Discovery of human antibodies against the C5aR target using ... - DCU

JOURNAL OF MOLECULAR RECOGNITION
J. Mol. Recognit. 2005; 18: 327–333
Published online 10 February 2005 in Wiley InterScience (www.interscience.wiley.com). DOI:10.1002/jmr.735
Discovery of human antibodies against the
C5aR target using phage display technology
Lili Huang*, Aaron K. Sato, Meena Sachdeva, Tony Fleming, Susan Townsend and Daniel T. Dransfield
Discovery Research, Dyax Corp., 300 Technology Square, Cambridge, MA 02139, USA
Phage display technologies have been increasingly utilized for the generation of therapeutic, imaging and
purification reagents for a number of biological targets. Using a variety of different approaches, we have
developed antibodies with high specificity and affinity for various targets ranging from small peptides to large
proteins, soluble or membrane-associated as well as to activated forms of enzymes. We have applied this
approach to G-protein coupled receptors (GPCRs), often considered difficult targets for antibody therapeutics
and targeting. Here we demonstrate the use of this technology for the identification of human antibodies
targeting C5aR, the chemoattractant GPCR receptor for anaphylatoxin C5a. The N-terminal region
(residues 1–31) of C5aR, one of the ligand binding sites, was synthesized, biotinylated and used as the target
for selection. Three rounds of selection with our proprietary human Fab phage display library were
performed. Screening of 768 isolates by phage ELISA identified 374 positive clones. Based on sequence
alignment analysis, the positive clones were divided into 22 groups. Representative Fab clones from each
group were reformatted into IgGs and tested for binding to C5aR-expressing cells, the differentiated U-937
cells. Flow cytometric analysis demonstrated that nine out of 16 reformatted IgGs bound to cells. Competition
with a C5aR monoclonal antibody S5/1 which recognizes the same N-terminal region showed that S5/1
blocked the binding of positive cell binders to the peptide used for selections, indicating that the identified cell
binding IgGs were specific to C5aR. These antibody binders represent viable candidates as therapeutic or
imaging agents, illustrating that phage display technology provides a rapid means for developing antibodies
to a difficult class of targets such as GPCRs. Copyright # 2005 John Wiley & Sons, Ltd.
Keywords: antibodies; phage display; GPCRs; C5aR
Received 2 November 2004; revised 10 December 2004; accepted 15 December 2004
INTRODUCTION
Over the past decade, antibodies, mainly used as research
reagents, have been developed into effective therapeutic
agents with broad applications in cancer, inflammation
and infectious diseases. Up to now, there are 15 monoclonal
antibodies approved for clinical use, more than 70 candidates
in late stage clinical trials, and greater than 1000 in
preclinical development (for review, see Stockwin and
Holmes, 2003). The majority of these antibody therapeutic
candidates are derived from classical hybridoma technology.
To reduce immunogenicity in humans, these mouse
monoclonal antibodies are often humanized. The development
of human antibody-based phage display libraries and
human immunoglobulin transgenic mice has allowed the
discovery of human antibodies. One human monoclonal
antibody, adalimumab, a TNF inhibitor, has recently
been approved for the treatment of rheumatoid arthritis.
*Correspondence to: L. Huang, Discovery Research, Dyax Corp., 300 Technology
Square, Cambridge, MA 02139, USA. E-mail: lhuang@dyax.com
Abbreviations used: BSA, bovine serum albumin; C5aR, C5a receptor; ELISA,
enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate; FOI,
fraction of input; GPCRs, G-protein coupled receptors; HRP, horseradish
peroxidase; IRES, eukaryotic internal ribosome entry site; mAb, monoclonal
antibody; ONCL, oligonucleotide-assisted cleavage and ligation; PBS,
phosphate-buffered saline; PBST, phosphate-buffered saline, 0.1% Tween-20;
PCR, polymerase chain reaction; RBS, ribosome binding site; RT, room
temperature; TMB, tetramethylbenzidine.
More human antibodies are currently under evaluation in
clinical trials and pre-clinical development. The use of these
new technologies has greatly shortened the time to develop
human antibodies with high specificity and affinity.
Phage display has become increasingly used for the
identification of therapeutic, affinity purification and imaging
agents (for review, see Azzazy and Highsmith, 2002).
