Patent application title: RECOMBINANT ALLERGEN WITH REDUCED lgE BINDING BUT UNDIMINISHED T-CELL ANTIGENICITY
Inventors:
Nicole Deweerd (Seaford, AU)
Mohan Bir Singh (Templestowe, AU)
Prem L. Bhalla (Templestowe, AU)
Ines Swobda (Klagenfurt, AT)
IPC8 Class: AA61K3935FI
USPC Class:
4241851
Class name: Drug, bio-affecting and body treating compositions antigen, epitope, or other immunospecific immunoeffector (e.g., immunospecific vaccine, immunospecific stimulator of cell-mediated immunity, immunospecific tolerogen, immunospecific immunosuppressor, etc.) amino acid sequence disclosed in whole or in part; or conjugate, complex, or fusion protein or fusion polypeptide including the same
Publication date: 2010-12-02
Patent application number: 20100303843
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Patent application title: RECOMBINANT ALLERGEN WITH REDUCED lgE BINDING BUT UNDIMINISHED T-CELL ANTIGENICITY
Inventors:
NICOLE DEWEERD
MOHAN BIR SINGH
PREM L. BHALLA
INES SWOBDA
Agents:
ARENT FOX LLP
Assignees:
Origin: WASHINGTON, DC US
IPC8 Class: AA61K3935FI
USPC Class:
Publication date: 12/02/2010
Patent application number: 20100303843
Abstract:
The present invention relates generally to reagents useful in the
immunotherapeutic or immunoprophylactic treatment of allergic diseases.
More particularly, the present invention provides modified allergens
exhibiting reduced IgE interactivity including reduced IgE
production-stimulatory activity, while retaining T-cell antigenicity,
which are useful in the immunomodulation of type I allergic disease
conditions. The present invention further contemplates a method of
immunomodulation of allergic diseases such as type I allergic disease
conditions by the administration of modified allergens exhibiting reduced
IgE interactivity while retaining T-cell antigenicity.Claims:
1-29. (canceled)
30. A protein allergen variant selected from the group consisting of an Lol p 5 protein allergen variant, a Phl p 5 protein allergen variant and a Poa p 5 protein allergen variant, wherein the variant comprises a naturally occurring Lol p 5, Phl p 5 or Poa p 5 protein allergen comprising (i) mutations K->N, F->L, T->A, V->A in amino acid residues homologous to the amino acid residues K172, F173, T174 and V175 of SEQ ID NO: 1 or (ii) mutations A->G, V->A, K->A in amino acid residues homologous to the amino acid residues A204, V205 and K206 of SEQ ID NO: 1.
31. The protein allergen variant of claim 30, wherein one or more further IgE epitopes are mutated or deleted thereby increasing the hypoallergenicity of the allergen variant as compared to a naturally occurring Lol p 5, Phl p 5, or Poa p 5.
32. The allergen variant of claim 30, wherein said protein allergen in naturally occurring form is associated with type I allergic disease in sensitive subjects.
33. The allergen variant of claim 32, wherein the type I allergic disease is sensitivity to rye grass pollen.
34. The allergen variant of claim 32, wherein said sensitive subject is a human, a primate, livestock animal, laboratory test animal or companion animal.
35. The allergen variant of claim 34, wherein said sensitive subject is a human.
36. A composition comprising the allergen variant according to claim 30 and one or more pharmaceutically acceptable carriers and/or diluents.
37. A method for the prophylaxis or treatment of an allergic disease or condition comprising administering the allergen variant of claim 30 to a human or animal in need thereof.
38. The protein allergen variant of claim 30, wherein the naturally occurring Lol p 5 protein allergen comprises the amino acids of SEQ ID NO: 1 or SEQ ID NO: 2, the naturally occurring Phl p 5 protein allergen comprises the amino acids of SEQ ID NO: 3 or SEQ ID NO: 4 and the naturally occurring Poa p 5 protein allergen comprises the amino acids of SEQ ID NO: 5 or SEQ ID NO: 6.
39. The protein allergen variant of claim 31, wherein the variant comprises mutations K->N, F->L, T->A, V->A of (i) and wherein the one or more mutations or deletions comprise a mutation K->A in an amino acid residue homologous to the amino acid residue K57 of SEQ ID NO: 1 and mutations G->A, K->A in amino acid residues homologous to the amino acid residues G273 and K275 of SEQ ID NO: 1.
40. The protein allergen variant of claim 31, wherein the variant comprises mutations A->G, V->A, K->A of (ii) and wherein the one or more mutations or deletions comprise a mutation K->A in an amino acid residue homologous to the amino acid residue K57 of SEQ ID NO: 1 and mutations G->A, K->A in amino acid residues homologous to the amino acid residues G273 and K275 of SEQ ID NO: 1.
41. The protein allergen variant of claim 31, wherein the variant comprises mutations K->N, F->L, T->A, V->A of (i) and wherein the one or more mutations or deletions comprise a mutation K->A in an amino acid residue homologous to the amino acid residue K57 of SEQ ID NO: 1 and a deletion of an amino acid residue homologous to the amino acid residue G272 of SEQ ID NO: 1.
42. The protein allergen variant of claim 37, wherein the variant comprises mutations A->G, V->A, K->A of (ii) and wherein the one or more mutations or deletions comprise a mutation K->A in an amino acid residue homologous to the amino acid residue K57 set forth in SEQ ID NO: 1 and a deletion of the amino acid residue homologous to amino acid residue G272 of SEQ ID NO: 1.
43. The protein allergen variant of claim 44 comprising amino acids 26-301 of SEQ ID NO: 11 or amino acids 26-301 of SEQ ID NO: 12.
Description:
[0001]This is a Divisional Application, which claims the benefit of
pending U.S. patent application Ser. No. 10/490,305 filed Sep. 22, 2004,
which is a 371 National Stage Entry of PCT/AU02/01261 filed Sep. 13,
2002, which claims priority to Australian Patent Application No. PR 7792
filed Sep. 20, 2001. The disclosures of these prior applications are
hereby incorporated by reference herein in their entirety.
FIELD OF THE INVENTION
[0002]The present invention relates generally to reagents useful in the immunotherapeutic or immunoprophylactic treatment of allergic diseases. More particularly, the present invention provides modified allergens exhibiting reduced IgE interactivity including reduced IgE production-stimulatory activity, while retaining T-cell antigenicity, which are useful in the immunomodulation of type I allergic disease conditions. The present invention further contemplates a method of immunomodulation of allergic diseases such as type I allergic disease conditions by the administration of modified allergens exhibiting reduced IgE interactivity while retaining T-cell antigenicity.
BACKGROUND OF THE INVENTION
[0003]Bibliographic details of references provided in the subject specification are listed at the end of the specification.
[0004]Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
[0005]Type I allergic diseases such as seasonal allergic rhinitis (hayfever), conjunctivitis, allergic asthma and allergic dermatitis represent a major health problem in industrialized countries (Wuthrich, Int. Arch. Allergy Immunol. 90: 3-10, 1989). It is currently estimated that 15-20% of the population in developed countries are afflicted with some form of allergy (Miyamaoto, Advances in Allergology and Clinical Immunology. Godard P, Bousquet J, Michel F B (eds) pp. 343-347. The Parthenon Publishing Group, Cornforth, UK, 1992). Therefore, the diagnosis and therapy of these diseases have become focal points of interest for scientific investigation.
[0006]The primary immunological and biochemical bases of type I allergic reactions are the interaction of allergenic substances (allergens) with IgE antibodies bound to high affinity Fc receptors on the surface of mast cells and basophils. This interaction results in cross linking of allergen-specific IgE antibodies which in turn stimulates an immediate release and cascade production of inflammatory mediators responsible for allergic symptoms. Allergens are present in airborne particles such as house-dust, pollen of grasses, weeds and trees, mould spores and animal dander.
[0007]At present, one form of therapeutic intervention of allergic diseases (such as rhinitis, conjunctivitis and allergic asthma) involves injection of the allergen assumed to be responsible for the allergic response. This is referred to as hypo-sensitization treatment. Extracts currently in use in this procedure are prepared from natural sources and contain, in addition to the allergens, components such as proteins to which patients are not allergic.
[0008]The development of recombinant techniques has provided the means to produce high levels of purified allergens for diagnostic and therapeutic purposes. However, the high level of purity of recombinant allergen preparations results in a high anaphylactogenic index even at very low doses. Accordingly, extreme care is required when they are administered to patients. There is a need, therefore, to develop recombinant allergens with a reduced risk of anaphylactic shock.
[0009]The major outdoor cause of seasonal hay-fever and allergic asthma is airborne grass pollen (Smart et al., Int. Arch. Allergy Immunol. 7: 243-248, 1983). Pollen calenders show that grass pollen is most abundant in spring and early summer when grasses flower and this is when allergic asthma peaks in incidence. The most important sources of grass pollen are common agricultural pasture grasses which have been widely introduced throughout the world, but vary in temperature and tropical climate zones. In cool temperature regions, grasses such as rye-grass, Kentucky bluegrass and timothy (all belonging to the subfamily Pooideae) are of clinical significance, whereas in warm temperature and subtropical environments pollen of Bermuda grass (subfamily Chloridoideae) becomes the most important source of allergens. The most comprehensive studies have been made on proteins from rye-grass pollen and to a lesser extent Kentucky bluegrass and timothy.
[0010]Individuals sensitive to allergens from one grass are often sensitive to those of a number of other grass genera. This is particularly true for pollen of grasses within the subfamily Pooideae (Smith et al., "Analysis of rye-grass pollen allergens using two dimensional electrophoresis and immunoblotting." In Kraft D (ed), Molecular Biology and Immunology of Allergens, CRC Press, Boca Raton, Fla., 1994), where immunological cross-reactivity has been demonstrated in inhibition experiments using an IgE-binding assay, the radioallergosorbent test (RAST). In these experiments, pollen extracts from one grass were able to inhibit binding of IgE to extracts from other grasses.
[0011]Allergenic components of grass pollen can be classified into different groups according to their physiochemical and immunological properties. The major allergens that elicit an allergic reaction to pollen from the Pooid grasses are group 1 and 5 allergens, as judged by both criteria of the number of allergic patients responding and relative amounts of IgE binding to the allergens (Singh et al., 1991, supra). In case of perennial rye-grass, Lolium perenne, pollen extracts contain more than 17 proteins which have the capacity to bind IgE from sera of grass pollen allergic patients (Smith et al., 1993, supra). However, it has been shown that allergens of group 1 and 5 together can inhibit most of the IgE binding to crude pollen extracts (Bond et al., J. Allergy Clin. Immunol. 91: 339, 1993).
