Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/53—DNA (RNA) vaccination
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to diagnosis and treatment of allergy. More specifically the invention provides ways of obtaining mutated allergen molecules suitable for these purposes.
• the invention furthermore relates to novel recombinant allergens, which are mutants of naturally occurring allergens as well as their use.
• the invention relates to a composition comprising a mixture of novel recombinant mutant allergens.
• the invention relates to a method of preparing such recombinant mutant allergens as well as to pharmaceutical compositions, including vaccines, comprising the recombinant mutant allergens.
• the present invention relates to methods of generating immune responses in a subject, vaccination or treatment of a subject as well as processes for preparing the compositions of the invention.
• Allergic responses range from hay fever, rhinoconductivitis, rhinitis and asthma to systemic anaphylaxis and death in response to e.g. bee or hornet sting or insect bite.
• the reaction is immediate and can be caused by a variety of atopic allergens such as compounds originating from grasses, trees, weeds, insects, food, drugs, chemicals and perfumes.
• the responses do not occur when an individual is exposed to an allergen for the first time.
• the initial adaptive response takes time and does usually not cause any symptoms. But when antibodies and T cells capable of reacting with the allergen have been produced, any subsequent exposure may provoke symptoms.
• allergic responses demonstrate that the immune response itself can cause significant pathological states, which may be life threatening.
• the antibodies involved in atopic allergy belong primarily to immunoglobulins of the IgE class.
• IgE binds to specific receptors on the surface of mast cells and basophils. Following complex formation of a specific allergen with IgE bound to mast cells, receptor cross-linking on the cell surface results in signalling through the receptors and the physiological response of the target cells. Degranulation of a mast cell results in the release of i.a. histamine, heparin, a chemotactic factor for eosinophilic leukocytes, leukotrienes C4, D4 and E4, which cause prolonged constriction of the bronchial smooth muscle cells. The resulting effects may be systemic or local in nature.
• the antibody-mediated hypersensitivity reactions can be divided into four classes, namely type I, type II, type III and type IV.
• Type I allergic reactions is the classic immediate hypersensitivity reaction occurring within seconds or minutes following antigen exposure. These symptoms are mediated by allergen specific IgE.
• allergic reactions are observed as a response to protein allergens present e.g. in pollens, house dust mites, animal hair and dandruff, venoms, and food products.
• allergy vaccines In order to reduce or eliminate allergic reactions, carefully controlled and repeated administration of allergy vaccines is commonly used. Allergy vaccination is traditionally performed by parenteral, intranasal, or sublingual administration in increasing doses over a fairly long period of time, and results in desensitisation of the patient. The exact immunological mechanism is not known, but induced differences in the phenotype of allergen specific T ceils is thought to be of particular importance.
• Vaccination will prime the immune system of the recipient, and upon repeated exposure to similar proteins the immune system will be in a position to respond more rigorously to the challenge of for example a microbial infection.
• Vaccines are mixtures of proteins intended to be used in vaccination for the purpose of generating such a protective immune response in the recipient.
• the protection will comprise only components present in the vaccine and homologous antigens.
• 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 mediating the release of allergic symptoms upon exposure to allergens.
• allergens from natural sources has an inherent risk of side effects being in the utmost consequence life threatening to the patient.
• First category of measures includes the administration of several small doses over prolonged time to reach a substantial accumulated dose.
• Second category of measures includes physical modification of the allergens by incorporation of the allergens into gel substances such as aluminium hydroxide. Aluminium hydroxide formulation has an adjuvant effect and a depot effect of slow allergen release reducing the tissue concentration of active allergen components.
• Third category of measures include chemical modification of the allergens for the purpose of reducing allergenicity, i.e. IgE binding.
• Th1 and Th2 determine the allergic status of an individual.
• Th1 cells Upon stimulation with allergen Th1 cells secrete interleukines dominated by interferon- ⁇ leading to protective immunity and the individual is healthy.
• Th2 cells o ⁇ the other hand secrete predominantly interleukin 4 and 5 leading to IgE synthesis and eosinophilia and the individual is allergic.
• IgE synthesis and eosinophilia In vitro studies have indicated the possibility of altering the responses of allergen specific T cells by challenge with allergen derived peptides containing relevant T cell epitopes.
• Current approaches to new allergy vaccines are therefore largely based on addressing the T cells, the aim being to silence the T cells (anergy induction) or to shift the response from the Th2 phenotype to the Th1 phenotype.
• B-cell epitopes Antibody-binding epitopes (B-cell epitopes)
• antibody-binding epitopes can be defined as a section of the surface of the antigen comprising atoms from 15-25 amino acid residues, which are within a distance from the atoms of the antibody enabling direct interaction.
• the affinity of the antigen-antibody interaction can not be predicted from the enthalpy contributed by van der Waals interactions, hydrogen bonds or ionic bonds, alone.
• the entropy associated with the almost complete expulsion of water molecules from the interface represent an energy contribution similar in size. This means that perfect fit between the contours of the interacting molecules is a principal factor underlying antigen-antibody high affinity interactions.
• WO 97/30150 a population of protein molecules is claimed, which protein molecules have a distribution of specific mutations in the amino acid sequence as compared to a parent protein. From the description, it appears that the invention is concerned with producing analogues which are modified as compared to the parent protein, but which are taken up, digested and presented to T cells in the same manner as the parent protein (naturally occurring allergens). Thereby, a modified T cell response is obtained. Libraries of modified proteins are prepared using a technique denoted PM (Parsimonious Mutagenesis).
• recombinant DNA molecules are described, which molecules comprise a DNA coding for a polypeptide having at least one epitope of an allergen of trees of the order Fagales, the allergen being selected from Aln g 1 , Cor a 1 and Bet v 1.
• the recombinant molecules described herein do all have an amino acid sequence or part of an amino acid sequence that corresponds to the sequence of a naturally occurring allergen.
• WO 90/11293 (ref. 3) relates i.a. to isolated allergenic peptides of ragweed pollen and to modified ragweed pollen peptides.
• the peptides disclosed therein have an amino acid sequence corresponding either to the sequence of the naturally occurring allergen or to naturally occurring isoforms thereof.
• allergens More recent approaches to chemical modification of allergens aim at a total disruption of the tertiary structure of the allergen thus eliminating IgE binding assuming that the essential therapeutic target is the allergen specific T cell.
• Such vaccines contain allergen sequence derived synthetic peptides representing minimal T cells epitopes, longer peptides representing linked T cells epitopes, longer allergen sequence derived synthetic peptides representing regions of immunodominant T cell epitopes, or allergen molecules cut in two halves by recombinant technique.
• Another approach based on this rationale has been the proposal of the use of "low IgE binding" recombinant isoforms.
• the article by Ferreira et al discloses the use of site directed mutagenesis for the purpose of reducing IgE binding.
• Bet v 1 the three- dimensional structure of Bet v 1 is mentioned in the article the authors do not use the structure for prediction of solvent exposed amino acid residues for mutation, half of which have a low degree of solvent exposure. Rather they use a method developed for prediction of functional residues in proteins.
• the authors do discuss conservation of ⁇ -carbon backbone tertiary structure this concept is not a part of the therapeutic strategy but merely included to assess in vitro IgE binding.
• the evidence presented is not adequate since normalisation of CD-spectra prevents the evaluation of denaturation of a proportion of the sample, which is a common problem.
• the therapeutic strategy described aim at inducing tolerance in allergen specific T cells and initiation of a new immune response is not mentioned.
• the article by Wiedemann et al. (ref. 12) describes the use of site directed mutagenesis and peptide synthesis for the purpose of monoclonal antibody epitope characterisation.
• the study demonstrates that substitution of a surface exposed amino acid has the capacity to modify the binding characteristics of a monoclonal antibody, which is not surprising considering common knowledge.
• the experiments described are not designed to assess modulation in the binding of polyclonal antibodies such as allergic patients' serum IgE.
• serum IgE One of the experiments does apply serum IgE and although this experiment is not suitable for quantitative assessment, IgE binding does not seem to be affected by the mutations performed.
• the article by Smith et al. (ref. 5) describes the use of site directed mutagenesis for the purpose of monoclonal antibody epitope mapping and reduction of IgE binding.
• the authors have no knowledge of the tertiary structure and make no attempt to assess the conservation of ⁇ -carbon backbone tertiary structure.
• the algorithm used does not ensure that amino acids selected for mutation are actually exposed to the molecular surface. Only one of the mutants described lead to a substantial reduction in IgE binding. This mutant is deficient in binding of all antibodies tested indicating that the tertiary structure is disrupted.
• the authors do not define a therapeutic strategy and initiation of a new immune response is not mentioned.
• the article by Colombo et al. (ref. 14) describes the study of an IgE binding epitope by use of site directed mutagenesis and peptide synthesis.
• the authors use a three dimensional computer model structure based on the crystal structure of a homologous protein to illustrate the presence of the epitope on the molecular surface.
• the further presence of an epitope on a different allergen showing primary structure homology is addressed using synthetic peptides representing the epitope.
• the therapeutic strategy is based on treatment using this synthetic peptide representing a monovalent IgE binding epitope.
• WO 01/83559 discloses a method of selecting a protein variant with modified immunogenicity by using antibody binding peptide sequences to localise epitope sequences on the 3-dimensional structure of the parent protein. An epitope area is subsequently defined and one or more of the amino acids defining the epitope area are mutated.
• the invention is exemplified by industrial enzymes that function as allergens.
• WO 99/47680 discloses the introduction of artificial amino acid substitutions into defined critical positions while retaining the ⁇ -carbon backbone tertiary structure of the allergen.
• WO 99/47680 discloses a recombinant allergen, which is a non-naturally occurring mutant derived from a naturally occurring allergen, wherein at least one surface-exposed, conserved amino acid residue of a B cell epitope is substituted by another residue which does not occur in the same position in the amino acid sequence of any known homologous protein within the taxonomic order from which said naturally occurring allergen originates, said mutant allergen having essentially the same ⁇ -carbon backbone tertiary structure as said naturally occurring allergen, and the specific IgE binding to the mutated allergen being reduced as compared to the binding to said naturally occurring allergen.
• the recombinant allergen disclosed in WO 99/47680 is obtainable by a) identifying amino acid residues in a naturally occurring allergen which are conserved with more than 70% identity in all known homologous proteins within the taxonomic order from which said naturally occurring allergen originates, b) defining at least one patch of conserved amino acid residues being coherently connected over at least 400 A 2 of the surface of the three- dimensional structure of the allergen molecule as defined by having a solvent accessibility of at least 20%, said at least one patch comprising at least one B cell epitope, and c) substituting at least one amino acid residue in said at least one patch by another amino acid being non-conservative in the particular position while essentially preserving the overall ⁇ -carbon backbone tertiary structure of the allergen molecule.
• Patent application PCT/DK 01/00764 relates to mutants of naturally occurring allergens.
• the following specific Bet v 1 mutants are disclosed therein: Mutant A: Asn28Thr, Lys32Gln, Asn78Lys, Lys103Val, Arg145Glu,
• Mutant B Tvr ⁇ Val, Glu42Ser, Glu45Ser, Asn78Lys, Lys103Val, Lys123lle,
• Mutant 2628 Tyr ⁇ Val, Glu45Ser, Lys65Asn, Lys97Ser, Lys134Glu. Mutant 2637: Ala16Pro, Asn28Thr, Lys32Gln, Lys103Thr, Pro108Gly,
• Mutant 2724 N28T, K32Q, N78K, K103V, P108G, R145E, D156H, +160N.
• Mutant 2733 Tyr ⁇ Val, Lys134Glu, Asn28Thr, Lys32Gin, Glu45Ser,
• Mutant 2744 Tyr ⁇ Val, Lys134Glu, Glu42Ser, Glu45Ser, Asn78Lys,
• Mutant 2744 + 2628 Y5V, E42S, E45S, K65N, N78K, K97S, K103V, K123I,
• Figure 1 shows a theoretical mode! of the reaction between an allergen and mast cells by IgE cross-linking.
