# Patent

## Title
Cloning of honey bee allergen

## Abstract

The present invention relates to a recombinant polypeptide capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera having a homology of more than 70% to the amino acid sequence of SEQ ID NO: 2, which is the honey bee allergen Api m3 (acid phosphatase). The invention further relates to nucleic acid encoding the polypeptide, expression vectors, host cells and methods of preparing the polypeptide, as well as diagnostic and pharmaceutical uses thereof.

## Background
RELATED APPLICATIONS
This application is a continuation-in-part of U.S. patent application Ser. No. 11/301,329 filed Dec. 13, 2005, which claims the benefit under 35 USC 119(e) of U.S. provisional application 60/635,479, filed Dec. 14, 2004.


FIELD OF THE INVENTION
The present invention relates to a recombinant polypeptide capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera having a homology of more than 70% to the amino acid sequence of SEQ ID NO: 2, which is the honey bee allergen Api m3 (acid phosphatase). The invention further relates to nucleic acid encoding the polypeptide, expression vectors, host cells and methods of preparing the polypeptide, as well as diagnostic and pharmaceutical uses thereof.
SUMMARY OF THE INVENTION
It has long been recognised that allergies against insect venoms are relatively common 4-5% of the German population react allergic to insect venoms. In Europe the relevant stinging insects are honey bees (Apis mellifera), wasps (Vespula spp.), bumble bees (Bombus spp.), hornets (Vespa crabo), midges, and horse flies (Helbing et al 2004, Eich-Wanger and Müller 1998). Bees, bumble bees, wasps, and hornets belong to the order Hymenoptera.
These social insects do not normally attack people, but will sting them in self defence if disturbed. Once stung, if the stinger remains in the skin, a honey bee is responsible, while, if no stinger is present, a wasp is likely to be the culprit. The female worker honey bee carries the stinger and dies soon after discharging a sting.
If a bee stings a vertebrate, the stinger will be detached from the insect, but the venom sack will still be attached to the stinger and if not removed, the whole venom volume (up to 50 μl) will be injected into the victim. Wasps can retract the stinger, and only inject about 20 μl venom.
The differences in stinging behaviour are based on natural evolution. Bees collect nectar, whereas wasps and hornets are insect hunters. Therefore, bees need to protect the hive, even against vertebrates like mice or larger animals. The insect dies upon the sting, but will inject the maximum volume of venom, if the stinger is not removed. Wasps and hornets do not have such natural enemies.
Since it is easy to obtain sufficient quantities of material, honey bee venom has been well studied. Honey bee venom contains at least 18 active substances. Melittin, the most prevalent substance, is one of the most potent anti-inflammatory agents known (100 times more potent than hydrocortisone). Adolapin is another strong anti-inflammatory substance, and inhibits cyclooxygenase; it thus has analgesic activity as well. Apamin inhibits complement C3 activity, and blocks calcium-dependent potassium channels, thus enhancing nerve transmission. Other substances, such as Compound X, Hyaluronidase, Phospholipase A2, Histamine, and Mast Cell Degranulating Protein (MSDP), are involved in the inflammatory response to venom, with the softening of tissue and the facilitation of flow of the other substances. Finally, there are measurable amounts of the neurotransmitters Dopamine, Norepinephrine and Serotonin. The water content varies between 55-70%. The pH range is between 4.5-5.5. A summary of the components of bee venom is given in Table 1 (Dotimas and Hider 1987).
The LD50 dose, i.e., the amount of bee venom which causes 50% of the tested individuals to die, is 6 mg venom/kg body weight for mice and rats. This equals 40 stings/kg body weight. For hornets, this factor is around 154-180 stings/kg body weight. Bee venom is 1.7-1.5 more effective than those of hornets (Habermann 1974, Kulike 1986).
Honey bees and wasps of the Hymenoptera order are by far the most frequent cause of serious allergic reactions. Normally, at least more than 50 stings of a bee per children or 100 per adult are necessary to induce life threatening conditions (see above). In case of allergic persons, one sting can be enough to cause death by adverse immunological reactions.
This type of allergy is mediated by IgE antibodies which react to venom components. The possibility, therefore, exists that desensitisation therapy by repeated and progressively increased doses of bee venom components would be successful. Several polypeptides from bee venom have been cloned and expressed as recombinant molecules (Sobotka et al 1974, Sobotka et al 1976, Hoffman and Shipman 1976, Kuchler et al 1989, Gmachl and Kreil 1993, Vlasak et al 1983, Hoffman et al 1977, Kettner et al 1999, King and Spangfort 2000). One component of bee venom, acid phosphatase, is one of the more potent allergic proteins (Arbesman et al 1976). Until now, no information about the complete gene sequence has been published and only initial studies on protein level have been made (Soldatova et al 2000, Barboni et al 1987, Hoffman 1996, Jacobsen and Hoffman 1995).
Barboni et al. (1987) describe two different proteins with acid phosphatase activity from honey bee venom, having a molecular weight of 45 and 96 kDa. Enzymatic activity is partly lost during purification in the gel filtration step. Other publications (Soldatova et al 2000, Barboni et al 1987, Jacobsen and Hoffman 1995) report contrasting data, teaching different fragments of the protein and the corresponding nucleic acid, and coming to different conclusions about the family of phosphatases that honey bee venom acid phosphatase might belong to, either prostatic phosphatases or lysosomal phosphatases. Soldatova et al. (2000) describe the incomplete cloning of a partial cDNA possibly encoding an acid phosphatase from honey bee venom. They report difficulties in cloning and obtaining the full length sequence and do not teach the sequence they seem to have cloned.
In light of the prior art, the person skilled in the art is therefore faced with the problem of providing a nucleic acid suitable for recombinant production of acid phosphatase (api m3) from the venom of an insect from the order Hymenoptera, in particular the honey bee, which can be used in such desensitisation therapy as well as in diagnostic tests for the detection of allergy.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the nucleotide sequence of the isolated cDNA for Api m 3 in FASTA format (SEQ ID NO: 1).
FIG. 2 shows the predicted restriction enzyme pattern of the isolated cDNA for Api m 3.
FIG. 3 shows the predicted translated amino acid sequence of the isolated cDNA for Api m 3 (SEQ ID NO:2). The underlined peptides can be aligned to prior published fragments. See FIGS. 5A, 5B, and 6.
FIG. 4A shows a vector map of a preferred insect cell expression vector, pIB/Mel opt-H10 Api m 3. The vector was modified to include a N-terminal 10× histidine-tag, cleavable with factor Xa protease as well as the signal sequence of bee melittin for secreted expression. The gene of interest was cloned between the EcoR V and Sac II site. The gene comprises a stop codon at the 3′-end. The expressed protein should be secreted and will have a factor Xa cleavable 10× histidine-tag at the N-terminus.
FIG. 4B shows optimisation of the Kozak sequence for insect cell expression. The former sequence a) was changed into b) to be in accordance with the preferred translation initial sequence (G/A)NNATGG adding an alanine to the N-terminal sequence.
FIG. 4C shows a vector map of a preferred bacterial expression vector, pET26b(+) api 3 pro. The vector was modified to contain the gene of interest between the Sac I and Nde I site. The protein sequence was taken from the verified mammalian expression vector pIB/Mel opt-H10 api m 3.
FIG. 5A shows the sequence information of potential peptide fragments of acid phosphatase publicly known prior to this invention. Peptide fragments are listed in order of alignment to human and rat prostate phosphatase as published by Hoffman (1996). The alignment order of fragments to derived sequence is given in the second column. Positions of aligned peptide segments can be taken from FIG. 3 and FIG. 5B, as well as FIG. 6. Highlighted sequence segments in the third column show amino acids present in the Api m 3 sequence. The forth column shows the length of the published peptide fragments.
FIG. 5B shows the corrected alignment of peptides originally postulated by Hoffmann (1996) to Api m 3 and as seen in SEQ ID NOs 17-23.
FIG. 6 shows a schematic alignment of peptides postulated by Hoffman et al. (1996) (A), in comparison to the corrected order after cloning and sequencing of the Api m 3 gene (B). It is obvious that the alignment differs from the published alignment with human and rat prostate phosphatase (Hoffman 1996). The published peptide fragments can not be aligned to match the sequence as would be expected. Firstly, the order of alignment positions is different from the publication. Secondly, some fragments, like fragments 1 and 7 partially align at different sites in the sequence, and therefore are not continuous peptides derived from a cDNA sequence. Furthermore, some published fragment sequences, like fragment 5, cannot be aligned at all. The scheme also shows the leader peptide and is not exact regarding the number of amino acids.
FIG. 7 depicts recombinant Api m 3 expression and purification. Shown is a 10% silver stained SDS-PAGE gel. Lane 1, protein molecular weight standards; lane 2, diluted bee venom; lane 3, purified recombinant Api m 3 derived from insect cell expression; lane 4, supernatant from cells stably transfected with recombinant Api m 3.
FIG. 8 Alignment of Api m 3 to acid phosphatase sequences. Shown is the alignment of cloned Api m 3 to different insect acid phosphatases with significant homology. The highest homology with 35% is found for Acph-1 from D. melanogaster. Amino acids necessary for acid phosphatase activity and for glycosylation are shaded in grey. The sequence motif ‘RHGXRXP’ is listed as SEQ ID NO: 41.
FIG. 9 Results from MALDI-TOF spectrometry in comparison with predicted tryptic fragments. Experimental data are in accordance with the prediction.
FIG. 10 shows the enzymatic activity of purified recombinant Api m 3. Shown is the acid phosphatase enzymatic activity of recombinant Api m 3 dependent on the amount of protein used. The experiment was performed according to Barboni et al (1987).
FIG. 11 shows an IgE immunoblot of pooled honey bee venom-reactive patient serum with recombinant Api m 3. Lane 1, protein molecular weight standards; lane 2, diluted bee venom; lane 3, purified recombinant Api m 3 derived from insect cell expression.
FIG. 12A Immunoreactivity of 59 individual patient sera with recombinant Api m 3. Shown are the results of an ELISA assay measuring the IgE antibody reactivity with Api m 3. FIG. 12A shows the results for an ELISA assay measuring the IgE antibody reactivity with Api m3 for 40 honey bee venom-sensitized patients (1-40; sIgE to honey bee venom >0.35 kU/L).
FIG. 12B shows the results for an ELISA assay measuring the IgE antibody reactivity with Api m 3 for 19 honey bee venom-negative patients (41-50; sIgE to honey bee venom <0.35 kU/L and to vespid venom >50 kU/L) (51-59; sIgE to honey bee and vespid venom <0.35 kU/L).
FIG. 12C shows an 8-point calibration ELISA standard for total human IgE (31.25; 62.5; 125; 250; 500; 1,000; 2,000; 4,000 pg/ml) for an ELISA assay measuring the IgE antibody reactivity with Api m 3.
FIG. 13 shows the detection of native Api m 3 with IgE from sera of honeybee venom-allergic individuals. Experimental conditions are described in Example 7. Shown are data after subtraction of background values.
FIG. 14 shows the detection of prokaryotically produced Api m 3 with IgE from sera of honeybee venom-allergic individuals. Experimental conditions are described in Example 6. Shown are data after subtraction of background values.
FIG. 15 shows the detection of Api m 3 produced in insect cells (HighFive insect cells in A and Sf9 insect cells in B) with IgE from sera of honeybee venom-allergic individuals. Experimental conditions are described in Example 6. Shown are data after subtraction of background values.
FIG. 16 shows the detection of Api m 3 produced in Sf9 and HighFive insect cells with IgE from sera of patients allergic to both honeybee and wasp venom. Experimental conditions are described in Example 6. Shown are data after subtraction of background values.



