ZA200303667B - Novel mutant allergens. - Google Patents

Description

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WO (2/40676 PCT/DK01/00764

NOVEL MUTANT ALLERGENS

FIELD OF THE INVENTION

The present invention relates to novel recombinant allergens, which are mutants of naturally occurring allergens. Also, the invention relates to a composition comprising a mixture of the novel recombinant mutant allergens. Further, 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. In further embodiments, 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.

BACKGROUND OF THE INVENTION

Genetically predisposed individuals become sensitised (allergic) to antigens originating from a variety of environmental sources, to the allergens of which the individuals are exposed. The allergic reaction occurs when a previously sensitised individual is re-exposed to the same or a homologous allergen. 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.

However, 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. Thus, allergic responses demonstrate that the immune response itself can . 5 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 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

IIT 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.

Commonly, 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.

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 cells is thought to be of particular importance.

Allergy vaccination

The concept of vaccination is based on two fundamental characteristics of the immune system, namely specificity and memory. 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.

Compared to other types of vaccination 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. Thus, allergy vaccination using allergens from natural sources has an inherent risk of side effects being in the utmost consequence life threatening to the patient.

Approaches to circumvent this problem may be divided in three categories. In practise measures from more than one category are often combined. 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.

The detailed mechanism behind successful allergy vaccination remains controversial. It is, however, agreed that T cells play a key role in the overall regulation of immune responses. According to current consensus the relation between two extremes of T cell phenotypes, Thl and Th2, determine the allergic status of an individual.

Upon stimulation with allergen Thl cells secrete interleukines dominated by interferon-y leading to protective immunity and the individual is healthy. Th2 cells on the other hand secrete predominantly interleukin 4 and 5 leading to IgE synthesis and eosinophilia and the individual is allergic. 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 Thl phenotype.

Antibody-binding epitopes (B-cell epitopes)

X-ray crystallographic analyses of Fyp-antigen complexes has increased the understanding of antibody-binding epitopes. According to this type of analysis 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 5 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.

In WO 97/30150 (ref. 1), 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).

In WO 92/02621 (ref. 2), 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.

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.

Chemical modification of allergens

Several approaches to chemical modification of allergens have been taken. Approaches of the early seventies include chemical coupling of allergens to polymers, and chemical cross-linking of allergens using formaldehyde, etc., producing the so-called ‘allergoids’. The rationale behind these approaches was random destruction of IgE binding epitopes by attachment of the chemical ligand thereby reducing IgE-binding while retaining immunogenicity by the increased molecular weight of the complexes. Inherent disadvantages of ‘allergoid’ production are linked to difficulties in controlling the process of chemical cross-linking and difficulties in analysis and standardisation of the resulting high molecular weight complexes. ‘Allergoids’ are currently in clinical use and due to the random destruction of IgE pinding epitopes higher doses can be administered as compared to conventional vaccines, but the safety and efficacy parameters are not improved over use of conventional vaccines.

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. In recent years it has become clear that natural allergens are heterogeneous containing isocallergens and variants having up to approximately 25% of their amino acids substituted. Some recombinant isoallergens have been found to be less efficient in IgE binding possibly due to irreversible denaturation and hence total disruption of tertiary structure.

In vitro mutagenesis and allergy vaccination

Attempts to reduce allergenicity by in vitro site directed mutagenesis have been performed using several allergens including Der f 2 (Takal et al, ref. 4), Der p 2 (Smith et al, ref. 5), a 39 kDa Dermatophagoides farinae allergen (Aki et al, ref. 6), bee venom phospholipase A2 (Forster et al, ref. 7), Ara h 1 (Burks et al, ref. 8), Ara h 2 (Stanley et al, ref. 9), Bet v 1 (Ferreira et al, ref. 10 and 11), birch profilin (Wiedemann et al, ref. 12), and Ory s 1 (Alvarez et al, ref. 13).

The rationale behind these approaches, again, is addressing allergen specific T cells while at the same time reducing the risk of IgE mediated side effects by reduction or elimination of IgE binding by disruption of the tertiary structure of the recombinant mutant allergen. The rationale behind these approaches does not include the concept of dominant IgE binding epitopes and it does not include the concept of initiating a new protective immune response which also involves B-cells and antibody generation.

The article by Ferreira et al (ref. 11) describes the use of site directed mutagenesis for the purpose of reducing

IgE binding. Although 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 different from the concept of structure based identification of conserved surface areas described here.

Although the authors do discuss conservation of a-carbon backbone tertiary structure this concept is not a part of the therapeutic strategy but merely included to assess in vitro IgE binding. Furthermore, 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 authors have knowledge of the tertiary structure of the antigen and they use this knowledge to select a surface exposed amino acid for mutation. The algorithm used can be said to be opposite to the one described by the present inventors since an amino acid differing from homologous sequences is selected. The study demonstrates that substitution of a surface exposed amino acid has the capacity to modify the binding characteristics of a monoclonal antibody, which i WO 02/40676 PCT/DK01/00764 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. One of the experiments contained do 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 o- 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 : 20 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. Conserved surface areas between homologous allergens as well as the therapeutic concept of initiating a new protective immune response are not mentioned.

The article by Spangfort et al. (ref. 15) describes the three-dimensional structure and conserved surface exposed patches of the major birch allergen. The article does not mention major IgE binding epitopes nor site directed mutagenesis, neither is therapeutic application addressed.

In none of the studies described above is IgE binding reduced by substitution of surface exposed amino acids while conserving a-carbon backbone tertiary structure.

The rationale behind above-mentioned approaches does not include the concept of dominant IgE binding epitopes and it does not include the therapeutic concept of initiating a new protective immune response.

WO 99/47680 discloses the introduction of artificial amino acid substitutions into defined critical positions while retaining the a-carbon backbone tertiary structure of the allergen. In particular, WO 29/47680 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 «a-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 3 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? of the surface of the three-dimensional 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 a-carbon backbone tertiary structure of the allergen molecule.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows mutant-specific oligonucleotide primers used for Bet v 1 mutant number 1. Mutated nucleotides are underlined.

Figure 2 shows two generally applicable primers (denoted "all-sense" and "all non-sense"), which were synthesised and used for all mutants.

Figure 3 shows the DNA and amino acid sequence of the naturally occurring allergen Bet v 1 as well as a number of Bet v 1 mutations.

Figure 4 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 Glud45Ser mutant.

Figure 5 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 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 Prol08Gly 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 Glu60Ser mutant.

Figure 8 shows the CD spectra of recombinant and

Triple-patch mutant, recorded at close to equal concentrations.

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 Triple-patch mutant.

Figure 10 shows solvent accessibility of individually aligned antigen 5 residues and alignment of Vespula antigen 5 sequences (left panel). On the right panel of

Figure 10 is shown the molecular surface of antigen 5 with conserved areas among Vespula antigen 5:s. : Figure 11 shows the sequence of the primer corresponding to the amino terminus of Ves v 5 derived from the sense : strand. The sequence of the downstream primer is derived from the non-sense strand. :

Figure 1Z shows two generally applicable primers (denoted

“all sense” and ‘all non-sense”, which were synthesised and used for all mutants.

Figure 13 shows the DNA and amino acid sequence of the naturally occurring allergen Ves v 5 as well as two Ves Vv 5 mutations.

Figure 14 shows the inhibition of the binding of biotinylated recombinant Ves v 5 to serum IgE from a pool of allergic patients by non-biotinylated Ves v 5 and by

Ves v 5 Lys72Ala mutant. oo

Figure 15 ‘shows a theoretical model of the reaction between an allergen and mast cells by IgE cross-linking.

Figure 16 shows the DNA and amino acid sequence of the naturally occurring allergen Der p 2.

Figure 17 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 18 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 19 shows introduced point mutations at the surface of Bet v 1 (2628) and Bet v 1 (2637). In mutant Bet v 1 (2628), five primary mutations were introduced in one half of Bet v 1 leaving the other half unaltered. In mutant Bet v 1 (2637), five primary and three secondary mutations were introduced in the other half, leaving the first half unaltered.

Figure 20 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 21 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 vl (2637) .

Figure 22 shows histamine release in human basophil cells of Bet v 1.2801 (wild type), Bet v 1 (2628), and Bet v 1 (2637).

Figure 23 shows histamine release in human basophil cells of Bet v 1.2801 (wild type), Bet v 1 (2628), and Bet wv 1 (2637) .

Figure 24 shows point mutations at the surface of Bet v 1 (2744) .

Figure 25 shows point mutations at the surface of Bet v 1 (2753).

Figure 26 shows point mutations at the surface of Bet v 1 (2744) and Bet v 1 (2753).

Figur 27 shows circular dichroism (CD) spectra of Bet V 1.2801 (wild type) and Bet v 1 (2744), recorded at nearly equal concentrations.

Figur 28 shows histamine release in human basophil cells of Bet v 1.2801 (wild type), and mutant Bet v 1 (2744).

Figur 29 shows histamine release in human basophil cells

© WO 02/40676 PCT/DK01/00764 of Bet v 1.2801 (wild type), and mutant Bet v 1 (2744).

Figur 30 shows point mutations at the surface of Bet v 1 (2733).

S

Figure 31 shows primers used for site-directed mutagenesis of Der p 2.

Figure 32 shows a sequence alignment of Der p 2 with other group 2 house dust mite allergens.

Figure 33 shows surface contours of Der p 2 from four different angles.

Figure 34 shows surface contours of a Der p 2 mutant from four different angles.

Figure 35A and B shows a sequence alignment of Der p 1 with other group 1 house dust mite allergens.

Figure 36 shows surface contours of Der p 1 from four different angles.

Figure 37 shows surface contours of a Der p 1 mutant from four different angles.

Figure 38A-D shows a sequence alignment of Phl p 5 with other group 5 grass allergens.

Figure 39A and B shows surface contours of Phl p 5 Model

A and Model B, respectively, from four different angles.

Figure 40A and B shows surface contours of a Phl p 9 mutant Model A and B, respectively, from four different angles.

Figure 41 shows the proliferation of Peripheral Blood

Lymphocytes expressed as Stimulation Index (SI) for various Bet v 1 preparations.

Figure 42-44 show the cytokine profile of T cells stimulated with various Bet v preparations. Figure 42 shows a patient with a ThO profile, Figure 43 a Thl profile and Figure 44 a Th2 profile.

OBJECT OF THE INVENTION

Rationale behind the present invention

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

Thl~-type immune response. This model is supported by the observation that levels of specific IgE are unaffected by successful vaccination treatment, and that successful treatment is often accompanied by a substantial rise in allergen specific IgG4. In addition, studies of nasal biopsies before and after allergen challenge do not show +a reduction in T cells with the Th2-like phenotype, but rather an increase in Thl-like T cells are observed. When the vaccine (or pharmaceutical compositions) is administered through another route than the airways, it is hypothesised, that the new immune response evolves in a location physically separated from the ongoing Th2 response thereby enabling the two responses to exist in parallel. :

Another important aspect of the immunological system is the assertion of the existence of so-called dominant IgE binding epitopes. It is proposed that these dominant IgE binding epitopes are constituted by tertiary structure dependent coherent surface areas large enough to accommodate antibody binding and conserved among iscallergens, variants, and/or homologous allergens from related species. The existence of cross-reactive IgE capable of Dbinding similar epitopes on homologous allergens is supported by the clinical observation that allergic patients often react to several closely related species, e.g. alder, birch, and hazel, multiple grass species, or several species of the house dust mite genus

Dermatophagoides. it is furthermore supported by laboratory experiments demonstrating IgE cross-reactivity between homologous allergens from related species and the capacity of one allergen to inhibit the binding of IgE to homologous allergens (Ipsen et al. 1992, ref. 16). It is well known that exposure and immune responses are related : in a dose dependent fashion. Based on the combination of these observations it is hypothesised that conserved surface areas are exposed to the immune system in higher dcses than non-conserved surface areas resulting in the generation of IgE antibodies with higher affinities, hence the term ‘dominant IgE binding epitopes’.

According to this rationale it is essential that the allergen has an a-carbon backbone tertiary structure which essentially is the same as that of the natural allergen, thus ensuring conservation of the surface topology of areas surrounding conserved patches representing targets for mutagenesis aimed at reducing

IgE binding. By fulfilling these criteria the allergen has the potential to be administered in relatively higher doses improving its efficacy in generating a protective immune response without compromising safety.

Furthermore, the invention is based on the finding that allergic symptoms are triggered by the cross-linking of allergen with two specific IgE's bound to the surface of effector cells, i.e. mast cells and basophils, via the

S high affinity IgE receptor, FceRI. For illustration, we refer to Fig. 15, which depicts a theoretical model of an allergen with 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 15A. In the situation shown in Fig. 15B 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.

However, ‘from the theoretical situation depicted in Fig. 15 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.

However, in order for a mutated allergen to be able to raise the new immune response, including the IgG response, the allergen must comprise at least one intact epitope. Preferably, the intact epitope is a dominant epitope, since such a mutated allergen will provide an improved protection when used for vaccination. . In conclusion, the inventive idea of the present invention is based on the recognition that a mutated allergen having IgE binding reducing mutations in multiple B cell epitopes, and at least one intact 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. Thus, the said mutated allergen would constitute a highly advantageous allergen in that the risk of anaphylactic reactions would be strongly reduced.

Also, 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, 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.

SUMMARY OF THE INVENTION

The present invention relates to the introduction of artificial amino acid substitutions into a number of defined critical positions, i.e. IgE binding epitopes, with the object of reducing the specific IgE binding capability of each mutated epitope.

The invention provides a recombinant allergen, characterised in that it is a mutant of a naturally occurring allergen, wherein the mutant allergen has 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 said naturally occurring allergen, wherein each primary mutation is a substitution of one surface-exposed amino acid residue with another residue, which does not occur in the same position in the amino acid sequence of any known homologous protein within the taxonomic species from which said naturally occurring allergen originates, wherein each primary mutation is spaced from each other primary mutation by at least 15 A, and wherein the primary mutations are placed in such a manner that at least one circular surface region with a area of 800 A?

comprises no mutation.

Without being bound by theory it is believed that the B cell epitopes can be distributed over almost the entire surface of the allergen. Furthermore, there is experimental evidence that at least some epitopes constitute a part of a cluster of epitopes comprising a large number of overlapping epitopes. Therefore, the a. theoretical basis for the present invention is that any surface-exposed amino acid constitutes a potential site of mutation, which may result in a reduced capability to bind specific IgE.

Accordingly, the primary mutations are defined by their : 15 location in respect to each other, i.e. they are spaced apart, to ensure that they are mutations in separate clusters of epitopes.

The present invention also provides a composition comprising two or more recombinant mutant allergen variants according to claim 1, wherein each variant is defined by having at least one principal mutation, which is absent in at least one of the other variants, wherein for each variant no secondary mutation is present within a radius of 15 A from each absent primary mutation. The composition preferably comprises 2-12, more preferably 3- 10, more preferably 4-8 and most preferably 5-7 variants.

The present invention also provides a method of preparing the recombinant allergen according to claim 1, characterised in a) identifying a number of amino acid residues in @ naturally occurring allergen, which has a solvent 3s accessibility of at least 20 %;

b) selecting at least four of the identified amino acid residues in such a manner that each selected amino acid is spaced from each other selected amino acid by at least 15 A, and that the selected amino acids are placed in such a manner that at least one circular surface region with a area of 800 A? comprises no selected amino acid; and c) effecting for each of the selected amino acids a primary mutation, which reduce the specific IgE binding capability of the mutated allergen as compared to the binding capability of the said naturally occurring allergen, wherein each primary mutation is a substitution of a selected amino acid residue with another amino acid, which does not occur in the same position in the amino acid sequence of any known homologous protein within the taxonomic species from which said naturally occurring allergen originates.

In an alternative aspect the invention relates to a method of preparing a recombinant allergen according to the invention, characterised in that the allergen is produced from a DNA sequence obtained by DNA shuffling (molecular breeding) of the DNA encoding the corresponding naturally occurring.

Furthermore, the invention relates to a recombinant allergen according to claim 1 for use as a pharmaceutical.

Also, the invention relates to use of the recombinant allergen according to claim 1 for preparing a pharmaceutical for preventing and/or treating allergy.

Furthermore, the invention relates to the composition according to claim 37 for use as a pharmaceutical.

Also, the invention relates to the use of a composition according to claim 37 for preparing a pharmaceutical for preventing and/or treating allergy.

Further, the invention relates to a pharmaceutical composition, characterised in that it comprises a recombinant allergen according to claim 1 or a composition according to «claim 37, optionally in combination with a pharmaceutically acceptable carrier and/or excipient, and optionally an adjuvant. The pharmaceutical composition according to the invention may be in the form of a vaccine against allergic reactions elicited by a naturally occurring allergen in patients suffering from allergy.

Also, the invention relates to a method of generating an immune response in a subject comprising administering to the subject a recombinant allergen according to claim 1, a composition according to claim 37 or a pharmaceutical composition according to claim 41-42 or 46.

Further, the invention relates to vaccination or treatment of a subject comprising administering to the subject a recombinant allergen according to claim 1, a composition according to claim 37 or a pharmaceutical composition according to claim 41-42 or 46.

Also, the invention relates to a process for preparing a pharmaceutical composition according to claim 41 or 42 comprising mixing a recombinant allergen according to claim 1 or a composition according to claim 37 with pharmaceutically acceptable substances and/or excipients.

Further, the invention relates to a pharmaceutical composition obtainable by the process according to claim

Also, the invention relates to a method for the treatment, prevention or alleviation of allergic reactions in a subject comprising administering to a subject a recombinant allergen according to claim 1, a composition according to claim 37 or a pharmaceutical : composition according to claims 41-42 or 46.

Further, the invention relates to a DNA sequence encoding an allergen according to invention, a derivative thereof, a partial sequence thereof, a degenerated sequence thereof or a sequence, which hybridises thereto under stringent conditions, wherein said derivative, partial sequence, degenerated sequence or hybridising sequence encodes a peptide having at least one B cell epitope.

Also, the invention relates to an expression vector comprising the DNA according to the invention.

Furthermore, the invention relates to a host cell comprising the expression vector according to the invention.

Additionally, the invention relates to a method of producing a recombinant mutant allergen comprising the step of «cultivating the host cell according to the invention.

Finally, the invention relates to a recombinant allergen according to the invention or encoded by the DNA sequence : according to the invention comprising at least one T cell epitope capable of stimulating a T cell clone or T cell line specific for the naturally occurring allergen.

The mutants according to invent should preferable be able to stimulate allergen specific T-cell lines in a similar manner/degree as measured by the T-cell stimulation index.

DETAILED DESCRIPTION OF THE INVENTION

In a preferred embodiment of the invention, the primary mutations are spaced 20 A, preferably 25 A and most preferably 30 A.

It is believed that an allergen comprises a number of potential binding regions for specific IgE's, wherein each region approximately has a size of 800 A?, each surface region comprising a large number of overlapping epitopes. Thus, an allergen has a number of potential primary mutations of the surface area divided by 800 A.

Preferably, the recombinant allergen according to the invention comprises from 5 to 20, preferably from 6 to 15, more preferably from 7 to 12, and most preferably from 8 to 10 primary mutations.

In a preferred embodiment of the invention, the surface region comprising no mutation has an area of 700 AZ, preferably 600 A?, more preferably 500 A? and most preferably 400 A®.

In a preferred embodiment of the invention, the recombinant allergen 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, wherein each secondary mutation is a substitution of one surface-exposed amino acid residue with another residue, which does not occur in the same position in the amino acid sequence of any known homologous protein within the taxonomic species from v . which said naturally occurring allergen originates, wherein the secondary mutations are placed outside the said circular region.

The secondary mutations may be located close to a primary mutation, i.e. a secondary mutation may well be an additional mutation for the same epitope, which is mutated by the primary mutation.

In a preferred embodiment of the invention, at least one of the surface-exposed amino acids to be substituted in the naturally occurring allergen has a solvent accessibility of above 20 %, preferably above 30 %, more preferably above 40 % and most preferably above 50 %.

In another preferred embodiment of the invention, at least one of the surface-exposed amino acids to be substituted in the naturally occurring allergen is conserved with more than 70 %, preferably 80 % and most preferably 90 % identity in all known homologous proteins within the species from which said naturally occurring . allergen originates.

Preferably, the recombinant allergen according to invention essentially has the same a-carbon backbone tertiary structure as said naturally occurring allergen.

When comparing the a-carbon backbone tertiary structures of the mutant and the naturally occurring allergen molecules, the average root mean square deviation of the atomic coordinates is preferably below 2A.

In a preferred embodiment of the recombinant allergen of the invention, each amino acid residue 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.

In a preferred embodiment of the invention the recombinant mutant allergen according to the invention is a non-naturally occurring allergen.

Specific IgE binding to the mutated allergen is preferably reduced by at least 5%, preferably at least 10% in comparison to naturally-occurring isoallergens or similar recombinant proteins in an immuno assay with sera from source-specific IgE reactive allergic patients or pools thereof.

Another way of assessing the reduced IgE binding and the reduced ability of mediating cross-linking of the mutant are the capability of the mutant to initiate Histamine

Release (HR). The release of Histamine can be measured in several Histamine releasing assays. The reduced Histamine release of the mutants originates from reduced affinity toward the specific IgE bound to the cell surface as well as their reduced ability to facilitate cross-linking. HR is preferably reduced by 5-100%, more preferably 25-100%, more preferably 50-100% and most preferably 75-100% for the mutants of the invention in comparison to the naturally occurring allergens.

Typically, the circular surface region with an area of 800 A’ comprising no mutation comprises atoms of 15-25 amino acid residues. :

A preferred recombinant allergen according to the invention is characterised in that the surface-exposed amino acid residues are ranked with respect to solvent accessibility, and that one or more amino acids among the more solvent accessible ones are substituted.

A further preferred recombinant allergen according to the invention 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 amino acids among the more conserved ones are substituted.

Preferably, the recombinant allergen according to the invention comprises from 1 to 4 secondary mutations per primary mutation.

A preferred embodiment of the invention is characterised in that one or more of the substitutions is carried out by site~directed mutagenesis.

Another preferred embodiment of the invention is characterised in that one or more of the substitutions is carried out by random mutagenesis.

A further preferred embodiment of the invention is characterised in that one or more of the substitutions is carried out by DNA shuffling.

Recombinant allergens according to the invention may suitably be a mutant of an inhalation allergen originating i.a. from trees, grasses, herbs, fungi, house dust mites, cockroaches and animal hair and dandruff.

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 Alternaria 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. Further, 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. Others are Cry J 1 (Pinales), Amb a 1 and 2, Art v 1 (Asterales), Par j 1 (Urticales), Ole e 1 {(Oleales), Ave e 1, Cyn d 1, Dac ¢g 1, Fes pl, Hol 1 1, Lol p 1 and 5, Pas nl, Phl p 1 and 5, Poa pl, 2 and 5, Sec ¢ 1 and 5, and Sor h 1 (various grass pollens), Alt a 1 and Cla h 1 (fungi), Der f 1 and 2, Der p 1 and 2 (house dust mites, D. farinae and D. pteronyssinus, respectively), Lep d 1 and 2 (Lepidoglyphus destructor; storage mite), Bla g 1 and 2,

Per a 1 (cockroaches, Blatella germanica and Periplaneta americana, respectively), Fel d 1 (cat), Can f 1 (dog).

Equ c¢ 1, 2 and 3 (horse), Apis m 1 and 2 (honeybee), Ves © v1, 2 and 5, Polia 1, 2 and 5 (all wasps) and Sol i 1, 2, 3 and 4 (fire ant).

