Active immunization for treating atopic dermatitis

Abstract

An immunogenic fusion protein for active immunization or a bivalent vaccine for treating atopic dermatitis (AD) in a subject and methods thereof. The immunogenic fusion protein comprises an IL31 polypeptide and at least one substance P polypeptide (SP). The present invention is particularly useful in the treatment and / or prevention of AD and symptoms associated with AD, including itching, in mammals.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims the benefit of and priority to U.S. Patent Application No. 63 / 527,735, filed July 19, 2023, the entire contents of which are hereby incorporated by reference in their entirety. BACKGROUND TECHNICAL FIELD

[0003] The present invention relates to vaccine technology, immunotherapy, veterinary and medical. The present invention relates to recombinant fusion proteins and immunogenic compositions, methods for engineering and producing recombinant fusion proteins and their use in immunotherapy against diseases involving inflammation and / or chronic pruritus associated with inflammatory skin conditions such as atopic dermatitis (AD) in mammals, including dogs, cats, horses. The present invention relates to the use of immunogenic fusion proteins and immunogenic compositions in active immunization or vaccination and in methods for treating and / or preventing inflammatory related skin conditions and / or AD and its associated symptoms, including pruritus and secondary skin lesions. BACKGROUND

[0004] Atopic dermatitis (AD) is a chronic skin disease common in companion animals including dogs, cats and horses, characterized by inflammatory and pruritic skin lesions. AD is considered a multifactorial allergic disease triggered by environmental allergens involving allergen-specific IgE, which appears to affect genetically predisposed animals [2; 3]. AD is characterized by widespread pruritus, scratching, hair loss and secondary infections, affecting the quality of life and health of companion animals and their owners [4; 5]. AD is a chronic lifelong condition with partial onset and remission, and is often complicated by secondary infections.

[0005] The estimated prevalence of canine AD (CAD) is variable, but can affect 10% to 15% of dogs depending on breed, living conditions, climate and geographical region [5; 6; 7]. CAD is prone to occur in dogs between 6 months and 2 years of age. Clinically, CAD is characterized by moderate to severe pruritus, usually accompanied by localized erythema, erythematous papules, scratch-induced alopecia, scratch marks, skin thickening [8; 9]. Depending on the breed, skin lesions can be localized to the face, pinnae, ear canal, paws, axillary, abdomen and groin. The disease can be perennial or seasonal

[10] . Secondary microbial infections in affected skin areas are a common complication. Considering that millions of dogs are chronically affected by AD, the challenge currently faced by veterinary medicine is to develop effective, easy to administer and affordable therapies to achieve disease-modifying effects of AD.

[0006] AD-like syndromes also occur in cats and horses, which include skin inflammation, skin lesions, and chronic recurrent pruritus as the main signs. Common clinical manifestations of feline atopic dermatitis include localized skin lesions on the neck and head, including self-induced alopecia, excoriations, erosions, and ulcers [11; 12]. Complications can lead to more complex lesions, including plaques, granulomas, and chronic ulcers. Atopic dermatitis in horses typically presents as recurrent pruritus on the face, legs, or torso, associated with different skin lesions such as erythema, urticaria, papules, and alopecia [2].

[0007] Systemic treatments for inflammatory skin conditions, pruritus, and atopic dermatitis include a range of medications aimed at reducing inflammation, immune response, and severity of symptoms. Corticosteroids are commonly used for their anti-inflammatory effects, while immunosuppressants such as cyclosporine and methotrexate help control severe cases by suppressing the immune system. Biologies targeting the interleukin 31 pathway target specific pathways in the immune response and are effective for moderate to severe atopic dermatitis. Antihistamines can relieve pruritus, although their effectiveness can vary. Additionally, newer oral treatment approaches like Janus kinase (JAK) inhibitors (such as upadacitinib, baricitinib, or oclacitinib) offer targeted immune modulation with good efficacy for atopic dermatitis. These treatment approaches are often used in combination with topical therapies to manage symptoms and improve patients' quality of life.

[0008] In veterinary medicine, currently approved immunotherapies for the treatment of atopic dermatitis primarily include monoclonal antibody treatments and Janus kinase (JAK) inhibitors. Lokivetmab (Cytopoint) is a monoclonal antibody that targets and neutralizes interleukin-31 (IL31), a cytokine involved in the pruritus signaling pathway, thereby providing relief from pruritus in dogs. These treatments offer improvements in managing atopic dermatitis, focusing on reducing symptoms and improving the quality of life for affected animals.

[0009] Developmental active immunization-based therapies for the treatment of atopic dermatitis in veterinary medicine focus on stimulating an animal's immune system to recognize and combat allergens or specific immune components that cause the condition. These therapies often involve vaccines designed to induce an immune response against cytokines or other molecules involved in the inflammatory process. For example, vaccine studies targeting IL31, a key cytokine involved in pruritus signaling, are underway with the aim of reducing pruritus and inflammation over a long period.

[0010] Therapies developed for substance P, a substance associated with pruritus or atopic dermatitis, focus on blocking the neuropeptide neurokinin-1 receptor, which is known to play a significant role in transmitting pruritus signals and promoting inflammation. Substance P interacts with neurokinin-1 receptors (NK-1R) on skin cells and nerve fibers, thereby inducing itching and an inflammatory response. Therapies under development include NK-1R antagonists, which aim to inhibit the effects of substance P, thereby reducing itching and inflammation. These antagonists can be administered topically or systemically, and the efficacy and safety of some antagonists in treating atopic dermatitis are being evaluated in clinical trials. No approved or developed treatments based on vaccines or antibodies for blocking or neutralizing substance P have been reported.

[0011] To date, no studies have evaluated therapies that simultaneously target both IL31 and SP to control pruritus, skin inflammation, and neurogenic inflammation and to effectively treat, alleviate, and / or prevent AD-related symptoms and / or secondary skin lesions. Summary of the Invention

[0012] This invention relates to an immunogenic fusion protein comprising at least one IL31 polypeptide (IL31) or an immunogenic fragment thereof; and at least one substance, a P peptide (SP). The immunogenic fusion protein is capable of inducing the production of neutralizing polyclonal antibodies against IL31 and SP.

[0013] The present invention also relates to active immunization, peptide vaccines and immunogenic compositions for treating inflammation associated with AD, said inflammation being mediated by neuroimmune factors such as the pro-inflammatory and pruritogenic cytokine IL31 and the neuropeptide substance P (SP) that causes neurogenic inflammation and pruritus.

[0014] The immunogenic compositions of the present invention may be derived from canine, cat, horse or human sequences encoding IL31 and SP expressed as fusion proteins.

[0015] The immunogenic composition comprises at least one immunogenic fusion protein containing at least one IL31 sequence or an immunogenic fragment thereof linked to at least one SP sequence (i.e., IL31-1SP or IL31-3SP) via a flexible linker. The immunogenic fusion protein is non-natural, may lack the physiological activity of IL31 and / or SP, may be able to overcome immune tolerance to self-antigens, and induce the production of neutralizing antibodies against endogenous IL31 and SP. Notably, the immunogenic fusion protein of the present invention can have a synergistic immune effect, possessing bivalent capabilities against inflammatory pathways of the immune system and peripheral nervous system, thereby providing therapeutic and / or preventative benefits against inflammatory skin conditions such as AD, specifically pruritus.

[0016] Advantageously, when administered to mammals such as dogs, cats, horses, or humans, the immunogenic compositions of the present invention alleviate skin inflammation-related symptoms such as itching and reduce the need for systemic drug treatment (including corticosteroids, cyclosporine, antihistamines, or tyrosine kinase inhibitors). The immunogenic compositions according to the invention are preferably administered as bivalent vaccines targeting IL31 and SP. The immunogenic compositions may include at least one adjuvant to enhance the therapeutic immune response in immunized mammals.

[0017] This invention relates to the design and production of a bivalent non-natural fusion protein that, when used for active immunization, stimulates the production of neutralizing antibodies against the pro-inflammatory immune mediator IL31 and the neurogenic mediator substance P.

[0018] The immunogenic fusion protein of any of the foregoing embodiments induces the production of neutralizing antibodies in mammals and modulates the function of IL31 and SP.

[0019] When administered to mammals, the immunogenic fusion proteins of any of the foregoing embodiments produced higher neutralizing antibody titers compared to the combination of non-fusion IL31 peptides and non-fusion SP peptides when administered as a single agent.

[0020] The immunogenic fusion protein of any of the foregoing embodiments comprises at least one IL31 polypeptide, the at least one IL31 polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or an immunogenic fragment thereof.

[0021] The immunogenic fusion protein of any of the foregoing embodiments comprises at least one SP peptide, the at least one SP peptide comprising an amino acid sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0022] The immunogenic fusion protein in any of the foregoing embodiments contains one, three, or five SP peptides.

[0023] The immunogenic fusion protein of any of the foregoing embodiments comprises an IL-31 polypeptide or an immunogenic fragment thereof fused with one, three or five SP peptides.

[0024] The immunogenic fusion protein of any of the foregoing embodiments comprises at least one SP peptide fused to at least one IL31 polypeptide or its immunogenic fragment via a flexible amino acid linker GSGS.

[0025] The immunogenic fusion protein of the aforementioned embodiments comprises an IL31 polypeptide fused with an SP peptide.

[0026] The immunogenic fusion protein in the foregoing embodiments comprises an amino acid sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with one of SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 23, or SEQ ID NO: 29.

[0027] The immunogenic fusion protein of any of the foregoing embodiments comprises an IL31 polypeptide and three SP peptides linked by a flexible peptide linker.

[0028] The immunogenic fusion protein in the foregoing embodiments comprises an amino acid sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with one of SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 26, or SEQ ID NO: 32.

[0029] The present invention also relates to a recombinant vector comprising at least one nucleotide encoding the immunogenic fusion protein of any of the foregoing embodiments.

[0030] The recombinant vector described in the foregoing embodiments comprises a nucleotide sequence encoding an immunogenic fusion protein, wherein the nucleotide sequence has at least 90%, at least 95%, at least 99%, or 100% sequence identity with one of SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, or SEQ ID NO: 31.

[0031] The recombinant vector described in the foregoing embodiments comprises a nucleotide sequence having at least 90%, at least 95%, at least 99%, or 100% identity with one of SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 27, or SEQ ID NO: 33.

[0032] The present invention relates to a method for generating immunogenic fusion proteins of any of the foregoing embodiments in a prokaryotic or eukaryotic expression system.

[0033] The method of the foregoing embodiments, wherein the immunogenic fusion protein is obtained from inclusion bodies soluble in high molar concentrations of urea.

[0034] The method of any of the foregoing embodiments, wherein the recombinant vector comprises a nucleotide sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with one of SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:15, SEQ ID NO:21, SEQ ID NO:27, or SEQ ID NO:33.

[0035] The present invention also relates to an immunogenic composition produced according to the method of any of the foregoing embodiments.

[0036] The present invention also relates to an immunogenic composition comprising at least one immunogenic fusion protein of any of the foregoing embodiments.

[0037] The immunogenic composition of any of the foregoing embodiments further comprises an acceptable carrier and / or adjuvant, the adjuvant being selected from the group consisting of: oil-in-water adjuvants, polymer and water adjuvants, water-in-oil adjuvants, aluminum hydroxide adjuvants, and combinations thereof.

[0038] The immunogenic compositions described in the foregoing embodiments comprise at least one adjuvant selected from the following: complete or incomplete Freund's adjuvant, polymeric adjuvants such as Montanide. TM Gels, aluminum salts (alum), oil emulsions, saponins, immunostimulatory complexes (ISCOM), liposomes, microparticles, nonionic block copolymers, derived polysaccharides, cytokines, or bacterial derivatives.

[0039] The immunogenic compositions of any of the foregoing embodiments can be used as pharmaceuticals, preferably vaccines.

[0040] The immunogenic compositions described in the foregoing embodiments can be used to treat and / or prevent pruritus-related skin conditions, preferably AD, and most preferably inflammation in chronic and / or refractory AD.

[0041] The immunogenic compositions described in the foregoing embodiments can be used to treat AD that is refractory to at least one anti-inflammatory compound selected from the group consisting of: corticosteroids, antihistamines, tyrosine kinase inhibitors, and cyclosporine.

[0042] The present invention also relates to a pharmaceutical composition for treating or preventing AD and / or AD-related symptoms in an individual, comprising at least one immunogenic fusion protein of any of the foregoing embodiments and a pharmaceutically acceptable carrier.

[0043] The pharmaceutical compositions described above can be used to relieve AD-related symptoms, such as itching and / or secondary skin lesions.

[0044] When AD is refractory to at least one anti-inflammatory compound selected from the group consisting of: corticosteroids, antihistamines, tyrosine kinase inhibitors, and cyclosporine, the pharmaceutical composition of the foregoing embodiments may be used.

[0045] The present invention also relates to a method for treating or preventing AD and / or AD-related symptoms in mammals, the method comprising administering an effective amount of the immunogenic composition of any of the foregoing embodiments.

[0046] In the methods of the foregoing embodiments, AD-related symptoms may include itching and / or secondary skin lesions.

[0047] In any of the methods described in the foregoing embodiments, applying an effective amount of the immunogenic composition reduces the pruritus score. The pruritus score is determined using methods such as... Figure 6A It is determined using a defined itch scale (e.g., scratching, biting, licking, and rubbing objects or the floor).

[0048] In any of the methods described in the foregoing embodiments, the AD-related symptoms may be refractory to at least one anti-inflammatory compound selected from the group consisting of: corticosteroids, antihistamines, tyrosine kinase inhibitors, and cyclosporine.

[0049] In any of the methods described in the foregoing embodiments, the immunogenic composition is administered in combination with at least one anti-inflammatory compound selected from the group consisting of: corticosteroids, antihistamines, tyrosine kinase inhibitors, and cyclosporine.

[0050] In any of the methods described in the foregoing embodiments, the immunogenic composition is administered in combination with at least one receptor antagonist that blocks nociceptive signaling pathways, preferably a neurokinin-1 receptor (NK1-R) antagonist, and most preferably the compound CP-96,345.

[0051] In any of the methods described in the foregoing embodiments, the immunogenic composition may be administered orally, subcutaneously, intramuscularly, or transdermally, preferably subcutaneously.

[0052] The method of any of the foregoing embodiments includes administering an immunogenic composition at an initial dose, followed by three booster doses at intervals of approximately 2 weeks, and optionally, one or more booster doses at intervals of 3 to 6 months for a period of time required to maintain high and neutralizing antibody titers.