In phage display libraries, peptides and proteins including
antibodies are displayed on the surface of filamentous phage
through phage coat proteins such as pIII. Target-specific
binders can be selected from phage display libraries through
bio-panning processes. We have developed a series of Fab
displaying phage libraries along with a procedure for rapidly
reformatting these Fabs into fully human IgGs (Hoet et al.,
2004; Jostock et al., 2004). Using this approach, we have
developed fully human antibodies with high specificity and
affinity for various targets ranging from small peptides to
large proteins, both soluble and membrane-associated, as
well as to activated forms of enzymes. Here we demonstrate
the use of this technology for the identification of human
antibodies targeting the C5a receptor (C5aR), a member of
the GPCR superfamily.
GPCRs are seven-transmembrane G protein-coupled receptors
involved in signal transduction cascades, contributing
to many biological activities. As the largest and most
diverse group of transmembrane proteins, GPCRs represent
the most druggable targets. However, due to the difficulty in
getting soluble and active proteins, GPCRs are difficult
Copyright # 2005 John Wiley & Sons, Ltd.

328 L. HUANG ET AL.
targets for any antibody-based technology. To conquer this
difficulty, we developed a strategy to fully identify human
antibodies targeting C5aR.
C5aR (CD88) is the receptor for C5a, a 74 amino acid
peptide derived from the complement system following
activation via the classical, alternative or lectin-binding
pathways (Gerard and Gerard, 1994). C5a and its receptor
C5aR mediate immunomodulatory and inflammatory activities
such as chemotaxis, degranulation, vascular permeability
changes and cytokine regulation, thus playing a central
role in host defense against microorganisms and a diverse
range of immunoinflammatory disorders (for a review, see
Makrides, 1998; Pellas and Wennogle, 1999). C5aR is highly
expressed on cells of myeloid origin including granulocytes,
monocytes, lymphocytes and tissue inflammatory cells, including
macrophages, microglia and mast cells. C5aR also is
expressed, albeit at a lower level, on non-myeloid cells such
as epithelial, endothelial and smooth muscle cells in the liver
and lung. Owing to the high expression of C5aR on blood
leukocytes and tissue inflammatory cells as well as the
central role that this ligand–receptor complex plays in a
number of inflammatory disorders, C5aR and C5a have been
targets for therapeutic intervention. A large focus of this
effort has been on the identification and development of
small molecule inhibitors to the receptor and neutralizing
antibodies to C5a. Antibodies targeting C5aR will have the
potential to be developed as agents for therapeutics and
infection/inflammation imaging.
With our Fab displaying phage library and rapid reformatting
procedure, we were able to discover fully human
anti-C5aR antibodies within a short period of time, demonstrating
that difficult targets such as GPCRs can be successfully
targeted using this approach.
EXPERIMENTAL PROCEDURES
Cell culture
The human embryonic kidney HEK293T cells (GenHunter,
catalog no. Q401) were cultured in DMEM supplemented
with 10% ‘ultralow IgG’ fetal calf serum (Invitrogen,
catalog no. 31966021). The promonocytic U-937 cells
(ATCC, catalog no. CRL-1593.2) were cultured in RPMI
1640 medium supplemented with 10% heat-inactivated fetal
calf serum (FCS), 2 mM L-glutamine, 0.1 mM non-essential
amino acids, 1 mM sodium pyruvate, 100 Unit/ml of penicillin,
and 100 mg/ml of streptomycin. To induce differentiation
of U-937 cells, cells were treated with the membranepermeable
cAMP analog, N 6 ,2 0 -o-dibutyryladenosine 3 0 ,5 0
cyclic monophosphate (Bt 2 cAMP) (Sigma, catalog no.
C5788) at 0.5 mM for 3 days. All cell cultures were incubated
at 37 C in a humidified atmosphere with 5% CO 2 .
Peptide synthesis and labeling
The C5aR N-terminal peptide (amino acid 1–31) with an
addition of lysine residue at the C-terminus (NH2-
MNSFNYTTPDYGHYDDKDTLDLNTPVDKTSNK-NH2)
was synthesized on solid phase using 9-fluorenylmethoxycarbonyl
protocols. A portion of the synthesized peptide was
labeled on resin with the dPEG4/Biotin derivative via the
lysine residue. Both labeled and unlabeled peptides were
cleaved from resin with trifluoroacetic acid, purified using
reversed-phase high-performance liquid chromatography,
confirmed by electrospray mass spectrometry, and lyophilized.
Peptide concentrations were determined by measuring
the absorbance at 280 nm. The purity of each peptide was
greater than 99.9%.
