AU2012205288B2 - DNA-sequence and recombinant production of group 4 major allergens from cereals - Google Patents

Abstract

C:\NRPortbl\DCC\GRS\4488542_1.DOC-23/07/2012 DNA sequence, and recombinant preparation of group 4 major allergens from cereals Abstract The invention relates to the preparation of DNA sequences of group 4 major allergens from cereals. The invention also includes fragments, novel combinations of partial sequences and point mutations with hypoallergenic effects. The recombinant DNA molecules and the derived polypeptides, fragments, novel combinations of partial sequences and variants can be used for therapy of pollen-allergy diseases. The proteins produced by recombination can be applied to in-vitro and in-vivo diagnosis of pollen allergies.

Description

Australian Patents Act 1990 - Regulation 3.2 ORIGINAL COMPLETE SPECIFICATION STANDARD PATENT Invention Title: DNA-sequence and recombinant production of group 4 major allergens from cereals The following statement is a full description of this invention, including the best method of performing it known to me: P/00/01 I WO 2005/059136 PCT/EP2004/013664 DNA sequence, and recombinant preparation of group 4 major allergens from cereals 5 Background of the invention 10 The present invention relates to the provision of DNA sequences of group 4 major allergens from cereals (Triticeae). The invention also encompasses fragments, new combinations of partial sequences and point mutants hav ing a hypoallergenic action. The recombinant DNA molecules and the derived polypeptides, fragments, new combinations of partial sequences 15 and variants can be utilised for the therapy of pollen-allergic diseases. The proteins prepared by recombinant methods can be employed for in vitro and in vivo diagnosis of pollen allergies. 20 Type I allergies are of importance worldwide. Up to 20% of the population in industrialised countries suffer from complaints such as allergic rhinitis, conjunctivitis or bronchial asthma. These allergies are caused by allergens present in the air (aeroallergens) which are released by sources of various 25 origin, such as plant pollen, mites, cats or dogs. Up to 40% of these type 1 allergy sufferers in turn exhibit specific IgE reactivity with grass pollen allergens, inter alia cereal pollen allergens (Freidhoff et al., 1986, J. Allergy Clin. Immunol. 78, 1190-2001). Of the cereal pollen allergens, the allergens of rye have particular importance. 30 The substances which trigger type 1 allergy are proteins, glycoproteins or polypeptides. After uptake via the mucous membranes, these allergens react with the IgE molecules bonded to the surface of mast cells in sensi 35 tised individuals. If two IgE molecules are crosslinked to one another by an allergen, this results in the release of mediators (for example histamine, WO 2005/059136 PCT/EP2004/013664 -2 prostaglandins) and cytokines by the effector cell and thus in the corres ponding clinical symptoms. 5 A distinction is made between major and minor allergens, depending on the relative frequency with which the individual allergen molecules react with the IgE antibodies of allergy sufferers. The allergens from the pollen of various species from the family of the 10 grasses (Poaceae) are divided into groups which are homologous amongst one another. In particular, the molecules of major allergen group 4 have high immuno logical cross-reactivity with one another both with monoclonal murine anti 15 bodies and also with human IgE antibodies (Fahlbusch et al., 1993 Clin. Exp. Allergy 23:51-60; Leduc-Brodard et al., 1996, J. Allergy Clin. Immunol. 98:1065-1072; Su et al., 1996, J. Allergy Clin. Immunol. 97:210; Fahlbusch et al., 1998, Clin. Exp. Allergy 28:799-807; Gavrovic-Jankulovic et al., 2000, 20 Invest. Allergol. Clin. Immunol. 10 (6):361-367; Stumvoll et al. 2002, Biol. Chem. 383:1383-1396; Grote et al., 2002, Biol. Chem. 383:1441-1445; Andersson and Lidholm, 2003, Int. Arch. Allergy Immunol. 130:87-107; Mari, 2003, Clin. Exp. Allergy, 33 (1):43-51). 25 A complete DNA sequence is hitherto not known for any of the group 4 major allergens. From the group 4 allergen from Dactylus glomerata, it has hitherto only 30 been possible for peptides to be obtained by enzymatic degradation and sequenced: DIYNYMEPYVSK (SEQ ID NO 13), VDPTDYFGNEQ (SEQ ID NO 14), 35 ARTAWVDSGAQLGELSY (SEQ ID NO 15) and GVLFNIQYVNYWFAP (SEQ ID NO 16, Leduc-Brodard et al., 1996, J. Allergy Clin. Immunol. 98: 1065-1072).