[0053] The methods of any of the foregoing embodiments can be performed in mammals (such as dogs, humans, cats, or horses).

[0054] A method for treating or preventing Alzheimer's disease (AD) or AD-related symptoms in dogs comprises subcutaneously administering a bivalent vaccine at an initial dose, followed by three booster doses at 2-week intervals, and optionally one or more booster doses at 3 to 6 months intervals or for a period of time required to maintain high and neutralizing antibody titers, wherein the bivalent vaccine comprises an immunogenic fusion protein IL31-SP or an immunogenic fusion protein IL31-3SP, wherein the immunogenic fusion protein IL31-SP comprises an amino acid sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO: 11, and wherein the immunogenic fusion protein IL31-3SP comprises an amino acid sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO: 14.

[0055] In any of the methods described in the foregoing embodiments, according to Figure 6A The pruritus scores (e.g., scratching, biting, licking, and rubbing objects or the floor) assessed by the pruritus scale defined in the study were reduced in treated mammals.

[0056] In the method of the foregoing embodiments, the pruritus score is in the range of 1 to 3.

[0057] This invention relates to a bivalent vaccine for the treatment or prevention of inflammation-related skin conditions, preferably Alzheimer's disease (AD) or AD-related symptoms, including itching, erythema, and / or related hair loss in dogs, wherein the vaccine comprises at least one immunogenic fusion protein of any of the foregoing embodiments.

[0058] The bivalent vaccine of the foregoing embodiments contains an immunogenic fusion protein IL31-SP or an immunogenic fusion protein IL31-3SP, wherein the immunogenic fusion protein IL31-SP contains an amino acid sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO: 11, and the immunogenic fusion protein IL31-3SP contains an amino acid sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO: 14. Attached Figure Description

[0059] Figure 1 is a schematic diagram illustrating the pathophysiological effects of IL-31 and substance P on pruritus and inflammation in Alzheimer's disease (AD), and explaining the mechanism by which vaccine-induced neutralization reduces chronic inflammation. (Adapted from Ständer and Yosipovitch, 2019 [1]). A) IL-31-induced pruritus perception pathway and the role of substance P. This figure illustrates the IL-31-induced pruritus perception pathway, highlighting the involvement of substance P and its receptors. Top small image: Diagram showing the epidermis and subcutaneous dermis, indicating the presence of keratinocytes, immune cells, and nerve endings. Allergen exposure and epidermal inflammation lead to the production and release of IL-31 by immune cells. Middle small image: Enlarged view detailing the interaction between IL-31, substance P, and its receptors. IL-31 produced by inflammatory immune cells acts on IL-31 receptors located on peripheral sensory nerve endings. This interaction stimulates the release of substance P from the sensory nerve endings. Bottom small image: Pathway for sensory signals to be transmitted from peripheral nerve endings to the brain. The released substance P binds to receptors on secondary neurons in the dorsal horn of the spinal cord. These neurons then transmit itch signals to the brain, generating the perception of itch. B) Schematic illustration of the vaccination process that neutralizes IL-31 and substance P in the host. The process begins with administration of a non-natural recombinant fusion protein to the host (1), which triggers the production of neutralizing antibodies against IL-31 and substance P (2). These antibodies then neutralize inflammatory mediators in the skin (3), resulting in a significant reduction in chronic inflammation, a reduction in itching and scratching behavior, and an overall improvement in the host's quality of life (4).

[0060] Figure 2 illustrates the construction, expression, and purification of the recombinant fusion proteins IL31-1SP and IL31-3SP. Inset A shows gene constructs linked by GSGS adapters, each containing a 6xHis tag, a TEV site, and the IL31 sequence, along with one or three SP fragments. Inset B outlines the expression process in *E. coli* BL21, followed by purification, endotoxin removal, and quality testing. Inset C presents SDS-PAGE analysis confirming the expected molecular weight of the purified protein. Inset D shows mass spectrometry data confirming the molecular weight and sequence coverage, validating the correct assembly of the IL31 and SP fragments.

[0061] Figure 3. This figure illustrates the immunogenic response in mice vaccinated with IL31-1SP and IL31-3SP. Inset A details the immunization regimen, where group 1 received IL31-1SP and group 2 received IL31-3SP, with three booster doses in each group. Inset B shows a significant increase in IgG titers specific to IL31-1SP, IL31-3SP, native IL31, and SP following vaccination in both groups. Inset C demonstrates that the presence of competitive antigens (non-convergent canine IL31 and SP) reduced IgG binding to IL31-3SP in a dose-dependent manner, confirming the specificity of antibodies against IL31 and SP (*p < 0.05, **p < 0.01).

[0062] Figure 4 illustrates the immunogenic response in mice vaccinated with IL31-3SP compared to a combination of non-fusion canine IL31 and non-fusion canine SP. Inset A outlines the immunization regimen, where group 1 received IL31-3SP and group 2 received both non-fusion IL31 and SP, with initial doses administered at 2-week intervals, followed by three booster doses. Inset B shows significantly higher IgG titers specific to native canine IL31, native canine SP, and IL31-3SP in the IL31-3SP vaccination group compared to the group receiving the non-fusion protein. This indicates that the immunogenic properties of the IL31-3SP fusion protein are superior to those of its components administered alone, demonstrating enhanced antibody responses against both native IL31 and SP. Statistical significance is indicated by *p < 0.05.

[0063] Figure 5 illustrates the immunogenic response in dogs vaccinated with IL31-1SP. Inset A outlines the immunization regimen in which dogs received a primary dose of 5 mg IL31-1SP, followed by three booster doses at 2-week intervals, and indicates blood collection and clinical evaluation points. Inset B shows the specific antibody titer against natural canine IL31, demonstrating a significant increase after vaccination in all dogs. Inset C shows the specific antibody titer against natural canine SP, also showing a significant increase after vaccination. This indicates that the IL31-1SP recombinant fusion immunogen effectively induced a strong immune response against both IL31 and SP in vaccinated dogs.

[0064] Figure 6 evaluates the effectiveness of IL31-1SP treatment in customer-owned dogs with atopic dermatitis. These results indicate that IL31-1SP effectively reduces pruritus and corticosteroid use in dogs with atopic dermatitis. The severity of pruritus and corticosteroid use were evaluated in dogs affected by atopic dermatitis who were vaccinated with the recombinant fusion immunogen IL31-1SP. A. Scoring system for measuring pruritus severity: A 1-10 scale was used to assess the level of pruritus in dogs with atopic dermatitis. The scale categorized symptom severity, minor symptoms, and recommended treatments. Scores ranged from 1 (non-invasive, sporadic scratching) to 10 (compulsive scratching requiring an E-collar). B. Scratch scores before and after IL31-1SP treatment: The figure shows the scratch scores of dogs before treatment with IL31-1SP and at 1 month, 1.5 months, and 2 months after treatment. Data showed that scratching scores decreased significantly over time, with the most significant reduction observed at 2 months post-treatment (*p < 0.05). C. Number of dogs receiving corticosteroids: The bar graph shows the number of dogs requiring corticosteroids before vaccination and 90 days after vaccination with IL31-1SP. The number of dogs receiving corticosteroids was significantly reduced 90 days post-vaccination, indicating symptom improvement and a reduced need for corticosteroid treatment. Detailed Implementation

[0065] This invention relates to a comprehensive method for treating Alzheimer's disease (AD) based on the synergistic effect of simultaneously controlling excess IL31 and SP-mediated neurogenic inflammation. Specifically, embodiments relate to an autoantigen vaccine based on an immunogenic fusion protein or immunogen containing IL31 linked to SP via a flexible linker. When administered as a vaccine to mammals, the immunogen triggers an immune response, including the production of antibodies against both native IL31 and SP, with the potential to neutralize excess levels of these two peptides in AD and / or pruritus. Advantageously, the use of the immunogenic fusion protein IL13-1SP or IL31-3SP in AD treatment leads to control of neurogenic inflammation and pruritus by inducing a synergistic antibody response against IL31 and SP. Specifically, it provides long-term relief of pruritus and scratching in immunized animals.

[0066] The pathophysiology of AD is complex and not fully understood. Canine AD is known to be a multifactorial disease involving a combination of genetic, environmental and immune factors

[10] . The immune system plays a key role in the development and progression of AD, with abnormal immune responses to environmental allergens being the main trigger. Abnormal immune responses are closely associated with functional defects of keratinocytes and the skin barrier, as well as with hypersensitivity and pruritus signals of the nerve fibers that innervate the skin

[13] . Keratinocytes influence the interaction between dendritic cells and lymphocytes, in which thymic stromal lymphopoietin plays a role in linking skin barrier damage with the regulation of T helper (Th)2 responses, and an imbalanced immune response characterized by elevated Th2, Th17 and CD4+CD25+ regulatory T cells has been documented

[14] .

[0067] The biological causes of pruritus and skin inflammation in AD are thought to be mediated by a complex interface between sensitized Th2 lymphocytes in the skin, keratinocytes, and pruritogenic nerve fibers projecting into the CNS. However, there are currently no specific therapeutic agents that address the neurogenic component of AD. In fact, an antagonist of the neurokinin-1 receptor (NK1-R), which blocks the nociceptive signaling pathway, has been shown to have no significant improvement in AD, except in one study using an animal model.

[59]

[0068] Treatment for AD in companion animals remains limited, given that the disease is a chronic allergic condition with no definitive cure. The main treatment goals include environmental management, allergen avoidance, hygiene measures, topical therapy, and allergen-specific immunotherapy. In recent years, with increasing complexity, drug treatment for canine AD has made progress. Systemic drug therapy, including corticosteroids, antihistamines, cyclosporine, or tyrosine kinase inhibitors targeting the JAK1-dependent pathway, has been shown to control skin inflammation and relieve itching and scratching symptoms, although long-term use may lead to side effects [4].

[0069] Given its role as a neuroimmune mediator, passive immunotherapy targeting cytokines such as IL31 has recently been developed for clinical veterinary use. Lokivitumab (Cetorum, Zoetis) is a canine IL31 monoclonal antibody that has been approved for the treatment of CAD. It provides durable relief of itching in dogs with AD and atopic dermatitis [49; 50; 51; 52; 53; 54; 55]. In human patients with AD, nemolizumab, a humanized monoclonal antibody targeting the IL31 receptor (IL31RA), was recently approved in Japan

[56] . Systemic immunization using IL31-based vaccines has also been shown to improve AD symptoms in dogs

[57] and allergic skin lesions from insect bites in horses

[58] , although these vaccines are still under development and use virus-like particle technology. Despite recent advances in the treatment of CAD, the management of the disease in dogs remains difficult and expensive. Furthermore, the significant cost of anti-IL31 monoclonal antibodies and the need for frequent and prolonged dosing limit their use in a small subset of affected animals worldwide. Unfortunately, the failure of low-cost, conventional therapies has led to decreased quality of life and medical complications for many patients.

[0070] IL31 signaling plays a pathogenic role in the development of pruritus and atopic dermatitis-like skin lesions

[17] . IL31 belongs to the cytokine IL-6 family and is primarily expressed during inflammatory and immune-related processes

[18] . Several studies have shown that IL31 is expressed by immune cells, including sensitized effector Th2 cells, eosinophils, basophils, mast cells, monocytes, macrophages, and dendritic cells, with effector memory Th2 cells representing the major source of IL31 [19; 20; 21; 22; 23]. Immunosensitization of IL31-producing effector Th2 cells in AD is a complex process involving the recognition of allergens by antigen-presenting cells from the skin, activation of T cells, production of IgE antibodies by B lymphocytes, and release of pro-inflammatory mediators upon re-exposure to the allergen

[24] . Upon release, IL31 binds to a heterodimer receptor composed of the IL31RA chain, which is expressed in dorsal root ganglion neurons that transmit pruritus and in their cutaneous nerve endings, but also in other cell types such as keratinocytes, fibroblasts, and other sensory neuronal subsets [25; 26]. In IL31RA+ neurons, IL31 activates the ion channels TRPV1 and TRPA1, which mediate intracellular Ca2+ mobilization

[23] . These neurons are thought to signal to the dorsal horn of the spinal cord and then to projection neurons that transmit information to the brain

[27] . In addition, IL31 also induces a unique transcriptional program in sensory neurons, leading to the sprouting and branching of nerve endings in the skin of AD [28; 29]. The ability of IL31 to increase neuronal network density and activate ion channels in nerve endings may explain the increased sensitivity to subtle stimuli that induce AD ​​pruritus and trigger neurogenic inflammation. Other cellular targets of IL31 include keratinocytes and dermal fibroblasts. IL31 prevents the normal differentiation of keratinocytes and thus leads to the disruption of the skin's barrier function

[30] . IL31 also stimulates keratinocytes and dermal fibroblasts to produce cytokines, chemokines and pruritus mediators, thereby exacerbating skin inflammation and tissue remodeling

[31] .

[0071] Neurogenic inflammation in AD is mediated by the release of neuropeptides, including SP, from the nerve endings of primary afferent sensory C-fibers innervating the skin [1; 32]. Upon release, SP interacts locally with surrounding endothelial cells, keratinocytes, macrophages, and mast cells, thereby inducing an inflammatory phenotype

[33] . Abundant SP-positive nerve fibers are observed in the damaged skin of patients with AD, but not in healthy controls [34; 35]. In pruritus skin, SP-positive nerve fibers near the skin-epidermal junction and SP receptor NK1R are overexpressed

[36] . In addition, SP-positive fibers are spatially associated with mast cells in AD and may induce mast cell degranulation, increasing the release of histamine and serotonin, proteolytic enzymes, and cytokines and trophic factors

[37] .