[0012]Lol p 5, a protein of 28-33 kDa, is the second most prevalent rye-grass allergen which causes allergy in 85-90% of grass pollen allergic individuals. Molecular cloning of cDNAs encoding this group 5 allergen (Singh et al., Proc. Natl. Acad. Sci. USA 88: 1384-1388, 1991; Ong et al., Gene 134: 235-240, 1993) has shown that Lol p 5 exists as a family of homologous but distinguishable isoforms which retain their IgE reactivity even after separation on denaturing SDS-PAGE gels and immunoblotting (Singh et al., 1991, supra).
[0013]A need, accordingly, exists to develop modified forms of recombinant allergens useful in immunotherapy and immunoprophylaxis of allergic conditions.
SUMMARY OF THE INVENTION
[0014]Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
[0015]Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>1 (SEQ ID NO:1), <400>2 (SEQ ID NO:2), etc. A summary of the sequence identifiers is provided in Table 1. A sequence listing is provided after the claims.
[0016]The present invention provides a modified recombinant allergen, wherein in naturally occurring form, the allergen is associated with allergic disease conditions in sensitive subjects. Conveniently, the modified recombinant allergen comprises an amino acid sequence modified from the naturally occurring amino acid sequence such that the allergen lacks or comprises reduced numbers of IgE epitopes and/or exhibits reduced binding capacity for IgE and/or exhibits reduced IgE production-stimulatory activity while retaining T-cell antigenicity.
[0017]Preferably, the allergic disease condition is a type I allergic disease condition.
[0018]Preferably, the recombinant allergen is a grass pollen allergen.
[0019]Most preferably, the grass allergen is a rye-grass pollen allergen such as but not limited to Lol p 5 or immunologically or botanically related allergens such as Phl p 5 and Poa p 5.
[0020]In a particularly preferred embodiment, the present invention provides a modified Lol p 5 allergen which lacks or comprises a reduced number of IgE epitopes and/or exhibits reduced IgE binding capacity and/or exhibits reduced IgE production-stimulatory activity while retaining T-cell antigenicity wherein said Lol p 5 variant is selected from a molecule having the amino acid sequence set forth in SEQ ID NOS:8 to 12 (see Table 1) or a modified allergen corresponding to an immunologically or botanically related allergen.
[0021]The present invention is further directed to a composition comprising a modified allergen such as a grass allergen (e.g. a rye-grass pollen allergen) which lacks or comprises reduced numbers of IgE epitopes and/or exhibits reduced binding capacity for IgE and/or exhibits reduced IgE production-stimulatory activity while retaining T-cell antigenicity. The composition further comprises one or more pharmaceutically acceptable carriers and/or diluents.
[0022]The present invention further contemplates a method for the prophylaxis or treatment of an allergic disease condition in a subject by administering to the subject, an effective amount of a modified allergen which lacks or comprises reduced numbers of IgE epitopes and/or exhibits reduced binding capacity for IgE and/or exhibits reduced IgE production-stimulatory activity while retaining T-cell antigenicity.
[0023]A summary of sequence identifiers used throughout the subject specification is provided in Table 1.
TABLE-US-00001 TABLE 1 Summary of sequence identifiers SEQUENCE ID NO: DESCRIPTION 1 Amino acid sequence of isoform A of Lol p 5 2 Amino acid sequence of isoform B of Lol p 5 3 Amino acid sequence of isoform A of Phl p 5 4 Amino acid sequence of isoform B of Phl p 5 5 Amino acid sequence of isoform Poa p 5 6 Amino acid sequence of isoform Poa p5 7 Amino acid sequence of Lol p 5 variant 8 Amino acid sequence of Lol p 5 variant D1 9 Amino acid sequence of Lol p 5 variant D2 10 Amino acid sequence of Lol p 5 variant D3 11 Amino acid sequence of Lol p 5 variant D4 12 Amino acid sequence of Lol p 5 variant D5 13 Nucleotide sequence of forward primer used to clone out D1 14 Nucleotide sequence of reverse primer used to clone out D1 15 Nucleotide sequence of forward primer used to clone out D2 16 Nucleotide sequence of reverse primer used to clone out D2 17 Nucleotide sequence of forward primer used to clone out D3 18 Nucleotide sequence of reverse primer used to clone out D3 19 Nucleotide sequence of forward primer used to clone out D4 20 Nucleotide sequence of reverse primer used to clone out D4 21 Nucleotide sequence of forward primer used to clone out D5 22 Nucleotide sequence of reverse primer used to clone out D5
BRIEF DESCRIPTION OF THE FIGURES
[0024]FIG. 1 is a representation showing a comparison of deduced amino acid sequences of group 5 allergens. Dashes indicate gaps which have been introduced to give maximum alignment. Residues identical to Lol p 5 A are indicated by asterisks.
[0025]FIG. 2 is a representation showing amino acid sequence of Lol p 5 A indicating the mutations introduced in the allergen to give D1 (SEQ ID NO: 8), D2 (SEQ ID NO: 9), D3 (SEQ ID NO: 10), D4 (SEQ ID NO: 11) and D5 (SEQ ID NO: 12) mutants. Amino acids of Lol p 5 A that were changed are indicated by boxes, whereas new sequences are given in bold.
[0026]FIG. 3 is a schematic representation of mutated Lol p 5 variants; e.g. mut 1 contains mutation D1.
[0027]FIG. 4 is a representation showing sequences of primers used to create mutations in Lol p 5 A.
[0028]FIG. 5 is a diagrammatic representation showing slot blot analysis of Lol p 5 (non-mutated) and the nine mutated variants (mut 1 to mut 9) of reactivities of the purified proteins to a polyclonal (p), a monoclonal (m) antibody and to sera of 7 rye-grass pollen-allergic patients.
[0029]FIG. 6 is a diagrammatic representation showing immunoblot analyses of Lol p 5 (non-mutated) and the nine mutated variants (mut 1 to mut 9) of reactivities of the purified proteins to a polyclonal (p), a monoclonal (m) antibody and to the serum of a rye-grass pollen-allergic patient (patient 2).
[0030]FIGS. 7 A, B and C are graphical representations of ELISA assays using purified non-mutated Lol p 5 and four of the mutated proteins (mut 3, mut 4, mut 6, mut 9) showing a reduction in reactivity of the mutated variants to a monoclonal (mAb A7) and to IgE of two patients (patient 4, patient 27).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0031]In accordance with the present invention, genetically engineered substantially hypoallergenic, variants of allergens with an inability or reduced capacity to interact with IgE are provided for use in immunotherapy and immunoprophylaxis. Certain types of modifications of the amino acid sequence are determined to result in a lack of or reduced numbers of IgE epitopes, reduced activity of IgE epitopes, reduced ability to interact with IgE and/or reduced IgE production-stimulatory activity.
[0032]Accordingly, one aspect of the present invention provides a modified recombinant allergen, wherein said allergen in naturally occurring form is associated with allergic disease conditions in sensitive subjects, wherein said modified recombinant allergen comprises an amino acid sequence modified from the naturally occurring amino acid sequence such that the allergen lacks or comprises reduced numbers of IgE epitopes and/or exhibits reduced binding capacity for IgE and/or exhibits reduced IgE production-stimulatory activity while retaining T-cell antigenicity.
[0033]The term "sensitive" subject is used in its broadest sense to include an individual exhibiting the symptoms of an allergic disease and more particularly a type I allergic disease in response to or associated with the allergen. An "individual" is preferably a human but also extends to a non-human primate, livestock animal (e.g. sheep, cow, pig, horse, donkey, goat), laboratory test animal (e.g. mouse, rat, rabbit, guinea pig) and a companion animal (e.g. dog, cat).
[0034]The present invention is particularly directed to grass pollen allergens.
[0035]Accordingly, another aspect of the present invention is directed to a modified recombinant grass pollen allergen, wherein said grass pollen allergen in naturally occurring form is associated with type I allergic disease conditions wherein said modified recombinant grass pollen allergen comprises an amino acid sequence modified from the naturally occurring amino acid sequence such that the allergen lacks and/or comprises reduced numbers of IgE epitopes or exhibits reduced binding capacity for IgE and/or exhibits reduced IgE production-stimulatory activity while retaining T-cell antigenicity.
[0036]In a particularly preferred embodiment, the grass pollen allergen is a rye-grass or immunologically related grass pollen allergen such as but not limited to Lol p 5, Phl p 5 and Pao p 5. Reference to "grass pollen allergen" includes all rye-grass or immunologically related grass pollen allergens or other grass allergens.
[0037]Accordingly, this aspect of the present invention contemplates a modified recombinant rye grass pollen allergen comprising an amino acid sequence modified from the naturally occurring amino acid sequence such that the allergen lacks or comprises reduced numbers of IgE epitopes and/or exhibits reduced binding capacity for IgE and/or exhibits reduced IgE production-stimulatory activity while retaining T-cell antigenicity.
[0038]The retention of T-cell antigenicity includes reference to the retention of T-cell epitopes or an otherwise capacity to interact with T cells to elicit a T-cell response.
[0039]The present invention is hereinafter described in relation to Lol p 5. This is done since Lol p 5, up to the present time, represents a particularly useful allergen in which to practice the present invention. This is done, however, with the understanding that the present invention extends to any allergen, especially an allergen involved in type I allergic diseases such as but not limited to a group 5 grass pollen allergen. The methods of the present invention are particularly applicable to any rye-grass or immunologically related grass pollen allergen in addition to Lol p 5 such as Phl p 5 and Poa p 5.
[0040]In work leading up to the present invention, the inventors expressed recombinant Lol p 5 in E. coli as a nonfusion protein and found that removal of the N-terminal signal peptide from the cDNA prior to cloning into the bacterial expression vector resulted in a soluble recombinant form of the allergen. This approach made it possible to avoid the harsh denaturing conditions for isolation of the allergen from bacterial cells. Recombinant Lol p 5 was tested for antigenic similarity with its natural counterpart by inhibition ELISA experiments and the inventors showed that the recombinant form fully inhibited IgE binding of an isolated form of its natural pollen counterpart. The fact that single recombinant isoforms can inhibit IgE binding to natural allergens further implied that different allergen isoforms were similar. The inventors used recombinant allergens in immunoblot inhibition studies where allergic sera reincubated with recombinant Lol p 5 were used to probe two dimensional immunoblots of rye-grass soluble protein. It was found in accordance with the present invention that preincubation with one form completely abolished binding to all the different forms encoded by different genes. This showed that even with sequence micro-heterogenities, different allergen isoforms were antigenically very similar.
[0041]The next step in the development of the present invention was to determine key amino acid residues of the allergenic proteins which could be changed which removed or reduced IgE interactivity while maintaining the general structure and functionality of the T-cell epitopes.
[0042]The inventors determined which amino acid residues on isoforms A and B of Lol p 5 were conserved. It was reasoned that such conserved amino acid residues would be important for IgE binding since there is cross-reactivity between a number of allergens from different grasses. By selectively mutating these conserved amino acid residues, mutants were identified which have no or reduced IgE interactivity while retaining T-cell antigenicity.
