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doi:10.1182/blood-2002-08-2480
Prepublished online March 20, 2003;
2003 102: 564-570
Haspot, Jérôme Tiollier and Jean-Paul Soulillou
Bernard Vanhove, Geneviève Laflamme, Flora Coulon, Marie Mougin, Patricia Vusio, Fabienne
1-antitrypsin fusion antibody α−Selective blockade of CD28 and not CTLA-4 with a single-chain Fv
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IMMUNOBIOLOGY
Selective blockade of CD28 and not CTLA-4 with a single-chain Fv–
␣1-antitrypsin fusion antibody
Bernard Vanhove, Genevie`ve Laflamme, Flora Coulon, Marie Mougin, Patricia Vusio, Fabienne Haspot, Je´roˆme Tiollier, and Jean-Paul Soulillou
B7-1 and B7-2 are costimulatory molecules
expressed on antigen-presenting cells. The
CD28/B7 costimulation pathway is critical
for T-cell activation, proliferation, and Th
polarization. Blocking both cytotoxic T-
lymphocyte–associated antigen 4 (CTLA-4)
and CD28 interactions with a CTLA-4/Ig
fusion protein inhibits various immune-
mediated processes in vivo, such as allo-
graft rejection and autoimmunity. However,
selective blockade of CD28 may represent a
better strategy for immunosuppression than
B7 blockade, because CTLA-4/B7 interac-
tions have been shown to participate in the
extinction of the T-cell receptor–mediated
activation signal and to be required for the
induction of immunologic tolerance. In addi-
tion, selective CD28 inhibition specifically
decreases the activation of alloreactive and
autoreactive T cells, but not the activation of
T cells stimulated by exogenous antigens
presented in the context of self major histo-
compatibility complex (MHC) molecules.
CD28 blockade cannot be obtained with
anti-CD28 dimeric antibodies, which cluster
their target and promote T-cell costimula-
tion, whereas monovalent Fab fragments
can block CD28 and reduce alloreactivity. In
this study, we report the construction of a
monovalent single-chain Fv antibody frag-
ment from a high-affinity antihuman CD28
antibody (CD28.3) that blocked adhesion of
T cells to cells expressing the CD28 recep-
tor CD80. Genetic fusion with the long-lived
serum protein ␣1-antitrypsin led to an ex-
tended half-life without altering its binding
characteristics. The anti-CD28 fusion mole-
cule showed biologic activity as an immuno-
suppressant by inhibiting T-cell activation
and proliferation in a mixed lymphocyte
reaction. (Blood. 2003;102:564-570)
©2003 by The American Society of Hematology
Introduction
Targeting T-cell costimulation has been widely investigated to control
T-cell reactivity in autoimmunity and transplantation. Inhibiting costimu-
lation through B7 (B7-1/B7-2 or CD80/86) blockade by cytotoxic
T-lymphocyte–associated antigen 4 (CTLA-4)–Ig has now become a
clinical reality.1Blocking B7, however, might have 2 opposite effects. In
addition to reducing T-cell costimulation through CD28, B7 blockade
can also prevent CTLA-4 from transmitting a negative/regulatory
signal. Indeed, the signal transmitted through CTLA-4 leads to the
recruitment of phosphatases that dephosphorylate activated second
messengers in the CD3 complex resulting in a reduction in the nuclear
translocation of Rel A needed for the production of multiple cytokines
produced by Th1 and Th2 cells.2,3 Moreover, CTLA-4 has been
implicated in the development of regulatory T cells in several models of
transplantation4and animals lacking CTLA-4 are resistant to tolerance
induction.5Therefore, inactivation of CTLA-4 might well be detrimen-
tal to the development of transplantation tolerance and may ultimately
oppose the effect of B7 blockade. This double-edged nature of B7
blockade suggests that an agent that would selectively block CD28
and not CTLA-4 may be more adapted to maneuvers aimed at
inducing tolerance.
Despite the clinical promise of anti-CD28–targeted therapy, antago-
nists of human CD28 are currently unavailable. So far, all antibodies
described as reacting against human CD28 are agonists. Indeed, CD28 is
a homodimeric receptor6whose degree of cross-linking is implicated in
signal transduction through its association with phosphatidylinositol
3-kinase (PI3-kinase) via the cytoplasmic domain.7However, monova-
lent fragments can inhibit CD28/B7 interactions without stimulating
CD28,7although they cannot be used therapeutically in vivo because of
their rapid elimination from the body. Extension of the half-life of
antibody fragments has been achieved by in vitro conjugation to one or
more molecules of polyethylene glycol8,9 or by molecular fusion with
serum albumin.10 Our group has recently investigated strategies aimed at
developing monovalent anti-CD28 reagents by cloning antigen-
combining portions and fusing them with monovalent carrier molecules.
In this paper we demonstrate that an scFv recombinant fragment from a
monoclonal antibody (mAb) directed against human CD28 (CD28.3
mAb11) fused with the monovalent and long-lived human serum protein
␣1-antitrypsin (HaaT), retains the avidity and the biologic properties of
the original monovalent Fab antibody fragment. In addition, this genetic
fusion extends the serum half-life of the recombinant protein further
than that of HaaT.
Materials and methods
Cells and antibodies
Mouse antihuman CD28 hybridoma CD28.1 (IgG1), CD28.2 (IgG1),
CD28.3 (IgG1), CD28.4 (IgM), CD28.5 (IgG1), and CD28.6 (IgG2a) and
mouse L fibroblasts expressing human CD80 (LB7 cells) were kindly
provided by D. Olive (INSERM U119).11,12 CD28.1, CD28.2, CD28.3, and
From the Institut de Transplantation et de Recherche en Transplantation, Institut
National de la Sante´ et de la Recherche Me´dicale (INSERM) U437, Centre
Hospitalier Universitaire (CHU) Hotel Dieu, Nantes, France; INSERM U463, Institut
de Biologie, Nantes, France; and Sangstat Europe, Lyon, France.