Library construction
The human Fab phage display library (Fab 400) was constructed
as described (Hoet et al., 2004). Briefly, V-genes
from autoimmune patients (V L C L and V H -CDR3) and
synthetic V H -CDR1/2 were cloned into a phage display
vector, using a completely novel cloning method, oligonucleotide-assisted
cleavage and ligation (ONCL). The library
has a diversity of 1 10 10 .
Library selection
The biotinylated 31 amino acid C5aR N-terminal peptide
(DX-1186) was used as the target for selection. Magnetic
Dynabeads M-280 streptavidin (Dynal Biotech Inc., catalog
no. 112.06) were used to immobilize DX-1186. In round 1,
input phage (1 10 12 PFU of Fab400 library per selection)
and beads were each blocked at room temperature (RT) for
1 h with phosphate buffered saline (PBS) containing 2% (w/
v) non-fat dry milk and 0.1% (v/v) Tween-20. Blocked
phage were allowed to bind the free target DX-1186 in
solution (liquid binding selection) or the target immobilized
on beads (bead binding selection). In liquid binding selection,
after incubation with free DX-1186 (1 mM) at RT for
1 h, the phage target mixture was applied to beads for 15 min
at RT. The beads were washed seven times with PBS
containing 0.1% (v/v) Tween-20 (PBST) to remove unbound
phage. The bound phage were then eluted with
10 mM non-biotinylated peptide DX-1185 in PBST by incubating
at RT for 1 h. Eluted phage and remaining phage
on beads were amplified by infecting E. coli cells, and
underwent two more rounds of selection and amplification.
The subsequent rounds of selection were essentially same as
round 1, except depletion steps were included in rounds 2
and 3 before incubating with the target. Depletion was
carried out in rounds 2 and 3 to remove non-specific binders
by incubating the input phage with beads for 15 min at RT on
a rotator. The supernatant was removed from beads and
subjected to two more depletion steps. The depleted input
phage underwent selections as in round 1.
Screening for C5aR peptide binders by phage ELISA
Phage enriched from the third round of selection were
screened by phage ELISA for C5aR peptide binding. Immulon
2HB 96-well plates (ThermoLabsystems, catalog no.
3455) were coated with 100 ng per well of streptavidin
(Pierce, catalog no. 21120) diluted in PBS for 1 h at
37 C. The target plates were further coated with 40 ng
per well of DX-1186 diluted in PBS for 1 h at RT. The
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HUMAN ANTIBODIES AGAINST THE C5aR TARGET 329
background plates were incubated with PBS. The coated
plates were blocked with 1% (w/v) bovine serum albumin
(BSA) in PBS for 1 h at 37 C, and washed five times with
PBST. The plates were then incubated for 1 h with 1:2
diluted overnight phage cultures that were produced by
inoculating phage from individual plaques into E. coli cells.
After washing seven times with PBST, the plates were
incubated with horseradish peroxidase (HRP)-conjugated
anti-M13 antibody (Amersham Pharmacia, catalog no.
27-9421-01) for 1 h, washed seven times, developed with
tetramethylbenzidine (TMB) peroxidase substrate solution
(Kirkegaard & Perry Laboratories, catalog no. 50-76-03)
and read at 630 nm on an ELISA plate reader.
Reformatting of Fabs into whole IgG antibodies
IgG reformatting was carried out as described (Jostock et al.,
2004). Briefly, the Fab cassette containing a prokaryotic
ribosome binding site (RBS) was lifted from the phage
display vector by PCR and inserted into the IgG expression
vector. The prokaryotic RBS was then replaced with a
eukaryotic internal ribosome entry site (IRES). To express
IgG antibodies, HEK293T cells were transiently transfected
with the reformatted IgG vectors using Lipofectamine 2000
reagent (Invitrogen, catalog no. 11668019). Conditioned
media were harvested 72 and 144 h after transfection,
pooled, and sterile-filtered. IgGs were purified using protein
A beads (Amersham Biosciences, catalog no. 17-1279-01).
The final eluted IgGs were dialyzed against PBS, and IgG
concentrations were determined by measuring the absorbance
at 280 nm.
RT. After washing seven times with PBST, the plate was
incubated with a secondary HRP-conjugated anti-human
IgG (Bethyl, catalog no. A80-104P-38) for 1 h at RT,
washed seven times, developed with TMB solutions, and
read at 630 nm on an ELISA plate reader.
