Mammalian il-4 / il-13 / flagellin constructs

Abstract

The present disclosure relates to IL-13 / IL-4 / Flagellin fusion proteins for the stimulation of the production of anti-IL-4 and anti-IL13 antibodies in a non-human mammal, in particular antibodies that reduce Interleukin (IL)-4 and Interleukin (IL)-13 in a subject.

Description

MAMMALIAN IL-4 / IL-13 / FLAGELLIN CONSTRUCTS FIELD OF THE INVENTION

[0001] The present disclosure relates a self-vaccine construct for neutralization of Interleukin(IL)-4 and Interleukin (IL) -13 in for the treatment of inflammation, including inflammation associated with atopic dermatitis and allergic dermatitis. REFERENCE TO SEQUENCE LISTING

[0002] Pursuant to 37 C.F.R. 1.821(c), a sequence listing is submitted herewith as an ASCIIcompliant text file named 2920951-523000-SEQ-LISTING 2_0, created on 25 July 2024 and having a size of 29,774 kilobytes. The content of the aforementioned file is hereby incorporated by reference in its entirety. BACKGROUND

[0003] Atopic dermatitis is an allergic skin disorder that is characterized by aberrant andexcessive Th2 cell and ILC2 activation, with robust expression of type 2 cytokines, including interleukin (IL)-4, IL-5, IL-13 and IL-31, and variable activation of other cytokines, in particular IL-22 and IL-33, but also IL-17, IL-9 and IFN-γ. Moyle et al. Experimental Dermatology.2019; 28:756–768; Renert-Yuval & Guttman-Yassky, Dermatol Clin.2019; 37:205–213. Atopic dermatitis is a frequent disorder in animals, particularly dogs. In fact, atopic dermatitis is the most common allergy in dogs and affects approximately 10% of the dog population, resulting in 15 million to 20 million dogs suffering from the disease in Europe and the United States alone Griffin, et al. “The ACVD task force on canine atopic dermatitis (XIV): clinical manifestations of canine atopic dermatitis.” Veterinary immunology and immunopathology. 2001; 81(3-4), 255- 269. The itching or pruritus which is caused by this allergic skin disease is usually recurrent or chronic. It deeply impacts the quality of life for both the dogs and their owners.

[0004] There exists a need in the art for the treatment of atopic dermatitis and allergic dermatitis.SUMMARY OF THE INVENTION

[0005] In an embodiment, a fusion protein can comprise:an IL-13 polypeptide; an IL-4 polypeptide; and a flagellin polypeptide.

[0006] In an embodiment, the N-terminal of the flagellin polypeptide is linked to the C-terminalof the IL-4 polypeptide. The N-terminal of the IL-4 polypeptide can be linked to the C-terminal of IL-13 polypeptide.

[0007] In an embodiment, the fusion protein can comprise: a flagellin polypeptide linked to anIL-4 polypeptide linked to an IL-13 polypeptide linked to an IL-4 polypeptide linked to an IL-13 polypeptide.

[0008] In an embodiment, the fusion protein can comprise: a flagellin polypeptide linked to anIL-13 polypeptide linked to an IL-4 polypeptide linked to an IL-13 polypeptide linked to an IL-4 polypeptide.

[0009] In an embodiment, the fusion protein can comprise: IL-4 polypeptide linked to an IL-13polypeptide linked to an IL-4 polypeptide linked to an IL-13 polypeptide linked to a flagellin polypeptide.

[0010] In an embodiment, the fusion protein can comprise: an IL-13 polypeptide linked to an IL-4 polypeptide linked to an IL-13 polypeptide linked to an IL-4 polypeptide linked to a flagellin polypeptide.

[0011] In an embodiment, the fusion protein can comprise: a flagellin polypeptide linked to anIL-4 polypeptide linked to an IL-13 polypeptide.

[0012] In an embodiment, the fusion protein can comprise: IL-13 polypeptide linked to an IL-4polypeptide linked to a flagellin polypeptide.

[0013] In an embodiment, the fusion protein can comprise: an IL-4 polypeptide linked to an IL-13 polypeptide to a flagellin polypeptide.

[0014] In an embodiment, the fusion protein can comprise: two IL-4 polypeptides.

[0015] In an embodiment, the fusion protein can comprise: two IL-13 polypeptides.

[0016] In an embodiment, the flagellin polypeptide and the IL-4 polypeptide are joined by alinker.

