EP2523685A1

EP2523685A1 - Variants of the group-5 allergens of the poaceae with reduced allergenicity by mutagenesis of proline residues

Info

Publication number: EP2523685A1
Application number: EP10795625A
Authority: EP
Inventors: Martin Wald; Andreas Nandy; Helmut Fiebig; Bernhard Weber; Helga Kahlert; Gerald Reese; Oliver Cromwell
Original assignee: Merck Patent GmbH
Current assignee: Merck Patent GmbH
Priority date: 2010-01-14
Filing date: 2010-12-17
Publication date: 2012-11-21
Also published as: CN105061571A; EP3170510A1; CA2786936A1; CN102791289B; CN102791289A; JP5871819B2; WO2011085783A1; AU2010342533B2; BR112012016768A2; AU2010342533A1; US20150140024A1; US9975932B2; RU2658767C1; JP2013516962A; RU2012134508A; US20180002387A1

Abstract

The present invention relates to the preparation and the use of recombinant variants of the group-5 allergens of the poaceae which are characterized by the fact that their IgE reactivity is reduced in comparison with the known wild-type allergens while simultaneously the reactivity with T-lyphocytes is largely retained.

Description

Variants of the group 5 allergens of sweet grasses with reduced allergenicity by mutagenesis of proline residues

Field of the invention

The present invention relates to the production and use of recombinant variants of the group 5 allergens of Poaceae (sweet grasses), which are characterized by a relation to the known wild-type allergens decreased IgE reactivity and at the same time a largely maintained reactivity with T lymphocytes.

The reduced IgE reactivity is achieved primarily by the substitution or deletion of certain proline residues, which are highly conserved in group 5 allergens. In addition, the amino acid proline at position 2 1 (Phl p 5.0109 numbering) was identified as a key position for the targeted influencing of the solubility of the variants in aqueous formulations.

These hypoallergenic allergen variants can be specific

Immunotherapy (hyposensitization) of patients with grass pollen allergy or for preventive treatment to avoid the expression of grass pollen allergies.

A preferred embodiment of the invention relates to variants of

Main allergen Phl p 5 from the pollen of meadow wattle grass {Phleum pratense).

Background of the Invention Type 1 allergies are of global importance. Up to 20% of the population in industrialized countries suffer from conditions such as allergic rhinitis, conjunctivitis or bronchial asthma. These allergies are caused by sources of various origins such as trees and grasses (pollen), fungi (spores), mites (feces), cats or dogs. The allergen sources are released directly into the air (pollen, spores) or can be bound to diesel soot particles (pollen) or house dust (mite feces, skin particles, hair) to get into the air. Because they are allergy-causing substances in the air, it is also called aerosol allergens.

The type 1 allergy-causing substances are proteins, glycoproteins or polypeptides. Upon ingestion via mucous membranes, these allergens react with the IgE molecules bound to the surface of mast cells in sensitized persons. If these IgE molecules are cross-linked by an allergen, this leads to the release of mediators (eg histamine, prostaglandins) and cytokines through the

Effector cell and thus the corresponding allergic symptoms.

Up to 40% of type 1 allergy sufferers show specific IgE reactivity

Pollen Extracts of Sweet Grasses (Burney et al., 1997, J. Allergy Clin.

Immunol. 99: 314-322; D'Amato et al., 1998, Allergy 53: 567-578; Freidhoff et al., 1986, J. Allergy Clin Immunology, 78, 1 190-2002). The family of

Sweet grasses (Poaceae) comprise over 10,000 species, of which well over 20 are known to cause allergic symptoms (Andersson & Lidholm, 2003, Int. Arch. Allergy Immunol., 130: 87-107, Esch, 2008, Allergens and Allergen Immunotherapy , Clinical Allergy and Immunology Series, 107-126).

Most of the allergy-causing grasses belong to the subfamily Pooideae. In addition to the grass species occurring as wild forms such as Holcus lanatus (Wolliges honey grass), Phalaris aquatica (Knolliges Glanzgras), Anthoxanthum odoratum (common smelling grass), Dactylis glomerata (common ball grass), Festuca pratensis Meadow fescue, Poa pratensis or Lolium perenne are also cultivated cereals such as Triticum aestivum (wheat), Seeale cereale (rye) and Hordeum vulgare (barley) known members of this subfamily.

One of the species of the Pooideae best studied for their allergens is the meadow worm grass (Phleum pratense), which is widespread worldwide as a wild plant and also plays an economic role as a pasture and hardy fodder grass.

Depending on the relative frequency in a population with which the individual allergen molecules react with the IgE antibodies of allergy sufferers, a distinction is made between major and minor allergens. Six allergens of meadowweed grass can be considered as major allergens: Phl p 1 (Petersen et al., 1993, J. Allergy Clin Immunol 92: 789-796), Phl p 5 (Matthiesen and Lowenstein, 1991, Clin Exp 21: 297-307, Petersen et al., 1992, Int. Arch. Allergy Immunol., 98: 105-109), Phl p 6 (Petersen et al., 995, Int. Arch. Allergy Immunol., 108, 49-54 Phl p 2/3 (Dolecek et al., 1993, FEBS 335 (3): 299-304), Phl p 4 (Haavik et al., 1985, Int. Arch. Allergy Appl. Immunol. 268; Valenta et al., 992, Int. Arch. Allergy Immunol. 97: 287-294; Nandy et al., Biochem. Biophys. Res.

Commun., 2005, 337 (2): 563-70) and Phl p13 (Suck et al., 2000, Clin. Exp. Allergy 30: 1395-1402).

The dominant major allergens of meadowweed grass are Phl p 1 and Phl p 5 (Andersson & Lidholm, 2003, Int. Arch. Allergy Immunol., 130: 87-107), where Phl p 5 in the two differ in molecular mass

Shapes 5a and 5b encoded by independent genes occur. Following the official nomenclature for allergens, Phl p 5a is named as Phl p 5.01 and Phl p 5b as Phl p 5.02 (WHO / IUIS Allergen Nomenclature Subcommittee, www.allergen.org). The amino acid sequences of both Phl p 5a as well as Phl p 5b could be deduced from the cloned cDNA sequences. Of both isoforms, natural variants were identified which differ from one another by point mutations and correspond to different allelic forms (Vrtala et al., 1993, J.

Immunol., 151: 4773-4781; Gelha et al., 1997, Eur. J. Biochem., 247: 217-23). These variants are listed in the WHO / IUIS database as Phl p 5.01xx and Phl p 5.02xx.

The natural Phl p 5a (nPhl p 5a) is a protein of about 32 kDa and reacts with IgE antibodies from 85-90% of grass pollen allergy sufferers (Rossi et al., 2000, Allergy Int., 49: 93-97).

The pollen of the related sweet grass species of the family Poaceae and in particular the subfamily Pooideae, such as Lolium perenne or Poa pratensis contain allergens that are homologous to the Phl p 5 and are collectively referred to as group 5 allergens. The high structural homology of these group 5 allergens requires correspondingly high cross-reactivity of the molecules with IgE antibodies (Lorenz et al., 2009, Int. Arch. Immunol., 148: 1-17). Ultimately, due to this cross-reactivity

Sensitization by a kind of grass sufficient to allergic one

Trigger reaction by other related grasses.

The high cross-reactivity of the group 5 allergens is ultimately based on a similar primary sequence of the homologous allergens. This shows

Amino acid sequence comparison of group 5 allergens from selected species of the Pooideae (Figure 1).

In addition to the cross-reactivity of the group 5 allergens with each other is also a cross-reactivity of Phl p 5 with another major allergen of the

Timothy grass known (Lowenstein, 1978, Allergy 33: 30-41; Petersen et al, 1995, Int Arch Allergy Immunol. 108: 55-59; Blume et ^'al, 2004 Proteomics 4:.... 1366-71). The polypeptide chain of the allergen Phl p 6 shows a very similar to the N-terminal half of the various Phl p 5 sequences (Figure 1). It is believed that the allergens on one

common origin gene. The similarity between the allergens of the two groups causes some of the Phl p 5-reactive IgE antibodies to also bind to Phl p 6 (Petersen et al., 1995, Int. Arch. Allergy Immunol., 108: 49-54, Andersson & Lidholm, 2003, Int. Arch. Allergy Immunol., 130: 87-107).

The 3D structure of many allergens has been elucidated in the past by NMR spectroscopy or X-ray crystallography and has been used, among other things, as a basis for the localization of IgE-binding epitopes on the protein surface. In the case of the group 5 allergens of grass pollen, it has not been possible to generate a model that covers the entire

Polypeptide chain (Rajashankar et al., 2002, Acta Cryst. D58: 1 75-1 181; Maglio et al., 2002, Protein Engineering 15: 635-642).

Based on 3D structures of the allergen Phl p 6 (RCSB protein data bank entry: 1 NLX) and a Phl p 5b half-molecule (RCSB Protein Data Bank entry: 1L3P), a homology model of Phl p 5a was generated (Wald et al , 2007, Clin Exp. Allergy 37: 441-450). According to this model, Phl p 5a consists of two helix bundles, but the exact position of the two bundles can not be clarified by the homology model (Figure 2). Specific immunotherapy (SIT) or hyposensitization is considered to be an effective approach to the therapeutic treatment of allergies (Fiebig 995 Allergo J. 4 (6): 336-339, Bousquet et al., 1998, J. Allergy Clin Immunol 102 (4)). : 558-562); Cox et al., 2007, J. Allergy Clin. Immunol. 120.S25-85; James & Durham, 2008, Clin. Exp. Allergy 38: 1074-1088).

The classic form of therapy for injection therapy (SCIT), in which the patient injects natural allergen extracts in increasing doses subcutaneously be used successfully for about 100 years. The immune system of the allergic person is repeatedly confronted with allergens, which results in a reprogramming of the immune system combined with a tolerance to the allergens. After uptake of the antigens from the allergen preparations by antigen-presenting cells, peptides of the antigens are presented on the cell surface. Some particular peptides, called T cell epitopes, are recognized by antigen-specific T cells. This bond leads among other things to the expression

different types of T cells with regulatory function. In the course of SIT, the regulatory T cell response leads to tolerance to the allergen, downregulation of T _H 2 cytokines, restoration of the T _H 1 / T _H 2 equilibrium, suppression of allergen-specific IgE, the Induction of IgG4, IgG1 and IgA Antibodies, Suppression of

Effector cells (astzellen, basophils and eosinophils) and the renewal of inflamed tissue (Akdis et al., 2007, J. Allergy Clin Immunol 119 (4): 780-789; Lärche et al., 2008, Nature Reviews 6: 761 -771). The T-cell epitopes are thus of crucial importance for the therapeutic effect of the allergen preparations in hyposensitization. Due to the existing at both IgE and T-cell level

Cross-reactivity of the major allergens of sweet grasses is a successful one

Therapy with an allergen extract from a single representative grass species is usually sufficient (Mailing et al., 1993, EAACI Position Paper:

Immunotherapy, Allergy 48: 9-35; Cox et al., 2007, J Allergy Clin Immunol 120: 25-85).

In addition to subcutaneous immunotherapy, a sublingual therapy in which the allergens or allergen derivatives are taken up via the oral mucosa comes as an alternative to injection therapy for clinical testing and application (James & Durham, 2008, Clin. Exp. Allergy 38: 1074-1088). Another possibility is treatment with expressive DNA encoding the relevant allergens (immunotherapeutic vaccination). Experimental evidence for the allergen - specific influence of the

Immune responses were provided to rodents by injection of allergen-encoding DNA (Hsu et al., 1996, Nature Medicine 2 (5): 540-544, Weiss et al., 2006, Int. Arch. Allergy Immunol., 139: 332-345).

In all these forms of therapy there is a fundamental risk of allergic reactions or even of anaphylactic shock (Kleine- Tebbe, 2006, Allergologie, 4.135-156). To minimize these risks, innovative preparations in the form of allergoids are used. These are chemically modified allergen extracts which have markedly reduced IgE reactivity but identical T cell reactivity compared to untreated extract (Fiebig 1995 Allergo J. 4 (6): 336-339, Kahlert et al., 1999, Int. Arch. Allergy Immunol, 120: 146-157).

Therapy optimization is possible with recombinant allergens. Defined, if necessary, to the individual

Sensitization patterns of patient-matched cocktails of high-purity, recombinantly-produced allergens could be extracts of natural extracts

Replace allergen sources, as these contain, in addition to the various allergens, a larger number of immunogenic, but not allergenic accompanying proteins. Initial clinical trials of recombinant allergens have been successful (Jutel et al., 2005, J. Allergy Clin Immunol., 116: 608-613, Valenta & Niederberger, 2007, J. Allergy Clin Immunol 119: 826- 830).

Realistic perspectives, which can lead to a safe hyposensitization with recombinant expression products, provide targeted mutant recombinant allergens in which IgE epitopes are altered without adversely affecting the T-cell epitopes essential for therapy (Schramm et al., 1999, J. Immunol 162: 2406-2414). These hypoallergenic proteins could be used in higher doses during the SIT without the

Increase probability of unwanted IgE-mediated side effects. In the past, many aeroallergens (including pollen and pollen) have been identified

House dust mite allergens) and food allergens such

"Hypoallergenic" variants with reduced IgE binding published., Starting from the DNA unchanged allergens could, inter alia, by

Fragmentation, oligomerization, deletions, point mutations, or recombining of individual portions of an allergen (DNA shuffling) to produce and express recombinant DNA (Ferreira et al., 2006, Inflamm. & Allergy - Drug Targets 5: 5-14, Bhalla & Singh , 2008, Trends in Biotechnology 26: 153-161). With respect to grass pollen allergens, hypoallergenic variants have been described for groups 1, 2, 5a, 5b, 6, 7 and 12 (Ferreira et al., 2006, Inflamm. & Allergy - Drug Targets 5: 5-14, Westritschnig et al. , 2007, J. Immunol., 179: 7624-7634). In several publications are so far approaches to development

hypoallergenic group 5 allergens. For Phl p 5a and Phl p 5b it could be shown that the combined deletion of two

Sequence sections a strong reduction of IgE binding and a reduced stimulability of basophilic effector cells result. However, the T cell reactivity of the deletion mutants was slightly altered from that of the unaltered allergen (Schramm et al., 1999, J. Immunol., 162: 2406-2414, Wald et al., 2007, Clin. Exp. Allergy 37: 441 -450).