RESULTS
Selection of C5aR binders
The 31 amino acid N-terminal extracellular domain of C5aR
was synthesized and biotinylated via the lysine residue
added at the C-terminus [Fig. 1(A)]. This biotinylated
peptide, DX-1186, was used as the target for selection of
C5aR binders from a Fab phage display library (Fab 400)
which was constructed using the novel ONCL method and
contained novel V-gene composition resulting from a combination
of natural and synthetic diversity (Hoet et al.,
2004). Two arms of selection were carried out: liquid
binding and bead binding selections. For the liquid binding
Flow cytometric analysis
U-937 cells were harvested and resuspended in cold staining
buffer [PBS, 0.1% (w/v) sodium azide, 1% (w/v) BSA]. For
indirect immunofluorescence staining, cells were incubated
with individual reformatted IgG antibody or the anti-C5aR
mouse monoclonal antibody (mAb) S5/1 (Serotec, catalog
no. MCA1283XZ) at 10 mg/ml at 4 C for 20 min, washed,
and further incubated at 4 C for 20 min with a secondary
fluorescein isothiocyanate (FITC)-conjugated goat anti-human
IgG (Jackson ImmunoResearch Laboratories, catalog
no. 109-096-098) or goat anti-mouse IgG (Jackson ImmunoResearch
Laboratories, catalog no. 115-096-072). Stained
cells were then washed and resuspended in staining buffer,
and analyzed on a FACScan flow cytometer (Becton
Dickinson).
Competition ELISA
The target wells in an ELISA plate were sequentially coated
with streptavidin and DX-1186 as described above for the
phage ELISA. The background wells were only coated with
streptavidin. The plate was blocked with 1% (w/v) BSA at
37 C for 1 h, and washed five times with PBST. Prior to
incubation with individual reformatted IgG at 1 mg/ml
for 1 h at RT, the target wells were pre-incubated with
mAb S5/1 or mouse IgG at 10-fold molar excess for 1 h at
Figure 1. Library selection against the C5aR N-terminal peptide
DX-1186 target. (A) Amino acid sequences of the target peptide
DX-1186 and the peptide DX-1185 for elution. A lysine residue
was added at the C-terminus of the peptide for labeling with
dPEG4-biotin for DX-1186 peptide. DX-1185 was the unlabeled
peptide for elution during selection. (B) Fraction of input (FOI)
recovered after each round of selection. Three rounds of selection
from the Fab phage library (Fab400) were carried out. FOI
from each round was calculated as the total amount of output
phage divided by the total amount of input phage. FOIs from
both selections (liquid binding and bead binding) are shown.
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330 L. HUANG ET AL.
Figure 2. Representative phage ELISA results. Phage enriched from the third round of selection
were screened for positive binders by phage ELISA. The target wells were sequentially coated
with streptavidin and biotinylated DX-1186 peptide, and the background wells were coated only
with streptavidin. Plotted in the histograms is absorbance at 630 nm (A630) of individual phage
clone. Solid bar: DX-1186 target signal; open bar: streptavidin background signal. Shown here are
representative ELISA data from two selections, liquid binding selection (A) and bead binding
selection (B).
selections, input phage were first incubated with the target
DX-1186 in solution, and then immobilized to streptavidincoated
magnetic beads via DX-1186. In the bead binding
selection campaigns, input phage were allowed to bind the
immobilized target DX-1186 on streptavidin-coated magnetic
beads. After removal of unbound phage by washing
extensively with PBST, the bound phage were eluted with
DX-1185, the non-biotinylated C5aR peptide [Fig. 1(A)].
Eluted phage and the remaining phage on beads were
subsequently amplified, and subjected to further rounds of
selection. In rounds 2 and 3, depletion steps were carried out
to remove non-specific phage binders. After three rounds of
selection, the fraction of input (FOI), which was calculated
as the total amount of output phage divided by the total
amount of input phage, increased from 10 7 –10 6 at the
first round to 10 3 –10 2 by the third round [Fig. 1(B)],
suggesting enrichment of phage binders to the target.
To identify positive phage binders, the eluted phage from
the third round of selection were screened by phage ELISA.
The target plates were sequentially coated with streptavidin
and DX-1186; and the background plates were coated only
with streptavidin. As shown in Fig. 2 for representative
ELISA data and Table 1 for summary of results, the hit rates
of positive isolates with ELISA signals greater than 5-fold
over background levels were high from both selection
campaigns: 44% for liquid binding selection and 53% for
bead binding selection. If phage isolates with ELISA signals
greater than 3-fold over background levels were considered
positive, then the hit rates were more than 80% from both
selections. As noted in Fig. 2, a significant number of phage
isolates showed no to very low ELISA signals (A630 < 0.2).