• Figure 3 shows mutant-specific oligonucleotide primers used for mutation of Bet v 1. Mutated nucleotides are underlined.
• Figure 4 shows two generally applicable primers (denoted “all-sense” and “all non-sense”), which were synthesised and used for all mutants.
• Figure 5 shows the DNA and amino acid sequence of the naturally occurring allergen Sef v 1 as well as a number of Bet v 1 mutations.
• Figure 6 shows the inhibition of the binding of biotinylated recombinant Bet v 1 to serum IgE from a pool of allergic patients by non-biotinylated Bet v 1 and by Bet v 1 Glu45Ser mutant.
• Figure 7 shows the inhibition of the binding of biotinylated recombinant Bet v 1 to serum IgE from a pool of allergic patients by non-biotinylated Bet v 1 and by Bet v 1 mutant Asn28Thr+Lys32Gln.
• Figure 8 shows the inhibition of the binding of biotinylated recombinant Bet v 1 to serum IgE from a pool of allergic patients by non-biotinylated Bet v 1 and by Bet v 1 Pro108Gly mutant.
• Figure 9 shows the inhibition of the binding of biotinylated recombinant Bet v 1 to serum IgE from a pool of allergic patients by non-biotinylated Bet v 1 and by Bet v 1 Glu ⁇ OSer mutant.
• Figure 10 shows the CD spectra of recombinant and the (Asn28Thr, Lys32Gln, Glu45Ser, Pro108Gly) mutant, recorded at close to equal concentrations.
• Figure 11 shows the inhibition of the binding of biotinylated recombinant Ser v 1 to serum IgE from a pool of allergic patients by non-biotinylated Bet v 1 and by the (Asn28Thr, Lys32Gln, Glu45Ser, Pro108Gly) mutant.
• Figure 12 shows a graphical illustration of the 2-step PCR mutation technique used for generating mutated Bet v 1 allergens.
• Figure 13 shows a graphical illustration of the PCR mutation events leading to the cloning of Bet v 1 (3004), (3005), (3007) and (3009). Primers used for introducing point mutations are listed.
• Figure 14 shows a graphical illustration of the PCR mutation events leading to the cloning of Bet v 1 (3031 ) to (3045). Degenerated primers used for introducing random mutations in position 10, 20, 36, 73, 87, 129 and 149 are listed. The possible outcome of mutation for each position is shown at the top.
• Figure 15 shows schematically the primers used to create the mutations.
• (I) shows the sense and antisense primers.
• (II) shows the final recombinant protein harbouring mutations at the indicated positions.
• Figure 16 shows an illustration of the construction of Bet v 1 mutants and a listing of the primers used. The mutants contain from five to nine amino acids.
• Figure 17 shows introduced point mutations at the surface of Bet v 1 (2628) and Bet v 1 (2637).
• mutant Bet v 1 (2628) five primary mutations were introduced in one half of Bet v 1 leaving the other half unaltered.
• mutant Bet v 1 (2637) five primary and three secondary mutations were introduced in the other half, leaving the first half unaltered.
• Figure 18 shows the circular dichroism (CD) spectra of recombinant Bet v 1.2801 (wild type) and the Bet v 1 (2637) mutant recorded at nearly identical concentrations.
• Figure 19 shows the inhibition of the binding of biotinylated recombinant Bet v 1.2801 (wild type) to serum IgE from a pool of allergic patients by non- biotinylated Bet v 1.2801 and by Bet v 1 (2628), Bet v 1 (2637), and a 1 :1 mix of Bet v 1 (2628) and Bet v1 (2637).
• Figure 20 shows histamine release in human basophil cells of Bet v 1.2801 (wild type), Bet v 1 (2628), and Bet v 1 (2637).
• Figure 21 shows histamine release in human basophil cells of Bet v 1.2801 (wild type), Bet v 1 (2628), and Bet v 1 (2637).
• Figure 22 shows point mutations at the surface of Bet v 1 (2744).
• Figure 23 shows point mutations at the surface of Bet v 1 (2753).
• Figure 24 shows point mutations at the surface of Bet v 1 (2744) and Bet v 1 (2753).
• Figure 25 shows circular dichroism (CD) spectra of Bet v 1.2801 (wild type) and Bet v 1 (2744), recorded at nearly equal concentrations.
• Figure 26 shows histamine release in human basophil cells of Bet v 1.2801 (wild type), and mutant Bet v 1 (2744).
• Figure 27 shows histamine release in human basophil cells of Bet v 1.2801 (wild type), and mutant Bet v 1 (2744).
• Figure 28 shows point mutations at the surface of Bet v 1 (2733).
• Figure 29 shows the proliferation of Peripheral Blood Lymphocytes expressed as Stimulation Index (SI) for various Bet v 1 preparations.
• SI Stimulation Index
• Figures 30-32 show the cytokine profile of T cells stimulated with various Bet v 1 preparations.
• Figure 30 shows a patient with a ThO profile, Figure 31 a Th1 profile and Figure 32 a Th2 profile.
• Figure 33 shows Circular dichroism (CD) spectroscopy of rBet v 1.2801 (•) (wildtype) and the rBet v 1 3007) mutant [ ⁇ ] with 12 mutations, recorded at equal concentrations. Overlay of circular dichroism (CD) spectra obtained at 15°C are shown.
• Figure 34 shows the inhibition of the binding of biotinylated rBet v 1.2801 to pooled IgE serum from birch allergic patients by rBet v 1.2801 (•) (wildtype) or mutated rBet v 1 (3007) [ ⁇ ] with 12 mutations.
• the object of the invention is to provide improved recombinant mutant allergen proteins.
• the current invention is based on a unique rationale. According to this rationale the mechanism of successful allergy vaccination is not an alteration of the ongoing Th2-type immune response, but rather a parallel initiation of a new immune response involving tertiary epitope recognition by B-cells and antibody formation. It is believed that this new immune response is partly a Th1-type immune response.
• the vaccine or pharmaceutical compositions
• the new immune response evolves in a location physically separated from the ongoing Th2 response thereby enabling the two responses to exist in parallel.
• the invention is based on the finding that allergic symptoms are triggered by the cross-linking of allergen with at least two specific IgE's bound to the surface of effector cells, i.e. mast cells and basophils, via the high affinity IgE receptor, Fc ⁇ RI.
• Fig. 1 depicts a theoretical model of an allergen with three IgE binding epitopes. Induction of mediator release from the mast cell and hence allergic symptoms is effected by allergen-mediated cross-linking of IgE bound to the surface of the mast cell, cf. Fig 1A. In the situation shown in Fig. 1 B two of the epitopes have been mutated so as to reduce their IgE binding ability, and hence the allergen-mediated cross-linking is prevented. In this connection it should be noted that allergens usually comprise more than three B cell epitopes.
• the mutant allergen In order for a mutant allergen to be able to raise the new immune response, including an IgG response, the mutant allergen must comprise at least one intact epitope or an epitope, which has been altered only moderately.
• the surface topography of a moderately altered epitope preferably resembles the original epitope, allowing new more numerous IgG antibodies to be raised.
• These new IgG antibodies have specificities which can compete and to some degree oust IgE binding to the natural occurring allergen. Further, it may be assumed that the more epitopes, which have been mutated so as to eliminate or reduce their IgE binding ability, the lower the risk of allergen- mediated cross-linking and resulting allergic symptoms upon administration of an allergen vaccine.
• the mutant allergen has an ⁇ - carbon backbone tertiary structure which is essentially the same as that of the natural allergen.
• positions suitable for mutation are located exclusively in areas consisting of conserved amino acid residues believed to harbour dominant IgE binding epitope.
• surface exposed amino acid residues suitable for mutation comprise both highly conserved residues and residues that are not conserved or only conserved to a smaller degree. Such amino acid residues appear to be distributed over the entire molecular surface with a tendency to form small groupings covering a defined area on the molecular surface.
• surface exposed amino acids suitable for mutation can be divided into groups as illustrated in Fig. 2.
• the groupings rely on the tendency of these amino acid residues to form separate areas and these groupings are furthermore independent of the degree of conservation of the amino acid residues.
• Each group represents a number of surface exposed amino acid residues that are found within a limited area on the surface of the allergen.
• Each individual group most likely comprises part of at least one epitope or at least one intact epitope.
• Each separate group may comprise as well amino acids positions that will result in a moderately altered epitope upon mutation as well as amino acid positions that will result in a more drastic alteration of the epitope upon mutation.
• a single amino acid residue typically results in a moderate alteration of an epitope if the original amino acid residue is substituted with an amino acid that posseses similar chemical features (E.g. exchanging a hydrophobic amino acid with another hydrophobic amino acid residue).
• exchanging a hydrophobic amino acid with another hydrophobic amino acid residue provides a tool for rendering it very likely that a mutant allergen according to the present invention is mutated in several B-cell epitopes and has a ⁇ -carbon backbone structure that is similar to the naturally occurring allergen.
• the mutated allergen retains a continous surface region with an area of about 400-800 A 2 that contains either no mutations or only moderate mutations. It is believed that an allergen comprises a number of potential binding regions for specific IgE's, wherein each region has an area of approximately 800 A 2 .
• the inventive idea of the present invention is based on the recognition that a mutated allergen having IgE binding reducing mutations in at least 4 defined groups, each group comprising surface exposed amino acids suitable for mutation, but retaining at least one intact or moderately altered epitope, would on the one hand reduce the allergen-mediated cross-linking and on the other hand allow the raising of an IgG response with a binding ability competitive with that of IgE.
• the said mutated allergen constitutes a highly advantageous allergen in that the risk of anaphylactic reactions is being strongly reduced.
• the mutant allergen has the potential to be administered in relatively higher doses improving its efficacy in generating a protective immune response without compromising safety.
• the present invention is based on the recognition that a vaccine comprising a mixture of different such mutated allergens, wherein ideally many or all epitopes are represented as intact epitopes or epitopes that are only moderately altered on different mutated allergens, would be equally efficient in its ability to induce protection against allergic symptoms as the natural occurring allergen from which the mutated allergens are derived.
• the present invention relates to the introduction of amino acid substitutions into allergens.
• the amino acid substitutions are chosen from at least four groups of amino acids suitable for amino acid substitution.
• the object being to reduce the specific IgE binding capability of each mutated epitope while retaining at least one intact or only moderately altered epitope on the mutated allergen.