In one embodiment, the recombinant allergen is a mutant of Bet v 1. Amino acids potentially suitable for substitution comprise amino acids V2, D72, E87, K-129, E- 60, N-47, K-65, P-108, N-159, D-93, K-123, K-32, D-125,

R-145, D-109, E-127, Q-36, E-131, 1-152, E-6, E-96, D- 156, P-63, H-76, E-8, K-134, E-45, T-10, V-12, K-20, S- 155, H-126, P-50, N-78, K-119, V-2, L-24, E-42, N-4, A- 153, I-44, E-138, G-61, A-130, R-70, N-28, P-35, S-149,

K-103, Y-150, H-154, N-43, A-106, K-115, P-14, Y-5, K- 137, E-141, E-87 and E-73. One or more of the primary and secondary substitutions may be selected from the group consisting of V2F, V2L, V2I, V2M, YS5V, T10P, T10A, K20N,

D25E, N28T, K32Q, Q36A, Q36K, E42S, E45S, N47S, KS5N,

K65N, D72H, D72Q, D72N, T77A, N78K, E87G, E96L, K97S,

K103Vv, P108G, D109N, K123I, D125Y, KI129N, KI134E, R145E,

S149R, 8149T, D156H and +160N, wherein + means that an additional amino acid is incorporated.

Examples of Bet v 1 mutants according to the present invention are as follows (parentheses, when used, indicate primary and secondary mutations):

Mutant A: (Asn28Thr, Lys32Gln), (Asn78Lys, Lysl03Val), ArgldSclu, (AsplS56His, +160Asn).

Mutant B:

Tyr5val, Glu42Ser, Glu4bSer, Asn78Lys, Lysl03val,

Lysl23Ile, Lys134Glu, Aspl56His.

Mutant 25385 (Example 2):

N28T, K32Q, E45S, P108G

Mutant 2628 (Example 4):

TyrSvVal, Glu4S5Ser, Lys65Asn, Lys97Ser, Lysl34Glu.

Mutant 2637 (Example 4):

Alalé6Pro, (Asn28Thr, Lys32Gln), Lysl03Thr, Prol08Gly, (Leul52Lys, Alal53Gly, Serl55Pro).

Mutant 2724:

N28T, K32Q, N78K, K103V, P108G, R145E, D156H, +160N.

Mutant 2733 (Example 4): (Tyr5val, Lysl134Glu), (Asn28Thr, Lys32Gln), GludSSer,

Lys65Asn, (Asn78Lys, Lysl03val), Lys97Ser, Prol08Gly,

Argl45Glu, (AsplS6His, +160Asn)

Mutant 2744: (Tyr5Val, Lys134Glu), (GludZSer, Glud5Ser), (Asn78Lys, Lys103vVal), Lysl23Ile, (Aspl56His, +160Asn) .

Mutant 2753 (Example 4): (Asn28Thr, Lys32Gln), Lys65Asn, (Glu96Leu, Lys97Ser), (Prol08Gly, AsplO09Asn), (Aspl25Tyr, Glul27Ser),

Argl45Glu.

Mutant 2744 + 2595:

Y5V, N28T, K32Q, E42S, E45S, N78K, K103V, P108G, K123I,

K134E, D156H, +160N.

Mutant 2744 + 2628:

YSV, E42S, E45S, K65N, N78K, K97S, K103V, K123I, KI134E,

D156H, +160N.

Mutant 2744 + 2595 + 2628:

Y5V, N28T, K320Q, E42S, E45S, K65N, N78K, K97TS§, K103V,

P108G, K123I, K134E, D156H, +160N.

Furthermore, all of the above mutants comprising one OI more of the following substitutions: V2F, Vv2L, V2I, V2M,

T10A, K20N, Q36A or Q36K, D72H, D72Q, D72N, E87G, KI129N and S149R or S149T.

In another embodiment, the recombinant allergen is derived from a venom allergen from the taxonomic order of

Vespidae, Apidae and Formicoidae.

In a further embodiment, the recombinant allergen is derived from Ves v 5. Amino acids potentially suitable for substitution comprise amino acids Amino acids potentially suitable for substitution comprise amino acids K-16, K-185, K-11, K-44, K-210, R-63, K-13, F-6, K- 149, K-128, E-184, K-112, F-157, E-3, K-29, N-203, N-34,

K-78, K-151, L-15, L-158, Y-102, W-186, K-134, D-87, K- 52, T-67, T-125, K-150, Y-40, (Q-48, L-65, K-81, Q-101, Q- 208, K-144, N-8, N-70, H-104, Q-45, K-137, K-159, E-205,

N-82, A-111, D-131, K-24, Vv-36, N-7, M-138, T-209, Vv-84,

K-172, v-19, D-56, P-73, G-33, T-106, N-170, L-28, T-43, 0-114, C-10, K-60, N-31, K-47, E-5, D-145, v-38, A-127,

D-156, E-204, P-71, G-26, Y-129%, D-141, ¥F-201, R-68, N- 200, D-49, s-153, K-35, s-39, Y-25, V-37, G-18, W-85 and

I-182. One or more of the primary and secondary substitutions may be selected from the group consisting of K29A, Te67A, K78A, V84S, Y102A, K112S, Kl44Aa, K202M and

N203G.

In a further embodiment, the recombinant allergen is derived from Der p 2. Amino acids potentially suitable for substitution comprise amino acids R-128, D-129, H-11,

H-30, Ss-1, K-77, Y-75, R-31, K-82, K-6, K-96, K-48, K-55,

K-89, Q-85, wW-92, 1-97, H-22, V-65, S-24, H-74, K-126, L- 61, P-26, N-93, D-64, I-28, K-14, K-100, E-62, I-127, E- 102, E-25, pP-66, L-17, G-60, P-95, E-53, Vv-81, K-51, N- 103, ©-2, N-46, E-42, T-91, D-87, N-10, M-111, C-8, EH- 124, I-68, P-79, K-109 and R-128, D-129, H-11, H-30, Ss-1, :

K-77, Y-75, R-31, K-82, K-6, K-96, K-48, K-55, K-89, Q- 85, W-92, I-97, H-22, V-65, S-24, H-74, K-~126, L-61, P- 26, N-93, D-64, I-28, K-14, K-100, E-62, I-127, E-102, E-

25, pP-66, L-17, G-60, P-95, E-53, V-81, K-51, N-103, 0-2,

N-46, E-42, T-91, D-87, N-10, M-111, C-8, H-124, I-68, P- 79, K-109, K-15. One or more of the primary and secondary substitutions may be selected from the group consisting of K6A, N10S, K1SE, S24N, H30N, K48A, E62S, HT4N, K77N,

K82N, K100N and R128Q.

Examples of Bet v 1 mutants according to the present invention are as follows:

Mutant A:

K6A, K15E, H30N, E62S.

Mutant B:

KGA, K15E, H30N, E62S, H74N, KB82N.

Mutant C:

K6A, N10S, K15E, S24N, H30N, K48Aa, E62S, H74N, K77IN,

K82N, K10ON and R128Q

Vaccines

Preparation of vaccines is generally well known in the art. 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 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 ng to 1000 ug.

As mentioned above, an increased effect may be obtained by adding adjuvants to the formulation. Examples of such adjuvants are 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. Mixture with bacterial cells such as C. parvum, endotoxins or lipopolysaccharide components of gram- negative bacteria, emulsion in physiologically acceptable 0il 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-59 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.

Most often, multiple administrations of the vaccine will be necessary to ensure an effect. Frequently, 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. :

Method of preparing a recombinant allergen according to the invention

As mentioned above, the present invention also relates to a method of preparing the recombinant mutant allergen of the invention, cf. claim 48.

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.

A second parameter, which may contribute to the identification of surface-exposed amino acids suitable : for substitution in accordance with the present invention is the extent in which an amino acid is conserved in all known homologous proteins within the species from which said naturally occurring allergen originates.

Alternatively, the extent in which in all known homologous proteins within the taxonomic genus, subfamily, family, superfamily, legion suborder or order from which said naturally occurring allergen originates is used as such a second parameter.

Accordingly, a preferred embodiment of the method of the invention is characterised in selecting identified amino acid residues, which are conserved with more than 70 %, preferably more than 80 % and most preferably more than 90 ¢ identity in all known homologous proteins within the species from which said naturally occurring allergen originates.

Furthermore, a particularly preferred embodiment of the method of the invention is characterised in ranking the said 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 a-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.

Criteria for substitution :

For molecules for which the tertiary structure has been determined (e.g. by x-ray crystallography, or NMR electron microscopy), the mutant carrying the substituted amino acid(s) should preferably fulfil the following criteria: 1. The overall a-carbon backbone tertiary structure of the molecule is preferably conserved. Conserved is defined as an average root mean square deviation of the atomic coordinates comparing the structures below 24.

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.

2. The amino acids to be substituted are preferably located at the surface, and thus accessible for antibody- binding. Amino acids located on the surface in the three- dimensional structure usually have a solvent (water) accessibility of at least 20%, suitably 20-80%, more suitably 30-80%. Solvent accessibility is defined as the area of the molecule accessible to a sphere with a radius comparable to a solvent (water, r = 1.4 A) molecule. 3. Each of the substituted amino acids is preferably located in conserved patches having an area larger than 400 £2. Conserved patches are defined as coherently connected areas of surface-exposed conserved amino acid residues and backbone. Conserved amino acid residues are defined by sequence alignment of all known (deduced) amino acid sequences of homologues proteins within the : same taxonomic species, genus, subfamily, family, : superfamily, legion, suborder or order. 2Amino acid positions having identical amino acid residues in more than 70% of the sequences are considered conserved.

Conserved patches are expected to contain epitopes to which the IgE of the majority of patients is directed.

Conservation of a-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 a-carbon backbone tertiary structure probable, if compared to the data obtained by analysis of a structurally determined molecule. 4. Within the conserved patches amino acids for mutagenesis should .preferentially be selected among the most solvent (water) accessible ones located preferably near the centre of the conserved patch. 5. Preferentially, a polar amino acid residue is substituted by another polar residue, and a non-polar amino acid residue is substituted by another non-polar residue.

With an object of essentially retaining the three- dimensional structure of the allergen, 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, 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.

DNA according to the invention

In a preferred embodiment, the DNA sequence of the invention is a derivative of the DNA sequence encoding the naturally occurring allergen.

Preferably, the DNA derivative 1s obtained by site- directed or random mutagenesis of the DNA encoding the naturally occurring allergen.

In a first particularly preferred embodiment, the DNA sequence is a derivative of the sequence shown in Fig. 3, wherein the DNA sequence is mutated so as to encode an allergen having at least four mutations selected from the group consisting of K-129, E-60, N-47, K-65, P-108, N- 159, D-93, K-123, K-32, D-125, R-145%5, D-109, E-127, Q-36,

E-131, 1-152, E-6, E-96, D-156, P-63, H-76, E-8, K-134,

E-45, T-10, V-12, K-20, s-155, H-126, P-50, N-78, K-119,

Vv-2, L-24, E-42, N-4, A-153, I-44, E-138, G-61, A-130, R- 70, N-~28, P-35, 5-149, K-103, Y-150, H-154, N-43, A-106,

K-115, p-14, Y-5, K-137, E-141, E-87, E-73.

In a second particularly preferred embodiment, the DNA sequence is a derivative of the sequence shown in Fig. 13, wherein the DNA sequence is mutated so as to encode an allergen having at least four mutations selected from the group consisting of K-16, XK-185, K-11, K-44, K-210, . R-63, K-13, F-6, K-149, K-128, E-184, K-112, F-157, E-3,

K-29, N-203, N-34, K-78, K-151, L-15, L-158, Y-102, W- 186, K-134, D-87, K-52, T-67, T-125, K-150, Y¥Y-40, Q-48, 1-65, K-81, Q-101, Q-208, K-144, N-8, N-70, H-104, Q-45,

K-137, K-159, E-205, N-82, A-111, D-131, K-24, V-36, N-7,

M-138, T-209, V-84, K-172, V-19, D-56, P-73, G-33, T-106,

N-170, L-28, T-43, 0-114, C-10, K-60, N-31, K-47, E-5, D- 145, v-38, A-127, D-156, E-204, P-71, G-26, Y-129, D-141,

F-201, R-68, N-200, D-49, S-153, K-35, s-39, Y-25, V-37,

G-18, W-85 and I-182.

In a third particularly preferred embodiment, the DNA sequence 1s a derivative of the sequence shown in Fig. 16, wherein the DNA sequence is mutated so as to encode an allergen having at least four mutations selected from the group consisting of R-128, D-129, H-11, H-30, S-1, K- 77, Y-75, R-31, K-82, K-6, K-96, K-48, K-55, K-89, (Q-85,

W-92, I-97, H-22, Vv-65, S-24, H-74, K-126, L-61, P-26, N- 93, D-64, I-28, K-14, K-100, E-62, I-127, E-102, E-25, P- 66, L-17, G-60, P-95, BE-53, V-81, K-51, N-103, Q-2, N-46,

E-42, T-91, D-87, N-10, M-111, C-8, H-124, I-68, P-79, K- 109 and R-128, D-129, H-11, H-30, S-1, K-77, ¥-75, R-31,

K-82, K-6, K-96, K-48, K-55, K-89, 0-85, W-92, I-97, H- 22, V-65, S-24, H-74, K-126, 1-61, P-26, N-93, D-64, I- 28, K-14, K-100, E-62, I-127, E-102, E-25, P-66, L-17, G- 60, P-95, E-53, V-81, K-51, N-103, Q-2, N-46, E-42, T-91,

D-87, N-10, M-111, C-8, H-124, 1-68, P-79, K-109, K-15.

DNA shuffling

The recombinant mutant allergen according to the present invention may be produced using a DNA sequence obtained by DNA shuffling (molecular breeding) of the corresponding naturally DNA. DNA shuffling may be carried out according to the procedures disclosed in the article by Punnonen et al. (ref. 25) as well as the procedures disclosed in the articles mentioned therein, which are all included herein by this reference.

Diagnostic assay

Furthermore, 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. Due to this heterogeneity of the specificities of the IgE population, some 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. . Thus, the mutants of the invention and the compositions of the invention intended to be administered therapeutically nay 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 sera.

Thus, 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.

Definitions

In connection with the present invention the expression "reduce the specific IgE binding capability as compared te the IgE binding capability of the said natural- occurring allergen" means that the reduction is measurable in a statistically significant manner (p <0.05) in at least ore immunoassay using serum from a subject allergic to the natural-occurring allergen.

Preferably, the IgE binding capability is reduced by at least 5 %, more preferably at least 10 %.

The expression "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 sclution at least a part of at least one atom of the amino acid residue 1s accessible for contact with the surrounding solvent. Preferably, 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 %.

The expression "solvent accessibility" is defined as the area of the molecule accessible to a sphere with a radius comparable to a solvent (water, r = 1.4 A) molecule.

The expressions "surface-exposed" and "solvent-exposed" are used interchangeably.

The expression "the taxonomic species from which said naturally occurring allergen originates" means species in the taxonomic system.

Furthermore, the expression "said mutant allergen having essentially the same a-carbon backbone tertiary structure as said naturally occurring allergen" means that when comparing the structures, the average root mean square deviation of the atomic coordinates is below 2 A.

In connection with the present invention the expression “substitution” means the deletion, substitution or addition of an amino acid in comparison to the amino acid sequence of the naturally occurring allergen.

The present invention is further illustrated by the following non-limiting examples. :

EXAMPLES

EXAMPLE 1

Example 1 describes the preparation of recombinant mutant allergens with one and three primary mutations.

Recombinant mutant allergens according to the invention, i.e. allergens comprising at least four primary mutations, may be prepared using the same procedures.

Identification of common epitopes within Fagales pollen allergens

The major birch pollen allergen 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.

In addition, 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.

Molecular modelling suggests that the structures of

Fagales and food allergens and PR-10 proteins are close to being identical with the Bet v 1 structure.

The structural basis for allergic Bet wv 1 cross- reactivity was reported in (Gajhede et al 1996, ref. 17) where three patches on the molecular surface of Bet v 1 could be identified to be common for the known major tree pollen allergens. Thus, any IgE recognising these patches on Bet v 1 would be able to cross-react and bind to other

Fagales major pollen allergens and give rise to allergic symptoms. The identification of these common patches was performed after alignment of all known amino acid sequences of the major tree pollen allergens in combination with an analysis of the molecular surface of

Bet v 1 revealed by the o-carbon backbone tertiary structure reported in ref. 17. In addition, the patches were defined to have a certain minimum size (>400 A?) based on the area covered by an antibody upon binding.

Selection of amino acid residues for site-directed mutagenesis

Amino acid residues for site-directed mutagenesis were selected among residues present in Bet v 1 specific areas and the common patches since modifications of these is expected to affect the binding of serum IgE from the majority of patients showing clinical tree pollen allergic cross-reactivity.

The relative orientation and percentage of solvent- exposure of each amino acid residue within respective patch 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.

Sequence alignment

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 Bet v 1 No. 2801 of which 57 sequences originates from taxonomically related species.

Cloning of the gene encoding Bet v 1

RNA was prepared from Betula verrucosa pollen (Allergon,

Sweden) by phenol extraction and LiCl precipitation. 0ligo (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 5'-ends to accommodate restriction sites (NcoI and HindIII) for directional cloning into pKK233-2.

Subcloning into pMAL-c

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)-Bet v 1 protein fusion operons in which MBP and Bet v 1 were separated by a factor X5 protease clevage site positioned to restore the authentic aminoterminal sequence of Bet v 1 upon cleavage, as described in ref. 15. In brief, 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, protease cleavage site. Both primers were furthermore extended in the 5'-ends to accommodate restriction sites (KpnI) for cloning. The

Bet v 1 encoding genes were subcloned using 20 cycles of

PCR to reduce the frequency of PCR artefacts.

In vitro mutagenesis

In vitro mutagenesis was performed by PCR using recombinant pMAL-c with Bet v 1 inserted as template.

Each mutant Bet v 1 gene was generated by 3 PCR reactions using 4 primers.

Two mutation-specific oligonucleotide primers were synthesised accommodating each mutation, one for each DNA strand, see Figs. 1 and 2. Using the mutated nucleotide(s) as starting point both primers were extended 7 nucleotides in the 5'-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.

Two generally applicable primers (denoted "all-sense" and "all non-sense" in Figure 2) 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. 2.

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.

Introduction of the four amino acid substitutions (Asn28Thr, Lys32Gln, Glud45Ser, Prol08Gly) in the Triple- patch mutant were performed like described above in a step by step process. First the Glud45Ser mutation then the Prol08Gly mutation and last the Asn28Thr, Lys32Gln mutations were introduced using pMAL-c with inserted Bet v 1 No. 2801, Bet v 1 (Glu45Ser), Bet v 1 (Glu45Ser,

Prol08Gly) as templates, respectively. ’

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 electrophoresis and electro-elution followed by ethanol precipitation, cut with restriction enzymes (BsiWI/EcoRI), and ligated directionally into pMAL-c with Bet wv 1 inserted restricted with the same enzymes.

Figure 3 shows an overview of all 9 Bet v 1 mutations, which are as follows

Thrl0Pro, Asp25Gly, Asn28Thr + Lys32Gln, GludSSer,

Asnd7Ser, Lys55Asn, Glub0Ser (non-patch), Thr77Ala and

Prol08Gly. An additional mutant with four mutations was also prepared (Asn28Thr, Lys32Gln, Glu45Ser, Prol08Gly) .

Of these, five mutants were selected for further testing:

Asn28Thr + Lys32Gln, Glu45Ser, Glu60Ser, Prol08Gly and the Triple-patch mutant Asn28Thr, Lys32Gln, Glu4SSer,

Prol08Gly.

Nucleotide sequencing

Determination of the nucleotide sequence of the Bet v 1 encoding gene was performed before and after subcloning, and following in vitro mutagenesis, respectively.

Plasmid DNA's from 10 ml of bacterial culture grown to saturation overnight in LB medium supplemented with 0.1 g/l ampicillin were purified on Qiagen-tip 20 columns and sequenced using the Sequenase version 2.0 DNA sequencing kit (USB) following the recommendations of the suppliers.

Expression and purification of recombinant Bet v 1 and mutants

Recombinant Bet v 1 (Bet v 1 No. 2801 and mutants) were over-expressed in Escherichia coli DH 5a 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. After sonication and additional centrifugation, 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 Vv 1 was concentrated by ultrafiltration to about 5 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.

We have previously shown (ref. 15) that recombinant Bet Vv 1 No. 2801 is immunochemically indistinguishable from naturally occurring Bet v 1.

Immunoelectrophoresis using rabbit polyclonal antibodies

The seven mutant Bet 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 o~carbon backbone tertiary structure.

In order to analyse the effect on human polyclonal IgE- response, the mutants Glu4SsSer, ProlO8Gly,

Asn28Thr+lLys32Gln and Glu60Ser were selected for further analysis.

Bet v 1 Glu4SSer mutant

Glutamic acid in position 45 show a high degree of solvent-exposure (40%) and is located in a molecular

JY ro

Ea i B4003/ 3667

A

WO 02/40676 PCT/DK01/00764 Co 50 surface patch common for Fagales allergens (patch I). A serine residue was found to occupy position 45 in some of the Bet v 1 homologous PR-10 proteins arguing for that glutamic acid «can be replaced by serine without distortion of the a-carbon backbone tertiary structure.

In addition, as none of the known Fagales allergen sequences have serine in position 45, the substitution of glutamic acid with serine gives rise to a non-naturally occurring Bet v 1 molecule.

T cell proliferation assay using recombinant Glu45Ser Bet v 1 mutant

The analysis was carried out as described in Spangfort et al 199%6a. It was found that recombinant Bet v 1 Glu4b5Ser mutant was able to induce proliferation in T cell lines from three different birch pollen allergic patients with stimulation indices similar to recombinant and naturally occurring.

Crystallisation and structural determination of recombinant Glué45Ser Bet v 1

Crystals of recombinant Glu45Ser Bet v 1 were grown by vapour diffusion at 25°C, essentially as described in {Spangfort et al 1996b, ref. 21). GludSSer 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 ref. 21, using crystals of recombinant wild-type Bet v 1 as a source of seeds. :

After about 2 months, crystals were harvested and

RR

To WO 02/40676 PCT/DK01/00764 analysed using X-rays generated from a Rigaku rotating anode as described in ref. 21 and the structure was solved using molecular replacement.

Structure of Bet v 1 Glud5Ser mutant

The structural effect of the mutation was addressed by growing three-dimensional Bet v 1 Glué45Ser protein crystals diffracting to 3.0 A resolution when analysed by

X-rays generated from a rotating anode. The substitution of glutamic acid to serine in position 45 was verified by the Bet v 1 GludS5Ser structure electron density map which also showed that the overall a-carbon backbone tertiary structure is preserved.

IgE-binding properties of Bet v 1 Glu45Ser mutant

The IgE-binding properties of Bet v 1 Glud4S5Ser nutant 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.

Recombinant Bet v 1 no. 2801 was biotinylated at a molar ratio of 1:5 (Bet v 1 no. 2801:biotin). The inhibition assay was performed as follows: a serum sample (25 ul) was incubated with solid phase anti IgE, washed, re- suspended and further incubated with a mixture of biotinylated Bet v 1 no. 2801 (3.4 nM) and a given mutant (0-28.6 nM). The amount of biotinylated Bet v 1 no. 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 4 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.

There is a clear difference in the amount of respective recombinant proteins necessary to reach 50% inhibition of the binding to serum IgE present in the serum pool.

Recombinant Bet v 1 reaches 50% inhibition at about 6.5 ng whereas the corresponding concentration for Bet v 1

Glud5Ser mutant is about 12 ng. This show that the point mutation introduced in Bet v 1 Glud5Ser mutant lowers the affinity for specific serum IgE by a factor of about 2.

The maximum level of inhibition reached by the Bet v 1

Glud5Ser mutant is clearly lower compared to recombinant

Bet v 1. This may indicate that after the Glu4d5Ser substitution, some of the specific IgE present in the serum pool are unable to recognise the Bet v 1 Glu4SSer mutant.

Bet v 1 mutant Asn28Thr+Lys32Gln

Aspartate and lysine in positions 28 and 32, respectively show a high degree of solvent-exposure (35% and 50%, respectively) and are located in a molecular surface patch common for Fagales allergens (patch II). In the structure, aspartate 28 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 28 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 a-carbon backbone tertiary structure. In addition, as none of the naturally occurring isoallergen sequences have threonine and glutamate in positions 28 and 32, respectively, the

—_— - substitutions gives rise to a non-naturally occurring Bet v 1 molecule.