These phage isolates were either Fab-displaying phage that
did not bind the target or phage with no Fab display, and thus
served as good internal negative controls for the assay.
Therefore, the selection campaigns on peptide target were
successful in identifying Fab-phage binders to the target
peptide.
Table 1. Summary of phage ELISA results
Selection arm Liquid binding Bead binding
Total isolates 384 384
Positive isolates a 170 204
Hit rate (%) 44 53
a Isolates with ELISA signals greater than 5-fold over streptavidin
background levels.
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HUMAN ANTIBODIES AGAINST THE C5aR TARGET 331
Sequencing of Fab-phage isolates and IgG reformatting
Selected ELISA positive isolates were subsequently sequenced.
Sequence alignment analysis of Fab amino acid
sequences of 77 isolates with full Fab sequence data available
resulted in the classification of the ELISA hits into five
major groups, based on heavy chain (HC) CDR1–3 usage.
Each major group has the same HC CDRs, but different light
chain (LC) CDRs. Variations of LC usage further divided
each group into subgroups, with a total number of groups
being 22. Besides the 22 subgroups, there were seven unique
sequences. Representative phage clones, a total of 29 isolates,
were chosen from each subgroup and unique isolates,
and reformatted into whole IgG molecules. Sixteen
Figure 3. FACS analysis of reformatted IgGs. U-937 cells were differentiated into macrophages by treatment with Bt 2 cAMP at
0.5 mM for 3 days. Differentiated cells were stained with IgGs by indirect immunofluorescence. Cells were first incubated with
reformatted IgGs and control human IgG (hIgG) in staining buffer. S5/1, which binds to the C5aR N-terminal region, was used
as a positive control. Mouse IgG (mIgG) was used as an isotype control for S5/1. After washing, cells were stained with a FITClabeled
secondary antibody, and then analyzed by flow cytometry. (A) Individual histograms of control hIgG, selected
reformatted IgGs (IgG-4, 32, 15, 28, 31, 33, 38, 43), control mIgG and S5/1. The x-axis depicts the fluorescence intensity of
individual cells, and the y-axis represents the cell number. (B) Summary of percentage positive cells for all IgGs tested. The y-
axis represents the relative percentages of positively stained cells, which were set based on negative controls. Data shown
are representative of three independent experiments.
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332 L. HUANG ET AL.
reformatted clones were expressed as IgGs (5–15 mg/ml) in
transiently transfected HEK293T cells. IgGs from these 16
clones were subsequently purified, and tested in cell binding
assays.
Testing reformatted IgGs for cell binding activities
Since synthetic peptides lack post-translational modifications
such as glycosylation and may adopt conformations different
to the same sequence in the native protein, antibodies
discovered from selection on peptide targets need to be
further evaluated in cell-based assays. Reformatted IgGs
were thus tested by flow cytometric analysis for their cell
binding activities on C5aR-expressing cells. C5aR was
induced in differentiated promonocytic U-937 cells (Gerard
and Gerard, 1990). U-937 cells were terminally differentiated
by treatment with 0.5 mM Bt 2 cAMP for 3 days, and then
stained with IgGs by indirect immunofluorescence. Cells
were first incubated with reformatted IgGs and control human
IgG (hIgG) in staining buffer. S5/1, amAb that recognizes
the C5aR N-terminal region (Oppermann et al., 1993)
was used as a positive control. Flow cytometric analysis
demonstrated that six reformatted IgGs (IgG-15, 28, 31, 33,
38 and 43) showed good binding to differentiated U-937
cells, with percentages of positive cells ranging from 11–30%
(Fig. 3). The positive control antibody S5/1 also showed
binding to cells with the percentage of positive cells being
approximately 17%. The other 10 reformatted IgGs (IgG-4,
11, 12, 16, 17, 18, 25, 29, 32 and 37) did not show significant
binding to cells, indicating that they were not cell binders.
Consistent with the C5aR expression profile that this
GPCR was not significantly expressed on undifferentiated
U-937 cells but was induced in differentiated U-937 cells,
one representative positive cell binder IgG-28 showed
binding in differentiated U-937 cells but not in undifferentiated
cells (Fig. 4), suggesting that IgG-28 is specific to
differentiated U-937 cells.
Figure 5. Specificity of positive cell binders to C5aR. Competition
ELISA was carried out as described in the Experimental
Procedures. The target wells were sequentially coated with
streptavidin and biotinylated DX-1186 peptide (white and striped
bars), and the background wells were coated just with streptavidin
(black bars). Prior to the addition of hIgG control and
positive IgGs, target wells were incubated with 10 mIgG (white
bars) or 10 S5/1 (striped bars). Plotted in the histograms are
ELISA signals as measured by A630.
