EP1620461A2 - Variants de polypeptides d'acariens du groupe 1 - Google Patents

Variants de polypeptides d'acariens du groupe 1

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Publication number
EP1620461A2
EP1620461A2 EP04729048A EP04729048A EP1620461A2 EP 1620461 A2 EP1620461 A2 EP 1620461A2 EP 04729048 A EP04729048 A EP 04729048A EP 04729048 A EP04729048 A EP 04729048A EP 1620461 A2 EP1620461 A2 EP 1620461A2
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EP
European Patent Office
Prior art keywords
group
substituted
variant
residue selected
polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP04729048A
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German (de)
English (en)
Inventor
Nanna Kristensen Soni
Esben Peter Friis
Erwin Ludo Roggen
Stina Thulesen Lyngstrand
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Novozymes AS
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Novozymes AS
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Publication of EP1620461A2 publication Critical patent/EP1620461A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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 present invention relates to variants of the group 1 mite polypeptide antigens allergens having an altered antigenic profile, compared to the parent group 1 polypeptide allergens, processes for making such variants, compositions comprising the variants and use of the variants in immuno-therapy such as allergy vaccination and/or desensitisation.
  • Antigenic polypeptides heterologeous to humans and animals such as the group 1 mite polypeptide allergens, present e.g., in excrements of dust mites Dermatophagoides pteronyssinus (Der p 1) or Dermatophagoides farinae (Der f 1), can induce immunological responses in susceptible individuals, such as an atopic allergic response, in humans and animals. Allergic responses may range from hay fever, rhinoconjunctivitis, rhinitis, and asthma, and in cases when the sensitised individual is exposed, e.g., to bee sting or insect bites, even to systemic anaphylaxis and death.
  • allergens an individual may become sensitised to such polypeptides, termed allergens, by inhalation, direct contact with skin or eyes, ingestion or injection.
  • the general mechanism behind an allergic response is divided into a sensitisation phase and a symptomatic phase.
  • the sensiti- sation phase involves a first exposure of an individual to an allergen. This event activates specific T- and B-lymphocytes, and leads to the production of allergen specific antibodies, such as immunoglobulin E (IgE).
  • IgE immunoglobulin E
  • the specific IgE antibodies bind to IgE receptors on mast cells and basophils, among others, and the symptomatic phase is initiated upon a second exposure to the same or a homologous allergen.
  • the allergen will bind to the cell-bound IgE, and the poly- clonal nature of the antibodies results in bridging and clustering of the IgE receptors, and subsequently in the activation of mast cells and basophils. This activation results in the release of various chemical mediators, such as histamine, heparin, proteases, prostaglandin D2 and leu- kotrienes, involved in the early as well as late phase reactions of the symptomatic phase of allergy.
  • various chemical mediators such as histamine, heparin, proteases, prostaglandin D2 and leu- kotrienes
  • SAV specific allergy vaccination
  • IT immuno therapy
  • SAV specific allergy vaccination
  • IT immuno therapy
  • allergy vaccination is complicated by the presence of an existing and ongoing immune response in the allergic patients.
  • effector cells such as mast cells and basophils
  • the inherent risk of adverse events or side effects limits the antigen dose, which can be administered, and has necessitated prolonged (12-36 months) and cumbersome treatment regimes where the delivered dose slowly is increased over time.
  • modified allergens with a lower inherent risk of inducing adverse events, which can be used for specific allergy vaccination.
  • modified aller- gens should have a reduced capacity for binding, and especially cross-linking, antigen-specific IgE molecules.
  • An epitope is the structural area on a complex antigen that can combine with an antibody, while the minimal epitope contains the amino acids involved directly in antibody binding.
  • B-cell epitopes can in nature be continuous, discontinuous or a combination thereof, but must contain around 10 amino acids in order to elicit an antibody response.
  • WO 99/47680 discloses that allergens may be modified to render these polypeptides less allergenic. This disclosure concerns mainly modification of the birch pollen protein, Bet v 1 and Venom allergen Ves V 5.
  • WO 02/40676 discloses modified allergens, said modifications alleg- edly causing the allergenicity of the allergen to be reduced.
  • amino acids suitable for modification are selected by virtue of their solvent accessibility, i.e. if they are present on the surface of the allergen or they are selected if they are conserved vis a vis homologeous allergens of the same taxonomic group.
  • WO 01/29078 HESKA d escribes recombinant expression of group 1 mite p roteins, nucleotide sequences encoding these proteins and nucleotide sequences modified to enable expression of the proteins in certain microorganisms.
  • the group 1 mite polypeptides of this disclosure are said to bind to IgE which also bind to native group 1 mite polypeptides.
  • EP-A-1 219 300 describes a method for administering an allergy vaccine.
  • the inadequacy of the epitope identification in for example Der p 1 may be the reason why very different potential epitopes on for example Der p 1 have been reported in different documents.
  • residues E13, P24, R20, Y50, S67, R78, R99, Q109, R128, R156, R161, P167 and W192 are selected as being important for the allergenicity of Der p 1, while in Pierson-Mullany et al. the epitopes are contemplated to be T1 to T21, E59 to Y93, Y155 to W187 and I209 to 1221, in Furmonaviciene et al.
  • the major epitope is deter- mined to be L147 to Q160, and in Jeannin et al, (where an almost identical approach as in WO 02/40676 is utilised), N52-C71, C117-Q133, G176-I187 and V188-Y199 are identified.
  • WO 99/25823 discloses variants of Der p 1 in which a) C34 is mutated, b) the pro-peptide site is modified, e.g. by deletion of NAET sequence or c) H170 is mutated.
  • WO 03/016340 discloses variants of Der p 1 in which either of six cysteinses (C4, C31, C65, C71, C103, or C117) are mutated.
  • the ambiguity of the art concerning epitopes means that presently no conclusive and reliable data is available on epitopes of Group 1 mite polypeptides and even less on amino acids comprised in said epitopes suitable for mutation with the purpose of reducing the antigenicity of these polypeptides.
  • modified group 1 mite polypeptides with improved properties as a vaccine agent, it is an advantage to first identify the minimal B cell epitopes on the molecule.
  • An epitope is the smallest structural area on a complex antigen that can bind an antibody.
  • B- cell epitopes can be continuous or discontinuous in nature.
  • the minimal epitope consists of the specific amino acids directly involved in antibody binding.
  • the present invention relates to variants of group 1 mite polypeptide antigens, including Der p 1 , comprising a mutation in a minimal epitope and thus having an altered immmunogenic profile in exposed animals, including humans.
  • the present in- vention provide in a first aspect a variant of a group 1 mite polypeptide, wherein the mature polypeptide of the variant comprises one or more mutations in the positions or corresponding to the positions consisting of A10, A12, E13, G29, G30, G32, A46, Y47, S54, L55, D64, A66, S67, G73, T75, I80, Q84, N86, G87, S92, Y93, Y96, A98-Q101 , R104-P106, Q109-1113, A132, I144-D146, D148, R151 , I158-Q166, N179, A180, G182-D184, A205, I208 of SEQ ID NO: 1 or alternatively 10, 12, 13, 29,
  • said variants have reduced IgE-binding, more particularly combined with preserved immunogenicity for inducing protective responses (vide supra). Still more preferably, the variants have an altered immmunogenic profile in exposed animals, including humans, as compared to the native group 1 mite polypeptide.
  • the invention provides a nucleotide sequence encoding the variant of the invention; a nucleotide construct comprising the nucleotide sequence encoding the variant, operably linked to one or more control sequences that direct the production of the variant in a host cell; a recombinant expression vector comprising the nucleotide construct of the invention and to a recombinant host cell comprising the nucleotide construct of the invention.
  • the invention provides a method of preparing a variant of the invention comprising:
  • the invention provides a composition comprising a variant of the invention and a pharmaceutically acceptable carrier and a method for preparing such a pharmaceutical composition comprising admixing the variant of the invention with an acceptable pharmaceutical carrier.
  • the invention provides a variant or a composition of the invention for use as a medicament.
  • the invention provides use of a variant or the composition of the invention for the preparation of a medicament for the treatment of an immunological disorder. In still further aspect the invention provides use of the variant or the composition of the invention for the treatment of a disease.
  • the invention provides use of the variant or the composition of the invention for the treatment of an immunological disorder.
  • the invention provides a kit comprising the variant of the inven- tion immobilized on a solid support.
  • Figure 1 shows histamine release in one representative donor, donor 1 , in response to stimulation with group 1 mite polypeptide (nDerpl) and the group 1 mite polypeptide variants, rec- proDer p 1 , rec-Der p 1 and DP070.
  • group 1 mite polypeptide nDerpl
  • group 1 mite polypeptide variants rec- proDer p 1 , rec-Der p 1 and DP070.
  • Figure 2 shows normalized histamine release
  • EC 50 was calculated for the group 1 mite polypeptide variants.
  • the dose response curves in nDer p 1-specific IgE serum isolated from 14 patients with dust-mite allergy were plotted and fitted to a sigmoid curve, and the EC 50 was calculated for group 1 mite polypeptide variants and normalized to group 1 mite polypeptide (nDer p l).
  • native polypeptide as used herein is to be understood as a polypeptide essentially in its naturally occurring form.
  • a native polypeptide may be for example be a wild type polypeptide, i.e. a polypeptide isolated from the natural source, a polypeptide in its natu- rally occurring form obtained via genetic engineering by expression in host organism different from the natural source or by polypeptide synthesis.
  • an “epitope” or a “B-cell epitope”, as used in this context, is an antigenic determinant and the structural area on a complex antigen that can combine with or bind an antibody. It can be discontinuous in nature, but will in general have a size of 1 kD or less (about 10 amino ac- ids or less). The size may be 3 to 10 amino acids or 5 to 10 amino acids or even 7 to 10 amino acids, depending on the epitope and the polypeptide.
  • epitope pattern as used herein is to be understood as a consensus sequence of antibody binding peptides.
  • An example is the epitope pattern A R R * R.
  • the sign "*" in this notation indicates that the aligned antibody binding peptides included a non-consensus moiety between the second and the third arginine. That moiety may be any amino acid or a few amino acids or no amino acid.
  • Epitope patterns are used to identify epitopes and minimal epitopes on complex antigens.
  • Anchor amino acid as used herein is to be understood as conserved individual amino acids of an epitope pattern recurring in all peptides bound by monospecific anti- bodies used to define that pattern. Anchor amino acid will usually also be the amino acid of a minimal epitope on the full polypeptide.
  • the "antigenicity" of a polypeptide indicates, in this context, its ability to bind antibodies e.g., of IgE and/or IgG and/or other immunoglobulin classes.
  • the 'IgE-antigenicity' of a polypeptide as used herein, indicates its ability to bind IgE antibodies.
  • the "immunogenicity" of a polypeptide indicates its ability to stimulate antibody production and immunological reactions in exposed animals, including humans.
  • the “allergenicity” of a polypeptide indicates its ability to stimulate IgE antibody production and allergic sensitization in exposed animals, including humans.
  • the term "parent” or “parent group 1 mite polypeptide” is to be understood as a group 1 mite polypeptide (also refered to as group 1 mite allergen) before introducing the mutations according to the invention.
  • the parent group 1 mite polypeptide is the native group 1 mite polypeptide.
  • Group 1 mite polypeptides As described in the art such as WO 01/29078, mites produce several classes or groups of allergens, one of which is known as Group 1 allergens. Group 1 allergens, displaying considerable cross-reactivity, have been found in Dermatophagoides pteronyssinus, Dermatophagoides farinae, Dermatophagoides siboney, Dermatophagoides microceaus, Blomia tropi- calis and Euroglyphus maynei, see for example, Thomas et al, 1998, Allergy 53, 821-832.
  • Group 1 mite allergens share significant homology with a family of cysteine proteases including actinidin, papain, cathepsin H and cathepsin B. which is why they often are referred to as Group 1 mite cysteine proteases.
  • the Group 1 mite allergens are commonly found in the feces of mites and are thought to function as digestive enzymes in the mite intestine.
  • Group 1 allergens from different mites are highly homologous, approximately 25 kilo- dalton (kD) secretory glycoproteins, that are synthesized by the cell as a pre-pro-protein that is processed to a mature form.
  • D. farinae, D. pteronyssinus, and E. maynei Group 1 proteins share about 80% identity.
  • Group 1 allergens from D. farinae and D. pteronyssinus also referred to as Der f 1 and Der p 1 proteins, respectively, show extensive cross-reactivity in binding IgE and IgG.
  • IgE In patients that are mite allergic, approximately 80% to 90% of the individuals have IgE that is reactive to Group 1 allergens (Thomas, Adv. Exp. Med. Biol., 409, pp. 85-93, 1996).
  • Group 1 mite allergens thus include native polypeptides known in the art as Der p 1 obtainable from Dermatophagoides pteronyssinus (NCBI accession number: P08176, SEQ ID NO:1), Der f 1 obtainable from Dermatophagoides farinae (NCBI accession number: P16311, SEQ ID NO:2), Eur m 1 obtainable from Euroglyphus maynei (NCBI accession number: P25780, SEQ ID NO: 3), Der m 1 obtainable from Dermatophagoides microceaus (NCBI ac- cession number: P16312, SEQ ID NO: 4), and Bio 1 1 obtainable from Blomia tropicalis (NCBI accession number: Q95PJ4, SEQ ID NO: 5).
  • Der p 1 obtainable from Dermatophagoides pteronyssinus
  • Der f 1 obtainable from Dermatophagoides farinae
  • Eur m 1 obtainable from Euroglyphus maynei NCBI acces
  • group 1 mite allergens includes in particular native group 1 mite allergens, but also includes ho- mologs to the native group 1 allergens, such as recombinant variants with disrupted N- glycosylation motifs, and hybrids of the above mentioned mite allergens, e.g. as created by family shuffling as described in the art (J.E. Ness, et al, Nature Biotechnology, vol. 17, pp. 893- 896, 1999).
