WO2006003815A1 - Modification de l’allergène d’acarien du groupe 1 - Google Patents

Modification de l’allergène d’acarien du groupe 1 Download PDF

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WO2006003815A1
WO2006003815A1 PCT/JP2005/011356 JP2005011356W WO2006003815A1 WO 2006003815 A1 WO2006003815 A1 WO 2006003815A1 JP 2005011356 W JP2005011356 W JP 2005011356W WO 2006003815 A1 WO2006003815 A1 WO 2006003815A1
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residue
der
allergen
rdfl
substituted
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PCT/JP2005/011356
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Toshiro Takai
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Juridical Foundation The Chemo-Sero-Therapeutic Research Institute
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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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43531—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from mites
    • 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
    • A61K39/0003—Invertebrate antigens

Definitions

  • the present invention relates to a modified tick group 1 allergen obtained by gene recombination technology. More specifically, a modified major mite allergen Der f 1 in which the cysteine residue at position 35 of the major mite allergen D er f 1 classified as a tick group 1 allergen is substituted with a serine residue, the modified tick major allergen Der f 1
  • the present invention relates to a production yeast and a production method thereof. Background art
  • mite allergen proteins in mite extracts and mite excreta have been identified and classified into multiple groups. Among them, mite allergens derived from Der f and Der p belonging to Group 1 (Der f 1 and Der p 1) and mite allergens derived from Der f and De rp belonging to Group 2 (Der f 2 and Der p 2) It is recognized as a major allergen. Most mite allergic patients are positive for skin tests against Der f 1 and Der f 2 and positive for specific IgE antibodies in serum and are known to be strongly sensitized to both allergens (eg, Non-patent document 1). In particular, it has been reported that 50% or more of IgE that recognizes mite crude antigen recognizes Der p 1, and the tick group 1 allergens Der f 1 and Der p 1 Has been shown to be deeply involved in tick allergic diseases.
  • Mite allergens belonging to Group 1 are abundant in mite excreta, have a protein with one asparagine-type glycosylation in a molecule with a molecular weight of about 25 kD, have protease activity, and are heated. It has been reported that IgE antibody binding ability is lost.
  • mite allergens belonging to Group 2 are present in mite bodies, and have a protein with a molecular weight of about 14 to 15 kD and three disulfides in the molecule. It has been clarified that it has binding and does not lose IgE binding ability by heat treatment (for example, see Non-Patent Document 1 and Non-Patent Document 2).
  • Desensitization therapy is a treatment method that eliminates allergic reactions in the body by injecting allergens in small doses to allergic patients. Its effective rate is over 70%, and is considered the only way to cure allergic diseases.
  • house dust allergen (Torii Pharmaceutical) manufactured by a method that extracts allergen from indoor dust is the only known mite allergen that is used in desensitization therapy.
  • House dust allergens produced by this method contain various antigens other than mites, such as mold bacteria, as well as difficulty in securing a sufficient amount of antigen.
  • Its biological activity (potency ) Cannot be clearly defined, (2) these antigens can trigger and cause other allergies, (3) phenols known to attack the skin are added as preservatives However, the dose is limited.
  • Tick Major allergen Der f 2 in which both cysteine residues at positions 8 and 119 are replaced with serine residues clearly shows that IgE binding ability and histamine release activity are significantly reduced.
  • Non-patent Document 1 and Patent Document 2 V and high therapeutic effects are expected in desensitization therapy.
  • Der p 1 induces IgE production in vivo in a protease-dependent manner in mice, enhances IgE production against another co-administered antigen, and shifts the T cell response from Thl to Th2,
  • Der p 1 protease activity disrupts intercellular tight junctions and directly activates airway epithelial cells to induce production of inflammatory site force-ins. It has been suggested that there is a relationship between protease activity and the development of allergies and exacerbations (for example, see Non-Patent Documents 3 and 5).
  • tick group 1 allergen protease activity has strong IgE-inducing activity and Th2-inducing activity, and this activity is thought to cause allergies and exacerbations.
  • Mite extract which has been used in desensitization therapy so far, is rich in tick group 1 allergens with protease activity. This reduces the effectiveness or instability of the treatment, and the risk of inducing IgE and Th2-type responses to other mite allergens that were not originally sensitized or to other antigens exposed during treatment. There is.
  • Patent Document 1 Japanese Patent No. 2657861
  • Patent Document 2 Japanese Patent Laid-Open No. 06-253851
  • Patent Document 3 Japanese Patent Publication No. 11-509543
  • Non-Patent Document 1 Toshiro Takai, Clinical Immunity, 2002, 38 (3): p. 298—305
  • Non-Patent Document 2 Piatt-Mills TAE et al., 1987, J. Allergy Clin. Immunol., 80: p.755-775
  • Non-Patent Document 3 Shak3 ⁇ 4 F et al., 1998, Immunol Today. 19: p.313-316
  • Non-patent literature 4 Takahashi K et al., 2001, Int. Arch Allergy Immunol. 124: p.454-460
  • Non-patent literature 5 Toshiro Takai, Asthma, 2004, 17 (1): p. 15-20
  • Der f 1 is one of the major allergens causing tick allergic diseases.
  • the modified tick major allergen Der f 1 modified so as not to have IgE-inducing ability and Th2-inducing ability can be an effective drug for treating tick allergy.
  • a greater therapeutic effect is also expected to be obtained in combination with the modified major mite allergen Der f 2 in which both cysteine residues at positions 8 and 119 of the amino acid are substituted with serine residues.
  • the present invention applies gene recombination technology to eliminate IgE-inducible ability and Th2-inducible ability by substituting the serine residue for the cysteine residue at position 35 of the major mite allergen Der f 1.
  • An object of the present invention is to provide a modified form of Der f 1. Means for solving the problem
  • the present invention provides the following modified mite group 1 allergen.
