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Receptor Binding and the Influence of CH1 and CH3 Domains on In Vivo Effector Function1
Department of Pathology, Immunology Division, University of Cambridge, Cambridge, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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R binding (glutamate 233 to
proline, leucine/phenylalanine 234 to valine, and leucine 235 to
alanine) completely prevented depletion. Mouse IgG2b was also
potent, and mutations to prevent complement activation did not impair
depletion with this isotype, as previously shown for human IgG1. In
contrast, a mutation that impaired binding to mouse FcRII (glutamate
318 to alanine) eliminated effector function of mouse IgG2b and also
reduced the potency of human IgG4. To reveal potential contributions of
domains other than CH2, domain switch mutants were created
between human IgG1 and rat IgG2a. Two hybrid mAbs were generated with
potencies exceeding anything previously seen in this model. While their
mechanism of depletion was not defined, their activity appeared
dependent upon interdomain interactions in the Fc
region. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In view of these limitations, it is important to be able to evaluate mAb function in vivo. This is not easily achieved in the clinical setting; what is needed is a preclinical model that might allow us to predict the biologic effects of mAb administration. One could then correlate in vivo and in vitro data so as to devise predictive in vitro tests of in vivo function. A sensitive system of this kind would also enable a fuller description of the structural basis of in vivo activity.
In a previous paper, we described a mouse model designed for this purpose (11). We compared a number of chimeric CD8 mAbs for their ability to deplete CD8+ PBL in CBA/Ca mice. We observed all human IgGs (hIgG) as well as rat IgG1 (rIgG1) and rIgG2b to be potent depleting agents, while rIgG2a was intermediate in potency, and hIgA2 and hIgE were inactive. With mutants of hIgG1, we were able to document the importance of the N-linked carbohydrate at residue N297 for in vivo effector function and also to show that complement was not necessary for in vivo killing. Thus, a mutant of hIgG1 that lacked the C1q binding motif (E318 K320 K322) (12) remained highly lytic both in complement-replete and in complement-depleted animals.
In the current paper, we build on these experiments using mutants of
hIgG1 and hIgG4 to demonstrate the importance of FcR interactions
for in vivo killing. We also investigate an entirely homologous system
using mouse IgG2b (mIgG2b) and mutants thereof. Finally, using
domain switch mutants between hIgG1 and rIgG2a, we generate a chimeric
protein of superior potency. These latter data suggest that interdomain
interactions may be an important factor in determining mAb effector
function.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Monoclonal Abs
Chimeric mAbs were created by transfection of the plasmid
pSV-V169 encoding chimeric CD8 heavy chains into the heavy chain loss
variant YTS 169.4L (11). Table I
lists
mutants of hIgG1, IgG4, and mIgG2b that were generated and
includes details of mutated amino acid residues. A mutated residue is
denoted AnB, where n signifies the position of the mutated residue, A
is the wild-type (WT), and B the mutant amino acid. The origin of WT
hIgG1 and hIgG4 C regions and the derivation of mutants by
site-directed mutagenesis was described previously (11). The
oligonucleotide used to mutate the C1q binding site of hIgG1 and hIgG4
was 5'-TTT GTT GGA GAC CGC GCA CGC GTA CGC CTT GCC ATT CA-3'. The lower
hinge FcR binding site was mutated using the oligonucleotide 5'-GAC
TGA CGG TCC CCC CGC GAC TGG AGG TGC TGA GGA-3'. The mIgG2b WT and
mutant constructs were described previously (12, 13).
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Transfectants were cloned once in soft agar, and high secreting clones (detected by isotype-specific ELISA) were grown to stationary phase in roller cultures. Supernatants were concentrated by ammonium sulfate precipitation and dialysed against PBS. hIgG1 and hIgG4 mAbs and mutants thereof were further purified on protein A, but other mAbs were used as crude precipitates.
mAb concentrations in all preparations were calculated by competitive binding assay using the WT rIgG2b mAb YTS 169.4 as previously described (11). SDS-PAGE analysis confirmed all mAbs to be H2L2 dimers (not shown), and therefore binding equivalents translated directly into µg/ml as used throughout this paper (11).
Staining of mouse peripheral blood
The preparation of PBL from peripheral blood and dual-color immunofluorescence has been described previously (14). mAbs used in the current study were biotin-coupled MTF 171 (aglycosyl hIgG1 anti-mouse CD8) (11) with either YTS 177.9 (rIgG2a anti-mouse CD4) (15) or KT3-1.1 (rIgG2a anti-mouse CD3) (16). Detection reagents were MARG2A-FITC (anti-rat IgG2a, Serotec MCA 278F, Oxford, U.K.) and streptavidin-phycoerythrin. Cells were fixed and stored after staining for subsequent analysis using a FACScan incorporating a FACSmate robotic sampler (Becton Dickinson, Mountain View, CA). The percentage of CD8+ PBL was calculated as the mean of CD4-CD8+ and CD3+CD8+ values.