Submitted August 13, 2002; accepted March 9, 2003. Prepublished online as
Blood First Edition Paper, March 20, 2003; DOI 10.1182/blood-2002-08-2480.
Supported in part by the “Fondation Progreffe,” by the Post-Genome Program,
grant 109 from the French government (Ministe`re de l’Education Nationale de
la Recherche et de la Technologie [MENRT]), and by the association “Vaincre
La Mucoviscidose.”
Reprints: Bernard Vanhove, ITERT, INSERM U437, CHU Hotel Dieu, 30 Bld
Jean Monnet, 44093 Nantes, France; e-mail: bvanhove@nantes.inserm.fr.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2003 by The American Society of Hematology
564 BLOOD, 15 JULY 2003 䡠VOLUME 102, NUMBER 2
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CD28.5 mAbs share a common target epitope, whereas CD28.4 and
CD28.6 mAbs bind to 2 other epitopes.11 Fab fragments were prepared
using the Immunopure IgG1 Fab preparation kit (Pierce, Rockford, IL).
In vivo infusion and blood sampling in macacus fascicularis
This part of the study was performed by Biomatech (Lyon, France) in
Cynomolgus maccaca fascicularis. Two infusions of 8 mg/kg antibodies
were performed on days 0 and 2.
Amplification and assembly of VHand VLby PCR
mRNA from CD28.3 hybridoma was prepared and retrotranscribed, and
cDNA for VHand VLwas amplified using primers for mouse V-gene
amplification of the mouse ScFv module of the recombinant phage antibody
system (Pharmacia Biotech, Guyancourt, France), according to the manufac-
turer’s instructions. The amplified V and H genes were cloned into a TA
vector (Invitrogen, Cergy Pontoise, France), followed by DNA sequencing.
The cloned VH gene was modified by polymerase chain reaction (PCR)
amplification by introducing a 5⬘BamHI site (primer sc28.3-5: 5⬘-
ATATGGATCCGTCAAGCTGCAGCAGTCAGG-3⬘)anda3⬘(SGGGG)3
synthetic linker primer (primer: 5⬘-GACTGGGTCATCTGGATGTCCGA-
TCCGCCACCGCCAGAGCCACCTCCGCCTGAACCGCCTCCACC-
TGAGGAGACGGTGACCATGG-3⬘). The VL gene was then assembled to
the modified VH gene by PCR using a 3⬘primer (primer sc28.3-3:
5⬘-ATATCTCGAGTTATTAGAATTCCCGTTTTATTTCCAGCTTGG-3⬘)
introducing EcoRI and XhoI cloning sites and using the modified VH gene
as the 5⬘primer. Assembled VH and VL genes were then reamplified using
a3⬘primer introducing a TVAAPS peptidic linker originating from the
natural Fab hinge region of immunoglobulins and EcoRI and XhoI restric-
tion sites (primer sc28.3-TV5: 5⬘-ATATCTCGAGTTATTAGAATTCA-
GATGGTGCAGCCACAGTCCGTTTTATTTCCAGCTT-3⬘). The con-
struct was then sequenced to verify its identity with the original VH and VL
genes. The construct was further subcloned into the BamHI/XhoI sites of
the pSecTag2B eukaryotic expression plasmid (Invitrogen), 3⬘to the Ig
leader and introduced into the EcoRI/EcoRV sites of the pIg6 prokaryotic
expression plasmid (a gift from Dr Jean-Charles Tellier, University of
Nantes, Nantes, France) 3⬘to the OmpA leader.
Genetic fusion of scFv with HaaT
The cDNA for human ␣1-antitrypsin (corresponding to the GenBank
sequence no. X01683) was a gift from Dr D. Favre (INSERM ERM-0105,
Nantes, France). Amino acids 48 to 425 were amplified by PCR with
primers that add flanking EcoRI restriction sites (5⬘primer: 5⬘-
ATATGAATTCAACAAGATCACCCCCAAC-3⬘;3⬘primer: 5⬘-ATAT-
GAATTCTTTTTGGGTGGGATTCAC-3⬘). The modified gene was in-
serted into the EcoRI site of the pSecTag2B-scFv28.3, 3⬘and into the
EcoRVsite of the pIg6-scFv28.3 in frame with the scFv cDNA.
Expression in Escherichia coli
JM83 cells were transformed with the pIg6-scFv28.3-HaaT plasmid.
Transfectants were allowed to grow in Luria-Bertani (LB) medium
containing 100 g/mL ampicillin until an OD600 of 0.5 was reached. Then,
1 mM isopropyl thiogalacto-pyranoside (IPTG) was added to the cultures
and the cells were incubated at 25°C for 3 hours. The cells were then
centrifuged and the periplasmic extracts were collected after hypotonic
lysis and ultracentrifugation at 10 000g.
Transient transfection of Cos cells and purification
of scFv28.3-HaaT
Cos cells were transfected using the Fugene lipofection kit (Roche
Diagnostics, Basel, Switzerland) according to the manufacturer’s instruc-
tions. Cultures were maintained for 3 days at 37°C, divided one third, and
put back into culture for an additional 3 days, after which time the medium
was collected. Supernatants were passed through NiNTA Sepharose col-
umns (Amersham Pharmacia Biotech, Saclay, France) at a rate of
0.5 mL/min. The columns were rinsed with 0.02 M imidazole in phosphate-
buffered saline (PBS) and proteins were eluted with 0.2 M imidazole in
PBS and then dialyzed extensively against PBS at 4°C.
