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(Received for publication, December 14, 1995) From the
To characterize human IgE antibodies with specificity for a
major allergen at the molecular level, we have constructed an IgE
combinatorial library from a grass pollen allergic patient. cDNAs
coding for IgE heavy chain fragments and for light chains were
reverse-transcribed and polymerase chain reaction-amplified from RNA of
peripheral blood lymphocytes and randomly combined in plasmid pComb3H
to yield a combinatorial library of 5 More than 20% of the population suffers from type I allergic
reactions (allergic rhinitis, conjunctivitis, and bronchial asthma).
The symptoms of type I allergy are due to release of mediators (e.g. histamine) resulting from the cross-linking of specific
IgE antibodies, which are bound to allergic effector cells (mast cells
and basophils). Studies on the primary structure of immunoglobulin E
were initially hampered by the extremely low concentration of IgE
(10-400 ng/ml) in the serum. Due to the availability of
IgE-secreting myeloma cells, it was, however, possible to characterize
IgE antibodies by immunochemical, protein chemical, and finally
molecular biological techniques (Bennich et al., 1968, 1973;
Ishizaka and Ishizaka, 1970; Terry et al., 1970; Kochwa et
al., 1971; Flanagan and Rabbitts, 1982; Kurokawa et al.,
1983; Seno et al., 1983). The cDNA sequence of human C To investigate the molecular interaction of
IgE antibodies and allergens, studies on the V regions of specific IgE
antibodies would be needed. Because of the low number of IgE-secreting
B-cells in the peripheral blood of allergic patients (McKenzie and
Dosch, 1989), a detailed study of allergen-specific IgE antibodies and
in particular of their V regions has proven to be extremely difficult.
In addition, it has been so far impossible to immortalize B-lymphocytes
that were switched to specific IgE production in vivo. Using PCR ( In the present
study, the isolation and characterization of human IgE Fabs with
specificity for a major allergen is reported. For this purpose we have
constructed an IgE combinatorial library from blood lymphocytes of a
grass pollen allergic patient by reverse transcription and PCR
amplification of cDNAs coding for IgE-Fd and L chains. The cDNAs were
randomly combined in the pComb3H vector (Barbas et al., 1991;
Kang et al., 1991a) and expressed on the surface of
filamentous phage to allow the selection of IgE Fab-expressing phage
clones by panning to given allergens. Purified recombinant timothy
grass pollen allergens Phl p 1 (Laffer et al., 1994), Phl p 2
(Dolecek et al., 1993), and Phl p 5 (Vrtala et al.,
1993a) were used to determine the IgE specificities of the allergic
patient. Recombinant human IgE Fabs with specificity for Phl p 5, a
major timothy grass pollen allergen (Vrtala et al., 1993a),
were isolated by panning and analyzed. The present approach may
contribute to the molecular analysis of allergen-IgE interactions and
may perhaps be useful to define recombinant Fabs, which, due to the
lack of the Fc
The detection of
nitrocellulose-blotted or ELISA plate-coupled allergens with the IgE
Fabs was done using an alkaline phosphatase coupled goat anti-human Fab
antiserum (Pierce).
Figure 1:
Serum IgE reactivity
of the grass pollen allergic donor who was used for the construction of
the IgE combinatorial library with natural grass pollen extracts and
recombinant timothy grass pollen allergens. Grass pollen extracts (rye
grass, L. perenne, lane 1; Kentucky Bluegrass, P.
pratense, lane 2; rye, S. cereale, lane
3; timothy grass, P. pratense: lane 4) as well
as recombinant timothy grass pollen allergens (rPhl p 1, lane
5; rPhl p 2, lane 6; rPhl p5, lane 7) were
separated by SDS-PAGE and blotted onto nitrocellulose. Nitrocellulose
strips were incubated with serum IgE, and bound IgE was detected with
Figure 2:
Agarose gel showing the PCR amplification
of IgE heavy chain cDNAs using primers specific for different VH-gene
families. RNA was isolated from peripheral blood mononuclear cells of a
grass pollen allergic patient, and cDNAs coding for IgE-heavy chain Fds
were reverse-transcribed and PCR-amplified using different V
Figure 3:
1% agarose gel showing the insertion of
IgE heavy chain fragments and light chains into plasmid pComb 3H.