[0043]Accordingly, another aspect of the present invention comprises a modified group 5 grass pollen allergen, wherein said group 5 grass pollen allergen comprises a substitution, deletion and/or addition at one or more amino acid residues which is/are conserved in at least two immunologically cross-reactive group 5 grass pollen allergens and wherein said modified group 5 grass pollen allergen lacks or comprises reduced numbers of IgE epitopes and/or exhibits reduced binding capacity for IgE and/or exhibits reduced IgE production-stimulatory activity compared to the corresponding naturally occurring form.
[0044]In accordance with the aspect of the present invention, one suitable reference amino acid sequence is SEQ ID NO:1 which is the amino acid sequence of Lol p 5, isoform A. An amino acid sequence comparison, such as in FIG. 1, shows the conserved amino acid residues in isoforms A and B of Lol p 5 and Phl p 5 and in isoforms of Poa p 5. Conserved residues in FIG. 1 are indicated by asterisks. Mutants are then readily introduced which alter one or more of these conserved residues.
[0045]Another aspect of the present invention provides a modified group 5 grass pollen allergen comprising an amino acid truncation or substitution, deletion and/or addition at a position corresponding to one or more of mutants 1 to 9 of Lol p 5 as depicted in FIG. 3 or a corresponding mutant in an immunologically related allergen.
[0046]In a particularly preferred embodiment, the present invention provides a modified Lol p 5 allergen which lacks or comprises a reduced number of IgE epitopes and/or exhibits reduced IgE binding capacity and/or exhibits reduced IgE production-stimulatory activity while retaining T-cell antigenicity wherein said Lol p 5 variant is selected from a molecule having the amino acid sequence set forth in SEQ ID NOs:8 to 12 or a modified allergen corresponding to an immunologically related allergen.
[0047]The Lol p 5 variants identified by SEQ ID NOs:8 to 12 are referred to herein as mutants D1 to D5, respectively.
[0048]The present invention further provides a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a modified recombinant allergen, wherein said allergen in naturally occurring form is associated with allergic disease conditions in sensitive subjects, wherein said modified recombinant allergen comprises an amino acid sequence modified from the naturally occurring amino acid sequence such that the allergen lacks or comprises reduced numbers of IgE epitopes and/or exhibits reduced binding capacity for IgE and/or exhibits reduced IgE production-stimulatory activity while retaining T-cell antigenicity.
[0049]Another aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a modified recombinant allergen, wherein said allergen in naturally occurring form is associated with type I allergic disease conditions in sensitive subjects, wherein said modified recombinant allergen comprises an amino acid sequence modified from the naturally occurring amino acid sequence such that the allergen lacks or comprises reduced numbers of IgE epitopes and/or exhibits reduced binding capacity for IgE and/or exhibits reduced IgE production-stimulatory activity while retaining T-cell antigenicity.
[0050]Yet another aspect of the present invention is directed to a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding modified recombinant grass pollen allergen, wherein said grass pollen allergen in naturally occurring form is associated with type I allergic disease conditions in sensitive subjects wherein said modified recombinant grass pollen allergen comprises an amino acid sequence modified from the naturally occurring amino acid sequence such that the allergen lacks or comprises reduced numbers of IgE epitopes and/or exhibits reduced binding capacity for IgE and/or exhibits reduced IgE production-stimulatory activity while retaining T-cell antigenicity.
[0051]Still yet another aspect of the present invention relates to a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding modified recombinant rye-grass pollen allergen comprising an amino acid sequence modified from the naturally occurring amino acid sequence such that the allergen lacks or comprises reduced numbers of IgE epitopes and/or exhibits reduced binding capacity for IgE and/or exhibits reduced IgE production-stimulatory activity while retaining T-cell antigenicity.
[0052]Thus, a particularly preferred aspect of the present invention provides purified nucleic acid molecules encoding a modified grass pollen allergen and more particularly a modified group 5 grass pollen allergen, or an antigenic fragment thereof, or derivative or homolog thereof, or the functional equivalent of such a nucleic acid sequence wherein the modified grass pollen allergen lacks or comprises reduced number of IgE epitopes and/or exhibits reduced binding capacity for IgE and/or exhibits reduced IgE production-stimulatory activity while retaining T-cell antigenicity. Preferred nucleic acid sequences encode group 5 allergen family members such as Lol p 5, Poa p 5 and Phl p 5. One particularly useful nucleic acid molecule encodes Lol p 5 mutants D1 to D5.
[0053]The nucleic acid molecule of the present invention may be genomic or cDNA molecules or a corresponding mRNA molecule and may be referred to as a gene. Reference to a "gene", in respect of the present invention, means any contiguous sequence of nucleotides, the transcription of which leads to a mRNA molecule or which sequence is a mRNA molecule, which mRNA molecule is capable of being translated into a protein. The gene encoding a group 5 grass pollen allergen family member means the nucleotide sequence encoding the protein or a derivative or a homolog of the protein which may contain single or multiple amino acid substitutions, deletions and/or additions relative to the corresponding naturally occurring molecule. A modified Lol p 5 gene also refers to cDNAs complementary to the mRNAs corresponding to the full or partial length of a Lol p 5 protein having at least one truncated or amino acid substitution, addition and/or deletion relative to the naturally occurring molecules.
[0054]The present invention further contemplates fusion molecules. For example, for some aspects of the present invention, it is desirable to produce a fusion protein comprising modified grass pollen allergen or a fragment thereof or a derivative thereof and an amino acid sequence from another peptide or protein, examples of the latter being enzymes such as β-galactosidase, phosphatase, urease and the like. Most fusion proteins are formed by the expression of a recombinant gene in which two coding sequences have been joined together such that their reading frames are in phase. Alternatively, proteins or peptides can be linked in vitro by chemical means. All such fusion protein or hybrid genetic derivatives of a grass pollen allergen or its encoding nucleotide sequences are encompassed by the present invention. Furthermore, by homologs and derivatives of a grass pollen allergen protein is meant to include synthetic derivatives thereof. The nucleotide sequences as elucidated herein, can be used to chemically synthesize the entire potein or generate any number of fragments (peptides) by chemical synthesis by well known methods (e.g. solid phase synthesis). All such chemically synthesized peptides are encompassed by the present invention. Accordingly, the present invention extends to isolated modified grass pollen allergen family members, fragments thereof and their derivatives, homologs and immunological relatives made by recombinant means or by chemical synthesis.
[0055]The terms "isolated" and "purified" are used interchangeably herein and refer to peptides, proteins, protein fragments and nucleic acid sequences substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when synthesized chemically. The term "naturally occurring" as used herein refers to proteins or fragments thereof purified from grass pollen or other plant part. It also includes reference to an amino acid sequence determined by a cDNA sequence but which is associated with allergic conditions in a similar way to an allergen purified from grass pollen.
[0056]Fragments of nucleic acid molecules within the scope of the invention include those coding for parts of grass pollen allergens that exhibit T-cell antigenicity but which lacks or exhibits reduced IgE interaction in mammals, preferably humans.
[0057]Fragments and mutants of recombinantly or synthetically produced modified grass pollen allergens which do not bind IgE and/or which have minimal IgE interacting ability and/or which have minimal capacity to stimulate IgE production are desirable. It is preferable that such minimal IgE interacting activity does not lead to histamine release. For example, it is preferable that the modified allergen does not cause cross linking of IgE on mast cells or basophils. Minimal IgE interacting activity refers to IgE interaction activity which is less than the amount of IgE interaction by recombinantly or synthetically produced "naturally occurring" grass pollen allergen protein or whole purified native grass pollen allergen. IgE interaction may also be measured as IgE production stimulating activity. Preferred fragments also include antigenic fragments which, when administered to a grass pollen-sensitive individual or an individual allergic to an allergen cross-reactive with grass pollen allergen, are capable of modifying the allergic response to grass pollen allergen of the individual.
[0058]Antigenic fragments of the present invention which have T-cell stimulating activity i.e. T-cell antigenicity, and thus comprise at least one T-cell epitope are particularly desirable. T-cell epitopes are believed to be involved in initiation and perpetuation of the immune response to a protein allergen which is responsible for the clinical symptoms of allergy. These T-cell epitopes are throught to trigger early events at the level of the T helper cell by binding to an appropriate HLA molecule on the surface of an antigen presenting cell and stimulating the relevant T-cell sub-population. These events lead to T-cell proliferation, lymphokine secretion, local inflammatory reactions, recruitment of additional immune cells to the site, and activation of the B cell cascade leading to production of antibodies. One isotype of these antibodies, IgE, is fundamentally important to the development of allergic symptoms and its production is influenced early in the cascade of events, at the level of the T helper cell, by the nature of the lymphokines secreted. A T-cell epitope is the basic element or smallest unit of recognition by a T-cell receptor, where the epitope comprises amino acids essential to receptor recognition. Amino acid sequences which mimic those of the T-cell epitopes and which modify the allergic response to protein allergens are within the scope of this invention.
[0059]Exposure of patients to purified modified protein allergens of the present invention or to the antigenic fragments of the present invention which comprise at least one T-cell epitope and are derived from protein allergens may tolerize or anergize appropriate T-cell subpopulations such that they become unresponsive to the protein allergen and do not participate in stimulating an immune response upon such exposure. In addition, administration of the protein allergen of the invention or an antigenic fragment of the present invention which comprises at least one T-cell epitope may modify the lymphokine secretion profile as compared with exposure to the naturally-occurring protein allergen or portion thereof (e.g. result in a decrease of IL-4 and/or an increase in IL-2). Furthermore, exposure to such antigenic fragment or protein allergen may influence T-cell subpopulations which normally participate in the response to the allergen such that these T-cells are drawn away from the site(s) of normal exposure to the allergen (e.g. nasal mucosa, skin and lung) towards the site(s) of therapeutic administration of the fragment or protein allergen. This redistribution of T-cell sub-populations may ameliorate or reduce the ability of an individual's immune system to stimulate the usual immune response at the site of normal exposure to the allergen, resulting in a diminution in allergic symptoms.
[0060]The present invention provides expression vectors and host cells transformed to express the nucleic acid sequences of the invention. Expression vectors of the present invention comprise a nucleic acid sequence coding for a modified grass pollen allergen, or an antigenic fragment thereof, or a derivative or homolog thereof, or the functional equivalent of such nucleic acid sequence, The nucleic acid sequences may be expressed in prokaryotic or eukaryotic host cells. Suitable host cells include bacterial cells such as E. coli, insect cells, yeast, or mammalin cells such as Chinese hamster ovary cells (CHO). Suitable expression vectors, promoters, enhancers, and other expression control elements may be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Suitable vectors for expression in yeast include YepSec1 (Baldari et al., EMBO J. 6: 229-234, 1987); pMF (Kurj an and Herskowtiz, Cell 30: 933-943, 1982); and JRY 88 (Schultz et al., Gene 54: 113-123, 1987).