  • Group 1 mite polypeptides may be epitope mapped using the proprietary in silico epi- tope mapping tool disclosed in detail in WO 00/26230 and WO 01/83559. jn brief, this tool comprises a database of epitope patterns (determined from an input of peptide sequences, known to bind specifically to anti-protein antibodies) and an algorithm to analyse 3-D structure of a given protein against the epitope pattern database. This will determine the possible epitopes on that protein, and the preference of each amino acid in the protein sequence to be part of epitopes.
  • Antibody-binding peptides can be identified by many different ways. One is to synthesize a number of peptides of known sequence, and test for their ability to bind antibodies of interest, e.g., in ELISA or other immunochemical assays. Such data are available in great abundance in the literature.
  • a particularly effective way is to prepare a library of many different random peptide sequences and select experimentally only the ones that bind antibodies well and specific (i.e., can be outcompeted by the protein towards which the antibodies were raised).
  • Phage display techniques are well suited for this way of finding antibody bidning peptides:
  • a sequence encoding a desired amino acid sequence is incorporated into a phage gene coding for a protein displayed on the surface of the phage.
  • the phage will make and display the hybrid protein on its surface, where it can interact with specific target agents.
  • an average phage display library can express 10 8 - 10 12 different random sequences. If the displayed sequence resembles an epitope, the phage can be selected by an epitope-specific antibody. Thus, it is possible to select specific phages from the bulk of a large number of phages, each expressing their one hybrid protein.
  • the amino acid sequence of the (oligo)peptides presented by the phage display system may have from 5 to 25 amino acids, preferably at least 8 amino acids, such as 9 amino acids.
  • the antibodies used for reacting with the oligopeptides can be polyclonal or monoclonal.
  • they may be IgE antibodies to ensure that the epitopes identified are IgE epitopes, i.e., epitopes inducing and binding IgE.
  • the antibodies may also be monospecific, meaning they are isolated according to their specificity for a certain protein.
  • Polyclonal antibodies are preferred for building up data on antibody-binding peptides to be used in the in silico mapping tool in order to obtain a broader knowledge about the epitopes of a polypeptide.
  • the reactive (oligo)peptides identified e.g. by phage display are compared and aligned in order to identify common epitope patterns, which then can be used for identification of antibody binding epitopes on a 3-dimensional polypeptide.
  • conservative alternatives to an amino acid such as aspartate and glu- tamate, lysine and arginine, serine and threonine are considered as one or equal.
  • the alignment results in a number of patterns, which depend on the chosen number of residues of the peptides.
  • the pattern may have the form:
  • the epitope patterns may be determined directly from the reactive peptides; if for example a library of 7-mer reactive peptides is made, one can use each different reactive 7 mer peptide, taking conservative alternatives into account, as an epitope pattern in the epitope mapping approach as described below.
  • anchor amino acids are conservative, called anchor amino acids.
  • the anchor amino acids recur in all or a majority of the reactive peptides.
  • any polypeptide for which a three-dimensional structure is known may be analysed for epitopes matching the epitope patterns. Finding an epitope on a polypeptide is achieved by searching the surface of the polypep- tide in the following way:
  • step 3 For all amino acids within a selected distance (e.g., 10 Angstroms) of the amino acids selected in step 2 it is examined if (a) the amino acid type matches the third amino acid of the pattern and (b) the surface accessibility greater than or equal to a chosen threshold allowing the amino acid to be immunological interactive. Those amino acid satisfying 3(a) and 3(b) are selected. This procedure (step 3) is repeated for all amino acids in the epitope pattern consensus sequence. The coordinates of its C-alpha atom define the spatial positioning of an amino acid. The surface solvent accessibility threshold is given in percent of an average for the particular residue type (see example 2). If matching amino acids for all amino acids in the epitope pattern can be found in the structure of the polypeptide it is a very strong indication that an epitope has been found.
  • a selected distance e.g. 10 Angstroms
  • the size of the epitope is satisfactory, i.e., the distance between any two residues is below a given threshold, usually 25 A.
  • the epitopes found may be ranked a nd weighted according to their total accessible surface area, in order to improve further the predictability of the tool.
  • the epitope mapping tool can be adjusted, such that only a subset of the known reactive peptides are included as data set for building epitope patterns, and thus for conducting epitope mapping. For instance, one may choose only to include peptides reactive to IgE anti- bodies (rather than to IgG or other antibodies), or one may include only peptides reactive to human antibodies etc. One may choose to involve only peptides reactive against the target protein in order to get a more specific response; however, in general, peptides reactive to antibodies that in turn were raised against any protein are included.
  • the in silico epitope mapping tool can be used to predict if mutating one amino acid residue will result in that the new variant overall will have fewer epitopes.
  • some or all 19 possible substitutions can be tested in a given position, the epitope mapping procedure repeated for a model structure of each of these proposed variants, and the best variant(s) can be constructed by mutation and tested experimentally.
  • variants of the polypeptide with modified antigenic properties can be made by mutating one or more of the amino acid residues comprised in the epitope.
  • mutation encompasses deletion and/or substitution of an amino acid residue and/or insertion of one or more amino acids before or after- that residue.
  • polypeptide variant suitable as a vaccine agent for treatment of allergies
  • the variant is capable of stimulating T-cells sufficiently, preferable at the level of the parent polypeptide or better. Still further it is desirable that the variant is capable of invoking an IgG response in human and animals.
  • the epitope identified may be mutated by substituting at least one amino acid of the epitope.
  • at least one anchor amino acid is mutated.
  • the mutation will often be a substitution with an amino acid of different size, hydrophilicity, polarity and/or acidity, such as a small amino acid in exchange of a large amino acid, a hydrophilic amino acid in exchange of a hydrophobic amino acid, a polar amino acid in exchange of a non-polar amino acid and a basic in exchange of an acidic amino acid.
  • Other mutations may be the insertion or deletion of at least one amino acid of the epitope, particularly deleting an anchor amino acid.
  • an epitope may be mutated by substituting some amino acids, and deleting and/or inserting others.
  • the mutation(s) performed may be performed by standard techniques well known to a person skilled in the art, such as site-directed mutagenesis (see, e.g., Sambrook et al. (1989), Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, NY).
  • the mutagenesis may be spiked mutagenesis which is a form of site-directed mutagenesis, in which the primers used have been synthesized using mixtures of oligonucleo- tides at one or more positions.
  • nucleotide substitution can be found in e.g., Ford et al., 1991 , Protein Expression and Purification 2, pp. 95-107.
  • the polypeptide variant of the invention concerns variants of parent group 1 mite polypeptides comprising one or more mutations in the parent polypeptide in the positions or corresponding to the positions consisting of A10, A12, E13, G29, G30, G32, A46, Y47, S54, L55, D64, A66, S67, G73, T75, I80, Q84, N86, G87, S92, Y93, Y96, A98, R99, E100, Q101 , R104, R105, P106, Q109, R110, F111 , G112, 1113, A132, 1144, K145, D146, D148, R151 , 1158, 1159, Q160, R161 , D162, N163, G164, Y165, Q166, N179, A180, G182, V183, D184, A205, I208 of SEQ ID NO: 1 or 10, 12, 13, 29, 30, 32, 46, 47, 54, 55, 64, 66, 67,
  • the variant polypeptide of the invention comprises one or more mutations in the parent polypeptide in the positions or corresponding to the positions consisting of A10, A12, G29, G30, G32, A46, Y47, S54, L55, D64, A66, G73, T75, I80, Q84, N86, G87, S92, Y93, Y96, A98, E100, Q101 , R104, R105, P106, R110, F111 , G112, 1113, A132, 1144, K145, D146, 1158, 1159, Q160, D162, N163, G164, Y165, Q166, N179, A180, G182, V183, D184, A205, I208 of SEQ ID NO: 1 or 10, 12, 29, 30, 32, 46, 47, 55, 64, 66, 73, 75, I80, 84, 86, 87, 92, Y93, 96, 98, 100, 101 , 104,
  • the variant has an altered antibody binding profile as compared the parent group 1 mite polypeptide, more particularly the variant has a reduced IgE-binding, more par- ticularly combined with preserved immunogenicity for inducing protective responses (vide supra). Still more preferably, the variant has an altered immmunogenic profile in exposed animals, including humans, as compared to the parent group 1 mite polypeptide. Further the variant has in particular an altered IgE-antigenicity as compared to the parent group 1 mite polypeptide.
  • the variant has in particular at least the same T-cell stimulatory effect compared to the parent group 1 mite polypeptide as measured by the procedure in example 6. Still further the variant induces an altered immunogenic response in exposed animals, including humans, as compared to the parent group 1 mite polypeptide.
  • the variant induces in particular an altered immunogenic response in humans, as compared to the parent group 1 mite polypeptide.
  • the variant polypeptide of the invention comprises one or more mutations in the parent polypeptide in the positions or corresponding to the positions consisting of A10, A12, G30, G32, A46, Y47, S54, L55, D64, A66, S67, G87, S92, A98, R99, E100, Q101 , R105, R110, F111 , G112, 1113, 1144, K145, D146, D148, R151 , 1159, Q160, R161 , D162, N163, G164, Y165, Q166, N179, A180, G182, V183, D184, A205, I208 of SEQ ID NO:1 or 10, 12, 30, 32, 46, 47, 54, 55, 64, 66, 67, 87, 92, 98, 99, 100, 101, 105, 110, 111 , 112, 113, 144, 145, 146, 148, 151 , 159, 160, 161 , 162, 16
  • the variant polypeptide of the invention comprises one or more mutations in the parent polypeptide in the positions or corresponding to the positions consisting of A10, A12, G32, S54, L55, A66, S67, G87, A98, R99, F111, G112, 1113, 1144, D 146, D 148, 1 159, R 161 , G 164, Q166, A 180, D 184, A205 and I 208 of S EQ I D NO:1 or 10, 12, 32, 54, 55, 66, 67, 87, 98, 99, 111 , 112, 113, 144, 146, 148, 159, 161 , 164, 166, 180, 184, 205 and 208 of the mature Der p 1 polypeptide.
  • the variant polypeptide of the invention comprises a mutation selected from the group consisting of
  • said numbering being positions of or corresponding to positions of the mature Der p1 polypeptide.
  • A10 substituted by a residue selected from the group consisting of V, Y, Q, N, E and D;
  • A12 substituted by a residue selected from the group consisting of V, Y, Q, N and F;
  • G32 substituted by a residue selected from the group consisting of V, Y, E, D, N and Q;
  • S54 substituted by a residue selected from the group consisting of N, A, T, V, and Q;
  • L55 substituted by a residue selected from the group consisting of V, N and Q;
  • A66 substituted by a residue selected from the group consisting of V, H, Y, D, E, N and Q;
  • G87 substituted by a residue selected from the group consisting of V, Y, D and E;
  • R99 substituted by a residue selected from the group consisting of H, Y, V, N, Q, E and D;
  • F111 substituted by a residue selected from the group consisting of V, H and W;
  • G112 substituted by a residue selected from the group consisting of V, H, N, Q, E, D and Y;
  • I208 substituted by a residue selected from the group consisting of A, G, V, W and H;
  • said numbering being positions of or corresponding to positions of SEQ ID NO:1.
  • the parent group 1 mite polypeptide has in its mature form a sequence which displays at least 80% identity to SEQ ID NO:1; in particular at least 90 % identity; in particular at least 95 % identity, more particularly 98 % identity, more particularly 100 % identity to SEQ ID NO:1 or 100 % identity to Der p 1.
  • the variant group 1 mite polypeptide has in its mature form a sequence which displays at least 80% identity to SEQ ID NO:1 ; in particular at least 90 % identity; in particular at least 95 % identity, more particularly 98 % identity, more particularly 100 % identity to SEQ ID NO:1 or 100 % identity to Der p 1.
  • the parent group 1 mite polypeptide has in its mature form a sequence which displays at least 80% identity to SEQ ID NO:2; in particular at least 90 % iden- tity; in particular at least 95 % identity, more particularly 98 % identity, more particularly 100 % identity to SEQ ID NO:2 or 100 % identity to Eur ml .
  • the variant group 1 mite polypeptide has in its mature form a sequence which displays at least 80% identity to SEQ ID NO:2; in particular at least 90 % identity; in particular at least 95 % identity, more particularly 98 % identity, more particularly 100 % identity to SEQ ID NO:2 or 100 % identity to Eur ml .
  • the parent group 1 mite polypeptide has in its mature form a sequence which displays at least 80% identity to SEQ ID NO:3; in particular at least 90 % identity; in particular at least 95 % identity, more particularly 98 % identity, more particularly 100 % identity to SEQ ID NO:3 or 100 % identity to Der l.
  • the variant group 1 mite polypeptide has in its mature form a sequence which displays at least 80% identity to SEQ ID NO:3; in particular at least 90 % iden- tity; in particular at least 95 % identity, more particularly 98 % identity, more particularly 100 % identity to SEQ ID NO:3 or 100 % identity to Der f1.
  • the parent group 1 mite polypeptide has in its mature form a sequence which displays at least 80% identity to Der ml; in particular at least 90 % identity; in particular at least 95 % identity, more particularly 98 % identity; more particularly 100 % identity to Der ml.
  • the variant group 1 mite polypeptide has in its mature form a sequence which displays at least 80% identity to Der ml; in particular at least 90 % identity; in particular at least 95 % identity, more particularly 98 % identity; more particularly 100 % iden- tity to Der ml
  • the parent group 1 mite polypeptide has in its mature form a sequence which displays at least 80% identity to SEQ ID NO:5; in particular at least 90 % identity; in particular at least 95 % identity, more particularly 98 % identity, more particularly 100 % identity to SEQ ID NO:5 or 100 % identity to Bio 1 1.