[0072] Neurogenic inflammation is associated with AD and is mediated by the release of neuropeptides such as substance P (SP) from sensory nerve endings in the skin [15; 16]. Therefore, increased IL31 expression from immune cells and keratinocytes, along with increased SP release from nerve endings, may synergistically mediate AD symptoms, including pruritus, skin inflammation, and remodeling of affected epithelium. While IL31 induces pruritus and inflammation, substance P, as a neuropeptide, participates in localized neurogenic inflammation and indirectly transmits pruritus signals by inducing mast cell degranulation (which contains pruritogenic histamine). There is evidence that the binding of IL31 to receptors on its sensory nerve fibers triggers the release of SP, which further enhances pruritus perception and promotes inflammatory processes in atopic dermatitis. It is likely that this interaction between IL31 and substance P leads to the persistent pruritus and chronic inflammation characteristic of the disease. In addition, there is evidence that the neural innervation patterns behind chronic pruritus and inflammation associated with AD are significantly altered

[38] . Repeated scratching and rubbing of the affected skin may lead to overgrowth or proliferation of nerve fibers, particularly those that transmit pruritus signals to the brain. Evidence suggests that increased levels of neuropeptide NGF and histamine in AD skin stimulate the growth and proliferation of nerve fibers, including the upregulation of ion channels and SP

[39] . Over time, these nerve fibers become more abundant and sensitive, leading to enhanced pruritus perception, which in turn leads to a vicious cycle of scratching and further skin damage. Thus, substance P is currently considered one of the key pruritogenic inflammatory factors in AD[40; 41]. As a mediator of pruritus signaling, SP acts on NK1R expressed in skin cells and the central nervous system. Antagonists of SP-NK1R signaling have been shown to disrupt pruritus behavior to some extent[1; 42]. SP and neurogenic inflammation are considered key mediators of stress-induced skin inflammation

[16] . In addition, SP is known to stimulate degranulation of skin mast cells via the receptor Mas-associated G protein-X2 (MRGPRX2), which may also play a pathogenic role in AD[43; 44].

[0073] In humans, topical or intradermal application of SP is sufficient to induce rash and pruritus [33; 45; 46; 47]. In mice, dermal injection of SP resulted in a dose-dependent increase in scratching at the injection site, and skin scratching led to an increase in the number of SP-immunoreactive cutaneous nerve fibers

[36] , further suggesting a link between sensitized cutaneous nerve endings and SP-mediated neurogenic inflammation. However, various reports have found little or no therapeutic effect from drug blocking of NK1R in AD [1], suggesting that alternative receptors, such as those expressed in mast cells, may be the primary signaling pathway for SP in AD [44; 48]. Thus, IL31 and SP depict a complex and interdependent pathological axis in AD. While IL31 acts on a variety of cells in the skin, including keratinocytes, immune cells, and sensory nerves, to induce pruritus, it also induces long-term changes in sensitized cutaneous nerve endings. In turn, SP released from nerve endings can activate mast cells, keratinocytes, endothelial cells, and immune cells to induce local inflammation and further infiltration of immune cells.

[0074] Synergistic effects of IL31 and SP in an AD pruritus and inflammation model (Figure 1). Following allergen exposure, immune cells in the skin release IL31, which leads to the transmission of pruritus signals to the CNS via sensory C-fibers. Additionally, IL31 increases the expression of other pruritus-related molecules, such as the TRPV1 ion channel and neuropeptides, which contribute to increased density of sensory nerve endings in AD skin. Activated C-fibers release substance P (SP) at nerve endings, thereby inducing neurogenic inflammation and vasodilation. Histamine and cytokines released by immune cells in response to SP amplify key AD triggers, perpetuating skin inflammation. Neurogenic and neuroendocrine mechanisms induced by psychosocial stress can further promote immune cell activation. Therefore, IL31 and SP work together to enhance pruritus signaling and perception, leading to scratching behavior and exacerbation of skin inflammation in AD. Neutralizing excess levels of IL31 and SP with therapeutic antibodies induced by IL31-1SP or IL31-3SP vaccines can improve AD symptoms and relapses, reduce scratching, and restore the skin's natural barrier to allergens. (Figure 1)

[0075] Some embodiments of the present invention are discussed in detail below. Specific terminology is used in the description of the embodiments for clarity. However, the invention is not intended to be limited to the specific terminology chosen. Those skilled in the art will recognize that other equivalent components and other methods can be used and developed without departing from the broad concept of the invention. All references cited anywhere in this specification, including the background and detailed description sections, are incorporated by reference as if each were individually incorporated.

[0076] Definitions are included herein for the purpose of understanding the subject matter and the appended claims. Abbreviations used herein have their conventional meanings within the chemical and biotechnological context.

[0077] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. See, for example, Singleton et al., *Dictionary of Microbiology and Molecular Biology*, 2nd ed., J. Wiley & Sons (New York, NY 1993); Sambrook et al., *Molecular Cloning, A Labours Manual*, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, apparatus, and materials similar to or equivalent to those described herein may be used to practice the invention. The following definitions are provided to facilitate understanding of certain terms frequently used herein and are not intended to limit the scope of this disclosure.

[0078] This specification identifies certain nucleotide and amino acid sequences (polynucleotides and polypeptides) as part of this invention. It should be understood that the specifically identified sequences adequately describe other sequences containing less than 100% sequence identity, but differ from the identified sequences that provide the same function. For example, a nucleotide sequence may have 90% or 95% sequence identity with a polynucleotide specifically disclosed herein and still encode a fully equivalent or functionally equivalent polypeptide. Similarly, a polypeptide may contain less than 100% sequence identity with a polypeptide specifically identified herein and provide the same function. For example, a polypeptide may have 90%, 95%, or 99% sequence identity with a polypeptide specifically disclosed herein and still retain the same or sufficiently similar activity or function as the specifically identified polypeptide.

[0079] As used throughout the text, the term "gene" refers to a nucleotide sequence or portion thereof that plays a functional role in protein coding, transcription, or regulation of the expression of other genes. A gene may consist of all the nucleotides encoding a functional protein or a portion of the nucleotides encoding or expressing said protein. The nucleotide sequence may include exons, introns, start or stop regions, promoter sequences, other regulatory sequences, or gene mutations in unique sequences adjacent to the gene.

[0080] As used throughout, the term "neurogenic mediator or factor" refers to neuropeptides, including but not limited to SP, that directly or indirectly promote inflammation. In the context of this invention, the release of neurogenic factors into the skin contributes to the development of inflammation that leads to atopic dermatitis and pruritus.

[0081] As used throughout, the term "immune factor or mediator" refers to a polypeptide whose biological activity affects the immune system and inflammation, specifically a cytokine such as IL31. In the context of this invention, immune factors, such as IL31, have pro-inflammatory and pruritus-inducing activities.

[0082] As used throughout the text, the term "immunogenic fragment or immunogen" refers to an amino acid sequence that has the ability to induce humoral and / or cell-mediated immune responses. For example, an immunogenic fragment derived from IL31 can trigger the production of antibodies against IL31.

[0083] As used throughout the text, the term "neurogenic inflammation" refers to the physiological process by which mediators are released directly from sensory nerves to trigger an inflammatory response. This results in a local inflammatory response, including erythema, swelling, fever, tenderness, and pain. The fine unmyelinated somatic C-fibers, which respond to low-intensity mechanical and chemical stimuli, are primarily responsible for the release of inflammatory mediators. When stimulated, these nerve fibers in cutaneous nerves rapidly release active neuropeptides such as SP into the microenvironment, thereby triggering a series of inflammatory responses.

[0084] As used throughout, the term "antibody or immunoglobulin" refers to a protein produced by B cells of the immune system that can identify, bind to, and neutralize antigens. In the context of this invention, antibodies are produced by the immune system and bind to endogenous proteins or peptides, such as neurogenic mediators or cytokines. Antibodies may have neutralizing properties and may be able to inhibit or reduce the biological activity of downstream pathways mediated by neurogenic mediators or cytokines, such as blocking their binding to specific receptors.

[0085] As used throughout, the term "autoantigen" refers to any molecule or chemical group of an organism that, as an antigen, induces antibody production in another organism, but is tolerated by the healthy immune system of the parent organism. Immunization / vaccination against autoantigens requires specific design of immunogens and formulations to allow vaccination to break the autotolerance of a particular organism. Inducing a durable immune response against autoantigens is extremely challenging due to several central and peripheral tolerance mechanisms. In the context of this invention, autoantigens are immune mediators such as endogenous IL31 and neurogenic mediators such as SP.

[0086] As used throughout, the term "active immunization" refers to immunity that stimulates the immune system to produce antibodies against self-antigens. Active immunization can be induced through vaccination. In the context of this invention, a bivalent vaccine or immunogenic composition comprises at least one immunogenic fusion protein containing at least two self-antigens or endogenous peptides, such as IL31 and SP. When injected into a mammal, the vaccine induces the production of polyclonal antibodies against different epitopes without causing any disease or uncontrolled side effects. Such antibodies may have neutralizing or therapeutic properties that capture self-antigens, thereby modulating their respective functions. Active immunization is typically durable and can be reactivated by repeated injections of boosters. In contrast, passive immunization occurs when antibodies against a specific antigen are administered to an individual.

[0087] As used throughout this text, the term "adjuvant" refers to a substance that, when co-administered with an immunogen, enhances the strength of the immune response. Adjuvants can act as immune enhancers, enabling immunogenic compositions or vaccines to induce an effective and sustained immune response while reducing the dosage and amount of enhancer. Adjuvants can also increase the stability of immunogenic compositions or vaccines.

[0088] As used throughout the text, the terms "recombinant protein" or "recombinant polypeptide" are used interchangeably and refer to a protein or polypeptide encoded by a recombinant nucleotide that has been cloned into an expression vector supporting gene expression and messenger RNA translation. *Escherichia coli* (bacteria) is one of the preferred organisms for producing recombinant proteins. Its use as a cell factory is established, and it has become the most popular expression platform. High-level expression of many recombinant proteins in *E. coli* leads to the formation of highly aggregated proteins commonly referred to as inclusion bodies. Inclusion bodies typically form in the cytoplasm. Bacterial inclusion bodies are common mesoscale protein aggregates in recombinant bacteria and are primarily formed from recombinant proteins. Other expression systems may include, but are not limited to, insect cells and yeast cells.

[0089] As used throughout the text, the term "fusion protein or fusion polypeptide" refers to a hybrid protein or polypeptide having an amino acid sequence comprising at least two partial or complete sequences derived from, obtained from, or isolated from non-naturally adjacent different polypeptides. The terms "fusion" and "chimerism" are used interchangeably throughout the text. A fusion protein or fusion polypeptide is a functional product of a fusion gene or fusion nucleotide sequence. Fusion genes can be further modified by mutation, deletion, insertion, or substitution of heterologous sequences or by any available method using recombinant DNA technology.

[0090] As used throughout, the term "fragment" refers to a peptide or polypeptide that is a chain polymer formed by at least six amino acid residues linked together by peptide bonds. The fragment may also comprise the complete amino acid sequence of a natural polypeptide or protein. The fragment may include amino acid sequences as conserved variations. The terms fragment, peptide, and polypeptide are used interchangeably. In the context of this invention, an immunogenic fragment is a peptide or polypeptide capable of evoking a cellular and / or humoral immune response, including the production of specific antibodies against the immunogenic fragment or a protein having the immunogenic fragment. Those skilled in the art can introduce substitutions to obtain immunogenic fragments with higher immunogenicity. For example, one aspect of this application discloses fragments corresponding to amino acid sequences (e.g., SEQ ID NOS: 1-9), as well as their analogs, homologues, isomers, derivatives, amidated variants, and conserved variants, provided that the fragment retains immunogenicity. The IL31 peptide may comprise at least one immunogenic fragment derived from the canine IL31 peptide defined as SEQ ID NO: 1, the equine IL31 peptide defined as SEQ ID NO: 2, the human IL31 peptide defined as SEQ ID NO: 3, or the feline IL31 peptide defined as SEQ ID NO: 4. The SP peptide may comprise one of the following: the canine SP peptide defined as SEQ ID NO: 5, the equine SP peptide defined as SEQ ID NO: 6, the mouse SP peptide defined as SEQ ID NO: 7, the human SP peptide defined as SEQ ID NO: 8, or the feline SP peptide defined as SEQ ID NO: 9. Minor modifications to the primary amino acid sequence disclosed herein, compared to the specific fragments described herein, can produce fragments with substantially equivalent or enhanced immunogenicity. Such modifications may be intentional (e.g., by site-directed mutagenesis) or spontaneous.

[0091] All peptides, polypeptides, or fragments can be synthesized using L-amino acids, but the D-form of all peptides can be synthesized. Additionally, according to one embodiment disclosed in this application, C-terminal derivatives, such as C-terminal methyl esters and C-terminal amide esters, can be generated to increase the immunogenicity of the peptide.

[0092] This invention relates to a fusion of the endogenous peptides IL31 and SP (i.e., autoantigens) as a peptide-based vaccine for active immunization of mammals suffering from inflammatory skin conditions, pruritus (itching), and dermatitis. The recombinant fusion protein is derived from inclusion bodies and is soluble in high molar concentrations of urea (4-8 M). Sequence and biophysical properties support the fact that the recombinant fusion immunogen exhibits a non-natural conformation not naturally occurring. These characteristics allow the recombinant fusion protein to possess high immunogenicity, exceeding natural immune tolerance to autoantigens such as native IL31 and SP, while also possessing the unexpected ability to elicit therapeutic immune responses, including antibodies that cross-react with IL31 and SP.

[0093] Compared to conventional vaccines targeting pathogens, the bivalent vaccines of this invention targeting autoantigens such as IL-31 and SP require a specific design, wherein the sequences of peptides and / or polypeptides are appropriately linked by flexible peptide linkers containing glycine (Gly) and serine (Ser) residues in short sequences such as GSGS within the recombinant fusion protein. Therefore, the recombinant fusion protein adopts a non-natural conformation, allowing recognition as non-self and evoking an immune response unaffected by central and peripheral tolerance. The bivalent immunogenicity of the recombinant fusion protein induces high anti-IL31 and anti-SP antibody titers, a situation not observed when the non-fusion components IL-31 and SP are administered as single agents.

[0094] As used in this article, "high neutralizing antibody titer" refers to a significantly higher titer of neutralizing antibodies compared to the therapeutic effect of IL31 and SP administered alone or in combination as single agents. High neutralizing antibody titer can also refer to a titer that provides longer-lasting immune memory by persisting throughout the therapeutic window compared to the therapeutic effect of IL31 and SP administered alone or in combination as single agents.

[0095] Immunogenic fusion protein targeting IL31 and SP

[0096] In the context of this invention, the terms "fusion protein" and "recombinant fusion protein" are used interchangeably, since fusion proteins are products of recombinant DNA and are produced by recombinant expression systems.

[0097] In some embodiments, the immunogenic fusion protein includes an immunogenic fragment derived from IL31 and SP, and induces the production of immunoglobulins capable of neutralizing endogenous IL31 and / or SP.