Single-color immunofluorescence (see Table III) employed YTS 177.9, KT3-1.1,
and YTS 105.18 (rIgG2a anti-mouse CD8) using MARG2A-FITC for
detection as previously described (11). Circulating lymphocytes coated
with mAb were detected using MAR 18.5 (anti-rat 
light chain).
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CBA/Ca mice were maintained at the animal facility of the Department of Pathology, University of Cambridge. They were thymectomized at 4 to 5 wk of age (17) and used for experiments in age- and sex-matched groups. When required, depletion of complement C3 component was achieved by administration of 75 µg cobra venom factor in four divided doses 48 to 24 h before mAb administration. This dose is at least six times larger than required to deplete mice of C3 for 96 h after the fourth dose of cobra venom factor (18).
Test mAb was administered via a tail vein at time zero. mAb preparations were centrifuged before injection (13,000 rpm for 20 min in a bench top microfuge), and only the top one-third of MAb was used, to avoid administering large aggregates. Mice were bled at intervals following mAb administration for staining of PBL. Each experimental group comprised four CBA/Ca mice, and data were pooled from more than one experiment for analysis. Intergroup statistical comparisons utilized the Mann-Whitney U test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R binding motif are impotent
in vivo
In previous work, we demonstrated that a hIgG1 mAb was very
efficient at killing mouse PBL, but an aglycosylated variant lost this
capacity. Additionally, we showed that complement activation was not
necessary for depletion with this mAb (11). A critical motif for the
interaction of Igs with the high affinity human FcR (hFcRI, CD64)
has been identified (13, 19), and the same motif has been shown to be
important for binding mFcRI (20). The motif (E233 L234 L235 G236
G237 P238) is shared by hIgG1 and hIgG3 (Table II). hIgG4 differs by one amino acid
(F234) and has a 10-fold reduced affinity for hFcRI, while hIgG2
(P233 V234 A235) does not bind (19). We created mutants of hIgG1 and
hIgG4 with the hIgG2 sequence at residues 233 to 235 and assessed them
in vivo. This manipulation completely abolished their depleting
capacity (Fig. 1), but targeted PBL were
coated with CD8 mAb for 4 to 7 days following 5 µg of mAb (Table III
)
and up to 14 days after 25 µg (not shown). Residues 233 to 235 thus
play a critical role in the effector function of these isotypes. A
motif implicated in Ig binding to mFcRII and probably to mFcRIII
has been localized to this same region (21), and while our data
strongly implicate FcR-mediated clearance mechanisms, they do not
denote a specific receptor subtype.
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hIgG4 does not bind C1q (22); mutations in the C1q binding motif
(E318A K320A K322A) (12) were therefore not predicted to influence
depleting potency. Surprisingly, depletion was reduced by 
50%
compared with WT but was not further compromised by pretreatment of
mice with cobra venom factor (Fig. 2). A
possible explanation is the involvement of residue 318 in interactions
with mFcRII. Thus, mIgG2b with the mutation E318A no longer bound
mFcRII (21), and the same may hold for hIgG4. The equivalent
mutation did not influence depletion by hIgG1 (11), suggesting either
lack of involvement of FcRII or redundancy of clearance mechanisms
with that isotype.
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RII-dependent mechanism
The data accumulated thus far suggest that in this model
depletion requires interaction with Fc receptors, and circumstantial
evidence implicates both FcRI and FcRII. To clarify these
findings we investigated the depleting capacity of mIgG2b and mutants
thereof. WT mIgG2b binds mFcRII but not mFcRI (20), and it
activates complement (12). It depleted CD8+ PBL with a
potency equivalent to hIgG1 and hIgG4 (Fig. 3): 70 to 80% depletion was achieved
with 5 µg mAb per mouse and 50% less when 0.5 µg was
administered. As with hIgG1, inactivating the C1q binding site (K322A)
did not impair this activity in either complement-sufficient or
-depleted mice. A mutation that enabled binding to mFcRI (E235L)
(20) did not enhance potency, but inactivation of the FcRII binding
motif (E318A) (21) significantly reduced depletion. These data clearly
demonstrate that depletion can occur via interaction with mFcRII.
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RI binding site
The data presented above suggest that hIgG4 may interact with both
mFcRI and mFcRII. Thus, the E233P L234V L235A mutant failed to
deplete, and the E318A K320A K322A mutant was less potent than WT.
Surprisingly, a double mutant mAb encompassing both sets of changes
retained weak activity (Fig. 4). This
result suggested that the two sets of mutations balanced each other in
some way, although an equivalent effect was not seen with hIgG1, a
double mutant of which failed to deplete (Fig. 4).