  • the variant group 1 mite polypeptide has in its mature form a sequence which displays at least 80% identity to SEQ ID NO:5; in particular at least 90 % identity; in particular at least 95 % identity, more particularly 98 % identity, more particularly 100 % identity to SEQ ID NO:5 or 100 % identity to Bio 1 1.
  • the risk linked to protein engineering in order to eliminate epitopes that new epitopes are made, or existing epitopes are duplicated is reduced by testing the planned mutations at a given position in the 3-dimensional structure of the protein of interest against the found epitope patterns thereby identifying the mutations for each position that are feasible for obtaining the desired properties of the polypeptide.
  • a diversified library can be established by a range of techniques known to the person skilled in the art (Reetz MT; Jaeger KE, in Biocatalysis - from Discovery to Application edited by Fessner WD, V ol. 200, p p. 31-57 ( 1999); Stemmer, N ature, vol. 370, p .389-391, 1 994; Zhao and Arnold, Proc. Natl. Acad. Sci., USA, vol. 94, pp.
  • substitutions are found by a method comprising the following steps: 1 ) a range of substitutions, additions, and/or deletions are listed encompassing several epitopes, 2) a library is designed which introduces a randomized subset of these changes in the amino acid sequence into the target gene, e.g., by spiked mutagenesis, 3) the library is expressed, and preferred variants are selected.
  • this method is s upplemented with additonal rounds of screening and/or family shuffling of hits from the first round of screening (J.E. Ness, et al, Nature Biotechnology, vol. 17, pp. 893-896, 1999).
  • the mutations are designed, such that recognition sites for post- translational modifications are introduced in the epitope areas, and the protein variant is expressed in a suitable host organism capable of the corresponding post-translational modification.
  • These post-translational modifications may serve to shield the epitope and hence lower the immunogenicity of the protein variant relative to the protein backbone.
  • Post-translational modifications include glycosylation, phosphorylation, N-terminal processing, acylation, ribosy- lation and sulfatation. A good example is N-glycosylation.
  • N-glycosylation is found at sites of the sequence Asn-Xaa-Ser, Asn-Xaa-Thr, or Asn-Xaa-Cys, in which neither the Xaa residue nor the amino acid following the tri-peptide consensus sequence is a praline (T. E. Creighton, 'Proteins - Structures and Molecular Properties, 2nd edition, W.H. Freeman and Co., New York, 1993, pp. 91-93). It is thus desirable to introduce such recognition sites in the sequence of the backbone protein.
  • the specific nature of the glycosyl chain of the glycosylated protein variant may be linear or branched depending on the protein and the host cells.
  • Another way of making mutations that will change the antigenic properties of a polypep- tide is to react or conjugate polymers to amino acids in or near the epitope, thus blocking or shielding the access to the anchor amino acids and thus the binding of antibodies and/or recp- tors to those amino acids.
  • a suitable mutation is the insertion of one or more amino acids being attachment sites and/or groups and/or amino acids for polymer conjugation. Which amino acids to substitute and/or insert depends in principle on the coupling chemistry to be applied. The chemistry for preparation of covalent bioconjugates can be found in "Bioconjugate Techniques", Hermanson, G.T. (1996), Academic Press Inc., which is hereby incorporated as reference.
  • activated polymers are conjugated to amino acids in or near the epitope area. It is preferred to make conservative substitutions in the polypeptide when the polypeptide has to be conjugated, as conservative substitutions secure that the impact of the substitution on the polypeptide structure is limited.
  • this may be done by substitution of Arginine to Lysine, both residues being positively charged, but only the Lysine having a free amino group suitable as an attachment groups.
  • the conservative substitution may for instance be an Asparagine to Aspartic acid or Glutamine to Glutamic acid substitution. These residues resemble each other in size and shape, except from the carboxylic groups be- ing present on the acidic residues.
  • the conservative substitution may be done by substitution of Threonine or Serine to Cysteine.
  • the mutation of amino acids, comprised in an epitope will cause the antigenic proper- ties of the polypeptide to change, as predicted by the in silico determination of the epitopes.
  • the quantitative effect of the mutation on the antigenicity, i.e., the antibodybinding, and the immunogenicity of the variant is suitably determined using various in vivo or in vitro model systems.
  • the polypeptide variant of interest can be expressed in larger scale and purified by conventional techniques. Then the functionality and specific activity may be tested by cysteine protease activity assays, in order to assure that the variant has retained three-dimensional structure.
  • In vitro systems include assays measuring binding to IgE in serum from dust mite allergic patients or exposed animals, cytokine expression profiles or proliferation responses of primary T-cells from dust mite allergic patients or T cell clones or T cell line generated from dust mite allergic patients (Current protocols in Immunology, chapter 7 and 9), or exposed animals, and histamine release from basophils from dust mite allergic patients.
  • the IgE antibody binding can be examined in detail using, e.g., direct or competitive ELISA (C- ELISA), histamine release assays on basophil cells from allergic patients, or IgE -stripped basophils from whole blood incubated with IgE-containing serum from allergic patients, or by other or other solid phase immunoassays or cellular assays (see Example 9).
  • C- ELISA direct or competitive ELISA
  • histamine release assays on basophil cells from allergic patients or IgE -stripped basophils from whole blood incubated with IgE-containing serum from allergic patients, or by other or other solid phase immunoassays or cellular assays (see Example 9).
  • C- ELISA direct or competitive ELISA
  • histamine release assays on basophil cells from allergic patients or IgE -stripped basophils from whole blood incubated with IgE-containing serum from allergic patients, or by other or other solid phase immunoassays or cellular as
  • the ability of the polypeptide variant to bind IgE is reduced at least 3 times as compared to the binding ability of the orginal or parent group 1 mite polypeptide, preferably 5 times reduced, more preferably 10 times reduced, or more preferably 50 times reduced.
  • the ability of the polypeptide variant to induce histamine release in basophil cells from subjects allergic to dust mites is reduced least 3 times, as compared to that of the parent group 1 mite polypeptide, preferably 10 times reduced, more preferably 50 times.
  • the ability of the polypeptide variant to invoke a recall T-cell response in lymphocytes from animals, including humans, previously exposed to the orginal or parent group 1 mite allergen is measured, preferably the strength of the response is comparable to or higher than that to the parent group 1 mite allergen.
  • the in vivo verification comprises skin prick testing (SPT), in which a dust mite allergic subject/indvidual is exposed to intradermal or subcutaneous injection of group 1 mite polypeptides and the IgE reactivity, measured as the diameter of the wheal and flare reaction, in response to a polypeptide variant of the invention is compared to that to the parent group 1 mite polypeptide (Kronquist et al., Clin. Exp. Allergy, 2000, vol. 30, pp. 670- 676).
  • SPT skin prick testing
  • the in vivo immunogenic properties of the polypeptide variant of the invention may suitably be measured in an animal test, wherein test animals are exposed to a vaccination allergen polypeptide and the responses are measured and compared to those of the target allergen or other appropriate references.
  • the immune response measurements may include com- paring reactivity of serum IgG, IgE or T-cells from a test animal with target polypeptide and the polypeptide variant.
  • Animal immunization can be conducted in at least two distinct manners: on naive animals and on pre-sensitized animals (to better simulate the vaccine situation). In the context of this invention affinity of immunoglobulins towards the target antigen is tested.
  • the affinity of animal IgG and/or lgG1 and/or lgG4 following administration of the variant molecule is tested.
  • test animals can either be naive animals or pre-sensitized animals.
  • the in vivo verification comprises exposing a mouse to a parent target allergen by the intranasal route.
  • Useful in vivo animal models include the mouse intranasal test (MINT) model (Robinson et al., Fund. Appl. Toxicol. 34, pp. 15-24, 1996).
  • the i n vivo verification comprises exposing a test animal to a polypeptide variant by the intratracheal route.
  • Useful in vivo animal models include the guinea pig intratracheal (GPIT) model (Ritz, et al. Fund. Appl. Toxicol., 21, pp.
  • the in vivo verification comprises exposing a test animal subcutaneously to the target allergen and the vaccination allergen variant.
  • a suitable model is the mouse subcutaneous (mouse-SC) model (WO 98/30682, Novo Nordisk).
  • the method comprises exposing the test animal in- traperitoneally.
  • ALK-Abell ⁇ disclose (WO02/40676) a method to assess the ability of allergen variants (of the birch pollen allergen bet v 1) to induce IgG antibodies upon immunization of mice: BALB/C mice were immunized intraperitoneally with the relevant allergy variant or controls, four times at dose intervals of 14 days. The proteins were conjugated to 1.25 mg/mL al- hydogel (AIOH gel, 1 ,3%, pH8-8.4, Superfos Biosector). The mice were immunized with either 1 or 10 ug protein/dose. Blood samples were drawn at day 0, 14, 21, 35, 49, and 63 and ana- lysed by direct ELISA using rBet v 1 coated microtiterplates and biotinylated rabbit anti mouse IgG antibodies as detecting antibodies.
  • the method comprise using transgenic mice capable of facilitating production of donor-specific immunity as test animals.
  • transgenic mice capable of facilitating production of donor-specific immunity as test animals.
  • Such mice are disclosed by Genencor International (WO 01/15521).
  • WO 01/15521 Genencor International
  • a number of studies have assessed the effect of allergy vaccination compositions in animal models, in which the animals were sensitized to the relevant allergen prior to exposure to the vaccination composition:
  • mice Li et al. (J. Allergy Clin. Immunol, vol. 112, pp159-167, 2003) disclose a mice- based system to assess efficacy of allergy vaccines. The mice are sensitized intra-gastrically with a food allergen, and the treatment is introduced as an intra-rectum injection. In a separate allegy vaccination system, Hardy et al. (AM J. Respir. Crit Care Med, vol 167, pp. 1393-1399, 2003) show that mice can be sensitized by i ntraperitonal injection, and that allergy vaccine compounds can be administered intrtracheally withthe animals anaestethized. Sudowe et al., (Gene T herapy, vol. 9, 147-156, 2002) show that intraperitoneal injection in mice could be made to produce either TH1 or TH2 responses.
  • Rats Wheeler et al., (Int. Arch. Allergy Immunol, vol. 126, pp. 135-139, 2001) disclose a rat allergy model in which rats are injected subcutaneously along with adjuvant. These 'allergic' rats can then be made to conduct an allergy-vaccine like response, when subjected to subsequent injections with trial vaccine compositions.
  • Guinea Pigs Nakamoto et al., (Clin Exp. Allergy, vol. 27, pp 1103-1108 1997) demonstrate the use of guinea pigs as model system for SIT. Guinea pigs were injected intraperitoneally and boosted twice, and then they were exposed to the vaccine compound to register decreases in allergenicity by measuring antibody titers as a function of the compound, formulation, or mode of application.
  • nucleotide constructs Preparation of nucleotide constructs, vectors, host cells, protein variants and polymers for conjugation
  • conventional molecular biology, microbiology, and recombinant DNA techniques well known to a person skilled in the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein "Sambrook et al., 1989") DNA Clon- ing: A Practical Approach, Volumes I and II /D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed.
  • the method may in a particular embodiment be carried out to express group 1 dust mite proteins as inclusion bodies in E.coli or in soluble form in methylotrophic yeasts such as Pichia pastoris, as described in WO 01/29078 (HESKA) describing recombinant expression of group 1 m ite p roteins i ncluding n ucleotide s equences m odified t o e nable e xpression o f t he polypeptides in microorganisms.
  • HESKA WO 01/29078
  • a preferred method is to express the group 1 dust mite proteins in S.cerevisiae cells, as described by Chua et al. (J. Allergy Clin Immunol. 1992, vol. 89, pp 95-102).
  • Another preferred method is to express group 1 dust mite proteins in insect cells such as Drosophila (Jacquet et al, Clin Exp. Allergy, 2000, vol. 30 pp. 677-84) or Spodoptera frugiperda Sf9 cells infected with a bacullovirus system (Shoji, et al., Biosci. Biotech. Biochem. 1996, vol. 60, pp. 621-25).
  • insect cells such as Drosophila (Jacquet et al, Clin Exp. Allergy, 2000, vol. 30 pp. 677-84) or Spodoptera frugiperda Sf9 cells infected with a bacullovirus system (Shoji, et al., Biosci. Biotech. Biochem. 1996, vol. 60, pp. 621-25).
  • the present invention also encompasses a nucleotide sequence encoding a polypeptide variant of the invention.
  • a description of standard mutation of nucleotide se- quences to encode polypeptide variants by nucleotide substitution can be found in e.g., Ford et al., 1991 , Protein Expression and Purification 2, p. 95-107.
  • Other standard methods, such as site-directed mutagenesis is described in e.g., Sambrook et al. (1989), Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, NY.
  • nucleotide sequence is a single- or double-stranded polymer of deoxyribonucleo- tide or ribonucleotide bases read from the 5' to the 3' end.
  • Nucleotide sequences include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules.
  • a nucleotide sequence alias a nucleotide sequence encoding a polypeptide
  • isolation from genomic DNA, preparation from cDNA, or a combination thereof.
  • the cloning of the nucleotide sequences of the present invention from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g. Innis et al., 1990, A Guide to Methods and Application, Academic Press, New York.
  • PCR polymerase chain reaction
  • nucleotide amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nuceic acid sequence-based amplification (NASBA) may be used.
  • LCR ligase chain reaction
  • LAT ligated activated transcription
  • NASBA nuceic acid sequence-based amplification
  • the nucleotide sequence may be cloned from a strain producing the polypeptide, or from another related organism and thus, for example, may be an allelic or species variant of the polypeptide encoding region of the nucleotide sequence.
  • isolated nucleotide sequence refers to a nucleotide sequence which is essentially free of other nucleotide sequences, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by agarose gel electorphoresis.
  • an isolated nucleotide sequence can be obtained by standard cloning procedures used in genetic engineering to relocate the nucleotide sequence from its natural location to a different site where it will be reproduced.
