Patent Grant
9844591
The present invention relates to novel polypeptides and uses thereof.
Type I allergy is an IgE-mediated hypersensitivity disease affecting almost 25% of the population. It is based on the recognition of harmless airborne, insect, venom, food allergen and contact allergen antigens derived from per se harmless antigen sources such as pollen, insects, mold and animal proteins by specific immunoglobulin E. The crosslinking of effector cell-bound IgE antibodies leads to a release of inflammatory mediators (e.g., histamine, leucotrienes) and thus to the immediate symptoms of allergy (e.g., rhinoconjunctivitis, asthma, dermatitis, anaphylaxis). T-cell activation via IgE-dependent as well as IgE-independent mechanisms contributes to chronic allergic inflammation.
The probably only causative form of allergy treatment is allergen-specific immunotherapy, which is based on the repeated administration of increasing amounts of allergen extracts for most sources. Numerous clinical studies have documented the clinical efficacy of injection immunotherapy and there is evidence for several immunological mechanisms underlying this treatment. Due to the difficulty to prepare high quality allergen extracts for certain allergen sources and the fact that the administration of allergens to patients can cause severe side effects, allergen-specific immunotherapy can only be recommended for certain patients groups and disease manifestations. It is especially difficult to treat patients with co-sensitizations to several different allergen sources and patients suffering from severe disease manifestations such as allergic asthma. Allergic asthma is one of the most vigorous manifestations of allergy, because it severely affects the quality of daily life, causes a high rate of hospitality admissions and can manifest itself in serious, life-threatening forms requiring intensive care of the patient.
Allergen extracts prepared from natural allergen-sources are crude in nature, and it is impossible to influence the quality and amounts of individual allergens in such preparations by technical means. They also contain numerous undefined non-allergenic components, and several recent studies indicate the poor quality of such extracts and document their great heterogeneity.
In the last decade great progress has been made in the field of molecular allergen characterization using recombinant DNA technology. A large number of the most important disease-eliciting allergens has been characterized down to the molecular level, and recombinant allergens mimicking the epitope complexity of natural allergen extracts have been produced. Moreover, several research groups have used the knowledge regarding allergen structures to develop defined new allergy vaccines. Genetic engineering, synthetic peptide chemistry and conjugation of allergens with immunostimulatory DNA sequences have been used to reduce the allergenic activity of the new vaccines and thus the rate of therapy-induced side effects. First promising clinical studies were conducted with such allergen derivatives. Interestingly, it turned out that although IgE-reactivity of genetically engineered recombinant allergens and allergen-derived synthetic T-cell epitope-containing peptides could be strongly reduced or even abolished, these derivatives still could induce systemic side effects appearing several hours after injection. For example, it was reported that T-cell epitope peptides of the major cat allergen, Fel d 1, induced asthma and bronchial hyper reactivity several hours after intracutaneous injection, and there is strong evidence that this effect is T-cell mediated and MHC-restricted.
These results indicate that the removal of IgE-reactivity diminishes IgE-mediated side effects since no immediate reactions were recorded in the course of these immunotherapy studies. However, the allergen-specific T-cell epitopes which have been preserved in the recombinant allergen derivatives as well as in the peptide mixtures are responsible for the late side effects (e.g. very problematic or atopic dermatitis, chronic T-cell-mediated allergic skin manifestation). The side effects caused in the case of recombinant allergen-derivatives were relatively mild and in the case of the T-cell peptide vaccines may be overcome by adequate dosing. Both of the two new approaches therefore seem very promising for immunotherapy of allergic rhinoconjunctivitis but may have limitations when it comes to the treatment of severe forms of allergic asthma, where the induction of late side effects in the lung may be very problematic.
In order to administer and consequently to provoke an efficient immune response against peptides, polypeptides and proteins, adjuvants and/or carriers are regularly used. Complete Freund's adjuvant (CFA), for instance, is one of the most potent adjuvants available. There exists a need for vaccine compositions able to induce strong immune responses against peptides and polypeptides derived from allergens and of course of other antigens with or without the use of complete Freund's adjuvant. Further, while BSA has been used successfully as a carrier in animal models it may not be appropriate for use in human vaccine compositions because of the risk of adverse reactions such as the risk of transmitting prion disease (variant Creutzfeldt-Jakob disease). A further challenge to the development of an effective vaccine against allergens is the need for an immune response able to rapidly decrease allergens in an individual or animal. Therefore, high concentrations of allergen-specific antibodies in the blood, which are mainly of the IgG subtype, are needed. In mucosal surfaces IgA antibodies are also important.
Cholera toxin, a known carrier protein in the art, is also used regularly as an adjuvant. However, cholera toxin increases total and specific IgE antibody levels and leads to IgE-associated inflammatory reactions.
Due to the side effects provoked by most carrier proteins used for vaccination, there exists a need for carrier systems which are able to stimulate immune responses against allergens or other antigens, without using toxic adjuvants, without using poorly tolerated carrier proteins and, in certain situations, without stimulation of potentially pathologic immune responses. Novel carrier systems meeting these specifications can be used towards the formation of novel conjugates and compositions suitable for the treatment or prevention of diseases like allergic diseases.
In Bohle B. et al. (J. Immunol. 172 (11) (2004): 6642-6648) a recombinant fusion protein comprising an S-layer protein moiety and Bet v 1 moiety is described. This molecule comprises the native Bet v 1 allergen including Bet v 1-specific T cell epitopes.
WO 2004/004761 relates to virus like particles which are fused to an immunogen and which may be used for immunisation.
In WO 2004/003143 the use of fusion proteins comprising a virus like particle and an allergenic molecule as immunogen for vaccination is disclosed.
In WO 2007/140505 and Edlmayr et al. (J. Immunol. 182 (10) (2009) 6298-6306) the use of fusion proteins comprising various carrier molecules fused to allergen-derived peptides are described to induce allergen-specific IgG antibodies but these constructs do not exhibit an immunomodulatory effect which may be considered advantageous for allergic patients such as the induction of IL-10 or Th1 immunity. FIG. 4 of Edlmayr et al shows that KLH-fused peptides induce the Th2 cytokine IL-5 and VP1 fusion proteins do not induce IL-10 or IFN-gamma.
In Niespodziana et al (J. Allergy Clin. Immunol. 127 (6) (2011) 1562-1570) the use of fusion proteins each comprising Hepatitis B-derived PreS and two peptides derived from the major cat allergen Fel d 1 are described to induce allergen-specific IgG antibodies. However, no regimen suitable for vaccination of humans has been described and the peptides contained allergen-specific T cell epitopes.
It is an object of the present invention to provide medicaments and carriers which overcome the aforementioned drawbacks and allow an allergen vaccination with reduced side effects.
Therefore, the present invention relates to a polypeptide comprising at least three peptide fragments consisting of 10 to 50 consecutive amino acid residues of at least one wild-type allergen fused to the N- and C-terminus of a surface polypeptide of a virus of the hepadnaviridae family or at least one fragment of said surface polypeptide or comprising a surface polypeptide of a virus of the hepadnaviridae family or at least one fragment thereof fused N- and/or C-terminally to at least three peptides derived from at least one wild-type allergen.
In order to provoke an enhanced immune response against a molecule, in particular against an allergenic or hypoallergenic molecule according to the present invention, at least three peptide fragments derived from at least one wild-type allergen are fused (by genetic engineering) to a surface polypeptide of a virus of the hepadnaviridae family, preferably of a Hepatitis B virus, more preferably of a Hepatitis B PreS polypeptide, or at least one fragment thereof. It turned surprisingly out that in contrast to conventionally and regularly employed carrier proteins like KLH (Keyhole limpet hemocyanin) a surface polypeptide of a virus of the hepadnaviridae family, preferably of a Hepatitis B virus, more preferably of a Hepatitis B PreS polypeptide, or fragments thereof lead to an enhanced formation of antibodies directed to those peptides which are bound thereto.
Moreover, it turned out that allergen specific IgG antibodies induced by immunization with more than three properly selected allergen derived peptide fragments fused to the Hepatitis B PreS polypeptide are better focused to the IgE epitopes of the allergen while immunization with the wild-type allergen leads to IgG which are directed to all parts of the allergen—also those which are not IgE reactive. In an experiment normalized for IgG titers this leads to a better blocking capacity of PreS/peptide induced IgG compared to wild-type allergen induced (
Also very surprisingly, it turned out that in cultures of human PBMCs fusion proteins of allergen derived peptide fragments to the Hepatitis B PreS polypeptide strongly induced the cytokines IL-10 and IFN-gamma, which have been attributed as positive indicators for a successful allergy immunotherapy. In contrast, induction of IL-10 and IFN-gamma was significantly lower with wild-type allergen, allergen derived peptide fragments alone or PreS alone (
“Fused to the N- and C-terminus”, as used herein, means that at least one peptide is fused to the N-terminus of a surface polypeptide of a virus of the hepadnaviridae family or at least one fragment of said surface polypeptide and at least one peptide is fused to the C-terminus of a surface polypeptide of a virus of the hepadnaviridae family or at least one fragment of said surface polypeptide. In a most simplest embodiment of the present invention a surface polypeptide of a virus of the hepadnaviridae family or at least one fragment of said surface polypeptide may comprise at the N-terminus one peptide and on the C-terminus two peptides or vice versa.
The polypeptide of the present invention preferably comprises at least four, more preferably at least five, even more preferably at least six, peptide fragments, preferably B cell binding peptides, derived from an allergen, whereby four peptides are most preferred.
According to a particularly preferred embodiment of the present invention the carrier protein is the Hepatitis B PreS polypeptide with the following amino acid sequence (SEQ ID No. 21):
It is also possible to use fragments Hepatitis B PreS1 or Hepatitis B PreS2 of the Hepatitis B PreS polypeptide. A fragment of the Hepatitis B PreS polypeptide preferably comprises or consists of at least 30, preferably at least 40, more preferably at least 50, consecutive amino acid residues of SEQ ID No. 21.
“Hypoallergenic” as used herein, refers to molecules with reduced or no allergenic potential (i.e. IgE reactivity determined with IgE binding assays known in the art). Such molecules have a decreased capacity to provoke allergic reactions in an individual compared to the wild-type protein from which these molecules are derived.
The at least three, preferably at least four, more preferably at least five, even more preferably at least six, peptide fragments fused to the N- and C-terminus of a surface polypeptide of a virus of the hepadnaviridae family or at least one fragment of said surface polypeptide comprise or consist of 10 to 50 consecutive amino acids, more preferably 15 to 50 consecutive amino acids, in particular 20-50 consecutive amino acids, of at least one wild-type allergen and exhibit preferably reduced IgE reactivity compared to the wild-type allergen from which the peptide fragments are derived from. These peptide fragments are preferably designed to exclude allergen-specific T-cell epitopes which may cause T-cell-mediated side effects. T-cell epitopes and molecules exhibiting reduced T-cell response may be determined and identified by methods known by the person skilled in the art (e.g., Bercovici N. et al. Clin Diagn Lab Immunol. (2000) 7:859-864).
The at least three peptide fragments comprising or consisting of 10 to 50 consecutive amino acids, more preferably 15 to 50 consecutive amino acids, in particular 20-50 consecutive amino acids, of at least one wild-type allergen can be derived from one and the same allergen. If two or more fragments are derived from the same allergen these two or more fragments are not adjacently located in the wild type allergen and/or have an order in the polypeptide of the present invention which does not correspond to the order in the wild type allergen.