[0061]Host cells can be transformed to express the nucleic acid sequences of the present invention using conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, or electroporation. Suitable methods for transforming the host, cells may be found in Sambrook et al., 1989, supra, and other laboratory textbooks. The nucleic acid sequences of the invention may also be synthesized using standard techniques.
[0062]Accordingly, another aspect of the present invention provides a method of producing a recombinant modified grass allergen or a fragment thereof, or a derivative or homolog thereof, or immunological relatives thereof comprising culturing an organism containing a replicable recombinant DNA molecule, said molecule comprising a promoter capable of expression in said organism, a gene encoding a modified grass pollen allergen or family member, a fragment or homolog or derivative thereof, or an immunological relative thereof, located downstream of and transcribed from said promoter, a selectable marker and a DNA vehicle containing a prokaryotic or eukaryotic origin of replication, under conditions and for a time sufficient for said recombinant DNA molecule to be stably maintained and direct the synthesis of the modified grass pollen allergen or fragment or derivative, homolog or immunological reative thereof and then optionally isolating same.
[0063]The grass pollen allergens and fragments (peptides) thereof can be purified from cell culture medium, host cells, or both using techniques known in the art for purifying peptides and proteins, including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis and immunopurification with antibodies specific for the modified grass pollen allergen. The terms "isolated" and "purified" are used interchangeably herein and refer to peptides, proteins, protein fragments, and nucleic acid sequences substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when synthesized chemically.
[0064]Another aspect of the invention provides protein preparations comprising Lol p5 D1, D2, D3, D4 and D5 or their functional or immunological equivalents, homologs or derivatives.
[0065]Thus, the present invention provides modified grass pollen allergens or their derivatives which, when administered to a grass pollen-sensitive individual, reduce the allergic response of the individual to grass pollen such as lye-grass pollen or pollen from immunologically related grasses. Preferred modified grass pollen allergens include modified Lol p5 protein or a derivative or homolog thereof. Other preferred allergens are Phl p 5 and Poa p 5.
[0066]In addition to inducing an amino substitution, addition and/or deletion or truncation, another example of a modification of proteins or peptides is substitution of cysteine residues preferably with alanine, serine, threonine, leucine or glutamic acid to minimize dimerization via disulfide linkages. Another example of modification of the proteins and peptides of the invention is by chemical modification of amino acid side chains or cyclization of the peptide.
[0067]In order to enhance stability and/or reactivity, proteins or peptides of the invention can also be modified to incorporate one or more polymorphisms in the amino acid sequence of the protein allergen resulting from natural allelic variation. Additionally, D-amino acids, non-natural amino acids or non-amino acid analogs can be substituted or added to produce a modified protein or peptide within the scope of this invention.
[0068]Another aspect of the present invention relates to recombinant vectors comprising DNA sequences encoding proteins displaying modified allergenic activity from pollen of a grass species. More particularly, the grass species belongs to the family Poaceae (Gramineae), and even more particularly, to the genus Lolium. Still even more particularly, the allergenic protein is characterized as being immunologically cross-reactive with antibody to Lol pIb protein of Lolium perenne pollen, namely: [0069]Pooid (festucoid) grasses. Group 1: Triticanea: Bromus inermis, smooth broom; Agropyron repens, English couch; A. cristatum; Secale cereale rye Triticum aestivum, wheat. Group 2: Poanae: Dactylis glomerata, orchard grass of perennial ryegrass; L. multiflorum, Italian ryegrass; Poa pratensis, Kentucky bluegrass; P. compressa, flattened meadow grass; Avena sativa, oat; Holcus lanatus, velvet grass or Yorkshire fog; Anthoxanthum odoratum; sweet vernal grass; Arrhenatherum elatius, oat grass; Agrostis alba, red top; Phleum pratense, timothy; Phalaris arundinacea, reed canary grass. Panicoid grass, Paspalum notatum, Bahia grass, Andropogonoid grasses: Sorghum halepensis, Johnson grass.
[0070]A variety of expression vectors can be constructed for the production of a modified grass pollen allergen or a fragment or derivative thereof.
[0071]The present invention extends to monoclonal and polyclonal antibodies to modified grass pollen allergens or fragments, derivatives or homologs thereof.
[0072]The monoclonal antibodies are useful to screen cDNA libraries or to purified recombinantly produced proteins or even in therapy to reduce the activity of an introduced protein. In the following discussion, reference to grass pollen protein allergens include their derivatives, homologs and immunological relatives and chemical synthetic derivatives thereof. The following discussion also includes antibodies specific for purified modified Lol p 5 and fragments, derivatives and homologs thereof. Such antibodies are contemplated to be useful in developing detection assays (immunoassays) for modified grass pollen allergens especially during the monitoring of a therapeutic or diagnostic regimen and in the purification of recombinantly or synthetically produced grass pollen family members and in particular group 5 grass pollen allergen. The antibodies may be monoclonal or polyclonal. Additionally, it is within the scope of this invention to include any second antibodies (monoclonal or polyclonal) directed to the first antibodies discussed above. The present invention further contemplates use of these first or second antibodies in detection assays and, for example, in monitoring the effect of a diagnostic or an administered pharmaceutical preparation. Furthermore, it is within the scope of the present invention to include antibodies to any molecules complexed with a modified grass pollen protein allergen. Accordingly, an antibody to a grass pollen protein allergen encompasses antibodies to such a protein allergen, or antigenic parts thereof, and to any associated molecules (e.g. lipid regions, carrier molecules, fused proteins, and the like).
[0073]The grass pollen family members, or fragments thereof, considered herein are purified then utilized in antibody production. Both polyclonal and monoclonal antibodies are obtainable by immunization with recombinant or synthetic modified grass pollen protein family members, and either type is utilizable for immunoassays. The methods of obtaining both types of sera are well known in the art. Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of a purified modified grass pollen allergen, or antigenic parts thereof, collecting serum from the animal, and isolating specific sera by any of the known immunoabsorbent techniques. Although antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favored because of the potential heterogeneity of the product.
[0074]The use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art. (See, for example, Kohler and Milstein, Nature 256: 495-499, 1975; Kohler and Milsten, Eur. J. Immunol. 6: 511-519, 1976).
[0075]Unlike preparation of polyclonal sera, the choice of animal is dependent on the availability of appropriate immortal lines capable of fusing with lymphocytes. Mouse and rat have been the animals of choice in hybridoma technology and are preferably used. Humans can also be utilized as sources for sensitized lymphocytes if appropriate immortalized human (or non-human) cell lines are available. For the purpose of the present invention, the animal of choice may be injected with from about 0.1 mg to about 20 mg of purified modified grass pollen allergen or parts thereof. Usually the injecting material is emulsified in Freund's complete adjuvant. Boosting injections may also be required. The detection of antibody production can be carried out by testing the antisera with appropriately labeled antigen. Lymphocytes can be obtained by removing the spleen or lymph nodes of sensitized animals in a sterile fashion and carrying out fusion. Alternatively, lymphocytes can be stimulated or immunized in vitro.
[0076]The presence of modified grass pollen allergens contemplated herein, or antibodies specific for same, in a patient's serum, plant or mammalian tissue or tissue extract, can be detected utilizing antibodies prepared as above, either monoclonal or polyclonal, in virtually any type of immunoassay. A wide range of immunoassay techniques are available as can be seen by reference to U.S. Pat. Nos. 4,015,043, 4,424,279 and 4,018,653. This includes both single-site and two-site, or "sandwich", assays of the non-competitive types, as well as in the traditional competitive binding assays. Sandwich assays are among the most useful and commonly used assays and are favored for use in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabeled antibody is immobilized in a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen secondary complex, a second antibody, labeled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of a tertiary complex of antibody-antigen-labeled antibody (e.g., antibody-modified grass pollen allergen protein-antibody). Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten. Variations on the forward assay include a simultaneous assay, in which both sample and labeled antibody are added simultaneously to the bound antibody, or a reverse assay in which the labeled antibody and sample to be tested are first combined, incubated and then added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent.
[0077]Although the following discussion is concerned with detecting modified grass pollen allergen, it is equally applicable to detecting antibodies to same and it is intended to be a sufficient description thereof.
[0078]In the typical forward sandwich assay a first antibody having specificity for modified grass pollen allergen, or antigenic parts thereof; contemplated in this invention, is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated from about room temperature to about 37° C. for a period of time sufficient to allow binding of any subunit present in the antibody. The incubation period will vary but will generally be in the range of about 2-40 minutes or overnight if more convenient. Following the incubation period, the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the hapten. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the hapten.
[0079]By "reporter molecule" as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes). In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, (3-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. For example, p-nitrophenyl phosphate is suitable for use with alkaline phosphatase conjugates, for peroxidase conjugates, 1,2-phenylenediamine, 5-aminosalicylic acid, or toluidine are commonly used. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labeled antibody is added to the first antibody hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the tertiary complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectophotometrically, to give an indication of the amount of hapten which was present in the sample. "Reporter molecule" also extends to use of cell agglutination or inhibition of agglutination such as red blood cells or latex beads, and the like.
[0080]Alternately, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labeled antibody adsorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the light at a characteristic color visually detectable with a light microscope. As in the ETA, the fluorescent labeled antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength, the fluorescein observed indicates the presence of the hapten of interest. Immunofluorescence and ETA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemilluminescent or bioluminescent molecules, may also be employed. It will be readily apparent to the skilled technician how to vary the procedure to suit the required purpose. It will also be apparent that the foregoing can be used to detect directly or indirectly (i.e. via antibodies) grass pollen allergen protein of this invention.
[0081]Accordingly, one aspect of the present invention provides a method of detecting a modified pollen allergen or a derivative or homolog thereof or an allergenic protein immunologically reactive with said modified grass pollen allergen or a derivative or homolog present in serum, tissue extract, plant extract or other biological fluid or composition comprising the steps of containing said fluid or composition to be tested with an antibody to said modified grass pollen protein allergen for a time and under conditions sufficient for a modified allergenic protein-antibody complex to form and subjecting said complex to a detecting means. For purification methods, an antibody to a native allergen may also be effective in order to purify a modified allergen and such an embodiment is encompassed by the present invention.
[0082]The present invention is also directed to a kit for the rapid and convenient assay for antibodies to modified grass pollen allergens or derivatives, homologs or immunological relatives thereof in mammalian body fluids e.g. serum, tissue extracts, tissue fluids), in vitro cell culture supernatants, and cell lysates. The kit is compartmentalized to receive a first container adapted to contain an antigenic component thereof, and a second container adapted to contain an antibody to the grass pollen allergen, said antibody being labeled with a reporter molecule capable of giving a detectable signal. If the reporter molecule is an enzyme, then a third container adapted to contain a substrate for said enzyme is provided. In an exemplified use of the subject kit, a sample to be tested is contacted with the contents of the first container for a time and under conditions for an antibody, if present in the sample, to bind to the grass pollen allergen in said first container.
