EA041116B1 - IMPROVED MODULAR ANTIGEN TRANSPORT MOLECULES AND THEIR APPLICATION IN ANIMALS - Google Patents

Description

The field of technology to which the invention belongs

The present invention relates to (isolated) recombinant proteins, also referred to as improved MAT (iMAT) molecules, comprising at least one translocation module, at least one targeting module and at least one antigenic module, where at least one cysteine residue is substituted with another amino acid residue. The iMAT molecules can be produced with substantially reduced manufacturing effort and are species-specific, safe and immunologically highly effective. Such (isolated) recombinant proteins are useful in particular as vaccines, for example for the therapy and/or prevention of allergies and/or infections and/or for the prevention of the transmission of infections in animals, preferably ruminants, pigs, humans, dogs and /or cats, but excluding horses.

Prerequisites for the creation of the invention

The prior art published by Crameri et al. describes the background of the invention in more detail (Crameri R. et al., Allergy 2007, 62: 197-206). Briefly, antigen processing by antigen presenting cells (APC) occurs in two distinct ways. Antigens that are intracellular are presented by MHC I (major histocompatibility complex class I, MHC class I) molecules on the cell surface, while extracellular antigens are presented by MHC II (major histocompatibility complex class II, MHC class II) molecules on the cell surface .

Both mechanisms initiate the host's immune response to the antigen. Major histocompatibility complex class II molecules are cell surface glycoproteins that present peptides to CD4+T cells. In the endoplasmic reticulum (ER), MHC-II molecules become associated with a type II transmembrane protein, referred to as invariant chain (Ii), preventing the peptide from binding to MHC-II in the ER. Ii homotrimers bind in the ER to αβ dimers of 3 MHC class II and this prevents endogenous peptides from binding to class II molecules. The N-terminal cytoplasmic domain Ii contains targeting motifs that result in its retention in the ER, or targeting of class II αβ dimers into the endosomal-lysosomal pathway via the Golgi. Subsequent proteolytic degradation of Ii leaves a small fragment of CLIP (Ii-associated class II peptide) associated with class II αβ dimers in the peptide binding groove. Interaction of class II αβ/CLIP complexes with HLA-DM, the αβ dimer associated with class II, in a specialized compartment, releases CLIP and allows class II molecules to bind peptides derived from exogenous proteins. It has been shown that endogenously synthesized proteins, generally excluded from the MHC-II presentation pathway, can be efficiently presented as peptide-MHC-II complexes when expressed as Ii fusion proteins. This property can be used to clone genes encoding MHC-II-restricted antigens from cell lines transfected with Ii cDNA fusion libraries. Efficient allergovaccines targeting the MHC-II pathway of processing and presentation are prepared using translocation-capable Ii-allergen fusions. The concept, referred to as Modular Antigen Translocation Technology (MAT), is based on a fusion protein consisting of a TAT-derived translocator peptide that converts extracellular to cytoplasmic proteins, the first 110 amino acids of Ii to target the fusion proteins to endosomal/lysosomal compartments, and an antigen for induction of specific immune responses.

The concept of providing modular antigen transport molecules (MATs) to modulate immune responses, their associated constructs, method and use is disclosed in WO 2004/035793 (equivalent to US 2005/0281816). This document describes the utility of a three-part MAT molecule for introducing antigen epitopes into cells, thus determining the immune response to be modulated by such a MAT. This document describes various translocation modules, targeting modules, and antigenic modules. This technology and the method underlying it make it possible, firstly, to effectively transfer antigens from the extracellular space to the intracellular space of the target cell, and, secondly, provide an opportunity for antigens, after they enter the cell, to effectively reach cell organelles in order to subsequently undergo processing for antigen presentation. Typically, this two-step process can be used to target, effectively modulate the immune response in a subject. The use of MAT molecules is disclosed, for example, in Martinez-Gomez JM et al. [Allergy 2009, 64(1): 172-178]; Rose H (Arb Paul Ehrlich Inst Bundesinstitut Impfstoffe Biomed Arzneim Langen Hess, 2009, 96, 319-327) and later in Senti G et al. [J Allergy Clin Immunol., 2012, 129(5): 1290-1296] . Based on MAT technology, the major feline allergen Fel dl was fused to the translocation domain of a protein that is derived from TAT and to a truncated human invariant chain to target the MHC class II pathway. Immunogenicity was assessed in mice, while potential safety concerns were assessed using appropriate tests based on basophil reactivity obtained from feline allergic patients.

- 1 041116 wool. The possible use of this model compound has been demonstrated. This reference describes that MAT molecules are expected to be safer and more effective in inducing the desired immune response, namely desensitization to antigens, than recombinant allergens or allergen extracts when using conventional allergen-specific immunotherapy (SIT). A recent publication by Senti G. et al. described intralymphatic immunotherapy for the treatment of cat hair allergy in humans, which induced tolerance after three injections. This method described the first human clinical study using MAT-Fel d1, which demonstrated safety and induction of allergen tolerance after only three intralymphatic injections. Additional prior art sources are as follows:

Gadermaier G. et al. (Molecular Immunology 2010, 47: 1292-1298) describe the targeting of cysteine-stabilized Art v1 fold for immunotherapy of mugwort pollen allergy. The authors used genetic engineering approaches to target post-translational modifications of Art v1 to create a hypoallergenic molecule: (i) the disulfide bridges of the defensin domain were broken using site-directed mutagenesis and (ii) the resulting mutant constructs were expressed in E. coli to obtain non-glycosylated proteins. However, the goal was only to manipulate the three-dimensional fold of the Art v1 defensin domain to eliminate IgE binding (i.e., create a hypoallergenic molecule) by replacing single cysteine residues with serine while maintaining intact (i.e., unmodified) epitopes that recognized by T cells (even if they contain cysteine residues).

The paper from the third Havemeyer Workshop on Equine Allergy Disease (H0lar, Iceland, June 2007, Veterinary Immunology and Immunotherapy 2008, 126: 351-361) focused on the immunological and genetic aspects of bug sting hypersensitivity (IBH) and recurrent airway obstruction ( RAO). This workshop discussed new approaches for SIT against IBH, in particular the use of viral vectors or protein vaccination using allergens fused to modular translocation antigen (MAT) molecules. In the annual reports of SIAF 2010 and 2011. Crameri R reports the use of MAT technology to vaccinate IBH-susceptible horses. Zhao et al. (Int J Clin Exp Med 2015;8(4):6436-6443) described the results of experiments with mosaic fusion proteins with the MAT structure described in WO 2004/035793 and 3 segments of the T cell epitope encoding Der p1 in as an antigen module. They reassembled these sequences in a linear manner to form a fusion gene for protein expression. They describe their construct as exhibiting greater allergenicity (hyperallergenicity) than the Der p1 protein.

However, major problems have arisen in the production and manufacture of the MAT molecules described in the prior art. In particular, the standard methods used in the development of the isolation and purification process (DSP) in the manufacture of MAT molecules using good existing manufacturing practices (GMP) cannot be applied. It was not possible to purify the homogeneous molecular species of the MAT molecule, which is obviously due to their abnormal physicochemical properties.

Some known purification methods cannot be applied (see example 4 in the present description) using the MAT molecules described in the prior art, despite the fact that various separation principles have been investigated (for example, size exclusion chromatography, reverse phase HPLC ). Methods used to determine the purity of recombinant proteins generally include chromatographic separation, eg, reverse phase HPLC, and electrophoretic separation (eg, capillary electrophoresis, zonal electrophoresis, isoelectric focusing, SDS-PAGE under reducing or non-reducing conditions). In addition, these analytical methods cannot be used for MAT molecules without molecule-specific adaptation. An adapted SDS-PAGE specific test procedure should be developed to evaluate purity. This testing procedure involves preparing a sample with a reducing agent and lithium dodecyl sulfate (LDS) and heating to 75° C., resulting in multiple, reproducible clear bands after electrophoretic separation. Staining with Coomassie blue results in linear quantitative behavior (densitometry) in gels. Using a monoclonal antibody that allows detection of the allergen module in the MAT molecule, a main band and several weak bands were shown. All bands migrated in a reproducible manner to the same position as in the original gel, also after reloading the second gel with the bands of the first gel cut out. Surprisingly, in all of these apparent lower and higher molecular weight MAT bands, full length molecules were identified by excision of the bands from the gel, digestion with trypsin and subsequent analysis by mass spectrometry (nanoLC/ERI-MS-MS). From these experiments, it can be concluded that there is an atypical, anomalous behavior of various variants of the folding of MAT molecules in DSNPAGE (gel shift). In addition, in all batches of MAT molecules, multidimensional forms of the protein can be detected, which were difficult to separate from monomeric forms.

For example, for economic aspects, as well as for regulatory requirements, it is necessary to improve

- 2 041116 sew (I) the manufacturing process of MAT molecules and (II) their suitability for standard analytical methods for determining purity. In addition, adaptation of MAT molecules to specific target species such as ruminants, pigs, dogs and/or cats requires adaptation of immunological targeting within MAT technology. This species specificity needs to be represented in the indicated iMAT molecules because there are differences in amino acid sequence homologies between mammalian species (FIG. 10). However, proper binding of the Ii fusion protein (iMAT) to the MHC class II αβ subunits, especially in the CLIP region, is essential for optimal immunological function towards antigen in these iMAT molecules. Proper binding to these class II MHC molecules is achieved if the Ii sequence in the iMAT bears as much resemblance to the original as possible.

In addition, MAT molecules are readily used in allergies that are caused by a known major allergen (eg cat hair allergies in humans caused by Fel d1). However, it is difficult to use prior art MAT molecules in clinical settings such as allergies, in which, for example, various non-cross-reacting allergens are known to be involved, but the importance of such allergens in stimulating the allergy is unknown (i.e., major allergens not known).

In addition, the prior art does not describe how more than one allergen (eg 2, 3, 4 or more) can be incorporated into (i) MAT molecules without exceeding a certain size of the fusion protein, which interferes with protein production.

The object that underlies the invention is to provide improved MAT molecules that are useful as active agents in pharmaceutical composition such as vaccines and their respective therapeutic and/or prophylactic uses in animals, excluding horses, which overcome the problems in the field of technology.

Disclosure of invention

Brief summary of the invention

In one aspect, the object underlying the invention has been unexpectedly solved by providing a (isolated) recombinant protein, preferably an improved MAT (iMAT) molecule, comprising:

(I) at least one first module that has an amino acid sequence that allows translocation of the iMAT molecule from the extracellular space into cells, (II) at least one second module that has an amino acid sequence that allows for species-specific intracellular targeting iMAT molecules on cell organelles that are involved in the processing of antigens and/or the loading of MHC molecules with antigens, preferably processed antigens, and (III) at least one third module as an antigenic module, which has an amino acid sequence that is of at least from one full or partial epitope, at least one antigen, preferably at least one allergen, which determines the specificity of the immune response modulated by such an iMAT molecule, which is characterized in that, at least in the antigenic(s) module ( yah) n at least one cysteine residue is substituted with another amino acid residue, preferably serine, leucine, isoleucine, arginine, methionine and/or aspartic acid, for use in a method for preventing and/or treating one or more allergies in animals other than horses, and/ or for use in a method for preventing and/or treating one or more infectious diseases in animals other than horses and/or for use in a method for preventing transmission of one or more infectious diseases in animals other than horses and/or for use in a method for preventing transmission one or more infectious diseases in animals, excluding horses, using vectors.

Appropriate methods for the prevention and/or treatment of animals other than horses in need thereof and uses for the preparation of a pharmaceutical composition/drug for the prevention and/or treatment of animals other than horses are also within the scope of the present invention.

It is preferred that in at least one antigenic module all cysteine residues are replaced with another amino acid residue, preferably serine, leucine, isoleucine, arginine, methionine, and/or aspartic acid. More preferably, in the whole iMAT molecule, all cysteine residues are replaced with another amino acid residue, preferably serine, leucine, isoleucine, arginine, methionine and/or aspartic acid.

Preferably, when not all cysteine residues are substituted with another amino acid residue, preferably serine, leucine, isoleucine, arginine, methionine, and/or aspartic acid, even some cysteine residues remain in the whole iMAT molecule.

Preferably, all such modules are covalently bonded to each other, optionally via additional spacer module(s) between two or more adjacent modules, optionally all of the first, second and/or third modules.

More preferably, all such modules are covalently bonded to each other and no additional spacer module(s) is present between two or more adjacent modules, such as the first, second and/or third modules, at all.

In another aspect, the object underlying the invention has been unexpectedly solved by providing an iMAT molecule relating to one or more amino acid sequences according to SEQ ID NOs: 2 or 3, preferably comprising one or more amino acid sequences according to SEQ ID NOs: 4 or 5. In a further aspect, the problem underlying the invention has been unexpectedly solved by providing an iMAT molecule comprising one or more amino acid sequences according to SEQ ID NOs: 14-23. In a preferred aspect, the problem underlying the invention has been surprisingly solved by providing an iMAT molecule comprising, preferably consisting of, one or more amino acid sequences according to SEQ ID NOs: 24-83.

In another aspect, the problem underlying the invention has been unexpectedly solved by providing a vaccine or immunogenic composition or pharmaceutical composition containing an (isolated) recombinant protein as disclosed or claimed in this application.

In another aspect, the object underlying the invention has been unexpectedly solved by providing an (isolated) recombinant protein as disclosed or claimed in this application, or a vaccine or immunogenic composition or pharmaceutical composition as disclosed or claimed in this application for use in a method preventing and/or treating one or more allergies in animals, preferably dogs and/or cats, but excluding horses; preferably flea bite allergies preferably in dogs and/or cats; allergies to certain food components, preferably in dogs and/or cats; atopic dermatitis, preferably in dogs and/or cats; allergic inflammation and/or airway obstruction, preferably in cats. Appropriate methods for the prevention and/or treatment of animals, preferably dogs and/or cats, but excluding horses in need thereof, and use for the preparation of a pharmaceutical composition/drug for the prevention and/or treatment of animals, preferably dogs and/or cats, but excluding horses are also covered by the scope of the present invention.

In another aspect, the object underlying the invention has been unexpectedly solved by providing a (isolated) recombinant protein as disclosed or claimed in this application, or a vaccine or immunogenic composition or pharmaceutical composition as disclosed or claimed in this application for use in a method preventing and/or treating one or more infectious diseases in animals, preferably ruminants, pigs, dogs and/or cats, but excluding horses, and/or preventing the transmission of one or more infectious diseases in animals, preferably ruminants, pigs, dogs and/or cats, but excluding horses, by vectors, preferably by blood-feeding insects, flies, midges, mites and/or mosquitoes. The infectious pathogen and/or infectious disease may be one or more selected from campylobacter, heartworm, ehrlichiosis, leishmaniasis, trypanomiasis, borreliosis, Schmallenberg virus, African bluetongue and/or West Nile virus, dermatophytosis, and/or infections of the digestive system, and/or other organs caused by viruses (eg rota-, coronavirus) and/or parasites (eg helminths) and/or protozoa (eg coccidiosis, cryptosporidiosis) and/or their stages of parasitic incubation. Appropriate methods for the prevention and/or treatment of animals, preferably ruminants, pigs, humans, dogs and/or cats, but excluding horses in need thereof, and use for the preparation of a pharmaceutical composition/drug for the prevention and/or treatment of animals, preferably ruminants , pigs, humans, dogs and/or cats, but excluding horses, are also covered by the scope of the present invention.

In a further aspect, the problem underlying the invention has been surprisingly solved by providing a nucleic acid that encodes a (isolated) recombinant protein as disclosed or claimed in this application.

In a further aspect, the problem underlying the invention has been unexpectedly solved by providing a vector that includes at least one nucleic acid as disclosed or claimed in this application.

In an additional aspect, the problem underlying the invention has been unexpectedly solved by providing a primary cell or cell line comprising at least one nucleic acid as disclosed or claimed in this application and/or at least one vector as disclosed or claimed in this application.

Surprisingly, iMAT molecules according to the present invention, as disclosed or claimed in this application, have been found to have physicochemical and/or immunological characteristics that provide them with advantages over prior art MAT molecules:

(I) Replacement of at least one cysteine residue, preferably all cysteine residues, with

- 4 041116 With another amino acid residue in the antigenic module(s), preferably a whole iMAT molecule, which is chosen in silico so as not to compromise the stability of the final iMAT molecule, provides an iMAT molecule in accordance with the present invention, which is surprisingly acceptable for applying standard purification procedures for biopharmaceuticals, as well as for standard analytical methods.

(II) The preferred direct covalent linkage of the modules of the iMAT molecule, i.e. the first translocation module, the first targeting module and the third antigenic module, without any additional spacer modules between such modules, i.e. without additional spacer modules between two or more adjacent modules, such as a first, second and/or third module, generally contributes to obtaining additional to the above improved characteristics of the iMAT molecules in accordance with the present invention: those iMAT molecules that are considered more rigid in terms of three-dimensional structural and, therefore, are not able to form conformational IgE epitopes are even more hypoallergenic - in fact, up to the complete absence of allergenicity.

(III) The preferred presence of an (n) (quasi) N-terminal or C-terminal His tag results in iMAT molecules of the present invention that can be used as a surrogate marker for monitoring immunity and/or immunity duration, since such the label module, optionally in conjunction with one or more adjacent amino acid residues from the translocation module, can be used to induce a specific signal that is immunologically detectable (e.g., an antibody) in the target subject, i.e., specific for the structure of the iMAT molecules (see example 1 below in present application). Additionally, the presence of such a label module can be used to separate proteins in a sample that includes iMAT molecules in accordance with the present invention, for example, using zinc - or cobalt-charged solid carriers, and therefore further enhances the possibility of obtaining iMAT molecules in accordance with the present invention. invention with no aggregation in the purification process. An (n) (quasi) N-terminal His-tag is preferred.

(IV) At least one targeting module in the iMAT molecules according to the present invention is preferably species-specific, i.e. in the case of intended use of such iMAT molecules in dogs, cats, cattle, sheep, goats or pigs, the targeting module, for example invariant chain of the dog, choose accordingly. With this species-specific targeting, the optimized characteristics of the iMAT molecules of the present invention for binding to class II MHC molecules can be successfully achieved.

(V) At least one antigenic module in the iMAT molecules according to the present invention is preferably an allergen. It may originate from food and/or molds (fungi and/or their spores), pollen, house dust or barn mites (and/or their faeces) and/or fleas, preferably from pollen from trees, grasses, herbaceous plants, ragweed and/or pollen from cruciferous plants and/or fungi and/or their spores of the genera aspergillus, alternaria, botrytis, cercospora, cladosporium, curvularia, drechslera, eurotium, helminthosporium, epicoccum, erysiphe/oidium, fusarium, lichtheimia, nigrospora, penicillium, periconia, peronospora, polythrincium, saccharopolyspora (also faenia or micropolyspora), thermoactinomyces, stemphylium, torula and/or mites (or their feces) of the genera acarus, glycophagus, tyrophagus, dermatophagoides, euroglyphus, lepidoglyphus, blomia and/or fleas of the genera Ceratophyllus, Ctenophyllus, Pulex , Archaeopsylla. At least one antigenic module in the iMAT molecules according to the present invention is more preferably a Dermatophagoides allergen. The allergen can be selected according to the following criteria: if the main allergens that cause allergy in subjects are not known, then at least one antigenic module in the iMAT molecules in accordance with the present invention can be selected using a bioinformatics approach, as described illustratively and in detail set forth in examples 5 and 6 in this application. In this way, improved Mab molecules can be obtained that can be used specifically as vaccines, for example, for the treatment and/or prevention of allergies in animals, preferably dogs and/or cats, but excluding horses. The at least one antigenic module in the iMAT molecules of the present invention may also be a pathogen antigen that is involved in one or more infectious disease(s). It may originate from the genera Campylobacter, Dirofilaria, Ehrlichia, Leishmania, Trypanosoma, Borrelia, Orthobunyavirus, Orbivirus, Flavivirus, Rotavirus, Coronavirus, Trichophyton, Microsporum; other helminths such as Cooperia, Haemonchus, Ostertagia, Trichostrongylus, Dictyocaulus, Metastrongylus; and/or protozoa with gastrointestinal manifestations such as Eimeria, Isospora, Cryptosporidium, Giardia - in the case of parasites, antigens derived from parasitic incubation steps can also be used. At least one antigenic module in the iMAT molecules according to the present invention may also be an antigen (e.g. a saliva component) of a vector involved in the transmission of one or more infectious disease(s), e.g. those belonging to the families Culicidae, Ceratopogonidae Phlebotominae Ixodidae

- 5 041116 and/or Cimicidae. Thus, improved Mab molecules can be obtained which can be used specifically as vaccines, for example, for the treatment and/or prevention and/or prevention of the transmission of infectious diseases in animals, preferably ruminants, pigs, dogs and/or cats, but excluding horses.

(VI) New iMAT molecules can be provided that contain sequence motifs of more than one allergen in only one of said iMAT molecules, i.e., mosaic fusion proteins. The choice of peptide sequences embedded in the allergen module is based on the polyvalent allergenic motifs found in the relevant major allergens using bioinformatic approaches. With this approach, the size of such iMAT molecules can be kept low enough to allow efficient production in suitable expression systems.

In particular, the present invention provides (isolated) recombinant proteins that exhibit improved solubility, that are readily amenable to chromatographic separation methods, and that exhibit improved stability.

Additionally, the (isolated) recombinant proteins according to the present invention preferably exhibit high activities and efficiencies with respect to inducing the desired immunological effects, namely, favorable species-specific modulation of the immune response to allergens in a subject, e.g., animals, more preferably dogs and/ or cats, but excluding horses, allergen-specific IgE-mediated hypersensitivity reactions can be modulated in various target organs such as the skin, respiratory and/or gastrointestinal system. It also becomes possible and/or prevention and/or treatment of infectious diseases and/or prevention of transmission of infectious diseases by means of vectors in animals, preferably ruminants, pigs, humans, dogs and/or cats, but excluding horses.

The allergenicity of a therapeutic allergen is extremely important and is indicative of the potential for inducing side effects, such as provoking an anaphylactic reaction. With respect to prior art MAT molecules, conflicting results regarding their allergenicity compared to corresponding native allergens have been described in the prior art. Senti G et al. (J Allergy Clin Immunol. 2012, 129(5): 1290-1296) have demonstrated the hypoallergenicity of MAT-Fel d1 in the cell antigen stimulation test (CAST) as well as intradermal and intradermal testing. The quantitative difference in sensitivity between the allergen and the MAT molecule containing Fel d1 was 100-, 23- and 16-fold, respectively. Although MAT-Fel d1 was clearly hypoallergenic, some allergenicity remained. In contrast, Zhao et al. (Int J Clin Exp Med 2015; 8(4): 6436-6443) have reported that their MAT-Der p1 construct exhibits even more allergenicity (hyperallergenicity) than native Der p1 protein. .

Surprisingly, the safety of the improved MAT molecules as described and claimed in this application is superior in this aspect. Unlike native allergens, which typically elicit a strong histamine release, unexpectedly, the iMAT molecules of the present invention actually elicit no histamine release at all. Thus, iMAT molecules are superior in terms of safety compared to the MAT molecule as described in the prior art (see above).