  • the cloning procedures may involve excision and isolation of a desired nucleotide fragment comprising the nucleotide sequence encoding the polypeptide, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into a host cell where multiple copies or clones of the nucleotide sequence will be replicated.
  • the nucleotide sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.
  • isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones.
  • Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, and may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316: 774-78, 1985).
  • nucleotide construct is intended to indicate any nucleotide molecule of cDNA, genomic DNA, synthetic DNA or RNA origin.
  • construct is intended to indicate a nucleotide segment which may be single- or double-stranded, and which may be based on a complete or partial naturally occurring nucleotide sequence encoding a polypeptide of interest.
  • the construct may optionally contain other nucleotide segments.
  • the DNA of interest may suitably be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the polypeptide by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., supra).
  • the nucleotide construct may also be prepared synthetically by established standard methods, e.g., the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859 - 1869, or the method described by Matthes et al., EMBO Journal 3 (1984), 801 - 805.
  • oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer, purified, annealed, ligated and cloned in suitable vectors.
  • nucleotide construct may be of mixed synthetic and genomic, mixed synthetic and cDNA or mixed genomic and cDNA origin prepared by ligating fragments of syn- thetic, genomic or cDNA origin (as appropriate), the fragments corresponding to various parts of the entire nucleotide construct, in accordance with standard techniques.
  • nucleotide construct m ay also be prepared by polymerase chain reaction using specific primers, for instance as described in US 4,683,202 or Saiki et al., Science 239 (1988), 487 - 491.
  • the term nucleotide construct may be synonymous with the term expression cassette when the nucleotide construct contains all the control sequences required for expression of a coding sequence of the present invention.
  • coding sequence as defined herein is a sequence which is transcribed into mRNA and translated into a polypeptide of the present invention when placed under the control of the above mentioned control sequences.
  • the boundaries of the coding sequence are generally determined by a translation start codon ATG at the 5'-terminus and a translation stop codon at the 3'-terminus.
  • a coding sequence can include, but is n ot limited to, DNA, cDNA, and recombinant nucleotide sequences.
  • control sequences is defined herein to include all components which are necessary or advantageous for expression of the coding sequence of the nucleotide sequence.
  • Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide.
  • control sequences include, but are not limited to, a leader, a polyadenyla- tion sequence, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator.
  • the control sequences include a promoter, and transcriptional and translational stop signals.
  • the control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding a polypeptide.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • polyadenylation signals are control sequences.
  • the nucleotide constructs of the present invention may also comprise one or more nucleotide sequences which encode one or more factors that are advantageous in the expression of the polypeptide, e.g., an activator (e.g., a trans-acting factor), a chaperone, and a processing protease. Any factor that is functional in the host cell of choice may be used in the present invention.
  • the nucleotides encoding one or more of these factors are not necessarily in tan- dem with the nucleotide sequence encoding the polypeptide.
  • the control sequence may also be a propeptide coding region, which codes for an amino acid sequence positioned at the amino terminus of a polypeptide.
  • the resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases).
  • a propolypeptide is generally inactive and can be converted to mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.
  • protease of the family of subtilisin-like serine proteases have proven useful for activation of propeptides. Subtilisin could be added to the crude cell supernatant, to filtron concentrated supernatant, or to material that had been purified by column chromatography.
  • the subtilisin could be removed by an extra chromatography step or inactivated with barley chy- motrypsin inhibitor (Ci-2A). The time of incubation could range from 1 to 21 or 24 hours.
  • the pro-der p 1 or corresponding propeptide of the variants were cleaved at the native processing site (as verified by Edman degradation and N-terminal sequencing) to give the N-terminal sequence TNACSIN.
  • the preferred Subtilisins are SavinaseTM (Subtilisin from Bacillus clausii.
  • BPN' Subtilisin Novo from Bacillus amyloliquefaciens, Swis- sProt:SUBT_BACAM see Siezen et al., Protein Engng. 4 (1991) 719-737
  • PD498 Subtilisin from a Bacillus sp., GeneSeqP:AAW24071 ; WO9324623A1
  • B34 Subtilisin from Bacillus alcalophilus, patent WO 0158275.
  • BPN' BASBPN
  • the propeptide coding region may be obtained from the Bacillus subtilis alkaline protease gene (aprE), the Bacillus subtilis neutral protease gene (nprT), the Saccharomyces cere- visiae alpha-factor gene, or the Myceliophthora thermophilum laccase gene (WO 95/33836).
  • Activators include Bacillus subtilis alkaline protease gene (aprE), the Bacillus subtilis neutral protease gene (nprT), the Saccharomyces cere- visiae alpha-factor gene, or the Myceliophthora thermophilum laccase gene (WO 95/33836).
  • An activator is a protein which activates transcription of a nucleotide sequence encoding a polypeptide (Kudla et al., 1990, EMBO Journal 9:1355-1364; Jarai and Buxton, 1994, Current Genetics 26:2238-244; Verdier, 1990, Yeast 6:271-297).
  • T he nucleotide sequence encoding an activator may be obtained from the genes encoding Bacillus stearothermophilus NprA (nprA), Saccharomyces cerevisiae heme activator protein 1 (hapl), Saccharomyces cer- evisiae galactose metabolizing protein 4 (gal4), and Aspergillus nidulans ammonia regulation protein (areA).
  • nprA Bacillus stearothermophilus NprA
  • hapl Saccharomyces cerevisiae heme activator protein 1
  • gal4 Saccharomyces cer- evisiae galactose metabolizing protein 4
  • areA Aspergillus nidulans ammonia regulation protein
  • a chaperone is a protein which assists another polypeptide in folding properly (Hartl et al., 1994, TIBS 19:20-25; Bergeron et al., 1994, TIBS 19:124-128; Demolder et al., 1994, Journal of Biotechnology 32:179-189; Craig, 1993, Science 260:1902-1903; Gething and Sambrook, 1992, Nature 355:33-45; Puig and Gilbert, 1994, Journal of Biological Chemistry 269:7764-7771 ; Wang and Tsou, 1993, The FASEB Journal 7:1515-11157; Robinson et al., 1994, Bio/Technology 1 :381-384).
  • the nucleotide sequence encoding a chaperone may be obtained from the genes encoding Bacillus subtilis GroE proteins, Aspergillus oryzae protein disulphide isomerase, Saccharomyces cerevisiae calnexin, Saccharomyces cerevisiae BiP/GRP78, and Saccharomyces cerevisiae Hsp70. For further examples, see Gething and Sambrook, 1992, supra, and Hartl et al., 1994, supra.
  • a processing protease is a protease that cleaves a propeptide to generate a mature biochemically active polypeptide (Enderlin and Ogrydziak, 1994, Yeast 10:67-79; Fuller et al., 1989, Proceedings of the National Academy of Sciences USA 86:1434-1438; Julius et al., 1984, Cell 37:1075-1089; Julius et al., 1983, Cell 32:839-852).
  • the nucleotide sequence encoding a processing protease may be obtained from the genes encoding Aspergillus niger Kex2, Saccharomyces cerevisiae dipeptidylaminopeptidase, Saccharomyces cerevisiae Kex2, and Yarrowia lipolytica dibasic processing endoprotease (xpr6).
  • the control sequence may be an appropriate promoter sequence, a nucleotide sequence which is recognized by a host cell for expression of the nucleotide sequence.
  • the promoter sequence contains transcription and translation control sequences which mediate the expression of the polypeptide.
  • the promoter may be any nucleotide sequence which shows transcriptional activity in the host cell of choice and may be obtained from genes encoding ex- tracellular or intracellular polypeptides either homologous or heterologous to the host cell.
  • promoter is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription.
  • Promoter sequences are commonly, but not always, found in the 5' non- coding regions of genes.
  • suitable promoters for directing the transcription of the nucleotide constructs of the present invention, especially in a bacterial host cell are the promoters obtained from t he E .
  • the Streptomyces coelicolor a garase gene (dagA), the Bacillus subtilis levansucrase gene (sacB), the Bacillus subtilis alkaline protease gene, the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus amylo- liquefaciens BAN amylase gene, the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and the prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences USA 75:3727-3731), as well as the tac promoter (DeBo
  • promoters for directing the transcription of the nucleotide con- structs of the present invention in a filamentous fungal host cell are promoters obtained from the genes encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic pro- teinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium oxysporum trypsin-like protease (as described in U.S.
  • Patent No. 4,288,627 which is incorporated herein by reference
  • Particularly preferred promoters for use in filamentous fungal host cells are the TAKA amylase, NA2-tpi (a hybrid of the promoters from the genes encoding Aspergillus niger neutral (-amylase and Aspergillus oryzae triose phosphate isomerase), and glaA promoters.
  • Further suitable promoters for use in filamentous fungus host cells are the ADH3 promoter (McKnight et al., The EMBO J. 4 (1985), 2093 - 2099) or the tpiA promoter.
  • promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255 (1980), 12073 - 12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1 (1982), 419 - 434) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals (Hollaender et al, eds.), Plenum Press, New York, 1982), or the TPI1 (US 4,599,311) or ADH2-4c (Russell et al., Nature 304 (1983), 652 - 654) promoters.
  • yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.
  • useful promoters include viral promoters such as those from Simian Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus, and bovine papilloma virus (BPV).
  • SV40 Simian Virus 40
  • RSV Rous sarcoma virus
  • BPV bovine papilloma virus
  • Suitable promoters for directing the transcription of the DNA encoding the polypeptide of the invention in mammalian cells are the SV40 promoter (Subramani et al., Mol. Cell Biol. 1 (1981), 854 -864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809 - 814) or the adenovirus 2 major late promoter.
  • a suitable promoter for use in insect cells is the polyhedrin promoter (US 4,745,051; Vasuvedan et al., FEBS Lett. 311, (1992) 7 - 11), the P10 promoter (J.M. Vlak et al., J. Gen. Virology 69, 1988, pp. 765-776), the Autographa californica polyhedrosis virus ba- sic protein promoter (EP 397 485), the baculovirus immediate early gene 1 promoter (US 5,155,037; US 5,162,222), or the baculovirus 39K delayed-early gene promoter (US 5,155,037; US 5,162,222).
  • the polyhedrin promoter US 4,745,051; Vasuvedan et al., FEBS Lett. 311, (1992) 7 - 11
  • the P10 promoter J.M. Vlak et al., J. Gen. Virology 69, 1988, pp. 765-776
  • the control sequence may also be a suitable transcription terminator sequence, a se- quence recognized by a host cell to terminate transcription.
  • the terminator sequence is oper- ably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.
  • Preferred terminators for filamentous fungal host cells are obtained from the genes encoding Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidu- lans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease for fungal hosts) the TPI1 (Alber and Kawasaki, op. cit.) or ADH3 (McKnight et al., op. cit.) terminators.
  • TPI1 Alber and Kawasaki, op. cit.
  • ADH3 McKnight et al., op. cit.
  • Preferred terminators for yeast host cells are obtained from the genes encoding Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), or Sac- charomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase.
  • Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.
  • the control sequence may also be a polyadenylation sequence, a sequence which is operably linked to the 3' terminus of the nucleotide sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the host cell of choice may be used in the present invention.
  • Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes encoding Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, and Aspergillus niger alpha-glucosidase.
  • Polyadenylation sequences are well known in the art for mammalian host cells such as SV40 or the adenovirus 5 Elb region.
  • the control sequence may also be a signal peptide coding region, which codes for an amino acid sequence linked to the amino terminus of the polypeptide which can direct the expressed polypeptide into the cell's secretory pathway of the host cell.
  • the 5' end of the coding sequence of the nucleotide sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted polypeptide.
  • the 5' end of the coding sequence may contain a signal peptide coding region which is foreign to that portion of the coding sequence which encodes the secreted polypeptide.
  • a foreign signal peptide coding region may be required where the coding sequence does not normally contain a signal peptide coding region.
  • the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to obtain enhanced secretion relative to the natural signal peptide coding region normally associated with the coding sequence.
  • the signal peptide coding region may be obtained from a glucoamylase or an amylase gene from an Aspergillus species, a li- pase or proteinase gene from a Rhizomucor species, the l gene for the alpha-factor from Sac- charomyces cerevisiae, an amylase or a protease gene from a Bacillus species, or the calf preprochy osin gene.
  • any signal peptide coding region capable of directing the expressed polypeptide into the secretory pathway of a host cell of choice may be used in the present invention.
  • a “secretory signal sequence” is a D NA sequence that e ncodes a polypeptide (a "secretory peptide” that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
  • the larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
  • An effective signal peptide coding region for bacterial host cells is the signal peptide coding region obtained from the maltogenic amylase gene from Bacillus NCIB 11837, the Bacillus stearothermophilus alpha-amylase gene, the Bacillus licheniformis subtilisin gene, the Bacillus licheniformis beta-lactamase gene, the Bacillus stearothermophilus neutral proteases genes (nprT, nprS, nprM), and the Bacillus subtilis PrsA gene. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.
  • An effective signal peptide coding region for filamentous fungal host cells is the signal peptide coding region obtained from Aspergillus oryzae TAKA amylase gene, Aspergillus niger neutral amylase gene, the Rhizomucor miehei aspartic proteinase gene, the Humicola lanugi- nosa cellulase or lipase gene, or the Rhizomucor miehei lipase or protease gene, Aspergillus sp. amylase or glucoamylase, a gene encoding a Rhizomucor miehei lipase or protease.
  • the signal peptide is preferably derived from a gene encoding A. oryzae TAKA amylase, A. niger neutral (-amylase, A. niger acid-stable amylase, or A. niger glucoamylase.
  • Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae a-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding regions are described by Romanos et al., 1992, supra.
  • the secretory signal sequence may encode any signal peptide which ensures efficient direction of the expressed polypeptide into the secretory pathway of the cell.
