Gene, 172(1996)295-298
0 1996 Elsevier Science B.V. All rights reserved.
295
037%1119/96/$15.00
GENE 09619
Recombinant rabbit Fab with binding activity to type-l plasminogen
activator inhibitor derived from a phage-display library against human
a-granules
(Recombinant DNA; recombinant antibody; immunoglobulin; phagemid; gene enrichment; prokaryotic expression)
Irene M. Lang”,*, Carlos F. Barbas, IIIb and Raymond R. Schleef”
Departments
of“Vascular Biology, and bMolecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
Received by M.J. Benedik:
11 September
1995; Revised/Accepted:
22 November/24
November
1995; Received at publishers:
12 January
1996
SUMMARY
The display of panels of antibody (Ab) fragments on the surface of filamentous bacteriophage offers a way of making
Ab with defined binding specificities. Because rabbit Ab are routinely utilized as immunologic probes in a variety of
biological techniques, the aim of this study was to design and utilize primers for the amplification of mRNAs encoding
rabbit K light and y heavy chains for the construction of an Ab library from this species. Using the polymerase chain
reaction, a diverse Ab library with a repertoire of 2 x lo7 clones was derived from the spleen and bone marrow of a
rabbit that had been immunized with purified human platelet cl-granules. From this library, specific clones were isolated
after three rounds of affinity selection with binding activity to type-l plasminogen activator inhibitor, a trace protein
contained in platelet a-granules. These data indicate that recombinant phage-displayed Ab libraries obtained after
immunization with complex biological antigens can be employed for the isolation of rabbit monoclonal Fab against
specific antigens contained in the biological sample.
INTRODUCTION
Antibodies (Ab) are a fundamental tool for the investigation of biological processes. Several strategies are availCorrespondence
to: Dr. CF.
Barbas,
III, Department
Biology
(MB1 I), or Dr. R.R. Schleef, Department
(VB-l),
The Scripps
Research
Institute,
10666 N. Torrey
Jolla, CA 92037, USA. Tel. (1-619) 554-9098;
e-mail: Carlos@Scripps.edu
*Present address: UniversitLtsklinik
Kardiologie,
(43-l)
Guertel
Fax (1-619) 554-6778;
ftir Innere
18-20,
Biology
Pines Rd., La
Medizin
1090 Vienna,
II, Abteilung
Austria.
Tel.
404004636.
Abbreviations:
BSA, bovine
region;
Waehringer
of Molecular
of Vascular
Ab, antibody(ies);
serum albumin;
cfu, colony-forming
B, cysteine, guanosine
CDR, complementarity
u; ELISA,
enzyme-linked
or thymidine;
determining
immunosorbent
assay; Fab, Ab-binding fragment(s); mAb, monoclonal Ab; PAGE, polyacrylamide-gel
electrophoresis;
PAI-1, type-l plasminogen
activator
inhibitor; PBS, phosphate-buffered
saline; PCR, polymerase chain reaction; PNPP, paranitrophenylphosphate;
re-, recombinant;
SDS, sodium
dodecyl sulfate; u, units.
PII SO378-lll9(96)00021-2
able for the preparation of Ab. Conventionally, because
an antigenic challenge in vertebrates elicits the production of approx. lo7 Ab molecules, animals are immunized with a purified target protein and the Ab isolated
from serum (Harlow and Lane, 1988). Moreover, spleen
cells of immunized animals can be fused with myeloma
cells to generate mAb molecules derived from hybridomas (Harlow and Lane, 1988; Kohler and Milstein, 1975).
In order to achieve satisfactory titers of Ab, the target
protein has to be prepared in sufficient quantities from a
biological sample or as a re-protein; this preparation is
usually time-consuming. Recently, recombinant Ab technology (Huse et al., 1989), combined with efficient selection techniques (Barbas et al., 1991), has allowed the
identification of Ab molecules with high affinity towards
antigens. In cases where the target protein is not available
in sufficient quantity, ‘naive’ or synthetic Ab libraries can
be used for screening (Marks et al., 1991; Gram et al.,
296
A
1992; Barbas et al., 1992). Because rabbit Ab are routinely
utilized as immunologic probes in a variety of biological
techniques, a system designed to generate specific rabbit
Fab mAb would provide reagents that readily complement current assays and protocols. The aim of the present
report was to construct a rabbit Fab library displayed on
the surface of phages by designing and utilizing specific
primers for amplification of the mRNA harvested from
the bone marrow and spleen of a rabbit immunized with
cl-granules, a platelet storage organelle that contains several key hemostatic proteins (Harrison and Martin
Cramer, 1995). We demonstrate that this library can be
employed to rapidly select for clones expressing Fab that
recognize one particular a-granule constituent (i.e., type-l
plasminogen activator inhibitor, PAI-1) in a protocol that
requires only small amounts of the purified molecule (i.e.,
approx. 1 pg).