The term “peptide fragment” as used herein means a part/fragment of a hypoallergenic polypeptide or fusion protein of the invention which is derived from the primary structure of a wild-type allergen and comprise or consist of 10 to 50 consecutive amino acids, more preferably 15 to 50 consecutive amino acids, in particular 20-50 consecutive amino acids, of this wild-type allergen.
The terms “derived from an allergen” and “derived from at least one wild-type allergen”, as used herein, mean that the peptide fragments according to the present invention are obtained directly from an allergen by fragmentation or truncation. The amino acid sequence of these peptide fragments is preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical, in particular 100% identical, to the amino sequence stretch of the wild-type allergen, from which the peptide fragments are derived from. However, the peptides which are not 100% identical to the wild-type allergen fragments should be able to bind with at least 60%, preferably at least 70%, more preferably at least 80%, most preferably at least 90%, strength to an antibody or to antibodies, preferably to IgG antibodies, which are directed to said wild-type allergen fragments. “At least one wild-type allergen” means that the polypeptide of the present invention may comprise B-cell binding peptides of more than one, preferably two, more preferably three, different wild-type allergens (i.e. sources) (e.g. one peptide is derived from Bet v 1, one from Amb a 1 and one from Phl p 1 or two peptides are derived from Bet v 1 and one from Amb a 1).
The degree of identity of a first amino acid sequence to a second amino acid can be determined by a direct comparison between both amino acid sequences using certain algorithms. Such algorithms are, for instance, incorporated in various computer programs (e.g. “BLAST 2 SEQUENCES (blastp)” (Tatusova et al. (1999) FEMS Microbiol. Lett. 174:247-25; Corpet F, Nucl. Acids Res. (1988) 16:10881-10890).
The polypeptides of the present invention may be obtained by recombinant methods or chemical synthesis. Alternatively, it is, of course, also possible to obtain the molecules by enzymatic or chemical cleavage of the wild-type allergen or a polypeptide/protein harbouring the molecule of interest.
It was now surprisingly found that peptide carrier fusion proteins with improved properties can be obtained by employing surface proteins from viruses of the hepadnaviridae class, more specifically the human hepatitis B virus. One up to 20, preferably 3 or 4 up to 20, more preferably 3 or 4 up to 15, even more preferably 3 or 4 up to 10 (i.e. 3, 4, 5, 6, 7, 8, 9, 10), peptide fragments, preferably hypoallergenic peptide fragments, can be fused to the C-terminus and the N-terminus of a surface polypeptide of a virus of the hepadnaviridae family or at least one fragment of said surface polypeptide. A preferred embodiment of the current invention are therefore fusion proteins composed of at least 3 up to 6 hypoallergenic peptide fragments with a carrier protein derived from the surface antigens of human hepatitis B virus. According to a particularly preferred embodiment of the present invention such fusion proteins use the preS protein as carrier. A most preferred embodiment of this invention are fusion proteins where 4 hypoallergenic peptide fragments are fused to the preS carrier protein or a fragment thereof. The (hypoallergenic) peptide fragments can be the same or different and can derived from one or several allergenic proteins and the locus of the peptides within the fusion protein is the C-terminus and the N-terminus of the carrier protein. One up to three (hypoallergenic) peptide fragments can be fused to each of the C-terminus and the N-terminus in such a way that the sum of the (hypoallergenic) peptide fragments will be, for instance, three or four to six. The terms “fused” or “fusion protein”, refer to a preferred embodiment of the invention, meaning that the non-allergenic carrier protein and the (hypoallergenic) peptide fragments at the carrier's C- and N-terminus are expressed and prepared as one singular recombinant polypeptide chain
A most highly preferred embodiment of the current invention are fusion proteins of the hepatitis B virus preS protein, which carry (hypoallergenic) peptide fragments derived from a specific allergen, such that one or two, preferably two, peptide fragments each are fused to the C-terminus and the N-terminus of the carrier. For illustration, the preferred polypeptides of the current invention may have the general molecular structure represented by the following generic structures:
It is understood that peptides A, B, C and D can be the same or different and may be derived from the same allergen for each individual fusion protein or will be derived from different allergens.
The (hypoallergenic) peptides to be fused to the N- and C-terminus of the surface polypeptide of a virus of the hepadnaviridae family or at least one fragment of said surface polypeptide, preferably the preS protein or a fragment thereof, are preferably selected from the group consisting of major birch pollen allergens, in particular Bet v 1 and Bet v 4, major timothy grass pollen allergens, in particular Phl p 1, Phl p 2, Phl p 5, Phl p 6 and Phl p 7, major house dust mite allergens, in particular Der p 1, Der p 2, Der p 5, Der p 7, Der p 21 and Der p 23, major cat allergen Fel d 1, the major ragweed allergen Amb a 1, the major Japanese cedar allergens Cry j 1 and Cry j 2, major bee allergens, major wasp allergens, profilins, especially Phl p 7, Phl p 12.
Other suited allergens to be used according to the present invention can be derived from the following table 2 without being restricted to said table.
Ambrosia artemisiifolia
Ambrosia trifida
Artemisia vulgaris
Helianthus annuus
Mercurialis annua
Chenopodium album
Salsola kali
Humulus japonicus
Parietaria judaica
Parietaria officinalis
Cynodon dactylon
Dactylis glomerata
Festuca pratensis
Holcus lanatus
Lolium perenne
Phalaris aquatica
Phleum pratense
Poa pratensis
Sorghum halepense
Phoenix dactylifera
Alnus glutinosa
Betula verrucosa
Carpinus betulus
Castanea sativa
Corylus avellana
Quercus alba
Fraxinus excelsior
Ligustrum vulgare
Olea europea
Syringa vulgaris
Plantago lanceolata
Cryptomeria japonica
Cupressus arisonica
Cupressus sempervirens
Juniperus ashei
Juniperus oxycedrus
Juniperus sabinoides
Juniperus virginiana
Platanus acerifolia
Acarus siro
Blomia tropicalis
Dermatophagoides farinae
Dermatophagoides microceras
Dermatophagoides pteronyssinus
Euroglyphus maynei
Glycyphagus domesticus
Lepidoglyphus destructor
Tyrophagus putrescentiae
Bos domesticus
Canis familiaris
Equus caballus
Felis domesticus
Cavia porcellus
Mus musculus
Rattus norvegius
Alternaria alternata
Cladosporium herbarum
Aspergillus flavus
Aspergillus fumigatus
Aspergillus niger
Aspergillus oryzae
Penicillium brevicompactum
Penicillium chrysogenum
Penicillium citrinum
Penicillium oxalicum
Fusarium culmorum
Trichophyton rubrum
Trichophyton tonsurans
Candida albicans
Candida boidinii
Psilocybe cubensis
Coprinus comatus
Rhodotorula mucilaginosa
Malassezia furfur
Malassezia sympodialis
Epicoccum purpurascens
Aedes aegyptii
Apis mellifera
Bombus pennsylvanicus
Blattella germanica
Periplaneta americana
Chironomus kiiensis
Chironomus thummi thummi
Ctenocephalides felis felis
Thaumetopoea pityocampa
Lepisma saccharina
Dolichovespula maculata
Dolichovespula arenaria
Polistes annularies
Polistes dominulus
Polistes exclamans
Polistes fuscatus
Polistes gallicus
Polistes metricus
Vespa crabo
Vespa mandarina
Vespula flavopilosa
Vespula germanica
Vespula maculifrons
Vespula pennsylvanica
Vespula squamosa
Vespula vidua
Vespula vulgaris
Myrmecia pilosula
Solenopsis geminata
Solenopsis invicta
Solenopsis saevissima
Triatoma protracta
Gadus callarias
Salmo salar
Bos domesticus
Cyprinus carpio
Gallus domesticus
Metapenaeus ensis
Penaeus aztecus
Penaeus indicus
Penaeus monodon
Todarodes pacificus
Helix aspersa
Haliotis midae
Rana esculenta
Brassica juncea
Brassica napus
Brassica rapa
Hordeum vulgare
Secale cereale
Triticum aestivum
Zea mays
Oryza sativa
Apium graveolens
Daucus carota
Corylus avellana
Malus domestica
Pyrus communis
Persea americana
Prunus armeniaca
Prunus avium
Prunus domestica
Prunus persica
Asparagus officinalis
Asparagus
Crocus sativus
Lactuca sativa
Vitis vinifera
Musa × paradisiaca
Ananas comosus
Citrus limon
Citrus sinensis
Litchi chinensis
litchi
Sinapis alba
Glycine max
Vigna radiata
Arachis hypogaea
Lens culinaris
Pisum savitum
Actinidia chinensis
Capsicum annuum
Lycopersicon esculentum
Solanum tuberosum
Bertholletia excelsa
Juglans nigra
Juglans regia
Anacardium occidentale
Ricinus communis
Sesamum indicum
Cucumis melo
Anisakis simplex
Argas reflexus
Ascaris suum
Carica papaya
papaya
Dendronephthya nipponica
Hevea brasiliensis
Homo sapiens
Triplochiton scleroxylon
According to a particularly preferred embodiment of the present invention at least one, preferably at least two, more preferably at least three, in particular all, of the at least three peptides derived from the at least one wild-type allergen is a B cell binding peptide.
“B cell binding peptides” to be used for allergy vaccination according to the invention are derived from or close to the IgE binding sites of allergens but per se show no or minimal IgE reactivity compared to the wild-type allergen (Focke M et al. Clinical & Experimental Allergy 40(2010):385-397). Requirements for their production and selection are the knowledge of the primary sequence of the allergen and regarding the IgE binding sites. Upon immunization, B cell binding peptides fused to a suitable immunogenic carrier, are capable of inducing the production of allergen-specific IgG which can block IgE binding to the allergen. Whether the IgG induced with the fusion protein can recognize the allergen can be determined, for instance, by testing the IgG for reactivity with the complete allergen. Suitable methods include ELISA, dot blot or Western blot assays. Those peptides are preferred which induce IgG that blocks patients IgE binding to the allergen.
The present invention shows that the use of suitable B cell binding peptides in particular when three or more are fused to a suitable carrier according to the present invention allows the induction of IgG responses which are better focused to the IgE epitopes than those induced by immunization even with a complete allergen. Furthermore, the invention shows that the combination of the appropriate peptides and their number with a suitable carrier can direct the allergen-specific immune response towards a favorable anti-allergic immune response (characterized by the induction of preferentially allergen-specific IgG and not IgE responses and tolerogenic (IL-10) and Th1 (Interferon gamma) cytokine responses.
Moreover, it surprisingly turned out that—despite the fact that they lack allergen-specific T-cell epitopes—polypeptides according to the invention containing 3 or more B cell binding peptides fused to an immunogenic carrier are able reduce allergen-specific T-cell reactions. This is shown by the fact that the presence of allergen-specific IgG induced by therapeutic vaccination with the hypoallergenic polypeptides of the present invention reduces allergen-specific T-cell activation caused by IgE facilitated antigen presentation in PBMCs from vaccinated human allergic individuals. (
According to a preferred embodiment of the present invention at least one of said at least three peptides exhibits no or reduced IgE-binding capacity compared to the wild-type allergen.
According to another preferred embodiment of the present invention at least one, preferably at least two, more preferably at least three, of said at least three B-cell binding peptides exhibits no or substantially no T-cell reactivity.