Twenty clones from the IgE combinatorial library were randomly picked
and analyzed for the presence of IgE Fd and light chain cDNAs. DNA from
the clones was cut with XhoI/SpeI in the upper
panel to release the heavy chain fragment (HC-Fd) and
with SacI/XbaI (lower panel) to liberate the
light chain (LC), respectively. Plasmid pComb3H migrated at 4
kilobase pairs, whereas the HC-Fd cDNA and light chain cDNA appeared at
approximately 650 base pairs.
Both strands of the DNA sequences coding for the Fd fragments and
light chains of the clones were determined according to Sanger by
primer walking, and the amino acid sequence was deduced. Fig. 4shows the DNA and deduced amino acid sequence of the IgE
heavy chain fragment, which was utilized by all four clones. It is
noteworthy that identical heavy chain fragments were obtained by using
two different PCR primers for the c
Figure 4:
cDNA and deduced amino acid sequence of
the heavy chain fragment of the Fabs with specificity for the major
timothy grass pollen allergens Phl p 5. The cDNA and deduced amino acid
sequence corresponding to the C
As can be seen in Fig. 5(A and B), different
Figure 5:
cDNA sequences and deduced amino acid
sequences of the light chains of four Phl p 5-specific IgE Fabs. cDNA
sequences of the light chains of four Phl p 5-specific IgE Fabs (clones
5, 14, 28, and 31) are aligned in Fig. 5A. The SacI site is printed in italics. In Fig. 5B, the alignment of the deduced amino acid
sequences is shown. Identical nucleotides and amino acids are indicated
by dashes. The constant region (C
Figure 6:
Cross-reactivity of Phl p 5-specific
recombinant IgE Fabs with natural group 5 allergens from different
grasses. Two Phl p 5-specific IgE Fabs (clone 5 and 28) and a
recombinant IgG Fab with specificity for the major birch pollen
allergen Bet v 1 (Co, negative control) were tested for
reactivity with nitrocellulose-blotted grass pollen extracts (rye
grass, L. perenne; Kentucky Bluegrass, P. pratense;
rye, S. cereale; timothy grass, P. pratense) and
birch pollen extract (B.
verrucosa).
No reactivity of the Phl p 5-specific IgE Fabs was observed
with birch pollen extract, which does not contain group 5 allergens. A
recombinant IgG Fab (Co) with specificity for the major birch pollen
allergen Bet v 1 was included as control and showed no reactivity with
grass pollen extracts, whereas it bound to Bet v 1 at 17 kDa in birch
pollen extract. Recombinant human IgE Fabs, which were isolated by
panning to purified recombinant Phl p 5, cross-reacted with natural
group 5 allergens from different grass species as is known for natural
IgE antibodies from grass pollen allergic patients.
Figure 7:
Purification of a recombinant Phl p
5-specific IgE Fab by affinity chromatography to immobilized
recombinant Phl p 5. The Coomassie Blue-stained SDS-PAGE in Fig. 7A shows purified recombinant Phl p 5-specific
Fabs from clone 5 and 31 separated under reducing conditions. A gel
containing samples of the total E. coli supernatant: Fabs (SN), the wash fraction (lane 1), and the elution
fractions (lanes 2-5) of the Phl p 5 affinity column,
was blotted onto nitrocellulose and Fabs were detected with a goat
anti-human Fab antiserum (Fig. 7B).
Figure 8:
Influence of the preincubation of
recombinant Phl p 5 with a Phl p 5-specific IgE Fab on the IgE binding
of grass pollen allergic patients. Nitrocellulose strips containing
blotted recombinant Phl p 5 (duplicates) were preincubated with Phl p
5-specific IgE Fabs (clones 5 and 31) or with Bet v 1 IgG Fabs
(negative control, Co) before serum IgE from two grass pollen
allergic patients (A and B) was applied. Bound IgE
was detected with
The cross-linking of effector cell-bound IgE antibodies by
allergens has been recognized as the key event leading to Type I
allergic reactions. Although IgE antibodies are present at extremely
low levels in serum (10-400 ng/ml), the release of mediators
triggered by the cross-linking event causes severe allergic reactions
(rhinitis, conjunctivitis, allergic asthma, and anaphylaxis). For this
reason immunoglobulin E has been characterized extensively by protein
chemical and molecular biological techniques (Terry et al.,
1970; Kochwa et al., 1971; Bennich et al., 1973;
Flanagan and Rabbitts, 1982; Kurokawa et al., 1983; Seno et al., 1983). Whereas considerable progress was achieved
regarding the characterization of the constant regions of IgE, in
particular the binding site for the high affinity receptor (Helm et
al., 1988; Nissim and Eshhar, 1992; Presta et al., 1994),
nothing was known about the V regions of IgE antibodies with
specifities for allergens. In the present study we have used the
filamentous phage display system to isolate human allergen-specific
recombinant IgE Fab fragments. To achieve this goal, a highly sensitive
PCR technique was established to allow the amplification of IgE Fd from
the peripheral blood of allergic patients (Steinberger et al.,
1995). An IgE combinatorial library was constructed in the pComb3H
plasmid, starting from peripheral blood lymphocytes from a grass pollen
allergic patient. Using purified recombinant timothy grass pollen
allergens for the panning procedure, human IgE Fabs with specificity
for the major timothy grass pollen allergen, Phl p 5 (Vrtala et
al., 1993a), could be isolated. Phl p 5 was used as a model
allergen, because it represents a major allergen for more than 80% of
grass pollen allergic individuals and cross-reacts with group 5
allergens from most grass species (van Ree et al., 1992).
