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(Received for publication, October 16,
1995; and in revised form, January 12, 1996) From the
This report describes the construction of leucine zipper-based
dimerization cassettes for the conversion of recombinant monomeric scFv
antibody fragments to bivalent and bispecific dimers. A truncated
murine IgG3 hinge region and a Fos or Jun leucine zipper were cloned
into four scFv fragments previously isolated from a synthetic antibody
phage display library. Cysteine residues flanking the zipper region
were introduced to covalently link dimerized scFv fragments. The
secreted fusion proteins were shown to spontaneously and efficiently
form stable Fos
Surface display of Fab or single chain Fv (scFv) antibody
fragments on filamentous phage particles in combination with an array
of versatile selection procedures has become a powerful approach to
obtain recombinant molecules with desired specificities and binding
properties from large libraries (reviewed by Winter et al. (1994) and Burton and Barbas(1994)). The Fab and scFv fragments
thus obtained are monovalent, whereas in many in vitro and in vivo applications, multivalency of antibody molecules is a
desirable property. In addition, linking two or more binding sites
efficiently increases the functional avidity of antibody molecules or
results in the construction of antibodies with dual specificities
(Plückthun, 1992). Several approaches have been
employed to generate genetically engineered, multimerized antibody
fragments. Bivalent (and bispecific) (scFv) In
designing strategies for dimerization of antibody fragments, several
issues need to addressed including stability and homogeneity of the
dimers, resistance to proteolytic cleavage during in vivo assembly, efficient production of preferably soluble protein,
simple engineering steps, and general applicability for the
construction of both bivalent and bispecific recombinant antibodies.
With these issues in mind, we designed dimerization cassettes that
allow the conversion of scFv antibodies from a number of published
phage display libraries to bivalent or bispecific reagents involving a
single cloning step. In this procedure, the flexible and
proteolysis-resistant truncated mouse IgG3 upper hinge region (Pack and
Plückthun, 1992) and either Fos or Jun leucine
zippers were fused to scFv proteins. Two cysteine residues were
engineered in the Fos and Jun zipper domains to produce
disulfide-stabilized homodimers. Using four scFv antibodies previously
isolated from a synthetic phage display library, we show that this
approach results in the efficient in vivo production of
stable, secreted homodimers that retain their specificity as assessed
in a number of assays. Furthermore, exploiting preferential
Fos
Figure 1:
Diagram of the Fos and Jun dimerization
cassettes cloned into scFv-containing pHEN1 phagemids. Cysteine
residues are underlined.
For sandwich ELISAs, 50 µl of 3F
Figure 2:
SDS-PAGE Western blot analysis of
expressed scFv and (scFv)
To investigate the antigen binding potential of the
closely spaced (scFv)
Figure 3:
SDS-PAGE Western blot analysis of
(scFv)
Figure 4:
Antigen
specificity of (scFv)
Figure 5:
Flow cytometric analysis of peripheral
blood leukocytes double stained with anti-CD8
Figure 6:
Immunohistochemical staining of COS7 cells
transfected with a cDNA encoding the CD22
Figure 7:
Formation of bispecific anti-IgG
Figure 8:
Detection of bispecific anti-IgG
We have constructed scFv antibody fragment dimerization
cassettes that can be readily introduced in the NotI
restriction sites of genes encoding scFvs isolated from a variety of
phage display libraries described in the literature (Hoogenboom and
Winter, 1992; Nissim et al., 1994; de Kruif et al.,
1995a). These cassettes add a truncated, flexible murine IgG3 hinge
region and either a Fos or Jun leucine zipper to the scFv proteins. To
increase stability of the bivalent antibodies, cysteine residues were
incorporated at the N and C termini of each of the leucine zippers,
facilitating disulfide bridge formation in the periplasmic space
(Crameri and Suter, 1993). The performance of zipper-linked
(scFv) Monomeric scFv molecules were detectable as
single bands in non-reducing SDS-PAGE, consistent with the notion that
proteins secreted into the periplasmic space of Gram-negative bacteria
refold properly with formation of the correct disulfide bonds (Huston et al.(1993) and references therein). In contrast, Fos or Jun
homodimers presented themselves as multiple closely spaced bands in
non-reducing SDS-PAGE. Immunoaffinity purification of (scFv) Previously, a tendency of GCN4 zipper-linked
``mini-antibodies'' to display nonspecific binding to
antigens coated to microtiter wells has been noted (Pack et al. 1993). We examined the binding specificities of our bivalent and
