AU684269B2 - Recombinant timothy grass pollen allergen Ph1 p II - Google Patents

Abstract

A recombinant DNA molecule codes for a peptide with the antigenicity of timothy grass pollen allergens Phl p II. The amino acid sequence (5) and the most important B cell and T cell epitopes of the molecule are derived therefrom. The recombinant Phl p II allergen is expressed in Escherichia coli and binds serum IgE of more than 60 % of all those allergic to grass pollen and may therefore be used in the same way as the naturally occurring Phl p II for processes based on antigen-antibody interaction, mediator release and T cell reactivity.

Description

WO 94/23035 PCT/AT94/00039 Recombinant timothy grass pollen allergen Phl p II Grass pollen allergies are among the most important plant allergies occurring during the summer. More than 20% of individuals allergic to pollens show allergic symptoms to grass pollen allergens. The most important grass pollen allergens include Group I Group V Group II/III and Group LV 5) allergens. Meanwhile profilin, too, was discovered to be a grass pollen allergen The aforementioned allergens may be found as immunologically or structurally closely related molecules in pollen of different grass species, and related allergens of one group show cross reactivity with patient IgE. So far, a number of these allergens have been isolated by recombinant techniques and expressed in E. coli Many of the allergens expressed in E. coli show properties similar to those of natural proteins, and can therefore be used for the diagnosis and therapy of allergic diseases (10, 11, 12). Now, described for the first time is a molecular characterization of a complete cDNA which codes for Phl p II, and the expression of this protein in E. coll. Until now a complete recombinant Group II/III grass pollen allergen was not available; and, as can be seen from the examples, it can be used like the natural protein for processes based on antigen-antibody interaction, such as are used for cellular processes, because all T-cell epitopes are present in the recombinant molecule, as in the natural molecule. Furthermore, the recombinant Phl p II is suitable for processes which result in measurable mediator release. The therapeutic use of recombinant Phl p II, based on an effect on immunoregulatory processek antigen-antibody interaction, T-cell reactivity and mediator release, i the consequence of the structural and biological similarity between tLne natural and the recombinant proteins. The present invention provides a complete cDNA which codes :or a recombinant Phl p II allergen. On the basis of the amino acid sequence deduced from the cDNA, B-cell a .d T-cell epitopes of the Phl p II allergens are determined. The recombinant Phl p II allergen was prepared in E. coli and has properties similar to those of natural Group II/III grass pollen allergens. From this it follows that the recombinant Phl p II allergen, like the natural Group II/III allergens, can be used for processes based on antigen-antibody interaction, antigen-dependent T-cell action or antigen-dependent e I I mediator release, where, however, the recombinant allergens have the additional advantage of greater purity and higher specificity.

SUMMARY OF THE INVENTION According to a first aspect the present invention consists in an isolated and purified nucleic acid sequence coding for a polypeptide which possesses the antigenicity of the allergen Phl p II, or for a peptide which has at least an epitope of Phi p II, wherein the nucleic acid sequence is homologous to the total sequence shown in Figure 1 or with partial regions thereof, or wherein the nucleic acid sequence is a degenerate sequence of the sequence shown in Figure 1 or a partial region thereof.

According to a second aspect the present invention consists in an isolated and purified nucleic acid sequence which hybridises under stringent conditions to a nucleic acid sequence according to the first aspect.

According to a third aspect the present invention consists in a recombinant DNA molecule comprising a nucleic acid sequence according to the first or the second aspect.

According to a fourth aspect the present invention consist, in a host system, which is transformed with a recombinant DNA molecule according to the third aspect.

According to a fifth aspect the present invention consists in a recombinant or synthetic protein or polypeptide derived from a nucleic acid according to the first aspect .0 or a molecule according to the third aspect, which possesses the antigenicity of Phi p II S: 20 or at least of an epitope thereof.

According to a sixth aspect the present invention consists in a diagnostic or therapeutic reagent, comprising a synthetic protein or polypeptide according to the fifth aspect.

o: According to a seventh aspect the present invention con, 'sts in a method for in vitro detection of a patient's allergy to the Phi p II allergen, comprising the step of measuring the ieaction of the IgE antibodies in the patient's serum with a recombinant or synthetic protein or polypeptide according to the fifth aspect.

