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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/02—Nasal agents, e.g. decongestants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
  • A61P27/14—Decongestants or antiallergics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents

Definitions

• the invention relates to variants of the major allergen Phl p 1 from lieschgrass, characterized in that a previously impossible production of monomeric, stable and soluble in physiological media molecules can be carried out with the help of prokaryotic expression systems and their subsequent purification.
• Type 1 allergies are of worldwide importance. Up to 25% of the population of industrialized countries suffer from complaints such as allergic rhinitis, conjunctivitis or bronchial asthma, which are caused by airborne allergens (aeroallergens) of various origins such as plant pollen, mites, cats or dogs. Up to 40% of these type 1 allergy sufferers in turn show specific IgE (immunoglobulin E) reactivity in grass pollen (Freidhoff et al., 1986, J. Allergy. Clin. Immunol. 78, 1190-201).
• IgE immunoglobulin E
• the type 1 allergy-triggering substances are proteins, glycoproteins or polypeptides. After absorption via the mucous membranes, these allergens react with the IgE molecules bound to the surface of mast cells in sensitized persons. If two IgE molecules are cross-linked by an allergen, this leads to the release of mediators (e.g. histamine, prostaglandins) and cytokines by the effector cell and thus to the corresponding clinical symptoms.
• mediators e.g. histamine, prostaglandins
• cytokines e.g. histamine, prostaglandins
• Antibodies against certain allergens are distinguished between major and minor allergens.
• Phl p 1 (Petersen et al., 1993, J. Allergy Clin. Immunol. 92, 789-796), Phl p 5 (Matthiesen and Löwenstein, 1991, Clin. Exp Allergy 21, 297-307), Phl p 6 (Petersen et al., 1995, Int. Arch. Allergy Immunol. 108, 49-54) and Phl p 2/3 (Dolecek et al., 1993) as major allergens and Phl p 4 (Löwenstein, 1978, Prog. Allergy 25, 1-62) and groups 10 and 11 from Lolium perenne (Ansah et. Al., 1987, J. Allergy Clin. Immunol. 80, 229-235) are characterized as minor allergens Service.
• Group 1 is classified as one of the most relevant allergen groups of grass pollen (Tamborini, E. et al., Eur. J. Biochem. 1997, 249: 886-894), to which Phl p 1 from lying grass belongs.
• Phl p 1 the other representatives of group 1 from other grasses have homologies of over 95% in some cases (Petersen, A., et al., J. Allergy Clin. Immunol. 1995, 95: 987-994). Due to the high homologies, sensitization to grass also leads to reactions to the allergens of other cross-reactive species. Therefore, these molecules are of paramount importance for appropriate diagnostic and therapeutic approaches.
• the therapeutic use of these allergens uses the reaction with T helper cells, which leads to a reorientation of the pathological
• TH2 cells come into the TH1 type. This results in a change in the cytokine profile in such a way that B cells are stimulated to form IgG instead of IgE.
• Phl p 1 is a protein of 240 amino acids and an N-glycosylation site. The proportion of glycosylation is 5% of the molecular weight, which is approximately 30-35 kDa for the natural protein (Petersen et al., Allergy Clin. Immunol. 1995, 95: 987-994; Suck et al., J. Immunol. Meth. 199.9, 229: 73-80).
• the nucleic acid sequence of Phl p 1 is known (Laffer et al., J. Allergy Clin Immunol. 1994, 94: 689-698; Petersen et. Al., J. Allergy Clin. Immunol., 1995, 95: 987-94) and can thus be used for the recombinant production of the molecule.
• Phl p 1 is deposited as inclusion bodies (Vrtala et al., J. Allergy Clin. Immunol, 1996; 97: 781-7) and must first be denatured before cleaning. The denaturant is then removed. A complete refolding of the protein into the naturally soluble conformation has not yet been achieved (Andersson, Lidholm, Int. Arch. Allergy Immunol. 2003; 130: 87-107). A possible obstacle to the formation of a stable conformation could have been the lack of glycosylation. However, even in eukaryotic systems in which glycosylation is possible, no stable Phl p 1 was obtained (K. Grobe, dissertation, 1998, University of Hamburg). Instead, the cause of the lack of solubility is assumed to be a proteolytic activity that leads to the self-degradation of the molecule
• the object on which the present invention was based was therefore to provide variants of the major allergen Phl p 1 from lying grass, which, with full preservation of the therapeutically and diagnostically important immunological properties, are distinguished by improved solubility and which can thus be purified in a pharmaceutically suitable form.