  • the signal peptide may be a naturally occurring signal peptide, or a functional part thereof, or it may be a synthetic peptide. Suitable signal peptides have been found to be the a- factor signal peptide (cf.
  • a sequence encoding a leader peptide may also be inserted downstream of the signal sequence and uptream of the DNA sequence encoding the polypeptide.
  • the function of the leader peptide is to allow the expressed polypeptide to be directed from the endoplasmic reticulum to the Golgi apparatus and further to a secretory vesicle for secretion into the culture medium (i.e. exportation of the polypeptide across the cell wall or at least through the cellular membrane into the periplasmic space of the yeast cell).
  • the leader peptide may be the yeast a-factor leader (the use of which is described in e.g., US 4,546,082, EP 16 201 , EP 123 294, EP 123 544 and EP 163 529).
  • the leader peptide may be a synthetic leader peptide, which is to say a leader peptide not found in nature. Synthetic leader peptides may, for instance, be constructed as described in WO 89/02463 or WO 92/11378.
  • the signal peptide may conveniently be derived from an insect gene (cf. WO 90/05783), such as the lepidopteran Manduca sexta adipokinetic hormone precursor signal peptide (cf. US 5,023,328).
  • insect gene cf. WO 90/05783
  • the signal peptide may conveniently be derived from an insect gene (cf. WO 90/05783), such as the lepidopteran Manduca sexta adipokinetic hormone precursor signal peptide (cf. US 5,023,328).
  • regulator seguences It may also be desirable to add regulatory sequences which allow the regulation of the expression of the polypeptide relative to the growth of the host cell.
  • regulatory systems are those which cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • Regulatory systems in prokaryotic systems would include the lac, tac, and trp operator systems.
  • yeast the ADH2 system or GAL1 system may be used.
  • filamentous fungi the TAKA alpha- amylase promoter, Aspergillus niger glucoamylase promoter, and the Aspergillus oryzae glucoamylase promoter may be used as regulatory sequences.
  • regulatory sequences are those which allow for gene amplification. In eukaryotic systems, these include the dihydrofolate reductase gene which is amplified in the presence of methotrexate, and the met- allothionein genes which are amplified with heavy metals. In these cases, the nucleotide sequence encoding the polypeptide would be placed in tandem with the regulatory sequence.
  • the present invention also relates to a recombinant expression vector comprising a nucleotide sequence of the present invention, a promoter, and transcriptional and translational stop signals.
  • the various nucleotide and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleotide sequence encoding the polypeptide at such sites.
  • the nucleotide sequence of the present invention may be expressed by inserting the nucleotide sequence or a nucleotide construct comprising the sequence into an appropriate vector for expression.
  • the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression, and possibly secretion.
  • “Operably linked”, when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.
  • An “Expression vector” is a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments may include promoter and terminator sequences, and optionally one or more origins of replication, one or more selectable markers, an enhan- cer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
  • the recombinant expression vector may be any vector (e.g., a plasmid or virus), which can be conveniently subjected to recombinant DNA procedures and can bring about the ex- pression of the nucleotide sequence.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the vectors may be linear or closed circular plasmids.
  • the vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.
  • the vector may contain any means for assuring self-replication.
  • the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • the vector system may be a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.
  • the vectors of the present invention preferably contain one or more selectable markers which permit easy selection of transformed cells.
  • a selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
  • Examples of bacterial selectable markers are the dal genes from Ba- cillus subtilis or Bacillus licheniformis, or markers which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, tetracycline, neomycin, hygromycin or methotrexate resistance.
  • a frequently used mammalian marker is the dihydrofolate reductase gene (DHFR).
  • Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.
  • a selectable marker for use in a filamentous fungal host cell may be selected from the group including, but not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), trpC (anthranilate synthase), and glufosinate resistance markers, as well as equivalents from other species.
  • amdS and pyrG markers of Aspergillus nidulans or Aspergillus oryzae and the bar marker of Streptomyces hygroscopicus.
  • selection may be accomplished by co-transformation, e.g., as described in WO 91/17243, where the selectable marker is on a separate vector.
  • the vectors of the present invention preferably contain an element(s) that permits stable integration of the vector into the host cell genome or autonomous replication of the vector in the cell independent of the genome of the cell.
  • the vectors of the present invention may be integrated into the host cell genome when introduced into a host cell.
  • the vector may rely on the nucleotide sequence encoding the polypeptide or any other element of the vector for stable integration of the vector into the genome by homologous or nonhomologous recombination.
  • the vector may contain additional nucleotide sequences for directing integration by homologous recombination into the genome of the host cell.
  • the additional nucleotide sequences enable the vector to be integrated into the host cell genome at a precise location(s) in the chromosome(s).
  • the integrational elements should preferably contain a sufficient number of nucleotides, such as 100 to 1 ,500 base pairs, preferably 400 to 1 ,500 base pairs, and most preferably 800 to 1 ,500 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination.
  • the integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell.
  • the integrational elements may be non-encoding or encoding nucleotide sequences.
  • the vector may be integrated into the genome of the host cell by non-homologous recombination.
  • These nucleotide sequences may be any sequence that is homologous with a target sequence in the ge- nome of the host cell, and, furthermore, may be non-encoding or encoding sequences.
  • the vector m ay f urther comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question.
  • bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, pACYC184, pUB110, pE194, pTA1060, and pAM ⁇ l
  • origin of replications for use in a yeast host cell are the 2 micron origin of replication, the combination of CEN6 and ARS4, and the combination of CEN3 and ARS1.
  • the origin of replication may be one having a mutation which makes its functioning temperature-sensitive in the host cell (see, e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75:1433).
  • More than one copy of a nucleotide sequence encoding a polypeptide of the present invention may be inserted into the host cell to amplify expression of the nucleotide sequence.
  • Stable amplification of the nucleotide sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome using methods well known in the art and selecting for transformants.
  • the present invention also relates to recombinant host cells, comprising a nucleotide sequence or nucleotide construct or recombinant expression vector of the invention, which are advantageously used in the recombinant production of the polypeptide variants of the inven- tion.
  • host cell encompasses a parent host cell and any progeny thereof, which is not identical to the parent host cell due to mutations that occur during replication.
  • the cell is preferably transformed with a vector comprising a nucleotide sequence of the invention followed by integration of the vector into the host chromosome.
  • Transforma- tion means introducing a vector comprising a nucleotide sequence of the present invention into a host cell so that the vector is maintained as a chromosomal integrant or as a self- replicating extra-chromosomal vector. Integration is generally considered to be an advantage as the nucleotide sequence is more likely to be stably maintained in the cell. Integration of the vector into the host chromosome may occur by homologous or non-homologous recombination as described above.
  • the choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.
  • the host cell may be a unicellular microorganism, e.g., a prokary- ote, or a non-unicellular microorganism, e.g., a eukaryote.
  • Useful unicellular cells are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans or Streptomyces murinus, or gram negative bacteria such as E.
  • a Bacillus cell e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis
  • the bacterial host cell is a Bacillus lentus, Bacil- lus licheniformis, Bacillus stearothermophilus or Bacillus subtilis cell.
  • the transformation of a bacterial host cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168:111-115), by using competent cells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnar and Davidoff- Abelson, 1971, Journal of Molecular Biology 56:209-221), by electroporation (see, e.g., Shi- awa and Dower, 1988, Biotechniques 6:742-751), or by conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169:5771-5278).
  • protoplast transformation see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168:111-115
  • competent cells see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnar and Davidoff
  • the host cell may be a eukaryote, such as a mammalian cell, an insect cell, a plant cell or a fungal cell.
  • Useful mammalian cells include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, COS cells, or any number of other immortalized cell lines available, e.g., from the American Type Culture Collection.
  • suitable mammalian cell lines are the COS (ATCC CRL 1650 and 1651), BHK (ATCC CRL 1632, 10314 and 1573, ATCC CCL 10), CHL (ATCC CCL39) or CHO (ATCC CCL 61) cell lines.
  • Methods of transfecting mammalian cells and expressing DNA sequences introduced in the cells are described in e.g., Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601 - 621; Southern and Berg, J. Mol. Appl. Genet. 1 (1982), 327 - 341; Loyter et al., Proc. Natl. Acad. Sci.
  • the host cell is a fungal cell.
  • Fungi as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et al., 1995, supra).
  • Basidiomycota include mushrooms, rusts, and smuts.
  • Representative groups of Chytridiomycota include, e.g., Allomyces, Blastocladiella, Coelomomyces, and aquatic fungi.
  • Representative groups of Oomycota include, e.g., Saprolegniomycetous aquatic fungi (water molds) such as Achlya. Examples of mitosporic fungi include Aspergillus, Penicil- lium, Candida, and Alternaria.
  • Representative groups of Zygomycota include, e.g., Rhizopus and Mucor.
  • the fungal host cell is a yeast cell.
  • yeast as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast be- longing to the Fungi Imperfecti (Blastomycetes).
  • the ascosporogenous yeasts are divided into the families Spermophthoraceae and Saccharomycetaceae. The latter is comprised of four subfamilies, Schizosaccharomycoideae (e.g., genus Schizosaccharomyces), Nadsonioideae, Lipomycoideae, and Saccharomycoideae (e.g., genera Pichia, Kluyveromyces and Saccharomyces).
  • Schizosaccharomycoideae e.g., genus Schizosaccharomyces
  • Nadsonioideae e.g., Lipomycoideae
  • Saccharomycoideae e.g., genera
  • the basidiosporogenous yeasts include the genera Leucosporidim, Rhodosporidium, Sporidiobolus, Filobasidium, and Filobasidiella.
  • Yeast belonging to the Fungi Imperfecti are divided into two families, Sporobolomycetaceae (e.g., genera Sorobolomyces and Bullera) and Cryptococcaceae (e.g., genus Candida). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F.A., Passmore, S.M., and Davenport, R.R., eds, Soc. App. Bacte- riol. Symposium Series No. 9, 1980.
  • yeast host cell may be selected from a cell of a species of Candida, Kluyveromyces, Saccharomyces, Schizosaccharomyces, Candida, Pichia, Hansehula, , or Yarrowia.
  • the yeast host cell is a Saccharomyces carlsbergensis, Saccharomy- ces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluy- veri, Saccharomyces norbensis or Saccharomyces oviformis cell.
  • Other useful yeast host cells are a Kluyveromyces lactis Kluyveromyces fragilis Hansehula polymorpha, Pichia pastoris Yar- rowia lipolytica, Schizosaccharomyces pombe, Ustilgo maylis, Candida maltose, Pichia guiller- mondii and Pichia methanolio cell (cf. Gleeson et al., J. Gen. Microbiol. 132, 1986, pp. 3459- 3465; US 4,882,279 and US 4,879,231).
  • the fungal host cell is a filamentous fungal cell.
  • filamentous fungi include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra).
  • the filamentous fungi are characterized by a vege- tative mycelium composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligated aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
  • the filamentous fungal host cell is a cell of a species of, but not limited to, Acremonium, Aspergillus, Fusarium, H umicola, M ucor, Myceliophthora, N eurospora, Penicillium, Thielavia, Tolypocladium, and Trichoderma or a teleomorph or synonym thereof.
  • the filamentous fungal host cell is an Aspergillus cell.
  • the filamentous fungal host cell is an Acremonium cell.
  • the filamentous fungal host cell is a Fusa- rium cell.
  • the filamentous fungal host cell is a Humicola cell.
  • the filamentous fungal host cell is a Mucor cell. In another even more preferred embodiment, the filamentous fungal host cell is a Myceliophthora cell. In another even more preferred embodiment, the filamentous fungal host cell is a Neurospora cell. In another even more preferred embodiment, the filamentous fungal host cell is a Penicillium cell. In another even more preferred embodiment, the filamentous fungal host cell is a Thielavia cell. In another even more preferred embodiment, the filamentous fungal host cell is a Tolypocladium cell. In another even more preferred embodiment, the filamentous fungal host cell is a Trichoderma cell.
  • the filamentous fungal host cell is an Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus niger, Aspergillus nidulans or Aspergillus oryzae cell.
  • the filamentous fungal host cell is a Fusarium cell of the section Discolor (also known as the section Fusarium).
  • the filamentous fungal parent cell may be a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sulphureum, or Fusarium trichothecioides cell.
  • Fusarium bactridioides Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarc
  • the filamentous fungal parent cell is a Fusarium strain of the section Elegans, e.g., Fusarium ox- ysporum.
  • the filamentous fungal host cell is a Humicola insolens or Humicola lanuginosa cell.
  • the filamentous fungal host cell is a Mucor miehei cell.
  • the filamentous fungal host cell is a Myceliophthora thermophilum cell.
  • the filamentous fungal host cell is a Neurospora crassa cell.
  • the filamentous fungal host cell is a Penicillium purpurogenum cell.
  • the filamentous fungal host cell is a Thielavia terrestris cell or an Acremonium chrysogenum cell.
  • the Trichoderma cell is a Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Tricho- derma reesei or Trichoderma viride cell.
  • Aspergillus spp. for the expression of proteins is described in, e.g., EP 272 277, EP 230 023.
  • the nucleotide sequences, DNA, of the invention may be modified such as to optimize the codon usage for a preferred particular host organism in which it will be expressed. Examples of this are published for yeast (Woo JH, et al, Protein Expression and Purification, Vol. 25 (2), pp. 270-282, 2002), fungi (Te'o et al, FEMS Microbiology Letters, Vol. 190 (1) pp. 13-19 (2000)), and other microorganisms, as well as for Der p 1 expressed in mammalian cells (Mas- saer M, et al, International Archives of Allergy and Immunology, Vol. 125 (1), pp. 32-43, 2001).
  • the host cell is an insect cell and/or insect cell line.
  • the insect cell line used as the host may suitably be a Lepidoptera cell line, such as Spodoptera frugiperda cells or Trichoplusia ni cells (cf. US 5,077,214).
