From CNRS UMR 5094, Institut de Biotechnologie et
Pharmacologie, Faculté de Pharmacie, 15 Avenue Charles Flahault,
34093 Montpellier Cedex 5, France, ¶ CNRS UMR 5121, Laboratoire
Infections Rétrovirales et Signalisation Cellulaire, Institut de
Biologie, 4 Boulevard Henri IV, 34060 Montpellier Cedex 2, France,
Institut National de la Recherche Agronomique-CNRS UMR
5087, Laboratoire de Pathologie Comparée, 30380 Saint-Christol-Lez-Alès, France, ** CNRS UPR 1086, Centre de Recherche en Biochimie Macromoléculaire, 1919 Route de
Mende, 34293 Montpellier Cedex 5, France, and
Synt:em, Parc Scientifique G. Besse,
30035 Nimes Cedex 1, France
Received for publication, October 18, 2002, and in revised form, January 27, 2003
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ABSTRACT |
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We analyzed antigen-binding
residues from the variable domains of anti-CD4 antibody 13B8.2 using
the Spot method of parallel peptide synthesis. Sixteen amino acids,
defined as Spot critical residues (SCR), were identified on the basis
of a 50% decrease in CD4 binding to alanine analogs of reactive
peptides. Recombinant Fab 13B8.2 mutants were constructed with alanine
residues in place of each of the 16 SCR, expressed in the baculovirus
cell system, and purified. CD measurements indicated that the mutated
proteins were conformationally intact, with a The transmembrane glycoprotein CD4 is a major molecular partner in
the immunological synapse that leads to optimal activation of T
lymphocytes during immune responses (1). CD4 also serves as the primary
receptor for the human immunodeficiency virus
(HIV),1 thereby allowing the
virus to enter cells (2). At present, redesigned recombinant anti-CD4
antibodies (3-6) and a fully human anti-CD4 antibody from human
immunoglobulin transgenic mice (7) are currently included in phase
I-III trials for the treatment of autoimmune diseases, allograft
rejection, and AIDS. We have expressed humanized anti-CD4 recombinant
Fab 13B8.2 in the baculovirus/insect cell system, which is directed
against the CDR3-like region of the D1 domain of CD4 (8). In
vitro, this antibody prevents HIV transcription in
CD4+ cells at a post-gp120 binding step (8, 9) and also
inhibits antigen presentation (8). Early phase I clinical trials
(10-12) using the murine 13B8.2 antibody led to clinical benefits for some HIV-infected patients, i.e. disappearance of
circulating p24 antigen (10, 12), no detectable reverse
transcriptase activity (10), and generation of serum antibodies to
gp120 and HIV-neutralizing antibodies (12).
The identification of critical residues from the antibody paratope
involved in CD4 binding is a prerequisite for the improvement of such a
recombinant antibody for future clinical trials. To this end, we
previously characterized CD4-immunoreactive peptides in the 13B8.2
variable regions using the Spot method of parallel peptide synthesis
(13). Whereas the Spot method has often been used to map interaction
sites in epitopes (14), study of the antibody paratope (15) by probing
overlapping peptides covering the variable regions of an antibody with
labeled antigen was contrary to accepted dogma, probably due to the
widely held view that the three-dimensional structure of the antibody
is absolutely required for antigen binding. Initial experiments on
defining residues critical for lysozyme binding in the HyHEL-5 antibody
paratope (16) by the Spot method indirectly suggested that Spot
critical residues (SCR) are involved in the "true" antibody
paratope because ~65% of the identified SCR correlated with
contributing residues previously defined by x-ray crystallography of
the lysozyme-HyHEL-5 complex (17, 18). Until now, no direct
demonstration by simultaneous investigation using site-directed
mutagenesis of the whole protein and Spot technology has shown that SCR
from a given antibody actually belong to the paratope.
In this study on the mapping of key residues of Fab 13B8.2 involved in
CD4 recognition, we first identified residues important for binding by
the Spot approach; then, we used this information to design recombinant
Fab 13B8.2 single mutants. These mutated recombinant molecules showed
far-UV CD spectra similar to that obtained for wild-type Fab 13B8.2.
Replacement of 11 of the 16 SCR with alanine resulted in impaired CD4
binding, thus confirming the results of the Spot antibody mapping. This
impaired CD4 binding led to a loss of biological function of the
recombinant Fab single mutants with respect to CD4-mediated immune
responses. Based on a predicted three-dimensional model, all of these
critical residues showed appropriate positioning inside the putative
CD4-binding pocket. These results led to a view of the functional
paratope of an anti-CD4 antibody and could further lead to a
Spot-guided reshaping of anti-CD4 recombinant Fab 13B8.2.
Reagents, Cell Lines, and Vectors--
CD4-inserted recombinant
baculovirus was constructed and further used for the production of
recombinant human soluble CD4 as described (8, 13). For Spot analysis,
recombinant human CD4 (Repligen Inc., Needham, MA) was biotinylated
using a commercial reagent (Amersham Biosciences) according to the
manufacturer's instructions. The murine hybridoma cell line that
produces mAb 13B8.2 (IgG1/ Alanine Scanning of CD4-binding Peptides from the Variable Heavy
and Light Chain Sequences of Fab 13B8.2--
The general protocol for
Spot peptide synthesis on cellulose membrane has been described (15).
In 202 overlapping dodecapeptides frameshifted by one residue,
corresponding to the deduced amino acid sequence of variable regions
from the 13B8.2 antibody, anti-CD4 immunoreactivity was previously
observed (13) for peptides including heavy chain sequences 31-41,
49-70, and 90-103 and light chain sequences 19-26, 32-40, and
85-96 (Kabat numbering). Seventeen hexapeptides covering those
immunoreactive amino acid sequences and the six alanine analogs of each
peptide were synthesized by the Spot method. Antigen reactivity of
cellulose-bound peptides was assayed with biotinylated CD4 (1 µg/ml)
under conditions that yielded a blue precipitate on reactive spots as
described (13). The reactivity of the spots was evaluated by scanning
the membrane and measuring the intensities of the spots with NIH Image
Version 1.61 software. The SCR of the 13B8.2 paratope were identified on the basis of decreased antigen-binding capacity equal or superior to
50% of that of the unmodified peptide sequence.