bispecific (scFv) Employing the much greater tendency of Fos and
Jun zipper peptides to form heterodimers over homodimers (O'Shea et al., 1989; Kostelny et al., 1992), bivalent Fos
and Jun leucine-zippered (scFv) The dimerization system
described here may be used to construct phage display libraries of
bispecific antibodies. Bispecific antibodies that simultaneously
recognize adjacent and non-overlapping epitopes on a target protein
have higher avidities than the single chain or Fab antibodies obtained
from conventional libraries (Neri et al., 1995). Thus, a
Fos-linked scFv with a desirable specificity may be cloned into a phage
library of Jun-scFv antibodies, permitting the direct recovery of high
avidity bispecific antibodies using stringent selection procedures. We
are currently performing experiments to assess the feasibility of this
approach. We show that using cysteine-modified Fos and Jun leucine
zipper peptides, scFv antibody fragments isolated from phage display
libraries can be simply converted to functional bivalent and bispecific
molecules involving only a single cloning step. It is important to note
that scFv molecules obtained from phage display libraries have been
through a stringent selection for correct expression, transport, and
folding in bacterial cells. This explains why these antibodies and the
derivatives described in this paper do not appear to suffer from many
of the problems associated with bacterially expressed scFvs derived
from hybridomas.
Volume 271,
Number 13,
Issue of March 29, 1996 pp. 7630-7634
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Fos or JunJun homodimers in the Escherichia coli periplasm at levels comparable to their
monovalent counterparts. The bivalent (scFv)
fragments
performed well in enzyme-linked immunosorbent assay, flowcytometric,
and immunohistochemical analysis. Fos and Jun homodimer
(scFv) antibodies with different specificities could be
reduced, reshuffled, and reoxidized to form preparations of functional
bispecific (scFv) FosJun heterodimers. These Fos and
Jun fusion protein cassettes provide a universal basis for the
construction of dimeric scFv antibodies with enhanced avidity or dual
specificity.
and (Fab) fragments have been successfully produced by association of two
molecules through flexible linker polypeptides, chemical cross-linking,
and dimerization domains (reviewed by Holliger and Winter(1993)). In
the latter approach, introduction of amphipathic helices or leucine
zippers was shown to mediate dimerization of scFv or Fab fragments in vivo (Pack and Plückthun, 1992; Pack et al., 1993, 1995; Kostelny et al., 1992). These
efforts have resulted in the production of higher valency antibody
fragments with widely varying physicochemical properties.Jun heterodimer over FosFos or JunJun homodimer
formation, we show that in vitro reduction, mixing, and
re-oxidation of Fos and Jun scFv antibodies with different
specificities results in the production of bispecific (scFv) molecules.
scFv Fragments
The scFv antibodies were selected
from a semi-synthetic antibody phage display library constructed in the
phagemid vector pHEN1. scFv clone 3 is specific for an IgG paraprotein,
scFv clones 22 and 23 are specific for dinitrophenol coupled to bovine
serum albumin (DNP-BSA), (
)scFv clone 35 is specific for the
chain of the CD8 molecule expressed on a subpopulation of human T
lymphocytes, and scFv clone 40 is specific for the 
chain of the
CD22 molecule expressed on mature human B lymphocytes. All scFv
molecules have been described in detail elsewhere under different names
(clone 3, IgG2; clone 22, DNP2; clone 23, DNP5 (de Kruif et al. 1995a); clone 35, T1; clone 40, B28 (de Kruif et al. 1995b)).Construction of scFv-Zipper
Proteins
Oligonucleotide primers (Table 1) were developed
to append NotI restriction sites and a murine IgG3 upper hinge
region (Pack and Plückthun, 1992) to modified Fos
and Jun leucine zipper regions. At the N and C termini, the modified
zipper regions contain cysteine residues added via Gly-Gly spacers
(Crameri and Suter, 1993). Template consisting of the plasmid pJuFo
(encoding both modified zipper genes; Crameri and Suter(1993)) was
polymerase chain reaction amplified with the appropriate primer set (30
s at 94 °C, 30 s at 58 °C, and 45 s at 72 °C for 25
cycles), digested with NotI, and cloned into phagemid vector
pHEN1 (Hoogenboom et al., 1991) to yield pHEN1-hFo and
pHEN1-hJu. Sequences were verified by the dideoxy chain termination
procedure. NotI digestion fragments from pHEN1-hFo and
pHEN1-hJu were ligated into NotI-digested pHEN1-scFv clones to
produce clones 3F (anti-IgG-hinge-Fos), 23J (anti-DNP-hinge-Jun), 35F
(anti-CD8-hinge-Fos), and 40J (anti-CD22-hinge-Jun). Diagrams
of the resulting fusion proteins are shown in Fig. 1.