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The data in the previous section imply that discrete loci within
an Ig molecule can interact and contribute to effector function. Domain
switch mAbs, in which an entire Fc domain is substituted by its
equivalent from an alternative isotype, can provide similar
information. In previous work, we showed the rIgG2a isotype to possess
unusual depleting properties. Not only did it have an intermediate
potency compared with other isotypes, but depletion occurred slowly
over 10 days (11). To further dissect these findings, we produced
rIgG2a/hIgG1 domain switch mAbs. Figure 5
illustrates these hybrids and the results of their in vivo
administration to mice. The 5.0-µg data broadly confirm the in vitro
work of other authors and demonstrate that the CH2 domain
is the dominant determinant of effector function (10, 23, 24): Any
hybrid possessing a hIgG1 CH2 domain achieved 80 to 90%
depletion, although there was more variability among hybrids with a
rIgG2a CH2 domain. Thus, adding a hIgG1 hinge to rat WT
(group G) substantially improved potency, as did the combination of
hIgG1 CH1 and CH3 domains (groups H and J). The
hinge has been implicated previously in mAb effector function (25), but
a contribution from CH1 or CH3 domains was not
predicted. Equivalent results were also seen with the administration of
0.5 µg of hybrid mAb per mouse. Most mAbs were unable to deplete
CD8+ PBL by >20 to 30% at this dose, but two were
consistently more potent, exceeding by two- to threefold the depleting
capacity of hIgG1 WT. These mAbs shared rat CH1 and
CH3 domains (groups B and C), and regardless of mechanism,
clearly demonstrated an influence of domains outside the hinge and
CH2 on effector function. These are the most potent mAbs
thus far tested in this model, and to our knowledge, this is the first
demonstration of bivalent mAbs with effector potencies in vivo superior
to the "best" WT isotypes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R binding for all isotypes
tested. Depleting isotypes cleared cells from the circulation with
rapid kinetics (Table III), presumably via interactions with
reticuloendothelial cells in the liver and spleen. In the current work,
PBL populations were monitored for 14 to 21 days and in our previous
work for several months. Depleted cells did not reappear during these
time periods, strongly suggesting that they had been killed and
not merely sequestered.
Care is necessary in interpreting the role of each FcR class, but
minimal conclusions are as follows. 1) mFcRII (or mFcRIII; see
below) mediates clearance with mIgG2b. This potent isotype does not
bind mFcRI (20), and preventing complement activation (K322A) had no
effect on its activity. However, blocking binding to FcRII (E318A)
reduced the level of depletion (Fig. 3). 2) Mutating residues
233 to 235 in both hIgG1 and hIgG4 abolished depletion by these
isotypes (Fig. 1), and these residues are critical for mFcRI (20)
and mFcRII (21) binding. The mutation E318A also impaired the
activity of hIgG4 (Fig. 2) but not hIgG1 (11). Thus, it seems likely
that both hIgG1 and hIgG4 use mFcRI to deplete, although hIgG4 may
additionally interact with mFcRII.
Unexpectedly, mutations toward the carboxyl terminus of IgG4
CH2 (residues 318, 320, and 322) partially restored the
activity of the impotent lower hinge (233–235) mutant (Fig. 4). Loci
that are distant in primary sequence have previously been shown to
interact in both complement activation (10, 23) and FcR binding
(24), purportedly by becoming close neighbors in Ig quaternary
structure. In the current example, both regions have been implicated in
FcR interactions, and it seems likely that we fortuitously restored
a FcR binding capability, although direct binding studies would be
required to confirm this. The spatial proximity of these two sets of
residues are illustrated in Figure 6, a
raster space-filling model of a hIgG1 mAb (26). There have been
no detailed studies of binding motifs on Igs for mFcRIII, but
mFcRII and III share >95% homology in their extracellular domains
and an identical hierarchy for binding to WT mouse and human Igs (27).
Therefore, mutations that affect binding to mFcRII may also affect
binding to mFcRIII. It should also be noted that in a single recent
report, the lower hinge was implicated in complement activation as well
as FcR binding (28). Overall, however, our data are not consistent
with a complement-mediated mechanism for mAb cytotoxicity in this
model.
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Table IV and Figure 6 highlight
nonconserved residues within the CH2 domains of hIgG2,
hIgG4, hIgG1FcR- and hIgG4FcR-, which
might account for these paradoxical data. Although several are located
close to previously defined effector motifs, no residues, either singly
or in combination, adequately resolve the disparate properties of these
four mAbs. Interpretation is slightly complicated by a single amino
acid truncation of the hIgG2 hinge, but an alternative explanation is
that mAb effector function is modulated by Ig regions outside the
CH2 domain, as suggested by our final set of experiments in
which we created domain switch mutants between hIgG1 and rIgG2a. These
confirmed CH2 as a critical determinant of effector
function, but also exposed influences from other domains. For example,
substitution of a human hinge on rIgG2a improved the latter’s potency,
but so too did exchange of CH1 and CH3 domains
for their human counterparts (but not either alone; Figure 5, right panel, hybrids G to L). Similarly, replacement of
human CH1 and CH3 with the equivalent rat
domains improved potency of hIgG1 (Fig. 5, middle panel,
hybrids A–F).