• the present invention relates to a recombinant Bet v 1 allergen, characterised in that it is a mutant of a naturally occurring Bet v 1 allergen where: the mutant retains essentially the same ⁇ -carbon backbone structure as the naturally occurring allergen, the mutant comprises at least four primary mutations, which each reduce the specific IgE binding capability of the mutated allergen as compared to the IgE binding capability of the naturally occurring Bet v 1 allergen, each primary mutation is a substitution of one surface-exposed amino acid residue with another residue, the mutations are placed in such a manner that at least one area of 400-800 A 2 comprises either no mutations or one or more moderate mutations, the primary mutations are selected from at least 4 of the following 10 groups, each group comprising surface exposed amino acid positions suitable for amino acid substitution: group 1 : A130, E131 , K134, A135, K137, E138, E141 , T142, R14 ⁇ ; group 2: V2, F3, N4, Y ⁇ , E6, T7, K119; group
• the present invention relates to a recombinant Bet v 1 allergen where the primary mutations are selected from at least 4 of the following 10 groups, each group comprising surface exposed amino acid positions suitable for the following amino acid substitutions: ⁇ group 1 : A130: A130V, A130G, A130I, A130L, A130S, A130H, A130T; E131 E131 D, E131 H, E131 K, E131 R, E131 S; K134: K134R, K134H, K134S K134Q, K134I, K134E; A13 ⁇ : A13 ⁇ V, A135G, A135I, A13 ⁇ L, A13 ⁇ S, A13 ⁇ H A13 ⁇ T; K137: K137R, K137H, K137S, K137Q, K137I, K137E; E138: E138D E138H, E138K, E138R, E138S, E138N; E141 : E141 D, E141 H, E141 K 0 E141 R
• S1 ⁇ S1 ⁇ T, S1 ⁇ L, S1 ⁇ V, S1 ⁇ D, S1 ⁇ K; D1 ⁇ 6: D1 ⁇ 6H, D1 ⁇ 6E, D1 ⁇ 6S,
• N28 N28H, N28K, N28M, N28Q, N28R, N28T;
• K32 K32Q, K32R, K32N, K32H, K32S, K32I, K32E; group 7: H76: H76W, H76F, H76S, H76D; T77: T77A, T77S, T77L, T77V, T77D, T77K, T77N; N78: N78H, N78K, N78M, N78Q, N78R; F79: F79H,
• E101 E101D, E101H, E101K, E101R, E101S; K103: K103R, K103H, K103S,
• K97 K97R, K97H, K97S, K97Q, K97I, K97E; group 9: G1: G1N, G1H, G1K, G1M, G1Q, G1R; G92: G92N, G92H, G92K,
• K129 K129R, K129H, K129S, K129Q, K129I, K129E, K129N; group 10: P3 ⁇ : P3 ⁇ G; Q36: Q36K, Q36R, Q36N, Q36H, Q36S, Q36I, Q36E;
• E60 E60H, E60K, E60M, E60Q, E60R; G61: G61N, G61H, G61K, G61M, G61Q, G61 R; P63: P63G; F64: F64H, F64W, F64S, F64D; K6 ⁇ : K6 ⁇ R, K65H, K6 ⁇ S, K6 ⁇ Q, K65I, K6 ⁇ E, K6 ⁇ N; Y66: Y66D, Y66G, Y66H, Y66I, Y66K, Y66V.
• the present invention further relates to a recombinant Bet v 1 mutant allergen comprising substitutions that are selected from at least four of the following 10 groups:
• Group 2 V2L, Y ⁇ V, E6S, K119N, 0
• Group 3 E42S, E4 ⁇ S, N47K, K ⁇ N, E73S, E73T, E73S,
• Group 4 E8S, T10P, P14G, P108G, D109N, K115N,
• Group ⁇ A16G, K20S, S149T L1 ⁇ 2A A1 ⁇ 3V, S1 ⁇ T, N1 ⁇ 9G, +160N,
• Group 6 L24A, D2 ⁇ E, N28T, K32Q,
• Group 7 T77A, T77N, N78K, K103V, 5 Group 8: R70N, E87A, E96S, K97S,
• Group 10 Q36N, E60S, G61 S, P63G.
• the present invention further relates to a recombinant Bet v 1 mutant 0 allergen comprising substitutions that are selected from at least four of the following 10 groups:
• Group 3 E4 ⁇ S, E42S, K ⁇ N, N47K, E73S, ⁇ Group 4: E96S, K97S, P108G, D109N, T10N, K115N, P14G,
• Group 7 K103V, T77N, N78K,
• Group 8 E96S, K97S, E87A, 0
• Group 9 K129N, D12 ⁇ Y, K123I, D93S,
• the present invention further relates to the following:
• Bet v 1 allergens variants that can be used as a pharmaceutical and for preparing a pharmaceutical for preventing and/or treating birch pollen allergy.
• a composition comprising two or more different recombinant mutant Bet v 1 allergen variants according to the present invention wherein each variant has at least one primary mutation, which is absent in at least one of the other variants.
• the composition comprises 2-12, preferably 3-10, more preferably 4-9 and most preferably 5-8 variants.
• a composition according to the present invention can be used as a pharmaceutical and for preparing a pharmaceutical for preventing and/or treating birch pollen allergy.
• the pharmaceutical composition preferably comprises a pharmaceutically acceptable carrier, and/or excipient, and optionally an adjuvant.
• Methods of generating an immune response in a subject comprising administering to a subject a recombinant allergen, a composition, or a pharmaceutical composition.
• Vaccination or treatment of a subject comprises administering to the subject a recombinant allergen, a composition, or a pharmaceutical composition.
• a method for preparing a pharmaceutical composition comprising mixing a recombinant allergen, or a composition with pharmaceutically acceptable substances, and/or excipients.
• a method for the treatment, prevention or alleviation of allergic reactions in a subject that comprises administering to a subject a recombinant Bet v 1 allergen, a composition, or a pharmaceutical composition.
• a method of preparing a recombinant Bet v 1 allergen characterised in that the substitution of amino acids is carried out by site-directed mutagenesis, or DNA shuffling (molecular breeding) (Punnonen et al., ref. 25).
• a DNA sequence which is a derivative of the DNA sequence encoding the naturally occurring allergen is obtained by site-directed mutagenesis of the DNA encoding the naturally occurring Bet v 1 allergen.
• An expression vector comprising DNA encoding a recombinant Bet v 1 variant, a host cell comprising the expression vector, and a method of producing a recombinant mutant Bet v 1 allergen comprising cultivating the host cell.
• a recombinant Bet v 1 allergen or a recombinant Bet v 1 allergen that is encoded by the DNA sequence comprises at least one T celle epitope capable of stimulating a T cell clone or T cell line specific for the naturally occurring Bet v 1 allergen.
• the expression "reduce the specific IgE binding capability as compared to the IgE binding capability of the naturally occurring allergen” means that the reduction is measurable in a statistically significant manner (p ⁇ 0.0 ⁇ ) in at least one immunoassay using serum from a subject allergic to the natural-occurring allergen.
• the IgE binding capability is reduced by at least 10%, more preferably at least 30%, more preferably at least 50%, and most preferably at least 70%.
• surface-exposed amino acid means that the amino acid residue is located at the surface of the three-dimensional structure in such a manner that when the allergen is in solution at least a part of at least one atom of the amino acid residue is accessible for contact with the surrounding solvent.
• the amino acid residue in the three-dimensional structure has a solvent (water) accessibility of at least 20%, suitably at least 30%, more suitably at least 40% and most preferably at least 50%.
• surface-exposed and “solvent-exposed” are used interchangeably.
• Group of amino acids should be understood as division of surface exposed amino acids suitable for mutation into groups. Each group represents a number of surface exposed amino acid residues that are found within a limited area on the surface of the allergen. An individual group comprises a number of amino acids that are part of at least one epitope. An individual group may also cover an area that comprises an entire epitope.
• One or more mutations within a single group is defined as one primary mutation.
• a mutated allergen with at least four primary mutations thus ensures that several epitopes will have a lowered IgE binding affinity.
• Mutation of amino acids from at least four groups may furthermore ensure an approximately even distribution of mutations on the molecular surface and ensure that several epitopes are mutated and thus resulting in a lowered IgE binding affinity of several epitopes compared to mutants with less than four mutations.
• the taxonomic species from which said naturally occurring allergen originates means species within the taxonomic genus, preferably the subfamily, more preferably the family, more preferably the superfamily, more preferably the legion, more preferably the suborder and most preferably the order from which said naturally occurring allergen originates.
• Moderately altered epitopes means epitopes that retain essentially the same tertiary structure and surface topography as the corresponding unmutated epitopes. Moderate alterations are, generally speaking, achieved by exchanging an amino acid with another amino acid with similar chemical characteristics as the original amino acid. One way of achieving this is by exchanging one or more surface exposed amino acids with amino acids that might be found within the taxonomic order wherein the naturally occurring allergen is found. A moderately altered epitope might also contain amino acid substitutions where one or more of the substituted amino acid is not found within the taxonomic order wherein the naturally occurring allergen is found, as long as the substitution only slightly affects the tertiary structure of the epitope and/or the IgE binding affinity.
• the mutated allergen can be evaluated with respect to e.g. structure and IgE binding affinity subsequently.
• epitopes that are altered in a more drastic manner, e.g. mutations that significantly reduce the IgE binding affinity.
• drastic alterations of epitopes comprise amino acid substitutions where one or more amino acids have been exchanged with amio acids with different chemical properties.
• the expression "the mutant allergen having essentially the same ⁇ -carbon backbone tertiary structure as the naturally occurring allergen” means that when comparing the structures of the mutant and the naturally occurring allergen, the average root mean square deviation of the atomic coordinates is preferably below 2 A.
• Conservation of ⁇ -carbon backbone tertiary structure is best determined by obtaining identical structures by x-ray crystallography or NMR before and after mutagenesis. In absence of structural data describing the mutant indistinguishable CD- spectra or immunochemical data, e.g. antibody reactivity, may render conservation of ⁇ -carbon backbone tertiary structure probable, if compared to the data obtained by analysis of a structurally determined molecule.
• mutation means the deletion, substitution or addition of an amino acid in comparison to the amino acid sequence of the naturally occurring allergen.
• the terms “mutation” and “substitution” are used interchangeably.
• a recombinant mutated Bet v 1 allergen according to the invention may furthermore comprise amino acid insertions or amino acid deletion in particular surface exposed regions of the molecules e.g. "loop regions". Loop regions connect secondary structure elements e.g. ⁇ -sheet, ⁇ -helixes and random coil structures.
• Loop regions in Bet v 1 are: Val 12 to ala16, val33 to ser40, glu45 to Thr ⁇ 2, ⁇ ro ⁇ 4 to tyr66, his76 to asn78, gly89 to glu96, vaMO ⁇ to gly111 , thr122 to glu131.
• Mutant variants may comprise 1-5, more preferable 1-3 most preferably 1-2 substitutions in a loop region.
• a primary mutation is defined as one or more mutations within a single group of surface exposed amino acids suitable for substitution. Each group of at least one mutated amino acids will have reduced IgE binding affinity as compared to the same group without mutations.
• the recombinant allergen according to the invention comprises from ⁇ to 10, preferably from 6 to 10, more preferably from 7 to 10, and most preferably from 8 to 10 primary mutations.
• Secondary mutations are defined as additional mutations within a single group.
• the recombinant allergen preferably comprises a number of secondary mutations, which each reduce the specific IgE binding capability of the mutated allergen as compared to the binding capability of the said naturally occurring allergen.
• a primary mutation that comprises several secondary mutations will in many cases have a more reduced IgE binding affinity than a primary mutation that has only one mutation.
• the recombinant allergen according to the invention comprises from 1 to 15, preferably 1-10 and most preferably 1-5 secondary mutations per primary mutation.
• conserved residues conserved residues in the naturally occurring allergen are conserved with more than 70 %, preferably 80 % and most preferably 90 % identity in all known homologous proteins within the species from which said allergen originates. Amino acid residues that are highly solvent exposed and conserved constitute targets for substitution.
• HR Histamine Release
• a surface region comprising no mutation or only moderate mutations has an area of 800 A 2 , preferably 600 A 2 , more preferably ⁇ OO A 2 and most preferably 400 A 2 .
• a surface region with an area of 800 A 2 comprising no mutation or only moderate mutations comprises atoms of 15-25 amino acid residues.
• At least one of the amino acid residues to be incorporated into the mutant allergen does not occur in the same position in the amino acid sequence of any known homologous protein within the taxonomic genus, preferably the subfamily, more preferably the family, more preferably the superfamily, more preferably the legion, more preferably the suborder and most preferably the order from which said naturally occurring allergen originates.
• the surface-exposed amino acid residues are ranked with respect to solvent accessibility, and at least four amino acids among the more solvent accessible ones are substituted.
• a recombinant allergen is characterised in that the surface-exposed amino acid residues are ranked with respect to degree of conversation in all known homologous proteins within the species from which said naturally occurring allergen originates, and that one or more surface exposed amino acids among the more conserved ones are substituted.