The presence of allergen-specific T cell epitopes may give rise to unwanted T cell mediated side effects. Therefore it is particularly preferred to use peptides exhibiting no or substantially no T-cell reactivity in order to obtain the polypeptides of the present invention.
However, also allergen fragments comprising at least one T-cell epitope may be used in the polypeptide according to the present invention.
“Exhibiting reduced IgE-binding capacity”, as used herein, means that the molecules according to the present invention show significantly reduced IgE-binding capacity or activity (at least 50% less, preferably at least 70% less, more preferably at least 80% less, even more preferably at least 90% less, most preferably at least 95% less, binding capacity compared to the wild-type allergen) or even lack IgE-binding at all.
IgE-binding activity/capacity of molecules like peptides and proteins can be determined by, for example, an enzyme linked immunosorbent assay (ELISA) using, for example, sera obtained from a subject, (i.e., an allergic subject) that has been previously exposed to the wild-type allergen. Briefly, a peptide to be tested is coated onto wells of a microtiter plate. After washing and blocking the wells, an antibody solution consisting of the plasma of an allergic subject, who has been exposed to the peptide being tested or the protein from which it was derived, is incubated in the wells. A labelled secondary antibody is added to the wells and incubated. The amount of IgE-binding is then quantified and compared to the amount of IgE bound by a purified wild-type allergen.
Alternatively, the binding activity of a peptide can be determined by Western blot analysis. For example, a peptide to be tested is run on a polyacrylamide gel using SDS-PAGE. The peptide is then transferred to nitrocellulose and subsequently incubated with serum from an allergic subject. After incubation with the labelled secondary antibody, the amount of IgE bound is determined and quantified.
Another assay which can be used to determine IgE-binding activity of a peptide is a competition ELISA assay. Briefly, an IgE-antibody pool is generated by combining plasma from allergic subjects who have been shown by direct ELISA to be IgE-reactive with wild-type allergen. This pool is used in ELISA competition assays to compare IgE-binding to wild-type allergen to the peptide tested. IgE-binding for the wild-type allergen and the peptide being tested is determined and quantified.
A “T-cell epitope” means a protein, peptide or polypeptide (e.g., allergen) or fragment thereof, for which a T-cell has an antigen specific binding site, the result of binding to said binding site activates the T-cell. The term “exhibiting reduced T-cell reactivity”, as used herein, refers to molecules which exhibit a T-cell reactivity which is significantly reduced compared to the stimulation induced by the wild-type allergen from which the hypoallergenic molecule is derived using equimolar amounts in standard assays known in the art (reduced T-cell reactivity means at least 30%, preferably at least 50%, more preferably at least 70%, most preferably at least 90%, less stimulation of hypoallergenic molecules compared to the wildtype allergen at equimolar amounts). In a particular preferred embodiment of this invention, the molecules may “lack” T-cell epitopes and thus molecule shows reduced T-cell reactivity in the individual(s) to be treated (i.e., who is to receive an epitope-presenting valency platform molecule). It is likely that, for example, an allergen-derived molecule may lack a T-cell epitope(s) with respect to an individual, or a group of individuals, while possessing a T-cell epitope(s) with respect to other individual(s). Methods for detecting the presence of a T-cell epitope are known in the art and include assays which detect T-cell proliferation (such as thymidine incorporation). Immunogens that fail to induce statistically significant incorporation of thymidine above background (i.e., generally p less than 0.05 using standard statistically methods) are generally considered to lack T-cell epitopes, although it will be appreciated that the quantitative amount of thymidine incorporation may vary, depending on the immunogen being tested (see, e.g., Zhen L. et al. (Infect Immun. (2003) 71:3920-3926)). Generally, a stimulation index below about 2-3, more preferably less than about 1, indicates lack of T-cell reactivity and epitopes. The presence of T-cell epitopes can also be determined by measuring secretion of T-cell-derived lymphokines according to standard methods. The stimulation index (SI) may be calculated by dividing the proliferation rate (Thymidine uptake) of stimulated cells through the proliferation rate of unstimulated cells in medium alone. SI=1 means no stimulation, and SI>1 indicates stimulation of cells. Location and content of T-cell epitopes, if present, can be determined empirically.
The cytokine secretion may be determined in addition to the stimulation of T cells. For example, IFN-gamma and IL-10 as biomarkers for increased activity of regulatory T cells have been recognized as cytokines accompanying a successful allergy immunotherapy.
The peptide fragments of the present invention are preferably composed or consist of amino acids 151 to 177, 87 to 117, 1 to 30, 43 to 70 or 212 to 241 of Phl p 1, amino acids 1 to 33, 8 to 39, 34 to 65 or 66 to 96 of Phl p 2, amino acids 93 to 128, 98 to 128, 26 to 53, 26 to 58, 132 to 162, 217 to 246, 252 to 283 or 176 to 212 of Phl p 5, amino acids 23 to 54, 56 to 90, 73 to 114 or 95 to 127 of Phl p 6, amino acids 1 to 34 or 35 to 70 of chain 1 of Fel d 1, amino acids 1 to 34, 35 to 63 or 64 to 92 of chain 2 of Fel d 1, amino acids 30 to 59, 50 to 79, 75 to 104, 30 to 74 or 60 to 104 of Bet v 1, amino acids 1 to 30, 52 to 84 or 188 to 222 of Der p 1, amino acids 1 to 33, 21 to 51, 42 to 73, 62 to 103 or 98 to 129 of Der p 2, amino acids 1 to 30, 20 to 50, 50 to 80, 90 to 125, 125 to 155 or 165 to 198 of Der p 7, amino acids 1-35, 36-70, 71-110, 111-145, 140-170, 175-205, 210-250 or 250-284 of Der p 10, amino acids 1 to 35, 35 to 72, 70 to 100 or 90 to 122 of Der p 21, amino acids 1 to 32, 15 to 48 or 32 to 70, 32 to 60, 52 to 84, 32 to 70 (Cys→Ser) of Der p 23, amino acids 19 to 58, 59 to 95, 91 to 120 or 121 to 157 of Alt a 1, amino acids 31 to 60, 45 to 80, 60 to 96 or 97 to 133 of Par j 2, amino acids 1 to 40, 36 to 66, 63 to 99, 86 to 120 or 107 to 145 of Ole e 1, amino acids 25 to 58, 99 to 133, 154 to 183, 277 to 307, 334 to 363, 373 to 402, 544 to 573, 579 to 608, 58 to 99, 125 to 165, 183 to 224, 224 to 261, 252 to 289, 303 to 340, 416 to 457, 460 to 500 or 501 to 542 of Fel d 2, amino acids 19 to 58, 52 to 91, 82 to 119, 106 to 144 or 139 to 180 of Can f 2, amino acids 19 to 56, 51 to 90, 78 to 118, 106 to 145 or 135-174 of Can f 1, amino acids 27 to 70, 70 to 100 or 92 to 132 of Art v 1, amino acids 31 to 70, 80 to 120, 125 to 155, 160 to 200, 225 to 263, 264 to 300 305 to 350 or 356 to 396 of Amb a 1, amino acids 1 to 34, 35 to 74, 74 to 115, 125 to 165, 174 to 213, 241 to 280, 294 to 333, 361 to 400 or 401 to 438 of Alt a 6, amino acids 1 to 40, 41 to 80, 81 to 120, 121 to 160 of Alt a 2 or fragments or sequence variations thereof.
The specific amino acid sequences of the above identified allergen-derived molecules are (peptides in the following table having an N- and/or C-terminal cysteine residue (C) being used in the polypeptide of the present invention may lack said cysteine residue):
The terms “fragments thereof” and “sequence variations thereof” refer to peptides which are deduced from the allergen-derived molecules disclosed herein and show biochemical properties (e.g. the capacity to prevent IgE binding to the allergen from which those molecules are derived from) which are comparable or identical to said allergen-derived molecules. The fragments of the present invention comprise at least 5, preferably at least 7, more preferably at least 10, successive and/or a maximum of 95%, preferably a maximum of 90%, more preferably a maximum of 80% amino acid residues of the allergen-derived molecule. The term “sequence variation” includes modifications of the peptides such as fragmentation (see above), amino acid substitutions (in particular cysteine or methionine residues may be exchanged with serine, alanine or other natural or non-natural amino acids or amino acid derivatives), deletions or additions. “Sequence variation” refers also to said allergen-derived molecules of the above table, wherein at least 1, preferably at least 2, more preferably at least 3, even more preferably at least 4 (5, 6, 7, 8, 9, 10, 15, 20) amino acid residues are added to the C- and/or N-terminus.
It is noted that the allergen referred to herein as “clone 30 allergen” is an allergen derived from the house dust mite Dermatophagoides pteronyssinus and consists of the following sequence: MANDNDDDPTTTVHPTTTEQPDDKFECPSRFGYFADPKDPHKFYICSNWEAVHKDCP GNTRWNEDEETCT (SEQ ID No. 140; see also WO 2007/124524). In the meantime, the allergen name Der p 23 has been assigned to clone 30 allergen. This means that Der p 23 and clone 30 allergen are synonyms.
According to the present invention also peptides are encompassed which are at least 80% identical, preferably 90% identical, to the amino sequences disclosed above.
According to a preferred embodiment of the present invention the surface polypeptide of the virus of the hepadnaviridae family or at least one fragment thereof comprises at least two B-cell binding peptide fragments derived from at least one wild-type allergen fused to its N-terminus and at least two B-cell binding peptide fragments derived from at least one wild-type allergen fused to its C-terminus.
In a particularly preferred embodiment of the present invention at least two of said at least three B-cell binding peptides are identical.
The polypeptide of the present invention can be used as vaccine in the treatment or prevention of an allergy in a human or animal.
The polypeptide is preferably administered to an individual in the amount of 0.01 microgram per kg body weight to 5 mg/kg body weight, pref-erably 0.1 microgram per kg body weight to 10 microgram per kg body weight.
According to a particularly preferred embodiment of the present invention the polypeptides of the present invention are administered to an individual in an amount of at least 10 μg, preferably at least 20 μg, per polypeptide. The maximum amount of polypeptides to be administered can be varied but is preferably below 100 μg, more preferably below 50 μg, even more preferably 40 μg or less, per polypeptide.
The amount of polypeptides that may be combined with excipients to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. The dose of the vaccine may vary according to factors such as the disease state, age, sex and weight of the individual, and the ability of antibody to elicit a desired response in the individual. Dosage regime may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. The dose of the vaccine may also be varied to provide optimum preventative dose response depending upon the circumstances. For instance, the polypeptides and vaccine of the present invention may be administered to an individual at intervals of several days, one or two weeks or even months depending always on the level of allergen-specific IgG induction.
In a preferred embodiment of the present invention the polypeptide/vaccine is applied between 2 and 10, preferably between 2 and 7, even more preferably up to 5 and most preferably up to 3 times. In a particularly preferred embodiment the time interval between the subsequent vaccinations is chosen to be between 2 weeks and 5 years, preferably between 1 month and up to 3 years, more preferably between 2 months and 1.5 years. The repeated administration of the peptide/vaccine of the present invention may maximize the final effect of a therapeutic vaccination.
According to a particularly preferred embodiment of the present invention three or more B-cell binding peptides selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 5, SEQ ID No. 9, SEQ ID No. 137, SEQ ID No. 139, SEQ ID No. 142 and SEQ ID No. 10 are bound N- and C-terminally to a surface polypeptide of the virus of the hepadnaviridae family, preferably the hepatitis PreS polypeptide or fragments thereof.
