The present invention relates to novel recombinant protein variants that are useful as immunotherapeutic components. Also the present invention relates to DNA sequences encoding said protein variants as well as compositions comprising said protein variants.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention relates to a recombinant protein variant with the ability to induce a protective immune response to a naturally occurring allergen, wherein the protein variant is a variant of a scaffold protein, said scaffold protein having a three-dimensional folding pattern that is structurally similar to that of the naturally occurring allergen, compared to the scaffold protein, comprises two or more primary mutations spaced by at least one non-mutated amino acid residue, each primary mutation introducing into the scaffold protein at least one amino acid residue identical or homologous to the corresponding amino acid residue or residues in the naturally occurring allergen, and has, compared to the scaffold protein, an increased affinity and/or binding capacity to IgE antibodies that are specific to the naturally occurring allergen.

The present invention furthermore relates to the use of one or more protein variants for preparing a pharmaceutical composition for treatment of allergic individuals.

The present invention also relates to DNA sequences encoding protein variants according to the present invention as well as to vectors and host-cells hosting such DNA sequences and methods of producing recombinant proteins.

Also, the present invention relates to diagnostic assays.

DETAILED DESCRIPTION OF THE INVENTION

The idea of the present invention is to use a "scaffold protein" with a similar three-dimensional folding pattern but with little or no antibody cross-reactivity with a naturally occurring allergen as the basis for creating novel allergen mutants.

This scaffold protein can be used for creating novel recombinant protein variants that possess surface areas with and without antibody cross-reactivity with the allergen specific antibodies. There are several important advantages by using the scaffold protein as the basis for creating allergen vaccine candidates. It is usually required to perform a large number of mutations of an allergen in order to lower its allergen specific IgE reactivity and thereby the risk of inducing adverse effects during vaccination. Also, as a large number of mutations tend to destabilize the protein, these mutated allergens are often not suitable for use as vaccine candidates. The idea behind the invention is thus that a relatively small number of mutations are generally required in order to partly or fully establish allergen specific IgE recognizing contours on the surface of an appropriate scaffold protein. Such molecules have the potential of inducing new protective immune responses that can compete with IgE binding upon allergen exposure leading to a reduced risk of inducing IgE-mediated allergic responses.

One aspect of the invention is to generate a mutated protein variant with one allergen homologous surface area. A preferred scaffold protein according to this aspect is a scaffold with little or no binding to IgE antibodies specific to the naturally occurring allergen as a stating point thereby minimizing the need for introduction of secondary mutations. A scaffold with some binding to to allergen specific antibodies may also be suitable. Introduction of one or more secondary mutations in such a scaffold protein may limit or eliminate potential cross-reactivity. The ability of scaffold proteins and scaffold protein variants to bind antibodies specific to the naturally occurring allergen can be tested by RAST inhibition using a panel of sera from patients allergic to the allergen in question (Nolte et al (1987) Allergy, 42:366-373). Devoid in antibody binding means a reduction in antibody affinity by a factor of 10.sup.3, more preferably 10.sup.4, most preferably 10.sup.5 or more.

Comparison of structural similarities between the scaffold protein and the allergen in question includes assessment of three-dimensional structures and/or amino acid sequence. Folding pattern similarity is preferably assessed by comparison of scaffold and allergen three-dimensional structures. In absence of a known three-dimensional structure of the scaffold protein and/or the naturally occurring allergen, proteins having sequence identity greater than 10%, preferably greater than 20% to the allergen can in general be anticipated to have homologous folding patterns and thus be applied as scaffold proteins. However, even protein with amino acid identity less than 10% (exemplified by accession no. AAH05642 and pdb entry 1jss) have been known to have the same folding pattern as the allergen to which is compared e.g. in this case Bet v 1.

The three-dimensional structure of the scaffold protein may also be modeled based on the known structure of the allergen in question, or vice versa. More preferably amino acid sequence identity between the scaffold molecule and the allergen in question should be below 67%, more preferably between 20 and 60%, more preferably between 20 and 50%, and even more preferable 30-50%.

A number of amino acids on the molecular surface of the scaffold molecule are identical with amino acids on the surface of the allergen in question, and positioned in corresponding positions in the homologous structure. This can be visualized by comparison of the three-dimensional models or predicted three-dimensional structures.