[0083]Because of the presence of allergens in the environment, hayfever and seasonal asthma continue to have significant morbidity and socio-economic impact on Western communities, despite advances made in their pharmacology and immunology. While the available spectrum of drugs, including antihistamines and steroids have resulted in improvement in the treatment of allergic disease, they have unfortunate side-effects associated with long term usage. Because of these problems, renewed interest has been shown in the immunotherapy of allergic disease. Immunotherapy involves the injection of potent allergen extracts to desensitize patients against allergic reactions. Unfortunately, the pollen preparations used as allergens are polyvalent and of poor quality. Consequently, concentrations used are frequently high in order to induce IgG responses, but may be lethal through triggering of systemic reactions, including anaphylaxis. The cloned gene product or synthetic peptides based on the sequence of allergens provides a safer medium for therapy since it can be quality controlled, characterized and standardized.
[0084]Accordingly, the present invention contemplates a method for desensitizing a mammal (e.g. human) allergic to grass pollen which comprises administering to said mammal a desensitizing-effective amount of a modified grass pollen allergen which lacks or comprises reduced numbers of and/or exhibits reduced IgE binding activity and/or exhibits reduced IgE production-stimulatory activity or a fragment or derivative, homolog, or immunological relative thereof, for a time and under conditions sufficient to effect desensitization of the mammal (e.g. human) to the grass pollen.
[0085]The present invention also provides a method of treating sensitivity to ryegrass pollen or pollen from an immunological relative of rye-grass in a mammal (e.g. human) sensitive to such pollen, comprising administering to the mammal a therapeutically effective amount of a therapeutic composition of the invention. The present invention further provides a method of treating sensitivity to ryegrass pollen allergen or an allergen immunologically cross-reactive with ryegrass pollen allergen comprising administering to a mammal a therapeutically effective amount of said protein preparation of the invention.
[0086]Through the use of the peptides and protein of the present invention, preparations of consistent, well-defined composition and biological activity can be made and administered for therapeutic purposes (e.g. to modify the allergic response of a L. perenne sensitive individual to pollen of such plant. Administration of such peptides or protein may, for example, modify IgE response to the grass pollen allergen. Purified peptides can also be used to study the mechanism of immunotherapy of L. perenne allergy and to design modified derivatives or analogs useful in immunotherapy.
[0087]The present invention is directed, therefore, to the use of a modified allergen in the manufacture of a medicament for the treatment or prophylaxis of allergen-sensitive
[0088]The present invention, therefore, provides a pharmaceutical composition comprising a desensitizing or therapeutically effective amount of modified grass pollen allergen and in particular group 5 grass pollen allergen or derivatives, homologs or immunological relatives thereof and one or more pharmaceutically acceptable carriers and/or diluents. The active ingredients of a pharmaceutical composition comprising the modified grass pollen allergen prophylactic is contemplated to exhibit excellent therapeutic or activity, for example, in the desensitization of humans allergic to grass pollen when administered in amount which depends on the particular case. For example, from about 0.5 Fg to about 20 mg per kilogram of body weight per day may be administered. Dosage regime may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. The activity compound may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intramuscular, subsutaneous, intranasal, intradermal or suppository routes or implanting (e.g. using slow release molecules). Depending on the route of administration, the active ingredients which comprise the pharmaceutical composition of the invention may be required to be coated in a material to protect said ingredients from the action of enzymes, acids and other natural conditions which may inactivate said ingredients. For example, the modified grass pollen allergen may be administered in an adjuvant, co-administered With enzyme inhibitors or in liposomes. Adjuvant is used in its broadest sense and includes any immune stimulating compound, such as interferon. Adjuvants contemplated herein include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin. Liposomes include water-in-oil-in-water CF emulsions as well as conventional liposomes. For purposes of inducing T cell anergy, the pharmaceutical composition if preferably administered in non-immunogenic form (e.g. it does not contain adjuvant).
[0089]The active compounds may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
[0090]The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions or sterile powders for preparation of injectable solutions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, iropylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
[0091]Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
[0092]When a modified grass pollen allergen, or a fragment thereof is suitably protected as described above, the active compound may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets or it may be incorporated directly with food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be carried and may conveniently be between about 5 to 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit fowl contains between about 10 Fg and 2000 mg of active compound.
[0093]The tablets, troches, pills, capsules and the like may also contain the ingredients listed below: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations.
[0094]As used herein "pharmaceutically acceptable carrier and/or diluent" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
[0095]The present invention is further described by the following non-limiting Examples.
Example 1
[0096]Generation of Lol p 5 Mutant Proteins
[0097]To engineer hypoallergenic, non-IgE reactive allergen variants, it was required to be determined whether key residues of the proteins can be selected which can be changed while keeping the general structure and T-cell epitopes intact. Since the highest frequency of IgE binding is observed in peptide fragments which span the C-terminal half of Lol p 5, the inventors introduced mutations predominantly in the C-terminus of the allergen. To identify amino acid positions in Lol p 5 likely to have an influence on the IgE intereactivity of the protein, protein sequences of isoform A and B of Lol p 5 were compared with group 5 allergens of other grasses (FIG. 1). Site-directed mutagenesis was employed to replace residues that are highly conserved among group 5 allergens (FIG. 2). Mutant proteins altered in one or three domains were generated (FIG. 3).
[0098]Non-mutated Lol p 5 and mutated variants of Lol p 5 were expressed in soluble forms in E. coli using the pQE expression vector system (Qiagen). Bacterial expression (see below) of proteins using this vector introduces a polyhistidine tag at the N-terminus of the molecules which is useful for purification of recombinant proteins by one step metal chelate affinity chromatography. Non-mutagenized control and mutated proteins were then tested for IgE reactivity as well as for reactivity with anti-Lol p 5 monoclonal antibody A7 (Mab A7) and polyclonal anti-Lol p 5 antiserum in slot blots (FIG. 5), Western blots (FIG. 6) and ELISA assays (FIG. 7). The results showed a substantial reduction in IgE binding activity in case of several of the mutated proteins (e.g. mut 4, mut 6, mut 9). Such engineered allergenic molecules are potentially useful for safer and more effective immunotherapy for type I allergic diseases and the described approach may be generally applied to produce non-IgE reactive variants of allergens.
Example 2
Expression and Purification of Recombinant Lol p 5 and Mutant Proteins
[0099]The coding sequences of Lol p 5 and the mutant proteins were introduced in-frame into the expression vector pQE31 (QIAGEN). The vector allows expression of recombinant proteins with an N-terminal, 6-residue histidine tag. Expression and harvesting of the proteins was carried out as outlined in the QIA expressionist manual. Histidine-tagged proteins were purified using TALON metal affinity resin (Clontech), following the procedure for batch/gravity flow column purification as outlined in the TALON Metal Affinity Resin user manual (Clontech).
Example 3
[0100]SDS-Page and Western Blotting
[0101]For SDS-PAGE, 1.3 Fg of Lol p5 and each of the mutant proteins were boiled for 5 minutes with 10× protein sample buffer. Samples were loaded onto a 15% w/v acrylamide mini gel at 200 V for 40 minutes in a buffer of 0.2 M glycine, 0.025 M Tris, 0.1% w/v SDS.
[0102]For staining, gels were shaken in 0.1% w/v Coomassie Brilliant Blue R250 for at least an hour. Gels were destained in 20% v/v methanol, 7% v/v glacial acetic acid, 3% v/v glycerol, overnight with two buffer changes.
[0103]Gels were western blotted in a BIORAD mini-Protean II cell western blot apparatus in a buffer of 0.025 M Tris, 0.2 M glycine, 20% v/v methanol onto Nytran 0.2 Fm nylon membrane (Schleicher & Schuell) at 100 V for 1 hour at 4° C.
Example 4
Slot Blot Analysis
[0104]For slot blot analysis, 0.7 Fg of mutant proteins and Lol p5 were added into the slots of a Hybri-Slot manifold slot blot apparatus (Life Technologies, Inc.) and blotted onto Nytran 0.2 Fm nylon membrane (Schleicher & Schuell) under suction from a water vacuum.
Example 5
[0105]Incubation of Blots with Antibodies and Patient Sera
[0106]Prior to incubation with antibodies or sera, all western and slot blots were blocked in 10% w/v skim mills powder in PBS (150 mM sodium chloride, 36 mM sodium phosphate monobasic, monohydrate, 7 mM sodium phosphate dibasic, dihydrate) for one hour with shaking. Blots were washed once with PBS, 0.5% v/v Tween 20, twice with PBS and incubated overnight with monoclonal antibodies (mAb A7: diluted 1:5), polyclonal antibodies (B1: diluted 1:50) or patient sera. All dilutions were prepared in PBS, 0.5% w/v BSA, 0.1% w/v sodium azide, and shaken with the blots overnight at room temperature. After washing as above blots were incubated with alkaline-phosphatase conjugated anti-mouse (mAb A7) or anti-rabbit (B1) secondary antibodies (Promega) diluted 1:5000 in PBS, 0.5% v/v Tween 20, 1% w/v BSA for 1 hour with shaking at room temperature. All blots were than washed as above. Bound anti-mouse and anti-rabbit antibodies were detected by a colour reaction--10 ml alkaline phosphatase buffer (0.1 M Tris, pH 9.5, 0.1 M sodium chloride, 0.05 M magnesium chloride) with 66 Fl of BCIP stock (5% w/v bromochloroindolyl phosphate in 100% v/v dimethylformamide). Blots incubated with patient sera were probed with I125-labeled anti-human antibody (Bioclone) diluted 1:5 in PBS, 0.5% v/v Tween 20, 1% w/v BSA (buffer B) overnight with shaking at room temperature. All blots were washed as above. After washing, bound I125-labeled anti-human IgE was detected by exposure to Kodak Biomax MS film at -70° C.
Example 6
Direct ELISA
[0107]The wells of an ELISA plate (Greiner) were coated with 50 Fl aliquots of 100, 500, 1000, 5000 and 10000 ng/ml dilutions of Lol p5 and the four mutant proteins, and incubated at 4° C. overnight. The wells were then washed four times with PBS, 0.5% v/v Tween 20. After blocking with buffer B at room temperature for one hour, the wells were washed again, and incubated with 50 Fl of an appropriate dilution of patient sera in buffer B at 4° C. overnight. Wells were washed as above before incubation with 50 Fl of a 1:2000 dilution of anti-human IgE antibody (Alkaline-Phosphatase conjugated: Sigma) in buffer B at room temperature for one hour. After a final series of washes, bound anti-human IgE was detected with Blue Phos microwell phosphatase substrate system (Kirkegaard & Perry Laboratories). Colour development was detected by a Spectracount plate reader at 630 nm (Packard).
