Vaccine for treating allergy

Abstract

Provided are peptides consisting of 20 to 30 amino acid residues derived from amino acids at positions 120 to 171 of a mature allergen Fel d 4, and fusion proteins comprising the peptides.

Description

Technical Field

[0001] The present invention relates to the field of allergies and means and methods for treating allergies. Background Art

[0002] Fur animals (especially cats) are one of the most important sources of indoor allergens in most regions of the world. Patients allergic to fur animals may present with a variety of allergic symptoms, including respiratory allergic symptoms (such as rhinitis and asthma), conjunctivitis, and different types of dermatitis manifestations.

[0003] Regarding cat allergy, Fel d 1 is the main allergen, showing high IgE reactivity and strong sensitizing activity in most patients allergic to cats. It is the main target of IgE antibodies in patients with cat respiratory allergy, and it has been shown that children with cat-related asthma symptoms have a strong sensitization reaction to Fel d 1. Therefore, the development of modern molecular allergen-specific therapies has mainly focused on Fel d 1.

[0004] One of these molecular methods for treating cat allergy is allergen-specific immunotherapy (AIT) using hypoallergenic T cell epitope-containing peptides derived from Fel d 1, which has been evaluated in a first clinical trial using a limited number of Fel d 1 peptides (PS Norman, Annals of Allergy, 71(1993):330-3). Subsequently, this method was further developed by adding additional peptides to achieve broad coverage of MHC class II diversity in patients (M Worm et al., J. Allergy Clin. Immunol. 127(2011):89-97). Other molecular methods for allergen-specific immunotherapy for cat allergy also target only the main cat allergen Fel d 1 (K Niespodziana et al., J allergy Clin Immunol. 127(2011):1562-70). Other allergen-specific treatment methods are based on the passive immunization with Fel d 1-specific monoclonal IgG antibodies that block the binding of IgE of allergic patients to Fel d 1 (MA Kamal et al., Clin TranslSci. 2021;14:2440-2449).

[0005] However, to date, allergen-specific treatment methods have not considered the possible relevance of IgE sensitization to cat allergen molecules other than Fel d 1, especially those allergen molecules that may show IgE cross-reactivity to allergens of other fur animals (such as Fel d 4, Fel d 7, and Can f 6). So far, no allergen-specific method for the combined treatment and prevention of allergies to several fur animals has been described.

[0006] Therefore, the object of the present invention is to provide methods and means for solving this problem. Summary of the Invention

[0007] The present invention relates to a peptide consisting of 20 to 30 amino acid residues derived from amino acids 120 to 171 (i.e., the C-terminus) of the mature allergen Feld 4.

[0008] Surprisingly, the peptides as defined herein are capable of inducing the formation of antibodies in humans or mammals that can inhibit the binding of Feld 4-specific IgE of allergic patients to the allergen. Preventing or reducing the binding of allergen-IgE results in the prevention or alleviation of allergic symptoms caused by the corresponding allergen (i.e., cat allergen, especially Feld 4). It was unexpectedly found that other Feld 4-derived fragments from amino acids 1 to 119 of the mature allergen cannot induce the formation of IgE-blocking antibodies. This property of the inventive peptide can be used for the production of vaccines for the treatment and / or prevention of cat allergy in humans and mammals.

[0009] In addition to forming Feld 4-specific antibodies, the peptides of the present invention are also capable of inducing the formation of antibodies against other allergens from fur-bearing animals other than cats (such as horses). The surprising finding is that the antibodies induced by the peptides of the present invention show binding to allergens from other fur-bearing animals. Therefore, the peptides of the present invention can also be used for the prevention and / or treatment of allergies generally caused by fur-bearing animals (especially by cats, horses, and dogs) and particularly allergies caused by cats, horses, and dogs.

[0010] Another aspect of the present invention relates to a fusion protein or conjugate comprising at least one peptide as described in the present invention.

[0011] In order to enhance the immune response induced by the administration of the peptides of the present invention, for example, these peptides can be part of fusion proteins and conjugates. In addition, the peptides of the present invention can also be fused or conjugated with other allergens and / or allergen fragments from homologous (i.e., cat) or any other allergen source. The resulting fusion proteins and conjugates can be used for the treatment of allergies caused by, for example, pollen, animal dander, dust mites, molds, peanuts, tree nuts, wheat, soybeans, fish, shellfish, eggs, milk and other foods, or insect stings such as bees or wasps.

[0012] Yet another aspect of the present invention relates to the use of a peptide, fusion protein or conjugate as described in the present invention for the prevention or treatment of cat allergy.

[0013] The peptides, fusion proteins and conjugates of the present invention induce the formation of allergen-specific antibodies. These antibodies (usually including IgG) bind to the allergens contacted by the subject (human or mammal). This binding prevents these allergens from binding to allergen-specific IgE and thus causing an allergic reaction.

[0014] Another aspect of the present invention relates to the use of the peptides, fusion proteins or conjugates as described in the present invention for the prevention or treatment of allergies in fur animals, preferably dog or horse allergies.

[0015] Surprisingly, the peptides, fusion proteins and conjugates of the present invention not only induce the formation of antibodies against a single allergen, but also induce the formation of antibodies against other allergens. This cross-reactivity is particularly advantageous because it allows the peptides, fusion proteins and conjugates of the present invention to also be used for the treatment of general fur animals.

[0016] Another aspect of the present invention relates to a nucleic acid molecule encoding the peptide or fusion protein of the present invention.

[0017] Another aspect of the present invention relates to a vector comprising the nucleic acid molecule of the present invention.

[0018] One aspect of the present invention relates to a host cell comprising the nucleic acid molecule or vector of the present invention.

[0019] Yet another aspect of the present invention relates to a vaccine preparation comprising the peptide, fusion protein, nucleic acid molecule and / or vector of the present invention. Description of the Drawings

[0020] Figure 1 Shows the prevalence of IgE responses to cat allergens and IgE levels (kUA / L) in cat allergy patient populations in Russia and Sweden detected by ImmunoCap.

[0021] Figure 2 Shows the inhibition of binding of patient IgE to allergens obtained with anti-peptide antiserum compared to antiserum against the complete allergen (see Example 2).

[0022] Figure 3 Shows that the combination of antisera against P3 and P5 in Fel d 7 has a better inhibitory effect on patient IgE binding compared to antisera against individual peptides.

[0023] Figure 4 Shows that IgG antibodies generated against peptides derived from Fel d 7 and Fel d 4 respectively prevent the binding of IgE to homologous allergens in dogs (Can f 1) and horses (Equ c 1).

[0024] Figure 5 Shows the PreS fusion protein (labeled "SuperCat") which contains two copies of peptides derived from Feld 1 (P1, P5), Feld 4 (P9) and Feld 7 (P3, P7) in different orders.

[0025] Figure 6Shows the comparison of the IgE-binding ability of SuperCat 1 to 5 with an equimolar mixture of Feld 1, Feld 4, and Feld 7 as determined by ImmunoCap.

[0026] Figure 7 Shows the comparison of the sensitizing activity of SuperCat with a cat allergen mixture in the RBL assay.

[0027] Figure 8 Shows the comparison of all SuperCat constructs in a competitive ELISA.

[0028] Figure 9 Shows the comparison of the ability of antibodies obtained by immunization with SuperCat 1, 3, and 5 and the previous Fel d 1-based vaccines PreS-P1-P5 and PreS-2xP1 to inhibit the binding of IgE from allergic patients (n = 11) to Fel d 1.

[0029] Figure 10 Shows a schematic diagram of the rabbit immunization experimental protocol as described in Example 7.