According to an eighth aspect the present invention consists in a method for the in vitro detection of the cellular reaction to the Phi p II allergen, comprising the step of 17729-00 DOC/jp I stimulating or inhibiting the cellular reaction with a recombinant or synthetic protein or polypeptide according to the fifth aspect.

According to a ninth aspect the present invention consists in a method for the treatment of a mammal suffering from a pollen allergy, compilsing the administration to said mammal of a recombinant or synthetic protein or polypeptide according to the fifth aspect.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Material and Methods 1. Construction of the cDNA gene library Timothy grass pollen (Allergon AB Engelholm, Sweden), which had been investigated for purity by means of light microscopy and electron microscopy, was used for the isolation of polyadenylated RNA cDNA synthesis was carried out with oligo-dT and random primers; the ends of the cDNA were digested with T4 polymerase and provided with EcoRI linkers. The linkered cDNA was ligated into dephosphorylated lambda gtl 1 arms and packaged. A cDNA gene library of 800,000 independent clones was obtained (13).

2. Screening of the cDNA gene library, subcloning. and DNA sequence analysis IgE screening of the timothy grass pollen cDNA gene library was carried out as described by Breiteneder et al. IgE-binding clones were enriched and phage-DNA was prepared from these clones By means of Kpn I and Sac I cuts two DNA fragments were obtained which contained both portions of the complete Phi p II cDNA and flanking lambda gtl 1 sequences. The resulting KpnI/SacI and Sad DNA fragments were subcloned into the plasmid pUC 18, and transformed into E. coli XL-1 Blue.

Suitable clones were identified by restriction analysis and both strands were sequenced by means of lambda gtl 1 forward sequencing primer (22-mer) and lambda gtl 1 reverse sequencing primer (22-mer), Clontech Laboratories, Palo Alto, USA, and by means of M13 pUC18 forward and reversed primers, Boehringer-Mannheim, Germany, according to Sanger (16).

3. RNA (Northern) blots 10 mog of total RNA from pollen of timothy grass (Phleum pratense) and rye grass (Lolium perenne) was separated by denaturing gel electrophoresis, and blotted onto 17729-00 DOC/jp p--~a nitrocellulose To isolate the cDNA coding for Phl p II, the corresponding DNA insert was amplified by PCR, starting from recombinant phages. 5 picomoles of primer (lambda gtl 1 forward sequencing primer (22-mer) and lambda gtl 1 reverse sequencing

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17729-00 DOC/jp C- C- primer (22-mer), Clontech Laboratories, USA), were used in each case.

The PCR product was applied to 1% agarose gel and the bands were eluted by means of a DEAE ion exchanger paper The DNA obtained was 31P labeled by random priming Prehybridization and hybridization were carried out by standard methods The blots were washed with x SSC (20 x SSC 3 M NaC1, 0.3 M Na citrate, pH 0.1% SDS (sodium dodecyl sulfate); 1.5 SSC, 0.1% SDS; and finally with 0.75% SSC, 0.1% SDS at 50"C, and then autoradiographed (Hyperfilm MP, Amersham, London, UK).

4. Expression of Phl p II cDNA in lysogenic E. coli Y 1089 as galactosidase fusion protein By means of IgE screening a complete cDNA clone was obtained which codes for a Phi p II allergen. The lysogenic E. coli strain Y 1089 was infected with recombinant lambda gtll and the p-galactosidase fusion protein was obtained from the batch The batch was separated electrophoretically in a 7.5% polyacrylamide gel (21) and blotted onto nitrocellulose The fusion protein was detected by means of serum IgE of patients allergic to grass pollen and an iodine-labeled rabbit antihuman IgE antibody (Pharmacia, Uppsala, Sweden).

The attached Figures serve to illustrate the usability of the recombinant Phl p II allergen for processes based on antigen-antibody interaction, antigen-dependent mediator release, and antigen-dependent cellular reactivity. Fig. 1 shows the cDNA of the recombinant Phl p II allergen and the latter's amino acid sequence deduced f;om said DNA.

The complete cDNA sequence of the Phl p II allergen is shown. A 24-base noncoding sequence was found at the 5' end of the cDNA, which is followed by a 78-base leader sequence, which codes for the signal peptide underlined in the Figure The amino acid sequen!ce of the mature protein can be deduced from base 78 on, and begins with valine.

The stop codon TAG, which breaks off the coding sequence at the 3' end, is denoted by an asterisk. The 3 noncoding sequence ends in the poly A tail.