Construction of Recombinant Baculoviruses Producing Anti-CD4
Wild-type or Alanine-mutated Fab 13B8.2--
The general procedures
concerning the cloning and sequencing of mAb 13B8.2 variable regions
have been described (8, 13, 23). Site-directed mutagenesis of heavy or
light chain genes from the 13B8.2 antibody was performed by overlapping
PCR (24). The sixteen amino acids demonstrated to be SCR by alanine
scanning analysis, two additional residues (Thr53 from the
light chain and Val61 from the heavy chain) as controls,
and Asn95 in the heavy chain were mutated to alanines. Each
mutation was confirmed by automated DNA sequencing. For the preparation
of heavy chain genes, the PstI/SacI-linearized
variable heavy chain fragments were cloned into the plasmid cassette
transfer vector pBHuFd Recombinant Fab Production, Purification, and
Characterization--
Each recombinant Fab 13B8.2 mutant was
protein G-immunopurified from a 400-ml supernatant of Sf9 cells
(American Type Culture Collection CRL 1711) infected with recombinant
baculovirus as previously described (8). Purified recombinant Fab
was quantified by a sandwich ELISA using sheep anti-human
Fd
Far-UV CD spectra were measured at 20 °C in 10 mM sodium
phosphate buffer (pH 7.0) with a Jasco J-810 spectropolarimeter (Tokyo, Japan). The spectra represent an average of two scans recorded at a
speed of 20 nm/min and a resolution of 0.1 nm. The antibody concentration was ~0.5 mg/ml. A cell with a 0.1-cm optical path length was used. Stability was characterized by further examination of
the temperature dependence of the CD spectra in the range of 20-80 °C.
CD4 Binding Studies of Wild-type or Mutated Recombinant Fab
13B8.2--
An ELISA method was performed to screen Fab 13B8.2 mutants
for their ability to bind soluble CD4. A 1:500 dilution of the baculovirus-expressed CD4 fraction (8) in 0.1 M
carbonate/bicarbonate buffer (pH 9.6) was coated overnight at 4 °C
onto 96-well enzyme immunoassay plates (Nunc, Paisley, UK). Four washes
with 160 mM PBS (pH 7.2) containing 0.1% Tween 20 (PBS/T)
were performed before and after saturating plates in 1% nonfat
powdered milk in PBS/T for 1 h at 37 °C. Thereafter, 100 µl
of 2-fold serial dilutions of a 2.5 µg/ml antibody solution was added
to each well. Following incubation for 2 h and four washes with
PBS/T, bound antibodies were detected by addition of 100 µl of a
1:1000 solution of peroxidase-conjugated anti-human
The kinetic parameters of the binding of CD4 to Fab 13B8.2 were
determined at 25 °C by surface plasmon resonance analysis using a
BIAcore 2000 instrument (BIAcore AB, Uppsala, Sweden). Baculovirus-expressed CD4 was covalently immobilized on a CM5 sensor
chip surface using the amine coupling method according to the
manufacturer's instructions. A control reference surface was prepared
using the same chemical treatment of the flow cell surface without
injection of CD4. Recombinant Fab mutants in buffer containing
10 mM Hepes (pH 7.4), 3 mM EDTA, 150 mm NaCl,
and 0.005% nonionic surfactant P20 (BIAcore AB) were then
injected at concentrations between 5 and 20 µg/ml over the flow cell,
and the dissociation phase was followed by a regeneration step with 5 mM HCl solution. The flow rate was 30 µl/min. All the
sensorgrams were corrected by subtracting the low signal from the
control reference surface. The data were globally fitted to a 1:1
Langmuir binding isotherm using BIAevaluation Version 3.2 software
(26).
The binding of wild-type or mutated Fab to membrane CD4 was evaluated
by flow cytometry. A2.01/CD4 T cells (1 × 106) were
incubated with PBS containing 0.2% BSA (PBS/BSA) or with PBS/BSA
supplemented with each recombinant Fab 13B8.2 mutant or irrelevant anti-digoxin Fab 1C10 expressed in the baculovirus/insect cell system. A similar experiment was performed with A2.01 T cells (a
CD4-negative cell line). After three washes with PBS/BSA, bound antibodies were revealed by incubation of 50 µl of a 1:1000 dilution of fluorescein isothiocyanate-conjugated anti-human IL-2 Secretion Assay following Antigen
Presentation--
Peptide-pulsed EBV-Lu antigen-presenting cells
(105/well) were co-cultured with pdb10F responder T cells
(2 × 104/well) as described (8, 13). Wild-type or
mutated Fab 13B8.2 (20 µg/ml) was then added to the cells, which were
subsequently cultured for 24 h at 37 °C. Thereafter, 100 µl
of supernatant was harvested and tested for IL-2 secretion using a
commercial ELISA kit (Pharmingen).