  • a modified mite dallop 1 allergen having the following characteristics (1) to (4), wherein the amino acid residue at the catalytic center is substituted.
  • Tick group 1 allergen is the major allergen Der f 1 or Der p 1 above! Any variant of Tanshi 4
  • the present invention also provides a host that produces a modified variant of the above mite group 1 allergen and a method for producing the same. Specifically, the present invention provides a method for producing a tick group 1 allergen variant, wherein protease activity is eliminated by a gene recombination technique, including the following steps (1) to (3).
  • a modified major mite allergen in which protease activity is eliminated is obtained by genetic recombination technology, and the modified product does not induce the production of IgE antibody and Th2-type cytodynamic in. Elimination of protease activity is performed by substituting the amino acid residue forming the catalytic center or the amino acid residue important for the catalytic reaction, and preferably removing the cysteine residue at position 35 of the major allergen Der f 1.
  • the modified major mite allergen of the present invention lacking protease activity can induce specific IgG without inducing specific IgE elevation. confirmed.
  • the modified major mite allergen of the present invention lacks protease activity at all, and thus it was considered that the formation of a microenvironment in vivo that contributes to IgE production was not induced. However, IgG production was induced.
  • the property of inducing blocking antibodies such as IgG other than IgE without inducing IgE is preferable for desensitization therapy.
  • FIG. 1 is a drawing showing the results of subjecting rDfl and rDfl-C35S to SDS-PAGE (15-25%) under non-reducing and reducing (2-mercaptoethanol) conditions.
  • rDfl Major tick allergen Der f 1 with broken glycosylation site, rDfl -C35S; Modification by replacing cysteine residue at position 35 of major tick allergen Der f 1 with broken glycosylation site with serine residue Body
  • FIG. 2 shows the results of measuring the CD spectrum of rDfl and rDfl-C35S with JASCO J-820 spectropolarimeter (JASCO Corporation) using a 0.1cm cell at 25 ° C and 190-260nm. It is drawing which shows.
  • rDfl Major tick allergen Der f 1 with destroyed glycosylation site, rDfl- C35S; Modification by replacing cysteine residue at position 35 of major tick allergen Der f 1 with destroyed glycosylation site with serine residue Body
  • FIG. 3 is a drawing showing the results of measuring the gel filtration elution volume for rDfl and rDfl-C35S.
  • rDfl Major tick allergen Der f 1 with disrupted glycosylation site, rDfl- C35S; Modified by replacing the cysteine residue at position 35 of major tick allergen Der f 1 with disrupted glycosylation site with a serine residue
  • Fig. 4 is a diagram showing the results of measurement of total IgE in the serum of mice immunized with the following antigens.
  • rDfl major tick allergen Der f 1, which disrupted glycosylation site, rDfl + E64; major tick allergen Der f 1, which disrupted glycosylation site after treatment with the protease inhibitor (E-64), rDfl-C35S ; A modified version in which the cysteine residue at position 35 of the major allergen Der f 1 in which the sugar chain-attached cocoon site was destroyed was replaced with a serine residue
  • FIG. 5 is a drawing showing the results of measuring antigen-specific IgE in the serum of mice immunized with the following antigens.
  • rDfl Major tick allergen Der f 1, which destroyed the glycosylation site Der f 1, rDfl + E64;
  • Major tick allergen Der f 1, rDfl- C35S Glycosylation which destroyed the glycosylation site after protease inhibitor (E-64) treatment
  • E-64 protease inhibitor
  • FIG. 6 shows that spleen cells of a mouse immunized with the following antigen were cultured in a medium alone, an rDfl-containing medium or an rDfl-C35S-containing medium, It is drawing which shows the result of having measured.
  • FIG. 7 shows that spleen cells of mice immunized with the following antigens were cultured in a medium alone, a rDfl-containing medium or a rDfl-C35S-containing medium, It is drawing which shows the result of having measured.
  • FIG. 8 is a drawing showing the results of measuring the protease activity of the major mite allelegen Der f 1 or its variant rDfl-C35S.
  • rDfl Major allergen Der f 1, which has destroyed the glycosylation site, rDfl- C35S; Cysteine residue at position 35 of major mite allergen Der f 1, which has destroyed the glycosylation site, has been replaced with a serine residue
  • FIG. 9 is a drawing showing the results of measuring total IgE (a) and antigen-specific IgE (b) in the serum of mice immunized with the following antigens.
  • rDfl Major tick allergen Der f 1 with destroyed glycosylation site, rDfl- C35S; Cysteine residue at position 35 of major tick allergen Der f 1 with destroyed sugar chain was replaced with serine residue Variant
  • FIG. 10 is a drawing showing the results of measuring antigen-specific IgG in the serum of mice immunized with the following antigens.
  • rDfl Major tick allergen Der f 1 with disrupted glycosylation site, rD H-C35S; Modified mite allergen Der f 1 with disrupted glycosylation site substituted with serine residue at position 35
  • FIG. 11 is a drawing showing the results of measurement of total IgE in the serum of mice immunized with the following antigens. rDpl; major tick allergen Der p 1 with disrupted glycosylation site, rDpl + E64; major tick allergen Der p 1 with destroyed glycosylation site after protease inhibitor (E-64) treatment
  • FIG. 12 shows the result of SDS-PAGE of the following modified allergen precursor and staining the gel with Coomassie Brilliant Blue.
  • Pro-rDfl Precursor of major tick allergen Der f 1 with disrupted glycosylation site, Pro-rDfl- C35S; Cysteine residue at position 35 of major tick allergen Der f 1 with disrupted glycosylation site
  • Pro-rDfl-C35A a modified version in which the cysteine residue at position 35 of the major mite allergen Der f 1 precursor with the glycosylation site destroyed is replaced with an alanine residue, Pro-rDfl-C35A rDpl-C34S; Major allergen of tick whose glycosylation site was destroyed Der p 1 precursor modified with cysteine residue at position 34, Pro-rDpl-C34A; Glycosylation site destroyed Major tick allergen Der p 1 precursor modified by replacing the cysteine residue at position 34 with alanine residue
  • FIG. 13 is a drawing showing the results of examining IL-8 production when normal keratinocytes and HaCaT cells were stimulated with rDfl or rDfl-C35S.