Expression of scFv Proteins
scFv and (scFv) proteins were expressed in Escherichia coli strain SF110
(Meerman and Georgiou, 1994), modified to contain the F` episome of E. coli XL1-Blue. Induction of protein synthesis and isolation
of scFv fragments from the periplasmic space was performed as described
(de Kruif et al., 1995a).SDS-PAGE and Western Blotting
Samples were
separated on 10% SDS-polyacrylamide gels followed by electroblotting to
nitrocellulose membranes. scFv proteins were visualized by staining
with undiluted hybridoma supernatant containing the Myc tag-specific
antibody 9E10 (9E10 SN), followed by a horseradish
peroxidase-conjugated goat anti-mouse antibody (DAKO, Denmark) diluted
1/1000 in 4% milk powder-PBS (MPBS). In non-reducing SDS-PAGE, samples
were preincubated in 60 mM iodoacetamide to block free
sulfhydral groups before boiling in SDS-containing sample buffer.Affinity Purification of Functional (scFv)
Tosil-activated paramagnetic beads (Dynal,
Norway) were coated overnight in 400 µg/ml IgG or DNP in borate
buffer, pH 9.5. The beads were then blocked for 4 h in 2% MPBS.
200-µl (scFv) Molecules periplasmic preparations were diluted 1/1
in 4% MPBS and added to the beads. After another 2-h incubation, the
beads were washed five times in PBS, 0.1% Tween 20 (PBST), boiled in
non-reducing sample SDS buffer, and subjected to SDS-PAGE analysis as
described.ELISA
Wells of microtiter plates (Nunc Maxisorp)
were coated overnight at room temperature with DNP-BSA, IgG
paraprotein, thyroglobulin, lysozyme, HMG-box protein, ovalbumin, or
non-fat milk at 10 µg/ml in 50 mM NaHCO
(pH
9.6). Excess antigen was removed, after which the wells were blocked
for 2 h in 2% MPBS. 100-µl scFv and (scFv) preparations
were diluted 1/1 in 4% MPBS and added to the wells. After a 2-h
incubation, wells were washed in PBST, incubated in undiluted 9E10 SN,
washed with PBST, and finally incubated with horseradish
peroxidase-conjugated polyclonal goat-anti-mouse antibody (1/1750 in 2%
MPBS). After a final wash in PBST, plates were developed using
2`,2`-amino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium as a
substrate.
23J (IgG
DNP) bispecific (scFv) preparation was blocked in
150 µl of 4% MPBS for 15 min before addition to IgG-coated wells.
