US20050123568A1 - Method for producing hypoallergenic major birch pollen allergen rbet v 1 - Google Patents

Method for producing hypoallergenic major birch pollen allergen rbet v 1 Download PDF

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Publication number
US20050123568A1
US20050123568A1 US10/505,897 US50589704A US2005123568A1 US 20050123568 A1 US20050123568 A1 US 20050123568A1 US 50589704 A US50589704 A US 50589704A US 2005123568 A1 US2005123568 A1 US 2005123568A1
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rbet
birch pollen
pollen allergen
eluent
process according
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US10/505,897
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Roland Suck
Helmut Fiebig
Oliver Cromwell
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Merck Patent GmbH
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Merck Patent GmbH
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Publication of US20050123568A1 publication Critical patent/US20050123568A1/en
Priority to US12/167,712 priority Critical patent/US9441021B2/en
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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
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the invention relates to a process for the preparation of birch pollen allergens which are distinguished by a lack of, but at least by reduced immunoglobulin E binding, i.e. by hypoallergeneity. These allergens completely retain therapeutically relevant T-cell stimulation. They can therefore be employed as low-side-effect therapeutic agents for specific immunotherapy.
  • Type 1 allergies have dramatically increased worldwide in recent decades. Up to 20% of the population in industrialised countries suffer from complaints, such as allergic rhinitis, conjunctivitis or bronchial asthma, which are caused by allergens present in the air (aeroallergens), which are released by various sources, such as plant pollen, mites, mammals (cats, dogs, horses) and mould fungi. Severe allergies can also be initiated by insect stings or bites, such as, for example, of bees and wasps.
  • the type 1 allergy-initiating substances are proteins, glycoproteins or polypeptides.
  • allergens react via the mucous membranes after ingestion or with the IgE antibodies bound to the surface of mast cells in sensitised people after stings or bites. If two or more IgE antibodies are crosslinked to one another by an allergen, this results in the release of mediators (for example histamine, prostaglandins) and cytokines by the effector cell and thus in initiation of the allergic symptoms.
  • mediators for example histamine, prostaglandins
  • Birch pollen are the most frequent initiators of allergic reactions amongst tree pollen (Jarolim E. et al., 1989, Allergy 44:385-95). More than 90% of sufferers from birch pollen allergies have IgE antibodies against the major allergen Bet v 1 (Elfman, L. et al., 1997, Int. Arch. Allergy Immunol., 113: 249-51).
  • a potential disadvantage in recombinant allergen variants is that the modification of the primary structure causes loss or a reduction in the reactivity of the T-cell epitopes which are necessary for therapeutic success. This possibility can only be excluded if the primary structure corresponding to the natural allergen serves as the basis for the preparation of the recombinant protein.
  • a suitable starting point for the preparation of a recombinant major allergen rBet v 1 which can be utilised for therapeutic purposes would accordingly be a molecule which corresponds to the wild type in the primary structure and is unrestricted in its T-cell stimulation, but has reduced IgE activity, i.e. is hypoallergenic.
  • the form of the preparation process of the recombinant allergens is of particular importance here inasmuch as the proteins are converted in the course of this process into a conformation which has no or greatly reduced affinity to IgE with constant T-cell stimulation.
  • FIG. 1A SDS-PAGE for characterisation of hypoallergenic rBet v 1
  • FIG. 1B Nitrocellulose blot of the SDS-PAGE from FIG. 1A
  • FIG. 2A Nitrocellulose blot for determination of the IgE activity with 20 individual patient sera
  • FIG. 2B Nitrocellulose blot for determination of the identity of Bet v 1 samples
  • FIG. 3 Enzyme allergo sorbent test (EAST) for quantification of IgE binding
  • the concentration of an inhibitor of IgE-Bet v 1 binding in ⁇ g/ml is plotted on the vertical axis, and the degree of inhibition in [%] is shown on the horizontal axis.
  • FIG. 4 Determination of T-cell stimulation by Bet v 1 variants
  • concentrations of natural nBet v 1, of conventionally purified recombinant rBet v 1 and of recombinant rBet v 1 purified in accordance with the invention and the respective stimulation indices (SI) obtained with various T-cell lines (TCLs) and T-cell clones (TCCs) are compared.