Another reason for selecting Phl p 5 was the fact that a high
percentage of grass pollen-specific IgE antibodies are directed against
this allergen in most patients (Vrtala et al., 1993a, 1996).
Serum from the patient who was used for the construction of the
combinatorial library contained high levels of Phl p 5-specific IgE
compared to Phl p 1-specific IgE (Fig. 1). In fact, many more
phage clones with specificity for Phl p 5 could be recovered from the
combinatorial library after five rounds of panning than clones that
reacted with Phl p 1. ( The
sequence analysis of four independent Phl p 5-specific IgE Fabs
revealed that all four clones used the same type of heavy chain
fragment originating from different PCR reactions, which had recombined
with different kappa light chains. The finding that different PCR
products of the same IgE Fd and similar light chains had combined to
form the Fabs that were selected by the panning procedure indicated
that the recombinant Fabs might closely reflect the structure of Phl p
5-specific natural IgE antibodies. The fact that, during 2 years of
continuous blood sampling and PCR amplifications of IgE Fds, PCR
products were most efficiently obtained during the grass pollen season
indicated that the IgE Fabs represented in the combinatorial library
most likely were produced in response to repeated allergen stimulation. Like the natural IgE antibodies, the Phl p 5-specific IgE Fabs
cross-reacted with group 5 allergens from rye grass, Kentucky
Bluegrass, and rye. Using immobilized recombinant Phl p 5, soluble
human IgE Fabs could be purified to homogeneity up to milligram
amounts. Due to a lack of the c The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
X95746[GenBank]-X95750[GenBank].
Volume 271,
Number 18,
Issue of May 3, 1996 pp. 10967-10972
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ISOLATION AND CHARACTERIZATION OF HUMAN IgE Fabs WITH SPECIFICITY
FOR THE MAJOR TIMOTHY GRASS POLLEN ALLERGEN, Phl p 5 (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
10
primary
clones. IgE Fabs with specificity for Phl p 5, a major timothy grass
pollen allergen, were isolated by panning. Sequence analysis showed
that the 4 of the Fabs used the same heavy chain fragments which had
combined with different kappa light chains. Soluble recombinant IgE
Fabs were purified by affinity chromatography to Phl p 5 and, like
natural IgE antibodies, cross-reacted with group 5 allergens from
different grass species. The described approach should facilitate
studies on the molecular interaction between IgE antibodies and
allergens and encourages the consideration of specific IgE Fabs that
are capable of interfering with allergen-IgE binding as potential
therapeutic tools.
could be determined (Flanagan and Rabbitts, 1982; Kurokawa et
al., 1983; Seno et al., 1983), and those portions of
C that interact with the high affinity Fc receptor were
characterized as possible targets for a therapy of Type I allergic
diseases (Helm et al., 1988; Nissim and Eshar, 1992; Presta et al., 1994).
)techniques, nucleotide sequences of epsilon
VH
transcripts from peripheral blood B-cells of atopic
patients were analyzed, suggesting that the molecular characteristics
of the V
regions argue for a selection process due to
recurrent or chronic stimulation of the immune system by antigens (e.g.. allergens), but nothing is known about their
specificities (van der Stoep et al., 1993). receptor binding site, may be envisaged as
potential therapeutic tools that can compete with natural IgE
antibodies for the allergen binding.