Enzyme-linked immunosorbent assay
Recombinant human CD28-Fc (R&D Systems, Abingdon, United King-
dom) was used at 1 g/mL in borate buffer (pH 9.0) to coat 96-well
microtiter plates (Immulon, Chantilly, VA) overnight at 4°C. Reactive sites
were blocked with 5% skimmed milk in PBS for 2 hours at 37°C and
supernatants or purified material were reacted for 2 hours at 37°C. Bound
scFV28.3-HaaT was revealed with successive incubations (1 hour, 37°C)
with rabbit antihuman ␣1-antitrypsin antibodies (1:500 dilution; Dako,
Trappes, France) and horseradish peroxidase (HRP)–conjugated donkey
antirabbit Ig antibodies (1:500 dilution; Jackson ImmunoResearch Labora-
tories, Bar Harbor, ME). Control scFv-M13 was revealed with an HRP-
conjugated anti-M13 antibody (Amersham Pharmacia Biotech), as de-
scribed.13 Bound antibody was revealed by colorimetry using the ABTS
substrate (Roche, Mannheim, Germany) read at 405 nm.
EliSpot assay
CD4⫹T cells (105) were mixed with irradiated allogeneic peripheral blood
mononuclear cells (PBMCs; 105) in RPMI 1640 medium (Sigma, St
Quentin Fallavier, France) supplemented with glutamine, nonessential
amino acids, sodium pyruvate, antibiotics, and 10% heat-inactivated fetal
calf serum (FCS) and plated in quadruplicate into microtiter plates coated
with anti–interferon-␥(anti–IFN-␥) mAb (AID, Strassberg, Germany).
After a 24-hour incubation at 37°C, wells were washed, incubated with a
biotin-labeled secondary anti–IFN-␥mAb, and revealed according to the
manufacturer’s instructions. Spots were counted with an EliSpot Reader
System (AID).
Detection of scFv28.3-HaaT by Western blotting
scFv28.3-HaaT fusion proteins were electrophoresed in 10% polyacryl-
amide gels and blotted onto nitrocellulose membranes. Blots were revealed
with an anti–c-myc mAb (prepared in our laboratory from 9E10 hybridoma)
and an HRP-conjugated donkey antimouse Ig antibody (Jackson Immuno-
Research Laboratories) and developed by chemiluminescence (Amersham
Pharmacia Biotech).
Adhesion assay
Adhesion assays were performed as previously described12 except that
Jurkat T cells were loaded with calcein AM (Molecular Probes, Eugene, OR)
instead of being radiolabeled. Jurkat T cells (6 ⫻104) were added to a
monolayer of 104LB7 cells (expressing human CD80) and incubated at
37°C for 1 hour.Adherent cells were analyzed by fluorometry (excitation at
485 nm and emission at 430 nm).
Cytofluorometry
Jurkat T cells or U937 cells were incubated for 1 hour at 4°C with
supernatants from control or transfected Cos cells, or with purified proteins.
Bound scFv28.3-HaaT fusion proteins were detected with a rabbit antihu-
man ␣1-antitrypsin antibody (1:200 dilution; Dako) and a fluorescein
isothiocyanate (FITC)–conjugated donkey antirabbit Ig antibody (dilution
1:200; Jackson ImmunoResearch Laboratories) for 30 minutes at 4°C. Cells
were then analyzed by fluorescence-activated cell sorting (FACS). Monkey
PBMCs were prepared by centrifugation of blood on Ficoll (Pharmacia
Biotech) and incubated on ice in PBS/0.1% NaN3with a mixture of
anti–CD2-phycoerythrin (PE) mAb (Immunotech, Marseille, France) and
FITC-labeled anti-CD28.6 mAb (prepared in our laboratory) for revelation
of CD28 on T cells from monkeys treated with CD28.1, CD28.2, CD28.3,
and with a mixture of anti–CD2-PE plus FITC-labeled anti-CD28.2 mAb
(prepared in our laboratory), for revelation of CD28 on T cells from
monkeys treated with CD28.6.
Mixed lymphocyte reactions
Human PBMCs were seeded in triplicate at a final concentration of
105cells/well in RPMI 1640 medium (Sigma) supplemented with glu-
tamine, nonessential amino acids, sodium pyruvate, antibiotics, and
ANTI-CD28 scFv–␣1-ANTITRYPSIN FUSION ANTIBODY 565
BLOOD, 15 JULY 2003 䡠VOLUME 102, NUMBER 2
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10% heat-inactivated FCS and cultured for 5 days with 105allogeneic
PBMCs irradiated at 30 Gy. Proliferation was measured by 3H-thymidine
incorporation after 16 hours of incubation with 10⫺6Ci (37 KBq).
Alternatively, CD4⫹T cells prepared from PBMCs using the Lymph-Kwik
Th system (One Lambda, Canoga Park, CA) were used as responding cells.
The results were expressed as specific counts per minute per triplicate: (cpm
assay ⫺cpm stimulating cells alone ⫺cpm responding cells alone).