Fig. 2 shows the similarity of the amino acid sequence of the recombinant Phi p II allergen to other Group II/III grass pollen allergens.

^/V'r Fig. 3 presents the description of B-cell epitopes of the recombinant Phl p II allergen, and Fig. 4 that of the T-cell epitopes.

Fig. 5 shows the reactivity of the recombinant Phl p II allergen with serum IgE of patients allergic to grass pollen. Recombinant Phi p II was expressed as P-galactosidase fusion protein in lysogenic E. coli Y 1089 by phage infection and induction with IPTG. The B. coli proteins, which contain the recombinant Phl p II, were electrophoretically separated and transferred onto nitrocellulose. Used in Lane 1 was serum IgE of a Group II/III-reactive patient; while in Lanes 2 and 3 the detection was done with IgE of allergic patients who show no reaction with Group II/III allergens; Lane 4 shows the control experiment with serum of a nonallergic control person.

Fig. 6 presents a table which lists the percentage of grass pollenallergic patients with IgE reactivity with specific grass pollen allergens. The frequency of reactivity of grass pollen-allergic patients with different grass pollen allergens is shown. The figures represent approximate values obtained by tests with natural and recombinant grass pollen allergens in a representative number of patients.

Fig. 7 proves that recombinant Phl p II carries the same IgE epitopes as [is the case with] natural Group II/III allergen/IgE-inhibition. The serum of a patient with grass pollen allergy, who shows IgE reactivity with Group II/III allergens (10-12 kD) and Group I allergens (at about kD), was preincubated with recombinant Phl p II (Lane 2),ecombinant Phl p I (Lane recombinant Bet v I (Lane 4) or E. coli proteins (Lane Preincubation with recombinant Phl p II makes the IgE binding to natural Phl p II almost completely disappear in the 12 kD region, whereas the reactivity with Phl p I at 30 kD I.s not affected.

Recombinant Phl p I reduces only the binding to natural Phl p I, but has no effect on the IgE binding to Phl p II. The preincubation with control proteins, recombinant Bet v I and E. coli proteins does not impair the IgE binding to natural Phl p I and Phl p II. From this it follows that recombinant Phl p II has epitopes that are similar or identical to those of natural Phl p II, but there is no antigenic relatedness to natural or recombinant Group I allergens.

Fig. 8 shows the hybridization of the cDNA coding for Phl p II with mRNA from timothy grass and rye grass. Total RNA was isolated from rye grass

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9> s I pollen (Lane 1) and timothy grass pollen (Lane 10 mcg was separated in denaturing agarose gel and blotted onto nitrocellulose. The 32

P-

labeled cDNA, coding for Phi p II, hybridizes with both rye grass RNA and timothy grass RNA at approximately the level of 18S RNA, but even distinctl, below that, at a transcript size of about 600 bases. The cross hybridization shows the structural similarity of the transcripts which code for Group II/III allergens in different grass species.

Figures 9 A and 9 B show the reactivity of the recombinant Phi p II allergen with an antibody specific to Group II/III grass pollen allergens.

A: Lambda gtll phages, which express timothy grass pollen allergens and the principal birch tree allergen, Bet v I as well as nonrecombinant phages were tested by the dot blot process with antibodies specific to grass pollen allergens. 4B 1 binds to Group V allergens, R4 detects Group I allergens, and R5 identifies Group V and Group II/III allergens.

B: The diagram illustrates the clone designation. Recombinant Phl p II is expressed by clone A.

Examples Sequence analysis of the recombinant Phl p II allergen Similarity to other Group II/III allergens The DNA sequence of the Phl p II allergen was determined by sequencing the cDNA according to the method of Sanger Fig. 1 shows the determined DNA sequence and the amino acid sequence deduced therefrom.

A signal peptide in front of the mature protein, which peptide shows significant homology with other eukaryotic signal peptides, unequivocally proves that Group II/III allergens of distinct gene fragments are coded and do not result from Group I allergens through proteolytic decomposition. The high degree of sequence homology (about 70% sequence identity) of the recombinant Phl p II with the homologous proteins from rye grass (Lol p II and Lol p III) (Fig. 2) shows the close structural relatedness of these proteins and thereby provides the molecular basis for the immunological relatedness of Group II/III allergens of different species.