  • rDfl Major allergen Der f 1, which has destroyed the glycosylation site, rDfl- C35S; Modification by replacing the cysteine residue at position 35 of the major mite allergen Der f 1, which has destroyed the glycosylation site, with a serine residue Body
  • FIG. 14 is a drawing showing the results of comparing the production of rDfl-specific IgE, IgGl and IgG2b when rDfl, rDfl-C35S, or buffer alone was administered to mice.
  • rDfl Major tick allergen Der f 1, rDfl-C35S; Glycosylation site destroyed A modified product in which the cysteine residue at position 35 of the major allergen Der f 1 was replaced with a serine residue.
  • FIG. 15 is a drawing showing the results of immunization with rDfl and the measurement of total IgE in serum when rDfl-C35S was administered intranasally to mice and when it was not administered.
  • rDfl Major tick allergen Der f 1 with disrupted glycosylation site, rDfl- C35S; Major modification of tick major allergen Der f 1 with glycosylation site substituted with serine residue at position 35
  • the present invention is characterized by a variant of the major mite allergen Der f 1 derived from Dermatophagoides farinae (abbreviation: Der f), which has eliminated protease activity.
  • the variant does not induce the production of IgE antibodies and Th2-type cytodynamic ins.
  • a Der p 1 recombinant belonging to the same tick group 1 allergen was treated with an irreversible cysteine protease inhibitor E-64 to inactivate the protease, and obtained by immunizing mice. No IgE antibody is detected in the serum.
  • IgE antibody and Th2-type site strength can be obtained by substituting the amino acid of recombinant Der p 1 to eliminate the protease activity in the same manner as recombinant Der fl. It is easily predicted that a variant that does not induce in production will be obtained.
  • the method of the present invention can be applied to Der p 1 from Dermatophagoides pteronyssinus (abbreviated as Der p) and other duni, which are classified into the tick group 1 allergen, which consists only of the two major allergens Der f 1. It can be used against the derived mite group 1 allergen.
  • Der p Dermatophagoides pteronyssinus
  • other duni which are classified into the tick group 1 allergen, which consists only of the two major allergens Der f 1. It can be used against the derived mite group 1 allergen.
  • Elimination of protease activity is performed by substituting amino acid residues that form the catalytic center or amino acids that are important for catalytic reaction (in the present invention, it is important for the amino acid residue at the catalytic center and catalytic reaction).
  • Amino acid residues that play a role are defined as “catalytic centers”).
  • the cysteine residue at the 35th position, the histidine residue at the 171st position, the asparagine residue at the 191st position important for the catalytic reaction, and the glutamine residue at the 29th position are formed. Replaced. More preferred is a cysteine residue at position 35.
  • any amino acid residue other than the original amino acid residue such as a serine residue or alanine residue, is possible.
  • a serine residue or alanine residue is preferable.
  • Tick other than Der f 1 the same substitution operation is performed on the amino acid residue at a position homologous to the residue of Der f 1 described above. For example, since the cysteine residue at position 35 of Der fl corresponds to the cysteine at position 34 of Derpi, this cysteine residue at position 34 is replaced with a serine residue or alanine residue.
  • modified mite major allergen Der f 1 a modified form of the major mite allergen Der f 1 in which the cysteine residue at position 35 is substituted with a serine residue (hereinafter referred to as "modified mite major allergen Der f 1"). Yes).
  • the modified major mite allergen of the present invention lacking protease activity, for example, does not induce IL-8 production which does not stimulate skin keratinocytes depending on protease activity. It has been reported that various cells other than skin keratinocytes are also activated depending on the protease activity of group 1 allergens, and the production of various site force-ins and chemokines including IL-8 is induced. These cell activities are thought to contribute to the formation of a microenvironment suitable for IgE / Th2 induction during the allergen sensitization process.
  • the major mite allergen of the present invention lacking protease activity does not induce the formation of a microenvironment that contributes to such allergic exacerbations upon administration.
  • the modified major mite allergen of the present invention lacking protease activity induces specific IgG without inducing specific IgE elevation. It was confirmed that in other words, in the administration system using a patch having noria destructive skin strength, the modified major mite allergen of the present invention lacks protease activity at all, and thus it was considered that the formation of a microenvironment in vivo that contributes to IgE production was not induced. However, IgG production was induced.
  • the property of inducing blocking antibodies such as IgG other than IgE without inducing IgE is preferable for desensitization therapy.
  • the modified tick major allergen of the present invention nasally, the increase in IgE originally induced by immunization with the tick major allergen was suppressed.
  • the induction of IgE / Th2 by new tick major allergen sensitization can be suppressed.
  • Such new suppression of IgE / Th2 induction is expected to be observed even in already sensitized individuals and is preferred over desensitization therapy.
  • the modified tick major allergen Der f 1 will be described in more detail.
  • reagents such as TRIzol reagent (Invitrogen), ISOGEN (-Tubon Gene), StrataPrep Total RNA Purification Kit (Toyobo), for mRNA purification, mRNA Purification Kit (Amersham Biosciences) , Poly (A) Quick mRNA Isolation Kit (Toyobo), mRNA Separator Kit (Clontech), etc., and conversion to cDNA [Superscript plasmid system for cDNA synthesis and plasmid cloning (Inhitlogin), cDNA Synthesis Kit (Takara Shuzo), SMART PCR cDNA Synthesis & Library Construction Kits (ClonAtsu), Directional cDNA Library Construction systems (Novagen), GeneAmp PCR Gold (Applied Biosystems), etc. are used.