The polypeptides of the present invention comprising the at least three B-cell binding peptides derived from at least one wild-type allergen are preferably selected from the group consisting of SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18 and SEQ ID No. 19.
Another aspect of the present invention relates to a nucleic acid molecule encoding a polypeptide according to the present invention.
Another aspect of the present invention relates to a vector comprising a nucleic acid molecule according to the present invention.
Said vector is preferably an expression vector.
The vector harbouring the nucleic acid molecule of the present invention may be used for cloning purposes or for the production of expression vectors. Said vector can be a plasmid, cosmid, virus, bacteriophage or any other vector commonly used in genetic engineering, and can include, in addition to the nucleic acid molecule of the invention, eukaryotic or prokaryotic elements for the control of the expression, such as regulatory sequences for the initiation and the termination of the transcription and/or translation, enhancers, promoters, signal sequences and the like.
According to a preferred embodiment of the present invention the vector is a bacterial, fungal, insect, viral or mammalian vector.
The vector of the present invention may preferably be employed for cloning and expression purposes in various hosts like bacteria, yeasts, filamentous fungi, mammalian cells, insect cells, plant cells or any other prokaryotic or eukaryotic cells. Therefore, said vector comprises besides a nucleic acid encoding for a hypoallergenic molecule or fusion protein according to the present invention host specific regulatory sequences.
Another aspect of the present invention relates to a host comprising a nucleic acid molecule or a vector according to the present invention.
The nucleic acid molecule and the vector according to the present invention may be introduced into a suitable host. Said molecule may be incorporated into the genome of the host. The vector may exist extrachromosomally in the cytoplasm or incorporated into the chromosome of the host.
Yet another aspect of the present invention relates to an antibody directed against a hypoallergenic molecule, hypoallergenic fusion protein or a fusion protein according to the present invention.
Another aspect of the present invention relates to a vaccine formulation comprising at least one, preferably at least two, more preferably at least three, even more preferably at least 4, polypeptide according to the present invention.
In a particularly preferred embodiment of the present invention the vaccine comprises at least one, preferably at least two, preferably at least three, preferably at least four, preferably at least 5, polypeptides having an amino acid sequence selected from the group consisting of SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 149, SEQ ID No. 150, SEQ ID No. 151 and SEQ ID No. 152.
Depending on the composition such a vaccine can be used in the treatment and/or prevention of grass pollen allergies, birch pollen allergies, house dust mite allergies or a combination of those allergies in individuals suffering from such allergies or being at risk to suffer therefrom.
The term “preventing”, as used herein, covers measures not only to prevent the occurrence of disease, such as risk factor reduction, but also to arrest its progress and reduce its consequences once established. “Preventing” means also to prevent sensitization of an individual being at risk to get an allergy.
As used herein, the term “treatment” or grammatical equivalents encompasses the improvement and/or reversal of the symptoms of disease (e.g., allergy). A compound which causes an improvement in any parameter associated with disease when used in the screening methods of the instant invention may thereby be identified as a therapeutic compound. The term “treatment” refers to both therapeutic treatment and prophylactic or preventative measures.
According to one of the most preferred embodiment of the present invention the vaccine comprises polypeptides having amino acid sequence SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16 and SEQ ID No. 17.
According to another preferred embodiment of the present invention the vaccine comprises polypeptides having amino acid sequence SEQ ID No. 18 and/or SEQ ID No. 19.
According to a particularly preferred embodiment of the present invention the vaccine comprises polypeptides of the present invention which comprise allergen fragments derived from house dust mite allergens. Particularly preferred are amino acid residues 1 to 33, 21 to 51, 42 to 73, 62 to 103 or 98 to 129 of Der p 2, amino acid residues 1 to 30, 20 to 50, 50 to 80, 90 to 125, 125 to 155 or 165 to 198 of Der p 7, amino acid residues 1 to 35, 35 to 72, 70 to 100 or 90 to 122 of Der p 21, amino acids 1 to 32, 15 to 48 or 32 to 70, 32 to 60, 52 to 84, 32 to 70 (Cys→Ser) of Der p 23, amino acid residues 1 to 30, 1 to 41, 27 to 57, 42 to 82, 52 to 84, 85 to 115, 99 to 135, 145 to 175, 155 to 187, 175 to 208 or 188 to 222 of Der p 1. Most preferably, the vaccine comprises at least one of the polypeptides SEQ ID No. 149 to 152 (shown in
In a particularly preferred embodiment the polypeptide/vaccine of the present invention is administered 4 times per treatment year over a total treatment period of 1 to 5 years, preferably over 2 to 3 years. Of said 4 yearly administrations 3 are applied within a period of 6 to 12, preferably 8, weeks having intervals of 3 to 6 weeks, preferably 4 weeks, between administration 1 and 2 and another 3 to 6 weeks, preferably 4 weeks, between administration 2 and 3. The fourth administration is applied 3 to 7 months after the third administration. If the total treatment period exceeds 1 year, the same dosing regimen is applied in the following treatment years.
For the treatment of seasonal allergies (e.g. pollen allergies such as grass pollen allergy or birch pollen allergy) administration 1, 2, and 3 are preferably scheduled before the respective season with allergen exposure (pollen season), and the fourth administration is scheduled after the season.
The vaccine formulation according to the present invention may be formulated as known in the art and necessarily adapted to the way of administration of said vaccine formulation.
Preferred ways of administration of the vaccine formulation (of the present invention) include all standard administration regimes described and suggested for vaccination in general and allergy immunotherapy specifically (orally, transdermally, intraveneously, intranasally, via mucosa, rectally, etc). However, it is particularly preferred to administer the molecules and proteins according to the present invention subcutaneously or intramusculary.
The vaccine formulation according to the present invention may only comprise a viral capsid protein or fragments thereof of a member of the genus of hepadnaviridae.
Said formulation preferably further comprises at least one adjuvant, pharmaceutical acceptable excipient and/or preservative.
In order to increase the immunogenicity of the hypoallergenic molecules according to the present invention, adjuvants, for instance, may be used in a medicament according to the present invention. An adjuvant according to the present invention is an auxiliary agent which, when administered together or in parallel with an antigen, increases its immunogenicity and/or influences the quality of the immune response. Hence, the adjuvant can, e.g., considerably influence the extent of the humoral or cellular immune response. Customary adjuvants are, e.g., aluminum compounds, lipid-containing compounds or inactivated mycobacteria.
Generally, adjuvants can be of different forms, provided that they are suitable for administration to human beings. Further examples of such adjuvants are oil emulsions of mineral or vegetal origin, mineral compounds such as aluminium phosphate or hydroxide, or calcium phosphate, bacterial products and derivatives, such as P40 (derived from the cell wall of Corynebacterium granulosum), monophosphoryl lipid A (MPL, derivative of LPS) and muramyl peptide derivatives and conjugates thereof (derivatives from mycobacterium components), alum, incomplete Freund's adjuvant, liposyn, saponin, squalene, etc. (see, e.g., Gupta R. K. et al. (Vaccine 11:293-306 (1993)) and Johnson A. G. (Clin. Microbiol. Rev. 7:277-289). The medicament of the present invention comprises most preferably alum as adjuvant.
Another preferred embodiment of the present invention is a combination of more than one fusion protein containing hypoallergenic peptides and the hepatitis B pre S protein. These combinations may be derived from peptides from a single allergen or from different allergens of the same allergen source or from several different allergen source.
A preferred embodiment of the present invention relates to a mixture of four fusion proteins containing hypoallergenic peptides from Phl p 1, Phl p 2, Phl p 5, and Phl p 6 and the hepatitis B virus preS protein.
Another preferred embodiment of the present invention relates to a fusion protein or a mixture of 2 fusion proteins containing hypoallergenic peptides from Bet v 1 and the hepatitis B virus PreS protein.
Yet another preferred embodiment of the present invention relates to a mixture of at least 2 fusion proteins containing hypoallergenic peptides from house dust mite allergens, most preferably selected from Der p 1, Der p 2, Der p 5, Der p 7, Der p 21 and Der p 23 and the hepatitis B virus PreS protein. Most preferably, the mixture contains 3 fusion proteins containing hypoallergenic peptides derived from Der p 1, Der p 2, and Der p 23. It is particularly preferred that the mixture comprises at least one, preferably at least two, more preferably at least three, of the polypeptides shown in SEQ ID No. 149 to 152 (see also
Generally, specific vaccine formulations according to the present invention can be prepared for the treatment or prevention of different allergies by combination of hypoallergenic polypeptides of the invention representing the clinically relevant allergens of an allergen source. Methods to determine the clinically relevant allergens of an allergen source are known in the art and have been described before (Valenta and Niederberger, 2007, J Allergy Clin Immunol, 119 (4): 826-830). In a preferred embodiment, the hypoallergenic polypeptides of said specific vaccine formulation are adsorbed to an adjuvant which can be used in human (e.g. aluminium hydroxide), and the mixture is administered 3-4 times per year for 1-3 years applying more than 10 μg of each polypeptide present in the vaccine formulation per dose.
According to another preferred embodiment of the present invention said formulations comprise 10 ng to 1 g, preferably 100 ng to 10 mg, especially 0.5 μg to 200 μg of said hypoallergenic molecule or antibody.
Another aspect of the present invention relates to the use of a hypoallergenic protein or an antibody according to the present invention for manufacturing a medicament for the treatment or prevention of a viral infection and/or an allergy in a human or animal.
Said medicament preferably further comprises at least one adjuvant, pharmaceutical acceptable excipient and/or preservative.
The medicament according to the present invention may be used for active (administration of the hypoallergenic protein and/or molecules of the invention) as well as for passive immunization (antibodies directed to the hypoallergenic protein and/or molecules of the invention).
According to a preferred embodiment of the present invention said medicament comprises 10 ng to 1 g, preferably 100 ng to 10 mg, especially 0.5 μg to 200 μg of said hypoallergenic molecule, nucleic acid molecule, vector, host or antibody.
The medicament is preferably administered to an individual in amount of 0.01 μg/kg body weight to 5 mg/kg body weight, preferably 0.1 μg/kg body weight to 10 μg/kg body weight.
In a particularly preferred embodiment, the medicament is administered in a dose containing an absolute amount of 5-200 μg, more preferably 10-80 μg, most preferably 20-40 μg of each included hypoallergenic polypeptide
The particular dosage regimen, i.e., dose, timing and repetition, will depend on the particular individual and that individual's medical history. Empirical considerations, such as the half life, will generally contribute to determination of the dosage. Frequency of administration may be determined and adjusted over the course of therapy.
Most preferably, the dosing regimen for the medicament will consist of 4 yearly subcutaneous injections of one and the same dose over a total treatment period of 2 to 3 years. Of said 4 yearly subcutaneous injections 3 are applied within a period of 6 to 12, preferably 8, weeks having intervals of 3 to 6 weeks, preferably 4 weeks, between injection 1 and 2 and another 3 to 6 weeks, preferably 4 weeks, between injection 2 and 3. The fourth injection is applied 4 to 6 months after the third administration. The same dosing regimen is applied in the following treatment years.
For the treatment of seasonal allergies (e.g. pollen allergies such as grass pollen allergy or birch pollen allergy) administration 1, 2, and 3 are preferably scheduled before the respective season with allergen exposure (pollen season), and the fourth administration is scheduled after the season.