The result of this unexpected safety property of iMAT molecules, as opposed to prior art MAT molecules, is the fact that iMAT molecules used as desensitizing proteins can be used similar to pathogen vaccines. There is no need to increase the dosage, as is the case with classical therapeutic allergens, since vaccines containing iMAT molecules do not show allergenic properties in relation to allergic side effects. Even the dose of the first injection of the iMAT molecule during the course of treatment is chosen only for reasons of effectiveness and does not take into account possible allergic side reactions. This cannot be done using the MAT molecules described in the prior art, since the allergenicity of the MAT, in comparison with the native allergen, is only reduced to a certain level. However, the MAT molecules described in the prior art are still allergens; iMAT molecules, in contrast, are not. The advantage of this improved property is a more efficient treatment regimen, possible, for example, with three subcutaneous or intralymphatic injections with a high content of the biopharmaceutical (for example, 3 times 1 μg to 100 μg, preferably 3 times 10 μg to 50 μg of iMAT protein).

The lack of allergenicity of iMAT molecules can be explained by the fact that, unlike the MAT molecules described in the prior art, there are no linker amino acid residues [i.e. spacer(s) module(s) between the first, second and/or third module(s) ] used to separate different modules in such iMAT molecules.

It is known in the art that engineered fusion proteins containing two or more

- 6 041116 functional polypeptides connected by a peptide or protein linker, such a linker is important for the functioning (for example, epitope recognition by the immune system) of proteins [Klein

JS et al., Protein Eng Des Sel. 2014, 27(10): 325-330]. The separation distance between functional units can influence epitope accessibility and ability to bind to avidity.

It is believed that in the iMAT molecules of the present invention, which lack amino acid linkers between modules, in particular between the target domain and the antigenic module, resulting in a more rigid structure, conformational epitopes of the allergen module cannot be formed due to misfolding. Cross-linking of antibodies bound to the surface of basophils (eg, IgE) by its high affinity receptors is necessary to induce histamine activation and release. However, misfolded allergens are unable to induce such cross-linking. Thus, iMAT molecules without additional spacer modules/linkers between the first, second and third module cannot form the conformational IgE epitopes that render the iMAT molecules of the present invention non-allergenic. Thus, in a specific embodiment, the iMAT molecules of the present invention lack any additional spacer modules or linkers between the first, second and third module.

Detailed description of the invention

Before embodiments in accordance with the present invention are described in detail, it should be noted that, as used in this application and in the appended claims, the singular forms of any and given include references to the plural, unless the context clearly indicates follows otherwise.

Unless otherwise indicated, all technical and scientific terms used in this application have the same meanings as commonly understood by one of ordinary skill in the art to which this invention pertains. All ranges and values given may vary from 1% to 5% unless otherwise noted or otherwise known to one of skill in the art, so the term approximately is generally omitted from the specification and claims. Although any methods and materials similar or equivalent to those described in this application can be used in the practice or testing of the present invention, the preferred methods, devices and materials will be described below. All publications mentioned in the present description are incorporated into this application by reference for the purpose of describing and disclosing substances, excipients, carriers and methods, as described in publications that can be used in connection with the present invention. Nothing in this application should be construed as an admission that the invention is not entitled to backdate such disclosure by virtue of prior invention.

The terms isolated recombinant protein, recombinant protein and/or improved MAT molecule (iMAT) are used interchangeably in the description of this invention. They all have the same meaning. The term module in the description of the present invention refers to a specific amino acid sequence, for example, a part, block or fragment of a polypeptide, usually short amino acid / peptide sequences that have a specific function. The term first module that has an amino acid sequence allowing the translocation of an (isolated) recombinant protein, preferably an improved MAT (iMAT) molecule, from the extracellular space into cells in this application can also be interchangeably formulated as a translocation module or a translocation sequence, and in the context of this The invention relates to a specific amino acid sequence that facilitates the transport of a carrier molecule, for example, an amino acid sequence, a peptide, a polypeptide, a protein, and substances of other classes, such as nucleic acids or pharmaceutically active ingredients (APIs), into cells, in particular eukaryotic cells, more specifically cells that express MHC class II molecules on their surface and/or MHC class I molecules on their surface, as is known in the literature.

By the presence of a translocation module, it is possible to promote the entry of said carrier molecule into the cells. Amino acid sequences useful as translocation modules are described in the prior art. For example, US 7,653,866 discloses several useful translocation sequences involving the HIV-tat molecule or VP22 protein, which is derived from the herpes simplex virus. This principle of promoting entry of a given target molecule into cells is described in a number of different studies in the relevant patent and non-patent literature. In addition, suitable translocation sequences include homeoprotein sequences, leucine zipper sequences or lysine-rich and arginine-rich sequences, as well as various other protein or polypeptide sequences that are secreted despite the absence of a secretion signal sequence. Particularly useful are viral peptide sequences, such as the HIV transcription activator protein (HIV tat). The sequence of a Tat or a Tat peptide has been described in the art, including various modifications. All variations that are described in the prior art for

- 7,041,116 Tat peptide sequences are generally acceptable as translocation modules. Other examples include the VP22 peptide as well as the Antennapedia peptides (a complex locus at which homeotic mutations are located) derived from the Drosophila homeotic antennapedia protein. In addition, other homeoproteins may be used. Various examples of acceptable homeoproteins are described in the prior art. In addition, leucine zipper proteins like human cFos-(139-164) or human cJun-(252-279) can be used. In addition, arginine-rich and/or lysine-rich peptides are suitable as translocation modules, including sequences like HIV-1 rev (34-50) or other peptides that are derived from viruses or yeast. Of course, peptides enriched in polyarginine and/or polylysine can be obtained synthetically. Said polyarginine and/or polylysine enriched peptides may include additional amino acids. Preferred examples are described in the relevant prior art.

In a preferred embodiment, the at least one translocation module comprises, preferably consists of, an amino acid sequence that does not consist of the entire protein sequence as illustrated above, but instead contains the minimum sequence that is still functional, i.e., which is capable of effectively promoting entry into cells. Acceptable minimum sequences are, for example, the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 1).

In another preferred embodiment, at least one translocation module comprises, preferably consists of, HIV-tat, VP22 and/or Antennapedia or partial sequences thereof, provided that such at least one translocation module is functional as a module for translocation from the extracellular spaces within cells.

The term is a second module that has an amino acid sequence that allows species-specific intracellular targeting of an (isolated) recombinant protein, preferably an improved MAT (iMAT) molecule, to cellular organelles that are involved in antigen processing and/or loading of MHC molecules with antigens, preferably processed antigens , in this application also interchangeably referred to as a targeting module or targeting sequence, and in the context of the present invention refers to a specific amino acid sequence that allows / facilitates intracellular transport of (isolated) recombinant proteins, as disclosed and claimed in this application, to such cellular organelles, which are involved in antigen processing and/or loading of MHC molecules with antigens.

In particular, such cellular organelles include the endoplasmic reticulum, the Golgi apparatus, the trans-Golgi network, lysosomes, endosomes, and class II MHC compartments. Such intracellular organelles are involved in processes such as, for example, transport and/or processing of antigens, production and/or loading of MHC II molecules with antigens or processed antigens, and/or transport of MHC II molecules loaded with such antigens onto their surface.

A number of sequences are known in the art. A notable example of useful targeting sequences includes the invariant chain of the MHC class II molecule, which is also known as the Ii invariant chain or MHC II gamma chain. Various variants of the invariant circuit are described in the patent and non-patent literature. In a preferred embodiment of the present invention, the invariant chain is selected from species in which the immune response is to be modulated and/or from species in which the iMAT molecule is to be targeted to the intracellular space.

This species specificity must be represented in these iMAT molecules, since there is homology of the Ii amino acid sequences between mammalian species (FIG. 10). However, proper binding of the Ii fusion protein (iMAT) to the MHC class II αβ subunits specifically at the CLIP site is required for optimal immunological function with respect to the antigen in these iMAT molecules. Proper binding to these MHC class II molecules is achieved if the Ii sequence in the iMAT bears as much resemblance to the original as possible.

For example, for dogs, cats, cattle, sheep, goats, or pigs, the preferred invariant chain selected is a dog, cat, bovine, sheep, goat, or porcine invariant chain. For dogs and cats, the preferred invariant chain is the amino acid sequence according to SEQ ID NO: 2 (canine) and SEQ ID NO: 3 (feline) or fragments thereof, provided that such fragments retain their intracellular transport function (e.g., the first 110 amino acids, as shown in Fig. 10).

Other suitable examples for targeting sequences include lysosomal membrane proteins, which include sequences suitable as targeting modules. There are a number of membrane proteins that are produced in lysosomes and have motif sequences that allow targeting to the lysosome. These groups of proteins include,

- 8 041116, among others, lamp 1, lamp 2, lamp 3, limp II and lap. In addition, tetraspanin proteins are known in the art as targeting modules. Additional proteins can be found in the endosomal/lysosomal compartments, these proteins show targeting properties. One of skill in the art knows how to determine acceptable targeting sequences, respectively.

In another embodiment, at least one targeting module comprises, preferably consists of, an invariant dog, cat, bovine, ovine, goat, or porcine chain or fragments thereof, provided that such fragments retain their intracellular transport function.

In a preferred embodiment, the at least one targeting module is an invariant canine chain, which comprises, preferably consists of, SEQ ID NO: 4 (canine). In another preferred embodiment, the at least one targeting module is a feline invariant strand, which comprises, preferably consists of, SEQ ID NO: 5 (feline).

The term third module as an antigenic module that has an amino acid sequence that is derived from at least one complete or partial epitope of at least one antigen, preferably at least one allergen, which determines the specificity of the immune response modulated by such (isolated) recombinant protein, preferably an improved MAT molecule (iMAT) (in a subject, preferably an animal, more preferably a ruminant, a pig, a dog and/or a cat, but excluding a horse), which is also interchangeably referred to in this application as an antigenic module or antigenic sequence, in the context of the present invention refers to a specific amino acid sequence that provides the ability to modulate an immune response against an epitope/antigen and that determines the specificity of the immune response in a subject, preferably an animal, more preferably a ruminant , pigs, dogs and/or cats, but excluding horses.

In this context, such(s) antigenic(s) module(s) include(s) at least one cysteine residue, which is substituted with another amino acid residue, preferably serine, leucine, isoleucine, arginine, methionine, and/or aspartic acid . Thus, the immune response is different from that of a subject, preferably an animal, more preferably a ruminant, a pig, a dog and/or a cat, but excluding a horse that is exposed to the unaltered amino acid sequence of the antigen.

There are no restrictions associated with antigens based on this method. This method can be used, for example, to activate the subject's immune system against pathogens such as, for example, viruses, bacteria, fungi, parasites, protozoa, etc., that is, in the most general form, as a vaccine. In addition, this method can be used not only directly against such pathogens, but also to activate the host's immune system to prevent the transmission of vector-borne diseases, including those caused by viruses, bacteria, fungi, parasites, protozoa, etc. In addition, this method can be used to activate the immune system against degenerative cells such as, for example, tumor cells, etc. However, it can also be used, on the other hand, to desensitize the subject's immune system against allergens, such as, for example, food allergens and/or aeroallergens, for example, allergens from molds (fungi and/or their spores), pollen, animal hair, house dust or barn mites (and/or their faeces), insect toxins, etc., or for targeted suppression of the immune system, e.g. if autoimmune reactions are present, such as e.g. arthritis, rheumatism, diabetes, SLE (systemic lupus erythematosus), etc., as well as to suppress transplant rejection reactions. Additional disorders that have not been mentioned and that are associated with an immune response that is too strong or too weak can also be treated with iMAT molecules as disclosed and claimed in this application.

It is possible to use as antigenic modules for the purposes of the present invention, in principle, all types of antigens capable of modulating an immune response. Both antigens that are currently known and antigens that will be discovered in the future are acceptable. In some cases, the antigens may also be those which do not elicit an immune response using conventional immunization methods currently known in the art, but which elicit an immune response in the subject using the new method described in the present invention. In addition, the term antigen includes antigenic fragments containing the antigenic determinant/antigenic determinants, which(s) is(are) known(s) as epitope(s). Thus, an antigenic module is an entire molecule, such as a protein, or is a part of a molecule, i.e. a fragment thereof such as a peptide encompassing at least one antigenic determinant or epitope. At least one antigenic determinant or epitope is capable of eliciting an immune response directed against the antigen. An epitope may contain one or more amino acids or peptides, or other structures capable of inducing an immune response, such as structures of sugars, phosphorylated amino acids, etc., or combinations thereof. An antigen can be a continuous-type determinant (i.e., one that is conformation-independent, such as present in native and denatured proteins) or a discontinuous type determinant (i.e., one that is conformation-dependent, such as present only in native, folded form, but not present in denatured proteins). It is possible to use not only proteins and peptides, but also structures of sugars, lipids, for example, lipopolysaccharides, lipoteichoic acids and other components of bacterial membranes (CD1b, for example, binds sugar structures and lipids), nucleic acids, such as, for example, DNA containing motifs CpG, various organic substances such as, for example, latex or pharmaceutically active substances such as antigen for the purposes of the present invention. The antigen can be obtained from all possible life forms, such as, for example, animals, plants, fungi, parasites, unicellular or multicellular microorganisms, viruses and other life forms. Antigens can be isolated from biological material, can be obtained as recombinant antigens, or obtained by synthesis, for example by peptide synthesis. Synthetically produced antigens may be substances that occur naturally, or those that do not occur in nature, but can be obtained by chemical synthesis. Examples of substances that do not occur naturally but are nevertheless suitable as an antigen under certain circumstances, such as synthetically derived substances that are present in drugs, or synthetic peptides containing amino acid sequences that do not occur naturally, or peptidomimetics etc. Naturally occurring or synthetic or recombinant antigens can be modified by molecular biology, enzymatic, chemical and/or other means to give them properties that are more beneficial for a particular application. These beneficial properties may in particular be greater or lesser activity as an antigen, broader or more specific action as an antigen, better solubility in hydrophilic or hydrophobic solvents, greater penetration of antigenic modules for cell membranes, for organelle membranes, for blood-brain barrier, etc., higher or lower half-life in vivo or in vitro, lower or higher toxicity, better ability to detect antigen in vivo or in vitro after applying the antigen as an iMAT molecule, etc. . In addition, for the purposes of the present invention, it is possible to combine multiple antigens in one antigen module. To this end, it is possible for identical antigens to be present in more than one copy in an antigenic module, or it is possible, for example, for different variants of the same antigen to be combined in an antigenic module.

A combination of antigens, eg antigen 1, and other antigens, eg antigen 2, in an antigen module is also possible, and so on. Additional combinations such as, for example, more than one copy of antigen 1 and a single copy of antigen 2 can also be combined in an antigen module, and so on. It is also additionally possible for one or more different and/or one or more identical antigenic modules to be present in an iMAT molecule. It is possible, in principle, for all possible combinations of one or many, that identical or altered copies of antigens are present, which are derived from one or more different antigens, to be combined for the purposes of the present invention. In a preferred embodiment, the antigenic module includes at least one full or partial epitope that is derived from at least one antigen, where such antigen is an allergen. At least one epitope may elicit an immune response against an allergen, whereby an epitope may comprise one or more structures, such as a peptide, capable of eliciting an immune response. The epitope may be a continuous epitope or a discontinuous epitope of the allergen. Epitopes may preferably be at least eight amino acids long, preferably at least ten amino acids long, more preferably at least 13 amino acids long. An antigenic module includes at least one complete or partial epitope, and may also contain two or more complete or partial epitopes, which may be the same or different from each other. In addition, the antigenic module may include additional amino acid sequences adjacent to at least one complete or partial epitope. An epitope may be a naturally occurring epitope or may be a modified epitope that is either modified in its amino acid sequence or using one or more post-translational modifications.

In one embodiment, at least one (third) antigenic module includes at least one complete or partial epitope that is derived from at least one pathogen antigen that is involved in one or more animal infectious disease(s). , more preferably ruminants, pigs, dogs and/or cats, but excluding horses. It may originate from the genera Campylobacter, Dirofilaria, Ehrlichia, Leishmania, Trypano

- 10 041116 soma, Borrelia, Orthobunyavirus, Orbivirus, Flavivirus, Rotavirus, Coronavirus, Trichophyton, Microsporum; other helminths such as Cooperia, Haemonchus, Ostertagia, Trichostrongylus, Dictyocaulus, Metastrongylus; and/or protozoa with gastrointestinal manifestations such as Eimeria, Isospora, Cryptosporidium, Giardia - in the case of parasites, antigens derived from parasitic incubation steps can also be used. At least one antigenic module in the iMAT molecules according to the present invention may also be an antigen (e.g. saliva component) of a vector involved in the transmission of one or more infectious disease(s), e.g. belonging to the families Culicidae, Ceratopogonidae , Phlebotominae, Ixodidae and/or Cimicidae.

In another preferred embodiment, at least one (third) antigenic module comprises at least one complete or partial epitope that is derived from at least one allergen that causes one or more allergies in animals, more preferably dogs and/or cats but excluding horses. It may represent at least one full or partial epitope of at least one allergen that is of food and/or mold origin (fungi and/or their spores), pollen, house dust or barn mites (and/or their faeces) preferably from pollen from trees, grasses, herbaceous plants, ragweed and/or pollen from cruciferous plants and/or fungi and/or their spores of the genera aspergillus, alternaria, botrytis, cercospora, cladosporium, curvularia, drechslera, eurotium, helminthosporium, epicoccum, erysiphe /oidium, fusarium, lichtheimia, nigrospora, penicillium, periconia, peronospora, polythrincium, saccharopolyspora (formerly also faenia or micropolyspora), thermoactinomyces, stemphylium, torula and/or mites (or their faeces) of the genera acarus, glycophagus, tyrophagus, dermatophagoides, euroglyphus , lepidoglyphus, blomia and/or fleas of the genera Ceratophyllus, Ctenocephalides, Pulex, Archaeopsylla. In a preferred embodiment, it is preferably at least one complete or partial epitope of at least one allergen which is of mite origin, more preferably of the genus dermatophagoides.

Examples of allergens of the genus dermatophagoides are shown in FIG. 9 (9A and 9B) identifying species, allergen and UNIPROT accession number.

In another preferred embodiment, such at least one allergen is Der f 15 according to SEQ ID NO: 11 (full) and SEQ ID NO: 18 (iMAT form). Preferred specific sequences of antigenic modules are amino acid sequences according to SEQ ID NOS: 7-23, preferably SEQ ID NOs: 14-23 (iMAT forms).

The term immune response that is modulated by or interchangeably immunomodulatory immune response in association with an (isolated) recombinant protein and/or iMAT molecule in the context of the present invention refers to immunogenic and/or tolerogenic immune responses.

The terms iMAT hybrid or iMAT hybrid or mosaic iMAT are used interchangeably. These terms refer to an iMAT molecule that includes, in its third module, more than one complete or partial epitope sequence from two or more antigens. Preferably, said antigens are two or more allergens, more preferably two or more short peptide sequences from different allergens, which determine the specificity of the immune response modulated by such an iMAT molecule (in a subject, preferably an animal).

The term allergen in the context of the present invention refers to a type of antigen that, in its native form, elicits an overly vigorous immune response in which the immune system fights off all identified threats that would otherwise be harmless to the subject. Typically, these types of reactions result in a phenotype known as allergies. Various types of allergens are described in the prior art, including foods, drugs, animal products, natural or synthetic materials. Preferably, a protein is considered an allergen when it elicits a specific IgE response in native form in at least five subjects, preferably animals, more preferably dogs and/or cats, but excluding horses. For the avoidance of doubt, an allergen in connection with at least one third antigenic module comprising at least one complete or partial epitope that is derived from at least one allergen no longer needs to be in its native form, which is also preferred, in other words, the term allergen in the context of the present invention also explicitly refers to non-native amino acid sequences within iMAT molecules, as described and claimed in this application, and which no longer elicit a specific IgE response in at least five subjects, preferably animals, more preferably dogs and/or cats, but excluding horses.

The term allergenicity of a therapeutic allergen is indicative of the potential for inducing side effects, such as provoking an anaphylactic reaction. For an example of allergy in mammals, an allergenicity test in dogs is described in Griffin et al. [Griffin CE. Diagnosis of canine atopic dermatitis DOI: 10.1002/9781118738818.ch10]. This document

- 11 041116 describes the measurement of allergen-specific IgE mediated hypersensitivity in procedures such as allergen provocation tests, in particular such tests aimed at the skin. Intradermal skin tests are used for the biological evaluation of recombinant allergens and for the validation of genetically engineered hypoallergenic derivatives. Intradermal testing in dogs is performed by injecting small amounts of allergen solutions directly into the dog's skin. This is done with small gauge needles (Gage 27 gauge needles) and 0.5-0.1 ml is injected into each site. Positive reactions are randomly interpreted by the presence of erythema, swelling, height and size of the allergic papule. The advantage of the intradermal test is its high sensitivity. It is extremely important if the test will deliver a quantitative indicator for allergenicity. When this test according to Griffin et al. is performed using iMAT molecules according to the present invention, these iMAT molecules exhibit a 10-, 100--1000-fold or even higher molar concentration of the allergenic component compared to the corresponding natural, native allergen. used in a similar test to achieve a positive reaction in sensitive individuals, such as cats and dogs.

The term epitope, which is also interchangeably referred to in this application as an antigenic determinant, in the context of the present invention refers to the portion of an antigen that is recognized by the immune system, either by B cells or T cells. Epitopes are displayed on the surface of antigen-presenting cells by the MHC molecules to which they are associated.

The term subject, which in this application is also interchangeably referred to as an individual and/or organism and/or host, in the context of the present invention preferably refers to animals and/or humans, for example, ruminants, pigs, more preferably dogs and/or cats, but excluding horses. The term ruminant, in the context of the present invention, embraces ruminant mammals, including cattle, goats and sheep. Therefore, members of the genera Bos, Capra, and/or Ovis are referred to interchangeably as bovine, goat, and/or sheep species. The term animal as used in this application includes mammals. The animal may be selected from the group consisting of ruminants or members of the genus Canis or interchangeably referred to as species of dogs (e.g. dogs, wolves, foxes, coyotes, jackals) or members of the genera Felis or interchangeably referred to as species of felines (e.g. lions, tigers). , domestic cats, feral cats, other big cats, and other felids, including cheetahs and lynx) or pigs, that is, members of the genera Sus are interchangeably designated as pig species.

The terms peptide and protein are used as equivalents in the context of the present invention. A peptide or protein for the purposes of the present invention means a covalent connection of at least two amino acids through a peptide bond. The term amino acid and the term amino acid residue are used as equivalents in this application, that is, the meaning of these two terms is the same. The terms amino acid/amino acid residue and peptide/protein are used in this application in the form of the broadest possible definition. In this regard, the term recombinant protein refers to a polypeptide that can be obtained through genetic engineering and expression in eukaryotic or prokaryotic systems. In addition, the specified term covers polypeptides obtained artificially (for example, using solid phase synthesis).