HIV-1 Promoter Activation Assay--
HeLa P4 indicator cells
(8 × 104/ml) were cultured in medium supplemented or
not with infectious HIV-1Lai in the presence or absence of
Fab (20 µg/ml) for 3 days, harvested, and lysed. Molecular Modeling of the Variable Regions of the 13B8.2
Antibody--
A three-dimensional model of the variable heavy and
light chains of the 13B8.2 antibody was obtained using the research
version of the antibody modeling software AbM
(Accelrys, Cambridge, UK) (28) running on an O2 R5000 Silicon
Graphics work station. The CDR-L2, -L3, -H1, and -H2 loops were
constructed using canonical class 1 frameworks and the CDR-L1 loop
using a canonical class 2 framework, as defined by AbM. The
CDR-H3 loop was built up using the combined data base/CONGEN
search, a conformational generator program implemented in
AbM, combined with a three-dimensional structural data base
search. Hydrogens were added to the model using SYBYL software (Tripos
Inc., St. Louis, MO), and the model was minimized during 100 iterations
with the Tripos force field and the conjugate gradient method to
eliminate all small steric conflicts. The solvent-accessible surface
areas of 13B8.2 residues were calculated on the three-dimensional model
by the SALVOL program (29) and implemented in SYBYL.
Identification by the Spot Method of Residues from the 13B8.2
Sequence Contributing to the Binding of CD4--
Seventeen
hexapeptides from previously identified antigen-binding sequences from
the variable regions of the 13B8.2 antibody (13) and sets of alanine
analogs were synthesized onto a cellulose membrane by the Spot method
to identify antibody residues critically involved in CD4 binding. A
detailed study of the reactivity of sequence 49-57 from the CDR-H2
region of the 13B8.2 antibody is shown as an example in Fig.
1 (A and B).
Substituting Trp52 of peptide
49GVIWRS54 (designated as control peptide 1)
with alanine led to a 50% decrease in CD4-binding capacity, whereas
changing Arg53 led to an almost complete loss of antigen
reactivity. The four other alanine replacements in peptide
49GVIWRS54 did not modify CD4-binding ability.
The contribution of Trp52 and Arg53 to CD4
binding was confirmed by alanine scanning of peptide
52WRSGIT57 (Fig. 1, A and
B), designated as control peptide 2. Similar
experiment using alanine analogs of hexapeptides
61VPFMSR66 and
65SRLSIT70 from the CDR-H2 region identified
Arg66 as another CD4-binding residue (data not shown).
Thus, the key residues from the H2 region were determined to be
Trp52 and Arg53 (belonging to the CDR) and
Arg66 (Fig. 1C).
In a similar manner, Phe32, His35,
Trp36, and Arg38 were determined by alanine
scanning analysis of the three hexapeptides
31TFGVHW36, 34VHWVRQ39,
and 36WVRQSP41 from the H1 region of the 13B8.2
antibody, with two residues (Phe32 and His35)
being involved in the H1 region (Fig. 1C). The CD4-binding
residues Cys92, Phe100K, Tyr102,
and Trp103 (Phe100K and Tyr102 from
CDR-H3 and the two other residues from frameworks) were determined from
the three hexapeptides 90YYCAKN95,
92CAKNDP97, and
99TGFAYW103 from the H3 region. Study of the
four hexapeptides 19VTFTCR24,
21FTCRAS26, 32YLAWYQ37,
and 35WYQQKQ40 from the L1 region identified
Arg24, Tyr32, Trp35,
Tyr36, and Lys39 as contributing to CD4
binding. Because no reactivity was observed in the L2 region (13), no
critical residue could be identified. The motif
88C- -HY92 contributed to CD4 binding, as
defined by Spot alanine scanning analysis of the three hexapeptides
85TYYCQH90,
88CQHHYG93, and
91HYGNPP96 from the L3 region, with
His91 and Tyr92 belonging to CDR-L3 (Fig.
1C). Taken together, 19 residues from the 13B8.2 variable
heavy and light chain sequences were identified as contributing to CD4
binding by alanine scanning of previously identified CD4-binding
peptides. These residues, defined on the basis of their importance in
the Spot assay, were named SCR. However, among them, Arg66
from the heavy chain and Arg24 and Lys39 from
the light chain were systematically found not to be accessible to the
solvent in the three-dimensional structure of antibodies and have never
been previously identified as critical residues for antigen binding (30,
31).2,3
Therefore, only 16 SCR from the paratope of the 13B8.2 antibody were
selected for further site-directed mutagenesis.
Characterization of Alanine-mutated Fab 13B8.2--
The variable
heavy and light chain genes from the 13B8.2 antibody were used as
templates for overlapping PCR/site-directed mutagenesis to replace each
identified SCR with an alanine residue in the Fab 13B8.2 context.
Following cloning in the appropriate pBHuFd CD4-binding Ability of Alanine-mutated and Wild-type Fab
13B8.2--
The ability of mutated versus wild-type Fab to
bind soluble CD4 was first assessed by an ELISA method (Fig.
3) and then quantified by BIAcore
analysis (Table I). In ELISA, the
CD4-binding activity of wild-type Fab 13B8.2 was detectable in the
19.5-1250 ng/ml range (Fig. 3), whereas no binding was observed with
irrelevant baculovirus-expressed anti-digoxin Fab 1C10. A CD4
dose-dependent reactivity similar to that found for
wild-type Fab was demonstrated for control mutants T53-L and V61-H and
also for alanine-mutated Fab C88-L, F32-H, W36-H, C92-H, and Y102-H. On
the other hand, alanine mutation of Tyr32,
His91, and Tyr92 from the CDR-L regions;
His35, Trp52, Arg53, and
Phe100K from the CDR-H regions; Trp35 and
Tyr36 from the light chain frameworks; and
Arg38 and Trp103 from their heavy chain
counterparts affected CD4 binding in a dose-dependent
manner (Fig. 3). These results were confirmed using BIAcore technology
because most of the Fab mutants demonstrating a loss of CD4 binding by
ELISA, i.e. Y32-L, H91-L, H35-H, R38-H, W52-H, R53-H,
F100K-H and W103-H, showed a 100-1000-fold decrease in their
KD values, whereas those that maintained their CD4-binding ability by ELISA exhibited KD values
similar to that found for wild-type Fab 13B8.2 (Table I). Finally,
mutants W35-L, Y36-L, and Y92-L, which showed a decrease in CD4 binding by ELISA, had KD values resembling that of wild-type Fab 13B8.2, although with a higher dissociation rate.