WO 2005/059136 PCT/EP2004/013664 -3 Peptides have also been obtained from the group 4 allergen of sub-tropical Bermuda grass (Cynodon dactylon) by proteolysis and sequenced: KTVKPLYIITP (SEQ ID NO 17), 5 KQVERDFLTSLTKDIPQLYLKS (SEQ ID NO 18), TVKPLYIITPITAAMI (SEQ ID NO 19), LRKYGTAADNVIDAKWDAQGRLL (SEQ ID NO 20), KWQTVAPALPDPNM (SEQ ID NO 21), VTWIESVPYIPMGDK (SEQ ID NO 22), 10 GTVRDLLXRTSNIKAFGKY (SEQ ID NO 23), TSNIKAFGKYKSDYVLEPIPKKS (SEQ ID NO 24), YRDLDLGVNQWG (SEQ ID NO 25), SATPPTHRSGVLFNI (SEQ ID NO 26) 15 and AAAALPTQVTRDIYAFMTPYVSKNPRQAYVNYRDLD (SEQ ID NO 27, Liaw et al., 2001, Biochem. Biophys. Research Communication 280: 738 743). 20 For Lolium perenne, peptide fragments having the following sequences have been described for the basic group 4 allergen: FLEPVLGLIFPAGV (SEQ ID NO 28) and GLIEFPAGV (SEQ ID NO 29, Jaggi et al., 1989, Int. Arch. Allergy Appl. Immunol. 89: 342-348). 25 As the first sequence of a group 4 allergen, the still unpublished sequence of Phl p 4 from Phleum pratense (SEQ ID NO 11) has been elucidated by the inventors of the present patent application and described in Interna tional Application WO 04/000881. 30 Nothing is hitherto known on the sequences of the group 4 major allergens from cereals (Triceae). 35 The object on which the present invention was based therefore consisted in the provision of DNA sequences of group 4 major allergens from cereals, in WO 2005/059136 PCT/EP2004/013664 -4 particular the allergen Sec c 4 from rye (Secale cerale) (SEQ ID NO 1, 3), Hor v 4 from barley (Hordeum vulgare) (SEQ ID NO 5) and Tri a 4 from wheat (Triticum aestivum) (SEQ ID NO 7, 9) and of corresponding recom 5 binant DNA molecules on the basis of which the allergens can be ex pressed as protein and made available, as such or in modified form, for pharmacologically significant exploitation. The sequence of Phi p 4 (SEQ ID NO 11) was the starting point for the present invention. 10 List of sequences according to the invention The DNA and protein sequences of the mature allergens in accordance 15 with SEQ ID NO 1-10 are preceded by a signal sequence. The encoding region ends with the TGA or TAG stop codons in the DNA sequences. - DNA sequence of Sec c 4. (a) Isoform Sec c 4.01 (SEQ ID NO 1), (b) 20 isoform Sec c 4.02 (SEQ ID NO 3). - Protein sequences (SEQ ID NO 2, 4) derived from the DNA sequences in accordance with SEQ ID NO 1 and 3. - DNA sequence of Hor v 4 (SEQ ID NO 5). - Protein sequence (SEQ ID NO 6) derived from the DNA sequence in 25 accordance with SEQ ID NO 5. - DNA sequence of Tri a 4. (a) Isoform Tri a 4.01 (SEQ ID NO 7), (b) iso form Tri a 4.02 (SEQ ID NO 9). - Protein sequences (SEQ ID NO 8, 10) derived from the DNA sequences 30 in accordance with SEQ ID NO 7 and 9. - DNA sequence of PhI p 4 (SEQ ID NO 11), in accordance with SEQ ID NO 5 from WO 04/000881. - Protein sequence of PhI p 4 (SEQ ID NO 12), in accordance with SEQ ID NO 6 from WO 04/000881. 35 H:\mm\Interwoven\NRPortbl\DCC\MM\7430977_1.doc-4/02/201S -5 Description of the invention According to a first aspect of the present invention there is provided an isolated DNA molecule corresponding to a nucleotide sequence of a cereal pollen major allergen comprising the sequence set forth in SEQ ID NO 5. According to a second aspect of the present invention there is provided an isolated DNA molecule which hybridises with a DNA molecule according to the first aspect under stringent conditions and originates from DNA sequences from Poaceae species. According to a third aspect of the present invention there is provided an isolated DNA molecule corresponding to a partial sequence or a combination of partial sequences according to the first or second aspects, which encodes an immunomodulatory, T-cell-reactive fragment of a group 4 allergen from the Poaceae species. According to a fourth aspect of the present invention there is provided a recombinant DNA expression vector or a cloning system comprising a DNA molecule according the first, second or third aspects, functionally linked to an expression control sequence. According to a fifth aspect of the present invention there is provided a host organism transformed with a DNA molecule according to the first, second or third aspects, or an expression vector according to the fourth aspect. According to a sixth aspect of the present invention there is provided a process for the preparation of a polypeptide encoded by a DNA sequence according to the first, second or third aspects by cultivation of a host organism according to the fifth aspect, and isolation of the corresponding polypeptide from the culture.