• the principle disclosed in the present invention comprises mutation of surface exposed amino acid residues selected from at least four groups of amino acids, wherein each group represents separate areas on the surface on the molecule. This principle may also be applied to allergens other than Bet v 1.
• a recombinant allergen according to the invention may suitably be a mutant of an inhalation allergen originating i.a.
• Important pollen allergens from trees, grasses and herbs are such originating from the taxonomic orders of Fagales, Oleales and Pinales including i.a. birch (Betula), alder (Alnus), hazel (Corylus), hornbeam (Carpinus) and olive (Olea), the order of Poales including i.a. grasses of the genera Lolium, Phleum, Poa, Cynodon, Dactylis and Secale, the orders of Asterales and Urticales including i.a. herbs of the genera Ambrosia and Artemisia.
• Important inhalation allergens from fungi are i.a. such originating from the genera Alternaha and Cladosporium.
• Other important inhalation allergens are those from house dust mites of the genus Dermatophagoides, those from cockroaches and those from mammals such as cat, dog and horse.
• recombinant allergens according to the invention may be mutants of venom allergens including such originating from stinging or biting insects such as those from the taxonomic order of Hymenoptera including bees (superfamily Apidae), wasps (superfamily Vespidea), and ants (superfamily Formicoidae).
• Specific allergen components include e.g. Bet v 1 (B. verrucosa, birch), Aln g 1 (Alnus glutinosa, alder), Cor a 1 (Corylus avelana, hazel) and Car b 1 (Carpinus betulus, hornbeam) of the Fagales order.
• Bet v 1 B. verrucosa, birch
• Aln g 1 Alnus glutinosa, alder
• Cor a 1 Corylus avelana, hazel
• Car b 1 Carpinus betulus, hornbeam
• Eur m 1 (mite, Euroglyphus maynei), (Lep d 1 and 2 (Lepidoglyphus destructor; storage mite), Bla g 1 and 2, Per a 1 (cockroaches, Blatella germanica and Periplaneta americana, respectively), Pel d 1 (cat), Can f 1 (dog), Equ c 1 , 2 and 3 (horse), Apis m 1 and 2 (honeybee), Ves v 1 , 2 and ⁇ , Po/ a 1 , 2 and ⁇ (all wasps) and So/ / ' 1 , 2, 3 and 4 (fire ant).
• Eur m 1 mite, Euroglyphus maynei
• Lep d 1 and 2 Lepidoglyphus destructor; storage mite
• Bla g 1 and 2 Per a 1 (cockroaches, Blatella germanica and Periplaneta americana, respectively)
• Pel d 1 cat
• Can f 1 dog
• mutant allergens further substitutions are added to mutant allergens in such a way that it is ensured that the substitutions of the final 0 mutant allergen are essentially evenly distributed on the molecular surface and that the groups contain essentially the same number of introduced mutations.
• mutants comprising specific substitutions preferably should have added further substitutions from a list where the succession of amino acids reflects the 5 preferred order of adding more substitutions. Without limiting the present invention, these examples represent one application of how to design mutants and the man skilled in the art might very well choose a somewhat different approach in order to ensure an even distribution of substitutions. Mutants may thus be designed comprising one or more substitutions from the 0 lists given below.
• Bet v 1 mutant (“3004A") allergens comprising the following substitutions: Y ⁇ V, E4 ⁇ S, N78K, K97S, K103V, K134E, +160N. Further substitutions may comprise one or more of the following: E8 or K115, D12 ⁇ or H126, E138 or ⁇ K137 or E141 , D2 ⁇ or N28, E87 or K ⁇ , S1 ⁇ or H1 ⁇ 4 or N159, N47 or P ⁇ O or H76 or N43 or I44 or R70, E73 or P ⁇ O or D72, A130, N28 or D2 ⁇ , P108, V2 or K119 or N4 or E6 or E96.
• Bet v 1 mutant (“3004B") allergens comprising the following substitutions: 0 Y ⁇ V, E4 ⁇ S, L62F, N78K, K97S, K103V, K134E, +160N. Further substitutions may comprise one or more of the following: T10P, K6 ⁇ N, N28 or D2 ⁇ or K32Q or E141X or K137X or E138X, D12 ⁇ X or K123I or H126, P108X or D109N, E42S or K ⁇ X or I44X or N43X, E73X or D72X, E87X, E96X or K119, A130X, V2X or E6X, E8X or K11 ⁇ , N47X or P ⁇ OX or R70X or H76X or T77A, S1 ⁇ X or D1 ⁇ 6H or N1 ⁇ 9X, E6X or V2X. ⁇
• Bet v 1 allergen mutants (“300 ⁇ A") comprising the following substitutions: Y ⁇ V, N28T, K32Q, E4 ⁇ S, L62F, N78K, K97S, K103V, K134E, +160N. Further substitutions may comprise one or more of the following: E8X or K11 ⁇ X, D12 ⁇ or H126, E138X or K137X or E141X, E87X or K ⁇ X, Sl ⁇ X or 0 H 1 ⁇ 4X or N 1 ⁇ 9X, N47X or P ⁇ OX or H76X or N43X or I44X or R70X, E73X or P ⁇ OX or D72X, A130X, D2 ⁇ X, P108X, V2X or K119X or N4X or E6X or E96X.
• Bet v 1 allergen mutants (“300 ⁇ B") comprising the following substitutions: ⁇ Y ⁇ V, N28T, K32Q, E4 ⁇ S, L62F, N78K, K97S, K103V, K134E, +160N.
• substitutions may comprise one or more of the following: T10P, K6 ⁇ N, E141X or K137X or E138X, D12 ⁇ X or K123I or H126X, P108X or D109N, E42S or K ⁇ X or I44X or N43X, E73X or D72X, E87X, E96X or K119X, A130X, V2X or E6X, E8X or K11 ⁇ X, N47X or P ⁇ OX or R70X or H76X 0 or T77A, Sl ⁇ X or D1 ⁇ 6H or N1 ⁇ 9X, E6X or V2X.
• Bet v 1 allergen mutants (“3006A") comprising the following substitutions: Y ⁇ V, N28T, K32Q, E4 ⁇ S, N78K, E87S, K97S, K103V, K134E, N1 ⁇ 9G, +160N. Further substitutions may comprise one or more of the following: ⁇ K ⁇ , A138 or K137 or E141 , D12 ⁇ or H126, P108, V2 or N4 or K119 or E6, S1 ⁇ or H1 ⁇ 4, N47 or P ⁇ O or H76, E73, R70, A130, E8 or K11 ⁇ , E96.
• Bet v 1 allergen mutants (“3006B") comprising the following substitutions:
• Bet v 1 allergen mutants (“3007A") comprising the following substitutions: Y ⁇ V, N28T, K32Q, E4 ⁇ S, L62F, N78K, K97S, K103V, P108G, D12 ⁇ Y, K134E, +160N. Further substitutions may comprise one or more of the following: E87, E141 , E138, K ⁇ , N47 or N43X or I44 or H76, S1 ⁇ or H1 ⁇ 4, A130, E8, E73, V2 or K119, D2 ⁇ .
• Bet v 1 allergen mutants (“3007B") comprising the following substitutions: Y ⁇ V, N28T, K32Q, E4 ⁇ S, L62F, N78K, K97S, K103V, P108G, D12 ⁇ Y, K134E, +160N. Further substitutions may comprise one or more of the following: K6 ⁇ N, T10P or E8, E87, S1 ⁇ or D1 ⁇ 6H, E138, E141 , E42S, A130, E8 or TI OP, N47, H76, R70, E96.
• Bet v 1 allergen mutants (“3008A") comprising the following substitutions: Y ⁇ V, N28T, K32Q, E45S, L62F, E73S, E96S, P108G, D12 ⁇ Y, N1 ⁇ 9G, +160N. Further substitutions may comprise one or more of the following: E134, N78, E87, K119, E8, K ⁇ , E138, E141 , S1 ⁇ , N47, E6, K103, D2 ⁇ , A130, V2, R70.
• Bet v 1 allergen mutants (“3008B") comprising the following substitutions: Y ⁇ V, N28T, K32Q, E4 ⁇ S, L62F, E73S, E96S, P108G, D12 ⁇ Y, N1 ⁇ 9G, +160N. Further substitutions may comprise one or more of the following: K6 ⁇ N or K ⁇ , T10P or E8 or E141 , E138 or K134, E87, E42S or K ⁇ or I44, S1 ⁇ or D1 ⁇ 6H, N78, K119 or V2 or N4, N47 or P ⁇ O, H76 or T77A, A130, D2 ⁇ , E6 or K11 ⁇ or K103.
• Bet v 1 allergen mutants (“3009A") comprising the following substitutions: Y ⁇ V, N28T, K32Q, E4 ⁇ S, L62F, E96S, P108G, +160N. Further substitutions may comprise one or more of the following: E134, N78, E87, K1 19, E8, K ⁇ , E138, E141 , S1 ⁇ , N47, E6, K103, D2 ⁇ , A130, V2, R70.
• Bet v 1 allergen mutants (“3009B") comprising the following substitutions: ⁇ Y ⁇ V, N28T, K32Q, E4 ⁇ S, L62F, E96S, P108G, +160N. Further substitutions may comprise one or more of the following: N78 or T77A, K103, E134 or E138, K6 ⁇ N or K ⁇ , T10P, D12 ⁇ or H126, S1 ⁇ or D1 ⁇ 6H or HIS1 ⁇ 4, K119 or V2, E87, N47 or P ⁇ O or H76, E42S or K ⁇ , I44 or N43, A130.
• mutant allergens according to the invention furthermore comprise amino acid insertions or amino acid deletion in particular surface exposed regions of the molecules e.g. loop regions.
• Loop regions connect secondary structure elements e.g. ⁇ -sheet, ⁇ -helixes and random coil structures.
• Loop regions in Bet v 1 are: val 12 to ala16, val33 to ser40, glu4 ⁇ to Thr ⁇ 2, pro ⁇ 4 to tyr66, his76 to asn78, gly89 to glu96, val10 ⁇ to gly111 , thr122 to glu131.
• Mutant variants according to this embodiment comprise 1 - ⁇ , more preferable 1-3 most preferably 1-2 0 substitutions in a loop region.
• mutant allergens comprise at least four mutations selected from the 10 groups as well as a number of additional "loop-mutations". Examples of such "loop mutations", wherein x represents an added amino acid residue, are:
• Bet v 1 (3007-L3) with amino acid insertion between residue V12 and residue 113: 0 GVFNVETETTSVxlPAARLFKAFILDGDTLFPQVAPQAISSVENISGNGGPGT IKKISFPEGFPFKYVKDRVDEVDHTKFKYNYSVIEGGPIGDTLESISNEIVIVA TGDGGSILKISNKYHTKGYHEVKAEQVEASKEMGETLLRAVESYLLAHSDA YNN
• Bet v 1 (3007-L4) with amino acid insertions between residue I56 and residue S ⁇ 7 and between residue K6 ⁇ and residue T66
• Bet v 1 (3007-L ⁇ ) with amino acid deletion of residue G111 GVFNVETETTSVIPAARLFKAFILDGDTLFPQVAPQAISSVENISGNGGPGTI KKISFPEGFPFKYVKDRVDEVDHTKFKYNYSVIEGGPIGDTLESISNEIVIVAT ⁇ GDGSILKISNKYHTKGYHEVKAEQVEASKEMGETLLRAVESYLLAHSDAYN N
• the surface-exposed amino acids suitable for substitution in accordance with the present invention may be identified on the basis of information of their solvent (water) accessibility, which expresses the extent of surface exposure.
• a preferred embodiment of the method of the invention is characterised in ranking the said identified amino acid residues with respect to solvent accessibility and substituting one or more amino acids among the more solvent accessible ones.