  • ISOGEN Nonsubon Gene
  • SEQ ID NO: 1 A primer corresponding to the 5 ′ side of the prepro Der f 1 gene (SEQ ID NO: 1) and a primer corresponding to the 3 ′ side (SEQ ID NO: 2) were reported by Yasuhara T et al. (Biosci. Biotechnol. Biochem. Vol. 65 , p.563-569, 2001).
  • Takara's te-Directed Mutagenesis System (Mutan- Super Express Km, Mutan-Express Km, Mutan-K, etc.), Stratagene's QuickChange Multi Site-Directed Mutagenesis Kit, QuickChange A commercially available kit such as XL Site-Directed Mutagenesis Kit or Invitrogen's GeneTailor Site-Directed Mutagenesis System is used according to the attached protocol.
  • QuickChange (Stratagene) is used.
  • the synthetic DNA described in SEQ ID NO: 5 and SEQ ID NO: 6 and the cysteine residue at position 35, which is the enzyme active center, are used.
  • the synthetic DNA described in SEQ ID NO: 7 and SEQ ID NO: 8 is used for the substitution of serine residue.
  • the preb-mouthed Der f 1 gene thus obtained or a modified gene in which the gene is mutated is incorporated into an appropriate expression vector, and the expression vector is introduced into a host, whereby the major tick allergen Der f 1 or The variant can be expressed in the host.
  • Bacteria, yeast, animal cells, plant cells, and insect cells commonly used for expression of foreign proteins can be used as hosts for expressing major tick allergens. It may be selected as appropriate in accordance with.
  • yeast is used as the host. Since various expression vectors for yeast expression have been developed and marketed, they can be appropriately selected from these. Examples of the method for introducing a gene into yeast include an electopore method and a spheroplast method, and any method can be used.
  • a plasmid DNA in which the prepro Der f 1 gene is cloned is used as a saddle, and PCR is performed using the synthetic DNAs described in SEQ ID NO: 3 and SEQ ID NO: 4, whereby prepro Der f 1 Amplify the gene.
  • These primers are obtained by adding a restriction enzyme cleavage site of BamHI to SEQ ID NO: 3 corresponding to the 5 ′ side of the prepro Der f1 gene and Notl to SEQ ID NO: 4 corresponding to the 3 ′ side.
  • the obtained cDNA fragment is deleted with restriction enzymes BamHI and Ncol. This is cloned into the yeast Pichia pastoris expression vector pPIC3.5 (Invitrogen) previously treated with the same restriction enzymes.
  • each of the rDfl expression vector and the rDfl-C35S expression vector is introduced into the yeast Pichia pa stris, and rDfl and rDfl-C35S producing cells are obtained by homologous recombination.
  • a medium for culturing yeast an agar medium, a YPD medium, a BMGY medium, a BMMY medium, and the like are used, and may be appropriately selected according to the culture purpose and the culture stage. In actual use, those supplemented with amino acids, vitamins, sugars, alcohol, yeast extract, antibiotics, pH adjusting buffer, etc. are used according to the protocol of each medium.
  • the pH of the culture medium is 5-8, and the culture temperature is 25 ° C-30 ° C.
  • the amount of medium, additives, and culture time are appropriately adjusted according to the culture scale.
  • rDfl and rDfl- C35S production Yeast cloning is the minimum agar medium in which glucose is added as a carbon source by picking up multiple single colonies that have been converted to histidine-free and grown on minimal agar as a result of homologous recombination.
  • the cells were transplanted to a minimum agar medium prepared with methanol as a carbon source, and clones that grew at a normal rate in the former medium but extremely decreased in the latter were screened. In this way, cells stably expressing rDfl and rDfl-C35S are established.
  • rDfl stable expression cells or rDfl-C35S stable expression cells are cultured in YPD medium at 30 ° C, and 1 ml of this culture solution is added to 400 ml of BMGY medium and further cultured at 30 ° C for 24 hours. To precipitate the cells. Add 80ml of BMMY to the sediment and incubate for 72-96 hours. At this time, 0.8 ml of methanol is added every 24 hours. The culture supernatant is collected by centrifugation, and 1/5 volume of 0.5M Tris-HCl (pH 9.0) is added to it, and then separated with 50% saturated ammonium sulfate.
  • Tris-HCl pH 9.0
  • the rDfl precursor (hereinafter also referred to as “Pro-rDfl”) or the rDfl-C35S precursor (hereinafter also referred to as “Pro-rDfl-C35S”) is recovered in the sediment. This is dissolved in 1/20 volume of 50 mM Tris-HCl (pH 9.0) or purified water of the first culture solution to make a precursor solution.
  • the Pro-rDfl and Pro-rDfl-C35S solutions described above were added to 10 to 100 volumes of 0.1M acetate buffer (pH 4.0) at room temperature for 0.5 to 12 hours (0 to 2 external dialysis solutions during this period).
  • the Pro-rDfl-C35S is matured by dialysis at 4 ° C for 1-14 days.
  • maturation can be promoted by adding protease from outside such as protease-immobilized beads. The progress of maturation can be confirmed by focusing the sample on SDS polyacrylamide gel electrophoresis (SDS-PAGE).
  • the pro-sequence is cleaved and removed, and the band corresponding to the pro-form at the position of about 32 kDa disappears under SDS-PAGE under reducing conditions. Instead, the pro-sequence is removed and cleaved to a short position at the position of about 27 kDa. A mature band appears.
  • rDfl Mature rDfl (hereinafter sometimes simply referred to as “rDfl”) or mature rDfl-C35S (hereinafter also simply referred to as “rDfl-C35S”) due to impurities such as unreacted substances and decomposition products in the processing solution
  • purification methods generally used in protein chemistry such as salting out, ultrafiltration, isoelectric precipitation, electrophoresis, ion exchange chromatography, gel filtration, etc. Methods such as chromatography, affinity chromatography, hydrophobic chromatography, and hydroxyapatite chromatography are used.