[0098] In some embodiments, the immunogenic fusion protein comprises an IL-31 peptide and one or more SP peptides, such as three or five SP peptides, each linked by a flexible amino acid linker, thereby potentially enhancing the immunogenicity of the recombinant fusion protein. The length of the linker ensures that the peptide linkage is substantially uninterrupted. In some embodiments, the linker can be selected to maintain structural flexibility while ensuring the non-native conformation of the recombinant fusion protein. In other embodiments, the linker may also provide additional beneficial properties to the protein, such as increased protein expression in the expression system, improved biophysical properties such as stability and solubility, improved protein purification and detection, and / or increased enzyme activity. Exemplary linkers may have residues of the formula Ser(Gly4Ser)n or (Gly-Ser)n, wherein some Glu or Lys residues are dispersed to increase solubility, wherein n can be an integer from 1 to 20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In another aspect of the invention, the at least one SP peptide is fused with at least one IL31 polypeptide or its immunogenic fragment via a flexible amino acid linker, preferably GSGS.

[0099] Antibodies offer significant advantages as therapeutic agents due to their higher specificity and reduced off-target effects. By binding to their respective autoantigens, polyclonal antibodies can inhibit or reduce the biological activity of neurogenic SP and immune IL31 mediators and / or their downstream signaling pathways, such as by blocking binding to their specific receptors or inhibiting mediator-induced cellular responses. In some embodiments, systemic active immunization of mammals (e.g., dogs, horses, humans, or cats) affected by Alzheimer's disease and pruritus with at least one immunogenic fusion protein of the examples (such as IL31-1SP or IL31-3SP) resulted in unexpectedly high titers of antibodies that cross-react with endogenous IL31 and SP.

[0100] In some embodiments, the recombinant fusion protein exhibits inherent immunogenicity due to its design and, when administered to mammals, overcomes immune tolerance against the self-antigens IL31 and / or SP. In some aspects of the embodiments, the immunogenic fusion protein can be recognized as non-self and triggers an immune response without immune tolerance, simultaneously generating neutralizing antibodies against IL-31 and / or SP. The fusion of multiple sequences from IL31 and SP results in the recombinant protein exhibiting enhanced immunogenicity. Notably, when compared to an equivalent amount of non-fusion IL31 and non-fusion SP administered as a single agent, the fusion protein induces higher antibody titers against endogenous IL31 and SP.

[0101] In some embodiments, the recombinant fusion protein comprises engineered sequences derived from at least one IL-31 peptide and at least one SP peptide, exhibiting enhanced immunogenicity. Support for the chimeric approach to conceiving non-natural recombinant fusion proteins stems from their unique immunogenic characteristics against natural IL-31 and SP, compared to individual IL-31 and SP peptides administered alone or as a single drug combination. In one aspect of the embodiments, the immunogenic fusion protein may comprise multiple copies of SP peptides, preferably one, three, or five SP peptides. Increasing the number of SP sequences within the fusion protein enhances SP exposure and immunogenicity. Without being bound by theory, several features are considered to contribute to enhanced immunogenicity. 1) Structure-function relationship: Substance P acts as a neurogenic mediator involved in the sensation of inflammation and pruritus. Amplifying the number of SP peptides within the fusion protein can increase the number of potential conformational epitopes presented and recognized by immune cells such as B cells, thereby enhancing humoral responses. 2) Multivalent immune recognition: The presence of multiple SP copies in the recombinant fusion protein increases local antigen concentration and provides multivalent antigen stimulation. Multivalent antigens have the potential to bind to multiple B cell receptors simultaneously, thereby enhancing immune activation and antibody production. This strategy leverages the concept of affinity, where increased binding interactions compared to monovalent antigens can lead to a stronger immune response. 3) Furthermore, extending the peptide length into longer polypeptides can overcome immune tolerance because the recombinant fusion protein is recognized as non-self and potentially triggers an immune cell response. Following vaccination, antigen-presenting cells (APCs) can process the long peptide for presentation and immune cell activation, thereby mitigating potential immune tolerance and enhancing vaccine efficacy. In some embodiments, the length and conformation of the immunogenic fusion protein are optimized to facilitate its internalization and processing by APCs, thereby enhancing the overall immune response to endogenous IL31 and / or SP.

[0102] In a preferred embodiment, the immunogenic fusion protein induces an immune response specific to IL31 and SP epitopes, thereby reducing the possibility of off-target effects.

[0103] In some embodiments, the immunogenic fusion protein IL31-1SP comprises an IL31 polypeptide or an immunogenic fragment thereof fused to an SP peptide (each separated by a flexible peptide linker (e.g., GSGS)). In other embodiments, the immunogenic fusion protein IL31-3SP comprises an IL31 polypeptide or an immunogenic fragment thereof fused to three SP peptides (each separated by a flexible linker). Similar methods can be implemented to prepare immunogenic fusion proteins having more than one IL31-derived immunogenic fragment and one or more SP peptides.

[0104] In some embodiments, the mammal is a dog, and the immunogenic fusion protein comprises at least one IL31 polypeptide having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 1, and at least one SP peptide having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 5. In one aspect of the embodiment, the at least one IL31 polypeptide is fused to the at least one SP peptide via a flexible linker (e.g., GSGS). In another aspect of the embodiment, the immunogenic fusion protein comprises an IL31 polypeptide fused to an SP peptide. In a preferred embodiment, the immunogenic fusion protein comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 11. In another embodiment, the immunogenic fusion protein comprises an IL31 polypeptide fused with three SP peptides. In a preferred embodiment, the immunogenic fusion protein comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 14.

[0105] In some embodiments, the mammal is a horse, and the immunogenic fusion protein comprises at least one IL31 polypeptide having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 2, and at least one SP peptide having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 6. In one aspect of the embodiments, the at least one IL31 polypeptide is fused to the at least one SP peptide via a flexible peptide linker (e.g., GSGS). In another aspect of the embodiments, the immunogenic fusion protein comprises an IL31 polypeptide fused to an SP peptide. In a preferred embodiment, the immunogenic fusion protein comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 17. In another embodiment, the immunogenic fusion protein comprises an IL31 polypeptide fused with three SP peptides. In a preferred embodiment, the immunogenic fusion protein comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 20.

[0106] In some embodiments, the mammal is a human, and the immunogenic fusion protein comprises at least one IL31 polypeptide having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 3, and at least one SP peptide having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 8. In one aspect of the embodiments, the at least one IL31 polypeptide is fused to the at least one SP peptide via a flexible peptide linker (e.g., GSGS). In another aspect of the embodiments, the immunogenic fusion protein comprises an IL31 polypeptide fused to an SP peptide. In a preferred embodiment, the immunogenic fusion protein comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 23. In another embodiment, the immunogenic fusion protein comprises an IL31 polypeptide fused to three SP peptides. In a preferred embodiment, the immunogenic fusion protein comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 26.

[0107] In some embodiments, the mammal is a cat, and the immunogenic fusion protein comprises at least one IL31 polypeptide having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 4, and at least one SP peptide having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 9. In one aspect of the embodiments, the at least one IL31 polypeptide is fused to at least one SP peptide via a flexible peptide linker (e.g., GSGS). In another aspect of the embodiments, the immunogenic fusion protein comprises an IL31 polypeptide fused to an SP peptide via a flexible peptide linker (e.g., GSGS). In a preferred embodiment, the immunogenic fusion protein comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 29. In another embodiment, the immunogenic fusion protein comprises an IL31 polypeptide fused to three SP peptides, each SP peptide being separated by a flexible linker, preferably GSGS. In a preferred embodiment, the immunogenic fusion protein comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 32.

[0108] In some embodiments, the mammal is a mouse, and the immunogenic fusion protein comprises at least one IL31 polypeptide and at least one SP peptide having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 7. In one aspect of the embodiment, the at least one IL31 polypeptide is fused to the at least one SP peptide via a flexible linker. In another aspect of the embodiment, the immunogenic fusion protein comprises one IL31 polypeptide fused to one SP peptide via a flexible peptide linker (e.g., GSGS). In yet another aspect of the embodiment, the immunogenic fusion protein comprises one IL31 polypeptide fused to three SP peptides, each SP peptide being separated by a flexible linker, preferably GSGS.

[0109] In some embodiments, the nucleotide sequence encoding the immunogenic fusion protein has at least 90%, at least 95%, at least 99%, or 100% nucleotide sequence identity with one of SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, or SEQ ID NO: 31.

[0110] Therapeutic uses

[0111] The immunogenic fusion protein of the present invention is an immunogen suitable for treating diseases such as Alzheimer's disease (AD) with inflammatory skin lesions and itching as the main symptoms.

[0112] In some embodiments, when administered in mammals, immunogenic compositions comprising the immunogenic fusion proteins IL31-1SP and / or IL31-3SP significantly enhance humoral immune responses against endogenous IL31 and SP, as demonstrated by higher levels of cross-reactivity and high-affinity antibodies against endogenous IL31 and SP compared to a combination of non-fusion IL31 and non-fusion SP administered as a single agent. In some aspects of the embodiments, immunogenic compositions combining at least one immunogenic fusion protein IL31-1SP and / or IL31-3SP with at least one suitable adjuvant exhibit a synergistic effect of further enhancing the immune response, resulting in the production of antibodies that cross-react with native IL31 and SP, which mediate pathological effects in treated mammals. The results presented herein support the synergistic effect of combining IL31 and SP in recombinant fusion proteins to reduce inflammation and pruritus.

[0113] In the context of active immunization, immunogenic compositions can be formulated as bivalent vaccines.

[0114] Advantageously, systemic immunization of mammals with the immunogenic fusion proteins of the present invention, such as IL31-1SP or IL31-3SP, provides long-term immune memory with sustained high antibody titers in the mammalian immune system.

[0115] Systemic immunization using a combination of non-fusion IL31 and non-fusion SP, administered as a single peptide, failed to produce significant antibody titers compared to systemic immunization using an immunogenic composition containing at least one recombinant fusion protein. Systemic immunotherapy using an immunogenic composition containing at least one immunogenic fusion protein targeting neurogenic SP and immune IL31 mediators improved clinical symptoms associated with pruritus and secondary skin lesions while reducing the use and / or dosage of conventional anti-inflammatory drugs such as corticosteroids, antihistamines, and tyrosine kinase inhibitors. This is particularly relevant because prolonged use of anti-inflammatory drugs can produce serious side effects.

[0116] The concomitant blocking of IL31 and SP mediators produces a synergistic effect, thereby reducing the dose of the immunogenic composition required to obtain effective therapeutic benefits while preventing side effects. Surprising and unexpected results (i.e., neutralizing antibody titers) showed a better effect than the additive effect expected from immunization with natural canine IL31 or natural canine SP as single agents.

[0117] It is noteworthy that the ability of IL31-1SP and IL31-3SP immunogens to generate cross-reactive and therapeutic antibodies that bind to native IL31 and SP is unexpected, because immunogenic fusion proteins are non-natural, non-physiological proteins that differ from native IL31 and SP. Immunogenic fusion proteins can contain structural epitopes different from those predicted for IL31 and SP. Because fusion proteins are molecularly heterologous, they can adopt conformations displaying multiple conformational epitopes. When used as vaccines, IL31-1SP or IL31-3PS immunogens elicit complex immune responses, including the production of antibodies against endogenous IL31 and SP, which is the cause of the therapeutic response.

[0118] Advantageously, systemic active immunization with recombinant fusion proteins IL31-1SP or IL31-3SP produced by inclusion bodies can effectively treat Alzheimer's disease (AD) in mammals with inflammatory and pruritic skin lesions.

[0119] Surprisingly, systemic immunization with a bivalent fusion protein as disclosed herein effectively relieves pruritus-related symptoms, including excessive scratching, itching, and / or reduces or prevents secondary skin damage. Unexpectedly, this type of active immunization enhances therapeutic efficacy, provides durable therapeutic effects (e.g., immune memory), and has virtually no side effects. Furthermore, systemic immunization with a bivalent fusion protein as disclosed herein reduces the intake of corticosteroids (pharmacological treatment) commonly used to manage pruritus symptoms.

[0120] In some embodiments, the method includes administering an immunogenic fusion protein (e.g., IL31-1SP or IL31-3SP) in combination with at least one therapeutic agent as an immunogenic composition or vaccine to enhance therapeutic benefits in mammals affected by or susceptible to disease-related symptoms mediated by IL31 and SP overexpression. Non-limiting examples of therapeutic agents are anti-inflammatory compounds selected from the group consisting of: corticosteroids, antihistamines, tyrosine kinase inhibitors, and cyclosporines or receptor antagonists that block nociceptive signaling pathways, preferably neurokinin-1 receptor (NK1-R) antagonists, and most preferably compound CP-96,345.

[0121] In some embodiments, an effective amount of an immunogenic fusion protein, such as IL31-1SP or IL31-3SP, is administered to mammals in need to treat and / or prevent inflammatory skin symptoms and pruritus mediated by IL31 and SP. In other embodiments, an effective amount of the immunogenic fusion protein IL31-1SP or IL31-3SP is administered to mammals in need to treat and / or prevent allergies mediated by IL31 and SP.

[0122] In some embodiments, the immunogenic composition is used to treat and / or prevent dermatitis associated with inflammation and itching, preferably Alzheimer's disease (AD), most preferably chronic and / or refractory AD. AD is refractory when AD-related symptoms cannot be treated with current standard of care centered on anti-inflammatory compounds such as corticosteroids, antihistamines, tyrosine kinase inhibitors, and cyclosporine.

[0123] Methods for treating Alzheimer's disease (AD) and pruritic skin lesions include administering to a mammal at least one immunogenic fusion protein designed and produced as a bivalent fusion protein that stimulates a humoral immune response and the production of specific antibodies that simultaneously bind to and neutralize the upregulated activity of neurogenic and immune mediators in the affected mammal.

[0124] In some embodiments, methods for treating AD and / or (or in other embodiments, preventing) symptoms associated with pruritus include administering an effective amount of a bivalent vaccine or immunogenic composition comprising at least one immunogenic fusion protein (e.g., IL31-1SP or IL31-3SP) to reduce excessive scratching, itching, and / or reduce or prevent secondary skin lesions. In some embodiments, up to four or more booster doses of immunization may be administered to alleviate AD-related symptoms, particularly pruritus.