• Another embodiment of the method of the invention is characterised in ranking the identified amino acid residues with respect to degree of conversation in all known homologous proteins within the species from which said naturally occurring allergen originates and substituting one or more amino acids among the more conserved ones.
• a further preferred embodiment of the method of the invention comprises selecting the identified amino acids so as to form a mutant allergen, which has essentially the same ⁇ -carbon backbone tertiary structure as said naturally occurring allergen.
• Another preferred embodiment of the method of the invention is characterised in that the substitution of amino acid residues is carried out by site-directed mutagenesis.
• An alternative preferred embodiment of the method of the invention is characterised in that the substitution of amino acid residues is carried out by DNA shuffling or by setting up a library comprising suitable positions and their preferred substitutents. Criteria for substitution
• the mutant carrying the 5 substituted amino acid(s) should preferably fulfil the following criteria:
• the overall ⁇ -carbon backbone tertiary structure of the recombinant mutant is preferably conserved. conserveed is defined as an average root mean square deviation of the atomic coordinates below 2A 0 when comparing the structures of the mutated allergen and the naturally occurring allergen. This is important for two reasons: a) It is anticipated that the entire surface of the natural allergen constitutes an overlapping continuum of potential antibody-binding epitopes. The majority of the surface of the molecule is not affected by the substitution(s), and thus retain its antibody-binding inducing properties, which is important for the generation of new protective antibody specificities being directed at epitopes present also on the natural allergen, b) Stability, both concerning shelf-life and upon injection into body fluids.
• the amino acids to be substituted are preferably located at the surface, and thus accessible for antibody-binding.
• Amino acids located on the 0 surface in the three-dimensional structure usually have a solvent (water) accessibility of at least 20%, suitably 20-80%, more suitably 30-80%.
• the substituted amino acids are selected from at least four groups.
• Each ⁇ group represents a number of preferred surface exposed amino acid residues that are found within a limited area on the surface of the allergen.
• One or more mutations within a single group is defined as one primary mutation.
• An individual group comprises a number of amino acids that are part of at least one epitope.
• An individual group may also comprise an entire 0 epitope.
• a mutated allergen with at least four primary mutations thus ensures that several epitopes will have a lowered IgE binding affinity.
• Mutation of amino acids from at least four groups furthermore ensures an approximately even distribution of mutations on the molecular surface and it ensures that several epitopes will become mutated and thus obtaining a lowered IgE ⁇ binding affinity of several epitopes.
• the amino acid to be incorporated may be selected on the basis of a comparison with a protein, which is a structural homologue to the allergen, 0 e.g. a protein, which belongs to the same taxonomic order as the allergen, and which does not have any cross-reactivity with the allergen.
• Vaccines are typically prepared as injectables either as liquid solutions or suspensions. Such vaccine may also be emulsified or formulated so as to enable nasal administration as well as oral, including buccal and sublingual, administration.
• the immunogenic component in question (the recombinant 0 allergen as defined herein) may suitably be mixed with excipients which are pharmaceutically acceptable and further compatible with the active ingredient. Examples of suitable excipients are water, saline, dextrose, glycerol, ethanol and the like as well as combinations thereof.
• the vaccine may additionally contain other substances such as wetting agents, emulsifying agents, buffering agents or adjuvants enhancing the effectiveness of the vaccine.
• Vaccines are most frequently administered parenterally by subcutaneous or intramuscular injection.
• Formulations which are suitable for administration by another route include oral formulations and suppositories.
• Vaccines for oral administration may suitably be formulated with excipients normally employed for such formulations, e.g. pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like.
• the composition can be formulated as solutions, suspensions, emulsions, tablets, pills, capsules, sustained release formulations, aerosols, powders, or granulates.
• the vaccines are administered in a way so as to be compatible with the dosage formulation and in such amount as will be therapeutically effective and immunogenic.
• the quantity of active component contained within the vaccine depends on the subject to be treated, i.a. the capability of the subject's immune system to respond to the treatment, the route of administration and the age and weight of the subject. Suitable dosage ranges can vary within the range from about 0.0001 ⁇ g to 1000 ⁇ g.
• adjuvants examples include aluminum hydroxide and phosphate (alum) or calcium phosphate as a 0.05 to 0.1 percent solution in phosphate buffered saline, synthetic polymers of sugars or polylactid glycolid (PLG) used as 0.25 percent solution.
• alum aluminum hydroxide and phosphate
• PLG polylactid glycolid
• Mixture with bacterial cells such as C. parvum, endotoxins or lipopolysaccharide components of gram-negative bacteria, emulsion in physiologically acceptable oil vehicles such as mannide monoaleate (Aracel A) or emulsion with 20 percent solution of a perfluorocarbon (e.g. Fluosol-DA) used as a block substitute may also be employed.
• Oil emulsions such as MF- ⁇ 9 may also be used.
• Other adjuvants such as Freund's complete and incomplete adjuvants as well as saponins, such as QuilA, Qs-21 and ISCOM, and RIBI may also be used.
• the vaccine is administered as an initial administration followed by subsequent inoculations or other administrations.
• the number of vaccinations will typically be in the range of from 1 to 50, usually not exceeding 35 vaccinations.
• Vaccination will normally be performed from biweekly to bimonthly for a period of 3 months to 5 years. This is contemplated to give desired level of prophylactic or therapeutic effect.
• the recombinant allergen may be used as a pharmaceutical preparation, which is suitable for providing a certain protection against allergic responses during the period of the year where symptoms occur (prophylaxis). Usually, the treatment will have to be repeated every year to maintain the protective effect. Preparations formulated for nasal, oral and sublingual application are particular suited for this purpose.
• the DNA sequence of the invention is a mutant of a DNA sequence encoding a naturally occurring Bet v 1 allergen.
• Naturally occurring Bet v 1 molecules are SEQ ID NO 1 (data base accession number Z80104) and SEQ ID NO 2 (data base accession number P15494).
• the DNA derivative is obtained by site-directed or random or semi random mutagenesis of the DNA encoding the naturally occurring allergen.
• a “mutant library” is a library of mutant allergens. This library is constructed using degenerated DNA oligonucleotide primers that allow introduction of none, a single or several different amino acid residues in each position. Such a library approach allows amino acid residues to be either conservatively or non-conservatively substituted. As structural integrity may be less affected by conserved mutations introduction of such "soft" or moderate mutations in certain positions may increase the changes of generating stable mutants. Construction of mutant libraries may be one way to overcome problems with protein stability of mutated allergens caused by a single or a certain combination of mutations.
• a "semi-random library” means that positions to be mutated are confined to amino acid residues, which are surface exposed.
• a library according to the invention comprises a number of rBet v 1 mutant allergens each having at least 4 amino acid substitutions compared to non-mutated Bet v 1.
• a semi-random library based on rBet v 1 (2744) (mutated in positions Y5, E42, E45, N78, K103, K123, K134, D156, +160) and rBet v1 (2628) (mutated in positions Y ⁇ , E4 ⁇ , K6 ⁇ , K97, K134) was constructed where an additional 7 target positions on the allergen surface were targeted: T10, K20, Q36, E73, E87, K129 and S149. These seven positions were selected from surface areas that are outside coherent surface areas that are ⁇ common among Fagales allergens.
• the library was based on the use of degenerated DNA oligonucleotide primers allowing introduction of several different amino acid residues in each position.
• several mutated amino acid residue positions in rBet v 1 (2744) and rBet v1 (2628) could either be maintained or mutated back to the residues found in WT rBet v 0 1.2801.
• a semi-random library based on rBet v 1 (2744) and rBet v1 (2628) and rBet v 1 (2 ⁇ 9 ⁇ ) i.e. N28, K32, E4 ⁇ , P108 was constructed where an additional 7 target positions on the allergen surface were targeted: ⁇ T10, K20, Q36, E73, E87, K129 and S149.
• Bet v 1 allergen mutants Examples of specific Bet v 1 allergen mutants according to the present 0 invention are listed below. Mutated amino acid positions are indicated in bold small print:
• Bet v l (“3004") (SEQ ID NO 3):
• Bet v 1 (“300 ⁇ ") (SEQ ID NO 4):
• Bet v 1 (3009) (SEQ ID NO 6):
• Bet v l (“3006") (SEQ ID NO 7):
• Bet v 1 (“3008") (SEQ ID NO 8):
• the present invention furthermore comprises the following specific mutants:
• Bet v 1 (“3006-7") (SEQ ID NO 9): ⁇ Y ⁇ V, N28T, K32Q, E4 ⁇ S, N78K, K97S, K103V, K134E, +160N, E8S, D12 ⁇ Y, E141 S, D2 ⁇ T, E87A, S1 ⁇ T, N47K, K ⁇ N.
• Bet v 1 (“3005-12”) (SEQ ID NO 10): Y ⁇ V, N28T, K32Q, E4 ⁇ S, N78K, K97S, K103V, K134E, +160N, E8S, D126Y, E141 S, D2 ⁇ T, E87A, S1 ⁇ T, N47K, K ⁇ N, E73T, A130V, P108G, V2L GIFNvETsTTSVIPAARLFKAFILtGDtLFPqVAPQAISSVENIsGkGGPGTIKnlS FPEGLPFKYVKDRVDtVDHTkFKYNYSVIaGGPIGDTLEslSNEIvlVATgDGGS ⁇ ILKISNKYHTKGyHEVKvEQVeASKEMGsTLLRAVESYLLAHtDAYNn
• Bet v 1 (“300 ⁇ -22") (SEQ ID NO 11 ):
• Bet v 1 (“300 ⁇ -27”) (SEQ ID NO 12): 5 Y ⁇ V, N28T, K32Q, E4 ⁇ S, N78K, K97S, K103V, K134E, +160N, T10K, K66N,
• Bet v 1 (“3007-6") (SEQ ID NO 13):
• Bet v 1 (“3007-10") (SEQ ID NO 14): 0 Y ⁇ V, N28T, K32S, E4 ⁇ S, N78K, K97S K103V, P108G, D12 ⁇ Y, K134E, +160N, E87A, E141 N, K ⁇ N, N47K, S1 ⁇ T, A130V, E8S, E73T, V2L. 4 ⁇
• Bet v 1 (“3007-22”) (SEQ ID NO 16):
• Bet v 1 (“3008-13") (SEQ ID NO 18):
• Bet v 1 (“3009-15”) (SEQ ID NO 22):
• Bet v 1 (“3009-22") (SEQ ID NO 23):
• Bet v 1 (“3009-28”) (SEQ ID NO 24):
• Bet v 1 clone (SEQ ID NO 25): GVFNVETETASVIPAARLFNAFILDGDTLFPQVAPQAISSVSNISGNGGPGTI KKISFPEGLPFNYVKDRVDSVDHTNFKYNYSVIEGGPIGDTLESISNEIVIVAT PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEPYLLAHSHAYN N
• Bet v 1 clone (SEQ ID NO 26):
• Bet v 1 clone (SEQ ID NO 27): GVFNVETETPSVIPAARLFHAFILDGDTLFPQVAPKAISSVSNISGNGGPGTI KKISFPEGLPFNYVKDRVDRVDHTKFKYNYSVIEGGPIGDTLESISNEIVIVAT PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEGYLLAHSHAYN N
• Bet v 1 clone (3034") (SEQ ID NO 28):
• Bet v 1 clone (SEQ ID NO 29):
• Bet v 1 clone (SEQ ID NO 30):
• Bet v 1 clone (SEQ ID NO 31 ): GVFNVETETPSVIPAARLFQAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI KKISFPEGLPFNYVKDRVDSVDHTNFKYNYSVIGGGPIGDTLESISNEIVIVAT PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEPYLLAHSHAYN N
• Bet v 1 clone (SEQ ID NO 32): GVFNVETETASVIPAARLFLAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI KKISFPEGLPFNYVKDRVDGVDHTKFKYNYSVIDGGPIGDTLESISNEIVIVAT PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVERYLLAHSHAYN N
• Bet v 1 clone (SEQ ID NO 33):
• Bet v 1 clone (SEQ ID NO 34):
• Bet v 1 clone "3041”) (SEQ ID NO 35):
• Bet v 1 clone (SEQ ID NO 36): GVFNVETETPSVIPAARLFKAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI KKISFPEGLPFNYVKDRVDRVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVAT PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVERYLLAHSHAYN N
• Bet v 1 clone (SEQ ID NO 37): ⁇ 0
• Bet v 1 clone (3044") (SEQ ID NO 38):
• Bet v 1 clone (SEQ ID NO 39):
• Bet v l (3010) (SEQ ID NO 40)
• the recombinant mutant allergens according to the invention have diagnostic possibilities and advantages.