A surface area is selected for "epitope grafting", preferentially an area comprising as many identically positioned amino acid residues in the scaffold and the allergen respectively as possible. The number of artificial mutations needed to fully or partly establish a surface area that mimicks an allergen on the surface of the scaffold protein is thereby minimized. Preferably, the generated surface area comprises between 1000 and 3600 .ANG..sup.2, and not larger than 5000 .ANG..sup.2. It has been estimated that around 2000 .ANG..sup.2 is masked upon binding by the CDR region alone. Furthermore there will be steric hindrance caused by the presence of the entire antibody. Thus, an area of up to 5000 .ANG..sup.2 is therefore inaccessible to binding by other antibody molecules.

Within the selected area of the scaffold protein, surface amino acids that are different between the scaffold protein and the allergen in question are mutated in such a way that most or preferably all of the surface exposed amino acid residues within the selected area become homologous, or identical to surface exposed amino acids of the allergen in question.

In a further aspect surface areas may be modified to become less identical or homologous to the allergen surface in order to avoid cross-reaction to these surfaces of allergen specific antibodies. In such surface areas, amino acid residues that are identical or homologous to amino acid residues in the corresponding position of the allergen are substituted by residues that are different or less homologous.

The three-dimensional structure of the resulting mutated scaffold protein, i.e. the recombinant protein variant may be verified with respect to its overall folding pattern as well as the conformation of the "grafted" epitope area. Methods of determining secondary and tertiary structures of a protein include X-ray crystallography, NMR spectroscopy, and circular dichroism spectroscopy. The stability of the resulting mutated molecule may be assessed by circular dichroism spectroscopy applying a range of variations in urea concentration, pH, ion strength, temperature, or other parameters.

In a vaccination scenario, protein variants or preferentially a mixture of protein variants with non-overlapping allergen specific surface areas, are expected to be safe, since two IgE antibodies specific for the allergen in question preferably cannot bind simultaneously, and hence do not trigger allergic reactions. Furthermore they are expected to be efficient in stimulating protective IgG antibody responses, wherein the IgGs are reactive with the natural allergen in question, within surface areas covered by the allergen specific surface areas.

According to the other aspect of the invention, mutated protein variant with several grafted areas distributed over parts of or the entire molecule are generated. Scaffold proteins may be mutated so as to reduce or increase antibody binding affinity or both, thereby eliminating fully or partly epitope areas and/or establishing epitopes areas on other parts of the scaffold protein. The idea is to achieve a general low affinity interaction over the entire surface with respect to binding of allergen specific IgE antibodies.

A number of amino acid residues on the surface of the scaffold protein are identical with amino acid residues on the surface of the allergen in question. This can be visualized by comparison of the three-dimensional structures of the allergen and the scaffold protein.

The surface areas that are selected for reducing IgE affinity of the scaffold protein are coherent surface areas comprising amino acid residues that are homologous or identical between the allergen and the scaffold protein. Such amino acid residues are then mutated to amino acid residues that are related or non-related to amino acid residues in the allergen in question.

In surface areas that are selected for increasing the allergen specific antibody cross-reactivity of the protein variant, surface amino acid residues, which are different comparing the scaffold molecule and the allergen are selected. These amino acid residues are then mutated to amino acids that are homologous, or more preferably identical, with the allergen in question.

The surface of a recombinant protein variant(-s) comprises areas of amino acids that are identical as well as areas of amino acids that are non-identical with the allergen in question. When the surface of the recombinant protein variant is graphically represented, it can be visualized that the surface comprises at least one essentially circular area that is homologous or identical with the corresponding area of the naturally occurring allergen. This area covers about 300-900 .ANG..sup.2, more preferably 500-800 .ANG..sup.2, and most preferably 500-600 .ANG..sup.2.

Accordingly, preferably the scaffold protein does not have any immunogenic reactivity with naturally occurring allergen specific antibodies. However, it is possible to use a scaffold protein having reactivity with natural occurring allergen specific antibodies, in which case preferable one or more of the epitopes are modified in order to eliminate or decrease the antibody binding affinity of these epitopes.

In other words, the main aspect of the present invention relates to a recombinant protein variant with the ability to induce a protective immune response to a naturally occurring allergen, wherein the protein variant is a variant of a scaffold protein, said scaffold protein has a three-dimensional folding pattern that is structurally similar to that of the naturally occurring allergen, compared to the scaffold protein, comprises two or more primary mutations spaced by at least one non-mutated amino acid residue, each primary mutation introducing into the scaffold protein at least one amino acid residue identical or homologous to the corresponding amino acid residue or residues in the naturally occurring allergen, and has, compared to the scaffold protein, an increased affinity and/or binding capacity to IgE antibodies that are specific to the naturally occurring allergen.