After a 1-h incubation, plates were washed in PBST, and 200 µl of 1
µg/ml DNP-BSA in MPBS was added to the wells. Following another 1-h
incubation, unbound DNP-BSA was removed by washing in PBST, and bound
DNP-BSA was detected using clone 22 phage antibodies recognizing a
different epitope on DNP than scFv clone 23. Binding of the clone 22
phage antibodies was visualized by incubation with a polyclonal sheep
anti-M13 horseradish peroxidase-conjugated antibody (Pharmacia,
Uppsala, Sweden) as described (de Kruif et al., 1995a). All
incubations were performed at room temperature.Immunohistochemical Staining
COS7 cells were
transfected with CD22 cDNA cloned into the CDM8 vector using
DEAE-dextran sulfate and plated in Petri dishes according to standard
protocols. 48 h after transfection, plates were washed in PBS, 1% BSA
(PBSB) and incubated for 1 h with Jun-linked clone 40 (scFv) antibodies diluted 1/2 in 4% MPBS. Plates were washed in PBSB and
incubated with 9E10 SN for 1 h, followed by washing in PBSB and
incubation in goat-anti-mouse antibodies coupled to horseradish
peroxidase diluted 1/1000 in PBSB. Plates were developed in
3-amino-9-ethyl-carbazol and photographed. All incubations were
performed at 4 °C.Fluorescence-activated Cell Sorter
Analysis
Peripheral blood leukocytes were isolated by
Ficoll-Hypaque density centrifugation and stained with clone 35 scFv
and Fos-linked clone 35 (scFv) proteins using the anti-Myc
monoclonal antibody and a phycoerythrin-conjugated goat anti-mouse IgG
polyclonal antibody as second and third step reagents as described (de
Kruif et al., 1995b). Double staining was performed with a
conventional fluorescein isothiocyanate-conjugated anti-CD8 monoclonal
antibody (Becton Dickinson). In control staining experiments,
incubations with scFv and (scFv) fragments were omitted
from the procedure.Formation of Anti-IgG
Periplasmic preparations of clones
3F and 23J containing approximately equal amounts of (scFv) Anti-DNP Bispecific
(scFv) Fragments homodimers were reduced in 32 mM 2-mercaptoethanol at 37
°C for 1 h, mixed, and dialyzed against redox buffer (50 mM Tris-HCL, pH 8.5, 1 mM EDTA, 500 µM reduced
glutathione, and 500 µM oxidized glutathione) for 24 h at
4 °C. Subsequently, the buffer was changed back to PBS by dialysis.
Construction of scFv-Leucine Zipper Fusion
Proteins
A polymerase chain reaction approach was used to append
restriction sites and the mouse IgG3 upper hinge region to modified Fos
and Jun leucine zippers. These ``dimerization cassettes''
were subsequently cloned into a unique NotI restriction site
present in the 3`-end of the genes encoding scFv antibody fragments
isolated from a semi-synthetic human phage antibody display library (Fig. 1).SDS-PAGE and Western Blot Analysis
Equal volumes
of periplasmic preparations containing expressed scFv fragments or
their Fos and Jun fusion protein derivatives were run on an SDS-PAGE
gel and analyzed by Western blotting using the anti-Myc tag antibody
9E10. Under reducing conditions, equal amounts of scFv fragments are
detectable in the periplasm of scFv, scFv-hinge-Fos (3F), and
scFv-hinge-Jun (23J) expressing bacteria, indicating that expression
levels of the scFv molecules are not affected by addition of the hinge
region and leucine zipper domains (Fig. 2). A shift in the gel
mobility of the scFv-zipper proteins is observed, corresponding to the
56 amino acids introduced by the hinge region and zipper domains. When
SDS-PAGE is performed under non-reducing conditions, a set of proteins,
with closely spaced bands, corresponding to approximately twice the
size of scFv-zipper proteins is detected solely in periplasmic
preparations containing zipper constructs. No protein bands
corresponding to the size of monomeric scFv zippers are present (Fig. 2).
proteins. The upper panel shows the migration of anti-IgG (lane 1) and anti-DNP (lane 2) scFv and their Fos and Jun fusion protein derivatives (lanes 3 and 4, respectively) run under reducing
conditions. In the lower panel, reducing agents were omitted
from the sample SDS buffer.
bands (Fig. 2), periplasmic
preparations of Fos-dimerized anti-IgG (3F) and Jun-dimerized anti-DNP
(scFv) (23J) were allowed to bind to DNP-BSA and IgG coated
to paramagnetic beads. After washing, bound proteins were eluted from
the beads and analyzed by non-reducing SDS-PAGE. In control
incubations, no binding of 23J (scFv) to IgG or 3F
(scFv) to DNP was observed (Fig. 3, lanes 2 and 6). In contrast, immunoaffinity selection of 3F and
23J scFv dimers on the corresponding antigen-coated beads resulted in
the purification of a set of proteins displaying the same
characteristic banding pattern as their non-purified counterparts (Fig. 3, lanes 1 and 3-5). These results
suggest that all (scFv) proteins formed in the bacterial
periplasm retained their specific antigen binding capacity.
proteins selected for binding to their target
antigen. (scFv) proteins were incubated with paramagnetic
beads coated with either IgG or DNP. After washing, the beads were
boiled in non-reducing SDS sample buffer, and the resulting protein
mixture was applied to the gel. Lane 1, 3F (scFv) periplasm; lane 2, 3F (scFv) selected on
DNP; lane 3, 3F (scFv) selected on IgG; lane
4, 23J periplasm; lane 5, 23J (scFv) selected
on DNP; lane 6, 23J (scFv) selected on
IgG.