• Prior art allergy vaccines are based on extracts of the naturally occurring allergen source, and thus represent a wide variety of isoforms.
• the allergic individual has initially been sensitised and has IgE to one or some of the isoforms present.
• Some of the isoforms may be relevant with respect to the allergic reactions of the allergic individual due to homology and subsequent cross-reactivity with the isoform to which the individual is allergic, whereas other isoforms may be irrelevant as they do not harbour any of the IgE binding epitopes to which the allergic individual has specific IgE.
• isoforms may therefore be safe to administer, i.e. they do not result in an allergic response via IgE, whereas other isoforms may be harmful causing undesirable side-effects.
• mutants of the invention and the compositions of the invention intended to be administered therapeutically may also be used for an in vivo or in vitro diagnostic assay to monitor the relevance, safety or outcome of a treatment with such mutants or compositions.
• Diagnostic samples to be applied include body samples, such as blood or sera.
• the invention also relates to a diagnostic assay for assessing relevance, safety or outcome of therapy of a subject using a recombinant mutant allergen according to the invention or a composition according to the invention, wherein an IgE containing sample of the subject is mixed with said mutant or said composition and assessed for the level of reactivity between the IgE in said sample and said mutant.
• the assessing of the level of reactivity between the IgE in the sample and the mutant may be carried out using any known immunoassay.
• This Example describes characterisation of recombinant mutant Bet v 1 mutant allergens with diminished IgE-binding affinity.
• the specific mutant allergens are also disclosed in PCT/DK 01/00764. The following represents an illustrating example of how to prepare mutants according to the present invention.
• Bet v 1 shows about 90% amino acid sequence identity with major allergens from pollens of taxonomically related trees, i.e Fagales (for instance hazel and hornbeam) and birch pollen allergic patients often show clinical symptoms of allergic cross-reactivity towards these Bet v 1 homologous proteins.
• Bet v 1 also shows about 50-60% sequence identity with allergic proteins present in certain fruits (for instance apple and cherry) and vegetables (for instance celery and carrot) and there are clinical evidence for allergic cross- reactivity between Bet v 1 and these food related proteins.
• Bet v 1 shares significant sequence identity (20-40%) with a group of plant proteins called pathogenesis-related proteins (PR-10), however there are no reports of allergic cross-reactivity towards these PR-10 proteins.
• Amino acid residues for site-directed mutagenesis were selected among surface exposed residues present in Ser v 1. The relative orientation and percentage of solvent-exposure of each amino acid residue was calculated based on their atomic coordinates. Residues having a low degree of solvent exposure ( ⁇ 20%) were not regarded relevant for mutagenesis due to the possible disruption of the structure or lack of antibody interaction. The remaining residues were ranked according to their degree of solvent- exposure.
• Sequences homologous to the query sequence (Bet v 1 No. 2801 , WHO IUIS Nomenclature Subcommittee on Allergens) were derived from GenBank and EMBL sequence databases by a BLAST search (Altschul et al., ref. 18). All sequences with BLAST reported probabilities less than 0.1 were taken into consideration and one list were constructed containing a non-redundant list of homologous sequences. These were aligned by CLUSTAL W (Higgins et al., ref. 19) and the percentage identity were calculated for each position in the sequence considering the complete list or taxonomically related species only. A total of 122 sequences were homologous to Ser v 1 No. 2801 of which 57 sequences originates from taxonomically related species.
• RNA was prepared from Betula verrucosa pollen (Allergon, Sweden) by phenol extraction and LiCI precipitation. Oligo(dT)-cellulose affinity chromatography was performed batch-wise in Eppendorph tubes, and double-stranded cDNA was synthesised using a commercially available kit (Amersham). DNA encoding Bet v 1 was amplified by PCR and cloned. In brief, PCR was performed using cDNA as template, and primers designed to match the sequence of the cDNA in positions corresponding to the amino terminus of Bet v 1 and the 3'-untranslated region, respectively. The primers were extended in the ⁇ '-ends to accommodate restriction sites ( ⁇ /col and rV/ndlll) for directional cloning into pKK233-2.
• the gene encoding Bet v 1 was subsequently subcloned into the maltose binding protein fusion vector pMAL-c (New England Biolabs). The gene was amplified by PCR and subcloned in frame with malE to generate maltose binding protein (MBP)-Bef v 1 protein fusion operons in which MBP and Bet v 1 were separated by a factor X a protease clevage site positioned to restore the authentic aminoterminal sequence of Bet v 1 upon cleavage, as described in ref. 15.
• MBP maltose binding protein
• Bet v 1 were separated by a factor X a protease clevage site positioned to restore the authentic aminoterminal sequence of Bet v 1 upon cleavage, as described in ref. 15.
• PCR was performed using pKK233-3 with Bet v 1 inserted as template and primers corresponding to the amino- and carboxyterminus of the protein, respectively.
• the promoter proximal primer was extended in the 5'-end to accommodate 4 codons encoding an in frame factor X a protease cleavage site. Both primers were furthermore extended in the ⁇ '-ends to accommodate restriction sites (Kpn ⁇ ) for cloning.
• the Bet v 1 encoding genes were subcloned using 20 cycles of PCR to reduce the frequency of PCR artefacts.
• Two mutation-specific oligonucleotide primers were synthesised accommodating each mutation, one for each DNA strand, see Figs. 3 and 4.
• both primers were extended 7 nucleotides in the ⁇ '-end and 15 nucleotides in the 3'-end.
• the extending nucleotides were identical in sequence to the Bet v 1 gene in the actual region.
• primers Two generally applicable primers (denoted “all-sense” and “all non-sense” in Figure 4) were furthermore synthesised and used for all mutants. These primers were 15 nucleotides in length and correspond in sequence to regions of the pMAL-c vector approximately 1 kilobase upstream and downstream from the Bet v 1. The sequence of the upstream primer is derived from the sense strand and the sequence of the downstream primer is derived from the non-sense strand, see Fig. 4.
• PCR reaction Two independent PCR reactions were performed essentially according to standard procedures (Saiki et al 1988, ref. 20) with the exception that only 20 temperature cycles were performed in order to reduce the frequency of PCR artefacts.
• Each PCR reaction used pMAL-c with Bet v 1 inserted as template and one mutation-specific and one generally applicable primer in meaningful combinations.
• the PCR products were purified by agarose gel electrophoresis and electro- elution followed by ethanol precipitation.
• a third PCR reaction was performed using the combined PCR products from the first two PCR reactions as template and both generally applicable primers. Again, 20 cycles of standard PCR were used.
• the PCR product was purified by agarose gel 0 electrophoresis and electro-elution followed by ethanol precipitation, cut with restriction enzymes (Ss/WI/EcoRI), and ligated directionally into pMAL-c with Bet v 1 inserted restricted with the same enzymes.
• Figure ⁇ shows an overview of all 9 Bet v 1 mutations, which are as follows ⁇
• Plasmid DNA's from 10 ml of bacterial culture grown to saturation overnight 0 in LB medium supplemented with 0.1 g/l ampicillin were purified on Qiagen- 63
• Bet v 1 (Bet v 1 No. 2 ⁇ 01 and mutants) were over-expressed in Escherichia coli DH ⁇ a fused to maltose-binding protein and purified as described in ref. 15. Briefly, recombinant E.coli cells were grown at 37°C to an optical density of 1.0 at 436 nm, whereupon expression of the Bet v 1 fusion protein was induced by addition of IPTG. Cells were harvested by centrifugation 3 hours post-induction, re-suspended in lysis buffer and broken by sonication.
• recombinant fusion protein was isolated by amylose affinity chromatography and subsequently cleaved by incubation with Factor Xa (ref. 15). After F Xa cleavage, recombinant Bet v 1 was isolated by gelfiltration and if found necessary, subjected to another round of amylose affinity chromatography in order to remove trace amounts of maltose-binding protein.
• Purified recombinant Bet v 1 was concentrated by ultrafiltration to about ⁇ mg/ml and stored at 4 °C. The final yields of the purified recombinant Bet v 1 preparations were between 2-5 mg per litre E. coli cell culture.
• the purified recombinant Bet v 1 preparations appeared as single bands after silver-stained SDS-polyacrylamide electrophoresis with an apparent molecular weight of 17.5 kDa.
• N-terminal sequencing showed the expected sequences as derived from the cDNA nucleotide sequences and quantitative amino acid analysis showed the expected amino acid compositions.
• the seven mutant Ser v 1 were produced as recombinant Bet v 1 proteins and purified as described above and tested for their reactivity towards ⁇ polyclonal rabbit antibodies raised against Bet v 1 isolated from birch pollen. When analysed by immunoelectrophoresis (rocket-line immunoelectrophoresis) under native conditions, the rabbit antibodies were able to precipitate all mutants, indicating that the mutants had conserved ⁇ - carbon backbone tertiary structure. 0
• mutants Glu4 ⁇ Ser, Prol O ⁇ Gly, Asn23Thr+Lys32Gln and Glu60Ser were selected for further analysis.
• Glutamic acid in position 4 ⁇ show a high degree of solvent-exposure (40%).
• a serine residue was found to occupy position 4 ⁇ in some of the Bet v 1 homologous PR-10 proteins arguing for that glutamic acid can be replaced by 0 serine without distortion of the ⁇ -carbon backbone tertiary structure.
• the substitution of glutamic acid with serine gives rise to a non- naturally occurring Bet v 1 molecule.
• Crystals of recombinant Glu4 ⁇ Ser Bet v 1 were grown by vapour diffusion at 2 ⁇ °C, essentially as described in (Spangfort et al 1996b, ref. 21 ).
• Glu4 ⁇ Ser ⁇ Bet v 1 at a concentration of 5 mg/ml, was mixed with an equal volume of 2.0 M ammonium sulphate, 0.1 M sodium citrate, 1 % (v/v) dioxane, pH 6.0 and equilibrated against 100x volume of 2.0 M ammonium sulfate, 0.1 M sodium citrate, 1 % (v/v) dioxane, pH 6.0. After 24 hours of equilibration, crystal growth was induced by applying the seeding technique described in 0 ref. 21 , using crystals of recombinant wild-type Bet v 1 as a source of seeds.
• the IgE-binding properties of Bet v 1 Glu4 ⁇ Ser mutant was compared with recombinant Bet v 1 in a fluid-phase IgE-inhibition assay using a pool of serum IgE derived from birch allergic patients.
• 0 Recombinant Bet v 1 no. 2S01 was biotinylated at a molar ratio of 1 :5 (Ser v 1 no. 2801 :biotin).
• the inhibition assay was performed as follows: a serum sample (25 ⁇ l) was incubated with solid phase anti IgE, washed, resuspended and further incubated with a mixture of biotinylated Ser v 1 no.
• Figure 6 shows the inhibition of the binding of biotinylated recombinant Bet v 1 to serum IgE from a pool of allergic patients by non-biotinylated Bet v 1 and by Bet v 1 Glu45Ser mutant.