A primary mutation according to the present invention might comprise a substitution a deletion and/or an addition. According to the invention, 2-50, preferably 2-40, more preferably 3-25, more preferably 4-15 and most preferably 5-12 primary mutations are performed. It is a very important aspect of the present invention that at least two primary mutations are performed, ensuring that three-dimensional epitope areas are established that can bind allergen specific antibodies are grafted onto the scaffold molecule.

The present invention furthermore comprises protein variants with one or more secondary mutations introducing into the scaffold protein amino acid residues, which are not present in the corresponding position in the natural occurring allergen. The purpose of introducing secondary mutations may e.g. be to stabilize the three-dimensional folding pattern of the protein variant or to decrease allergen specific IgE cross-reactivity.

According to the invention, 0-20, preferably 1-10, or most preferably 2-5 secondary mutations are performed.

In general, secondary mutations according to the present invention may be performed as a mutation of one or more amino acid residues with any amino acid residue that is not present in the particular position in either the scaffold protein or the naturally occurring allergen. Preferably the secondary mutation is a substitution of an amino acid residue that is not present in the naturally occurring allergen but is homologous to the scaffold amino acid in order to minimize the risk of destabilizing the protein variant.

A primary or secondary mutation according to the present invention may comprise mutations carried out by site-directed mutagenesis (insertion, deletion or substitution), DNA shuffling, and/or by gene library methods.

Any primary or secondary mutation according to the invention may consist of a number of consecutively mutated amino acids. 1-15, preferably 1-10 and most preferably 1-5 consecutive amino acids are mutated in a primary or secondary mutation according to the present invention. In many cases the surface exposed amino acids that are part of an epitope area are present as single amino acid "points" within the primary structure of an allergen. In such cases it is usually sufficient to point mutate such amino acids. However, in other cases it is necessary to mutate several consecutive amino acids in order to increase or decrease antibody binding affinity.

An aspect of the invention comprises a method of producing a recombinant protein variant with the ability to induce a protective immune response to a naturally occurring allergen, comprising the steps of: selecting a scaffold protein, said scaffold protein having a three-dimensional folding pattern that is structurally similar to that of the naturally occurring allergen, introducing two or more primary mutations, that are spaced by at least one non-mutated amino acid residue, into the scaffold protein, each primary mutation introducing into the scaffold protein at least one amino acid residue identical or homologous to the corresponding amino acid residue or residues in the naturally occurring allergen, and thereby producing a protein variant that has, compared to the scaffold protein, an increased affinity and/or binding capacity to IgE antibodies that are specific to the naturally occurring allergen.

A product obtainable by the above method is also included within the scope of the present invention.

In one embodiment, a protein variant according to the present invention compared with the scaffold protein has an increased binding capacity with respect to antibodies specific to the naturally occurring allergen. Said binding capacity is preferably increased to at least 10%, preferably to least 50%, and up to 100% of the antibody binding capacity of the natural allergen. Said protein variant furthermore preferably has a reduced ability to induce histamine release compared to the naturally occurring allergen. The reduction in histamine release potential of the protein variant is preferably reduced by 2 fold, more preferably by 10 fold, even more preferably by 100 fold, even more preferably by 1000 fold and most preferably by a 10,000 fold or more reduction compared with the ability of the naturally occurring allergen.

By ensuring that the ability of the protein variant to induce histamine release is greatly reduced i.a. the ability of the variant to cross-link the IgE receptor the protein variant thus constitutes a more safe alternative to the natural allergen as the active component of an allergen vaccine, since the ability to cause histamine release is largely associated with and responsible for adverse effects associated with the immediate type allergy response and allergy vaccination.

According to a second embodiment, a protein variant according to the present invention has a three-dimensional folding pattern or .alpha.-carbon backbone tertiary structure that closely resembles that of the scaffold protein as measured as described in Example 4. The surface contour making up an epitope is supported by the folding pattern of the peptide .alpha.-carbon backbone.

According to a preferred embodiment a protein variant according to the invention is therefore preferably derived from a scaffold protein that has a level of amino acid identity with the naturally occurring allergen of between 20 and 60%, preferably between 30 and 50%.