In another work, the group 5 allergen was isolated from Lolium perenne (Lol p 5) by amino acid substitutions and / or short deletions at the C-

Terminus changed (Swoboda et al., 2002, Eur. J. Immunol., 32: 270-280). The mutations were not limited to a particular type of amino acid. The substituted amino acids were lysine, phenylalanine, threonine, valine or alanine. The conceptual approach to generate hypoallergenic mutants by targeted mutation of multiple residues of a single amino acid has also been described. Gelhar et al. described the generation of a recombinant Phl p 5b fragment by substitution of ten lysine residues localized on the protein surface with alanine (Gelhar et al., 2006, Int. Arch. Allergy Immunol. 140: 285-294). One on point mutations in

Proline residue-based mutation strategy, but has not yet been published for grass pollen allergens of group 5.

The object underlying the present invention was now to provide novel recombinant variants of the group 5 allergens of the Poaceae on protein and DNA level, which are characterized by a reduced IgE reactivity with largely retaining the T cell reactivity and therefore are suitable for curative and preventive specific immunotherapy as well as immunotherapeutic DNA vaccination.

Description of the invention

Surprisingly, it has been found that variants of group 5 allergens of the grass family (Poaceae), in which the proline, in a

Alignment corresponded to the prolines of positions 57, 58, 17, 146, 155, 211, 229 in the amino acid sequence of the wild-type Phl p 5.0 09, mutated individually or in combinations, compared to the wild-type allergens decreased IgE reactivity and simultaneously a largely preserved

Reactivity with T lymphocytes and are therefore hypoallergenic.

The present invention accordingly relates to hypoallergenic variants of group 5 allergens of the family of sweet grasses (Poaceae), in which the proline, which in an alignment with the proles of the positions 57, 58, 117, 146, 155, 211, 229 in the amino acid sequence of wild-type Phl p 5.0109

correspond, individually or in combinations are mutated.

Particularly preferred are allergen variants according to the invention, characterized in that the prolines are deleted or substituted.

Preference is given to hypoallergenic variants according to the invention of group 5 allergens from the subfamily Pooideae, preferably from the groups Poodae and Triticodae, preferably represented by Phleum pratense, Holcus lanatus, Phalaris aquatica, Anthoxanthum odoratum, Dactylis glomerata,

Lolium perenne, Poa pratensis, Festuca pratensis, Hordeum vulgare, Seeale cereale and Triticum aestivum. It is preferable

hypoallergenic variants of tri a 5, lake c 5 and aud v 5 of Triticum aestivum, Seeale cereale and Hordeum vulgare according to the invention. Particular preference is given to hypoallergenic variants of group 5 allergens of the Poodae according to the invention. Preferably, these group 5 allergens are Poa p 5, Dac g 5, Hol p 5, Lol p 5 and Pha a 5 from Poa pratensis, Dactylis

glomerata, Holcus lanatus, Lolium perenne and Phalaris aquatica, and most preferably Phl p 5 is phleum pratense. Also included in the invention are all naturally occurring isomers, polymorphs and variants of the aforementioned allergens, as well as their precursor proteins.

In the case of the hypoallergenic variants according to the invention, the prolines are preferably mutated which, in an alignment (FIG. A), include the prolines of positions 57, 58, 117, 146, 155, 211 or 229 in the amino acid sequence of the mature Phl p 5.0109 (Phl p 5 a, UniProtKB entry: Q84UI2) Fig. 3 and 4, SEQ ID NO: 1, SEQ ID NO: 2) or its variants (Fig. 1 b) or the mature Phl p 5.0201 (Phl p5 b; Swiss-Prot: Q40963 Fig. 24, SEQ ID NO: 5) or its

Variants (FIG. 1 b), particularly preferably the mature Phl p 5.0109,

correspond. Although it was known that proline could exert an influence on the protein structure, targeted point mutations of prolestesten were used as a starting point for the generation of hypoallergenic mutants of allergens only in the group 2 major allergens of house dust mite

Dermatophaogides farinae (Der f 2, replacement of proline residues by alanine) (Takai et al., 2000, Eur. J. Biochem. 267: 6650-6656). However, the IgE-binding ability and ability to stimulate basophilic cells was only slightly reduced in three point mutants, while the remaining three behaved like the unchanged allergen. The proline mutations thus showed no or only a very slight reduction in Der f 2

Allergenicity. Other strategies for the production of hypoallergenic mutants by proline exchange mutations have not been published. Thus, it was not expected for the skilled person that proline mutations would be successful as a starting point for the generation of hypoallergenic mutants of allergens.

Moreover, no allergen has yet been tested for how a specific deletion of proline residues affects the overall IgE binding ability of the protein

Expression product and what effects on the activation of allergy-relevant effector cells.

The amino acid sequence of Phl p 5.0109 (UniProtKB: Q84UI2) contains 16 proline residues (Figure 1). The six proline residues P7, P10, P13, P19, P22 and P27 are located in the low-conserved N-terminal region within the group 5 allergens. The proline amino acid positions 57, 58, 85, 117, 146, 155, 211, 229, and 256 are at the beginning or end of a helices or loops connecting the a helices and are highly conserved except for amino acid P85 ( Fig. 1, 2).

Starting from the amino acid sequence of the Phl p 5a isoform Phl p 5.0109, the recombinant unmodified wild-type allergen (rPhl p 5a wt, Figure 4, SEQ ID NO: 2) and genetically engineered variants are prepared. Analogous to the manufacturing method shown below can also be the wild-type proteins and the hypoallergenic variants according to the invention of the further group 5 allergens of sweet grasses according to the invention, for example Lol p 5; Poa p 5, Pha a 5, Dac g 5; Make I 5, Tri a 5, and Listen v 5. These are the Proline, which in an alignment the prolines of the positions of positions 57, 58, 117, 146, 155, 21 1, 229 in the

Amino acid sequence of the wild-type Phl p 5 correspond individually or in

Combinations are mutated, preferably by substitution or deletion.

Of course, in addition to the described variations of group 5 allergens, other changes in other positions - such as to increase hypoallergenicity - are possible. These modifications may be, for example, amino acid insertions, deletions, exchanges and splits of the protein into fragments and fusions of the protein or its fragments with other proteins or peptides, as well as multimers by fusions of like proteins or fragments.

Fragments of the invention preferably comprise 20-109 amino acids, preferably 30-100 amino acids, more preferably 40-90 amino acids. Variants according to the invention also include precursor proteins such as, for example, ProPhl p 5 with an upstream, natural or artificial signal sequence. Also contemplated by the invention are fusion proteins having N- or C-terminal fusion tags (e.g., His tag as in Figures 5 and 6, MBP tag, expression control sequences, etc.), hybrid molecules such as

For example, fusions with other allergens or their hypoallergenic variants or fusions of fragments in any order. Moreover, the variants of the invention also include homologous sequences (polymorphisms (SNPs), isoforms) with an amino acid sequence identity of at least 80% to the respective group 5 wild-type allergen, preferably of at least 90% to the respective group 5 wild-type allergen, more preferably of at least 95% to the respective group 5 wild-type allergen. In these variants, one or a few amino acids are preferably conservatively exchanged, for example a polar amino acid by a other polar amino acid or neutral neutralized by another neutral, but variants by non-conservative substitution according to the invention are also. Multimers preferably comprise dimers or trimers of the hypoallergenic variants of the invention joined by a

Linker sequence or directly fused.

Examples of isoforms are the allergens Phl p 5a and Phl p 5b, in which individual amino acids which are not relevant for the effect according to the invention are exchanged, or in which regions in the amino acid sequence are absent or added (see FIG. 1a). For example, these wild-type allergen isoforms have an amino acid sequence identity of 63% -71%. Further examples of variants according to the invention are variants of the wild-type allergen isoforms Phl p 5a and Phl p 5b, for example Phl p 5.0109, Phl p 5.0201, Phl p 5.0204, Phl p 5.0206, Phl p 5.0207 and

the like and other variants with replacement of one or more amino acids, lack of one or more amino acids at the N and / or C terminus or with corresponding deletion gaps within the

Amino acid sequence. Likewise according to the invention are variants with

Insertions of single or multiple amino acids isolated in different positions or as a group in a position within the

Amino acid sequence or at the N and / or C-terminus.

Hypoallergenic variants of group 5 allergens of sweet grasses (Poaceae) are therefore also subject matter according to the invention, characterized in that they are a fragment or a variant of a hypoallergenic variant according to the invention or a multimer of one or more

According to the invention, hypoallergenic variants or characterized in that one or more inventive, hypoallergenic variants or fragments thereof, variants or multimers are part of a recombinant fusion protein. The invention also relates to a DNA molecule coding for a hypoallergenic variant according to the invention.

Another object of the invention is a recombinant

Expression vector containing such a DNA molecule of the invention operably linked to an expression control sequence. By expression control sequence is meant, for example, a promoter or a sequence segment by means of which the expression of the target protein is influenced and which is functionally connected to the target gene, but does not necessarily have to be located directly adjacent to the target gene.

An article according to the invention is also a non-human one

Host organism transformed with a DNA molecule of the invention or an expression vector of the invention.

An article according to the invention is a process for producing a hypoallergenic variant according to the invention by culturing a non-human host organism according to the invention and recovering the corresponding allergen variant from the culture.

Suitable non-human host organisms may be pro- or

eukaryotic, single or multicellular organisms such as bacteria or yeasts. A preferred host organism according to the invention is E. coli.

The effect of the deletion of one or two closely spaced proline on the IgE-binding ability of Phl p 5a can be achieved by deletion of proline 57 + 58, proline 85, proline 117, proline 146 + 155, proline 180, proline 221, proline 229 and of Proline 256 will be investigated. These proline are localized in the Phl p 5 wild-type protein in the Schletfen regions at the beginning or at the end of a helices (Figure 3). Similarly, the influence of

Proline mutations in the corresponding homologous positions of the others Group 5 allergens of sweet grasses according to the invention, for example Poa p 5 and Lol p 5 are examined for the IgE binding ability.

For faster, high-yield purification, the coding DNA for these studies is provided with a sequence coding for an N-terminal hexa-histidine fusion fraction (+ 6His) (FIG. 5, SEQ ID NO: 3, FIG. 6, SEQ ID NO: 4 ). The tag-free, usable for pharmaceutical purposes, variants of the invention and wild-type proteins are also purified by standard methods and confirm the results of His-tag proteins.

Accordingly, sequences encoding the proteins rPhl p 5a d [P57, P58] + 6His, rPhl p 5a d [P85] + 6His, rPhl p 5a d [P117] + 6His, rPhl p 5a d [P146, P155] + 6His , rPhl p 5a d [P180] + 6His, rPhl p 5a d [P211] + 6His, rPhl p 5a d [P229] + 6His, and rPhl p 5a d [P256] + 6His. The

Sequences can be expressed in all known eukaryotic and prokaryotic expression systems, preferably in E. coli. Subsequently, the proteins are purified as soluble monomers by standard methods. Finally, purity can be analyzed through denaturing

Polyacrylamide gel (SDS-PAGE) should be checked.

Analytical gel filtration (SEC) coupled with a refractometer (RI detector) and a multi-angle light scattering detector (MALS detector) allows the on-line determination of the molecular weight of the eluted proteins

(S EC / MALS / RI method).

Moreover, the recombinant variants according to the invention can be investigated for their binding ability to human IgE antibodies by IgE inhibition tests (EAST). In this procedure, the allergen / IgE interaction in solution can be studied, bringing disturbing

Masking of epitopes of the test substance, as by immobilization to a membrane, can be excluded. There may be a decreased IgE binding of mutants rPhl p 5a d [P57, P58] + 6His, rPhl p 5a d [P1 17] + 6His, rPhl p 5a d [P146, P155] + 6His, rPhl p 5a d [P180 ] + 6His, rPhl p 5a d [P211] + 6His and rPhl p 5a d [P229] + 6His are detected in comparison to the unmodified rPhl p 5a wt (Figure 7).

This basically proves that the deletion of proline residues of group 5 allergens reduces the IgE-binding ability of these allergens. On the other hand, only deletion of certain prolines results in decreased IgE binding.

Another object of the present invention are therefore

hypoallergenic variants of group 5 allergens of the grasshopper family (Poaceae), in which the prolines aligned in alignment with the proliferation sites 57, 58, 117, 146, 155, 21 1 or 229 in the amino acid sequence of the wild-type Phl p 5,0109, mutated individually.

A preferred subject of the present invention are hypoallergenic variants of Phl p 5, Poa p 5, Lol p 5, Dac g 5, Hol I 5, Pha a 5, in which the prolines, which in alignment to the positions 57, 58 , 117, 146, 155, 211 or 229 in the amino acid sequence of the wild-type Phl p 5.0109 are individually mutated.

A particularly preferred subject of the present invention are hypoallergenic variants of Phl p 5 in which the prolines aligned in alignment with the proliferation sites 57, 58, 117, 146, 155, 21 1 or 229 in the amino acid sequence of the wild-type Phl p 5,0109, mutated individually.

Another preferred subject matter of the present invention are hypoallergenic variants of group 5 allergens of the grass family (Poaceae) according to the invention, in which the prolines which are present in a

Alignment of the prolines of positions 57, 58, 117, 146, 155, 21 1 or 229 in correspond to the amino acid sequence of the wild-type Phl p 5.0109, are individually removed.

Another object of the present invention are also

inventive, hypoallergenic variants of group 5 allergens of the family of sweet grasses (Poaceae), in which the prolines, which in a

Alignment of the prolines of positions 57, 58, 117, 146, 155, 211 or 229 in the amino acid sequence of the wild-type Phl p 5.0109 are individually substituted. Here proline is exemplarily replaced by leucine (L). According to the invention, however, the prolines of the invention may be replaced by any amino acid.