[0030] Figure 11 Shows the inhibition of the binding of IgE from cat allergic patients to Feld 1 by antisera obtained by immunizing rabbits with SuperCat and a commercially available allergen extract-based vaccine.

[0031] Figure 12 Shows the inhibition of the binding of IgE from cat allergic patients to Feld 4 (left) and Feld 7 (right) by antisera obtained by immunizing rabbits with SuperCat and a commercially available allergen extract-based vaccine. Detailed Description

[0032] The peptides of the present invention derived from the mature allergen Feld 4 are fragments of said allergen (i.e., allergen fragments) consisting of 20 to 30 amino acid residues. As used herein, "allergen fragment" refers to a peptide or polypeptide fragment derived from an allergen by fragmentation.

[0033] Peptides derived from the C-terminal portion after the 120th amino acid residue of Fel d 4 unexpectedly are able to induce the formation of Fel d 4-specific antibodies in humans and mammals and exhibit low allergenicity. In addition, these peptides have no or substantially no IgE-binding ability. The latter property is crucial for a safe vaccine with no or substantially no side effects.

[0034] As used herein, the terms "mature allergen-derived" and "derived from mature allergen" refer to an amino acid sequence of a fragment according to the invention obtained from the amino acid sequence of an allergen by fragmentation or truncation. Thus, the peptides according to the invention consist of 20 to 50 consecutive amino acid residues of the mature allergen from which they are derived. "Mature allergen-derived" and "derived from mature allergen" also include substitutions of individual amino acid residues in the above-mentioned fragments. In a particularly preferred embodiment of the invention, one or more cysteine residues in amino acids 120 to 171 of mature allergen Feld 4 (especially SEQ ID No. 1) are deleted or replaced by other amino acid residues, preferably selected from the group consisting of serine, threonine and methionine, most preferably serine. For example, the removal or replacement of cysteine residues is particularly advantageous for avoiding potential disulfide bond formation within or between fragments.

[0035] As defined herein, "mature allergen" refers to the amino acid sequence of an allergen that has been processed and is free of a signal peptide. The allergen itself is encoded by the corresponding gene that still contains the signal peptide. The signal peptide of an allergen can be identified by methods known in the art, including sequence alignment (see, for example, Bendtsen JD et al., J Mol Biol. 340 (2004): 783-95; AK et al., Bioinformatics 21 (2005): 39-50). A simple method for identifying the amino acid sequence of a mature allergen is to isolate the mature allergen from the allergen source and sequence its N-terminus. These sequence data can be compared with the sequence of the gene or mRNA encoding the same allergen to identify the amino acid sequences of the mature allergen and the signal peptide. Thus, the sequence of a "mature allergen" is encoded by its mRNA and / or gene and does not contain cleavage products that may be produced by post-translational modification of the primary translated mRNA or gene product (except for the potential cleavage of the signal peptide). A "mature allergen" reflects the amino acid sequence encoded by an mRNA molecule that is free of a signal peptide.

[0036] Feld 4 may have the amino acid sequence under UniProt database accession number Q5VFH6:

[0037]

[0038] The signal peptide is indicated in italics and underlined)

[0039] Mature Feld 4 contains 171 amino acid residues and is free of the signal peptide indicated in italics and underlined above. The term "Feld 4" also includes its isomers.

[0040] According to a preferred embodiment of the present invention, the peptide derived from the mature allergen Feld 4 consists of 22 to 28 amino acid residues, preferably 24 to 28, more preferably 25 to 27, and even more preferably 26.

[0041] According to a preferred embodiment of the present invention, the peptide of the present invention is derived from amino acids 130 to 171 of mature Feld 4, preferably amino acids 140 to 171, more preferably amino acids 145 to 171, and even more preferably amino acids 146 to 171.

[0042] According to another preferred embodiment of the present invention, mature Fel d 4 comprises SEQ ID No.1 or consists of:

[0043] HEEENVVRSNIDISKISGEWYSILLASDVKEKIEENGSMRVFVKHIKA

[0044] LDNSSLSFVFHTKENGKCTEIFLVADKTKDGVYTVVYDGYNVFSIVE

[0045] TVYDEYILLHLLNFDKTRPFQLVEFYAREPDVSQKLKEKFVKYCQE

[0046] HGIVNILDLTEVDRCLQARGSEVAQDSSVE

[0047] According to another preferred embodiment of the present invention, the peptide consisting of 20 to 30 amino acid residues derived from amino acids 120 to 171 of mature allergen Fel d 4 comprises the amino acid sequence SEQ ID No.3 (NILDLTEVDRCLQARGSEVAQDSSVE) or consists of it.

[0048] Another aspect of the present invention relates to a fusion protein or conjugate comprising at least one peptide according to the present invention.

[0049] The peptide of the present invention as defined above, consisting of 20 to 30 amino acid residues derived from amino acids 120 to 171 of mature allergen Feld 4, may be part of a fusion protein or conjugate. The fusion partner or conjugate partner may be another allergen or a fragment thereof, or any other peptide, polypeptide or protein. The peptide of the present invention may be fused to another peptide molecule at the C-terminus and / or N-terminus.

[0050] As used herein, "fusion protein" refers to a protein or polypeptide expressed and prepared as a single recombinant polypeptide chain comprising the allergen fragment and / or other protein, polypeptide or peptide described therein.

[0051] As used herein, the term "conjugate" refers to a molecule formed by covalently linking at least two coupling partners to each other. The "conjugate" of the present invention comprises at least the peptide described in the present invention. For example, the conjugation between two or more peptides, polypeptides or proteins is achieved by adding an N-terminal cysteine or C-terminal cysteine amide residue to one of the coupling partners to produce a molecule containing a free thiol group. The terminal cysteine residue can be conjugated to any maleimide-activated polypeptide or protein. If the coupling partner does not contain a cysteine residue at the end, the EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) chemistry can be used to conjugate an amino group (lysine) or a carboxylic acid (glutamic acid, aspartic acid or 5'-phosphate group) to the coupling partner. Subsequently, crosslinking between the peptide and the carrier is achieved, for example, using an MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester) coupling agent.

[0052] According to a preferred embodiment of the present invention, the peptide described in the present invention is conjugated or fused to at least one (additional) allergen fragment and / or at least one carrier protein.

[0053] At least one peptide of the present invention can be fused or conjugated to at least one allergen fragment that can be different from the peptide, and / or fused or conjugated to at least one carrier protein.

[0054] The peptide of the present invention can be conjugated to a carrier protein to obtain a conjugate product capable of more effectively inducing the formation of allergen-specific IgG antibodies. For example, Keyhole limpet hemocyanin (KLH) or bovine or human serum albumin can be used as the carrier protein. Other carrier proteins can also be used, such as ovalbumin, thyroglobulin, tetanus toxoid or diphtheria toxoid. In another embodiment of the present invention, the peptide described in the present invention can be fused to a carrier protein. Most preferably, a fusion protein comprising the peptide described in the present invention and at least one carrier protein is provided.

[0055] As used herein, the term "at least one allergen fragment" refers to another allergen fragment beside the peptide described in the present invention, which can also be regarded as an allergen fragment. This means that the "at least one allergen fragment" should be "at least one other allergen fragment".

[0056] According to another preferred embodiment of the present invention, the at least one allergen fragment is derived from at least one fur animal allergen, preferably from at least one cat allergen.

[0057] At least one allergen fragment fused or conjugated to the peptide according to the present invention can be from any allergenic source. The at least one fragment can be derived from at least one allergen of one or more plants, insects, arthropods such as mites, mammals, etc., and mammalian allergens are particularly preferred.