IgE-binding properties of Bet v 1 mutant

Asn28Thr+Lys32Gln

The IgE-binding properties of mutant Asn28Thr+Lys32Gln 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 5 shows the inhibition of the binding of biotinylated recombinant Bet Vv 1 to serum IgE from a pool of allergic patients by non-biotinylated Bet v1 and by

Bet v 1 mutant Asn28Thr+Lys32Gln.

There is a clear difference in the amount of respective recombinant proteins necessary to reach 50% inhibition of the binding to serum IgE present in the serum pool.

Recombinant Bet v 1 reaches 50% inhibition at about 6.5 ng whereas the corresponding concentration for Bet v 1 autant Asn28Thr+Lys32Gln is about 12 ng. This show that the point mutations introduced in Bet v 1 nutant

Asn28Thr+Lys32Gln lowers the affinity for specific serum

IgE by a factor of about 2.

The maximum level of inhibition reached by the Bet v 1 mutant Asn28Thr+Lys32Gln mutant is clearly lower compared to recombinant Bet v 1. This may indicate that after the

Asn28Thr+Lys32Gln substitutions, some of the specific IgE present in the serum pool are unable to recognise the Bet v 1 mutant Asn28Thr+Lys32Gln.

Bet v 1 mutant Prol08Gly proline in position 108 show a high degree of solvent-

exposure (60%) and is located in a molecular surface patch common for Fagales allergens (patch III). 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 glycine without distortion of the a-carbon backbone tertiary structure. In addition, as none of the naturally occurring iscallergen sequences have glycine in position 108, the substitution of proline with glycine gives rise to a non-naturally occurring Bet v 1 molecule.

IgE-binding properties of Bet v 1 ProlO8Gly nutant

The IgE-binding properties of Bet v 1 ProlO8Gly 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 Prol08Gly mutant.

There is a clear difference in the amount of respective recombinant proteins necessary to reach 50% inhibition of the binding to serum IgE present in the serum pool.

Recombinant Bet v 1 reaches 50% inhibition at about 6.5 ng whereas the corresponding concentration for Bet v 1

Prol08Gly is 15 ng. This show that the single point mutation introduced in Bet Vv 1 Prol08Gly lowers the affinity for specific serum IgE by a factor of about 2,

The maximum level of inhibition reached by the Bet v 1

Prol08Gly mutant is somewhat lower compared to recombinant Bet v 1. This may indicate that after the

Prol08Gly substitution, some of the specific IgE present in the serum pool are unable to recognise the Bet v 1

Prol08Gly mutant.

Bet v 1 mutant Glu60Ser (non-patch mutant)

Glutamic acid in position 60 show a high degree of solvent-exposure (60%) however, it is not located in a molecular surface patch common for Fagales allergens. A serine residue was found to occupy position 60 in some of . the Bet v 1 homologous PR-10 proteins arguing for that glutamic acid can be replaced with serine without distortion of the a-carbon backbone tertiary structure.

In addition, as none of the naturally occurring isoallergen sequences have serine in position 60, 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 Glu60Ser mutant

The IgE-binding properties of Bet v 1 Glu60Ser 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 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 Glu60Ser mutant. In contrast to the Glu4b5sSer,

Prol08Gly and Asn28Thr+Lys32Gln mutants, the substitution glutamic acid 60 to serine, does not shown any significant effect on the IgE-binding properties of. This indicates that substitutions outside the defined Fagales common patches only have a marginal effect on the binding of specific serim IgE supporting the concept that conserved allergen molecular surface areas harbours dominant IgE-binding epitopes.

WQ 02/40676 PCT/DK01/00764

Bet v 1 Triple-patch mutant

In the Triple-patch mutant, the point mutations (Glu45Ser, Asn28Thr+Lys32Gln and Prol08Gly) introduced in the three different common Fagales patches, described above, were simultaneously introduced in creating an artificial mutant carrying four amino acid substitutions.

Structural analysis of Bet v 1 Triple-patch mutant }

The structural integrity of the purified Triple-patch mutant was analysed by circular dichroism (CD) spectroscopy. Figure 8 shows the CD spectra of recombinant and Triple-patch 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 a-carbon backbone tertiary structure is not affected by the introduced amino acid substitutions.

IgE-binding properties of Bet v 1 Triple-patch mutant

The IgE-binding properties of Bet v 1 Triple-patch 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 Triple-patch mutant. In contrast to the single mutants described above, the inhibition curve of the

Triple-patch mutant 1s no longer parallel relative to recombinant. This shows that the substitutions introduced in the Triple-patch mutant has changed the IgE-binding properties and epitope profile compared to recombinant.

The lack of parallellity makes it difficult to quantify the decrease of the Triple-patch mutant affinity for specific serum IgE.

Recombinant Bet v 1 reaches 50% inhibition at about 6 ng whereas the corresponding concentration for Bet v 1

Triple-patch mutant is 30 ng, i.e a decrease in affinity by a factor 5. However, in order to reach 80% inhibition the corresponding values are 20 ng and 400 ng, respectively, i.e a decrease by a factor 20.

T cell proliferation assay using recombinant Bet Vv 1

Triple-patch mutant

The analysis was carried out as described in ref. 15. It was found that recombinant Bet v 1 Triple-patch mutant was able to induce proliferation in T cell lines from three different birch pollen allergic patients with stimulation indices similar to recombinant and naturally occurring. This suggests that the Triple-patch mutant can initiate the cellular immune response necessary for antibody production.

EXAMPLE 2

Example 2 describes the preparation of recombinant mutant allergens with one primary mutation. Recombinant mutant allergens according to the invention, i.e. allergens comprising at least four primary mutations, may be prepared using the same procedures.

Identification of common epitopes within Vespula vulgaris venom major allergen antigen 5 oo WO 02/40676 PCT/DK01/00764

Antigen 5 is one of the three vespid venom proteins, which are known allergens in man. The vespids include hornets, vyellow-jacket and wasps. The other two known allergens of vespid venoms are phospholipase A; and hyaluronidase. Antigen 5 from Vespula vulgaris (Ves v 5) has been cloned and expressed as recombinant protein in the yeast system (Monsalve et al. 1999, ref. 22). The three-dimensional crystal structure of recombinant Ves v 5 has recently been determined at 1.8 A resolution (in preparation). The main features of the structure consist of four pB-strands and four a-helices arranged in three stacked layers giving rise to a “a-f-a sandwich”. The sequence identity between Antigen 5 homologous allergens from different Vespula species is about 90% suggesting presence of conserved molecular surface areas and B cell epitopes.

The presence and identification of common patches was performed after alignment of all known amino acid sequences, as previously described for tree pollen allergens, of the Vespula antigen 5 allergens in combination with an analysis of the molecular surface of

Antigen 5 revealed by the three-dimensional structure of

Ves v 5. Figure 10 shows solvent accessibility of individually aligned antigen 5 residues and alignment of

Vespula antigen 5 sequences (left panel). On the right panel of figure 10 is shown the molecular surface of antigen 5 with conserved areas among Vespula antigen 5:s coloured.

Selection of "amino acid residues for site-directed mutagenesis

Amino acid residues for site-directed mutagenesis were selected among residues present the patches common for

Vespula since modifications of these is expected to affect the binding of serum IgE from the majority of patients showing clinical Vespula allergic Cross- reactivity.

The relative orientation and percentage of solvent- exposure of each amino acid residue within respective patch was calculated based on their atomic coordinates.

Residues having a low degree of solvent exposure were not regarded suitable 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.

Cloning of the gene encoding Ves v §

Total RNA was isclated from venom acid glands of Vespula vulgaris vespids as described in (Fang et al. 1988, ref. 23).

First-strand cDNA synthesis, PCR amplification and cloning of the Ves v 5 gene was performed as described in (Lu et al. 1993, ref. 24)

Subcloning into pPICZoA

The gene encoding Ves v 5 was subsequently sub-cloned : into the pPICZaA vector (Invitrogen) for secreted expression of Ves v 5 in Pichia pastoris. The gene was amplified by PCR and sub-cloned in frame with the coding 30. sequence for the o-factor secretion signal of

Saccharomyces cerevisiae. In this construct the a-factor is cleaved off, in vivo, by the Pichia pastoris Kex2 protease system during secretion of the protein,

In brief PCR was performed using Ves v 5 as template and primers corresponding to the amino- and carboxyterminus of the protein, respectively. The primers were extended in the 5'-end to accommodate restriction sites for cloning, EcoRI and XbaI, respectively. Nucleotides encoding the Kex2 cleavage site was in this construct positioned 18 nucleotides upstream to the amino terminus of the protein, resulting in the expression of Ves v 5 with six additional amino acids, Glu-Ala-Glu-Ala-Glu-Phe, at the amino terminus.

Insertion of pPICZaA-Ves v 5 into P. pastoris

The pPICZoA vectors with the Ves v 5 gene inserted was linearised by Sac I restriction and inserted into the

AOX1 locus on the Pichia pastoris genome. Insertion was performed by homologous recombination on Pichia pastoris

KM71 cells following the recommendations of Invitrogen.

In vitro mutagenesis

In vitro mutagenesis was performed by PCR using recombinant pPICZoA with Ves v 5 inserted as template.

Each mutant Ves v 5 gene was generated by 3 PCR reactions using 4 primers.

Two mutation-specific oligonucleotide primers were synthesised accommodating each mutation, one for each DNA strand, see Figures 11 and 12. Using the mutated nucleotide (s) as starting point both primers were extended 6-7 nucleotides in the 5'-end and 12-13 nucleotides in the 3'-end. The extending nucleotides were identical in sequence to the Ves v 5 gene in the actual region.

Two generally applicable primers (denoted “all sense” and “all non-sense” in Figure 12) were furthermore synthesised and used for all mutants. To insure expression of Ves v 5 mutants with authentic amino terminus, one primer corresponding to the amino terminus of the protein was extended in the 5 -end with a Xho I site. Upon insertion of the Ves v 5 mutant genes into the pPICZaA vector, the Kex2 protease cleavage site was regenerated directly upstream to the amino terminus of

Ves v 5. The second primer was corresponding in sequence to a region of the pPICZaA vector positioned approximately 300 bp downstream from the Ves v 5 gene.

The sequence of the primer corresponding to the amino terminus of Ves v 5 is derived from the sense strand and the sequence of the downstream primer is derived from the non-sense strand, see Figure 11.

Two independent PCR reactions were performed essentially according to standard procedures (Saiki et al 1988) with the exception that only 20 temperature cycles were performed in order to reduce the frequency of PCR artefacts. Each PCR reaction used pPICZaA with Ves v 5 inserted as template and one mutation-specific and one generally applicable primer in meaningful combinations.

The PCR products were purified by using “Concert, Rapid

PCR Purification System” (Life Technologies). 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 with the “Concert, Rapid PCR Purification System” (Life

Technologies), cut with restriction enzymes {(XhoI/Xbal). and ligated directionally into pPICZaA vector restricted with the same enzymes. Figure 13 shows an overview of all

Ves v 5 mutations. ,

Insertion of pPICZaA-Ves v 5 mutants into P. pastoris

The pPICZaA vectors with the Ves v 5 mutant denes inserted were linearised by Sac I restriction and inserted into the A0X1 locus on the Pichia pastoris genome. Insertions were performed by homologous recombination on Pichia pastoris KM71 cells following the recommendations of Invitrogen.

Nucleotide sequencing

Determination of the nucleotide sequence of the Ves v 5 encoding gene was performed before and after subcloning, and following in vitro mutagenesis, respectively.

Plasmid DNA's from 10 ml of bacterial culture grown to saturation overnight in LB medium supplemented with 0.1 g/l ampicillin were purified on Qiagen-tip 20 columns and sequenced using the Sequenase version 2.0 DNA sequencing kit (USB) following the recommendations of the suppliers. © 20 Expression and purification of recombinant Ves v 5

Recombinant yeast cells of Pichia pastoris strain KM71 were grown in 500 ml bottles containing 100 ml of pH 6.0 phosphate buffer containing yeast nitrogen base, biotin, glycerol and histidine at 30°C with orbital shaking at 225 rpm until Age nm of 4-6. Cells were collected by centrifugation and re-suspended in 10 ml of similar buffered medium containing methanol in place of glycerol.

Incubation was continued at 30°C for 7 days with daily addition of 0.05 ml methanol.

Cells were harvested by centrifugation and the collected culture fluid was concentrated by ultrafiltration. After dialysis against 50 mM ammonium acetate buffer, pH 4.6, the sample was applied to a FPLC (Pharmacia) SE-53 cation exchange column equilibrated in the same buffer. The column was eluated with a 0-1.0 M NaCl, 50 mM ammonium acetate linear gradient. The recombinant Ves Vv 5 peak eluting at about 0.4 M NaCl was collected and dialysed against 0.02 N acetic acid. After concentration to about mg/ml, the purified Ves v 5 was stored at 4°C.

Crystallisation of recombinant Ves v 5

Crystals of Ves v 5 was grown by the vapour diffusion 10 technique at 25°C. For crystallisation, 5 pl of 5 mg/ml

Ves v 5 was mixed with 5 nl of 18% PEG 6000, 0.1 M sodium citrate, pH 6.0 and equilibrated against 1 ml of 18% PEG 6000, 0.1 M sodium citrate, pH 6.0.

X-ray diffraction data was collected at 100K from native

Ves v 5 crystals and after incorporation of heavy-atom derivatives and used to solve the three-dimensional structure of Ves v 5, see Figure 10 (manuscript in preparation).

Immunoelectrophoresis using rabbit polyclonal antibodies

The two Ves v 5 mutants were produced as recombinant Ves v 5 proteins and tested for their reactivity towards polyclonal rabbit antibodies raised against recombinant

Ves v 5. When analysed by rocket immunoelectrophoresis under native conditions, the rabbit antibodies were able to precipitate recombinant Ves v 5 as well as both mutants, indicating that the mutants have conserved o- carbon backbone tertiary structure.

Inhibition of specific serum IgE

The IgE-binding properties of Ves v 5 mutants were compared to recombinant Ves v 5 in a fluid-phase IgE- inhibition assay using a pool of serum IgE derived from vespid venom allergic patients.

The inhibition assay was performed as described above using biotinylated recombinant Ves v 5 instead of Bet v 1.

Ves v 5 Lys72Ala mutant

Lysine in position 72 show a high degree of solvent- exposure (70%) and is located in a molecular surface patch common for Vespula antigen 5. The relative orientation and high degree of solvent exposure argued for that lysine 72 can be replaced by an alanine residue without distortion of the a-carbon backbone tertiary structure. In addition, as none of the naturally occurring isocallergen sequences have alanine in position 72, the substitution of lysine with alanine gives rise to a non-naturally occurring Ves v 5 molecule.

IgE-binding properties of Ves v 5 Lys72Ala mutant

The IgE-binding properties of Ves v 5 Lys72Ala mutant was compared with recombinant Ves v 5 in a fluid-phase IgE- inhibition assay using the pool of serum IgE derived from birch allergic patients described above.

Figure 14 shows the inhibition of the binding of biotinylated recombinant Ves v 5 to serum IgE from a pool of allergic patients by non-biotinylated Ves v 5 and by

Ves v 5 Lys72Ala mutant.

There is a clear difference in the amount of respective recombinant proteins necessary to reach 50% inhibition of the binding to serum IgE present in the serum pool.

Recombinant Ves v 5 reaches 50% inhibition at about 6 ng whereas the corresponding concentration for Ves v 5

Lys72Ala mutant is 40 ng. This show that the single point mutation introduced in Ves v 5 Lys72Ala mutant lowers the affinity for specific serum IgE by a factor of about 6.

The maximum level of inhibition reached by the Ves v 5

Lys72Ala mutant significantly lower compared to recombinant Ves v 5. This may indicate that after the

Lys72Ala substitution, some of the specific IgE present in the serum pool are unable to recognise the Ves v 5

Lys72Ala mutant.

Ves v 5 Tyr96Ala mutant

Tyrosine in position 96 show a high degree of solvent- exposure (65%) and is located in a molecular surface patch common for Vespula antigen 5. The relative orientation an high degree of solvent exposure argued for that tyrosine 96 can be replaced by an alanine residue without distortion of the three-dimensional structure. In addition, as none of the naturally occurring isoallergen sequences have alanine in position 96, the substitution of tyrosine with alanine gives rise to a non-naturally occurring Ves v 5 molecule.

IgE-binding properties of Ves v 5 Tyr96Ala mutant

The IgE-binding properties of Ves v 5 Tyr96Ala mutant was compared with recombinant Ves v 5 in a fluid-phase IgE- inhibition assay using the pool of serum IgE derived from birch allergic patients described above.

Figure 14 shows the inhibition of the binding of biotinylated recombinant Ves v 5 to serum IgE from a pool of allergic patients by non-biotinylated Ves v 5 and by

Ves v 5 Tyr96Ala miitant.

There is a clear difference in the amount of respective recombinant proteins necessary to reach 50% inhibition of the binding to serum IgE present in the serum pool.

Recombinant Ves v 5 reaches 50% inhibition at about 6 ng whereas the corresponding concentration for Ves v 35

Tyr96Ala mutant is 40 ng.

This show that the single point mutation introduced in

Ves v 5 Tyr96Ala mutant. lowers the affinity for specific serum IgE by a factor of about 6.

The maximum level of inhibition reached by the Ves v 5

Tyr96Ala mutant significantly lower compared to recombinant Ves v 5. This may indicate that after the

Tyr96Ala substitution, some of the specific IgE present in the serum pool are unable to recognise the Ves v 5

Tyr96Ala mutant.

EXAMPLE 3

Identification and selection of amino acids for substitution

The parameters of solvent accessibility and conservation degree were used to identify and select surface-exposed amino acids suitable for substitution for the ellergens

Bet v 1, Der p 2 and Ves Vv 5.

Solvent accessibility

Solvent accessibility was calculated using the software

InsightII, version 97.0 (MSI) and a probe radius of 1.4 A (Connolly surface).

Internal cavities were excluded from the analyses by filling with probes using the software PASS (Putative

Active Sites with Spheres). Probes on the surface were subsequently removed manually.

Conservation

Bet v 1; 3-D structure is based on accession number 280104 (1bvl.pdb). 38 other Bet v 1 sequences included in the analysis of conserved residues comprise accession numbers:

P15494=X15877=280106, 280101, AJ002107, 272429, AJ002108, z80105, 280100, 280103, AJO01555, 280102, AJ002110, 272436, P43183=X77271, 272430, AJ002106, P43178=X77267,

P43179=X77268, P43177=X77266, 272438, P43180=X77269,

AJ0O01551, P43185=X77273, AJ001557, 272434, AJ001556, 272433=pP43186, AJO01554, X81972, 272431, P45431=X77200,

P43184=X77272, P43176=X77265, S47250, S47251, 272435, 272439, 272437, S47249.

Der p 2: 3-D structure is based on accession number P49278 {(la9v.pdb). 6 other Der p 2 sequences included in the analysis of conserved residues comprise the following substitutions:

ALK-G: V40L, T47s, M111L, D114N.

ALK-101: M76V.

ALK-102: V40L, T47sS.

ALK-104: T47S, M111I, D114N.

ALK-113: T47S.

ALK-120: VA40L, T47S, D114N.

Ves v 5: : 3~D structure is based on accession number Q05110 (pdb :

coordinates unpublished).

Another Ves v 5 sequence in the analysis of conserved residues contains one amino acid substitution: M202K.

Results

Bet v 1 59 amino acids highly solvent exposed:

K-129, E-60, N-47, K-65, P-108, N-159, D-93, K-123, K-32,

D-125, R-145, D-109, T-77, E-127, (0-36, E-131, L-152, E- 6, E-9%6, D-156, P-63, H-76, E-8, K-134, E-45, T-10, V-12,

K-20, L-62, S-155, H-126, P-50, N-78, K-119, V-2, L-24,

E-42, N-4, A-153, I-44, E-138, G-61, A-130, R-70, N-28,

P-35, 5-149, K-103, Y-150, H-154, N-43, A-106, K-115, P- 14, Y-S, K-137, E-141, E-87, E-73. : 57 amino acids highly solvent exposed and conserved (>70%) :

K-129, E-60, N-47, K-65, P-108, N-159, D-93, K-123, K-32,

D-125, R-145, D-109, E-127, (Q-36, E-131, L-152, E-6, E- 86, D-156, P-63, H-76, E-8, K-134, E-45, T-10, Vv-12, K- 20, 5-155, H-126, P-50, N-78, K-119, Vv-2, L-24, E-42, N- 4, A-153, I-44, E-138, G-61, A-130, R-706, N-28, P-35, S- 149, K-103, Y-150, H-154, N-43, A-106, K-115, P-14, Y-5,

K-137, E-141, E-87, E-73. 23 mutations performed:

Y5V, T10P, D2S5E, N28T, K32Q0, E42S, EA45S, N47S, KS55N,

K65N, T77A, N78K, ES6l, K97S, K103V, P108G, D1OSN, K123I,

D125Y, K134E, R145E, D156H, +160N.

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 amino-terminal, 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.

Table 1: Bet v 1

NO AA Solv_exp Cons % 129K 1,000 90 60E 0,986 97 47N 0,979 100 65K 0,978 100 108P 0,929 100 159N 0,869 100 93D 0,866 100 123K 0,855 100 32K 0,855 100 125D 0,821 74 145R 0,801 80 109D 0,778 82 777 0,775 56 127E€ 0,760 100 36Q 0,749 95 131E 0,725 100 1521 0,718 97 6E 0,712 100 96E 0,696 100 156D 0,693 97 63P 0,692 97 76H 0,683 90 8E 0,638 97 134K 0,630 100 45E 0,623 100 10T 0,613 97 12V 0,592 100 20K 0,584 100 62L 0,575 5 1658 0,568 97 126H 0,551 95 50P 0,541 100 78N 0,538 100 119K 0,529 100 2V 0,528 100 24L 0,528 100 42E 0,519 100 4N 0,517 a5 153A 0,513 100 44] 0,508 97 138E 0,496 100 61G 0,488 100

130A 0,479 97 70R 0,474 100 28N 0,469 90 35P 0,467 100 1498 0,455 92 103K 0,447 100 160Y 0,438 100 154H 0,436 100 43N 0,412 100 106A 0,411 95 115K 0.411 100

14P 0,410 97 : 5Y 0,410 100 137K 0,396 100 141E 0,387 95 87E 0,385 100 73E 0,384 100 16A 0.367 100 79F 0,362 100 3F 0,355 100 158Y 0,346 100 105V 0,336 100 101E 0,326 100 64F 0,325 100 86 0.322 100 398 0,314 100 124G 0,310 100 72D 0,308 07 1427 0,293 67 66Y , 0,289 100 55K 0,288 100 77 0,279 67 408 0,274 95 25D 0.271 87 135A 0,267 92 68K 0,262 100 97K 0,247 100 46G 0,235 100 27D 0,232 97 16 0,227 100 1131 0,225 77 51G 0,220 100 92G 0,218 100 80K 0,212 100 110G 0.211 100 107T 0,203 85 94T 0,202 92 41v 0,201 97 48G 0,198 100 911 0,182 18 31P © 0.188 100 75D 0,188 97 33V 0,183 100

49G 0,176 100 17R 0,172 100 99S 0,158 64 89G 0,154 100 53} 0,154 100 121H 0,153 100 oT 0,150 72 74V 0,148 97 132Q 0,146 72 57S 0,137 49 148E 0,135 100 82N 0,133 41 128V 0,125 64 117s 0,124 87 90P 0,117 67 1161 . 0112 100 122T 0,107 100 139M 0,104 62 95L 0,104 97 54K 0,096 100 146A 0,095 100 59P 0,088 97 187A 0,088 100 133V 0,077 44 : 88G 0,068 100 140G 0,053 85 37A 0,042 95 81Y 0,041 100 231 0,036 95 1041 0,036 92 15A 0,036 97 58F 0,029 100 29L 0,028 100 19F 0,027 100 100N 0,022 97 22F 0,021 97 71V 0,014 100 111G 0,014 100 13} 0,014 100 18L 0,014 97 114L 0,014 100 118 0,007 100 151L 0,007 97 1441 0,007 90 52T 0,007 100 84S 0,007 97 118N 0,007 97 1021 0,007 100 21A 0,000 97 26G 0,000 97 30F 0,000 44 34A 0,000 100 asl 0,000 87

561 0,000 100 67V 0,000 a7 69D 0,000 62 83Y 0,000 95 85V 0,000 72 981 0,000 95 1128 0,000 77 120Y 0,000 95 1368 0,000 67 143L 0,000 100 -147V 0,000 100

Der p 2 55 amino acids highly solvent exposed:

R-128, D-129, H-11, H-30, S-1, K-77, Y-75, R-31, K-82, K- 6, K-96, K-48, K-55, K-89, (0-85, W-92, I-97, H-22, V-65, s-24, H-74, K-126, L-61, P-26, N-93, D-64, I-28, K-14, K- 100, E-62, I-127, E-102, E-25, Pp-66, D-114, L-17, G-60, p-95, E-53, Vv-81, K-51, N-103, Q-2, N-46, E-42, T-91, D- 87, N-10, M-111, C-8, H-124, I-68, P-79, K-109, K-15. 54 anino acids highly solvent exposed and conserved (>70%) :

R-128, D-129, H-11, H-30, s-1, K-77, Y-75, R-31, K-82, K- 6, K-96, K-48, K-55, K-89, (Q-85, W-92, I-97, H-22, vV-65,

S-24, H-74, K-126, L-61, P-26, N-93, D-64, I-28, K-14, K- 100, E-62, I-127, E-102, E-25, P-66, L-17, G-60, P-85, E- 53, v-81, K-51, N-103, Q-2, N-46, E-42, T-91, D-87, N-10,

M-111, C-8, H-124, I-68, P-79, K-109, K-15. 6 mutations performed:

K6A, K15E, HK30N, E6235, H74N, KB82N

Table 2 shows a listing in descending order of solvent exposure of Der p 2 amino acids. Column 1 lists the amino acid number starting from the amino-terminal, 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.