Capping and immunofluorescence microscopy
Capping experiments were performed using a modified procedure of
Gassmann et al.14 Jurkat T cells were incubated either at 37°C for 1 hour
with mouse mAb CD28.3 (10 g/mL), CD28.3 Fab fragments, scFv28.3-
HaaT, or scFv28.3 recombinant proteins in RPMI 1640 medium (Sigma)
containing 10% FCS, or at 0°C for 1 hour in the same medium containing
0.1% NaN3. After washing, cells were immediately fixed in 0.5%
paraformaldeyde (wt/vol) for 30 minutes at room temperature, centrifuged
onto glass slides, and allowed to dry overnight. Slides were immunolabeled
with either FITC-labeled goat antimouse IgG antibodies (Jackson Immu-
noResearch Laboratories), rabbit antihuman ␣1-antitrypsin followed by
FITC-labeled donkey antirabbit IgG antibodies, or mouse anti–c-myc 9E10
mAb followed by goat antimouse IgG antibodies. After washing, the slides
were mounted in Moviol and examined using the ⫻63 immersion lens of a
Zeiss microscope equipped for epifluorescence.
Biosensor affinity measurement
CD28-Fc recombinant protein (R&D Systems) was immobilized onto a
CM5 sensor chip. Analysis was performed with a BIAcore 2000 (Biacore,
Paris, France). Antibodies or recombinant proteins were applied at concen-
trations ranging from 0.67 to 12.5 nM with a flow rate of 30 L/min. An
association period of 300 seconds was followed by a dissociation period of
600 seconds. Analyses and binding constants were deduced using the
BIAevaluation software (Biacore).
Pharmacokinetic analysis of the conjugate
Proteins (30-60 g) were labeled with 100 g iodogen and 3.10⫺6Ci (110
KBq)/mg 125I as described.15 Free iodine was eliminated by chromatogra-
phy on Sephadex G-25M using a PD10 column (Amersham Pharmacia
Biotech). Via thin layer chromatography with 10% trichloroacetic acid it
was determined that 91% to 96% of the radioactivity was associated with
the proteins. The specific activity of each preparation was calculated from
estimates of protein concentration (measured by absorption at 280 nm) and
radioactivity and was typically in the range of 0.1 to 0.3 ⫻10⫺6Ci (4 to 10
KBq)/g. The radiolabeled samples were used directly after labeling.
Thirty or 60 g125I-labeled protein in PBS was injected into the tail vein of
10-week-old male Balb/c mice. Blood samples were collected periodically
from the orbital sinus. Blood samples were weighed and radioactivity
detected using a gamma counter (Canberra Packard Topcount, Mississauga,
ON, Canada). Percent injected dose (%ID) was calculated for each
individual mouse and expressed as %ID/mL total blood volume. The data
were analyzed by Siphar software (Simed, Utrecht, The Netherlands) with
the use of a 2-compartment model.
Results
The antihuman CD28 mAbs tested do not down-regulate
their target in vivo
In the rat species, one mAb is known to induce CD28 capping and
complete down-regulation in vivo.16 Thus, we first screened a
series of antihuman CD28 mAbs for cross-reaction with macaques
to investigate a possible down-regulating effect in vivo. Cross-
reacting mAbs (CD28.1, CD28.2, CD28.3, and CD28.6 mAb12)
were then injected intravenously into macacus fascicularis and
CD28⫹CD2⫹cell counts and CD28 expression intensity were
monitored in the blood (Figure 1). No reduction in CD28 expres-
sion intensity was noted. Conversely, it was increased, together
with an increase in the mean forward scatter (FSC) signal in flow
cytometry (not shown). CD28⫹cell counts were unchanged
(CD28.3) or reduced by 30% for CD28.2, 50% for CD28.1, and
60% for CD28.6.
Selection of an anti-CD28 Fab fragment inhibiting
CD28/B7 interaction
In the absence of modulating activity of the anti-CD28 antibodies
tested, we screened the CD28 blocking ability of their monovalent
fragments. Fab fragments were prepared and tested by flow
cytometry, inhibition of adhesion of Jurkat T cells to CD80-
transfected fibroblasts, and inhibition of mixed lymphocyte reac-
tions (MLRs; Figure 2). Reactivity was compared with initial
divalent IgG molecules. As determined by FACS, the CD28.1 and
CD28.2 antibodies reduced their binding activity at least 100-fold
after digestion into monovalent Fab fragments. In contrast, the
reduction in reactivity of Fab fragments from CD28.3 was below a
factor of 10 (Figure 2A). An evaluation of binding parameters
performed by Biacore confirmed that Fab monovalent fragments
from the CD28.3 mAb had a higher association rate (Ka, 9650/
mole-second) than Fab fragments from the CD28.2 and CD28.1
mAb (Ka, 920/mole-second and below the detection limit, respec-
tively). Fab fragments from the CD28.3 antibody fully inhibited the
adhesion of CD28⫹T cells to CD80-expressing cells (Figure 2B),
whereas fragments from the other antibodies only partially reduced
or had no effect on the adhesion of T cells. In MLRs, anti-CD28
antibodies are expected to costimulate (cross-linking of CD28) or,
alternatively, to reduce T-cell proliferation (CD28/B7 interaction
inhibition). In contrast, monovalent fragments are expected to
inhibit T-cell proliferation. The anti-CD28 antibodies in their
dimeric configuration showed no inhibition of proliferation in
MLRs and some of them promoted T-cell proliferation at high
doses (CD28.1, Figure 2C). Monovalent Fab fragments from the
CD28.3 antibody showed a dose-response inhibition of prolifera-
tion, whereas Fab fragments from the other antibodies did not,
probably as a result of their lower binding avidity. Finally,
observations by immunofluorescence revealed that the divalent
anti-CD28 mAb CD28.3 induced a capping of CD28 molecules
on T-cell surfaces at 37°C, whereas monovalent Fab fragments
did not.