H:\mm\lnterwoven\NRPortbl\DCC\MM\7430977_1.doc-4/02/2015 - 5a According to a seventh aspect of the present invention there is provided an isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO 6, or which is encoded by a nucleotide sequence as defined in the first, second or third aspects. According to an eighth aspect of the present invention there is provided an isolated polypeptide corresponding to a mature allergen comprising an amino acid sequence according to the seventh aspect, comprising the amino acid sequence set forth in SEQ ID NO 6, beginning with the amino acid residue at position 23. According to a ninth aspect of the present invention there is provided a medicament comprising a polypeptide according to the seventh or eighth aspects. According to a tenth aspect of the present invention there is provided a pharmaceutical composition comprising at least one polypeptide according to the seventh or eighth aspects when used for the diagnosis and/or treatment of allergies triggered by group 4 allergens from the Poaceae species. According to an eleventh aspect of the present invention there is provided use of at least one polypeptide according to the seventh or eighth aspects, for the preparation of a medicament for the diagnosis, treatment and/or prevention of allergies triggered by group 4 allergens from the Poaceae species. According to a twelfth aspect of the present invention there is provided a medicament comprising a DNA molecule according to the first, second or third aspects. According to a thirteenth aspect of the present invention there is provided a medicament comprising a recombinant expression vector according to the fourth aspect.

H:\mm\lnterwoven\NRPartbl\DCC\MM\7430977_1.doc-4/02/2015 - 5b According to a fourteenth aspect of the present invention there is provided a pharmaceutical composition comprising at least one DNA molecule according to the first, second or third aspects, or at least one expression vector according to the fourth aspect, when used for the immunotherapeutic DNA vaccination of patients with allergies triggered by group 4 allergens from the Poaceae species, and/or for the prevention of such allergies. According to a fifteenth aspect of the present invention there is provided use of at least one DNA molecule according to the first, second or third aspects, or at least one expression vector according to the fourth aspect, for the preparation of a medicament for the immunotherapeutic DNA vaccination of patients with allergies triggered by group 4 allergens from the Poaceae species. According to a sixteenth aspect of the present invention there is provided a method for the diagnosis, treatment and/or prevention of allergies triggered by group 4 allergens from Poaceae species, comprising administering to a subject in need thereof at least one polypeptide according to the seventh or eighth aspects, or a medicament according to the ninth, twelfth or thirteenth aspects. The present invention now provides for the first time DNA sequences of the cereal pollen major allergens Sec c 4, Hor v 4 and Tri a 4, in accordance with SEQ ID NO 1, 3, 5, 7, and 9. The present invention therefore relates to DNA molecules selected from the nucleotide sequences in accordance with SEQ ID NO 1, 3, 5, 7, and 9. The invention furthermore relates to sequences homologous to the DNA sequences according to the invention and corresponding DNA molecules of group 4 allergens from other Poaceae, such as, for example, Lolium perenne, Dactylis glomerata, Poa pratensis, Cynodon dactylon and Holcus lanatus, which, owing to the sequence homology that exists, hybridise with the DNA sequences according to the invention under stringent conditions, or have immunological H:\mm\interwoven\NRPortbI\DCC\MM\74309771.dc-4/02/2015 - 5c cross-reactivity with respect to the allergens according to the invention. The invention also encompasses fragments, new combinations of partial sequences and point mutants having a hypoallergenic action The invention therefore furthermore relates to corresponding partial sequences, a combination of partial sequences, or replacement, elimination or addition mutants which encode an immunomodulatory, T-cell-reactive fragment of a group 4 allergen from the Poaceae. With knowledge of the DNA sequence of the naturally occurring allergens, it is now possible to prepare these allergens as recombinant proteins which can be used in the diagnosis and therapy of allergic diseases (Scheiner and Kraft, 1995, Allergy 50: 384-391).

