected
binding to Fc 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 Fc
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 Fc
Class 1 variants (in addition to the hinge residues, which were not
investigated in this study) compose the entire binding site on IgG1 for
Fc
Class 2 consists of three variants with reduced binding to Fc
Class 3 consists of two variants with improved binding to Fc
Class 4 variants were characterized by improved binding only to
Fc
Class 5 variants exhibited improved binding to Fc
Class 6 residues show diminished binding to Fc
Class 7 is composed of S298A that reduced binding to Fc
Reduced binding only to Fc
At position Lys338, altering the side chain to Ala or Met
affected reduction in binding to Fc
Class 9 is characterized by improved binding only to Fc
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 Fc 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 Fc
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
Fc 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 Fc
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-
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 Fc Binding to Allotypic Forms of Fc
For Fc 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 Fc
A second set of assays was performed in which the Fc The Set of Human IgG1 Residues Involved in Binding to All Human
Fc
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
Fc
In the co-crystal structure of IgG1 Fc:Fc Human IgG1 Binding to Fc
It has been noted that the presence of the
Since Fc Human IgG1 Binding to Human Fc
A previous study elucidated the residues in murine IgG2b involved in
binding to murine Fc
In contrast to Fc
Recently the crystal structures of human Fc Human IgG1 Binding to Human Fc
Variants that improved binding to Fc
Several residues that influenced binding, albeit modestly, to
Fc 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 Fc Human IgG1 Binding to Fc
Human Fc
Some of the IgG1 variants exhibited better binding to
Fc
Although the influence of FcRI, Fc
RIIA, Fc
RIIB, Fc
RIIIA, and
FcRn receptors has been determined. A common set of IgG1 residues is
involved in binding to all Fc
R; Fc
RII and Fc
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
Fc
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/Fc
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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
R, are expressed on
leukocytes and are composed of three distinct classes as follows:
Fc
RI (CD64), Fc
RII (CD32), and Fc
RIII (CD16). In humans, the
latter two classes can be further divided into Fc
RIIA and Fc
RIIB,
Fc
RIIIA and Fc
RIIIB. Structurally, the Fc
R are all members of
the immunoglobulin superfamily, having an IgG-binding
-chain with an
extracellular portion composed of either two (Fc
RII and Fc
RIII)
or three (Fc
RI) Ig-like domains. In addition, Fc
RI and Fc
RIII
have accessory protein chains (
and
) associated with the
-chain that function in signal transduction. The receptors are also
distinguished by their affinity for IgG. Fc
RI exhibits a high
affinity for IgG, Ka = 108-109
M
1 (14), and can bind monomeric
IgG. In contrast, Fc
RII and Fc
RIII show a weaker affinity for
monomeric IgG, Ka
107
M
1 (14), and hence can only
interact effectively with multimeric immune complexes.
R could provide for alternative methods of either
abrogating or enhancing immune recruitment via Fc
R. Previous studies
mapped the binding site on human and murine IgG for Fc
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 Fc
RI (21),
Lys274-Arg301 and
Tyr407-Arg416 for human Fc
RIII (23, 24), or
found few specific residues outside the lower hinge, e.g.
Asn297 and Glu318 for murine IgG2b interacting
with murine Fc
RII (25). The very recent report of the 3.2-Å crystal
structure of the human IgG1 Fc fragment with human Fc
RIIIA
delineated IgG1 residues Leu234-Ser239,
Asp265-Glu269,
Asn297-Thr299, and
Ala327-Ile332 as involved in binding to
Fc
RIIIA (26).
R receptors (Fc
RI, Fc
RIIA, Fc
RIIB, and
Fc
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 Fc
R; Fc
RII and
Fc
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 Fc
R and reduced binding to another type.
Notably, for both Fc
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
Fc
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.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RIIA (CD32A; His131 allotype), Fc
RIIB (CD32B), and
Fc
RIIIA (CD16A; Val158 allotype) were provided by Dr. J. Ravetch (Rockefeller University, New York).