As used in this application, the term distinct amino acid residue refers to a known amino acid residue other than cysteine, unless otherwise indicated. For example, said amino acid residue may be a naturally occurring amino acid residue such as serine or isoleucine. As used in this application, the term linear shape refers to proteins in accordance with the present invention, which lack a secondary structure. Such proteins are often assumed to have a random conformation in which the only fixed relationship is the joining of adjacent amino acid residues via a peptide bond.

In a preferred embodiment, the (isolated) recombinant protein as disclosed or claimed in this application is present in monomeric form and/or linear form.

As used in this application, the term treatment refers to the administration of an (isolated) recombinant protein as disclosed and claimed in this application and/or appropriate vaccines and/or immunogenic compositions and/or pharmaceutical compositions in order to obtain the desired clinical results, including prophylactic and/or therapeutic treatment.

As used in this application, the term immunotherapy refers to the therapeutic and/or prophylactic treatment of a subject, for example, by prophylactic and/or therapeutic vaccination.

As used in this application, the term vector in connection with the transmission of one or more infectious disease(s) refers to a living organism and is used interchangeably in this application with the term biological vector, biological carrier and/or disease carrier, such as beetles. , flies, midges, mites and / or mosquitoes that feed on blood. In addition, it is

- 12 041116 possible and preferred that the (isolated) recombinant protein, as described and claimed in this invention, additionally contains at least one tag module. That is, it is possible and preferred that one or more different and/or identical tag modules are part of an (isolated) recombinant protein as described and claimed in this application. Tag modules can be short peptides that often include up to 20 amino acids or functional groups that do not consist of amino acids, such as, for example, biotin or digoxigenin. Acceptable tag modules include the well-known and preferred His tag containing a histidine sequence in an amount of 4 to 12 or more, preferably immediately adjacent histidine residues, preferably 5, 6 or 7 consecutive histidine residues. Other acceptable modules include a HA tag, a FLAG tag, a GST tag, or a Strep tag. While the label may be present anywhere on the (isolated) recombinant protein as described and claimed herein, in a preferred embodiment, the label module is present at the (quasi) N-terminal end and/or the C-terminal end of the (isolated) ) of the recombinant protein.

Label modules are useful for isolating (isolated) recombinant proteins as described and claimed herein, and additionally allow the presence of such (isolated) recombinant proteins to be detected in vitro or in vivo. In addition, a label module, optionally together with one or more adjacent amino acid residues from an adjacent module, or a linker that separates the various modules from each other, can be used to induce a specific immunologically detectable signal (e.g., an antibody) in a subject. , of interest, which can be used as a surrogate marker of immunity and/or duration of immunity.

Immunotherapy using (isolated) recombinant proteins as described and claimed herein elicits an antigen-specific, preferably allergen-specific, immune response in test subjects that is qualitatively indistinguishable from the natural immune response following exposure to naturally occurring antigens, preferably allergens . Thus, antibodies that bind to the antigenic module are unsuitable as a surrogate marker for the efficacy of the immune modulating effect induced by the iMAT molecule. This obstacle can be overcome by identifying a unique antigen-specific immunological signal produced by the C-terminal and/or (quasi) N-terminal tag module - optionally together with adjacent amino acid residues. Therefore, it is possible to provide suitable surrogate markers.

In a preferred embodiment, the (isolated) recombinant protein as disclosed and claimed herein further comprises at least one tag module, preferably at least one His tag, where such at least one tag module is preferably the N-terminal and/or the C-terminal end of the (isolated) recombinant protein, more preferably the N-terminal end after one methionine residue.

In addition, modules of an (isolated) recombinant protein as disclosed and claimed in this application, in particular at least one translocation module, at least one targeting module and at least one antigenic module, may optionally be separated by one or more spacer modules. located between at least two such modules.

The spacer modules may in particular be peptide sequences or organic molecules. Numerous spacer molecules that can be used for the purposes of this invention are known in the art. In addition, it is also possible to use spacer molecules for the purposes of this invention, which may be improved or disclosed in the future. Suitable spacer modules are, among others, peptide spacers, crosslinkers, natural or synthetic polymers such as, for example, nucleic acids, substituted or unsubstituted hydrocarbons, and the like.

The connection can be carried out both through covalent (preferably) and non-covalent bonds. The spacer modules have the task, in particular, of separating the various modules of the (isolated) recombinant protein as described and claimed in this application from each other in space in such a way that they do not adversely affect each other in terms of their functionality. The (isolated) recombinant protein modules for the purposes of the present invention may be connected by one or more spacer modules, which may be cleaved by chemical and/or enzymatic reactions, eg by proteases. Thus, it is possible to separate the modules of the (isolated) recombinant protein, as described and claimed in this invention, which are connected by spacer modules, from each other, as needed.

However, in a preferred embodiment, in particular when the antigenic module is an amino acid sequence that is derived from at least one complete or partial epitope of at least one antigen that is

- 13 041116 at least one allergen, no such additional spacer modules, that is, no modules between two or more adjacent modules of such first, second and/or third modules are present at all.

Any desired change in the placement of individual modules of the (isolated) recombinant protein, as disclosed and claimed in this application, is generally possible. Each module may be present one or more times in an (isolated) recombinant protein. The minimum requirement is the presence of at least one translocation module, at least one targeting module, and at least one antigenic module. Additional modules such as label modules, spacer modules, etc. may optionally be present, but this is not required. All modules may be present one or more times in an (isolated) recombinant protein as disclosed and claimed in this application. If modules are present more than once, then they may be present as identical copies or as different versions of the module in each case in a single instance or in more than one instance. It is also possible for completely different modules of the same class of modules, for example, a His tag module and a biotin tag module, to be present in an (isolated) recombinant protein as disclosed and claimed in this application. Both modules functionally perform the same task (label module) in an (isolated) recombinant protein, but should have nothing in common in terms of their molecular structure.

In a preferred embodiment, it is possible for two or more copies of one of the modules to be present in the (isolated) recombinant protein as disclosed and claimed in this application. That is, two or more copies of identical or different antigenic modules may be present. Alternatively, the (isolated) recombinant protein may contain two different antigenic modules to modulate an immune response in a subject.

Two or more identical copies of an antigenic module in a recombinant protein may, for example, elicit an improved immune response to that relevant antigen. Two or more different antigenic modules can, for example, be combined into one (isolated) recombinant protein in order to simultaneously modulate the immune response to two or more different antigens. Two or more different translocation modules can be used in an (isolated) recombinant protein as disclosed and claimed in this application. For example, the Tat sequence and the VP22 sequence can serve to make translocation more efficient, since the (isolated) recombinant protein is then efficiently translocated in a wider range of different cell types or tissue types. It is also possible, for example, to use two or more tag modules in one (isolated) recombinant protein, such as a His tag and a FLAG tag, accordingly, for example, a His tag is used for a recombinant protein, and, for example, a FLAG the label serves to identify the (isolated) recombinant protein. It is also possible to use two or more different modules in a single (isolated) recombinant protein, for example a sequence from the invariant strand of the MHC II molecule and an additional targeting module of the mannose 6-phosphate group. For example, the invariant strand acts as a targeting module to MIIC, and the mannose 6-phosphate moiety mediates targeting to the liposome. Thus, it is possible to increase the efficiency of presentation of an antigen or a number of different epitopes of an antigen presented by antigen presenting cells in general. In addition, the iMAT molecule of the present invention may span two or more different invariant strands that originate from the same or different species, thus allowing the proteins of the present invention to be used in different species.

The position of individual modules in (isolated) recombinant proteins, as disclosed and claimed in this application, can also be varied as desired, as long as at least one translocation module, at least one targeting module and at least one antigenic module are present. . It is also possible for all or some of the modules in an (isolated) recombinant protein, for example, to be present not as a linear sequencing of modules, but as a circular or branched module structure, either in the form of dendrimers, or as a combination of linear and/or branched and /or circular and/or dendritic part of the molecule. There are a number of commercial expression vector vendors who supply specific vectors that enable the production of circular fusion proteins using mechanisms such as, for example, the IMPACT™-TWIN system from New England Biolabs, Beverly, MA, USA. Branched modules can be obtained by synthesizing, for example, peptides in which, starting from poly-L-lysine, a new lysine residue is attached to each free amino group of each of the subsequent lysine residues. Thus, it is possible to create a peptide structure with almost any degree of branching. Subsequently, it is possible to synthesize, for example, translocation modules and/or targeting modules on a branched peptide backbone. Additional modules can also be connected on a linear, circular or branched main peptide structure by protein ligation. In addition, biotin groups can be introduced, for example, into the peptide of the main structure in

- 14 041116 during peptide synthesis, and modules can then be attached to these biotin groups using, for example, streptavidin, the Strep tag system, or using the PinPoint™ system (respectively, IBA GmbH, Gottingen, Germany and Promega Biosciences Inc., San Louis Obispo, CA, USA). Modules attached in this way are then connected via non-covalent bonds to the main structure of the peptide.

The antigenic module of iMAT molecules in accordance with the present invention can be selected using a bioinformatics approach, as described illustratively and detailed in examples 5 and 6 in this application. Thus, iMAT molecules may be useful specifically as vaccines, for example, for the treatment and/or prevention of allergic diseases based on the involvement of allergen-specific IgE-mediated hypersensitivity reactions in various target organs such as the skin, the respiratory tract - and also the gastrointestinal tract. tract, such as atopic dermatitis (AD), food allergies and/or allergic asthma in subjects, preferably animals, more preferably dogs and/or cats, but excluding horses.

In a preferred embodiment, the (isolated) recombinant protein refers to, contains, preferably consists of one or more amino acid sequences according to SEQ ID NOs: 24-83. The (isolated) recombinant proteins as described and claimed herein are particularly useful in a method that specifically relates to therapy and/or prophylaxis in subjects such as animals, more preferably dogs and/or cats, but excluding horses, suffering from allergic diseases based on the involvement of allergen-specific IgE mediated hypersensitivity reactions in various target organs, such as the skin, respiratory tract - and also the gastrointestinal tract.

In veterinary medicine, allergies are extremely important for pets.

Dogs and cats suffer from diseases such as canine atopic dermatitis or feline atopic dermatitis or feline asthma. Canine or feline atopic dermatitis (AD) is defined as a genetically predisposed inflammatory and pruritic allergic skin disease with characteristic clinical manifestations. It is most commonly associated with IgE antibodies to environmental allergens. An atopic phenotype can be observed in animals with an IgE-mediated skin disease, food allergy, or a condition called non-atopic dermatitis (ALD). ALD is defined as an pruritic skin disease in dogs with characteristic features of AD but negative IgE antibody tests. Feline atopic dermatitis is similar in many ways to canine atopic dermatitis. Common clinical signs for canine atopic dermatitis include a history of seasonal or non-seasonal pruritus, otitis externa, recurrent and chronic inflammatory dermatitis, especially in the axillary, groin, and flexor skin surfaces, recurrent bacterial infections, facial desquamation, and/or foot licking and chewing.

The etiology and pathogenesis of AD is complex and involves genetic predisposition, disruption of normal skin barrier function, and immunological abnormalities. Animals with AD are believed to be genetically predisposed to sensitization to environmental allergens. Allergens are proteins that, when inhaled or absorbed through the skin, respiratory tract, or gastrointestinal tract, cause allergen-specific IgE production. These allergen-specific IgE molecules attach themselves to tissue mast cells or basophils via Fcε receptors on these cells. When these primed cells come into contact with a specific allergen again, mast cell degranulation results in the release of proteolytic enzymes, histamine, bradykinins, and other vasoactive amines, resulting in inflammation (redness, swelling, and itching). The skin is the primary target organ in dogs and cats, but rhinitis and asthma can also occur in ~15% of affected animals. Ticks are known to be one of the main causes of allergic diseases such as atopic dermatitis and asthma, for example, in dogs and cats. It is generally accepted that desensitization therapy, which uses substances that cause allergic reactions as therapeutic agents for allergic diseases, is regarded as the most important basic drug. In particular, desensitization therapy is widely used for diseases such as hay fever, house dust allergy, and fungal allergies that are induced by antigens such as inhaled allergens that are difficult to avoid. However, for desensitizing therapy, there is a risk of side effects, especially anaphylactic reaction, due to exposure to sensitizing antigens, therefore, the introduction of safe therapeutic antigen-like molecules iMAT is required.

With regard to allergic diseases caused by mites, several species have been described as relevant: Dermatophagoides pteronyssinus, Dermatophagoides farinae, Euroglyphus maynei, Dermatophagoides siboney, Dermatophagoides microceras, Lepidoglyphus destructor, Blomia tropicalis, Tyrophagus putrescentiae, Glycophagus domesticus, Acarus siro. However, two types of mites, Dermatophagoides pteronyssinus and Dermatophagoides farinae, have been described as major sources of allergens in house dust (Thomas, WR. et al., Chang Gung Med J 2004; 27:563-569). The main tick allergens were

- 15 041116 fractionated from these mites.

Group 1 and 2 allergens of Dermatophagoides sp. induces high titers of IgE and Th2 cytokines in 80% of allergic patients. Allergens Der p 3, 5, 6, 7 and 8 induce IgE in approximately 50% of subjects, usually in low titers. Allergens 92/98 kDa paramyosin (Group 11) bind IgE in 80% of allergic subjects and chitinase enzymes 98 and 60 kDa (Der f 15 and 18) bind IgE from approximately 70 and 54% of allergic subjects and are important allergens for allergic dogs (McCall C et al., Vet Immunol Immunopath 2001; 78: 231-247).

In general, various environmental allergens such as grass pollen, tree pollen, weeds, house dust, dust and barn mites and mold and/or mold spores, as well as epidermal and insect antigens, have been described as sensitizing dogs to canine atopic dermatitis ( Hill et al. Vet Immunol Immunopathol, 2001;81(3-4):169-186).

As described above, the atopic phenotype can also be induced by food allergies, hypersensitivity with various clinical manifestations. In addition to gastrointestinal changes (eg, gastroenteritis, diarrhea, or vomiting), food hypersensitivity often manifests in animals as pruritic dermatitis and/or dermatosis of the face and neck, miliary dermatitis, generalized scaling, or symmetrical alopecia. Especially in cats, all forms of eosinophilic granuloma may result from hypersensitivity to certain food allergens.

The most common food allergens come from meat, milk, fish, and also from soy and/or, most generally, canned foods and dehydrated foods. In particular, the latter listed are also reported to be involved in allergic reactions to additives and/or barn mites (Guaguere E et al. EJCAP, 2009, 19(3), 234-241; Jackson HA, EJCAP, 2009, 19(3 ), 230-233).

Current therapeutic options in veterinary medicine are limited to symptomatic (eg, corticoids) treatment and/or exclusion of the underlying nutritional component(s). However, special attention should be paid to the absence of a negative impact on the rational combination of nutrients in the diet. Recently, nutrition containing hydrolysed proteins has been available. Proteins are broken down - therefore, are less allergenic, which is more effective and well tolerated. However, this food is expensive and unappetizing, which can be a major limitation for animal compliance.

Allergen-specific immunotherapy for the treatment of food allergies in animals, preferably dogs and/or cats, has not been used to date.

In a further embodiment, the (isolated) recombinant proteins as described and claimed herein are particularly useful in a method that particularly relates to therapy and/or prophylaxis in cats suffering from allergic inflammation and/or airway obstruction. (allergic asthma).

Cats spontaneously develop eosinophilic airway inflammation and airway hyperresponsiveness that is very similar to human allergic asthma, i.e., feline allergic asthma is a chronic inflammatory disorder of the lower airways that can present with acute, life-threatening clinical symptoms.

Typical treatment will include only palliative care (eg, bronchodilators and/or corticosteroids), but there is currently no treatment directed at the cause of the disease. Some pilot studies led by Carol Reinero have developed targeted allergen-specific immunotherapy in an animal model of bermuda grass-induced allergic asthma in cats. Feline allergic asthma is a complex disease, but exposure to airborne allergens undoubtedly plays a critical role in the etiology of the disease. Clinical remission can be achieved by eliminating exposure to aeroallergens. It is believed that the major antigens involved in triggering feline allergic asthma have not yet been identified. Various potential agents are present in a cat's environment, such as pollen, mold, litter dust, perfumes, room air fresheners, carpet deodorants, hairspray, aerosol cleaners, or cigarette smoke. When screened using serum or intradermal skin tests, naturally allergic house cats have reactive IgE to many allergens similar to those seen in allergic asthma in humans, i.e. mainly to house dust and barn mites and/or or pollen (Prost C, Rev Fr Allergol Immunol Clin, 2008, 48(5), 409-413).

In a further embodiment, the (isolated) recombinant proteins as described and claimed herein are particularly useful in a method that specifically relates to therapy and/or prophylaxis in cats and/or dogs suffering from flea bite AD (FAD). ). FAD is one of the most severe skin allergies caused by flea attacks in dogs and cats. FAD may present with both immediate hypersensitivity and

- 16 041116 delayed hypersensitivity reactions. Typically, an immediate type hypersensitivity reaction in an FAD sensitive animal involves the formation of an allergic papule at the site of a flea bite. Such allergic papules may develop into a scaly papule, which is characteristic of delayed-type hypersensitivity reactions. Hypersensitivity reactions to flea bites can occur in genetically predisposed animals, as well as in animals sensitized by prior exposure to block bites. In addition, flea bites can cause scratch-related secondary infections due to inflammatory irritation of the host's erythema, papules, scales, and alopecia. Previously published work has found that pets sometimes become desensitized to flea bites even though they were previously allergic to these flea bites. Thus, in addition to the destruction of fleas, alleviating the distress of animals becomes an extremely important problem. Current therapies for this disease include desensitization therapy or the use of some type of pharmacological intervention. However, each of these types of approaches has certain drawbacks. For example, antihistamine drugs can cause drowsiness, dry mouth, difficulty urinating, and constipation; while classical desensitizing therapy can cause life-threatening anaphylactic shock. Other disadvantages of these therapies are the high potential for recurrence and the need for long-term treatment. Thus, new therapeutic approaches are needed and should be developed to overcome unwanted side effects. Effective treatment of FAD is very difficult, if not impossible. FAD infects about 15% of cats and dogs in flea endemic areas. In a geographic area, all animals must be treated to effectively control fleas. One therapeutic approach proposed by the researchers involves animal desensitization using flea allergens. However, reliable, well-defined flea allergen preparations are needed for such treatment. Whole flea antigen preparations are used to diagnose and desensitize animals with FAD. Commercially available whole flea extracts, however, contain only a minor fraction of saliva proteins and thus are unpredictable in their content of specific allergens and therefore of limited utility. Prior art US Pat. No. 7,629,446 and McDermott MJ et al. (Molecular Immunology 2000, 37: 361-375) describe the discovery of the Cte f 1 allergen as a major allergen in FAD. In this publication, they described cDNA cloning and characterized the flea saliva protein, Cte f 1, the major allergen for flea allergy in dogs and cats. Native Cte f 1 has a calculated molecular weight of 18 kDa and a pI of 9.3. Mass spectrometric analysis indicates that there are no post-translational modifications in the native molecule and that all 16 cysteines are involved in intramolecular disulfide bonds. However, in the same publication, the authors described several isoforms of the recombinant protein. The 16 cysteines in the secreted protein results in these isoforms, which makes stonecrop, and therefore the preparation of such a product, extremely difficult or even impossible according to GMP.

In experimental dogs experimentally sensitized to flea bites, Cte f 1 is the main allergen. Using E. coli-produced rCte f 1 as the antigen in the intradermal skin test and in the solid phase ELISA, IgE can be detected in 100% of these FAD experimentally sensitized dogs. Additionally, a competitive ELISA performed using sera from 14 sensitized dogs demonstrated that rCte f 1 produced in three different expression systems (E. coli, P. pastoris, and baculovirus-infected insect cells) can inhibit approximately 95% of antigen-specific IgE binding to native Cte f 1.

The therapeutic potential of immunotherapeutic approaches containing Cte f 1 has been demonstrated by J. Jin et al. (Jin J. et al. Vaccine 28 (2010) 1997-2004). They described that simultaneous co-immunization with a DNA vaccine and its cognate encoded protein antigen (Cte f 1) showed the potential to protect animals from FAD in a mouse model. In addition, in their study, they clinically tested this protocol for the treatment of confirmed FAD in cats following a bed bug attack. They presented data indicating a therapeutic improvement in dermatitis in these FAD cats after two co-immunizations.

iMAT molecules containing a modified Cte f 1 sequence with substituted cysteines for other amino acid residues can be incorporated into the iMAT molecule as an allergenic module in conjunction with either a feline or canine invariant chain to achieve an optimized species-specific immuno-modulatory effect. Surprisingly, these molecules (I) allow efficient production of the recombinant protein in suitable expression systems, (II) have a significantly reduced safety risk because effector cells as well as mast cells are activated at significantly higher concentrations compared to unmodified Cte f 1 and (III ) induce a long-term immunological effect and long-term clinical improvement after only three injections.

In a further embodiment, the (isolated) recombinant proteins as described and claimed herein are particularly useful in a method that specifically relates to

- 17 041116 therapy and / or prophylaxis in animals, more preferably ruminants, pigs, dogs and / or cats, but excluding horses suffering from infectious diseases caused by bacteria.

The Gram-negative bacterium Campylobacter is the most common cause of gastroenteritis in pets such as dogs, cats, pigs, and ruminants. Clinical symptoms are generally more severe in young mammals. In addition to enteritis, miscarriages and infertility in various species have also been described. The infection enters the body by the oral route - for penetration, bacteria must overcome the host's defense mechanisms with the expression of various colonizing and virulent determinants. Many of these are antigenic surface or outer membrane proteins, and their interaction with, for example, epithelial cells of the gastrointestinal tract is essential for colonization, ie host infection. To solve this medical problem, at least one antigenic module in the Campylobacter specially bred (isolated) recombinant protein, as described and claimed in this application, can be selected from antigens that are derived from Campylobacter species, for example, flagellin, surface-lying proteins ( CadF and PEB1) or other surface proteins. The improved MAT molecules may be specifically suitable as vaccines, for example for the treatment and/or prevention of campylobacteriosis. Treatment according to the present invention may include administering the iMAT molecule to offspring and/or may also include treatment of the pregnant female.

In a further embodiment, the (isolated) recombinant proteins as described and claimed herein are particularly useful in a method that specifically relates to therapy and/or prophylaxis in animals, more preferably ruminants, pigs, dogs and/or cats, but excluding horses suffering from infectious diseases caused by viruses.

For example, West Nile virus (WNV) is a positive single-stranded RNA virus that is transmitted by mosquitoes and is grouped within the Flavivirus Japanese encephalitis virus serocomplex in the Flaviviridae family. WNV is the causative agent of the disease syndrome, also known as West Nile fever. Birds are a natural source and WNV is maintained in nature in a mosquito-bird-mosquito transmission cycle. However, humans, horses, dogs, cats, and ruminants have been described as susceptible. Although most WNV infections remain clinically latent - WNV is a potential neuroinvasive virus that can cause meningitis or encephalitis. In animals, WNV often goes unrecognized, but animals can be euthanized due to the severe neurological symptoms caused by WNV—including paresis, ataxia, rollover, and muscle fasciculation—while others present with mild to severe polyencephalomyelitis.