In particular, according to the invention, the hypoallergenic variants rPhl p 5a d [P57], rPhl p 5a d [P58], rPhl p 5a d [P57, P58], rPhl p 5a d [P117], rPhl p 5a d [P146], rPhl p 5a d [P155], rPhl p 5a d [P146, P155], rPhl p 5a d [P180], rPhl p 5a d [P211], rPhl p 5a d [P229], rPhl p 5a P57L, rPhl p 5a P58L, rPhl p 5a P57, P58L, rPhl p 5a P117L, rPhl p 5a P146L, rPhl p 5a P155L, rPhl p 5a P146L, P155L, rPhl p 5a P180L, rPhl p 5a P211L, and rPhl p 5a P229L, and the like, including all those described below .

Hypoallergenic variants according to the invention, wherein the numbering of the sequence of Phl p 5.0 09 follows.

Furthermore, these examples are not limited to variants of Phl p 5a but also relate in particular to Phl p 5b, Poa p 5, Hol I 5, Pha a 5, Ant o 5, Dac g 5, Lol p 5, Fes p 5, Hör v 5, lake c 5, tri a 5 and group 5 allergens of all other grasses. Particularly noteworthy are

corresponding variants of Poa p 5, Lol p 5, Dac g 5, Hol I 5, Pha a 5 and Phl p 5b. Surprisingly, variants with combinations of deletions from the group of mutations d [P57, P58], d [P17], d [P146, P155], d [P180], d [P2 1] and d [P229] show a significantly stronger reduced IgE-binding ability in the EAST method, as shown by the example of the variant rPhl p 5a d [P117, 180] + 6His (Figure 8).

Another object of the present invention are therefore

hypoallergenic variants of group 5 allergens of the grasshopper family (Poaceae), in which the prolines aligned in an alignment with the proliferation of positions 57, 58, 117, 146, 155, 180, 21, 229 in the amino acid sequence of the wild-type Phl p 5.0109, mutated in combinations.

Preference is given to mutations by deletion and substitution by other amino acids. Each amino acid can be chosen for exchange with proline.

A preferred subject of the present invention are hypoallergenic variants of Phl p 5, Poa p 5, Lol p 5, Dac g 5, Hol I 5, Pha a 5, in which the prolines, which in alignment to the positions 57, 58 , 17, 146, 155, 180, 211, 229 in the amino acid sequence of wild-type Phl p 5.0109, mutated in combinations.

A particularly preferred subject matter of the present invention are hypoallergenic variants of Phl p 5, in which the prolines which, in an alignment with the proliferation of the positions 57, 58, 117, 146, 155, 180, 211, 229 of the amino acid sequence of the wild-type Phl p 5.0109, in

Combinations are mutated. Furthermore, these examples are not limited to variants of Phl p 5 but also relate in particular Poa p 5, Hol I 5, Pha a 5, Ant o 5, Dac g 5, Lol p 5, Fes p 5, Listen v 5, See c 5, Tri a 5 and the group 5 allergens of all other grasses. Particular emphasis should be given to corresponding variants of Phl p 5a, Phl p 5b, Poa p 5, Lol p 5, Dac g 5, Hol I 5, Pha a 5.

Particular preference is given to hypoallergenic variants according to the invention in which the prolines 57, 58, 117, 146, 155, 180, 211, 229 are in combinations are removed. Particularly preferred are also inventive,

Hypoallergenic variants in which the prolines 57, 58, 117, 146, 155, 180, 211, 229 are substituted in combinations. Also particularly preferred are hypoallergenic variants according to the invention in which the prolines 57, 58, 117, 146, 155, 180, 211, 229 are removed and / or substituted in combinations. According to the invention, for example, the hypoallergenic variants rPhl p 5a d [57, 58, 117], rPhl p 5a d [57, 58, 146], rPhl p 5a d [57, 58, 150], rPhl p 5a d [57, 58, 180], rPhl p 5a d [57, 58, 211], rPhl p 5a d [57, 58, 229], rPhl p 5a d [117, 146, 155], rPhl p 5a d [117, 146, 155, 180], rPhl p 5a d [117, 146, 155, 229], rPhl pa 5 1 17L, P146L, P155L, P211 L, rPhl p 5a d [117, 180, 229] P21 1 L, rPhl p 5a [117, 180, 229] P211 L, rPhl p 5a d [57, 58, 117, 180, 229] P211 L and the like, including all the hypoallergenic variants of the invention described below, the numbering of which follows the sequence of Phl p 5.0109 ,

Here proline is exemplarily replaced by leucine (L). According to the invention, however, the prolines of the invention can be replaced by any amino acid. Accordingly, according to the invention, all listed or conceivable hypoallergenic variants are those in which one or more

Proline invention are substituted by another amino acid. Furthermore, these examples are not limited to variants of Phl p 5 but also relate in particular Phl p 5b, Poa p 5, Hol I 5, Pha a 5, Ant o 5, Dac g 5, Lol p 5, Fes p 5, Hör v 5, lake c 5, tri a 5 and group 5 allergens of all other grasses. However, all listed hypoallergenic variants according to the invention of phleum pratense Phl p 5a and Phl p 5b are particularly preferred, in particular based on Phl p 5.0109. Based on this finding, the variants rPhl p 5a d [P57, P58, P85, P117, P146, P155, P180, P211, P229, P256] + 6His (short form: MPV.1 + 6His) and rPhl p 5a d [P57 , P58, P117, P146, P155, P180, P21 1, P229] + 6His (Short: MPV.2 + 6His) containing as many combined deletions as possible.

These proteins become insoluble aggregates upon expression

(inclusion bodies) are stored in the E. coli cells. Inclusion bodies are usually solubilized in denaturing agents such as 6-8 molar guanidinium hydrochloride or urea solution and later transferred to a non-denaturing aqueous solution. This transition to a non-denaturing solvent environment is a crucial step in the

Protein purification. Usually, only such proteins can be in

Final formulations of therapeutics that are soluble after this process. An industrially used routine method of transferring the denatured-solubilized proteins to the aqueous solvent is the "rapid dilution" method, in which the proteins dissolved in a denaturing agent are placed in a large volume of the non-denaturing solvent, the denaturing agent is greatly diluted.

The solubility behavior of the mutants described here can

be systematically investigated. The inclusion bodies prepared in E. coli and dissolved in guanidinium hydrochloride are diluted in ten different aqueous buffered solutions, and the degree of solubility is then determined by UV-Vis spectroscopy. In UV-Vis spectroscopy, an absorption spectrum of the protein solution in the wavelength range of 240-800 nm is recorded. insoluble

Aggregates in protein solutions absorb in the> 300 nm wavelength range, while highly soluble proteins in this range do not absorb. The test solutions cover a wide pH range (4.5 - 9.0) and contain partly stabilizing additives. The additives Glyzerin, Tween 80 and L-arginine monohydrochloride are very often used as an aid in the Solubilization difficult-to-produce, recombinant proteins used. They represent the three most important groups of such co-solvents: glycerin as the polyalcohol, tween as the nonionic surfactant, and L-arginine monohydrochloride as the amino acid derivative.

The variants MPV.1 + 6His and MPV.2 + 6His can indeed be solubilized by using the denaturing agent guanidinium hydrochloride, but the systematic investigation of the solubility shows that a subsequent transfer of the proteins into a guanidinium hydrochloride-free formulation always with the formation of insoluble protein aggregates and is therefore not possible (Tab. 1, 2).

During the PCR experiments carried out for the DNA synthesis, in the present example a DNA coding for the protein rPhl p 5a d [P57, P58, P229] K61E, E205K, P21 1 L + 6His is produced by several polymerase errors (short form: MPV. 3 + 6His). This DNA is also expressed in E. coli. Like the variants MPV.1 and MPV.2, the protein is deposited as inclusion bodies in the E. coli cell.

However, the protein MPV.3 + 6His can surprisingly be converted into a non-denaturing buffer solution (Table 3).

EAST IgE inhibition tests show a markedly reduced ability of the protein to bind to IgE antibodies compared to the variants rPhl p 5a d [P57, P58] + 6His and rPhl p 5a d [P229] + 6His, which have single-loop proline deletions (Fig. 9).

For the further characterization of the mutations K61 E, E205K and P211 L of variant MPV.3 + 6His, the DNA of the three variants rPhl p 5a K61 E + 6His, rPhl p 5a E205K + 6His and rPhl p 5a P211 L + 6His are prepared and described in EAST examined. All three mutants show a slightly reduced IgE binding ability, suggesting a synergistic influence of all mutations the reduced allergenicity of MPV.3 + 6His indicates (Figure 10). Both the deletion and the replacement of proline 211 show a much stronger single effect than the mutations K6 E or E205K (Figure 0). By means of a test with basophil granulocytes obtained from the whole blood of grass pollen allergy sufferers, the effect of the reduced IgE binding ability of MPV.3 + 6His on the activation of human effector cells in vitro is investigated. At the same concentration of rPhl p 5a wt and MPV.3 + 6His, the latter shows a lower activation of basophilic granulocytes. This indicates a functionally reduced allergenicity of MPV.3 + 6His (Figure 11).

The randomly generated DNA of variant MPV.3 + 6His now serves as

Starting point for the targeted construction of further variants. The aim was to maintain the high solubility of MPV.3 + 6His and to further reduce the IgE binding capacity by incorporating additional proline mutations.

First, the DNA of the variants rPhl p 5a d [P57, P58, P117, P146, P 55, P180, P229] K61 E, E205K, P2 1L (short form: MPV.4) and rPhl p 5a d [P57, P58, P117, P180, P229] K61 E, E205K, P211 L (short: MPV.5). This DNA is constructed both with and without histidine fusion and expressed in E. coli. The proteins can be purified from the inclusion bodies of the E. coli cells as soluble proteins by a simple purification procedure.

Since both the proteins with and without fusion moiety can be solubilized, the solubility is not dependent on the presence of the histidine fusion moiety. The histidine fusion fraction also has no effect on IgE binding ability as shown by IgE inhibition tests with proteins with and without fusion (Figure 12). The DNA variants rPhl p 5a d [P57, P58, P117, P146, P155, P180, P229] P21 1 L (short: MPV.6) and rPhl p 5a d [P57, P58, P117, P180, P229] P21 1 L (short: MPV.7) are generated exclusively without fusion components and expressed in E. coli. They can also be purified from inclusion bodies by a simple purification procedure as highly soluble proteins.

The variant MPV.6 differs from the variant MPV.2 only by the change at position 211. Instead of the deletion of P211 there is an amino acid exchange to leucine in MPV.6. The extremely different

Solubility thus depends directly on heading 211. All mutants carrying the mutation P211 L (MPV.3-MPV.7) are characterized by high solubility in various aqueous formulations, while the mutants with a deletion at position 211 remain insoluble (Table 1 - Tab. 5) ,

The present invention therefore relates to hypoallergenic variants of the group 5 allergens of sweet grasses according to the invention, in which the proline residue corresponds to that of mature Phl p 5.0109

Amino acid position 211 is not deleted, but by another

Amino acid is replaced.

The present invention therefore relates to inventive hypoallergenic variants of the group 5 allergens of sweet grasses, in which the proline residue, which corresponds in an alignment proline 21 1 in the amino acid sequence of the wild-type Phl p 5 and the mature Phl p 5.0109, not deleted but is replaced by leucine.

In analytical gel filtration (SEC), protein species do not

separated exclusively according to their molecular weight (molar mass), here the conformation, the specific hydrodynamic radius and possible interactions with the matrix also play an important role. A true online determination of molecular weight can be achieved by coupling a refractometer (RI detector) and a multi-angle light scattering detector (MALS detector) to the SEC chromatography system (SEC / MALS / RI method). The given at the time of measurement

Concentration of a particle determined by the Rl detector and the

Light scattering of the particle was recorded by the MALS detector (Wen et al., 1996, Anal. Biochem. 240: 155-166). By SEC / MALS / RI can

Monomers, dimers, multimers and aggregates clearly detected. The result of SEC / MALS / Rl of MPV.5 and MPV.7 shows that these variants are pure and are soluble monomers (Figure 13, Figure 14, Table 6). The two variants MPV.4 and MPV.6 are each detected as a mixture of soluble monomers and dimers (Figure 15, Figure 16, Table 6). The variants MPV.5 and MPV.7 differ from the variants MPV.4 and MPV.6 only at positions 146 and 155. The Proline P146 and P155 are available at MPV.5 and MPV.7. This shows that the deletion d [P146, P155] causes a strong tendency to dimerization of the corresponding variants. The IgE-binding ability of the optimized variants MPV.4 to MPV.7 is first investigated by a method in which the proteins are immobilized on a nitrocellulose membrane and incubated with IgE antibody-containing sera from individual grass pollen allergy. The

Allergen variant antibody complexes are then stained by an enzymatic reaction. In this procedure, a very reduced IgE binding ability of all four proteins is shown (Figure 17).

This result is confirmed by an EAST IgE inhibition test with a representative human allergy seropool, wherein the variants MPV.4 and MPV.6 each have a slightly lower IgE binding than the corresponding mutants MPV.5 and MPV.7 without the Mutation d [P146, P155] (Figure 18, Figure 19). The lower IgE binding in existing Mutation d [P146, P155] can probably be attributed to the dimerization tendency of these variants, which may be one

Obstruction of IgE epitopes at the contact surfaces of the

Dimerisierungspartner leads.

The test results for the activation of basophilic granulocytes show a functionally greatly reduced allergenicity for all four variants (Fig. 20, Fig. 21, Fig. 22, Fig. 23).

The present invention therefore relates to hypoallergenic variants of the group 5 allergens of sweet grasses according to the invention, characterized in that the proline corresponding in an alignment to the proline 211 of the wild-type Phl p 5.0109, not deleted, but is replaced by another amino acid ,

A preferred subject of the invention are thus also

Hypoallergenic variants of the group 5 allergens of the grasses according to the invention, in which the prolines which correspond in an alignment to the proliferation of the positions 57, 58, 17, 180 and 229 in the amino acid sequence of the wild-type Phl p 5.0109, individually or in combinations mutated, preferably deleted and proline 211 is replaced by leucine.

