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Treatment and prevention of house dust mite allergies

A technology for dust mites and allergens, applied in the directions of allergen antigen components, antibody medical components, chemical instruments and methods, etc., can solve the problems of HDM allergy patients who cannot be treated and cannot be induced.

Pending Publication Date: 2021-02-02
WORG PHARM (HANDZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this context, it was found that current HDM vaccines based on allergen extracts do not induce sufficient protective IgG antibodies against Der p 5, Der p 7, Der p 21 and Der p 23 and render many HDM allergens Responsive patients were not treated (Selection of house dust mite-allergic patients by molecular diagnosis may enhance success of specific immunotherapy. Chen KW, Zieglmayer P, Zieglmayer R, Lemell P, Horak F, Bunu CP, Valenta R, Vrtala S. J Allergy Clin Immunol , March 2019;143(3):1248-1252.e12.doi:10.1016 / j.jaci.2018.10.048

Method used

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  • Treatment and prevention of house dust mite allergies
  • Treatment and prevention of house dust mite allergies
  • Treatment and prevention of house dust mite allergies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0150] Example 1: Design of allergen fragments for fusion proteins of the invention

[0151] Peptides spanning the target allergen sequence were determined based on amino acid surface exposure predictions determined from the ProtScale bioinformatics tool of the ExPASY server (http: / / web.expasy.org / protscale / ). If the peptides do not contain a cysteine ​​residue in their sequence, a cysteine ​​is added to their N- or C-termini to allow them to be conjugated to keyhole limpet hemocyanin (KLH) . Peptides were synthesized using an Applied Biosystems Model 433A peptide synthesizer (Foster City, USA) and then purified by preparative high-performance liquid chromatography (HPLC) (Dionex, Thermofischer Scientific, USA) (Focke et al., FASEB J.2001; 15(11): 2042-4). The size and identity of the peptides were confirmed by mass spectrometry (Bruker, Austria).

[0152] Table I lists allergen fragments derived from the house dust mite allergen Der p 1

[0153]

[0154]

[0155] ...

Embodiment 2

[0168] Embodiment 2: recombinant production of protein

[0169] Genes encoding the fusion proteins Der p 1-2C3 and Der p 5 7 21 23_P6 (large) were synthesized (codon-optimized for E. coli expression) (ATG: Biosynthesis, Merzhausen, Germany and GenScript, Piscataway, USA), and It was inserted into the NdeI / XhoI site of pET-27b (Novagen, Germany). Recombinant proteins were expressed in E. coli strain BL21-Gold (DE3). Der p 1-2C3 with an OD600 of 0.4 was induced by adding 0.5 mM IPTG to the bacterial culture and incubating at 37°C for 3 hours (see figure 1 ; SEQ ID No.27) expression. Der p 5 7 21 23_P6 (large) with an OD600 of 0.6 was induced by adding 1 mM IPTG and incubating the culture at 37 °C for 2.5 h (see figure 1 ; SEQ ID No. 28).

[0170] After harvest and addition of protease inhibitors, an inclusion body preparation is performed to remove soluble bacterial proteins. The pellet containing partially purified Der p 1-2C3 protein was dissolved in 6M urea, 10 mM Tris...

Embodiment 3

[0173] Example 3: IgE reactivity assay

[0174]The IgE reactivity of the construct of Example 2 was determined by immunoblotting (Curin M, et al., Sci Rep. 22(2017):12135). Contains 0.5 μg of purified nDer p 1 (natural isolate; accession number: PDB: 3RVW_A; reference method: Hales, B.J. et al., Clin Exp Allergy. 30(2000): 934–943), rDer p 2 (recombinantly produced Der p 2; sequence published in Chen KW et al., Allergy.67(2012): 609-21; reference method: Chen, K, et al., MolImmunol.45(2008): 2486–2498.), rDer p 5( GeneBank: X17699; Reference method: Weghofer M et al., Int Arch Allergy Immunol.147(2008):101-9.), rDer p 7 (GeneBank: U37044; Reference method: Resch Y et al., Clin Exp Allergy.41( 2011): 1468-77), rDer p 21 (GeneBank: DQ354124; reference method: Weghofer M et al., Allergy.63(2008): 758-67), rDer p 23 (GeneBank: EU414751.1; reference method: Weghofer Aliquots of M et al., J Immunol.190 (2013): 3059-67), Der p 1-2C3 (see Example 2), Der p 5 7 21 23_P6 (large) (se...

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Abstract

The present invention relates to a fusion protein having formula (I): X1-Y-X2, wherein X1 and X2 comprise each four to six allergen fragments or variants thereof fused to each other, wherein said allergen fragments are derived from at least two allergens of the genus Dermatophagoides, and wherein Y is a carrier protein.

Description

technical field [0001] The present invention relates to the field of immunotherapy of patients suffering from allergies, in particular house dust mite allergies. Background technique [0002] In industrialized countries, more than 25% of the population suffers from IgE-mediated allergies. Allergic patients are characterized by increased production of IgE antibodies to antigens that are not themselves harmful (ie, allergens). Immediate symptoms of type I allergy (allergic rhinoconjunctivitis, asthma, dermatitis, anaphylactic shock) are induced by allergens through the crosslinking and bioactive mediators of mast cell-bound IgE antibodies (eg, histamine , leukotrienes) release. [0003] WO 2012 / 168487 describes the use of surface polypeptides of viruses of the Hepadnaviridae family (eg hepatitis B virus) as carrier proteins for allergen fragments. [0004] Proteins comprising two or four identical Der p 23 allergen fragments fused to the N- and C-terminus of PreS are disclo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435A61K39/35A61K39/00A61P37/08
CPCC07K14/43531C07K2319/00A61K39/35A61K2039/55566A61K2039/6075A61K2039/6081A61P37/08C12N2730/10134C07K14/005C12N15/62A61K39/385C12N2730/10122
Inventor R·瓦伦察M·邱林K-W·陈S·威塔拉
Owner WORG PHARM (HANDZHOU) CO LTD
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