In a further embodiment, the (isolated) recombinant proteins as described and claimed herein are particularly useful in a method specifically aimed at preventing the transmission of infectious diseases in animals, more preferably ruminants, pigs, dogs and/or cats, but excluding horses, using vectors such as insects that feed on blood, flies, midges, ticks and/or mosquitoes.

Pathogens are delivered to the skin of the host, which is a mammal, along with arthropod saliva, which contains a wide range of biologically active molecules. These salivary components are capable of altering hemostasis and immune responses and may contribute to the pathogen's ability to cause infection. The presence of infectious microorganisms in the salivary glands of arthropods, which themselves feed on blood, alters the composition of saliva, for example, causing changes in the concentration of, for example, apyrase or antithrombinase in infected mosquitoes. Vector-associated or salivary components, for example, can alter vascular activity and/or modulate the host's immune response and are extremely important in the transmission of infectious diseases. Vaccination of the host against arthropod saliva components may interfere with transmission of the virus, as shown for mosquito saliva proteins and transmission of Leishmania species. However, to date, these vaccines have not passed preclinical trials (WHO PD-VAC 2014 - Vaccine Development and Research Statute for Vaccines for Leishmaniasis).

Arthropod saliva antigens are well suited for use in the antigen module of iMAT molecules according to the present invention and can be used specifically as vaccines to activate the host's immune system to prevent transmission of infectious pathogens by means of vectors, e.g. viruses, fungi and/or parasites and/ or their stages of parasitic incubation in animals, more preferably ruminants, pigs, dogs and/or cats, but excluding horses.

In a further embodiment, the (isolated) recombinant proteins as described and claimed herein are particularly useful in a method that specifically relates to therapy and/or prophylaxis in animals, more preferably ruminants, pigs, dogs and/or cats, but excluding horses suffering from infectious diseases caused by fungi.

Dermatomycosis or lichen is a fungal infection of the hair and superficial layers of keratinized skin cells that occurs in animals and humans. Several species of the genus Microsporum

- 18 041116 or the genus Trichophyton, belonging to the groups of zoophilic and geophilic dermatophytes, can cause clinical infections in mammals. A variety of fungal surface antigens and/or their spores are suitable for use in the antigenic module of the iMAT molecule according to the present invention and can be used specifically as vaccines, for example, for the treatment and/or prevention of dermatophytosis in animals, more preferably ruminants, pigs, dogs and/or cats, but excluding horses.

In a further embodiment, the (isolated) recombinant proteins as described and claimed herein are particularly useful in a method that specifically relates to therapy and/or prophylaxis in animals, more preferably ruminants, pigs, dogs and/or cats, but excluding horses suffering from infectious diseases caused by parasites.

Mammalian parasites are ubiquitous and clinically significant throughout the world. The main parasitic threats to ruminants, pigs, dogs and/or cats are, for example, Cooperia, Haemonchus, Ostertagia, Trichostrongylus, Dictyocaulus, Metastrongylus. Increasing levels of helminth resistance in parasites have been reported worldwide. For protozoa, for example, Cryptosporidium, several drugs consistently inhibit parasitic infestation and/or reproduction in the host. Neonates or young mammals are mainly affected and outcome depends on innate and adaptive immune responses.

Antigens derived from adult parasites as well as parasitic incubation steps are another example in the antigen module of iMAT molecules according to the present invention and may be useful specifically as vaccines, for example, to treat and/or prevent parasitic infection in animals, more preferably ruminants, pigs, dogs and/or cats, but excluding horses.

Treatment according to the present invention may include administering the iMAT molecule to offspring and/or may also include treatment of the pregnant female.

Particularly in the case of immune response modulation in dog, cat, bovine, ovine, goat or porcine species, at least one targeting module is preferably a corresponding invariant chain.

In a preferred embodiment, at least one targeting module is a dog invariant chain according to SEQ ID NO: 2 or 4. In another preferred embodiment, at least one targeting module is a cat invariant chain according to SEQ ID NO. : 3 or 5. In a preferred embodiment of the invention, the (isolated) recombinant protein as described and claimed in this application is present in monomeric form, since, for example, recombinant allergens tend to form aggregates, in particular if obtained using inclusion bodies . By replacing at least one, preferably all, cysteine residues in the entire sequence of the (isolated) recombinant protein, preferably by replacing with serine, leucine, isoleucine, arginine, methionine and/or aspartic acid, the formation of intermolecular disulfide bonds can be prevented, thus eliminating , any aggregation, in particular any unnatural formation of intermolecular and/or intramolecular bonds. That is, the (isolated) recombinant protein, being completely devoid of cysteine residues, does not undergo aggregation. Therefore, such a protein is easily expressed and exhibits improved targeting and MHC presentation.

In addition, such cysteine-free variants, such as allergens, in which cysteine residues in the amino acid sequence of wild-type allergens have been mutated individually or in combination, have reduced IgE reactivity compared to the corresponding wild-type allergens, while at the same time significantly retaining reactivity towards T-lymphocytes and thus are hypoallergenic.

Thus, the invention relates to such hypoallergenic variants of allergens, causing, for example, allergies to flea bites; to certain dietary components and/or atopic dermatitis and/or allergic inflammation and/or airway obstruction in animals, more preferably ruminants, pigs, dogs and/or cats, but excluding horses, where in the variants the cysteine residues of the allergens have been mutated individually or in combination.

In addition, the presence of a labeling module for separating proteins in a sample comprising iMAT molecules according to the present invention, for example when using a charged zinc or cobalt based solid support, further improves the possibility of obtaining fusion proteins without aggregation during the purification process. Typically, the label module includes a polyhistidine label of five to six consecutive histidine residues.

(Isolated) recombinant proteins as described and claimed in this application are useful in a pharmaceutical composition. For example, (isolated) recombinant proteins are intended for use in a vaccine. Thus, the present invention provides vaccine compositions containing one or more (isolated) recombinant proteins as described and claimed in this application. Such a vaccine composition may be used therapeutically and/or prophylactically.

- 19 041116 in animals, more preferably dogs and/or cats, but excluding horses, suffering from allergic diseases, based on the involvement of allergen-specific IgE-mediated hypersensitivity reactions in various target organs, such as the skin, respiratory and gastrointestinal systems; or such vaccine composition can be used therapeutically and/or prophylactically in animals, more preferably ruminants, pigs, dogs and/or cats, but excluding horses suffering from infectious diseases induced by pathogens, for example, viruses, fungi and/or parasites and/or their stages of parasitic incubation. Additionally, the method can be used not only against such pathogens, but can also activate the host's immune system to prevent transmission of the disease by vectors such as blood-feeding insects, flies, midges, mites and/or mosquitoes.

Thus, in a preferred embodiment of the present invention, a vaccine is provided for subjects such as animals, more preferably dogs and/or cats, but excluding horses, for the treatment and/or prevention of atopic dermatitis and/or allergic asthma in response to, for example, on exposure to dermatophagoides mites. In a further embodiment of the present invention, a vaccine is provided for subjects such as animals, more preferably cats, but excluding horses, for the treatment and/or prevention of allergic asthma that is caused by a response to, for example, molds (fungi and/or their spores), pollen, house dust or barn mites (and/or their faeces).

In a further embodiment of the present invention, a vaccine is provided for subjects such as animals, more preferably ruminants, pigs, dogs and/or cats, but excluding horses, for the treatment and/or prevention of infectious diseases that involve, for example, the genera Campylobacter, Dirofilaria , Ehrlichia, Leishmania, Trypanosoma, Borrelia, Orthobunyavirus, Orbivirus, Flavivirus, Rotavirus, Coronavirus, Trichophyton, Microsporum; other helminths such as Cooperia, Haemonchus, Ostertagia, Trichostrongylus, Dictyocaulus, Metastrongylus; and/or protozoa with gastrointestinal manifestations such as Eimeria, Isospora, Cryptosporidium, Giardia - in the case of parasites, antigens can also be used which are derived from parasitic incubation steps. Additionally, a vaccine may be provided to activate the host's immune system to prevent disease transmission by vectors such as those belonging to the families Culicidae, Ceratopogonidae, Phlebotominae Ixodidae and/or Cimicidae and/or other insects that feed on blood.

The pharmaceutical composition, for example in the form of a vaccine, based on (isolated) recombinant proteins is preferably intended for sublingual administration, subcutaneous and/or intradermal injection, injection into the lymph nodes and/or for administration through the mucous membranes, in particular the mucous membranes of the gastrointestinal tract or respiratory system.

In a preferred embodiment of the present invention, the pharmaceutical compositions are administered parenterally.

The iMAT molecules according to the present invention can be used as a pharmaceutical preparation or as a vaccine to modify, for example, allergic disorders. For example, atopic dermatitis and/or allergic asthma can be treated with such iMAT molecules.

Low amounts (from 1 to 1000 µg, based solely on the weight of one or more antigenic modules) of recombinant iMAT molecules containing atopic dermatitis and/or allergic asthma allergens derived from mites of the genus dermatophagoides, for example, which are administered 1-5 times subcutaneously, intradermally or directly to the lymph node, induce a strong and prolonged immune response in cats and/or dogs, leading to the prevention of disease and/or the reduction of clinical symptoms.

In one preferred embodiment, the iMAT molecules of the present invention are administered in combination with at least one adjuvant. The adjuvant includes, but is not limited to, one or more of the following: alum, BCG, aluminum hydroxide, aluminum phosphate, calcium phosphate, lipid emulsions, lipid or polymer nano- or microspheres, micelles, liposomes, saponin, lipid A, or dipeptide, bacterial products, chemokines, cytokines and hormones, chitosan, starch, alginate, cellulose derivatives (eg ethylcellulose, hydroxypropyl methylcellulose), nucleic acids or nucleic acid constructs. One or more of these components may be added to enhance or modify the immune response. Alternatively, the iMAT molecule can be administered without adjuvant or in aqueous form.

The iMAT molecules can be administered at a dose of from about 1 to 1000 μg (this and subsequent doses refer solely to the weight of one or more antigenic modules) and more preferably at a dose of from about 10 to about 100 μg and even more preferably at a dose of from about 20 to about 50 μg, although the optimal dose may vary depending on the antigen administered, preferably the allergen, the subject's weight, the subject's immune system, and so on. Effective treatment in many cases can be achieved using a single injection. In some embodiments, the implementation of the processing includes from 1 to 15 introductions. In preferred

- 20 041116 embodiments, the treatment includes from 1 to 5 injections, and more preferably from 1 to injections. For the initial stages of treatment, administration may be intermittent, for example over a course of several days, once or twice a month or year, or several times a year. In order to maintain the immune response, administration can be carried out at intervals of several months to several years.

In a preferred embodiment of the present invention, the (isolated) recombinant proteins as disclosed and claimed herein are for administration to the lymph nodes. In the process of direct injection into a lymph node, the corresponding lymph node can be visualized during the injection procedure, for example, using ultrasound, in order to monitor the location of the needle and changes in the lymph nodes, such as swelling. Injections into the mandibular, axillary, inguinal, and/or popliteal lymph nodes are preferred due to the ease of ultrasound locating and injection.

It is known in the art that certain identified proteins from ticks in faeces and from the bodies of whole ticks induce IgE reactivity and abnormal skin and respiratory reactions in dogs and/or cats [Allergome (www.allergome.org)]. Thus, it is believed that treatment can only be successful if most of the relevant allergens are included in the specific immunotherapy drug. However, surprisingly only 1, 2, 3 or 4 of these iMAT molecules according to the present invention, each containing different antigenic modules, e.g. ticks or epitope mosaics, is sufficient to induce immunomodulation and/or clinical improvement in diseased subjects. if such iMAT molecules are injected 1-5 times subcutaneously, intradermally and/or directly into the lymph node.

In a preferred embodiment, a single iMAT molecule is used and is sufficient to induce a therapeutic effect and/or prevent the development of allergen-specific IgE mediated hypersensitivity reactions in various target organs such as the skin, respiratory tract and/or gastrointestinal system, for example, in animals, more preferably dogs and/or cats, but excluding horses. Prevention and/or treatment of infectious diseases and/or prevention of transmission of infectious diseases using vectors can be carried out in animals, preferably ruminants, pigs, dogs and/or cats, but excluding horses.

In another preferred embodiment, a combination of 2, 3 or 4 iMAT molecules is used with simultaneous, sequential and/or chronologically offset co-administrations.

In a preferred embodiment, a single iMAT molecule is used and is sufficient to induce a therapeutic effect and/or prevent and/or prevent the transmission of infectious diseases in animals, more preferably ruminants, pigs, dogs and/or cats, but excluding horses, e.g. fungi and/or parasites and/or their stages of parasitic incubation and/or transmission of such infectious diseases, for example by blood-feeding insects, flies, midges, mites and/or mosquitoes.

In another preferred embodiment, a combination of 2, 3 or 4 iMAT molecules is used with simultaneous, sequential and/or chronologically offset co-administrations.

Depending on the thermodynamic evaluation of the (isolated) recombinant protein, as disclosed or claimed in this application, its stability depends on the mutation of cysteine, in particular, the replacement of different(s) amino acid(s) residue(s). For example, it may be the replacement of Cys with Ser. However, stability can be improved by using an amino acid residue other than Ser in order to achieve the desired stability and solubility. That is, while the first choice may be to replace cysteine with Ser, in case of instability, an amino acid residue other than Ser should replace Cys.

In order to select amino acid residues for stabilization as replacements for cysteine residues in target sequences, a three-step approach is chosen:

1. Modeling the tertiary structure of the target protein, including terminal hexahistidine tags, the iMAT sequence, and the primary amino acid sequence of the protein of interest. Modeling can be done using a native sequence and include cysteine substitutions.

2. Iterative determination of protein stability based on point substitutions, such as replacing a cysteine residue with a different amino acid residue, eg Ser and/or Ile, and categorization to determine stabilizing substitutions by analyzing all available 3D structures.

3. Re-simulate the stabilized structure and confirm the stability by repeating steps 1 and 2.

Three-dimensional protein structures are critical to understanding protein function at the molecular level and are of great interest for the rational design of many different

- 21 041116 types of biological experiments, such as site-directed mutagenesis. However, the number of structurally characterized proteins is small compared to the number of known protein sequences. It is possible to identify a homologous protein with a known structure (template) for a given protein sequence (target sequence). In these cases, homology modeling has proven to be the method of choice for generating a reliable 3D model of a protein from its primary amino acid sequence. The homology building model includes four main steps: (1) identification of the structural matrix(es), (2) alignment of the target sequence and structure(s) of the matrices (matrices), (3) model building, and (4) qualitative evaluation of the model. These steps can be repeated until a satisfactory simulation result is achieved. In cases where an exact homology model cannot be determined, computational approaches are used to determine the protein structure from the primary structure (amino acid sequences). De novo or ab initio methods are based on physical principles and attempt to mimic the folding process. Such methods must explore a large number of conformations and require very precise energy functions to identify structures at the global free energy minimum. Many methods use a combination of these described principles. The availability of computational tools provides fairly accurate estimates of the effect of amino acid substitutions on protein stability and is critical in a wide range of applications. In particular, such tools have the potential to stimulate and support protein engineering and design projects dedicated to the creation of modified proteins. Protein stability can be viewed in terms of the thermodynamic stability of a protein, which rapidly and reversibly unfolds and folds the protein. In these cases, protein stability is the difference in Gibbs free energy between the folded and unfolded states. The only factors affecting stability are the relative free energies of the folded and unfolded states. The larger and more positive the difference in folding free energy, the more stable the protein is for denaturation. The difference in free energy of folding is usually small, on the order of 5-15 kcal/mol for a globular protein. However, on the other hand, protein stability can be thought of as the property of a protein to withstand high temperatures or dissolution conditions. This is due to thermodynamic stability, as well as the reversibility or irreversibility of folding / unfolding (kinetic stability).

To predict thermodynamic changes in stability caused by point substitutions in proteins, several different approaches can be applied to study the effect of such substitutions on protein structure and function [Pires DE et al., Bioinformatics 2014, 30(3): 335-342]. Such approaches can be broadly classified into those that seek to understand the effect of substitutions in the amino acid sequence of the protein itself, and those that use extensive structural information. Structure-based approaches typically attempt to predict either the direction of change in protein stability during substitutions or the actual amount of free energy (AAG).

The results for each particular protein and its respective models are statistically analyzed in terms of the number of occurrences of specific substitutions using a scoring system that eliminates substitution based on occurrences in the models used and changes in protein stability free energy (AAG), thereby identifying the most destabilizing residues (if any) in the structure into which they are introduced, and possible substitutions. The score is calculated by determining the lowest AAG (AAG < 0) at each position of interest in each model and assigning the appropriate linear and cumulative AAG values for each potential replacement position, and the results are then evaluated to determine inter-model consistency. Since the estimated protein models have different properties (probability of predicting the correct 3D structure), a weighting factor can be applied to prioritize results from more accurate models. The residue that has a significant (AAG based) destabilizing effect (if any) is replaced; get new models and resubmit them to iterative analysis until a steady state is reached. In addition, the generated models are evaluated by analyzing xyz coordinates using principal component analysis to identify potential structural misfoldings due to amino acid substitutions at specific positions.

In yet another additional aspect, the problem underlying the invention has been unexpectedly solved by providing a method for identifying amino acid substitutions in a predetermined amino acid sequence, allowing stabilization of such a predetermined amino acid sequence, which includes the following steps:

(I) modeling the 3D/tertiary structure of the target predetermined amino acid sequence;

(II) iterative determination of protein stability based on point substitutions, such as replacing cysteine residues with different amino acid residues, preferably serine and/or iso-

- 22 041116 leucine, and categorization based on changes in the free energy of protein stability (AAG) to determine stabilizing substitutions by analyzing all available three-dimensional/tertiary structures, (III) re-modeling the substituted three-dimensional/tertiary structure of the amino acid sequence and calculating its stability by repetition (II ) and (III) until a steady state is reached;

In yet another additional aspect, the problem underlying the invention has been unexpectedly solved by providing an (isolated) recombinant protein as disclosed or claimed in this application, where the full or partial amino acid sequence is stabilized by a method for identifying amino acid substitutions in a predetermined amino acid sequence. , as disclosed or claimed in this application (see example 5 and also example 6 in this application).

Sequences

The following protein sequences are described in detail and thus disclosed in the present invention (ak or AK is an abbreviation for amino acids):

SEQ ID NO: 1 refers to a suitable minimum amino acid sequence for one translocation module in accordance with the present invention, which is still functional, ie able to effectively promote cell entry -> TAT sequence;

SEQ ID NOs: 2-5 Invariant chains (full sequences and POacs):

SEQ ID NO: 2 refers to the complete amino acid sequence of the invariant dog chain;

>gi|545496086|Canis_lupus_familiaris|XP_536468.5|

PREDICTION: HLA class II histocompatibility gamma chain antiten isoform XI [Canis lupus familiaris]

SEQ ID NO: 3 refers to the complete amino acid sequence of the cat invariant chain;

>gi|41094965 l|Felis_catus|XP_003981534.11 PREDICTION: HLA class II histocompatibility gamma chain antiten isoform XI [Felis catus]

SEQ ID NO: 4 refers to the first 110 aa amino acid sequence of the dog invariant chain;

SEQ ID NO: 5 refers to the first 110 aa amino acid sequence of the cat invariant chain;

SEQ ID NO: 6 is for a 22 aa N-terminal marker

SEQ ID NOs: 7-13 Allergens, full sequences: SEQ ID NO: 7 refers to the complete amino acid sequence of Der f 1 allergen; >Q58A71 Der f 1 pre-enzyme allergen Dermatophagoides farinae (American house dust mite) SEQ ID NO: 8 refers to the complete amino acid sequence of Der f 2 allergen; >Q00855 Der f 2 allergen group 2 mites (Der f II allergen) (Der f 2 allergen) Dermatophagoides farinae (American house dust mite) SEQ ID NO: 9 refers to the complete amino acid sequence of Der f 23 allergen; >A0A088SAW7 Der f 23 allergen Dermatophagoides farinae (American house dust mite)

- 23 041116

SEQ ID NO: 10 refers to the complete amino acid sequence

Der f 18p allergen; >Q86R84 60 kDa allergen

Der f 18p Dermatophagoides farinae (American house dust mite)

SEQIDNO:11 refers to the complete amino acid sequence of Der f 15 allergen;

>Q9U6R7 98 kDa HDM allergen (Der f 15 allergen) (Group 15 Der f 15 allergen) Dermatophagoides farinae (American house dust mite)

SEQ ID NO: 12 refers to the complete amino acid sequence of the allergen Zen 1 protein;

>I7HDR2 Zen 1 protein Dermatophagoides farinae (American house dust mite)

SEQ ID NO: 13 refers to the complete amino acid sequence of Cte f 1 allergen;

>Q94424 Saliva antigen 1 (FS-I) (Cte f 1 allergen) Ctenocephalides felis (Cat flea)

SEQ ID NOs: 14-20 Allergens, IMAT Forms (short):

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 refers to iMAT Form (short) sequence Der f 1 allergen; refers to iMAT Form (short) sequence Der f 2 allergen; refers to iMAT Form (short) sequence Der f 23 allergen; refers to iMAT Form (short) sequence Der f 18p allergen; refers to iMAT Form (short) sequence Der f 15 allergen; refers to iMAT Form (short) sequence Zen 1 allergen protein; refers to iMAT Form (short) sequence Cte f 1 allergen a;

amino acid amino acid amino acid amino acid amino acid amino acid amino acid

- 24 041116

SEQ ID NOs: 21-23 Allergens from Hybrids 1, 2, and 3 - IMAT Forms (short):

SEQ ID NO: 21 refers to iMAT Form (short) Hybrid 1 allergen

SEQ ID NO: 22 is for iMAT Form (short) Hybrid 2 allergen

SEQ ID NO: 23 is for iMAT Form (short) Hybrid 3 allergen

SEQ ID NOs: 24-44 iMAT for cats (with N-terminal or C- SEQID NO: SEQID NO: SEQID NO: 24 25 26 terminal Hexa-Histidine or without Hexa- Histidine / Methionine (1MAT_Pure)): With With N- WITH- refers to Der f 1 iMAT molecule (cats) terminal Hex-Histidine label; refers to Der f 1 iMAT molecule (cats) terminal Hex-Histidine label; refers to Der f 1 iMAT (cat) molecule without label; SEQID NO: 27 refers to the Der f 2 iMAT (cat) molecule with a terminal Hexa-Histidine tag; With N- SEQID NO: SEQID NO: 28 29 refers to the Der f 2 iMAT (cat) molecule with a terminal Hexa-Histidine tag; refers to Der f 2 iMAT (cat) molecule without label; With WITH- SEQID NO: thirty refers to the Der f 23 iMAT (cat) molecule with a terminal Hexa-Histidine tag; With N- SEQID NO: SEQID NO: 31 32 refers to the Der f 23 iMAT (cat) molecule with a terminal Hexa-Histidine tag; refers to Der f 23 iMAT (cat) molecule without label; WITH- SEQID NO: 33 refers to the Der f 18p iMAT (cat) molecule with a terminal Hexa-Histidine tag; With N- SEQ ID NO: 34 SEQ ID NO: 35 refers to the Der f 18p iMAT (cat) molecule with a terminal Hexa-Histidine tag; refers to Der f 18p iMAT (cat) molecule without label; WITH-