[0058] According to another preferred embodiment of the present invention, the at least one cat allergen is selected from the group consisting of: Fel d 1 chain 1 (P30438 UniProt), Fel d 1 chain 2 (P30440 UniProt), Fel d 2 (P49064 UniProt), Fel d 3 (Q8WNR9 UniProt), Fel d 5, Fel d 6, Fel d 7 (E5D2Z5 UniProt), and Fel d 8 (F6K0R4 UniProt); preferably selected from the group consisting of: Fel d 1 chain 1, Fel d 1 chain 2, and Fel d 7; more preferably selected from the group consisting of: Fel d 1 chain 1, Fel d 1 chain 2, and Fel d 7.

[0059] According to a preferred embodiment of the present invention, the at least one allergen fragment derived from the allergens Fel d 1 chain 1, Fel d 1 chain 2, Fel d 2, Fel d 3, Fel d 5, Fel d 6, Fel d 7, and Fel d 8 comprises or consists of 20 to 40 amino acid residues.

[0060] According to another preferred embodiment of the present invention, at least one allergen fragment of the at least one cat allergen is derived from and comprises the N-terminus or C-terminus of the at least one cat allergen.

[0061] It has been found to be advantageous to use (hypoallergenic) cat allergen fragments that are derived from and comprise the N-terminus or C-terminus of the allergen. If the at least one allergen fragment comprises these parts of the allergen, the immune response against the corresponding cat allergen is even higher than that of fragments obtained from other parts of the cat allergen.

[0062] According to a preferred embodiment of the present invention, the at least one allergen fragment derived from Fel d 1 chain 1 comprises or consists of amino acid residues 1 to 34 of mature Fel d 1 chain 1.

[0063] According to another preferred embodiment of the present invention, the at least one allergen fragment derived from Fel d 1 chain 2 comprises or consists of amino acid residues 81 to 109 of mature Fel d 1 chain 2.

[0064] According to another preferred embodiment of the present invention, said at least one allergen fragment derived from Fel d 7 comprises and / or consists of amino acid residues 61 to 97 and / or amino acid residues 124 to 162 of mature Fel d 7.

[0065] According to a preferred embodiment of the present invention, said carrier protein is a viral protein or a fragment thereof, said fragment consisting of 50 to 300 amino acid residues (preferably 60 to 250, more preferably 80 to 200, more preferably 100 to 200).

[0066] According to another preferred embodiment of the present invention, said viral protein is a capsid protein.

[0067] According to another preferred embodiment of the present invention, said viral protein is derived from a hepadnavirus.

[0068] According to a preferred embodiment of the present invention, said hepadnavirus is hepatitis B virus.

[0069] According to a preferred embodiment of the present invention, said viral protein of hepatitis B virus is PreS, PreS1 or PreS2.

[0070] A fragment of the hepatitis B PreS polypeptide preferably consists of at least 30, preferably at least 40, more preferably at least 50 consecutive amino acid residues, and may comprise PreS1 and / or PreS2 of said hepatitis B PreS polypeptide.

[0071] The hepatitis B virus PreS polypeptide used as a carrier protein and as a fusion or conjugation partner for the peptides of the present invention may comprise the following amino acid sequence (SEQ ID No.4) or consist thereof:

[0072] GGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFN

[0073] PIKDHWPAANQVGVGAFGPGLTPPHGGILGWSPQAQGILTTVSTIPP

[0074] PASTNRQSGRQPTPISPPLRDSHPQAMQWNSTAFHQALQDPRVRGL

[0075] YFPAGGSSSGTVNPAPNIASHISSISARTGDPVTN

[0076] The hepatitis B PreS polypeptide may be composed of an amino acid sequence having at least 70% (preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99%, especially 100%) sequence identity with SEQ ID No. 4.

[0077] According to a preferred embodiment of the present invention, the fusion protein comprises at least two peptides consisting of 20 to 30 amino acid residues from amino acid position 120 to the C-terminus of the mature allergen Feld 4 of the present invention and at least two allergen fragments as defined above.

[0078] A combination of such allergen fragments is particularly preferred because, for example, it enables the construction of a fusion protein capable of inducing the production of antibodies against more cat allergens. If multiple peptides and allergen fragments from the same source are combined in a single fusion protein, the order of these peptides and allergen fragments is not the same as that in the naturally occurring allergen.

[0079] According to another preferred embodiment of the present invention, two to eight, preferably two to six of the at least one peptide and two to eight, preferably two to six of the at least one allergen fragment are fused to the N-terminus and C-terminus of at least one carrier protein.

[0080] According to another preferred embodiment of the present invention, 2 to 8 (preferably 2 to 6) of the at least one peptide and 2 to 8 (preferably 2 to 6) of the at least one allergen fragment are fused to the N-terminus and C-terminus of the at least one carrier protein.

[0081] The peptides and allergen fragments of the present invention may be fused to the N- and / or C-terminus of the carrier protein. It is particularly preferred to fuse the peptides and allergen fragments to the N- and C-terminus of the carrier protein because it has been found that this results in a higher level of allergen-specific antibody induction in humans and mammals.

[0082] According to a preferred embodiment of the present invention, two of the at least one peptide are adjacent to each other within the fusion protein.

[0083] According to another preferred embodiment of the present invention, the fusion protein comprises two peptides comprising or consisting of the amino acid residues 146 to 171 of mature Feld4, two allergen fragments comprising or consisting of the amino acid residues 1 to 34 of mature Fel d 1 chain 1, two allergen fragments comprising or consisting of the amino acid residues 81 to 109 of mature Fel d 1 chain 2, two allergen fragments comprising or consisting of the amino acid residues 81 to 109 of mature Fel d 1 chain 2, two allergen fragments comprising or consisting of the amino acid residues 61 to 97 of mature Feld 7, and two allergen fragments comprising or consisting of the amino acid residues 124 to 162 of mature Fel d 7.

[0084] According to a preferred embodiment of the present invention, the fusion protein comprises the amino acid sequence SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13 or SEQ ID No.14 (preferably SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11 or SEQ ID No.13), or consists of the same.

[0085] Surprisingly, it has been found that these fusion proteins are capable of inducing the production of cat allergen-specific antibodies in humans and mammals, thereby covering the most widely present allergens. SEQ ID No.5, SEQ ID No.9 and SEQ ID No.13 are particularly preferred fusion proteins.

[0086] Another aspect of the present invention relates to the use of the peptides, fusion proteins or conjugates according to the present invention for the prevention or treatment of cat allergy.

[0087] Yet another aspect of the present invention relates to the use of the peptides, fusion proteins or conjugates according to the present invention for the prevention or treatment of fur animal allergy, preferably dog or horse allergy.

[0088] The fusion proteins and conjugates according to the present invention induce the production of antibodies which are also capable of binding allergens from other sources than cats. Thus, the fusion proteins and conjugates according to the present invention can be used for the prevention or treatment of allergy to another fur-bearing animal. Particularly preferred are dog and horse allergy.

[0089] A further aspect of the present invention relates to a nucleic acid molecule encoding a peptide or fusion protein according to the present invention.

[0090] The nucleic acid molecule of the present invention can be an RNA or DNA molecule.

[0091] Another aspect of the present invention relates to a vector comprising the nucleic acid molecule according to the present invention.

[0092] The vector according to the present invention can be an expression or cloning vector. The vector can be a bacterial, fungal, insect, viral or mammalian vector.