Testing the specificities of positive cell binding IgGs
To further determine if the positive cell binding IgGs were
specific to C5aR, the positive IgGs (IgG-15, 28, 31, 33, 38
and 43) were tested in a peptide-based ELISA. Each of the 6
IgGs bound to DX-1186 (Fig. 5). Competition ELISA with
mAb S5/1 known to bind the same C5aR N-terminal region
showed that all six IgGs were competed with by S5/1
(Fig. 5), indicating that these positive IgG cell binders
were specific to C5aR. This was further confirmed by
FACS analysis of C5aR-expressing cells showing that the
IgG binding to cells was competed by S5/1 (data not
shown).
DISCUSSION
Figure 4. Specific binding of positive IgG binders to differentiated
U-937 cells. U-937 cells treated with Bt 2 cAMP at 0.5 mM for
3 days were used as differentiated cells, and untreated cells were
used as undifferentiated cells. Both differentiated and undifferentiated
cells were stained by indirect immunofluorescence and
analyzed by flow cytometry. Shown here are histograms of
representative positive IgG binder, IgG-28.
We have used phage display technology to identify human
antibodies to the chemoattractant receptor C5aR, a member
of the GPCR superfamily. GPCRs have proved successful
drug targets and a large proportion of existing drugs target
these receptors. However, owing to the complex structures
of these seven transmembrane domain-based proteins,
GPCRs are often considered difficult targets for antibodybased
therapeutics and targeting. We have designed a
strategy using a synthetic peptide encompassing the N-
terminal region of C5aR as the selection target. After three
rounds of selection with a Fab phage library, we were able to
obtain very high hit rates (50% of isolates with ELISA
signals greater than 5-fold over background levels) utilizing
two selection strategies: liquid binding and bead binding. In
liquid binding selection, the input phage were incubated
with the biotin-conjugated target in solution before capture
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HUMAN ANTIBODIES AGAINST THE C5aR TARGET 333
on streptavidin beads; in bead binding selection, the input
phage were incubated with the target immobilized on beads.
There are advantages and disadvantages for both selection
strategies. In liquid binding selection, high-affinity binders
may be enriched due to the lack of avidity effect, but the
target concentration should be well controlled so that the
target is well below the saturating concentration and potential
binders will not be lost after immobilizing to beads. In
bead binding selection, low-affinity binders also may be
enriched due to avidity effect, but the target concentrations
did not need to be well-controlled as in liquid binding
selection. In this study, we included both selection strategies
to ensure maximum success. Our results showed that both
selections worked very well, resulting in similar hit rates.
Representative Fab clones were reformatted into IgGs, and
tested for cell binding activities by flow cytometry. Approximately
40% of the reformatted IgGs showed binding to
differentiated U-937 cells in which C5aR expression was
induced. Flow cytometric analysis of HepG2 cells known to
express C5aR (Haviland et al., 1995) paralleled the results
from U-937 cells: positive binders to differentiated U-937
cells were also positive binders to HepG2 cells, and negative
binders to differentiated U-937 cells were also negative
binders to HepG2 cells (data not shown). These positive
cell binders were specific for C5aR since they bound to
C5aR N-terminal peptide DX-1186 in peptide-based
ELISA, and their binding was competed by S5/1, an
anti-C5aR mAb known to bind the same C5aR N-terminal
region (Oppermann et al., 1993). The cell binding activities
of these positive binders also were competed by
S5/1, suggesting specific binding to the C5aR N-terminal
region.
The library used here for selection is a Fab phage display
library with combined natural and synthetic diversity (Hoet
et al., 2004). The advantage of using a Fab phage library for
selection is that there is no need to do phagemid rescue
during selection and phage ELISA, thus greatly reducing the
amount of time for selection and screening. The human Fab
library has been used for selection with many soluble
protein targets, yielding target-specific human antibodies
with high affinity (Hoet et al., 2004). With the Fab library
and the IgG reformatting procedure (Jostock et al., 2004),
we were able to obtain specific human IgGs to a GPCR
within a short period of time (8 weeks), demonstrating
that our phage display approach has utility for rapid development
of antibody therapeutic and imaging agents to
GPCRs.
Acknowledgments
We thank Greg Conley for peptide synthesis and Gary Bassil for DNA
sequencing. We also thank Albert Edge for helpful discussions and Clive
Wood for critical reading of the manuscript.
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