A preferred subject of the invention are thus also

Hypoallergenic variants of the group 5 allergens of the grasses according to the invention, in which the prolines which correspond in an alignment to the proliferation of the positions 57, 58 and 229 in the amino acid sequence of the wild-type Phl p 5.0109 are deleted and proline 211 is replaced by leucine ,

The subject matter of the present invention accordingly also

hypoallergenic variants according to the invention in which the prolines which are aligned with the prolines of positions 146 and 155 in the Amino acid sequence of the wild-type Phl p 5.0109 correspond, not mutated. These are preferably present as a monomer.

According to the invention, all abovementioned hypoallergenic variants according to the invention, in which additionally the amino acids which correspond in a lysine alignment of position 61 and glutamic acid of position 205 in the amino acid sequence of the wild-type Phl p 5, are present individually or in each case

Combinations are mutated. Preferably, these amino acids are substituted.

The T cell reactivity underlying the efficacy of specific immunotherapy is tested in vitro by a proliferation test with Phl p 5-specific T lymphocytes from grass pollen allergy sufferers. By way of example, the results of the allergen variants MPV.4 and MPV.7 are described here. The dimerizing variant MPV.4 carries all mutations investigated here (d [P57, P58, P117, P146, P155, P180, P229] K61 E, E205K, P211 L) and thus represents a molecule with a maximum modified amino acid sequence. The mutant MPV.7 is chosen as an example of a monomeric form with pure proline mutations. Both mutants show a comparable T cell reactivity with the unchanged allergen (Table 7, Table 8). The receipt of the

crucial T-cell epitopes allows an immunotherapeutic

Use of the variants described.

T helper lymphocytes react with peptide fragments of the allergens that are generated by degradation processes in antigen-presenting cells (APC) and are presented bound to MHC class II molecules on the surface of the APC. The peptides are usually 13-18 amino acids in length, but may be longer due to the MHC class 2 side-open binding site. The main contact points of the peptide with the MHC class molecule can be found in a core sequence of about 7-10 amino acids. The allergen-specific activation of T helper lymphocytes is the

Prerequisite for their proliferation and functional differentiation (eg Treg, TH1 and TH2). The ability of an allergen or an allergen variant for the stimulation of allergen-specific T-lymphocytes is called a

Key considered for their therapeutic efficacy.

All based on the allergen variants generated by Phl p 5 and Phl p 5.0109 and shown here show in experiments a substantial preservation of crucial T-cell epitopes.

Thus, for the first time, variants of the group 5 allergens of the Poaceae are described, which are novel due to the modification of proline residues

Have protein properties. The affected proline residues are in

Loop regions isolated. Only the modification of certain proline residues leads to the novel variants, which are characterized by a reduced IgE reactivity when the T cell reactivity is largely preserved and are therefore suitable for curative and preventive specific immunotherapy. Appropriate DNA molecules are suitable for immunotherapeutic vaccination.

The present invention therefore relates to the described allergen variants according to the invention, DNA molecules and recombinant expression vectors as medicaments.

According to the invention, hypoallergenic variants, DNA molecules and

Recombinant expression vectors or medicaments according to the invention can be used in particular for the prophylaxis and / or treatment of diseases and disease states.

Medicaments according to the invention are particularly suitable for the treatment and / or prophylaxis of type 1 allergies, that is to the specific one

Immunotherapy (hyposensitization) of patients with grass pollen allergy or for the preventive immunotherapy of grass pollen allergies in their triggering group 5 allergens of species of the Poaceae are involved.

DNA molecules according to the invention and recombinant expression vectors can be used for the corresponding immunotherapeutic and prophylactic DNA vaccination.

The invention also provides the use of at least one hypoallergenic variant according to the invention for the preparation of a

Medicament for the prevention and / or therapeutic treatment of type 1 allergies, the induction of which is caused by group 5 allergens of grasses.

According to the invention, the use of at least one

DNA molecule according to the invention and / or a recombinant expression vector according to the invention including mixtures thereof in all ratios for the preparation of a medicament for immunotherapeutic DNA vaccination.

The invention further provides a pharmaceutical preparation comprising at least one hypoallergenic variant according to the invention, a DNA molecule according to the invention and / or a recombinant expression vector according to the invention, including mixtures thereof in all ratios and optionally further excipients and / or adjuvants for prevention and / or therapeutic treatment of type 1 allergies.

In particular, pharmaceutical according to the invention own

Preparations for the prevention and / or therapeutic treatment of type 1 allergies, in the initiation of which group 5 allergens of grasses are causally involved.

Pharmaceutical preparations in the sense of this invention can be described as

Therapeutics can be used in human or veterinary medicine and therefore administered to humans and animals, in particular mammals such as monkeys, dogs, cats, rats or mice and in the therapeutic

Treatment of the human or animal body as well as in the Combating the diseases listed above. They can still be used as diagnostic agents or as reagents.

When using preparations or medicaments according to the invention, the hypoallergenic variants, DNA molecules or recombinant expression vectors according to the invention are generally used analogously to known, commercially available preparations or preparations, preferably in dosages between 0.001 and 500 mg, in hypoallergenic variants about 1-500 μg , preferably 5-200 pg per dose in the maintenance phase. The preparation may be administered one or more times a day, e.g. two, three or four times a day. Typically, the doses are increased in an overdose phase to the maintenance dose. For this purpose, different schemes of dosing and conservation are possible. These can be used, for example, in subcutaneous immunotherapy (SCIT).

Short-term therapies (limited number of injections prior to the onset of seasonal complaints, typically 4-7 injections), preseasonal therapies (beginning of pollen season, typically with weekly injections during the boost phase and monthly injections with the maintenance dose until the onset of the pollen season) or year-round Therapies (uptake typically with up to 16 weekly injections followed by monthly injections with the

Maintenance dose, if necessary reduced dose during the pollen season). In sublingual immunotherapy with aqueous or solid preparations (tablets, wafers, etc.), the initiation of therapy may take place with or without an uptake phase. The therapy is preferably carried out with daily doses all year round, but can also pre-seasonally or with other application schemes (eg every other day, weekly, monthly) are performed. The term "effective amount" means the amount of a drug or pharmaceutical agent that produces a biological or medical response in a tissue, system, animal or human which is sought or desired, for example, by a researcher or physician.

In addition, the term "therapeutically effective amount" means an amount which, compared to a corresponding subject who has not received this amount, results in: improved curative treatment, cure, prevention or elimination of a disease, a disease, a disease state, suffering, interference or prevention of side effects, or even reducing the progression of a disease, condition or disorder. The term "therapeutically effective amount" also includes the amounts effective to increase normal physiological function.

Drugs may be administered by any suitable route, for example oral (including buccal or sublingual), rectal, pulmonary, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral

(including subcutaneous, intramuscular, intravenous or

intradermal) routes. Such drugs can be prepared by any method known in the pharmaceutical art, such as, for example, the active ingredient with the carrier (s) or

Excipient (s) is brought together.

For parenteral administration, in particular solutions, preferably oily or aqueous solutions, furthermore suspensions, emulsions or implants are used. The allergen variants according to the invention can also

lyophilized and the resulting lyophilisates e.g. for production of

Injection preparations are used. The preparations indicated may be sterilized and / or contain adjuvants such as lubricants, preservatives, stabilizers and / or wetting agents, emulsifiers, salts for influencing the osmotic pressure, buffer substances and / or several further active ingredients. Furthermore, by appropriate formulation of Inventive allergen variants Depotpräparate, for example, be obtained by adsorption on aluminum hydroxide, calcium phosphate or tyrosine.

Suitable carriers are organic or inorganic substances which are suitable for parenteral administration and which do not react with group 5 allergen variants according to the invention. Examples include carriers such as water, vegetable oils, benzyl alcohols, polyethylene glycols,

Glycerol triacetate, gelatin, carbohydrates, such as lactose or starch,

Magnesium stearate, talc, lanolin or Vaseline.

For administration of the medicaments according to the invention is suitable

preferably parenteral administration. In the case of parenteral

Application are the intravenous, subcutaneous, intradermal or

intralymphatic administration is particularly preferred. In the case of intravenous administration, the injection may be direct or as an adjunct to

Infusion solutions take place.

Medicaments adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions containing anti-oxidants, buffers, bacteriostats and solubilizers through which the

Formulation is made isotonic with the blood of the recipient to be treated, as well as aqueous and non-aqueous sterile

Suspensions which may contain suspending agents and thickeners. The formulations may be administered in single or multiple dose containers, e.g. sealed ampoules and vials, presented and in

lyophilized state, so that only the addition of the sterile carrier liquid, e.g. Water for injections, needed immediately before use. Prescribed

Injection solutions and suspensions may be sterile powders,

Granules and tablets are produced. If desired, preparations or medicaments according to the invention may contain one or more further active ingredients and / or one or more

Amplifiers (adjuvants) included. Another object of the present invention are thus

pharmaceutical preparations containing further active ingredients and / or

Excipients. A preferred subject matter of the invention are pharmaceutical preparations according to the invention, characterized in that the other active substances are allergens or variants thereof.

Examples of suitable further active ingredients are other allergens,

in particular allergens of the grasses, more preferably allergens from the subfamily Pooideae, preferably from the groups Poodae and Triticodae, preferably represented by Phleum pratense, Holcus lanatus, Phalaris aquatica, Dactylis glomerata, Lolium perenne, Poa pratensis,

Hordeum vulgare, Seeale cereale and Triticum aestivum, for example group 1, 2, 3, 4, 5, 6, 7, 10, 12 or 13 allergens and variants thereof, for example hypoallergenic variants, fragments , Multimers, hybrid molecules or recombinant fusion proteins. Another

The present invention relates to pharmaceutical preparations containing at least one further excipient, particularly preferably so-called potentiators. Examples of active enhancers are

Aluminum hydroxide, monophosphoryl lipid A, activators of Toll-like

Receptors such as e.g. Lipopolysaccharides and CpG oligonucleotides, vitamin D3, mycobacterial antigens, and molecules from parasites (e.g., schistosomes or filariae), e.g. Cystatin or ES-62.

An object of the invention are also kits consisting of

separate packs of

a) a pharmaceutical preparation according to the invention comprising an effective amount of a hypoallergenic variant, DNA molecule or recombinant expression vector according to the invention b) a pharmaceutical preparation containing an effective amount of another active pharmaceutical ingredient and / or potentiator.

The kit contains suitable containers, such as boxes or boxes, individual bottles, bags or ampoules. The set may e.g. containing separate ampoules each containing a formulation according to the invention containing an effective amount of a hypoallergenic variant of the invention, a DNA molecule or a recombinant expression vector and a formulation of another drug in dissolved or in lyophilized form.

Explanation of the illustrations: Fig. 1a: Alignment of deduced amino acid sequences of the group 5- allergens of the Poaceae: alignment of mature group 5 sequences of different species. The boxes indicate the location of the helices of Phl p 6 (PDB entry 1 LX) and the C-terminal half of a Phl p 5.02 fragment (PDB entry 1L3P). The amino acid positions of the proline residues are labeled according to their position in the mature Phl p 5.0109.

Sequence references: Phl p 5.0101 (Phleum pratense lUIS sequence,

UniProtKB Q40960), Phl p 5.0104 (Phleum pratense lUIS sequence,

UniProtKB P93467), Phl p 5.0109 (Phleum pratense lUIS sequence,

UniProtKB Q84UI2), Phl p 5.0201 (Phleum pratense lUIS sequence,

UniProtKB Q40963), Phl p 6.0101 (Phleum pratense lUIS sequence,

UniProtKB P432 5), Lol p 5.0101 (Lolium perenne lUIS sequence, UniProtKB Q40237), Pha a 5.0101 (Phalaris aquatica lUIS sequence, UniProtKB P56164), Dac g 5 (Dactylis glomerata, UniProtKB Q93XD9), Hol I 5.0101 (Holcus lanatus lUIS sequence, UniProtKB 023972), Hol I 5.0201 (Holcus lanatus IUIS sequence, UniProtKB 23971), Poa p 5.0101 (Poa pratensis lUIS sequence,

UniProtKB Q9FPR0), Tri a 5 (Triticum aestivum, UniProtKB Q70JP9), Listen v 5 (Hordeum vulgare, EST TC 190653). Fig. 1 b: Preservation of proline residues

Fig. 2: Working models for the position of proline residues in the 3D structure of Phl p 5a

3D homology models of the N-terminal (amino acids 31-139 of Phl p 5.0109; template molecule: Phl p 6, PDB entry 1 LX, shown on the left) and C-terminal 4-helix bundles (amino acids 155-255 ;

Template molecule: Phl p 5b fragment, PDB entry 1 L3P; right).

a. Very simplified models of the two 4-helix bundles. H1-H8: Helices 1-8. Proline are shown with their position designation and atomic structure. The proline residues P146 and P256 are located in areas that could not be displayed in the 3D models of the template molecules due to missing structural data (dotted lines). The loop between helix 2 and 3 is speculative because the template molecule Phl p 6 has no region homologous to this Phl p 5a region.

b. Surface models. All representable proline residues are exposed on the surface (colored black).

Fig. 3: rPhl p 5a wt (IUIS entry Phl p 5.0109); cDNA sequence (GenBan entry: AJ555152; 855 bp), SEQ ID NO: 1

Fig. 4: rPhl p 5a wt (IUIS entry Phl p 5.0109); deduced

Amino acid sequence (UniProtKB entry: Q84UI2; 284 aa), SEQ ID NO: 2

Fig. 5: N-terminal histidine fusion portion; DNA sequence (57 bp), SEQ ID NO: 3

Fig. 6: N-terminal histidine fusion fraction; Amino acid sequence (19 aa) SEQ ID: 4 FIG. 7: Results of the IgE inhibition tests with Phl p 5a variants with proline deletions in individual loops when using a serum pool

(a) + (b): Data from a single experiment with duplicate determination. The

Symbols represent the average of the duplicates measured at eight

Concentrations of inhibitors. The horizontal lines of the error bars show the single values of the double determination.