[0093] The vector according to the present invention is preferably useful for cloning and expression purposes in various hosts such as bacteria, yeast, filamentous fungi, mammalian cells, insect cells, plant cells or any other prokaryotic or eukaryotic cells. Thus, the vector contains, in addition to the nucleic acid encoding the fusion protein of the present invention, host-specific regulatory sequences.

[0094] One aspect of the present invention relates to a host cell comprising the nucleic acid molecule or vector according to the present invention.

[0095] Another aspect of the present invention relates to a vaccine preparation comprising the peptide, fusion protein, nucleic acid molecule and / or vector of the present invention.

[0096] According to a preferred embodiment of the present invention, the vaccine preparation of the present invention can be used for preventing or treating allergic reactions in fur animals, preferably cat, dog and / or horse allergies.

[0097] According to another preferred embodiment of the present invention, the preparation comprises 10 ng to 1 g, preferably 100 ng to 10 mg, particularly 0.5 μg to 200 μg of the fusion protein, nucleic acid molecule or vector.

[0098] According to a particularly preferred embodiment of the present invention, the fusion protein, nucleic acid molecule or vector of the present invention is administered to an individual at a dose of 0.01 pg / kg body weight to 5 mg / kg body weight at least once, preferably 0.1 pg / kg body weight to 2 mg / kg body weight.

[0099] According to another preferred embodiment of the present invention, the fusion protein, nucleic acid molecule or vector is administered to a patient at a dose of 5 to 100 μg, preferably 10 to 80 μg, regardless of body weight (i.e., the dose can include 15, 20, 25, 30 or 80 μg) or per kg body weight.

[0100] The amount of the fusion protein, nucleic acid molecule or vector that can be combined with excipients to produce a single dosage form will vary depending on the host being treated and the specific mode of administration. The dosage of the fusion protein, nucleic acid molecule or vector may vary depending on factors such as the individual's disease state, age, gender and body weight, as well as the ability to elicit the desired antibody response in the individual. The dosage regimen can be adjusted to provide an optimal therapeutic response. The dosage of the fusion protein, nucleic acid molecule or vector can also vary according to circumstances to provide an optimal prophylactic dosage response. For example, the fusion protein, nucleic acid molecule or vector of the present invention can be administered to an individual at intervals of several days, one or two weeks or even several months depending on the level of allergen-specific IgG induction.

[0101] In a preferred embodiment of the present invention, the fusion protein, nucleic acid molecule or vector of the present invention is administered 2 to 10 times (preferably 2 to 7 times, more preferably at most 5 times), and the dosing interval is 2 to 60 days (preferably 5 to 40 days, more preferably 14 to 28 days). In another preferred embodiment of the present invention, a single booster vaccination is carried out 3 months to 5 years after the first dosing regimen. These booster vaccinations can be repeated 2 to 10 times (preferably 2 to 5 times, most preferably 2 to 3 times) to maintain a high IgG level. In a particularly preferred embodiment, the interval between subsequent vaccinations is selected to be 2 weeks to 5 years, preferably 3 weeks to 3 years, more preferably 3 weeks to 1 year. The repeated administration of the fusion protein, nucleic acid molecule or vector of the present invention can maximize the final therapeutic effect.

[0102] In a particularly preferred embodiment of the present invention, the fusion protein, nucleic acid molecule or vector of the present invention can be administered by injection 3 to 6 times per month, preferably 5 times; subsequently, booster injections are carried out every 1 to 6 months (preferably 3 to 4 months) according to the above method; for at least 1 year, preferably at least 2 years, more preferably 2 to 6 years, more preferably 3 to 5 years.

[0103] According to another preferred embodiment of the present invention, the vaccine preparation further comprises at least one adjuvant, pharmaceutically acceptable excipient and / or preservative.

[0104] The fusion protein, nucleic acid molecule, vector and pharmaceutical preparation of the present invention can be administered subcutaneously, intramuscularly, mucosally, etc. Depending on the dosage form and route of administration, the fusion protein, nucleic acid molecule or vector of the present invention can be combined with excipients, diluents, adjuvants and / or carriers. A preferred adjuvant is aluminum hydroxide. Suitable protocols for the production of vaccine preparations are known to those skilled in the art, for example, see "Vaccine Protocols" (A. Robinson, M. P. Cranage, M. Hudson; Humana Press Inc., USA; 2nd Edition, 2003).

[0105] The fusion protein described in the present invention can also be formulated with other adjuvants commonly used in vaccines. For example, suitable adjuvants can be MF59, aluminum phosphate, calcium phosphate, cytokines (such as IL2, IL-12, GM-CSF), saponins (such as QS21), MDP derivatives, CpG oligonucleotides, LPS, MPL, polyphosphazenes, emulsions (such as Freund's adjuvant, SAF), liposomes, virosomes, immunostimulating complexes (iscoms), spirochetes, PLG microparticles, poloxamer particles, virus-like particles, heat-labile enterotoxin (LT), cholera toxin (CT), mutant toxins (such as LTK63 and LTR72), microparticles and / or polymeric liposomes. Suitable adjuvants can be commercially available, such as ASO1B (MPL and QS21 in a liposomal formulation), ASO2A, AS15, AS-2, AS-03 and their derivatives (GlaxoSmithKline, USA); CWS (cell wall skeleton), TDM (trehalose-6,6'-dimycolate), LeIF (Leishmania elongation initiation factor), aluminum salts such as aluminum hydroxide gel (alum), or aluminum phosphate; salts of calcium, iron or zinc; insoluble suspensions of acylated tyrosine; acylated sugars; cationic or anionic derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and Quillaja saponaria saponin A. Cytokines such as GM-CSF or interleukin-2, -7 or -12 can also be used as adjuvants. Preferred adjuvants for eliciting a predominantly Th1-type response include, for example, a combination of monophosphoryl lipid A (preferably 3-O-deacylated monophosphoryl lipid A (3D-MPL)) optionally with an aluminum salt. An aqueous formulation containing monophosphoryl lipid A and a surfactant is described in WO 98 / 43670.

[0106] Another preferred adjuvant is a saponin or saponin mimetic or derivative, preferably QS21 (Aquila Biopharmaceuticals, Inc.), which can be used alone or in combination with other adjuvants. For example, an enhanced system involves a combination of monophosphoryl lipid A and a saponin derivative, such as a combination of QS21 and 3D-MPL. Other preferred formulations include oil-in-water emulsions and tocopherol. A particularly effective adjuvant formulation is QS21, 3D-MPL and tocopherol in an oil-in-water emulsion. Other saponin adjuvants for use in the present invention include QS7 (described in WO 96 / 33739 and WO 96 / 11711) and QS17 (described in US 5057540 and EP 0 362279 B1).

[0107] The vaccine formulation comprising the fusion protein described in the present invention most preferably comprises aluminum hydroxide as an adjuvant.

[0108] Examples

[0109] Example 1: Identification of cat allergens recognized by IgE antibodies from most cat-allergic patients with high allergenic activity

[0110] To determine the most relevant cat allergens in cat allergy vaccines, two parameters were evaluated. The first parameter was the frequency of IgE recognition of cat allergens by cat allergic patients and the second parameter was the sensitizing activity, which corresponds to the potency of a specific allergen molecule to induce allergic inflammation in sensitized patients.