Construction and expression of sc28.3HaaT from
the CD28.3 hybridoma
VHand VLchain fragments were amplified from CD28.3 hybrid-
oma cDNA and assembled using a (GGGGS)4linker. The nucleo-
tide sequence of this scFv was registered in GenBank under the
Figure 1. Assessment of a possible in vivo modulating activity of anti-CD28
antibodies. Anti-CD28 mAb (8 mg/kg) was infused intravenously into macacus fascicu-
laris on days 0 and 2. Blood samples were collected before injection and every 2 days for
10 days. PBMCs were then analyzed by flow cytometry after gating of CD2⫹cells. Mean
fluorescence intensity (MFI) of CD28 expression (A) as well as percent of CD2⫹CD28⫹
double-positive cells within mononuclear cells (B) are represented; 䉬indicates CD28.1; Œ,
CD28.2; f, CD28.3; F, CD28.6; and E, isotype control. Similar data were found in 2
monkeys per antibody, one of them being shown here.
566 VANHOVE et al BLOOD, 15 JULY 2003 䡠VOLUME 102, NUMBER 2
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accession no. AF451974. To load the scFv with a carrier molecule
that would increase the molecular weight of the complex and give
an extended bioavailability in vivo, we selected a series of
monovalent macromolecules that are abundant in plasma. We first
fused the coding sequence of serum albumin to the C-terminus of
the scFv because this had already been used successfully in a rabbit
system.17,18 In the meantime, feasibility was reported with human
albumin.10 In our hands, the genetic fusion of scFv 28.3 with serum
albumin resulted in the production of a misfolded, insoluble
complex without antibody activity in prokaryotic and eukaryotic
expression systems (not shown). In contrast, the genetic fusion of
scFv28.3 with a TVAAPS Fab hinge sequence and with amino
acids 53 to 425 (referring to GenBank accession no. K01396) of the
human ␣1-antitrypsin resulted in the production of a soluble and
active molecule, as illustrated in Figure 3. The scFv28.3-HaaT
cDNA was first subcloned into the EcoRV/XhoI sites of the pIg6
prokaryotic expression plasmid (a generous gift from Dr Jean-
Charles Tellier, University of Nantes, Nantes, France) 3⬘to an
ompA signal sequence, enabling accumulation of recombinant
proteins in the periplasm of JM83 E coli cells, under the control of
the lac promoter (Figure 4A). However, periplasmic extracts
contained only low levels of soluble protein. The scFv28.3-HaaT
cDNA was subsequently subcloned into the BamHI/EcoRI sites of
the pSecTag2B pCMV-based eukaryotic expression plasmid, en-
abling a fusion at the C-terminus with the Igleader sequence and
at the N-terminus with a 6-HIS tag. After transfection in Cos cells,
anti-CD28 activity was found in the supernatant and the recombi-
nant protein was purified on NiNTAaffinity columns with a yield of
1.5 mg/L supernatant (Figure 4B-D).
Detection of sc28.3HaaT binding activity by flow cytometry,
ELISA, and biosensor
The binding of the scFv28.3-HaaT construct was then analyzed by
flow cytometry using CD28⫹Jurkat T cells and control U937 cells.
The binding activity was tested by adding the scFv28.3-HaaT
construct to Jurkat cells followed by staining with a rabbit
anti–␣1-antitrypsin antibody and an FITC-conjugated donkey anti-
rabbit Ig antibody as a third-step reagent and analysis by flow
cytometry. The results indicated that scFv28.3-HaaTbound specifi-
cally to CD28 expressed on Jurkat cells with no binding to U937
cells, which do not express CD28 (Figure 4). Binding was also
confirmed in an enzyme-linked immunosorbent assay (ELISA),
performed by adding various concentrations (25-400 ng/mL) of
scFv28.3-HaaT to plates coated with recombinant CD28-Fc mol-
ecules (Figure 4C). Negative controls where plates were coated
with irrelevant molecules (gelatin, bovine serum albumin, ovalbu-
min) gave no signal (not shown). In addition, an unrelated scFv
(fused with the G3p protein from M13 bacteriophage) and ␣1-
antitrypsin alone did not bind to recombinant CD28-Fc. From this
analysis, the inhibitory concentration of 50% (IC50) was estimated
as being 0.4 nM. The avidity of the scFv28.3-HaaT was evaluated
via a biosensor analysis. Adetector chip was coated with CD28-Fc
Figure 2. Characterization of the Fab fragments of anti-CD28 antibodies.
(A) CD28⫹Jurkat T cells were incubated with the indicated amounts of anti-CD28
mAb or their Fab fragments and stained with antimouse-FITC antibody. MFI was
determined by flow cytometry. (B) Inhibition of CD28/CD80 interactions was as-
sessed by incubating calcein-labeled CD28⫹Jurkat T cells on monolayers of mouse
fibroblasts expressing human CD80 in the presence of saturating doses of anti-CD28
Fab fragments or control Fab fragments. After washing, adherent cells were collected
and associated fluorescence was measured by fluorometry. (C) PBMCs (105) were
mixed with 105allogeneic irradiated PBMCs in the presence of the indicated amount
of anti-CD28 mAb and Fab fragments or with 10 g/mL control antibody. Proliferation
on day 5 was measured by addition of 10⫺6Ci (37 KBq) 3H-thymidine and
measurement of incorporation after 16 hours; 䉬indicates IgG CD28.1; Œ, IgG
CD28.2; f, IgG CD28.3; 䉫, Fab CD28.1; ‚, Fab CD28.2; 䡺, Fab CD28.3; and ⫻,
isotype control. The results shown are representative of more than 3 independent
experiments.