[0125] In one embodiment, the immunogenic composition or bivalent vaccine is used in an active immunization regimen. Active immunization with the IL31-1SP or IL31-3SP immunogen is safe and therapeutic in dogs affected by chronic atopic dermatitis (which typically requires systemic corticosteroid therapy). Following administration of a bivalent vaccine containing the immunogen and at least one adjuvant (initiator and three boosters), immunized dogs showed significant improvement in skin lesions, as assessed by clinical observation.

[0126] In one embodiment, administration of the immunogenic composition reduced AD-related symptoms such as pruritus and reduced the need for corticosteroid treatment. (Figure 6) Vaccination with the immunogenic fusion protein (including IL31-1SP) significantly reduced the frequency of scratching in treated dogs within one month after completion of the immunization cycle. (Figure 5) This therapeutic effect is durable, lasting for several months. Furthermore, the number of dogs taking corticosteroids was significantly reduced long after vaccination, indicating symptom improvement and reduced need for corticosteroid treatment. In some aspects of the embodiment, dogs immunized with IL31-1SP could discontinue corticosteroid use three months post-immunization, further confirming the effectiveness of the bivalent vaccine in effectively reducing skin inflammation and pruritus-related symptoms.

[0127] use Figure 6A The pruritus scale defined herein indicates that effective treatment can be defined as achieving a pruritus score in the range of 1 to 3, where the dog experiences occasional or mild pruritus that does not significantly affect its overall quality of life. The severity of pruritus and the use of corticosteroids in dogs with atopic dermatitis before and after treatment with the recombinant fusion immunogen (preferably IL31-1SP or IL31-3SP) described herein (i.e., immunization regimen) can be evaluated using [the relevant technology / method]. Figure 6A The scale defined in [the text] was used. A. The scoring system provides a comprehensive assessment of symptoms such as the severity of itching and appropriate treatment. A 1-10 scale was used to assess the level of itching in dogs with atopic dermatitis. The scale categorizes symptoms by severity, minor symptoms, and recommended treatments. Scores range from 1 (sporadic scratching without injury) to 10 (compulsive scratching requiring an E-collar).

[0128] As shown in Figure 6, IL31-1SP treatment was effective in reducing the severity of itching and corticosteroid use in dogs with atopic dermatitis. Dogs treated with IL31-1SP showed a significant reduction in scratching scores over a two-month period, with fewer dogs requiring corticosteroids 90 days after vaccination. These results suggest that IL31-1SP is a promising treatment option for managing canine atopic dermatitis.

[0129] Notably, active immunization using the immunogenic fusion protein yielded promising results, reducing pruritus scores for several months after treatment completion. Furthermore, active immunization significantly reduced corticosteroid consumption, highlighting the efficacy and safety of the immunogenic fusion protein of this invention. The immunogenic fusion protein provides therapeutic benefits and has an impact on pruritus management.

[0130] Potential mechanisms of skin inflammation and pruritus in AD include increased expression of IL31 and SP, with IL31 and SP synergistically contributing to pruritus and neurogenic inflammation [1; 29; 31; 32; 33; 34; 35; 36]. In the current study, there is evidence that vaccination with the IL31-1SP immunogen induced an immune response in both mice and dogs, characterized by the production of neutralizing antibodies that recognize endogenous IL31 and SP. Therefore, concomitant immune neutralization of IL31 and SP may explain the effective and durable therapeutic effect of IL31-1SP or IL31-3SP immunization in canine AD.

[0131] Many authors have reported the safety and efficacy of systemic immunization with IL31 or as its major epitope sequence associated with virus-like particles in dogs with CAD and horses with dermatitis secondary to hypersensitivity to insect bites [57; 58]. These vaccines generate (therapeutic) neutralizing antibodies against IL31 in the host, which reduces itching and inflammatory lesions secondary to scratching. These treatments remain experimental and have not been tested in clinical controlled trials or chronic treatment regimens. However, the proposed mechanism of action appears to be the neutralization of endogenous IL31, which is upregulated in atopic dermatitis.

[0132] The therapeutic efficacy of the IL31-1SP vaccine in dogs with Alzheimer's disease (AD) suggests that neutralization of IL31 by specific antibodies is sufficient to alleviate major symptoms of AD, regardless of how the immune system is stimulated. However, the fact that the IL31-1SP vaccine also produces antibodies against SP makes it a different immunization strategy. Antibodies that neutralize SP may enhance the effects of anti-IL31 antibodies and downregulate the synergistic effect of two mediators that induce skin inflammation and pruritus. Neutralizing antibodies against SP may reduce the reported effects of SP-mediated neurogenic inflammation in AD, including increased skin vascular permeability, immune cell infiltration, and mast cell degranulation. Assuming that SP activates the mast cell MRGPRX2 receptor, which mediates the release of inflammatory mediators, including pruritogenic monoamine histamine [43; 44], neutralizing SP through systemic immunization may become an important therapeutic mechanism in several pathological conditions involving neurogenic inflammation.

[0133] The safety and efficacy of systemic immunization with the IL31-1SP immunogen in mice and dogs with Alzheimer's disease (AD) have been established. The recombinant immunogen elicited an immune response in mammals, including the production of neutralizing antibodies against endogenous IL31 and SP, thereby alleviating skin inflammation and pruritus-related symptoms.

[0134] In some embodiments, the immunogenic composition or bivalent vaccine is administered at an initial dose, followed by three booster doses at intervals of approximately 2 weeks, and optionally one or more booster doses at intervals of 3 to 6 months or for a period of time required to maintain high and neutralizing antibody titers.

[0135] In some embodiments, treatment or prevention of inflammatory skin conditions, preferably Alzheimer's disease (AD) or AD-related symptoms (such as pruritus), in mammals (such as dogs) of need comprises subcutaneous administration of an initial dose, followed by three booster doses at approximately 2-week intervals, and optionally one or more booster doses at intervals of 3 to 6 months or for a period of time required to maintain high and neutralizing antibody titers. The bivalent vaccine comprises an immunogenic fusion protein IL31-SP or an immunogenic fusion protein IL31-3SP, wherein the immunogenic fusion protein IL31-SP comprises an amino acid sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO: 11, and the immunogenic fusion protein IL31-3SP comprises an amino acid sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO: 14.

[0136] Vaccines can be administered to mammals in need via a variety of suitable routes. For example, vaccines can be administered orally, subcutaneously, intramuscularly, or transdermally, together or alone, and / or simultaneously and / or sequentially. In some embodiments, the present invention provides a pharmaceutical composition for treating Alzheimer's disease (AD) comprising an effective amount of a bivalent or multivalent vaccine and a pharmaceutically acceptable carrier.

[0137] In one embodiment, a drug comprising at least one immunogenic fusion protein is able to induce high therapeutic antibody titers against endogenous IL31 and SP.

[0138] In one embodiment, the immunogenic fusion protein is used to prepare a medicament for treating or preventing Alzheimer's disease (AD) and / or pruritus in mammals.

[0139] In the treatment methods according to the embodiments, the immunogenic composition comprising an immunogenic fusion protein as an active ingredient can be administered via intravenous, intra-arterial, intraperitoneal, intramuscular, intrasternal, percutaneous, local, intraocular, or subcutaneous routes.

[0140] In one embodiment, the immunogenic composition is capable of inducing therapeutic antibodies that synergistically act in drug preparation to mediate the in vivo inhibition of the bioactivity of IL31 and SP in AD.

[0141] Methods for generating immunogenic fusion proteins

[0142] In some embodiments, the immunogenic fusion protein is generated as an inclusion body in a genetically modified microorganism. In a preferred embodiment, the immunogenic fusion protein comprises multiple epitopes targeting IL31 and / or SP.

[0143] In some embodiments, a method for generating an immunogenic fusion protein includes: i) transfecting *E. coli* cells with an expression vector containing a nucleotide sequence encoding an immunogenic fusion protein as described herein; ii) isolating inclusion bodies containing the generated immunogenic fusion protein; and iii) purifying and dissolving the immunogenic fusion protein in a solubilizing solvent. In some aspects of the embodiments, the solubilizing solvent used to maintain the solubility of the immunogenic fusion protein is urea 4 M-8 M.

[0144] Sequence and biophysical properties support the fact that immunogenic fusion proteins have artificial, non-naturally occurring structures, and are therefore products of human ingenuity. These features allow immunogenic fusion proteins to possess high immunogenicity, overcome natural immune tolerance to self-antigens IL-31 and SP, and have the unexpected ability to elicit therapeutic immune responses, including the production of high-affinity antibodies that cross-react with IL-31 and SP.

[0145] In some embodiments, a method for producing an immunogenic composition or bivalent vaccine comprising the immunogenic fusion protein IL31-1SP or IL31-3SP includes the steps of cloning, expressing, purifying the fusion protein, and reconstituted the fusion protein with a suitable adjuvant. Choosing an appropriate adjuvant can enhance the versatility of the therapeutic formulation. For example, depending on the species being treated (e.g., horse, human, cat, dog) and / or the route of administration, a particular adjuvant may be more effective.

[0146] Surprisingly, the immunogenic fusion protein generated as an inclusion body triggers a humoral immune response, in which therapeutic antibodies against endogenous IL31 and / or SP are produced, which act synergistically under pathological conditions.

[0147] Inclusion bodies are readily available (as a source of immunogenic species) and induce strong immune responses. Immunogenic compositions or bivalent vaccines containing at least one immunogenic fusion protein are easy to administer and do not present the common challenges associated with the preparation and production of monoclonal antibodies, such as high production costs.

[0148] In some embodiments, the autoantigens IL31 and SP are expressed as fusion proteins in bacterial cells. The homogeneity of the resulting composition, achieved through simple design of the immunogenic fusion protein and further isolation and purification of inclusion bodies, ensures precise dosing.

[0149] In some embodiments, the recombinant expression vector has a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with one of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, or SEQ ID NO: 33.

[0150] In some embodiments, the recombinant expression vector comprises a nucleotide sequence encoding an immunogenic fusion protein, said nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with one of SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, or SEQ ID NO: 31.

[0151] In some embodiments, a recombinant expression vector having a nucleotide sequence encoding at least one immunogenic fusion protein may be inserted into or recombined in the host cell genome.

[0152] In other embodiments, the nucleotide sequence encoding at least one immunogenic fusion protein refers to any nucleotide, including nucleotide sequences capable of spontaneous replication as episomes. Such vectors may include linear nucleotides, plasmids, phage particles, granules, RNA vectors, viral vectors, etc.

[0153] In some embodiments, the vector can be genetically engineered to incorporate a nucleotide sequence encoding IL31 in the correct reading frame orientation at the N-terminus and / or C-terminus of the nucleotide sequence encoding SP, thereby enabling expression of the immunogenic fusion protein. A flexible linker encoding a Gly and Ser-rich flexible linker (such as GSGS) may be used, for example, as a linker sequence. For example, the expression vector may include SEQ ID NO: 10 encoding the canine polypeptide IL31-1SP, or SEQ ID NO: 13 encoding the canine polypeptide IL31-3SP. It should be understood that the expression vector may comprise any combination of at least one immunogenic fragment derived from IL31 and at least one immunogenic fragment derived from SP.

[0154] The expression vector can also be selected from those vectors that are readily available for prokaryotic or eukaryotic expression systems.

[0155] Standard recombinant nucleotide methods can be used to express genetically engineered immunogenic fusion proteins. In some embodiments, the nucleotide sequence encoding the immunogenic fusion protein can be cloned into an expression vector, for example, having appropriate signaling and processing sequences as well as regulatory sequences for transcription and translation. In other embodiments, the immunogenic fusion protein can be synthesized using automated organic synthesis methods.

[0156] To obtain high-level expression of the immunogenic fusion protein, the cDNA encoding the immunogenic fusion protein can be subcloned into an expression vector comprising a strong promoter for initiating transcription, a transcription / translation terminator, and, in the case of nucleotides encoding the protein, a ribosome-binding site for initiating translation. Suitable bacterial promoters are well known in the art. Bacterial expression systems for expressing recombinant fusion proteins are available, for example, from *Escherichia coli*, *Bacillus*, and *Salmonella*. Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available. The eukaryotic expression vector may preferably be an adenovirus vector, an adeno-associated vector, or a retroviral vector.

[0157] In some embodiments, the nucleotide sequence encoding the immunogenic fusion protein may be present in a vector, wherein the nucleotide sequence is operatively linked to a regulatory sequence capable of providing nucleotide sequence expression via a suitable host cell.

[0158] Within the expression vector, the term "operably ligated" is intended to mean that the nucleotide sequence of the immunogenic fusion protein is ligated to a regulatory sequence in a manner that allows the nucleotide sequence to be transcribed. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements. Such operably ligated to the expression vector can be achieved using conventional gene recombination techniques known in the art, while site-directed DNA cleavage and ligation are performed using conventional enzymes known in the art.

[0159] The expression vector may contain signal sequences or leader sequences for membrane targeting or secretion, as well as regulatory sequences such as promoters, operons, start codons, stop codons, polyadenylation signals, enhancers, etc. The promoter may be a constitutive promoter or an inducible promoter. Furthermore, the expression vector may include one or more selectable marker genes for selecting host cells containing the expression vector, and may further include polynucleotide sequences that enable the vector to replicate in the host cells.

[0160] The expression vector constructed according to the embodiments can be a vector in which a polynucleotide encoding a recombinant fusion protein is inserted into the multiple cloning site (MCS) of the pT7 vector.

[0161] Recombinant fusion proteins can be introduced into suitable host cells (e.g., bacterial cells, yeast cells, insect cells, or tissue culture cells). They can also be introduced into embryonic stem cells to produce transgenic organisms. A wide range of suitable vectors and promoters are known to those skilled in the art and are commercially available for producing recombinant proteins.

[0162] Known methods can be used to construct vectors comprising a polynucleotide sequence according to an embodiment disclosed in this application and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / gene recombination.

[0163] Another aspect of the embodiments provides a transformant converted using a recombinant expression vector.

[0164] As used in this article, the term “introducing” proteins, peptides, or organic compounds into cells can be used interchangeably with the expression “carrying,” “penetrating,” “transporting,” “delivering,” “permeating,” or “transferring.”

[0165] It should be understood that a host cell refers to a eukaryotic or prokaryotic cell in which one or more DNA or vectors have been introduced, as well as its offspring or potential offspring. Such offspring may differ in fact from the parent cell due to mutations or environmental influences that may result in certain modifications in the offspring, but are still included within the scope of the terminology used herein.