[0111] The frequency of IgE responses was evaluated by detecting the IgE reactivity of cat allergic patients to the following allergens using the ImmunoCAP technique (van Hage M et al., Journal of Allergy and Clinical Immunology 140(2017):974 - 977): Feld 1 (Feld1.A.0101 (chain 1), Feld 1.B.0101 (chain 1)), Feld 2 (Fel d 2.0101), Feld 3 (Feld 3.0101), Feld 4 (Feld 4.0101), Feld 6 (Feld 6.0101), Feld 7 (Feld 7.0101), and Feld 8 (Feld8.0101). The above cat allergens were biotinylated and bound to streptavidin CAP and used for the determination of IgE reactivity in two groups of cat allergic patients by ImmunoCAP, one group from Sweden (n = 72) and one group from Russia (n = 73). The sensitizing activity of the cat allergens was performed by an eosinophil activation assay using rat basophilic leukemia cells, which express the high - affinity receptor for human IgE and thus can be loaded with the serum IgE of cat allergic patients.

[0112] As described by Rodríguez - Domínguez A et al. (Journal of Allergy and Clinical Immunology 146(2020):1097 - 1108), basophil activation and the release of β - hexosaminidase were induced by adding allergen - crosslinked IgE, and its activity could be measured in the cell culture supernatant.

[0113] The allergen - specific IgE recognition rates of Swedish and Russian patients were as follows: Fel d 1, 88% vs 97%; Feld 2, 22% vs 30%; Feld 3, 39% vs 51%; Feld 4, 51% vs 52%; Feld 6, 18% vs 33%; Feld 7, 53% vs 55%; and Feld 8, 46% vs 42% (see Figure 1 ). Thus, the cat allergens most frequently recognized by patients' IgE were Feld 1, Feld 7, Feld 4, and Feld 3 (see Figure 1 ).

[0114] The relationship between the cat allergen-specific IgE levels (in kUA / L, y-axis) of two groups of patients reactive to cat extract (Group A Swedish, Group B Russian) and cat extract (x-axis) is shown in a scatter plot ( Figure 1 ). The horizontal line represents the cut-off value of 0.1 kUA / L, and the median IgE levels were calculated only for values > 0.1 kUA / L. The percentage of IgE-positive sera for each allergen is marked above the figure. For the Russian group, IgE equal to or higher than 100 kUA / L was counted as 100.

[0115] The sensitizing activity of cat allergens was determined using the basophil activation test. RBL (rat basophilic leukemia) cells were loaded with sera from 17 Swedish cat-allergic patients and 1 non-allergic subject, and then stimulated with decreasing concentrations of each allergen. According to the basophil activation test, Fel d 1, Fel d 4, and Fel d 7 were identified as the most common and most sensitizing cat allergen molecules that should be included in the cat allergy vaccine, while other cat allergen molecules were less frequently recognized and / or showed weaker sensitizing activity.

[0116] Example 2: The Feld 4-derived peptide P9 and the Fel d 7-derived peptides P3 and P5 are each part of the region containing the major IgE epitopes in Fel d 4 and Fel d 7

[0117] To study the IgE epitopes on Fel d 4 and Fel d 7, several fragments of these allergens have been chemically synthesized.

[0118] Table 1. Amino acid sequences of Feld 4- and Feld 7-derived peptides (cysteine residues can be added to the N- or C-terminus to facilitate coupling reactions)

[0119]

[0120]

[0121] Subsequently, rabbits were immunized with KLH-conjugated peptides, and the complete allergens (i.e., rFeld 4, rFeld 7) were used as controls. A total of three injections were given, with the first booster at 4 weeks and the second booster at 3 weeks later. Then, a competitive ELISA experiment was performed using anti-peptide-specific antiserum to determine the inhibitory effect of peptide-specific IgG on the binding of allergens to IgE in allergic patients. The method is described in (Curin M et al., Frontiers in Immunology 12 (2021): 687294).

[0122] The lowest average inhibition rate of the antiserum against Fel d 4-derived peptides was P3, at 6.46%. Some inhibitory effects were observed for the antisera against P1 and P6; 22.70% and 14.06% respectively. The best inhibitory effect (i.e., 65.95%) was obtained for the antibody specifically against P9, which was comparable to the inhibitory effect obtained with the anti-serum against the complete Feld 4 Figure 2 ). Figure 2 The histograms in Figure 2 A and Figure 2 C show the mean inhibition percentages + / - SD of patients grouped according to the peptide and the results of the anti-serum against the complete allergen. Figure 2 The graphs in

[0123] B and Figure 3 D show the binding of the allergen bound to the plate, pre-incubated with pre-immune serum or the anti-serum generated against the peptide or the complete allergen (x-axis), to the IgE of allergic patients (n = 15) (optical density - OD value). (The number of patients N = 15). The dots represent the OD values, and the paired values are connected by a straight line. The paired-sample Student's correlated t-test was used to compare the differences between groups. A P-value < 0.05 was considered significant. Figure 3 The lowest average inhibition rate of the antiserum against Fel d 7-derived peptides was P4, at 7.47%. Some inhibitory effects were observed for the antisera against P1 and P2; 17.44% and 19.01% respectively. When a combination of specific antisera against Fel d 7-derived peptides P3 and P5 was used to inhibit the binding of patient IgE, the mean inhibition percentage was 72.53%, which was similar to the inhibition rate achieved with the antibody generated against the complete Feld 7 (i.e., 87.61%) Figure 3 )

[0124] The antisera against Feld 7-derived peptides P3 and P5 could also inhibit the binding of IgE from allergic patients (n = 6) to the cross-reactive allergen Can f 1 Figure 4 A). Similarly, the antibody against Feld 4-derived peptide P9 could inhibit the binding of IgE from allergic patients (n = 4) to the homologous allergen Equ c 1 of horse Feld 4 Figure 4 B). The histograms show the inhibition percentages of the anti-peptide antisera against the cross-reactive allergens Can f 6 (A) and Equ c 1 (B) detected by competitive ELISA.

[0125] These results indicate that the C-terminal portion of Fel d 4 serves as the major IgE epitope. In addition, the Fel d 7-derived peptides P3 and P5 are derived from the major IgE-reactive regions of Fel d 7 (Table 1). Thus, these newly identified peptides can be used to construct feline vaccines that induce protective IgG antibodies against Fel d 4 and Fel d 7.

[0126] Example 3: Recombinant PreS fusion proteins containing hypoallergenic peptides from Fel d 1, Fel d 4, and Fel d 7

[0127] Previous studies (Niespodziana K et al., Journal of Allergy and Clinical Immunology 127 (2011): 1562-70) have described the construction, expression, and purification of fusion proteins containing the PreS protein from hepatitis B virus and hypoallergenic peptides of the major feline allergen Fel d 1 for allergen-specific immunotherapy of cat allergy. In the above study, a fusion protein containing PreS and two copies of Fel d 1-derived peptide 1, designated PreS-2xP1, a fusion protein containing PreS and two copies of Fel d 1-derived peptide 5, designated PreS-2xP5, and a fusion protein containing PreS, one copy of peptide 1, and one copy of peptide 5, designated PreS-P1-P5, were used. When tested in cat allergy patients, the three recombinant fusion proteins showed reduced IgE reactivity and allergenic activity, and it was found that after immunization in mice and rabbits, PreS-2xP1 and PreS-P1-P5 were superior to PreS-2xP5 in inducing IgG antibodies that blocked the binding of IgE from allergic patients to Fel d 1. Since the Fel d1-derived peptides P1 and P5 play an important role in inducing IgG antibodies that block the binding of IgE from cat allergy patients to Fel d 1, both of these peptides were used to construct a combined vaccine for Fel d 1, Fel d 4, and Fel d 7.