To determine the binding of Fab 13B8.2 to membrane-bound CD4,
antibody-labeled CD4+ A2.01/CD4 T cells were analyzed by
indirect immunofluorescence staining and flow cytometry (Fig.
4). Dose-dependent T cell
staining was obtained with wild-type Fab 13B8.2, whereas control Fab
1C10 or fluorescein isothiocyanate-conjugated anti-human Biological Activities of Alanine-mutated Versus Wild-type Fab
13B8.2 on CD4-mediated Responses--
pep24-pulsed EBV-Lu
antigen-presenting cells co-cultured with pdb10F responder T
cells led to IL-2 secretion following antigen presentation (20). As
shown in Fig. 5, a
dose-dependent inhibition of IL-2 secretion was
demonstrated following incubation with anti-CD4 wild-type Fab 13B8.2 in
this T cell activation model. At the same concentrations, control Fab
1C10 did not display any inhibitory activity. Mutants C88-L, F32-H,
W36-H, and C92-H, which exhibited in vitro CD4-binding
ability similar to that of wild-type Fab, also retained the wild-type
inhibitory capacity in the antigen presentation test. In contrast,
other mutations completely or partially abrogated the inhibition of the
antigen-presenting function of the Fab (Fig. 5). This result correlated
with those obtained in the study of the inhibitory property of Fab
mutants H91-L, F32-H, H35-H, W52-H, and R53-H versus
wild-type Fab on HIV-1 promoter activity (Fig.
6). In this case, Fab mutants showing
impaired CD4-binding capacity (H91-L, H35-H, W53-H, and R53-H) were not able to block HIV-1 long terminal repeat-driven Positioning Critical Residues on a Computer Model of the Variable
Regions of the 13B8.2 Antibody--
A three-dimensional model of the
antibody paratope was obtained following the alignment of the
amino acid sequence of mAb 13B8.2 with the AbM antibody
sequence library (Fig. 7). The structure of 1fdl and 1nld antibodies provided the template for the 13B8.2 light
and heavy chain frameworks, respectively. CDR conformations, except for
CDR-H3, were predicted based on canonical classes. The most
sequence-homologous known loops of the same canonical classes were
used. CDR-L1, CDR-L2, and CDR-L3 were built using the three-dimensional
1fdl antibody structure, and CDR-H1 and CDR-H2 were built using the
1nld structure. The flexible CDR-H3 loop was not described as a
canonical class and was built using a data base search combined with a
conformational search (CONGEN). As expected, the structure presented
two disulfide bridges, between Cys22 and Cys92
of the
When positioned on the 13B8.2 model, the SCR were found to fall into
four groups depending on their location, solvent accessibility, and
side chain orientation in the model, together with their true involvement as defined by site-directed mutagenesis (Fig. 7): group 1 (red) for well exposed SCR and group 2 (pink) for
less accessible SCR, whose mutation affected CD4 reactivity; and group 3 (blue) for well exposed residues and group 4 (turquoise) for buried amino acids, whose mutant reactivity
was not affected by site-directed mutagenesis.
Group 1 SCR (Tyr32, His91, and
Tyr92 from the light chain and His35,
Trp52, Arg53, and Phe100K from the
heavy chain) are exclusively composed of basic and aromatic residues
and are well located with great solvent accessibility and side chains
oriented inside the CD4-binding pocket. Trp52 and
Arg53 from the CDR-H2 loop and Tyr32 and
Tyr92 from the CDR-L1 and CDR-L3 loops, respectively, are
placed at the entrance of the antigen-binding site and are correctly
positioned for CD4 binding. The bottom of the CD4-binding site is
composed of a cluster of aromatic or positively charged residues:
His35 and Phe100K from the CDR-H1 and CDR-H3
loops, respectively, and His91 from the CDR-L3 region.
Group 2 SCR (Trp35 and Tyr36 from the light
chain and Arg38 and Trp103 from the heavy
chain) belong to the framework and are probably not directly involved
in CD4 interaction (Fig. 7). These four additional residues, with less
solvent accessibility, probably stabilize the backbone conformation of
regions involved in the CD4-binding pocket or shape the variable
heavy/light chain interface of the antibody. Although well located in
the antibody paratope, one SCR (Phe32 from the
heavy chain) belonging to group 3 points in a direction opposite to the
main orientation of the CD4-binding pocket and was not confirmed by
site-directed mutagenesis. Finally, group 4 SCR (Trp36 and
Cys88 from the light chain and Cys92 and
Tyr103 from the heavy chain) are clearly not involved in
CD4 interaction because an alanine mutation in their Fab fragments did
not affect CD4 binding, and they showed a side chain inaccessible to
the solvent (Cys92, Trp36, and
Cys88) and/or were at a distance from the CD4-binding
pocket, as for Tyr102 (Fig. 7). More specifically, the two
cysteines are not accessible to the solvent and are involved in
disulfide bridges, which might decrease their probability of
interacting with CD4. Most of the amino acids mapped by the Spot
approach and confirmed by site-directed mutagenesis are aromatic and/or
charged residues. The presence of three critical positively charged
residues lying at the entrance (Arg53 in CDR-H2) and the
center (His35 in CDR-H1 and His91 in CDR-L3) of
the CD4-binding site suggests that electrostatic interactions could be
one major element of the binding between the 13B8.2 antibody and its
epitope in the CDR3-like region of CD4. Indeed, CD4 binding to
wild-type Fab 13B8.2 exhibited a pH dependence (Fig.