Table 2: Der p 2

NO AA Solv_exp Cons % 128R 1,000 100 1280 0,965 100 ’ 11H 0,793 100 30H 0,712 100 18 0,700 100 77K 0,694 100 75Y 0,681 100 31R 0,677 100 82K 0,658 100 6K 0,645 100 96K 0,643 100 48K 0,642 100 55K 0,641 100 ’ 89K 0,627 100 85Q 0,624 100 92W 0,610 100 971 0,581 100 22H 0,568 100 esV 0,559 100 24S 0,557 100 74H 0,542 100 126K 0,542 100 e1L 0,539 100 26P 0,516 100 93N 0,513 100 64D 0,509 100 281 0,504 100 14K 0,493 100 100K 0,489 100 62E 0,454 100 1271 0,439 100 102E 0,428 100 25E 0,428 100 66P 0,427 100 114D 0,418 57 170 0,412 100 60G 0,390 100 95P 0,388 100 : 53E 0,377 100 81v 0,377 100 51K 0,370 100 103N 0,369 100 2Q 0,366 100 : 46N 0,360 100 42E 0,357 100

91T 0,340 100 87D 0,334 100 10N 0,333 100 1M1M 0,325 71 8C 0,323 100 124H 0,315 100 681 0,313 100 79P ' 0,307 100 109K 0,307 100 15K 0,302 100 49T 0,292 100 44N 0,291 100 113D 0,290 100 63V 0,286 100 105V 0,280 100 19P 0,270 100 84Q 0,264 100 76M 0,262 86 7D 0,251 100 116V 0,244 100 78C 0,238 100 : 36Q 0,235 100 45Q 0,233 100 40V 0,223 57 57S 0,212 100 : 38E 0,205 100 69D 0,203 100 9A 0,196 100 71N 0,190 100 98A 0,186 100 115G 0,180 100 13) 0,179 100 1237 0,179 100 34P 0,178 100 : 4D 0,157 100 : 20G 0,150 100 107T 0,143 100 12E 0,137 100 94V 0,137 100 1211 0,136 100 83G 0,128 100 70P 0,128 100 73C 0,120 100 3V 0,116 100 35F 0,111 100 59D 0,099 100 29] 0,098 100 23G 0,085 100 54] 0,075 100 5v 0,075 100 1018S 0,074 100 ) 72A 0,069 100 27C 0,060 100

32G 0,059 100 99pP 0,058 100 86Y 0,056 100 16V 0,052 100 50A 0,040 100 20Y 0,039 100 18V 0,035 100 33K 0,033 100 521 0,029 100 581 0,029 100 104V 0,024 100 112G 0,023 100 21C 0,023 100 88| 0,023 100 117L 0,016 100 56A 0,011 100 : 41F 0,011 100 120A 0,006 100 119C 0,006 100 67G 0,005 100 122A 0,005 100 37L 0,000 100 39A 0,000 100 43A 0,000 100 477 0,000 29 80L 0,000 100 108V 0,000 100 108V 0,000 100 ) 110V 0,000 100 118A 0,000 100 125A 0,000 100

Ves v 5 89 amino acids highly solvent exposed:

K-16, K-185, K-11, K-44, K-210, R-63, K-13, F-¢6, K-149,

K-128, E-184, K-112, K-202, F-157, E-3, K-29, N-203, N- 34, K-78, K-151, L-15, L-158, Y-102, W-186, K-134, D-87,

K-52, T-67, T-125, K-150, Y-40, (Q-48, L-65, K-81, Q-101, 0-208, K-144, N-8, N-70, H-104, Q-45, K-137, K-159, E- 205, N-82, A-111, D-131, K-24, V-36, N-7, M-138, T-209, v-84, K-172, v-19, D-56, P-73, G-33, T-106, N-170, 1-28,

T-43, 0-114, C-10, K-60, N-31, K-47, E-5, D-145, V-38, A- 127, D-156, E-204, :P-71, G-26, Y-129, D-141, F-201, R-68,

N-200, D-49, s-153, K-35, s$-39, Y-25, V-37, G-18, W-85,

I-182. :

88 amino acids highly solvent exposed and conserved (>70%)

K-16, K-185, K-11, K-44, K-210, R-63, K-13, F-6, K-149,

K-128, E-184, K-112, F-157, E-3, K-29, N-203, N-34, K-78,

K-151, L-15, L-158, Y-102, W-186, K-134, D-87, K-52, T- 67, T-125, K-150, Y-40, Q-48, L-65, K-81, (Q-101, Q-208,

K-144, N-8, N-70, H-104, Q-45, K-137, K-159, E-205, N-82,

A-111, D-131, K-24, V-36, N-7, M-138, T-209, Vv-84, K-172,

Vv-19, D-56, P-73, G-33, T-106, N-170, L-28, T-43, Q-114,

Cc-10, K-60, N-31, K-47, E-5, D-145, Vv-38, A-127, D-156,

E-204, P-71, G-26, Y-129, D-141, F-201, R-68, N-200, D- 49, 5-153, K-35, s-39, vY-25, v-37, G-18, W-85, I-182. 9 mutations performed:

K29A, T67A, K78A, V84S, Y102A, K112S, K144A, K202M, N203G

Table 3 shows a listing in descending order of solvent exposure of Ves v 5 amino acids. Column 1 lists the amino acid number starting from the amino-terminal, 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.

Table 3: Ves v 5 :

NO AA Solv_exp 16K 1,000 100 185K 0,989 100 11K 0,978 100 44K 0,978 100 . 210K 0,962 100 63R 0,956 100 13K 0,951 100 6F 0,868 100 149K 0,868 100 128K 0,857 100 184E 0,841 100 112K 0,824 100

202K 0,824 50 157F 0,819 100 3E 0,802 100 29K 0,797 100 203N 0,797 100 34N 0,775 100 78K 0,775 100 151K 0,753 100 15L 0,714 100 158L 0,714 100 102Y 0,687 100 186W 0,665 100 134K 0,654 100 87D 0,621 100 52K 0,615 100 67T 0,610 100 125T 0,610 100 150K 0,604 100 40Y 0,593 100 48Q 0,593 100 65L 0,593 100 81K 0,588 100 101Q 0,577 100 208Q 0,566 100 144K 0,560 100 8N 0,555 100 -_ 70N 0,549 100 104H 0,549 100 45Q 0,538 100 137K 0,538 100 159K 0,533 100 205E 0,511 100 82N 0,500 100 111A 0,500 100 131D 0,495 100 24K 0,489 100 36V 0,489 100 7N 0,484 100 138M 0,473 100 2007 0,473 100 84V 0,462 100 172K 0,451 100 19V 0,445 100 56D 0,445 100 73P 0,440 100 33G 0,429 100 106T 0,429 100 170N 0,429 100 28L 0,423 100 43T 0,423 100 114Q 0,423 100 10C 0412 100 60K 0,407 100

Co WO 02/40676 PCT/DK01/00764

31N 0,396 100 47K 0,396 100 5E 0,390 100 1450 0,390 100 38Vv 0,379 100 : 127A 0,379 100 156D 0,379 100 204E 0,374 100 71P 0,363 100 26G 0,352 100 .129Y 0,352 100 141D 0,341 100 201F 0,341 100 68R 0,335 100 200N 0,308 100 49D 0,302 100 1638 0,302 100 35K 0,297 100 398 0,291 100 25Y 0,280 100 : 37V 0,280 100 oo 18G 0,275 100 85wW 0,275 100 : 1821 0,275 100 46E 0,264 100 126A 0.253 100 : 88E 0,247 100 76P 0,236 100 79N 0,236 100 124S 0,236 100 30P 0,231 100 123G 0,231 100 162H 0,231 100 183Q 0,231 100 121 0,225 100 197P 0.225 100 130D 0,220 100 . 148P 0,214 100 180K 0,214 100 23C 0,209 100 75P 0,208 100 113Y 0,209 100 108R 0,203 100 188K 0,203 100 51L 0.198 100 59Q 0,198 100 121L 0,198 100 1227 0,198 100 154G 0,192 100 §3E 0,170 100 72G 0,170 100 41G 0,165 100 86N 0,165 100

147N 0,165 100 173E 0,165 100 278 0,159 100 94Q 0,159 100 187H 0,159 100 142E 0,154 100 64G 0,148 100 17G 0,143 100 133V 0,137 100 421 0,121 100 155N 0,121 100 55N 0,115 100 01Y 0,115 100 69G 0,110 100 103G 0,110 100 1908S 0,110 100 1090 0,093 100 207Y 0,082 100 96W 0,077 100 161G 0,077 100 140E 0,071 100 152F 0,071 100 80M 0,066 100 117Q 0,066 100 4A 0,060 100 32c 0,055 100 90A 0,055 100 206L 0,055 100 22A 0,049 100 110V 0,044 100 148Y 0,044 100 14C 0,038 100 oY 0,033 100 62A 0,033 100 132P 0,033 100 57F 0,027 100 29Q 0,027 100 100C 0,027 100 199G 0,027 100 77A 0,022 100 105D 0,022 100 119V 0,022 100 20H 0,016 100 83L 0,016 100 120A 0,016 100 139W 0,016 100 176C 0,016 100 178s 0,016 100 : 181Y 0,016 100 95V 0,011 100 115V 0,011 100 116G 0,011 100 165Q 0,011 100

169A 0,011 100 189H 0,011 100 66E 0,005 100 74Q 0,005 100 89L 0,005 100 92Vv 0,005 100 . 98N 0,005 100 118N 0,005 100 168W 0,005 100 217 0,000 100 501 0,000 100 54H 0,000 100 58R 0,000 100 61! 0,000 100 93A 0,000 100 97A 0,000 100 107C 0,000 100 135L 0,000 100 136V 0,000 100 143V 0,000 100 160T 0,000 100 163Y 0,000 100 164T 0,000 100 166M 0,000 100 167V 0,000 100 1717 0,000 100 174V 0,000 100 175G 0,000 100 177G 0,000 100 1791 0,000 100 190Y 0,000 100 191L 0,000 100 192V 0,000 100 193C 0.000 ~ 100 194N 0,000 100 195Y 0,000 100 196G 0,000 100

EXAMPLE 4

This Example describes preparation and characterisation ) of recombinant mutant Bet v 1 allergens according to the invention, i.e. allergens with diminished IgE-binding affinity comprising at least four primary mutations.

Selection of amino acid residues for site-directed mutagenesis of Bet v 1

Solvent accessibility of amino acid residues of Bet v 1 is shown in Example 3, table 1. The rate of amino acid conservation is based on sequence alignment performed at the ExPaSy Molecular Biology Server (http://www.expasy.ch/) using the ClustalW algorithm on a

BLAST search where the Bet V 1.2801 wild type amino acid sequence is used as input sequence. The alignment includes 67 allergen sequences (39 Bet v 1 sequences, ll

Car b 1 sequences, 6 Cor a 1 sequences, and 13 Aln g 1 sequences) from species within the order Fagales (Bet wv 1: Betula verrrucosa; Car b 1: Carpinus betulus; Cor a 1:

Corylus avellana; Aln g 1: alnus glutinosa). In respect to the mutated recombinant Bet v 1 allergens shown in the examples, target residues for substitution was based on 295% amino acid identity.

As described in Example 1, amino acid residues with a high degree of . solvent-exposure and a high degree of conservation between pollen allergens from related species, were selected for site-directed mutagenesis.

Residues having a low degree of solvent exposure (<20%) were not regarded relevant for mutagenesis due to the possible disruption of the tertiary structure or lack of antibody interaction.

The introduced residues were all present in corresponding positions in isoforms of a group of plant proteins called pathogenesis related (PR-10) proteins. Molecular modelling suggests that the tertiary structures of

Fagales allergens and PR-10 proteins are close to being identical. Bet v 1 shares significant sequence identity (20-40%) with PR-10 proteins. However, there are no reports of allergic cross-reactivity towards these PR-10 proteins. Thus, ' exchange of a highly conserved and solvent exposed amino acid from Bet v 1 with an amino acid in the corresponding position in a PR-10 protein,

results in a mutated Bet v 1 protein with an unaltered a- carbon backbone tertiary structure but with diminished

IgE-binding affinity.

In vitro mutagenesis

In vitro mutagenesis was performed by PCR using recombinant pMAL-c with Bet v 1 inserted as template.

Preparation of recombinant mutant allergens comprising five to nine primary mutations included two PCR steps; step I and II. First, 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 or Bet v 1.2801 derivatives using sense and anti-sense mutation-specific oligonucleotide primers accommodating each mutation(s) along with sense and anti- sense oligonucleotide primers accommodating elther upstream or downstream neighbour mutations or the N- terminus/C-terminus of Bet v 1, respectively as schematically illustrated in Figure 17 (I). Secondly, 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 17 (II). The PCR products were purified by agarose gel electrophoresis and PCR gel purification (Life

Techhnologies) followed by ethanol precipitation, cut with restriction enzymes (SacI/EcoRI) or (SacI/ XbaI), and ligated directionally into pMAL-c restricted with the same enzymes.

Figure 18 shows synthesised oligonucleotide primers and schematically illustrations for the construction of Bet Vv 1 mutants with five to nine 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 the following primary and secondary mutations stated in parenthesis:

Mutant Bet v 1 (2628): Tyr5val, Glud45Ser, Lys 65Asn,

Lys97Ser, Lysl134Glu.

Mutant Bet v 1 (2637): AlaléPro, (Asn28Thr, Lys32Gln),

Lys103Thr, Prol08Gly, (Leul52Lys, Alal53Gly, SerlS55Pro).

Mutant Bet v 1 (2733): (TyrS5val, Lys134Glu), (Asn28Thr,

Lys32Gln), Glué4S5Ser, Lys65Asn, (Asn78Lys, Lysl03Vval),

Lys97Ser, Prol08Gly, Argl4d5Glu, (AsplS56His, +160Asn)

Mutant Bet v 1 (2744): (Tyr5val, Lysl134Glu), (Glud2Ser,

Glu45Ser), (Asn78Lys, Lysl03Val), Lysl23Ile, (Aspl56His, +160Asn).

Mutant Bet v 1 (2753): (Asn28Thr, Lys32Gln), Lys65Asn, : (Glu96Leu, Lys97Ser), (Prol08Gly, Aspl09Asn), (Aspl25Tyx,

Glul27Ser), Argld5Glu.

Nucleotide sequencing

Determination of the nucleotide sequence of the Bet v 1 encoding gene was performed before and after subcloning, and following in vitro mutagenesis, respectively. plasmid DNA's from 10 ml of bacterial culture grown to saturation overnight in LB medium supplemented with 0.1 g/l ampicillin were purified on Qiagen-tip 20 columns and sequenced using the Ready reaction dye terminator cycle i sequencing kit and-a Fluorescence Sequencer AB PRISM 377, : both from (Perkin Elmer), following the recommendations of the suppliers.

Expression and purification of recombinant Bet v 1 and mutants

Recombinant Bet v 1 (Bet v 1.2801 and mutants) were over- expressed in Escherichia coli DH 5a 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 0.8 at 600 nm, whereupon expression of 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. After sonication and additional centrifugation, 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 wv 1 was isolated by gelfiltration and 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 tec about 5 mg/ml and stored at 4. °C. The final yields of the purified recombinant Bet vv 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.

We have previously shown (ref. 15) that recombinant Bet v 1 No. 2801 is immunochemically indistinguishable from naturally occurring Bet v 1.

Bet v 1 (2628) and Bet v 1 (2637) mutants :

Figure 19 shows introduced point mutations at the molecular surface of Bet v 1 (2628) and Bet v 1 (2637).

In mutant Bet v 1 (2628) five primary mutations were introduced in one half of Bet v 1 leaving the other half unaltered. In mutant Bet v 1(2637) five primary and three secondary mutations were introduced in the other half leaving the first half unaltered. In this way, mutations in mutant Bet v 1 (2628) and mutant Bet v 1(2637) affects different halves of the Bet v 1 surface, respectively.

Crystallisation and structural determination of recombinant Bet v 1(2628) mutant protein.

Crystals of recombinant Bet v 1 (2628) were grown by vapour diffusion at 25°C, essentially as described in (Spangfort et al 1996b, ref. 21). Bet v 1 (2628), at a concentration of 5 mg/ml, was mixed with an equal volume of 2.2 M ammonium sulphate, 0.1 M sodium citrate, 1% (v/v) dioxane, pH 6.3 and equilibrated against 100x volume of 2.2 M ammonium sulfate, 0.1 M sodium citrate, 1% (v/v) dioxane, pH 6.3. After 24 hours of equilibration, crystal growth was induced by applying the seeding technique described in ref. 21, using crystals of recombinant wild-type Bet v 1 as a source of seeds.

After about 4 months, crystals were harvested and analysed using X-rays generated from a Rigaku rotating anode as described in ref. 21 and the structure was solved using molecular replacement. ) Structure of Bet v 1 (2628) mutant

The structural effect of the mutations was addressed by growing three-dimensional Bet v 1 (2628) protein crystals diffracting to 2.0 A resolution when analysed by X-rays generated from a rotating anode. The substitutions

TyrSvVal, Glu45Ser, Lys65Asn, Lys97Ser, Lysl34Glu were verified by the Bet v 1 (2628) structure electron density map which also showed that the overall a-carbon backbone tertiary structure is preserved.

Structural analysis of Bet v 1 (2637) mutant

The structural integrity of the purified Bet v 1 (2637) mutant was analysed by circular dichroism (CD) spectroscopy. Figure 20 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 a-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 (2628) and Bet v 1 (2637) as well as a 1:1 mix of Bet v 1 (2628) and Bet v 1 (2637) was compared with recombinant wild type Bet Vv 1.2801 in a fluid-phase IgE-inhibition assay using a pool of serum IgE derived from birch allergic patients.

As described in Example 1, recombinant Bet v 1.2801 was biotinylated at a molar ratio of 1:5 (Bet wv 1 no. 2801:biotin). The inhibition assay was performed as follows: a serum sample (25 pl) 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

S obtained using buffer and mutant as inhibitor.

Figure 21 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 Vv 10 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).

There is a clear difference in the amount of respective recombinant proteins necessary to reach 50% inhibition of 15 the binding to serum IgE present in the serum pool.

Recombinant Bet v 1.2801 reaches 50% inhibition at about ng whereas the corresponding concentration for Bet v 1 (2628) mutant is about 15-20 ng. This show that the point mutation introduced in the Bet v 1 (2628) mutant lowers the affinity for specific serum IgE by a factor of about 3-4.

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 : 35 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 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.

In addition to the inhibition assays with single modified allergens a 1:1 mix of Bet 1 (2628) and Bet v 1 (2637) having same molar concentration of Bet v 1 as each of the samples with Bet 1 (2628) or Bet v 1 (2637), respectively was tested and showed full (100%) capacity to inhibit

IgE-binding to rBet wv 1.2801... The capacity to fully inhibit 1IgE-binding is a clear indication that all reactive epitopes present on Bet v 1.2801 were present in the 1:1 allergen mix. Further support comes from the comparable inclination of the two inhibition curves for

Bet v 1.2801 and the allergen mix. Reduced IgE-reactivity of the mixed allergen sample is demonstrated by the need of a four-fold higher concentration of the allergen mix, : when compared to Bet v 1.2801, for obtaining 50% inhibition of IgE-binding.

T cell proliferation assay using mutated recombinant Bet v 1 allergens.

The analysis was carried out as described in ref. 15. 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 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 assays with human basophil. }

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 25 microlitres of allergen or anti-IgE 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 properties of Bet v 1 (2628) and Bet v 1 (2637) mutant protein.

Histamine release data is shown in Figure 22 and Figure 23. 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. In both cases the release curve of the mutated allergens to induce histamine release is clearly shifted to the right compared to the release curve of Bet Vv 1.2801. The shift indicate that the potency of Bet v 1 (2628) and Bet v 1 (2637) is reduced 3 to 10-fold.

Mutant Bet v 1 (2744) and mutant Bet v 1 (2753)

Bet v 1 (2744) and Bet vv 1 (2753) was likewise constructed for use as a mixed allergen vaccine. In these mutated allergens point mutations were distributed in an all surface arranged fashion as shown in Figure 24 and

Figure 25 and was again designed to affect different surface areas in the two molecules, respectively, as shown in Figure 26. However these modified allergens might individually be used as single allergen vaccines as well.

Structural analysis of Bet v 1 (2744) mutant protein

The structural integrity of the purified Bet v 1 (2744) mutant was analysed by circular dichroism (CD) spectroscopy. Figure 27 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 a-carbon backbone tertiary structure is not affected by the introduced amino acid substitutions. :

Histamine release properties of Bet v 1 (2744)

Histamine release data from five experiments with basophil leucocytes from five different birch pollen allergic patients is shown in Figure 28 and Figure 29A-D.

The potency of Bet v 1 (2744) mutant protein to induce histamine release in human basophil has been tested. The release curves of the mutated allergens are clearly shifted to the right compared to the release curve of Bet v 1.2801 indicating that the potency of Bet v 1 (2744) to release histamine is reduced 3 to 5-fold.

Mutant Bet v 1 (2733)

A Mutant Bet v 1 (2733) with nine primary mutations has been constructed and recombinantly expressed. The distribution of point mutations in Bet v 1 (2733) leave several surface areas constituting >400A? unaltered.

Figure 30 show introduced point mutations at the molecular surface of Bet v 1 (2733).

EXAMPLE 5

This Example describes cloning of the gene encoding Der p 2 from Dermatophagoides pteronyssinus and construction of : mutants with reduced IgE-binding affinity.

PCR amplified products from first strand cDNA synthesis of Dermatophagoides pteronyssinus total RNA was obtained from Dr. Wendy-Anne Smith and Dr. Wayne Thomas (TVW

Telethon Institute for Child Health Research, 100 Roberts

Rd, Subiaco, Western Australia 6008). During the amplification of the first strand cDNA library, Der p 2 had been selectively amplified using Der p 2 specific primers. PCR fragments were subsequently cloned into the

Bam HI site of pUCl9 (New England Biolabs). DNA sequencing of Der p 2 was performed using vector specific sense (5'-GGCGATTAAGTTGGGTAACGCCAGGG-3’) and anti-sense (5’ -GGAAACAGCTATGACCATGATTACGCC-3’) primers.