Characterization of the Grass Pollen Allergic
Patient
For the construction of the IgE combinatorial library,
peripheral blood mononuclear cells were obtained during the grass
pollen season from a grass pollen allergic patient after informed
consent was given. The patient suffered from allergic conjunctivitis
and rhinitis during the grass pollen season, was skin test-positive for
timothy grass, and had received no hyposensitization treatment. Total
serum IgE was determined by radioimmunoassay with a RIACT kit
(Pharmacia, Uppsala, Sweden) to be 500,000 units/liter and a RAST class
of >4 for timothy grass pollen was measured. The patient was further
tested for IgE reactivity to purified recombinant birch (Betv 1
(Breiteneder et al., 1989) and Bet v 2 (Valenta et
al., 1991)) and timothy grass pollen allergens (Phl p 1, Phl p 2,
and Phl p 5) to determine his allergogram (Laffer et al.,
1994; Dolecek et al., 1993; Vrtala et al., 1993a).Preparation of RNA and PCR Amplification of cDNAs Coding
for IgE Heavy Chain Fragments and Light Chains from Peripheral Blood
Mononuclear Cells of the Grass Pollen Allergic Patient
150 ml of
heparinized blood were obtained from the allergic donor during the
grass pollen season (with informed consent). Peripheral blood
mononuclear cells were prepared by Ficoll-Paque density gradient
centrifugation (Pharmacia) (Steinberger et al., 1995). RNA was
prepared by a guanidinium isothiocyanate method (Davis et al.,
1986). Several independent cDNA synthesis and PCR amplification
reactions were carried out using a RNA PCR kit (Perkin-Elmer). In
brief, total RNA (20-60 µg) was mixed with 10-20 pmol
of oligonucleotide primers specific for the constant region of the
epsilon chains (C1, 5`-GCT ACT AGT TTT GTT GTC GAC CCA GTC;
C2, 5`-CGA CTG TAA ACT AGT CAC GGT GGG CGG GGT G) and for the
light chains (C
1a, 5`-GCG CCG TCT AGA ACT AAC ACT CTC CCC TGT TGA
AGC TCT TTG TGA CGG GCA AG; C1d, 5`-GCG CCG TCT AGA ATT AAC ACT
CTC CCC TGT TGA AGC TCT TTG TGA CGG GCG AAC TCA G; C
2, 5`-CGC CGT
CTA GAA TTA TGA ACA TTC TGT AGG), heated at 65 °C for 5 min and
then used in a 2-h reverse transcription reaction according to the
suppliers protocol. The reverse transcription reactions and
oligonucleotide primer specific for variable regions of the heavy
chains: V
, 5`-CAC TCC CAG GTG CAG CTG CTC GAG TCT GG;
V
, 5`-GTC CTG TCC CAG GTC AAC TTA CTC GAG TCT GG;
V
, 5`-GTC CAG GTG GAG GTG CAG CTG CTC GAG TCT GG;
V
, 5`-GTC CTG TCC CAG GTG CAG CTG CTC GAG TCG GG;
V
, 5`-GTC TGT GCC GAG GTG CAG CTG CTC GAG TCT GG;
V
, 5`-GTC CTG TCA CAG GTA CAG CTG CTC GAG TCA GG;
V
, 5`-AG GTG CAG CTG CTC GAG TCT GG; V
,
5`-CAG GTG CAG CTG CTC GAG TCG GG; and the - or -chains
(V1, 5`-GAG CCG CAC GAG CCC GAG CTC CAG ATG ACC CAG TCT CC;
V1a, 5`-GAC ATC GAG CTC ACC CAG TCT CCA; V2a, 5`-GAG CCG CAC
GAG CCC GAG CTC GTG ATG AC(C/T) CAG TCT CC; V3a, 5`-GAA ATT GAG
CTC ACG CAG TCT CCA; V3, 5`-GAG CCG CAC GAG CCC GAG CTC GTG
(A/T)TG AC(A/G) CAG TCT CC; V1, 5`-AAT TTT GAG CTC ACT CAG CCC
CAC; V3, 5`-TCT GTG GAG CTC CAG CCG CCC TCA GTG) were then used in
a 100-µl hot start PCR amplification at the following conditions: 1
cycle of 5 min at 95 °C for denaturation, 50 s annealing at 54
°C, and 50 s elongation at 72 °C followed by 40 cycles: 1 min
denaturation at 92 °C, 50 s annealing at 54 °C, and 50 s
elongation at 72 °C. PCR reactions were done using a combination of
each constant region and variable region primer and pooled for the
construction of the library. The sequences of oligonucleotide primers
of the epsilon chains and light chains were synthesized according to
(Kabat et al., 1987). Oligonucleotide primers specific for the
variable region of the heavy chains and variable and constant regions
of the - and -chains were synthesized according to Persson et al.(1991) and Kang et al. (1991b).Construction of the IgE Combinatorial Library in Plasmid
pComb3H
The PCR products coding for IgE Fds and light chains
were ethanol-precipitated, gel-purified, and cut with SpeI/XhoI and SacI/XbaI,
respectively (Boehringer Mannheim). The digested PCR products were
again ethanol-precipitated and gel-purified. For the construction of
the IgE combinatorial library, the light chains were first ligated into
the SacI/XbaI site of pComb3H and transformed into Escherichia coli XL-1Blue to yield a light chain library of 3
10 independent clones. The plasmid DNA containing
the light chain library was then isolated, cut with SpeI/XhoI to release the heavy chain stuffer, and