According to a third embodiment, a protein variant according to the present invention has all the primary mutations located in a surface region having an area of about 600-900 .ANG..sup.2 so as to ensure that the primary mutations constitute an epitope area that can be recognized by the complementarity determining region of an allergen specific antibody. Furthermore, a protein variant according to the invention preferably comprises primary mutations of surface-exposed amino acids. A surface exposed amino acid has a solvent accessibility of above 20%, preferably above 30%, more preferably above 40%, and most preferably above 50%.

According to a fourth embodiment of the present invention, the naturally occurring allergen is an inhalation allergen, preferably originating from the taxonomic order of Fagales, Oleales or Pinales, and most preferably Bet v 1 (database accession number: Z80104). A preferred Bet v 1 scaffold protein is Mal d 1.

In other preferred embodiments, scaffold proteins of the major birch pollen allergen Bet v 1 are e.g. isoforms of Aln g 1 (alder) exemplified by accession number S50892, isoforms of Car b 1 (hornbeam) exemplified by Q96382, Q96381, CAA47367, Q96377, Q96378, Q96379, Q96503, Q96501, CAA47366, Q96380, isoforms of Fag s 1 (beech) exemplified by AJ130889, isoforms of Cas s 1 [Castanea sativa] exemplified by AJ417550, isoforms of Mal d 1 (apple) exemplified by accession numbers AJ488060, Q43550, Q43551, Q43552, Q43549, AAK13027, AAK13028, Q40280, AAD13683, Q9SYV2, Q9SYV3, Q9SYV4, Q9SYV6, Q9SYV9, Q9SYW3, Q9SYV5, AAK13029, Q9SYV7, Q9SYV8, JC4276, S51119, isoforms of Pru av 1 (cherry) exemplified by accession numbers O22521 O24248, O50001 and gi13787043, isoforms of Pyr c 1 (pear) exemplified by accession numbers AF057030, isoforms of Api g 1 (celery) exemplified by accession numbers p49372, p92918, isoforms of Dau c 1 (carrot) exemplified by accession numbers 004298, t14322, t14325, isoforms of Lupin exemplified by accession number p52779 (pdb entry 1IFV_A), isoforms of other Patogenesis Related proteins exemplified by accession numbers CAA10719, S20517, P93333, AB17_PEA, AB18_PEA, T14817 and other proteins that contains the same arrangement of secondary structure elements exemplified by accession number AAH05642 (pdb entry 1jss).

In an embodiment, where rMal d 1 (2620) (database accession number: AJ488060) is the Bet v 1 scaffold protein, a protein variant comprises at least two primary mutations selected from the group consisting of: (E12V, E12I, E12M, E12L), P16A, (H40S, H40T), I43N, L44I, D47N, G65K, K70R, (E76H, E76R, E76K, E76Q), S107T, G108P, +109D, S110G, E129A, K152L, (P154S, P154T), P155S and optionally one or more secondary mutations are selected from the group consisting of: N28X, preferably N28T, K32X, preferably K32Q, E45S, E96X, +159X. In this context, as well as in the following examples, the first letter and number corresponds to the original amino acid code in a particular amino acid position. The following letter is the amino acid that can be substituted with the original amino acid. Parenthesis means that there are a number of different options when choosing to mutate in a given amino acid position. One of these can be chosen.

In a specific embodiment, a Mal d 1 protein variant (rMal d 1 (2760)) comprises the sequence defined in SEQ ID NO 1 -- see Original Patent.

In another specific embodiment, a protein variant of a Mal d 1 protein variant (rMal d 1 (2781)) comprises the sequence defined in SEQ ID NO 2 -- see Original Patent.

In yet another specific embodiment, a protein variant of a Mal d 1 protein variant (rMal d 1 (2762)) comprises the sequence as defined in SEQ ID NO 3 -- see Original Patent.

In an embodiment demonstrating that a protein variant of Mal d 1 may comprise both primary and secondary mutations, such a protein variant comprises at least two primary mutations selected from the group consisting of: (E12V, E12I, E12M, E12L), (H40S, H40T), (E76H, E76R, E76K), E129A, (P154S, P154T), and optionally one or more secondary mutations selected from the group consisting of: E8X, N28X, K32X, E96X, +159X.