• Lane 1 Molecular weight standard (10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 160, 220 kDa, Bench Mark Protein Ladder, Invitrogen, Düsseldorf, Germany)
• Lane 2 Extract from phleum p guess se pollen lane 3: nPhl p 1 lane 4: rPhl p 1-LM lane 5: rPhl p 1-HM
• Figure 2 Gel filtration with rPhl p 1-HM and rPhl p 1-LM on a Sephacryl S100 column.
• the figure shows that the two folding variants have different apparent molecular weights.
• FIG. 3 Enzyme Allergy Sorbent Test (EAST) to quantify the IgE binding of the folding variants rPhl p 1-A236C -LM and -HM.
• EAST Enzyme Allergy Sorbent Test
• concentration of an inhibitor of the IgE-nPhl p 1 binding in mol is on the horizontal axis / l, the degree of inhibition is given in [%] on the vertical axis.
• the measurement was carried out with nPhl p 1 on the solid phase and a typical serum of a grass pollen allergy sufferer.
• the invention therefore relates to variants of the major allergen Phl p 1 from grass, which have an additional Cys residue compared to the wild type, and to fragments and variants derived from the base molecules and which have the same or similar advantageous properties.
• the invention relates to a method for producing the variants of the recombinant major allergen rPhl p 1 according to the invention, characterized in that a base triplet coding for a Cys residue is introduced into the Phl p 1 gene by insertion or exchange according to methods known per se , the gene thus modified is overexpressed in a host organism and the allergen variant obtained by overexpression is purified.
• the prokaryotic recombinant production and purification can be carried out with or without a genetically introduced fusion fraction and always leads to the same products. If a fusion component is used, it is preferably a His tag.
• the cleaning procedures vary depending on the expression vector or system.
• the present invention accordingly comprises a specifically modified primary sequence of the recombinant allergen rPhl p 1, which is its recombinant
• the invention thus also relates to DNA molecules which code for the allergen variants according to the invention.
• the recombinant proteins are autoproteolytically inactive and can therefore in stable monomeric form in physiological, buffered or each be stored in other solutions after application. T cell stimulation shows no significant differences between recombinant and natural Phl p 1.
• Variants can thus be used to treat grass pollen-induced allergic diseases.
• the present invention also relates to the new allergen variants in their properties as medicaments.
• the recombinantly produced allergen variants and fragments can be used to diagnose pollen allergies.
• the amino acid exchange is effected by directed nucleotide exchange, for example by means of PCR.
• the mutant rPhl p 1-A236C according to SEQ ID NO 2 the alanine at position 236 is replaced by cysteine.
• the exchange site can also be located anywhere else in the molecule. As a rule, however, it will be located in the C-terminal region of the molecule, preferably from position 140, in particular between positions 230 and 240.
• rPhl p 1-LM low molecular weight
• rPhl p 1-HM high molecular weight
• IgE reactivity also differs. While rPhl p 1-LM has a reactivity comparable to that of natural proteins, rPhl p 1-HM is from
• IgE antibodies are less well bound and, because of their hypoallergenicity, are particularly suitable for specific immunotherapy (see Fig. 3). In principle, however, both forms of folding are suitable for both therapeutic and diagnostic applications.
• the invention thus furthermore relates to different forms of folding of the rPhl p 1 allergen variant according to the invention and their use for therapeutic and diagnostic purposes.
• Both folding variants are easily soluble, stable monomer and have no detectable proteolytic / autoproteolytic activity.
• allergen variants according to the invention can be obtained, for example, using the two production processes outlined below - with or without an artificial fusion fraction:
• the initially insoluble crude protein is purified via several biochemical separation steps, comprising one or more metal ion chelate affinity chromatography steps and the His tag being split off.