8), arguing in favor of the role of
positively charged residues in the 13B8.2 binding site. Finally,
molecular modeling analysis of the CDR-L2 region showed that the L2
loop is relatively well exposed to the solvent, but structurally
outside the antigen-combining site, thus explaining the reason why no CD4-binding activity was previously demonstrated by the Spot method (13).
The definition of critical residues involved in antigen binding
from a given antibody paratope is a prerequisite for guiding the
construction of a variant with improved activities. X-ray crystallography, sometimes combined with site-directed mutagenesis and/or molecular modeling, is the method of choice for delineating the
antigen/antibody interface. Such analysis of the structural paratope
is, however, limited to certain favorable cases, depending on the
antibody and antigen availability, the level of antigen post-translational modifications, and the crystal quality of the complex, with these prerequisites being particularly critical for large
proteins. Because of these difficulties, the atomic coordinates of
antigen-antibody complexes from only ~20 different large proteins
were available in December 2002 in the Protein Data Bank.
In this work, we used an approach based on the identification of
residues critical for antigen binding by methods of parallel peptide
synthesis combined with site-directed mutagenesis. Alanine scanning of
previously defined CD4-binding peptides (13) from the 13B8.2 sequence
mapped 16 amino acids whose mutation to alanine decreased peptide
reactivity by >50%. Eleven CD4-binding residues as defined in the
Spot format (SCR) were further confirmed to be directly or indirectly
involved in the functional antibody paratope by site-directed
mutagenesis experiments. Among them, 10 residues (Tyr32,
Trp35, Tyr36, His91, and
Tyr92 from the 13B8.2 light chain and His35,
Trp52, Arg53, Phe100K, and
Trp103 from the 13B8.2 heavy chain), which we defined as
belonging to SCR groups 1 and 2, are in fact localized to positions of
the antibody sequence corresponding to statistically frequent antigen contact residues (30, 31); four residues from SCR group 4, of the five
excluded from the CD4 paratope by the results of site-directed mutagenesis experiments, do not belong to this contact group. Interestingly, although located at a position belonging to the group of
antigen contact residues (30, 31), Phe at position 32 of the heavy
chain has never been retrieved as a critical amino acid (31). As we
suggested in a previous study (15), the occurrence of false-positive
SCR can be explained by the fact that the peptide format used to map
the SCR allowed antibody residues normally buried in the folded
paratope to be exposed. This is emphasized by the 13B8.2 modeling
study, in which most of the SCR with no binding role were
inappropriately oriented in the antigen-binding pocket, in accordance
with the general view that the position and orientation of a residue
relative to the center of the combining site are key points with
respect to its propensity to bind antigen (30). Four critical residues
from group 2 were found to be less accessible in the antigen-combining
site model, but their mutations by site-directed mutagenesis affected
CD4 binding, suggesting that they have an indirect influence on CD4
binding to the paratope. Among them, Trp35 and
Tyr36 from the light chain and Trp103 from the
heavy chain are located at positions belonging to the Vernier zone
(32), which contains residues that adjust the CDR structure and
fine-tune the fitting to antigen. Furthermore, amino acids at position
36 in the light chain and 103 in the heavy chain show a reduction of
side chain-accessible surface upon formation of the dimer interface
between the variable heavy and light chains (31), suggesting that they
may be important for determining the shape of the antigen-binding
pocket at the variable heavy/light chain interface.
One major question is how can interactions between an antigen and its
combining site on the antibody be predicted from studies based on
antigen interaction with cellulose-bound peptide fragments of the
paratope? We can assume that probing such an overall interaction can be
assimilated with the mapping of discontinuous epitopes by the Spot
method (33, 34). In our case, the binding sites corresponding to the
antigen-binding pocket were found to be distributed over most if not
all of the CDR, located far apart in the primary structure, but brought
together in the folded antibody to form the paratope. The complete
paratope binds the antigen with high affinity, but individual binding
sites from each CDR, corresponding to a peptide sequence, are probably
characterized by a "degenerated" weak antigen interaction,
which is much more difficult to identify. The Spot method can overcome
such difficulty because high peptide density at each spot, in the range
of 200-500 nmol/cm2 (35), coupled with a very sensitive
electrochemiluminescence detection system facilitates identification of
weak binding peptides due to increased avidity for the probed antigen
(36). In addition, several studies have demonstrated that some local
flexibility contributes to antigen/antibody recognition, leading to
conformational adaptation (37-40). C-terminally bound peptides on the
cellulose membrane are unconstrained, as evidenced for peptides
mimicking an interleukin-10 epitope (41), thus allowing high
conformational flexibility, which could facilitate optimal positioning
of key residues for binding. The binding conformation is thus
"frozen" by the binding of the antigen. Finally, it has been
reported (42) that the Spot method facilitates identification of
binding site segments located in Our observation that 13B8.2 mutants C88-L and C92-H expressed in the
baculovirus/insect cell system still recognized CD4 and maintained
biological properties deserves comments. Less than 1% of all variable
region sequences have been described as lacking Cys at position 88 in
the light chain or at position 92 in the heavy chain. Together with
Cys23 from the light chain and Cys22 from the
heavy chain, they form disulfide bonds between We cannot exclude that other residues, mainly located in the CDR-H3 and
CDR-L3 regions and not identified by the Spot method, contribute to CD4
binding. As suggested by computer-assisted 13B8.2 modeling analysis,
Asn95 and Thr99 from the heavy chain and
Asn94 from the light chain show a side chain orientation
toward the center of the CD4-combining site (data not shown). In
addition, they are located at positions defined as antigen contacts for the antibody (31). Preliminary ELISA experiments (Fig.
9) using alanine-mutated Fab N95-H argue
in favor of such a contribution because no CD4 binding was observed for
this mutant (<10% ELISA activity compared with wild-type Fab). Taken
together, these observations outline the need to combine molecular
modeling of the variable regions of a given antibody with 6-mer/12-mer
Spot alanine scanning analysis of the paratope to accurately define
critical residues for antigen binding.
