(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 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.
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
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).
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 ()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).
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 receptor binding site, may be envisaged as
potential therapeutic tools that can compete with natural IgE
antibodies for the allergen binding.
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 I labeled anti-human IgE monoclonal antibodies. The
position of group 1 and group 5 allergens at approximately 30 kDa is
indicated.
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 family primers (lane 1, V
1; lane 2,
V
2; lane 3, V
3; lane 4,
V
4; lane 5, V
5; lane 6,
V
6).
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 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).
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 C1 and C
2 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.
As can be seen in Fig. 5(A and B), different 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.
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), framework
regions (FR), and CDRs are
indicated.
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 I-labeled anti-human IgE monoclonal
antibodies.
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. ()It appeared hence that the
repertoire represented in the IgE combinatorial library closely
reflected the natural IgE antibody response of the patient.
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 c2-c
4 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).
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].