(c): Summary of the results of several individual experiments.

Left bar (dark gray): IgE inhibition relative to rPhl p 5a wt at 5 pg / ml

Inhibitor concentration.

Right bar (light gray): IgE inhibition relative to rPhl p 5a wt at 1.25 pg / ml inhibitor concentration.

The bar height indicates the average, which consists of a number (n) of

Single experiments was collected. The error bars show the maximum (horizontal line of the upper error bar) and the minimum (horizontal line of the lower error bar) values that were collected in a number (n) of evaluated individual experiments.

Solid phase: rPhl p 5a wt. Serum pool: Bor 18/100, Allergopharma.

Fig. 8: Comparison of IgE inhibition of rPhl p 5a d [P117, 180] + 6His with the single-site proline mutants rPhl p 5a d [P117] + 6His and d [P180] + 6His

Summary of the results of several individual experiments:

Left bar (dark gray): IgE inhibition relative to rPhl p 5a wt at 5 pg / ml

Inhibitor concentration.

Right bar (light gray): IgE inhibition relative to rPhl p 5a wt at 1.25 g / ml inhibitor concentration.

The bar height indicates the average, which consists of a number (n) of

Solid phase: rPhl p 5a wt. Serum pool: Bor 18/100, Allergopharma. Fig. 9: Comparison of IgE inhibition of MPV.3 + 6His with the

Single-site proline mutants rPhl p 5a d [P57, 58] + 6His and d [P229] + 6His

Summary of the results of several individual experiments:

Left bar (dark gray): IgE inhibition relative to rPhl p 5a wt at 5 pg / ml

Inhibitor concentration.

The bar height indicates the average, which consists of a number (n) of

Single experiments was collected.

The error bars show maximum (horizontal line of the upper

Error bar) and minimum (horizontal line of lower error bar) values collected in a number (n) of evaluated individual experiments.

Solid phase: rPhl p 5a wt. Serum pool: Bor 18/100, Allergopharma.

Fig. 10: Comparison of IgE inhibition of MPV.3 + 6His with the

Single-site proline mutants rPhl p 5a d [P211] + 6His, P211L + 6His, K61E + 6His, and E205K + 6His

Summary of the results of several individual experiments:

Left bar (dark gray): IgE inhibition relative to rPhl p 5a wt at 5 pg / ml

Inhibitor concentration.

The bar height indicates the average, which consists of a number (n) of

Single experiments was collected.

The error bars show maximum (horizontal line of the upper

Solid phase: rPhl p 5a wt. Serum pool: Bor 18/100, Allergopharma. Fig. 11: Functional allergenicity test of MPV.3 + 6His

Demonstration of reduced functional allergenicity of MPV.3 + 6His by basophil activation test with basophils from four clinically defined grass pollen allergy sufferers (P).

Horizontal line: level of stimulant through a negative control.

Fig. 12: IgE inhibition test with MPV.4 + 6His and MPV.4

Left bar (dark gray): IgE inhibition relative to rPhl p 5a wt at 5 pg / ml inhibitor concentration.

The bar height indicates the average, which consists of a number (n) of

Single experiments was collected.

The error bars show maximum (horizontal line of the upper

Solid phase: rPhl p 5a wt. Serum pool: Bor 18/100, Allergopharma.

Fig. 13: Determination of the molecular mass of MPV.5

Chromatogram of an analytical SEC with online molecular weight determination.

Shown is the relative UV signal at 280 nm (right Y-axis) and the molecular weight (left Y-axis, measurement point line in the peak region), plotted against the elution time (X-axis).

The refractive index detector OptilabrEX (Wyatt, Santa Barbara, USA) was used to determine the protein concentration online. The

Light scattering of the particles was determined with the multi-angle detector MiniDAWN Treos (Wyatt). The particle mass was calculated using the software ASTRA 5.3.2.17 (Wyatt) via Debeye formalism with a refractive index increment of 0.180 ml / g. Column: Superdex 2O0 GL 10/300 (GE Healthcare, Uppsala, Sweden). The size exclusion (t ₀ ) is 20.45 min (equivalent to -670 kD).

Eluent: 20 mM Tris 8.0, 150 mM NaCl

Fig. 14: Determination of the molecular mass of MPV.7

Chromatogram of an analytical SEC with online molecular weight determination.

Light scattering of the particles was determined with the multi-angle detector MiniDAWN Treos (Wyatt). The particle mass was calculated using the software ASTRA 5.3.2.17 (Wyatt) via Debeye formalism with a refractive index increment of 0.180 ml / g.

Column: Superdex 200 GL 10/300 (GE Healthcare, Uppsala, Sweden). The size exclusion (fo) is 20.45 min (equivalent to -670 kD).

Eluent: 20 mM Tris 8.0, 150 mM NaCl

Fig. 15: Determination of the molecular mass of the MPV.4

Chromatogram of an analytical SEC with online molecular weight determination.

Light scattering of the particles was determined with the multi-angle detector MiniDAWN Treos (Wyatt). The particle mass was calculated using the software ASTRA 5.3.2.17 (Wyatt) via Debeye formalism with a refractive index increment of 0.180 ml / g. Column: Superdex 200 GL 10/300 (GE Healthcare, Uppsala, Sweden). The size exclusion (fo) is 20.45 min (equivalent to -670 kD).

Eluent: 20 mM Tris 8.0, 150 mM NaCl

Fig. 16: Determination of the molecular mass of MPV.6

Chromatogram of an analytical SEC with online molecular weight determination.

Column: Superdex 200 GL 10/300 (GE Healthcare, Uppsala, Sweden). The size exclusion (t ₀ ) is 20.45 min (equivalent to -670 kD).

Eluent: 20 mM Tris 8.0, 150 mM NaCl

Fig. 17: IgE binding to immobilized MPV.4, MPV.5, MPV.6 and MPV.7

Test for binding of IgE to nitrocellulose membrane strips

immobilized Phl p 5 proteins.

rPhl p 5b wt: Recombinant Phl p 5b isoform

rPhl p 5a wt: Recombinant Phl p 5a isoform

nPhl p 5a / b: Phl p 5 allergen of natural origin consisting of protein of a and b isoform (allergopharma)

MPV.4: d [P57, P58, P117, P146, P155, P180, P229] K61 E E205K P211 L MPV.5: d [P57, P58, P117, P180, P229] K61 E E205K P21 1 L

MPV.6: d [P57, P58, P117, P146, P155, P180, P229] P21 1 L

MPV.7: d [P57, P58, P117, P180, P229] P211 L HSA: albumin from human serum (negative control).

Total: Control for even loading of the strips. Staining with reagent DB71 (Sigma, USA)

SP: Serum pool Bor18 / 100 (Allergopharma)

Fig. 18: IgE inhibition test with MPV.5 and MPV.4

Left bar (dark gray): IgE inhibition relative to rPhl p 5a wt at 5 g / ml inhibitor concentration.

The bar height indicates the average, which consists of a number (n) of

Single experiments was collected.

The error bars show maximum (horizontal line of the upper

Solid phase: rPhl p 5a wt. Serum pool: Bor 18/00, Allergopharma.

Fig. 19: IgE inhibition test with MPV.6 and MPV.7

Left bar (dark gray): IgE inhibition relative to rPhl p 5a wt at 5 μg / ml inhibitor concentration.

The bar height indicates the average, which consists of a number (n) of

Single experiments was collected.

The error bars show maximum (horizontal line of the upper

Solid phase: rPhl p 5a wt. Serum pool: Bor 18/100, Allergopharma. Fig. 20: Functional allergenicity test of MPV.4

Detection of reduced functional allergenicity of MPV.4 by basophil activation test with basophils from four clinically defined grass pollen allergy sufferers (P).

Horizontal line: level of stimulant through a negative control.

Fig. 21: Functional allergenicity test of MPV.5

Demonstration of reduced functional allergenicity of MPV.5 by basophil activation test with basophils from four clinically defined grass pollen allergy sufferers (P).

Horizontal line: level of stimulant through a negative control.

Fig. 22: Functional allergenicity test of MPV.6

Demonstration of reduced functional allergenicity of MPV.6 by basophil activation test with basophils of clinically defined grass pollen allergy sufferers (P).

Horizontal line: level of stimulant through a negative control.

Fig. 23: Functional allergenicity test; MPV.7

Demonstration of reduced functional allergenicity of MPV.7 by basophil activation test with basophils from four clinically defined grass pollen allergy sufferers (P).

Horizontal line: level of stimulant through a negative control.

Fig. 24: rPhl p 5b wt precursor (IUIS entry Phl p 5.0201); Reduced amino acid sequence 284 aa (Swiss-Prot: Q40963.2), SEQ ID NO: 5

Even without further embodiments, it is assumed that a person skilled in the art can make the most of the above description. The preferred embodiments are therefore to be considered as illustrative, but not as a limiting revelation in any way

specific.

The following examples are thus intended to illustrate the invention without limiting it. Unless otherwise indicated, percentages are by weight. All temperatures are in degrees Celsius.

"Usual workup": Water is added, if necessary, adjusted to pH values between 2 and 10, if necessary, depending on the constitution of the final product.

The following hypoallergenic variants according to the invention were prepared and characterized by biotechnology. The production and

However, characterization of the substances is also feasible in other ways for the skilled person. For example, the hypoallergenic variants of the invention can also be chemically synthesized. The

The hypoallergenic variants according to the invention described below are likewise provided by the invention.

Example 1: Variants of Ph) p 5a with one or two adjacent

Prolindeletionen

Preparation of the variants rPhl p 5a d [P57, P58] + 6His, rPhl p 5a d [P85] + 6His, rPhl p 5a d [P117] + 6His, rPhl p 5a d [P146, P155] + 6His, rPhl p 5a d [P180] + 6His, rPhl p 5a d [P211] + 6His, rPhl p 5a d [P229] + 6His and rPhl p 5a d [P256] + 6His and their immunological characterization is shown below. Analogously, the recombinant unchanged allergen (rPhl p 5 wt + 6His) is prepared and tested and analogously, the hypoallergenic variants of the further group 5- allergens of the grasses according to the invention and their wild-type proteins, in particular Lol p 5 and Poa p 5, prepared and to be examined.

Genetic Engineering:

The synthesis of the DNA of the variants takes place by the connection of long overlapping DNA oligonucleotides and the amplification of the DNA by a PCR standard method. The codons are chosen so that the deduced amino acid sequence is based on that of Phl p 5.0109 (Figures 3, 4). The mutations for the proline deletions are introduced by using specific oligonucleotides in the PCR reactions lacking the corresponding codons for proline. The oligonucleotides are chosen so that the deduced protein at the 5 ^' end of a hexa-histidine fusion! carries (Fig. 5, 6). The cDNAs are ligated into the expression vector pTrcHis2 Topo (Invitrogen, Carlsbad, USA). The correctness of the DNA is confirmed by sequencing.

Expression, Purification and Biochemical Analysis:

Expression takes place in Escherichia coli (strain Top10, Invitrogen). rPhl p 5a wt and the variants are obtained by the specific binding of the N-terminal histidine residues to a Ni2 + chelate matrix (Immobilized Metal Ion Affinity Chromatography, IMAC, Material: HiTrap, GE Healthcare, Uppsala, Sweden) cleaned. The absence of insoluble protein aggregates is confirmed by UV-Vis spectroscopy.

Detection of reduced IgE binding:

The study of the IgE binding ability of the test substances is carried out with an EAST IgE inhibition test (Enzyme Allergosorbent Test). In this method, the allergen / IgE interaction in solution can be examined, which interfering masking of epitopes of the test substance can be excluded, for example by immobilization to a membrane.

The EAST inhibition test is carried out as follows. Microtiter plates are coated with the allergens, here recombinant wild-type Phl p 5.0109 (rPhl p 5a wt). After removal of the unbound allergen molecules by washing, the plate is blocked with bovine serum albumin to avoid later nonspecific binding. Allergic IgE antibodies are incubated in the form of a representative pool of single allergy sera (serum pool) in appropriate dilution with the allergen-coated microtiter plates. The amount of allergen-bound IgE antibodies is photometrically quantified via an anti-human IgE / alkaline phosphatase conjugate by the reaction of a substrate to a colored end product.

The binding of the IgE antibodies is inhibited by a soluble allergen or the substance to be tested (recombinant modified allergen) depending on the concentration substance-specific. The results of IgE inhibition tests with the recombinant allergen variants of Phl p 5a shown in FIG. 7 show that deletion of the proline residues P57 and P58; P1 17; P146 and P155; P180; P211 or P229, but not by deletion of P85 or P256, a diminished IgE-binding ability of Phl p 5a is caused. A lower inhibitory effect indicates a loss of IgE epitopes. Example 2: Variants of Phl p 5a with combinations of proline deletions

The production and immunological characterization of the variant rPhl p 5a d [P117, P180] + 6His is shown below by way of example for hypoallergenic variants of the group 5 allergens of the Poaceae according to the invention

Combinations of multiple proline deletions according to the

Amino acid positions of 57 and 58, 117, 146, and 155, 180, 211 and 229 relative to Phl p 5.0109. Analogously, the recombinant unmodified allergen (rPhl p 5 wt + 6His) is prepared and investigated, and analogously, the hypoallergenic variants of the other

Group 5 allergens of sweet grasses and their wild-type proteins according to the invention, in particular Lol p 5 and Poa p 5, are prepared and investigated.

Genetic Engineering:

The synthesis of the variants was made longer by the compound

overlapping DNA oligonucleotides and the amplification of the DNA by PCR. The codons are chosen so that the deduced

Amino acid sequence based on the Phl p 5.0109. The mutations for the proline deletions are introduced by using specific oligonucleotides in the PCR reactions lacking the corresponding codons for proline. The oligonucleotides are selected so that the deduced protein ^'end carries a hexa-histidine fusion Contribution. 5 The cDNAs are inserted into the expression vector pTrcHis2 Topo (Invitrogen) and in

Escherichia coli transformed. The correctness of the DNA is through

Sequencing confirmed.