[0166] The host cell can preferably be a bacterial cell, and there are no restrictions on the type of bacterial cell. They can be eubacteria (Gram-positive or Gram-negative) or archaea, as long as they allow for gene manipulation to insert genes of interest, preferably site-specific integration, and they can be cultured on a production scale. Preferably, the host cell can have properties that allow for culture to high cell densities.

[0167] Examples of bacterial host cells that can be used to prepare recombinant fusion proteins include *Escherichia coli*, *Bacillus subtilis*, *Pseudomonas fluorescens*, and various *Corynebacterium* and *Lactococcus lactis* strains. Preferably, the host cell is *Escherichia coli* cell.

[0168] In one embodiment, the host cell may include an RNA polymerase capable of binding to the promoter of a gene of interest. The RNA polymerase may be endogenous or exogenous to the host cell.

[0169] In one embodiment, a host cell with a strong exogenous RNA polymerase can be used. For example, an *E. coli* strain engineered to carry an exogenous RNA polymerase integrated into its genome can be used (e.g., a T7-like RNA polymerase in a so-called "T7 strain," as in the case of using a T7 promoter). Examples of T7 strains, such as BL21(DE3), HMS174(DE3), and their derivatives or relatives (see Novagen, pET System manual, 11th edition), are widely available and commercially available. Preferably, BL21-CodonPlus(DE3)-RIL or BL21-CodonPlus(DE3)-RIPL can be used. These strains are DE3 lysogens containing a T7 RNA polymerase gene under the control of the lacUV5 promoter. Induction with IPTG can produce a T7 RNA polymerase, which then directs the expression of the gene of interest under the control of the T7 promoter.

[0170] The host cell strains *Escherichia coli* BL21 (DE3) or HMS174 (DE3) received their genome-based T7 RNA polymerase via phage DE3, and are lysogenic. Preferably, the T7 RNA polymerase contained in the host cell has been integrated by means of avoiding or preferably excluding the insertion of residual phage sequences into the host cell genome, because lysogenic strains have the disadvantage of potentially exhibiting lytic properties, leading to undesirable phage release and cell lysis.

[0171] Methods for preparing recombinant fusion proteins include preparing a recombinant expression vector; preparing a transformant using the recombinant expression vector; culturing the transformant; and recovering the recombinant fusion protein expressed by culture.

[0172] For fed-batch, semi-continuous, or continuous modes, culture may employ fed-batch media, and the bacterial expression host cell may include a DNA construct integrated into its genome, the construct carrying a DNA sequence encoding the protein of interest, the DNA sequence being under the control of a promoter capable of expressing the protein.

[0173] There are no restrictions on the type of culture medium. The medium can be semi-deterministic, containing complex culture medium compounds (e.g., yeast extract, soybean peptone, casein amino acids), or it can be chemically defined without any complex compounds. Preferably, a compositionally defined medium can be used. Compositionally defined media (also called basal media or synthetic media) are specifically composed of chemically defined substances, i.e., carbon sources such as glucose or glycerol, salts, vitamins, and, considering possible auxotrophic strains, specific amino acids or other substances such as thiamine. Most preferably, glucose can be used as the carbon source. Typically, the carbon source in fed media is used as a growth-limiting component to control the specific growth rate.

[0174] Host cells can be lysed by any convenient method, including freeze-thaw cycles, sonication, mechanical disruption, or the use of cell lysis agents. Many general methods for purifying recombinant (and non-recombinant) proteins are known in the art. These methods may include, for example, ion exchange chromatography, size exclusion chromatography, affinity chromatography, selective precipitation, dialysis, and hydrophobic interaction chromatography. These methods can be adapted to design purification strategies for cell-permeable recombinant fusion proteins. If the cell-permeable recombinant fusion protein includes purification treatments (such as epitope tags or metal chelate sequences), then affinity chromatography can be used to readily purify the protein.

[0175] The amount of protein produced can be assessed by detecting higher macromolecular transduction domains directly (e.g., using protein analysis) or indirectly (e.g., by measuring the specific DNA-binding activity of cellular materials, such as by electrophoretic mobility shift assays). Proteins can be detected before purification, during any stage of purification, or after purification. In some embodiments, purification or complete purification may not be necessary.

[0176] Pharmaceutical compositions and formulations

[0177] In some embodiments, the pharmaceutical composition comprises at least one immunogenic fusion protein as an active ingredient. The pharmaceutical composition may further comprise at least one pharmaceutically acceptable carrier. The pharmaceutical composition may be used to treat or prevent Alzheimer's disease (AD) and / or AD-related symptoms, such as itching or secondary skin lesions, in mammals (preferably dogs). The pharmaceutical composition is effective in treating AD refractory to anti-inflammatory compounds such as corticosteroids, antihistamines, tyrosine kinase inhibitors, and cyclosporine.

[0178] In some embodiments, the pharmaceutical composition may be injectable (e.g., subcutaneous, intraperitoneal, intramuscular) and may include an active ingredient in an amount of 0.001 mg / kg to 1000 mg / kg, preferably 0.01 mg / kg to 100 mg / kg, more preferably 0.1 mg / kg to 20 mg / kg for humans, more preferably 0.001 mg / kg to 10 mg / kg for dogs, more preferably 0.001 mg / kg to 5 mg / kg for cats, and more preferably 0.001 mg / kg to 20 mg / kg for horses.

[0179] For example, the dosage is typically in the range of about 0.001 mg / kg body weight to about 1000 mg / kg body weight. In adult treatment, a single dose or fractions in the range of about 0.1 mg / kg / day to about 50 mg / kg / day are particularly preferred. However, it should be understood that the actual concentration of the recombinant fusion protein administered will be determined by a physician or veterinarian based on relevant circumstances (including the condition to be treated, the chosen route of administration, the individual's age, weight and response, and the severity of the patient's symptoms), and therefore the above dosage range is not intended to limit the scope of the invention in any way. In some cases, dosage levels below the lower limit of the aforementioned range may be sufficient, while in others, larger doses may be used without causing any harmful side effects, provided that such larger doses are first divided into several smaller doses for administration throughout the day.

[0180] In addition to the active ingredient, the pharmaceutical composition according to the embodiments can be prepared by using pharmaceutically suitable and physiologically acceptable additives, and the additives may include excipients, disintegrants, sweeteners, binders, coating agents, foaming agents, lubricants, flow aids, flavoring agents, etc.

[0181] To formulate immunogenic compositions or bivalent vaccines into liquid formulations, sterile and biocompatible pharmaceutically acceptable carriers such as saline, sterile water, Ringer's solution, buffered saline, albumin infusion solution, glucose solution, maltodextrin solution, glycerol, and ethanol can be used, and these materials can be used alone or in any combination thereof. If desired, other commonly used additives such as antioxidants, buffers, and antibacterial agents can be added. Furthermore, diluents, dispersants, surfactants, binders, and lubricants can be added additionally to prepare injectable formulations such as aqueous solutions, suspensions, and emulsions. Moreover, depending on the disease and composition, the composition can preferably be formulated using any suitable method known in the art.

[0182] In one embodiment, an effective amount is the amount of the immunogenic fusion protein or the pharmaceutical composition disclosed herein as an active ingredient sufficient to achieve such treatment or prevention of the disease when administered to a mammal for the treatment or prevention of Alzheimer's disease and / or pruritus. Any improvement in the mammal is considered sufficient to achieve treatment. The effective amount of the active ingredient or pharmaceutical composition disclosed herein for the treatment of Alzheimer's disease and / or pruritus can vary depending on the method of administration, age, weight, and the overall health condition of the mammal. Ultimately, the prescribing physician will determine the appropriate dosage and dosing regimen.

[0183] In one embodiment, the immunogenic composition or bivalent vaccine comprises an immunogenic fusion protein in an effective amount between 0.5 µg and 10,000 µg.

[0184] In some embodiments, pharmaceutical compositions comprising immunogenic fusion proteins (e.g., IL31-1SP or IL31-3SP) are intended for veterinary use and are also formulated with veterinary adjuvants suitable for administration in animals suffering from symptoms associated with IL31 and SP. The optimal adjuvant will depend solely on the animal species (e.g., dog, horse, cat) and the route of immunization (e.g., by injection or mucosa).

[0185] In some embodiments, the adjuvant includes an oil-in-water adjuvant, a polymer and water adjuvant, an oil-in-water adjuvant, an aluminum hydroxide adjuvant, or a combination thereof.

[0186] Example

[0187] Specific embodiments will now be illustrated with reference to the following examples. It should be understood that these examples are disclosed only by way of illustrating the invention and should not be construed as limiting the scope of the invention in any way.

[0188] Materials and Methods

[0189] Recombinant IL31-1SP and IL31-3SP immunogenic proteins were expressed and purified from inclusion bodies.

[0190] According to the manufacturer's instructions, the IL31-1SP and IL31-3SP fusion proteins, as defined in SEQ ID NO: 11 and SEQ ID NO: 14, were expressed by transforming plasmid pT7 containing the IL31-1SP gene into competent One Shot™ BL21 Star™ (DE3) chemically competent E. coli (Thermo Fisher Scientific C601003).

[0191] To generate the immunogenic fusion proteins IL31-1SP and IL31-3SP, transformed *E. coli* cells were cultured overnight at 37°C and 220 rpm in Lauria-Bertany broth supplemented with 100 µg / mL ampicillin (Sigma A0166). Then, 1 liter of premium broth was inoculated with 10 mL of pre-culture and incubated at 37°C and 220 rpm until an optical density of 2 was reached at 600 nm. Induction was performed by adding 1 mM IPTG (Euromedex EU0008-B) and incubating at 37°C and 220 rpm for 4 hours. After 4 hours, cells were harvested by centrifugation at 10,000 xg and 4°C for 10 minutes, and then resuspended in a buffer containing 30 mM Tris (pH 8), 150 mM NaCl, and 0.5 mg / mL lysozyme. The cell suspension was sonicated using a 1:20 timer cycle (20 seconds on, 1 minute off at 50%) and centrifuged at 20,000 xg and 4°C for 40 minutes. The resulting precipitate was resuspended in Wash Buffer I (WBI) consisting of 50 mM Tris (pH 8), 50 mM NaCl, 0.5% Triton X-100, 1.5 mM β-mercaptoethanol, and 1.6 M urea. The resuspended precipitate was then centrifuged at 20,000 xg and 4°C for 20 minutes. This washing step was repeated three times. Subsequently, the precipitate was resuspended in Wash Buffer II (WBII) containing 30 mM Tris (pH 8) and 150 mM NaCl, followed by centrifugation at 20,000 xg and 4°C for 20 minutes. This step was repeated twice. Finally, the sample was resuspended in a final buffer (FB) consisting of 20 mM Tris (pH 8), 500 mM NaCl, 30 mM imidazole and 8 M urea.

[0192] Purification by Ni²⁺ affinity chromatography was performed at room temperature. Proteins in the final buffer were applied at a maximum flow rate of 2 mL / min to a pre-equilibrated 5 mL HisTrap chelated HP column packed with Ni²⁺ (GE17-5248-02, Cytiva). The column was then washed with 5 column volumes (CV) of the final buffer. Proteins were eluted in three steps using an elution buffer consisting of 20 mM Tris (pH 8), 500 mM NaCl, 500 mM imidazole, and 8 M urea, 10 mL per step.

[0193] All fractions were obtained by SDS-PAGE analysis, and imidazole was removed by dialysis.

[0194] To deplete endotoxins, endotoxin removal beads from Miltenyi Biotec (130-093-657) were used according to the manufacturer's instructions. The sample was passed through the magnetic beads five times consecutively to reduce the endotoxin level to below 10 UE / mg protein.

[0195] Following the manufacturer's instructions, use the Pierce™ Chromogenic Endotoxin Quantitative Kit (Thermo Fisher Scientific A39553) to measure endotoxin levels.

[0196] Protein quantification was performed using the Bradford protein assay, and a standard curve was generated using final buffer (FB). Protein quantity and purity were determined by mass density assay using acrylamide gel electrophoresis and 1D electrophoresis. Sample identification was confirmed by mass spectrometry.

[0197] Mass spectrometry (MS) analysis.

[0198] Bottom-up LC-MS Analysis. For bottom-up LC-MS analysis, the corresponding purified recombinant fusion proteins IL31-1SP and IL31-3SP were alkylated with 50 mM iodoacetamide (IAM), then digested with sequencing-grade modified trypsin in carbonate buffer at pH 7 at 37°C for 16 h. The samples were then concentrated and desalted using a ZipTip C18. Finally, 3 μL of each sample at a concentration of 1 μg / L was injected into a Thermo Fisher Scientific Q-Exactive Plus (Q-orbittrap) LC-MS system. LC gradients used: HPLC gradients were used under the following conditions: Time (min): 0–110, Flow rate (µL / min): 0.2 constant % B: 0 min: 1%, 15 min: 1%, 75 min: 35%, 90 min: 99%, 100 min: 99%, 102 min: 1%, 110 min: 1%. Both the guard column and column were from Thermo Fisher Scientific. The theoretical sequences of the proteins of interest were used to identify IL31-1SP and IL31-3SP using Patternlab V software.

[0199] Direct injection MS1. For MS1 analysis, the protein was precipitated in phosphate buffer, centrifuged, and washed. The final precipitate was dissolved in 1% acetic acid. The protein was directly injected and analyzed by MS1 on an Agilent 6545XT Advanced Bio LC / Q-TOF mass spectrometer. Software from Agilent: ExDviewer version 4.6.12.

[0200] Mouse Immunization. To assess the safety of the product, three B6 mice were immunized every 15 days with a subcutaneous administration of 5 mg / kg of protein. The immunization regimen consisted of a primary injection and three subsequent booster injections. Each dose was prepared using Tris 20 mM pH 8, NaCl 500 mM, urea 4 M, and 3% adjuvant Montanide GEL 01 PR (Seppic). Submandibular blood samples were collected before each injection and 7 days later. The blood samples were incubated at room temperature for 2 hours and then centrifuged at 3000 x g for 10 minutes at 4°C to separate the serum.