According to a fifth embodiment a protein variant of the present invention is a protein variant wherein Dau c 1 (database accession number: T14325) is a scaffold protein of Bet v 1. According to this embodiment, a protein variant of Dau c 1 comprises at least two primary mutations selected from the group consisting of: (S12V, S12L, S12I, S12M), S14P, E16A, P105A, A107P, (A148S, A148T), (I151L, I151V, I151M), (N153H, N153K, N153R), (+154S, +154T), (+155D, +155E), +156A, (+157Y, +157F), (+158N, +158Q), (K39S, K39T), (K44E, K44D), (V52I, V52M, V52L), (I54K, I54R, I54H), (T64K, T64R, T64H), (T65Y, T65F, T65W), (T67K, T67R, T67H), D86E, L91G, (G92D, G92E) and optionally one or more secondary mutations are selected from the group consisting of: K32X, E42X, E59X, R69X, E95X, K122X, E8X, T10X, D25X, D46X, D108X.

In a specific embodiment, a Dau c 1 protein variant comprises at least two primary mutations selected from the group consisting of: (S12V, S12L, S121, S12M), S14P, E16A, P105A, A107P, (A148S, A148T), (I151L, I151V, I151M), (N153H, N153K, N153R), (+154S, +154T), (+155D, +155E), +156A, (+157Y, +157F), (+158N, +158Q) and optionally one or more secondary mutations selected from the groups consisting of: K32X, E42X, E59X, R69X, E95X, K122X. This embodiment is a demonstration of establishment of a single epitope area on the surface of a scaffold protein while other areas have been mutated so as to decrease antibody affinity.

In another specific embodiment, a Dau c 1 variant comprises comprises at least two primary mutations selected from the group consisting of: (K39S, K39T), (K44E, K44D), (V52I, V52M, V52L), (154K, 154R, 154H), (T64K, T64R, T64H), (T65Y, T65F, T65W), (T67K, T67R, T67H), D86E, L91G, (G92D, G92E) and optionally at least one secondary mutation is selected from the group consisting of: E8X, T10X, D25X, K32X, D46X, E59X, E95X, D108X, K122X. This embodiment is another example of establishment of a single epitope area on the surface of a scaffold protein and decrease of antibody affinity of other parts of the molecule.

In a sixth embodiment of the present invention, the naturally occurring allergen originates from the taxonomic order of Poales, Asterales or Urticales. Examples of scaffold proteins of the major grass pollen allergen Phl p 1 are isoforms of Zea m 1 exemplified by accession number Q07154, isoforms of Gly m 1 exemplified by accession number U03860, isoforms of Ory s 1 exemplified by accession number U31771, isoforms of .beta.-expansins exemplified by accession numbers: U95968, AC001229, U95967, s53082, U30477, U85246, Y07782, U30479, U30481, U30480, U30478, U30476, U30460, U30382, U64890, U64891, U64892, U64893, U82123, D26459, D88415, isoforms of Group 2 and group 3 grass allergens exemplified by accession numbers: P14947, X73363, A48595, P43214, P14948, U25343, Z50867. Phl p 5 is another example of a grass pollen allergen. Examples of Phleum pratense group 5 allergen, Phl p 5 scaffold proteins include: P93466, O81342, Q9SBE0, O81343, O81344, 2023228A, S38584. Examples of Lolium perenne group 5 allergen, Lol p 5 scaffold proteins include CAB64344, Q9XF24, a38582. Examples of Poa pratensis group 5 allergen, Poa p 5 scaffold proteins include: Q9FPR0, B39098. Isoforms of Holcus lanatus group 5 allergen, Hol 15 exemplified by (O23971, O23972, Q9FPQ9). Isoforms of Phalaris aquatica group 5 allergen, Pha a 5 exemplified by (MP51_PHAAQ). Isoforms of Dactylis glomerata group 5 allergen, Dac g 5 exemplified by (Q93XE0, Q93XD9). Isoforms of Hordeum vulgare group 5 allergen, Hor v 5 also referred to as Hor v 9 exemplified by (O04828). Isoforms of Phleum pratense group 6 allergen, Phl p 6 exemplified by (O65868, CAA76557).

In a seventh embodiment of the present invention, the naturally occurring allergen is a dust mite allergen, preferably originating from Dermatophagoides, and preferably Der p 2 (database accession number: P49278). Preferred Der p 2 scaffold proteins are Eur m 1 (database accession number: P25780), Gly d 2 (database accession number: AJ272216), and Lep d 2 (database accession number: S66499).