gel-purified. The ligation of the cDNAs coding for the IgE Fds into the
light chain plasmid yielded a library of 5 10 independent primary clones. All molecular biological
manipulations used for the construction of the IgE combinatorial
library followed the protocols of the Cold Spring Harbor Course on
Monoclonal Antibodies from Combinatorial Libraries by Carlos F. Barbas
and Dennis R. Burton.Isolation of Phage Clones Expressing Fab Fragments with
Specificity for the Major Timothy Grass Pollen Allergen Phl p 5 by
Panning
ELISA plates (Costar 3690, Cambridge, MA) were coated
with purified recombinant timothy grass pollen allergens rPhl p 1, rPhl
p 2, and rPhl p 5, respectively (0.2 µg/well). The wells were
blocked with phosphate-buffered saline containing 3% (w/v) bovine serum
albumin. Freshly prepared phage suspension (approximately 10
plaque-forming units) was added to each well and incubated at
room temperature for 2 h. The phage were then removed, and the wells
were washed with Tris-buffered saline containing 0.05% (v/v) Tween 20
once. Phage were eluted with 0.1 M glycine-HCl, pH 2.2,
containing 1 mg/ml bovine serum albumin, and the eluent was neutralized
with 2 M Tris. Freshly grown E. coli XL-1Blue were
then infected with the eluted phage. An aliquot was used to determine
the titer of infected E. coli. The culture was grown in SB
medium containing 50 µg/ml ampicillin and 10 µg/ml tetracyclin.
By infection with helper phage VCS M13, filamentous phage were produced
for the next round of panning as described (Barbas et al.,
1991). The panning was repeated four times. During the subsequent
pannings, additional washing of the wells was done and individual
clones were then analyzed for the production of Phl p 5-specific Fabs
by ELISA.Sequence Analysis of the cDNAs Coding for IgE Fds and
Light Chains
Clones 5, 14, 28, and 31 were checked for the
production of Phl p 5-specific Fabs by ELISA and for the correct
insertion of cDNAs coding for heavy chain fragments and light chains by
restriction analysis before sequencing. Plasmid DNA was prepared from
recombinantE. coli XL-1 Blue using Qiagen tips (Hilden,
Germany). Both DNA strands were sequenced using 
S-dCTP
(DuPont NEN) and a T7 polymerase sequencing kit (Pharmacia) by primer
walking (Sanger et al., 1977). Sequencing primers were
obtained from Pharmacia. The DNA and deduced amino acid sequence of the
heavy and light chains were compared with the GenBank and Swissprot
library.Production of Soluble Recombinant Fab Fragments with
Specificity for Phl p 5
For the production of soluble Fab
fragments, DNA was isolated from several independent clones after the
fifth round of panning. The plasmid DNA was digested with SpeI
and NheI, recovered from a 1% agarose gel, self-ligated, and
retransformed into E. coli XL-1 Blue. E. coli containing the correctly religated plasmid were then used to
produce soluble Fab fragments. In brief, single colonies were
inoculated into SB medium containing 20 mM MgCl
and 50 µg/ml carbenicillin. The cultures were grown at 37
°C for 6 h and then induced by adding
isopropyl-1-thio-
-D-galactopyranoside to a final
concentration of 4 mM. Induced E. coli were then
grown at 30 °C overnight, and cells were harvested by
centrifugation at 3000 g for 10 min at 4 °C. The E. coli supernatants were used for ELISA assays,
immunoblotting, and for the affinity purification of Phl p 5-specific
Fabs.Purification of Phl p 5-specific IgE Fabs by Affinity to
Purified Recombinant Phl p 5
2.5 mg of purified recombinant Phl
p 5 was coupled to an AminoLink(TM) column (Pierce) according to the
manufacturer's advice. Approximately 200 ml of E. coli supernant containing Phl p 5-specific Fabs were centrifuged at
20,000 g and subsequently filtered through folded
filters (Macherey-Nagel, Düren, Germany) to remove
debris from the solution. The supernatants were applied to the column
at 4 °C, and the column was then washed extensively with
phosphate-buffered saline until no protein could be detected by
photometry at 280 nm in the wash fractions. Bound Phl p 5-specific Fabs
were eluted with 100 mM glycine-HCl, pH 2.7, and neutralized
in 3 M Tris, pH 9.Immunoblotting: IgE Competition Studies
Natural
grass pollen extracts were prepared from rye grass (Lolium
perenne), rye (Secale cereale), Kentucky Bluegrass (Poa pratensis), timothy grass (Phleum pratense), and
birch (Betula verrucos) pollen purchased from Allergon,
Välinge, Sweden (Vrtala et al., 1993b).