  • the present invention relates to a biochemical purification process which results in the preparation of proteins having the properties modified in accordance with the invention via efficient purification, using specific eluents, of, for example, allergens prepared by recombinant methods. These properties consist in a lack of, but at least a greatly reduced IgE activity, with simultaneous maintenance of T-cell stimulation.
  • the invention thus relates to a process for reducing the IgE activity of the major birch pollen allergen rBet v 1 which consists in the use of soluble recombinant major birch pollen allergen rBet v 1 and in carrying out the chromatography steps described below for the purification thereof and the subsequent neutralisation step.
  • the invention furthermore relates to a process for the preparation of hypoallergenic major birch pollen allergen rBet v 1 by means of a plurality of chromatographic purification steps using essentially unbuffered aqueous bases as eluent and subsequent neutralisation, where the starting material employed is a soluble rBet v 1 crude protein prepared by recombinant methods.
  • the chromatographic purification steps preferably include anion exchange chromatography, hydrophobic interaction chromatography and gel filtration and can be carried out once, a number of times one after the other or a number of times alternately.
  • the chromatographic purification steps are preferably carried out in the following sequence: first gel filtration, anion exchange chromatography, hydrophobic interaction chromatography, second gel filtration.
  • the chromatographic purification is usually carried out with a base concentration of from 5 to 100 mM, but preferably with from 5 to 40 mM and particularly preferably with from 10 to 30 mM, where the basic substance employed is preferably NaOH.
  • a base concentration of from 5 to 100 mM, but preferably with from 5 to 40 mM and particularly preferably with from 10 to 30 mM, where the basic substance employed is preferably NaOH.
  • a base concentration of from 5 to 100 mM, but preferably with from 5 to 40 mM and particularly preferably with from 10 to 30 mM, where the basic substance employed is preferably NaOH.
  • a mixed system comprising, for example, water and methanol.
  • a non-aqueous, for example methanol system is also conceivable. However, preference is given to the use of an aqueous system.
  • a neutral salt preferably NaCl
  • concentrations of a neutral salt up to about 5 M can be added to the eluent.
  • the intention of establishing physiological conditions at the end of the purification can also be a reason for the presence of sodium hydrogen-carbonate during the chromatography steps. Concentrations of up to 100 mM are basically possible here. However, work is preferably carried out in the physiological range below 20 mM, particularly preferably at 11 mM.
  • the invention thus relates to a process for the preparation of hypoallergenic major birch pollen allergen rBet v 1 in which essentially unbuffered basic eluents maintain the proteins to be purified in solution under mild conditions and thus in a chromatographable state.
  • the rBet v 1 crude protein is prepurified before the actual purification by chromatography, for example hydrophobic interaction chromatography or ion exchange chromatography, and/or salt precipitation, where, in contrast to the subsequent principal purification, a buffered eluent or a buffered solution is used.
  • chromatography for example hydrophobic interaction chromatography or ion exchange chromatography, and/or salt precipitation, where, in contrast to the subsequent principal purification, a buffered eluent or a buffered solution is used.
  • the starting materials for the process are soluble, recombinant allergens expressed in bacteria or other suitable host cells (such as, for example, yeasts). Since the process represents a general application for these expression products, soluble allergens of different origin can also, in accordance with the invention, be purified by the process, renatured and formulated. In particular in the case of corresponding similarity of these allergens of different origin to the major birch pollen allergen Bet v 1, it can be expected that the properties according to the invention will be achieved. However, the process is particularly suitable for obtaining major birch pollen allergen rBet v 1 prepared by recombinant methods. However, natural major birch pollen allergen nBet v 1 is also basically suitable as starting material.
  • the pharmaceutical active ingredients can be used directly after neutralisation as parenteral products.
  • the reproducible and standardisable process can be carried out under the conditions of Good Manufacturing Practice (GMP), which is necessary for pharmaceuticals.
  • GMP Good Manufacturing Practice
  • the invention thus serves for the preparation of improved preparations for the specific immunotherapy of allergies, which is achieved by the process according to the invention.