[0108]Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
BIBLIOGRAPHY
[0109]Bond, J. F, Segal, D. B, Yu X-B, Theriault, K. A, Pollock, M. S, Yeung H. J. Allergy Clin. Immunol. 91: 339, 1993. [0110]Baldari et al., EMBO J. 6: 229-234, 1989. [0111]Kohler and Milstein, Nature 256: 495-499, 1975. [0112]Kohler and Milstein, Eur. J. Immunol. 6: 511-519, 1976. [0113]Kurjan and Herskowitz, Cell 30: 933-943, 1982. [0114]Miyamoto T: Advances in Allergology and Clinical Immunology. Godard. P, Bousquet J, Michel F B (eds) pp. 343-347. The Parthenon Publishing Group, Cornforth, UK, 1992. [0115]Ong, E. K, Griffith, I. J., Knox, R. B., Singh, M. B. Gene 134: 235-240, 1993. [0116]Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. [0117]Schultz et al., Gene 54: 113-123, 1987. [0118]Singh, M. B., Hough, T., Theerakulpisut, P., Avjioglu, A., Davies, S., Smith, P. M., Taylor, P., Simpson, R. J., Ward. L. D., McCluskey, J., Puy, R., Knox, R. B. Proc. Natl. Acad. Sci. USA 88: 1384-1388, 1991. [0119]Smart, I. J., Heddle, R. J., Zola, H., Bradley, J., Int. Arch. Allergy Immunol. 72: 243-248, 1983. [0120]Smith, P. M., Ong, E. K, Avjioglu, A., Singh, M. B., Knox, R. B. Analysis of rye-grass pollen allergens using two dimensional electrophoresis and immunoblotting. In Kraft D (ed), Molecular Biology and Immunology of Allergens, CRC Press, Boca Raton, Fla., 1993. [0121]Smith, P. M., Ong, E. K., Knox, R. B., Singh, M. B. Mol. Immunol. 31: 491-498, 1994. [0122]Wuthrich, B., Int. Arch. Allergy Immunol. 90: 3-10, 1989.
Sequence CWU
1
221276PRTLolium perenne 1Ala Asp Ala Gly Tyr Thr Pro Ala Ala Ala Ala Thr
Pro Ala Thr Pro1 5 10
15Ala Ala Thr Pro Ala Ala Ala Gly Gly Lys Ala Thr Thr Asp Glu Gln
20 25 30Lys Leu Leu Glu Asp Val Asn
Ala Gly Phe Lys Ala Ala Val Ala Ala 35 40
45Ala Ala Asn Ala Pro Pro Ala Asp Lys Phe Lys Ile Phe Glu Ala
Ala 50 55 60Phe Ser Glu Ser Ser Lys
Gly Leu Leu Ala Thr Ser Ala Ala Lys Ala65 70
75 80Pro Gly Leu Ile Pro Lys Leu Asp Thr Ala Tyr
Asp Val Ala Tyr Lys 85 90
95Ala Ala Glu Gly Ala Thr Pro Glu Ala Lys Tyr Asp Ala Phe Val Thr
100 105 110Ala Leu Thr Glu Ala Leu
Arg Val Ile Ala Gly Ala Leu Glu Val His 115 120
125Ala Val Lys Pro Ala Thr Glu Glu Val Pro Ala Ala Lys Ile
Pro Thr 130 135 140Gly Glu Leu Gln Ile
Val Asp Lys Ile Asp Ala Ala Phe Lys Ile Ala145 150
155 160Ala Thr Ala Ala Asn Ala Ala Pro Thr Asn
Asp Lys Phe Thr Val Phe 165 170
175Glu Ser Ala Phe Asn Lys Ala Leu Asn Glu Cys Thr Gly Gly Ala Tyr
180 185 190Glu Thr Tyr Lys Phe
Ile Pro Ser Leu Glu Ala Ala Val Lys Gln Ala 195
200 205Tyr Ala Ala Thr Val Ala Ala Ala Pro Glu Val Lys
Tyr Ala Val Phe 210 215 220Glu Ala Ala
Leu Thr Lys Ala Ile Thr Ala Met Thr Gln Ala Gln Lys225
230 235 240Ala Gly Lys Pro Ala Ala Ala
Ala Ala Thr Gly Ala Ala Thr Val Ala 245
250 255Thr Gly Ala Ala Thr Ala Ala Ala Gly Ala Ala Thr
Ala Ala Ala Gly 260 265 270Gly
Tyr Lys Ala 2752314PRTLolium perenne 2Ala Asp Ala Gly Tyr Ala Pro
Ala Thr Pro Ala Thr Pro Ala Ala Pro1 5 10
15Ala Thr Ala Ala Thr Pro Ala Thr Pro Ala Thr Pro Ala
Thr Pro Ala 20 25 30Ala Val
Pro Ser Gly Lys Ala Thr Thr Glu Glu Gln Lys Leu Ile Glu 35
40 45Lys Ile Asn Ala Gly Phe Lys Ala Ala Val
Ala Ala Ala Ala Val Val 50 55 60Pro
Pro Ala Asp Lys Tyr Lys Thr Phe Val Glu Thr Phe Gly Thr Ala65
70 75 80Thr Asn Lys Ala Phe Val
Glu Gly Leu Ala Ser Gly Tyr Ala Asp Gln 85
90 95Ser Lys Asn Gln Leu Thr Ser Lys Leu Asp Ala Ala
Leu Lys Leu Ala 100 105 110Tyr
Glu Ala Ala Gln Gly Ala Thr Pro Glu Ala Lys Tyr Asp Ala Tyr 115
120 125Val Ala Thr Leu Thr Glu Ala Leu Arg
Val Ile Ala Gly Thr Leu Glu 130 135
140Val His Ala Val Lys Pro Ala Ala Glu Glu Val Lys Val Gly Ala Ile145
150 155 160Pro Ala Ala Glu
Val Gln Leu Ile Asp Lys Val Asp Ala Ala Tyr Arg 165
170 175Thr Ala Ala Thr Ala Ala Asn Ala Ala Pro
Ala Asn Asp Lys Phe Thr 180 185
190Val Phe Glu Asn Thr Phe Asn Asn Ala Ile Lys Val Ser Leu Gly Ala
195 200 205Ala Tyr Asp Ser Tyr Lys Phe
Ile Pro Thr Leu Val Ala Ala Val Lys 210 215
220Gln Ala Tyr Ala Ala Lys Gln Ala Thr Ala Pro Glu Val Lys Tyr
Thr225 230 235 240Val Ser
Glu Thr Ala Leu Lys Lys Ala Val Thr Ala Met Ser Glu Ala
245 250 255Glu Lys Glu Ala Thr Pro Ala
Ala Ala Ala Thr Ala Thr Pro Thr Pro 260 265
270Ala Ala Ala Thr Ala Thr Ala Thr Pro Ala Ala Ala Tyr Ala
Thr Ala 275 280 285Thr Pro Ala Ala
Ala Thr Gly Thr Ala Thr Pro Ala Ala Ala Thr Ala 290
295 300Thr Pro Ala Ala Ala Gly Gly Tyr Lys Val305
3103285PRTPhleum pratense 3Ala Asp Leu Gly Tyr Gly Pro Ala Thr
Pro Ala Pro Ala Ala Gly Tyr1 5 10
15Thr Pro Ala Thr Pro Ala Ala Pro Ala Gly Ala Asp Ala Ala Gly
Lys 20 25 30Ala Thr Thr Glu
Glu Gln Lys Leu Ile Glu Lys Ile Asn Ala Gly Phe 35
40 45Lys Ala Ala Leu Ala Gly Ala Gly Val Gln Pro Pro
Ala Asp Lys Tyr 50 55 60Arg Thr Phe
Val Ala Thr Phe Gly Pro Ala Ser Asn Lys Ala Phe Ala65 70
75 80Glu Gly Leu Ser Gly Glu Pro Lys
Gly Ala Ala Glu Ser Ser Ser Lys 85 90
95Ala Ala Leu Thr Ser Lys Leu Asp Ala Ala Tyr Lys Leu Ala
Tyr Lys 100 105 110Thr Ala Glu
Gly Ala Thr Pro Glu Ala Lys Tyr Asp Ala Tyr Val Ala 115
120 125Thr Leu Ser Glu Ala Leu Arg Ile Ile Ala Gly
Thr Leu Glu Val His 130 135 140Ala Val
Lys Pro Ala Ala Glu Glu Val Lys Val Ile Pro Ala Gly Glu145
150 155 160Leu Gln Val Ile Glu Lys Val
Asp Ala Ala Phe Lys Val Ala Ala Thr 165
170 175Ala Ala Asn Ala Ala Pro Ala Asn Asp Lys Phe Thr
Val Phe Glu Ala 180 185 190Ala
Phe Asn Asp Glu Ile Lys Ala Ser Thr Gly Gly Ala Tyr Glu Ser 195
200 205Tyr Lys Phe Ile Pro Ala Leu Glu Ala
Ala Val Lys Gln Ala Tyr Ala 210 215
220Ala Thr Val Ala Thr Ala Pro Glu Val Lys Tyr Thr Val Phe Glu Thr225
230 235 240Ala Leu Lys Lys
Ala Ile Thr Ala Met Ser Glu Ala Gln Lys Ala Ala 245
250 255Lys Pro Pro Pro Leu Pro Pro Pro Pro Gln
Pro Pro Pro Leu Ala Ala 260 265
270Ala Gly Ala Ala Thr Ala Ala Thr Gly Gly Tyr Lys Val 275
280 2854262PRTPhleum pratense 4Ala Asp Ala Gly
Tyr Ala Pro Ala Thr Pro Ala Ala Ala Gly Ala Ala1 5
10 15Ala Gly Lys Ala Thr Thr Glu Glu Gln Lys
Leu Ile Glu Lys Ile Asn 20 25
30Val Gly Phe Lys Ala Ala Val Ala Ala Arg Gln Pro Ala Ala Asp Lys
35 40 45Phe Lys Thr Phe Glu Ala Ala Ser
Pro Arg His Pro Arg Pro Leu Arg 50 55
60Gln Gly Ala Gly Leu Val Ser Lys Leu Asp Ala Ala Tyr Ser Val Ala65
70 75 80Tyr Lys Ala Ala Val
Gly Ala Thr Pro Glu Ala Lys Phe Asp Ser Phe 85
90 95Val Ala Ser Leu Thr Glu Ala Leu Arg Val Ile
Ala Gly Ala Leu Glu 100 105
110Val His Ala Val Lys Pro Val Thr Glu Glu Pro Gly Met Ala Lys Ile
115 120 125Pro Ala Gly Glu Leu Gln Ile
Ile Asp Lys Ile Asp Ala Ala Phe Lys 130 135
140Val Ala Ala Thr Ala Ala Ala Thr Ala Pro Ala Asp Asp Lys Phe
Thr145 150 155 160Val Phe