WO 2005/059136 PCT/EP2004/013664 -6 A classical approach to effective therapeutic treatment of allergies is spe cific immunotherapy or hyposensitisation (Fiebig, 1995, Allergo J. 4 (6): 336-339, Bousquet et al., 1998, J. Allergy Clin. Immunol. 102 (4): 558-562). 5 In this method, the patient is injected subcutaneously with natural allergen extracts in increasing doses. However, there is a risk in this method of allergic reactions or even anaphylactic shock. In order to minimise these risks, innovative preparations in the form of allergoids are employed. These are chemically modified allergen extracts which have significantly reduced 10 IgE reactivity, but identical T-cell reactivity compared with the untreated extract (Fiebig, 1995, Allergo J. 4 (7): 377-382). Even more substantial therapy optimisation would be possible with aller gens prepared by recombinant methods. Defined cocktails of high-purity 15 allergens prepared by recombinant methods, optionally matched to the individual sensitisation patterns of the patients, could replace extracts from natural allergen sources since these, in addition to the various allergens, contain a relatively large number of immunogenic, but non-allergenic sec 20 ondary proteins. Realistic perspectives which may result in reliable hyposensitisation with expression products are offered by specifically mutated recombinant aller gens in which IgE epitopes are specifically deleted without impairing the T-cell epitopes which are essential for therapy (Schramm et al., 1999, 25 J. Immunol. 162: 2406-2414). A further possibility for therapeutic influencing of the disturbed TH cell equilibrium in allergy sufferers is immunotherapeutic DNA vaccination, 30 which involves treatment with expressible DNA which encodes the relevant allergens. Initial experimental evidence of allergen-specific influencing of the immune response has been furnished in rodents by injection of aller gen-encoding DNA (Hsu et al., 1996, Nature Medicine 2 (5): 540-544). 35 WO 2005/059136 PCT/EP2004/013664 -7 The present invention therefore also relates to a DNA molecule described above or below as medicament and to a corresponding recombinant expression vector as medicament. 5 The corresponding proteins prepared by recombinant methods can be employed for therapy and for in vitro and in vivo diagnosis of pollen aller gies. For preparation of the recombinant allergen, the cloned nucleic acid is 10 ligated into an expression vector, and this construct is expressed in a suit able host organism. After biochemical purification, this recombinant aller gen is available for detection of IgE antibodies by established methods. 15 The present invention therefore furthermore relates to a recombinant expression vector comprising a DNA molecule described above or below, functionally linked to an expression control sequence, and a host organism transformed with said DNA molecule or said expression vector. 20 The invention also relates to the use of at least one DNA molecule de scribed above or at least one expression vector described above for the preparation of a medicament for the immunotherapeutic DNA vaccination of patients with allergies in the triggering of which group 4 allergens from 25 the Poaceae, preferably Triticeae, in particular Sec c 4, Hor v 4, Tri a 4, are involved and/or for the prevention of such allergies. As already stated, the invention can be used as an essential component in 30 a recombinant allergen- or nucleic acid-containing preparation for specific immunotherapy. A number of possibilities arise here. On the one hand, the protein with an unchanged primary structure may be a constituent of the preparation. On the other hand, a hypoallergenic (allergoid) form can be 35 used in accordance with the invention for therapy in order to avoid unde sired side effects by specific deletion of IgE epitopes of the molecule as a whole or the production of individual fragments which encode T-cell epi- WO 2005/059136 PCT/EP2004/013664 topes. Finally, the nucleic acid per se, if ligated with a eukaryotic expres sion vector, gives a preparation which, when applied directly, modifies the allergic immune state in the therapeutic sense. 5 The present invention furthermore relates to the polypeptides encoded by one or more of the DNA molecules described above, preferably in their property as medicament. These are proteins corresponding to an amino acid sequence in accor 10 dance with SEQ ID NO 2, 4, 6, 8 or 10. In particular, these are mature pro teins (without signal sequence component), beginning with amino acid 23 (SEQ ID NO 2, 4 and 6) and with amino acid 22 (SEQ ID NO 8, 10). The invention furthermore relates to proteins which contain these amino acid 15 sequences or parts of these sequences. The invention accordingly also relates to a process for the preparation of such polypeptides by cultivation of a host organism and isolation of the 20 corresponding polypeptide from the culture. The invention likewise relates to the use of at least one polypeptide de scribed above for the preparation of a medicament for the diagnosis and/or treatment of allergies in the triggering of which group 4 allergens from the 25 Poaceae, preferably Triticeae, in particular Sec c 4, Hor v 4, Tri a 4, are involved and for the prevention of such allergies. 30 When determining the protein and DNA sequences according to the inven tion, the following procedure was followed: Sec c 4 from rye 35 1. Starting from the DNA sequence of Phi p 4 (SEQ ID NO 12, WO 04/000881), specific primers (Table 1) derived from the Phi p 4 sequence WO 2005/059136 PCT/EP2004/013664 -9 were generated. Five clones were obtained from rye pollen DNA by PCR with primers #87 and #83. The amplified Sec c 4 gene fragment 1 corre sponding to these clones encodes a polypeptide corresponding to amino 5 acids 68-401 of PhI p 4 (SEQ ID NO 12). 2. An EST database search was carried out with the partial Sec c 4 se quence. However, no homologous sequences were found in EST data bases specialising in rye. Instead, individual, homologous, non-overlapping 10 EST fragments were found in EST databases specialising in barley and wheat. Individual EST fragments extend into the 5'-UTR region and others into the 3'-UTR region (UTR = untranslated region) of the corresponding genes. 15 3. However, a complete group 4 gene from wheat or barley cannot be con structed from the EST sequences found in the databases since these se quences do not overlap and a homologous group 4 gene is not known. 20 However, it was possible to assign these EST sequences with reference to the Phi p 4 sequence (SEQ ID NO 11) and the Sec c 4 fragment obtained in step I and these served as template for the preparation of PCR primers. 4. With the aid of primers #195 and #189 prepared in this way, three clones 25 were obtained by PCR. Primer #195 was derived from a barley EST sequence, primer #189 is a Phi p 4-specific primer and overlaps the Phi p 4 stop codon and the codons of the 10 C-terminal PhI p 4 amino acids. The Sec c 4 gene fragment 2 amplified in this way encodes a polypeptide, 30 beginning within the signal sequence and ending with the position corresponding to position 490 of Phi p 4. This polypeptide covers the N terminal of Sec c 4. 35 5a. Three further clones were obtained by PCR with primers #195 and #202. Both primers were derived from barley EST sequences. The ampli- WO 2005/059136 PCT/EP2004/013664 -10 fied Sec c 4 gene 3 encodes the corresponding amino acids beginning within the signal sequence and ending at the C terminal of Sec c 4. The complete sequence of mature Sec c 4 is thus present in the sequence determined. 5 The next two steps 5b and 5c serve to double-check the result obtained in step 5a: 10 5b. A further clone was obtained by PCR with primers #195 and #203. Primer #195 was derived from a barley EST sequence, primer #203 from a wheat EST sequence. The amplified Sec c 4 gene encodes the corre sponding amino acids beginning within the signal sequence and ending at 15 the C terminal of Sec c 4. The complete sequence of mature Sec c 4 is therefore present in the sequence determined. 5c. A further clone was obtained by PCR with primers #195 and #198. Also 20 primer #198 The amplified Sec c 4 gene encodes the corresponding amino acids beginning within the signal sequence and ending at the C terminal of Sec c 4. The complete sequence of mature Sec c 4 is therefore present in the sequence determined. 25 Two isoforms Sec c 4.01 and 4.02 were found. The mature allergens begin with amino acid 23 of the sequences in accordance with SEQ ID NO 2, 4 and 6. 30 Hor v 4 from barley With the aid of the Sec c 4 sequences obtained as described above, homo logous EST fragments were found in EST databases of Hordeum vulgare. 35 These fragments overlap, but not to give a complete gene. With reference to the EST sequences found, however, it was possible to generate Hor v WO 2005/059136 PCT/EP2004/013664 - 11 4-specific primers, which were used for amplification of the Hor v 4 gene from genomic DNA. 5 In total, 15 clones were analysed. 4 clones were obtained by PCR with primers #195 and #198. 4 clones were obtained by PCR with primers #195 and #202. 3 clones were obtained by PCR with primers #194 and #198. 4 clones were obtained by PCR with primers #194 and #202. 10 The derived protein sequence begins within the signal sequence of Hor v 4 and extends to the C-terminal end of the protein (from amino acid 23 of SEQ ID NO 6). 15 Tri a 4 from wheat With the aid of the Sec c 4 sequences obtained as described above, homo 20 logous EST fragments were found in EST databases of Triticum aestivum. These fragments overlap, but not to give a complete gene. With reference to the EST sequences found, however, it was possible to generate the Tri a 4-specific primers #199, #203, #204 and #206, which were used for ampli fication of the Tri a 4 gene from genomic DNA. 25 In total, 13 clones were analysed. 4 clones were obtained by PCR with primers #204 and #203. 4 clones were obtained by PCR with primers #204 and #199. 30 3 clones were obtained by PCR with primers #206 and #203. 4 clones were obtained by PCR with primers #206 and #199. The derived protein sequences begin within the signal sequence of Tri a 4 and extend to the C-terminal end of the protein. 35 Two variants Tri a 4.01 (from amino acid 22 of SEQ ID NO 8) and Tri a 4.02 (from amino acid 22 of SEQ ID NO 10) were found.