  • the above maturation treatment solution is dialyzed against 50 mM Tris-HCl buffer (pH 8.0) and then equilibrated with the same buffer (HiTrap QXL). , 5mL, Amersham Biosciences Co., Ltd.). Elution with a 0 to 400 mM sodium chloride concentration gradient. Perform SDS-PAGE for each fraction and collect the fraction containing rDfl or rDfl-C35S. The obtained fraction containing the target product is collected and concentrated, and further applied to a molecular sieve column (Superdex 75, 25 mL, Amersham Biosciences) to increase the degree of purification.
  • a molecular sieve column Superdex 75, 25 mL, Amersham Biosciences
  • a cation exchange column chromatography purification step is inserted between the anion exchange column chromatography and the molecular column to remove the remaining precursor.
  • anion The purified fraction from the exchange column was dialyzed against 50 mM acetate buffer (pH 4.0), then applied to a cation exchange column (HiTrap SPHP, 5 mL, Amersham Biosciences) equilibrated with the same buffer, and a sodium chloride concentration gradient Elution with Perform SDS-P AGE on each fraction and collect fractions containing only mature rDfl-C35S. Fractions containing the desired product are collected, concentrated, and purified using a molecular sieve column.
  • the purity of the rDfl and rDfl-C35S-containing solutions thus obtained is determined by the methods generally used for protein analysis, such as EIA and Western plots using specific antibodies, SDS-polyacrylamide gels in reduced and non-reduced states. It is measured by subjecting it to analysis such as electrophoresis (SDS-PAGE), high performance liquid chromatography (HPLC), absorbance measurement, isoelectric focusing, and densitometry.
  • the protein concentration is measured using a protein analysis kit (Protein Assay kit: Bio-Rad) based on the method of Bradford.
  • the rDfl and rDfl-C35S solutions that have passed through the final step were subjected to Coomassie brilliant blue staining of the gel after SDS-PAGE. As a result, no band other than the target product was confirmed, and the purity was below the detection limit.
  • the usefulness of the rDfl-C35S thus obtained as a desensitizer can be obtained by immunizing the rDfl-C35S to various animals such as mice, rats, guinea pigs, mice, musters, nu, monkeys, etc. This can be shown by examining the amount of IgE in the serum or the amount of spleen cells produced by cytodynamic force.
  • aluminum hydroxide for example, ImjectAlum (Pierce)
  • the administration method for example, subcutaneous, intradermal, intraperitoneal, nasal drop, etc. It can be administered as a desensitizing agent by administering it to mice induced with an asthma model or directly treating the disease.
  • CBA / J mice (Charles River Inc.) are used, and 0.5 microgram of rDfl or rDfl-C35S mixed with ImjectAlum (Pierce) is used per mouse, It is performed by administering to the abdominal cavity once a week. Blood is collected from the orbital venous plexus 4 weeks after the first immunization, and the serum is collected. Serum total IgE concentration is determined by sandwich ELI using specific antibodies. Measured by SA method. After blocking a microtiter plate coated with anti-HgE monoclonal antibody (Pharmingen) with Block Ace (Snow Brand), add diluted serum and react.
  • Der f 1-specific IgE was measured by destroying the glycosylation site and recombining with Der f 1 (FEBS Lett, vol.531, p.265-272, 2002 This is done using a plate coated with After blocking the microtiter plate with Derfl-WT, 10-fold diluted mouse serum is incubated with Protein G-immobilized beads, and after IgG is absorbed, it is added to the plate and allowed to react. After washing, react with peroxidase-labeled anti-HgE monoclonal antibody (Cerotech), wash, perform color reaction, stop reaction, measure absorbance, and compare Der f 1 specific IgE amount .
  • cytodynamic force produced by spleen cells is performed as follows. The day after the blood is collected 4 weeks after the first immunization, the mouse is sacrificed, the spleen is removed, and homogenized through a stainless steel mesh.
  • ACK buffer (NH CI 8.3g, K HCO lg, EDTA-2NA 37.2mg
  • Protease activity is measured according to the report by Takai et al. (FEBS Lett, vol.531, p.265-272, 2002).
  • rDfl-C35S and substrate butyloxycarbony ⁇ Gin-Ala-Arg-MCA; peptide laboratory
  • the reaction buffer 50m
  • Fluoroscan Ascent (Lab System) is used for fluorescence measurement.
  • rDfl-C35S which does not have highly purified protease activity obtained by the method of the present invention, has a total IgE, antigen-specificity when administered compared to rDfl with protease activity. Almost no production of IgE or cytodynamic ins is induced. The absence of IgE-inducing activity and Th2-inducing activity means that efficient Th2 to Thl shift induction is expected. Therefore, commonly used additives such as stabilizers (arginine, polysorbate 80, macrogol 4000, etc.), excipients (mannitol, sorbitol, sucrose), etc.
  • stabilizers arginine, polysorbate 80, macrogol 4000, etc.
  • excipients mannitol, sorbitol, sucrose
  • sterile filtration, dispensing, freezing Can be clinically used in the same way as the mite allergen extract or the lawn dust extract as a preparation that has been processed by drying, etc., and is administered as an injection or transmucosally (nasally, orally, sublingually) is there.
  • the modified tick major allergen Der fl was prepared according to a report by Takai et al. (FEBS Lett, vol.531, p.2 65-272, 2002).
  • the 5 ′ and 3 ′ primers were prepared by referring to the sequence described in the report of Yasuhara T et al. (Biosci. Biotechnol. Biochem. Vol. 65, p.563-569, 2001).
  • the synthetic DNA described in 2 and 2 was used. These primers have BamHI added to SEQ ID NO: 1 corresponding to the 5 ′ side of the prep mouth Der f1 gene, and a Pstl restriction enzyme cutting site added to SEQ ID NO: 2 corresponding to the 3 ′ side. Nucleotide sequence of the obtained cDNA fragment was determined by DNA sequencing (ABI Prism 377, Applied Biosystems) after cloning into pBluescript II SK + (Stratagen) or pCR2.1-TOPO (Invitrogen).