Fc
RIIA-Arg131 allotype and Fc
RIIIA-Phe158
allotype were generated by site-directed mutagenesis (31). The cDNA
for Fc
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
-chain
extracellular domain. The cDNAs encoding human neonatal Fc receptor
(FcRn)
-chain,
2-microglobulin subunit, and human Fc
R
-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).
R and the FcRn
-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
-chain fused at their carboxyl termini to Gly/His6/GST
at amino acid positions as follows: Fc
RI, His292;
Fc
RIIA, Met216; Fc
RIIB, Met195;
Fc
RIIIA, Gln191; FcRn, Ser297 (residue
numbers include signal peptides).
RI), IV.3 (anti-Fc
RII), 3G8
(anti-Fc
RIII) (Medarex, Annandale, NJ), and B1G6
(anti-
2-microglobulin) (Beckman Coulter, Palo Alto, CA).
Receptor concentrations were determined by amino acid analysis.
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
light chain.
R--
Fc
RIIA,
Fc
RIIB, and Fc
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 Fc
RIIA and Fc
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 Fc
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 Fc
RII and Fc
RIIIA was not apparent based on the lack of
binding of several Ala variants (Class 1, Table I).
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 Fc
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).
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 Fc
RII and Fc
RIIIA and 2 µg/ml for
Fc
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.
R--
The expression of Fc
RI
on THP-1 cells (38) was confirmed using the anti-Fc
RI antibody 32.2 (Medarex). Binding of IgG to Fc
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.
RIIIA-Phe158 and
Fc
RIIIA-Val158 with human
-chain were generated by
subcloning the
-chain and
-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-Fc
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.
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.
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
R--
IgG1 binding to Fc
RI
is of sufficient affinity to allow detection of monomeric IgG1. In
contrast, monomeric IgG1 does not bind well to Fc
RII and Fc
RIII,
and these receptors require a multimeric complex for assay. Previous
studies investigating Fc
RII and Fc
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 Fc
RII and Fc
RIII. Fig.
1 shows that these complexes bind to
Fc
RII and Fc
RIII in a concentration-dependent, saturable manner.
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Fig. 1.
Binding of anti-IgE E27 IgG1 to human
Fc R. A, binding of E27
monomers (solid circles), dimeric (solid
squares), and hexameric (open squares) complexes to
Fc
RIIA-Arg131. B, binding of E27 monomers
(solid circles), dimeric (solid squares), and
hexameric (open squares) complexes to
Fc
RIIIA-Phe158. Dimers were formed by mixing E27 IgG1
and a F(ab')2 fragment of goat anti-human
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).
R have
been previously determined (14-16). The pattern of binding of human
and murine subclasses to human Fc
RI, Fc
RIIA, and Fc
RIIIA was
verified using the ELISA format assays (data not shown). Human
Fc
RIIA has two known, naturally occurring allotypes that are
determined by the amino acid at position 131 (52). In this study, the
Fc
RIIA-Arg131 allotype was primarily used, but some IgG
variants were tested against the Fc
RIIA-His131 form.
Human Fc
RIIIA has naturally occurring allotypes at position 48 (Leu,
His, or Arg) and at position 158 (Val or Phe). For the Fc
RIIIA
assays in this study, the
Fc
RIIIA-Arg48/Phe158 allotype was used.
Binding to the Fc
RIIIA-Arg48/Val158 allotype
was also evaluated for selected variants.
R on cells, selected variants were tested in cell-based
assays. The binding of variants to THP-1 cells, which express Fc
RI,
was the same as for the ELISA-based assays; indeed, for Fc
RI the
cell-based results were included in the mean values cited in Table
I. For Fc
RIIIA, a select panel of variants was assayed on a stable-transfected CHO cell line expressing human FcgRIIIA-Phe158 (
- and
-chains) and the pattern
of binding reflected that found in the ELISA-based format (data not
shown).