- 25 041116

SEQ ID NO: 36 refers to the Der f 15 iMAT molecule (cat) with an N-terminal Hexa-Histidine tag; SEQ ID NO: 37 refers to the Der f 15 iMAT molecule (cat) with a Terminal Hexa-Histidine tag; SEQ ID NO: 38 refers to Der f 15 iMAT (cat) molecule without label; SEQ ID NO: 39 refers to the iMAT Zen 1 protein (cat) molecule with an N-terminal Hexa-Histidine tag; SEQ ID NO: 40 refers to the iMAT Zen 1 protein molecule (cat) with the Terminal Hexa-Histidine tag; SEQ ID NO: 41 refers to the unlabeled iMAT Zen 1 protein (cat) molecule; SEQ ID NO: 42 refers to the iMAT Cte f 1 protein (cat) molecule with an N-terminal Hexa-Histidine tag; SEQ ID NO: 43 refers to the iMAT Cte f 1 protein molecule (cat) with a Terminal Hexa-Histidine tag; SEQ ID NO: 44 refers to the unlabeled iMAT Cte f 1 protein (cat) molecule;

SEQ ID NOs: 45-65 iMAT for dogs (N-terminal or C-terminal)

Hex-Histidine or without Hex-Histidine/Methionine (IMAT Pure)):

SEQ ID NO: 45 refers to a Der f 1 iMAT (dog) molecule with an N-terminal Hexa-Histidine tag;

SEQ ID NO: 46 refers to Der f 1 iMAT (dog) molecule with a Terminal Hex-Histidine tag;

SEQ ID NO: 47 refers to Der f 1 iMAT (dog) molecule without label;

SEQ ID NO: 48 refers to the Der f 2 iMAT (dog) molecule with an N-terminal Hexa-Histidine tag;

SEQ ID NO: 49 refers to a Der f 2 iMAT (dog) molecule with a Terminal Hex-Histidine tag;

SEQ ID NO: 50 refers to the Der f 2 iMAT (dog) molecule without label;

- 26 041116

SEQ ID NO: 51 refers to the Der f 23 iMAT (dog) molecule with an N-terminal Hexa-Histidine tag;

SEQ ID NO: 52 refers to the Der f 23 iMAT molecule (dog) with a C-terminal Hexa-Histidine tag;

SEQ ID NO: 53 refers to Der f 23 iMAT (dog) molecule without label;

SEQ ID NO: 54 refers to a Der f 18p iMAT (dog) molecule with an N-terminal Hexa-Histidine tag;

SEQ ID NO: 55 refers to the Der f 18p iMAT (dog) molecule with a C-terminal Hex-Histidine tag;

SEQ ID NO: 56 refers to the unlabeled Der f 18p iMAT (dog) molecule;

SEQ ID NO: 57

SEQ ID NO: 58

SEQ ID NO: 59 refers to the Der f 15 iMAT (dog) molecule with an N-terminal Hexa-Histidine tag;

refers to the Der f 15 iMAT (dog) molecule with a Terminal Hexa-Histidine tag;

refers to Der f 15 iMAT (dog) molecule without label;

SEQ ID NO: 60 refers to the iMAT Zen 1 protein (dog) molecule with an N-terminal Hex-Histidine tag;

SEQ ID NO: 61 refers to the iMAT Zen 1 protein (dog) molecule with a Terminal Hex-Histidine tag;

SEQ ID NO: 62 refers to the unlabeled iMAT Zen 1 protein (dog) molecule;

SEQ ID NO: 63 refers to the iMAT Cte f 1 protein (dog) molecule with an N-terminal Hex-Histidine tag;

SEQ ID NO: 64 refers to the Cte f 1 protein (dog) iMAT molecule with a Terminal Hex-Histidine tag;

SEQ ID NO: 65 refers to the unlabeled iMAT Cte f 1 protein (dog) molecule;

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SEQ ID NOs: 66-74 hybrid/mosaic cat iMATs (N-terminal or C-terminal Hexa-Histidine or no Hexa-Histidine/Methionine (1MAT_Clear)):

SEQ ID NO: 66 refers to the iMAT molecule of Hybrid 1 (cat) with an N-terminal Hexa-Histidine tag;

SEQ ID NO: 67 refers to the iMAT molecule of Hybrid 1 (cat) with a Terminal Hexa-Histidine tag;

SEQ ID NO: 68 refers to an unlabeled iMAT molecule of Hybrid 1 (cat) (consisting of SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 21);

SEQ ID NO: 69 refers to an iMAT molecule of Hybrid 2 (cat) with an N-terminal Hexa-Histidine tag;

SEQ ID NO: 70 refers to the iMAT molecule of Hybrid 2 (cat) with a Terminal Hexa-Histidine tag;

SEQ ID NO: 71 refers to an unlabeled Hybrid 2 (feline) iMAT molecule (consisting of SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 22);

SEQ ID NO: 72 refers to an iMAT molecule of Hybrid 3 (cat) with an N-terminal Hexa-Histidine tag;

SEQ ID NO: 73 refers to the iMAT molecule of Hybrid 3 (cat) with a Terminal Hexa-Histidine tag;

SEQ ID NO: 74 refers to an unlabeled iMAT molecule of Hybrid 3 (cat) (consisting of SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 23);

SEQ ID NOs: 75-83 hybrid/mosaic dog iMATs (N-terminal or C-terminal Hexa-Histidine or no Hexa-Histidine/Methionine (1MAT_Clear)):

SEQ ID NO: 75 refers to the iMAT molecule of Hybrid 1 (dog) with an N-terminal Hexa-Histidine tag;

SEQ ID NO: 76 refers to the iMAT molecule of Hybrid 1 (dog) with a Terminal Hex-Histidine tag;

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SEQ ID NO: 77 refers to an unlabeled Hybrid 1 (dog) iMAT molecule (consisting of SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 21); SEQ ID NO: 78 refers to a Hybrid 2 (dog) iMAT molecule with an N-terminal Hexa-Histidine tag; SEQ ID NO: 79 refers to the iMAT molecule of Hybrid 2 (dog) with a Terminal Hexa-Histidine tag; SEQ ID NO: 80 refers to an unlabeled Hybrid 2 (dog) iMAT molecule (consisting of SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 22); SEQ ID NO: 81 refers to the iMAT molecule of Hybrid 3 (dog) with an N-terminal Hexa-Histidine tag; SEQ ID NO: 82 refers to the iMAT molecule of Hybrid 3 (dog) with a Terminal Hexa-Histidine tag; SEQ ID NO: 83 refers to an unlabeled Hybrid 3 (dog) iMAT molecule (consisting of SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 23);

SEQ ID NOs: 84-88 Non Excessive Hybrid Allergens:

SEQ ID NO: 84 refers to A1KXC1DERFA DFP1 (UNIPROT database); SEQ ID NO: 85 refers to A0A088SAS1DERFA Der f 28 allergen (UNIPROT database); SEQ ID NO: 86 refers to B7U5T1DERFA Der f 6 allergen (UNIPROT database); SEQ ID NO: 87 refers to T2B4F3DERPT LytFM (UNIPROT database); SEQ ID NO: 88 refers to A7XXV2 DERFA Der f 2 allergen (UNIPROT database);

SEQ ID NOs: 89-90 Various sequences:

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SEQ ID NO: 89 refers to the Cui o2 iMAT molecule with N-terminal HEXA-HISTIDINE;

SEQ ID NO: 90 refers to the Cui o3 iMAT molecule with N-terminal HEXA-HISTIDINE.

SEQ ID NOs: 91-102 Peptide components of Hybrids: SEQ ID NO: 91 refers to the peptide portion of the hybrid, which has origin SEQ ID NO: 10, SEQ ID NO: 92 refers to the peptide portion of the hybrid, which has origin SEQ ID NO: 85, SEQ ID NO: 93 refers to the peptide portion of the hybrid, which has origin SEQ ID NO: 88, SEQ ID NO: 94 refers to the peptide portion of the hybrid, which has origin SEQ ID NO: 86, SEQ ID NO: 95 refers to the peptide portion of the hybrid, which has origin SEQ ID NO: 87, SEQ ID NO: 96 refers to the peptide portion of the hybrid, which has origin SEQ ID NO: AND, SEQ ID NO: 97 refers to the peptide portion of the hybrid, which has origin SEQ ID NO: 7, SEQ ID NO: 98 refers to the peptide portion of the hybrid, which has origin SEQ ID NO: 10, SEQ ID NO: 99 refers to the peptide portion of the hybrid, which has origin SEQ ID NO: 12, SEQ ID NO: 100 refers to the peptide portion of the hybrid, which has origin SEQ ID NO: 9, SEQ ID NO: 101 refers to the peptide portion of the hybrid, which has origin SEQ ID NO: 11, SEQ ID NO: 102 refers to the peptide portion of the hybrid, which has

origin SEQ ID NO: 8.

Hybrid/mosaic iMAT molecules:

In a specific aspect of the present invention, the iMAT molecules are further improved if the components (amino acid sequences/epitopes) of more than one allergen are included in the antigenic module. For this purpose, the basic principle of the bioinformatic selection approach described above (Example 5) can be applied in another way. Instead of selecting complete allergens based on allergen peptide hit counts found in the allergen database, only the most frequently occurring peptides of several of these allergens are used to construct the iMAT antigen module (see, for example, example 6). Thus, such an iMAT molecule consists of an antigenic module of peptides that are derived from several allergens. This makes it possible to broaden the spectrum of a single iMAT molecule with respect to its target immunological profile, and thus it is useful for pharmaceutical drug development.

As a further step in constructing the hybrid iMAT molecule, TAT and a targeting domain, and optionally a His tag, are added. Finally, cysteine residues are replaced with more stabilizing residues as described in Examples 5 and 6.

Hybrid I.

The protein precursor is selected from a list of protein precursors according to the top ranking peptides and is used as a scaffold protein (SEQ ID NO: 84) to insert other top ranking peptides from other antigens (SEQ ID NOS: 10 , 11, 85, 86, 87, 88). The signal peptide sequence is removed from the scaffold protein. Optionally, additional adjacent N- or C-terminal amino acids are inserted within the original scaffold protein sequence and replace portions of the original scaffold protein sequence as described below.

Components of the following proteins are used to construct fusion 1:

SEQ ID NO: 84 (A1KXC1DERFA DFP1 OS=Dermatophagoides farina)

SEQ ID NO: 10 (Q86R84DERFA 60 kDa allergen Der f 18p

OS=Dermatophagoides farinae GN=Der f 18 PE=2 SV=1)

SEQ ID NO: 85 (A0A088SAS1DERFA Der f 28 allergen OS=Dermatophagoides farinae PE=2 SV=1)

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SEQ ID NO: 88: (A7XXV2_DERFA Der f 2 allergen OS=Dermatophagoides farinae

PE=4 SV=1)

SEQ ID NO: 86 (B7U5T1DERFA Der f 6 allergen OS=Dermatophagoides farinae

PE=2 SV=1)

SEQ ID NO: 87 (T2B4F3DERPT LytFM OS=Dermatophagoides pteronyssinus

GN=lytFM PE=4 SV=1)

SEQ ID NO: 11 (Q9U6R7DERFA 98 kDa HDM allergen OS=Dermatophagoides farinae PE=2 SV=1)

Hybrid Skeleton 1:

SEQ ID NO: 84 (A1KXC1 18-400, no replacement parts below)

[Replacement 1 for: SEQ ID NO: 84 AA 39-52]:

SEQ ID NO: 10 (Q86R84AA 97-110) i.e. GNAKAMIAVGGSTM (SEQ ID NO:

91)

[Replacement 2 for: SEQ ID NO: 84 AA 261-274]:

SEQ ID NO: 85 (A0A088SASl_AA 611-624) i.e. MMKIYQQQQQHHP (SEQ

ID NO: 92)

[Replacement 3 for: SEQ ID NO: 84 AA 234-246]:

SEQ ID NO: 88 (A7XXV2 AA 48-61) i.e. FLVYIHIANNEIKK (SEQ ID NO: 93)

[Replacement 4 for: SEQ ID NO: 84 AA 53-65]:

SEQ ID NO: 86 (B7U5T1AA 166-178) i.e. IVDGDKVTIYGWG (SEQ ID NO:

94)

[Replacement 5 for: SEQ ID NO: 84 AA 276-289]:

SEQ ID 87: (T2B4F3AA 134-147) i.e. REENIWSDHIANVA (SEQ ID NO: 95)

[Replacement 6 for: SEQ ID NO: 84 AA 203-216]:

SEQ ID NO: 11 (Q9U6R7AA 469-482) i.e. TPTTPTPAPTTSTP (SEQ ID NO: 96)

After the cysteine residues have been replaced with the most stabilizing residues, the biotechnological method described in Examples 5 and 6 is used, which results in SEQ ID NO: 21.

The skeleton of hybrid 1 is formed by the amino acid sequence of SEQ ID NO: 84. Further peptide components of the hybrid antigen module 1 of SEQ ID NOs: 91-96 are inserted into a framework sequence which is derived from SEQ ID NO: 84. These additional peptide components of the hybrid antigenic module 1 SEQ ID NOs: 91-96 may be rearranged in any order/other order (compared to the replacement order described above). Any such rearranged peptide order based on the antigens/allergens described above is within the scope of the present invention.

Hybrid 2.

Complete allergens and/or top-ranked peptides are selected and spliced together to produce an allergen module for a given iMAT molecule, described below.

Components of the following proteins are used to construct fusion 2 in the preferred order shown, wherein moiety 1 is the N-terminus. Further parts are added, in the order given below, to the C-end of the previous batch. In the case of hybrid 2, 5 further parts are presented. There are 6 parts in total. Part 1: SEQ ID NO: 7 (Q58A71 Der f 1 allergen pre-enzyme Dermatophagoides farinae (American house dust mite) AK 99321) i.e.

TSACRINSVNVPSELDLRSLRTVTPIRMQGGCGSCWAFSGVAATESAYLAYRNT

(SEQ ID NO: 97)

Part 2: SEQ ID NO: 10 (Q86R84 60 kDa allergen Der f 18p Dermatophagoides farinae (American house dust mite) AK 277-304) i.e. FTQTDGFLSYNELCVQIQAETNAFTITR (SEQ ID NO: 98)

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Part 3: SEQ ID NO: 12 (I7HDR2 Zen 1 protein Dermatophagoides farinae (American house dust mite) AK 181-220) i.e. EPTTPTPEPTTKTPEPTTKTPEPSTPTPEPTTKTPEPTTK (SEQ ID NO:

99)

Part 4: SEQ ID NO: 9 (A0A088SAW7 Der f 23 allergen Dermatophagoides farinae (American house dust mite) AK 22-91) i.e. DIDHDDDPTTMIDVQTTTVQPSDEFECPTRFGYFADPKDPCKFYICSNWEAIHKSCPGNTRWNEKELTCT (SEQ ID NO: 100)

Part 5: SEQ ID NO: 11 (Q9U6R7 98 kDa HDM allergen (Der f 15 allergen) (Group 15 Der f 15 allergen) Dermatophagoides farinae (American house dust mite) AK 437-463) i.e. SPTTPTTTPSPTTTTTTPSPTTPTTTP(SEQ ID NO: 101 )

Part 6: SEQ ID NO: 8 (Q00855 allergen Der f 2 group 2 mites (Der f II allergen) (Der f 2 allergen) Dermatophagoides farinae (American house dust mite) AK 18-146) i.e.

DQVDVKDCANNEIKKVMVDGCHGSDPCIIHRGKPFTLEALFDANQNTKTAKIEI

KASLDGLEIDVPGIDTNACHFMKCPLVKGQQYDIKYTWNVPKIAPKSENVVVT

VKLIGDNGVLACAIATHGKIRD (SEQ ID NO: 102)

After the cysteine residues have been replaced with the most stabilizing residues, the biotechnological method described in Examples 5 and 6 is used, this results in SEQ ID NO: 22.

However, the above-described peptide components of the hybrid 2 antigenic module can be rearranged in any order/other order (compared to the order of substitution described above). Any such rearranged peptide order based on the antigens/allergens described above is within the scope of the present invention.

Hybrid 3.

Components of the following proteins are used to construct fusion 3 in the preferred order shown, wherein moiety 1 is the N-terminus. Further parts are added, in the order given below, to the C-end of the previous batch. In the case of hybrid 3, 4 further parts are presented. There are 5 parts in total. Part 1: SEQ ID NO: 7 (Q58A71 Der f 1 allergen pre-enzyme Dermatophagoides farinae (American house dust mite) AK 99-321) i.e.

TSACRINSVNVPSELDLRSLRTVTPIRMQGGCGSCWAFSGVAATESAYLAYRNT

(SEQ ID NO: 97)

Part 2: SEQ ID NO: 10 (Q86R84 60 kDa allergen Der f 18p Dermatophagoides farinae (American house dust mite) AK 277-304) i.e. FTQTDGFLSYNELCVQIQAETNAFTITR (SEQ ID NO: 98)

Part 3: SEQ ID NO: 11 (Q9U6R7 98 kDa HDM allergen (Der f 15 allergen) (Group 15 Der f 15 allergen) Dermatophagoides farinae (American house dust mite) AK 437-463) i.e. SPTTPTTTPSPTTTTTTPSPTTPTTTP (SEQ ID NO: 101 )

Part 4: SEQ ID NO: 8 (Q00855 allergen Der f 2 group 2 mites (Der f II allergen) (Der f 2 allergen) Dermatophagoides farinae (American house dust mite) AK 18-146) i.e.

DQVDVKDCANNEIKKVMVDGCHGSDPCIIHRGKPFTLEALFDANQNTKTAKIEI

KASLDGLEIDVPGIDTNACHFMKCPLVKGQQYDIKYTWNVPKIAPKSENVVVT

VKLIGDNGVLACAIATHGKIRD (SEQ ID NO: 102)

After the cysteine residues have been replaced with the most stabilizing residues, the biotechnological method described in Examples 5 and 6 is used, this results in SEQ ID NO: 23.

However, the above-described peptide components of the hybrid 3 antigenic module can be rearranged in any order/other order (compared to the order of parts described above). Any such rearranged peptide order based on the antigens/allergens described above is within the scope of the present invention.

Further specific aspects of the present invention:

The invention provides an amino acid sequence/improved MAT molecule (iMAT) comprising:

(I) at least one first module that has an amino acid sequence that allows translocation of the iMAT molecule from the extracellular space into cells, (II) at least one second module that has an amino acid sequence that allows for species-specific intracellular targeting iMAT molecules on cell organelles that are involved in antigen processing and/or loading of MHC molecules with antigens, preferably processed antigens, and (III) at least one third module as an antigenic module that has an amino acid sequence that is of at least from one complete or partial amino acid sequence, preferably an epitope, of at least one an-

- 32 041116 tigen, preferably at least one allergen, which determines the specificity of the immune response modulated using such an iMAT molecule, which is characterized in that, at least in antigenic modules, at least one cysteine residue is replaced with another amino acid residue , preferably serine, leucine, isoleucine, arginine, methionine and/or aspartic acid, for use in a method for the prevention and/or treatment of one or more allergies in animals other than horses, and/or for use in a method for the prevention and/or treatment of one or several infectious diseases in animals, excluding horses, and/or for use in a method for preventing the transmission of one or more infectious diseases in animals, excluding horses, and/or for use in a method for preventing the transmission of one or more infectious diseases in animals, excluding horses, with using vectors.

The invention further provides a method for preventing and/or treating one or more allergies in animals, excluding horses, and/or a method for preventing and/or treating one or more infectious diseases in animals, excluding horses, and/or a method for preventing transmission of one or more infectious diseases in animals other than horses, preferably by vectors, which comprises administering a therapeutically effective amount of an amino acid sequence/improved MAT molecule (iMAT) comprising:

(I) at least one first module that has an amino acid sequence that allows translocation of the iMAT molecule from the extracellular space into cells, (II) at least one second module that has an amino acid sequence that allows for species-specific intracellular targeting iMAT molecules on cell organelles that are involved in antigen processing and/or loading of MHC molecules with antigens, preferably processed antigens, and (III) at least one third module as an antigenic module that has an amino acid sequence that is of at least from one complete or partial amino acid sequence, preferably an epitope, of at least one antigen, preferably at least one allergen, which determines the specificity of the immune response modulated by such an iMAT molecule, which is characterized in that at least in antigenic modules, at least one cysteine residue is substituted with another amino acid residue, preferably serine, leucine, isoleucine, arginine, methionine and/or aspartic acid.

In a specific aspect, at least one antigenic module is all cysteine residues replaced with another amino acid residue, preferably serine, leucine, isoleucine, arginine, methionine and/or aspartic acid. Preferably all cysteine residues in the whole iMAT molecule are substituted with another amino acid residue, preferably serine, leucine, isoleucine, arginine, methionine and/or aspartic acid.

In a further aspect, all such modules are covalently bonded to each other, optionally via additional spacer(s) module(s) between two or more adjacent modules, optionally between all such first, second and/or third modules.

In a preferred aspect, all such modules are covalently bonded to each other and no additional spacer module(s) between two or more adjacent modules, such as the first, second and/or third modules, is present at all.

In another aspect, at least one second module includes an invariant circuit selected from canine, feline, bovine, ovine, porcine, and/or porcine species, or a partial sequence thereof, provided that such at least one second module functions as a module , which allows species-specific intracellular targeting of the iMAT molecule to cell organelles that are involved in antigen processing and/or loading of MHC molecules with antigens, preferably processed antigens. A preferred dog invariant strand sequence is SEQ ID NO: 2. A preferred feline invariant strand sequence is SEQ ID NO: 3.

In a preferred aspect, at least one second module comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 4 (canine) or SEQ ID NO: 5 (feline). SEQ ID NOs: 4 and 5 represent the 110 amino acid residues of the invariant chains from SEQ ID NOs: 2 and 3, respectively.

In a further preferred aspect, at least one second module comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 4 (canine) or SEQ ID NO: 5 (feline) or fragments thereof, provided that such fragments retain their intracellular transport function.

In a further aspect, at least one antigenic module includes at least one complete or partial amino acid sequence, preferably an epitope, which is derived from at least one allergen that causes allergy in animals, excluding horses, preferably at least one full or partial amino acid sequence, preferably an epitope, of at least one allergen, which is derived preferably from flea bite allergies, preferably in dogs and/or cats; allergies to certain food components, preferably in dogs and/or cats; atopic dermatitis, preferably in dogs and/or cats; allergic inflammation and/or airway obstruction, preferably in cats.