  • the plasmid DNA in which the pre-pro Der f 1 gene of the sequence was cloned was used as a saddle type, and the pre-pro Der f 1 gene was amplified using the synthetic DNAs described in SEQ ID NOs: 3 and 4. These primers have BamHI added to SEQ ID NO: 3 corresponding to the 5 ′ side of the prepro Derfl gene and a Notl restriction site cut to SEQ ID NO: 4 corresponding to the 3 ′ side.
  • the obtained cDNA fragment was cloned into the yeast Pichia pastoris expression vector pPIC3.5 (Invitrogen), and the nucleotide sequence was determined with a DNA sequencer.
  • rDfl expression vector As a saddle type, the synthetic DNA shown in SEQ ID NO: 7 and SEQ ID NO: 8 was used to substitute the serine residue for the cysteine residue at position 35 as the enzyme active center. .
  • the nucleotide sequence was determined using a DNA sequencer.
  • the obtained vector is referred to as a modified Der f 1 (hereinafter sometimes referred to as “rDfl-C35SJ” expression vector.
  • rDfl-C35SJ modified Der f 1
  • rDfl-C35SJ modified Der f 1
  • rDfl stable expression cells or rDfl-C35S stable expression cells were cultured in YPD medium at 30 ° C. After adding 1 ml of this culture solution to 400 ml of BMGY medium and culturing at 30 ° C. for 24 hours, the cells were precipitated by low speed centrifugation, and 80 ml of BMMY was added to the sediment, and further cultured for 72 to 96 hours. At this time, 0.8 ml of methanol was added every 24 hours.
  • rDfl rDfl precursor
  • Pro-rDfl-C35SJ rDfl- C 35S precursor
  • Example 1 The Pro-rDfl and Pro-rDfl-C35S solutions from (2) were added to 100 volumes of 0.1M acetate buffer (PH4.0) at room temperature for 3 hours, and at 4 ° C for 24-48 hours. Dialysis was performed to mature the precursor. Then, after dialyzing against 50 mM Tris-HCl buffer (pH 8.0), it was applied to an anion exchange column (HiTrap QXL, 5 mL, Amersham Biosciences) equilibrated with the same buffer, and 0 to 400 mM salt solution was added. Elution was performed with a sodium concentration gradient. Proteins in each fraction were detected by SDS-PAGE, and mature fractions were collected.
  • PH4.0 0.1M acetate buffer
  • the purified fraction from the anion exchange column was dialyzed against 50 mM acetate buffer (pH 4.0) and then cation exchange equilibrated with the same buffer to remove the remaining precursor.
  • the column HisTrap SPHP, 5 mL, Amersham Biosciences
  • elution was performed using a sodium chloride concentration gradient.
  • the fraction containing matured body was collected.
  • the obtained mature rDfl and mature rDfl-C35S were further applied to a molecular sieve column (Superdex 75, 25 mL, Amersham Bioscience).
  • rDf 1 mature rDfl-C35S
  • rDfl-C35S mature rDfl-C35S
  • Non-reduced and 2-mercaptoethanol-reduced rDfl and rDfl-C35S were subjected to SDS-PAGE (15-25%) and stained with Coomassie brilliant blue. The results are shown in Fig. 1.
  • the mobility of rDfl and rDfl-C35S was equivalent.
  • the mobility of the reduced sample was lower than that of non-reduced because of intramolecular disulfide bonds. This is because the structure of rDfl and rDfl-C35S retained in the pact has been loosened by reduction, and this also reflects the equivalence of rDfl with the higher-order structure of rDfl-C35S.
  • CD spectra were measured for rDfl and rDfl-C35S.
  • the CD spectrum was measured with a JA SCO J-820 spectropolarimeter (JASCO Corporation) at 25 ° C and 190-260 nm using a 0.1 cm cell.
  • a 0.1 mg / mL sample was dialyzed against 10 mM potassium phosphate buffer (pH 7.5).
  • the experimental parameters were as follows: bandwidth 0.5 nm; sensitivity 100 millidegree s; 0.5 nm / data step analysis; 50 nm / min scan rate; and 10 scans.
  • the protein concentration was measured using a protein analysis kit (Protein Assay kit: Bio-Rad) based on the Bradford method, based on the absorbance at 280 nm using BSA (Bio-Rad) as a standard.
  • the gel filtration elution volume was measured using rDfl and rDfl-C35S! Protein-Pak using purified rDfl and rDf 1- C35S (0.1 mL, 0.4 mg / mL) and 0.1 M potassium phosphate buffer (PH6.5) containing 0.01% sodium azide at 0.5 mL / min
  • the sample was subjected to gel filtration chromatography using 125 (0.78 cm ⁇ 30 cm, Waters) and the absorbance at 280 nm was monitored.
  • Example 1 Mice were immunized with rDfl and rDfl-C35S obtained in (3) as antigens.
  • ImjectAlum Pieris
  • 0.5 g of antigen per mouse was administered intraperitoneally once a week to CBA / J mice (Charles River Inc.).
  • blood was collected from the orbital venous plexus and the serum was collected.
  • Der f 1-specific IgE is described as recombinant Der f 1 without disrupting the glycosylation site (FEBS Lett, vol.531, p.265-272, 2002 as Der f 1-WT This was carried out using a plate coated with a material. A microtiter plate coated with Derfl-WT was blocked. Mouse serum diluted 10 times was incubated with Protein G-immobilized beads, and after IgG was absorbed, it was added to the plate and allowed to react. After washing, it was reacted with peroxidase-labeled anti-human IgE monoclonal antibody (Serotech). After washing, a color reaction was performed. After the reaction was stopped, the absorbance was measured, and the amount of Der f 1-specific IgE was compared. The results are shown in Fig. 5.