Binding of human IgG1 variants to human FcRn and FcR
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 Fc
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 Fc
R. In
the IgG1 Fc:Fc
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
Fc R. A, IgG1 residues
comprising the binding site for Fc
RI and Fc
RII. The two Fc heavy
chains are in light and medium gray; carbohydrate
is in dark gray. Residues that affected binding to all
Fc
R are in red; the Fc
RI binding site is composed only
of red residues. Residues that showed improved binding to
Fc
RII and Fc
RIIIA are in magenta. Residues that showed
reduced binding only to Fc
RII are in yellow. Residues
that showed
50% improved binding only to Fc
RII are in
green. B, IgG1 residues comprising the binding
site for Fc
RI and Fc
RIIIA. The two Fc heavy chains are in
light and medium gray; carbohydrate is in
dark gray. Residues that affected binding to all Fc
R are
in red; the Fc
RI binding site is composed only of
red residues. Residues that showed improved binding to
Fc
RII and Fc
RIIIA are in magenta. Residues that showed
reduced binding only to Fc
RIIIA are in yellow. Residues
that showed
25% improved binding only to Fc
RIIIA are in
green. Glu430, involved in a salt-bridge with
Lys338, is shown in blue.
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 Fc
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 Fc
RIIIA suggesting that this
receptor requires the presence of a small amino acid side chain at this position, whereas the other receptors do not.
RI. Residues in the F(ab) portions of the IgG1 do not contribute
to Fc
RI binding as evidenced by both a CD4-immunoadhesin (58) and an
Fc fragment binding to Fc
RI as effectively as did intact IgG1 (data
not shown). Notably, no variants were found that reduced binding only
to Fc
RI.
RII and
Fc
RIII but not Fc
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-
atom to the backbone nitrogens of Lys326
and Ala327 and to the side chain N-
of
Asn325. Disruption of these interactions by D270A could
cause a local conformational perturbation that affected the severe
reduction in binding to Fc
RII and Fc
RIIIA. However, D270A did not
affect binding to Fc
RI or FcRn. Furthermore, D270N, which could
maintain the aforementioned hydrogen bonds, also abolished binding to
Fc
RII and Fc
RIIIA, and D270E bound to Fc
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 Fc
RII and
Fc
RIIIA.
Binding of human IgG1 non-Ala variants to human FcRII and Fc
RIIIA
RIIA,
Fc
RIIB, and Fc
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).
RIIA and Fc
RIIB. Those that improved binding to Fc
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:Fc
RIIA crystal structure (26). S267A improved binding only to
Fc
RII, S267G abolished binding only to Fc
RIIIA, and S267T reduced
binding to Fc
RII and Fc
RIIIA (Tables I and II). D280N (Table II),
like D280A (Table I), improved binding only to Fc
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.
RIIA and Fc
RIIB
but, in contrast to Class 4, also showed reduced binding to Fc
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 Fc
RIIB and a pronounced reduction in
binding to Fc
RIIIA (Table I); the R301M variant, which may maintain
the aliphatic interaction of the Arg301 side chain, showed
improved binding to Fc
RIIA and Fc
RIIB and a less pronounced
reduction of binding to Fc
RIIIA compared with R301A (Table II).
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 Fc
RII binding, suggesting that it may
play only a minor role in binding (discussed below).
RII but
improved binding to Fc
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
Fc
RIIIA as well as Fc
RII (Table II).
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 Fc
RII (25), and in the IgG1 Fc:Fc
RIIIA crystal
structure (26), the Ser239 in one of the two heavy chains
forms a hydrogen bond to the Lys117 side chain of
Fc
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:Fc
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 Fc
RIIIA. Note, however, that Tyr296 was changed to Phe (not Ala) and
the 50% reduction in binding to Fc
RIIIA was due only to removal of
the side chain hydroxyl group.
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 Fc
RI, Fc
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
Fc
RIIIA, E430A (Class 4) improved binding, suggesting that the
Lys338:Glu430 salt bridge is not essential in
maintaining binding. Another CH3 residue affecting Fc
RIIIA is
Asp376 that interacts with the CH2 domain.