In a preferred aspect, such at least one antigen, preferably at least one allergen, is Der f 15 allergen according to SEQ ID NO: 11 or 18. In a preferred aspect, such at least one antigenic module comprises, preferably consists of SEQ ID NO: 18.

In a specific aspect, such at least one antigen, preferably at least one allergen, is one that is of food origin and/or mold (fungi and/or their spores), pollen, house dust or barn mites (and/or their feces) and/or fleas, preferably from pollen from trees, grasses, herbaceous plants, ragweed and/or pollen from cruciferous plants and/or fungi and/or their spores of the genera aspergillus, alternaria, botrytis, cercospora, cladosporium, curvularia, drechslera, eurotium , helminthosporium, epicoccum, erysiphe/oidium, fusarium, lichtheimia, nigrospora, penicillium, periconia, peronospora, polythrincium, saccharopolyspora (formerly also faenia or micropolyspora), thermoactinomyces, stemphylium, torula and/or mites (or their feces) of the genera acarus, glycophagus , tyrophagus, dermatophagoides, euroglyphus, lepidoglyphus, blomia and/or fleas of the genera Ceratophyllus, Ctenocephalides, Pulex, Archaeopsylla., and more preferably is a Dermatopha allergen goides.

In yet another specific aspect, at least one antigenic module comprises at least one complete or partial amino acid sequence, preferably an epitope, which is derived from at least one antigen of a pathogen that causes one or more infectious diseases in animals, excluding horses, preferably from at least one complete or partial amino acid sequence, preferably an epitope, of at least one antigen of a pathogen causing one or more infectious diseases in animals, excluding horses, selected from the genus Campylobacter, Dirofilaria, Ehrlichia, Leishmania, Trypanosoma, Borrelia, Orthobunyavirus, Orbivirus, Flavivirus, Rotavirus, Coronavirus, Trichophyton, Microsporum; Cooperia, Haemonchus, Ostertagia, Trichostrongylus, Dictyocaulus, Metastrongylus; Eimeria, Isospora, Cryptosporidium, Giardia, where preferably at least one antigenic module can also be an antigen of a vector involved in the transmission of one or more infectious diseases in animals, excluding horses, preferably an antigen selected from saliva components of vectors selected from insects that feed on blood, flies, midges, ticks and/or mosquitoes.

In a preferred aspect, the iMAT molecule further comprises at least one tag module, preferably at least one His tag. Preferably, such at least one label module is present at the N-terminal end. In another preferred specific aspect, such at least one label module is present at the C-terminal end. In another preferred specific aspect, such at least one label module is present at the N-terminal end and the C-terminal end.

In a specific preferred aspect, the iMAT molecule includes one tag module, preferably one His tag, at the N-terminal end after one methionine residue.

In another specific preferred aspect, there is no label module in the iMAT molecules.

In a further aspect, at least one first module comprises, preferably consists of, the amino acid sequence of HIV-tat, VP22 and/or Antennapedia or a partial sequence thereof, provided that such at least one first module is functional as a module for translocation of the molecule iMAT from the extracellular space into the cells, most preferably is the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 1).

In another aspect, the iMAT molecule is in monomeric form and/or in linear form.

In a further aspect, at least one third module comprises, preferably consists of, any of the sequences shown in SEQ ID NOs: 14-23.

In a preferred aspect, the iMAT molecule comprises, preferably consists of, one or more amino acid sequences according to SEQ ID NOS: 24-83.

A particularly preferred iMAT molecule comprises, preferably consists of, SEQ ID NO: 36 (Der f 15 feline iMAT molecule) or SEQ ID NO: 57 (Der f 15 dog iMAT molecule). Further preferred iMAT molecules (containing an N-terminal His tag) are selected from the group consisting of SEQ ID NOs: 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63 , 66, 69, 72, 75, 78, 81. Further preferred iMAT molecules (containing a C-terminal His tag) are selected from the group consisting of SEQ ID NOs: 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82. Further preferred iMAT molecules (without His tag) are selected from the group consisting of SEQ ID NOs: 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83.

- 34 041116

Further preferred iMAT molecules (hybrid/mosaic iMATs) are selected from the group consisting of SEQ ID NOs: 66-83. Further preferred iMAT fusion molecules are 66, 69, 72, 75, 78, 81 (with an N-terminal His tag). Particularly preferred is SEQ ID NO: 66 and/or 75. Further preferred specific iMAT fusion molecules are 67, 70, 73, 76, 79, 82 (with a C-terminal His tag). Particularly preferred is SEQ ID NO: 67 and/or 76. Further preferred specific iMAT fusion molecules are 68, 71, 74, 77, 80, 83 (no His tag). Especially preferred is SEQ ID NO: 68 and/or 77.

In a further aspect, the animal is selected from ruminants, including cattle, goats, sheep, such as members of the genera Bos, Capra and/or Ovis, members of the genera Canis, such as dogs, wolves, foxes, coyotes, jackals, members of the genera Felis, such as lions, tigers, domestic cats, feral cats, other big cats, and other felines, including cheetahs and lynxes, and/or members of the Sus genera such as pigs, where the animal is preferably selected from cats and/or dogs.

The invention provides an amino acid sequence containing:

(I) at least one first module that has an amino acid sequence that allows translocation of the iMAT molecule from the extracellular space into cells, preferably said first module comprises, more preferably consists of SEQ ID NO: 1, (II) at least one a second module that has an amino acid sequence that allows species-specific intracellular targeting of the iMAT molecule to cell organelles that are involved in antigen processing and/or loading of MHC molecules with antigens, preferably processed antigens, preferably said second module comprises, more preferably consists of , SEQ ID NO: 4 or 5;

(III) at least one third module as an antigenic module which has an amino acid sequence which is derived from at least one full or partial amino acid sequence, preferably an epitope, of at least one antigen, preferably at least one allergen, which determines the specificity of the immune response modulated by such an iMAT molecule, which is characterized in that at least one cysteine residue in antigenic modules is replaced by another amino acid residue, preferably serine, leucine, isoleucine, arginine, methionine, and /or aspartic acid. The third module preferably includes, more preferably consists of any of the sequences shown in SEQ ID NOs: 14-23.

The invention provides an improved MAT molecule (iMAT) comprising:

(I) at least one first module that has an amino acid sequence that allows translocation of the iMAT molecule from the extracellular space into cells, preferably said first module comprises, more preferably consists of SEQ ID NO: 1, (II) at least one a second module that has an amino acid sequence that allows species-specific intracellular targeting of the iMAT molecule to cell organelles that are involved in antigen processing and/or loading of MHC molecules with antigens, preferably processed antigens, preferably said second module comprises, more preferably consists of , SEQ ID NO: 4 or 5;

(III) at least one third module as an antigenic module which has an amino acid sequence which is derived from at least one full or partial amino acid sequence, preferably an epitope, of at least one antigen, preferably at least one allergen, which determines the specificity of the immune response modulated by such an iMAT molecule, which is characterized in that at least one cysteine residue in antigenic modules is replaced by another amino acid residue, preferably serine, leucine, isoleucine, arginine, methionine and/ or aspartic acid. The third module preferably includes, more preferably consists of any of the sequences shown in SEQ ID NOs: 14-23.

In a specific aspect, at least one antigenic module, all cysteine residues are substituted with another amino acid residue, preferably serine, leucine, isoleucine, arginine, methionine, and/or aspartic acid, preferably all cysteine residues in the whole iMAT molecule are substituted with another amino acid residue , preferably serine, leucine, isoleucine, arginine, methionine, and/or aspartic acid.

In a further aspect, all such modules are covalently bonded to each other, optionally via additional spacer(s) module(s) between two or more adjacent modules, optionally between all such first, second and/or third modules.

In a preferred aspect, all such modules are covalently bonded to each other, and no additional spacer(s) module(s) between two or more adjacent modules, such as the first, second and/or third modules. , is not present at all.

In a further preferred aspect, at least one second module comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 4 (canine) or SEQ ID NO: 5 (feline) or fragments thereof, provided that such fragments retain their intracellular transport function. SEQ ID NOs: 4 and 5 represent the 110 amino acid residues of the invariant chains from SEQ ID NOs: 2 and 3, respectively. In a more preferred aspect, at least one second module comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 4 (canine) or SEQ ID NO: 5 (feline).

In a further aspect, at least one antigenic module includes at least one complete or partial amino acid sequence, preferably an epitope, which is derived from at least one allergen that causes allergy in animals, excluding horses, preferably at least one complete or partial an amino acid sequence, preferably an epitope, of at least one allergen which is derived preferably from flea bite allergies, preferably in dogs and/or cats; allergies to certain food components, preferably in dogs and/or cats; atopic dermatitis, preferably in dogs and/or cats; allergic inflammation and/or airway obstruction, preferably in cats.

In a specific aspect, such at least one antigen comprises the amino acid sequence according to SEQ ID NO: 11 or 18. In a preferred aspect, such at least one antigen, preferably at least one allergen, is Der f 15 allergen according to SEQ ID NO: 11 or 18. In a further preferred aspect, such at least one antigenic module comprises, preferably consists of SEQ ID NO: 18. In a further preferred aspect, the iMAT molecule comprises, preferably consists of (one) amino acid sequence according to SEQ ID NOs: 24-83 (one of these alternatives). In a further preferred aspect, the iMAT molecule comprises, preferably consists of any of the sequences shown in SEQ ID NOs: 36-38 (Der f15 feline iMAT molecule), most preferably SEQ ID NO: 36, or the iMAT molecule contains, preferably consists of any of sequences shown in SEQ ID NOs: 57-59 (Der f15 canine iMAT molecule), most preferably SEQ ID NOs: 57.

In a preferred aspect, the iMAT molecule further comprises at least one tag module, preferably at least one His tag. Preferably, such at least one label module is present at the N-terminal end. In a specific preferred aspect, the iMAT molecule includes one tag module, preferably one His tag, at the N-terminal end after one methionine residue. Preferred N-terminal His-tagged iMAT molecules comprise, preferably consist of, any of the sequences shown in SEQ ID NOs: 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63 , 66, 69, 72, 75, 78, 81.

In another specific preferred aspect, such at least one label module is present at the C-terminal end. In another specific preferred aspect, such at least one label module is present at the N-terminal end and the C-terminal end. Preferred C-terminal His-tagged iMAT molecules comprise, preferably consist of, any of the sequences shown in SEQ ID NOs: 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61 , 64, 67, 70, 73, 76, 79, 82.

In another specific preferred aspect, there is no label module. Preferred iMAT molecules completely unlabeled comprise, preferably consist of, any of the sequences shown in SEQ ID NOs: 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83.

In a further aspect, at least one first module comprises, preferably consists of the amino acid sequence of HIV-tat, VP22 and/or Antennapedia or a partial sequence thereof, provided that such at least one first module is functional as a module for translocation of the iMAT molecule from the extracellular space into the cells, most preferably is the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 1).

In another aspect, the iMAT molecule is in monomeric form and/or in linear form.

In a further specific aspect, the iMAT molecule is a hybrid iMAT molecule (mosaic iMAT molecule). Preferably, such a hybrid iMAT molecule comprises an amino acid sequence according to any of the sequences shown in SEQ ID NOs: 21-23. More specifically, such a hybrid iMAT molecule contains, preferably consists of an amino acid sequence according to any of the sequences shown in SEQ ID NOs: 66-83.

The invention further provides an amino acid sequence that contains, preferably consists of one or more amino acid sequences according to

- 36 041116 with SEQ ID NOs: 24-83. The invention further provides an iMAT molecule which contains, preferably consists of, one or more amino acid sequences according to SEQ ID NOs: 24-83.

The invention further provides an iMAT amino acid sequence/molecule that comprises, preferably consists of, one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 24-83.

A particularly preferred amino acid sequence/iMAT molecule comprises, preferably consists of SEQ ID NO: 36 (Der f15 feline iMAT molecule) or SEQ ID NO: 57 (Der f 15 dog iMAT molecule). Further preferred amino acid sequences/iMAT molecules (containing an N-terminal His tag) are selected from the group consisting of SEQ ID NOs: 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81. Further preferred iMAT amino acid sequences/molecules (containing a C-terminal His tag) are selected from the group consisting of: SEQ ID NOs: 25, 28, 31, 34 , 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82. Further preferred iMAT amino acid sequences/molecules (without His tag) are selected from the group consisting of SEQ ID NOs: 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83.

Further preferred amino acid sequences/iMAT molecules (hybrid/mosaic iMATs) are selected from the group consisting of SEQ ID NOs: 66-83. Further preferred iMAT fusion molecules are 66, 69, 72, 75, 78, 81 (with an N-terminal His tag).

Further preferred iMAT fusion molecules are 67, 70, 73, 76, 79, 82 (with a C-terminal His tag). Further preferred iMAT fusion molecules are 68, 71, 74, 77, 80, 83 (no His tag).

The invention provides an amino acid sequence/improved MAT molecule (iMAT) comprising:

(I) at least one first module that has an amino acid sequence that allows translocation of the iMAT molecule from the extracellular space into cells, preferably said first module comprises, more preferably consists of SEQ ID NO: 1, (II) at least one a second module that has an amino acid sequence that allows species-specific intracellular targeting of the iMAT molecule to cell organelles that are involved in antigen processing and/or loading of MHC molecules with antigens, preferably processed antigens, preferably said second module comprises, more preferably consists of , SEQ ID NO: 4 or 5 (III) at least one third module as an antigenic module that has an amino acid sequence that is derived from at least one complete or partial amino acid sequence, preferably an epitopic sequence value, any combination of two or more antigens selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, 11, 12, 84, 85, 86, 87 and 88, which determines the specificity of the immune response modulated by such an iMAT molecule, which is characterized in that at least one cysteine residue in the antigenic modules is replaced with another amino acid residue, preferably serine, leucine, isoleucine, arginine, methionine and/or aspartic acid. In a preferred aspect, an antigenic module is an amino acid sequence that is derived from at least one complete or partial amino acid sequence, preferably an epitope sequence, from any combination of two or more antigens selected from the group consisting of: SEQ ID NOs: 10, 11, 84, 85, 86, 87 and 88 (Hybrid 1). In a specific aspect, an antigenic module is an amino acid sequence that is derived from at least one complete or partial amino acid sequence, preferably an epitope sequence, from any combination of two or more antigens selected from the group consisting of SEQ ID NOs: 7, 8, 9 ,10, 11 and 12 (Hybrid 2).

In a specific aspect, an antigenic module is an amino acid sequence that is derived from at least one complete or partial amino acid sequence, preferably an epitope sequence, from any combination of two or more antigens selected from the group consisting of SEQ ID NOs: 7, 8, 10 and 11 (Hybrid 3).

Thus, the invention specifically provides an amino acid sequence/improved MAT molecule (iMAT) comprising:

(I) at least one first module that has an amino acid sequence that allows translocation of the iMAT molecule from the extracellular space into cells, preferably said first module comprises, more preferably consists of SEQ ID NO: 1, (II) at least one the second module, which has an amino acid sequence that allows species-specific intracellular targeting of the iMAT molecule to

- 37 041116 cell organelles that are involved in the processing of antigens and/or the loading of MHC molecules with antigens, preferably processed antigens, preferably said second module comprises, more preferably consists of SEQ ID NO: 4 or 5, (III) at least one third module as an antigenic module that has an amino acid sequence based on a framework that has the origin of SEQ ID NOs: 84, containing any combination of one or more peptides according to SEQ ID NOs: 91-96 (in any order) inserted into the specified framework a sequence that determines the specificity of the immune response modulated by such an iMAT molecule, which is characterized in that at least in antigenic modules at least one cysteine residue is substituted with another amino acid residue, preferably serine, leucine, isoleucine, arginine, methionine and/or aspartic acid.

In a preferred aspect, the order of the peptide sequences in the backbone that originates from SEQ ID NO: 84 in the third module is: SEQ ID NO: 91 followed by SEQ ID NO: 92 followed by SEQ ID NO: 93 followed by followed by SEQ ID NO: 94 followed by SEQ ID NO: 95 followed by SEQ ID NO: 96 starting at the N-terminus. In a most preferred aspect, the third module comprises, preferably consists of, SEQ ID NO: 21.

In a further specific aspect, the order of the peptide sequences in the backbone that originates from SEQ ID NO: 84 in the third module is: SEQ ID NO: 93 followed by SEQ ID NO: 91 followed by SEQ ID NO: 92 followed by followed by SEQ ID NO: 94 followed by SEQ ID NO: 96 followed by SEQ ID NO: 95 starting at the N-terminus. Further aspects of the present invention relate to any other combination and order of peptides according to SEQ ID NOs: 91-96 inserted into a framework sequence that is derived from SEQ ID NO: 84.

Thus, the invention further provides an amino acid sequence/improved MAT molecule (iMAT) comprising:

(I) at least one first module that has an amino acid sequence that allows translocation of the iMAT molecule from the extracellular space into cells, preferably said first module comprises, more preferably consists of SEQ ID NO: 1, (II) at least one a second module that has an amino acid sequence that allows species-specific intracellular targeting of the iMAT molecule to cell organelles that are involved in antigen processing and/or loading of MHC molecules with antigens, preferably processed antigens, preferably said second module comprises, more preferably consists of SEQ ID NOs: 4 or 5, (III) at least one third module as an antigenic module that has an amino acid sequence that is derived from any combination of two or more peptides according to SEQ ID NOs: 97-102 (in any order), which is determined represents the specificity of the immune response modulated by such an iMAT molecule, which is characterized in that at least one cysteine residue in antigenic modules is replaced by another amino acid residue, preferably serine, leucine, isoleucine, arginine, methionine and/ or aspartic acid. In a preferred aspect, the order of the peptide sequences in the third module is: SEQ ID NO: 97 followed by SEQ ID NO: 98 followed by SEQ ID NO: 99 followed by SEQ ID NO: 100 followed by SEQ ID NO: 101 followed by SEQ ID NO: 102 starting at the N-terminus. In a most preferred aspect, the third module comprises, preferably consists of, SEQ ID NO: 22.

In a further specific aspect, the order of the peptide sequences in the third module is: SEQ ID NO: 99 followed by SEQ ID NO: 98 followed by SEQ ID NO: 102 followed by SEQ ID NO: 100 followed by SEQ ID NO: 101 followed by SEQ ID NO: 97 starting at the N-terminus. Further aspects of the present invention relate to any other combination and order of peptides according to SEQ ID NOs: 97-102.

Thus, the invention further provides an amino acid sequence/improved MAT molecule (iMAT) comprising: (i) at least one first module that has an amino acid sequence that allows translocation of the iMAT molecule from the extracellular space into cells, preferably said first module comprises , more preferably consists of SEQ ID NO: 1, (II) at least one second module that has an amino acid sequence that allows species-specific intracellular targeting of the iMAT molecule to cell organelles that are involved in antigen processing and/or loading MHC molecules with antigens, preferably processed antigens, preferably said second module comprises, more preferably consists of SEQ ID NO: 4 or 5, (III) at least one third module as an antigenic module, which has an amino acid sequence which it is derived from any combination of two or more

- 38 041116 peptides according to SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 101 and SEQ ID NO: 102 (in any order), which determines the specificity of the immune response modulated by such an iMAT molecule, which characterized in that at least in the antigenic modules at least one cysteine residue is substituted with another amino acid residue, preferably serine, leucine, isoleucine, arginine, methionine and/or aspartic acid. In a preferred aspect, the order of the peptide sequences in the third module is SEQ ID NO: 97 followed by SEQ ID NO: 98 followed by SEQ ID NO: 101 followed by SEQ ID NO: 102 starting at the N terminus. In a most preferred aspect, the third module comprises, preferably consists of, SEQ ID NO: 23.

In a further specific aspect, the order of the peptide sequences in the third module is: SEQ ID NO: 102 followed by SEQ ID NO: 98 followed by SEQ ID NO: 101 followed by SEQ ID NO: 97 starting with N- end. Further aspects of the present invention relate to any other combination and order of peptides according to SEQ ID NOs: 97, 98, 101 and 102.

The invention further provides a vaccine or immunogenic composition or pharmaceutical composition containing the amino acid sequence/iMAT molecule in accordance with the present invention.

The invention further provides a nucleic acid encoding an amino acid/iMAT molecule in accordance with the present invention.

Additionally, the invention provides a vector that includes at least one nucleic acid in accordance with the present invention.

In addition, the invention provides a primary cell or cell line that includes at least one nucleic acid in accordance with the present invention and/or at least one vector in accordance with the present invention.

The invention further provides a method for identifying improved MAT molecules, which includes the steps of:

a) selection of a protein as an allergenic module in iMAT molecules and

b) constructing an iMAT molecule with said allergens that is thermodynamically stable and can be efficiently obtained using protein engineering.

The invention specifically provides a method for identifying improved MAT molecules, which includes the steps:

a) selecting a protein as an allergenic module in iMAT molecules that is an allergen and thus has a high potential to induce hypersensitivity in affected subjects and thus also be a target for tolerance induction, and

b) designing an iMAT molecule with the indicated allergens that is thermodynamically stable and can be efficiently obtained using protein engineering and can be further analyzed using standard methods in order to provide a sufficiently high quality (i.e. identity, purity and potency).

In a specific aspect of the method, step a) comprises selecting the allergen(s) based on searches for local peptide homologies that have these proteins relative to known allergenic proteins, exporting the amino acid sequences of the proteins that are suspected to have allergenic properties from public databases (e.g., UNIPROT), determination of redundancies by analysis of sequence homologies in the exported dataset, elimination of highly homologous sequence analogues (the remaining sequences served as a canonical sequence database of likely valid antigens for subsequent analysis), in silico cleavage of proteins into peptides of 6 to 15 amino acids in length (co displacement of one amino acid), performing local pairwise alignment of proteins and corresponding peptides, scaling the matches obtained during the alignment by setting the self-alignment score of this protein at the level of one and matches when matching corresponding peptides, respectively, counting the number of matches in an alignment that exceeds a given threshold for each peptide, comparing local pairwise alignments for randomly generated databases of protein sequences with unknown allergenic properties, scaling and counting matches, subtracting the obtained values for a non-allergic protein from such results for the allergen, calculating cumulative match scores for each protein based on the number of matches for all relevant peptides, selecting the proteins with the highest scores as candidates for the iMAT antigen module.

In a specific aspect of the method, step b) comprises identifying amino acid substitutions in a predetermined amino acid sequence that allows the predetermined amino acid sequence to be stabilized, preferably, which comprises the steps of:

(I) modeling the 3D/tertiary structure of the target predefined amino-

- 39 041116 acid sequence, (II) iterative determination of protein stability based on single-point substitutions, such as the replacement of cysteine residues with other amino acid residues, preferably serine and/or isoleucine residues, and categorization based on changes in the free energy of protein stability (AAG) to determine stabilizing substitutions by analyzing all available 3D/tertiary amino acid sequence structures, (III) remodeling the substituted 3D/tertiary amino acid sequence structure and calculating its stability by repeating steps (II) and (III) until a stable state is reached.