A specific embodiment of a Lep d 2 protein variant is a protein variant that comprises at least two primary mutations selected from the group consisting of: D17L, D17I, D17V, D17M, S19P, Q32K, Q32R, Q32H, K33P, T35Q, T35N, N88K, N88R, N88H, T92N, T92Q, A95K, A95R, A95H and optionally one or more secondary mutations selected from the group consisting of: K6X, S22X, R30X, K76S, K81X, V114X. This is an example of a Lep d 2 protein variant with a single epitope area established on the surface of the protein, optionally with downregulation of Der p 2 antibody cross-reactivity at other parts of the surface.

Another specific embodiment of a Lep d 2 protein variant is a protein variant that comprises at least two primary mutations within a single epitope area selected from the group consisting of: D45N, D45Q, N47K, N47R, N47H, K48T, K48S, T50K, T50R, T50H, K52E, K52D, L54K, L54R, L54H, E107K, E107R, E107M, H112D, H112E, T119I, T119L, T119V, T199M and optionally one or more secondary mutations selected from the group consisting of: K6X, S22X, K29X, R30X, K76X, K81X.

A specific embodiment of a Gly d 2 protein variant is a protein variant that comprises at least two primary mutations selected from the group consisting of: K2Q, K2N, K4D, K4E, K10N, K10Q, T14K, T14R, S22H, S22R, S22K, K39V, K39L, K39I, K39M, D45N, D45Q, T60L, T60V, T60I, T60M, Q6three-dimensional, Q63E, K80V, K80L, K80I, K80M, T91S, H112D, H112E, R122I, R122V, R122L, R122M and optionally one or more secondary mutations selected from the group consisting of: K6X, preferably K6R or K6H, R30X, preferably R30K or R30H, F74X, preferably F74Y or, F74W, K81X, preferably K81Ror K81H, K88X, preferably K88R or K88H, T90X, and V114X, preferably V114L, V114I or V114M. This is an example of a protein variant with increased IgE reactivity in comparison with Der p 2 and optionally with secondary mutations where amino acids are substituted with homologous amino acids in order to avoid destabilization of the protein variant.

Other specific embodiments of Der p 2 scaffold proteins are isoforms of group 2 dust mite Tyrophagus putrescentiae allergens Tyr p 2 exemplified by accession number O02380, isoforms of storage mite Lepidoglyphus destructor group 2 allergens Led d 2 (mistakenly referred to as Led d 1 in some publications) exemplified by accession numbers: P80384, S48727, 2118249B, isoforms of Gly d 2 exemplified by accession numbers: CAB76459, Q9U5P7, isoforms from the group of homologous proteins exemplified by accession numbers 097763, P79345, Q9VQ62, Q9Z0J0, AAF99719, Q28895.

In a further preferred embodiment scaffold proteins of the major house dust mite allergen Der p 1 is Derf 1 (accession numbers P16311).

In an eighth embodiment of the present invention the naturally occurring allergen is a insect allergen from cockroach (Blatella germanica (allergens: Bla g 1 or Bla g 2), Periplenata Americana (allergen: Per a 1) or midges (Chironimus Spp.--allergen: Chi t 1)

In a ninth embodiment of the specific invention, the naturally occurring allergen is an animal allergen, preferably a mammalian allergen, preferably originating from a cat, dog, rat, mouse or horse, preferably the Fel d 1 allergen from cat.

In a tenth embodiment of the present invention, the naturally occurring allergen is a venom allergen, preferably originating from the taxonomic order of Hymenoptera, Vespidae, Apidae, or Formicoidae. A preferred venom allergen is Ves v 5. Possible Ves v 5 scaffold proteins include: P35783, P35760, P35785, P35784, P35787, P35736, P35786, P10737, Q05108, P35781, P35782, P81657, P81656, P35780, P35759, Q05109, P35779, P35778). Furthermore other low homologous proteins exemplified by (P54108, O19010, P16562, Q16937, P48060, Q60477, P35795, P35795, Q09566, P47033, Q40374, P12020, Q41359, P47032, Q91055, Q034401, P08299, P11670, P36110, P16563,P54107, Q08697, Q04108, P79845, P07053, P09042, P35794, P04284, Q03402, P33154, P35792, Q05968, P35793, Q00008, Q41495, P16547, P13390, O22456, Q15335, Q62871, 088487, Q13409, P54131, Q9LRZ5, P18859, O04067.

In an eleventh embodiment of the present invention, the naturally occurring allergen is a food allergen from peanut, soya, cows milk, hens egg white or yolk, shrimp or cod.