RIIIA and
includes E333A, K334A, and A339T. A previous study found that A339T
improved binding to Fc
RI (62); in this study the A339T variant bound
better than native IgG1 to Fc
RIIIA but not Fc
RI (Table I).
Several non-Ala variants were tested at Glu333 and
Lys334. E333D also improved binding to Fc
RIIIA, whereas
E333N reduced binding to Fc
RII as well as Fc
RIIIA (Table II). At
position 334, changing Lys to Gln, Glu, or Val maintained the improved binding to Fc
RIIIA (Table II). Surprisingly, the K334R variant reversed the receptor preference, i.e. this variant bound
better to Fc
RIIB and not Fc
RIIIA as for the K334A variant. Taken
together these data suggest that Fc
RIIIA interacts with
Lys334 even though this residue is not among the IgG1
residues found to interact with Fc
RIIIA in the co-crystal structure
(26).
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.
RIIA,
and Fc
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 Fc
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 Fc
RIIA and Fc
RIIB similar to the S298A variant
even though the T256A change effected better binding to both these
receptors (Class 3).
Binding of human IgG1 combination variants to human FcRn and FcR
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).
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.
Percent of total oligosaccharide area by glycan type
atom, which forms a hydrogen bond to the Asn297 O-
, 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.
R. For the E258A, D265A, R301A, and R301M variants, it is
difficult to discern whether reduction or improvement in Fc
R binding
is due to the change in amino acid side chain or from differences in glycosylation.
RIIA and
Fc
RIIIA--
Selected variants were tested for binding to the
Fc
RIIA-His131 and Fc
RIIIA-Val158
allotypic receptor forms based on their improved or reduced binding to
the allotypic forms used for the assays (i.e.
Fc
RIIA-Arg131 and Fc
RIIIA-Phe158). Table
V shows that most of the
variants bound equivalently to the Fc
RIIA-Arg131
and Fc
RIIA-His131 receptors. The exceptions were the
S267A, H268A, and S267A/H268A variants that displayed binding to
Fc
RIIA-His131 that was reduced compared with
Fc
RIIA-Arg131 but still equivalent to native IgG1. The
related S267G variant, however, showed a 50% reduction in binding to
the Fc
RIIA-His131 receptor compared with native IgG1. In
contrast to S267A and H268A, D270A reduced binding to
Fc
RIIA-His131 by 50% but completely abrogated binding
to Fc
RIIA-Arg131. This suggests that Ser267,
His268, and Asp270 interact with Fc
RIIA in
the vicinity of Fc
RIIA residue 131.
Binding of human IgG1 variants to human FcRIIA-R131 and
Fc
RIIA-H131 polymorphic receptors
RIIIA, the selected variants were assayed in the ELISA format
as well as on stable-transfected CHO cell lines expressing the
-chains (Fc
RIIIA-Phe158 or
Fc
RIIIA-Val158) with the associated human
-chain. For
Fc
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
-chain associated with the
-chain enhancing binding of the
IgG to Fc
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 Fc
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.
Binding of human IgG1 variants to human FcRIIIA-Phe158 and
Fc
RIIIA-Val158 polymorphic receptors
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.
RIIIA allotype
of the donors was determined. By using three
Fc
RIIIA-Val158/Val158 and three
Fc
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 Fc
RIIIA-Phe158/Phe158 and
Fc
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 Fc RIIIA-Phe158 and
Fc
RIIIA-Val158 allotypes.
ADCC was performed using p185HER2-expressing SK-BR-3 cells
as target, and NKs isolated from three
Fc
RIIIA-Val158/Val158 donors or three
Fc
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 Fc
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
Fc
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 Fc
RIIIA-Phe158/Phe158
donors showed a significant increase in ADCC over the increase seen for
Fc
RIIIA-Val158/Val158 donors
(p < 0.0001 for all variants using paired t
test).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
R--
The set of IgG1 residues involved in binding to all human
Fc
R is represented by Class 1 (Table I). Indeed, this set comprises the entire binding site on IgG1 for Fc
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
Fc
R include Leu234-Pro238,
Ala327, and Pro329 (and possibly
Asp265); 2) positions that influence carbohydrate nature or
position include Asp265 and Asn297.