DETAILED DESCRIPTION
This problem is solved by the subject matter of the claims.
Specific immunotherapy (desensitization) approaches are well known in the state of the art. In principle, repeated treatments of allergic individuals with suitable, normally progressively increased doses of allergen diverts the immune response to one dominated by T cells that favour the production of IgG and IgA antibodies over production of IgE antibodies. The IgG and IgA antibodies are thought to desensitise the subject by binding to the small amounts of allergen normally encountered, and preventing the allergen from binding to IgE. Desensitisation to honeybee venom is relatively successful (e.g., Hunt et al 1978).
However, there are serious limitations to the use of currently available allergen preparations for specific immunotherapy. While multiple studies have demonstrated that successful SIT requires administration of high doses of allergens, effective dosages are limited by potential systemic reactions. As a result, specific immunotherapy usually requires a treatment period of 2 to 3 years, over which the allergen preparation is administered at slowly increasing dosages followed by several injections of the final maintenance dose. Since the compliance of patients and doctors is very low due to the tedious and potentially harmful procedure, there is a need in the field for modified allergens capable of providing protection without the danger of serious side-effects.
In order to avoid undesirable systemic reactions on specific immunotherapy with natural allergens, there has been continued interest in the development of modified allergens with reduced allergenic activities for immunotherapy. In one approach T cell epitopes are used to modulate allergen-specific immune responses. It has been observed in vivo in mice for the allergen Fel d 1 (cat hair), Der p 1 (acarian: Dematophagoides pterissimus) and Bet v 1 (birch pollen) that the nasal, oral or subcutaneous administration of peptides carrying T cell epitopes of these allergens inhibits the activation of the specific T lymphocytes (Briner et al 1993; Hoyne et al 1993; Bauer et al 1997). Based on these results allergen peptide fragments capable of stimulating T lymphocytes in allergic patients were evaluated in clinical sudies. In the case of the major honeybee venom allergen Api m 1 fragments 50-69 and 83-97 have been described as being active during a study comprising a single patient (Dhillon et al. 1992). In a study comprising forty patients (Carballido et al 1993) Api m 1 fragments 45-62, 81-92 and 113-124 proved to be active. However, these three fragments proved to be T cell epitopes for only 25 to 45% of the patients, pointing to the existence of other epitopes. Nevertheless, the three peptides have been used successfully for desentization of five allergic patients whose T lymphocytes proliferated in the presence of these peptides (Müller et al 1998). No serious systemic effect was observed and the patients became tolerant to honeybee stings. This demonstrates the benefit of using peptides for desensitization. Therefore, there is a need in the field to identify peptide fragments of Api m 3 capable of stimulating T lymphocytes in patients allergic to honeybee venom.
In another approach, B cell epitopes of allergens are modified to decrease the risk of potential systemic reactions. B cell epitopes of poteinaceous allergens can include native protein structures (conformational or discontinuous or topographic epitopes), linear peptides (linear epitopes) and carbohydrates. The conformational type consists of amino acid residues which are spatially adjacent but may or may not be sequentially adjacent. The vast majority of IgE epitopes has been reported to be of the conformational type (King 1990). The linear type consists of only sequentially adjacent residues. However, even linear B cell epitopes are often conformation-dependent, and antibody-antigen interactions are improved when the epitope is displayed in the context of the folded protein. It is believed that the entire accessible surface of a protein molecule can be recognized as epitopes by the antigen receptor of B cells, although all epitopes are not necessarily recognized with equal likelihood (Benjamin et al 1984).
The aim of modification of B cell epitopes is to decrease the allergenicity while retaining its immunogenicity. Since allergenicity depends on the interaction of a multivalent allergen with basophil- or mast cell-bound IgE antibodies, allergenicity can be reduced by decreasing the density of B cell epitopes. One approach is by partial or complete denaturation of allergens on chemical modification because the vast majority of B cell epitopes are of the discontinuous type, being dependent on the native conformation of proteins. However, there are serious limitations to the use of such molecules. While linear T cell epitopes are preserved, the surface structure is not maintained and, thereby, the capacity of such molecules to stimulate an allergen-specific non IgE antibody response is severely limited. Similar considerations apply to an approach in which the accessibility of B cell epitopes is reduced by polymerization on formaldehyde or glutaraldehyde treatment or by attachment of non-immunogenic polymers. Usually near-complete loss of the discontinuous B cell epitopes occurs when allergens are modified with >100-fold reduction in allergenicity.
A more promising approach is to modify by site-directed mutagenesis identified discontinuous B cell epitopes recognized by IgE antibodies. While several IgE epitopes could be determined by mapping with synthetic overlapping peptides synthesized according to the allergen amino acid sequence, many relevant IgE epitopes could not be identified because peptides frequently fail to display conformations mimicking discontinuous epitopes. Programs have been developed for the prediction of both linear and conformational B cell epitopes (Zhang et al 2008). For example, DiscoTope is a method for discontinuous epitope prediction that uses protein 3D structural data as input. It is based on amino acid statistics, spatial information and surface accessibility for a set of discontinuous epitopes determined by X-ray crystallography of antibody-proteinaceous antigen-complexes. However, available data are limited and not suited for a reliable identification of epitopes of the conformational type on the Api m 3 molecule. There is no doubt that naturally occurring IgE antibodies represent ideal tools for structural analyses of IgE epitopes. However, the number of monoclonal allergen-specific IgE antibodies isolated from blood lymphocytes of allergic patients so far is extremely limited. In an alternative approach, animal derived monoclonal allergen-specific antibodies can be useful to identify IgE epitopes. For example, from a panel of mouse monoclonal antibodies that effectively inhibited binding of birch pollen allergen Bet v 1 to specific IgE, several monoclonal antibodies identified a continuous epitope within an exposed surface area of Bet v 1 that could be part of a discontinuous IgE epitope (Lebecque et al 1997) Provided such antibodies bind to Bet v 1 with high affinity, they represent useful tools for further structural analyses by X-ray diffraction of crystals obtained from allergen-antibody complexes. Although the surface area recognized by animal-derived allergen-specific antibodies may not be identical with that recognized by human IgE antibodies, both areas are closely related as indicated by the inhibition experiments. Therefore, structural information obtained from the analysis of such allergen-antibody complexes provide a valuable basis for the modification of IgE epotopes by site-directed mutagenesis. One problem of this approach, however, is the need of a panel of high affinity antibodies with different epitope specificities for each allergen to allow for a detailed analysis of the total spectrum of potential IgE epitopes. Assuming that a B cell epitope takes up an area of approximately 900 A2, the vast majority of allergens is likely to display more than one IgE epitope. Therefore, there is a need in the field to develop high affinity Api m 3-specific antibody panels that are capable of inhibiting IgE binding.
Another serious problem associated with the design of a hypoallergenic Api m 3 molecule for an improved immunotherapy is the lack of understanding of the immune response that guarantees a lasting protection after specific immunotherapy. The aim to decrease the allergenicity of a given allergen while retaining its immunogenicity is widely accepted, but the term immunogenicity remains to be defined. Evaluation of modified recombinant allergens with a strongly reduced IgE reactivity that display the full spectrum of linear T cell epitopes but a different surface structure as compared to the corresponding natural allergen, have demonstrated that such molecules are capable of reducing specific IgE development towards the native allergen (Niederberger et al 2004; Karamloo et al 2005) However, a long lasting protective effect after treatment with these molecules has not been demonstrated. Apparently, the capacity of recombinant allergens to stimulate a long lasting protective allergen-specific non IgE antibody response requires also a surface structure that is closely related to that of the corresponding natural allergen. Since disruption of IgE epitopes is associated with a significant alteration of the surface structure, there is a need in the field to identify those surface structures of allergens that mediate an appropriate non-IgE response for a long lasting protection. There is a particular need in the field to identify those surface structures of Api m 3 that mediate an appropriate non-IgE response for a long lasting protection.
In particular, the present invention provides a nucleic acid encoding a polypeptide capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera wherein the polypeptide has a homology of more than 70% to the amino acid sequence of SEQ ID NO: 2 (note: “SEQ ID NO” relates to code <400> in the attached sequence listing under WIPO standard ST.25).
In one embodiment, the nucleic acid comprises a sequence homologous to the sequence of SEQ ID NO: 1 (derived from Apis mellifera), which is a naturally occurring homologous sequence from an other insect from the order Hymenoptera. The invention also refers to the recombinant proteins encoded by the nucleic acids of the invention.
Preferentially, the degree of homology to the amino acid sequence of SEQ ID NO: 2 is more than 75%, more than 80%, more than 85%, more than 90%, more than 95% or more than 99%. The sequence homology is determined using the clustal computer program available from the European Bioinformatics Institute (EBI). Most preferentially, the polypeptide encoded by the nucleic acid has the amino acid sequence of SEQ ID NO: 2. This polypeptide is designated Api m 3. In particular, the nucleic acid comprises or has the nucleotide sequence of SEQ ID NO: 1.
In the context of this application, sequence identity is used interchangeably with homology.
In the context of the present invention, the terms “polypeptide” and “protein” are used interchangeably, without any limitation as to the number of amino acids linked. The polypeptides may also comprise non-naturally occurring amino acids.
Throughout this specification, the polypeptides encoded by the nucleic acid of the invention have to be capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera. Of course, the skilled person understands that this binding takes place in the area of sequence identity or homology.
Following unsuccessful attempts to clone the full length sequence of Api m 3 following the common deduced primer strategy (cf. Soldatova et al., 2000) based on the peptide fragments found by Hoffmann, 1996, and their postulated sequence (Hoffmann, 1996). A completely different approach was chosen in the present invention. Using nucleic acid sequences derived from the peptide sequences published by Hoffman, small virtual probes of partial deduced sequences were constructed, which were used to scan the published bee genome for targets. Two regions on different truncated chromosome 16 sequences matched the probes. Scanning of these regions with bioinformatic tools revealed possible open reading frames and gene sequences. Scanning for a potential sequence cleavage site led to the putative N-terminus of the Api m 3 gene. Primers were then designed according to the proposed protein N- and C-terminus. These primers were used to amplify the gene from bee venom gland cDNA synthesized from total RNA with oligodT(20) primers. The amplification was successful and resulted in a DNA fragment of the expected size. The identity of the DNA was verified by sequencing, molecular weight calculation and alignment to the homologous acid phosphatases of human as well as rat sequences and the proposed peptide fragments. The protein identity was also verified.
From the resulting full length cDNA sequence, it is clear the classical approach had to fail, as Hoffman erred in several points. For example, he chose an incorrect alignment of the peptide fragments (cf. FIG. 5), erroneously thought that several peptides that are in fact separated by other peptides were contiguous, and postulated existence of a fragment that does not belong to the acid phosphatase. In fact, using primers based on peptides 1 and 7, as proposed by Hoffman, one would expect to amplify a short fragment covering only the peptide sequence around amino acids 200 and 230 of the consensus sequence.
The social insects from the order Hymenoptera that commonly interact with man are members of the superfamilies Apoidea and Vespoidea, bees and wasps (Hoffman 1996). The Vespoidea include the social wasps and hornets, Vespidae, as well as ants, Formicidae. Important wasps comprise yellowjackets of the genus Vespula, hornets of the genera Dolichovespula and Vespa and paper wasps of the genus Polistes. Bees comprise, e.g., honey bees, Apis mellifera, and bumble bees of the species Bombus terrestris. In the context of the present invention, an insect from the order Hymenoptera can be from any of these species, but according to a particular embodiment, the insect is a bee from the genus Apis. Most preferably, the bee is the honey bee, Apis mellifera. 
Other species from the order Hymenoptera produce similar allergens with antigenic cross reactivity and a high degree of amino acid homology (Wypych et al 1989, Castro et al 1994, Hoffman et al 1988). Thus the present invention not only extends to the Api m3 allergen from Apis mellifera but also to homologous Hymenoptera allergens.
In particular, the polypeptides encoded by the nucleic acids of the invention have to be capable of binding to IgE from subjects allergic to venom of Apis mellifera. The subjects are commonly reactive to the Api m3 antigen, acid phosphatase from bee venom. For the purpose of testing, serum or purified IgE from such allergic subjects are contacted with the polypeptide, and specific binding of the polypeptide to the antibodies is detected. Such a test can, e.g., be an ELISA. For verifying the reactivity of the polypeptides with IgE antibodies, serum or IgE from several subjects are pooled, preferentially, from 5 to 20 subjects.
The nucleic acids of the invention can be either DNA or RNA.
In one embodiment, the invention also provides a nucleic acid, which is a fragment having a length of more than 255 nucleotides of a nucleic acid encoding a polypeptide having a homology of more than 70% to the amino acid sequence of SEQ ID NO: 2, wherein the fragment encodes a polypeptide capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera. Preferably, the nucleic acid is a fragment having a length of more than 255, more preferably of more than 600, more than 700 or more than 800 nucleotides of a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
In another embodiment, a nucleic acid fragment (polynucleotide) is provided that comprises at least 15 contiguous nucleotides of the nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2, wherein the polynucleotide is selected from the group consisting of nucleotides 78 to 299, 348 to 437, 459 to 476, 555 to 671, 696 to 830 or 1086 to 1121 of said nucleic acid, wherein the numbering corresponds to the region encoding said polypeptide. Specifically, said nucleic acid has the nucleotide sequence shown in SEQ ID NO: 1. Preferentially, the nucleotides are from the region of nucleotides 555 to 671 or 696 to 830. Alternatively, the nucleic acids encode polypeptides that are capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera, and comprise at least 15, preferably at least 18, 21, 24, 27, 30, 45, 60 or more nucleotides of a nucleic acid more than 70%, more than 80% or more than 90% homologous or identical to the nucleic acid shown in SEQ ID NO: 1, except for the nucleic acids from the group consisting of nucleotides 1 to 104, 189 to 142, 300 to 347, 426 to 449, 504 to 530, 672 to 719 and 774 to 1031 of the nucleic acid shown in SEQ ID NO: 1 or except for the nucleic acids encoding the polypeptides shown in FIG. 5. Additionally, such nucleic acids consisting of nucleotides 1 to 77, 438 to 458, 477 to 494, 504 to 554, 672 to 695, and 831 to 1085 of the nucleic acid shown in SEQ ID NO: 1 are provided.
Preferentially, the nucleic acid comprises 15 to 240, 15 to 90, 18 to 60, 21 to 30, more preferably at least 18, 21, 24, 27, 30, 60, 90 or more contiguous nucleotides from the above regions.
Alternatively, a nucleic acid is provided which encodes a polypeptide having more than 70% homology to the polypeptide encoded by said at least 15 contiguous nucleotides, wherein the polypeptide is capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera. In particular, this polypeptide comprises at least 5, preferably at least 6, 7, 8, 9, 10, 15, 20 or more amino acids of a polypeptide more than 70%, more than 80% or more than 90% homologous or identical to a polypeptide selected from the group consisting of amino acid 26 to 99, 116 to 145, 153 to 158, 185 to 223, 232 to 276 or 362 to 373 of the polypeptide shown in SEQ ID NO: 2. Alternatively, the polypeptides encoded by the nucleic acids are capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera, and comprise at least 5, preferably at least 6, 7, 8, 9, 10, 15, 20 or more amino acids of a polypeptide more than 70%, more than 80% or more than 90% homologous or identical to the polypeptide shown in SEQ ID NO: 2, except for the polypeptides from the group consisting of amino acids 1 to 34, 63 to 80, 100 to 115, 142 to 149, 168 to 176, 224-239 and 258 to 343 of the polypeptide shown in SEQ ID NO: 2 or except for the polypeptides shown in FIG. 5. Additionally, such polypeptides consisting of amino acids 1 to 25, 146 to 152, 159 to 164, 168 to 184, 224 to 231, and 277 to 361 are encoded by the nucleic acids.
In one embodiment, the invention also provides a polypeptide encoded by a nucleic acid of the invention. Preferentially, the polypeptide is a full length acid phosphatase from the venom of an insect from the order Hymenoptera. In particular, the polypeptide has an homology of more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95% or more than 99% to the amino acid sequence of SEQ ID NO: 2. Most preferred is a polypeptide having the amino acid sequence of SEQ ID NO: 2. Although not essential, it is preferred that the polypeptide has acid phosphatase activity. This activity can be tested, e.g., according to the method described by Barboni et al 1987.