-sheet secondary
structure similar to that of wild-type Fab. Compared with the
CD4-binding capacity of wild-type Fab 13B8.2, 11 light (Y32-L, W35-L,
Y36-L, H91-L, and Y92-L) and heavy chain (H35-H, R38-H, W52-H, R53-H, F100K-H, and W103-H) Fab single mutants showed a decrease in CD4 recognition as demonstrated by enzyme-linked immunosorbent assay, BIAcore, and flow cytometry analyses. The five remaining Fab mutants showed antigen-binding properties similar to those of wild-type Fab.
Recombinant Fab mutants that showed decreased CD4 binding also lost
their capacity to inhibit human immunodeficiency virus promoter
activation and the antigen-presenting ability that wild-type Fab
displays. Molecular modeling of the 13B8.2 antibody paratope indicated that most of these critical residues are
appropriately positioned inside the putative CD4-binding pocket,
whereas the five SCR that were not confirmed by mutagenesis show an
unfavorable positioning. Taken together, these results indicate that
most of the residues defined by the Spot method as critical matched with important residues defined by mutagenesis in the whole protein context. The identification of critical residues for CD4 binding in the
paratope of anti-CD4 recombinant Fab 13B8.2 provides the opportunity
for the generation of improved anti-CD4 molecules with more efficient
pharmacological properties.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) (10, 19) was kindly provided by Drs. D. Olive and C. Mawas (INSERM U119, Marseilles, France). The human
lymphoblastoid B cell line EBV-Lu expressing HLA DR5/6, DRB52, DQ6/7,
and A2 molecules and the murine T cell line pdb10F expressing human CD4 and pep24 (PAGFAILKCNNKTFNY)-specific chimeric T cell receptor (20) were a kind gift from Dr. P. De Berardinis (Consiglio Nazionale delle Ricerche, Napoli, Italy). The HeLa P4 HIV-1 long terminal repeat
-galactosidase indicator cell line (21) was provided by Dr. O. Schwartz (Institut Pasteur, Paris, France). The previously described
(22) A2.01/CD4 T cell line expressing human wild-type CD4 was provided
by Dr. D. Littman (New York University, New York).
1, which contains the upstream
pre-installed first domain of human C
1
(Fd
1), allowing the insertion and expression of the
13B8.2 heavy chain under the control of the polyhedrin promoter (8,
25). For the preparation of light chain genes, the
XhoI/KpnI-linearized variable light chain
fragments were cloned into the plasmid cassette transfer vector
pBHuC
, which contains the upstream pre-installed human C
gene (8,
25), allowing the insertion and expression of the 13B8.2 light chain
under the control of the p10 promoter. A two-step recombination
procedure (8, 25) was carried out to construct the recombinant
baculoviruses, expressing both the heavy and light chains of wild-type
or mutated Fab 13B8.2. Irrelevant anti-digoxin Fab 1C10 was expressed
similarly in the baculovirus/insect cell system.
1 antibody (The Binding Site, Birmingham, UK) as the
capture reagent and peroxidase-conjugated anti-human
light chain
antibody (Sigma) as the detection reagent. Antibody samples were
further checked for purity by electrophoresis and Western blot analysis.
light
chain antibody, followed by subsequent addition of peroxidase
substrate. Absorbance was measured at 490 nm. A similar ELISA
experiment was performed to study the pH dependence of CD4 binding to
wild-type Fab 13B8.2, except that the antibody was diluted in 0.2 M sodium phosphate buffer at various pH values, obtained by
mixing different volumes of 0.2 M sodium dihydrogen phosphate and 0.2 M disodium hydrogen phosphate stock solutions.
light
chain antibody (Sigma) for 1 h at 4 °C. After three subsequent
washes with PBS/BSA, the fluorescence intensity was measured on an
EPICS cytofluorometer (Beckman-Coulter, Fullerton, CA).
-Galactosidase activity was then determined as previously described by measuring the
absorbance at 410 nm (27).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (61K):
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Fig. 1.
Determination by Spot alanine scanning of
residues from the paratope of the 13B8.2 antibody contributing to CD4
binding. A, scan of the CD4-probed membrane
corresponding to control peptides 1 (49GVIWRS54) and 2 (52WRSGIT57) covering sequence 49-57 from the
CDR-H2 region (Kabat numbering) and their respective sets of alanine
hexapeptide analogs. B, quantitative analysis of the Spot
reactivities of control peptides 1 ( ) and 2 (
) covering sequence
49-57 from the CDR-H2 region. Each bar represents the
reactivity of a hexapeptide whose sequence comprises Ala in place of
the indicated amino acid. C, SPR from each CDR of the 13B8.2
variable heavy and light chains, measured by percent inhibition of CD4
binding (vertical bars). The percent inhibition is indicated
by the size of the character. The CDR identification according to the
Kabat nomenclature is boxed.
1 or pBHuC
vector, expression in the baculovirus/insect cell system, and
purification, each Fab alanine mutant (designated as F32-H, H35-H,
W36-H, R38-H, W52-H, R53-H, C92-H, F100K-H, Y102-H, and W103-H for the
heavy chain and Y32-L, W35-L, Y36-L, C88-L, H91-L, and Y92-L for the
light chain) was further quantified by ELISA and characterized by
Western blotting and far-UV CD. As exemplified for mutants Y36-L,
C88-L, F32-H, H35-H, W52-H, and R53-H (Fig.
2A) using a sandwich ELISA
format, Fab production could be demonstrated for each mutant. Coomassie
Blue/SDS-PAGE of 1 µg of loaded protein for each recombinant revealed
a single band at 50 kDa (data not shown), corresponding to the expected size of a correctly processed Fab fragment under nonreducing
conditions. The identity of the 50-kDa band was confirmed by Western
blotting using anti-human
chain antibody (Fig. 2B).