RI (8, 19) and Leu234 and Gly237 as the
two most important for Fc
RII (25, 56). Of the two residues in the
lower hinge investigated in this study, P238A affected binding to all
Fc
R, whereas S239A affected binding only to Fc
RIIIA.
RIIIA (26),
Pro329 interacts with two Trp side chains from the
receptor, and a similar interaction may occur with the other Fc
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 Fc
RI. Inspection
of the IgG1 Fc:Fc
RIIIA crystal structure shows that
Ala327 is near the IgG1:Fc
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.
RI--
For Fc
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 Fc
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 Fc
RI by 3-fold (62);
in this study the A339T variant was only equivalent in binding to
native IgG1 (Class 3).
-chain may augment the
binding affinity of the Fc
RI
-chain (63). Since it is conceivable
that some residues in the human IgG1 might interact directly with the
-chain, binding of the IgG1 variants to Fc
RI on THP-1 cells was
tested as well to the Fc
RI
-chain coated on a plate. The results
of the two assay formats were the same for the entire panel of
variants, suggesting that the
-chain augments binding by the
-chain through a mechanism other than direct interaction with the IgG1.
RI binds monomeric IgG1 about 100-fold more strongly than do
Fc
RII and Fc
RIII, one might expect that Fc
RI would utilize
either different or additional IgG1 residues to effect the tighter
binding. However, the set of IgG1 residues that control binding to
Fc
RI are a subset of those effecting binding to Fc
RII and
Fc
RIII (Class 1). This suggests that the comparatively strong binding of IgG1 to Fc
RI results from either 1) utilization of only
two Ig-like domains of Fc
RI (analogous to the two Ig-like domains of
Fc
RII and Fc
RIII) but with interaction of different amino acids
on Fc
RI than are used by Fc
RII and Fc
RIII, and 2) utilization
of the same amino acids on all three receptors but with additional
direct interaction of amino acids in the third Fc
RI domain, or 3)
the third domain of Fc
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 Fc
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.
RIIA and Human Fc
RIIB--
In
contrast to Fc
RI, Fc
RII requires the presence of two identical
IgG heavy chains (67), suggesting that residues from both heavy chains
may form the Fc
RII-binding site in IgG. The set of IgG1 residues, in
addition to the common Class 1 residues, that affect binding to
Fc
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.
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 Fc
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.
RI, several variants exhibited improved binding to
Fc
RIIA and Fc
RIIB (Classes 3-5). Of special interest are Class 4 containing residues which, when changed to Ala, improved binding only
to Fc
RII and Class 5 containing residues which, when changed to Ala,
simultaneously improved binding to Fc
RII and reduced binding to
Fc
RIIIA. These can be used to make IgG1 with improved specificity
for Fc
RII over Fc
RIIIA.
RIIA (68) and Fc
RIIB
(69) have been solved. In the Fc
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 Fc
RII lie outside of the residues at the Fc:Fc
RIIIA interface in the co-crystal structure (26). One of these, Asp280, is
not only distant from the Fc:Fc
RIIIA interface but is distant from
other Fc
RII-influencing residues (Fig. 2A). However, both D280A and D280N improved binding to Fc
RII, suggesting that this residue does indeed interact with Fc
RII.
RIIIA--
In addition to the
Class 1 residues, positions that reduced binding to Fc
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 Fc
RIIIA binding; note that changing these two residues did
not significantly reduce binding to Fc
RI, Fc
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:Fc
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
Fc
RIIIA.
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:Fc
RIIIA crystal structure report (26). Although
Glu333 and Lys334 do not interact with
Fc
RIIIA in the co-crystal structure, their interaction with
Fc
RIIIA is supported by four lines of evidence. First, murine IgG2b
E333A exhibited a modest reduction in binding to murine Fc
RII (25).