Alternatively, the polypeptide is a fragment of the full length protein capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera having a length of more than 85, more than 200 or more than 250 amino acids. Other fragments are provided, wherein the polypeptides are capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera, and comprise at least 5, preferably at least 6, 7, 8, 9, 10, 15, 20 or more amino acids of a polypeptide more than 70%, more than 80% or more than 90% homologous or identical to a polypeptide selected from the group consisting of amino acid 26 to 99, 116 to 145, 153 to 158, 185 to 223, 232 to 276 or 362 to 373 of the polypeptide shown in SEQ ID NO: 2. Alternatively, the polypeptides are capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera, and comprise at least 5, preferably at least 6, 7, 8, 9, 10, 15, 20 or more amino acids of a polypeptide more than 70%, more than 80% or more than 90% homologous or identical to the polypeptide shown in SEQ ID NO: 2, except for the polypeptides from the group consisting of amino acids 1 to 34, 63 to 80, 100 to 115, 142 to 149, 168 to 176, 224-239 and 258 to 343 of the polypeptide shown in SEQ ID NO: 2 or except for the polypeptides shown in FIG. 5. Additionally, such polypeptides consisting of amino acids 1 to 25, 146 to 152, 159 to 164, 168 to 184, 224 to 231, and 277 to 361 are provided.
In a preferred embodiment, the invention provides T-cell epitope-containing oligopeptides of at least 9 amino acids corresponding to a consecutive amino acid sequence within the Api m 3 molecule wherein the peptides are capable of stimulating T-cells of subjects allergic to Api m 3. Such peptides of the invention are preferably immunomodulatory peptides as well in that they induce T-cell anergy when administered to a subject allergic to Api m 3, or otherwise affect the immune response of the subject. Preferably, the amino acid sequence of the T-cell epitope-containing oligopeptide corresponds to a consecutive amino acid sequence of a polypeptide having the amino acid sequence of SEQ ID NO: 2, wherein the T-cell epitope-containing oligonucleotide is selected from the group consisting of 15 contiguous amino acid residues as defined in Tables 3 and 4 of said polypeptide, wherein the numbering corresponds to the region of said polypeptide.
T-cell stimulating activity can be tested by culturing T-cells obtained from an individual sensitive to the Api m 3 polypeptide, fragments, and analogs thereof described herein, with the Api m 3 polypeptide, fragments, and analogs thereof, and determining the presence or absence of proliferation by the T-cells in response to the peptide as measured by, for example, uptake of tritriated thymidine. Stimulation indices for responses by T-cells to peptides useful in methods of the invention can be calculated as the maximum counts per minute (cpm) taken up in response to the peptide divided by the cpm of the control medium. For example, a peptide derived from a protein allergen may have a stimulation index of about 2.0. As a result, a stimulation index of at least 2.0 is generally considered positive for purposes of defining peptides useful as immunotherapeutic agents. Preferred peptides have a stimulation index of at least 2.5, more preferably at least 3.5 and most preferably at least 5.0.
Preferably, the polypeptide of the invention is recombinantly expressed. This has the advantage, e.g., that the polypeptide can be expressed as a fusion protein linked to an additional polypeptide. For example, the polypeptide or fusion protein is attached to a signal sequence ensuring its secretion into the extracellular space or supernatant of the cultured cells, where appropriate. Due to novel techniques in molecular biology, the use of recombinant proteins in therapy and diagnostics is expected to increase the efficiency and diagnostic value in these medical applications (King 1990, Müller 2001, Müller 2002).
Depending on the host cell producing the recombinant protein, the protein is glycosylated (after expression in mammalian or yeast cells) or non-glycosylated (after expression in bacterial cells). The glycosylation pattern can vary depending on the host cell used, and can thus differ from the glycosylation pattern of natural acid phosphatase isolated from bee venom. In one alternative, the glycosylation pattern is identical to the glycosylation pattern of acid phosphatase isolated from bee venom. Glycosylation can have profound effects on the binding of specific antibodies.
When expressed in bacterial cells, the polypeptide of the invention lacks glycosylation. The protein thus differs from the native protein in respect to epitope presentation, and potentiality for folding and functionality. It was shown that carbohydrates can represent IgE epitopes and contribute to observed non-specific cross-reactivity of allergens, e.g., between bee and wasp proteins, due to similar features of the carbohydrate chains (Huby et al 2000, Tretter et al 1993, Hemmer et al 2004). The cross-reactivity is one reason for false positive results in in vitro immunological tests (Petersen and Mundt 2001). Expression of the non-glycosylated polypeptide eliminates these false positives, and can therefore be used to advantage in diagnostic and therapeutic applications.
The glycosylation pattern in eukaryotic cells other than insect cells, e.g., in mammalian cells, also varies from the glycosylation pattern of the native protein (Jenkins et al 1996). Even in insect cells, the glycosylation pattern is likely to be different due to overexpression of the protein.
Sequence analysis of Api m 3 shows that the protein comprises three putative glycosylation sites of the sequence Asn-Xaa-Ser/Thr. In one embodiment, the polypeptides of the invention comprise mutated glycosylation sites instead of glycosylation sites. In particular, in a mutated glycosylation site, the Asparagine (Asn) in the glycosylation site(s) can be exchanged against any other amino acid, preferably against Glutamine (Gln) (Elbein et al 1991). Alternatively, in a mutated glycosylation site, the Serine (Ser) can be exchanged against another amino acid or deleted. Accordingly, the invention also provides a nucleic acid encoding a polypeptide of the invention comprising at least one, preferably 2, or 3 mutated glycosylation sites instead of glycosylation sites. Most preferably, all glycosylation sites are mutated.
Using native Api m 3 in diagnostic assays for detecting allergy, e.g., to bee or wasp venom, cross-reactivity is a big problem. Based on the state of the art using native purified Api m 3 as an antigen in diagnostic tests, the skilled person was unable to differentiate between patients that had been sensitized to bee venom, patients that had been sensitized to wasp venom, and patients that had been sensitized to both. This differentiation is important, because, in case, e.g., a bee allergy is incorrectly diagnosed, a desensibilization therapy might be prescribed which then in fact serves to sensitize the patient to epitopes of bee allergen he was not previously allergic to.
The present invention now allows preparation of recombinant proteins that are useful in diagnostic tests to differentiate between such patients, because it provides recombinant antigens that are not bound by sera of some patients previously diagnosed as allergic to native Api m3 of honey bee venom. The expressed proteins exhibit epitopes that react with IgE antibodies to native Api m 3, but they do not react with all IgE antibodies that bind to native Api m 3.
As mentioned above, cross-reactivity is mainly due to the glycosylation of the bee protein, the sugar patterns being similar to glycosylation of e.g., wasp proteins.
The inventors have shown that unglycosylated Api m 3, e.g., expressed in procaryotes, provides IgE epitopes as the proteinaceous part of native Api m 3. Furthermore, as shown in FIG. 14, both prokaryotic Api m 3-fusion constructs exhibit a different reactivity to IgE in sera from patients with honeybee venom allergy. Based on these data both constructs provide a different set of IgE epitopes indicating a different folding structure. Such fusion proteins are extremely valuable in assessing sensitization of patients to the proteinaceous part of Api m 3. The differential reactivity of both fusion proteins to IgE antibodies as compared to the reactivity of native Api m 3 purified from bee venom, clearly demonstrates that recombinant, e.g., non-glycosylated Api m 3 fusion proteins provide novel means to eliminate carbohydrate mediated cross-reactivity, thereby eliminating potentially false positives in the diagnosis of honeybee venom allergy.
The results shown in the examples also demonstrate, that, similarly, recombinant Api m 3 molecules expressed in HighFive and SF9 insect cells are recognized to a different extent by IgE in sera from patients allergic to both honeybee and wasp venom.
As explained above, recombinant Api m 3 molecules expressed in insect cells (e.g., HighFive cells and SF9 cells) are glycosylated, but the glycosylation pattern provided by both insect cell lines to Api m 3, exhibits significant differences. As shown in FIG. 15, the two glycosylated Api m 3 molecules expressed in HighFive insect cells and SF9 insect cells exhibit a different reactivity to IgE in sera from patients with honeybee venom allergy. Furthermore, FIG. 16 demonstrates that both molecules are recognized to a different extent by IgE in sera from patients allergic to both honeybee and wasp venom. This observation is important, since both molecules allow an improved evaluation of carbohydrate based cross-reactivity of IgE antibodies.
In contrast to the data obtained with Api m 3 produced in HighFive insect cells, IgE antibodies in sera from patients allergic to both honeybee and wasp venom recognize Api m 3 produced in SF9 insect cells to a much lesser extent (see FIG. 16). Although Api m 3 produced in SF9 insect cells is also recognized by IgE in 15 of 23 (65%) of these sera, the IgE reactivity is very low as compared to the IgE reactivity towards Api m 3 produced in HighFive insect cells. The residual reactivity of IgE antibodies in sera from patients allergic to both honeybee and wasp venom could be due to those patients possessing IgE antibodies recognizing the proteinaceous part of Api m 3. The recombinant proteins produced according to the invention thus for the first time allow differentiation between allergic patients having antibodies binding to different epitopes of the antigen, which can lead to clearer diagnosis of allergies and potential cross-reactivity.
The present invention also relates to an expression vector comprising a nucleic acid of the invention operationally linked to an expression control sequence. In one alternative, the nucleic acid is linked in frame to a nucleic acid encoding an additional polypeptide, so the expression vector can be used for expression of a fusion protein. The additional polypeptide can be selected from the group comprising a poly-Histidine tag (His tag), glutathione-S-transferase, β-galactosidase, a cytokine, and an IgG-Fc. In particular, tags that simplify purification of the recombinant protein, e.g., a His tag, are employed. Such a tag may be cleaved off after purification of the protein.
Alternatively, it can be beneficial for therapeutic applications to express the polypeptide of the invention linked to a therapeutic polypeptide, e.g. a cytokine. For example, a fusion protein with a cytokine enhancing TH1 and downregulating TH2 responses or inducing class switch to IgG, such as IFN-γ, IL-10, IL-12 or TGF-β, can improve efficiency of desensitisation. If the expression vector is used for gene therapy, it is envisaged to use sequences rich in CpG (unmethylated cytosine guanidine dinucleotides), which promote TH1 responses. Additionally or alternatively, the polypeptide of the invention can be linked to another polypeptide or protein, such as in the form of a fusion protein or as separate proteins expressed by the same vector. Preferably, the further polypeptides or proteins are other Hymenoptera venom proteins or antigenic fragments thereof.
The expression vector can be suitable for expression in different cell types, such as bacterial, yeast or mammalian cells. Preferentially, the vector is suitable for expression in insect cells, e.g., HighFive insect cells (Invitrogen, Karlsruhe, Germany). Alternatively, especially for gene therapy applications, the vector is suitable for expression in human cells. In this context, the expression of the encoded polypeptide can be directed by the choice of a suitable expression control sequence, e.g., an expression control sequence mainly or specifically operational in different cell types, such as lymphoid cells, for example dendritic cells, B cells or macrophages.
In one embodiment of the invention, the expression vector is pIB/V5-His (Invitrogen, Karlsruhe, Germany, Invitrogen Manual: InsectSelect BSD System with pIB/V5-His, Version G, 30 May 2003).
In particular, the vector can be pIB/Mel opt-H10-Api m3, comprising the Api m3 cDNA sequence (SEQ ID NO: 1), which was modified to facilitate isolation and purification. A melittin signal sequence for secretion of the recombinant protein was added and the Kozak sequence was optimised for higher expression rates in insect cells (see FIG. 4 and Example 2). Alternatively, other signal sequences can be used for secretion of the protein. The expression vector can also be a different plasmid or a viral, e.g., baculoviral or adenoviral, vector. The expression vector further comprises a stop codon and a polyadenylation signal.
The present invention further relates to a host cell comprising said expression vector. This host cell can be a bacterial, yeast or mammalian cell, in particular an insect cell.
A method of producing a polypeptide encoded by a nucleic acid of the invention is provided, wherein the host cell is cultured under appropriate conditions for expression of said polypeptide and said polypeptide is purified. If the polypeptide is a fusion protein with a fusion partner facilitating purification, e.g., a His Tag or a GST-tag, a corresponding affinity column can be used for purification, e.g., a Ni2+ or glutathione affinity column. For purification of an IgG fusion protein, a protein A or protein G column is suitable.
The expression vector of the invention can be used for the preparation of a pharmaceutical composition for treating subjects allergic to the venom of an insect from the order Hymenoptera. Treatment regimens using gene therapy approaches to desensitisation are known in the state of the art (e.g., Sudowe et al 2002).
The present invention also relates to a mutant Api m 3 molecule comprising a reduced IgE binding capacity with limited impairment of the residual surface structure important for IgG and IgA immunological responses.
In one embodiment, the present invention provides methods for identification and modification via site-directed mutagenesis of those amino acid residues involved in the interaction of the polypeptides of this invention with human IgE, IgG and IgA antibodies. In particular, the present invention provides compositions comprising recombinant antibodies wherein each composition is capable of binding to all epitopes recognized by human IgE, IgG (including IgG4) and IgA antibodies, a method of obtaining such a composition and the use of individual antibodies of such a composition as tools for the design of a hypoallergenic Api m 3 molecule for specific immunotherapy.
In a specific embodiment, antibody compositions capable of binding to all epitopes of the Api m 3 polypeptide, fragments and analogs thereof that are recognized by human IgE antibodies, are utilized to identify and modify by site-directed mutagenesis those amino acid residues involved in the interaction with allergen-specific human IgE antibodies, thereby eliminating or decreasing the allergenicity of the Api m 3 polypeptide, fragments and analogs thereof in a structure-based approach. By site-directed mutagenesis of amino acid residues essential for the allergen-IgE antibody interaction, IgE epitopes are eliminated with minimal impairment of the residual surface structure important for a non-IgE immunological response.
In another specific embodiment, antibody compositions capable of binding to all epitopes of the Api m 3 polypeptide that are recognized by human IgG antibodies, including IgG4 antibodies, and IgA antibodies are utilized to maintain those structures that mediate an appropriate non IgE response for a long lasting protection after specific immunotherapy (SIT). This rational is based on the recent observation that immune deviation towards T regulatory (Treg) cells is an essential step in successful SIT (for a review, see Jutel et al 2006). Treg cells are defined by their ability to produce high levels of IL-10 and TGF-β and to suppress naive and memory T helper type 1 and 2 responses. There is now clear evidence that IL-10- and/or TGF-β-producing type 1 T regulatory cells are generated in humans during the early course of SIT. Since Treg cells have been shown to differentiate from naive T cells in the periphery upon encountering antigens present at high concentrations, it can be assumed that Treg cells are also induced by high and increasing doses of allergens. Most important is the fact that IL-10 and TGF-β suppress directly or indirectly effector cells of allergic inflammation such as basophils and mast cells, induce the production of non-inflammatory immunoglobulin isotypes (IgG and IgA) and suppress IgE production. Based on these observations, antibody compositions capable of binding to all epitopes of the Api m 3 polypeptide, fragments and analogs thereof that are recognized by human IgG, particularly by human IgG4, and by IgA antibodies are utilized to identify and maintain those amino acid residues involved in the interaction with allergen-specific human IgG and IgA antibodies.
In the context of this invention, the term “epitope recognized by human IgE (IgE epitope), human IgG (IgG epitope), including human IgG4 (IgG4 epitope), or human IgA (IgA epitope)”, or relates to the surface area of an allergen that is in contact to these antibodies upon binding to the allergen. It also relates to the surface area of the allergen that is in contact with an antibody construct comprised in the composition of the invention, that overlaps with the first-mentioned IgE epitope, IgG epitope, including IgG4 epitope, or IgA epitope”, so binding of the antibody construct can inhibit binding of the human IgE, human IgG, including human IgG4; or human IgA from the sera of patients allergic to the allergen (IgE related epitopes, IgG-related epitopes, IgG4 related epitopes, IgA-related epitopes). Preferably, the epitopes overlap by 20% or more, 50% or more, 60% or more, 70% or more, or 80% or more. Most preferably, the epitopes overlap by 90 or 95% or more or are identical. With reference to the number of epitopes of an allergen, the first-mentioned epitopes and the related epitopes are considered to represent the same epitopes.
For an estimation of the number of antibodies sufficient for binding to all epitopes recognized by human IgE, human IgG (including human IgG4), or human IgA antibodies on the Api m 3 polypeptide, it is important to know the approximate number of possible B cell epitopes per allergen. Therefore, methods for estimating the number of B cell epitopes per allergen have been developed. These methods are based on the following parameters:
a) Calculation of the surface of structurally characterized allergens in A2: The solvent accessible surfaces of proteins can be calculated with the aid of POPS (parameter optimized surfaces) according to Fraternali and Cavallo (2002).
b) Surface area of B-cell epitopes in A2: At the moment, one co-crystallization of allergen and antibody is available only, namely for the allergen Bet v 1 and a murine allergen-specific Fab-fragment. The surface area of this discontinuous epitope is 931 A2 (Mirza et al 2000). This correlates well with the area of other B cell epitopes (circa 2×3 nm).