As exemplified for alanine-mutated Fab H91-L and R53-H, whose
CD4-binding activities were the most drastically affected, a
far-UV CD spectrum similar to that obtained for wild-type Fab was
obtained for all mutants, with one negative peak at ~216 nm and one
positive peak at ~200 nm (Fig. 2C). These results indicate
that the mutated proteins we produced were conformationally intact,
with a main
-sheet secondary structure similar to that of wild-type
Fab 13B8.2. Furthermore, no modification of the spectra obtained with
mutated proteins could be detected in the 20-80 °C temperature
range (data not shown) in comparison with that obtained with wild-type
Fab 13B8.2, suggesting that antibody stability was not affected by the
mutations.
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Fig. 2.
Characterization of alanine-mutated
recombinant Fab 13B8.2 following protein G immunopurification from a
baculovirus supernatant. A, ELISA antibody
titration curves for mutants Y36-L, C88-L, F32-H, H35-H, W52-H, and
R53-H. Inset, the immunoglobulin standard curve.
B, Western blot analysis of the 50-kDa Fab band revealed by
peroxidase-conjugated anti-human light chain antibody.
C, far-UV CD spectra of the H91-L and R53-H mutants
versus wild-type (wt) Fab 13B8.2 in 10 mM phosphate buffer. deg, degrees.
View larger version (43K):
[in a new window]
Fig. 3.
CD4 binding curves of various concentrations
of alanine-mutated Fab 13B8.2 in comparison with wild-type Fab 13B8.2
and irrelevant Fab 1C10. Each value represents the mean ± S.D. of triplicate determinations in an ELISA format. Results are
representative of three independent experiments. wt,
wild-type.
BIAcore determination of the binding kinetics of the interaction
between sensor chip-bound CD4 and 13B8.2 antibodies
light chain antibody did not bind to the cells. Similar experiments
conducted with CD4-negative A2.01 cells failed to show specific binding either with wild-type Fab 13B8.2 or its alanine mutants (data not
shown). Alanine-mutated Fab Y36-L, C88-L, F32-H, W36-H, R38-H, and
C92-H and control mutants T53-L and V61-H showed CD4+
staining of A2.01/CD4 cells similar to that obtained with wild-type Fab. In contrast, a defect in CD4 binding was observed for mutants bearing alanine replacement of Tyr32, Trp35,
His91, and Tyr92 in the light chain and
His35, Trp52, Arg53,
Phe100K, and Trp103 in the heavy chain.
View larger version (53K):
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Fig. 4.
Flow cytometry analysis of the binding of
each recombinant Fab 13B8.2 mutant versus wild-type
Fab 13B8.2 and irrelevant Fab 1C10 to A2.01/CD4 T cells. Results
are representative of two different experiments. Antibody
concentrations are indicated. FITC, fluorescein
isothiocyanate.
-galactosidase reporter gene expression, whereas F32-H, with CD4-binding capacity similar to that of wild-type Fab 13B8.2, inhibited reporter gene expression. Taken together, mutations leading to impaired CD4 binding
correlated with a defect in the biological activities of the Fab
mutants.
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Fig. 5.
Inhibition of IL-2 secretion by pdb10f T
cells sensitized with pep24-stimulated EBV-Lu antigen-presenting cells
and co-cultured with recombinant Fab 13B8.2. A,
dose-response inhibition of IL-2 secretion by various concentrations of
wild-type (wt) Fab 13B8.2 in comparison with irrelevant Fab
1C10. B, IL-2 secretion inhibition of each recombinant Fab
13B8.2 mutant. Mean absorbance at 450 nm varied from 0.015 for pdb10F
cells co-cultured with unstimulated EBV-Lu antigen-presenting cells to
1.28 for pdb10F cells co-cultured with pep24-stimulated EBV-Lu
antigen-presenting cells. A positive control for IL-2 secretion carried
out by incubating murine anti-CD3 antibody (Pharmingen) with pdb10F T
cells gave an absorbance of 2.30. nd, not determined.
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Fig. 6.
Inhibition of long terminal
repeat-driven -galactosidase gene expression
induced by HIV-1Lai following incubation with recombinant
Fab 13B8.2. A, dose-response inhibition of long
terminal repeat (LTR)-driven
-galactosidase
(
-gal) gene expression in
HIV-1Lai-infected HeLa P4 cells cultured in the
presence of various concentrations of wild-type (wt)
recombinant Fab 13B8.2. B, inhibition of
-galactosidase
gene expression by recombinant Fab mutants H91-L, F32-H, H35-H, W52-H,
and R53-H. Mean absorbance at 410 nm varied from 0.01 for uninfected
indicator cells to 0.40 for HIV-1Lai-infected indicator
cells.
-sheet of the heavy chain and between Cys23 and
Cys88 of the
-sheet of the light chain.
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Fig. 7.
Three-dimensional model of the variable heavy
and light chains of the 13B8.2 antibody generated using the
AbM software, based on homology modeling (side
(A) and top (B) views).
The model is shown with side chains for each selected SCR from
the 13B8.2 paratope, using different colors relative to the SCR groups
(red for group 1, pink for group 2, blue for group 3, and turquoise for group 4). SCR
confirmed by site-directed mutagenesis experiments are in
red and pink, whereas those not confirmed are in
blue and turquoise. VH, variable heavy
chain; VL, variable light chain.
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Fig. 8.
pH dependence of the binding of CD4 to Fab
13B8.2. CD4 binding of a 10 µg/ml solution of wild-type Fab
13B8.2 ( ) was analyzed by diluting the antibody fragment in 0.2 M sodium phosphate buffer at various pH values between 6.0 and 7.8. Each value represents the mean ± S.D. of triplicate
determinations in an ELISA format.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheets inside the folded protein.