Glu Ala Ala Phe Asn Lys Ala Ile Lys Glu Ser Thr Gly Gly
165 170 175Ala Tyr Asp Thr Tyr Lys Cys
Ile Pro Ser Leu Glu Ala Ala Val Lys 180 185
190Gln Ala Tyr Ala Ala Thr Val Ala Ala Ala Ala Glu Val Lys
Tyr Thr 195 200 205Val Phe Glu Ala
Ala Leu Thr Lys Ala Ile Thr Ala Met Ser Glu Val 210
215 220Gln Ser Lys Val Gln Pro Ala Thr Gly Ala Ala Thr
Val Ala Ala Gly225 230 235
240Ala Ala Thr Thr Ala Ala Gly Ala Ala Ser Gly Ala Ala Thr Val Ala
245 250 255Ala Gly Gly Tyr Lys
Val 2605309PRTPoa pratensis 5Ala Asp Val Gly Tyr Gly Ala Pro
Ala Thr Leu Ala Thr Pro Ala Thr1 5 10
15Pro Pro Ala Ala Pro Ala Ala Gly Tyr Thr Pro Ala Ala Pro
Ala Gly 20 25 30Ala Ala Pro
Lys Ala Thr Thr Asp Glu Gln Lys Leu Ile Glu Lys Ile 35
40 45Asn Ala Gly Phe Lys Ala Ala Val Ala Ala Ala
Ala Gly Val Pro Ala 50 55 60Val Asp
Lys Tyr Lys Thr Phe Val Ala Thr Phe Gly Thr Ala Ser Asn65
70 75 80Lys Ala Phe Ala Glu Ala Leu
Ser Thr Glu Pro Lys Gly Ala Ala Ala 85 90
95Ala Ser Ser Asn Ala Val Leu Thr Ser Lys Leu Asp Ala
Ala Tyr Lys 100 105 110Leu Ala
Tyr Lys Ser Ala Glu Gly Ala Thr Pro Glu Ala Lys Tyr Asp 115
120 125Ala Tyr Val Ala Thr Leu Ser Glu Ala Leu
Arg Ile Ile Ala Gly Thr 130 135 140Leu
Glu Val His Ala Val Lys Pro Ala Gly Glu Glu Val Lys Ala Ile145
150 155 160Pro Ala Gly Glu Leu Gln
Val Ile Asp Lys Val Asp Ala Ala Phe Lys 165
170 175Val Ala Ala Thr Ala Ala Asn Ala Ala Pro Ala Asn
Asp Lys Phe Thr 180 185 190Val
Phe Glu Ala Ala Phe Asn Asp Ala Ile Lys Ala Ser Thr Gly Gly 195
200 205Ala Tyr Gln Ser Tyr Lys Phe Ile Pro
Ala Leu Glu Ala Ala Val Lys 210 215
220Gln Ser Tyr Ala Ala Thr Val Ala Thr Ala Pro Ala Val Lys Tyr Ala225
230 235 240Val Phe Glu Thr
Ala Leu Lys Lys Ala Ile Thr Ala Met Ser Gln Ala 245
250 255Gln Lys Ala Ala Lys Pro Ala Ala Ala Val
Thr Ala Thr Ala Thr Gly 260 265
270Ala Val Gly Ala Ala Thr Gly Ala Val Gly Ala Ala Thr Gly Ala Ala
275 280 285Thr Ala Ala Ala Gly Gly Tyr
Lys Thr Gly Ala Ala Thr Pro Thr Ala 290 295
300Gly Gly Tyr Lys Val3056283PRTPoa pratensis 6Ala Asp Leu Ser Tyr
Gly Ala Pro Ala Thr Pro Ala Ala Pro Ala Ala1 5
10 15Gly Tyr Thr Pro Ala Ala Pro Ala Gly Ala Ala
Pro Lys Ala Thr Thr 20 25
30Asp Glu Gln Lys Met Ile Glu Lys Ile Asn Val Gly Phe Lys Ala Ala
35 40 45Val Ala Ala Ala Gly Gly Val Pro
Ala Ala Asn Lys Tyr Lys Thr Phe 50 55
60Val Ala Thr Phe Gly Ala Ala Ser Asn Lys Ala Phe Ala Glu Ala Leu65
70 75 80Ser Thr Glu Pro Lys
Gly Ala Ala Ala Asp Ser Ser Lys Ala Ala Leu 85
90 95Thr Ser Lys Leu Asp Ala Ala Tyr Lys Leu Ala
Tyr Lys Ser Ala Glu 100 105
110Gly Ala Thr Pro Glu Ala Lys Tyr Asp Asp Tyr Val Ala Thr Leu Ser
115 120 125Glu Ala Leu Arg Ile Ile Ala
Gly Thr Leu Glu Val His Gly Val Lys 130 135
140Pro Ala Ala Glu Glu Val Lys Ala Thr Pro Ala Gly Glu Leu Gln
Val145 150 155 160Ile Asp
Lys Val Asp Ala Ala Phe Lys Val Ala Ala Thr Ala Ala Asn
165 170 175Ala Ala Pro Ala Asn Asp Lys
Phe Thr Val Phe Glu Ala Ala Phe Asn 180 185
190Asp Ala Ile Lys Ala Ser Thr Gly Gly Ala Tyr Gln Ser Tyr
Lys Phe 195 200 205Ile Pro Ala Leu
Glu Ala Ala Val Lys Gln Ser Tyr Ala Ala Thr Val 210
215 220Ala Thr Ala Pro Ala Val Lys Tyr Ala Val Phe Glu
Thr Ala Leu Lys225 230 235
240Lys Ala Ile Thr Ala Met Ser Gln Ala Gln Lys Ala Ala Lys Pro Ala
245 250 255Ala Ala Ala Thr Gly
Thr Ala Thr Ala Ala Val Gly Ala Ala Thr Gly 260
265 270Ala Ala Thr Ala Ala Ala Gly Gly Tyr Lys Val
275 2807301PRTLolium perenne 7Met Ala Val Gln Lys Tyr
Thr Val Ala Leu Phe Leu Ala Val Ala Leu1 5
10 15Val Ala Gly Pro Ala Ala Ser Tyr Ala Ala Asp Ala
Gly Tyr Thr Pro 20 25 30Ala
Ala Ala Ala Thr Pro Ala Thr Pro Ala Ala Thr Pro Ala Ala Ala 35
40 45Gly Gly Lys Ala Thr Thr Asp Glu Gln
Lys Leu Leu Glu Asp Val Asn 50 55
60Ala Gly Phe Lys Ala Ala Val Ala Ala Ala Ala Asn Ala Pro Pro Ala65
70 75 80Asp Lys Phe Lys Ile
Phe Glu Ala Ala Phe Ser Glu Ser Ser Lys Gly 85
90 95Leu Leu Ala Thr Ser Ala Ala Lys Ala Pro Gly
Leu Ile Pro Lys Leu 100 105
110Asp Thr Ala Tyr Asp Val Ala Tyr Lys Ala Ala Glu Gly Ala Thr Pro
115 120 125Glu Ala Lys Tyr Asp Ala Phe
Val Thr Ala Leu Thr Glu Ala Leu Arg 130 135
140Val Ile Ala Gly Ala Leu Glu Val His Ala Val Lys Pro Ala Thr
Glu145 150 155 160Glu Val
Pro Ala Ala Lys Ile Pro Thr Gly Glu Leu Gln Ile Val Asp
165 170 175Lys Ile Asp Ala Ala Phe Lys
Ile Ala Ala Thr Ala Ala Asn Ala Ala 180 185
190Pro Thr Asn Asp Lys Phe Thr Val Phe Glu Ser Ala Phe Asn
Lys Ala 195 200 205Leu Asn Glu Cys
Thr Gly Gly Ala Tyr Glu Thr Tyr Lys Phe Ile Pro 210
215 220Ser Leu Glu Ala Ala Val Lys Asp Ala Tyr Ala Ala
Thr Val Ala Ala225 230 235
240Ala Pro Glu Val Lys Tyr Ala Val Phe Glu Ala Ala Leu Thr Lys Ala
245 250 255Ile Thr Ala Met Thr
Gln Ala Gln Lys Ala Gly Lys Pro Ala Ala Ala 260
265 270Ala Ala Thr Gly Ala Ala Thr Val Ala Thr Gly Ala
Ala Thr Ala Ala 275 280 285Ala Gly
Ala Ala Thr Ala Ala Ala Gly Gly Tyr Lys Ala 290 295
3008301PRTLolium perenne 8Met Ala Val Gln Lys Tyr Thr Val
Ala Leu Phe Leu Ala Val Ala Leu1 5 10
15Val Ala Gly Pro Ala Ala Ser Tyr Ala Ala Asp Ala Gly Tyr
Thr Pro 20 25 30Ala Ala Ala
Ala Thr Pro Ala Thr Pro Ala Ala Thr Pro Ala Ala Ala 35
40 45Gly Gly Lys Ala Thr Thr Asp Glu Gln Lys Leu
Leu Glu Asp Val Asn 50 55 60Ala Gly
Phe Lys Ala Ala Val Ala Ala Ala Ala Asn Ala Pro Pro Ala65
70 75 80Asp Ala Phe Lys Ile Phe Glu
Ala Ala Phe Ser Glu Ser Ser Lys Gly 85 90
95Leu Leu Ala Thr Ser Ala Ala Lys Ala Pro Gly Leu Ile
Pro Lys Leu 100 105 110Asp Thr
Ala Tyr Asp Val Ala Tyr Lys Ala Ala Glu Gly Ala Thr Pro 115
120 125Glu Ala Lys Tyr Asp Ala Phe Val Thr Ala
Leu Thr Glu Ala Leu Arg 130 135 140Val
Ile Ala Gly Ala Leu Glu Val His Ala Val Lys Pro Ala Thr Glu145
150 155 160Glu Val Pro Ala Ala Lys
Ile Pro Thr Gly Glu Leu Gln Ile Val Asp 165
170 175Lys Ile Asp Ala Ala Phe Lys Ile Ala Ala Thr Ala
Ala Asn Ala Ala 180 185 190Pro
Thr Asn Asp Lys Phe Thr Val Phe Glu Ser Ala Phe Asn Lys Ala 195
200 205Leu Asn Glu Cys Thr Gly Gly Ala Tyr
Glu Thr Tyr Lys Phe Ile Pro 210 215
220Ser Leu Glu Ala Ala Val Lys Asp Ala Tyr Ala Ala Thr Val Ala Ala225
230 235 240Ala Pro Glu Val
Lys Tyr Ala Val Phe Glu Ala Ala Leu Thr Lys Ala 245
250 255Ile Thr Ala Met Thr Gln Ala Gln Lys Ala
Gly Lys Pro Ala Ala Ala 260 265
270Ala Ala Thr Gly Ala Ala Thr Val Ala Thr Gly Ala Ala Thr Ala Ala
275 280 285Ala Gly Ala Ala Thr Ala Ala
Ala Gly Gly Tyr Lys Ala 290 295
3009301PRTLolium perenne 9Met Ala Val Gln Lys Tyr Thr Val Ala Leu Phe Leu
Ala Val Ala Leu1 5 10
15Val Ala Gly Pro Ala Ala Ser Tyr Ala Ala Asp Ala Gly Tyr Thr Pro
20 25 30Ala Ala Ala Ala Thr Pro Ala
Thr Pro Ala Ala Thr Pro Ala Ala Ala 35 40
45Gly Gly Lys Ala Thr Thr Asp Glu Gln Lys Leu Leu Glu Asp Val