Brief description of the figures

Fig. 1: shows DNS-PAGE for MAT-Fel d1 (IVN201) under reducing conditions; A) original DNS-PAGE (10 μg of protein per lane, Coomassie staining) and B) DNS-PAGE with bands that were excised from Gel A and reloaded on DNS-PAGE (silver stain);

Fig. 2: shows DNS-PAGE for Fel d1 under reducing conditions; NuPAGE® 4-12% BisTris gel. Lane: 1) Marker: SeeBlue Plus2 pre-stained standard 2) 5 µg Fel d1; 3) 5 μg Fel dl;

Fig. 3: shows reverse phase HPLC gradient 0.1% TFA/acetonitrile a) native protein (MAT-Fel d1) without additives (left column); b) MAT-Fel d1 denatured by additions of guanidine chloride plus DTT (right column);

Fig. 4: shows the MAT-Fel d1 molecule and its Kite Doolittle hydrophobicity diagram;

Fig. 5: Shows the NuPAGE® DNS-PAGE System (4-12% Bis-Tris Gels, 1x MES Run Buffer, 35 min, 200 V). Tracks: 1) Lysozyme, 1 µg protein oxid. 2) PageRuler Prestained marker protein; 3) MAT-Fel d1 (5 μg) oxidized with iodoacetamide (oxidized); 4) iMAT-Culo4 (oxidized); 5) PageRuler Prestained marker protein; 6) MAT-Fel d1 (5 μg) reconstituted; 7) iMAT-Cul o4 reconditioned; 8) lysozyme restored;

Fig. 6: Shows a reverse phase HPLC gradient of 0.1% TFA/acetonitrile. The peak shows native (oxidized) protein (iMAT-Cul o4) without additives;

Fig. 7: shows the results of the following experiments: proteins and Adju-Phos® which were incubated at room temperature for 30 minutes with gentle agitation. After incubation, samples were centrifuged for 3 min and analyzed sequentially with LTOPS. Lane 1) PageRuler Prestained marker protein; 2) iMAT-Cul o3 supernatant; 3) Precipitate of iMAT-Cul o3 in Urea 4) Supernatant of iMAT-Cul o3; 5) Precipitate iMAT-Cul o3 in Urea; 6) empty track; 7) iMAT-Cul o2 supernatant 8) iMAT-Cul o2 precipitate in Urea 9) iMAT-Cul o2 supernatant 10) iMAT-Cul o2 precipitate in Urea; 11) empty track; 12) iMAT-Cul o4 supernatant; 13) iMAT-Cul o4 precipitate in Urea 14) iMAT-Cul o4 supernatant; 15) Precipitate iMAT-Cul o4 in Urea; (2, 3, 7, 8, 12, 13 w/v freeze/thaw); (4, 5, 9.10, 14.15 after the double freeze/thaw process);

Fig. 8: shows a Kite-Doolittle hydrophobicity diagram for the MAT molecule relative to the iMAT molecule in the generic portion of the respective fusion proteins [ie, without their respective antigenic module(s)]. The hydrophobicity index is shown on the y-axis relative to the position of the amino acid along the x-axis. Positive numbers in the index indicate hydrophobicity. The shift of the diagram of the iMAT molecule at the beginning of the hydrophobicity diagram with respect to the MAT molecule is due to the additional presence of the N-terminal His-tag and one methionine residue in the iMAT molecule, as well as the absence of any spacer(s) module(s);

Fig. 9: shows examples of allergens of the genus dermatophagoides, identifying species, allergen and deposit number of uniprot. (a) Allergens from D. pteronyssinus, (b) Allergens from D. farinae;.

Fig. 10: shows the alignment of the N-terminal sequence of invariant strands from dog and cat. CLIP sequence is shaded in grey;

Fig. 11: shows histamine release analysis (analysis similarities are shown in example 2) 5 concentrations of iMAT-Cul o2 and iMAT-Cul o3 and corresponding allergens in polysensitized horses (Horse 1).

Examples

The following examples serve to further illustrate the present invention, but they should not be construed as limiting the scope of the invention, which is disclosed in this application.

Example 1 Surrogate Marker for Immunity/Duration of Immunity

Administration of an (isolated) recombinant protein as described and claimed herein to an animal, e.g. ruminants, pigs, more preferably a dog and/or cat, but excluding a horse, elicits an immune response to an allergen and/or epitope that is present on the antigenic module. In addition, a C- or N-terminal tag, e.g., a His tag, a TAT module, together with adjacent amino acid residues from an adjacent module, is used to detect a unique immunological signal for a specific product (e.g., antibody production or TAT).

- 40 041116 cellular response) in the target subject, which is used as a surrogate marker of immunity or duration of immunity. This surrogate marker, as a treatment-specific immunological parameter, allows an assessment of immunity or immunity modulation, or the duration of immunity, or the duration of immune modulation after administration. Thus, a particular measure of the immune response that is triggered by the (isolated) recombinant protein of the present invention is the induction of the terminal label (optionally adjacent amino acid residues) of specific antibodies, such as IgG antibodies. Alternatively or in addition to this, this indicator is the induction of antibodies specific to the connection of the spacer and the module, as described and claimed in this application, or to the connection between the two modules.

A single iMAT molecule, such as SEQ ID NOs: 57 (Der f 15 iMAT molecule (dogs) with an N-terminal Hexa-Histidine tag) or a combination of one or more iMAT molecules selected from SEQ ID NOs: 45, 51, 54, 57, 60 , 63, 66, 69, 72 containing various antigenic modules according to the present invention are used prophylactically or therapeutically in a dog suffering from or at risk of developing allergic diseases, in particular atopic dermatitis. In such dogs, the above-described iMAT molecules are administered as described in Example 3. In serum samples that originate from blood collected from these iMAT-treated dogs, the immunological response during iMAT treatment to the N-terminus of the iMAT proteins can be determined by the ratio to antibody production, more specifically, specific IgG. For the change, standard ELISA (Enzyme-linked Immunosorbent Assay) techniques are used, for this, sufficient amounts of the synthesized peptide containing SEQ ID NO: 6 are coated on the surface of the ELISA plate. Thereafter, serum samples from treated animals were incubated on these plates and specific IgG binding to the indicated peptide was determined using a secondary biotinylated antibody specific for IgG in cats or dogs, respectively, followed by the use of an appropriate detection system, for example, streptavidin-peroxidase and 3,3' ,5,5'-tetramethylbenzidine (TMB) as a substrate.

With this ELISA assay, the onset of the immune response elicited by the iMAT immunotherapy as well as the duration of immunity are determined and monitored over time. The therapeutic vaccination schedule, ie the number and schedule of booster injections, is determined using this surrogate parameter in clinical trials.

Example 2. Hypoallergenic

The allergenicity of a therapeutic allergen is extremely important and is indicative of the potential for inducing side effects, such as provoking an anaphylactic reaction. For the example of allergy in mammals, allergen-specific IgE mediated hypersensitivity is investigated using procedures such as allergen provocation tests, in particular such tests that target the skin [Griffin CE. Diagnosis of canine atopic dermatitis DOI: 10.1002/9781118738818.ch10]. Intradermal skin tests are used for the biological evaluation of recombinant allergens and for the validation of genetically engineered hypoallergenic derivatives.

Intradermal testing in dogs is performed by injecting small amounts of allergen solutions directly into the dog's skin. This is usually done with small gauge needles (Gage 27 gauge needles) and 0.5-0.1 ml is injected into each site. Positive reactions are randomly interpreted by the presence of erythema, swelling, height and size of the allergic papule. The advantage of intradermal tests is high sensitivity. It is extremely important if the test will provide a quantitative indicator for hypoallergenicity. In these tests, the MAT molecules exhibit a 10-, 100-1000-fold or even higher molar concentration of the allergenic component compared to the natural, native allergen used in a similar test to achieve a positive reaction in susceptible individuals, such as cats. and dogs.

With respect to MAT molecules, conflicting results regarding their allergenicity compared to corresponding native allergens have been described in the prior art. Senti G et al. (J Allergy Clin Immunol. 2012, 129(5):1290-1296) have demonstrated the hypoallergenicity of MAT-Fel d1 in a cell antigen stimulation test (CAST) as well as intradermal and intradermal testing. The quantitative difference in sensitivity between the allergen and the MAT molecule containing Fel d1 was 100-, 23- and 16-fold, respectively. Although MAT-Fel d1 was clearly hypoallergenic, some allergenicity remained. In contrast, Zhao et al. Int J Clin Exp Med 2015; 8(4):6436-6443 have described that their MAT-Der p1 construct exhibits even more allergenicity (hyperallergenicity) than native Der p1 protein.

Surprisingly improved MAT molecules, as described and claimed in this application, show a clear superiority in this aspect. The safety of 2 iMAT molecules prepared in accordance with the present invention containing Cul o2 and Cul o3 in the antigenic module, respectively, was tested. Freshly obtained blood from a horse sensitized to these allergens and suffering from insect sting hypersensitivity (IBH) was used in the histamine release test (HRT) as described below. As shown in FIG. 11, native allergens stimulate strong histamine release, while, unexpectedly, two different iMAT molecules (iMAT-Cul o3 and iMAT-Cul o2) show virtually no response at all.

Thus, iMAT molecules show a clear superiority in terms of safety compared to the MAT molecule, as described in the prior art (see above).

Histamine Release Test (HRT): Freshly obtained blood from subjects was prepared to test the reactivity of basophils to (isolated) recombinant iMAT proteins/molecules as described and claimed herein. Briefly, serial 10-fold dilutions (eg, ranging from 10 to 0.001 nM final allergen concentration) of iMAT molecules and/or recombinant allergens were prepared in PIPES buffer (AppliChem, Darmstadt, Germany), pH 7.4. Washed red and white blood cells obtained from blood by inhibition of coagulation using Na-EDTA were incubated with individual dilutions for 1 hour at 37°C. The reaction was stopped by incubation on ice for 20 minutes and the supernatant containing released histamine was collected from each sample after centrifugation. The maximum histamine content was obtained by boiling blood cells for 10 min in a water bath (maximum recovery). Incubation of release buffer with washed blood cells served as a negative control (spontaneous release). Histamine concentration was determined using a competitive radioimmunoassay (LDN Nordhorn, Germany) according to the manufacturer's instructions.

Alternatively, another basophil activation test is the ELISA CAST® cellular antigen stimulation assay, which can also be considered an in vitro allergy provocation test. This analysis was carried out according to the manufacturer's instructions (Buhlmann Laboratories AG, Allschwil, Switzerland). In the CAST® assay, blood-precipitated leukocytes from allergic subjects were both primed with the cytokine IL-3 and stimulated with iMAT molecules and/or recombinant allergens. Basophil cells, along with others, produce the allergic mediator, sulfidoleukotriene LTC4, and its metabolites LTD4 and LTE4. These freshly synthesized sulfidoleukotrienes (SLTs) were subsequently evaluated in an ELISA test (enzyme-linked immunosorbent assay).

The potential of iMAT molecules to induce adverse events, such as provoking anaphylaxis as a side effect of administration, can be assessed in vitro using these assays by comparing the effects of the iMAT molecule (containing the allergen) versus the corresponding recombinant allergen taken alone.

Reduced degranulation of basophils, eg release of histamine and/or sulfidoleukotrienes, with iMAT molecules compared to the recombinant allergen indicates a lower potential for side effects, i.e. a better safety profile of the iMAT molecules. Said HRT (or CAST®) can be used as an in vitro provocation test for type 1 allergic reactions in a subject. Allergen-specific histamine release is indicative of the relevant allergen's relevance to basophil cell activation and thus can be used as a quantitative parameter for allergen-specific sensitization of a subject.

It can be expected that no allergen-related adverse reactions will be observed if subjects suffering from allergen-specific IgE mediated hypersensitivity are treated with appropriate iMAT molecules containing relevant allergens in the iMAT antigen module. This makes desensitization therapy using iMAT proteins particularly suitable for the treatment of life-threatening diseases.

The result of this unexpected safety property of iMAT molecules as opposed to MAT molecules is that iMAT molecules used as desensitizing proteins can be used similar to pathogen vaccines. There is no need to increase the dosage, as is the case with classical therapeutic allergens, since vaccines containing iMAT molecules do not show allergenic properties in relation to allergic side effects. Even the dose of the first injection of the iMAT molecule during the course of treatment is chosen only for reasons of effectiveness and does not take into account possible allergic side reactions. This cannot be done using the MAT molecules described in the prior art, since the allergenicity of the MAT, compared to the native allergen, is only reduced to a certain level. However, MAT molecules are still allergens; iMAT molecules, unlike them, are not. The advantage of this improved property is a more efficient treatment regimen, possible, for example, with three subcutaneous or intralymphatic injections with a high content of the biopharmaceutical (for example, 3 times 1 to 100 μg, preferably 3 times 10 to 50 μg of iMAT protein).

The lack of allergenicity of iMAT molecules can be explained by the fact that, unlike the MAT molecules described in the prior art, there are no linker amino acid residues

- 42 041116

[ie spacer(s) module(s) between the first, second and/or third module(s)] used to separate different modules in such iMAT molecules. It is known in the art that engineered fusion proteins containing two or more functional polypeptides connected by a peptide or protein linker are essential for the function (eg, epitope recognition by the immune system) of proteins [Klein JS et al., Protein Eng Des Sel. 2014, 27(10): 325-330]. The separation distance between functional units can influence epitope accessibility and ability to bind to avidity. If the missing amino acid residues of the linker between the modules, in particular between the target domain and the antigenic module, results in a more rigid structure, conformational epitopes of the allergen module cannot be formed due to misfolding. Cross-linking of antibodies bound to the surface of basophils (eg, IgE) with its high affinity receptors is necessary to induce histamine activation and release. However, misfolded allergens are unable to induce such cross-linking. Thus, an iMAT molecule without a linker cannot form conformational IgE epitopes, which makes iMAT molecules non-allergenic.

Example 3 Therapeutic Vaccine/Prophylaxis of Atopic Dermatitis in Dogs and/or Cats

A single iMAT molecule or a combination of iMAT molecules containing different antigenic modules according to the present invention has been used prophylactically or therapeutically in a dog and/or cat suffering from or at risk of developing atopic dermatitis (AD).

In the first example, the iMAT molecule according to SEQ ID NO: 36 (Der f 15) was injected into the popliteal lymph node of cats suffering from atopic dermatitis.

In a second example, an iMAT molecule according to SEQ ID NO: 66 (Hybrid 1) was injected into the popliteal lymph node of cats suffering from atopic dermatitis.

In a third example, an iMAT molecule according to SEQ ID NO: 57 (Der f 15) was injected into the popliteal lymph node of dogs suffering from atopic dermatitis.

In a fourth example, an iMAT molecule according to SEQ ID NO: 81 (Hybrid 3) was injected into the popliteal lymph node of dogs suffering from atopic dermatitis.

In each case, the hair over the affected animal's lymph node was shaved off and surgically prepared. Using palpation and/or under 25 G ultrasound guidance, the needle was inserted into the lymph node. The injectable iMAT molecule is adsorbed to the adjuvant. The adjuvant consists, for example, of aluminum phosphate (Adju-Phos®, Brenntag Biosector, Denmark). The stock solution of the iMAT molecule was a frozen solution, for example, at a concentration of 375 μg/ml of protein in vials containing 500 μl each, to be thawed before use.

After thawing the iMAT molecule solution, 400 µl of the solution was mixed with, for example, 200 µl of the adjuvant. This final preparation was left at room temperature, for example, for 60 min before intralymphatic injection, to allow absorption of the iMAT molecule, for example, on Adju-Phos®, for example, 50 μl of a mixture containing 12.5 μg of the iMAT molecule was removed into a 500 syringe µl for injection into the lymph node. This drug was initially administered typically on day 0, day 28 and day 56 at a dose ranging from 10 to 50 μg (based on the weight of one or more antigenic modules alone) per injection and iMAT molecule. Throughout the treatment period and / or after it, the effectiveness of treating or preventing AD was studied clinically using quantitative, semi-quantitative or qualitative assessment of pruritus, skin lesions and drugs in balls (Hobi S, Mueller RS; Tierarztliche Praxis. Ausgabe K, Kleintiere / Heimtiere 2014, 42(3):167-173).

These clinical parameters were compared with the clinical symptoms of the individual dog and/or cat before the start of the therapeutic intervention. Alternatively, comparison with dogs and/or cats that suffer from AD but are untreated or treated with placebo may demonstrate efficacy in treating and/or preventing the clinical signs of AD mediated by the iMAT molecule.

Alternatively or additionally, an intradermal challenge test with certain dermatophagoides allergens may be used in said dogs and/or cats. A reduced response (immediate and/or late reactivity phase) is indicative of a therapeutic and/or prophylactic effect of administration of the iMAT molecule.

In addition, the modulation of various components of the immune system is monitored, for example, changes in allergen-specific IgE and IgG antibody titers are indicative of therapeutic and/or prophylactic effects. In addition to changes in IgE levels, an increase in allergen-specific IgG was unexpectedly found when treating a dog and/or cat for AD using the iMAT molecule. These antibodies block IgE-mediated anaphylaxis in vivo and are expected to inhibit not only allergen-induced release of inflammatory mediators from basophils and mast cells, but also IgE-mediated allergen presentation to T cells. Among iMAT-induced IgG antibodies that specifically bind to an allergen, some allergen-specific subtypes are thought to play an important protective role because they compete with allergen-specific IgE antibodies and can prevent CD4 T cell activation by inhibition of IgE mediated antigen presentation. In addition, the subset of IgG that is secreted contributes to a significant decrease in the number of mast cells and eosinophils, which is accompanied by a decrease in the release of inflammatory mediators.

Allergen-specific immunotherapy can modulate various components of the immune system. Cellular changes include a decrease in allergen-induced T cell proliferation, indicating the induction of peripheral tolerance in allergen-specific T cells and a decrease in the antigen-specific Th2-dominant immune response in favor of a Th1 response with increased production of IFN-γ. The key cell type that is responsible for coordinating this immunological switch is a heterogeneous population of T cells called regulatory T cells (T _reg ). At the cellular level, a decisive factor for successful allergen immunotherapy is the peripheral induction of type 1 T _reg cells. Functional studies that have been performed on type 1 T _reg cells specific for antigen recognition have shown that the modulation of Th1 and Th2 responses to type 1 T reg cells is mainly dependent on the secretion of the cytokine IL-10, which has immunosuppressive properties. Indeed, IL-10 inhibits the proliferative response of peripheral T cells against specific allergens and plays a central role in the induction of T cell anergy. In vitro, IL-10 upregulates expression of the regulatory factor FoxP3, modulates eosinophil function, and reduces pro-inflammatory mediators released by mast cells.

Another possible marker of the exceptionally good clinical efficacy of said iMAT-mediated immunotherapy is the detection of a change in the number or pattern of allergen-specific T cells. Accordingly, for example, examination of Bet v1 tetramer staining, levels and characteristics of circulating birch pollen-specific CD4+ T cells could potentially be compared to those before and after SIT. Recently, transforming growth factor (TGF)-e has also been identified as a key cytokine in the successful implementation of SIT. Many actions can be taken into account to determine its relevance, such as the suppression of specific Th1, Th2 and Th17 cells, the induction of FoxP3 and suppressor function of T _reg . In addition, TGF-β downregulates FcεRI expression on Langerhans cells and downregulates IgE synthesis. These immunological responses mediated by immunologically relevant T cells can be measured in ex vivo experiments using peripheral blood mononuclear cell (PBMC) cultures and detection of their cytokines as described by Nuttall et al. (TJ Nuttall et al., Veterinary immunology and Immunopathology 84 (2002); 143-150).

Example 4 Comparison of an iMAT molecule according to the present invention with a prior art MAT molecule according to WO 2004/035793 (equivalent to US 2005/0281816)

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to assess the purity of the MAT protein (Thompson J et al., J Biol Chem 2002, 277: 34310-343). A method involving preparation of samples in the presence of a reducing agent, lithium dodecyl sulfate (LDS) and heating at 75° C. results in reproducible several clear bands after electrophoretic separation. Staining using Coomassie blue gives linear quantitative (densitometry) characterization of gels loaded with 200-1000 ng of protein. Using a monoclonal antibody that detects the allergenic module in the MAT molecule with Fel d1 as the allergenic module (MAT-Fel d1), it was shown that the main band and 13 minor bands all contain the MAT-Fel d1 protein. Small bands migrate to the same position as on the original gel, also after reloading onto the gel (FIG. 1). Several different methods, such as different temperature settings and formulation of the sample preparation buffer prior to starting PAGE, provide the same band image.

In all of these bands, the presence of full-length (whole) MAT-Fel d1 protein and only traces of host cell proteins can be demonstrated after each band is excised from the trypsin-digested gel and then analyzed by mass spectrometry (nano LC). /ERI-MS). From these experiments, it can be concluded that there is an anomalous feature (gel shear), for example, of different folding patterns of MAT-Fel d1 in SDS-PAGE. This means that MAT-Fel d1 in the analyzed preparation is not acceptable as a biopharmaceutical molecule, in particular for clinical and/or commercial biopharmaceutical production, since its purity cannot be determined using standard methods (for example, SDS-PAGE) , but only using a modified procedure described above.

In contrast to this MAT-Fel d1 gel shear phenomenon, Fel d1 per se does not exhibit such an abnormal function in SDS-PAGE (FIG. 2, lanes 2 and 3). Fel d1 shows one clear band at the expected molecular weight (19.6 kDa).

Another anomalous feature can be observed when analyzing using reverse phase HPLC. No MAT-Fel d1 peak was observed in this analytical method (FIG. 3) and no protein was observed either in native conformation or in denatured form induced by chaotropic and reducing conditions. However, for GMP-certified biopharmaceutical manufacture, the presence of a single isoform of a biomolecule in marketed pharmaceuticals is mandatory.

These observations in SDS-PAGE and reverse phase HPLC analysis can be explained by physicochemical properties based on the amino acid sequence. Analysis of the hydrophobicity diagram by Kyte and Doolittle [Kyte J, Doolittle RF, Journal of Molecular Biology 1982, 157(1), 105-132] revealed adjacent hydrophobic and hydrophilic end domains (FIG. 4) that may be responsible for this anomalous behavior.

In particular, the hydrophobic region of the targeting domain of the fusion protein is similar to transmembrane segments of membrane proteins that are known in the art to cause such abnormal function [Rath A et al., Proc Natl Acad Sci USA. 2009, 106(6): 1760-1765].