In Table 5, the surface of allergens for which structural data is available in the protein data bank (PDB) was calculated with the aid of a molecule of water. Under the assumption that a B cell epitope takes up an area of 950 A2, the maximal possible number of B cell epitopes for an allergen (without differentiation for IgE epitopes, IgG epitopes, or IgA epitopes) was determined. The number calculated in this way is much too high, but can be considered to provide an upper limit for the number of necessary antibody constructs for an allergen. On the basis of this data, an approximate relation between molecular weight and potential B cell epitopes was calculated. The mean value of the upper limit for potential B cell epitopes is approx. 0.5 B cell epitopes per 1 kDa.
Table 6 summarizes allergens that have been examined for IgE binding structures with overlapping oligopeptides. Utilizing overlapping oligopeptides (e.g., decapeptides), more potential IgE epitopes are identified than exist in reality, as the majority of IgE epitopes are discontinuous epitopes composed of at least two different areas of the molecule brought together by folding. Different relevant allergens, such as Phospholipase A2 and the birch pollen allergens Bet v1, Bet v3 and Bet v4 exclusively have discontinuous epitopes (Valenta et al 1998). Since the identified linear epitopes probably only form part of these discontinuous epitopes, for estimation of the number of epitopes it is supposed that at least three linear IgE binding epitopes are, as partial structures, involved in forming a discontinuous IgE epitope. Therefore, the number of identified IgE binding peptides has been divided by three and related to the molecular weight of the allergen. A number of 0.06 to 0.19 epitopes per 1 kDa calculated on the basis of linear IgE binding peptides is preferred. The best estimation is possible on the basis of the number of 0.12 IgE epitopes per 1 kDa, which is possibly still too high but could be considered realistic. The preferred compositions correlate well with known data for Bet v 2 (17.4 kDa), which can be bound by three different monoclonal Fab fragments (without differentiating between IgG epitopes, IgE epitopes, or IgA epitopes; Valenta et al 1998). Bet v 2 has at least two IgE epitopes, since it can induce in vivo cross-linking of surface-bound IgE antibodies.
The plurality of Api m 3-specific monoclonal antibodies can be generated from different sources. Naturally occurring IgE antibodies represent ideal tools for structural analyses of IgE epitopes, but their availability is limited. Cloning and selecting allergen-specific IgE antibodies from the immune repertoire of peripheral blood mononuclear cells of allergic donors is extremely difficult. The low number of IgE-secreting B cells in the peripheral blood of allergic patients (MacKenzie and Dosch 1989) seriously hampers this approach for generating monoclonal IgE antibodies. Cloning and selecting Api m 3-specific IgG antibodies, including IgG4 antibodies, or IgA antibodies from the immune repertoire of peripheral mononuclear cells of allergic donors may be less difficult due to the significantly higher number of IgG- or IgA-secreting B cells in the peripheral blood of allergic patients as compared to IgE-secreting B cells. Currently, however, the availability of human monoclonal allergen-specific IgG4 or IgA antibodies is limited.
Semisynthetic or synthetic immunolibraries (e.g., scFv or Fab format) provide a high degree of variability and, thereby, a valuable alternative for generating the required plurality of Ves v 4-specific monoclonal antibody fragments. However, newly generated immunolibraries derived from animals (mammalian species as well as avian species) after immunization with the Api m 3 polypeptide or fragments thereof provide a significantly higher number of Api m 3-specific variable antibody domains and, thereby, an increased probability for the selection of the required plurality of Api m 3-specific high affinity monoclonal antibody fragments. In a preferred embodiment a combination of immunolibraries derived from avian and mammalian species after immunization with the Api m 3 polypeptide or fragments thereof are used. The phylogenetic difference between avian and mammalian species provides access to a different antibody repertoire than the traditional mammalian antibodies. IgY antibodies recognize other epitopes than mammalian antibodies. Therefore, a combination of immunolibraries from avian and mammalian species provides a significant advantage for generating a plurality of Ves v 4-specific high affinity monoclonal antibodies. If it should—unexpectedly—be found that the combination of all antibodies capable of binding to the Api m 3 polypeptide is not sufficient to effect essentially complete inhibition of binding of Api m 3 to antibodies in a pool serum of patients allergic to said allergen or obtained from said sera, it is recommended to additionally use further antibodies from a different library. Methods for generating immunolibraries are known in the art (e.g., Steinberger et al 1996; Edwards et al 2002; Powers et al 2001; Boel et al 2000).
Each antibody composition is obtainable by a method for generating a composition comprising recombinant antibodies, comprising steps of
a) Generation a plurality of allergen-specific antibodies capable of binding to the Api m 3 polypeptide,
b) combining all generated antibodies and testing whether essentially complete inhibition of binding of the Api m 3 polypeptide to IgE, IgG (including IgG4), and IgA antibodies; in a pool serum of patients allergic to said allergen or obtained from said serum is achieved,
c) in case essentially complete inhibition is not achieved in step b), steps a) and b) are repeated;
d) in case essentially complete inhibition is achieved, the number of antibodies is reduced to the minimal number of antibodies sufficient for essentially complete inhibition by a method wherein
i) groups of the antibodies obtained in step a) are generated, comprising different numbers and combinations of antibodies;
ii) said groups are tested for essentially complete inhibition of binding of the Api m 3 polypeptide to IgE, IgG (including IgG4), and IgA antibodies, in a pool serum of patients allergic to said allergen or obtained from said sera;
iii) wherein, in case one or more group effects essentially complete inhibition in step ii), steps i) and ii) are repeated with sub-combinations of the antibodies from said group or groups until the minimal number of antibodies in said group or groups is identified which effects essentially complete inhibition;
iv) wherein, in case essentially complete inhibition is not achieved in step ii) or iii), steps i), ii) and iii) are repeated with different groups of antibodies;
and wherein the group identified in step d), iii) is said composition. It is preferred that the composition comprises the minimal number of antibodies necessary and sufficient for binding to all epitopes recognized by human IgE, human IgG (including human IgG4), and human IgA antibodies; on the Api m 3 polypeptide. Additional antibodies may, however, be added.
In the method of the invention, inhibition of binding of the Api m 3 polypeptide to IgE can be determined by incubating IgE antibodies in a pool serum of patients allergic to the Api m 3 polypeptide or antibodies obtained from said serum with human basophils after stripping of said basophils, and with or without preincubation of the Api m 3 polypeptide with the recombinant antibodies or recombinant antibody fragments or, for comparison, antibodies in a pool serum of patients allergic to said allergen or obtained from said serum, contacting said basophils with said allergen, and detecting release of histamine.
Alternatively, inhibition can be determined by contacting anti IgE antibodies immobilized on a carrier with antibodies in a pool serum of patients allergic to the Api m 3 polypeptide or obtained from said serum, and, with or without preincubation of labelled Api m 3 polypeptide with the recombinant antibodies or recombinant antibody fragments or, for comparison, antibodies in a pool serum of patients allergic to said allergen or obtained from said serum, contacting the carrier with said allergen and detecting binding of the labelled Api m 3 polypeptide to the carrier. For this purpose, the Api m 3 polypeptide can be labelled with an enzyme, a radioisotope, biotin or a fluorescent marker.
In the method of the invention, inhibition of binding of the Api m 3 polypeptide to IgG can be determined by contacting anti IgG antibodies immobilized on a carrier with antibodies in a pool serum of patients allergic to the Api m 3 polypeptide or obtained from said serum, and, with or without preincubation of labelled Api m 3 polypeptide with the recombinant antibodies or recombinant antibody fragments or, for comparison, antibodies in a pool serum of patients allergic to said allergen or obtained from said serum, contacting the carrier with said allergen and detecting binding of the labelled Api m 3 polypeptide to the carrier. For this purpose, the Api m 3 polypeptide can be labelled with an enzyme, a radioisotope, biotin or a fluorescent marker.
In the method of the invention, inhibition of binding of the Api m 3 polypeptide to IgG4 can be determined by contacting anti IgG4 antibodies immobilized on a carrier with antibodies in a pool serum of patients allergic to the Api m 3 polypeptide or obtained from said serum, and, with or without preincubation of labelled Api m 3 polypeptide with the recombinant antibodies or recombinant antibody fragments or, for comparison, antibodies in a pool serum of patients allergic to said allergen or obtained from said serum, contacting the carrier with said allergen and detecting binding of the labelled Api m 3 polypeptide to the carrier. For this purpose, the Api m 3 polypeptide can be labelled with an enzyme, a radioisotope, biotin or a fluorescent marker.
In the method of the invention, inhibition of binding of the Api m 3 polypeptide to IgA can be determined by contacting anti IgA antibodies immobilized on a carrier with antibodies in a pool serum of patients allergic to the Api m 3 polypeptide or obtained from said serum, and, with or without preincubation of labelled Api m 3 polypeptide with the recombinant antibodies or recombinant antibody fragments or, for comparison, antibodies in a pool serum of patients allergic to said allergen or obtained from said serum, contacting the carrier with said allergen and detecting binding of the labelled Api m 3 polypeptide to the carrier. For this purpose, the Api m 3 polypeptide can be labelled with an enzyme, a radioisotope, biotin or a fluorescent marker.
The inhibition by recombinant antibodies or recombinant antibody fragments is considered essentially complete if it is comparable to the inhibition by IgE, IgG (including IgG4), or IgA antibodies in a pool serum of patients allergic to the Api m 3 polypeptide or obtained from said serum, i.e. if it varies from that inhibition by 20% or less, preferably by 10% or less, or most preferably, by 5% or less.
The pool serum used in the present invention comprises serum from several patients allergic to the Api m 3 polypeptide. Preferably, said pool serum comprises the antibodies from the sera of at least 5 patients, at least 10 patients or at least 15 patients allergic to said allergen. For IgE inhibition experiments, patients are preferred that are highly sensitized to the allergen. For IgG, IgG4, and IgA inhibition experiments, patients after successful SIT are preferred.
IgE antibodies can be obtained from the pool serum, e.g., by affinity chromatography using anti-human IgE antibodies. Preferably, IgG antibodies are removed from the pool serum, e.g. by pre-treatment with a protein A matrix, such as a protein A column. This step, however, is not essential, as sera of allergic patients in all probability only contain relatively low amounts of allergen-specific IgG antibodies, even though the serum level of IgG is about 10.000 times higher than the serum level of IgE. For example, serum obtained fom a birch pollen allergic patients which was purified by affinity chormatography on immobilized Bet v 1, did not contain significant quantities of allergen specific IgG antibodies (Ganglberger et al 2000). Human IgG4 and human IgA antibodies can also be obtained from the pool serum by affinity chromatography using anti-human IgG4 or anti-human IgA antibodies.
The individual antibodies of a generated composition are used for structural analyses of IgE, IgG (including IgG4), and IgA epitopes. Since each composition effects essentially complete inhibition of binding of the Api m 3-polypeptide to patient-derived IgE, IgG (including IgG4), and IgA antibodies, the individual antibodies of each generated composition are capable of identifying all epitopes on the Api m 3 polypeptide that are accessible for patient-derived IgE, IgG (including IgG4), and IgA antibodies.
According to the present invention the most potent Api m 3-related hypoallergenic molecule for specific immunotherapy is an allergen that does not exhibit allergenicity, contains an array of T cell epitpes that is comparable to that of the corresponding natural allergen, and displays a surface structure that is recognized by human IgG, particularly by human IgG4, and IgA antibodies with specificity for the corresponding natural allergen. For the design of such a molecule, the individual antibodies of the different antibody compositions are essential to maintain IgG epitopes and IgA epitopes upon modification of the IgE epitopes by a structure-based approach.
In specific embodiments, the present invention provides methods for decreasing the allergenicity (IgE reactivity) of the polypeptides of this invention in a structure-based approach via mutagenesis of IgE epitopes with limited impairment of the residual surface structure important for IgG and IgA immunological responses. In a preferred embodiment, the allergenicity of the polypeptides of this invention is reduced by at least 50% while at least 50% of IgG epitopes and IgA epitopes are maintained. In a more preferred embodiment, the allergenicity of the polypeptides of this invention is reduced by at least 70% while at least 50% of IgG epitopes and IgA epitopes are maintained. In a most preferred embodiment allergenicity is reduced by at least 90% while at least 50% of IgG epitopes are maintained.
In the context of this invention, allergenicity is defined as the capability of a proteinaceous allergen to bind human IgE antibodies. Antigenicity in the context of this invention is defined as the capability of a proteinaceous allergen to bind human IgG (including IgG4) and IgA antibodies.
The invention thus also provides a method of treating subjects allergic to the venom of an insect from the order Hymenoptera comprising administering to a subject with such an allergy a protein/polypeptide of the invention.
As used herein, “subject” encompasses human subjects (patients), grown-ups as well as children, and animals.
A pharmaceutical composition comprising a protein of the invention, and, optionally, comprising a suitable adjuvant or expedient, can be employed for this purpose.
The polypeptide of the invention is used for the preparation of a pharmaceutical composition for treating subjects allergic to the venom of an insect from the order Hymenoptera. The invention thus provides a method of treating subjects allergic to the venom of an insect from the order Hymenoptera, comprising administering a polypeptide of the invention to a subject having such an allergy.
Desensitisation approaches are well known in the state of the art. In principle, repeated treatments of allergic individuals with suitable, normally progressively increased doses of allergen diverts the immune response to one dominated by T cells that favour the production of IgG and IgA antibodies over production of IgE antibodies. The IgG and IgA antibodies are thought to desensitise the subject by binding to the small amounts of allergen normally encountered, and preventing the allergen from binding to IgE. Desensitisation to insect or bee venom is almost universally successful (Hunt et al 1978). Different protocols and time schedules can be used, from traditional protocols, rush protocols to ultrarush protocols (e.g., Schiavino et al 2004), all of which are incorporated herein by reference. The efficacy of such protocols can be evaluated by testing the adjustment of IgE and IgG (different isotypes) and/or IgA levels in the subject's blood or by challenging the subject in a controlled manner and determining the allergic response.
The Api m 3 polypeptide, a fragment, a derivative or an analog thereof is administered over a period of time in gradually increasing doses effective to reduce the allergic response of the individual to the protein allergen. Examples of routes of administration include parenteral (e.g., intravenous), intradermal, subcutaneous, oral (e.g., sublingual or via inhalation), transdermal (topical), and rectal administrations. The effective amount of the Api m 3 polypeptide, a fragment, a derivative and an analog thereof will vary according to factors such as the degree of sensitivity of the individual to Api m 3, the age, sex, and weight of the individual, and the ability of the fragment, derivative, or analog thereof to elicit an antigenic response in the individual. In one embodiment, the amount of Api m 3 polypeptide administered to an individual corresponds to the amount of Api m 3 in the venom of vespids that is injected into an individual by a sting. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily.
The polypeptide of the invention can be administered alone or combination with other allergens, e.g. other Hymenoptera venom proteins or fragments thereof. In particular, combinations with bee or Hymenoptera venom phospholipase A2, hyaluronidase, glucosidase and/or mellitin are suitable, as this therapy induces generation of IgG/IgA antibodies to several venom allergens and can thus lead to full protection. The identified bee allergens are shown in Table 2.
In a specific embodiment, the present invention features a method of modulating an immune response by administering T cell epitope-containing peptides of at least 9 amino acids corresponding to a consecutive amino acid sequence within Api m 3 to a subject in need thereof in an amount sufficient to inhibit an immune reaction by the subject against the Api m 3 polypeptide. If desired, T cell epitope-containing peptides of at least 9 amino acids corresponding to a consecutive amino acid sequence within one or more additional polypeptides, e.g., within a second, third, fourth, or more honeybee venom polypeptide or polypeptides can be comprised in such peptides. The additional honeybee venom polypeptides can include, e.g., the Api m 1 polypeptide (phospholipase A2), the Api m 2 polypeptide (hyaluronidase), the Api m 4 oligopeptide (mellitin), the Api m 5 polypeptide, (allergen C, dipeptidylpeptidase), and other glycosylated or non-glycosylated IgE-binding honeybee venom proteins, or analogs or derivatives thereof.
As used herein, a decrease or modification of the T cell response of a mammal sensitive to a protein allergen is defined as non-responsiveness or diminution in symptoms to the protein allergen in the mammal, as determined by standard clinical procedures (see e.g., Varney et al 1991). As referred to herein, a diminution in symptoms to an allergen includes any reduction in the allergic response of a mammal (such as a human) to the allergen following a treatment regimen with a polypeptide as described herein. This diminution in symptoms may be determined subjectively in humans (e.g., the patient feels more comfortable upon exposure to the allergen), or clinically, such as with a standard skin test.
The polypeptide of the invention can also be used for the preparation of a diagnostical composition for diagnosing or identifying subjects allergic to the venom of an insect from the order Hymenoptera. A method of diagnosing an allergy to venom of an insect from the order Hymenoptera is thus provided, comprising the steps of