Expression, Purification and Biochemical Analysis:

Expression takes place in Escherichia coli (strain Top10, Invitrogen). The variants are cleaned by IMAC. The purity of the eluted proteins is controlled by SDS-PAGE and the absentness is more insoluble

Protein aggregates confirmed by UV-Vis spectroscopy. Detection of reduced IgE binding:

The testing of the IgE-binding capacity of the test substances is carried out with an EAST inhibition test with IgE antibodies from allergic persons, which were used in the form of a representative serum pool. The results shown in Fig. 8 show that IgE inhibition of the variant rPhl p 5a d [P117, P180] + 6His is significantly lower than that of the variants with only one of the proline deletions rPhl p 5a d [P117] + 6His and rPhl p 5a d [P180] + 6His. This result shows that the combination of single proline deletions can lead to an increased reduction of the IgE binding ability of Phl p 5a.

Example 3: Hypoallergenic variant rPhl p 5a d [P57, P58, P85, P117, P146, P155, P180, P211, P229, P256] (PV.1 + 6His)

The allergen variant MPV.1 + 6His shows a combination of deletions of all investigated proline residues of Phl 5a. The aim of the preparation is maximum reduced IgE binding ability with acceptable protein solubility.

Genetic Engineering:

The synthesis of the variants takes place by the compound of long

Amino acid sequence based on the Phl p 5.0109. The mutations for the proline deletions are introduced by using specific oligonucleotides in the PCR reactions lacking the corresponding codons for proline.

The oligonucleotides are chosen such that the deduced protein carries a hexa-histidine fusion moiety at the 5 ^' end. The cDNA are transformed into the expression vector pTrcHis2 Topo (Invitrogen) and into Escherichia coli. The correctness of the DNA is confirmed by sequencing. expression:

Expression takes place in Escherichia coli (strain Top10, Invitrogen). The proteins are deposited by the host cell as insoluble inclusion bodies.

Solubility test:

The protein aggregates are isolated by cell disruption with a standard method in a purity of about 80% and denatured by treatment with a 6 molar solution of guanidinium hydrochloride denaturing.

The denatured proteins are diluted 1: 50 in a volume of 25 ml at 4 ° C for this assay and kept at 4 ° C overnight. On the following day, any visible precipitate formation is organoleptically checked. After concentration, the samples are centrifuged and the clear supernatants are analyzed by UV-Vis spectroscopy for the presence of insoluble micro-aggregates.

The systematic investigation of the solubility behavior of the variant MPV.1 + 6His shows that the subsequent transfer of the proteins into a guanidinium hydrochloride-free formulation is always accompanied by the formation of P red inagga regates and is therefore not possible (Table 1).

Example 4: Hypoallergenic variant rPhl p 5a d [P57, P58, P1 7, P146, P155, P180, P211, P229] (MPV.2 + 6His)

The preparation and immunological characterization of variant MPV.2 + 6His is exemplified below for hypoallergenic variants of the group 5 allergens of Poaceae with combinations of proline deletions

corresponding to amino acid positions 57, 58, 17, 146, 155, 180, 211 and 229 relative to Phl p 5.0109. Analogously, the recombinant unmodified allergen (rPhl p 5 wt + 6His) is prepared and investigated, and analogously, the hypoallergenic variants of the other Group 5 allergens of sweet grasses and their wild-type proteins according to the invention, in particular Lol p 5 and Poa p 5, are prepared and investigated.

Genetic Engineering:

The synthesis of the variants takes place by the compound of long

Amino acid sequence based on the Phl p 5.0109.

The mutations for the proline deletions are introduced by using specific oligonucleotides in the PCR reactions lacking the corresponding codons for proline.

The oligonucleotides are chosen such that the deduced protein carries a hexa-histidine fusion moiety at the 5 ^" end The cDNA is ligated into the expression vector pTrcHis2 Topo (Invitrogen) and transformed into Escherichia coli The correctness of the DNA is confirmed by sequencing.

expression:

Solubility test:

MPV.2 + 6His shows poor solubility throughout the pH range studied, 4.5-9.0. Only with a solution containing Tween 80 and L-arginine monohydrochloride can the formation be visually more visible

Precipitates are prevented, however, are also below in this approach micro-aggregates detected (Table 2). Example 5: Hypoallergenic variant rPhl p 5a d [P57, P58, P229] K61E, E205K, P2L (MPV.3 + 6His)

The preparation and immunological characterization of variant MPV.3 + 6His is exemplified below for hypoallergenic variants of the Poaceae group 5 allergens with combinations of deletions of proline residues 57, 58, 117, 146, 155, 180 or 229 corresponding to the amino acid positions of the Phl p 5 5.0109, in which the proline residue 211 is mutated into any other amino acid or in which additionally lysine 61 in

Glutamate or glutamate 205 is converted to lysine. Analogously, the recombinant unchanged allergen (rPhl p 5 wt + 6His) is prepared and tested and analogously, the hypoallergenic variants of the other group 5 allergens of the grasses according to the invention and their wild-type proteins, in particular Lol p 5 and Poa p 5, prepared and

to be examined.

The effect of the mutations K61 E, E205K and P21 1L, which are described in the

The IgE-binding ability is investigated by preparing the variants rPhl p 5a K61 E + 6His, rPhl p 5a E205K + 6His and rPhl p 5a P211 L + 6His and the MPV.3 + 6His variants

be characterized immunologically.

Genetic Engineering:

The nucleotide sequence encoding MPV.3 + 6His is surprisingly generated in this example by multiple polymerase errors in a cDNA synthesis. The DNA is ligated into the expression vector pTrcHis2 Topo (Invitrogen). The DNA encoding the variants rPhl p 5a K61E + 6His, rPhl p 5a E205K + 6His and rPhl p 5a P211 L + 6His are selectively produced and ligated into the expression vector pTrcHis2 Topo. The correctness of the generated cDNA sequences is checked by DNA sequencing.

expression Expression takes place in Escherichia coli (strain Top10, Invitrogen). The proteins are deposited by the host cell as insoluble inclusion bodies.

Solubility test of MPV.3 + 6His:

The protein aggregates are isolated after cell disruption in a purity of about 80% and by treatment with a 6 molar solution

Guanidinium hydrochloride denaturing solubilized. The serial testing of various solutions to verify the solubility properties of MPV.3 + 6His in a non-denaturing environment is analogous to the experiments with variants MPV.1 + 6His and MPV.2 + 6His

carried out.

It is surprisingly found that variant MPV.3 + 6His has a high solubility at slightly basic pH during the dilution process (Table 3). This behavior is in contrast to the previously studied variants MPV.1 + 6His and MPV.2 + 6His and represents a decisive advantage for the representation of the protein in a non-denaturing environment. Purification of MPV.3 + 6His, as well as the mutants rPhl p 5a K61 E + 6His, rPhl p 5a E205K + 6His and rPhl p 5a P211 L + 6His:

The denatured proteins are diluted 1:50 in 20 mM Tris, 150 mM NaCl, pH 9.0 and kept at 8 ° C overnight. The chromatographic

Cleaning is performed by IMAC (HiTrap Material, GE Healthcare) and SEC (Superdex 75 Material, GE Healthcare). Finally, the proteins are stable and soluble in 25 mM sodium phosphate buffer with 150 mM NaCl, pH 7.5.

Biochemical analysis:

The purity is controlled by SDS-PAGE. The absence of insoluble protein aggregates is confirmed by UV-Vis spectroscopy. Evidence of reduced IgE binding of MPV.3 + 6His:

The study of the IgE binding ability of MPV.3 + 6His was carried out with an EAST inhibition test with IgE antibodies from allergic persons, which are used in the form of a representative serum pool. The results shown in Fig. 9 show that the IgE inhibition of MPV.3 + 6His is significantly lower than that of the variants rPhl p 5a d [P57, P58] + 6His and rPhl p 5a d [P229] + 6His.

To further characterize mutations K61E, E205K and P211 L of variant MPV.3 + 6His, the three variants rPhl p 5a K61 E + 6His, rPhl p 5a E205K + 6His and rPhl p 5a P211L + 6His are investigated by EAST.

The variant rPhl p 5a P211 L + 6His shows a comparably low level

Inhibition behavior as the previously tested point mutant rPhl p 5a d [P211] + 6His. The variants rPhl p 5a K61E + 6His and rPhl p 5a E205K + 6His show a slightly decreased IgE binding (Figure 10).

It can be concluded that the three point mutations K61 E, E205K and P2 1 L contribute to the markedly decreased IgE binding of MPV.3 + 6His. Evidence of the reduction of functional allergenicity of MPV.3 + 6His: The functional effect of MPV.3 + 6His in the cross-linking of

membrane-bound IgE of effector cells and their activation is examined in vitro. For the basophil activation test heparinized whole blood of

Grass pollen allergy sufferers with different concentrations of

Test substances incubated. Allergenic substances can bind specific IgE antibodies, which are associated with the high-affinity IgE receptors of basophilic granulocytes. The crosslinking of the IgE / receptor complexes triggered by the allergen molecules leads to a

Signal transduction, which results in the degranulation of effector cells and thus the triggering of allergic reactions in vivo. Allergen-induced activation of basophilic immunocytes can be detected in vitro by quantifying the expression of a surface protein (CD203c) coupled to signal transduction of IgE receptor crosslinking (Kahlert et al., Clinical Immunology and Allergy in Medicine Proceedings of the EAACI 2002 ( 2003) Naples, Italy 739-744). The number of expressed surface proteins on a cell and the percentage of activated cells of a cell pool is determined by the binding of a cell

fluorescently labeled monoclonal antibody to the surface protein and subsequent analysis by fluorescence-activated flow cytometry highly sensitive measured. PV.3 + 6His shows a reduced activation of basophils here

Granulocytes of grass pollen allergy sufferers compared to rPhl p 5a wt and thus a functionally reduced allergenicity (Fig. 1 1)

Example 6: Hypoallergenic variant rPhl p 5a d [P57, P58, 117, 146, 155, 180, P229] K61 E, E205K, P211 L (MPV.4)

The preparation and immunological characterization of the variant MPV.4 is below by way of example for hypoallergenic variants of the group 5 allergens of the Poaceae with combinations of deletions of the proline residues 57, 58, 17, 146, 155, 180 or 229 corresponding to the amino acid positions of the Phl p 5.0109), in which the proline residue 21 1 is mutated into any other amino acid or in which lysine 61 is additionally converted into glutamate or glutamate 205 into lysine. Analogously, the recombinant unmodified allergen (rPhl p 5 wt + 6His) is prepared and investigated, and analogously, the hypoallergenic variants of the other

Group 5 allergens of sweet grasses and their wild-type proteins according to the invention, in particular Lol p 5 and Poa p 5, are prepared and investigated. Genetic engineering of MPV.4 (+ 6His):

To produce the cDNA of the histidine fusion protein of MPV.4 (MPV.4 + 6His), a fragment of the already cloned cDNA of MPV.1 + 6His is recloned into the already existing plasmid MPV.3 + 6His / pTrcHis2 topo.

To prepare the fusion-share-free protein (MPV.4), the DNA is ligated without the section coding for the histidine fusion portion in the vector pTMP (Allergopharma, Reinbek). The correctness of the sequence is checked by DNA sequencing.

Expression of MPV.4 (+ 6His):

Expression is either as a histidine fusion protein

(Expression vector pTrcHis2Topo; Invitrogen) in Escherichia coli (strain Top 0, Invitrogen) or without fusion fraction (expression vector pTMP) in Escherichia coli (strain BL21, Merck, Darmstadt). In both cases, the recombinant proteins are deposited as inclusion bodies. The proteins are solubilized with a 6 molar solution of guanidinium hydrochloride.

Solubility test of MPV.4 + 6His.

Row testing of various solutions to verify the

Solubility properties of MPV.4 + 6His in non-denaturing

Environment is carried out analogously to the experiments with the variants MPV.1 + 6His. It is found that the variant MPV.4 + 6His has solubility at slightly basic pH (Table 4)

Purification of MPV.4 + 6His:

The fusion protein is purified on a preparative scale by IMAC (HiTrap material, GE Healthcare) and SEC (Superdex 75 material, GE Healthcare) and is finally in 25 mM sodium phosphate buffer with 150 mM NaCl, pH 7.5.

Purification of the non-fusion protein MPV.4: The denatured non-fusion protein solution is first diluted 1: 50 with 20 mM Tris, 150 mM NaCl, pH 9.0 analogously to the purification scheme of the fusion protein and kept at 4 ° overnight. In a first purification step, the target protein is enriched by hydrophobic interaction chromatography (HIC).

Proteins bind to a HIC column at high ionic strength, depending on their surface hydrophobicity, with specific strength, so that they can be selectively eluted by gradually reducing the salt concentration.

The diluted protein solution is first 1: 2 with 20 mM Tris, 2 M

Ammonium sulfate, 150mM NaCl, pH 9.0, to give a final ammonium sulfate concentration of 1M. This protein solution is then passed over a HiTrap Butyl S FF HIC column (GE Healthcare, Uppsala, Sweden) and gradually eluted after binding of the target protein with 20 mM Tris, 150 mM NaCl, pH 9.0.

The HIC eluates are rebuffered via a Sephadex G25 column (GE Healthcare) in 20mM Tris, 50mM NaCl, pH 8.0, in preparation for the second purification step.

As a second purification step, the eluates are applied to an anion exchange chromatography column (HiTrap Q HP, GE Healthcare) and the run collected. The target protein is in the process while foreign proteins bind to the column and are effectively purified.