Asn 50 55 60Ala Gly Phe Lys Ala Ala
Val Ala Ala Ala Ala Asn Ala Pro Pro Ala65 70
75 80Asp Lys Phe Lys Ile Phe Glu Ala Ala Phe Ser
Glu Ser Ser Lys Gly 85 90
95Leu Leu Ala Thr Ser Ala Ala Lys Ala Pro Gly Leu Ile Pro Lys Leu
100 105 110Asp Thr Ala Tyr Asp Val
Ala Tyr Lys Ala Ala Glu Gly Ala Thr Pro 115 120
125Glu Ala Lys Tyr Asp Ala Phe Val Thr Ala Leu Thr Glu Ala
Leu Arg 130 135 140Val Ile Ala Gly Ala
Leu Glu Val His Ala Val Lys Pro Ala Thr Glu145 150
155 160Glu Val Pro Ala Ala Lys Ile Pro Thr Gly
Glu Leu Gln Ile Val Asp 165 170
175Lys Ile Asp Ala Ala Phe Lys Ile Ala Ala Thr Ala Ala Asn Ala Ala
180 185 190Pro Thr Asn Asp Lys
Phe Thr Val Phe Glu Ser Ala Phe Asn Lys Ala 195
200 205Leu Asn Glu Cys Thr Gly Gly Ala Tyr Glu Thr Tyr
Lys Phe Ile Pro 210 215 220Ser Leu Glu
Ala Ala Val Lys Asp Ala Tyr Ala Ala Thr Val Ala Ala225
230 235 240Ala Pro Glu Val Lys Tyr Ala
Val Phe Glu Ala Ala Leu Thr Lys Ala 245
250 255Ile Thr Ala Met Thr Gln Ala Gln Lys Ala Gly Lys
Pro Ala Ala Ala 260 265 270Ala
Ala Thr Gly Ala Ala Thr Val Ala Thr Gly Ala Ala Thr Ala Ala 275
280 285Ala Gly Ala Ala Thr Ala Ala Ala Gly
Ala Tyr Ala Ala 290 295
30010300PRTLolium perenne 10Met Ala Val Gln Lys Tyr Thr Val Ala Leu Phe
Leu Ala Val Ala Leu1 5 10
15Val Ala Gly Pro Ala Ala Ser Tyr Ala Ala Asp Ala Gly Tyr Thr Pro
20 25 30Ala Ala Ala Ala Thr Pro Ala
Thr Pro Ala Ala Thr Pro Ala Ala Ala 35 40
45Gly Gly Lys Ala Thr Thr Asp Glu Gln Lys Leu Leu Glu Asp Val
Asn 50 55 60Ala Gly Phe Lys Ala Ala
Val Ala Ala Ala Ala Asn Ala Pro Pro Ala65 70
75 80Asp Lys Phe Lys Ile Phe Glu Ala Ala Phe Ser
Glu Ser Ser Lys Gly 85 90
95Leu Leu Ala Thr Ser Ala Ala Lys Ala Pro Gly Leu Ile Pro Lys Leu
100 105 110Asp Thr Ala Tyr Asp Val
Ala Tyr Lys Ala Ala Glu Gly Ala Thr Pro 115 120
125Glu Ala Lys Tyr Asp Ala Phe Val Thr Ala Leu Thr Glu Ala
Leu Arg 130 135 140Val Ile Ala Gly Ala
Leu Glu Val His Ala Val Lys Pro Ala Thr Glu145 150
155 160Glu Val Pro Ala Ala Lys Ile Pro Thr Gly
Glu Leu Gln Ile Val Asp 165 170
175Lys Ile Asp Ala Ala Phe Lys Ile Ala Ala Thr Ala Ala Asn Ala Ala
180 185 190Pro Thr Asn Asp Lys
Phe Thr Val Phe Glu Ser Ala Phe Asn Lys Ala 195
200 205Leu Asn Glu Cys Thr Gly Gly Ala Tyr Glu Thr Tyr
Lys Phe Ile Pro 210 215 220Ser Leu Glu
Ala Ala Val Lys Asp Ala Tyr Ala Ala Thr Val Ala Ala225
230 235 240Ala Pro Glu Val Lys Tyr Ala
Val Phe Glu Ala Ala Leu Thr Lys Ala 245
250 255Ile Thr Ala Met Thr Gln Ala Gln Lys Ala Gly Lys
Pro Ala Ala Ala 260 265 270Ala
Ala Thr Gly Ala Ala Thr Val Ala Thr Gly Ala Ala Thr Ala Ala 275
280 285Ala Gly Ala Ala Thr Ala Ala Ala Gly
Tyr Lys Ala 290 295 30011301PRTLolium
perenne 11Met Ala Val Gln Lys Tyr Thr Val Ala Leu Phe Leu Ala Val Ala
Leu1 5 10 15Val Ala Gly
Pro Ala Ala Ser Tyr Ala Ala Asp Ala Gly Tyr Thr Pro 20
25 30Ala Ala Ala Ala Thr Pro Ala Thr Pro Ala
Ala Thr Pro Ala Ala Ala 35 40
45Gly Gly Lys Ala Thr Thr Asp Glu Gln Lys Leu Leu Glu Asp Val Asn 50
55 60Ala Gly Phe Lys Ala Ala Val Ala Ala
Ala Ala Asn Ala Pro Pro Ala65 70 75
80Asp Lys Phe Lys Ile Phe Glu Ala Ala Phe Ser Glu Ser Ser
Lys Gly 85 90 95Leu Leu
Ala Thr Ser Ala Ala Lys Ala Pro Gly Leu Ile Pro Lys Leu 100
105 110Asp Thr Ala Tyr Asp Val Ala Tyr Lys
Ala Ala Glu Gly Ala Thr Pro 115 120
125Glu Ala Lys Tyr Asp Ala Phe Val Thr Ala Leu Thr Glu Ala Leu Arg
130 135 140Val Ile Ala Gly Ala Leu Glu
Val His Ala Val Lys Pro Ala Thr Glu145 150
155 160Glu Val Pro Ala Ala Lys Ile Pro Thr Gly Glu Leu
Gln Ile Val Asp 165 170
175Lys Ile Asp Ala Ala Phe Lys Ile Ala Ala Thr Ala Ala Asn Ala Ala
180 185 190Pro Thr Asn Asp Asn Leu
Ala Ala Phe Glu Ser Ala Phe Asn Lys Ala 195 200
205Leu Asn Glu Cys Thr Gly Gly Ala Tyr Glu Thr Tyr Lys Phe
Ile Pro 210 215 220Ser Leu Glu Ala Ala
Val Lys Asp Ala Tyr Ala Ala Thr Val Ala Ala225 230
235 240Ala Pro Glu Val Lys Tyr Ala Val Phe Glu
Ala Ala Leu Thr Lys Ala 245 250
255Ile Thr Ala Met Thr Gln Ala Gln Lys Ala Gly Lys Pro Ala Ala Ala
260 265 270Ala Ala Thr Gly Ala
Ala Thr Val Ala Thr Gly Ala Ala Thr Ala Ala 275
280 285Ala Gly Ala Ala Thr Ala Ala Ala Gly Gly Tyr Lys
Ala 290 295 30012301PRTLolium perenne
12Met Ala Val Gln Lys Tyr Thr Val Ala Leu Phe Leu Ala Val Ala Leu1
5 10 15Val Ala Gly Pro Ala Ala
Ser Tyr Ala Ala Asp Ala Gly Tyr Thr Pro 20 25
30Ala Ala Ala Ala Thr Pro Ala Thr Pro Ala Ala Thr Pro
Ala Ala Ala 35 40 45Gly Gly Lys
Ala Thr Thr Asp Glu Gln Lys Leu Leu Glu Asp Val Asn 50
55 60Ala Gly Phe Lys Ala Ala Val Ala Ala Ala Ala Asn
Ala Pro Pro Ala65 70 75
80Asp Lys Phe Lys Ile Phe Glu Ala Ala Phe Ser Glu Ser Ser Lys Gly
85 90 95Leu Leu Ala Thr Ser Ala
Ala Lys Ala Pro Gly Leu Ile Pro Lys Leu 100
105 110Asp Thr Ala Tyr Asp Val Ala Tyr Lys Ala Ala Glu
Gly Ala Thr Pro 115 120 125Glu Ala
Lys Tyr Asp Ala Phe Val Thr Ala Leu Thr Glu Ala Leu Arg 130
135 140Val Ile Ala Gly Ala Leu Glu Val His Ala Val
Lys Pro Ala Thr Glu145 150 155
160Glu Val Pro Ala Ala Lys Ile Pro Thr Gly Glu Leu Gln Ile Val Asp
165 170 175Lys Ile Asp Ala
Ala Phe Lys Ile Ala Ala Thr Ala Ala Asn Ala Ala 180
185 190Pro Thr Asn Asp Lys Phe Thr Val Phe Glu Ser
Ala Phe Asn Lys Ala 195 200 205Leu
Asn Glu Cys Thr Gly Gly Ala Tyr Glu Thr Tyr Lys Phe Ile Pro 210
215 220Ser Leu Glu Ala Gly Ala Ala Asp Ala Tyr
Ala Ala Thr Val Ala Ala225 230 235
240Ala Pro Glu Val Lys Tyr Ala Val Phe Glu Ala Ala Leu Thr Lys
Ala 245 250 255Ile Thr Ala
Met Thr Gln Ala Gln Lys Ala Gly Lys Pro Ala Ala Ala 260
265 270Ala Ala Thr Gly Ala Ala Thr Val Ala Thr
Gly Ala Ala Thr Ala Ala 275 280
285Ala Gly Ala Ala Thr Ala Ala Ala Gly Gly Tyr Lys Ala 290
295 3001324DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 13cctccggcgg acgcgttcaa gatc
241424DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
14gatcttgaac gcgtccgccg gagg
241529DNAArtificial SequenceDescription of Artificial Sequence Synthetic
primer 15gctgctggtg cctacgcagc ctgatcagc
291629DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 16gctgatcagg ctgcgtaggc accagcagc
291728DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 17ccaccgccgc tgcttgaggc tacaaagc
281828DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 18gctttgtagc ctcaagcagc
ggcggtgg 281934DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
19ccaccaacga taacttggcc gccttcgaga gtgc
342034DNAArtificial SequenceDescription of Artificial Sequence Synthetic
primer 20gcactctcga aggcggccaa gttatcgttg gtgg
342129DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 21cctcgaggcc ggggccgcgc aggcctacg
292229DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 22cgtaggcctg cgcggccccg gcctcgagg
29
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