Specificity of Bivalent (scFv)
ELISA assays were performed to assess the
specificity of Fos- and Jun-linked bivalent (scFv) Antibody
Molecules in ELISA molecules. E. coli SF110 cells were transformed with
constructs encoding the 3F and 23J (scFv) antibodies.
Periplasmic preparations of cells containing approximately similar
concentrations of antibody were incubated with DNP-BSA, IgG, and six
control antigens coated to wells of a microtiter plate. Bivalent
antibodies 3F and 23J bound to IgG and DNP, respectively, whereas no
significant binding to any of the control antigens was observed (Fig. 4).
antibody fragments. Microtiter plates
were coated with IgG, DNP, or a panel of control antigens including
lysozyme, thyroglobulin, ovalbumin, HMG-box protein, bovine serum
albumin, and milk powder. (scFv) molecules were allowed to
bind and were detected using the 9E10
antibody.
Performance of the Anti-CD8
Peripheral blood mononuclear cells were
incubated with periplasmic preparations of bacteria-producing
(scFv) (scFv) in
Flow Cytometric Analysis antibodies against the CD8 chain expressed on a
subpopulation of human T lymphocytes. Costaining with a fluorescein
isothiocyanate-labeled CD8 monoclonal antibody shows that the
(scFv) molecules brightly and specifically stain the CD8
positive cells (Fig. 5). Note that some nonspecific staining of
lymphocytes is caused by the second and third step antibodies.
scFv or (scFv) antibody fragments and a conventional fluorescein
isothiocyanate-conjugated anti-CD8 monoclonal antibody. Peripheral
blood leukocytes were incubated with periplasmic preparations of scFv (middle panel) and (scFv) (right panel)
secreting bacteria, and bound fragments were detected using the 9E10
monoclonal antibody followed by a goat-anti-mouse phycoerythrin-labeled
polyclonal antibody. As a control, the incubation step with scFv
fragments was omitted (left panel). Only cells with a forward
scatter/side scatter profile corresponding to lymphocytes are shown. Boxed area, CD8+ T cells as detected with a conventional
monoclonal antibody.
Immunohistochemical Staining of CD22
COS cells were
transfected with the CD22 Transfected COS
Cells with Bivalent (scFv) Antibodies cDNA cloned into the CDM8 vector. A
periplasmic preparation from E. coli SF110 transformed with
Jun-linked anti-CD22 (scFv) (40J) was used to stain
transfected cells. Results show an intense staining of approximately
10% of the transfected cells (Fig. 6), corresponding to the
transformation efficiency in this particular experiment. No staining
was observed when non-relevant (scFv) molecules with
anti-DNP specificity were used to stain CD22-transfected COS cells
(results not shown).
chain. COS7 cells were
stained with a periplasmic preparation of bacteria transformed with the
40J construct encoding a Jun dimerized anti-CD22
(scFv). Bound antibodies were detected using the 9E10
anti-Myc antibody followed by goat-anti-mouse antibodies coupled to
horse-radish peroxidase.
Formation and Performance of Anti-IgG
The formation of
bispecific (scFv) Anti-DNP
Bispecific (scFv) Molecules molecules was examined using anti-DNP and
anti-IgG scFv clones 3F and 23J. Periplasmic preparations from bacteria
transformed with these constructs were reduced in 2-mercaptoethanol.