The individual to whom the medicament according to the present invention is administered is preferably an individual or animal which is having or is at risk of having an allergy.
Subjects having or at risk of having an allergy, allergic condition, allergic disorder or allergic disease include subjects with an existing allergic condition or a known or a suspected predisposition towards developing a symptom associated with or caused by an allergic condition. Thus, the subject can have an active chronic allergic condition, disorder or disease, an acute allergic episode, or a latent allergic condition, disorder or disease. Certain allergic conditions are associated with seasonal or geographical environmental factors. Thus, at risk subjects include those at risk from suffering from a condition based upon a prior personal or family history, and the season or physical location, but which the condition or a symptom associated with the condition may not presently manifest itself in the subject.
The administration of the medicament according to the present invention, which comprises at least one hypoallergenic molecule as described herein, to an individual may prevent sensitization of said individual or may induce an appropriate immune response to allergens. If the medicament of the present invention is used to prevent sensitization, it should be administered to an individual prior to the first contact with said allergen. Therefore, it is preferred to administer the medicament according to the present invention to neonates and children. It turned out that also the administration of the medicament according to the present invention to pregnant individuals will induce the formation of antibodies directed against allergens in the unborn child. It is especially beneficiary to use hypoallergenic molecules according to the present invention for such therapies, because due to the lack of allergen-specific T-cell epitopes side effects occurring in the course of allergen immunotherapy can significantly be reduced or even be completely avoided.
Another aspect of the present invention relates to the use of a viral capsid protein from a virus of the family of hepadnaviridae as a carrier in medicaments or vaccines.
One of the advantages of such a carrier is that not only the antigen fused or conjugated thereon may be exposed to the immune system, but also an immune response against the capsid protein of a hepadnavirus is induced. Consequently, such a vaccination may lead to the prevention and/or treatment of diseases caused by hepadnaviruses. The virus is preferably of the species of human hepatitis B virus.
Another aspect of the present invention relates to a hypoallergenic molecule derived from Phl p 5 (Genbank Nr. X7435) having a C- and/or N-terminal truncation and lacking substantially IgE-binding capacity.
Grass pollen is one of most potent outdoor seasonal sources of airborne allergens responsible for hay fever and allergic asthma.
More than 40% of allergic individuals display IgE-reactivity with grass pollen allergens, which are divided into more than 11 groups. More than 80% of the grass pollen allergic patients react with group 5 allergens.
Group 5 allergens are non-glycosylated, highly homologous proteins with a molecular mass range from 25-33 kD. Several group 5 allergens have been cloned and/or immunologically characterized.
The trial to reduce the allergenic activity by introducing point mutations, mutations of several amino acids in row or deletions showed no effect (Schramm G, et al. J Immunol 1999; 162: 2406-1435). IgE-binding regions of Phl p 5 (Flicker S, et al. J Immunol 2000; 165: 3849-3859) have already been described and the three-dimensional structure has been solved (Maglio O, et al. 2002. Protein Eng. 15:635-642).
It turned out that in particular the Phl p 5 peptides according to the present invention, which are C- and/or N-terminally truncated and lack IgE-binding capacity, may be employed for the active vaccination of individuals.
According to a preferred embodiment of the present invention the truncated molecule substantially lacks T-cell epitopes and, thus lacks Phl p 5-specific T-cell reactivity.
As already outlined above, late side effects of allergen immunotherapy can be significantly reduced or even be avoided if the hypoallergenic molecules substantially lack allergen-specific T-cell epitopes.
Truncated Phl p 5 molecules lacking T-cell epitopes are composed of amino acids 93 to 128, 98 to 128, 26 to 53, 26 to 58 or 252 to 283 of Phl p 5 or fragments or sequence variations thereof.
In particular these truncated molecules substantially show low or no allergen-specific T-cell reactivity and are, nevertheless, able to provoke an appropriate immune response directed against the wild-type allergen.
According to another preferred embodiment of the present invention the hypoallergenic truncated Phl p 5 is composed of amino acids 132 to 162, 217 to 246 or 176 to 212 of Phl p 5 or sequence variations thereof.
These hypoallergenic molecules comprise one or more T-cell epitopes but lack IgE-binding capacity.
Another preferred embodiment of the present invention are truncated Phl p 1 molecules lacking T-cell epitopes, which are composed of amino acids 1 to 30, 43 to 70, 87 to 117, 151 to 171 or 214 to 241 of Phl p 1 or sequence variations thereof fused to a viral carrier protein, preferable the Hep B pre S protein.
Another preferred embodiment of the present invention are truncated Phl p 2 molecules lacking T-cell epitopes, which are composed of amino acids 1 to 33, 8 to 39, 34 to 65 or 66 to 96 of Phl p 2 or sequence variations thereof fused to a viral carrier protein, preferrably the Hep B pre S protein.
Another preferred embodiment of the present invention are truncated Phl p 6 molecules lacking T-cell epitopes, which are composed of amino acids 23 to 54, 56 to 90, 73 to 114 or 95 to 127 of Phl p 6 or sequence variants thereof fused to a viral carrier protein, preferrably the Hep B pre S protein.
Another preferred embodiment of the present invention refers to truncated Bet v 1 molecules lacking T-cell epitopes, which are composed of amino acids 30 to 59, 50 to 79, 75 to 104, 30 to 74 or 60 to 104 of Bet v 1.
Another preferred embodiment of the present invention are combinations or mixtures of truncated Phleum pratense molecules lacking T-cell epitopes, fused to a viral carrier protein, preferrably the Hep B pre S protein, as described above.
A preferred embodiment of the present invention are combinations or mixtures of truncated Phleum pratense molecules lacking T-cell epitopes, which are composed of one each such fusion protein from truncated Phl p 1, Phl p 2, Phl p 5, and Phl p 6, as described above.
Another aspect of the present invention relates to a hypoallergenic molecule derived from Fel d 1 (Genbank Nr. X62477 and X62478) having a C- and/or N-terminal truncation and lacking IgE-binding capacity.
Allergies to animals affect up to 40% of allergic patients. In the domestic environment, allergies to the most popular pets, cats and dogs, are particularly prevalent and connected with perennial symptoms. Animal allergens are present in dander, epithelium, saliva, serum or urine. Exposure to the allergens can occur either by direct skin contact or by inhalation of particles carrying the allergens. The major cat and dog allergens were shown to be present widespread and could even be detected in non-pet owning households and in public places, e.g., schools. This can be attributed to the high and increasing number of households keeping pets in industrialized countries (about 50%) and the high stability of the allergens, which are carried off and distributed.
Fel d 1 was identified as the major cat allergen, which is recognized by more than 90% of cat allergic patients. Fel d 1 represents a 38 kDa acidic glycoprotein of unknown biological function. It consists of two identical non-covalently linked heterodimers, which, again, are composed of two polypeptide chains antiparallely linked by three disulfide bonds. Chain 1 and chain 2 are encoded on different genes, each consisting of 3 exons. Recombinant Fel d 1 (rFel d 1), expressed as a chain 2- to chain 1 fusion protein, has been generated in E. coli. This recombinant Fel d 1 is able to completely mimic the immunological properties of the wild-type allergen.
Peptides derived from the major cat allergen Fel d 1, and lacking IgE-binding capacity are suitable, e.g., for immunotherapy and prophylactic allergy vaccination. The Fel d 1-derived synthetic peptides—like the Phl p 5 and allergen-derived peptides disclosed herein—are capable of inducing an IgG response, i.e., the production of so called “blocking antibodies” or “protective antibodies”. These antibodies prevent IgE-binding to the allergen Fel d 1. A significant reduction in allergic symptoms may thus be achieved.
According to a preferred embodiment of the present invention the truncated molecule exhibits reduced T-cell reactivity.
In order to avoid or to significantly reduce late side effects the Fel d 1 derived hypoallergenic molecule exhibits reduced T-cell reactivity as defined in the present invention.
The truncated Fel d 1 is preferably composed of amino acids 1 to 34 or 35 to 70 of chain 1 of Fel d 1, amino acids 1 to 34, 35 to 63 or 64 to 92 of chain 2 of Fel d 1 or sequence variations thereof.
Another aspect of the present invention relates to hypoallergenic molecules being composed of or comprising amino acids 1 to 33, 21 to 51, 42 to 73, 62 to 103 or 98 to 129 of Der p 2, amino acids 1 to 30, 20 to 50, 50 to 80, 90 to 125, 125 to 155 or 165 to 198 of Der p 7, amino acids 1 to 35, 35 to 72, 70 to 100 or 90 to 122 of Der p 21, amino acids 1 to 32, 15 to 48 or 32 to 70, 32 to 60, 52 to 84, 32 to 70 (Cys→Ser) of Der p 23, amino acids 19 to 58, 59 to 95, 91 to 120 or 121 to 157 of Alt a 1, amino acids 31 to 60, 45 to 80, 60 to 96 or 97 to 133 of Par j 2, amino acids 1 to 40, 36 to 66, 63 to 99, 86 to 120 or 107 to 145 of Ole e 1, amino acids 25 to 58, 99 to 133, 154 to 183, 277 to 307, 334 to 363, 373 to 402, 544 to 573, 579 to 608, 58 to 99, 125 to 165, 183 to 224, 224 to 261, 252 to 289, 303 to 340, 416 to 457, 460 to 500 or 501 to 542 of Fel d 2, amino acids 19 to 58, 52 to 91, 82 to 119, 106 to 144 or 139 to 180 of Can f 2, amino acids 19 to 56, 51 to 90, 78 to 118, 106 to 145 or 135-174 of Can f 1, amino acids 27 to 70, 70 to 100 or 92 to 132 of Art v 1, amino acids 31 to 70, 80 to 120, 125 to 155, 160 to 200, 225 to 263, 264 to 300 305 to 350 or 356 to 396 of Amb a 1, amino acids 1 to 34, 35 to 74, 74 to 115, 125 to 165, 174 to 213, 241 to 280, 294 to 333, 361 to 400 or 401 to 438 of Alt a 6, amino acids 1 to 40, 41 to 80, 81 to 120, 121 to 160 of Alt a 2 or fragments or sequence variations thereof.
Methods for the production of fusion proteins are well known in the art and can be found in standard molecular biology references such as Sambrook et al. (Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory Press, 1989) and Ausubel et al. (Short Protocols in Molecular Biology, 3rd ed; Wiley and Sons, 1995). In general, a fusion protein is produced by first constructing a fusion gene which is inserted into a suitable expression vector, which is, in turn, used to transfect a suitable hosT-cell. In general, recombinant fusion constructs are produced by a series of restriction enzyme digestions and ligation reactions which result in the desired sequences being incorporated into a plasmid. If suitable restriction sites are not available, synthetic oligonucleotide adapters or linkers can be used as is known by those skilled in the art and described in the references cited above. The polynucleotide sequences encoding allergens and native proteins can be assembled prior to insertion into a suitable vector or the sequence encoding the allergen can be inserted adjacent to a sequence encoding a native sequence already present in a vector. Insertion of the sequence within the vector should be in frame so that the sequence can be transcribed into a protein. It will be apparent to those of ordinary skill in the art that the precise restriction enzymes, linkers and/or adaptors required as well as the precise reaction conditions will vary with the sequences and cloning vectors used. The assembly of DNA constructs, however, is routine in the art and can be readily accomplished by a person skilled in the art.