In a twelfth embodiment of the present invention, the scaffold protein is homologous to a plant, grass, food, or mite allergens. Protein variants according to this embodiment comprise at least one primary mutation. Protein variant furthermore have deconvoluted CD-spectra that deviate less than 30%, preferably less than 20%, and even more preferably less than 10% compared to the deconvoluted CD-spectra of the naturally occurring allergen. Preferably, the scaffold protein has a level of sequence identity with the naturally occurring allergen of between 30 and 50%, and preferably, the .alpha.-carbon backbone tertiary structures of the scaffold protein and the natural occurring allergen resemble each other to the extent that the deviation between their circular dichroism spectra should deviate by less than 30%, preferably less than 20%, and even more preferably less than 10 (Example 4).

In a thirteenth preferred embodiment of the present invention, the naturally occurring allergen is a fungal protein.

Preferably, a protein variant of the invention comprises at least one T-cell epitope capable of stimulating a T-cell clone or T-cell line specific for the naturally occurring allergen.

Accordingly, a protein variant preferably induces a new allergen specific Th0/1 T-cell immune response on top of the Th2 T-cell response or the scaffold protein variants induce T-cells anergy or a shift of the response of the T-cells from the Th2-type to the Th1-type

The invention further relates to the use of protein variants according to the present invention for use as a pharmaceutical as well the manufacture of a medicament for the treatment or prevention of allergy. Preferably, a pharmaceutical composition comprises two or more different recombinant protein variants according to the present invention, wherein each variant is defined by having at least one primary mutation, which is absent in at least one of the other variants. In this way, presentation of different epitope areas that mimick epitopes of the naturally allergen can be achieved while ensuring that the risk of IgE cross-binding on the surface of e.g. mast-cells is minimized. A composition according to the present invention preferably comprises 2-12, preferably 3-10, more preferably 4-8, and most preferably 5-7 different protein variants. Such compositions can be used for preparing a pharmaceutical for preventing and/or treating allergy.

Pharmaceutical compositions according to the present invention preferably further comprise a physiological or a pharmaceutically acceptable carrier and/or excipient and/or an adjuvant. "Carriers" or excipients include any carrier or excipient commonly used with pharmaceuticals such as oils, starch, sucrose and lactose. The compositions according to the present invention may be in a form suited for oral intake including, capsules, pills, tablets and syrups. Alternatively, compositions according to the present invention may be in the form of a parenteral formulation suitable for e.g. intravenous, subcutaneous, etc. administration.

"Adjuvants" refers to a substance that stimulates and prolongs antibody synthesis when injected together with an antigen. An example of an adjuvant includes Freunds complete/incomplete adjuvans and aluminium hydroxide gels. A pharmaceutical composition according to the present invention is preferably in the form of a vaccine against allergic reactions elicited by a naturally occurring allergen in patients suffering from allergy.

Included in the scope of the present invention is methods of vaccination or treatment of an individual by generating an immune response in the individual comprising administering to the subject a recombinant protein variant or a pharmaceutical composition according to the invention.

Also comprised in the scope of the present invention are processes for preparing a pharmaceutical composition mixing a recombinant protein variant or a composition according to the present invention with pharmaceutically acceptable substances and/or excipients and/or adjuvants.

A further aspect of the present invention includes methods of preparing a recombinant protein variant wherein the recombinant protein variant is produced from a DNA sequence obtained by DNA shuffling (molecular breeding) of DNA sequences encoding the scaffold protein and the naturally occurring allergen.

Finally the present invention relates to DNA sequences encoding recombinant protein variants. Likewise, the present invention comprises expression vectors and host-cells that contain DNA sequences according to the present invention and are capable of producing recombinant protein variants. Also, methods of producing recombinant protein variants comprising the step of cultivating such host-cells with vectors are included in the present invention. Diagnostic assays for assessing relevance, safety, or outcome of therapy of a subject using a recombinant protein variant are also included in the invention, wherein an IgE containing sample of the subject is mixed with said protein variant or said composition and assessed for the level of reactivity between the IgE in said sample and said protein variant.
 

Claim 1 of 5 Claims

1. A recombinant protein variant (rMal d 1 (2781)), comprising the sequence defined in SEQ ID NO: 2 with the ability to induce a protective immune response to a naturally occurring allergen, wherein the naturally occurring allergen is Bet v 1.

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