[0201] The synergistic immunogenicity of IL31-3SP compared to non-fusion IL31 and SP administered alone as antigens was demonstrated. In other experiments, three B6 mice in two groups were immunized every 15 days with a subcutaneous administration of 1 mg / kg of recombinant fusion protein IL31-3SP, with the total immunization regimen including a primary immunization and three booster immunizations. Each dose was prepared using IL31-3SP antigen, Tris 20 mM pH 8, NaCl 500 mM, urea 4 M, and 3% adjuvant Montaned GEL 01 PR (Sebic). For comparison, another group of three B6 mice were immunized with non-fusion recombinant canine IL31 (CYT 604, Bio-Propec) and synthetic substance P (RP10178 Genscript) as antigens in a solution prepared with Tris 20 mM pH 8, NaCl 500 mM, and 3% adjuvant Montaned GEL 01 PR (Sebic). Submandibular blood samples were collected before immunization and 7 days after the third booster. The blood samples were incubated at room temperature for 2 hours and then centrifuged at 3000 x g for 10 minutes at 4°C to separate the serum.

[0202] Antibody titers in serum samples were determined by ELISA.

[0203] The following ELISA assay for measuring antibody titers was performed: Plates were coated with 2.5 µg / mL IL31-1SP, IL31-3SP, 2.5 µg / mL IL31 (CYT 604, Bayer), or 5 µg / mL Material P (RP10178, GenScript) in 0.05 M pH 9.6 carbonate buffer and incubated overnight at 37°C. The plates were then blocked in PBS with 1% gelatin (G9382, Sigma). The wells were then incubated with the specified serum dilutions in PBS-Tween 20 0.1% and gelatin 0.5% for 1 hour at 37°C. The wells were then washed five times with PBS-Tween 20 0.1% and incubated for 1 hour at 37°C with secondary anti-mouse and anti-canine IgG antibodies diluted (1 / 5000) in PBS-Tween 20 0.1% and gelatin 0.5%.

[0204] Competitive ELISA

[0205] For competitive ELISA, plates were coated with 2.5 µg / mL IL31-3SP dissolved in 10 mM Na2CO3 pH 8.0 buffer for 24 hours. After incubation, the plates were blocked with 3% gelatin dissolved in 50 mM PBS at 37°C for 1 hour. After blocking, the plates were incubated with mouse serum diluted 1 / 32000 at 37°C for 1 hour. The mouse serum had previously been incubated at 37°C for 1 hour with one of the following different competing antigens: 5 µg / mL recombinant IL31-3SP (canine sequence), 5 µg / mL recombinant canine IL31 (CYT 604, Bayer), and 20 µl / mL canine SP (RP10178 GenScript). The plates were then washed with 1.5% gelatin, 0.1% Tween, and 50 mM PBS buffer and incubated with a secondary antibody (goat anti-mouse IgG). Next, the plate was washed six times with 1.5% gelatin and 0.1% Tween dissolved in 50 mM PBS, developed with TMB substrate, and the reaction was terminated with 1 MH2SO4. A Multiskan FC (Thermo Fisher Scientific) was used to measure the absorbance at OD 450. The results were analyzed using a paired two-tailed t-test (P < 0.05).

[0206] Dogs with atopic dermatitis were immunized with recombinant IL31-1SP protein.

[0207] Following an open-label, non-placebo-controlled design, a total of five customer-owned dogs were selected for a proof-of-concept clinical trial. These five dogs suffered from refractory atopic dermatitis. All selected dogs were currently receiving some form of medication for dermatitis treatment and had not discontinued medication prior to initiating an immunization cycle with an additional regimen of ongoing medication.

[0208] The formulation and administration of IL31-1SP vaccine in dogs with atopic dermatitis.

[0209] Each dose of the vaccine for dogs was prepared using IL31-1SP as the antigen (5 mg per dose), dissolved in Tris 20 mM pH 8, NaCl 500 mM, urea 4 M, and 20% Montaned Gel 01 PR (Sebik) to a total volume of 1 ml. The components were vortexed for 5 minutes.

[0210] Five dogs received active immunization therapy with the IL31-1SP vaccine. Each dog was given a total of four doses, one dose every 15 days. Venous blood samples were collected before each injection and 15 days after the last dose to analyze antibody titers.

[0211] Dog's itching score.

[0212] To assess the progression of the skin condition and its evolution during an immunization regimen with the IL31-1SP immunogen, a panel of three trained veterinarians used a scratching and pruritus scale on a scale ranging from 1 to 10, as shown in Figure 6. The scale was designed to measure the severity of pruritus in dogs. Pruritus may include scratching, biting, licking, rubbing against objects or the floor

[60] . At diagnosis, the scores were applied to each recruited dog and repeated 15 days after each booster.

[0213] Ethical considerations

[0214] All procedures involving laboratory animals were conducted in accordance with national and international guidelines and approved by the Animal Committee of the Laboratory Animals (CEUA approved protocol: #012-16, Dr. Martina Crispo). This study was conducted strictly in accordance with the requirements of the Pasteur Institute Committee in Montevideo and complied with the current ethical regulations of Uruguay Law 18.611 concerning animal experimentation, which follows the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (USA). All procedures involving a client-owned dog with atopic dermatitis were conducted in accordance with national and international guidelines and approved by the Animal Committee of the Laboratory Animals (CEUA approved protocol: #008-20, Dr. Luis Barbeito). This study was conducted strictly in accordance with the requirements of the Pasteur Institute Committee in Montevideo and complied with the current ethical regulations of Uruguay Law 18.611 concerning animal experimentation, which follows the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (USA). Three veterinary experts supervised the entire process. Prior to the screening process, dog owners received a detailed description of the protocol and signed informed consent forms. Owner participation incurred no cost. Following screening of potential candidates, a total of five dogs were recruited for the study. Systemic subcutaneous immunization using the IL31-1SP immunogen began on day 0, with booster injections repeated on days 15, 30, and 45, as shown in Figure 3. Blood was collected before the initial immunization and each booster. Additionally, blood was collected 2 and 12 weeks after immunization.

[0215] sequence

[0216] Table 1.

[0217]

[0218] plasmid pT7

[0219] In one embodiment, plasmid pT7 was used as an expression vector for cloning recombinant DNA. Correa et al. described T7-modified plasmids in 2014. Gene synthesis of the gene and its cloning in the pT7 vector were performed by GenScript (project U407GHG110). All sequences were optimized for expression in *E. coli* and inserted into the T7 plasmid between BamHI / BamHI restriction sites.

[0220] In one embodiment, the nucleotide sequences of certain recombinant pT7 plasmids are disclosed in Table 2.

[0221] Table 2. Sequences of the immunogenic fusion proteins IL31-1SP and IL31-3SP

[0222]

[0223] For example, the amino acid sequences of certain immunogenic fusion proteins are SEQ ID NO: 11, 17, 23, 29, 14, 20, 26, 32-5. IL31-1SP contains an IL31-derived immunogenic fragment fused to an SP peptide via a specific amino acid linker. IL31-3SP contains an IL31-derived immunogenic fragment fused to three SP peptides, each peptide separated via an amino acid linker. Similar methods can be implemented to prepare recombinant fusion peptides having more than one IL31-derived immunogenic fragment and one or more SP peptides. Similar methods can be implemented to prepare recombinant proteins having IL31 and SP-derived immunogenic fragments having corresponding cat, horse, or human sequences (see Table 2).

[0224] Results

[0225] Example 1. Construction, recombination, purification, and analysis of recombinant fusion immunogenic proteins of IL3-1SP and IL31-3SP.

[0226] Two plasmid constructs, pT7-IL31-1SP and pT7-IL31-3SP, were successfully designed and generated to express recombinant fusion immunogens IL31-1SP and IL31-3SP, which have different signal peptides separated by a 4-amino acid linker. A flexible GSGS linker separates the coding sequences for IL31 and substance P in both constructs. Both constructs include a 6xHis tag for purification and a TEV protease cleavage site, ensuring efficient downstream processing. Figure 2AThe IL31-1SP and IL31-3SP fusion proteins were expressed in *E. coli* BL21 (DE3) cells. Induction with IPTG induced high levels of expression, as evidenced by inclusion bodies observed in cell lysates. The inclusion bodies were dissolved and purified by Ni-NTA affinity chromatography. The purified protein was further processed to remove endotoxins, thus ensuring its suitability for in vivo administration. Figure 2B The purified proteins were insoluble in saline buffer but soluble in 4–8 M urea, further demonstrating their anomalous folding and unnatural sequences and conformations.

[0227] Figure 2C SDS-PAGE analysis of IL31-1SP and IL31-3SP after isolation and purification is shown, with distinct bands observed at approximately 22 kDa and 25.2 kDa, respectively. Lanes 1 and 2 show IL31-1SP and IL31-3SP, both purified by IMAC chromatography. An asterisk indicates the protein of interest, and arrows indicate the theoretical molecular weights of IL31-1SP (22 kDa) and IL31-3SP (25.2 kDa). Figure 2C The protein purity was assessed as high, with minimal contamination bands, indicating effective purification. Further analysis using mass spectrometry provided a detailed characterization of the purified protein. Figure 2D MS1 analysis confirmed the expected molecular weights (22011 Da and 25249 Da) of IL31-1SP and IL31-3SP, respectively. Spectroscopic analysis showed that both proteins matched the indicated theoretical molecular weights, and species with poorly representative post-translational carbamoyl modification sites were also identified (arrows). Figure 2D Top-down MS2 analysis of IL31-1SP and IL31-3SP. MS analysis of the peptides after trypsin digestion. The analysis confirmed the conservation of the predicted amino acid sequences of the two immunogens, as well as the sequences of IL-31 and SP. The sequence coverage of IL31-1SP was 49%, and that of IL31-3SP was 83%, indicating the extent of protein sequence identification by mass spectrometry.

[0228] The results demonstrated the successful construction, expression, and purification of IL31-1SP and IL31-3SP fusion proteins with the expected molecular weight and high purity. The endotoxin removal step ensured that the purified proteins were suitable for subsequent in vivo studies. Overall, these findings validate the method used to generate the IL31-1SP fusion protein and lay the foundation for its application in immunological studies aimed at investigating its potential therapeutic benefits.

[0229] Example 2. Immunization with recombinant fusion IL31-1SP and IL31-3SP immunogens induced antibodies against native IL31 and SP in mice.

[0230] Immunogenicity of recombinant fusion IL31-1SP and IL31-3SP immunogenic proteins in mice. Figure 3A The immunization protocol for mice was described: Mice were divided into two groups for immunization. Group 1 was immunized with the recombinant fusion immunogenic protein IL31-1SP, and Group 2 was immunized with IL31-3SP. Both groups received an initial dose, followed by three booster doses (1 mg / kg) at two-week intervals. Blood samples were collected before the initial vaccination (week 0) and after the final booster (week 7) to assess the immune response. Figure 3B IgG titers specific to IL31, IL31-1SP, IL31-3SP, and substance P: Group 1 (vaccinated with IL31-1SP): The left inset shows the IgG titers specific to IL31-1SP before and after vaccination. The middle inset indicates the IgG titers specific to natural canine IL31, and the right inset shows the IgG titers specific to natural canine substance P (SP). A significant increase in IgG titers was observed after vaccination for both IL31-1SP and natural IL31, indicating a strong immune response. IgG titers specific to natural SP also increased, but to a lesser extent. Group 2 (vaccinated with IL31-3SP): The left inset shows the IgG titers specific to IL31-3SP before and after vaccination. The middle inset indicates the IgG titers specific to natural canine IL31, and the right inset shows the IgG titers specific to natural canine SP. Following vaccination, IgG titers of IL31-3SP and natural IL31 increased significantly, with a specific increase in IgG titers against natural SP, indicating an effective immune response. Figure 3C Competitive ELISA for IgG binding to IL31-3SP. The figure illustrates the binding of IgG to IL31-3SP in the presence of a competitive antigen. The binding of mouse serum to IL31-3SP was tested in the presence of different concentrations of the competitive antigen (0.5 µg IL31-3SP, 0.5 µg non-fusion canine IL31, and 2 µg non-fusion canine SP). Absorbance at 450 nm indicates the level of IgG binding. The results showed that IL31-3SP binding was significantly higher than the sham control (p < 0.05, **p < 0.01), and the competitive antigen reduced IgG binding to IL31-3SP in a dose-dependent manner.

[0231] like Figure 3AAs shown, mice were immunized subcutaneously with the corresponding antigens. Upon completion of the immunization protocol, both antigens IL31- elicited strong immune responses against the vaccine antigens IL31-1SP or IL31-3SP in all mice, with seroconversion rates of 100% and titers >1:10000 and 1:50000, respectively. Vaccination was safe and no side effects were observed. Surprisingly, as... Figure 3B As shown, both antigens also elicited antibodies against the natural species of IL31 and SP. Mice vaccinated with IL31-1SP showed IL31 and SP titers > 1:10000 and > 1:50, respectively. In contrast, mice vaccinated with IL31-3SP showed IL31 and SP titers > 1:10000 and > 1:2000, respectively. Figure 3C The diagram illustrates a competitive ELISA study in which the binding of IgG to the vaccine antigen induced by mouse immunization with IL31-3SP can be significantly competitive with either natural recombinant IL31 or natural SP. The figure shows the binding of IgG to IL31-3SP in the presence of a competitive antigen.

[0232] In summary, Figure 3 demonstrates that both IL31-1SP and IL31-3SP induced strong antigen-specific IgG responses in mice. Compared to IL31-1SP, the IL31-3SP construct elicited a higher immune response, as evidenced by higher IgG titers against the fusion protein, native canine IL31, and native canine substance P. Competitive ELISA confirmed the specificity of the immune responses generated by the recombinant fusion immunogen IL31-3SP against native canine IL31 and SP, with significant competition observed against both non-fusion canine IL31 and substance P.

[0233] Example 3: Comparison of the immunogenicity of recombinant fusion immunogen IL31-3SP with that of single non-fusion native peptides.

[0234] To determine whether the immunogenicity of the IL31-1SP peptide depends on the intrinsic non-natural properties of the recombinant fusion protein, an experiment was conducted in mice to compare the immunogenicity of IL31-3SP with that of a similar vaccine formulation containing equal amounts of single non-fusion canine IL31 and SP. Figure 4A ).

[0235] Comparison of IgG titers in the two groups of mice by ELISA showed that the recombinant fusion IL31-3SP antigen elicited a strong immune response, with antibody titers cross-reactive with canine IL31 and SP ranging from 1:64000 to 1:2000. Figure 4BIn contrast, immunization of mice with a mixture of recombinant canine IL31 and SP alone, along with the same adjuvant, resulted in virtually ineffective immunogenicity. For example, with IL31, the non-fusion peptide induced seroconversion in only 1 out of 3 mice, while with SP, no seroconversion was induced in mice. Figure 4B These results indicate the low immunogenicity of non-fusion peptides and their unique ability to elicit antibodies that cross-react with natural IL31 and SP.