Because the antibody structure is composed mainly of
-sheet
elements, the Spot method is particularly appropriate for the
identification of peptides and, in particular, their key residues
involved in antigen binding.
-sheets to maintain
thermodynamic stability and folding of the antibody. Whereas most of
the cysteine-lacking recombinant antibodies expressed in bacteria
demonstrated a defect in antigen-binding capacity, some active
antibodies have been described, however, in eukaryotic systems (43).
Our results might be explained by glutathione derivatization of the
residual cysteine as described for yeast expression of the
Cys-defective mutant lysozyme (44) or suggested for expression of
Cys-defective whole antibodies (45).
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Fig. 9.
CD4 binding curves of various concentrations
of alanine-mutated Fab N95-H on adsorbed CD4 in comparison with
wild-type Fab 13B8.2. Each value represents the mean ± S.D.
of triplicate determinations in an ELISA format. Data are
representative of three independent experiments. wt,
wild-type.
Although no structure of any CD4-antibody complex has yet been described, molecular modeling of the OKT4A antibody, which recognizes an epitope in the CDR2-like loop of domain 1 of CD4, has permitted the study of its antibody combining site (4). Interestingly, some similarities between the binding pockets of the OKT4A and 13B8.2 antibodies can be noted. Two charged residues (Lys95 and Asp100A) from the OKT4A heavy chain are centered in the binding site, which is also the case for the charged His35 and His91 residues from the heavy and light chains of mAb 13B8.2, respectively. The role of such positively charged residues from the 13B8.2 antibody must be underscored because (i) CD4 binding is increased by Fab 13B8.2 incubation at pH 6.0, at which 50% of the histidine residues are positively charged, but only 5% at pH 7.2; and (ii) the epitope of the 13B8.2 antibody mainly involves the negatively charged residues Glu87 and Asp88 on the CD4 molecule (46), suggesting that electrostatic interactions are of great importance in the 13B8.2 combining site. In a similar way, the two negatively charged critical residues from the OKT4A paratope (4) can interact with the positively charged Arg59 residue on the CD4 molecule, previously characterized as belonging to the epitope of the OKT4A antibody. Surrounding those residues, a cluster of aromatic side chain residues either from the 13B8.2 or OKT4A antibody essentially contribute to the binding. The bottom of the CD4-binding pocket involves more buried residues mainly belonging to the framework, such as Trp35, Tyr36, Arg38, and Trp103 for the 13B8.2 antibody and Ala34, Leu89, Ser35, and Ala50 for the OKT4A antibody, that may be critical for appropriately shaping the antigen-combining site (4). In addition, hydrogen bonding interactions between the main chain of His91 and the side chain of Tyr32 from the 13B8.2 light chain are suggested from our computational model to stabilize the CDR-L3 region, like those assessed by modeling analysis of the OKT4A antibody (between His49 from the light chain and Asp99 from the heavy chain) for stabilizing the CDR-H3 structure (4). On the basis of these arguments, we can speculate that similar interaction rules govern the binding of the 13B8.2 and OKT4A antibodies to their respective epitopes on the CD4 molecule, even if the induced biological responses are largely different.
The 13B8.2 antibody promotes post-entry inhibition of HIV transcription
and T cell activation (2), such biological effects being abrogated
using recombinant Fab harboring mutations impairing CD4 binding. In
other words, these results indicate that at least His35,
Arg38, Trp52, Arg53,
Phe100K, and Trp103 from the 13B8.2 heavy chain
and Tyr32, Trp35, Tyr36,
His91, and Tyr92 from the 13B8.2 light chain
seem to be particularly critical in maintaining the desired biological
effects of the 13B8.2 antibody. This set of data should be taken into
consideration for the rational design of new anti-CD4 ligands of
clinical value, derived either from recombinant Fab 13B8.2 (8) or from
the CB1 paratope-derived peptide from the CDR-H1 region of the 13B8.2
antibody (13). A Spot-guided, site-directed mutagenesis of recombinant
Fab 13B8.2 aimed at improving the activity of the 13B8.2 antibody can
now be envisaged.
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ACKNOWLEDGEMENTS |
---|
The skillful assistance of Verane Palumbo in the Spot synthesis of peptides is acknowledged. We thank Annick Ozil, Nicole Bres, Marylene Ozil, Sophie Boussinesq, Carine Charron, and Frederique Escuret for excellent technical assistance. We gratefully acknowledge Drs. D. Olive and C. Mawas for providing the mAb 13B8.2-producing cell line. We also thank Dr. Q. J. Sattentau for providing the CD4 cDNA-encoding plasmid, Dr. O. Schwartz for the HeLa P4 cell line, Dr. D. Littman for the A2.01/CD4 T cell line, and Dr. P. De Berardinis for the human lymphoblastoid B cell line EBV-Lu and the murine T cell line pdb10F. We are indebted to Dr. S. L. Salhi for editorial revision of the manuscript.
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FOOTNOTES |
---|
* This work was supported in part by CNRS.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.
§ Supported by fellowships from the Ministère de l'Education Nationale et de la Recherche and the Ensemble Contre le Sida.
§§ To whom correspondence should be addressed. Tel.: 33-467-548-604; Fax: 33-467-548-610; E-mail: thierry.chardes@ibph.pharma.univ-montp1.fr.
Published, JBC Papers in Press, February 3, 2003, DOI 10.1074/jbc.M210694200
2 Available at bioinf.org.uk/abs.
3 Available at biochem.unizh.ch.
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ABBREVIATIONS |
---|
The abbreviations used are: HIV, human immunodeficiency virus; CDR, complementarity-determining region; SCR, spot critical residue(s); mAb, monoclonal antibody; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; BSA, bovine serum albumin; IL-2, interleukin-2; L, variable region of the light chain; H, variable region of the heavy chain.
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REFERENCES |
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