Recombinant timothy grass pollen allergens were purified as described
(Vrtala et al., 1996). Approximately 100 µg of natural
pollen extract/cm of preparative 12% SDS-PAGE (Fling and Gregerson,
1986) were applied, whereas 1 µg/cm gel of the purified recombinant
allergens was separated. Proteins were electroblotted to nitrocellulose
(Towbin et al., 1979), and nitrocellulose strips were
incubated with 1:10 diluted sera from allergic patients followed by 
I-labeled anti-human IgE antibodies (Pharmacia). For IgE
inhibition studies, nitrocellulose-blotted recombinant Phl p 5 was
preincubated with the Phl p 5-specific Fabs. Fab preparations with
specificity for a different allergen (Bet v 1) were included as
negative controls. IgE binding was performed as described above and
visualized by autoradiography.
Determination of the Patients' IgE Reactivity
Profile to Grass Pollen Allergens
Serum from the donor who was
used for the construction of the IgE combinatorial library was tested
for the presence of grass pollen-specific IgE. Fig. 1shows that
the patient displayed IgE cross-reactivity to natural grass pollen
extracts from rye grass, Kentucky Bluegrass, rye, and timothy grass.
Most of the grass pollen-specific IgE bound to proteins of
approximately 30 kDa, which represent group 1 and group 5 allergens
(Laffer et al., 1994; Vrtala et al., 1993a). The
testing with recombinant Phl p 1 (lane 5) and Phl p 5 (lane 7) indicated that most of this binding was due to the
reactivity to group 5 allergens.
I labeled anti-human IgE monoclonal antibodies. The
position of group 1 and group 5 allergens at approximately 30 kDa is
indicated.
PCR Amplification of cDNAs Coding for IgE Fds and Light
Chains from Peripheral Blood Lymphocytes of a Grass Pollen Allergic
Individual
Starting from RNA of peripheral blood lymphocytes of
the grass pollen allergic donor, cDNAs coding for IgE Fds could be
amplified. Fig. 2shows the successful PCR amplification of IgE
Fds using primers specific for different V families
(V1-V6) and a primer located in the first
constant region of human IgE. RNA was isolated at different times of
the year during 2 years, and it is noteworthy that the efficacy of the
PCR amplification was best during the grass pollen season. Since the
patient did not receive hyposensitization therapy, it was assumed that
the increased IgE production during the grass pollen season was mostly
due to stimulation by allergen contact. The obtained PCR product
therefore may contain a high proportion of allergen-specific IgE Fds.
The PCR products were confirmed to represent IgE-Fd encoding fragments
by differential hybridization using synthetic oligonucleotides specific
for IgE and IgG as described (Steinberger et al., 1995).
family primers (lane 1, V1; lane 2,
V2; lane 3, V3; lane 4,
V4; lane 5, V5; lane 6,
V6).