  • ACK buffer (NH CI 8.3g, K HCO lg, ED
  • Red blood cells were removed using TA-2NA 37.2mg in 1L aqueous solution, and RPMI1640 medium (Shida) was supplemented with 10% fetal calf serum medium, 0.05mM 2-mercaptoethanol and antibiotics. After washing, 4 ⁇ 10 5 cells were seeded per well in a 96-well plate for round bottom culture. The cells were cultured in the medium alone or in a medium supplemented with rDfl or rDfl-C35S at a concentration of 10 / zg / mL. After 3 days, the culture supernatant was collected. IL-4 and IL-5 in the culture supernatant were measured using a cyto force-in measurement kit (Genzyme). The results are shown in Figs.
  • the protease activity was measured according to a report by Takai et al. (FEBS Lett, vol.531, p.265-272, 2002).
  • RDfl or rDfl-C35S (with buthyloxycarbony ⁇ Gin-Ala-Arg-MCA (O.lmM) at 37 ° C in the reaction buffer (50 mM phosphate buffer, ImM EDTA, ImM dithiothreitol, pH 7.0)
  • the substrate degradation reaction by 100 nM was followed by measuring fluorescence over time. Fluoroscan Ascent (Lab System) was used for fluorescence measurement. The results are shown in Fig. 8.
  • Example 2- (1) The same procedure as in Example 2- (1) was performed except that the amount of antigen administered per animal increased.
  • Example 2- (2) The total IgE was measured in the same manner as in Example 2- (2). Specific IgE was measured in the same manner as in Example 2- (3) for mice receiving rDfl and mice receiving only alum, and for mice receiving rDH-C35S, rDfl-C35S was coated. The same procedure as in Example 2- (3) was performed except that a plate was used. The results are shown in Fig. 9 (a) and Fig. 9 (b).
  • the measurement of specific IgG antibody was performed by a method similar to the above-described measurement of specific IgE, except for the following points. Serum was not treated with Protein G. Serum diluted 30,000 times was used for IgGl measurement, and peroxidase-labeled anti-HgGl monoclonal antibody (Pharmingen) was used as the secondary antibody for detection. For measurement of IgG2a, serum diluted 100-fold was used, and peroxidase-labeled anti-HgG2a polyclonal antibody (Southern Biotechnology) was used as a secondary antibody for detection.
  • Fig. 10 For measurement of IgG2b, 100-fold diluted serum was used, and peroxidase-labeled anti-HgG2b polyclonal antibody (Zymet) was used as a secondary antibody for detection. The results are shown in Fig. 10.
  • rDpl Der p 1
  • SEQ ID NOs: 9 and 10 Amplification of prepro Der p 1 gene fragment described in SEQ ID NOs: 9 and 10 prepared with reference to the sequence described in Chua KY et al. (Int Arch Allergy Immunol, Vol. L01: p3 64-368, 1993) Synthetic DNA was used and the amplified fragment was inserted into the vector without any restriction enzyme treatment.
  • Synthetic DNAs described in SEQ ID NOs: 11 and 12 were used for amplification of the prepro Der p 1 gene to be inserted into the expression vector.
  • Synthetic DNAs described in SEQ ID NOs: 13 and 14 were used to replace the asparagine residue at position 52, which is an N-type sugar chain binding site, with a glutamine residue.
  • Example 2 Shame as in (1) except that the amount of antigen administered per animal is
  • Example 1- (1) the same procedure as in Example 1- (1) was performed except for the following points. That is, the synthetic activity described in SEQ ID NOs: 15 and 16 was used in place of SEQ ID NOs: 7 and 8, and the cysteine residue at the 35th position, which is the enzyme activity center, was replaced with an alanine residue.
  • an rDpl expression vector was prepared according to Example 6- (1), and then the same as in Example 1- (1) except for the following points. Replacement of the active center residue and establishment of a stable expression yeast strain were performed. Since the amino acid sequence near the cysteine residue at position 34, which is the enzyme active center, is conserved between Der f 1 and Der p 1, the same synthetic DNA can be used for mutagenesis. That is, in the case of rDpl-C34S, the synthetic DNA described in SEQ ID NOs: 7 and 8 is used to substitute the serine residue at position 34, which is the enzyme active center, and in the case of rDpl-C34A. To the alanine residue of the 34th cysteine residue, which is the enzyme active center, using the synthetic DNAs shown in SEQ ID NOS: 15 and 16 Was replaced.
  • Example 1 The culture supernatant was collected in the same manner as described in (2). Gels after SDS-PAGE were stained with Coomassie Brilliant Blue, and the concentration of each pro-form band was compared. The result is shown in FIG. Compared with Pro-rDfl and Pro-rDpl, the expression level of each modified allergen precursor was increased.
  • Normal human keratinocytes from infant foreskin were purchased from Cascade Biologies, 0.1 ng / mL epidermal growth factor, 10 ⁇ g / mL insulin, 0.5 ⁇ g / mL hydrocortisone, 50 ⁇ g / mL gentamicin, 50 ng /
  • the cells were cultured in serum-free HuMedia-KG2 keratinocyte growth medium (Kurashiki Spinning) containing mL amphotericin B and 0.4% vol / vol pus pituitary extract. Normal keratinocytes were used for the third to fifth passage experiments.
  • Naturally immortalized human keratinocyte cellular HaCat cells were cultured in Dulbecco's modified Eagle medium (Sigma) supplemented with 10% FCS and antibiotics. Keratinocytes were passaged by incubating monolayer cells with pre-warmed trypsin-EDTA solution for 5-10 minutes at room temperature. A trypsin neutralization solution (Cambrex) was added to the cell suspension, and the cells were pelleted by centrifuging at lOOOrpm for 5 minutes. This was then resuspended in the medium and counted by dead cell exclusion by trypan blue staining to determine cell number and survival, and 0.5 mL volume on a 24 well plate (Corning) with 5 x 10 4 cells per well. Plated. Then 5% CO / 95% empty
  • Confluent keratinocyte cultures were washed and incubated in basal medium for 12 hours before stimulation, then washed and incubated with 100 nM rDfl or rDfl-C35S or buffer. All additives were prepared in basal medium pre-warmed to 37 ° C. After pre-incubation with 5 mM L-cysteine at neutral pH, 37 ° C for 10 minutes, rDfl and RDfl-C35S was diluted with basal medium. Cells were incubated for 48 hours and supernatants were collected to determine IL-8 production and subjected to an ELISA kit (DuoSet; R & D Systems).