WO 2005/059136 PCT/EP2004/013664 -12 In order to prepare the recombinant allergens according to the invention, the DNA sequences in accordance with SEQ ID NO 1, 3, 5, 7 and 9 were 5 incorporated into expression vectors (for example pProEx, pSE 380). E. coli-optimised codons were used for the N-terminal amino acids known from the protein sequencing. After transformation in E. coil, expression and purification of the recombi 10 nant allergens according to the invention by various separation techniques, the proteins obtained were subjected to a refolding process. Both allergens can be employed for highly specific diagnosis of grass pol len allergies. This diagnosis can be carried out in vitro by detection of spe 15 cific antibodies (IgE, IgG1 - 4, IgA) and reaction with IgE-loaded effector cells (for example basophiles from blood) or in vivo by skin test reactions and provocation at the reaction organ. 20 The reaction of the allergens according to the invention with T-lymphocytes from grass pollen allergy sufferers can be detected by allergen-specific stimulation of the T-lymphocytes for proliferation and cytokine synthesis both with T-cells in freshly prepared blood lymphocytes and also on estab lished nSec c 4, nHor v 4 or nTri a 4-reactive T-cell lines and clones. 25 The triplets encoding the cysteines were modified by site-specific muta genesis in such a way that they encode other amino acids, preferably ser ine. Both variants in which individual cysteines have been replaced and 30 those in which various combinations of 2 cysteine residues or all cysteines have been modified were prepared. The expressed proteins of these cys teine point mutants have greatly reduced or zero reactivity with IgE anti bodies from allergy sufferers, but react with the T-lymphocytes from these 35 patients.