  • Culture conditions may suitably be as described in, for instance, WO 89/01029 or WO 89/01028, or any of the aforementioned references.
  • the present invention also relates to a transgenic plant, plant part, or plant cell which has been transformed with a nucleic acid sequence encoding a polypeptide (i.e. variant) of the present invention so as to express and produce the polypeptide in recoverable quantities.
  • the polypeptide may be recovered from the plant or plant part.
  • the plant or plant part containing the recombinant polypeptide may be used as such for vaccine purposes.
  • the polypeptide is targeted to the endosperm storage vacuoles in seeds.
  • This can be obtained by synthesizing it as a precursor with a suitable signal peptide, see Horvath et al in PNAS, Feb. 15, 2000, vol. 97, no. 4, p. 1914-1919.
  • the transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a monocot) or engineered variants thereof.
  • monocot plants are grasses, such as meadow grass (blue grass, Pba), forage grass such as festuca, lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, and maize (corn).
  • dicot plants are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organism Arabidopsis thaliana.
  • Low-phytate plants as described e.g. in US patent no. 5,689,054 and US patent no. 6,111,168 are examples of engineered plants.
  • plant parts are stem, callus, leaves, root, fruits, seeds, and tubers. Also specific plant tissues, such as chloroplast, apoplast, mitochondria, vacuole, peroxisomes, and cytoplasm are considered to be a plant part. Furthermore, any plant cell, whatever the tissue origin, is considered to be a plant part.
  • the transgenic plant or plant cell expressing a polypeptide of the present invention may be constructed in accordance with methods known in the art. Briefly, the plant or plant cell is constructed by incorporating one or more expression constructs encoding a polypeptide of the present invention into the plant host genome and propagating the resulting modified plant or plant cell into a transgenic plant or plant cell.
  • the expression construct is a nucleic acid construct which comprises a nucleic acid sequence encoding a polypeptide of the present invention operably linked with appropriate regulatory sequences required for expression of the nucleic acid sequence in the plant or plant part of choice.
  • the expression construct may comprise a selectable marker useful for identifying host cells into which the expression construct has been integrated and DNA sequences necessary for introduction of the construct into the plant in question (the latter depends on the DNA introduction method to be used).
  • regulatory sequences such as promoter and terminator sequences and optionally signal or transit sequences are determined, for example, on the basis of when, where, and how the polypeptide is desired to be expressed.
  • the expression of the gene encoding a polypeptide of the present invention may be constitutive or inducible, or may be developmental, stage or tissue specific, and the gene product may be targeted to a specific tissue or plant part such as seeds or leaves.
  • Regulatory sequences are, for example, described by Tague et al., 1988, Plant Physiology 86: 506.
  • the 35S-CaMV promoter may be used (Franck et al., 1980, Cell 21: 285-294).
  • Organ-specific promoters may be, for example, a promoter from storage sink tissues such as seeds, potato tubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or from metabolic sink tissues such as meristems (Ito et al., 1994, Plant Mol. Biol.
  • a seed specific promoter such as the glutelin, prolamin, globulin, or albumin promoter from rice (Wu et al., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoter from the legumin B4 and the unknown seed protein gene from Vicia faba (Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), a promoter from a seed oil body protein (Chen et al., 1998, Plant and Cell Physiology 39: 935-941), the storage protein napA promoter from Brassica napus, or any other seed specific promoter known in the art, e.g., as described in WO 91/14772.
  • a seed specific promoter such as the glutelin, prolamin, globulin, or albumin promoter from rice (Wu et al., 1998, Plant and Cell Physiology 39: 885-889)
  • the promoter may be a leaf specific promoter such as the rbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiology 102: 991- 1000, the chlorella virus adenine methyltransferase gene promoter (Mitra and Higgins, 1994, Plant Molecular Biology 26: 85-93), or the aldP gene promoter from rice (Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or a wound inducible promoter such as the potato pin2 promoter (Xu et al., 1993, Plant Molecular Biology 22: 573-588).
  • a promoter enhancer element may also be used to achieve higher expression of the variant of the present invention in the plant.
  • the promoter enhancer element may be an intron which is placed between the promoter and the nucleotide sequence encoding a polypeptide of the present invention.
  • Xu et al., 1993, supra disclose the use of the first intron of the rice actin 1 gene to enhance expression.
  • codon usage may be optimized for the plant species in question to improve expression (see Horvath et al referred to above).
  • the selectable marker gene and any other parts of the expression construct may be chosen from those available in the art.
  • the nucleic acid construct is incorporated into the plant genome according to conventional techniques known in the art, including Agrobacterium-mediated transformation, virus- mediated transformation, microinjection, particle bombardment, biolistic transformation, and electroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990, Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).
  • Agrobacterium tumefaciens-mediated gene transfer is the method of choice for generating transgenic dicots (for a review, see Hooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38). However it can also be used for transforming monocots, although other transformation methods are generally preferred for these plants.
  • the present invention also relates to methods for producing a polypeptide of the present invention comprising (a) cultivating a transgenic plant or a plant cell comprising a nucleic acid sequence encoding a variant of the present invention under conditions conducive for production of the variant; and (b) recovering the variant.
  • the polypetide variants of the invention may be prepared by (a) transforming a suitable host cell with a nucleotide construct of the invention, (b) cultivating the recombinant host cell of the invention comprising a nucleotide construct of the invention under conditions conducive for production of the variant of the invention and (c) recovering the variant.
  • the method may in a particular embodiment be carried out as described in WO 01/29078 (HESKA) describing recombinant expression of group 1 mite proteins including nucleotide sequences modified to en- able expression of the polypeptides in microorganisms.
  • Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus host cells are described in EP 238 023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81:1470-1474. A suitable method of transforming Fusarium species is described by Malardier et al., 1989, Gene 78:147- 156 or in copending US Serial No. 08/269,449. Examples of other fungal cells are cells of filamentous fungi, e.g., Aspergillus spp., Neurospora spp., Fusarium spp.
  • Trichoderma spp. in particular strains of A. oryzae, A. nidulans or A. niger.
  • the use of Aspergillus spp. for the ex- pression of proteins is described in, e.g., EP 272 277 and EP 230 023.
  • the transformation of F. oxysporum may, for instance, be carried out as described by Malardier et al., 1989, Gene 78: 147-156.
  • Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N. and Simon, M.I., editors, Guide to Yeast Genetics and Molecular Biology, Meth- ods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153:163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75:1920. Mammalian cells may be transformed by direct uptake using the calcium phosphate precipitation method of Graham and Van der Eb (1978, Virology 52:546).
  • the transformed or transfected host cells described above are cultured in a suitable nutrient medium under conditions permitting the production of the desired molecules, after which these are recovered from the cells, or the culture broth.
  • the medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g., in catalogues of the American Type Culture Collection). The media are prepared using procedures known in the art (see, e.g., references for bacteria and yeast; Ben- nett, J.W. and LaSure, L., editors, More Gene Manipulations in Fungi, Academic Press, CA, 1991).
  • the polypeptide variant of the invention is in an isolated and purified form.
  • the polypeptide variant of the invention is provided in a preparation which more than 20 %w/w pure, particularly more than 50% w/w pure, more particularly more than 75% w/w pure, more particularly more than 90% w/w pure and even more particularly more than 95% w/w pure.
  • the purity in this context is to be understood as the amount of polypeptide variant of the invention present in the preparation of the total polypeptide material in the preparation.
  • isolated indicates that the polypeptide is found in a condition other than its native environment, such as apart from blood and animal tissue.
  • the isolated polypeptide is substantially free of other proteins, particularly other proteins of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e., greater than 95% pure, more preferably greater than 99% pure. If the molecules are secreted into the nutrient medium, they can be recovered directly from the medium. If they are not secreted, they can be recovered from cell lysates. The molecules are recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g., ammonium sulphate.
  • a salt e.g., ammonium sulphate.
  • the proteins may be matured in vitro e.g., by acidification to induce autocatalytic processing (Jac- quet et al., Clin Exp Allergy, 2002, vol. 32 pp 1048-53), and they may be purified by a variety of chromatographic procedures, e.g., ion exchange chromatography, gelfiltration chromatography, affinity chromatography, or the like, dependent on the type of molecule in question (see, e.g., Protein Purification, J-C Janson and Lars Ryden, editors, VCH Publishers, New York, 1989). Activation of polymers
  • the variant of the invention is to be conjugated to one or more polymers and if the polymeric molecules to be conjugated with the polypeptide i n q uestion a re n ot active it must be activated by the use of a suitable technique. It is also contemplated according to the invention to couple the polymeric molecules to the polypeptide through a linker. Suitable linkers are well-known to the skilled person.
  • the functional groups being amino, hydroxyl, thiol, carboxyl, aldehyde or sulfydryl on the polymer and the chosen attachment group on the protein must be considered in choosing the activation and conjugation chemistry which normally consist of i) activation of polymer, ii) conjugation, and iii) blocking of residual active groups.
  • Coupling polymeric molecules to the free acid groups of polypeptides may be performed with the aid of diimide and for example amino-PEG or hydrazino-PEG (Pollak et al., (1976), J. Amr. Chem. Soc, 98, 289D291) or diazoacetate/amide (Wong et al., (1992), “Chemistry of Protein Conjugation and Crosslinking", CRC Press).
  • Coupling polymeric molecules to hydroxy groups are generally very difficult as it must be performed in water. Usually hydrolysis predominates over reaction with hydroxyl groups.
  • Coupling polymeric molecules to free s ulfhydryl groups can be reached with special groups like maleimido or the ortho-pyridyl disulfide.
  • vinylsulfone US patent no. 5,414,135, (1995), Snow et al.
  • Accessible Arginine residues in the polypeptide chain may be targeted by groups comprising two vicinal carbonyl groups.
  • Lysines may also be useful. Many of the usual leaving groups for alcohols give rise to an amine linkage. For instance, alkyl sulfonates, such as tresylates (Nilsson et al., (1984), Meth- ods in Enzymology vol. 104, Jacoby, W. B., Ed., Academic Press: Orlando, p. 56-66; Nilsson et al., (1987), Methods in Enzymology vol. 135; Mosbach, K., Ed.; Academic Press: Orlando, pp. 65-79; Scouten et al., (1987), Methods in Enzymology vol.
  • tresylates such as tresylates (Nilsson et al., (1984), Meth- ods in Enzymology vol. 104, Jacoby, W. B., Ed., Academic Press: Orlando, p. 56-66; Nilsson et al., (1987), Methods in Enzymology vol
  • Organic sulfonyl chlorides e.g., Tresyl chloride
  • Tresyl chloride effectively converts hydroxy groups in a number of polymers, e.g., PEG, into good leaving groups (sulfonates) that, when reacted with n ucleophiles I ike a mino g roups i n polypeptides a Now stable l inkages to be formed between polymer and polypeptide.
  • the reaction conditions are in general mild (neutral or slightly alkaline pH, to avoid denaturation and little or no disruption of activity), and satisfy the non-destructive requirements to the polypeptide.
  • Tosylate is more reactive than the mesylate but also more unstable decomposing into PEG, dioxane, and sulfonic acid (Zalipsky, (1995), Bioconjugate Chem., 6, 150-165). Epoxides may also been used for creating amine bonds but are much less reactive than the above mentioned groups.
  • the derivatives are usually made by reacting the chloroformate with the desired leaving group. All these groups give rise to carbamate linkages to the peptide. Furthermore, isocyanates and isothiocyanates may be employed yielding ureas and thioureas, respectively.
  • Amides may be obtained from PEG acids using the same leaving groups as mentioned above and cyclic imid thrones (US patent no. 5,349,001, (1994), Greenwald et al.). The reactivity of these compounds is very high but may make the hydrolysis to fast.
  • PEG succinate made from reaction with succinic anhydride can also be used.
  • the hereby comprised ester group make the conjugate much more susceptible to hydrolysis (US patent no. 5,122,614, (1992), Zalipsky). This group may be activated with N-hydroxy succinimide.
  • polypeptides do not comprise many Lysines it may be advantageous to attach more than one PEG to the same Lysine. This can be done e.g., by the use of 1 ,3-diamino-2- propanol.
  • PEGs may also be attached to the amino-groups of the polypeptide with carbamate linkages (WO 95/11924, Greenwald et al.). Lysine residues may also be used as the back- bone.
  • the coupling technique used in the examples is the N-succinimidyl carbonate conju- gaion technique descried in WO 90/13590 (Enzon).
  • the present invention also relates to a composition
  • a composition comprising a variant of the inven- tion a nd o ptionally a p harmaceutically a cceptable carrier and/or adjuvant a nd a method for preparing such a composition comprising admixing the variant of the invention with an acceptable pharmaceutical carrier and/or adjuvant.
  • the composition is suitable for treating an immunological disorder, such as allergy in animals or humans, such as a vaccine.
  • Pharmaceutical carriers and/or adjuvants includes saline, glycerol, aluminium hydroxide, aluminium phosphate, calcium phosphate, saponins (e.g., Q21 and Quill A), squalene based emulsions (e.g., MF59), monophosphoryl lipid A (and synthetic mimics), polylactide co- glycolid (PLG) particles, ISCOMS, liposomes, chitosan, bacterial DNA (e.g., unmethylated CpG containing sequences).
  • S also i nclude p harmaceutically a cceptable s ol- vents and/or tabletting aids/auxilliaries.
  • the invention provide use of the variant or the composition of the invention as a medicament, particularly for the treatment of an immunological disorder, such as allergy in animals and humans and/or for the preparation of a medicament for the treatment of such immunological disorder.
  • allergy vaccination is performed by parenteral, i ntranasal, or sublingual administration in increasing doses over a fairly long period of time, and results in, so called, desensitisation of the patient.
  • the exact immunological mechanism is not known, but induced differences in the phenotype of allergen specific T and B cells are thought to be of particular importance.