Upon this treatment, all detected proteins are present as scFv-zipper
monomers (Fig. 7). To allow formation of heterodimeric
(scFv) molecules, the proteins were mixed and incubated in
a redox buffer. After subsequent dialysis in PBS, a set of proteins
corresponding to the size of (scFv) molecules was observed (Fig. 7). Binding properties of these reshuffled
(scFv)s was first examined in ELISA. The 3F 23J
protein preparation detects both the DNP and the IgG antigen (Fig. 4). No binding to other antigens is detected. To test the
bispecific properties of the reshuffled proteins, a sandwich ELISA was
performed. The proteins were allowed to bind to IgG-coated ELISA
plates. After washing, DNP was added to the wells. Bound DNP was
detected by an anti-DNP phage antibody followed by a horseradish
peroxidase-conjugated anti-M13 antibody. A strong signal developed in
the wells incubated with the 3Fx23J bispecific antibody (Fig. 8). No signal was observed in wells incubated with 3F or
23J homodimers or when DNP is omitted in the procedure.
anti-DNP (scFv) fragments in vitro, visualized by
non-denaturing SDS-PAGE and Western blotting. Periplasmic preparations
of scFv clones 3F and 23J were reduced in 2-mercaptoethanol. Reduced
proteins were mixed in a redox buffer followed by dialysis against PBS,
resulting in the generation of bispecific antibody 3F
23J.
anti-DNP fragments in a sandwich ELISA. IgG-coated plates were
incubated with (scFv) proteins. After washing, DNP was
added to the wells. Bound DNP was detected using an anti-DNP phage
antibody followed by a horseradish peroxidase-coupled anti-M13
antibody. 3F 23J, anti-DNP anti-IgG bispecific
(scFv) fragment; 3F and 23J, anti-IgG and anti-DNP dimers.
-DNP and -(scFv), no DNP or scFv protein
added.
molecules was assessed using four different scFv
antibodies selected from a synthetic phage display library as starting
material. All scFv-zipper molecules were secreted as soluble proteins
into the periplasmic space, obviating tedious refolding procedures
associated with the formation of insoluble inclusion bodies (Kurucz et al., 1995). In each instance, the level of expression did
not appear to be significantly affected by addition of the hinge and
Fos or Jun zipper regions. In Western blotting under non-denaturing
conditions, periplasmic preparations of Fos or Jun scFv zippers solely
contained dimeric molecules, indicating that the formation of
(scFv) homodimers from scFv-zipper monomeric precursors is
extremely efficient. The homodimers were resistant to boiling in sample
buffer containing 4% SDS and could only be dissociated to monomers
using reducing agents. We conclude that the monomers in a (scFv) complex are covalently linked via disulfide bridges connecting
the leucine zippers. containing periplasmic preparations on antigen-coated beads
showed that each of the closely spaced bands corresponded to functional
protein retaining the capacity to specifically bind antigen. Others
have also observed multiple scFv bands under non-reducing SDS-PAGE
conditions (Neuberger et al., 1984; Kostelny et al.,
1992; Huston et al., 1993), and it has been suggested that
this results from anomalies associated with SDS binding to unreduced
proteins. fragments in a number of assays,
including ELISA, flow cytometry and immunohistochemistry. In none of
these assays, significant nonspecific binding was observed. A reason
for this apparent discrepancy between GCN4 zippers and Fos/Jun zippers
may be a better shielding of the hydrophobic regions in the latter
and/or the more stable configuration caused by covalently cross-linking
the zipper regions. can be rapidly converted to
bispecific (scFv) molecules by simple reduction, mixing,
and reoxidation steps. Using this approach, the anti-IgG and anti-DNP
binding activities of two (scFv) homodimers were shown to
be combined in a single heterodimeric molecule. A major advantage of
this strategy is that only a single straightforward cloning step is
required to produce bispecific antibodies obviating the need for
extensive polymerase chain reaction and cloning efforts (Holliger et al., 1993; Mallender and Voss, 1994; Kurucz et
al., 1995; Mack et al., 1995).
)
-We thank Drs. M. Suter and H.C. Åsheim
for the kind gifts of the pJuFo vector and CD22 cDNA, respectively.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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K. Els Conrath, M. Lauwereys, L. Wyns, and S. Muyldermans Camel Single-domain Antibodies as Modular Building Units in Bispecific and Bivalent Antibody Constructs J. Biol. Chem., March 2, 2001; 276(10): 7346 - 7350. [Abstract] [Full Text] [PDF]
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