WO 2005/059136 PCT/IEP2004/013664 -13 The present invention therefore furthermore relates to a DNA molecule described above or below in which one, a plurality of or all the cysteine resi dues of the corresponding polypeptide have been replaced with another 5 amino acid by site-specific mutagenesis. The immunomodulatory activity of hypoallergenic fragments which corres pond to polypeptides having T-cell epitopes and that of the hypoallergenic point mutants (for example cysteine replacements) can be detected by their 10 reaction with T-cells from grass pollen allergy sufferers. Such hypoallergenic fragments or point mutants of the cysteines can be employed as preparations for hyposensitisation of allergy sufferers since 15 they react with the T-cells with equal effectiveness, but result in reduced IgE-mediated side effects owing to the reduced or entirely absent IgE reac tivity. 20 If the nucleic acids encoding the hypoallergenic allergen variants according to the invention or the unmodified DNA molecules according to the inven tion are ligated with a human expression vector, these constructs can likewise be used as preparations for immunotherapy (DNA vaccination). 25 Finally, the present invention relates to pharmaceutical compositions com prising at least one DNA molecule described above or at least one expres sion vector described above and optionally further active ingredients and/or adjuvants for the immunotherapeutic DNA vaccination of patients with 30 allergies in the triggering of which group 4 allergens from the Poaceae, preferably Triticeae, in particular Sec c 4, Hor v 4, Tri a 4, are involved and/or for the prevention of such allergies. 35 A further group of pharmaceutical compositions according to the invention comprises at least one polypeptide described above instead of the DNA and is suitable for the diagnosis and/or treatment of said allergies.