rabbit
RVKl
RVK2
rabbit
RCKl
RCK2
rabbit
k light chain variable domain 5’ primers:
= 5’-GCGCCGGAGCTCGTGATGACCCAGACTCCA-3
= 5’-GCGCCGGAGCTCGATATGACCCAGACTCCA-3
k light chain constant domain 3’ orimers:
= 5’-GCGCCGTCTAGACTAACAGTCACCCCTA~TGAAGC-3,
= 5’-GCGCCGTCTAGACTAACAGTE~TCCTACTGAAGC-Y
heavy chain variable domain 5’ primers:
RVHl = S-CAGTCGBTGCTCGAGTCCGGGGGTCGCCT-3
RVHP = 5’-CAGTCGBTGCTCGAGTCCGGGGGAGGC-3
RVM = 5’-CAGTCGBTGCTCGAGTCCGGGGGAGAC-3
rabbit heavy chain constant domain 3’ primer:
RCG = 5’-TGGGCAACTAGTCFrGCTGCATGTCGAGGG-3
B
9
2
s
EXPERIMENTAL
AND DISCUSSION
1
(a) Construction of a rabbit Fab library directed against
human platelet a-granules
0
1
2
3
4
5
6
7
6
9
101112131415161716192021222324
clones
Fig. 1. Construction
of a rabbit
Fab library
directed
against
human
of
platelet cc-granules. (A) Sequence of primers for the amplification
cDNA prepared from mRNA extracted from hyperimmune
rabbit bone
and spleen. (B) Phage displaying
marrow
a-granule
duction
proteins
of soluble rabbit
proteins
PAI-
and the resulting
Fab on microtiter
(open bars), dense granule
(shaded
incubating
of sonicated
from the donor
dense granules,
human
platelets
rabbit.
white rabbit
with isolated
reverse transcribed
a-granules,
restriction
(forward
chain:
sites (shown
light chain:
XhoI; reverse
previously
(Barbas
light chain library
(Gogstad,
marrow
chain:
injected
that also contained
protein)
light chain:
was
rabbit-
the following
in their 5’ overhang
XbaI;
SpeI) and conditions
et al., 1991). After PCR, the cDNA
was electroeluted
of a
(i.e., 16 week immunization
by underlining)
SacI; reverse
heavy
organelles
by PCR, using the indicated
specific light and heavy chain primers
internal
gradient
and bone
20 mg total
and amplified
by
by sub-fractionation
on a Metrizamide
hyperimmune
protocol
obtained
sera (1:2000 dilu-
Methods: Platelet
etc.) were isolated
from the spleen
New Zealand
with cc-granule
the signal
or immune
1981). The RNA extracted
on
for the pro-
(closed bars) or purified
bars). Bars 23 and 24 indicate
tion, respectively)
Fab were enriched
wells coated
proteins
wells with either pre-immune
(i.e., a-granules,
rabbit
clones were analyzed
from an agarose
forward
heavy
as described
encoding
gel, digested
the
with
excess Sac1 and XbaI (i.e., 35 u/pg and 70 u/pg of DNA, respectively)
and ligated into the vector pComb3H.
The PCR-amplified
cDNA
encoding the heavy chain library was digested with SpeI and XhoI (17
u/kg and 70 u/kg of DNA) and ligated into the light chain construct.