• the maximum level of inhibition reached by the Bet v 1 Glu4 ⁇ Ser mutant is clearly lower compared to recombinant Bet v 1. This may indicate that after the Glu4 ⁇ Ser substitution, some of the specific IgE present in the serum pool are unable to recognise the Bet v 1 Glu4 ⁇ Ser mutant.
• 2 ⁇ and lysine 32 are located close to each other on the molecular surface and most likely interact via hydrogen bonds.
• a threonine and a gluatamate residue were found to occupy positions 26 and 32, respectively in some of the Bet v 1 homologous PR-10 proteins arguing for that aspartate and lysine can be replaced with threonine and glutamate, respectively without distortion of the ⁇ -carbon backbone tertiary structure.
• the substitutions gives rise to a non- naturally occurring Ser v 1 molecule.
• Figure 7 shows the inhibition of the binding of biotinylated recombinant Bet v 1 to serum IgE from a pool of allergic patients by non-biotinylated Bet v 1 and by Bet v 1 mutant Asn23Thr+Lys32Gln. 0
• the maximum level of inhibition reached by the Bet v 1 mutant 0 Asn2 ⁇ Thr+Lys32Gln mutant is clearly lower compared to recombinant Bet v 1. This may indicate that after the Asn23Thr+Lys32Gln substitutions, some of the specific IgE present in the serum pool are unable to recognise the Bet v 1 mutant Asn2 ⁇ Thr+Lys32Gln.
• Proline in position 106 shows a high degree of solvent-exposure (60%).
• a glycine residue was found to occupy position 108 in some of the Bet v 1 homologous PR-10 proteins arguing for that proline can be replaced with 0 glycine without distortion of the ⁇ -carbon backbone tertiary structure.
• the substitution of proline with glycine gives rise to a non-naturally occurring Ser v 1 molecule.
• the IgE-binding properties of Bet v 1 Prol O ⁇ Gly mutant was compared with recombinant Bet v 1 in a fluid-phase IgE-inhibition assay using the pool of serum IgE derived from birch allergic patients described above.
• Figure 6 shows the inhibition of the binding of biotinylated recombinant Bet v 1 to serum IgE from a pool of allergic patients by non-biotinylated Bet v 1 and by Bet v 1 Prol O ⁇ Gly mutant.
• Recombinant Sef v 1 reaches 50% inhibition at about 6.5 ng whereas the corresponding concentration for Bet v 1 Prol O ⁇ Gly is 15 ng. This show that the single point mutation introduced in Bet v 1 Prol O ⁇ Gly lowers the affinity for specific serum IgE by a factor of about 2.
• the maximum level of inhibition reached by the Bet v 1 ProlO ⁇ Gly mutant is somewhat lower compared to recombinant Bet v 1. This may indicate that after the Prol O ⁇ Gly substitution, some of the specific IgE present in the serum pool are unable to recognise the Bet v 1 ProlO ⁇ Gly mutant.
• Glutamic acid in position 60 show a high degree of solvent-exposure (60%).
• a serine residue was found to occupy position 60 in some of the Sef v 1 homologous PR-10 proteins arguing for that glutamic acid can be replaced with serine without distortion of the ⁇ -carbon backbone tertiary structure.
• the substitution of glutamic acid with serine gives rise to a non-naturally occurring Bet v 1 molecule. IgE-binding properties of Bet v 1 Glu ⁇ OSer mutant
• the IgE-binding properties of Bet v 1 Glu ⁇ OSer mutant was compared with recombinant Bet v 1 in a fluid-phase IgE-inhibition assay using the pool of serum IgE derived from birch allergic patients described above.
• Figure 9 shows the inhibition of the binding of biotinylated recombinant Bet v 1 to serum IgE from a pool of allergic patients by non-biotinylated Bet v 1 and by Bet v 1 Glu60Ser mutant.
• the substitution glutamic acid 60 to serine does not shown any significant effect on the IgE-binding properties of.
• FIG. 10 shows the structural integrity of the purified recombinant protein, recorded at close to equal concentrations.
• the overlap in peak amplitudes and positions in the CD spectra from the two recombinant proteins shows that the two preparations contain equal amounts of secondary structures strongly suggesting that the ⁇ -carbon backbone tertiary structure is not affected by the introduced amino acid substitutions.
• the IgE-binding properties of the mutant was compared with recombinant Bet ⁇ / 1 in a fluid-phase IgE-inhibition assay using the pool of serum IgE derived from birch allergic patients described above.
• Figure 11 shows the inhibition of the binding of biotinylated recombinant Bet v 1 to serum IgE from a pool of allergic patients by non-biotinylated Bet v 1 and by the Bet v 1 mutant.
• the inhibition curve of the mutant is no longer parallel relative to recombinant. This shows that the substitutions introduced in the mutant have changed the IgE-binding properties and epitope profile compared to recombinant. The lack of parallellity makes it difficult to quantify the decrease of the mutants affinity for specific serum IgE.
• Recombinant Bet v 1 reaches 50% inhibition at about 6 ng whereas the corresponding concentration for Bet v 1 (Asn28Thr, Lys32Gln, Glu45Ser, ProlO ⁇ Gly) mutant is 30 ng, i.e a decrease in affinity by a factor ⁇ . However, in order to reach ⁇ 0% inhibition the corresponding values are 20 ng and 400 ng, respectively, i.e a decrease by a factor 20.
• each single mutation (or several mutations if located closely together in the DNA sequence) was introduced into sequential DNA sequences of Bet v 1.2801 derivatives i.e. Bet v 1 (2595) or Bet v 1 (2628) or Bet v 1 (2733) using sense and anti-sense mutation- specific oligonucleotide primers accommodating each mutation(s) along with sense and anti-sense oligonucleotide primers accommodating either upstream or downstream neighbour mutations or the N-terminus/C-terminus of Bet v 1 , respectively as schematically illustrated in Figure 12 (I).
• Bet v 1.2801 derivatives i.e. Bet v 1 (2595) or Bet v 1 (2628) or Bet v 1 (2733)
• sense and anti-sense mutation- specific oligonucleotide primers accommodating each mutation(s) along with sense and anti-sense oligonucleotide primers accommodating either upstream or downstream neighbour mutations or the N-terminus/C-terminus of Bet v 1
• PCR products from PCR reaction I were purified, mixed and used as templates for an additional PCR reaction (II) with oligonucleotide primers accommodating the N-terminus and C-terminus of Bet v 1 as schematically illustrated in Figure 13 (II).
• the PCR products were purified by agarose gel electrophoresis and PCR gel purification (Life Techhnologies) followed by ethanol precipitation, cut with restriction enzymes (Sacl/EcoRI) or (Sad/ Xbal), and ligated directionally into pMAL-c restricted with the same enzymes.
• FIG. 13 shows synthesised oligonucleotide primers and schematically illustrations for the construction of Bet v 1 mutants.
• the following Bet v 1 mutants were cloned and sequenced (sequencing of nucleic acid molecules is described in Example 1 ):
• Bet v 1 mutant library containing Bet v 1 mutants with 17-20 point mutations of which amino acid substitutions were randomly substituted in 7 positions.
• the library contained hundreds of different clones. Fifteen Bet v 1 mutants named 0 Bet v 1 (3031 ) to (3046) were obtained from this Bet v 1 mutant library generated using degenerated oligonucleotide primers.
• the cloning procedure was the same as illustrated in figure 12 except that 0 the primers used in the first PCR round were degenerated in certain positions as indicated in figure 16 by letters other than G, C, T or A. Use of other letters than G, C, T or A indicates that the primers contain several different nucleotides in these positions.
• Eight PCR products spanning the Bet v 1 gene were produced and purified in the first PCR round and then assembled ⁇ using end-primers (3076s and 3067a) in a second PCR reaction where the eight PCR products from the first PCR round were used as a template.
• Bet v 1 mutants 3031 to 3045 were DNA sequenced as described for the Bet v 1 3004, 3005, 3007 and 3007 mutants in order to verify the number and 0 nature of the introduced point mutations: Bet v 1 clone ("3031 ”) (SEQ ID NO 25):
• Bet v 1 clone (SEQ ID NO 26):
• Bet v 1 clone (SEQ ID NO 27):
• Bet v 1 clone (SEQ ID NO 26) (SEQ ID NO 26): 0 GVFNVETETTSVIPAARLFHAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI KKISFPEGLPFNYVKDRVDSVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVAT PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVERYLLAHSHAYN N
• Bet v 1 clone (SEQ ID NO 30): GVFNVETETPSVIPAARLFLAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI KKISFPEGLPFNYVKDRVDT /DHTKFKYNYSVIGGGPIGDTLESISNEIVIVAT PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVERYLLAHSHAYN N
• Bet v 1 clone (SEQ ID NO 31 ):
• Bet v 1 clone (SEQ ID NO 32):
• Bet v 1 clone (SEQ ID NO 33):
• Bet v 1 clone (SEQ ID NO 34): GVFNVETETPSVIPAARLFKAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI KKISFPEGLPFNYVKDRVDSVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVAT PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVETYLLAHSHAYN N
• Bet v 1 clone "3041”) (SEQ ID NO 36): GVFNVETETPSVIPAARLFKAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI KKISFPEGLPFNYVKDRVDRVDHTKFKYNYSVIGGGPIGDTLESISNEIVIVAT PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVERYLLAHSHAYN N ⁇
• Bet v 1 clone (SEQ ID NO 36):
• Bet v 1 clone (SEQ ID NO 37):
• Bet v 1 clone (3044") (SEQ ID NO 38):
• Bet v 1 clone (SEQ ID NO 39): ⁇ GVFNVETETPSVIPAARLFMAFILDGDNLFPKVAPPAISSVSNISGNGGPGTI KKISFPEGLPFNYVKDRVDGVDHTKFKYNYSVIDGGPIGDTLESISNEIVIVAT PDGGSILKISNKYHTIGDHEVEAEQVEASKEMGETLLRAVEGYLLAHSHAYN N
• Solvent accessibility was calculated using the software Insightll, version 97.0 0 (MSI) and a probe radius of 1.4 A (Connolly surface).
• 0 3-D structure is based on accession number Z80104 (I bvLpdb).
• Table 1 shows a listing in descending order of solvent exposure of Bet v 1 amino acids. Column 1 lists the amino acid number starting from the aminoterminal, column 2 lists the amino acid in one letter abbreviation, column 3 lists the normalised solvent exposure index, column 4 lists the percent of known sequences having the concerned amino acid in this position.
• This Example describes preparation and characterisation of recombinant mutant Bet v 1 allergens with more than four mutations and diminished IgE- binding affinity according to prior art PCT/DK 01/00764. Mutants according to the present invention are prepared and assayed accordingly.
• PCR products from PCR reaction I were purified, mixed and used as templates for an additional PCR reaction (II) with oligonucleotide primers accommodating the N-terminus and C-terminus of Bet v 1 as schematically illustrated in Figure 16 (II).
• the PCR products were purified by agarose gel electrophoresis and PCR gel purification (Life Techhnologies) followed by ethanol precipitation, cut with restriction enzymes (Sacl/EcoRI) or (Sad/ Xbal), and ligated directionally into pMAL-c restricted with the same enzymes.
• ⁇ 2 restriction enzymes
• FIG. 16 shows synthesised oligonucleotide primers and schematically illustrations for the construction of Bet v 1 mutants with more than four primary mutations.
• the mutated amino acids were preferably selected from the group consisting of amino acids that are characterised by being highly solvent exposed and conserved as described in Example 3.
• the Bet v 1 mutants are as follows:
• Bet v 1 (2637): Ala16Pro, Asn28Thr, Lys32Gln, Lys103Thr, Pro1 O ⁇ Gly, Leu162Lys, Ala163Gly, Ser1 ⁇ Pro.