A total of seven unique Der p 2 isoforms designated ALK- : 101, ALK=-102, ALK-103, ALK-104, ALK-113, ALK-114, and

ALK120 were identified. The clone entitled ALK-114 was chosen as starting point for generation of low-affinity

IgE-mutants because of its high sequence identity with the Der p 2 NMR structure with the data base accession 25 . number 1ASV. Compared to ALK-114, the 6 other naturally occurring isoforms comprise the following substitutions:

ATK-101: M76V.

ALK-102: VvV40L, T47S. :

ALK-103: M111L, D114N.

ALK-104: T47S, M111I, DI114N.

ALK-113: T47S.

ALK-120: V40L, T47S, D114N.

Insertion of Der pi 2 into pGAPZo-A

The gene encoding Der p 2 (ALK-114) was subsequently inserted into the pGAPZa-A vector (Invitrogen) for secreted expression of Der p 2 in the yeast, Pichia pastoris. The gene was amplified using sense primer OB27 {S'- GGAATTCCTCGAGAAAAGAGATCAAGTCGMATGTCARRGATTGTGCC-3') and anti-sense primer OB28 (57 -

CGTTCTAGACTATTAATCGCGGATTTTAGCATGAGTTIGC-3’) corresponding to the amino- and the carboxytermi of the Der p 2 polypeptide, respectively. The primers were extended in the 5’-end to accomodate the restriction sites Xho I and

Xba I, respectively. The Xho I restriction site fuses the first codon of Der p 2 in frame with the nucleic acid sequence encoding the KEX2 cleavage site (LYS-ARG) of pGAPZa-A. A single round of PCR amplification was performed in a 100 microliter (pl) volume: 0.1 mg of template ALK-114 DNA, 1 X Expand polymerase buffer (available from Boehringer Mannheim), 0.2 millimolar {mM) each of the four dNTPs, 0.3 micromolar (uM) each of the sense and anti-sense primers and 2.5 Units of Expand polymerase (available from Boehringer Mannheim). The DNA was amplified following 25 «cycles of: 95°C for 15 seconds, 45°C for 30 seconds, 72°C for 1 minute, followed by 1 cycle of 72°C for 7 minutes. The resulting 475 base pair ALK-114 PCR fragment was purified using a QIAquick spin purification procedure (available from Qiagen). The purified DNA fragment was then digested with Xho I and

Xba I, gel purified and ligated into similarly digested

PGAPZa-A. The ligation reaction was trasformed into

E.coli strain DH5a, resulting in plasmid, pCBo06.

The nucleotide sequence of Der p 2 was confirmed by DNA sequencing before and after cloning and following in vitro mutagenesis (see below).

Der p 2 sequences

SEQ ID NO 1 corresponds to the nucleic acid sequence of

Der p 2 (ALK-114): 1 gatcaagtcgatgtcaaagattgtgccaatcatgaaatcaaaaaagttttggtaccagga 61 tgccatggttcagaaccatgtatcattcatcgtggtaaaccattecaattggaagoegtt 121 ttegaagccaaccaaaacacaaaaaccgctaaaattgaaatcaaagcctcaatcgatggt 181 ttagaagttgatgttcccggtatcgatccaaatgcatgecattacatgaaatgcccattg 241 gttaaaggacaacaatatgatattaaatatacatggaatgttccgaaaattgcaccaaaa 301 tctgaaaatgttgtcgtcactgttaaagttatgggtgatgatggtgttttggectgtget 361 attgctactcatgctaaaatccgcgattaa

SEQ ID NO 2 corresponds to the deduced amino acid sequence of Der p 2 (ALK-114): 1 dqudvkdeanheikkvlvpgchgsepciihrgkpfqleavfeangntktakieikasidg ol levdvpgidpnachynkeplvkgggydikytwnvpkiapksenvvvtvkvmgddgvlaca 121 iathakird

Insertion of pGAPZa-A-Der p 2 into P. pastoris

The vector, pCBo06 was linearized using Avr IT restriction enzyme and transformed into competent P. pastoris strain, X-33, as described in the Invitrogen manual. Recombinant cells resistant to 100 micrograms per milliliter (ug/ml) of Zeocin were selected, and colony purifed on fresh YPD plates containing 100 pg/ml Zeocin.

Expression and purification of recombinant Der p 2 2A 250 ml of YPD medium (1% yeast extract, 2% peptone, 2% glucose) containing 100 ug/ml Zeocin was inoculated with an overnight culture of recombinant yeast cells expressing Der p 2. The culture was grown at 30°C for 72 hours to obtain optimal Der p 2 expression. Cells were harvested by centrifugation and the resulting culture supernatant was saturated with 50% ammonium sulfate.

Following centrifugation at 3000x g for 30 minutes, the supernatant was saturated with 80% ammonium sulfate.

Following centrifugation, the pellet was resuspended in 150 millimolar (mM) NHHCO; and fractionated on a

Superdex 75 gel filtration column, equilibrated with the same buffer. Der p 2 was eluted as a major peak corresponding to its expected molecular weight. The elution of Der p 2 was monitered both by SDS page electrophoresis, followed by silver staining and by immunoblot analysis using Der p 2 specific polyclonal antibodies.

Selection of amino acid residues for site-directed mutagenesis

Selection of amino acid residues for mutagenesis was based on identification of residues that are highly solvent exposed and highly conserved among allergens from

House Dust Mites (Der p 2/f 2 and Eur m 2) and storage mites (Tyr p 2, Lep d 2, Gly d 2). Highly solvent exposed amino acid residues were identified visually by analysis of the molecular surface of the Der p 2 NMR structure (#1.9, 1A9V.pdb). Twelve amino acid residues were selected for mutagenesis: K6A, N10S, KI15E, S24N, H3O0N,

K48A, E623, H74N, K77N, K82N, K100N and R128Q.

Site-directed mutagenesis

Construction of recombinant mutant allergens with single primary mutations and multiple combinations thereof, are described in the following.

Expression plasmids encoding Der p 2 mutants were produced using pCBo06 as DNA template. PCR reactions were performed using sense and anti-sense primers incorporating the specified mutations. Primer pairs used in the PCR reactions to generate the specified mutations are listed in Figure 31. The mutations are designated in bold and the restriction sites used in the subsequent cloning step are underlined in the figure. For the construction of mutants K6A, K15E, H30N, H74N and K82N,

PCR reactions were performed essentially as described in the section “Cloning of Der p 2 into pGAPZa-A”. The purified PCR fragments were digested with the designated : restriction enzyme sites (see Figure 31), gel purified, ligated into similarly digested pCBo0O6 and transformed into E.coli DH5«.

The mutation E62S was generated using an alternative PCR mutagenesis methodology described for the generation of :

Bet v 1 mutants in Example 1. Two mutation specific oligonucleotide primers were synthesized covering the specified mutations (OB47 and OB48, listed in Figure 31).

TWO additional primers used for the secondary amplification step were 0B27 and OB28 as described in the section: “Insertion of Der p 2 into pGAPZo-A”.

The mutant allergens produced are characterised using the same methods as described in example 4, e.g. circular dichroism (CD) spectroscopy, crystallisation, measurements of IgE binding properties, histamin-release,

T-cell proliferation stimulation capacity, etc.

EXAMPLE 6

Mutated recombinant mite allergens (Der p 2) with improved safety for specific allergy vaccination

In this example the application of the concept of the current invention on house dust mite allergens is exemplified by one allergen, Der p 2. Manipulation of other house dust mite allergens may be performed by equivalent procedures.

Design of mutated recombinant Der Pp 2 molecules.

SEQ ID NO. 3 shows the nucleotide and deduced amino acid

Sequence of Der p 2-ALK-G clone, which is a wild type isoform.

SEQ ID NO. 3: Nucleotide and deduced amino acid sequence of Der p 2-ALK-G.

GAT CAA GTC GAT GTC AAA GAT TGT GCC AAT CAT GAA ATC AAA AAA 45 b oo v D Vv XK pp ¢ A N H E I K K 15

GTT TTG GTA CCA GGA TGC CAT GGT TCA GAA CCA TGT ATC ATT CAT 90 v LL Vv Pp 6 Cc H GG Ss E P € I I H 30

CGT GGT AAA CCA TTC CAA TTG GAA GCT TTA TTC GAA GCC AAT CAA 135

R 6 XK P F Q L E A L F E A N 9 45 ~~ AAC TCA AAA ACA GCT AAA ATT GAA ATC AAA GCT TCA ATC GAT GGT 180

N Ss XK T A K I E I K A S I D GG 60

TTA GAA GIT GAT GTT CCC GGT ATC GAT CCA AAT GCA TGC CAT TAT 225 0 L E Vv D \" P G I D Pp N A Cc H Y 75 3

ATG AAA TGT CCA TTG GTT AAA GGA CAR CAA TAT GAT ATT AAA TAT 270

M K ¢ P L V K G © Q@ Y D I XK ¥Y 90

ACA TGG AAT GTT CCA AAA ATT GCA CCA AAA TCT GAA AAT GIT GTC 315 3% rT w N V P XK I A P K S E N V V 105

GTC ACT GIT ARA GTT TTG GGT GAT AAT GGT GTT TTG GCC TGT GCT 360

Vv T V XK Vv L 66 D N G6 V L A Cc A 120 40 ATT GCT ACT CAT GCT AAA ATC CGC GAT 387

I A T H A K I R D 129

Fig. 32 shows a sequence alignment performed at the

ExPaSy Molecular Biology Server (http://www.expasy.ch/) 45 using the ClustalW algorithm on a BLAST search using the

Der p 2-ALK-G amino acid sequence shown in SEQ ID NO. 3 as input sequence. The alignment includes sequences from house dust mite species, i.e. Der p 2, Der f 2 and Eur m 2. In Fig. 32 amino acid residues identical to amino 50 acids in the same position in the Der p 2-ALK-G protein sequence are highlighted using black letters on grey background. Non-identical amino acids are printed in black on a white background.

Surface structure images

Amino acid sequences representing the house dust mite group 2 allergens have a similarity greater than 85 % and some of the molecular surface is conserved (grey-coloured zones), see Fig. 33.

Fig. 33 shows surface contours viewed from 4 different angles when superimposing the Der p 2-ALK-G protein sequence on to the published PDB:1ASV NMR structure, structure number 1 of 10 contained in the PDB file.

Conserved and highly solvent exposed amino acid spatially distributed over the entire surface within distances in the range of 25-30 A are selected for mutation. In the sections below the following information is given: A list of amino acids considered to be appropriate for mutation (A), a 1list of the mutants designed (B) and the DNA sequences representing the mutants designed (C). Fig. 34 shows surface contours of mutant number 1 as an example.

Grey colour indicetes conserved amino acid residues.

Black colour indicates amino acid residues selected for mutation.

A. List of amino acids selected for mutation

K15, S24, H30, R31, K48, E62, H74, K77, K82, K100, R128

B. List of mutants designed

Mutant 1:

K15E, S24N, H30G, K48A, E62S, K77N, K82N, K100N

Mutant 2:

K15E, S24N, H30G, K48A, E62S, K77N, KB82N, R128Q

Mutant 3:

K15E, S24N, H30G, K48A, K77N, KB82N, KI100N, R128Q

Mutant 4: :

K15E, S24N, H30G, E®62S, K77N, KB82N, K100N, R128Q

Mutant 5:

K15E, H30G, K48A, E62S, K77N, K82N, K100N, R1280Q

Mutant 6:

S24N, H30G, K48A, E62S, K77N, K82N, K100N, R128Q

Mutant 7:

K15E, S24N, R31S, K48A, E62S, H74N, K82N, K100N

Mutant 8:

K15E, S24N, R31S, K48A, E62S, H74N, KB82N, R128Q

Mutant 9:

K15E, S24N, R31S, Kd48Aa, H74N, K82N, K100N, R128Q

Mutant 10:

K15E, S24N, R31S, E62S, H74N, K82N, K100N, R128Q

Mutant 11:

K15E, R31S, K4¢B8A, E6238, H74N, KB82N, K100N, R128Q

Mutant 12:

S24N, R31S, K4BA, E625, H74N, K82N, K100N, R128Q

C. Nucleotide sequence of mutants

Mutant 1:

K15E, S24N, H30G, K48A, E62S, K77N, K82N, KI1O00N

GatcaagtcgatgtcaaagattgtgccaatcatgaaatcaaaGAAgttttggtacca ggatgccatggtAACgaaccatgtatcattGGCcgtggtaaaccattccaattggaa gctttattcgaagccaatcaaaactcaGCGacagctaaaattgaaatcaaagettca atcgatggtttaAGCgttgatgttcccggtatcgatccaaatgecatgccattatatyg

AACtgtccattggttAACggacaacaatatgatattaaatatacatggaatgttcca aaaattgcaccaBACtctgaaaatgttgtcgtcactgttaaagttttgggtgataat ggtgttttggecctgtgectattgectactcatgctaaaatccgegat

Mutant 2:

K15E, S24N, H30G, K4BA, E62S, K77N, K82N, R128Q

GatcaagtcgatgtcaaagattgtgccaatcatgaaatcaaaGARgttttggtacca ggatgccatggtAACgaaccatgtatcattGGCcgtggtaaaccattccaattggaa gctttattcgaagccaatcaaaactcaGCGacagctaaaattgaaatcaaagettcea atcgatggtttaAGCgttgatgttcccggtatcgatccaaatgcatgecattatatg

AACtgtccattggttAACggacaacaatatgatattaaatatacatggaatgttcca aaaattgcaccaaaatctgaaaatgttgtcgtcactgttaaagttttgggtgataat ggtgttttggecctgtgectattgctactcatgctaaaatcCAGgat

Mutant 3:

K15E, S24N, HG30G, K48a, K77N, K82N, K100N, R128Q

GatcaagtcgatgtcaaagattgtgccaatcatgaaatcaaaGAAgttttggtacca ggatgccatggtAACgaaccatgtatcattGGCcgtggtaaaccattccaattggaa gctttattcgaagccaatcaaaactcaGCGacagctaaaattgaaatcaaagettcea atcgatggtttagaagttgatgttcccggtatcgatccaaatgcatgecattatatyg

AACtgtccattggttAACggacaacaatatgatattaaatatacatggaatgttcca aaaattgcaccaBACtctgaaaatgttgtcgtcactgttaaagttttgggtgataat ggtgttttggcctgtgctattgctactcatgctaaaatcCAGgat

Mutant 4:

K15E, S24N, H30G, E62S, K77N, K82N, K100N, R128Q

GatcaagtcgatgtcaaagattgtgccaatcatgaaatcaaaGAAgttttggtacca ggatgccatggthAACgaaccatgtatcattGGCegtggtaaaccattccaattggaa gctttattcgaagccaatcaaaactcaaaaacagctaaaattgaaatcaaagcttca atcgatggtttaAGCgttgatgttcccggtatcgatccaaatgeatgecattataty

AACtgtccattggttAACggacaacaatatgatattaaatatacatggaatgttcca aaaattgcaccaRACtctgaaaatgttgtcgtcactgttaaagttttgggtgataat ggtgttttggcctgtgctattgectactcatgctaaaatcCAGgat

Mutant 5:

K15E, H30G, K48A, E62S, K77N, K82N, K1O00N, R128Q

GatcaagtcgatgtcaaagattgtgccaatcatgaaatcaaaGAAgttttggtacca ggatgccatggttcagaaccatgtatcattGGCegtggtaaaccattccaattggaa gctttattcgaagccaatcaaaactcaGCGacagctaaaattgaaatcaaagettea atcgatggtttaAGCgttgatgttcccggtatcgatccaaatgecatgecattataty

AACtgtccattggttAACggacaacaatatgatattaaatatacatggaatgttcea aaaattgcaccahACtctgaaaatgttgtcgtcactgttaaagttttgggtgataat ggtgttttggectgtgctattgctactcatgctaaaatcCAGgat

Mutant 6:

S24N, H30G, K48A, E62s, K77N, K82N, KI1O0ON, R128Q

Gatcaagtcgatgtcaaagattgtgccaatcatgaaatcaaaaaagttttggtacca ggatgccatggtAACgaaccatgtatcattGGCcgtggtaaaccattccaattggaa gctttattcgaagccaatcaaaactcaGCGacagctaaaattgaaatcaaagettca atcgatggtttaAGCgttgatgttcccggtatcgatccaaatgcatgeccattatatg

AACtgtccattggttAACggacaacaatatgatattaaatatacatggaatgttcca aaaattgcaccahACtctgaaaatgttgtcgtcactgttaaagttttgggtgataat ggtgttttggectgtgctattgetactcatgctaaaatcCAGgat

¥ 101

Mutant 7:

K15E, S24N, R31S, K48A, E62S, H74N, K82N, KI1OON

GatcaagtcgatgtcaaagattgtgccaatcatgaaatcaaaGARgttttggtacca ggatgccatggtAACgaaccatgtatcattcatAGCggtaaaccattccaattggaa gctttattcgaagccaatcaaaactcaGCGacagctaaaattgaaatcaaagettcea atcgatggtttaAGCgttgatgttcccggtatcgatccaaatgcatgcAACtatatyg aaatgtccattggttAACggacaacaatatgatattaaatatacatggaatgttcca aaaattgcaccaBACtctgaaaatgttgtcgtcactgttaaagttttgggtgataat ggtgttttggcctgtgctattgectactcatgectaaaatccgegat

Mutant 8:

KI15E, S24N, R31S, K48A, E62S, H74N, K82N, R128Q

GatcaagtcgatgtcaaagattgtgccaatcatgaaatcaaaGArgttttggtacca ggatgccatggtAACgaaccatgtatcattcatAGCggtaaaccattccaattggaa gctttattcgaagccaatcaaaactcaGCGacagctaaaattgaaatcaaagettca atcgatggtttaAGCgttgatgttccecggtatcgatccaaatgcatgchAACtatatyg aaatgtccattggttAACggacaacaatatgatattaaatatacatggaatgttcca aaaattgcaccaaaatctgaaaatgttgtcgtcactgttaaagttttgggtgataat ggtgttttggectgtgctattgctactcatgctaaaatcCAGgat

Mutant 9: .

K15E, S24N, R31S, K48A, H74N, KB82N, K100N, R128Q

GatcaagtcgatgtcaaagattgtgccaatcatgaaatcaaaGAAgttttggtacca ggatgccatggtAACgaaccatgtatcattcatAGCggtaaaccattccaattggaa gctttattcgaageccaatcaaaactcaGCGacagctaaaattgaaatcaaagettca atcgatggtttagaagttgatgttcccggtatcgatccaaatgcatgcAACtataty aaatgtccattggttAACggacaacaatatgatattaaatatacatggaatgttcca aaaattgcaccaAACtctgaaaatgttgtcgtcactgttaaagttttgggtgataat ggtgttttggcctgtgectattgctactcatgctaaaatcChAGgat

Mutant 10:

K15E, S24N, R31S, E62S, H74N, K82N, K100N, R128Q

GatcaagtcgatgtcaaagattgtgccaatcatgaaatcaaaGhAgttttggtacca ggatgccatggtAACgaaccatgtatcattcatAGCggtaaaccattccaattggaa gctttattcgaagccaatcaaaactcaGCGacagctaaaattgaaatcaaagcttca atcgatggtttagaagttgatgttcccggtatcgatccaaatgcatgchACtatatg aaatgtccattggttAACggacaacaatatgatattaaatatacatggaatgttcca aaaattgcaccahACtctgaaaatgttgtcgtcactgttaaagttttgggtgataat ggtgttttggcctgtgctattgctactcatgctaaaatcCAGgat

Mutant 11:

K15E, R31S, K48A, E62S, H74N, K82N, K100N, R128Q

GatcaagtcgatgtcaaagattgtgccaatcatgaaatcaaaGAAgttttggtacca ggatgccatggttcagaaccatgtatcattcatAGCggtaaaccattccaattggaa gctttattcgaageccaatcaaaactcaGCGacagctaaaattgaaatcaaagcettca atcgatggtttaAGCgttgatgttcccggtatcgatccaaatgcatgcAACtatatg aaatgtccattggttAACggacaacaatatgatattaaatatacatggaatgttcca aaaattgcaccaBACtctgaaaatgttgtcgtcactgttaaagttttgggtgataat ggtgttttggcctgtgctattgectactcatgctaaaatcCAGgat

Mutant 12:

S24N, R31S, K48A, E62S, H74N, K82N, KI100N, R128Q

Gatcaagtcgatgtcaaagattgtgeccaatcatgaaatcaaaaaagttitggtacca ggatgccatggtAACgaaccatgtatcattcatAGCggtaaaccattccaattggaa gctttattcgaagccaatcaaaactcaGCGacagctaaaattgaaatcaaagcecttca atcgatggtttaAGCgttgatgttcccggtatcgatccaaatgcatgcAACtatatyg aaatgtccattggttAACggacaacaatatgatattaaatatacatggaatgttcca : aaaattgcaccaPACtctgaaaatgttgtcgtcactgttaaagttttgggtgataat ggtgttttggcctgtgctattgetactcatgcectaaaatcCAGgat

EXAMPLE 7

Mutated recombinant mite allergens (Der p 1) with improved safety for specific allergy vaccination

In this example the application of the concept of the current invention on house dust mite allergens is exemplified by one allergen, Der p 1. Manipulation of other house dust mite allergens may be performed by equivalent procedures.

Design of mutated recombinant Der p 1 molecules.

SEQ ID NO. 4 shows the nucleotide and deduced amino acid sequence of Der p 1-ALK clone, which is a wild-type isoform.

SEQ ID NO. 4: Nucleotide and deduced amino acid sequence of Der p 1-ALK

ACT AAC GCC TGC AGT ATC RAT GGA AAT GCT CCA GCT GAA ATC GAT 45

T N a Cc Ss I N G N A Pp A E I D 15

TTG CGA CAA ATG CGA ACT GTC ACT CCC ATT CGT ATG CAA GGA GGC 90

L R Q M R T \ T P I R M Q G G 30

TGT GGT TCA TGT TGG GCT TTC TCT GGT GTT GCC GCA ACT GAA TCA 1358

Cc 6 Ss €C W A F § G6 Vv A A T E Ss 45

GCT TAT TTG GCT TAC CGT AAT CAA TCA TTG GAT CTT GCT GAR CAA 180

AY L A Y R N @ § L D L A E Q 60

GAA TTA GTC GAT TGT GCT TCC CARA CAC GGT TGT CAT GGT GAT ACC 225

E L \' D Cc A Ss Q H G Cc H G D T 75

ATT CCAR CGT GGT ATT GAA TAC ATC CAA CAT AAT GGT GTC GTC CAA 270

I Pp R G I E Y I Q H N G Vv \' Q 90 40

GAA AGC TAC TAT CGA TAC GTT GCA CGA GAR CAA TCA TGC CGA CGA 315

BE Ss Y Y R Y Vv A R £ Q Ss Cc R R 10%

CCA AAT GCA CAA CGT TTC GGT ATC TCA AARC TAT TGC CAA ATT TAC 360 45 P ¥ A © R F G6 1 SS NN ¥Y CC Q I Y 120

CCA CCA AAT GTA AAC AAA ATT CGT GAR GCT TTG GCT CAR ACC CAC 405

P P N V N K I R E A L A @ T H 135 50 AGC GCT ATT GCC GTC ATT ATT GGC ATC AAR GAT TTA GAC GCA TTC 450 $s A I A Vv 1 1 6 I K D L DOD A F 150

CGT CRT TAT GAT GGC CGA ACA ATC RTT CAA CGC GAT AAT GGT TAC 495 ss R H Y D 6 R T I I © R D N G Y 165

CAM CCA ARC TAT CAC GCT GTC AAC ATT GIT GGT TAC AGT AAC GCA 540 v [of P N Y H A Vv N I v G Y S N A 180

CRRA GGT GTC GAT TAT TGS ATC GTA CGA AAC AGT TGG GAT ACC AAT 588

Q G Vv b Y Ww I v R N S w D T N 195 ee oor SRT or ser re er or or ser see he re SAT Tre 210 630

Re we rr ad ohn oT oh hae or ore mr ore 222 666

Fig. 35 shows a sequence alignment performed at the

ExPaSy Molecular Biology Server (http://www.expasy.ch/) using the ClustalW algorithm on a BLAST search using the

Der p 1-ALK amino acid sequence shown in SEQ ID NO. 4 as input sequence. The alignment includes sequences from house dust mite species, i.e. Der p 1, Der £ 1 and Eur m 1. In Fig. 35 amino acid residues identical to amino acids in the same position in the Der p 1-ALK protein sequence are highlighted using black letters on grey background. Non-identical amino acids are printed in black on a white background.