  • allergy vaccination is complicated by the existence of an ongoing immune response in allergic patients. This immune response is characterised by the presence of allergen specific IgE, that will mediate the release of allergic mediators, thereby inducing allergic symptoms upon exposure to allergens.
  • allergen specific IgE allergen specific IgE
  • First category of measures includes the administration of several small and increasing doses over a long period to reach a substantial accumulated dose. The theory being, that the protective immune response slowly is allowed to be initiated, before potentially anaphylactic doses of allergen is administrated.
  • Second category of measures includes physical modification of the allergen by incorporation of the allergen into e.g., a gel formulation such as a aluminium hydroxide. Aluminium hydroxide has an adjuvant effect and a depot effect of slow allergen release, thus reducing the the tissue concentration of the allergen.
  • Third category of measures include as described herein modification of the allergen for the purpose of reducing allergenicity.
  • the immunotherapeutic effect of an allergy vaccine can be assessed in a number of different ways.
  • several cellular assays could be employed to show the modified immunere- sponse indicative of good allergy vaccine potential as shown in several publications, all of which are hereby incorporated by reference (van Neerven et al., "T lymphocyte responses to allergens: Epitope-specificity and clinical relevance", Immunol Today, 1996, vol. 17, pp. 526- 532; Hoffmann et al., Allergy, 1999, vol. 54, pp. 446-454, WO99/07880).
  • Basophil histamine release e.g., Swoboda et al., Eur. J. Immunol., vol. 32, pp 270-280, 2002.
  • Sandwich ELISA Immunoplates (Nunc Maxisorb; Nunc-Nalgene) are coated overnight at 4 degree C with at suitable dose of polyclonal rabbit anti Der p 1 antibody. The plates are then washed thoroughly with 0.15 M Phosphate Buffered Saline (PBS) containing 0.05 % Tween 20 (PBST), and remaining binding sites are blocked with PBS with 2 % skim milk powder, 1 h at room temperature. Samples, it can be purified, semi-purified recombinant group 1 mite polypeptide variant allergen or crude culture broth containing protein of interest, are added in a suitable dose or dose-range.
  • PBS Phosphate Buffered Saline
  • PBST 0.05 % Tween 20
  • the plates are then washed thoroughly with 0.15 M PBST before the allergens are detected by incubation with biotinylated monoclonal anti Der p 1 antibody (INDOOR) 1 h at room temperature. Wash again in 0.15 M PBST. Conjugate with complexes of Streptavidin: Horse Radish Peroxidase (Kierkegaard & Perry) for 1 h at room temperature. Repeat washing step and develop by adding 3,3',5,5'-tetramethylbenzidine hydrogen peroxide (TMB Plus, Kem-En-Tec) and stop reaction by addition of 0.2 M H2S04.
  • TMB Plus 3,3',5,5'-tetramethylbenzidine hydrogen peroxide
  • OD450 will reflex allergen binding to the immunoglobin, and it is thus possible to detect and also determine the amount of allergen bound if natural Der p 1 (available from Indoor biotechnologies, product number: NA-DP1) in known concentrations is included in the experiment in a dose rage.
  • natural Der p 1 available from Indoor biotechnologies, product number: NA-DP1
  • Example 1 Finding of epitope patterns within oligo peptides with antibody reactivity
  • the phage libraries were obtained from Schafer-N, Copenhagen, Denmark.
  • Antibody samples were raised in animals (Rat, Rabbits or Mice) by parenteral or muco- sal administration of each of the proteins listed below.
  • the antibodies were dissolved in phos- phate buffered saline (PBS).
  • PBS phos- phate buffered saline
  • antibodies of specific subclasses were purified from the serum of immunised animals by capryilic acid precipitation (for total IgG) or by affinity chromatography using paramagnetic immunobeads (Dynal AS) loaded with one of the following antibodies: mouse anti-rat lgG1 or rat anti-mouse IgE.
  • alpha-amylase of Bacillus halmapalus W096/23873
  • amylase SP722 amylase SP722
  • T. lanuginosus lipase (LipolaseTM) (Rat IgG and Rabbit IgG), 6) family 45 cellulase from Humicola insolens (CarezymeTM) (Rabbit IgG),
  • Bacillus lentus protease (SavinaseTM) (Rat IgG, Mouse IgG, Mouse IgE, and Rabbit IgG),
  • Subtilisin 147 (EsperaseTM) (Rat IgG)
  • Bacillolysin from Bacillus amyloliquefaciens (NeutraseTM) (Rat IgG and Rat lgG1)
  • Subtilisin PD498 (WO 93/24623) (Rat IgG and Rabbit IgG).
  • the phage libraries were incubated with the antibody coated beads.
  • E.g. phages ex- pressing oligo-peptides with affinity for mouse IgE antibodies were captured onto rat anti- mouse IgE-coated beads and collected by exposing these paramagnetic beads to a magnetic field.
  • the collected phages were eluted from the immobilised antibodies by mild acid treatment, or by elution with intact protein antigen specific for the respective antibody sample (e.g., Savinase for anti-Savinase antibodies).
  • the isolated phages were amplified using methods known in the art.
  • immobilised phages were directly incubated with E.coli for infection. In short, F-factor positive E.
  • coli e.g., XL-1 Blue, JM101 , TG1
  • M13- derived vector in the presence of a helper phage (e.g., M13K07), and incubated, typically in 2xYT containing glucose or IPTG, and appropriate antibiotics for selection. Finally, cells were removed by centrifugation. This cycle of events was repeated on the respective cell super- natants, minimum 2 times and maximum 5 times. After selection round 2, 3, 4 and 5, a fraction of the infected E.coli was incubated on selective 2xYT agar plates, and the specificity of the emerging phages was assessed immunologically: Phages were transferred to a nitrocellulase (NC) membrane.
  • NC nitrocellulase
  • NC-replicas were made. One replica was incubated with the selection antibodies, the other replica was incubated with the selection antibodies and the immunogen used to obtain the antibodies as competitor. Those plaques that were absent in the presence of immunogen, were considered specific, and were am-plified according to the procedure described above.
  • the specific phage-clones were isolated from the cell supernatant by centrifugation in the presence of polyethylenglycol. DNA was isolated, the DNA sequence coding for the oli- gopeptide was amplified by PCR, and its DNA sequence was determined, all according to standard procedures known in the art. The amino acid sequence of the corresponding oli- gopeptide was deduced from the DNA sequence.
  • each of the the 658 reactive (oligo)peptide sequences represented an epitope p attern.
  • H owever, i n the 658 reactive ( oligo)peptide s equences s ome e pitope patterns were redundant and to remove redundency among the epitope patterns, the reactive (oligo)peptides sequences were subjected to computerised data analysis.
  • the frequency of each peptide combination among the 658 reactive (oligo)peptide sequences were then ranked and relevant epitope patterns were selected by a procedure where reactive ranked and relevant epitope patterns were selected by a procedure where reactive peptides covered by the most frequent combination were first selected and separated from the group of the 658 reactive peptides. Then reactive peptides covered by the second most frequent combination were selected and separated from the remaining group.
  • the Der p 1 model was built using the following three-dimensional structures as templates:
  • Modeler 5.0 The "Modeler 5.0" program (Accelrys Software, San Diego, CA, USA) was used to build the three-dimensional model of Der p 1. "Modeler 5.0” was started from the "Insightll” molecular modelling software (Accelrys Software, San Diego, CA, USA) using the following parameters: Number of models: 1 , Optimize level: None, More options: Yes, Optimize loop: Yes, Number of loop models: 2, Loop optimite level: Low, Build hydrogens: None.
  • P_1PP0 struct reX:P 1PP0:1 : 2 16 : : unknow : unknown : -1.00 : -1.00
  • Epitopes were predicted by a computer program on a 3-dimensional model of Der p 1 (SEQ ID NO:1) by using the epitope patterns found in example 1 as follows:
  • a limit of 25 A was set as the maximum distance between any two epitope residues.
  • Der p 1 holds an 18 amino acids signal peptide and an 80 amino acids propeptide while the mature Der p 1 is a 222 amino acid molecoule. In the alignement a gap has been made in po- sition 8 of Der p 1 because it lacks an amino acid here compared to other group 1 mite polypeptides. Similar descriptions may be made for Eur ml , while for Der f 1 only the mature polypeptide is shown and for Der m 1 only a fraction of the sequence has been identified.
  • Example 4 Construction and Expression of Enzyme Variants Der p 1 variants of the invention comprising specific substitutions were made by cloning of DNA fragments (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989) produced by PCR with oligos containing the desired mutations.
  • the template plasmid DNA may be pSteD212, or an analogue of this containing Der p 1 or a variant of Der p 1. Mutations were introduced by oligo directed mutagenesis to the construction of variants.
  • the Der p 1 plasmid constructs were transformed into S. cerevisiae, strain JG169, as described by Becker and Guarente (1991 , Methods Enzymology, 194: 182- 187).
  • the Cystein protease or variants hereof of the present invention were located in vector pSteD212, which is derived from yeast expression vector pYES 2.0 (Kofod et al. 1994 J. Biol. Chem. 269: 29182-29189 and Christgau et al. 1994, Biochem. Mol. Biol. Int. 33: 917-925).
  • This plasmid replicated both in E. coli and in S. cerevisiae. In S. cerevisiae Der p 1 or variants hereof according to the invention were expressed from this plasmid.
  • the transformation solution was plated on SC-agar plates for colony formation at 30 degree C, 3 days. Colonies were inoculated in 96 micro-well plates, each well containing 200 microL SC medium. The plates were fermented at 30 degree C, 250 r.p.m. for 5 days.
  • a part of the variants were concentration determined d irectly i n culture b roth by the sandwich ELISA technique with natural Der p 1 as a standard.
  • Ployclonal antibodies were raised in Rabbits against Native Der P1 bought from Indoor technologies.
  • the polyconal antibodies were purified by ammonium sulphate precipitation and on Protein a column as described in literature and finally dialyzed against 50 mM Borate pH 8 buffer.
  • the purified antibodies against Der P1 were labelled with Biotin using NHS-Biotin as described in Product sheet described by Pierce Chemicals 3747 N. Meridian Rd.PO Box117. Rockford, 1161105 USA, and the labelled antibodies were used as detecting antibodies.
  • Method for fast qualitative detection of Der P1 or Pro Der P1 was as follows. Immunosrop microtiter plates were bought from NUNC and microtiter wells were coated with 100 microlitres of 10 microgram per ml unlabelled polyclonal antibodies against Der P1 for overnight at 4 degree centigrade. The microtiter wells were then washed with PBS Tween buffer as described in literature. Microtiter wells were then saturated with 200 microlitres of PBS buffer containing 10 milligrams per millilitres BSA and 0.05 % Tween 20 and incubated for 30 minutes at room temperature.
  • Microtiter wells were then washed thrice with PBS buffer containing 0.05 % Tween 20.
  • Microtiter wells were then coated with 100 microlitres fractions containing Der P1 or Pro-Der P1 and incubated for 20 minutes with gentle shaking. Microtiter wells were then washed thrice with PBS buffer containing 0.05 % Tween 20. Microtiter wells were then coated with 100 microlitres of biotin labelled polyclonal antibodies around 1 microgram per millilitres diluted in PBS buffer with 0.05 % Tween 20 and incubated for 20 minutes at room temperature with gentle shaking.
  • Microtiter wells were then washed thrice with PBS buffer and coated with 100 micro- litres of properly diluted Immunopure Avidin Horse radish peroxidase conjugate which was purchased from Pierce chemicals. After 20 minutes incubation wait room temperature the wells were then washed with PBS buffer containing 0.05 % Tween 20.
  • This method can be used as qualitative assay for detection of Der P1 or Pro Der P1.
  • Sterile filtered cell supernatant containing the desired protein was then concentrated using Ultra filtration technique using 10 kDa cut off membrane purchased from from Millipore Corporation, Bedford. MA 01730 USA: The small molecules under 10 kDa were then removed by dia filtration using 50 mM Borate pH 8 as buffer.
  • the concentrated fermentation supernatant was then applied on the column with a flow of 20 mL per minute. Unbound material was then washed out using 1 M ammonium sulphate dissolved in the borate pH 8 buffer (Buffer A). When all the unbound material was washed out from the column which was monitored using UV detector attached to fraction collector from Amesham Pharmacia.
  • Bound proteins were then eluted with buffer B which contained 50 mM Borate pH 8 without any other salt and 10 ml fractions were collected. Fractions contain desired protein were checked by SDS-PAGE. Fractions containing Protein with molecular weights between 33 kDa and 22 kDa and found immunoreactive in the qualitative as described above were then pooled and further purified on anion exchange chromatography.
  • Bound proteins were then eluted with linear gradient using buffer B containing 50 mM Borate pH 8 with 1 M salt as Sodium chloride. Total buffer used was 20 column volumes All the fractions were then analysed by SDS-PAGE and qualitative ELISA assay. Proteins with molecular weight around 30 kDa were then pooled as Pro-Der P1 and mature Der P1 due to slight processing was observed as 20 kDa Protein. The purified proteins were then analysed for N-terminal after SDS-PAGE and blotting on PVDF membrane by Using applied Bio system equipment.
  • Example 8 In vivo assessment of allergenicity of an enzyme variant (MINT assay)
  • Mouse intranasal (MINT) model (Robinson et al., Fund. Appl. Toxicol. Vol. 34, pp. 15- 24, 1996) can be used to verify allergenicity of group 1 mite polypeptide variants.
  • mice are dosed intranasally with the group 1 mite polypeptide variant on the first and third day of the experiment and from thereon on a weekly basis for a period of 6 weeks. Blood samples are taken 15, 31 and 45 days after the start of the study, and the serum can subsequently be analysed for IgE levels.