Figure 3. Construction for expression of scFv28.3-HaaT in eukaryotic cells.
cDNA fragments coding for variable regions of VHand VLchains of the CD28.3
antibody were assembled with a (GGGGS)4linker. AcDNA coding for the TVAAPS hinge
peptide from the IgG heavy chain was added 3⬘and fused in frame with a cDNA coding for
amino acids 53 to 425 of HaaT cDNA. The complex was then introduced into the
pSecTag2B expression plasmid by adding an Igleader to the N-terminus and a stretch
sequence containing c-myc and 6HIS flag plus a stop codon to the C-terminus.
Figure 4. Expression and activity of scFv28.3-HaaT. (A) Prokaryotic expression:
10 g protein extract from the periplasmic, soluble (S), and insoluble (I) fractions of
E coli JM83 expressing scFv28.3-HaaT were resolved under reducing conditions on 10%
polyacrylamide electrophoresis gels and blotted onto nylon membranes. Recombinant
proteins were revealed by incubation of the membranes with HRP-conjugated anti–c-myc
antibody and by enhanced chemiluminescence (ECL). (B) Fifty nanograms scFv28.3-
HaaT produced in Cos cells, under native (N) or reducing (R) conditions, was analyzed by
Western blotting as described in panel A. Molecular size markers (kDa) are shown on the
left. (C) CD28⫹Jurkat T cells were incubated with supernatant from transfected Cos cells
containing 2 g/mL scFv28.3-HaaT (black histogram, first row) or with supernatant from
control Cos cells (white histogram, second row), revealed with anti-HaaT antibody plus
FITC-conjugated donkey antirabbit Ig antibody (DAR) and analyzed by cytofluorometry.
CD28⫺U937 cells were analyzed in parallel with supernatant containing scFv28.3-HaaT
(dark gray histogram, third row) or with control supernatant (light gray histogram, last row).
(D) The reactivity of Cos-produced scFv28.3-HaaT was examined by ELISA. CD28-Fc
molecules were coated onto microtiter plates. Purified scFv28.3-HaaT at different dilutions
in PBS-Tween was added and incubated for 1 hour at 37°C. After washing, bound
scFv28.3-HaaT molecules were revealed with a rabbit anti-HaaT antibody followed by a
peroxidase-conjugated DAR as a third-step reagent. Colorimetric analysis was performed
after reaction of ABTS with peroxidase. X indicates signal without sc28.3-HaaT; 䉬and 䉫,
signals obtained with incubations of HaaT alone and with an irrelevant scFv-M13 fusion
antibody, revealed with a peroxidase-labeled anti-M13 antibody. Data are presented as
means of triplicates.
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at high and low densities. Recombinant proteins were applied at
concentrations ranging from 0.67 to 12.5 nM with a flow rate of 30
L/min, an association period of 300 seconds followed by a
dissociation period of 600 seconds. The analysis of the avidity,
deduced from the BIA evaluation software from 6 determinations
at different doses, revealed a Kd(M) of 7.1 ⫾1.8 10⫺9. Similar
values were obtained in 3 independent analyses using either Cos
cell supernatants or purified material. A similar analysis of the
initial CD28.3 Fab fragments revealed avidity in the same order of
magnitude (Kdof 8.5 ⫻10⫺9).
Detection of sc28.3HaaT aggregation by Western blotting
and absence of capping induction
Recombinant scFv antibodies often tend to aggregate or form
dimers in solution19; thus, even after the fusion with a monovalent
carrier such as HaaT, our scFv28.3 may dimerize and induce
clustering of CD28 molecules on T-cell membranes. To character-
ize the nature of the recombinant protein produced in Cos cells,
supernatants were analyzed by electrophoresis under native and
denaturant conditions. After reduction and denaturation in sodium
dodecyl sulfate (SDS), the protein had an apparent molecular mass
of 90 kDa (Figure 4B). The fact that the same protein produced in
E coli had an apparent molecular mass of 78 kDa (Figure 4A)
indicated that approximately 12 kDa could be attributed to
glycosylation after production in eukaryotic cells. Under native
conditions, without SDS, the protein was mostly monomeric with
an apparent molecular weight similar to that of the denatured
protein. Material with a molecular weight of approximately 180
kDa was also detected, indicating the presence in the solution of
dimeric proteins (Figure 4). The capping assay, however, revealed
that the scFv28.3-HaaT produced in Cos cells, unlike divalent IgG
molecules, did not induce formation of CD28 clusters on T-cell
membranes, and neither did an scFv28.3 nonfused to the HaaT
(Figure 5). This suggests that the dimeric proteins contained in the
preparation could not stimulate CD28 either due to their associa-
tion being too weak or due to competition for binding with
quantitatively dominant monovalent fragments.
scFv28.3-HaaT modulates T-cell allogeneic responses
Our group20 has previously demonstrated that monovalent Fab
fragments from CD28 antibodies reduce proliferation of alloreac-
tive T cells but are ineffective in modulating proliferation of Tcells
stimulated by heterologous antigens presented in the context of self
major histocompatibility complex (MHC) molecules. To investi-
gate whether scFv28.3-HaaT could similarly reduce T-cell allore-
sponses, we first checked whether this construct inhibited CD28/B7
interactions and inhibited an MLR. As shown in Figure 6,
scFv28.3-HaaT dose dependently inhibited adhesion of Jurkat T
cells to CD80-expressing fibroblasts (Figure 6A) and reduced
proliferation of allogeneic T cells in vitro (Figure 6B). CD28
blockade also reduced the frequency of IFN-␥–producing cells in
MLRs 24 hours after stimulation (Figure 6E).