Surface structure images

Amino acid sequences representing the house dust mite group 1 allergens are similar to a certain degree and some of the molecular surface is conserved (grey-coloured zones), see Fig. 36. Fig. 36 shows surface contours viewed from 4 different angles when superimposing the Der p 1-ALK protein sequence on to a Der p 1 molecular structure model.

Conserved and highly solvent exposed amino acid spatially distributed over the entire surface within distances in the range of 25-30 A are selected for mutation. In the sections below the following information is given: A list of amino acids considered to be appropriate for mutation 40 (A), a list of the mutants designed (B) and the DNA sequences representing the mutants designed (C). Fig. 37 .shows surface contours of mutant number 11 as an example.

N WQ0 02/40676 PCT/DK01/00764

Grey colour indicates conserved amino acid residues.

Black colour indicates amino acid residues selected for mutation.

A. List of amino acids selected for mutation

E13, P24, R20, Yso, S67, R78, R99, 109, R128, R156,

R161, P167, W192

B. List of mutants designed

Mutant 1: -

P24T, Y50V, R78E, R99Q, R156Q, R161E, P1l67T

Mutant 2: . P24T, Y50V, R78Q, R3S9E, R1S6E, R161Q, P167T

Mutant 3:

R20E, Y50V, R78Q, R99Q, R156E, R161E, P167T

Mutant 4:

R20Q, Y50V, R78E, R9%E, R156Q, R161Q, P167T

Mutant 5:

P247T, Y50V, S67N, R99E, R156Q, R161Q, P167T

Mutant 6:

R20E, Y50V, S67N, RO99E, R156Q, R161E, P167T

Mutant 7:

R20Q, Y50V, S67N, R98Q, R156E, R161E, Pl67T

Mutant 8:

E13s, P24T, Y50V, R78E, R99(Q, Q109D, R128E, R156Q, R161E,

P167T

Mutant 9: £13, P24T, YS0V, R78Q, R99E, 0108D, R128Q, R156E, R161Q,

P167T

Mutant 10:

E13s, P24T, Y50V, R78E, R99Q, Q109D, R1Z8E, R156Q, R161lE,

P167T, W192F

Mutant 11:

E13S, P24T, Y50V, R78Q, R99E, (Q109D, R128Q, R156E, R161Q,

P167T, W192F

C. Nucleotide sequences of mutants

Mutant 1:

P24T, Y50V, R78E, R99Q, R156Q, R16lE, P1677

ACT AAC GCC TGC AGT ATC AAT GGA AAT GCT CCA GCT GAA ATC GAT TTG CGA CAA ATG CGR 60

ACT GTC ACT ACC ATT CGT ATG CAA GGA GGC TGT GGT TCA TGT TGG GCT TTC TCT GGT GTT 120

GCC GCA ACT GAA TCA GCT TAT TTG GCT GTG CGT AAT CAA TCA TTG GAT CTT GCT GAR CAA 180

GAA TTE GTC GAT TGT GCT TCC CAA CAC GGT TGT CAT GGT GAT ACC ATT CCA GAA GGT ATT 240

GAA TAC ATC CAA CAT AAT GGT GTC GTC CAA GAA AGC TAC TAT CGA TAC GTT GCA CAG GAA 300

CAA TCA TGC CGA CGA CCA AAT GCA CAA CGT TTC GGT ATC TCA AAC TAT TGC CAR ATT TAC 360

CCA CCK AAT GTA AAC AAA ATT CGT GAA GCT TTG GCT CAA ACC CAC AGC GCT ATT GCC GTC 420

ATT ATT GGC ATC AAA GAT TTA GAC GCA TTC CGT CAT TAT GAT GGC CAG ACA ATC ATT CAA 480

GAA GAT AAT GGT TAC CAA ACC AAC TAT CAC GCT GTC AAC ATT GTT GGT TAC AGT AAC GCR 540

CAR GGT GTC GAT TAT ‘TGG ATC GTA CGA ARC AGT TGG GAT ACC AAT TGG GGT GAT ART GGT 600

TAC GGT TAT TTT GCT GCC AAC ATC GAT TTG ATG ATG ATT GRA GAA TAT CCA TAT GTT GTC 660

ATT CTC 666

Mutant 2:

P24T, Y50V, R78Q, R99E, R156E, R161Q, P1l67T

ACT AAC GCC TGC AGT ATC AAT GGA AAT GCT CCA GCT GAA ATC GAT TTG CGA CAA ATG CGA 60 o

ACT GTC ACT ACC ATT CGT ATG CARR GGA GGC TGT GGT TCA TGT TGG GCT TTC TCT GGT GTT 120

GCC GCR ACT GAA TCA GCT TAT TTG GCT GTG CGT AAT CAA TCA TTG GAT CTT GCT GAA CAA 180

GAA TTA GTC GAT TGT GCT TCC CAA CAC GGT TGT CAT GGT GAT ACC ATT CCA CAG GGT ATT 240

GRA TAC ATC CAA CAT AAT GGT GTC GTC CAA GAA AGC TAC TAT CGA TAC GTT GCA GAR GAR 300

CAA TCA TGC CGA CGA CCR ART GCA CAA CGT TTC GGT ATC TCA AAC TAT TGC CAA ATT TAC 360

CCA CCA RAT GTA RAC AAA ATT CGT GAA GCT TTG GCT CAA ACC CRC AGC GCT ATT GCC GTC 420

ATT ATT GGC ATC ARA GAT TTA GAC GCA TTC CGT CAT TAT GAT GGC GAA ACA ATC ATT CAA 480

CAG GAT AAT GGT TAC CAA ACC AAC TAT CAC GCT GTC AAC ATT GTT GGT TAC AGT ARC GCA 540

CAA GGT GTC GAT TAT TGG ATC GTA CGA AAC AGT TGG GAT ACC AAT TGG GGT GAT AAT GGT 600

TAC GGT TAT TTT GCT GCC ARC ATC GAT TTG ATG ATG ATT GAR GAA TAT CCA TAT GTT GTC 660

ATT CTC 666

Mutant 3:

R20E, Y50V, R78(Q, R99Q, R156E, R16lE, P167T

ACT ARC GCC TGC AGT ATC AAT GGA AAT GCT CCA GCT GAA ATC GAT TTG CGA CAR ATG GAA 60

ACT GTC ACT CCC ATT CGT ATG CAA GGA GGC TGT GGT TCA TGT TGG GCT TTC TCT GGT GTT 120

GCC GCA ACT GAA TCA GCT TAT TTG GCT GTG CGT AAT CAA TCA TTG GAT CTT GCT GAR CAA 180

GAA TTR GTC GAT TGT GCT TCC CAA CAC GGT TGT CAT GGT GAT RCC ATT CCA CAG GGT ATT 240

GAA TAC ATC CAA CAT AAT GGT GTC GTC CAA GAA AGC TAC TAT CGA TAC GTT GCA CAG GAA 300

CAA TCA TGC CGA CGA CCA AAT GCA CAA CGT TTC GGT ATC TCA AAC TAT TGC CAA ATT TAC 36C

CCA CCA ART GTA AAC AAA ATT CGT GAA GCT TTG GCT CAR ACC CAC AGC GCT ATT GCC GTC 420

ATT ATT GGC ATC ARA GAT TTA GAC GCA TTC CGT CAT TAT GAT GGC GAA ACA ATC ATT CAA 480

CAG GAT AAT GGT TAC CAA ACC AAC TAT CRC GCT GTC ARC ATT GTT GGT TAC AGT AAC GCA 540

CAR GGT GTC GAT TAT TGG ATC GTA CGA AAC AGT TGG GAT ACC AAT TGG GGT GAT AAT GGT 600

TAC GGT TAT TTT GCT GCC AAC ATC GAT TTG ATG ATG ATT GAA GAA TAT CCA TAT GIT GTC 660

ATT CTC 666

Mutant 4:

R20Q, ¥50V, R78E, R99E, R1560Q, R161Q, P167T

ACT ARC GCC TGC AGT ATC AAT GGA AAT GCT CCA GCT GAA ATC GAT TTG CGA CAR ATG CAG 60

ACT GTC ACT CCC ATT CGT ATG CAA GGA GGC TGT GGT TCA TGT TGG GCT TTC TCT GGT GTT 120 :

GCC GCA ACT GAA TCA GCT TAT TTG GCT GTG CGT AAT CAA TCA TTG GAT CTT GCT GAA CAA 180

.

GAA TTA GTC GAT TGT GCT TCC CAA CAC GGT TGT CAT GGT GAT ACC ATT CCA GAR GGT ATT 240

GAR TAC ATC CAA CAT AAT GGT GTC GTC TAR GAA AGC TAC TAT CGA TAC GTT GCA GAA GAA 300

CAA TCA TGC CGA CGA CCA AAT GCA CAA CGT TTC GGT ATC TCA AAC TAT TGC CAA ATT TAC 360

CCA CCA AAT GTA AAC ARR ATT CGT GAA GCT TTG GCT CAA ACC CAC AGC GCT ATT GCC GTC 420

ATT ATT GGC ATC ARMA GAT TTA GAC GCA TTC CGT CAT TAT GAT GGC CAG ACA ATC ATT CAR 480

CAG GAT AAT GGT TAC CAA ACC AAC TAT CAC GCT GTC AAC ATT GTT GGT TAC AGT AARC GCA 540

CAA GGT GTC GAT TAT TGG ATC GTA CGA ARC AGT TGG GAT ACC AAT TGG GGT GAT AAT GGT 600

TAC GGT TAT TTT GCT GCC AAC ATC GAT TTG ATG ATG ATT GAA GAA TAT CCA TAT GTT GTC 660

ATT CTC 666

Mutant 5:

P24T, YS50V, S67N, R99E, R156Q, R16lQ, P167T

ACT AAC GCC TGC AGT ATC AAT GGA AAT GCT CCA GCT GAA ATC GAT TTG CGA CAR ATG CGA 60

ACT GTC ACT ACC ATT CGT ATG CAR GGA GGC TGT GGT TCA TGT TGG GCT TTC TCT GGT GTT 120

GCC GCA ACT GAR TCA GCT TAT TTG GCT GTG CGT AAT CAR TCA TTG GAT CTT GCT GAA CRRA 180

GAR TTA GTC GAT TGT GCT AAC CAA CAC GGT TGT CAT GGT GAT ACC ATT CCA CGT GGT ATT 240

GAR TAC ATC CAA CAT AAT GGT GTC GTC CAA GAA AGC TAC TAT CGA TAC GTT GCA GAA GRA 300

CAR TCA TGC CGA CGA CCA AAT GCA CAA CGT TTC GGT ATC TCA ARC TAT TGC CAR ATT TRC 360

CCA CCA AAT GTA AAC AAA ATT CGT GAA GCT TTG GCT CAR ACC CAC AGC GCT ATT GCC GTC 420

ATT ATT GGC ATC AAA GAT TTR GAC GCA TTC CGT CRT TAT GAT GGC CAG RCA ATC ATT CRA 480

CAG GAT AAT GGT TAC CAA ACC AAC TAT CAC GCT GTC ARC ATT GTT GGT TAC AGT ARC GCA 540

CARR GGT GTC GAT TAT TGG ATC GTA CGA RAC AGT TGG GAT RCC AAT TGG GGT GAT AAT GGT 600

TAC GGT TAT TTT GCT GCC AAC ATC GAT TTG ATG ATC ATT GAA GAA TAT CCA TAT GTT GTC 660

ATT CTC i 666

Mutant 6:

R20E, Y50V, S67N, RS99E, R156Q, R161E, P167T

ACT AAC GCC TGC AGT ATC AAT GGA AAT GCT CCA GCT GAA ATC GAT TTG CGA CAA ATG GRR 60

ACT GTC ACT CCC ATT CGT ATG, CAA GGA GGC TGT GGT TCA TGT TGG GCT TTC TCT GGT 6TT 120 {

GCC GCA ACT GAA TCA GCT TAT TTG GCT GTG CGT RAT CAA TCA TTG GAT CTT GCT GARR CAR 180

GAA TTA GTC GAT TGT GCT ARC CAA CAC GGT TGT CAT GGT GAT ACC ATT CCA CGT GGT ATT 240

GRA TAC ATC CAA CAT AAT GGT GTC GTC CAA GAR AGC TAC TAT CGA TAC GTT GCA GAR GAA 300

.

CAA TCA TGC CGA CGA CCA AAT GCA CAA CGT TTC GGT ATC TCA AAC TAT TGC CAA ATT TAC 360

CCA CCA AAT GTA AAC AAA ATT CGT GAA GCT TTG GCT CAA ACC CAC AGC GCT ATT GCC GTC 420

ATT ATT GGC ATC AAA GAT TTA GAC GCA TTC CGT CAT TAT GAT GGC CAG ACA ATC ‘ATT CAA 480

GAA GAT AAT GGT TAC CAA ACC AAC TAT CAC GCT GTC AAC ATT GTT GGT TAC AGT AAC GCA 540

CARA GGT GTC GAT TAT TGG ATC GTA CGA ARC AGT TGG GAT ACC AAT TGG GGT GAT AAT GGT 600

TAC GGT TAT TTT GCT GCC AAC ATC GAT TTG ATG ATG ATT GAR GAR TAT CCA TAT GTT GTC 660

ATT CTC 666

Mutant 7: ‘

R20Q, Y50V, S67N, R99Q, R156E, R161E, P167T

ACT AAC GCC TGC AGT ATC AAT GGA AAT GCT CCA GCT GAA ATC GAT TTG CGR CRA ATG CAG 60

ACT GTC ACT CCC ATT CGT ATG CAA GGA GGC TGT GGT TCA TGT TGG GCT TTC TCT GGT GTT 120

GCC GCA ACT GAA TCA GCT TAT TTG GCT GTG CGT AAT CAA TCA TTG GAT CTT GCT GAR CAA 180

GAA TTA GTC GAT TGT GCT ARC CAA CAC GGT TGT CAT GGT GAT ACC ATT CCA CGT GGT ATT 240

GAA TAC ATC CAA CAT AAT GGT GTC GTC CAA GAR AGC TAC TAT CGA TAC GTT GCA CAG GAA 300

CAR TCR TGC CGA CGA CCA AAT GCA CAA CGT TTC GGT ATC TCA AAC TAT TGC CAA ATT TAC 360

CCA CCA AAT GTA AAC ARA ATT CGT GAR GCT TTG GCT CAA ACC CAC AGC GCT ATT GCC GTC 420

ATT ATT GGC ATC ARA GAT TTA GAC GCA TTC CGT CAT TAT GAT GGC GAA ACA ATC ATT CAA 480

GAA GAT AAT GGT TAC CARA ACC AAC TAT CAC GCT GTC AAC ATT GTT GGT TAC AGT AAC GCA 540

CAA GGT GTC GAT TAT TGG ATC GTA CGA AAC AGT TGG GAT ACC PAT TGG GGT GAT AAT GGT 600

TAC GGT TAT TTT GCT GCC RAC ATC GAT TTG ATG ATG ATT GAA GAA TAT CCA TAT GTT GTC 660

ATT CTC 666

Mutant 8:

E135, P24T, Y50V, R78E, R99Q, Q109D, R128E, R156Q, RI161lE,

P167T

ACT AAC GCC TGC AGT ATC RAT GGA AAT GCT CCA GCT AGC ATC GAT TTG CGA CAA ATG CGA 60

ACT GTC ACT ACC RATT CGT ATG CAA GGA GGC TGT GGT TCA TGT TGG GCT TTC TCT GGT GTT 120

GCC GCA ACT GAR TCA GCT TAT TTG GCT GTG CGT AAT CAA TCA TTG GAT CTT GCT GAA CAA 180

GAA TTA GTC GAT TGT act TCC CAA CAC GGT TGT CAT GGT GAT ACC ATT CCA GAA GGT ATT 240

GAA TAC ATC CAA CAT AAT GGT GTC GTC CAA GAA AGC TAC TAT CGA TAC GIT GCA CAG GAA 300

CAA TCA TGC CGA CGA CCA RAT GCA GAT CGT TTC GGT ATC TCA AAC TAT TGC CAA ATT TAC 360

CCA CCA AAT GTA AAC AAA ATT GAA GAA GCT TTG GCT CAA ACC CAC AGC GCT ATT GCC GTC 420

ATT ATT GGC ATC AAA GAT TTA GAC GCA TTC CGT CAT TAT GAT GGC CAG ACA RTC ATT CAR 48C

GAA GAT AAT GGT TAC CAA ACC AAC TAT CAC GCT GTC AAC ATT GTT GGT TAC AGT AAC GCA 540

CAR GGT GTC GAT TAT TGG ATC GTA CGA AARC AGT TGG GAT ACC BAT TGG GGT GAT AAT GGT 600

TAC GGT TAT TTT GCT GCC AAC ATC GAT TTG ATG ATG ATT GAA GAA TAT CCA TAT GTT GTC 660

ATT CTC 666

Mutant 9:

E13S, P24T, Y50V, R78Q, R9%E, Q109D, R128Q, R156E, R1610Q,

Pl1677T

ACT AAC GCC TGC AGT ATC AAT GGA AAT GCT CCA GCT AGC ATC GAT TTG CGA CAA ATG CGA 60

ACT GTC ACT ACC ATT CGT ATG CAA GGA GGC TGT GGT TCA TGT TGG GCT TTC TCT GGT GTT 120

GCC GCA ACT GAA TCA GCT TAT TTG GCT GTG CGT AAT CAA TCA TTG GAT CTT GCT GAA CAA 180

GAR TTA GTC GAT TGT- GCT TCC CAA CAC GGT TGT CAT GGT GAT ACC ATT CCA CAG GGT ATT 240

GAR TAC ATC CAA CAT AAT GGT GTC GTC CAA GAA AGC TAC TAT CGA TAC GTT GCA GAA GAR 300

CAA TCA TGC CGA CGA CCA RAT GCA GAT CGT TTC GGT ATC TCA AAC TAT TGC CAA ATT TAC 360

CCA CCA AAT GTA AAC ARA ATT CAG GRA GCT TTG GCT CAA ACC CAC AGC GCT ATT GCC GTC 420

ATT ATT GGC ATC ARA GAT TTA GAC GCA TTC CGT CAT TAT GAT GGC GAA ACA ATC ATT CAA 480

CAG GAT AAT GGT TAC CAA ACC ARC TAT CAC GCT GTC AAC ATT GTT GGT TAC AGT AAC GCA 540

CAR GGT GTC GAT TAT TGG ATC GTA CGR ARC AGT TGG GAT ACC AAT TGG GGT GAT AAT GCT 600

TAC GGT TAT TTT GCT GCC AAC ATC GAT TTG ATG ATG ATT GAA GAA TAT CCA TAT GTT GTC 660 }

ATT CTC 666

Mutant 10: £138, P24T, Y5QV, R78E, R89Q, Q109D, R128E, R156Q, R161E,

P167T, W192F

ACT AAC GCC TGC AGT ATC AAT GGA AAT GCT CCA GOT AGC ATC GAT TTG CGA CAA ATG CGR 60

ACT GTC ACT ACC ATT CGT ATG CAA GGA GGC TGT GGT TCA TGT TGG GCT TTC TCT GGT GTT 120

GCC GCA ACT GAA TCA GGT TAT TTG GCT GTG CGT AAT CAR TCA TTG GAT CTT GCT GAA CRA 180

GAR TTA GTC GAT TGT GCT rc CAR CAC GGT TGT CAT GGT GAT ACC ATT CCA GAA GGT ATT 240

GAA TAC ATC CAR CAT AAT GGT GTC GPC CAA GAA AGC TAC TAT CGA TAC GTT GCA CAG GAR 300

CAA TCA TGC CGA CGA CCA AAT GCA GAT CGT TTC GGT ATC TCA ARC TAT TGC CAA ATT TAC 360

CCA CCA AAT GTA AAC AAA ATT GAA GAA GCT TTG GCT CAA ACC CAC AGC GCT ATT GCC GTC 420

ATT ATT GGC ATC AAA GAT TTA GAC GCA TTC CGT CAT TAT GAT GGC CAG ACA ATC ATT CAR 480

GAA GAT AAT GGT TAC CAA RCC AAC TAT CAC GCT GTC AARC ATT GIT GGT TAC AGT AAC GCA 540

CAA GGT GTC GAT TAT TGG ATC GTA CGA AAC AGT TTT GAT ACC AAT TGG GGT GAT AAT GGT 600

TAC GGT TAT TTT GCT GCC AAC ATC GAT TTG ATG ATG ATT GAA GAA TAT CCA TAT GIT GTC 660

ATT CTC 666

Mutant 11:

E13S, P24T, Y50V, R78Q, R99E, Q109D, R128Q, R156E, R161Q,

P1677, W192F

ACT ARC GCC TGC AGT ATC AAT GGA AAT GCT CCA GCT AGC ATC GAT TTG CGR CAA ATG CGA 60

ACT GTC ACT ACC ATT CGT ATG CAA GGA GGC TGT GGT TCA TGT TGG GCT TTC TCT GGT GTT 120

GCC GCA ACT GAR TCA GCT TAT TTG GCT GTG CGT AAT CAA TCA TTG GAT CTT GCT GRA CAA 180

GAA TTA GTC GAT TGT GCT TCC CAA CAC GGT TGT CAT GGT GAT ACC ATT CCA CAG GGT ATT 240 :

GAA TAC ATC CAA CAT AAT GGT GTC GTC CAA GAR AGC TAC TAT CGA TAC GTT GCA GAA GAA 300

CAA TCA TGC CGA CGA CCR AAT GCA GAT CGT TTC GGT ATC TCA AAC TAT TGC CAA ATT TAC 360

CCA CCAR AAT GTA ABC AAR ATT CAG GAA GCT TTG GCT CAR ACC CAC AGC GCT ATT GCC GTC 420

ATT ATT GGC ATC ARA GAT TTA GAC GCA TTC CGT CAT TAT GAT GGC GAA ACA ATC ATT CAA 480

CAG GAT RAT GGT TAC CAA ACC AAC TAT CAC GCT GTC AAC ATT GTT GGT TAC AGT AAC GCA 540

CAA GGT GTC GAT TAT TGG ATC GTA CGA BAC AGT TTT GAT ACC AAT TGG GGT GAT AAT GGT 600

TAC GGT TAT TTT GCT GCC AAC ATC GAT TTG ATG ATG ATT GAA GAA TAT CCA TAT GTT GTC 660

ATT CTC 666

EXAMPLE 8

Mutated recombinant grass allergens (Phl p 5) with improved safety for specific allergy vaccination

In this example the application of the concept of the current invention on grass pollen allergens is exemplified by one allergen, Phl p 5. Manipulation of other grass pollen allergens may be performed by equivalent procedures.

WQ 02/40676 PCT/DK01/00764

Design of mutated recombinant Phl p 5 molecules

SEQ ID NO. 5 shows the nucleotide and deduced amino acid sequence of the Phl p 5.0103 clone, which is a wild-type isoform.