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RI binding (24). In
contrast, both CH2 and CH3 domains of hIgG1
were needed to fully restore hFcRI and hFcRII binding activity to
a hapten-binding mIgE (29). Such effects presumably arise from improved
binding to complement components or FcR, either by provision of a
more stable binding motif or perhaps through stabilization of a
favorable quaternary structure. Alternatively, synergy between domains
may result from overlapping but distinct functions. For example, ADCC
via hFcRIII requires an IgG CH3 domain to bind
hFcRIII and a CH2 motif to trigger cytotoxicity (30).
Similarly, our in vivo readout may reflect more than one effector
function. For example, a C3b bridge between effector cell and target
could facilitate binding to Fc receptors and, since
CH1/CH2 interactions have been invoked in CML
(10) and CH2/CH3 in ADCC (30), this could also
explain a multidomain influence on effector function. Finally,
multimeric mAbs display improved effector functions via clustering of
Ig Fc regions at the target cell surface. This is achieved naturally in
IgM and IgA but can also be created artificially. Thus, dimerizing mAb
by means of a C-terminal disulfide bridge improved effector function
substantially (31, 32), and addition of a µ-tailpiece to IgG4 even
permitted CML with this isotype (33). Although we quantitated our mAb
preparations by competitive binding to CD8, demonstrated that they were
H2L2 tetramers by PAGE, and centrifuged them before injection, we
cannot exclude the possibility that interdomain interactions resulted
in multimerization of our domain switch chimeras, leading to
increased potency. In future work, different mAb preparations should be
analyzed by size exclusion to appraise a potential role for multimers
in dictating effector function.
Our data provide important, generalizable messages for mAb engineers.
Nondepleting mAbs are very powerful tools for modulating immune
responses (2, 34) and are fashionable agents for manipulation of human
autoimmunity. The best route to creating such mAbs has not been
identified, however. It had been assumed that "impotent" isotypes
such as hIgG4 could be used, but we previously argued that in vitro
activity may not predict in vivo function (11), and furthermore,
population FcR polymorphisms may lead to interindividual variations
in biologic activity (10). In contrast, aglycosylated forms of mAb are
generally devoid of effector function in vivo (11) and should not
provoke cytokine release reactions (35). Furthermore, fears
concerning their immunogenicity appear unfounded (36). Similarly, our
current work suggests that if a mAb depletes via FcRs, the
mutation of one or two amino acids may completely abolish effector
function. The immunogenicity of such agents is likely to be
similar to the humanized mAbs from which they are derived (37, 38). At
the other extreme, the more cytolytic a mAb, the more effective
it should be at targeting and killing tumor cells (1). The domain
switch mAbs described above have highlighted interactions between the
Ig constant domains and include hybrids of exceptional potency,
which should guide additional experiments in this area.
These data again emphasize the importance of an in vivo model for the investigation of mAb effector function. Although largely derived from a heterologous system, they have clearly demonstrated the in vivo effects of specific mutations and revealed complexities of interaction that would be extremely difficult to reproduce in vitro. We would argue that such a model provides a critical bridge between in vitro and clinical studies, although at our present state of knowledge, no model can currently substitute for small in vivo pilot trials (39).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to (current address) Dr. John D. Isaacs, Senior Lecturer in Rheumatology, Molecular Medicine Unit, Clinical Sciences Building, St James’s University Hospital, Leeds LS9 7TF, U.K. E-mail address:
3 Current address: Peptide Therapeutics Ltd., 321 Cambridge Science Park, Milton Road, Cambridge CB4 4WG, U.K.
4 Current address: Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, U.K.
5 Abbreviations used in this paper: CML, complement-mediated lysis; ADCC, Ab-dependent cell-mediated cytotoxicity; m, mouse (e.g., mIgG); h, human; r, rat; FcR, Fc receptor for IgG; WT, wild type.
Received for publication February 24, 1998. Accepted for publication June 5, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1, 2a, 2b, 2c, 
, and 
. J. Immunol. 142:3145.[Abstract]
receptor class I binding site in human IgG through the use of recombinant IgG1/IgG2 hybrid and point-mutated antibodies. Proc. Natl. Acad. Sci. USA 88:9036.RII. Mol. Immunol. 29:53.[Medline]
RI and FcRIII binding. Immunlogy 86:319.
receptors. J. Immunol. 145:3842.[Abstract]
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