Phagemids
were transformed
were infected with VCSM13
into E. coli XLl-Blue
helper phage (Barbas
Total RNA was extracted from the spleen and bone
marrow of a New Zealand white rabbit that had been
immunized with a-granules isolated from human platelets. The RNA was reverse transcribed and amplified utilizing primers in separate reactions for rabbit K light
chains or for rabbit y heavy chains that were designed
according to published rabbit sequences (Kabat et al.,
1991). More specifically, two 5’primers were designed to
anneal in the framework 1 region of rabbit K light chains
and were combined with two reverse primers designed to
anneal to the 3’end of the rabbit K light chain CL1 region
(Fig. 1A). Three 5’y heavy chain primers were designed
to anneal to the framework 1 variable region and were
combined in the reactions with a reverse primer that cor-
cells and the cultures
et al., 1991) for the
production of phage-displaying
Fab. Enrichment of phages (= panning)
was performed on microtiter
plates coated with proteins (1 pg/well)
extracted from u-granules and their membranes utilizing lithium diiodisalicylate (Marchesi and Andrews, 1971). Coated wells were washed,
blocked with 3% w/v bovine serum albumin (BSA) and incubated (2
h, 37°C) with 50 ~1 of phage (typically
10”-10’2
cfu). Phage were
removed and the wells were washed once (first round of panning), five
(second round
TBS/Tween
of panning)
solution
or ten times (third round
(50 mM TrisHCl,
of panning)
with
pH 7.5/150 mM NaCl/O.OS%
Tween 20). The adherent phage were eluted with 50 ~1 of elution buffer
(0.1 M HCl adjusted to pH 2.2 with glycine and supplemented
with
1 mg BSA/ml)
described
and used to infect logarithmic
previously
(Barbas
phase XLI-Blue
et al., 1991). For the preparation
cells as
and
analysis of soluble Fab, phagemid DNA from the panned library was
isolated, digested with @I, ligated into the arabinose-inducible
expression vector pAraHA for the preparation
of soluble Fab in which the C
terminus
of the heavy chain is fused to a decapeptide
tion (i.e., YPYDVPDYAS;
tag for identifica-
Huse et al., 1989). E. coli DHl2S
cells were
transformed
with these constructs,
single clones were grown, induced
with 1% (w/v) arabinose, lysed by four freeze-thawing
cycles and the
Fab-containing
supernatant
clarified by centrifugation.
Soluble Fab
were analyzed in an ELISA for their ability to bind to microtiter wells
coated with proteins extracted either from platelet a-granules
or from
platelet dense granules and the bound Fab detected by incubation with
alkaline phosphatase-labeled
monoclonal
Ab directed against the decapeptide
tag followed
by paranitrophenylphosphate
(PNPP).
297
/y&
# of washes
1
I-
-
I
Perm&3;;$id 1
Output
1.6 x 10’
12345678
12345678
12345678
RlGBantlPAl-1
ROlLntiPACI
R012antlPAC1
responded to a nt stretch within the hinge region of y
rabbit heavy chain. The 5’overhangs of each primer contained internal restriction sites (i.e., Sac1 and XbaI for
light chain primers, XhoI and SpeI for heavy chain primers; Fig. 1, Methods) to allow directional cloning into the
vector pComb3H (Barbas and Wagner, 1995); this vector
is a variant of the phagemid pComb3 (Barbas et al.,
1991), in which the heavy chain site and the region encoding the gene III fragment have been exchanged with the
light chain site to ease cloning for the expression of soluble Fab. A small fraction of inserts was found to be cut
with SpeI and was not included in the final library, which
consisted of 2 x lo7 clones. Phage displaying Ab combining sites was prepared by overnight infection of
phagemid-containing cells with VCSM 13 helper phage;
the resulting supernatant yielded titers of lo’* cfu/ml.
Enrichment of phage using microtiter plates coated with
a-granule proteins followed by subcloning into a prokaryotic expression system (i.e., pAraHA) resulted in a
series of Fab-producing clones that preferentially recognized proteins derived from a-granules as opposed to
proteins derived from another platelet-specific organelle
(i.e., dense granules) (Fig. 1B).
(b) Identification of Fab clones to PAI-
1
10.0:
2
kDa
5.05
E
a
$
1.01
0.5
lH
1
“‘i
PA&l (@ml)
Fig. 2. Selection and identification
of Fab displaying
of Fab clones to PAI-1. (A) Selection
phage from an a-granule
library
under conditions
of
increased washing stringency on PAI-l-coated
wells. (B) Sequences of
CDR3 domains of three selected anti-PAIFab clones. (C) Specificity
of Fab for PAI-1. (D) Two-site
ELISA for PAI-
antigen.
Numerous proteins are either stored within platelet
a-granules or associated with the a-granule membrane.
For example, important structural/functional
a-granule
constituents include fibrinogen, von Willebrand factor,
fibronectin, etc., and these molecules are associated with
platelets at levels ranging between 1% and 12% of total
platelet protein (Harrison and Martin Cramer, 1995).