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I- Determination of B-cell and T-cell epitopes of the recorr' ,nt Phl p II allergen On the basis of the deduced amino acid sequence of the Phi p II allergel the B-cell and T-cell epitopes were determined with the aid of appropriate computer programs (24, 25). The relevant B-cell and T-cell epitopes are compiled in Figurea 3 and 4. Synthetic peptides which correspond to B-cell epitopel3, bind IgE of grass pollen-allergic patients, whereas synthetic peptides, which correspond to T-cell epitopes, stimulate T-cells of grass pollen-allergic patients to proliferate, and result in an increased H3-thymidine uptake.

Expression of the cDNA coding for Phl p II in E. coli as recombinant Phi p II allergen Recombinant phages containing the cDNA for Phl p II were used for infection with lysogenic E. coll Y 1089. Recombinant P-galactosidase fusion protein was obtained by induction with IPTG (isopropyl-pthiogalactoside) in liquid culture The control protein 0galactosidase showed no IgE binding in the Western blot test, whereas the recombinant Phl p II fusion protein showed distinct IgE binding specifically to serum IgE of patients who reacted with Group II/III allergens of different grass species.

IqE-binding capacity of the recombinant Phi p II allergen Serum obtained from a grass pollen-allergic patient who reacts with natural Group II/III allergens, was preincubated with recombinant Phl p II, whereby Group II/III-specific IgE was bound. Fig. 6 shows that the binding to natural Group II/III allergens is almost completely annulled by the preincubation, .ereas the binding to Group I and Group V allergens was not affected. This shows that the IgE epitopes of natural Group II/III allergens are blocked by recombinant Phi p II, and that there is hardly any relevant cross reactivity between Group I, Group V and Group II/III allergens.

Cross hybridization of Phl p II CDNA with Lol p II/III mRNA Total RNA from rye grass and timothy grass pollen was isolated, separated in denaturing agarose gel and transferred onto nitrocellulose membranes. The membrane was hybridized with a complete cDNA which codes G LU -o

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7 for Phl p II. Hybridizing bands are always found at the level of the 18S ribosomal RNA and also considerably below that. The hybridization is clearly more intensive with timothy grass RNA than with rye grass RNA, although, according to the gel staining with ethidium bromide, about the same amounts of total RNA were used. Nevertheless, cross hybridization could be attained under stringent conditions. Under certain circumstances the larger transcript represents a larger and still immature RNA, since the hybridization withstands even stringent washing. This example is intended to document the homology of the Group II/III grass pollen allergens of different species.

WO 9412.3035 WO 943035PTIAT94/0 0 03 9 Bibliography 1 .Freidhoff, Ehrlich-Kautzky.. E.,Grant, Meyers, and Marsh, D.G. (1986) J Allergy Guin Immunol 78, 1190-1201 2. Matthiesen, Lowenstein, H. Clin Exp Allergy 21, 309-320.

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264, 11181-11185 4. Kisil, F. Jaggi, K. Lin, Z. Ekramodullah, A. K. M. (1989) in Sehon A. Kraft, Kunkel, G. eds. Epitopes of Atopic Allergens, 22-25.

Jaggi, K. Ekramodullah, A. K. Kisil, F. Dzuba-Fischer, J. M.

Rector, E. and Sehon, A. H. (1989) J Allergy Clin Immunol 83, 845-852.

6. van Ree, Driessen, N. B. van Leeuwen, W. Stapel, S. and Aalberse, R. C. (1992) Clin Exp Allergy.

7. Valenta, Duch~ne, Ebner, Valent, Sillaber, Deviller, Ferreira, Tejkl, Edeimann, Kraft, and Scheiner, 0. (1992) 3 Exp Med 175, 377-385.

S. Valenta, Duch~ne, Vrtala, Valent, Sillaber, Ferreira, Tej1l, Hirschwehr, Ebner, Kraft, and Schemner, 0. (1993) Tnt Arch Allergy Appl Immunol 99, 271-273.

9. Scheiner, Bohle, Breitenbach, Breitene-der, Duch~ne, M., Ebner, Ferreira, Hirschwehr, Hoffmann-Sommergruber, K., Pettenburger, Rumpold, Steiner, Tejkl, Valenta, and Kraft, D.

(1992) in: Advances in Allergology and Clinical Immunology, Godard, P., Bousquet, and Michel, F. B. eds. The Parthenon Publishing Group.