Although the same might not occur for murine Fc
RIII, this shows that
residues distant from the hinge region can influence binding to Fc
R.
Second, several non-Ala variants at Glu333 and
Lys334 either improved or reduced binding to Fc
RIIIA
(Table II). Third, binding of E333A and K334A to Fc
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).
RIIIA, Fc
RIIA, and Fc
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 Fc
RIIA and Fc
RIIB (Class 6), Arg416 a
30% reduction for Fc
RIIA and Fc
RIIIA, Gln419 a 30%
reduction to Fc
RIIA and a 40% reduction to Fc
RIIB, and Lys360 a 23% improvement to Fc
RIIIA (Class 10). Taken
together, their effect on binding of IgG1 to Fc
RIIA, Fc
RIIB, and
Fc
RIIIA suggests that the bottom of IgG1 may indeed be involved in
the IgG1-Fc
R interaction, although it may play only a minor role.
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 Fc
RI (56, 57). In human IgG1, D265A (Class 1) the
carbohydrate also differed from that of native IgG1 (Table IV), and
binding to Fc
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
Fc
RIIB, a decrease in binding to Fc
RIIIA, and no effect on
binding to Fc
RI or FcRn (Class 5). D265A (Class 1) showed decreased
binding to all Fc
R, whereas E258A (Class 4) showed increased binding
to Fc
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 Fc
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 Fc
R exhibited by these variants are
unlikely to be the result of glycosylation differences.
RIIA and Fc
RIIIA Polymorphic
Forms--
Human Fc
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
Fc
RIIA-Arg131 and Fc
RIIA-His131, variants
S267A, S267G, H268A, and D270 could discriminate between the
polymorphic forms. This suggests that these IgG1 residues interact with
Fc
RIIA in the vicinity of Fc
RIIA residue 131, and in the IgG1
Fc:Fc
RIIIA crystal structure (26), Ser267 is adjacent to
His131.
RIIIA has naturally occurring allotypes at position 48 (Leu,
His, or Arg) and at position 158 (Val or Phe). The
Fc
RIIIA-Val158 allotype binds human IgG better than the
Fc
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:Fc
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 Fc
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.
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 Fc
RIIIA-Val158 in the ELISA format assay.
However, when tested on cells expressing Fc
RIIIA-Val158
or in ADCC assays using
Fc
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 Fc
RIIIA, the variants exhibited a significant
improvement in ADCC for both Fc
RIIIA-Phe158/Phe158 and
Fc
RIIIA-Val158/Val158 donors (Fig. 4).
Indeed, using the S298A/E333A/K334A variant, the
Fc
RIIIA-Phe158/Phe158 donor ADCC could be
increased over 100% (i.e. >2-fold) compared with native
IgG1 (Fig. 4C).
RIIA polymorphic forms in various human
diseases has been investigated for many years (reviewed in Ref. 77),
the possible correlation between Fc
RIIIA polymorphic forms and human
disease has only recently been investigated (76, 78, 79). Given the
possible involvement of Fc
R in the mechanism of action of
therapeutic antibodies (4-6), human IgG1 variants with improved
binding capacity to human Fc
R, especially variants with better
binding to Fc
RIIIA and simultaneous abrogation of binding to the
inhibitory Fc
RIIB, could be used to provide more efficacious
therapeutic antibodies. In addition, a recent report on the occurrence
of polymorphic Fc
R forms in control populations showed that the
Fc
RIIIA-Phe158 allele is more prevalent than the
Fc
RIIIA-Val158 allele (77). Since the
Fc
RIIIA-Phe158 receptor binds human IgG1 less well than
the Fc
RIIIA-Val158 receptor, therapeutic antibodies with
variant Fc portions that improve binding to
Fc
RIIIA-Phe158 at least to the level seen for
Fc
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 FcR 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;
FcR, IgG Fc
-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.
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