It is a specific and unexpected advantage, that the fusion proteins derived from truncated hypoallergenic allergen molecules and the human hepatitis B pre S protein can be reproducibly expressed in standard expression systems and easily be manufactured produced in high yield with processes and reproducibly in standard expression systems known to a person skilled in the art, most particularly by using in an Escherichia coli as expression system. Such manufacturing process typically comprise the expression of the molecules according to the invention by the cultivation of cells in a bioreactor (e.g. in a fermenter, shake flask), followed by cell harvest (e.g. by filtration, centrifugation, etc.) and cell disruption (e.g. by high-pressure homogenization, sonication, freeze/thaw cycles, enzymatic or chemical cell lysis, etc.), purification of the molecules (e.g. by chromatography, filtration, precipitation, ultra/diafiltration, etc.) and final product formulation. In order to obtain a high yield of the molecules according to the invention, preferably high-cell density cultivation processes are employed, by application of fed-batch fermentation.
Another aspect of the present invention relates to a nucleic acid molecule coding for a hypoallergenic molecule and a fusion protein according to the present invention.
The nucleic acid molecule of the present invention may be employed, e.g., for producing said molecules recombinantly.
Said nucleic acid molecule may—according to another aspect of the present invention—be comprised in a vector.
This vector is preferably an expression vector.
The present invention is further illustrated by the following figures and examples, however, without being restricted thereto.
(A) In
(B) In
(C) In
(A) In
(B1)
(B2) In
(C) In
The synthetic BM321 gene were assembled from synthetic oligo-nucleotides and/or PCR products and was cloned into an appropriate standard vector (pMK-RQkanR). The plasmid was purified from a transformed E. coli K12 strain (DH10B-T1R) and concentration was determined by UV spectroscopy. The final synthetic and codon-optimized BM321 DNA-sequence was further cloned into the expression vector pET28b(+) using appropriate restriction sites (NcoI site at the 5′-end and EcoRI at the 3′-end). The plasmid DNA was purified from transformed E. coli K12 DH10B (dam+dcm+) and concentration determined by UV spectroscopy. The final construct was verified by sequencing of the insert. A summary of plasmid data and a plasmid map of final expression vector “pBM-321” is shown below.
Summary of BM321 sequence cloned into final expression vector pET-28b(+).
Chemically competent E. coli BL21(DE3) cells were transformed with the expression plasmid by heat shock method. Transformed cells were plated on LB-agar-plates consisting of 0.5% sodium chloride 1% soy peptone, 0.5% yeast extract, 1.5% agar and 50 μg/mL kanamycin for selection. Cells on LB plates were grown by over-night cultivation at 37° C. Single colonies of transformed BL21(DE3) E. coli cells were isolated, cultured in LB-medium and screened for growth and expression of BM321. The best performing clone was selected for the further establishment of a Master Cell Bank.
An aliquot of the selected clone was used for inoculation of 150 mL culture medium (composition: 0.5% sodium chloride, 1% soy peptone, 0.5% yeast extract, 50 μg/mL kanamycin). The Master Cell Bank (MCB) culture was incubated at 37° C. under constant agitation at 200 rpm until the culture reached an optical density of OD600=1-2. Glycerol was added in order to obtain a final glycerol concentration of 15% v/v and the MCB was aliquoted into 1 mL vials and stored in an ultra deep freezer at −75±10° C.
Synthetic culture medium (100 mL, pH=6.8, salts and trace elements, 10 g/L glucose as carbon source) was inoculated with 1 mL of Master Cell Bank (E. coli BL21(DE3)/pBM321) and cultured in a shake flask (37° C., 200 rpm) until an optical density target value of OD=1 was reached. A 22 L stainless steel fermenter was used to perform the fed-batch fermentation. For automatic and reproducible feed control, a recipe was programmed allowing to pre-define specific growth-rate, feed rate, duration of batch-phase and duration of exponential feed-phase. In order to increase the oxygen transfer rate of the fermenter, back-pressure was controlled and set to 1 bar. The fermenter was in-situ sterilized with the synthetic culture medium as mentioned above and the fermentation was started by inoculation with preculture. After depletion of glucose, the exponential feeding phase was started in order to maintain a specific growth rate of μ=0.25 h−1. At an OD=45, the expression of recombinant BM321 was induced by the bolus addition of IPTG (0.8 mM final concentration). The culture was harvested at OD600=73. BM321 product titer obtained from the fed-batch fermentation was 1.2 g per L culture broth. Afterwards, the bacterial culture broth was cooled down to ≦20° C. and centrifuged at 7,000 rpm (5,500 g) at 4° C. for 15 min. Wet cell biomass was aliquoted and stored at −75° C.
For cell disruption, 748 gram biomass from Example 6 were thawed and subdivided into aliquots á 125 gram and resuspended in a homogenization buffer (20 mM Tris, 1 mM EDTA, 0.1% Triton X-100, pH 11.0) under mechanical agitation at room temperature for 30 min. For cell disruption, a freeze/thaw procedure was applied by freezing −75° C. and subsequent thawing, followed by mechanical homogenisation. The pH of the homogenate was adjusted to pH=10.0. The crude cell homogenate was subjected to a centrifugation step at 7,000 rpm (5,500 g) at 4° C. for 30 min. The supernatants were subjected to precipitation with PEI (polyethyleneimine) under mechanical agitation. Insoluble matters were separated by a subsequent centrifugation step. The clarified supernatants were subjected to the following chromatography step.
A total of 1840 mL of the PEI precipitation supernatant from the clarification step as described in Example 7 were loaded on a 5×30 cm Q-Sepharose FF column and equilibrated with buffer A (TrisHCl, EDTA). Unbound material was removed by washing with buffer A, followed by a wash with buffer C (1 sodium phosphate, EDTA, pH 7.0). Elution of the product fraction was accomplished by a linear gradient elution with 0-100% BM32 buffer E (sodium phosphate, EDTA, NaCl pH 7.0) in BM32 buffer C. Selection of product-containing fractions for pooling was performed according to SDS-PAGE analysis, by densitometric evaluation of fraction purity and by product band intensity.
The pooled fractions from the capture step were adjusted to a conductivity of 115 mS/cm by the addition of 2.5 M sodium chloride, and this feedstock was loaded on a Phenyl Sepharose HP column equilibrated with buffer D (sodium phosphate, EDTA, NaCl pH 7.0). Unbound material was removed by washing with buffer D. Elution of the product fraction was accomplished by a gradient elution from 40-100% buffer C (sodium phosphate, EDTA, pH 7.0) in buffer D. Selection of product-containing fractions for pooling was performed according to SDS-PAGE analysis, by densitometric evaluation of fraction purity and by product band intensity.
The pooled fractions from the intermediate step were adjusted to a conductivity of 80 mS/cm by the addition of 2.5 M sodium chloride, and this feedstock was loaded on a Toyopearl Butyl 650-S column equilibrated with a mixture buffer F (sodium phosphate, EDTA, NaCl pH 7.0). Unbound material was removed by a gradient wash with 80-0% BM32 buffer F in buffer C (sodium phosphate, EDTA, pH 7.0). Elution of the fraction was accomplished by a gradient elution from 0-1 buffer G (sodium phosphate, EDTA, isopropanol, pH 7.0) in buffer C. Selection of product-containing fractions for pooling was performed according to SDS-PAGE analysis, by densitometric evaluation of fraction purity and by product band intensity.
For expression and manufacturing of the recombinant molecules according to the invention, namely HBV_Phlp2_4xP3 (BM322), HBV_Phlp5_V2 (BM325), and HBV_Phlp6_4xP1 (BM326), the same, similar or comparable methods and procedures as described in Example 1, Example 2, Example 3, Example 4, Example 5 and Example 6 were applied.
Each of the recombinant purified proteins was dissolved in an isotonic buffer containing 0.9% sodium chloride and 2 mM sodium phosphate and to each protein solution an appropriate amount of aluminium hydroxide was added. A mixture containing equal parts of the four resulting suspensions was prepared and aliquoted under sterile condition into sealed vials. The injectable formulation obtained by this procedure contained 0.4 mg/mL of each HBV_PhlP1_4xP5; HBV_PhlP2_4xP3, HBV_PhlP5_V2 and HBV_PhlP6_4xP1.
The gene coding for fusion proteins consisting of PreS fused with Bet v 1-derived peptide PA twice at the N- and C-terminus (i.e. 4PA-PreS) was synthesized by ATG:biosynthetics, Merzhausen, Germany and inserted into the NdeI/XhoI sites of the vector pET-17b (Novagen, Germany). The DNA sequences were confirmed by means of automated sequencing of both DNA strands (Microsynth, Balgach, Switzerland).
The fusion protein was expressed in E coli strain BL21 (DE3; Stratagene, La Jolla, Calif.). Cells were grown in Luria Bertani-medium containing 50 μg/mL kanamycin to an OD of 0.6. Protein expression was induced by adding isopropyl-β-D-thiogalactopyranoside to a final concentration of 1 mmol/L over night at 37° C. Cells were harvested by centrifugation at 3500 rpm for 10 minutes. The protein product was mainly detected in the inclusion body fraction. It was solubilized in 6M GuHCl, 100 mM NaH2PO4, 10 mM TRIS, pH 8.0 over night. The homogenate was centrifuged at 14,000 g for 18 minutes. Supernatants of were incubated with 2 mL of a previously equilibrated Ni-NTA resin for 4 hours (Qiagen, Hilden, Germany) and the suspensions were subsequently loaded onto a column, washed with 2 column volumes of washing buffer (8 mol/L urea, 100 mmol/L NaH2PO4, and 10 mmol/LTris-HCl [pH=6.1]), and eluted with the same buffer (pH=3.5). The purified protein was dialyzed against water.
The purity of recombinant proteins was analyzed by Coomassie-stained SDS-PAGE (12.5%) under reducing conditions.
The identity of the fusion protein was confirmed by the means of dot blot using monoclonal antibodies, specific for Bet v 1-derived peptides P2′ (mAb2) and P4′ (mAb12) and PreS-specific rabbit antibodies as well as corresponding rabbit preimmune IgGs. One μg of PreS fusion proteins, PreS and HSA (control) have been immobilized on nitrocellulose and were incubated with monoclonal as well as rabbit sera diluted 1:1000 have at 4° C. Bound antibodies were detected with iodine 125-labelled rabbit anti-mouse IgG (mAb2, mAb12) or 125I-goat anti-rabbit IgG (rabbit anti-PreS, rabbit preimmune) (Perkin-Elmer, Waltham, Mass.) diluted 1:500 for 2 hours and visualized by autoradiography. Furthermore ELISA plates (Maxisorp, Nunc, Denmark) were coated with 2 μg of PreS fusion protein and PreS, diluted in 0.1 mol/L carbonate buffer, pH 9.6 washed with PBS containing 0.05% vol/vol Tween 20 (PBST) 3 times and blocked for 2 hours with 1% BSA-PBST. Subsequently plates were incubated with mAb2, mAb12, anti-PreS rabbit serum and rabbit anti-Bet v 1 antibodies in a dilution of 1:5000 (dilution buffer: 0.5% wt/vol BSA in PBST) overnight at 4° C. After washing 5 times, bound IgG antibodies have been detected with a HRP-labelled sheep anti-mouse antibody (for mAb2, mAb12) or HRP-labelled donkey anti-rabbit antibody (rabbit sera) (both GE Healthcare, Uppsala, Sweden) and colour reaction was developed.