• Various other chromatographies as well as de-and renaturation steps can be used for pre- and post-cleaning.
• the denaturation batch is preferably added to a multiple of its volume (approximately 10 to 80 times, preferably 20 to ⁇ O times the volume) of the preferably stirred or otherwise mixed buffer solution - for example by decanting, pipetting or pumping; however, the buffer solution can also be added to the denaturation batch.
• the speed of the addition is not critical: the total amount can be added all at once within a few seconds or - preferably (but not necessarily) evenly - spread over several hours.
• the alternative stable folding forms LM and HM can be obtained in a targeted manner with minimal cross-contamination through different incubation times in the denaturation step.
• To produce the LM form it is basically possible to incubate between about 1 and 50 hours. As a rule, however, one will move in a range of 0 to 40 hours, preferably 15 to 30, an interval of 18 to 22 hours being particularly preferred.
• the HM variant requires significantly longer incubation times. They are of the order of 60 to 120 hours. Usually, however, one will incubate for 70 to 100 hours, particularly preferably in the range of 80 to 90 hours.
• the denaturation step is followed by the previously described dilution step with buffer solution.
• the intermediate interval (approx. 50 to 60 hours) represents the reorganization phase.
• the folding process is apparently fixed by the dilution step, no further kinetics take place.
• the fine cleaning for the separation of LM and HM is possible via hydrophobic interaction chromatography or is achieved by gel filtration, in which the forms have clearly different retention times.
• the folding variants LM and HM according to the invention can be obtained in high purity and have valuable pharmaceutically relevant properties. However, they can also be used individually as a mixture for the diagnosis (in particular rPhl p 1-LM because of the maintenance of the IgE activity) and therapy (in particular rPhl p 1-HM because of the reduced IgE activity) of allergic diseases.
• the invention therefore also relates to the use of the allergen variants and / or their pharmaceutically usable derivatives, including their mixtures in all ratios, for the production of a medicament for specific immunotherapy (hyposensitization) and the diagnosis of allergies, from the triggering of which the major allergen Phl p 1 Lieschgras is involved
• the invention furthermore relates to a pharmaceutical preparation comprising an allergen variant according to the invention and / or its pharmaceutically usable derivatives, including their mixtures in all ratios, and, if appropriate, carriers and / or auxiliaries.
• the active compounds according to the invention can be used together with at least one solid, liquid and / or semi-liquid carrier or auxiliary optionally in combination with one or more other active ingredients in a suitable dosage form.
• DNA molecules on which the allergen variants according to the invention are ligated are ligated with a suitable expression vector, these constructs can also be used as preparations for immunotherapy (DNA vaccination).
• the invention therefore also relates to a recombinant DNA expression vector, containing a DNA molecule according to the invention, for the treatment of allergies, the major allergen Phl p 1 of which is involved in lying grass, by immunotherapeutic DNA vaccination.
• the invention furthermore relates to the use of said expression vector and / or its derivatives, including their mixtures in all ratios, for the production of a medicament for the treatment of allergies, the major allergen Phl p 1 of which is involved in lying grass, by immunotherapeutic DNA -Vakzintechnik.
• the invention relates to a pharmaceutical preparation containing the said expression vector and / or its pharmaceutically usable derivatives, including their mixtures in all ratios, and, if appropriate, carriers and / or auxiliaries, for the treatment of allergies, the trigger of which is the major allergen Phl p 1 from Lieschgras is involved through immunotherapeutic DNA vaccination.
• Suitable carrier substances are organic or inorganic substances which are suitable for parenteral administration and which do not react with allergen variants according to the invention of the major allergen Phl p 1 from lying grass. Ren.
• solutions preferably oily or aqueous solutions, furthermore suspensions, emulsions or implants are used for parenteral use.
• the allergen variants according to the invention can also be lyophilized and the lyophilizates obtained can be used, for example, for the production of injection preparations.
• the specified preparations can be sterilized and / or contain auxiliary substances such as lubricants, preservatives, stabilizers and / or wetting agents, emulsifiers, salts for influencing the osmotic pressure, buffer substances and / or several other active substances.