  • the greatly reduced or lack of IgE activity provides advantageous properties for specific immunotherapy.
  • Recombinant hypoallergenic allergens prepared in this way can thus contribute to improved therapy of allergic diseases.
  • the invention therefore relates to a hypoallergenic major birch pollen allergen rBet v 1 obtainable by the process according to the invention, in particular in its use as medicament.
  • modifications can on the one hand be genetic modifications at the DNA level, where, for example, amino acid insertions, deletions and replacements, cleavage of the protein into fragments and fusion of the protein or fragments thereof with other proteins or peptides are suitable.
  • the modifications can also be of a chemical nature and take place at the protein level.
  • the invention thus relates to the use of hypoallergenic major birch pollen allergen rBet v 1 according to the invention and/or pharmaceutically usable derivatives thereof, including mixtures thereof in all ratios, for the preparation of a medicament for the specific immunotherapy of allergies in the initiation of which major birch pollen allergen Bet v 1 is involved.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising hypoallergenic major birch pollen allergen rBet v 1 according to the invention and/or pharmaceutically usable derivatives thereof, including mixtures thereof in all ratios.
  • the active ingredients according to the invention can be converted here into a suitable dosage form together with at least one solid, liquid and/or semi-liquid excipient or adjuvant and optionally in combination with one or more further active ingredients.
  • compositions can be used as therapeutic agents in human or veterinary medicine.
  • Suitable excipients are organic or inorganic substances which are suitable for parenteral administration and do not react with hypoallergenic major birch pollen allergen rBet v 1.
  • Suitable for parenteral administration are, in particular, solutions, preferably oil-based or aqueous solutions, furthermore suspensions, emulsions or implants.
  • Hypoallergenic major birch pollen allergen rBet v 1 according to the invention can also be lyophilised and the resultant lyophilisates used, for example, for the preparation of injection preparations.
  • compositions indicated can be sterilised and/or comprise adjuvants, such as lubricants, preservatives, stabilisers and/or wetting agents, emulsifiers, salts for modifying the osmotic pressure, buffer substances and/or a plurality of further active ingredients.
  • adjuvants such as lubricants, preservatives, stabilisers and/or wetting agents, emulsifiers, salts for modifying the osmotic pressure, buffer substances and/or a plurality of further active ingredients.
  • hypoallergenic major birch pollen allergen rBet v 1 enables depot preparations to be obtained, for example through adsorption onto aluminium hydroxide.
  • a first pre-purification step for removal of nucleic acids can consist of hydrophobic interaction chromatography carried out under physiological conditions (at a pH of 6-8, non-denaturing), where the target protein is simultaneously focused.
  • salt precipitation or ion exchange chromatography can also be carried out.
  • the next purification step serves to convert the proteins into weakly saline eluent in the concentration range 10-100 mM, for example 20 mM NaCl, for example by means of gel filtration on a Sephadex G-25 column. Conditions are thus created which facilitate the performance of ion exchange chromatography using alkaline eluents.
  • the protein solution prepared in this way is subsequently employed for anion exchange chromatography, for example using a Source Q column. Most allergens are bound to the support here.
  • the alkaline eluent causes even previously sparingly soluble or insoluble proteins to remain in solution. NaCl gradient elution results in partial removal of bacterial impurities and active-ingredient fragments.
  • the pre-purified and equilibrated allergens are essentially separated from bacterial impurities still remaining.
  • the allergens can be bound to the column in hydrophobic interaction chromatography using, for example, up to 5 M NaCl, 20 mM NaOH and 11 mM NaHCO 3 and subsequently eluted using low-salt or salt-free alkaline solution, for example 20 mM NaOH.
  • an eluent change is carried out in such a way that the purified recombinant proteins are obtained in soluble, ready-to-use form by simple neutralisation of the base present in the eluent using a corresponding acid.
  • concentrations of the eluent additives Given a suitable choice of the concentrations of the eluent additives, a physiological solution which is suitable for parenteral products is formed.
  • the purified allergens are identified via their known physical, chemical or biological properties, in particular by means of SDS-PAGE and specific monoclonal antibodies.