Construction and Characterization of an IgE Combinatorial
Library from a Grass Pollen Allergic Patient
Subcloning of light
chain cDNAs into plasmid pComb3H yielded a plasmid library consisting
of 3 10 primary clones. The subsequent ligation of
the IgE-Fd cDNAs led to the construction of a combinatorial library of
approximately 5 10 primary clones which had
contained heavy and light chain cDNAs. Fig. 3shows the
restriction analysis of 20 randomly isolated clones of the IgE
combinatorial library. Only seven clones did not contain both cDNAs
coding for the heavy chain fragment and light chain, indicating that
65% of the clones had combined correctly both cDNAs.
Isolation and Characterization of Recombinant Human IgE
Fabs with Specificity for the Major Timothy Grass Pollen Allergen Phl p
5
Filamentous phage expressing IgE Fabs on their surface were
panned five times using ELISA plate-immobilized recombinant Phl p 5,
Phl p 1, Phl p 2, and bovine serum albumin as a control. After the last
round of panning, clones were converted by restriction with SpeI and NheI to produce soluble Fabs. Supernatants
from 20 clones were then tested by ELISA for the presence of Phl p
5-specific Fabs, among which four Phl p 5-specific clones designated
clone 5, 14, 28, and 31 were characterized in detail. The specificity
of the supernatants for Phl p 5 was confirmed by testing them for
binding to recombinant Phl p 1, Phl p 2, Phl p 5, timothy grass pollen
extract, and birch pollen extract. Supernatants from these clones bound
to recombinant Phl p 5 and timothy grass pollen extract but not to any
of the other proteins tested. No differences in the binding to native
ELISA plate-immobilized, nitrocellulose-blotted Phl p 5 were observed. constant region, indicating a
positive selection for these particular IgE Fds during the panning
process. The parts of the C domain were completely identical with
known human IgE sequences (Flanagan and Rabbitts, 1982; Kurokawa et
al., 1983). A molecular mass of 24.2 kDa could be predicted for
the Fd-fragments of clones 5 and 28, whereas a molecular mass of 22.5
kDa could be deduced for clones 14 and 31, which were generated by a
constant region primer located closer to the variable region (Fig. 4).
1 and C2 portion, the
framework regions (FR), and the complementarity determining
regions (CDR1-CDR3) are indicated. The SpeI and XhoI sites are printed in italics, and the regions
corresponding to the constant region primers are underlined.
The cDNA sequences of the heavy chain fragments from clones 5, 14, 28,
and 31 were found to be identical, although they were generated with
two different primers.
light chains were used by the four clones.
Most of the differences in the nucleotide sequences of the framework
regions were silent. Regarding the CDRs of the four light chains, most
differences were found in the CDR3 and CDR1. In conclusion, the panning
procedure with recombinant Phl p 5 had enriched different IgE Fabs,
which used identical heavy chain fragments that had combined with
different light chains.
), framework
regions (FR), and CDRs are
indicated.
Recombinant Human IgE Fabs Specific for Phl p 5
Cross-react with Group 5 Allergens from Three Different Grass
Species
More than 80% of grass pollen allergic patients display
IgE cross-reactivity to group 5 allergens (Vrtala et al.,
1993a). cDNAs coding for functional recombinant group 5 allergens were
isolated from these species (Singh et al., 1991; Silvanovic et al., 1991; Vrtala et al., 1993a) and shown to be
highly homologous. In order to investigate whether the recombinant
human IgE Fabs that were isolated by panning to recombinant Phl p 5
cross-react with natural group 5 allergens from different grass
species, grass pollen extracts from rye grass, Kentucky Bluegrass, rye,
and timothy grass were probed. Fig. 6shows that supernatants
containing soluble Fabs from clones 5 and 28 reacted with
nitrocellulose blotted rye grass (L. perenne), Kentucky
Bluegrass (P. pratensis), rye (S. cereale), and
timothy grass (P. pratense) pollen extract at approximately 30
kDa, which corresponds to the molecular mass of group 5 allergens.
Additional weak binding to a 17-20 kDa component was observed in
Kentucky Bluegrass and rye. Group 6 allergens, which share a high
degree of sequence homology with group 5 allergens, were described to
migrate at that molecular mass; however, the question whether group 6
allergens represent cleavage products of group 5 allergens or
cross-reactive allergens is not yet clear (Matthiesen et al.,
1993).