[0236] These results demonstrate that, compared to IL31 and SP used alone as immunogens in vaccine formulations, the recombinant bivalent fusion immunogenic protein IL31-3SP not only enhances the strength and duration of the immune response but also unexpectedly optimizes the quality of the response, producing antibodies with higher affinity for natural IL31 and SP that cross-react with them, which is crucial for vaccine efficacy.

[0237] Comparative immunogenicity of recombinant fusion immunogen IL31-3SP and non-fusion native protein. A. Mouse immunization protocol: Two groups of mice were immunized to compare the immune responses induced by the combination of recombinant fusion immunogen IL31-3SP and non-fusion native canine IL31 and canine substance P (SP). Group 1 was immunized with IL31-3SP, and group 2 was immunized with non-fusion IL31 and non-fusion SP. Both groups received an initial dose, followed by three booster doses (1 mg / kg) at two-week intervals. Blood samples were collected before the initial vaccination (week 0) and after the final booster (week 7) for analysis. B. Specific IgG titers for recombinant antigen IL31-3SP, natural canine IL31, and natural canine SP: Specific IgG titers for natural IL31: The top inset shows a significant increase in specific IgG titers for natural IL31 after vaccination in group 1 (IL31-3SP) compared to group 2 (non-fusion IL31 + non-fusion SP), indicating that the IL31-3SP fusion protein induced a stronger immune response (*p < 0.05). Specific IgG titers for natural SP: The middle inset shows specific IgG titers for natural SP, which significantly increased after vaccination in group 1 (IL31-3SP) compared to immunization with the non-fusion natural protein, demonstrating cross-immune responses induced by the fusion protein (*p < 0.05). IL31-3SP-specific IgG titers: The bottom inset shows IL31-3SP-specific IgG titers, demonstrating a significant immune response in Group 1 (IL31-3SP) compared to Group 2 (non-fusion IL31 + non-fusion SP) after vaccination (*p < 0.05).

[0238] Compared to the single peptide administered as a non-fusion recombinant protein, the recombinant bivalent fusion immunogen IL31-3SP exhibited unexpectedly enhanced immunogenicity. This surprising enhancement is likely due to several factors that promote a robust and specific immune response when injected into a host animal. Key characteristics of this immunogen include: a) a non-natural protein, multiple conformational epitopes, and a bivalent fusion structure: fusing two peptides into an engineered single recombinant molecule containing linkers and molecular tags unexpectedly enhanced the protein's immunogenicity, including the generation of antibodies that cross-react with the naturally occurring physiological species IL31 and SP in the host. b) Evidence was provided that the IL31-3SP protein can break immune tolerance to self-antigens and is more easily captured and processed by APCs due to its optimized size and conformation, which facilitates its internalization and processing within lysosomes. This unexpected advantage enhances the humoral immune response. c) Furthermore, the recombinant bivalent fusion protein IL31-3SP can prolong antigen persistence in the host immune system, providing sustained stimulation of the immune system and promoting the formation of long-term immune memory. This unexpected persistence is crucial for a sustained immune response.

[0239] Example 4: Immunogenicity of the recombinant fusion immunogen IL31-1SP in dogs with atopic dermatitis. Figure 5A As described, IL31-1SP immunogen was used to immunize IL31-customer-owned dogs with Alzheimer's disease (AD). Following the initial immunization, three booster doses of 5 mg antigen in 4 M urea and 20% Montaned (SEPPIC) adjuvant were administered every two weeks. Blood samples were collected before and after each immunization. Dog immunization protocol: Dogs were immunized with the IL31-1SP fusion protein. Each dog received a 5 mg initial dose, followed by three booster doses at two-week intervals (weeks 2, 4, and 6). Blood samples were collected before the initial vaccination (week 0), at the final booster (week 8), and at the follow-up time point (week 20) ​​for clinical evaluation and assessment of the immune response. Two weeks after the final booster, mean antibody titers against native IL31 and SP were detected to be 1:30000 and 1:600, respectively. Figure 5B -C). The most significant peak in antibody titer occurred two weeks after the last booster.

[0240] Figure 5BThe figure shows antibody titers against canine IL31 in individual dogs before vaccination (pre-vaccination) and after the vaccination regimen (post-vaccination). It demonstrates a significant increase in specific IgG titers against canine IL31 observed in all dogs after vaccination, indicating that the recombinant IL31-1SP fusion protein elicited a strong immune response. In contrast, antibody titers against canine SP also increased after vaccination, demonstrating a cross-immune response induced by the IL31-1SP fusion protein. This increase in specific IgG titers after vaccination highlights the potential of IL31-1SP to generate targeted and effective immune responses against self-antigens such as IL31 and SP, further validating its efficacy as an immunogen in mammals. Figure 5C The figure shows the antibody titers against canine substance P (SP) in individual dogs before vaccination (pre-vaccination) and after the vaccination regimen (post-vaccination). An increase in specific IgG titers against canine SP was observed after vaccination, demonstrating a cross-immune response induced by the IL31-1SP fusion protein.

[0241] In summary, Figure 5 illustrates the immunogenicity of the IL31-1SP fusion protein in customer-owned dogs with atopic dermatitis. The vaccination regimen successfully induced a robust antibody response specific to both canine IL31 and SP. The increase in specific IgG titers after vaccination highlights the potential of IL31-1SP to generate a targeted and effective immune response, further validating its efficacy as an immunogen in mammals.

[0242] Example 5: Vaccination with recombinant fusion IL31-1SP antigen improved the severity of itching and reduced corticosteroid use in dogs with atopic dermatitis.

[0243] To assess the therapeutic potential of IL31-1SP vaccination in dogs with Alzheimer's disease (AD), a 1-10 scale was used to quantify the level of pruritus. The scale categorized the severity of symptoms and secondary symptoms. Figure 6A Scratch scores before and after IL31-1SP immunization showed a significant decrease in scratch scores in immunized dogs over time, with the most significant decrease observed at 2 months post-treatment (*p < 0.05). Figure 6B Furthermore, 90 days after vaccination, the number of dogs taking corticosteroids was significantly reduced, indicating symptom improvement and a decreased need for corticosteroid treatment. Figure 6C ).

[0244] These results demonstrate the effectiveness of IL31-1SP treatment in reducing the severity of itching and corticosteroid use in dogs with atopic dermatitis. The scoring system provides a comprehensive assessment of symptoms and appropriate treatment. Dogs treated with IL31-1SP showed a significant reduction in scratching scores over a two-month period, with fewer dogs requiring corticosteroids 90 days after vaccination. IL31-1SP is a promising treatment option for managing atopic dermatitis in dogs.

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Claims

1. An immunogenic fusion protein comprising: At least one IL31 polypeptide (IL31) or its immunogenic fragment; and At least one substance, P-peptide (SP), The immunogenic fusion protein described therein can induce the production of neutralizing polyclonal antibodies against IL31 and SP.

2. The immunogenic fusion protein of claim 1, wherein the neutralizing antibody titer produced by administration of the immunogenic fusion protein to mammals is higher than the neutralizing antibody titer of a combination of non-fusion IL31 peptide and non-fusion SP peptide administered as a single agent.

3. The immunogenic fusion protein according to claim 1, wherein the at least one IL31 polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or an immunogenic fragment thereof.

4. The immunogenic fusion protein according to any one of claims 1 to 3, wherein the at least one SP peptide comprises an amino acid sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO:

9.

5. The immunogenic fusion protein according to claim 4, wherein the at least one SP peptide is one, three, or five SP peptides.

6. The immunogenic fusion protein according to claim 1, comprising an IL-31 polypeptide or an immunogenic fragment thereof fused with one, three or five SP peptides.

7. The immunogenic fusion protein of claim 6, comprising an IL31 polypeptide fused to an SP peptide and optionally linked to a peptide linker.

8. The immunogenic fusion protein according to claim 7, wherein the at least one SP peptide and the at least one IL31 polypeptide or their immunogenic fragments are fused via a flexible peptide linker GSGS.

9. The immunogenic fusion protein according to claim 7, comprising an amino acid sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with one of SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 23, or SEQ ID NO:

29.

10. The immunogenic fusion protein of claim 6, comprising an IL31 polypeptide and three SP peptides, wherein the substances are optionally linked to peptide linkers.

11. The immunogenic fusion protein of claim 10, comprising an amino acid sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with one of SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 26, or SEQ ID NO:

32.

12. The immunogenic fusion protein of claim 6, comprising an IL31 polypeptide and five SP peptides, wherein the substances are optionally linked to peptide linkers.

13. A recombinant vector comprising at least one nucleotide encoding the immunogenic fusion protein according to any one of claims 1 to 12.

14. The recombinant vector of claim 13, comprising a nucleotide sequence encoding the immunogenic fusion protein, said nucleotide sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with one of SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, or SEQ ID NO:

31.

15. The recombinant vector of claim 13, comprising a nucleotide sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with one of SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 27, or SEQ ID NO:

33.

16. A method for producing an immunogenic fusion protein according to any one of claims 1 to 12, wherein the immunogenic fusion protein is produced in a prokaryotic or eukaryotic expression system.

17. The method of claim 16, wherein the immunogenic fusion protein is generated as an inclusion body in a prokaryotic expression system, and the immunogenic fusion protein is dissolved in a high molar concentration of urea.

18. The method of claim 16, wherein the recombinant vector comprises a nucleotide sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with one of SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 27, or SEQ ID NO:

33.

19. An immunogenic composition comprising at least one immunogenic fusion protein according to any one of claims 1 to 12.

20. An immunogenic composition produced by the method according to any one of claims 16 to 18.

21. The immunogenic composition of claim 19, further comprising an acceptable carrier and / or adjuvant, said adjuvant being selected from the group consisting of: oil-in-water adjuvants, polymeric and aqueous adjuvants, water-in-oil adjuvants, aluminum hydroxide adjuvants, and combinations thereof.

22. The immunogenic composition according to claim 21, wherein the adjuvant is a complete or incomplete Freund's adjuvant, Montanide, or other similar adjuvant. TM Gel 01 PR, aluminum salts (alum), oil emulsions, saponins, immunostimulatory complexes (ISCOM), liposomes, microparticles, nonionic block copolymers, derived polysaccharides, cytokines, or bacterial derivatives.

23. The immunogenic composition according to any one of claims 19 to 22, which is used as a pharmaceutical, preferably a vaccine.

24. The immunogenic composition according to claim 23, for the treatment and / or prevention of pruritus-related skin conditions, preferably Alzheimer's disease (AD), most preferably inflammation in chronic and / or refractory AD.

25. The immunogenic composition for use according to claim 24, wherein AD is refractory to at least one anti-inflammatory compound selected from the group consisting of: corticosteroids, antihistamines, tyrosine kinase inhibitors, and cyclosporine.

26. A pharmaceutical composition for treating or preventing AD and / or AD-related symptoms in an individual, said pharmaceutical composition comprising at least one immunogenic fusion protein according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier.

27. The pharmaceutical composition of claim 26, wherein AD-related symptoms are itching and / or secondary skin lesions.

28. The pharmaceutical composition of claim 27, wherein AD is refractory to at least one anti-inflammatory compound selected from the group consisting of: corticosteroids, antihistamines, tyrosine kinase inhibitors, and cyclosporine.

29. A method for treating or preventing AD and / or AD-related symptoms in mammals, the method comprising administering an effective amount of the immunogenic composition according to any one of claims 19 to 22.

30. The method of claim 29, wherein the AD-related symptoms are itching and / or secondary skin lesions.

31. The method of claim 29, wherein the application of the effective amount of the immunogenic composition results in a reduction in the pruritus score, the pruritus score being determined using a pruritus scale (e.g., scratching, biting, licking, and rubbing an object or floor) as defined in FIG6A.

32. The method of claim 29, wherein the AD-related symptoms are refractory to at least one anti-inflammatory compound selected from the group consisting of: corticosteroids, antihistamines, tyrosine kinase inhibitors, and cyclosporine.

33. The method according to any one of claims 29 to 31, wherein the immunogenic composition is administered in combination with at least one anti-inflammatory compound selected from the group consisting of: corticosteroids, antihistamines, tyrosine kinase inhibitors, and cyclosporine.

34. The method according to any one of claims 29 to 33, wherein the immunogenic composition is administered in combination with at least one receptor antagonist that blocks nociceptive signaling pathways, preferably a neurokinin-1 receptor (NK1-R) antagonist, and most preferably compound CP-96,345.

35. The method according to any one of claims 29 to 34, wherein the immunogenic composition is administered orally, subcutaneously, intramuscularly, or transdermally, preferably subcutaneously.

36. The method according to any one of claims 29 to 35, wherein the immunogenic composition is administered at an initial dose, followed by three booster doses at intervals of approximately 2 weeks, and optionally one or more booster doses at intervals of 3 to 6 months or for a period of time required to maintain high and neutralizing antibody titers.

37. The method according to any one of claims 29 to 36, wherein the mammal is a dog, human, cat, or horse.

38. A method for treating or preventing AD or AD-related symptoms in dogs, the method comprising subcutaneous administration of a bivalent vaccine at an initial dose, followed by three booster doses at intervals of approximately 2 weeks, and optionally one or more subsequent booster doses at intervals of 3 to 6 months or for a period of time required to maintain high and neutralizing antibody titers. The bivalent vaccine comprises an immunogenic fusion protein IL31-SP or an immunogenic fusion protein IL31-3SP, wherein the immunogenic fusion protein IL31-SP comprises an amino acid sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO: 11, and the immunogenic fusion protein IL31-3SP comprises an amino acid sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO:

14.

39. A bivalent vaccine for the treatment or prevention of inflammatory-associated skin conditions, preferably Alzheimer's disease (AD) or AD-related symptoms in mammals, said bivalent vaccine comprising an immunogenic fusion protein according to any one of claims 1 to 12.

40. A bivalent vaccine for treating or preventing Alzheimer's disease (AD) and / or pruritus in dogs, the bivalent vaccine comprising an immunogenic fusion protein IL31-1SP or an immunogenic fusion protein IL31-3SP, wherein the immunogenic fusion protein IL31-1SP comprises an amino acid sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO: 11, and the immunogenic fusion protein IL31-3SP comprises an amino acid sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO: 14.

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