  • an EAST inhibition assay (EAST denotes enzyme allergo sorbent test), with which the specific IgE binding of a protein compared to a reference can be determined, and/or a T-cell proliferation assay, for example, can be carried out.
  • the solvent is tested by pH measurement and quantification of the Na + and Cl ⁇ and, if desired, CO 3 2 ⁇ concentration.
  • the yield of the allergens prepared in accordance with the invention is generally 75-95%, based on the starting protein.
  • the process thus involves minimal sample treatments, short sample standing times, preferably the use of exclusively pharmacologically compatible substances, compatibility of a single eluent with diverse separation princepies, and the avoidance of lengthy and under certain circumstances nonvalidatable methods, such as dialysis.
  • the sodium hydroxide solution preferably employed as base which is known as an effective bacteriostatic, prevents the proteins present therein from being degraded or contaminated by microorganisms. Endotoxins, which can cause problems in bacterial expressions, other foreign proteins and DNA are likewise effectively removed or degraded.
  • FIG. 1 A particularly preferred embodiment of the process is shown in the following scheme (Tab. 1): Tab 1 Overview of the preparation process according to the invention 1.
  • Eluent 1 20 mM Tris/HCl, 1 M ammonium sulfate, pH 8.0
  • Eluent 2 dist. water
  • Eluent change for the main purification gel filtration (Sephadex 25)
  • Eluent 20 mM NaCl 3.
  • an E. coli lysate containing soluble rBet v 1 allergen is prepared by standard methods (Breiteneder H., et al., EMBO J. 1989, 8: 1935-8; Hoffmann-Sommergruber et al., Protein Exp. Purif. 9 (1), 1997: 33-39).
  • hydrophobic interaction chromatography is then carried out using phenyl-Sepharose in a Tris/ammonium sulfate buffer (20 mM Tris/HCl, 1 M ammonium sulfate, pH 8.0). The elution is carried out with distilled water.
  • the residual ammonium sulfate is replaced by 20 mM NaCl by means of gel filtration through Sephadex G-25.
  • the protein solution pre-purified in this way is employed for anion exchange chromatography using Source 15Q, where the support material is equilibrated with an alkaline solution (20 mM NaOH, 11 mM NaHCO 3 and 20 mM NaCl).
  • the relatively high pH of the starting solution causes virtually all target proteins to bind to the anion exchanger.
  • the subsequent elution is carried out with increasing NaCl gradient (from 20 mM NaOH; 11 mM NaHCO 3 ; 20 mM NaCl to 20 mM NaOH; 11 mM NaHCO 3 ; 0.5 M NaCl) and causes removal of impurities (host-cell proteins) and active-ingredient fragments.
  • the next chromatography step is hydrophobic interaction chromatography by means of Source PHE.
  • the eluate from the ion exchange chromatography is adjusted to 3 M NaCl, 20 mM NaOH, 11 mM NaHCO 3 by addition of corresponding amounts of a 5 M NaCl stock solution, a 2 M NaOH stock solution and sodium hydrogencarbonate.
  • rBet v 1 binds to the column material.
  • the elution of the bound target protein is carried out using 20 mM.NaOH.
  • gel filtration is carried out through Superdex 75 under alkaline conditions.
  • the chromatography solution is selected in such a way that neutralisation of the base added to the eluent results in the desired final formulation: 10 mM NaOH, 11 mM NaHCO 3 and 148.4 mM NaCl, which corresponds to the concentrations of physiological saline solution.
  • the eluate from the gel filtration is finally neutralised using the acid HCl corresponding to the base NaOH used, resulting in a neutral pH and at the same time achieving the desired salt content of physiological saline solution. This is achieved through addition of 1/10 (v/v) 100 mM HCl.
  • the SDS-PAGE from Example 2 is blotted on nitrocellulose. After a blood serum pool from birch pollen allergy sufferers has been added to the blot, the blot is incubated with a conjugate consisting of an anti-IgE antibody and alkaline phosphatase.
  • the colour reaction promoted by the alkaline phosphatase shows an IgE activity of natural nBet v 1 and of recombinant, conventionally purified rBet v 1, but not of rBet v 1 -MF purified in accordance with the invention.
  • nBet v 1 position 3
  • recombinant conventionally purified rBet v 1
  • rBet v 1 purified in accordance with the invention position 4
  • FIG. 2A shows that, with the exception of serum 5, where rBet v 1 purified in accordance with the invention has weak IgE activity, only natural nBet v 1 and recombinant, conventionally purified rBet v 1, but not rBet v 1 purified in accordance with the invention have IgE activity.
  • the nitrocellulose membrane was incubated with various polyclonal rabbit anti-Bet v 1 antibodies (samples 21 to 26) and with monoclonal mouse anti-Bet v 1 antibody 6B6 (sample 27) and subsequently treated analogously to Example 3 ( FIG. 2B ).
  • T-cell lines TCLs
  • T-cell clones TCLs
  • SI stimulation indices