Purification of Soluble Recombinant IgE Fabs Specific for
Phl p 5
For the purification of soluble IgE Fabs with
specificity for Phl p 5, we used a single-step affinity purification
procedure starting from E. coli supernatants that contained
soluble Fabs. Purified recombinant Phl p 5 was coupled to an
AminoLink(TM) column (Pierce), and E. coli supernatants
containing soluble Phl p 5-specific Fabs were applied to the column. Fig. 7A shows a Coomassie Blue-stained SDS-PAGE of
purified Fab preparations from clone 5 and 31 under reducing
conditions. Heavy and light chains comigrated at approximately 25 kDa,
and two weak bands were observed at approximately 15 and 16 kDa. The
slight difference in the molecular masses of clones 5 and 31 is due to
the fact that the cDNAs coding for the Fds were generated with
different PCR primers. The Western blot in Fig. 7B containing the purified recombinant Fabs was probed with a goat
anti-human Fab antiserum. The supernatant fraction contained low
amounts of Fab, and no Fab was detected in the column wash fraction. In
the elution fractions, a prominent 25-kDa band and bands at 15 and 16
kDa were bound by the anti-Fab antiserum, indicating that the lower
molecular mass components observed in the Coomassie gel in Fig. 7A represented cleavage products of the Fab. The
purified IgE Fab preparation contained approximately 0.1 mg/ml purified
Fab and could be used to detect nitrocellulose blotted recombinant Phl
p 5 up to dilutions of 1:50000 (data not shown). A single purification
procedure starting from about 200 ml E. coli supernatant
yielded approximately 0.5 mg of pure and soluble recombinant Fab.
Recombinant IgE Fabs Specific for Phl p 5 Compete with
Allergic Patients IgE Binding
Using competition experiments, it
was investigated whether recombinant Phl p 5-specific IgE Fabs might be
able to compete with grass pollen allergic patient IgE antibodies.
Pairs of nitrocellulose strips containing equal amounts of blotted
recombinant Phl p 5 were preincubated with supernatants from two Phl p
5-specific Fab clones (Fig. 8, 5 and 31) or
with supernatants from E. coli expressing Bet v 1-specific
Fabs (Fig. 8, Co). The nitrocellulose strips were then
incubated with serum IgE from two patients (A and B), and bound IgE was
detected. Preincubation of Phl p 5 with the Phl p 5-specific IgE Fabs
led to a weak but clearly visible reduction of IgE binding compared to
the control. The weak competition was not unexpected in view of the
fact that Phl p 5 harbors multiple IgE epitopes (Bufe et al.,
1994).
I-labeled anti-human IgE monoclonal
antibodies.
)It appeared hence that the
repertoire represented in the IgE combinatorial library closely
reflected the natural IgE antibody response of the patient.2-c4 domain, the
recombinant IgE Fabs were ineffective to trigger basophil degranulation
in combination with purified Phl p 5 (data not shown). In addition it
could be shown that the IgE Fabs were able to compete with the IgE
binding to Phl p 5 using sera from grass pollen allergic individuals.
The inhibitory effect was, however, very weak, which was not surprising
in view of the fact that Phl p 5 bears a number of different IgE
epitopes (Bufe et al., 1994) and on the basis that the Phl p
5-specific IgE-response in patients is polyclonal. Despite this, we
believe that the use of the combinatorial approach to define
recombinant Fabs with specificity for major allergens may have possible
therapeutic implications, as were discussed for antibodies directed
against tumor or viral antigens (Waldmann, 1991). In the case of the
major birch pollen allergen, Bet v 1, human and mouse monoclonal
antibodies could be defined that strongly inhibited the binding of
patients IgE to the allergen(
)(
)so that a local
application of such blocking antibodies for a passive therapy in the
allergic effector organs (nose, eyes, and lung) might be envisaged
(Valenta et al., 1994). Using the cDNAs coding for
allergen-specific Fabs, procedures such as in vitro affinity
maturation might be used to ``improve'' the antibodies for
such therapeutic applications (Barbas et al., 1994). Apart
from the possible therapeutic implications, we believe that the
combinatorial approach will allow the study of the interaction of human
IgE antibodies and allergens at the molecular level by using purified
recombinant allergens and human IgE Fabs for structural analysis (x-ray
crystallography and NMR).
))))
We thank Dr. Markus Susani (Advanced Biological
Systems, Institute of Molecular Biology) for the purification of
recombinant Phl p 5. We are grateful to D. Burton (The Scripps Research
Institute, La Jolla, CA) for the generous gift of several primers and
thank C. Barbas (The Scripps Research Institute) and Monique Vogel
(Institute of Clinical Immunology, Bern, Switzerland) for useful
discussions.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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