• auxiliary substances such as lubricants, preservatives, stabilizers and / or wetting agents, emulsifiers, salts for influencing the osmotic pressure, buffer substances and / or several other active substances.
• Depot preparations for example by adsorption on aluminum hydroxide, can furthermore be obtained by appropriate formulation of the allergen variants according to the invention.
• modifications can be, for example, chemical modifications of the allergen extract (Fiebig, 1995, Allergo J. 4 (7), 377-382).
• modifications can also be genetically engineered at the DNA level, e.g. Amino acid insertions, deletions and exchanges, splitting of the protein into fragments and fusions of the protein or its fragments with other proteins or peptides are possible.
• Example 1 Obtaining rPhl p 1-A236C-L and -HM - Cleaning Variant with His Tag
• the sequence coding for the Phl p 1 was amplified with 5'- and 3'-specific oligonucleotides by means of PCR and converted into a pProEx vector ( GIBCO, La Jolla, USA) ligated over the Marriage I and Hind III interface.
• the 3 'primer was changed at base position 706/707 from GC to TG in such a way that a triplet coding for alanine is converted into a triplet coding for cysteine (Essential Molecular Biology; TA Brown ed., IRL Press, Oxford, 1994). The transformation took place in E.
• the selected starting vector pProEx provides the N-terminal end localized 6xHis sequence, followed by a recognition sequence for the TEV protease.
• the recombinant, primarily 6xHIS-tagged rPhl p 1-A236C molecules present as insoluble aggregates after bacterial expression are dissolved in 6 M guanidium chloride (GdmCI), 50 mM Tris / HCl, 500 mM NaCI, pH 8.0) after pre-cleaning.
• the proteins bound to chelating Sepharose under denaturing conditions are composed of the denaturing solution in a buffer over a gradient of 90 min 50 mM phosphate buffer and 500 mM NaCI (pH 7.4) transferred. This is followed by a step elution with 500 mM imidiazole in phosphate buffer.
• the renatured fusion protein is cleaved into rPhl p 1 and the 6xHis fusion portion using a specific TEV protease.
• Ni-chelate affinity chromatography 2 run: rPhl p 1-LM eluate: rPhl p 1-HM, uncleaved His-rPhI p 1 fusion protein
• the first procedure is as in Example 1, but the vector chosen does not provide any fusion protein as the primary product. Now the aggregates are insoluble after bacterial expression
• inclusion bodies present recombinant, primarily rPhl p 1-A236C molecules, after pre-cleaning in 6 M guanidium chloride (GdmCI), 50 mM Tris / HCl, pH 8.0) dissolved. This is followed by a 40-fold dilution in 20 mM Tris pH 8.0. For this purpose, the denaturing solution is decanted into the dilution solution stirred with a magnetic stirrer
• the inclusion bodies are incubated in the denaturing solution for 20 h and then diluted as described above.
• the inclusion bodies are incubated in the denaturing solution for 85 h and then diluted as described above.
• 500 mM NaCl are added to the respective dilution (renaturation) approach.
• the dissolved molecules in the renaturation batch are concentrated by means of a Cu chelate affinity chromatography and eluted with 2 ⁇ 0 mM imidazole in phosphate buffer as a step (or gradually by 500 mM imidazole in phosphate buffer).
• chelate affinity chromatography can be used for conditioning prior to elution of the protein, such as using a 3 M NaCl solution for washing and a 3 M NaCl, 200 mM imidazoil solution for elution.
• the highly saline eluate could then be used directly for hydrophobic interaction chromatography.
• gel filtration with Superdex 75 is carried out for final cleaning and transfer to a desired solvent.
• Example 2 Different IgE bindings of the wild type and of the folding variants LM and HM of the allergen variant rPhl p 1-A236C
• the natural nPhl p 1 and the recombinant rPhl p 1 variants HM and LM are compared with respect to the strength of their IgE binding (Fig. 3). It can be seen that the variant HM has a significantly reduced IgE binding compared to the natural Phl p 1 protein, while the variant LM has an IgE binding comparable to the natural Phl p 1 protein.

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