[0128] Table 2. Amino acid sequences of Fel d 1, Fel d 4, and Fel d 7-derived peptides used in the fusion proteins shown in Table 3

[0129]

[0130] Five fusion proteins were designed that contain two copies each of Fel d 1-derived peptides P1 and P5, two copies each of Feld 4-derived peptides P9, and two copies each of Feld 7-derived peptides P3 and P9 ( Figure 5 ). These five fusion proteins, named SuperCat 1 to SuperCat 5, contain the same allergen-derived peptides but in different orders.

[0131] Table 3. Amino acid sequences of the fusion proteins (His-tag (italicized and bolded) added to the N-terminus to facilitate purification)

[0132]

[0133]

[0134] The recombinant SuperCat 1 to SuperCat 5 PreS fusion proteins were co-expressed with a C-terminal hexahistidine tag as described by Niespodziana K et al., Journal of Allergy and Clinical Immunology 127 (2011): 1562 - 70, to facilitate purification by nickel affinity chromatography as follows: The His-tagged PreS fusion proteins were purified under denaturing conditions. To break the bacterial cell wall, the pellet was first subjected to freeze-thaw (3 cycles) and then resuspended in 30 ml of lysis buffer B (8 M urea, 100 mM NaH2PO4, 10 mM Tris-Cl, pH 8). After centrifugation, the pellet was discarded, and the supernatant was incubated with 2 ml of nickel agarose (Quiagen) on a shaker at room temperature for 2 hours. Subsequently, the mixture was transferred to a polypropylene tube, and impurities were eluted with 10 ml of wash buffer C (8 M urea, 100 mM NaH2PO4, 10 mM Tris-Cl, pH 6.3). The SuperCat1 - 5 were eluted from the nickel agarose using two elution buffers with decreasing pH: elution 1 - 3 times with buffer D (8 M urea, 100 mM NaH2PO4, 10 mM Tris-Cl, pH 5.9) and elution 4 - 7 times with buffer E (8 M urea, 100 mM NaH2PO4, 10 mM Tris-Cl, pH 4.5). Finally, urea was removed by gradient dialysis, first gradually reducing the urea concentration from 8 M to 1 M with PBS (pH 7.4), and then dialyzing 3 times with PBS (pH 7.4) for at least 8 hours.

[0135] Example 4: The SuperCats 1 to 5 showed a similar degree of reduction in IgE reactivity and sensitizing activity compared to the mixture of Fel d 1, Fel d 4, and Fel d 7

[0136] Serum from cat-allergic patients (N = 19 and N = 29 from Austrian and Russian cohorts, respectively) and sera from 2 non-allergic controls were tested by ImmunoCap to compare the IgE reactivity of recombinant SuperCat 1 to 5 proteins with an equimolar mixture of Fel d 1, Feld 4, and Fel d 7. ImmunoCAPs containing Fel d 1, 4, 7 allergens and SuperCat fusion proteins were prepared using streptavidin ImmunoCAP (o212 ImmunoCAP, Thermo Fisher Scientific / Phadia) as described in (Huang HJ et al., Journal of Allergy and Clinical Immunology 142(2018):1656 - 1659). Specific IgE levels were measured according to the manufacturer's instructions on an ImmunoCAP100 instrument (Thermo Fisher Scientific / Phadia). Compared to the allergen mixture, the IgE reactivity of SuperCats 1 to 5 was significantly reduced, and the degree of reduction was comparable for each Supercar protein ( Figure 6 ). The IgE reactivity of the SuperCat antigens was significantly reduced compared to the mixture of Fel d 1, Fel d 4, and Fel d 7 allergens. IgE levels are presented on a logarithmic scale in units of kUA / L (y-axis). Dots represent IgE levels, and paired values (i.e., when patients were tested with the allergen mixture and SuperCat antigen) are connected by straight lines. Paired-sample student correlation t-tests were used to compare differences between groups. A P-value < 0.05 was considered significant. (Abbreviations Figure 6 : mix is the mixture of Fel d 1, 4, and 7; SC1-5 are SuperCats 1 to 5).

[0137] Next, a basophil activation test was performed in cat-allergic subjects using RBL cells expressing the human high-affinity IgE receptor Fc∈RI to detect the sensitizing activity of SuperCat 1 to 5 proteins. We performed RBL assays using a series of diluted antigens (100 ng / ml, 10 ng / ml, 1 ng / ml, 0.1 ng / ml of the mixture of Fel d 1, 4, and 7, or equimolar concentrations of SuperCats 1-5) and obtained the following results: The cat allergen mixture induced β-hexosaminidase release from basophils in most of the tested patients at very low concentrations (i.e., 0.1 ng / mL); while SuperCat 1-5 did not cause basophil degranulation in most patients ( Figure 7)。RBL cells were loaded with sera from cat-allergic patients (patients numbered 3, 6, 10, 12, 14, 15, 16, 17, 18, and 19), and then stimulated with decreasing antigen concentrations (x-axis; Feld mixture (Feld 1 + Fel d 7 + Fel d 4), SC1-5: SuperCat1, Supercat2, SuperCat3, SuperCat4, SuperCat5). The β-hexosaminidase release was expressed as a percentage of the total amount in the medium after cell lysis with detergent (y-axis). The horizontal critical line corresponded to the percentage of basophil release after medium exposure (negative control). Abbreviations: SC1-5 are SuperCat 1-5. Slight induction of β-hexosaminidase release by SuperCat protein at the highest concentration of 100 ng / ml was observed only in one extremely sensitive patient. In this patient, the allergen mixture induced strong basophil activation at all tested concentrations ( Figure 7 )。

[0138] Based on the comprehensive results of the IgE reactivity test and the sensitization activity assessment of SuperCat protein compared with the wild-type allergen mixture, both the IgE reactivity and the sensitization activity of SuperCat protein were significantly reduced. Therefore, it can be expected that cat-allergic patients can tolerate higher doses of SuperCat protein during allergen-specific immunotherapy (AIT), which enables a vaccination schedule with fewer doses and higher dosages for the SuperCat protein-based vaccine compared to the allergen immunotherapy vaccine based on natural wild-type allergens, making the AIT based on SuperCat protein safer and more convenient for patients.

[0139] Example 5: Inhibition of the binding of IgE from allergic patients to cat allergens by antisera obtained by immunization with SuperCats 1 to 5

[0140] To study the inhibitory effect of the blocking IgG induced by vaccination with SuperCat 1-5, a competitive ELISA experiment was performed according to the method of Focke-Tejkl M et al. (Journal of Allergy and Clinical Immunology 135 (2015): 1207-17). For this purpose, plates were coated with 1 μg / ml of rFeld 1, rFeld 4, or rFeld 7, and then incubated with pre-immune sera and specific rabbit antisera diluted 1:20 collected 4 weeks after the second or fifth injection. Subsequently, the plates were incubated with sera from allergic patients diluted 1:10. Figure 8 Shows the difference in the binding degree of patient sera IgE to different allergens after treatment with pre-immune sera and specific immune sera.