However, other proteins are associated with subcellular
organelles at either trace or low levels. To demonstrate
the applicability of this technique for the rapid identification of rabbit Fab clones directed against a specific granule molecule, PAI- was selected because it is a key
regulator of the fibrinolytic cascade (Fay et al., 1992) that
is present only in low amounts within the platelet
a-granule. More specifically, the average amount of the
inhibitor has been reported to be 0.3 fg per platelet, which
Inset: 0.1%
SDS-12% PAGE analysis and silver staining of a representative
Fab
(i.e., R013 anti-PAI-1) under non-reducing
(lane 1) and reducing condi-
iterwells:
tions (lane
vitronectin,
2). Methods:
PAI-
was isolated
as described
previously
an EIA against
the following
ovalbumin,
proteins
(Sigma)
a,-antitrypsin,
tissue-type
plasminogen
coated
antithrombin
activator,
III,
urokinase
onto
microt-
fibrinogen,
(bars
2-8,
(Schleef et al., 1990). The rabbit heavy and light chain library in
pComb3H-transformed/XL-l
Blue cells was infected with VCSM13
respectively). Fab were purified from bacterial lysates (6 l/preparation)
on affinity columns composed of goat anti-rabbit
IgG [F(ab),-specific]
helper phage, grown overnight, and the panning,
firmation of clones were performed as described
(Pierce) conjugated
to Gamma Bind Plus (Pierce) and eluted from the
columns utilizing acidic pH. The purified material was employed in a
with the modification
that incubations
subcloning, and reconin the legend to Fig. 1
were carried
out on microtiter
competitive
ELISA
for the determination
of a Fab’s dissociation
rate
plates coated with PAI- (100 ng/well). Accession numbers for the light
chain and heavy chain sequences of the Fab clones are listed respec-
constant (Barbas et al., 1992). A quantitative
ELlSA for solution-phase
PAIwas developed utilizing R013antiPAI-1
coated plates as the
tively: R012antiPAI-1
(U21884, U21883); R013antiPAI-1
(U21886,
U21885); R166antiPAI-1
(U21900, U21899). The reactivity of three Fab
secreting clones (C) for PAI- (100 ng/well; bar 1) were compared in
immunoabsorbent
phosphatase-labeled
PNPP.
for a dose-response
curve of PAIand alkaline
R166anti-PAIas the detecting Ab followed by
298
represents 4000 molecules per platelet and thus, only
about 0.01% of total platelet protein (Kruithof et al.,
1987). Fig. 2A indicates that by increasing the stringency
from one to five washes in the second round of panning,
a sub-population of Fab-displaying phage particles was
selected which was subsequently enriched in spite of ten
washing steps in the third round of panning. Previous
strategies utilized a constant number of washes, which
results in a constant increase in the yield (Barbas et al.,
1991). Subcloning of this heavy and light chain library
into the pAraHA expression system followed by confirmation of positive clones in an ELISA utilizing immobilized PAI- resulted in a series of clones (i.e., 46 out of
200) that produced soluble Fab directed against PAI-1.
Sequence analysis of the light and heavy chain complementarity determining regions (CDRs) l-3 of several
clones revealed 13 distinct re-clones. Three clones were
selected for further analysis and the predicted protein
sequences of their CDR3 regions are shown in Fig. 2B.
Competition ELISA revealed dissociation constants
of 5 x lo-’ M for clone RO12antiPAI-1, 1 x lop9 M
for clone R013antiPAI-1, and 9 x lo-’ M for clone
R166antiPAI-1. These clones bound specifically to PAIin comparison to their lack of reactivity to several other
members of the serine protease inhibitor superfamily,
other cl-granule proteins, and proteases that react against
PAI- (Fig. 2C). PAI- is known to exist in a number of
different conformations (Mottonen et al., 1992) and our
Fab were observed to react with the solution-phase form
of this protein in an ELISA format (Fig. 2D) but only
weakly with the denatured
conformation
present
following SDS-PAGE and transfer to nitrocellulose (data
not shown).
In summary, the ability to rapidly sort this Fab library
against individual a-granule proteins (e.g., PAI-1) suggests that this strategy can be readily adapted for the
identification and development of panels of rabbit
Fab mAb.
and the American Heart Association 93-83 (to I.M.L.),
an Investigator Award from the Cancer Research
Institute (to C.F.B.), and in part by grants from the
National Institutes of Health HL45954 and HL49563 (to
R.R.S.). The authors appreciate the technical assistance
of Theresa M. Jones and Trinette Ackerman, and thank
Dr. Dennis Burton for helpful discussions.
REFERENCES
Barbas
III, C.F. and Wagner,
and evolving
Barbas
functional
antibody
ACKNOWLEDGMENTS
This research was supported by fellowships from the
Tobacco Related Disease Research Program 3FT-0194
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