Genes coding for fusion protein consisting of PreS fused twice with Bet v 1-derived peptides at the N- and C-terminus 2xPA2xPB-PreS) was synthesized by GenScript Piscataway, N.J., USA, 2PAPB-Pres) and inserted into the NdeI/XhoI sites of the vector pET-17b (Novagen, Germany). The DNA sequences were confirmed by means of automated sequencing of both DNA strands (Microsynth, Switzerland).
The recombinant PreS fusion proteins was expressed in E coli strain BL21 (DE3; Stratagene, Calif.). Cells were grown in Luria Bertani-medium containing 50 μg/mL kanamycin to an OD of 0.6. Protein expression was induced by adding isopropyl-β-D-thiogalactopyranoside to a final concentration of 1 mmol/L over night at 37° C. Cells were harvested by centrifugation at 3500 rpm for 10 minutes. Proteins were mainly detected in the inclusion body fraction. The resulting protein was solubilized in 6M GuHCl, 100 mM NaH2PO4, 10 mM TRIS, pH 8.0 over night. The homogenate was centrifuged at 14,000 g for 18 minutes. Supernatants of were incubated with 2 mL of a previously equilibrated Ni-NTA resin for 4 hours (Qiagen, Hilden, Germany) and the suspensions were subsequently loaded onto a column, washed with 2 column volumes of washing buffer (8 mol/L urea, 100 mmol/L NaH2PO4, and 10 mmol/LTris-HCl [pH=6.1]), and eluted with the same buffer (pH=3.5). Protein was dialyzed against 10 mM NaH2PO4.
The purity of recombinant proteins was analyzed by Coomassie-stained SDS-PAGE (12.5%) under reducing conditions. The identity of the fusion proteins was confirmed by the means of dot blot using monoclonal antibodies, specific for Bet v 1-derived peptides P2′ (mAb2) and P4′ (mAb12) and PreS-specific rabbit antibodies as well as corresponding rabbit preimmune IgGs. One μg of PreS fusion protein, PreS and HSA (control) have been immobilized on nitrocellulose and were incubated with monoclonal as well as rabbit sera diluted 1:1000 have at 4° C. Bound antibodies were detected with iodine 125-labelled rabbit anti-mouse IgG (mAb2, mAb12) or 125I-goat anti-rabbit IgG (rabbit anti-PreS, rabbit preimmune) (Perkin-Elmer, Waltham, Mass.) diluted 1:500 for 2 hours and visualized by autoradiography. Furthermore ELISA plates (Maxisorp, Nunc, Rosklide, Denmark) were coated with 2 μg of PreS fusion protein and PreS, diluted in 0.1 mol/L carbonate buffer, pH 9.6 washed with PBS containing 0.05% vol/vol Tween 20 (PBST) 3 times and blocked for 2 hours with 1% BSA-PBST. Subsequently plates were incubated with mAb2, mAb12, anti-PreS rabbit serum and rabbit anti-Bet v 1 antibodies in a dilution of 1:5000 (dilution buffer: 0.5% wt/vol BSA in PBST) overnight at 4° C. After washing 5 times, bound IgG antibodies have been detected with a HRP-labelled sheep anti-mouse antibody (for mAb2, mAb12) or HRP-labelled donkey anti-rabbit antibody (rabbit sera) (both GE Healthcare, Uppsala, Sweden) and colour reaction was developed.
IgE binding in comparison to the complete allergen was tested by IgE dot-blot assay. Sera from grass pollen allergic patients were incubated with dotted proteins and bound IgE was detected with 125I-labelled anti-human IgE. No IgE binding was detected for HBV_Phlp1_4xP5 (BM321) as shown in
IgE binding in comparison to the complete allergen was tested by IgE dot-blot assay. Sera from grass pollen allergic patients were incubated with dotted proteins and bound IgE was detected with 125I-labelled anti-human IgE. No IgE binding was detected for HBV_Phlp2_4xP3 (BM321) as shown in
IgE binding in comparison to the complete allergen was tested by IgE dot-blot assay. Sera from grass pollen allergic patients were incubated with dotted proteins and bound IgE was detected with 125I-labelled anti-human IgE. No IgE binding was detected for HBV_Phlp5_V2 (BM325) as shown in
IgE binding in comparison to the complete allergen was tested by IgE dot-blot assay. Sera from grass pollen allergic patients were incubated with dotted proteins and bound IgE was detected with 125I-labelled anti-human IgE. No IgE binding was detected for HBV_Phlp1_4xP1 (BM326) as shown in
IgE binding in comparison to the complete allergen was tested by IgE dot-blot assay. Sera from grass pollen allergic patients were incubated with dotted proteins and bound IgE was detected with 125I-labelled anti-human IgE. No IgE binding was detected for both fusion proteins as shown in
To determine the ability of peptide-induced rabbit Ig to inhibit the binding of allergic patients' IgE antibodies to rPhl p 1, ELISA plates were coated with 1 μg/ml rPhl p 1, washed and blocked. The plates were preincubated with 1:100-diluted rabbit anti-peptide (HBV_Phlp1_4xP5, KLHP5), a rabbit anti rPhl p 1 and, for control purposes, with the corresponding preimmune sera. After washing, plates were incubated with human sera from Phl p 1-allergic patients (1:3 diluted) and bound IgE was detected with mouse anti-human IgE (Pharmingen 1:1000) and then with sheep anti-mouse IgG POX-coupled (Amersham Bioscience) 1:2000. The percentage of inhibition of IgE-binding achieved by preincubation with the anti-peptide antisera was calculated as follows: 100-ODi/ODP×100.
ODi and ODP represent the extinctions after preincubation with the rabbit immune and preimmune serum, respectively. Table 1 shows the capacity of anti-Phl p 1 peptide antibodies to inhibit the binding of 13 allergic patients' IgE to complete rPhl p 1. Anti-fusion protein sera blocked the IgE-binding to the same extent as sera against rPhl p 1 and KLHP5. Table 2 shows the inhibition (in %) of all 13 patients.
To determine the ability of peptide-induced rabbit Ig to inhibit the binding of allergic patients' IgE antibodies to rPhl p 2, ELISA plates were coated with 1 μg/ml rPhl p 2, washed and blocked. The plates were preincubated with 1:100-diluted rabbit anti-peptide (HBV_Phlp2_4xP3, KLHP3), a rabbit anti rPhl p 2 and, for control purposes, with the corresponding preimmune sera. After washing, plates were incubated with human sera from Phl p 2-allergic patients (1:3 diluted) and bound IgE was detected with mouse anti-human IgE (Pharmingen 1:1000) and then with sheep anti-mouse IgG POX-coupled (Amersham Bioscience) 1:2000. The percentage of inhibition of IgE-binding achieved by preincubation with the anti-peptide antisera was calculated as follows: 100−ODi/ODP×100.
ODi and ODP represent the extinctions after preincubation with the rabbit immune and preimmune serum, respectively. Table 2 shows the capacity of anti-Phl p 2 peptide antibodies to inhibit the binding of 19 allergic patients' IgE to complete rPhl p 2. Anti-fusion protein sera blocked the IgE-binding to the same extent as sera against rPhl p 2 and KLHP3. Table 2 shows the inhibition (in %) of all 19 patients.
To determine the ability of peptide-induced rabbit Ig to inhibit the binding of allergic patients' IgE antibodies to rPhl p 5, ELISA plates were coated with 1 μg/ml rPhl p 5, washed and blocked. The plates were preincubated with 1:100-diluted rabbit anti-peptide (HBV_Phl p2_V2), a rabbit anti rPhl p 5 and, for control purposes, with the corresponding preimmune sera. After washing, plates were incubated with human sera from Phl p 5-allergic patients (1:3 diluted) and bound IgE was detected with mouse anti-human IgE (Pharmingen 1:1000) and then with sheep anti-mouse IgG POX-coupled (Amersham Bioscience) 1:2000. The percentage of inhibition of IgE-binding achieved by preincubation with the anti-peptide antisera was calculated as follows: 100−ODi/ODP×100.
ODi and ODP represent the extinctions after preincubation with the rabbit immune and preimmune serum, respectively. Table 3 shows the capacity of anti-Phl p 5 peptide antibodies to inhibit the binding of 16 allergic patients' IgE to complete rPhl p 5. Anti-fusion protein sera blocked the IgE-binding to the same extent as sera against rPhl p 5 and better than KLH peptide mix. Table 3 shows the inhibition (in %) of all 16 patients.
To determine the ability of peptide-induced rabbit Ig to inhibit the binding of allergic patients' IgE antibodies to rPhl p 6, ELISA plates were coated with 1 μg/ml rPhl p 6, washed and blocked. The plates were preincubated with diluted rabbit anti-peptide (HBV_Phlp6_4xP1, KLHP1), a rabbit anti rPhl p 6 and, for control purposes, with the corresponding preimmune sera. After washing, plates were incubated with human sera from Phl p 6-allergic patients (1:3 diluted) and bound IgE was detected with mouse anti-human IgE (Pharmingen 1:1000) and then with sheep anti-mouse IgG POX-coupled (Amersham Bioscience) 1:2000. The percentage of inhibition of IgE-binding achieved by preincubation with the anti-peptide antisera was calculated as follows: 100−ODi/ODP×100. ODi and ODP represent the extinctions after preincubation with the rabbit immune and preimmune serum, respectively. Table 4 shows the capacity of anti-Phl p 6 peptide antibodies to inhibit the binding of 21 allergic patients' IgE to complete rPhl p 6. Anti-fusion protein sera blocked the IgE-binding to the same extent as sera against rPhl p 6 and KLHP1. Table 4 shows the inhibition (in %) of all 21 patients.
Purified rBet v 1, recombinant fusion proteins 4xPA-PreS, 2xPA2xPB-PreS were tested for their IgE reactivity by RAST-based, non-denaturing dot blot assays. Two μg of the purified proteins and, for control purposes, HSA were dotted onto nitrocellulose membrane strips (Schleicher & Schuell, Dassel, Germany).
Nitrocellulose strips were blocked in buffer A (Vrtala, J Clin Invest, 1997) and incubated with sera from birch pollen allergic patients (n=50), sera from non-allergic persons (n=3) diluted 1:10, buffer control and positive control (1:1000 diluted rabbit anti-rBet v 1 antiserum). Bound IgE antibodies were detected with 125I-labelled anti-human IgE antibodies (BSM Diagnostica, Vienna, Austria), bound rabbit antibodies with a 125I-labeled goat anti-rabbit antiserum (Perkin-Elmer) and visualized by autoradiography (Valenta et al., 1992). Additionally, ELISA plates were coated with rBet v 1 and the purified PreS fusion proteins (5 μg/mL). After washing and blocking as described above, plates were incubated with sera of birch pollen allergic patients (n=21) and three non-allergic control sera diluted 1:5. Bound IgE was detected by purified mouse anti human IgE (BD Pharmingen) diluted 1:1000 overnight and visualized with HRP-labelled sheep anti mouse IgG (GE Healthcare) diluted 1:2000. After washing, colour reaction was determined as described above.
Heparinized blood samples were obtained from birch allergic patients after informed consent was given and were incubated with increasing concentrations of rBet v 1, 4PA-PreS, 2PAPB-PreS ranging from 0.001 to 1 mg/mL, a monoclonal anti-IgE antibody (Immunotech, Marseille, France) as positive control, or PBS (negative control) for 15 min (37° C.). CD 203c expression was determined as previously described.