SEQ ID NO. 5: Nucleotide and deduced amino acid sequence of Phl p 5.0103. gccgatctcggttacggeccecgecaceccagetgeccececggecgecggetacacceecgee 60 ) A DL GY G PAT PAA AP AMA AGYT PA 20 accccecgecgcecccggeccggagcggagecagcaggtaaggecgacgaccgaggagecagaag 120

T P AAP A G A E P A G X A T T EE Q K 40 ctgatcgagaagatcaacgeccggcttcaaggcggecettggecgetgecgecggegteccg 180

L I E K I N A GF KA AL A A A A G V P 60 ccagcggacaagtacaggacgttegtcgcaacctteggegeggectccaacaaggectte 240

P AD K Y RTF FV ATF GAA S NK AF 80 gcggagggcctctcgggecgagcccaagggegeccgecgaateccageteccaaggeegegcete 300

A EG L S GE PK GAA AES Ss S KA ATL 100 acctccaagctcgacgecgectacaagctegectacaagacagccgagggegegacgect 360

T S K L D A AY KL AY KT AEG A T P 120 gaggccaagtacgacgcctacgtcgeccacecgtaagegaggcgctecgeatcategecgge 420

E A KY DAY V ATV Ss EAL RI I A GG 140 accctecgaggtccacgeccgtcaageecgeggecgaggaggtcaaggtcateccegeegge 480

T L EV HA V KP A A EE V KV I P A G 160 gagctgcaggtcatcgagaaggtcgacgccgecttcaaggtcecgetgecacegecgecaac 540

EL Q VI E KV DAA ATF KV A ATA AN 180 gccgcccccgecaacgacaagttcacegtcttcgaggecgecttcaacgacgecatcaag 600

A AP A NDI KZ FTV F EAA ATFUNDA ATI K 200 gcgagcacgggcggcgectacgagagctacaagttcatecceccgeactggaggecgeegte 660

A 5 T G GA Y ES Y KF I PA L EA AV 220 aagcaggcctacgeccgecaccgtegecacegegecggaggtcaagtacactgtetttgag 720

K QQ AY A A T V A T A P E V K Y TT V F BE 240 accgcactgaaaaaggccatcaccgccatgtccgaagcacagaaggctgccaageccgcee 780

T AL K K A I TA MS E A Q KA A K P A 260 gccgetgccaccgccaccgeaaccgecgecgttggecgeggeccaccggegecgecaccgee 840

A AA TATA ATA AAV GA AT G6 A AT A 280 gctactggtggctacaaagte ‘ 86l

A T G G ¥Y K Vv 40 i

Fig. 38 shows a sequence alignment performed at the

ExPaSy Molecular Biology Server (http: //www.expasy.ch/) using the ClustalW algorithm on a BLAST search using the

Phl p 5.0103 amino acid sequence shown in SEQ ID NO. 5 as input sequence. The alignment includes group 5 allergen sequences from grass species, i.e. Phl p 5, Poa p 5, Lol p 5 Hol 1 5, Pha a 5, Hor v 9 and Hor v 5. In Fig. 38 amino acid residues identical to amino acids in the same position in the Phl p 5.0103 protein sequence are highlighted using black letters on grey background. Non- identical amino acids are printed in black on a white background. surface structure images

Amino acid sequences representing the grass pollen group 5 allergens are similar to a certain degree and some of the molecular surface is conserved (grey-coloured zones), see Fig. 39. Fig. 39 shows surface contours viewed from 4 different angles when superimposing the Phl p 5.0103 protein sequence on to a Phl p 5 molecular structure model. The structure model encompass the molecule in two halves, Model A (amino acid 34-142) shown in Fig. 393, and Model B (amino acid 149-259) shown in Fig. 39B.

Highly solvent exposed amino acid spatially distributed over the entire surface within distances in the range of 25-30 A are selected for mutation. In the sections below, the following information is given: A list of amino acids considered to be appropriate for mutation (A), a list of the mutants designed (B) and the DNA sequences representing the mutants designed (C). Fig. 40 A and B shows surface contours of mutant number 1 Model A and

Model B, respectively, as an example. Grey colour indicates conserved amino acid residues. Black colour indicates amino acid residues selected for mutation.

A: List of amino acids selected for mutation

I45, R66, E133, R136, I137, D186, F188, K211, P214, Q222,

P232, L243, 0254 ‘

B. List of mutants designed

Mutant 1:

I45K, E1338, F188I, Q222K, L243E, Q254K

Mutant 2:

R66N, E133S, F188I, (Q222K, L243E, Q254K

Mutant 3:

I45K, R136S, F188I, Q222K, L243E, Q254K

Mutant 4:

I45K, TI137K, F188I, Q222K, L243E, Q254K

Mutant 5: 145K, E1335, D186H, Q222K, L243E, (Q254K

Mutant 6:

I45K, E133S, Q222K, P232G, L243E, Q254K

Mutant 7: ’

I45K, E133S, F188I, P214G, L243E, Q254K

Mutant 8: 145K, E133S, F188I, K211N, L243E, Q254K

Mutant 9: } }

R66N, R136S, F188I, Q222K, L243E, Q254K :

Mutant 10:

R66N, I137K, F188I, Q222K, L243E, Q254K

Mutant 11: 145K, E133S, D186H, P214G, L243E, Q254K

Mutant 12: 145K, E133s, D186H, K211N, L243E, Q254K

Mutant 13: 145K, E133S, P214G, P232G, L243E, Q254K

Mutant 14:

I45K, E1335, K211N, P232G, L243E, Q254K

C. Nucleotide sequence of mutants

Mutant 1: 145K, E133S, F188I, Q222K, L243E, Q254K: gccgatcteggttacggeececgecacceccagetgececcggecgecggctacaccccegece 60 acccccgeegeeccggecggagecggagecagcaggtaaggecgacgaccgaggagcagaag 120 ctgatcgagaagARRaacgccggcttcaaggeggecttggecgetgeecgecggegteccy 180 ccagcggacaagtacaggacgttecgtecgcaacctteggegecggectccaacaaggecette 240 gcggagggcctctegggegageccaagggegecgecgaatccagetccaaggecgegete 300 acctccaagctcgacgecgectacaagctegectacaagacagecgagggegcegacgect 360 gaggccaagtacgacgecctacgtegecaccgtaageAGCgegetececgeatcatecgecgge 420 accctecgaggtccacgecgtcaagececgecggecgaggaggtcaaggtcateccegeegge 480 gagctgcaggtcatcgagaaggtcgacgccgecttcaaggtcgetgecacecgecgccaac 540 gccgeccccgecaacgacaagATTaccgtcttcgaggecgecttcaacgacgecatcaag 600 gcgagcacgggcggcgectacgagagctacaagttcatcccecgeectggaggecgeegte 660 aagAPAgcctacgccgccaccgtegecacegegecggaggtcaagtacactgtetttgag 720 accgcaGAAaaaaaggccatcaccgccatgtccgaagcahABAaaggctgeccaagcecgec 780 gccgetgceccacecgccaccgcaaccgecgecgttggegeggeccaccggegecgeccaccgec 840 gctactggtggctacaaagte 861

Mutant 2:

R66N, E1335, F188I, Q222K, L243E, QZ54K: 40 gcecgatcteggttacggecceecgecacecccagetgececggeegecggetacacceecgee 60 acccccgecgecceggecggageggagecagecaggtaaggegacgacegaggageagaag 120 ctgatcgagaagatcaacgceggettcaaggeggecttggeegetgecgecggegtecey 180 ccagcggacaagtachACacgttcgtegecaacctteggegeggectecaacaaggectte 240 45 gcggagggectetegggegageccaagggegecgecgaatccagetecaaggeegegete 300 acctccaagctcgacgecgectacaagctcgectacaagacagecgagggegegacgect 360 gaggccaagtacgacgcctacgtcgecaccgtaageAGCgegetccgecatcategeegge 420 accctcgaggtccacgccgtcaageecgeggccgaggaggtcaaggtcatceccgeegge 480 gagctgcaggtcatcgagaaggtegacgecgecttcaaggtegetgecaccgeecgecaac 540 gccgccccegccaacgacaaghTTaccgtcttegaggeecgecttcaacgacgecatcaag 600 gcgagcacgggcggcgectacgagagetacaagttcatecccgeectggaggeegecgte 660 aagAAAgcctacgecgccacegtcgecaccgegecggaggtcaagtacactgtetttgag 720 accgcaGAAhaaaaaggccatcaccgecatgtccgaagcahRRaaggctgecaagecegec 780 gccgetgecacegecaccgcaaccgecgeegttggegeggecaccggegecgecaccgec 840 gctactggtggctacaaagtc 861

Mutant 3: 145K, R136S, F188I, Q222K, L243E, Q254K: gccgatcteggttacggeccegecaccecagetgecceggecgecggctacaceccegec 60 accccegecgecceggccggageggagecagecaggtaaggegacgaccgaggageagaag 120 ctgatcgagaagARAaacgccggct tcaaggeggecttggecgetgecgecggegtecey 180 ccagcggacaagtacaggacgttegtegeaaccttceggegeggectecaacaaggectte 240 gcggagggoctctcgggegageccaagggegecgecgaatccagetecaaggecgegete 300 acctccaagctcgacgccgectacaagetcgectacaagacagecgagggcgegacgect 360 gaggccaagtacgacgcctacgtogecaccgtaagegaggegctchGCateategeegge 420 accctcgaggtccacgccgtcaageeccgeggecgaggaggtcaaggtcatececgeegge 480 gagctgcaggtcatcgagaaggtcgacgecgecttcaaggtegetgecacegeegecaac 540 gcegeccccgccaacgacaagATTaccgtcttegaggecgectteaacgacgecateaag 600 gcgagcacgggcggcgectacgagagetacaagttcatccecgecctggaggecgecgte 660 aagAAAgcctacgccgccaccgtcgccaccgegecggaggtcaagtacactgtctttgag 720 accgcaGRAaaaaaggccatcaccgccatgtccgaagcaARhaaggctgccaagecegee 780 gccgetgeccaccgecaccgecaaccgecgeegttggegeggecaccggegecgecacegec 840 gctactggtggctacaaagte gel

Mutant 4: 145K, I137K, F188I, Q222K, L243E, Q254K: gccgatctcggttacggecccgecaccecagetgocceggecgceggetacaceccegec 60 acccccgecgecceggccggageggagecageaggtaaggegacgacegaggageagaagd 120 ctgatcgagaagAARaacgccggcettcaaggeggecttggecgetgeegecggegtecey 180 ccagcggacaagtacaggacgttcegtcogcaacctteggegeggectccaacaaggectte 240 gcggagggectcetegggegagececaagggogecgecgaatccagetecaaggeegegete 300 40 acctccaagctcgacgccgectacaagectcgecctacaagacageccgagggegegacgect 360 gaggccaagtacgacgectacgtcgecaccgtaagegaggegeteccgechhhatcegeegge 420 accctcgaggtccacgccgtcaageccgeggecgaggaggtcaaggtcateccegeecgge 480 gagctgcaggtcatcgagaaggtcgacgccgecttcaaggtcgetgecaccgecgeeaac 540 gcecgeccccgeccaacgacaaghTTaccgtcttegaggecgecttcaacgacgecatcaag 600 45 gcgagcacgggcggegectacgagagotacaagtteateccegeectggaggecgecgte 660 aaghAAgcctacgccgccaccgtegecaccgcgacggaggtcaagtacactgtetttgag 720 accgcaGAhRaaaaaggccatcaccgeccatgteccgaagecahhhaaggctgccaageccgecr 780 gcegectgccaccgacaccgcaaccgecgecgttggegeggecaccggegecgeeacegeo 840 gctactggtggctacaaagte 861 50

Mutant 5:

X

145K, E1333, D186H, Q222K, L243E, Q254K: 55 gcegatcteggttacggecccgecaccccagetgecccggeacgecggetacaceeecegee 60 acccccgecgeccecggccggagecggageccagecaggtaaggcgacgaccgaggageagaag 120 ctgatcgagaagAAAaacgccggcttcaaggecggecttggecgectgeecgecggegteccyg 180 ccagcggacaagtacaggacgttcgtcgcaacctteggegeggcetecaacaaggectte 240 gcggagggcectctcgggcgagecccaagggecgecgecgaatecagecteccaaggecgegete 300 acctccaagctcgacgccgectacaagetecgectacaagacagecegagggegegacgect 360 gaggccaagtacgacgcctacgtcgccaccgtaagcAGCgegetecgecatcatcecgeecgge 420 accctcgaggtccacgecgtcaageccecgeggecgaggaggtcaaggtcatecececgeegge 480 gagctgcaggtcatcgagaaggtcgacgccgecttcaaggtcgetgeccaccgeecgecaac 540 gccgccecccgecaacCATaagttecacegteottegaggecgecttcaacgacgececatcaag 600 gcgagcacgggcggecgectacgagagectacaagttcatccececgeectggaggeecgecgte 660 aagARAgcctacgecgceccaccgtecgeccaccgegecggaggtcaagtacactgtetttgag 720 accgcaGAAaaaaaggccatcaccgecatgtccgaagcaAhhaaggetgccaageecgee 780 gcegcetgecaccgecaccgecaaccgecgecgttggegeggeccaccggegecgeccacegec 840 gctactggtggctacaaagte 861

Mutant 6:

I45K, E1I33S, Q222K, P232G, L243E, Q254K: gccgatctcggttacggecaccgeccacceccagetgecceggecgeecggectacaccecegee 60 acccccgecgecceggecggagecggageccagcaggtaaggcgacgaccgaggagcagaag 120 } ctgatcgagaagAARaacgccggettcaaggecggecttggecgectgecgecggecgteccy 180 ’ ccagcggacaagtacaggacgttegtcgecaaccttecggecgeggectcraacaaggectte 240 gcggagggcectctecgggecgagecccaagggegecgecgaatccagctecaaggecgegete 300 acctccaagctcgacgccgecctacaagctegecctacaagacagccgagggegegacgect 360 gaggccaagtacgacgcctacgtecgeccaccgtaagecAGCgegetececgecatcatecgecgge 420 accctegaggteccacgecgtcaagecegeggecgaggaggtcaaggtcatececegecgge 480 gagctgcaggtcatcgagaaggtecgacgeccgecttcaaggtecgectgecacecgecgecaac 540 gccgcecccccgcecaacgacaagttcaccgtettecgaggecegecttcaacgacgecatcaag 600 gcgagcacgggcggcgecctacgagagctacaagttcateccececgecctggaggecgeegte 660 aagAAAgcctacgeccgeccaccgtecgecaccgecgGGCgaggtcaagtacactgtetttgag 720 accgcaGARaaaaaggccatcaccgcecatgtecgaagecaAAAaaggctgeccaagecegee 780 gccgetgceccaccgecaccgcaaccgecgecgttggegeggecaccggegecgeccaccgec 840 gctactggtggctacaaagtc 861

Mutant 7:

I45K, E133S, F188I, P214G, L243E, Q254K: 40 gccgatctcggttacggeccecgeccacceccagetgeocceggecgecggctacaceececegee 60 acccccgeegecceggeccggageggagccagcaggtaaggecgacgaccgaggagcagaag 120 ctgatcgagaagAhAaacdccggcttcaaggeggecttggecgetgecgecggegteceg 180 ccagcggacaagtacaggacgttcgtcecgecaaccttcggecgeggecteccaacaaggectte 240 gcggagggcctctecgggecgageccaagggegecgecgaatccagcteccaaggecgegete 300 45 acctccaagctcgacgccgcctacaagctcgectacaagacageccgagggcgcgacgect 360 gaggccaagtacgacgcctacgtcgccaccgtaagcAGCgecgetccgecatecategecgge 420 accctecgaggtceccacgecegtcaagececgeggeocgaggaggtcaaggtcateceegecgge 480 gagctgcaggtcatcgagaaggtecgacgccgecttcaaggtecgectgecaccgeegecaac 540 gccgceecccgeccaacgacaagATTaccgtcttecgaggecgecttcaacgacgecatcaag 600 50 gcgagcacgggcggcgcctacgagagctacaagttcatcGGCgecetggaggcegecgte 660 aagcaggcctacgccgecaccgtegecaccegegecggaggtcaagtacactgtetttgag 720 accgcaGhAhaaaaaggccatcaccgeccatgtccgaagcahAhaaggctgccaageecgee 780 gccgetgecaccgecaccgcaacegecgecgttggegeggecaccggegecgecaccgec 840 gctactggtggctacaaagte 861 55

Mutant 8:

145K, E133S, F188I, K211N, L243E, Q254K: gccgatctcggttacggecccegecaccccagetgecceggecgecggetacacceecgee 60 accccegecgeeccggccggageggagccageaggtaaggegacgaccgaggageagaag 120 ctgatcgagaaghAAaacgccggcttcaaggeggecttggecgetgeegecggegteccy 180 ccagcggacaagtacaggacgttcgtegeaaccttcggegeggectecaacaaggecttc 240 gcggagggectctcgggegageccaagggegecgcecgaatecagetccaaggeegegete 300 acctccaagctcgacgccgectacaagctegectacaagacagecgagggegegacgect 360 gaggccaagtacgacgcctacgtecgecaccgtaagcAGCgegetecgeatcategeegge 420 accctcgaggtccacgccgtcaagececgeggecgaggaggtcaaggteateccegeegge 480 gagctgcaggtcatcgagaaggtcgacgecgecttcaaggtegetgecaccgecgecaac 540 gccgeccccgecaacgacaaghTTaccgtcttcgaggoegecttcaacgacgecatcaag 600 gcgagcacgggcggcegcctacgagagctachACttcatecccgecetggaggecgecgte 660 aagcaggcctacgccgccaccgtcgecaccgegecggaggtcaagtacactgtetttgag 720 accgcaGARaaaaaggccatcaccgccatgtccgaagcahRAaaggcetgecaageccegec 780 gcegetgecaccgecaccgeaacegecgecgttggegeggecaccggegecgecacegece 840 gctactggtggctacaaagtc 861

Mutant 9:

R66N, R136S, F188I, 0222K, L243E, Q254K: gccgatctceggttacggecccgecacecccagetgecceggecgecggetacaceececegee 60 acccecgeegecceggecggageggagecagcaggtaaggcgacgaccgaggageagaag 120 ctgatcgagaagatcaacgceggcttcaaggeggecttggecgetgecgeeggegteceg 180 ccagcggacaagtacPACacgttegtcgcaaccttcggegeggectccaacaaggectte 240 gcggagggectctcggygcgageecaagggegecgecgaatecagetccaaggecgegete 300 acctceaageotcgacgccgectacaagetegoctacaagacageegagggegegacgect 360 gaggccaagtacgacgcctacgtcgccaccgtaagegaggegctcAGCateategecgge 420 accctegaggtccacgccgtcaageccgeggecgaggaggtcaaggtcatceccgecgge 480 gagctgcaggtcatcgagaaggtcgacgccgecttecaaggtegetgecaccgecgeeaac 540 gccgcecccgecaacgacaagATTaccgtcttegaggoegecttcaacgacgecatcaag 600 gcgagcacgggcggegectacgagagetacaagttcateccegeectggaggecgeegte 660 aaghAAgcctacgccgccaccgtecgocaccgegecggaggtcaagtacactgtctttgag 720 accgcaGARaaaaaggccatcaccgccatgtccgaagcahARaaggetgecaageecgec 780 gccgetgecaccgecaccgcaaccgecgeegttggegeggecaccggegecgecaceygea 840 gctactggtggctacaaagte 861 40 Mutant 10:

R66N, I137K, F188I, Q222K, L243E, Q254K: gccgatcteggttacggecccegecaccccagetgecceggecgeeggcetacacceccegee 60 45 acccccgecgceccggeccggageggagecagcaggtaaggegacgaccgaggageagaag 120 ctgatcgagaagatcaacgccggcttcaaggeggecttggecgetgeegecggegteeey 180 ccagcggacaagtacAACacgttcgtcgcaacctteggegeggectecaacaaggectte 240 geggagggectctcgggegageccaagggcgecgecgaatecagetccaaggecgegete 300 acctccaagctcgacgccgectacaagetcgectacaagacagecgagggegegacgect 360 50 gaggccaagtacgacgcctacgtcgeccacegtaagegaggegctccgcAhhategecgge 420 accctcgaggtccacgcecgtcaagecegeggecgaggaggtcaaggtcateceegecgge 480 gagctgcaggtcatcgagaaggtcgacgecgecttcaaggtegetgecacegecgecaac 540 gccgceccecgccaacgacaagATTacegtcttcgaggecgecttcaacgacgecatcaag 600 gcgagcacgggcggcgectacgagagetacaagttcateccegecctggaggecgecgte 660 55 aagAMAgcctacgccgccacegtegeeacegegecggaggtcaagtacactgtetttgag 720 accgcaGAAaaaaaggccatcaccgccatgtccgaagcahhrAaaggetgecaageccgee 780 gccgetgccaccgemaccgcaaccgecgecgttggecgeggecaccggegecgecacecgec 840 gctactggtggctacaaagtc 861

Mutant 11:

I45K, E1335, D186H, P214G, L243E, Q254K: gccgatctecggttacggececgecaccccagetgecceggecgecggctacaceceecgee 60 acccecegecgceccggeceggagcggagecagcaggtaaggegacgaccgaggagceagaag 120 ctgatcgagaagARRaacgccggettcaaggecggecttggecgetgecgecggegteceg 180 ccagcggacaagtacaggacgttcgtegecaacctteggegeggecteccaacaaggectte 240 gecggagggcctetegggcgageccaagggegecgecgaatccageteccaaggecgegete 300 acctccaagctcgacgeecgectacaagctecgectacaagacagecgagggcgegacgect 360 gaggccaagtacgacgectacgtegececacegtaagecAGCgegeteccgeatcategecgge 420 accctcgaggtccacgcegtcaagecegecggeccgaggaggtcaaggtcatccecegececgge 480 gagctgcaggtcatcgagaaggtcgacgccgccttcaaggtcgetgccaccgecgecaac 540 gccgcecccgecaacCATaagttcacegtettecgaggecgecttcaacgacgecatcaag 600 gcgagcacgggcggecgcctacgagagetacaagttcateGGCgecectggaggeccgecgte 660 aagcaggcctacgccgeccaccgtcgeccacegegecggaggtcaagtacactgtetttgag 720 accgcaGAhaaaaaggccatcaccgcecatgteccgaagcaAAhaaggctgeccaagecegee 780 gccgetgecaccgceccaccgcaaccgeecgeegttggegeggecacecggecgecgecaccgcec 840 gctactggtggctacaaagte 86l

Mutant 12:

I45K, E133S, D186H, K211N, L243E, Q254K: gccgatctecggttacgaccccgecaccccagectgecececggeccgeceggectacacceceegec 60 acccececgccgecccggeceggageggagecagcaggtaaggegacgaccgaggagcagaag 120 ctgatcgagaagARAaacgceggcttcaaggeggecttggeccgetgeecgeeggegtceeg 180 ccagcggacaagtacaggacgttecgtecgecaaccttecggegeggecteccaacaaggectte 240 gcggagggectctecgggcgageccaagggcgecgecgaatccagetccaaggeecgegete 300 acctccaagctcgacgccgecctacaagctcgectacaagacageccgagggcgcgacgect 360 gaggccaagtacgacgcctacgtcgccaccgtaagcAGCgegectceccgecatcategecgge 420 accctcgaggtccacgecgtcaageccegeggecgaggaggtcaaggtcatececeegecgge 480 gagctgcaggtcatcgagaaggtcgacgeccgecttcaaggtcgectgcecacecgecgecaac 540 gccgcecceccgecaacCATaagttcacegtcttegaggeecgecttcaacgacgeccatcaag 600 gcgagcacgggcggcgectacgagagctacAACttcatcecegecectggaggeecgeegte 660 aagcaggcctacgccgccacegtcgecaccgecgecggaggtcaagtacactgtetttgag 720 40 accgcaGARaaaaaggccatcaccgccatgtccgaagcaPRAaaggctgeccaagececgcec 780 gccgetgeccaccgccaccgecaacegeecgecgttggegeggeccaceggegecgecaccgec 840 gctactggtggctacaaagte 861

Mutant 13: 45 145K, E133S, P214G, P232G, L243E, Q254K: gccgatcteggttacggececgecaccccagetgecccggecgecggctacacceeegece 60 acccocgecgecccggecggagecggageccagecaggtaaggecgacgaccgaggagcecagaag 120 50 ctgatcgagaagAARaacgccggcttcaaggeggecttggecgetgecgecggegticecy 180 ccagcggacaagtacaggacgttcgtcgecaacctteggegeggecteccaacaaggectte 240 gcggagggectctegggegageccaagggegecgecgaatccagetccaaggecgegete 300 acctccaagctcgacgccgectacaagectcgectacaagacagccgagggegegacgect 360 gaggccaagtacgacgcctacgtcgeccaccgtaagcAGCgegctecegeatcategeegge 42Q 55 accctegaggtecacgeccgtcaageccgeggecgaggaggtcaaggtcateccegecgge 480 gagctgcaggtcatcgagaaggtcgacgccgecttcaaggtegetgeccaccgeegecaac 540 gccgcccccgecaacgacaagttcaccgtcttecgaggecgecttcaacgacgecatcaag 600 gcgagcacgggeggegectacgagagetacaagttcatcGGCgecctggaggeegecgte 660 aagcaggcctacgccgccaccgtcgecaccgegGGCgaggtcaagtacactgtettgag 720 accgcaGAAaaaaaggccatcaccgccatgteccgaagecaAhhaaggctgececaageeegec 780 gccgctgccaccgecaccgeaaccgecgecgttggegeggecaccggegecgecaccgec 840

Mutant 14:

I45K, E133S, K211N, P232G, L243E, 0254K: gccgatctcggttacggccecgecaccecagectgecceggecgecggectacaccecegec 60 accceccgecgecccggecggageggagecagcaggtaaggegacgaccgaggageagaag 120 ctgatcgagaagAPAaacgccggettcaaggeggecttggecgetgcegeeggegteccg 180 ccagcggacaagtacaggacgttcgtcgeaacctteggegeggectecaacaaggecttc 240 gcggagggectctcgggegageccaagggcgecgecgaatecageteccaaggeegegete 300 acctccaagctcgacgccgectacaagctegoctacaagacagecgagggegegacgect 360 gaggccaagtacgacgcctacgtcgecaccgtaagcAGCgegetecgeateategeegge 420 accctcgaggtccacgccgtcaageccgeggccgaggaggtcaaggtecateccegeegge 480 gagctgcaggtcatcgagaaggtegacgecegecttcaaggtegetgecacegecgeeaac 540 gccgcccecgecaacgacaagttcaccgtettegaggeegecttcaacgacgocatcaag 600 gcgagcacgggeggegectacgagagetachACttcatcccegecctggaggecgeegte 660 aagcaggcctacgocgecacegtcgecacegegGGCgaggtcaagtacactgtetitgag 720 . accgcaGAhaaaaaggccatcaccgccatgtccgaagcaAhhaaggectgccaageccgec 780 gccgctgecaccgecaccgecaaccgecgecgttggegeggecaceggegecgecaccgee 840 gctactggtggctacaaagtc 861 }

EXAMPLE 9

T-cell reactivity of recombinant and mutant Bet v 1:

Purpose:

To investigate an in vitro T-cell response to the mutated allergens in terms of proliferation and cytokine production.