    
    
        a) contacting a subject with a polypeptide of the invention and
        b) detecting an allergic reaction, wherein detecting an allergic reaction indicates said allergy.
    
    


In vivo tests for diagnosis of an allergy can easily be adapted to the polypeptide of the invention. Typically, a suitable amount of allergen is injected subcutaneously into a subject's limb, and, after a certain amount of time, the degree of localised inflammation in comparison to controls is determined (skin prick test). Such tests are well known in the art (Hamilton 2002, Poulsen 2001, Schmid-Grendelmeier 2001, Williams et al 1999, Barbee et al 1976).
An allergy to the venom of an insect from the order Hymenoptera can also be diagnosed by an in vitro method comprising the steps of

    
    
        a) in vitro contacting a blood sample from a subject with a polypeptide of the invention and
        b) detecting binding of IgE antibodies to the polypeptide, wherein detecting IgE antibodies binding to the polypeptide indicates said allergy.
    
    


Binding of IgE antibodies to the polypeptide can, e.g., be detected in an ELISA or by an in vitro release assay employing stripped mast cells and measuring the amount of released mediator, e.g., histamine. To determine specific binding, the results are compared with a specificity control, e.g., with an unrelated antibody. The diagnostic tests can in parallel be carried out to determine the levels of specific IgG (in particular IgG1 and/or IgG4) and/or IgA. For this, an ELISA with specific secondary antibodies recognising the different isotypes can be employed. Parallel testing is particularly useful for following and evaluating a course of specific immunotherapy.
In another embodiment, the present invention provides in vitro diagnostic assays on peripheral blood lymphocytes useful for obtaining information on Api m 3-specific T cell responses, the phenotype of the T cell response, and preferably the T cell epitope(s) of Api m 3 involved in T cell responses. The immunodominant epitope(s) and the epitope(s) involved in IgE isotype class switch events can be detected, if they are not identical. In particular, the T cell epiotope(s) of Api m 3 that stimulate proliferation and/or lymphokine secretion of T cells of a phenotype associated with IgE isotype class switching events can be identified for a specific individual, or for a class of individuals who share MHC haplotype or a predominant T cell receptor variable region expression, or both.
For the therapeutic and diagnostic uses and methods, it is preferred to employ the fusion polypeptides of the invention, non-glycosylated proteins or polypeptides that are capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera, and comprise at least 5, preferably at least 6, 7, 8, 9, 10, 15, 20 or more amino acids of a polypeptide more than 70%, more than 80% or more than 90% homologous or identical to a polypeptide selected from the group consisting of amino acid 26 to 99, 116 to 145, 153 to 158, 185 to 223, 232 to 276 or 362 to 373 of the polypeptide shown in SEQ ID NO: 2. Alternatively, the employed polypeptides are capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera, and comprise at least 5, preferably at least 6, 7, 8, 9, 10, 15, 20 or more amino acids of a polypeptide more than 70%, more than 80% or more than 90% homologous or identical to the polypeptide shown in SEQ ID NO: 2, except for the polypeptides from the group consisting of amino acids 1 to 34, 63 to 80, 100 to 115, 142 to 149, 168 to 176, 224-239 and 258 to 343 of the polypeptide shown in SEQ ID NO: 2 or except for the polypeptides shown in FIG. 5. Additionally, such polypeptides consisting of amino acids 1 to 25, 146 to 152, 159 to 164, 168 to 184, 224 to 231, and 277 to 361 can be used.
In one embodiment, the Api m 3 polypeptide, fragments, derivatives and/or analogs thereof, are incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the Api m 3 polypeptide, fragments, derivatives or analogs thereof, and a pharmaceutically acceptable carrier. As used herein, a ‘pharmaceutically acceptable carrier’ is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and adsorption delaying systems, and the like, compatible with the active compound and pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active compounds can also be incorporated into the composition. As used herein, the phrases ‘pharmaceutical composition’ and ‘medicament’ are interchangeable.
In another embodiment, the pharmaceutical composition includes an additional polypeptide, e.g., a second, third, fourth, or more honeybee venom polypeptide or polypeptides. The additional honeybee venom polypeptides can include, e.g., the Api m 1 polypeptide (phospholipase A2), the Api m 2 polypeptide (hyaluronidase), the Api m 4 oligopeptide (mellitin), the Api m 5 polypeptide, (allergen C, dipeptidylpeptidase), and other glycosylated or non-glycosylated IgE-binding honeybee venom proteins, or analogs or derivatives thereof.
In another embodiment, the present invention features a pharmaceutical composition comprising Api m 3 polypeptide fragments of the invention, preferably between 20-150 amino acids in length, wherein each fragment contains one or more B cell epitopes and one or more T cell epitopes, and a pharmaceutically acceptable carrier.
In another embodiment, the pharmaceutical composition includes polypeptide fragments derived from an additional polypeptide, e.g., a second, third, fourth, or more honeybee venom polypeptides or oligopeptides including, but not limited to, the Api m 1 polypeptide (phospholipase A2), the Api m 2 polypeptide (hyaluronidase), the Api m 4 oligopeptide (mellitin), the Api m 5 polypeptide, (allergen C, dipeptidylpeptidase), and other glycosylated or non-glycosylated IgE-binding honeybee venom proteins, or analogs or derivatives thereof.
In another embodiment, the pharmaceutical composition includes Api m 3 polypeptide fragments of the invention, fused to polypeptide fragments derived from an additional polypeptide, e.g., a second, third, fourth, or more honeybee venom polypeptides or oligopeptides including, but not limited to, the Api m 1 polypeptide (phospholipase A2), the Api m 2 polypeptide (hyaluronidase), the Api m 4 oligopeptide (mellitin), the Api m 5 polypeptide, (allergen C, dipeptidylpeptidase), and other glycosylated or non-glycosylated IgE-binding honeybee venom proteins, or analogs or derivatives thereof.
In another embodiment, the present invention features a pharmaceutical composition comprising T cell epitope containing peptides of at least 9 amino acids corresponding to a consecutive amino acid sequence within Api m 3 wherein the peptides are capable of stimulating T cells of subjects allergic to Api m 3. In a preferred embodiment, the composition comprises a set of T cell epitope-containing peptides capable of stimulating T cells of the great majority of subjects allergic to Api m 3.
In another embodiment, the pharmaceutical composition includes T cell epitope-containing peptides of at least 9 amino acids corresponding to a consecutive amino acid sequence within an additional polypeptide, e.g., a second, third, fourth, or more honeybee venom polypeptides or oligopeptides including, but not limited to, the Api m 1 polypeptide (phospholipase A2), the Api m 2 polypeptide (hyaluronidase), the Api m 4 oligopeptide (mellitin), the Api m 5 polypeptide, (allergen C, dipeptidylpeptidase), and other glycosylated or non-glycosylated IgE-binding honeybee venom proteins, or analogs or derivatives thereof.
Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents, antioxidants such as ascorbic acid or sodium bisulfate, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetates, citrates or phosphates and agents for the adjustment of toxicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. The composition should be fluid to the extent that easy syringability exists. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case dispersion and by use of surfactants. The composition should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimoseral, and the like. Delayed absorption of the injectable compositions can be achieved by including in the composition an agent such as aluminium monostearate and gelatin. In all cases, the composition must be sterile. Sterile injectable solutions can be prepared by filtered sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatine capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as mouthwash, wherein the active compound in the fluid carrier is swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth, or gelatine; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavouring agent such as peppermint, methyl salicylate, or orange flavouring.
For administration by inhalation, the active compounds are delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g. a gas such as carbon dioxide, or a nebulizer.
For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include for transmucosal administration, for example; detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. Suppositories can be prepared using conventional suppository base such as cocoa butter or other glycerides. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. For rectal delivery the compounds can also be prepared in the form of retention enemas.
In a further embodiment, the active compounds are prepared with carriers that will protect the active compounds against rapid elimination from the body, such as controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polyacetic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.
For oral and parenteral applications it is advantageous to formulate the compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated. Each unit contains a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in association with the included pharmaceutical carrier. The specification for the dosage unit forms of the invention are dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
The present invention also relates to a method of diagnosing an allergy to venom of an insect from the order Hymenoptera, comprising the steps of

    
    
        a) performing the method of producing a polypeptide encoded by the nucleic acid of the invention, wherein the host cell comprising the expression vector of the invention is cultured under appropriate conditions for expression of said polypeptide, and wherein said polypeptide is purified,
        b) contacting the polypeptide obtained by the method of step a) in vitro with a blood sample,
        c) and detecting binding of IgE antibodies to the polypeptide, wherein detecting IgE antibodies binding to the polypeptide indicates said allergy.
    
    


Furthermore, a method of diagnosing an allergy to venom of an insect from the order Hymenoptera is provided, comprising the steps of

    
    
        a) performing the method of producing a polypeptide encoded by the nucleic acid of the invention, wherein the host cell comprising the expression vector of the invention is cultured under appropriate conditions for expression of said polypeptide, and wherein said polypeptide is purified,
        b) contacting a subject with the polypeptide obtained by the method of step a) and detecting an allergic reaction, and
        c) detecting an allergic reaction, which is indicative of the allergy.
    
    


The invention also provides a method of preparing a composition for diagnosing an allergy to venom of an insect from the order Hymenoptera comprising the step of producing a polypeptide encoded by the nucleic acid of the invention, wherein the host cell comprising the expression vector of the invention is cultured under appropriate conditions for expression of said polypeptide and said polypeptide is purified and can be used as such for diagnosis. Optionally, the polypeptide is further formulated with stabilizers, such as a neutral protein (e.g., BSA) or detergents to give said composition.
In another embodiment, the invention teaches a method of preparing a composition for treating subjects allergic to the venom of an insect from the order Hymenoptera, comprising the step of performing the method of producing a polypeptide encoded by the nucleic acid of the invention, wherein the host cell comprising the expression vector of the invention is cultured under appropriate conditions for expression of said polypeptide and said polypeptide is purified and can be used as such for therapy. Optionally, the polypeptide is further formulated with appropriate excipient and/or carriers in order to provide said composition. Correspondingly, a method of treating subjects allergic to the venom of an insect from the order Hymenoptera is disclosed, comprising the steps of

    
    
        a) performing the method of producing a polypeptide encoded by the nucleic acid of the invention, wherein the host cell comprising the expression vector of the invention is cultured under appropriate conditions for expression of said polypeptide and said polypeptide is purified, and
        b) administering the polypeptide obtained by the method of step a) to a subject having such an allergy.
    
    


The present invention thus for the first time satisfies the need for a recombinantly produced Hymenoptera venom acid phosphatase or the cDNA encoding this polypeptide, which can be used for diagnostic and therapeutic applications.
EXAMPLES
Example 1
Cloning of cDNA
1.1 Total RNA Isolation
Total RNA was isolated from the separated stinger of a honey bee with attached venom sack and additional glands. The isolation of total RNA was performed using a kit according to the manual (peqGold TriFastTM, peqlab Biotechnologie GmbH, Erlangen, Germany) The organ was weighed and homogenised in a solution containing guanidiniumisothiocyanate and phenol. Phase separation was induced by addition of chloroform. The aqueous phase was separated after centrifugation, and the containing RNA precipitated with isopropyl alcohol. After washing with diluted ethanol the RNA was dissolved in RNase-free sterile water and used directly in RT-PCR experiments. To prepare RNase-free sterile water cell-culture suitable water was treated with 0.1% (v/v) diethylpyrocarbonate (DEPC) overnight, and then autoclaved for 20 minutes to destroy DEPC by causing hydrolysis of DEPC.
1.2 cDNA First Strand Synthesis
Reverse transcriptase was used to synthesise first strand cDNA from the isolated RNA. For this 5 μl of total bee RNA was mixed with 2 μl (20 pmol) oligonucleotide primer and 4 μl DEPC water. An universal oligo-dT of 20 base pair length was used for the purpose of transcribing the poly-adenylated portion of mRNA in the total RNA sample. The reaction mix was incubated at 70° C. for 5 minutes to break secondary structures. After this, the reaction was chilled on ice. Subsequently, 1.5 μl DEPC water, 4 μl 5× reaction buffer, 2 μl dNTP mix (10 mM), and 0.5 μl RNaseOutTM recombinant ribonuclease inhibitor (Invitrogen GmbH, Karlsruhe, Germany) were added. The reaction mix was incubated at 37° C. for 5 minutes. Then 1 μl (200 units) RevertAidTM M-MuLV Reverse Transcriptase (RT, Fermentas GmbH, St. Leon-Rot, Germany) was added and the reaction was incubated at 42° C. for 60 minutes. After this the reaction was stopped by heating to 70° C. for 10 minutes and chilled on ice.
1.3 RT-PCR
First strand cDNA from bee venom gland tissue was used as template for PCR amplification of Api m3 DNA sequences.
Known peptide fragments, public databases and bioinformatics were used to design the specific primers for Api m3. These primers have been designed to allow 5′-end blunt subcloning for native N-terminal expression and 3′-end directed Sac II restriction site subcloning. The nucleotide sequences of the oligonucleotides are:







Api m3 for, 21 mer, blunt end (SEQ ID NO: 3):


5′-GAA CTT AAA CAA ATA AAT GTG


 


Api m3 back 32 mer, Sac II site (SEQ ID NO: 4):


5′-AAC CGC GGT TAC TTA CTT ATT CTC AGT ACC CG.