• Bet v 1 (2733): Tyr ⁇ Val, Lys134Glu, Asn28Thr, Lys32Gln, Glu4 ⁇ Ser, Lys65Asn, Asn73Lys, Lys103Vai, Lys97Ser, ProlO ⁇ Gly, Arg14 ⁇ Glu, Aspl ⁇ His, +160Asn.
• Bet v 1 (2744): Tyr ⁇ Val, Lys134Glu, Glu42Ser, Glu4 ⁇ Ser, Asn7 ⁇ Lys, Lys103Val, Lys123lle, Aspl ⁇ His, +160Asn.
• Bet v 1 (2628) and Bet v 1 (2637) mutants Figure 17 shows introduced point mutations at the molecular surface of Bet v 1 (2628) and Bet v 1 (2637).
• FIG. 18 shows the CD spectra of recombinant Bet v 1.2801 (wildtype) and Bet v 1 (2637) mutant, recorded at close to equal concentrations.
• the overlap in peak amplitudes and positions in the CD spectra from the two recombinant proteins shows that the two preparations contain equal amounts of secondary structures strongly suggesting that the ⁇ -carbon backbone tertiary structure is not affected by the introduced amino acid substitutions.
• IgE-binding properties of Bet v 1 (2628. and Bet v 1 (2637) mutants.
• the IgE-binding properties of Bet v 1 (262 ⁇ ) and Bet v 1 (2637) as well as a 1 :1 mix of Bet v 1 (2626) and Bet v 1 (2637) was compared with recombinant wild type Bet v 1.2801 in a fluid-phase IgE-inhibition assay using a pool of serum IgE derived from birch allergic patients.
• recombinant Bet v 1.2801 was biotinylated at a molar ratio of 1 :6 (Bet v 1 no. 2801 :biotin).
• the inhibition assay was performed as follows: a serum sample (2 ⁇ ⁇ l) was incubated with solid phase anti IgE, washed, re-suspended and further incubated with a mixture of biotinylated Bet v 1.2801 and a given mutant or 1 :1 mix of the two mutants.
• the amount of biotinylated Bet v 1.2801 bound to the solid phase was estimated from the measured RLU after incubation with acridinium ester labelled streptavidin. The degree of inhibition was calculated as the ratio between the RLU's obtained using buffer and mutant as inhibitor.
• Figure 19 shows the inhibition of the binding of biotinylated recombinant Bet v 1.2801 to serum IgE from a pool of allergic patients by non-biotinylated Bet v 1.2801 and by Bet v 1 (2628), Bet v 1 (2637) and a 1 :1 mix of Bet v 1 (2628) and Bet v 1 (2637).
• the maximum level of inhibition reached by the Bet v 1 (2628) mutant protein is clearly lower compared to recombinant Bet v 1.2801. This may indicate that some of the specific IgE present in the serum pool are unable to recognise the Bet v 1 (2628) mutant protein due to the introduced point mutations.
• Bet v 1 (2637) reaches 50% inhibition at about 400-500 ng showing that the ⁇ point mutation introduced in the Bet v 1 (2637) mutant lowers the affinity for specific serum IgE by 80 to 100-fold compared to Bet v 1.2801.
• the large difference in IgE-binding is further supported by a clear difference in inclination of the inhibition curve obtained with Bet v 1 (2637) mutant protein compared to the inhibition curve for Bet v 1.2801.
• the different inclination 0 provide evidence that the reduction in IgE-binding is due to a distinctly different epitope pattern of the mutant compared to Bet v 1.2801.
• Bet v 1 (2628) and Bet v 1 (2637) mutant protein were both able to induce proliferation in T cell lines from birch pollen allergic patients with stimulation indices similar to 0 recombinant and naturally occurring. This suggests that both of Bet v 1 (2628) and Bet v 1 (2637) mutant protein can each initiate the cellular immune response necessary for antibody production.
• Histamine release from basophil leucocytes was performed as follows. Heparinized blood (20 ml) was drawn from each birch pollen patient, stored at room temperature, and used within 24 hours. Twenty-five microlitres of heparinized whole blood was applied to glass fibre coated microtitre wells (Reference Laboratory, Copenhagen, Denmark) and incubated with 26 microlitres of allergen or anti-lgE for 1 hour at 37°C. Thereafter the plates were rinsed and interfering substances were removed. Finally, histamine bound to the microfibres was measured spectrophotofluometrically.
• Histamine release data is shown in Figure 20 and Figure 21.
• the potency of Bet v 1 (2628) and Bet v 1 (2637) mutant protein to induce histamine release in human basophil from two birch pollen allergic patients has been tested.
• the release curve of the mutated allergens to induce histamine release is clearly shifted to the right compared to the release curve of Bet v 1.2801.
• the shift indicate that the potency of Bet v 1 (2628) and Bet v 1 (2637) is reduced 3 to 10-fold.
• Bet v 1 (2744) and Bet v 1 (2763) was likewise constructed for use as a mixed allergen vaccine.
• point mutations were distributed in an all surface arranged fashion as shown in Figure 22 and Figure 23 and was again designed to affect different surface areas in the two molecules, respectively, as shown in Figure 24.
• modified allergens might individually be used as single allergen vaccines as well.
• FIG. 25 shows the CD spectra of recombinant Bet v 1.2801 (wildtype) and Bet v 1 (2744) mutant, recorded at close to equal concentrations.
• the overlap in peak amplitudes and positions in the CD spectra from the two recombinant proteins shows that the two preparations contain equal amounts of secondary structures strongly suggesting that the ⁇ -carbon backbone tertiary structure is not affected by the introduced amino acid substitutions. 8 ⁇
• a Mutant Bet v 1 (2733) has been constructed and recombinantly expressed.
• the distribution of point mutations in Bet v 1 (2733) leave several surface areas constituting >40 ⁇ A 2 unaltered.
• Figure 28 show introduced point mutations at the molecular surface of Bet v 1 (2733).
• This Example describes characterisation of recombinant mutant Bet v 1 allergens with more than four mutations and diminished IgE-binding affinity according to prior art PCT/DK 01/00764. Mutants according to the present invention are prepared and assayed accordingly.
• PBL Peripheral blood lymphocytes
• Ten PBL and eight T-cell lines were stimulated with birch extract (Bet v), naturally purified bet v 1 (nBet v 1), recombinant Bet v 1 (rBet v 1 or wt; 27) and four different mutated forms of rBet v 1 (described elsewhere): 2596, 2628, 2637, 2744, 2773.
• the 2637 mutant was later found to be partly unfolded and will not be discussed.
• T-cells were added in 3x10 4 T-cells per well and stimulated with irradiated autologous PBL (1x10 5 cells/well) and 3 different concentrations of the different birch samples. After 1 day cells from each well with the highest concentration birch were harvested for cytokine production. Radioactive labelled thymidine were added to the wells. At day 2 the cells were harvested onto a filter and counted as described for PBL. Supernatant from the quadroplicates were pooled and cytokines were measured using a CBA (cytokine bead array) kit from Becton Dickinson.
• CBA cytokine bead array
• Fig. 29 shows the Stimulation Index for the above- mentioned Bet v 1 preparations.
• the Stimulation Index (SI) is calculated as proliferation (cpm: count per minute) of the stimulated sample (highest concentration) divided with the proliferation (cpm) of the medium control.
• PPD designates purified protein derivative from mucobacterium tuberculosis, which serves as a positive control.
• Cytokine production was dominated by IFN-gamma and increased proportionally with PBL proliferation. No signs of a Th1/Th2 shift were apparent (Fig. 30-32).
• Figure 30 shows a patient with a ThO profile, Figure 31 a Th1 profile and Figure 32 a Th2 profile. Cytokine production is measured in pg/ml indicated as the bars and the ratio between IL-5/IFN-gamma is the lower dashed line (Y-axis to the right). Proliferation is measured in cpm seen on the Y-axis to the right as a solid line measured in cpm. Medium and MBP (maltose bindig protein) are included as background controls.
• MBP maltose bindig protein
• T-cell lines established on nBet v 1 and all, except one, proliferated equally well to all birch samples.
• Four T-cell lines were secreting ThO like cytokines based on the IL- ⁇ and IFN-gamma ratio (Th2 > 5, ⁇ > ThO > 0.2, 0.2 > Th1 ).
• Three T-cell lines were secreting Th1 cytokines and one T-cell line was secreting Th2 cytokines.
• the IL-5/IFN-gamma ratio was not affected by the different birch samples.
• This Example describes characterisation of recombinant mutant Bet v 1 allergens with more than four mutations and diminished IgE-binding affinity according to prior art PCT/DK 01/00764. Mutants according to the present invention are be prepared and assayed accordingly.
• blocking antibodies is defined as antibodies, different from human IgE antibodies, that are able to bind to an antigen and prevent the binding of human IgE antibodies to that antigen.
• mice The ability of recombinant Bet v1 2227 wild type protein (rBet v 1 ) and Bet v 1 2595, 2628, 2744 and 2773 mutant proteins to induce Bet v 1 specific IgG antibodies and blocking antibodies was tested in immunization experiments in mice.
• BALB/cA mice (8 in each group) were immunized by intraperitoneal injections with recombinant Bet v1 2227 wild type protein or the four mutant proteins. The mice were immunized four times with a dose interval of 14 days.
• the different proteins were conjugated to 1 ,25 mg/ml Alhydrogel, (Aluminium Hydroxide gel, 1 ,3 % pH 8.0 - 8.4, Superfos Biosector). The mice were immunized with either 1 ug protein/dose or 10 ug protein/dose. Blood samples were drawn by orbital bleed at day 0,14,35, 21 , 49 and 63.
• IgG antibody levels was analyzed by direct ELISA using rBet v 1 coated microtiterplates and biotinylated rabbit anti mouse IgG antibodies (Jackson) as detection antibody. Immunization with recombinant Bet v1 2227 wild type protein or the four mutant proteins induced a strong r Bet v 1 specific IgG response. This finding demonstrates that the four mutated proteins are able to induce antibodies that are highly cross reactive to the Bet v 1 2227 wild type protein
• This example describes the structural characterization and IgE-binding properties of a mutant according to the invention having 12 point mutation.
• the mutations introduced in mutant 3007 are described in example 2. 0
• FIG. 33 shows 5 the CD spectra of recombinant Bet v 1.2801 (wildtype) and Bet v 1 (3007) mutant, recorded at equal concentrations as previously described in example 1.
• the overlap in amplitude-positions in the CD spectra from the two recombinant proteins indicates that the two preparations contain roughly equal amounts of secondary structures, strongly suggesting that the ⁇ - 0 carbon backbone tertiary structure is not or affected by the introduced amino acid substitutions.
• Figure 34 shows the inhibition of the binding of biotinylated recombinant Bet 5 v 1.2801 to serum IgE from a pool of allergic patients by non-biotinylated Bet v 1.2801 (wildtype) and the Bet v 1 (3007) mutant according to methods described in example 4.
• Recombinant Bet v 1.2801 0 reaches 50% inhibition at about 5 ng whereas the corresponding concentration for Bet v 1 (3007) mutant is about 200 ng.
• the level of inhibition reached by the Bet v 1 (3007) mutant protein is clearly lower compared to recombinant Bet v 1.2801. This show that the 12 point mutations introduced in the Bet v 1 (3007) mutant lowers the affinity for specific serum IgE.
• WO 90/11293 Immunologic Pharmaceutical Corporation, The University of North Carolina at Chapel Hill, Allergenic proteins from ragweed and uses thereof
• Lu G Villalba M, Coscia MR, Hoffman DR and King TP: "Sequence Analysis and Antigenic Cross-reactivity of a Venom Allergen, Antigen 5, from Hornets, Wasps, and Yellow Jackets". Journal of Immunology 160, 2823- 2630 (1993).

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