WO 2005/059136 PCT/EP2004/013664 -14 Pharmaceutical compositions in the sense of the present invention com prise, as active ingredients, a polypeptide according to the invention or an 5 expression vector and/or respective pharmaceutically usable derivatives thereof, including mixtures thereof in all ratios. The active ingredients according to the invention can be brought into a suitable dosage form here together with at least one solid, liquid and/or semi-liquid excipient or adju vant and optionally in combination with one or more further active ingredi 10 ents. Particularly suitable adjuvants are immunostimulatory DNA or oligonucleo tides having CpG motives. 15 These compositions can be used as therapeutic agents or diagnostic agents in human or veterinary medicine. Suitable excipients are organic or inorganic substances which are suitable for parenteral administration and do not adversely affect the action of the active ingredient according to the 20 invention. Suitable for parenteral use are, in particular, solutions, preferably oil-based or aqueous solutions, furthermore suspensions, emulsions or implants. The active ingredient according to the invention may also be lyophilised and the resultant lyophilisates used, for example, for the prepa ration of injection preparations. The compositions indicated may be steril 25 ised and/or comprise adjuvants, such as preservatives, stabilisers and/or wetting agents, emulsifiers, salts for modifying the osmotic pressure, buffer substances and/or a plurality of further active ingredients. Furthermore, sustained-release preparations can be obtained by corres 30 ponding formulation of the active ingredient according to the invention - for example by adsorption on aluminium hydroxide. The invention thus also serves for improving in vitro diagnosis as part of 35 allergen component-triggering identification of the patient-specific sensiti sation spectrum. The invention likewise serves for the preparation of sig- WO 2005/059136 PCT/EP2004/013664 -15 nificantly improved preparations for the specific immunotherapy of grass pollen allergies. Table 1 Primers used 5 a) Sec c 4 Primer SEQ ID NO Sequence number 10 #0083 30 GGCTCCCGGGGCGAACCAGTAG #0087 31 ACCAACGCCTCCCACATCCAGTC #0189 32 GATAAGCTTCTCGAGTGATTAGTACTTTTTGAT CAGCGGCGGGATGCTC #0195 33 GCTCTCGATCGGCTACAATGGCG #0198 34 CACGCACTACAAATCTCCATGCAAG 15 #0202 35 CATGCTTGATCCTTATTCTACTAGTTGGGC #0203 36 TACGCACGATCCTTATTCTACTAGTTGGGC a) Hor v 4 Primer SEQ ID NO Sequence 20 number #0194 37 GCCTTGTCCTGCCACCACGCCGCCGCCACC #0195 38 GCTCTCGATCGGCTACAATGGCG #0198 39 CACGCACTACAAATCTCCATGCAAG #0202 40 CATGCTTGATCCTTATTCTACTAGTTGGGC 25 c) Tri a 4 Primer SEQ ID NO Sequence number 30 #0199 41 CACGCACTAAATCTCCATGCAAG #0203 42 TACGCACGATCCTTATTCTACTAGTTGGGC #0204 43 AAGCTCTATCGCCTACAATGGCG #0206 44 GGTGCTCCTCTTCTGCGCCTTGTCC 35 C:\NRPotbl\DCC\GRS\4488542_1.DOC-23/07/2012 - 15a Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as, an acknowledgement or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims (19)