[0141] After pre-incubation with pre-immune and immune sera from rabbits against SuperCats (SC1 - SC5) (x-axis) obtained 4 weeks after the second injection of Fel d 1 and 4 weeks after the fifth injection of Fel d 4 and 7, the IgE reactivity of cat-allergic patients to cat allergens was reduced ( Figure 8 above figure). The experiment was conducted with 11 patients allergic to Feld 1 and 4 patients allergic to Feld 4 or Feld 7. Allergen-specific IgE binding was expressed on a logarithmic scale of OD values (y-axis). The dots represent the ELISA results of IgE binding after pre-incubation with pre-immune rabbit sera, the squares represent the results after pre-incubation with immune sera, and paired values are connected by straight lines. Paired-sample Student's correlated t-test was used to compare the differences between groups.. Abbreviations: SC1 - 5 are SuperCats 1 - 5

[0142] Antisera against SuperCats 1 to 5 (100 μg / injection) showed a significant inhibitory effect on the binding of IgE of allergic patients to Fel d 1 after the second injection. Among them, the specific antisera against SuperCat 1, 3, and 5 had a slightly better blocking effect on the binding of Fel d1 to IgE than the antisera against SuperCat 2 and 4 ( Figure 8 ). Similarly, the antisera against SuperCats 1, 3, or 5 (50 μg or 100 μg / injection) had a slightly better blocking effect on the binding of IgE of allergic patients to Fel d 4 and 7 than the antisera against SuperCat 2 and 4 ( Figure 8 ).

[0143] These data clearly indicate that SuperCats 1 to 5 reduced IgE reactivity and sensitizing activity. However, SuperCats 1, 3, and 5 were even superior to SuperCats 2 and 4 in inducing allergen-specific protective antibodies.

[0144] Example 6: Compared with two previously reported PreS-based Fel d 1-specific vaccines (i.e., PreS-2xP1, PreS-P1-P5), SuperCats showed a better inhibitory effect on the binding of IgE of cat-allergic patients to Fel d 2

[0145] In this example, the inhibitory effects of IgG induced by vaccination with two previously reported PreS-based Fel d 1 vaccines (i.e., PreS-P1P5 and PreS-2xP1) (Niespodziana K et al. Journal of Allergy and Clinical Immunology 127(2011):1562-70) were compared with those induced by vaccination with SuperCats 1, 3, and 5. Competitive ELISA analysis was performed using rabbit antiserum samples obtained 4 weeks after the second vaccination after the same immunization with equimolar amounts of the vaccines ( Figure 9 ).

[0146] After pre-incubation with pre-immune and immune sera from rabbits obtained 4 weeks after the second injection of SuperCats (SC1, 3, and 5) or the previous Fel d1-based vaccines PreS-P1-P5 and PreS-2xP1, the specific IgE reactivity of IgE binding Fel d 1 in cat allergic patients (n = 11) decreased. The OD values (y-axis) corresponded to the bound IgE and were represented as box plots, where the box represented the interquartile range containing 50% of the data, the horizontal line in the box represented the median, and the lines outside the box represented the maximum and minimum values. The critical value was 0.5 (abbreviations: P1P5 and 2xP1 were the previous Fel d 1-based vaccines PreS-P1-P5 and PreS-2xP1; SC1, 3, 5 were SuperCat 1, 3, 5).

[0147] It was found that the antibodies induced after the second injection of SuperCats were more inhibitory of IgE binding in allergic patients than those induced by the PreS-P1P5 or PreS-2xP1 vaccines. The average inhibition percentages of the antibodies induced by SuperCat were 37.9 - 44.9%, while those of PreS-P1-P5 were 13.4% and those of PreS-2xP1 were 21.3% ( Figure 9 ).

[0148] These results obtained by direct comparison in the same experiment showed that, in terms of inducing protective antibodies, Supercats, especially Supercats 1, 3, and 5, were superior to the two previously described PreS-based Fel d1 vaccines (i.e., PreS-P1P5 and PreS-2xP1).

[0149] Example 7: SuperCats 1, 3, or 5 are far superior to commercially available allergen extract-based vaccines in inducing Fel d 4 and Fel d 7 specific IgG antibodies

[0150] SuperCats 1, 3, and 5 were compared with commercial vaccines available for cat allergy AIT from different companies in Europe (i.e., Alutard, ALK Abellò, Bencard, Allergie GmbH, and Roxall ) and two previously reported PreS-based Fel d 1 vaccines (i.e., PreS-P1-P5 and PreS-2xP1) for their induction of IgG antibodies against Fel d 4 and Fel d 7. The specific IgG levels of Fel d 4 and Fel d 7 in rabbit sera immunized with commercial vaccines according to the instructions and SuperCat vaccines at monthly intervals for a total of 5 times were detected by ELISA( Figure 10 ). Pre-immune serum samples were collected shortly before the first immunization, immune samples were collected monthly during immunization (samples 1 - 4), and immune samples were collected 4 weeks after the last injection of each vaccine (i.e., the last sample).

[0151] The study found that SuperCats 1, 3, and 5 induced high levels of Fel d 4 and Fel d 7 specific IgG antibodies, while Alutard and Roxall only induced low levels of Fels d 4 and Fel d 7 specific IgG levels, and it was observed that Bencard and the previously reported PreS-based Fel d1 vaccines (i.e., PreS-P1-P5 and PreS-2xP1) failed to induce Fel d 4 and Fel d 7 specific IgG antibodies after vaccination.

[0152] Example 8: SuperCats 1, 3, or 5 are far superior to commercially available allergen extract-based vaccines in inducing Fel d 4 and Fel d 7 specific IgG antibodies capable of blocking the binding of IgE from allergic patients to Fel d 4 and Fel d 7

[0153] To study the blocking activity of specific IgG, an IgE binding inhibition experiment was performed using sera from allergic patients according to the method described in (Niespodziana K et al., Journal of Allergy and Clinical Immunology 127(2011):1562 - 70). ELISA plates were coated with 1 μg / ml of various allergens; then pre-incubated with rabbit sera obtained before injection and 4 weeks after the 2nd or 5th injection of SuperCats (100 μg per injection). The inhibition experiment using antisera obtained from allergen extracts was performed using immune sera obtained 4 weeks after the last injection, with Alutard, Bencard, and Roxall injected 15, 9, and 6 times, respectively. Figure 11 and Figure 12Shows the inhibition of the binding of IgE from allergic patients to Feld 1, Feld 4, and Feld 7 after pre-incubation with specific rabbit antiserum. Surprisingly, after the second injection, the strongest inhibition of the binding of IgE to Fel d 1 was observed with SuperCats 1, 3, and 5. The inhibitory effect after the second injection of SuperCats was almost as good as that after 6 and 15 injections of Roxall and Alutard, respectively( Figure 11 ), while Bencard failed to induce any relevant blocking antibodies even after 9 injections( Figure 11 ).

[0154] Figure 11 OD values (y-axis) showing the binding of IgE from allergic patients (n = 22) to Fel d 1. These data were measured after pre-incubation of Fel d1 with pre-immune serum and serum after the second and fifth injections of SuperCat and after a complete immunization course with allergen extract-based vaccines (i.e., 15 injections of Alutard, 9 injections of Bencard, or 6 injections of Roxall). The dots represent IgE levels (i.e., paired values are connected by a straight line). The mean percentage inhibition is shown below. Paired-sample Student's correlated t-tests were used to compare differences between groups (P values < 0.05 were considered significant. * is for p value < 0.01, ** is for p value ≤ 0.001, *** is for p value ≤ 0.0001; ns is not statistically significant; abbreviation: SC is SuperCat).

[0155] Regarding Feld 4 and Feld 7, after 15 injections of Alutard, only a slight inhibition of the IgE reactivity to Feld 4 was observed, while no relevant inhibitory effects on the binding of Feld 4 and Feld 7 to IgE were found with any other allergen extract-based vaccines( Figure 12 ). Only Supercats 1, 3, and 5 produced a strong inhibition of more than 40% of the binding of patient IgE to Feld 4 and Feld 7, and the best inhibitory effect also occurred after the second injection, indicating that it may be necessary to reduce the injections of Supercats to establish a protective IgG antibody response in patients( Figure 12 ).