PBMCs from birch pollen allergic patients (n=6) have been isolated by Ficoll (Amersham Biosciences, Uppsala, Sweden) density gradient centrifugation. Subsequently PBMCs were resuspended in AIM V medium (Life Technologies, Grand Island, N.Y.) to a final concentration of 2×105 cells/well and stimulated with decreasing antigen doses (equimolar amounts of 5 μg/well rBet v 1, PA, PB, PreS, 2PA-PreS, 2PB-PreS, 4PA-PreS, 2PAPB-PreS), with medium alone (negative control) or with IL-2 (4 IE/well) (positive control). After 6 days, proliferative responses were measured by [3H] thymidine incorporation and are expressed as stimulation indices (SI).
Furthermore cytokine production of 17 different cytokines (i.e. IL-113, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, IFN-γ, TNF-α, G-CSF, GM-CSF, MIP-1β, MCP-1) has been measured after 6 days of stimulation with Bio-plex Pro Human Cytokine 17-Plex Panel (Bio-Rad Laboratories) according the manufacturer's instructions. Briefly, the undiluted supernatants were mixed with anti-cytokine/chemokine mouse monoclonal antibodies coupled to different beads as capture antibodies (Bio-Rad). An 8-point standard curve was used to achieve low-end sensitivity. After washing, anti-cytokine biotinylated detection antibody was added. The reaction was visualized by adding Streptavidin-labelled Phycoerythrin (PE) and assay buffer. The samples were analyzed on a Luminex 100 instrument (Biosource, Nivelles, Belgium) and the data were acquired using the Bio-Plex Manager 6.0 software. All samples were analyzed in one run. Results are shown in
ELISA plates (Maxisorp, Nunc) were coated either with 1 μg/ml rBet v 1 or homologous allergens in alder (rAln g 1), hazel (rCor a 1), apple (rMal dl) and additionally with several Bet v 1-derived peptides in a concentration of 1 μg/ml overnight at 4° C. After washing and blocking as described above sera from rabbits immunized with rBet v 1 and the PreS fusion proteins conjugated to alum or CFA, were incubated in serial 1:2 dilutions ranging from 1:500 to 1:1 280 000 and in a concentration of 1:1000. Bound rabbit IgG was detected with HRP-labelled donkey anti-rabbit antibodies (GE Healthcare) and colour reaction was determined as described above.
An inhibition ELISA was used to study the inhibition of the binding of birch pollen allergic patients' IgE to rBet v 1. ELISA plates were coated with rBet v 1 in a concentration of 1 μg/m at 4° C. overnight. After washing and blocking plates were pre-incubated with rabbit sera directed against the PreS fusion protein 2PAPB-PreS and anti-Bet v 1 rabbit serum in a dilution of 1:80 and 1:160 in comparison with rabbit preimmune sera overnight at 4° C. After an additional washing step sera of birch pollen allergic patients diluted 1:5 were added overnight at 4° C. and bound human IgE were detected with a 1:1000 diluted alkaline phosphatase-conjugated mouse monoclonal anti human IgE antibody (BD Pharmingen). The percentage of inhibition of IgE binding to rBet v 1 after pre-incubation with 2PAPB-PreS rabbit antisera and Bet v 1 rabbit antisera was calculated as follows: percent inhibition=100−(ODi×100/ODp). ODp and ODi represent the extinctions after pre-incubation with specific rabbit IgG (ODi) or preimmune sera (ODp), respectively. (
An injectable formulation of hypoallergenic fusion proteins SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, and SEQ ID No. 17 with aluminum hydroxide was prepared as described in example 8. In the course of a clinical study, the vaccine was administered 3 times subcutaneously to 69 grass pollen allergic human subjects. (
Vaccination with the vaccine formulation led to a robust IgG immune response. Induction of allergen-specific IgG following s.c. injection of the 3 different dose levels of the vaccine and placebo was determined by ELISA in the sera collected from the study participants before and after treatment with 3 s.c. injections of the vaccine formulation. (
For this purpose, ELISA plates (Nunc Maxisorp, Roskilde, Denmark) were coated with 5 μg/ml of the antigens Phl p 1, Phl p 2, Phl p 5, and Phl p 6 or human serum albumin (HSA) as control over night at 4° C. After washing with PBS containing 0.5% Tween 20 (PT) and blocking with 2% w/v BSA in PT, plates were subsequently incubated with 1:10 to 1:100 diluted sera from patients, serum from a non-atopic individual or buffer alone in triplicates overnight at 4° C. Bound IgE antibodies were detected with HRP-coupled anti-human IgE antibodies diluted in PT, 0.5% w/v BSA. The colour development was performed by addition of staining solution ABTS (2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt; Sigma-Aldrich, St. Louis, Mo., USA) (100 μl/well). The optical density was measured using an ELISA Reader at 405 nm. The results of IgG assessments are shown in
The vaccine did not provoke any relevant T-cell reactivity towards the hypoallergenic fusion proteins present in the vaccine formulation as determined by in-vitro T-cell proliferation assay (
T-cell proliferation assays were performed using the following procedure: Peripheral blood mononuclear cells (PBMC) were isolated from heparinised blood samples of the grass pollen allergic patients by Ficoll (Amersham Pharmacia Biotech, Little Chalfont, UK) density gradient centrifugation. PBMC (2×105) were then cultured in triplicates in 96-well plates (Nunclone; Nalge Nunc International, Roskilde, Denmark) in 200 μl serum-free Ultra Culture medium (BioWhittaker, Rockland, Me.) supplemented with 2 mM L-glutamin (SIGMA, St. Louis, Mo.), 50 μM b-mercaptoethanol (SIGMA) and 0.1 mg gentamicin per ml (SIGMA) at 37° C. and 5% CO2 in a humidified atmosphere. Cells were stimulated with a mixture containing 0.25 μg of each polypeptide component of the vaccine and for comparison an equimolar concentrations of grasspollen extract or for control purposes with 4 U Interleukin-2 per well (Boehringer Mannheim, Germany) or medium alone. After 6 d culture 0.5 μCi per well [3H]thymidine (Amersham Pharmacia Biotech) was added and 16 h thereafter incorporated radioactivity was measured by liquid scintillation counting using a microbeta scintilllation counter (Wallac ADL, Freiburg, Germany). Mean cpm were calculated from the triplicates and stimulation indices (SI) were calculated as the quotient of the cpm obtained by antigen or interleukin-2 stimulation and the unstimulated control. Results of proliferation assays are shown in
Treatment with the vaccine induced IgG antibodies with the capability to modulate the allergen-specific T-cell response as demonstrated by a reduced proliferative response upon stimulation with grass pollen allergens in the presence of treatment-induced IgG. (
For this purpose, T-cell proliferation assays were performed with PBMCs isolated from study participants after treatment as described above with the exception that the stimulation was done with a mixture of the 4 grass pollen allergens Phl p 1, Phl p 2, Phl p5, and Phl p 6 (0.25 μg per allergen) together with serum collected from the same participant before and after the treatment. The experimental set-up and results are shown in
Reduction of nasal allergy symptoms induced by provocation in a pollen chamber and reduction of skin reactivity as determined by titrated skin prick testing was observed in patients having received 3 injections containing either 20 μg or 40 μg of each of the 4 polypeptides while there was no reduction in those parameters after treatment with doses of 10 μg of each polypeptide. (see
The 5 non IgE binding Der p 2 derived peptides—Der p2 Pep1 (SEQ ID No. 96), Der p2 Pep2 (SEQ ID No. 97), Der p2 Pep3 (SEQ ID No. 98), Der p2 Pep4 (SEQ ID No. 99), and Der p2 Pep5 (SEQ ID No. 100)—were screened with respect to
For that purpose, each of the peptides was chemically coupled to KLH. KLH and chemical coupling of the peptides was used in this screening experiment because it is an easy-to-use and well established and straight forward model system allowing initial comparison of the different peptides.
IgE binding of the Der p 2 derived peptides in comparison to the complete allergen was tested by IgE dot-blot assay. Sera from 26 house dust mite allergic patients were incubated with dotted KLH-conjugated peptides and bound IgE was detected with 125I-labelled anti-human IgE. No IgE binding was detected for any of the 5 peptides as shown below.
To identify peptides which induce a low lymphoproliferative response in PBMC from house dust mite allergic patients PBMCs isolated from 10 patients were stimulated with the 5 Der p 2 derived peptides alone, the KLH-conjugated peptides, and wild-type Der p 2 for comparison.
PBMCs from all 10 patient were stimulated by the wild-type Der p 2, and there was no or only very low proliferation upon stimulation with Der p2 Pep1, Der p2 Pep2, and Der p2 Pep4. Stimulation with Der p2 Pep3 and Der p2 Pep5 however, resulted in significant proliferation of the PBMCs in 4 out of 10 and 3 out of 10 cases, respectively, indicating that peptides 3 and 5 contain important T-cell epitopes.
To identify the ability of the peptides to induce blocking IgG, rabbits were immunized with the 5 individual KLH-peptide conjugates. Subsequently, the ability of peptide-induced rabbit IgG to inhibit the binding of allergic patients' IgE antibodies to rDer p 2 was investigated by ELISA. ELISA plates were coated with 1 μg/ml rDer p 2, washed and blocked. The plates were preincubated with 1:100-diluted rabbit anti-peptide (KLH-P1, KLH-P2, KLH-P3, KLH-P4, and KLH-P5), a rabbit anti rDer p 2 and, for control purposes, with the corresponding preimmune sera. After washing, plates were incubated with human sera from house dust mite allergic, Der p 2 sensitized patients (1:3 diluted) and bound IgE was detected with mouse anti-human IgE (Pharmingen 1:1000) and then with sheep anti-mouse IgG POX-coupled (Amersham Bioscience) 1:2000. The percentage of inhibition of IgE-binding achieved by preincubation with the anti-peptide antisera was calculated as follows: 100−ODi/ODP×100.
The ability of Der p 1 derived peptides to induce IgE-blocking IgG antibodies was determined using rabbit-anti-peptide KLH antisera and sera from 6 house dust mite allergic patients in an inhibition ELISA as described in example 26 with the exception that the ELISA plates were coated with wild-type Der p 1 instead of Der p 2.
| Number | Date | Country | Kind |
|---|---|---|---|
| 11169365 | Jun 2011 | EP | regional |
This application is a divisional of U.S. application Ser. No. 14/124,925 filed Dec. 9, 2013, now U.S. Pat. No. 9,308,251, which is a National Stage of PCT/EP2012/061040 filed Jun. 11, 2012 and claims the benefit of EP 11169365.1 filed Jun. 9, 2011.
| Number | Name | Date | Kind |
|---|---|---|---|
| 8258275 | Sallberg | Sep 2012 | B2 |
| 9296828 | Valenta | Mar 2016 | B2 |
| 9308251 | Niespodziana | Apr 2016 | B2 |
| 20090324501 | Valenta et al. | Dec 2009 | A1 |
| 20120207788 | Valenta et al. | Aug 2012 | A1 |
| Number | Date | Country |
|---|---|---|
| WO 2007140505 | Dec 2007 | WO |
| WO 2011029869 | Mar 2011 | WO |
| Entry |
|---|
| International Search Report dated Oct. 8, 2012 in PCT/EP2012/061040. |
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| Number | Date | Country | |
|---|---|---|---|
| 20150335735 A1 | Nov 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14124925 | US | |
| Child | 14701848 | US |