The PCR reaction contained 41 μl DEPC water, 5 μl 10× complete Pfu PCR buffer, 1 μl Api m3 for primer (100 pmol), 1 μl Api m3 back primer (100 pmol), 1 μl dNTP mix (10 mM), 0.5 μl bee venom gland tissue cDNA, and 0.5 μl recombinant Pfu DNA polymerase (Fermentas GmbH, St. Leon-Rot, Germany), to give a total reaction volume of 50 μl.
The PCR temperature program for amplification was:
Step 1: 96° C., 1 minute
Step 2: 95° C., 30 seconds
Step 3: 55° C., 30 seconds
Step 4: 72° C., 2 minutes
Repeat steps 2-4×29 times
Step 5: 72° C., 10 minutes
Step 6: 4° C., until end
Part of the PCR reaction was run on a 1% agarose (peqGOLD universal agarose, peqlab GmbH, Erlangen, Germany) gel in 0.5×TAE buffer and amplified DNA products visualised with ethidium bromide and UV illumination. A band at the expected size was visible.
1.4 Subcloning for Sequencing
DNA from the PCR reaction was isolated using the QIAEX II gel extraction kit (Qiagen GmbH, Hilden, Germany). Subcloning for sequencing was done using the TOPO TA Cloning® Kit (Invitrogen GmbH, Karlsruhe, Germany) with pCR®2.1-TOPO® vector according to the manual. Due to use of Pfu DNA polymerase an initial TA-elongation reaction step with AGS Gold Taq DNA Polymerase (AGS Hybaid, Heidelberg) was introduced. The ligated DNA was transformed into E. coli of the strain TG1 by electroporation (2 mm cuvettes, EasyJect+, Hybaid, Heidelberg, Germany) and selected on ampicillin agar plates.
1.5 Sequencing
The sequencing reaction was done with BigDye® Terminator Cycle Sequencing Kit from ABI (Applied Biosystems Applera Deutschland GmbH, Darmstadt, Germany) according to the manual. 25 cycles were run with a 30 seconds denaturation step at 96° C., 15 seconds annealing step at 50° C., and 4 minutes elongation step at 57° C. Sequencing primer were:









M13/Uni for (SEQ ID NO: 5):



5′-GTA AAA CGA CGG CCA GTG CCA A


 



M13/Uni rev (SEQ ID NO: 6):



5′-CAG GAA ACA GCT ATG ACC ATG A






The resulting sequence is shown in FIG. 1.
Example 2
Construction of Expression Vector
2.1 Modification of the Insect Expression Vector
For expression of recombinant Api m 3 with potential for native folding and posttranslational modification, the expression in insect cells was chosen. The expression vector pIB/V5-His. (Invitrogen GmbH, Karlsruhe, Germany) was modified to facilitate isolation and purification. A melittin signal sequence for secretion of the recombinant protein was added and the Kozak sequence was optimised for higher expression rates in insect cells. The melittin signal sequence was amplified from total bee RNA, synthesised as described above, using the primers:







melt leader for (SEQ ID NO: 7):


5′-GGA AAG CTT TCC GCC ATG GCG AAA TTC TTA GTC


 


melt leader back (SEQ ID NO: 8):


5′-CGG GAT CCC GCA TAG ATG TAA GAA ATG.






Underlined are the Hind III and, respectively, BamH I restriction sites in the corresponding primer. The sequence containing the 10× histidine-tag and factor Xa cleavage site has been cloned between the BamH I and EcoR V site of the parent vector. As first template, a tag containing vector was used with the following primers:









10xHis for (SEQ ID NO: 9):



5′-CTG AAT AGC GCC GGA TCC GAC CAT


 



10xHis back (SEQ ID NO: 10):



5′-CCC TCT AGA CTC GAG CCA ATG ATG






Underlined are the bases for the introduction of the BamH I restriction site. The resulting fragment was used as second template and further modified to contain a EcoR V site at the 3′-end by use of overlapping primers and PCR extension of the sequence (splice-overlap-extension, SOE). The extension primer used was:









SOE Xa (SEQ ID NO: 11):



5′-GGG ATA TCC CTT CCC TCG ATC CCT CTA GAC TC






Underlined is the newly introduced EcoR V restriction site for cloning and generation of the expression vector construct. For all PCR steps Pfu DNA polymerase (Fermentas GmbH, St. Leon-Rot, Germany) was used with standard reaction conditions. The annealing temperature was 55° C. for the 10× Histidin fragment amplification and 45° C. for the SOE reaction.
2.2 Re-PCR and Subcloning
After sequencing of selected subcloned cDNA clones and verification of the sequence, the clone was used for secondary amplification with Pfu DNA polymerase. The PCR product was subcloned into the EcoR V/Sac II digested expression vector after restriction digest with Sac II.
2.3 Modification of the Bacterial Expression Vector
The verified mammalian expression vector pIB/Mel opt-H10 was used as template for the construction of insert for subcloning into the prokaryontic expression vector pET26(+) (Novagen). The PCR program was done according to the temperature gradient given in 1.3. Pfu polymerase was used with the primers:







(SEQ ID NO: 12)








Api 3 for pro-his
AGAATTTCATATGAAATTCTTAGTCAACG


 







(SEQ ID NO: 13)








Api 3 back pro
AAGAGCTCTTACTTACTTATTCTCAG






The amplicon was digested with Sac I and Nde I. The partly digested fragment of correct size was isolated and ligated into the pre-digested vector.
Example 3
Expression of Recombinant Protein
3.1 Transfection
HighFive insect cell (Invitrogen GmbH, Karlsruhe, Germany) were used as hosts for the recombinant expression of Api m 3. DNA was purified from bacterial cultures using the E.Z.N.A. Plasmid Miniprep Kit II (peqlab GmbH, Erlangen, Germany) according to the manual. For transfection of purified DNA into cells the reagent Cellfectin® (Invitrogen GmbH, Karlsruhe, Germany) was used according to the manual.
3.2 Transformation
Vectors have been transformed into prokaryontic cell by electroporation. Cells have been prepared by standard procedures. Electroporation was done with an EasyJecT+ instrument (EquiBio, Maidstone, UK) with standard settings according to the manual of the manufacturer.
3.3 Isolation of Recombinant Protein
The protein was purified according to standard procedures.
In brief, prokaryotic cells were disrupted by sonication. Cell membranes etc. were sedimented by ultracentrifugation. The His-tagged protein was then purified from the extract by Ni2T affinity chromatography following the manufacturer's recommendations (e.g., His TrapTM HP Kit, Amersham Biosciences). Purification was controlled by SDS-PAGE. In the case of eukaryotic expression the supernatant medium was collected from confluent stably transfected insect cell expression cultures. The supernatant was adjusted to pH 7.8 and centrifuged at 4000×g for 5 minutes. Aliquots of 10-20 ml medium were applied to a nickel-chelating affinity matrix (NTA-agarose, Qiagen). The column was washed with 10 ml NTA-binding buffer (50 mM sodium phosphate, pH 7.6, 500 mM NaCl) and pre-eluted with NTA-binding buffer containing 20 mM imidazole. The recombinant protein was finally eluted from the matrix with 10 ml NTA-binding buffer containing 400 mM imidazole. Purification was controlled by SDS-PAGE and silver staining of protein.
Example 4
Analysis of Recombinant Api m 3
4.1 Sequence Alignment and Motif Analysis
Sequence databases were screened with BLAST algorithms for related sequences of the cloned Api m 3 in other organisms. Sequence alignment was performed with four homologous sequences found in the organisms Drosophila melanogaster and Drosophila subobscura coding for acid phosphatases. The sequences show significant homologies. The highest homology with 35% is found for Acph-1 from D. melanogaster Amino acids necessary for acid phosphatase activity (RHGXRXP motif) are highly conserved in the sequence. In addition, four potential N-glycosylation sites (NXS/T motif) have been identified.
4.2 Tryptic Fragment Prediction
To verify the cloned sequence matches the expressed recombinant protein a prediction of tryptic fragments was done based on the nucleic acid sequence. The purified protein was digested with sequence grade Trypsin (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) according to the instructions of the manufacturer and the resulting peptide fragments were analysed by MALDI-TOF spectrometry using standard protocols. The predicted fragments matched the data acquired by MALDI-TOF and therefore verified the identity of the recombinant protein.
4.3 Enzymatic Activity Assay
Enzymatic activity of the recombinant enzyme was confirmed according to a described method (Barboni et al 1987).
Example 5
Immunoreactivity of Recombinant Api m 3
Recombinant Api m 3 isolated from stably transfected insect cells was used in an immunoprinting experiment with serum from honey bee venom allergic patients to evaluate IgE reactivity. Diluted honey bee venom and purified recombinant Api m 3 were examined in the same experiment. Proteins were separated on 10% SDS-PAGE gels under reducing conditions. Transfer to nitrocellulose membrane (Protran, Schleicher & Schuell BioScience GmbH, Dassel, Germany) and subsequent immunostaining for sIgE reactive allergens was done using a kit according to the manual (AlaBLOT kit, DPC Biermann GmbH, Bad Nauheim, Germany) showing the immunoreactivity of recombinant Api m 3.
Example 6
Patient Screening with Recombinant Api m 3
Immunoreactivity Assays with Sera from Individual Patients
To detect specific IgE immunoreactivity of human sera with purified recombinant Api m 3, ELISA plates (NUNC GmbH & Co. KG, Wiesbaden, Germany) were coated with 100 μl of purified recombinant Api m 3 (1 μg/ml) or, as a positive control, purified natural Api m 1 (1 μg/ml) (Latoxan, Valence, France) at 4° C. overnight. For all reaction steps, an ELISA buffer reagent set was used according to the manual (BD Biosciences, Heidelberg, Germany). Appropriate dilutions (1:2; 1:5; 1:10) of the sera were made in assay diluent. Bound IgE was detected with a biotinylated mouse anti-human IgE (BD Biosciences) together with horseradish peroxidase-conjugated avidin, both diluted 1:250 in assay diluent. Color was developed with 100 μl substrate solution per well for 30 minutes in the dark. Finally, 50 μl stop solution were added and plates were read at 450/570 nm. For quality control of the assay, an 8-point human IgE standard curve was run on each plate using murine anti-IgE (10 μg/ml) as capture antibody and human myeloma IgE (Calbiochem-Merck, Darmstadt, Germany) over a concentration range of 31.25 to 4,000 pg/ml (100 μl per well, diluted in assay diluent). Secondary antibody and detection system for total IgE were identical to the one described above for the detection of Api m 1/rApi m 3 sIgE. It could be shown that approximately 37.5% (15/40) of the patient sera that were characterized by a positive sIgE test to honeybee venom had detectable sIgE to recombinant Api m 3. Of 19 patients lacking serologic reactivity to honeybee venom (sIgE <0.35 kU/L), 10 patients were highly sensitized to Vespula spp. venom but non-reactive towards honeybee venom (sIgE >50 kU/L, FIG. 6B) and 9 were individuals lacking serologic IgE reactivity to both hymenoptera venoms (sIgE <0.35 kU/L to both, vespid and honeybee venom). Only one serum out of the 19 sera lacking serologic reactivity to honeybee venom showed reactivity with recombinant Api m 3. This patient had a clearcut positive sIgE result in the recombinant Api m 3 ELISA. He reported to the allergy service with a history of a severe anaphylactic reaction after a hymenoptera sting. The offending insect was not identified by the patient. Despite a negative “classical” serologic result and a negative intradermal skin test, the patient was finally classified as an honey bee venom allergic patient. It can be assumed that he reacts strongly to native Api m 3 with is likely to be underrepresented in clinical test kits and therefore his allergy was not noticed.
Example 7
Improved differentiation between sera binding to different epitopes based on recombinantly expressed Api m 3
a) Api m3 Expressed in Bacterial Cells:
The different structural features of recombinant Api m 3 expressed in bacterial cells are documented by following experiments (experimental conditions as described in Example 6):
As shown in FIG. 13, native Api m 3 purified according to document Dl is recognized by IgE in 6 of 9 (66%) sera from patients with honeybee venom allergy. This result is in excellent accordance with data published by Kemeny et al. (1983). Using purified native Api m 3, Kemeney and coworkers demonstrated serum IgE to Api m 3 in 60% of the sera from patients with honeybee venom allergy.
In contrast, recombinant Api m 3 expressed in bacterial cells (E. coli) is recognized by IgE in a significantly lower number of sera from patients with honeybee venom allergy. When expressed as fusion protein with bacterial maltose bindin protein (MBP), Api m 3 is recognized by IgE in 3 of 9 (33%) sera from patients with honeybee venom allergy (see FIG. 14A). When expressed as fusion protein with eukaryotic glutathion-S-transferase (GST), Api m 3 is recognized by IgE in only 2 of 9 (22%) sera from patients with honeybee venom allergy (see FIG. 14B).
b) Api m3 Expressed in Insect Cells:
Recombinant Api m 3 molecules expressed in insect cells (HighFive cells or SF9 cells) are glycosylated, but the glycosylation pattern provided by both insect cell lines to Api m 3, exhibits significant differences. As a result, both Api m 3 molecules are recognized by IgE in different sera from patients with honeybee venom allergy.
The profound effects of different glycosylation patterns of Api m 3 expressed in different insect cells, on the binding of IgE antibodies are documented by following experiments:
Api m 3 expressed in HighFive insect cells is recognized by IgE in 6 of 9 (66%) sera from patients with honeybee venom allergy and, however, partly by different sera than native Api m 3 (see FIG. 15A). Api m 3 expressed in SF9 insect cells is recognized by IgE in only 3 of 9 (33%) sera from patients with honeybee venom allergy (see FIG. 15B).
Furthermore, FIG. 16 demonstrates that both molecules are recognized to a different extent by IgE in sera from patients allergic to both honeybee and wasp venom, which allows for an improved evaluation of carbohydrate based cross-reactivity of IgE antibodies. The data in FIG. 16 show that recombinant Api m 3 produced in HighFive insect cells is recognized by IgE in 19 of 23 (82%) sera from patients allergic to both honeybee and wasp venom. Ten of these sera contain IgE that is highly reactive with Api m 3 produced in HighFive insect cells. It should be stressed that the sera tested in FIG. 16 are obtained from patients allergic to both honeybee and wasp venom and, therefore, cannot be compared to those sera tested in FIG. 13-15 which are obtained from patients allergic only to honeybee venom.
In summary, the structural features of recombinant Api m 3 expressed in E. coli and insect cells differ significantly from those of native Api m 3.
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TABLE 1