[0156] Figure 12OD values (y-axis) showing the binding of serum IgE from allergic patients (n = 22) to Feld 1, measured after pre-incubation with sera before immunization and sera after the second and fifth injections of SuperCat and sera after a complete immunization course with allergen extract-based vaccines (i.e., 15 injections of Alutard, 9 injections of Bencard, or 6 injections of Roxall, respectively). Dots represent IgE levels (i.e., paired values are connected by a straight line). Mean inhibition percentages are shown below. Paired-sample Student's correlated t-tests were used to compare differences between groups (P values < 0.05 were considered significant. * is for p value < 0.01, ** is for p value ≤ 0.001, *** is for p value ≤ 0.0001; ns is not statistically significant; abbreviation: SC is SuperCat).

Claims

1. A peptide, characterized in that, The peptide consists of 20 to 30 amino acid residues derived from amino acids 120 to 171 of mature allergen Fel d 4.

2. The peptide according to claim 1, characterized in that, The peptide is derived from amino acids 130 to 171 of mature Fel d 4, preferably from amino acids 140 to 171, more preferably from amino acids 145 to 171, and even more preferably from amino acids 146 to 171.

3. The peptide according to claim 1 or 2, characterized in that, The Fel d 4 comprises SEQ ID No.1 or consists of the same.

4. The peptide according to any one of claims 1 to 3, characterized in that, The peptide comprises the amino acid sequence SEQ ID No.3 or consists of the same.

5. A fusion protein or conjugate, characterized in that, Comprises at least one peptide as described in any one of claims 1 to 4.

6. The fusion protein or conjugate according to claim 5, wherein, The at least one peptide is conjugated or fused with at least one allergen fragment and / or at least one carrier protein.

7. The fusion protein or conjugate according to claim 6, wherein, The at least one allergen fragment is derived from at least one fur animal allergen, preferably from at least one cat allergen.

8. The fusion protein or conjugate according to claim 7, wherein, The at least one cat allergen is selected from the group consisting of: Fel d 1 chain 1, Fel d 1 chain 2, Fel d 2, Fel ed 3, Fel d 5, Fel f 6, Fel d 7, and Fel d 8, preferably selected from the group consisting of: Fel d 1 chain 1, Fel d 1 chain 2, and Fel d 7, more preferably selected from the group consisting of: Fel d 1 chain 1, Fel d 1 chain 2, and Fel d 7.

9. The fusion protein or conjugate according to any one of claims 6 to 8, characterized in that, The at least one allergen fragment comprises 20 to 40 amino acid residues or consists of the same.

10. The fusion protein or conjugate according to claim 8 or 9, characterized in that, The at least one allergen fragment of the at least one cat allergen is derived from and comprises the N-terminus or C-terminus of the at least one cat allergen.

11. The fusion protein or conjugate according to any one of claims 8 to 10, characterized in that, The at least one allergen fragment derived from Fel d 1 chain 1 comprises or consists of amino acid residues 1 to 34 of mature Fel d 1 chain 1.

12. The fusion protein or conjugate according to any one of claims 8 to 11, characterized in that, The at least one allergen fragment derived from Fel d 1 chain 2 comprises or consists of amino acid residues 81 to 109 of mature Fel d 1 chain 2.

13. The fusion protein or conjugate according to any one of claims 8 to 12, characterized in that, The at least one allergen fragment derived from Fel d 7 comprises and / or consists of amino acid residues 61 to 97 and / or amino acid residues 124 to 162 of mature Fel d 7.

14. The fusion protein or conjugate according to any one of claims 6 to 13, characterized in that, The carrier protein is a viral protein or a fragment thereof consisting of 50 to 300 amino acid residues, preferably 60 to 250, more preferably 80 to 200, and even more preferably 100 to 200.

15. The fusion protein or conjugate according to claim 14, characterized in that, The viral protein is a capsid protein.

16. The fusion protein or conjugate according to claim 14 or 15, characterized in that, Wherein the viral protein is derived from a hepadnavirus.

17. The fusion protein or conjugate according to claim 16, wherein, The hepadnavirus is hepatitis B virus.

18. The fusion protein or conjugate according to claim 17, wherein, The viral protein of the hepatitis B virus is PreS, PreS1, or PreS2.

19. The fusion protein or conjugate according to any one of claims 6 to 18, characterized in that, The carrier protein comprises the amino acid sequence SEQ ID No.4 or consists of the same.

20. The fusion protein or conjugate according to any one of claims 6 to 19, characterized in that, The fusion protein comprises at least two peptides as described in any one of claims 1 to 4 and at least two allergen fragments as described in any one of claims 7 to 13.

21. The fusion protein or conjugate according to any one of claims 6 to 20, characterized in that, 2 to 8 (preferably 2 to 6) of the at least one peptide and 2 to 8 (preferably 2 to 6) of the at least one allergen fragment are fused to the N-terminus and C-terminus of the at least one carrier protein.

22. The fusion protein or conjugate according to any one of claims 6 to 20, characterized in that, Two of the at least one peptide are adjacent to each other within the fusion protein.

23. The fusion protein or conjugate according to any one of claims 6 to 22, characterized in that, The fusion protein comprises two peptides comprising or consisting of amino acid residues 146 to 171 of mature Fel d 4, two allergen fragments comprising or consisting of amino acid residues 1 to 34 of mature Fel d 1 chain 1, two allergen fragments comprising or consisting of amino acid residues 81 to 109 of mature Fel d 1 chain 2, two allergen fragments comprising or consisting of amino acid residues 81 to 109 of mature Fel d 1 chain 2, two allergen fragments comprising or consisting of amino acid residues 61 to 97 of mature Fel d 7, and two allergen fragments comprising or consisting of amino acid residues 124 to 162 of mature Fel d 7.

24. The fusion protein or conjugate according to any one of claims 6 to 23, characterized in that, The fusion protein comprises or consists of the amino acid sequences SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13 or SEQ ID No.14 (preferably SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11 or SEQ ID No.13).

25. The peptide according to any one of claims 1 to 4 or the fusion protein or conjugate according to any one of claims 5 to 24 for preventing or treating cat allergy.

26. The peptide according to any one of claims 1 to 4 or the fusion protein or conjugate according to any one of claims 5 to 24 for preventing or treating fur animal allergy, preferably dog or horse allergy.

27. A nucleic acid molecule, characterized in that, Encoding the peptide according to any one of claims 1 to 4 or the fusion protein according to any one of claims 5 to 24.

28. Carrier, characterized in that, Comprising the nucleic acid molecule according to claim 27.

29. A host cell, characterized in that, Comprising the nucleic acid molecule according to claim 27 or the vector according to claim 28.

30. A vaccine preparation, characterized in that, Comprising the peptide according to any one of claims 1 to 4, the fusion protein according to any one of claims 5 to 24, the nucleic acid molecule according to claim 27 and / or the vector according to claim 28.

31. The vaccine preparation according to claim 30 for preventing or treating fur animal allergy, preferably cat, dog and / or horse allergy.

32. The vaccine preparation according to claim 30 or 31, characterized in that, The preparation comprises 10 ng to 1 g, preferably 100 ng to 10 mg, in particular 0.5 μg to 200 μg of the fusion protein, nucleic acid molecule or vector.

33. The vaccine preparation according to any one of claims 30 to 32, characterized in that, The preparation further comprises at least one adjuvant, a pharmaceutically acceptable excipient and / or a preservative.

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