To determine whether the reduced alloreactivity in MLRs with
CD28 blockade was due to immune ignorance, anergy, or deletion
of alloreactive T cells, we measured secondary responses of cells
initially primed in the presence of sc28.3-HaaT. No modifications
in the frequency of IFN-␥–secreting cells (Figure 6E) or in
proliferation (Figure 6C) were noted after secondary stimulation,
indicating that alloreactive cells had not been deleted in the primary
stimulation. Inhibiting CD28 in primary MLRs with Fab fragments
from the CD28.3 antibody instead of sc28.3HaaT led to similar
conclusions (data not shown). However, proliferation in recall
stimulation was maximal on day 2, whereas it peaked on day 3
when third-party stimulating PBMCs were used (Figure 6D),
indicating that donor-specific proliferating cells had been primed in
the primary reaction despite CD28 blockade.
Pharmacokinetics in murine plasma
To address the question of whether the genetic fusion of the scFv
fragment with HaaT actually resulted in an extended half-life in
vivo, we followed the distribution in mice of iodinated scFv28.3-
HaaT, Fab fragments from the CD28.3 antibody, and HaaT. Figure 7
shows that the distribution and elimination half-lives of the Fab
fragments were rapid, as expected for a 50-kDa protein (14.5 ⫾3.3
minutes and 7.4 ⫾1.7 hours, respectively). HaaT distribution was
similar (17.4 ⫾10.8 minutes) but it was eliminated much more
slowly (10.8 ⫾0.6 hours) despite having a slightly lower molecu-
lar weight (46.6 kDa). The distribution and the elimination
half-lives were significantly extended for the scFv28.3-HaaT
recombinant molecule (26.6 ⫾2.3 minutes and 11.8 ⫾0.5 hours,
respectively). Finally, an analysis of the biodistribution of the
labeled molecules 48 hours after injection confirmed the accumula-
tion of scFv28.3-HaaT in the reticuloendothelial compartment
(liver, kidneys, intestine, spleen; not shown), whereas Fab frag-
ments were lost, probably through renal elimination. Furthermore,
the distribution volume increased from 3 mL for Fab and HaaT to 8
mL for scFv28.3-HaaT, indicating that the conjugate left the blood
to penetrate organs and tissues more rapidly.
Discussion
Antibody-induced modulation of CD28 prolongs allograft survival
in rats16 and administration of anti-CD28 Fab fragments reverses
the induction of experimental autoimmune encephalomyelitis21 and
uveoretinitis22 in mice. In a murine model of graft-versus-host
Figure 5. Immunofluorescent analysis of antibody-induced capping of CD28.
Jurkat T cells expressing CD28 were incubated in culture medium with 2 g/mL
divalent CD28.3 IgG antibody (A-B) or 2 g/mL CD28.3 Fab fragments, with 2 g/mL
scFv28.3-HaaT (E) or with 50% Cos cell supernatant containing approximately
4g/mL scFv28.3 (D). Cells were incubated for 1 hour at 0°C (A) or at 37°C (B-E),
washed in ice-cooled PBS containing NaN3, and centrifuged onto microscope slides.
After fixation with 0.5% paraformaldehyde (PFA), CD28 molecules were revealed
using FITC-labeled CD28.6, a mAb reacting against an epitope on CD28 other than
CD28.3.11 Slides were observed by fluorescence microscopy, and magnification is
⫻63 for all panels.
568 VANHOVE et al BLOOD, 15 JULY 2003 䡠VOLUME 102, NUMBER 2
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disease (GVHD), the selective blockade of CD28 was reported as
being more immunosuppressive than blocking B7 with CTLA-4/Ig.23
CD28 blockade also synergizes with cyclosporine in the rat
resulting in immune tolerance in transplantation.24 In CD28⫺/⫺
mice, immune responses to viral antigens or autoantigens are
impaired,25,26 whereas responses to exogenous antigens remain
normal.27 In rats treated with the modulating anti-CD28 antibody
JJ319, allogeneic T-cell responses are blunted but responses to
exogenous antigens are unaffected.20 Collectively, these data
suggest that T-cell responses to autoantigens or alloantigens are
more dependent on costimulation through CD28 than T-cell
responses to exogenous antigens. Thus, the blockade of CD28 may
represent an immunosuppression for the selective inhibition of
pathologic T cells in autoimmunity and in transplantation without
inhibition of other, protective, T-cell responses.
Costimulation through CD28 in conjunction with a triggering of
the T-cell receptor (TCR) leads to high-level production of
interleukin 2 (IL-2) and provides an essential survival signal for T
cells. Signals through CD28 also regulate T-cell cycle entry and
progression through the G1phase in an IL-2–independent man-
ner,28 resulting in the activation of cyclines.29 At the molecular
level, a selective blockade of CD28 with an unmodified CTLA-
4/B7 interaction would theoretically allow signals through the TCR
and CTLA-4 to be transmitted to antigen-challenged T cells.
Resting T cells express a relatively low level of CTLA-4; however,
when engaged on resting T cells, CTLA-4 transmits a signal that
blocks transition from G0to G1.30 Once activated, T cells further
increase their membrane expression of CTLA-4, which transmits a
signal leading to a Fas-independent cell death,31 contributing to the
down-regulation of the immune response. Therefore, allowing
CTLA-4/B7 interactions to proceed in the absence of CD28
costimulation will combine the effect of the lack of costimulation
with a cell growth arrest, or a clonal deletion, depending on
whether blockade is initiated before or after the initiation of T-cell
activation. In addition, as agonistic anti-CD28 antibodies promote
Th1 differentiation in vitro,32 inhibition of CD28 is likely to modify
the Th1/Th2 balance.