  • FIG. 13 IL-8 production by normal keratinocytes and HaCaT cells was the same as that with medium alone when rDfl-C35S was added, with the force increased by rDfl supplementation (FIG. 13). From this, it was confirmed that rDfl stimulates skin keratinocytes depending on protease activity and induces IL-8 production, but rDfl-C 35S lacking protease activity has no skin keratinocyte stimulating activity. .
  • Various cells other than cutaneous keratinocytes are also activated depending on the protease activity of group 1 allergen, and it is reported that production of various cytodynamic ins and chemokines including IL-8 is induced. Being sung. These cell activities are considered to contribute to the formation of a microenvironment suitable for IgE / Th2 induction during the allergen sensitization process. LDfl-C35S does not have any protease activity. It is thought that it will not induce the formation of a microenvironment that contributes to drought!
  • rDfl-specific IgE in the rDfl-C35S administration group was equivalent to that in the buffer only administration group, and there was no increase as seen in the rDfl administration group, while specific IgGl and IgG2b Increased in the rDfl-C35S administration group as in the rDfl administration group (Fig. 14).
  • Specific IgG2a did not increase in both rDfl-C35S and rDfl groups.
  • rDfl-C35S was used in the administration system using patches from Noria-destructed skin. was confirmed to be able to induce specific IgG without inducing an increase in specific IgE.
  • rDfl- C 35S lacks protease activity in the administration system with Noria-destroying skin force patch, and it is thought that the formation of in vivo microenvironment that contributes to IgE production was not induced. Production was induced.
  • the property of inducing blocking antibodies such as IgG other than IgE without inducing IgE is preferable in desensitization therapy. Even with other administration methods, it is thought that similar properties can be achieved by optimizing the conditions.
  • mice were given 1 g of rDfl-C35S per mouse nasally once daily for 3 consecutive days. After 2 weeks of breeding, immunization with rDfl and measurement of total serum IgE were performed by the method of Example 2. Comparison was made with the group immunized with rDfl after nasal administration of only nofa.
  • the variant of the major tick allergen Der f 1 of the present invention has lost IgE-inducing ability and Th2-inducing ability, and is more efficient when used for the prevention of tick allergic diseases and desensitization therapy. It can be a mite allergen preparation that exhibits a therapeutic effect. In addition, because it is manufactured using genetic recombination technology, a large amount of products of a certain quality can be supplied semipermanently. Furthermore, since its constituent components are also amino acids present in the living body, it is considered that there are no adverse health effects due to unknown causes. Therefore, the conventional house dust allergens produced by the method of extracting allergens from indoor dust (1) It is possible to obtain a preparation of constant quality. Difficult, (2) Various antigens may coexist and other allergies may be induced by these antigens, (3) It is difficult to secure a sufficient amount of antigens, (4) No effect is obtained or Problems such as low may be solved.
  • the highly purified rDfl-C35S of the present invention can be used as a material for constructing a detection system for mite allergens and antibodies, and loses protease activity, IgE antibody and Th2-type cyto force-in production ability. Therefore, it can be effectively used as a research material for elucidating the mechanism of the occurrence of mite allergy symptoms.

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Abstract

Cette invention a pour objet de procéder à une modification de l’allergène d’acarien du groupe I au sein duquel un acide aminé a été remplacé au centre catalytique. Cette modification de l’allergène d’acarien du groupe I, présentant les caractéristiques ci-dessous (1) à (4), au sein duquel un résidu d’acide aminé a été remplacé au centre catalytique à l’aide d’une technique de recombinaison génétique, d’une levure produisant ce changement et d’un procédé de production dudit changement : (1) aucune activité protéasique ; (2) pas d’induction de la production d’anticorps immunoglobulines E ; (3) pas d’induction de la production de cytokine de type Th2 ; et (4) induction de la production d’un anticorps spécifique de l’antigène immunoglobuline G.
PCT/JP2005/011356 2004-06-30 2005-06-21 Modification de l’allergène d’acarien du groupe 1 WO2006003815A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
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JP2016193929A (ja) * 2010-06-03 2016-11-17 アルク−アベッロ エイ／エスＡｌｋ−Ａｂｅｌｌｏ Ａ／Ｓ ダニアレルゲン抽出物を含んでなる医薬品及びその製造方法
CN117362453A (zh) * 2023-12-08 2024-01-09 山东硕景生物科技有限公司 一种Derf1重组抗原、其制备方法及应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TAKAHASHI K. ET AL.: "Effects of site-directed mutagenesis in the cysteine residues and the N-glycosylation motif recombinant Der f 1 on secretion and protease activity.", INTERNATIONAL ARCHIVES OF ALLERGY AND IMMUNOLOGY, vol. 124, no. 4, 2001, pages 454 - 460, XP008035576 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016193929A (ja) * 2010-06-03 2016-11-17 アルク−アベッロ エイ／エスＡｌｋ−Ａｂｅｌｌｏ Ａ／Ｓ ダニアレルゲン抽出物を含んでなる医薬品及びその製造方法
CN117362453A (zh) * 2023-12-08 2024-01-09 山东硕景生物科技有限公司 一种Derf1重组抗原、其制备方法及应用
CN117362453B (zh) * 2023-12-08 2024-02-27 山东硕景生物科技有限公司 一种Derf1重组抗原、其制备方法及应用

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