In the AIEX method, negative surface charge proteins are bound to the column material at low ionic strength while

do not bind uncharged or positively charged proteins. In the final step, the AIEX run is post-purified on a SEC column (Superdex 75, GE Healthcare) such that the target protein is finally in 20 mM Tris, 150 mM NaCl, pH 8.0. Biochemical analysis of MPV.4 (+ 6His):

The purity of the proteins is controlled by SDS-PAGE. The identity of the fusion proportion-free MPV.4 is determined by determination of the molecular mass by means of mass spectroscopy (MALDI-TOF) and sequencing of the N-

Terminal amino acids proved (sequence: A-D-L-G-Y-G-P-A-T) (Table 6). The absence of insoluble protein aggregates is confirmed by UV-Vis spectroscopy. The molecular weight analysis of MPV.4 using SEC / MALS / RI surprisingly shows that the mass of eluted proteins is between 28.2 and 49.3 kD (mean mass 36.9 kD). Since the theoretical mass of the monomer is 27.6 kD, it can be assumed that a mixture of monomers and dimers is present (Figure 15, Tab. 6). The qualitatively same result is obtained with the fusion protein.

Evidence of reduced IgE binding of MPV.4 (+ 6His):

The study of IgE-binding ability is carried out with an EAST inhibition test with IgE antibodies from allergic persons, in the form of a

representative serum pools are used.

The results shown in Fig. 12 show that MPV.4 shows equally reduced IgE binding both with and without fusion. It is thus shown that the reduced IgE reactivity does not depend on the

Presence of the histidine tag depends.

A simple test method for determining the IgE reactivity of specific IgE from allergy sera to membrane-bound test proteins is the streak test. For this, the test substances are bound in the same concentration and amount side by side to a strip of nitrocellulose membrane under non-denaturing conditions. A number of such membrane strips can be incubated in parallel with different types of allergic sera become. After a washing step, the specifically bound IgE antibodies are transformed by a color reaction mediated by an anti-human

IgE / alkaline phosphatase conjugate, visualized on the membrane.

The results of variant MPV.4 using individual

Grass pollen allergy sera are shown in Fig. 17. Sera of allergy sufferers with antibodies to natural Phl p 5 (nPhl p 5a / b,

Mixture of Phl p 5a- and b-isoform). The IgE antibodies also react with the recombinant rPhl p 5a wt and also the recombinant wild type of the b-isoform (rPhl p 5b wt), which was also investigated.

It becomes clear that the Phl p 5-specific IgE antibodies of all

Allergic sera bind the variant MPV.4 strongly reduced, while the recombinant wild-type proteins rPhl p 5a wt and rPhl p 5b wt are bound as strongly as nPhl p 5a / b.

Evidence of the reduction of functional allergenicity of MPV.4:

The functional effect of MPV.4 in the cross-linking of

membrane-bound effector cell IgE and its activation is being studied in vitro with both fusion proteins and non-fusion proteins. Both the fusion protein and the fusion portion of free MPV.4 show a strongly reduced activation of basophils

Granulocytes of grass pollen allergy sufferers compared to rPhl p 5a wt and thus a strongly functionally reduced allergenicity (Fig. 20).

Example 7: Hypoallergenic variant rPhl p 5a d [P57, P58, P117, P180, P229] K61E, E205K, P21 1L (MPV.5)

The production and immunological characterization of the variant MPV.5 is below by way of example for hypoallergenic variants of the group 5 allergens of the Poaceae with combinations of deletions of the proline residues 57, 58, 17, 180 or 229 corresponding to the amino acid positions of Phl p 5.0109 in which proline residue 211 has been mutated into any other amino acid or in which lysine 61 is additionally converted to glutamate or glutamate 205 to lysine. Analogously, the recombinant unchanged allergen (rPhl p 5 wt + 6His) is prepared and tested and analogously, the hypoallergenic variants of the other group 5 allergens of the grasses according to the invention and their wild-type proteins, in particular Lol p 5 and Poa p 5, prepared and to be examined. Genetic engineering of MPV.5 (+ 6His):

To produce the cDNA of the histidine fusion protein (MPV.5 + 6His), a fragment of the already cloned cDNA of rPhl p 5a d [P117, P180] + 6His is recloned into the already existing plasmid MPV.3 + 6His / pTrcHis2 topo. To produce the fusion-share-free protein (MPV.5), the DNA is ligated without the histidine fusion portion coding section in the vector pTMP (Allergopharma, Reinbek). The correctness of the sequence is checked by DNA sequencing.

Expression of MPV.5 (+ 6His):

Expression is either as a histidine fusion protein

(Expression vector pTrcHis2Topo; Invitrogen) in Escherichia coli (strain Top10; Invitrogen) or without fusion fraction (expression vector pTMP;

Allergopharma) in Escherichia coli (strain BL21, Merck, Darmstadt). In both cases, the recombinant proteins are deposited as inclusion bodies. The proteins are made by means of a 6 molar solution

Guanidinium hydrochloride solubilized.

Purification of MPV.5 + 6His:

The fusion protein is prepared on a preparative scale by IMAC (HiTrap-

Material, GE Healthcare) and SEC (Superdex 75 material, GE Healthcare) and is finally in 25 mM sodium phosphate buffer with 150 mM NaCl, pH 7.5 before.

Purification of the non-fusion protein:

The IB solution of the denatured non-fusion protein, analogously to the purification scheme of the fusion protein, is first diluted 1:50 with 20 mM Tris, 150 mM NaCl, pH 9.0 and kept at 4 ° overnight.

In a first purification step, the target protein is enriched by hydrophobic interaction chromatography (HIC). The diluted protein solution is first further diluted 1: 2 with 20mM Tris, 2M ammonium sulfate, 150mM NaCl, pH 9.0 to achieve a final ammonium sulfate concentration of 1M. This protein solution is then passed over a HiTrap Butyl S FF HIC column (GE Healthcare, Uppsala, Sweden) and gradually eluted after binding of the target protein with 20 mM Tris, 150 mM NaCl, pH 9.0.

The HIC eluates are rebuffered via a Sephadex G25 column (GE Healthcare) in 20mM Tris, 50mM NaCl, pH 8.0, in preparation for the second purification step. As a second purification step, the eluates are applied to an anion exchange chromatography column (HiTrap Q HP, GE Healthcare) and the run-through with the target protein is collected.

In the final step, the AIEX run is post-purified on a SEC column (Superdex 75, GE Healthcare) such that the target protein is finally in 20 mM Tris, 50 mM NaCl, pH 8.0.

Biochemical analysis of MPV.5 (+ 6His):

The purity of the proteins is controlled by SDS-PAGE. The

Absence of insoluble protein aggregates is confirmed by UV-Vis spectroscopy. The identity of the fusion proportion-free MPV.5 is determined by molecular mass measurements by mass spectroscopy (MALDI-TOF) and Sequencing of the N-terminal amino acid sequence proved (sequence: ADLGYGPAT) (Table 6).

The analysis of MPV.5 by SEC / MALS / RI shows that the protein exists exclusively as a monomer (Figure 13, Tab. 6). A comparable result is obtained with the fusion protein. The high tendency to

Dimerization, which is found in variant MPV.4, thus depends on the presence of mutation d [P146, P155]. Evidence of reduced IgE binding of MPV.5 (+ 6His):

representative serum pools are used. MPV.5 shows an equally reduced IgE binding capacity both with and without fusion. The IgE binding capacity is slightly higher than that of variant MPV.4, which may be based on a better accessibility of IgE epitopes in the exclusively monomeric MPV.5 (Figure 18).

The results of variant MPV.5 in the strip test using individual grass pollen allergy sera are shown in FIG. 17. It becomes clear that the Phl p 5-specific IgE antibodies of all types of allergy sufferers strongly diminish the variant MPV.5.

Evidence of the reduction of functional allergenicity of MPV.5 (+ 6His): The functional effect of MPV.5 in the cross-linking of

membrane-bound effector cell IgE and its activation is being studied in vitro with both fusion proteins and non-fusion proteins. MPV.5 shows a strongly reduced activation of basophilic granulocytes of grass pollen allergy sufferers compared to rPhl p 5a wt and thus a strongly functionally reduced allergenicity (Figure 21). Example 8: Hypoallergenic variant rPhl p 5a d [P57, P58, P117, P146, P155, P180, P229] P211 L (MPV.6)

The production and immunological characterization of the variant MPV.6 is below by way of example for hypoallergenic variants of the group 5 allergens of the Poaceae with combinations of deletions of the proline residues 57, 58, 117, 146, 155, 180 or 229 corresponding to the amino acid positions of the Phl p 5.0109, in which the proline residue 21 1 is mutated into any other amino acid. Analogously, the recombinant is unchanged

Allergen (rPhl p 5 wt + 6His) prepared and tested and analogously, the hypoallergenic variants of the other group 5 allergens of the grasses according to the invention and their wild-type proteins, in particular Lol p 5 and Poa p 5, prepared and investigated.

Genetic Engineering:

To prepare the DNA, a DNA fragment of MPV.2 + 6His is ligated into the vector rPhl p 5a d [57, 58, 117, 180, 229] P21 LJ pTMP (allergopharma) present at this time. The correctness of the sequence is checked by DNA sequencing.

expression:

The expression takes place exclusively as fusion-share-free protein in the expression vector pTMP (Allergopharma) in Escherichia coli (strain BL21, Merck, Darmstadt). The recombinant proteins are deposited as inclusion bodies (IB).

Cleaning:

The denatured non-fusion protein IB solution is first diluted 1:50 with 20 mM Tris, 150 mM NaCl, pH 9.0 and kept at 4 ° overnight.

In a first purification step, the target protein is enriched by hydrophobic interaction chromatography (HIC). The diluted protein solution is first 1: 2 with 20 mM Tris, 2 M

The HIC eluates are rebuffered via a Sephadex G25 column (GE Healthcare) in 20mM Tris, 50mM NaCl, pH 8.0, in preparation for the second purification step. As a second purification step, the eluates are applied to an anion exchange chromatography column (HiTrap Q HP, GE Healthcare) and the run-through with the target protein is collected. In the final step, the AIEX run is post-purified on a SEC column (Superdex 75, GE Healthcare) such that the target protein is finally in 20 mM Tris, 150 mM NaCl, pH 8.0.

Biochemical analysis:

The purity of the proteins is controlled by SDS-PAGE. The identity of the fusion fraction-free MPV.6 is proved by determination of the molecular mass by mass spectroscopy (MALDI-TOF) and sequencing of the N-terminal amino acid sequence (sequence: A-D-L-G-Y-G-P-A-T) (Table 6).

The absence of insoluble protein aggregates is caused by UV-Vis

Spectroscopy confirmed. In the analysis of MPV.6 by SEC / MALS / RI, the purified proteins elute into two peaks. Peak 1 represents the monomeric and peak 2 the dimeric form (Figure 16, Table 6).

The Dimerisierungsfähigkeit is, as well as the variants MPV.4 and

MPV.5 is due to the presence of the mutation d [P146, 155]. Detection of reduced IgE binding:

The study of IgE binding is carried out with an EAST inhibition test with IgE antibodies from allergic persons, which are used in the form of a representative serum pool. The results shown in Fig. 19 show that MPV.6 has greatly reduced IgE binding. The results in

Strip tests using individual grass pollen allergy sera are shown in Fig. 17. It becomes clear that the Phl p 5-specific IgE antibodies of all types of allergy sufferers strongly diminish the variant MPV.6. Evidence of the reduction of functional allergenicity:

The functional effect of MPV.6 in the cross-linking of

membrane-bound IgE and the activation of basophilic granulocytes is strongly reduced compared to rPhl p 5a wt, as shown by the whole blood of two grass pollen allergic patients (Fig. 22).

Example 9: Hypoallergenic variant rPhl p 5a d [P57, P58, P117, P180, P229] P211 L (MPV.7) The production and immunological characterization of the variant MPV.7 is below by way of example for hypoallergenic variants of the group 5 allergens the Poaceae with combinations of deletions of the proline residues 57, 58, 117, 180 or 229 corresponding to the amino acid positions of the Phl p 5a wild type or of the Phl p 5.0109, in which the proline residue 211 is mutated into any other amino acid. Analogously, the recombinant unmodified allergen (rPhl p 5 wt + 6His) is prepared and investigated, and analogously, the hypoallergenic variants of the other

Group 5 allergens of sweet grasses and their wild-type proteins according to the invention, in particular Lol p 5 and Poa p 5, are prepared and investigated. Genetic Engineering:

The DNA is produced by preparing a DNA fragment by means of specific oligonucleotides by PCR methods and then in the already existing at this time vector rPhl p 5a d [P57, P58, P117, P180, P211, P229] / pTMP (Allergopharma ) is recloned. The correctness of the sequence is checked by DNA sequencing.

expression:

The expression takes place as a fusion-share-free protein in the

Expression vector pTMP (Allergopharma) in Escherichia coli (strain BL21, Merck, Darmstadt). The recombinant proteins are deposited as inclusion bodies (IB).

Solubility test:

The series test for checking the solubility properties of the recombinant protein in non-denaturing environment shows a high solubility of the protein in the slightly basic pH range (Table 5).

Cleaning:

The I B solution of the denatured non-fusion protein is first diluted with 20 mM Tris, 50 mM NaCl, pH 9.0: 50 and kept at 4 ° overnight. In a first purification step, the target protein is enriched by hydrophobic interaction chromatography (HIC). The diluted protein solution is first further diluted 1: 2 with 20mM Tris, 2M ammonium sulfate, 150mM NaCl, pH 9.0 to achieve a final ammonium sulfate concentration of 1M. This protein solution is then passed over a HiTrap Butyl S FF HIC column (GE Healthcare, Uppsala, Sweden) and gradually eluted after binding of the target protein with 20 mM Tris, 150 mM NaCl, pH 9.0. The HIC eluates are rebuffered via a Sephadex G25 column (GE Healthcare) in 20mM Tris, 50mM NaCl, pH 8.0, in preparation for the second purification step. As a second purification step, the eluates to a Anion exchange chromatography column (HiTrap Q HP, GE Healthcare) is applied and the run with the target protein collected. In the final step, the AIEX run is post-purified on a SEC column (Superdex 75, GE Healthcare) such that the target protein is finally in 20 mM Tris, 150 mM NaCl, pH 8.0.