  • the relative concentrations of specific IgE antibodies in serum samples from mice are measured by a three layer sandwich ELISA according to the following procedure: 1) The ELISA-plate (Nunc Maxisorp) is coated with 100 microlitre/well rat anti-mouse IgE
  • Specific polyclonal anti-group 1 mite polypeptide variant antiserum serum (pig) for detecting bound antigen is diluted in 0.15 M PBS buffer with 0.15% skim milk and 0.05% Tween20. 100 microlJwell and incubated for 1 hour at 4°C. The plates are washed as before.
  • the dose response curves are graphed, and fitted to a sigmoid curve using non-linear regression, and the EC50 is calculated for the group 1 mite polypeptide variant.
  • the relative concentrations of specific lgG1 antibodies in serum samples from mice are measured by a three layer sandwich ELISA according to the following procedure:
  • the ELISA-plate (Nunc Maxisorp) is coated with 100 microlitre/well of group 1 mite polypeptide variant diluted in PBS to 10 microg/ml. Incubated over night at 4°C.
  • TMB Plus (Ready-to-go substrate; Kem-En-Tec, Cat. No.: 4390A) is added, and the reaction is allowed to run for 10 min.
  • Example 9 In vitro assessment of IgE-antigenicity of an enzyme variant
  • Immunoplates (Nunc Maxisorp; Nunc-Nalgene) were coated overnight at 4°C with a suitable dose, or dose-range, of group 1 mite polypeptide allergen or with recombinant group 1 mite polypeptide variant allergen. The plates were then washed thoroughly with Phosphate Buffered Saline (PBS) containing 0,05% Tween 20 (PBST), and remaining binding sites were blocked with PBS containing 2% Skim Milk Powder (SMP).
  • PBS Phosphate Buffered Saline
  • PBST Phosphate Buffered Saline
  • SMP Skim Milk Powder
  • Sera from patients allergic to dust mites were then diluted 1/4 in PBST and added to the plates and incubated at room temperature (RT) for 1 hour or overnight at 4degree C. Following a thorough wash with PBST, the allergen-lgE complexes were detected, by incubation with a rabbit anti-human I gE antibody (DakoCytomation), and swine anti-rabbit Ig coupled to horseradish peroxidase.
  • the enzymatic activity was measured by adding TMB from Kem-En-Tec, and the reaction was stopped by adding an equal volume of 0.2 M H2S04, and quantitaing colour development by measuring optical density at 450 nm (OD450) in an ELISA reader.
  • OD450 optical density at 450 nm
  • nDer p 1 -specific IgE-binding in serum isolated from 23 patients allergic to dust mite allergens were analysed in a dose response curve to nDer p 1 in a direct ELISA.
  • OD450 values obtaining a coating concentration of 500 n g/well a llergen were d etermined, a nd sera that d id not reach a n OD450 equal or higher than 0.5 were excluded from further analysis in the competitive ELISA.
  • Sera from donor 1 , 3, 6, 7, 9, 13, 14, 22 and 23 were chosen for analysis in competitive ELISA
  • OD450 titer obtaining a coating concentration of 500 ng/well group 1 mite wild type polypeptide was determined in sera isolated from 23 patients allergic to dust mite and are shown in the table below.
  • nDer p 1 -specific IgE-binding in serum isolated from 23 patients allergic to dust mite allergens were analysed in a dose response curve to nDer p 1 in a direct ELISA.
  • OD450 values correlating to a coating concentration of 500 ng/well allergen was determined and sera that did not reach an OD450 equal or higher than 0.5 were excluded from further analysis in the competitive ELISA.
  • sera from donor 1, 3, 6, 7, 9, 13, 14, 22 and 23 were chosen for analysis of the individual variants by competitive ELISA. Data from the full dose-response curve for different variant concentrations confirmed this selection (data not shown).
  • Table 2 Dose-response curves in nDer p 1 -specific IgE serum isolated from 9 dust mite allergic patients were plotted and fitted to a sigmoid curve, and the EC50 was calculated for the group 1 mite polypeptide variants.
  • Table 3 Dose response curves in nDer p 1 -specific IgE serum isolated from 6 patients with dust-mite allergy were plotted and fitted to a sigmoid curve, and the EC50 was calculated for group 1 mite polypeptide variants.
  • the data disclosed in Table 2 and Table 3 show that the three variants DP024, DP026, and DP070 have a highly reduced IgE binding as compared to the group 1 mite polypeptide.
  • the mutations introduced in the DP024 variant lowers the affinity for specific serum IgE by a factor of about 8 to 22-fold
  • the mutations introduced in DP026 lowers the affinity for specific serum IgE by a factor of 3 to 39-fold
  • the mutations introduced in the DP070 lowers the affinity for specific serum IgE by a factor of about 23-46, compared to the response of nDer p 1.
  • the data further indicates that the variants DP065 and DP071 have a reduced IgE binding compared to the response of nDer p 1.
  • Histamine release from basophil leukocytes was performed as follows. Heparinized blood (40 mL) was drawn from each dust-mite allergic patient, stored at room temperature, and used within 24 hours. Twenty-five microliters of heparinized blood was applied to glass fibre coated microtitre wells (Reference Laboratory, Copenhagen, Denmark) and incubated with 25 microliters of a dose-range of wild type polypeptide, recombinant wild type, variant allergen, House Dust mite (HDM) extract or anti-lgE for 1 hour at 37degree C. All serial-dilutions of al- lergen were made in PIPES-buffer (Reference Laboratory, Denmark). T hereafter the plates were rinsed with water and interfering substances were removed. Finally, histamine bound to the microfibres was measured spectrophotofluometrically. The results are interpreted using the following formula:
  • Basophile cells from 23 patients allergic to dust-mite and 3 negative donors (negative to histamine release on stimulation with house d ust-mite ( HDM) extract (ALK-Abello)) were analysed in a histamine release assay on stimulation with group 1 mite polypeptide and group 1 mite polypeptide variants.
  • group 1 mite polypeptide and group 1 mite polypeptide variants Of the 23 dust mite allergic patients, only 14 patients were found to induce histamine release in response to stimulation with nDer p 1 , demonstrating that ap- proximately 61 % o f t he p atients a llergic t o d ust-mite h ave n Der p 1 -specific I gE a ntibodies (data not shown).
  • FIG. 1 shows the potency of rec-proDer p 1, rec-Der p 1 and DP070 variants to induce histamine release in a human basophile cell assay from one dust-mite allergic patient. It is seen that the release curve of DP070 variant is clearly shifted to the right compared to the release curve of nDer p 1, rec-proDer p 1 and rec-Der p 1. The shift indicates that the potency of DP070 variant to induce histamine release is reduced about a 22-fold fold relative to nDer p 1. The shift of the DP070 release curve to the right was found in all the 14 pa- tients with nDer p 1 -specific IgE-mediated responses with the corresponding potency reductions ranging from 2 to 22-fold.
  • Basophile cells from the 9 remaining dust mite allergic patients did not respond to stimulation with concentrations of the group 1 mite polypeptide up to 1.67 ⁇ g/ml (data not shown). How- ever, at the highest concentration of group 1 mite polypeptide (20 ⁇ g/ml), histamine release was observed from basophile cells from these patients. This induction of histamine release in high concentration of nDer p 1 may be due to low levels of impurities of commercial nDer p 1 and thus, the presence of other dust mite allergens. No histamine release was observed in basophile cells from these 9 patients in response to stimulation with group 1 mite polypeptide variants (data not shown).
  • Basophile cells from the 3 negative donors did not respond to stimulation with group 1 mite polypeptide or to group 1 mite polypeptide variants, demonstrating no unspecific stimulation of the crude extract.
  • the data disclosed in Figure 2 demonstrate the overall reduction in IgE antigenicity as measured by histamine release assay.
  • the ratio of EC50 value for the variant to the EC50 value of nDer p 1 was calculated.
  • all donors show a normalized value of 1 (left column).
  • a control series of nDer p 1 samples were included and treated as a normal sample.
  • the result for this se- ries is shown in the rightmost column, and demonstrates a relatively low variability, considering this is a rather sensitive biological response assay.
  • the variants DP024 and DP070 show average improvement factors of around 5 and around 6,7, respectively.
  • the variants DP071, DP065, DP033, and DP026 show improvents in IgE-based antigenicity, as measured by the increase in EC50 value, in most of the donors.
  • basophil histamine release is described in Nolte H, Schiotz O, Skov PS.
  • Basophil histamine release, IgE, eosinophil counts, ECP, and EPX are related to the severity of symptoms in seasonal allergic rhinitis. Allergy. 1999 May;54(5):436-45.
  • recombinant allergens e.g., 1 mg protein/mL
  • suitable serial dilutions e.g., from 100 ⁇ g to 0.1 microg/mLI
  • sterile physiologic saline solution e.g., from 100 ⁇ g to 0.1 microg/mLI
  • These dilutions are selected ac- cording to the concentration allergens, which elicited significant histamine release by sensitized basophils. It has been shown that the thresholds of positivity for histamine release tests and intradermal reactions are in the same range; and it is assumed that the sensitivity of prick tests is 10 2 to 10 3 times lower than that of intradermal tests.
  • a negative control test is performed with saline solution, and a positive control test is done with histamine at e.g., 1 mg/mL.
  • the diameter of the weal is used as a measure of allergenic reactivity towards that variant, and this allows for comparison of the variant allergens to the parent or native type allergen.
  • lymphocyte fraction from heparinized blood from patients allergic to group 1 mite polypeptides was purified by density gradient centrifugation on Lymphoprep (Axix-Shield PoC, Norway) and resuspended in AIM-V (Invitrogene) and plated at a cellular density of 2.5 x 105 cells/well in a 96 sterile tissue-culture plate (Nunclon Delta). Serial dilution of wild type group 1 mite polypeptide and group 1 mite polypeptide variant allergens were made up in growth media and added to the cells, together with a media-only control. The plates were then incubated for 7 days at 37degree C, 5% C02, 100% humidity.
  • T cell prolif- eration was measured by the incorporation of 3[H]-thymidine.
  • 3[H]- thymidine 0.5 ⁇ Ci per well was added.
  • the cells were harvested onto glass fiber filters, and 3[H]-thymidine incorporation was measured in scintillate counter.
  • Proliferation was expressed as mean counts per miute (cpm) of 3[H]-thymidine incorporation of triplicate or duplicate wells.
  • the stimulation index (SI) was calculated as the quotient of the cpm obtained by allergen stimulation and the unstimulated control (media-only control). SI is shown for each donor and a selection of group 1 mite polypeptide variants in table 4.
  • Donor 1 - 23 represents the 23 dust mite allergic patients, whereas donor 24-26 represents the 3 negative donors.
  • Table 4 Stimulation indexes for T cell proliferation in response to stimulation with group 1 mite polypeptide and group 1 mite polypeptide variants. Unless otherwise stated in the table, analysis of T cell proliferation was carried out on stimulation with 1.0 ⁇ g/ml group 1 mite polypeptide or group 1 mite polypeptide variants.
  • Example 11 Epitope Mapping based on human anti-Der p 1 antiserum
  • Anti-hlgE antibodies were covalently linked to commercially available tosyl-activated paramagnetic beads. After inactivation of the remaining linkage sites and washing of the beads according to manufacturer's specification, the anti-hlgE-beads were incubated overnight at 4°C with pooled sera from patients sensitized to Der p 1 (the sera were 3x diluted in dilution buffer (PBS at pH 6.6)). 200 microlitre beads were washed 3x with 40ml of washing buffer (PBS at pH6.6 plus 0.05% Tween20), then incubated with PBS supple- mented with 2 % skim milk for an hour at room temperature and washed 3x as before.
  • PBS washing buffer
  • Epitope mimicking peptides were isolated from commercially available phage display libraries of either 7mer, constrained 7mer or 12mer peptide libraries (New England Biolabs). The anti-Der p 1 -beads were incubated with 2*10 11 library phages for 4h at room temperature, after which unbound phages were removed by extensive washing. To avoid the enrichment of peptides that were bound to either plain beads or to the anti-hlgE antibody, a specific elution procedure was implemented: After washing, beads were first incubated with PBS supplemented with 0.5% skim milk for an hour at room temperature.
  • phages were eluted from the beads by incubation with 25 microM purified Der p 1 in PBS supplemented with 0.5% skim milk, again lasting an hour at room temperature. Only phages in the supernatant of this elution were propagated further. Selected phages were amplified according to the guidelines of the library manufacturer (NEB user manual) after a first round of selection using ER2738 cells. After a second round, cells were infected and spread out for iso- lation of phages, which were subsequently tested for binding and sequenced.
  • Phage ELISA to test binding to serum IgE Phage ELISA to test binding to serum IgE:
  • HRP mouse anti M13-phage antibody-horseradish peroxidase

Abstract

L'invention concerne des variants de polypeptides d'acariens du groupe 1. Le polypeptide mature du variant comprend une ou plusieurs mutations situées dans les positions ou correspondant aux positions incluant A10, A12, E13, G29, G30, G32, A46, Y47, S54, L55, D64, A66, S67, G73, T75, I80, Q84, N86, G87, S92, Y93, Y96, A98, R99, E100, Q101, R104, R105, P106, Q109, R110, F111, G112, I113, A132, I144, K145, D146, D148, R151, I158, I159, Q160, R161, D162, N163, G164, Y165, Q166, N179, A180, G182, V183, D184, A205, I208 de SEQ ID NO: 1 ou 10, 12, 13, 29, 30, 32, 46, 47, 54, 55, 64, 66, 67, 73, 75, I80, 84, 86, 87, 92, Y93, 96, 98, 99, 100, 101, 104, 105, 106, 109, 110, 111, 112, 113, 132, 144, 145, 146, 148, 151, 158, 159, 160, 161, 162, 163, 164, 165, 166, 179, 180, 182, 183, 184, 205, 208 du polypeptide Der p 1 mature.
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