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US10/505,897 2002-02-27 2003-02-07 Method for producing hypoallergenic major birch pollen allergen rbet v 1 Abandoned US20050123568A1 (en)

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US12/167,712 US9441021B2 (en) 2002-02-27 2008-07-03 Process for the preparation of hypoallergenic major birch pollen allergen rBet v 1

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EP02004567 2002-02-27
EP02004567.0 2002-02-27
PCT/EP2003/001246 WO2003072601A1 (de) 2002-02-27 2003-02-07 VERFAHREN ZUR HERSTELLUNG VON HYPOALLERGENEM BIRKENPOLLENHAUPTALLERGEN rBet v 1

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DE (1) DE50311945D1 (es)
ES (1) ES2333951T5 (es)
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Cited By (1)

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US20090324501A1 (en) * 2006-06-09 2009-12-31 Biomay Ag Vaccine Carrier

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JP5279238B2 (ja) * 2006-11-16 2013-09-04 森川健康堂株式会社 血圧降下作用を有する花粉の製造方法
RU2658767C1 (ru) * 2010-01-14 2018-06-22 Мерк Патент Гмбх Варианты группы 5 аллергенов злаковых со сниженной аллергенностью вследствие мутагенеза остатков пролина
EP2974741A1 (en) * 2014-07-16 2016-01-20 Stallergenes Composition of allergen extracts having reduced toxicity and method of production thereof
BR102017026619A2 (pt) 2017-02-15 2018-10-30 Euroimmun Medizinische Labordiagnostika Ag ensaio melhorado para a diagnose de alergia a amendoim
RU2761431C9 (ru) 2020-10-26 2022-04-18 Федеральное государственное бюджетное учреждение "Государственный научный центр "Институт иммунологии" Федерального медико-биологического агентства России (ФГБУ "ГНЦ Институт иммунологии" ФМБА России) Рекомбинантный полипептид на основе аллергена пыльцы березы и аллергена яблока в качестве вакцины от аллергии

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US20030170815A1 (en) * 2000-09-08 2003-09-11 Roland Suck Method for purifying recombinant proteins expressed as insoluble aggregates

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030170815A1 (en) * 2000-09-08 2003-09-11 Roland Suck Method for purifying recombinant proteins expressed as insoluble aggregates

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090324501A1 (en) * 2006-06-09 2009-12-31 Biomay Ag Vaccine Carrier
US9296828B2 (en) 2006-06-09 2016-03-29 Biomay Ag Vaccine carrier

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CA2477409C (en) 2015-01-27
PL373526A1 (en) 2005-09-05
WO2003072601A8 (de) 2005-08-18
DE50311945D1 (de) 2009-11-05
WO2003072601A1 (de) 2003-09-04
RU2311424C2 (ru) 2007-11-27
EP1478662B2 (de) 2017-07-05
PL206709B1 (pl) 2010-09-30
EP1478662B1 (de) 2009-09-23
JP2005536454A (ja) 2005-12-02
CA2477409A1 (en) 2003-09-04
AU2003208817A1 (en) 2003-09-09
ATE443716T1 (de) 2009-10-15
US20090069236A1 (en) 2009-03-12
PT1478662E (pt) 2009-12-29
JP4343702B2 (ja) 2009-10-14
US9441021B2 (en) 2016-09-13
RU2004129282A (ru) 2005-06-10
EP1478662A1 (de) 2004-11-24
ES2333951T3 (es) 2010-03-03
ES2333951T5 (es) 2017-11-16

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