Biochemical analysis:

The purity of the proteins is controlled by SDS-PAGE. The identity of the fusion proportion-free MPV.7 is determined by molecular mass measurements by mass spectroscopy (MALDI-TOF) and sequencing of the N

Terminal amino acid sequence proved (sequence: A-D-L-G-Y-G-P-A-T) (Table 6). Absence of insoluble protein aggregates is confirmed by UV-Vis spectroscopy. Analysis by SEC / MALS / RI shows that the eluted proteins are present exclusively as monomers (Figure 14, Tab. 6). This is due to the lack of mutation d [P146, P155].

Detection of reduced IgE binding:

The study of IgE binding is carried out with an EAST inhibition test with IgE antibodies from allergic persons, which are used in the form of a representative serum pool. The results shown in Fig. 19 show that MPV.7 has greatly reduced IgE binding. The results in

Strip tests using individual grass pollen allergy sera are shown in Fig. 7. It becomes clear that the Phl p 5-specific IgE antibodies of all types of allergy sufferers bind strongly to the variant MPV.7.

Evidence of the reduction of functional allergenicity:

The functional effect of MPV.7 in the cross-linking of

membrane-bound IgE and the activation of basophilic granulocytes of grass pollen allergy is greatly reduced compared to rPhl p 5a wt (Figure 23).

T cell reactivity of the hypoallergenic Phl p 5a variants: To investigate the T-cell reactivity, oligoclonal T-cell lines of grass pollen allergy sufferers under stimulation with natural nPhl p 5α or rPhl p 5a wt olecules are established by conventional methods. In one

Proliferation test were the different T-cell lines with the

Reference allergen rPhl p 5a wt and the modified recombinant

Allergen variants stimulated. The proliferation rate was determined by the incorporation of [3H] thymidine by the usual methods.

Exemplary of the modified allergen variants described here are the results of the proliferation tests of MPV.4 and MPV.7 with T-cell lines of 12 grass pollen allergy sufferers.

The T cell reactivity of variant MPV.4, which carries the most mutations of all molecules studied, is despite the changes in the

Amino acid sequence compared to the unchanged rPhl p 5a wt not lowered, which demonstrates the receipt of crucial T-cell epitopes (Table 7).

The mutant MPV.7 contains the least number of altered

Amino acid positions of all investigated multi-proline mutants. As expected, the molecule stimulates the human T-lymphocytes comparably well, like the unchanged allergen rPhl p 5a (Table 8).

Tab. 1: Solubility behavior of MPV.1 + 6His

NaCl buffer substance evaluation

Solution pH Additive OK ¹

³

1 4.5 0.15 M 0.02 M Na acetate without - n.d. -

2 5.5 0.15 M 0.02 Na citrate without - n.d. -

3 6.5 0.15 M 0.02 Ka phosph. without - n.d. -

4 7.5 0.15 0.02 M Na phosph. without - n.d. -

5 8.0 0.15 M 0.02 M Tris without - n.d. -

6 9.0 0.15 M 0.02 M Tris without - n.d. -

7 8.0 0.075 M 0.02 Tris 0.5 M L-arginine HCl; + 12.5 (-)

0.005% (w / v) Tween 80

8 8.0 without 0.02 Tris 0.005% (w / v) Tween 80 + 10.2 (-)

9 8.0 without 0.02 Tris 0.5 L-arginine HCl - n.d. -

10 7.5 without 0.02 Na Phosph. 10% (w / v) glycerin - nd - The proteins isolated from "Inclusion" / ^" it" were first labeled with 6 M

Guanidinium hydrochloride denatured, then diluted 1:50 in a non-denaturing solution (Lsg. 1 -10) and kept at 4 ° C overnight. On the following day, an organoleptic control (OK) was carried out with respect to turbidity by visible macro-aggregates or precipitates. (-) turbidity; (+) clear solution.

² UV / Vis spectral analysis for the detection of insoluble micro-aggregates in clear solutions by determining the ratio of absorbance at 280 and 330 nm. Test of the batches after centrifugation. A280 / A330 s 20: precipitation (-); A280 A330 2 30:

Precipitation tendency (o); > 30: no precipitation (+). (nd) not performed.

³ Evaluation of the solubility behavior based on the organoleptic and

spectrophotometric analysis. (-) tends to be insoluble; (+) soluble.

Tab. 2: Solubility behavior of MPV.2 + 6His

The proteins isolated from inclusion bodies were first labeled with 6 M

Guanidinium hydrochloride, then diluted 1:50 in a non-denaturing solution (Ls. 1-10) and kept at 4 ° C overnight. On the following day, an organoleptic control (OK) was carried out with respect to turbidity by visible macro-aggregates or precipitates. (-) turbidity; (+) clear solution.

² UV / Vis spectral analysis for the detection of insoluble micro-aggregates in clear solutions by determining the ratio of absorbance at 280 and 330 nm. Test of the batches after centrifugation. A280 / A330 s 20: precipitation (-); A _2S0 l 3zo ^ 30:

Precipitation tendency (o); A ₂₈₀ / A ₃₃ o> 30: no precipitation (+). (nd) not performed.

³ Evaluation of the solubility behavior based on the organoleptic and

spectrophotometric analysis. (-) tends to be insoluble; (+) soluble.

Tab. 3: Solubility behavior of MPV.3 + 6His

The proteins isolated from inclusion bodies were first labeled with 6 M

² UV / Vis spectral analysis for the detection of insoluble micro-aggregates in clear solutions by determining the ratio of absorbance at 280 and 330 nm. Test of the batches after centrifugation. A ₂ 8o / A ₃₃₀ <20: precipitation (-); A280 A330 ^ 30:

Precipitation tendency (o); A ₂₈₀ / A ₃₃₀ > 30: no precipitation (+). (nd) not performed.

³ Evaluation of the solubility behavior based on the organoleptic and

spectrophotometric analysis. (-) tends to be insoluble; (+) soluble.

Tab. 4: Solubility behavior of MPV.4 + 6His

¹ The proteins isolated from inclusion bodies were first labeled with 6 M

² UV / Vis spectral analysis to detect insoluble ikro aggregates in clear solutions by determining the ratio of absorbance at 280 and 330 nm. Test Approaches after centrifugation. A ₂₈ c 33o s 20: precipitation (-); A ₂₈₀ lA ₃₃ o <30:

Precipitation tendency (o); A ₂ 8o / A ₃₃₀ > 30: no precipitation (+). (nd) not performed.

³ Evaluation of the solubility behavior based on the organoleptic and

spectrophotometric analysis. (-) tends to be insoluble; (+) soluble.

Tab. 5: Solubility behavior of PV.7

The proteins isolated from inclusion bodies were first labeled with 6 M

² UV / Vis spectral analysis for the detection of insoluble micro-aggregates in clear solutions by determining the ratio of absorbance at 280 and 330 nm. Test of the batches after centrifugation. A A330 <20: precipitation (-); Α _2Β ο / Α ₃₃₀ <30:

Precipitation tendency (o); A ₂₈ o / ₃ 3o> 30: no precipitation (+). (nd) not performed.

³ Evaluation of the solubility behavior based on the organoleptic and

spectrophotometric analysis. (-) tends to be insoluble; (+) soluble.

Table 6: Results of the molecular weight analysis of MPV.4, MPV.5, MPV.6 and MPV.7 in comparison to rPhl p 5a wt

¹ Calculated molecular weight (MW _calc .) Without starting ethionine on the basis of

Amino Acid Sequence (Software: DNA Star, Laser Genes, USA)

² Determination of particle mass by SEC-MALS. Indicated is the average of the mass of eluted protein particles in the set peak window, except for the measurement of MPV.4 ( ^* ), where the scattering range of the masses is given in the total peak.

The refractive index detector (RL) OptilabrEX (Wyatt, Santa Barbara, USA) was used to determine the protein concentration online. The light scattering of the particles was determined with the multi-angle detector MiniDAWN Treos (Wyatt). The calculation of

Particulate mass was measured using ASTRA 5.3.2.17 (Wyatt) software via Debeye formalism with an assumed refractive index increment of 0.180 ml / g.

Column: Superdex 200 GL 10/300 (GE Healthcare, Uppsala, Sweden). Of the

Size exclusion (f ₀ ) is 20.45 min (equivalent to -670 kD).

Eluent: 20 mM Tris 8.0, 150 mM NaCl Tab. 7: Detection of T cell reactivity of MPV.4

Stimulation Index ¹

Reactivity of MPV.4

Donor ² T cell line rPhl p 5a wt MPV.4

relative to rPhl p 5a wt ³

3 3.10 3.0 4.0 1, 33

8 8.2 13 12.7 0.98

8 8.3 2.9 4.0 1, 38

19.17 7.2 9.9 1, 38

19 19.2 3.5 4.9 1, 40

21 21,210 4.5 5.8 1, 29

23 23.22 4.3 2.2 0.51

55 55,184 26,7 28,5 1, 07

55 55,193 49.9 46.2 0.93

59 59.57 4,1 4,4 1, 07

59 59.91 7,7 9,0 1, 17

60 60,162 4.9 2.6 0.53

65 65,115 6.7 9.1 1, 36

116 116.34 9.4 2.8 0.30

128 128.40 8,8 8,9 1, 01

137 137.41 2.8 6.5 2.32

137 137.43 2.6 2.5 0.96

1, 12 average

0.45 SD

¹ stimulation index (Sl) calculated from [ ³ H] measurements of the proliferation test. cpm measurements of allergen-stimulated cell cultures / cpm measurements of unstimulated cell cultures.

² Donor: clinically defined grass pollen allergy sufferer.

³ Calculated with Sl (MPV.4) / Sl (rPhl p 5a wt). SD: standard deviation.

Tab. 8 Detection of T cell reactivity of MPV.7

Stimulation Index ¹

Reactivity of

Donor ² T cell line rPhl p 5a wt MPV.7 MPV.7 relative to rPhl p 5a wt ³

3 3.10 2.0 2.2 1, 10

8 8.2 27.7 23.6 0.85

8 8.3 5,2 8,2 1, 58

11 11.2 2,1 2,7 1, 29

11 1 1.3 2.4 2.4 1, 00

19 19.1 4,8 6,5 1, 35

21 21.2 3.7 3.4 0.92

21 21 .210 3.9 5.4 1, 38

55 55,181 12,4 14,8 1, 19

55 55,184 123.6 149.5 1, 21

59 59.91 4.5 5.0 1, 11

60 60,162 2.9 2.6 0.90

65 65,115 4,6 4,7 1, 02

70 70,126 6.5 6.5 1, 00

116 116.34 8.5 2.5 0.29

128 128.40 4.8 3.5 0.73

1, 06 Mean 0.30 SD

Stimulation index (SI), calculated by ^[3 H] metrics family of proliferation assays. cpm measurements of allergen-stimulated cell cultures / cpm measurements of unstimulated cell cultures.

² Donor: clinically defined grass pollen allergy sufferer.

³ Calculated with Sl (MPV.7) / Sl (rPhl p 5a wt). SD: standard deviation.

Claims

Patent claims

1. Hypoallergenic variant of a group 5 allergen of the family of

Sweet grasses (Poaceae), in which the prolines, in an alignment with the prolines of positions 57, 58, 117, 146, 155, 180, 211 in the

Amino acid sequence of the wild-type PhI p 5.0109, mutated individually or in combinations.

2. Hypoallergenic variant according to claim, characterized in that the proline, which corresponds in an alignment to proline 211 of the wild-type PhI p 5.0109, is not deleted but is replaced by another amino acid.

3. Hypoallergenic variant according to claim 1 or 2, in which the prolines which correspond in an alignment to the prolines of positions 146 and 155 in the amino acid sequence of the wild-type PhI p 5.0109 are not mutated.

4. Hypoallergenic variant according to one or more of claims 1 to

3, in which the additional amino acids in an alignment are lysine at position 61 and glutamic acid at position 205 in the

5. Hypoallergenic variant according to one or more of claims 1 to

4, characterized in that the group 5 allergen is a group 5 allergen of the subfamily Pooideae.

6. Hypoallergenic variant according to one or more of claims 1 to 5, characterized in that the Group 5 allergen is selected from the group consisting of Phl p 5, Lol p 5, Poa p 5, Hol I 5, Dac g 5, Pha a 5, Ant o 5, Fes p 5, Hör v 5, See c 5 and Tri a 5.

7. Hypoallergenic variant according to one or more of claims 1 to 6, characterized in that it is a fragment or a variant of a hypoallergenic variant according to one or more of claims 1 to 6 or a multimer of one or more

hypoallergenic variants according to one or more of claims 1 to 6 or characterized in that one or more hypoallergenic variants according to one or more of claims 1 to

6 or whose fragments, variants or multimers are part of a recombinant fusion protein.

8. DNA molecule that codes for a hypoallergenic variant according to one or more of claims 1 to 7.

9. Recombinant expression vector containing a DNA molecule according to claim 8, functionally linked to an expression control sequence.

10. Non-human host organism transformed with a DNA molecule according to claim 8 or a recombinant expression vector according to claim 9.

11. A method for producing a hypoallergenic variant according to one or more of claims 1 to 7 by cultivating a non-human host organism according to claim 10 and obtaining the corresponding allergen variant from the culture. 2. Hypoallergenic variant according to one or more of claims 1 to

7 as medicine.

3. DNA molecule according to claim 8 as a drug.

14. Recombinant expression vector according to claim 9 as a drug.

15. Use of at least one hypoaliergenic variant according to one or more of claims 1 to 7 for the production of a medicament for the prevention and / or therapeutic treatment of type 1 allergies, in the triggering of which Group 5 allergens from sweet grasses are causally involved.

16. Use of at least one DNA molecule according to claim 8 and / or a recombinant expression vector according to claim 9 for

Production of a drug for immunotherapeutic DNA vaccination.

17. Pharmaceutical preparation containing at least one hypoallergenic variant according to one or more of claims 1 to 7, at least one DNA molecule according to claim 8 and / or at least one

recombinant expression vector according to claim 9 and optionally further active ingredients and/or auxiliary substances for the prevention and/or therapeutic treatment of type 1 allergies. 8. Pharmaceutical preparation according to claim 17, thereby

characterized in that the other active ingredients are sweet grass allergens or variants thereof.