Migration on SDS-PAGE that does not correlate with formula molecular weight, called gel shear, appears to be common to membrane proteins. This means that the prescribed SDS-PAGE method, which is the separation of molecules solely according to their molecular weight, regardless of their native 2D or 3D structure, does not apply in these cases. In the above paper (PNAS paper), the authors investigate abnormal gel mobility for helical membrane proteins using a wild-type library and helix-loop-helix (hairpin) mutant sequences that are derived from human cystic fibrosis transmembrane conductance regulator (CFTR) transmembrane segments 3 and 4. ), including disease-associated phenotypic residue substitutions. They found that these hairpins migrated at rates of minus 10 to plus 30% of the molecular weights according to actual SDS-PAGE based formulas and loaded detergent at a ratio of 3.4-10 g SDS/g protein. In addition, they showed that mutant gel shears correlated strongly with changes in SDS hairpin loading capacity and with hairpin helicity, suggesting that gel shear behavior occurs with altered detergent binding. In some cases, this differential solvation using SDS may result from the replacement of protein-detergent contacts with protein-protein contacts, implying that detergent binding and folding are closely related to each other. SDS-PAGE (FIG. 5) as well as reverse phase HPLC analysis (FIGS. 3 and 6) of MAT and iMAT proteins revealed significant differences in migration or elution patterns, respectively. Oxidized forms of MAT-Fel d1 did not show any clear band on the SDS-PAGE gel (lane 3), but showed several smeared bands with higher and lower apparent molecular weights than the actual value of 32.2 kDa for MAT-Fel d1. In contrast, as an example, the iMAT molecule with the antigenic module of the culicoides obsoletus allergen (iMATCul o4) showed one clear band (M = 41.6 kDa) under oxidized conditions. In addition, the reverse phase HPLC chromatogram showed a single peak (FIG. 6). Under reducing conditions, the MAT-Fel d1 molecule in SDS-PAGE revealed a major band that migrated at approximately known molecular weight, but in addition to this, several minor bands, as described in FIG. 1 which were known to contain the complete MAT-Fel d1 sequence (FIG. 5, lane 6). This is a feature that is characteristic of the anomalous feature of MAT-Fel d1. In addition, the reverse phase HPLC chromatogram for MATFel d1 under reducing conditions (FIG. 3, right graph) shows at least 3 different isoforms of MAT-Fel. In contrast, iMAT molecules exhibit characteristics that appear to be indicative of a single isoform in SDS-PAGE (FIG. 5, lane 7) as well as in reverse phase HPLC (FIG. 6).

Reducing conditions lead to cleavage of the disulfide bridges in the MAT molecule, and thus the MAT and iMAT molecules should behave similarly under reducing conditions if the disulfide bridges are solely responsible for the abnormal characteristics of the MAT. However, this is not the case because the abnormal gel shift and isoform appearance in reverse phase HPLC when examining the MAT molecule is still present under reducing conditions. However, the iMAT molecule exhibits no such gel shift and peaks in the reverse phase HPLC chromatogram in the native (oxidized) form of the protein. In addition, the hydrophobicity diagrams of Kite and Dolittle of MAT and iMAT are almost identical at the N-terminal end spanning the sequence of the His-tag, TAT and targeting domain (FIG. 8). Thus, one skilled in the art would not be motivated to design a MAT molecule according to the prior art with cysteine residues substituted with other amino acid residues in order to overcome the shortcomings of the prior art.

The iMAT molecules can be designed according to the bioinformatics engineering procedures of Example 5 below and produced using recombinant expression technology in E. coli. As an example, three iMAT molecules, as shown in FIG. 7 were stable in buffer (20 mM citrate, 1 M arginine, pH 6.0) after freezing and thawing twice and can be adsorbed onto Adju-Phos® (Brenntag, Denmark) as an adjuvant so that

- 45 041116 iMAT molecules can be used as a vaccine. These proteins can be desorbed from

Adju-Phos® in the absence of degradation in the same buffer system (Fig. 7).

Example 5 Bioinformatic Engineering of iMAT Molecules: Selection of Proteins for the Antigenic Module and Optimization of the Whole iMAT Molecule

In order to effectively treat, for example, dogs and/or cats with allergic disorders with iMAT technology, it is additionally advisable:

a) select a protein as an allergenic module in iMAT molecules that is an allergen and thus has a high potential to cause hypersensitivity in affected subjects and thus also be a target for tolerance induction, and

b) design an iMAT molecule with the indicated allergens that is thermodynamically stable and can be efficiently obtained using protein engineering and can be further analyzed using standard methods in order to ensure a sufficiently high quality (i.e. identity, purity and potency).

To fulfill these requirements, a bioinformatic approach to the selection of the allergen to be included in the iMAT molecule in accordance with the present invention has been chosen. The purpose of selection is (i) to select one or more allergens that are assumed to be relevant for a given allergic disorder, i.e., that the majority of individuals suffering from an allergic disorder be sensitized to the relevant allergen, and (ii) to select the allergen with the highest the probability of including the characteristics of the linear epitopes of the allergen, i.e. inclusion of a large number of short peptide sequences (from 7 to 13 amino acid residues) homologous to those of published allergens.

In order to select appropriate antigens for pharmaceutical formulations, homology comparison based on local alignments with sequences of known allergens was chosen. Often the detection of an epitope for recognition by an antibody (mainly conformational epitopes) is achieved by functional analysis (eg, microarrays), and for T cell epitopes (linear epitopes) by calculating the probability of peptide binding to MHC molecules. The therapeutic principle of iMAT technology, among others, is based on endocytosis and degradation by acid-dependent proteases in endosomes, followed by MHC class II binding and antigen presentation. Thus, another non-experimental but bioinformatics approach is chosen for allergen selection, which is based on local homology searches for peptides that are derived from these proteins with respect to known allergenic proteins, most of which are known to cause allergies in humans. Amino acid sequences of proteins that are suspected to have allergenic properties are exported from public databases (eg UNIPROT) and redundancy is determined by sequence homology analysis in the exported data set. Highly homologous sequence analogs were eliminated and the resulting remaining sequences served as a canonical sequence database of likely valid antigens for further analysis. To identify proteins with a putatively high allergenic potential, the proteins were digested in silico into 6 to 15 amino acid long peptides with a single amino acid offset.

Subsequent pairwise alignments were then performed, for example, for dermatophagoides proteins and corresponding peptides with a database of canonical sequences. After that, the scaling of the matches obtained by alignment was carried out by setting the self-alignment score of this protein at the level of one, and matches when aligning the corresponding peptides, respectively. Next, the number of alignment matches that exceeded a predetermined threshold were counted for each and the peptide was compared by local pairwise alignment for randomly generated databases of protein sequences with no known allergenic properties, and then subjected to scaling and counting. The obtained values for the non-allergic protein were subtracted from those for the allergen and cumulative match scores for each protein were calculated based on the number of matches for all relevant peptides. Proteins with the highest values were chosen as candidates for the iMAT antigenic module.

Each of the selected allergens was integrated into a single iMAT molecule as an antigenic module and thereafter the complete iMAT molecule was optimized for thermodynamic stability by iteratively modeling the 3D structure of proteins and calculating free energy changes after single amino acid substitution. Physico-chemical properties and stability were influenced by the substitution of various amino acid residues within the primary amino acid sequence.

The results of the analyzes described in this application (search for antigen and modeling) were transformed into iMAT amino acid sequences acceptable for pharmacological production and use. In a specific example, this bioinformatics engineering approach identified Zen 1 and Der f 15 as being relevant for an allergic disorder, atopic dermatitis, caused by proteins that originate from the mite species dermatophagoides farinae. In addition, bioinformatic analysis revealed stable iMAT molecules with cysteines replaced by other amino acid residues. Examples of such stable iMAT molecules are SEQ ID NO: 39 (dog Zenl) and SEQ ID NO: 57 (dog Der f 15).

Example 6 Construction of Mosaic iMAT Molecules According to the Invention

It is expected that the iMAT molecule according to the invention is further improved if a component of more than one allergen is included in the antigenic module. For this purpose, the basic principle of the bioinformatic selection approach described above (Example 5) can be applied in another way. Instead of selecting complete allergens based on peptide match counts of allergens found in the allergen database, only the most frequently occurring peptides of several of these allergens are used to construct the iMAT antigen module. Thus, such an iMAT molecule consists of antigenic module peptides that are derived from several allergens. This allows the spectrum of a single iMAT molecule to be broadened with respect to its target allergic profile and thus is useful for drug development.

In order to find short peptide sequences that meet the criteria for such a mosaic iMAT molecule, proteins from eg dermatophagoides were analyzed by homology comparison as described above. Briefly, in silico cleaved proteins with a peptide length of 6-15 amino acid residues were locally aligned with a database of canonical sequences of allergen-related proteins and a randomly generated database of non-allergy-related proteins. A number of significant homology differences were determined for each peptide found in the canonical database. Subsequently, each peptide was subjected to local alignment with a randomly generated database that triples the size of the canonical database to reduce false positives. The upper portion (eg, tenth percentile) of the remaining homologies for each peptide length is particularly suitable to serve as a basis for constructing mosaic or hybrid iMAT molecules that carry the allergen. To construct the iMAT protein mosaic molecule, a precursor (eg, from a list of top ranked precursor proteins) was selected as the scaffold protein for introducing the top ranked peptides. This removes the signal peptide sequence from the scaffold protein, and top-ranked peptides, optionally with additional adjacent N- or C-terminal amino acids, can be inserted into the original scaffold protein sequence or can replace parts of the original scaffold protein sequence. The position for insertion or substitution is determined by using the similarity or reference position of the peptide in the corresponding precursor protein. As a next step, the His-tag, TAT and targeting domain are added. Finally, cysteine residues are replaced with the most stabilizing residues, as described above.

In a specific example (hybrid 1), the combination of SEQ ID NO: 84 (A1KXC1_DERFA DFP1 OS=Dermatophagoides farina), SEQ ID NO: 10 (Q86R84_DERFA 60 kDa allergen Der f 18p OS=Dermatophagoides farinae GN=Der f 18 PE=2 SV=1), SeQ ID NO: 85 (A0A088SAS1DERFA Der f 28 allergen OS=Dermatophagoides farinae PE=2 SV=1), SEQ ID NO: 88: (A7XXV2_DERFA Der f 2 allergen OS=Dermatophagoides farinae PE =4 SV=1), SEQ ID NO: 86 (B7U5T1_DERFA Derf 6 allergen OS=Dermatophagoides farinae PE=2 SV=1), SEQ ID NO: 87 (T2B4F3_DERPT LytFM OS=Dermatophagoides pteronyssinus GN=lytFM PE=4 SV= 1), SEQ ID NO: 11 (Q9U6R7_DERFA 98 kDa HDM allergen OS=Dermatophagoides farinae PE=2 SV=1) which is relevant for the allergic disorder atopic dermatitis caused by proteins that originate from the mite species dermatophagoides farinae. In addition, bioinformatic analysis revealed stable iMAT molecules with cysteines replaced by other amino acid residues. Examples of such stable iMAT mosaic molecules are SEQ ID NOS: 75 (Canine Hybrid 1) and SEQ ID NO: 66 (Feline Hybrid 1).

Example 7 Therapeutic Vaccine/Prophylaxis of Allergic Asthma in Cats

A single iMAT molecule or a combination of iMAT molecules containing different antigenic modules according to the present invention can be used prophylactically or therapeutically in a cat suffering from or at risk of developing allergic asthma. In cats, the iMAT molecules of the present invention are administered as described in Example 3.

Adult cats with a known history of allergic asthma were included in the study, that is, cats described for clinical signs such as spasmodic coughing fits, wheezing, and dyspnea with difficulty exhaling.

Bronchoalveolar lavage fluid (BALF) was collected before treatment and, for example, 2, 3, and 6 months during/after the study. BALF was used for cytology and scoring of nucleated cells. Cats were euthanized, for example, with intravenous ketamine NI. Bronchoalveolar lavage fluid was collected by gently inserting, for example, a 7 Fr (French) polypropylene catheter through the endotracheal tube. After a sense of resistance was achieved, up to a 20 ml aliquot of heated sterile saline was lavaged through the catheter and withdrawn by manual suction. After centrifugation and resuspension, a cytological smear of collected BALF cells was prepared, the presence of a significant amount of eosinophils confirming the diagnosis of feline asthma. By counting individual cell types, the ratio (%) of eosinophils in BAL fluids can be quantified.

Alternatively or additionally, using certain recombinant allergens, an intradermal provocation test, a skin prick test, or also an allergen-specific IgE and/or IgG determination in BAL fluid or serum can be monitored in these cats (Norris et al., Vet Immunol Immunopathol. 2003, 96(3-4): 119-127). Decreased response (immediate and/or late phase reactivity) and/or changes in antibody titers indicate the effectiveness of therapy and/or prevention when treated with iMAT molecules.

Clinical symptoms such as respiratory rate and respiratory effort/difficulty scores were used. This respiratory score system can also be used, for example, in response to an inhalation challenge. Briefly, spontaneously breathing and conscious cats were exposed to aerosolized recombinant allergens in a pressurized chamber for various time periods and/or various concentrations. Alternatively, airway hyperresponsiveness can be quantified in anesthetized cats.

Pneumotachograph measurements can be made at the time of entry into the study and in the broncho-provocation protocol, for example, the dose-dependent effect of pulmonary resistance on methacholine and/or selected recombinant allergens.

Thus, throughout the treatment period and/or after it, the effectiveness of the treatment or prevention of allergic asthma was investigated clinically using a quantitative, semi-quantitative or qualitative assessment.

Parameters can be compared for an individual cat with severity before starting a therapeutic intervention. Alternatively, comparison with untreated or placebo treated cats with allergic asthma demonstrates efficacy in treating and/or preventing the clinical symptoms of iMAT-mediated feline allergic asthma.

Example 8 Therapeutic Vaccine/Prophylaxis of Small Insect Allergy in Cats and/or Dogs

A single iMAT molecule or a combination of iMAT molecules containing different antigenic modules according to the present invention can be used prophylactically or therapeutically in a cat and/or dog suffering from or at risk of developing atopic dermatitis from flea bites.

In a first example, an iMAT molecule according to SEQ ID NO: 42 (Cte f 1 cats) was injected into the popliteal lymph node of cats suffering from or at risk of developing atopic dermatitis from flea bites. In a second example, an iMAT molecule according to SEQ ID NO: 63 (Cte f 1 dogs) was injected into the popliteal lymph node of dogs suffering from or at risk of developing atopic dermatitis from flea bites. Other details of the treatment were as described in Example 3 above. The efficacy of said iMAT treatment was assessed by intradermal test (IDT), T-cell assays, and measurement of flea allergen-specific IgE and IgG (Gerber J.D. Vaccine 1990-128(6):536-542) in treated cats and/or dogs before and after iMAT treatment as described by Jin (Jin J et al., Vaccine 28 (2010) 1997-2004). Intradermal tests (IDTs) were performed according to the protocol of Hillier and DeBoer, (DeBoer D.J., Hillier, A. Veterinary Immunology and Immunopathology 2001, 81 (3-4), 271-276). 4 weeks after the last immunization, cats and/or dogs were injected with 100 μl of PBS containing 100 μg of flea extract into the skin in the lateral thoracic region of cats and/or dogs intradermally; histamine was used as a positive control, BSA was used as an irrelevant stimulant, and saline was used as a negative control. The size of the reactive blister on the skin was marked with a marker pencil and measured perpendicularly and horizontally 20 minutes after provocation with a micrometer. The results were calculated as the average of three measurements. Decreased response (immediate and/or late phase reactivity) and/or changes in antibody titers or Th1 or Treg skew T cell responses are indicative of therapeutic and/or prophylactic effects of treatment with the iMAT molecule.

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Claims (15)

References (1) ^3rd Havemeyer workshop, Holar, Iceland, June 2007, Veterinary Immunology and Immunotherapy 2008, 126: 351-361 (2) Allergome (www.allergome.org) (3) Crameri R. et al., Allergy 2007 , 62: 197-206 (4) DeBoer, DJ, Hillier, A. Veterinary Immunology and Immunopathology 2001, 81(3-4): 271-276 (5) Gadermaier G et al., Molecular Immunology 2010, 47: 1292- 1298 (6) Gerber JD Vaccine 1990-12-8(6):536-542 (7) Griffin CE. Diagnosis of canine atopic dermatitis in Veterinary Allergy DOI: 10.1002/9781118738818. chlO (8) Guaguere E et al. EJCAP, 2009, 19(3), 234-241(9) Hill et al. Vet Immunol Immunopathol, 2001;81(3-4): 169-186(10) Hobi S, Mueller RS; Tierarztliche Praxis. Ausgabe K, Kleintiere/Heimtiere 2014, 42(3):167-173(11) Jackson HA, EJCAP, 2009, 19(3), 230-233(12) Jin J. et al. Vaccine 2010, 28: 1997-2004(13) Klein JS et al., Protein Eng Des Sei 2014, 27(10): 325-330 (14) Kyte J, Doolittle RF, Journal of Molecular Biology 1982, 157(1): 105-132 (15) Martinez-Gomez JM et al. ., Allergy 2009, 64(1): 172-178 (16) McCall C et al., Vet Immunol Immunopath 2001; 78:231-247 (17) McDermott MJ et al. Molecular Immunology 2000, 37: 361-375 ( 18) Norris et al., Vet Immunol Immunopathol. 2003, 96(3-4): 119-127(19) Nuttall T.J. et al., Veterinary immunology and Immunopathology 2002; 84: 143-150(20) Pires DE et al. Bioinformatics 2014,30(3): 335-342(21) Prost C, Rev Fr Allergol Immunol Clin, 2008, 48(5), 409-413(22) Rath A and et al., PNAS 2009, 106(6): 1760-1765 (23) Rose H, Arb Paul Ehrlich Inst Bundesinstitut Impfstoffe Biomed Arzneim Langen Hess 2009, 96: 319-327 (24) Senna G et al., Curr Opin Allergy Clin Immunol. 2011, 11(4):375-380(25) Senti G et al., J Allergy Clin Immunol. 2012, 129(5): 1290-1296 (26) SIAF Annual Report 2010 (27) SIAF Annual Report 2011 (28) Thomas WR et al., Chang Gung Med J 2004; 27: 563-569 (29) Thompson J et al., J Biol Chern 2002, 277: 34310-34331 (30) US 2005/0281816 (31) US 7,629,446 (32) US 7,653,866 (33) WHO PD-VAC 2014 - Status of Vaccine Research and Development of Vaccines for Leishmaniasis (34) WO 2004/035793 (35) Zhao et al. Int J Clin Exp Med 2015;8(4):6436-6443 CLAIM 1. A modular antigen transporter (MAT) molecule for the treatment or prevention of one or more allergies in animals other than horses, comprising: (I) one first module that has an amino acid sequence that allows translocation of the MAT molecule from the extracellular space into cells, (II) one second module that has an amino acid sequence that allows species-specific intracellular targeting of the MAT molecule to cell organelles, which are involved in the processing of antigens, and/or the loading of MHC molecules with antigens, (III) one third module as an antigenic module, which has an amino acid sequence that is derived from at least one epitope of at least one allergen, which determines the specificity of the immune response modulated by such a MAT molecule, - 49 041116 where the specified first module includes SEQ ID NO: 1; where the second module consists of SEQ ID NOs: 4 or 5 and where the third module consists of any sequence presented in SEQ ID NOs: 14-23. 2. A MAT molecule according to claim 1, wherein the third module comprises SEQ ID NO: 18. 3. The MAT molecule according to claim 1, where all modules are covalently linked to each other and where no additional(s) spacer(s) module(s) between two or more adjacent modules from the specified first, second and / or third modules are not present. 4. The MAT molecule according to claim 1, which further comprises one His tag module at the N-terminal end and/or C-terminal end, preferably one His tag at the N-terminal end after one methionine residue. 5. The MAT molecule according to claim 1, which includes any of the sequences of SEQ ID NO: 24-83. 6. The MAT molecule according to claim 5, wherein said sequence comprises SEQ ID NO: 36 (Der f15 feline MAT molecule), SEQ ID NO: 37 (Der f15 feline MAT molecule), SEQ ID NO: 38 (Der f15 feline MAT molecule ), SEQ ID NO: 57 (Der f15 dog MAT molecule), SEQ ID NO: 58 (Der f15 dog MAT molecule), SEQ ID NO: 59 (Der f15 dog MAT molecule). 7. The MAT molecule according to claim 5, which contains SEQ ID NO: 68 or SEQ ID NO: 77. 8. A modular antigen transporter (MAT) molecule for the treatment or prevention of one or more allergies in non-equine animals, comprising: (I) one first module that has an amino acid sequence that allows the translocation of the MAT molecule from the extracellular space into cells, containing SEQ ID NO: 1; (II) one second module that has an amino acid sequence that allows species-specific intracellular targeting of the MAT molecule to cell organelles that are involved in antigen processing and/or loading of MHC molecules with antigens, comprising SEQ ID NO: 4 or 5; and (III) one third module as an antigenic module that has an amino acid sequence that is derived from at least one complete or partial amino acid sequence of any combination of two or more allergens selected from the group consisting of SEQ ID NOs: 7, 8 , 9, 10, 11, 12, 84, 85, 86, 87 and 88, which determine the specificity of the immune response modulated by such a MAT molecule, where, at least in antigenic modules, all cysteine residues are replaced by various amino acid residues. 9. The MAT molecule according to claim 8, where the antigenic module is an amino acid sequence that is derived from at least one full or partial amino acid sequence of any combination of two or more allergens selected from the group consisting of SEQ ID NOs: 10, 11 , 84, 85, 86, 87 and 88 (Hybrid 1), or where the antigenic module is an amino acid sequence that is derived from at least one complete or partial amino acid sequence of any combination of two or more allergens selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, 11 and 12 (Hybrid 2), or where the antigenic module is an amino acid sequence that is derived from at least one full or partial amino acid sequence of any combination of two or more allergens selected from the group consisting of SEQ ID NOs: 7, 8, 10 and 11 (Hybrid 3). 10. The MAT molecule according to claim 9, where the antigenic module is an amino acid sequence based on the framework, which has the origin of SEQ ID NO: 84, containing any combination of one or more peptides in accordance with SEQ ID NOs: 91-96, built into the specified framework sequence, or where the antigenic module is an amino acid sequence that is derived from any combination of two or more peptides according to SEQ ID NOs: 97-102, or where the antigenic module is an amino acid sequence that is derived from any combination of two or more more peptides according to SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 101 and SEQ ID NO: 102. 11. A modular antigen transporter (MAT) molecule for the treatment or prevention of one or more allergies in animals other than horses, comprising an amino acid sequence selected from SEQ ID NO: 36 (Der f15 feline MAT molecule), SEQ ID NO: 37 ( Der f15 feline MAT molecule), SEQ ID NO: 38 (Der f15 feline MAT molecule), SEQ ID NO: 57 (Der f15 dog MAT molecule), SEQ ID NO: 58 (Der f15 dog MAT molecule), SEQ ID NO: 59 (Der f15 dog MAT molecule), SEQ ID NO: 66 (feline MAT 1 hybrid), SEQ ID NO: 67 (feline MAT 1 hybrid), SEQ ID NO: 68 (feline MAT 1 hybrid), SEQ ID NO: 75 (dog 1 MAT hybrid), SEQ ID NO: 76 (dog 1 MA hybrid) and/or SEQ ID NO: 77 (dog 1 MA hybrid). 12. An immunogenic composition containing a MAT molecule according to any one of claims 1-11. 13. A vaccine for the treatment or prevention of one or more allergies in animals, excluding horses, containing a MAT molecule according to any one of claims 1 to 11. 14. A nucleic acid which encodes a MAT molecule according to any one of claims 1-11. 15. The vector that provides the expression of the MAT molecule according to any one of claims 1-11, which contains -