Bee venom components











% weight of


Component type
name
dry mass





Proteins
Phospholipase A2 (Api m 1)
10-12



Hyaluronidase (Api m 2)
1-3



Phosphatase, Glucosidase
1-2


Peptides
Melittin (Api m 4)
50-55



Secapin, MCD-peptide
1.5-4  



Tertiapamin, Apamin, Procamin
2-5



Other small peptides
13-15


Biogene amines
Histamine
0.5-2  



Dopamine
0.2-1  



Norepinephrine
0.1-0.5



Sugars (Glucose, Fructose)
2


Phospholipids

5


Amino acids

—


Volatile
Pheromones
4-8


substances


Minerals

3-4
















TABLE 2







Identified bee allergens












Allergen
Common name
Size (processed)
Weight
SwissProt
Reference





Api m 1
Phospholipase A2
134 aa
15.2 kDa
P00630
Kuchler et al 1989


Api m 2
Hyaluronidase
349 aa
40.7 kDa
Q08169
Gmachl and Kreil 1993


Api m 3
Acid Phosphatase
nd
  45 kDa
—
Barboni et al 1987


Api m 4
Melittin
 26 aa
 2.8 kDa
P01501
Vlasak et al 1983


Api m 5
Allergen C
nd
 105 kDa
—
Hoffman et al 1977


Api m 6
—
 71 aa
 7.5 kDa
P83563
Kettner et al 2001
















TABLE 3







NetMHCII 1.0 predicted T cell epitopes in Api m 3 (only strong and weak binders)














Peptide
Start No.*
End No.*
Start No.**
End No.**
Length


Allele
No.
nucleic acid
nucleic acid
amino acid
amino acid
amino acid
















DRB1*0101
1
307
351
103
117
15



2
310
354
104
118
15



3
313
357
105
119
15



4
316
360
106
120
15



5
319
363
107
121
15



6
523
567
175
189
15



7
526
570
176
190
15



8
514
558
172
186
15



9
517
561
173
187
15



10
520
564
174
188
15



11
655
699
219
233
15



12
661
705
221
235
15



13
658
702
220
234
15



14
652
696
218
232
15



15
664
708
222
236
15



16
889
933
297
311
15



17
880
924
294
308
15



18
883
927
295
309
15



19
886
930
296
310
15



20
892
936
298
312
15



21
532
576
178
192
15



22
529
573
177
191
15



23
322
366
108
122
15



24
325
369
109
123
15



25
667
711
223
237
15



26
670
714
224
238
15



27
622
666
208
222
15



28
535
579
179
193
15



29
184
228
62
76
15



30
190
234
64
78
15



31
187
231
63
77
15



32
193
237
65
79
15



33
247
291
83
97
15



34
253
297
85
99
15



35
895
939
299
313
15



36
250
294
84
98
15



37
625
669
209
223
15



38
628
672
210
224
15



39
244
288
82
96
15



40
181
225
61
75
15



41
241
285
81
95
15



42
898
942
300
314
15



43
1066
1110
356
370
15



44
619
663
207
221
15



45
1069
1113
357
371
15



46
616
660
206
220
15



47
1063
1107
355
369
15



48
1060
1104
354
368
15



49
256
300
86
100
15



50
793
837
265
279
15



51
259
303
87
101
15



52
541
585
181
195
15



53
754
798
252
266
15



54
796
840
266
280
15



55
751
795
251
265
15



56
775
819
259
273
15



57
784
828
262
276
15



58
802
846
268
282
15



59
778
822
260
274
15



60
745
789
249
263
15



61
748
792
250
264
15



62
1072
1116
358
372
15



63
787
831
263
277
15



64
781
825
261
275
15



65
799
843
267
281
15



66
805
849
269
283
15



67
742
786
248
262
15



68
235
279
79
93
15



69
238
282
80
94
15



70
196
240
66
80
15



71
631
675
211
225
15



72
538
582
180
194
15



73
634
678
212
226
15



74
1075
1119
359
373
15



75
199
243
67
81
15



76
610
654
204
218
15



77
547
591
183
197
15



78
613
657
205
219
15



79
550
594
184
198
15



80
1057
1101
353
367
15



81
553
597
185
199
15



82
544
588
182
196
15



83
301
345
101
115
15



84
304
348
102
116
15



85
262
306
88
102
15



86
385
429
129
143
15



87
388
432
130
144
15



88
673
717
225
239
15



89
382
426
128
142
15



90
379
423
127
141
15



91
685
729
229
243
15



92
265
309
89
103
15



93
679
723
227
241
15



94
808
852
270
284
15



95
562
606
188
202
15



96
760
804
254
268
15



97
757
801
253
267
15



98
682
726
228
242
15



99
688
732
230
244
15



100
91
135
31
45
15



101
232
276
78
92
15



102
94
138
32
46
15



103
676
720
226
240
15



104
556
600
186
200
15



105
790
834
264
278
15



106
229
273
77
91
15



107
877
921
293
307
15



108
226
270
76
90
15



109
1021
1065
341
355
15



110
394
438
132
146
15



111
1015
1059
339
353
15



112
1018
1062
340
354
15



113
1024
1068
342
356
15



114
811
855
271
285
15



115
511
555
171
185
15



116
391
435
131
145
15



117
691
735
231
245
15



118
1012
1056
338
352
15



119
508
552
170
184
15



120
958
1002
320
334
15



121
568
612
190
204
15



122
565
609
189
203
15



123
574
618
192
206
15



124
571
615
191
205
15



125
955
999
319
333
15



126
82
126
28
42
15



127
85
129
29
43
15



128
874
918
292
306
15



129
88
132
30
44
15



130
505
549
169
183
15


DRB1*0401
131
616
660
206
220
15



132
622
666
208
222
15



133
619
663
207
221
15



134
613
657
205
219
15



135
610
654
204
218
15



136
625
669
209
223
15



137
628
672
210
224
15



138
190
234
64
78
15



139
187
231
63
77
15



140
193
237
65
79
15



141
181
225
61
75
15



142
184
228
62
76
15



143
745
789
249
263
15



144
742
786
248
262
15



145
748
792
250
264
15



146
754
798
252
266
15



147
196
240
66
80
15



148
199
243
67
81
15



149
751
795
251
265
15



150
880
924
294
308
15



151
883
927
295
309
15



152
514
558
172
186
15



153
886
930
296
310
15



154
889
933
297
311
15



155
517
561
173
187
15



156
511
555
171
185
15



157
520
564
174
188
15



158
892
936
298
312
15


DRB1*0404
159
703
747
235
249
15



160
697
741
233
247
15



161
691
735
231
245
15



162
700
744
234
248
15



163
694
738
232
246
15



164
667
711
223
237
15



165
679
723
227
241
15



166
676
720
226
240
15



167
670
714
224
238
15



168
673
717
225
239
15



169
706
750
236
250
15



170
709
753
237
251
15



171
562
606
188
202
15



172
685
729
229
243
15



173
682
726
228
242
15



174
565
609
189
203
15



175
568
612
190
204
15



176
841
885
281
295
15



177
838
882
280
294
15


DRB1*0405
178
301
345
101
115
15



179
556
600
186
200
15



180
553
597
185
199
15



181
967
1011
323
337
15



182
289
333
97
111
15



183
292
336
98
112
15



184
307
351
103
117
15



185
304
348
102
116
15



186
559
603
187
201
15



187
970
1014
324
338
15



188
973
1017
325
339
15



189
562
606
188
202
15



190
76
120
26
40
15



191
73
117
25
39
15



192
475
519
159
173
15



193
514
558
172
186
15



194
79
123
27
41
15



195
82
126
28
42
15



196
976
1020
326
340
15



197
295
339
99
113
15



198
478
522
160
174
15



199
517
561
173
187
15



200
979
1023
327
341
15



201
511
555
171
185
15



202
298
342
100
114
15



203
508
552
170
184
15



204
565
609
189
203
15



205
310
354
104
118
15



206
481
525
161
175
15



207
484
528
162
176
15



208
883
927
295
309
15



209
313
357
105
119
15



210
880
924
294
308
15



211
487
531
163
177
15



212
70
114
24
38
15



213
394
438
132
146
15



214
397
441
133
147
15



215
886
930
296
310
15



216
391
435
131
145
15



217
889
933
297
311
15



218
385
429
129
143
15



219
388
432
130
144
15



220
625
669
209
223
15



221
505
549
169
183
15


DRB1*0701
222
622
666
208
222
15



223
625
669
209
223
15



224
628
672
210
224
15



225
619
663
207
221
15



226
616
660
206
220
15



227
631
675
211
225
15



228
634
678
212
226
15



229
553
597
185
199
15



230
316
360
106
120
15



231
313
357
105
119
15



232
556
600
186
200
15



233
319
363
107
121
15



234
559
603
187
201
15



235
598
642
200
214
15



236
601
645
201
215
15



237
604
648
202
216
15



238
595
639
199
213
15



239
562
606
188
202
15


DRB1*0901
240
514
558
172
186
15



241
622
666
208
222
15



242
517
561
173
187
15



243
628
672
210
224
15



244
625
669
209
223
15



245
511
555
171
185
15



246
814
858
272
286
15



247
817
861
273
287
15


DRB1*1101
248
514
558
172
186
15



249
517
561
173
187
15



250
520
564
174
188
15



251
523
567
175
189
15



252
526
570
176
190
15



253
565
609
189
203
15



254
562
606
188
202
15



255
568
612
190
204
15



256
574
618
192
206
15



257
571
615
191
205
15


DRB1*1302
258
622
666
208
222
15



259
625
669
209
223
15



260
628
672
210
224
15



261
619
663
207
221
15



262
616
660
206
220
15



263
631
675
211
225
15



264
1066
1110
356
370
15



265
1069
1113
357
371
15



266
634
678
212
226
15



267
1063
1107
355
369
15



268
1072
1116
358
372
15



269
1075
1119
359
373
15



270
1060
1104
354
368
15



271
610
654
204
218
15



272
613
657
205
219
15



273
247
291
83
97
15



274
253
297
85
99
15



275
250
294
84
98
15



276
964
1008
322
336
15



277
259
303
87
101
15



278
568
612
190
204
15



279
256
300
86
100
15



280
562
606
188
202
15



281
571
615
191
205
15



282
565
609
189
203
15



283
1057
1101
353
367
15



284
574
618
192
206
15



285
967
1011
323
337
15



286
679
723
227
241
15



287
970
1014
324
338
15



288
682
726
228
242
15



289
958
1002
320
334
15



290
637
681
213
227
15



291
676
720
226
240
15



292
961
1005
321
335
15



293
871
915
291
305
15



294
955
999
319
333
15



295
874
918
292
306
15



296
973
1017
325
339
15



297
670
714
224
238
15



298
673
717
225
239
15



299
877
921
293
307
15



300
106
150
36
50
15



301
103
147
35
49
15


DRB1*1501
302
880
924
294
308
15



303
883
927
295
309
15



304
886
930
296
310
15



305
889
933
297
311
15



306
892
936
298
312
15



307
874
918
292
306
15



308
877
921
293
307
15



309
253
297
85
99
15


DRB4*0101
310
553
597
185
199
15



311
556
600
186
200
15



312
559
603
187
201
15



313
565
609
189
203
15



314
562
606
188
202
15



315
334
378
112
126
15



316
331
375
111
125
15



317
337
381
113
127
15



318
340
384
114
128
15



319
343
387
115
129
15



320
568
612
190
204
15



321
571
615
191
205
15



322
256
300
86
100
15



323
262
306
88
102
15



324
259
303
87
101
15



325
265
309
89
103
15



326
784
828
262
276
15



327
787
831
263
277
15



328
793
837
265
279
15



329
796
840
266
280
15



330
349
393
117
131
15



331
346
390
116
130
15



332
268
312
90
104
15



333
790
834
264
278
15



334
826
870
276
290
15



335
829
873
277
291
15



336
832
876
278
292
15



337
835
879
279
293
15


DRB5*0101
338
316
360
106
120
15



339
310
354
104
118
15



340
319
363
107
121
15



341
313
357
105
119
15



342
553
597
185
199
15



343
82
126
28
42
15



344
802
846
268
282
15



345
805
849
269
283
15



346
307
351
103
117
15





*Numbering according to SEQ ID NO: 1


**Numbering according to SEQ ID NO: 2













TABLE 4







NetMHCIIPAN predicted T cell epitopes in Api m 3 (only strong binders)