Methods: 40

PBL (Peripheral blood lymphocytes) from allergic patients were used in the following investigation. :

Eight bet v 1 specific T-cell lines were established from 45 the PBL with naturally purified bet v 1 in order to . ; }

sustain the variety of bet v 1 isoforms the T-cells are presented to, as described in a previously published protocecl (26).

Ten PBL and eight T-cell lines were stimulated with birch extract (Bet Vv), 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 vv 1 {described elsewhere): 2595, 2628, 2637, 2744, 2773. The 2637 mutant was later found to be partly unfolded and will not be discussed.

In brief: In a round-bottomed 96 well plate PBL were added in 2 x 10° per well. The different birch samples were added in three different concentrations in quadroplicates and allowed to grow for 6 days. At day 6 cell half of volume (100 ul) from each well with the highest concentration of birch were harvested for cytokine production. Radioactive labelled thymidine was added to the wells. Next day (day 7) the cells were harvested on a filter. Scintilation fluid was added to the filter and the radioactivity was measured in a scintilation counter.

Likewise in a 96 well round-bottomed 96 well plate T- cells were added in 3x10* T-cells per well and stimulated with irradiated autologous PBL (1x10° 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.

Results:

Ten PBL cultures showed specific stimulation to birch. In general proliferation of the PBL to the different birch samples were similar, although variations could be seen.

In 3 PBL, nBet v 1 stimulated proliferation better than rBet v 1 and the mutants. The mutant birch samples stimulated PBL almost identical to rBet v 1 (Fig. 41).

Fig. 41 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) i5 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 Thl/Th2 shift were apparent (Fig. 42-44). Figure 42 shows a patient with a ThO profile, Figure 43 a Thi profile and Figure 44 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.

Eight 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-5 and IFN-gamma ratio (Th2 > 5, 5 > ThO > 0.2, 0.2 > Thl). Three T-cell lines were secreting Thl cytokines and one T-cell line was secreting

Th2 cytokines. The IL-5/IFN-gamma ratio was not affected by the different birch samples.

Conclusion:

All PBL cultures and 7/8 T-cell lines that showed specific stimulation to nBet v 1 did also respond to rBet v 1 and the mutants. These data suggests that for T-cell stimulation a single isoform of Bet v 1 or these 4 mutants can substitute for the mixture of individual isoforms found in the natural allergen preparations.

Thus, vaccines based on recombinant allergens or these 4 mutants will address the existing Bet v 1 specific T-cell population.

EXAMPLE 10

Induction of Bet v 1 specific IgG antibodies and blocking antibodies following immunization with recombinant and mutant Bet v 1 proteins:

In this section the term "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.

The ability of recombinant Bet vl 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 vl 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.

Specific 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 vil 2227 wild type protein or the four mutant proteins induced a strong r Bet wv 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

To assess the induction of blocking antibodies, serum samples from birch pollen allergic patients were incubated with paramagnetic beads*® coated with a monoclonal mouse anti-human IgE antibody. After incubation, the beads were washed and resuspended in buffer or diluted samples (1:100) of mouse serum from un- immunized mice (control) or mice immunized as described above. Biotinylated r Bet v 1 was then added to this mixture of beads and mouse serum antibodies. After incubation, the beads were washed and bound biotinylated rBet v 1 was detected using acridinium labeled streptavidine. Incubation of beads with serum from un- immunized mice did not change the binding of r Bet v 1 to the beads. In contrast, incubation of the beads with serum from mice immunized with the recombinant Bet vl 2227 wild type protein or the four mutant proteins significantly reduced binding of r Bet v 1 to the beads demonstrating the presence of Bet v 1 specific blocking antibodies in the serum samples.

Thus, at day 63 one Or more serum samples from all high dose (10 ug/dose) immunization groups were able to reduce binding of r Bet vl to the beads with more than 80%. These findings demonstrate that the four mutated proteins are able to induce antibodies that can act as Bet v 1 specific blocking antibodies.

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Claims (89)

1. A recombinant allergen that is a mutant of a naturally occurring allergen, wherein the mutant allergen has at least four mutations, which each reduce the specific IgE binding capability of the mutated allergen as compared to the IgE binding capability of the said naturally occurring allergen, wherein each mutation is a substitution of one surface-exposed amino acid residue with another residue, which does not occur in the same position in the amino acid sequence of any known homologous protein within the taxonomic species from which said naturally occurring allergen originates, characterized in that at least four mutations (primary mutations) are mutually spaced by at least 15 A and wherein said primary mutations are placed in such a manner that at least one circular surface region with a area of 800 A? comprises no mutation.

2. A recombinant allergen according to claim 1, wherein the primary mutations are spaced 20 A.

3. A recombinant allergen according to claim 1, wherein the primary mutations are spaced 25 A.

4. A recombinant allergen according to claim 1, wherein the primary mutations are spaced 30 A.

5. A recombinant allergen according to claim 1 or 2 which, in addition to the at least four primary mutations, comprises at least one further mutation (secondary mutation) that is spaced less than 15 A from any primary mutation, wherein each secondary mutation reduces the specific IgE binding capability of the mutated allergen as compared to the binding capability of the said naturally occurring allergen, wherein each secondary mutation is a substitution of one surface-exposed amino acid residue with another residue, which does not occur in the same position in the amino acid sequence of any known homologous protein within the taxonomic species from which said AMENDED SHEET naturally occurring allergen originates, wherein the secondary mutations are placed outside the said circular region.

6. A recombinant allergen according to any of claims 1-5, wherein at least one of the surface-exposed amino acids to be substituted in the naturally occurring allergen has a solvent accessibility of above 20 %.

7. A recombinant allergen according to any of claims 1-5, wherein at least one of the surface-exposed amino acids to be substituted in the naturally occurring allergen has a solvent accessibility of above 30 %.

8. A recombinant allergen according to any of claims 1-5, wherein at least one of the surface-exposed amino acids to be substituted in the naturally occurring allergen has a solvent accessibility of above 40 %.

9. A recombinant allergen according to any of claims 1-5, wherein at least one of the surface-exposed amino acids to be substituted in the naturally occurring allergen has a solvent accessibility of above 50 %.

10. A recombinant allergen according to any of claims 1-6, wherein at least one of the surface-exposed amino acids to be substituted in the naturally occurring allergen is conserved with more than 70 % identity in all known homologous proteins within the species from which said naturally occurring allergen originates.

11. A recombinant allergen according to any of claims 1-6, wherein at least one of the surface-exposed amino acids to be substituted in the naturally occurring allergen is conserved with more than 80 % identity in all known homologous proteins within the species from which said naturally occurring allergen originates.

12. A recombinant allergen according to any of claims 1-6, wherein at least one of the surface-exposed amino acids to be substituted in the naturally AMENDED SHEET j 32 occurring allergen is conserved with more than 90 % identity in all known homologous proteins within the species from which said naturally occurring allergen originates.

13. A recombinant allergen according to any of claims 1-12, which essentially has the same a-carbon backbone tertiary structure as said naturally occurring allergen.

14. A recombinant allergen according to any of claims 1-13, wherein each amino acid residue 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.

15. A recombinant allergen according to any of claims 1-13, wherein each amino acid residue 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 subfamily.

16. A recombinant allergen according to any of claims 1-13, wherein each amino acid residue 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 family.

17. A recombinant allergen according to any of claims 1-13, wherein each amino acid residue 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 superfamily.

18. A recombinant allergen according to any of claims 1-13, wherein each amino acid residue 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 legion. AMENDED SHEET

A

19. A recombinant allergen according to any of claims 1-13, wherein each amino acid residue 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 suborder.

20. A recombinant allergen according to any of claims 1-13, wherein each amino acid residue 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 order form which the said allergen originates.

21. A recombinant allergen according to any of claims 1-14, characterised in that the specific IgE binding to the mutated allergen is reduced by at least 5%.

22. A recombinant allergen according to any of claims 1-14, characterised in that the specific IgE binding to the mutated allergen is reduced by at least

10%.

23. A recombinant allergen according to claim 13, characterised in that when comparing the a-carbon backbone tertiary structures of the mutant and the naturally occurring allergen molecules, the average root mean square deviation of the atomic coordinates is below 2A.

24. A recombinant allergen according to any of claim 1-23, characterised in that said circular surface region comprises atoms of 15-25 amino acid residues.

25. A recombinant allergen according to any one of claims 1-24, characterised in that the surface-exposed amino acid residues are ranked with respect to solvent accessibility, and that one or more amino acids among the more solvent accessible ones are substituted. AMENDED SHEET

/3¥ &

26. A recombinant allergen according to any one of claims 1-25, 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 amino acids among the more conserved ones are substituted.

27. A recombinant allergen according to any of claims 1-26, wherein the mutant allergen is a non-naturally occurring allergen.

28. A recombinant allergen according to any of claims 1-27 comprising from 5 to 20 primary mutations.

29. A recombinant allergen according to any of claims 1-27 comprising from 16 6 to 15 primary mutations.

30. A recombinant allergen according to any of claims 1-27 comprising from 7 to 12 primary mutations.

31. A recombinant allergen according to any of claims 1-27 comprising from 8 to 10 primary mutations.

32. A recombinant allergen according to any one of claims 1-31 characterised in that the mutant allergen comprises from 1 to 4 secondary mutations per primary mutation.

33. A recombinant allergen according to any one of claims 1-32, characterised in that one or more of the substitutions is carried out by site- directed mutagenesis.

34. A recombinant allergen according to any one of claims 1-33, characterised in that one or more of the substitutions is carried out by DNA shuffling. AMENDED SHEET po

35. A recombinant allergen according to any one of claims 1-34 characterised in that it is a mutant of an inhalation allergen.

36. A recombinant allergen according to claim 35, characterised in that it is a mutant of a pollen allergen.

37. A recombinant allergen according to claim 36 characterised in that it is a mutant of a pollen allergen originating from the taxonomic order of Fagales, Oleales or Pinales.

38. A recombinant allergen according to claim 37, characterised in that it is a mutant of Bet v 1. 16

39. A recombinant allergen according to claim 38, characterised in that one or more of the substitutions is selected from the group consisting of V2, D72, E87, K-129, E-60, N-47, K-65, P-108, N-159, D-93, K-123, K-32, D- 125, R-145, D-109, E-127, Q-36, E-131, L-152, E-6, E-96, D-156, P-63, H- 76, E-8, K-134, E-45, T-10, V-12, K-20, S-155, H-126, P-50, N-78, K-119, V- 2, L-24, E42, N-4, A-153, 1-44, E-138, G-61, A-130, R-70, N-28, P-35, S- 149, K-103, Y-150, H-154, N-43, A-106, K-115, P-14, Y-5, K-137, E-141, E- 87 and E-73.

40. A recombinant allergen according to claim 36, characterised in thatit is a mutant of a pollen allergen originating from the taxonomic order of Poales.

41. A recombinant allergen according to claim 36, characterised in that it is a mutant of a pollen allergen originating from the taxonomic order of Asterales or Urticales.

42. A recombinant allergen according to claim 35, characterised in that it is a mutant of a house dust mite allergen. AMENDED SHEET re

43. A recombinant allergen according to claim 42, characterised in that it is a mutant of a mite allergen originating from Dermatophagoides.

44. A recombinant allergen according to claim 35, characterised in that it is a mutant of a cockroach allergen.

45. A recombinant allergen according to claim 35, characterised in that it is a mutant of an animal allergen.

46. A recombinant allergen according to claim 45, characterised in that it is a mutant of an animal allergen originating from cat, dog or horse.

47. A recombinant allergen according to any one of claims 1-34 characterised in that it is a mutant of a venom allergen.

48. A recombinant allergen according to claim 47, characterised in that it is a mutant of a venom allergen originating from the taxonomic order of Hymenoptera.

49. A recombinant allergen according to claim 48, characterised in that is a mutant of a venom allergen from the taxonomic order of Vespidae, Apidae and Formicoidae.

50. A recombinant allergen according to any one of claims 47-49 characterised in that it is a mutant of Ves v 5.

51. A recombinant allergen according to claim 50 characterised in that one or more of the substitutions is selected from the group consisting of K-16, K- 185, K-11, K-44, K-210, R-63, K-13, F-6, K-149, K-128, E-184, K-112, F-157, E-3, K-29, N-203, N-34, K-78, K-151, L-15, L-158, Y-102, W-186, K-134, D- 87, K-52, T-67, T-125, K-150, Y-40, Q-48, L-65, K-81, Q-101, Q-208, K-144, N-8, N-70, H-104, Q-45, K-137, K-159, E-205, N-82, A-111, D-131, K-24, --V- AMENDED SHEET

F 36, N-7, M-138, T-209, V-84, K-172, V-19, D-56, P-73, G-33, T-106, N-170, L-28, T43, Q-114, C-10, K-60, N-31, K-47, E-5, D-145, V-38, A-127, D-156, E-204, P-71, G-26, Y-129, D-141, F-201, R-68, N-200, D-49, S-153, K-35, S- 39, Y-25, V-37, G-18, W-85 and 1-182.

52. A recombinant allergen according to any of claims 1-51 for use as a pharmaceutical.

53. Use of the recombinant allergen according to any of claims 1-51 for preparing a pharmaceutical for preventing and/or treating allergy.

54. A composition comprising two or more recombinant mutant allergen variants according to any of claims 1-51, wherein each variant is defined by having at least one primary mutation, which is absent in at least one of the other variants, wherein for each variant no secondary mutation is present within a radius of 15 A from each absent primary mutation.

55. A composition according to claim 54 comprising 2-12 variants.

56. A composition according to claim 54 comprising 3-10 variants.

57. A composition according to claim 54 comprising 4-8 variants.

58. A composition according to claim 54 comprising 5-7 variants.

59. A composition according to claim 54 or 55-58 for use as a pharmaceutical.

60. Use of a composition according to claim 54 or 55-58 for preparing a pharmaceutical for preventing and/or treating allergy.

61. A pharmaceutical composition, characterised in that it comprises a recombinant allergen according to any one of claims 1-51. AMENDED SHEET

62. A pharmaceutical composition, characterised in that it comprises a recombinant allergen according to claim 54 or any one of claims 55-58 in combination with a pharmaceutically acceptable carrier and/or excipient.

63. A pharmaceutical composition, characterised in that it comprises a recombinant allergen according to claim 54 or any one of claims 55-58 in combination with a pharmaceutically acceptable carrier and/or adjuvant.

64. A pharmaceutical composition according to claim 61-63, characterised in that it is in the form of a vaccine against allergic reactions elicited by a naturally occurring allergen in patients suffering from allergy.

65. Use of recombinant allergen for a method of generating immune response in a subject comprising administering to the subject a recombinant allergen according to any one of claims 1-51, a composition according to claim 54 or claims 55-58 or a pharmaceutical composition according to clams 61-64.

66. Use of recombinant allergen for a method of vaccination or treatment of a subject comprising administering to the subject a recombinant allergen according to any one of claims 1-51, a composition according to claim 54 or claims 55-58 or a pharmaceutical composition according to claims 61-64.

67. A process for preparing a pharmaceutical composition according to claim 61-63 or 64 comprising mixing a recombinant allergen according to any one of claims 1-51 or a composition according to claim 54 or 55-58 with pharmaceutically acceptable substances and/or excipients.

68. A pharmaceutical composition obtainable by the process according to claim 67. AMENDED SHEET a9

69. Use of recombinant allergen for a method of treatment, prevention or alleviation of allergic reactions in a subject comprising administering to a subject a recombinant allergen according to claim 1-51, a composition according to claim 54 or claims 55-58 or a pharmaceutical composition according to claims 61-64.

70. A method of preparing a recombinant allergen according to any one of claims 1-51, characterised in a) identifying a number of amino acid residues in a naturally occurring allergen, which has a solvent accessibility of at least 20 %; b) selecting at least four of the identified amino acid residues in such a manner that each selected amino acid is spaced from each other selected amino acid by at least 15 A, and that the selected amino acids are placed in such a manner that at least one circular surface region with a area of 800 A? comprises no selected amino acid; and c) effecting for each of the selected amino acids a primary mutation, which reduce the specific IgE binding capability of the mutated allergen as compared to the binding capability of the said naturally occurring allergen, wherein each primary mutation is a substitution of a selected amino acid residue with another amino acid, which does not occur in the same position in the amino acid sequence of any known homologous protein within the taxonomic species from which said naturally occurring allergen originates.

71. A method according to claim 70, 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.

72. A method according to claim 70 or 71, characterised in selecting identified amino acid residues, which are conserved with more than 70 % AMENDED SHEET identity in all known homologous proteins within the species from which said naturally occurring allergen originates.

73. A method according to claim 72, characterised in ranking the said 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.

74. A method according to any of claims 70-73 comprising selecting the identified amino acids so as to form a mutant allergen, which has essentially the same a-carbon backbone tertiary structure as said naturally occurring allergen.

75. A method according to any of claims 70-74 characterised in that the substitution of amino acid residues is carried out by site-directed mutagenesis.

76. A method of preparing a recombinant allergen according to any one of claims 1-51, characterised in that the allergen is produced from a DNA sequence obtained by DNA shuffling (molecular breeding) of the DNA encoding the corresponding naturally occurring.

77. A DNA sequence encoding a recombinant allergen according to any of claims 1-51, a derivative thereof, a partial sequence thereof, a degenerated sequence thereof or a sequence, which hybridises thereto under stringent conditions, wherein said derivative, partial sequence, degenerated sequence or hybridising sequence encodes a peptide having at least one B cell epitope.

78. A DNA sequence according to claim 77, which is a derivative of the DNA sequence encoding the naturally occurring allergen. AMENDED SHEET

79. A DNA sequence according to claim 77, wherein the derivative is obtained by site-directed mutagenesis of the DNA encoding the naturally occurring allergen.

80. A DNA sequence according to any of claims 77-79, wherein the sequence is a derivative of the sequence shown in Fig. 3, wherein the DNA sequence is mutated so as to encode an allergen having at least four mutations selected from the group consisting of V2, D72, E87, K-129, E-60, N-47, K-65, P-108, N-159, D-93, K-123, K-32, D-125, R-145, D-109, E-127, Q-36, E-131, L-152, E-6, E-96, D-156, P-63, H-76, E-8, K-134, E-45, T-10, V- 12, K-20, S-155, H-126, P-50, N-78, K-119, V-2, L-24, E-42, N4, A-153, |-44, E-138, G-61, A-130, R-70, N-28, P-35, S-149, K-103, Y-150, H-154, N-43, A- 106, K-115, P-14, Y-5, K-137, E-141, E-87 and E-73.

81. A DNA sequence according to any of claims 77-79, wherein the sequence is a derivative of the sequence shown in Fig. 13, wherein the DNA sequence is mutated so as to encode an allergen having at least four mutations selected from the group consisting of K-16, K-185, K-11, K-44, K- 210, R-63, K-13, F-6, K-149, K-128, E-184, K-112, F-157, E-3, K-29, N-203, N-34, K-78, K-151, L-15, L-158, Y-102, W-186, K-134, D-87, K-52, T-67, T- 125, K-150, Y-40, Q-48, L-65, K-81, Q-101, Q-208, K-144, N-8, N-70, H-104, Q-45, K-137, K-159, E-205, N-82, A-111, D-131, K-24, V-36, N-7, M-138, T- 209, V-84, K-172, V-19, D-56, P-73, G-33, T-106, N-170, L-28, T-43, Q-114, C-10, K-60, N-31, K-47, E-5, D-145, V-38, A-127, D-156, E-204, P-71, G-26, Y-129, D-141, F-201, R-68, N-200, D-49, S-153, K-35, S-39, Y-25, V-37, G- 18, W-85 and 1-182.

82. A DNA sequence according to any of claims 77-79, wherein the sequence is a derivative of the sequence shown in Fig. 16, wherein the DNA sequence is mutated so as to encode an allergen having at least four mutations selected from the group consisting of R-128, D-129, H-11, H-30, S- 1, K-77, Y-75, R-31, K-82, K-6, K-96, K-48, K-55, K-89, Q-85, W-92, I-97, H- 22, V-65, S-24, H-74, K-126, L-61, P-26, N-93, D-64, 1-28, K-14, K-100, E-62, AMENDED SHEET

1-127, E-102, E-25, P-66, L-17, G-60, P-95, E-53, V-81, K-51, N-103, Q-2, N- 46, E-42, T-91, D-87, N-10, M-111, C-8, H-124, 1-68, P-79, K-109 and R-128, D-129, H-11, H-30, S-1, K-77, Y-75, R-31, K-82, K-6, K-96, K-48, K-55, K-89, Q-85, W-92, I-97, H-22, V-65, S-24, H-74, K-126, L-61, P-26, N-93, D-64, |- 28, K-14, K-100, E-62, 1-127, E-102, E-25, P-66, L-17, G-60, P-95, E-53, V- 81, K-51, N-103, Q-2, N-46, E-42, T-91, D-87, N-10, M-111, C-8, H-124, |-68, P-79, K-109 and K-15.

83. An expression vector comprising the DNA according to any of claims 77-

82. :

84. A host cell comprising the expression vector of claim 83.

85. A method of producing a recombinant mutant allergen comprising the step of cultivating the host cell according to claim 84.

86. A recombinant allergen according to any of claims 1-51 or encoded by the DNA sequence according to any of claims 77-82 comprising at least one T cell epitope capable of stimulating a T cell clone or T cell line specific for the naturally occurring allergen.

87. A diagnostic assay for assessing relevance, safety or outcome of therapy of a subject using a recombinant mutant allergen according to any of claims 1-51 or a composition according to claim 54 or 55-58, 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.

88. A recombinant mutant allergen substantially as herein described with reference to and as exemplified in any one of Examples 1-5. AMENDED SHEET

89. Method for preparing a recombinant mutant allergen substantially as herein described with reference to and as exemplified in any one of Examples 1-5.

AMENDED SHEET