This study was undertaken to obtain an anti-CD28 antagonist
that would allow an in vivo investigation into whether recognition
of alloantigens or autoantigens by CD4⫹T cells in the absence of
CD28 costimulation, but with an unmodified CTLA-4/B7 interac-
tion, induces an immune regulation. The available anti-CD28
antibodies appear not to be antagonistic in vivo in monkeys, as
shown by the absence of CD28 down-regulation. On the contrary, 3
of the 4 antibodies we tested induced a strong up-regulation of
CD28 expression on CD2⫹cells (Figure 1), a phenomenon that has
previously been associated with T-cell activation.33
In the absence of identified modulating anti-CD28 antibodies,
we constructed a recombinant monovalent antibody from the
hybridoma of CD28.3. This clone was chosen because (1) it retains
its high affinity even after digestion into monovalent Fab frag-
ments, (2) it prevents CD28 from binding CD80, (3) its Fab
fragments dose dependently reduce T-cell proliferation in MLRs,
and (4) it retains its binding properties after recombinant expres-
sion in scFv format. The extension of its molecular weight through
a genetic fusion with ␣1-antitrypsin resulted in a soluble macromol-
ecule with an extended bioavailability and a preserved activity.
␣1-Antitrypsin is an abundant (3 g/L) monovalent plasma protein
that has already been used for genetic fusion.34 In humans it has a
half-life of about 8 days,35 although its half-life is much shorter in
rodents.36 To demonstrate the immunosuppressive potential of
scFv28.3-HaaT, we reproduced the biologic activities of the
CD28.3 antibody Fab fragments in vitro. This included a preserved
avidity for CD28, the absence of induction of CD28 capping on
target cells at 37°C, a competition with CD80 for binding to CD28,
and a dose-dependent inhibition in MLRs. In addition, sc28.3HaaT
reduced the frequency of allogeneic cells producing IFN-␥24 hours
after the initiation of an MLR.
Figure 6. Biologic activity of scFv28.3-HaaT in a binding assay and MLRs.
(A) CD28⫹Jurkat T cells stained with calcein AM dye were incubated on a monolayer
of CD80-expressing fibroblasts, as in Figure 2B, in the presence of dilutions of
supernatant from Cos cells transfected with pSecTag2B-scFv28.3-HaaT (f)or
pSecTag2B-scFv28.3 (䉬), in which recombinant protein was quantified by ELISA.
Control-transfected supernatant (E) was used at similar dilutions. The percentage of
inhibition of adhesion after washing was evaluated by luminescence. (B) CD4⫹T cells
from healthy volunteers were incubated in microtiter plates with irradiated allogeneic
PBMCs for 5 days in the presence of dilutions of purified sc28.3-HaaT. Proliferation was
measured by incorporation of 3H-thymidine for the last 16 hours of culture; m indicates
proliferation obtained after addition of a control mAb (mouse IgG1) to the cultures. Data
shown here are means ⫾SDs of triplicates and are representative of 5 experiments.
(C-E) MLRs similar to panel B were performed with (filled bars and symbols) or without
(empty bars and symbols) saturating amounts of sc28.3-HaaT.After 5 days an aliquot of
cells was assessed for proliferation (“primary”in panel C) and for frequency of IFN-␥
secretion (“primary”in panel E). The remaining cells were washed and cultured for a resting
period of 7 days, after which time cells were collected and restimulated (“secondary”) with
the same (C, circles in panels D and E) or third-party (triangles in panel D) stimulatory cells.
Proliferation was measured 2 (C-D), 3, and 4 (D) days later. The frequency of IFN-␥–
secreting cells during secondary stimulation was measured 24 hours after restimulation
(“secondary”in panel E).
Figure 7. Pharmacokinetics of 125I-labeled HaaT, CD28.3Ab Fab fragments, and
scFv28.3-HaaT in mice. Purified sc28.3-HaaT, HaaT, and CD28.3 Fab fragments
were labeled with 125I and injected intravenously into mice. Blood samples were
collected periodically and radioactivity was measured with a gamma counter. %ID
indicates percent injected dose; f, scFv28.3-HaaT; E, HaaT; and 䉬, Fab. For each,
n⫽3. Data are presented as means ⫾SDs.
ANTI-CD28 scFv–␣1-ANTITRYPSIN FUSION ANTIBODY 569
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Restimulation of T cells primed in the presence of the CD28
inhibitor resulted in intact secondary responses, suggesting that one
mechanism of action of sc28.3-HaaT in MLRs that results in an
inhibition of T-cell proliferation and activation is related to immune
ignorance rather than anergy or alloreactive cell deletion. However,
the earlier kinetics of T-cell proliferation following stimulation by
isogeneic antigen-presenting cells (APCs) as compared with third-
party APCs in secondary responses (proliferation peak on day 2
instead of day 3) suggests that T cells were primed in the primary
reaction whether or not CD28 had been blocked. Additional
mechanisms to ignorance might, however, play a role in vivo
because a short-course administration of Fab fragments or of a
modulating anti-CD28 mAb in rodents increased apoptosis of
alloreactive cells20 and, in the long term, improved disorders
related to alloreactivity23 and autoimmunity.21,22 Thus, deletion of
alloreactive cells or the development of regulatory mechanisms
might also result from selective CD28 blockade. The possibility
that a selective blockade of CD28 in humans could improve
disorders such as GVHD or allograft survival, however, remains to
be demonstrated.
In summary, we have produced a reagent that, for the first time,
can specifically inhibit CD28/B7 interactions without interfering
with those of CTLA-4/B7. This agent is efficient at inhibiting
several CD28-mediated processes in vitro. We believe that this new
strategy may have clinical applications in humans.
Acknowledgments
The authors thank J. Naulet and J. Lafond for assistance in protein
production and purification, S. Iyer for the preparation of Fab fragments,
and Alain Faivre-Chauvet for assistance in protein iodination.
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