1. An isolated DNA molecule corresponding to a nucleotide sequence of a cereal pollen major allergen comprising the sequence set forth in SEQ ID NO 5.

2. An isolated DNA molecule which hybridises with a DNA molecule according to Claim 1 under stringent conditions and originates from DNA sequences from Poaceae species.

3. An isolated DNA molecule corresponding to a partial sequence or a combination of partial sequences according to Claim 1 or 2, which encodes an immunomodulatory, T-cell-reactive fragment of a group 4 allergen from the Poaceae species.

4. A recombinant DNA expression vector or a cloning system comprising a DNA molecule according to any one of Claims 1 to 3, functionally linked to an expression control sequence.

5. A host organism transformed with a DNA molecule according to any one of Claims 1 to 3, or an expression vector according to Claim 4.

6. A process for the preparation of a polypeptide encoded by a DNA sequence according to any one of Claims 1 to 3 by cultivation of a host organism according to Claim 5, and isolation of the corresponding polypeptide from the culture.

7. An isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO 6, or which is encoded by a nucleotide sequence as defined in any one of Claims 1 to 3.

8. An isolated polypeptide corresponding to a mature allergen comprising an H:\mm\Interwoven\NRPortbl\DCC\MM\7430977_1.doc-4/02/2015 -17 amino acid sequence according to Claim 7, comprising the amino acid sequence set forth in SEQ ID NO 6, beginning with the amino acid residue at position 23.

9. A medicament comprising a polypeptide according to Claim 7 or 8.

10. A pharmaceutical composition comprising at least one polypeptide according to Claim 7 or 8, when used for the diagnosis and/or treatment of allergies triggered by group 4 allergens from the Poaceae species.

11. A pharmaceutical composition according to Claim 10, further comprising at least one additional active ingredient and/or at least one adjuvant.

12 Use of at least one polypeptide according to Claim 7 or 8, for the preparation of a medicament for the diagnosis, treatment and/or prevention of allergies triggered by group 4 allergens from the Poaceae species.

13. A medicament comprising a DNA molecule according to any one of Claims 1 to 3.

14. A medicament comprising a recombinant expression vector according to Claim 4.

15. A pharmaceutical composition comprising at least one DNA molecule according to any one of Claims 1 to 3, or at least one expression vector according to Claim 4, when used for the immunotherapeutic DNA vaccination of patients with allergies triggered by group 4 allergens from the Poaceae species, and/or for the prevention of such allergies.

16. A pharmaceutical composition according to Claim 15, further comprising at least one additional active ingredient and/or at least one adjuvant. H:\SZFInterwoven\NRPortbl\DCC\SZF\74309771.doc-4/02/2015 -18

17. Use of at least one DNA molecule according to any one of Claims I to 3, or at least one expression vector according to Claim 4, for the preparation of a medicament for the immunotherapeutic DNA vaccination of patients with allergies triggered by group 4 allergens from the Poaceae species.

18. A method for the diagnosis, treatment and/or prevention of allergies triggered by group 4 allergens from Poaceae species, comprising administering to a subject in need thereof at least one polypeptide according to claim 7 or 8, or a medicament according to Claim 9, 13 or 14.

19. An isolated DNA molecule according to Claim 1, an isolated polypeptide according to Claim 7, and uses thereof, substantially as herein described with reference to the Examples. 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012 2012205288 24 Jul 2012