Peptide
Start No.*
End No.*
Start No.**
End No.**
Length


Allele
No.
nucleic acid
Nucleic acid
amino acid
amino acid
amino acid
















DRB1*0101
1
523
567
175
189
15



2
625
669
209
223
15



3
511
555
171
185
15



4
316
360
106
120
15



5
889
933
297
311
15



6
652
696
218
232
15



7
1066
1110
356
370
15



8
751
795
251
265
15



9
961
1005
321
335
15



10
250
294
84
98
15



11
535
579
179
193
15



12
742
786
248
262
15



13
1018
1062
340
354
15



14
871
915
291
305
15



15
610
654
204
218
15



16
676
720
226
240
15



17
664
708
222
236
15



18
190
234
64
78
15



19
772
816
258
272
15


DRB1*0102
20
523
567
175
189
15



21
652
696
218
232
15



22
529
573
177
191
15



23
889
933
297
311
15



24
511
555
171
185
15



25
625
669
209
223
15



26
1075
1119
359
373
15



27
1066
1110
356
370
15



28
316
360
106
120
15



29
250
294
84
98
15



30
742
786
248
262
15



31
262
306
88
102
15



32
751
795
251
265
15



33
877
921
293
307
15



34
772
816
258
272
15



35
664
708
222
236
15


DRB1*0103
36
316
360
106
120
15



37
625
669
209
223
15



38
652
696
218
232
15



39
1066
1110
356
370
15



40
511
555
171
185
15



41
529
573
177
191
15



42
676
720
226
240
15



43
250
294
84
98
15



44
961
1005
321
335
15


DRB1*0104
45
523
567
175
189
15



46
529
573
177
191
15



47
889
933
297
311
15



48
652
696
218
232
15



49
625
669
209
223
15



50
250
294
84
98
15



51
511
555
171
185
15



52
1075
1119
359
373
15



53
751
795
251
265
15



54
1066
1110
356
370
15



55
742
786
248
262
15



56
262
306
88
102
15



57
316
360
106
120
15


DRB1*0105
58
523
567
175
189
15



59
625
669
209
223
15



60
511
555
171
185
15



61
316
360
106
120
15



62
889
933
297
311
15



63
652
696
218
232
15



64
1066
1110
356
370
15



65
751
795
251
265
15



66
961
1005
321
335
15



67
250
294
84
98
15



68
535
579
179
193
15



69
742
786
248
262
15



70
1018
1062
340
354
15



71
871
915
291
305
15



72
610
654
204
218
15



73
676
720
226
240
15



74
664
708
222
236
15



75
190
234
64
78
15



76
772
816
258
272
15


DRB1*0106
77
529
573
177
191
15



78
652
696
218
232
15



79
316
360
106
120
15



80
523
567
175
189
15



81
511
555
171
185
15



82
889
933
297
311
15



83
625
669
209
223
15



84
742
786
248
262
15



85
1066
1110
356
370
15



86
250
294
84
98
15



87
1075
1119
359
373
15



88
880
924
294
308
15



89
262
306
88
102
15


DRB1*0107
90
523
567
175
189
15



91
625
669
209
223
15



92
511
555
171
185
15



93
316
360
106
120
15



94
889
933
297
311
15



95
652
696
218
232
15



96
1066
1110
356
370
15



97
751
795
251
265
15



98
961
1005
321
335
15



99
250
294
84
98
15



100
535
579
179
193
15



101
742
786
248
262
15



102
1018
1062
340
354
15



103
871
915
291
305
15



104
610
654
204
218
15



105
676
720
226
240
15



106
664
708
222
236
15



107
190
234
64
78
15



108
772
816
258
272
15


DRB1*0108
109
523
567
175
189
15



110
625
669
209
223
15



111
511
555
171
185
15



112
316
360
106
120
15



113
889
933
297
311
15



114
652
696
218
232
15



115
1066
1110
356
370
15



116
751
795
251
265
15



117
961
1005
321
335
15



118
250
294
84
98
15



119
535
579
179
193
15



120
742
786
248
262
15



121
1018
1062
340
354
15



122
871
915
291
305
15



123
610
654
204
218
15



124
676
720
226
240
15



125
664
708
222
236
15



126
190
234
64
78
15



127
772
816
258
272
15


DRB1*0109
128
523
567
175
189
15



129
316
360
106
120
15



130
511
555
171
185
15



131
625
669
209
223
15



132
529
573
177
191
15



133
652
696
218
232
15



134
532
576
178
192
15



135
889
933
297
311
15



136
1066
1110
356
370
15



137
535
579
179
193
15



138
751
795
251
265
15



139
1018
1062
340
354
15



140
742
786
248
262
15



141
250
294
84
98
15



142
961
1005
321
335
15



143
610
654
204
218
15



144
676
720
226
240
15



145
871
915
291
305
15



146
664
708
222
236
15


DRB1*0110
147
523
567
175
189
15



148
625
669
209
223
15



149
511
555
171
185
15



150
316
360
106
120
15



151
889
933
297
311
15



152
619
663
207
221
15



153
652
696
218
232
15



154
751
795
251
265
15



155
1066
1110
356
370
15



156
1018
1062
340
354
15



157
535
579
179
193
15



158
961
1005
321
335
15



159
250
294
84
98
15



160
676
720
226
240
15



161
871
915
291
305
15



162
193
237
65
79
15



163
739
783
247
261
15



164
190
234
64
78
15



165
664
708
222
236
15


DRB1*0111
166
523
567
175
189
15



167
889
933
297
311
15



168
625
669
209
223
15



169
751
795
251
265
15



170
511
555
171
185
15



171
652
696
218
232
15



172
535
579
179
193
15



173
316
360
106
120
15



174
1018
1062
340
354
15



175
1066
1110
356
370
15



176
250
294
84
98
15



177
961
1005
321
335
15



178
610
654
204
218
15



179
190
234
64
78
15



180
676
720
226
240
15



181
871
915
291
305
15



182
193
237
65
79
15



183
91
135
31
45
15



184
1075
1119
359
373
15


DRB1*0112
185
523
567
175
189
15



186
625
669
209
223
15



187
511
555
171
185
15



188
316
360
106
120
15



189
889
933
297
311
15



190
652
696
218
232
15



191
1066
1110
356
370
15



192
751
795
251
265
15



193
961
1005
321
335
15



194
250
294
84
98
15



195
535
579
179
193
15



196
742
786
248
262
15



197
1018
1062
340
354
15



198
871
915
291
305
15



199
610
654
204
218
15



200
676
720
226
240
15



201
664
708
222
236
15



202
190
234
64
78
15



203
772
816
258
272
15


DRB1*0113
204
523
567
175
189
15



205
889
933
297
311
15



206
625
669
209
223
15



207
619
663
207
221
15



208
751
795
251
265
15



209
511
555
171
185
15



210
316
360
106
120
15



211
535
579
179
193
15



212
652
696
218
232
15



213
1066
1110
356
370
15



214
742
786
248
262
15



215
250
294
84
98
15



216
1075
1119
359
373
15



217
190
234
64
78
15



218
676
720
226
240
15



219
661
705
221
235
15



220
955
999
319
333
15



221
961
1005
321
335
15



222
1021
1065
341
355
15



223
871
915
291
305
15



224
208
252
70
84
15



225
847
891
283
297
15



226
91
135
31
45
15


DRB1*0114
227
523
567
175
189
15



228
625
669
209
223
15



229
316
360
106
120
15



230
511
555
171
185
15



231
652
696
218
232
15



232
751
795
251
265
15



233
889
933
297
311
15



234
1018
1062
340
354
15



235
535
579
179
193
15



236
1066
1110
356
370
15



237
961
1005
321
335
15



238
250
294
84
98
15



239
1075
1119
359
373
15



240
871
915
291
305
15



241
739
783
247
261
15



242
823
867
275
289
15



243
610
654
204
218
15



244
193
237
65
79
15



245
676
720
226
240
15



246
385
429
129
143
15



247
772
816
258
272
15


DRB1*0115
248
316
360
106
120
15



249
625
669
209
223
15



250
652
696
218
232
15



251
511
555
171
185
15



252
529
573
177
191
15



253
523
567
175
189
15



254
1066
1110
356
370
15



255
1018
1062
340
354
15



256
751
795
251
265
15



257
250
294
84
98
15



258
742
786
248
262
15



259
889
933
297
311
15



260
676
720
226
240
15



261
961
1005
321
335
15



262
610
654
204
218
15


DRB1*0116
263
625
669
209
223
15



264
529
573
177
191
15



265
523
567
175
189
15



266
1021
1065
341
355
15



267
751
795
251
265
15



268
889
933
297
311
15


DRB1*0117
269
523
567
175
189
15



270
625
669
209
223
15



271
511
555
171
185
15



272
316
360
106
120
15



273
652
696
218
232
15



274
889
933
297
311
15



275
751
795
251
265
15



276
1066
1110
356
370
15



277
1018
1062
340
354
15



278
250
294
84
98
15



279
535
579
179
193
15



280
961
1005
321
335
15



281
610
654
204
218
15



282
871
915
291
305
15



283
739
783
247
261
15



284
193
237
65
79
15



285
676
720
226
240
15



286
91
135
31
45
15



287
550
594
184
198
15


DRB1*0118
288
523
567
175
189
15



289
511
555
171
185
15



290
652
696
218
232
15



291
316
360
106
120
15



292
625
669
209
223
15



293
889
933
297
311
15



294
1066
1110
356
370
15



295
619
663
207
221
15



296
742
786
248
262
15



297
250
294
84
98
15



298
1075
1119
359
373
15



299
961
1005
321
335
15



300
751
795
251
265
15



301
535
579
179
193
15



302
871
915
291
305
15



303
664
708
222
236
15



304
1018
1062
340
354
15



305
676
720
226
240
15


DRB1*0119
306
523
567
175
189
15



307
625
669
209
223
15



308
511
555
171
185
15



309
316
360
106
120
15



310
889
933
297
311
15



311
652
696
218
232
15



312
1066
1110
356
370
15



313
751
795
251
265
15



314
961
1005
321
335
15



315
250
294
84
98
15



316
535
579
179
193
15



317
742
786
248
262
15



318
1018
1062
340
354
15



319
871
915
291
305
15



320
610
654
204
218
15



321
676
720
226
240
15



322
664
708
222
236
15



323
190
234
64
78
15



324
772
816
258
272
15





*Numbering according to SEQ ID NO: 1


**Numbering according to SEQ ID NO: 2













TABLE 5







Calculation of putative surface epitopes per protein size ratio














Surface
Surface


Antigen
Size
Surface (Å2)
epitopes*
epitopes/kDa















Amb t 5
4.3
kDa
2438.3
2.57
0.6


Api m 1
16-20
kDa
7606.4
8.0
0.4


Api m 2
43
kDa
15905.5
16.74
0.39


Api m 4
3
kDa
3885.7
4.09
1.36


Ara t 8
14.2
kDa
7080.1
7.45
0.52


Asp f 1
16.8
kDa
16037.6
16.88
1.0


Asp f 6
23.3
kDa
8793.2
9.26
0.4


Bet v 1
17.4
kDa
5215.3
5.49
0.32


Bet v 2
14.3
kDa
6493.9
6.84
0.48


Bos d 4
14.2
kDa
7246.9
7.63
0.54


Bos d 5
18.2
kDa
9546.5
10.05
0.55


Bos d 5
18.2
kDa
9618.4
10.12
0.56


Der f2
15.8
kDa
7785.2
8.19
0.52


Der p2
16
kDa
7588.8
7.99
0.5


Equ c 1
20
kDa
8907.4
9.38
0.47


Gal d 3
75.8
kDa
15952.9
16.79
0.22


Gal d 4
16.2
kDa
6951.3
7.32
0.45


Hev b 8
14
kDa
11982
12.61
0.9


Mus m 1
18.7
kDa
8943.5
9.41
0.5


Phl p 1
26.1
kDa
12145.6
12.78
0.49


Phl p 2
10.8
kDa
6099.5
6.42
0.59


Phl p 6
11.8
kDa
5429.5
5.72
0.48


Pru av 1
17.7
kDa
9742.8
10.26
0.58


Ves v 5
25.8
kDa
11657.1
12.27
0.47


Zea m14
11.7
kDa
5099.5
5.37
0.46








Average value
0.55 +/− 0.23





*Estimated IgE epitope area: 950 Å2













TABLE 6







Calculation of the average number of IgE epitopes on allergens

























Identified










Possible
IgE






PDB
Size
Surface
Size/
B-cell
binding


Antigen
Protein
Organism
Common
code
(kDa)
(Å2)
Surface
epitopes
peptides



















Alt a 1
—
Alt. alternata
Fungi
—
15.2
—
—
—
2


Ara h 1
Vicilin
Arachis
Peanut
—
67.7
—
—
—
21




hypogaea


Ara h 2
Conglutin
Arachis
Peanut
—
17.5
—
—
—
10




hypogaea


Asp f 1
Mitogillin
Asp.
Fungi
1AQZ
16.8
16037.6
1.0
16-17
13




fumigatus


Asp f 2
—
Asp.
Fungi
—
31.2
—
—
—
9




fumigatus


Asp f 3
Peroximal
Asp.
Fungi
—
18.4
—
—
—
7



protein
fumigatus


Asp f 13
Oryzin
Asp.
Fungi

28.7
—
—
—
5




fumigatus


Bet v 1
PR10
Betulla
Birch
1BV1
17.4
5215.3
3.3
5-6




verrucosa


Bet v 2
Profilin
Betulla
Birch
1CQA
14.3
6493.9
2.2
6-7
3




verrucosa


Bos d 5
b-
Bos
Cow
1B8E
18.2
9546.5
1.9
 9-10
7



Lactoglobulin
domesticus


Bos d 5
b-
Bos
Cow
1QG5
18.2
9618.4
1.9
 9-10
7



Lactoglobulin
domesticus


Cry j 2
Pectinase
Cryp.
Sugi
—
42.2
—
—
—
4




japonica


Gal d 1
Ovomucoid
Gallus
Chicken
—
20.1
—
—
—
9 (8 IgG)




domesticus


Hev b 1
Elongaton
Hevea
Latex
—
14.6
—
—
—
8



factor
brasiliensis


Hev b 3
SRPP
Hevea
Latex
—
22.3
—
—
—
11




brasiliensis


Hev b 5
—
Hevea
Latex
—
15.9
—
—
—
11




brasiliensis


Jun a 1
Pectate lyase
Juniperus
Cedar
—
37.6
—
—
—
4




ashei


Jun a 3
—
Juniperus
Cedar
—
21
—
—
—
5




ashei


Par j 1
Lipid transfer
Parietaria
Weed
—
15
—
—
—
5



prot. 1
judaica


Par j 2
Lipid transfer
Parietaria
Weed
—
11.3
—
—
—
8



prot. 2
judaica


Pen n18
Serine
Pen. notatum
Fungi
—
52.4
—
—
—
9



protease

## Claims

1. A recombinant polypeptide capable of binding to an IgE from subjects allergic to venom of an insect from the order Hymenoptera, wherein said polypeptide is expressed in E. Coli, High5 or Sf9 cells and has the amino acid sequence of SEQ ID NO: 2.

2. The polypeptide of claim 1, which is encoded by a naturally occurring nucleic acid of an insect from the order Hymenoptera.

3. The polypeptide of claim 1, wherein one or more glycosylation sites of the sequence Asn-Xaa-Ser/Thr has been mutated to a non-glycosylation site.

4. The polypeptide of claim 1, wherein the insect is a bee from the genus Apis.

5. The polypeptide of claim 4, wherein the bee is Apis mellifera.

6. The polypeptide of claim 1 having acid phosphatase activity.

7. The polypeptide of claim 1, wherein the polypeptide is non-glycosylated.

8. The polypeptide of claim 1, wherein the polypeptide is expressed in bacterial or insect cells.

9. The polypeptide of claim 1, wherein the glycosylation pattern of said polypeptide differs from the glycosylation pattern of natural acid phosphatase isolated from bee venom.

10. A pharmaceutical or diagnostic composition comprising the polypeptide of claim 1.

11. The composition of claim 10, further comprising a suitable adjuvant or excipient or further polypeptides from the venom of an insect from the order Hymenoptera.

12. A polypeptide selected from the group consisting of polypeptides consisting of amino acids 1 to 25, 146 to 152, 159 to 164, 168 to 184, 224 to 231, and 277 to 361 of the polypeptide of SEQ ID NO: 2.

13. A method of treating a subject allergic to the venom of an insect from the order Hymenoptera, comprising the step of administering the polypeptide of claim 1 to said subject.

14. A method of diagnosing an allergy to the venom of an insect from the order Hymenoptera, comprising the steps of
a) in vitro contacting a blood sample from a subject with the polypeptide of claim 1, and
b) detecting binding of IgE antibodies to the polypeptide, wherein specific binding can be determined by comparing with a specificity control, e.g., with an unrelated antibody; and wherein detecting IgE antibodies binding to the polypeptide indicates said allergy.