Patent Grant
9446121
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.
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.
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.
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
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
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
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
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
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
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
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
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
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
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
Furthermore, a method of diagnosing an allergy to venom of an insect from the order Hymenoptera is provided, comprising the steps of
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
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.
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 TriFast™, 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 RNaseOut™ 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) RevertAid™ 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:
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:
The resulting sequence is shown in
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:
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:
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:
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:
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.
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 Trap™ 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.
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).
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.
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,
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
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
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
Furthermore,
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.
Alt. alternata
Arachis
hypogaea
Arachis
hypogaea
Asp.
fumigatus
Asp.
fumigatus
Asp.
fumigatus
Asp.
fumigatus
Betulla
verrucosa
Betulla
verrucosa
Bos
domesticus
Bos
domesticus
Cryp.
japonica
Gallus
domesticus
Hevea
brasiliensis
Hevea
brasiliensis
Hevea
brasiliensis
Juniperus
ashei
Juniperus
ashei
Parietaria
judaica
Parietaria
judaica
Pen. notatum
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.
| Number | Name | Date | Kind |
|---|---|---|---|
| 5304631 | Stewart et al. | Apr 1994 | A |
| 6812339 | Venter et al. | Nov 2004 | B1 |
| 7365185 | Boukharov et al. | Apr 2008 | B2 |
| 20040023291 | Spertini | Feb 2004 | A1 |
| 20040034888 | Liu et al. | Feb 2004 | A1 |
| Number | Date | Country |
|---|---|---|
| 2341389 | Mar 2000 | GB |
| WO 0055174 | Sep 2000 | WO |
| WO 0277183 | Mar 2002 | WO |
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| Number | Date | Country | |
|---|---|---|---|
| 20100015122 A1 | Jan 2010 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60635479 | Dec 2004 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11301329 | Dec 2005 | US |
| Child | 12404168 | US |