Valenta, Duch~ne, Vrtala, Birkner, Ebner, C., Hirschwehr, Breitenbach, Rumpold, Schemner, and Kraft, D. (1991) J Allergy Guin Immunol 88, 889-894.

11. Valenta, Vrtala, Ebner, Kraft, and Scheiner, 0. (1992) Tnt Arch Allergy Appl Immunol 97, 287-294 WO 94123035 PCT/AT94/00039 12. Valenta, Sperr, W. Ferreira, Valent, Sillaber, Tejkl, Duch~ne, Ebner, Lechner, Kraft, and Schemner, 0. (1993). j Allergy Guin Immunol 91, 88-97.

13. Vrtala, Sperr, W. Reimitzer, vanRee, Laffer, Mfiller, Valent, Lechner, Rumpold, Kraft, Schemner, and Valenta, R. (1993) 1 Immunol. (accepted provided revision).

14. Breiteneder, Pettenburger, Bito, Valenta, Kraft, D., Rumpold, Schemner, and Breitenbach, M. (1989) EMBO J 8, 1935-1938 Ausubel, F. M. (1990) in Current protocols in molecular biology, Wiley, New York.

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Claims (9)

1. An isolated and purified nucleic acid sequence coding for a polypeptide which possesses the antigenicity of the allergen Phi p II, or for a peptide which has at least an epitope of Phl p II, wherein the nucleic acid sequence is homologous to the total sequence shown in Figure 1 or with partial regions thereof, or wherein the nucleic acid sequence is a degenerate sequence of the sequence shown in Figure 1 or a partial region thereof.

2. An isolated and purified nucleic acid sequence which hybridises under stringent conditions to a nucleic acid sequence according to claim 1.

3. An isolated and purified nucleic acid according to claim 1 or claim 2, wherein the allergen is derived from a monocotyledonous plant.

4. An isolated and purified nucleic acid sequence according to claim 3, wherein the allergen is derived from timothy grass pollen.

A recombinant DNA molecule comprising a nucleic acid sequence according to any one of claims 1 to 4.

6. A recombinant DNA molecule according to claim 5, wherein the nucleic acid is functionally combined with an expression control sequence to form an expression construct.

7. Host system, which is transformed with an expression construct according to claim 6.

8. Recombinant or synthetic protein or polypeptide derived from a nucleic acid according to any one of claims 1, 3 or 4, or a DNA molecule according to claim 5 or claim 6, which possesses the antigenicity of Phl p II or at least of an epitope thereof.

9. Recombinant or synthetic protein or polypeptide according to claim 8, having an amino acid sequence which fully corresponds to the sequence shown in Figure 1. Recombinant or synthetic protein or polypeptide according to claim 8 or claim 9, which is a fusion product possessing the antigenicity of the Phl p II allergen of timothy grass and has an additional polypeptide part, the whole fusion product being coded by the DNA of an expression construct according to claim 6. *u 0 0 0 00 0 D 0

17729-00.DC/jp I i I 11. Recombinant or synthetic protein or polypeptide according to claim 10, wherein said additional polypeptide part is P-galactosidase or another polypeptide suitable for fusion. 12. Diagnostic or therapeutic reagent, comprising a synthetic protein or polypeptide according to any one of claims 8 to 11. 13. A method for in vitro detection of a patient's allergy to the Phl p II allergen, comprising the step of measuring the reaction of the IgE antibodies in the patient's serum with a recombinant or synthetic protein or polypeptide according to any one of claims 8 to 11. 14. A method for the in vitro detection of the cellular reaction to the Phl p II allergen, comprising the step of stimulating or inhibiting the cellular reaction with a recombinant or synthetic protein or polypeptide according to any one of claims 8 to 11. A method for the treatment of a mammal suffering from a pollen allergy, comprising the administration to said mammal of a recombinant or synthetic protein or polypeptide according to any one of claims 8 to 11. 16. An isolated and purified nucleic acid sequence coding for a polypeptide which possesses the antigenicity of the allergen Phi p II, substantially as herein described with reference to the Examples or any one of Figures 1 or 3 to S' DATED this 14th day of August 1997 20 BIOMAY PRODUKTIONS-UND HANDELSGESELLSCHAFT M.B.H. I to. Attorney: IAN T. ERNST Fellow Institute of Patent Attorneys of Australia of SHELSTON WATERS I* *eg. 17729-00 DOC/jp ij c