[0017] In an embodiment, the IL-4 polypeptide and IL-13 polypeptide are joined by a linker. Thelinker can be an oligopeptide comprising about 4 to 20 amino acids. The oligopeptide can be a polyglycine oligopeptide. The oligopeptide can comprise the amino acid sequence of SEQ ID NO: 8 or 9. The oligopeptide can be a polyglycine / serine oligopeptide. IThe oligopeptide can comprise the amino acid sequence of SEQ ID NO: 10, 11, or 12.

[0018] In an embodiment, the flagellin polypeptide is a B. subtilis sequence. The flagellinpolypeptide can comprise an amino acid sequence with at least 75% homology to the amino acidsequence of SEQ ID NO: 5. The flagellin polypeptide can comprise an amino acid sequence withat least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 5.

[0019] In an embodiment, the IL-4 polypeptide is a canine IL-4 polypeptide. The IL-4 cancomprise an amino acid sequence with at least 75% homology to the amino acid sequence ofSEQ ID NO: 4. The IL-4 can comprise an amino acid sequence with at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 4.

[0020] In an embodiment, the IL-13 is a canine IL-13 polypeptide. The IL-13 can comprise anamino acid sequence with at least 75% homology to the amino acid sequence of SEQ ID NO: 3. The IL-13 can comprise an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 3.

[0021] In an embodiment, the fusion protein comprises an amino acid sequence with at least75% homology to the amino acid sequence of SEQ ID NO: 1. The fusion protein can comprise an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 1.

[0022] In an embodiment, the fusion protein can further comprise a Tetanus toxin T-cell epitope.The Tetanus toxin T-cell epitope can be between the Flagellin and IL-14, between the Flagellin and IL-4, between the IL-4 and IL-13, or a combination thereof. The Tetanus toxin T-cell epitope can be a Tetanus toxin T cell epitope p2, Tetanus toxin T cell epitope p30, or a combination. The Tetanus toxin T-cell epitope can comprise an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 6 or 7.

[0023] In an embodiment, the fusion protein can further comprise an ER import signal sequenceon the N-terminal.

[0024] In an embodiment, the fusion protein can further comprise an affinity tag on the C-terminal. The affinity tag can be a hexahistidine-tag.

[0025] In an embodiment, a method for treating or preventing inflammation can compriseadministering to a mammal in need thereof an effective amount of the fusion protein described herein.

[0026] In an embodiment, a method for treating or preventing inflammation can compriseadministering to a mammal in need thereof a composition comprising an effective amount of the fusion protein described herein.

[0027] The dermatological condition can comprise at least one skin disorder selected frompsoriasis, atopic dermatitis, skin rash, skin irritation, skin sensitization, allergic reactions, pruritus, and combinations thereof. The inflammation can be associated with atopic dermatitis. The inflammation can be associated with allergic dermatitis.

[0028] In an embodiment, the effective amount is between about 0.1 and 10 mg / kg. Theeffective amount can be between about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg. The effective amount can be between about 0.1 and 1 mg / kg, 0.5 and 2 mg / kg, 0.75 and 5 mg / kg, or 1 and 10 mg / kg. The effective amount can be between about 100 μg and 300 μg. The effective amount can be between about 100 μg and 200 μg, 150 μg and 250 μg, 175 μg and 225 μg, 195 μg and 205 μg, or 150 μg and 300 μg. The effective amount can be about 100 µg, 105 µg, 110 µg, 115 µg, 120 µg, 125 µg, 130 µg, 135 µg, 140 µg, 145 µg, 150 µg, 151 µg, 152 µg, 153 µg, 154 µg, 155 µg, 156 µg, 157 µg, 158 µg, 159 µg, 160 µg, 161 µg, 162 µg, 163 µg, 164 µg, 165 µg, 166 µg, 167 µg, 168 µg, 169 µg, 170 µg, 171 µg, 172 µg, 173 µg, 174 µg, 175 µg, 176 µg, 177 µg, 178 µg, 179 µg, 180 µg, 181 µg, 182 µg, 183 µg, 184 µg, 185 µg, 186 µg, 187 µg, 188 µg, 189 µg, 190 µg, 191 µg, 192 µg, 193 µg, 194 µg, 195 µg, 196 µg, 197 µg, 198 µg, 199 µg, 200 µg, 201 µg, 202 µg, 203 µg, 204 µg, 205 µg, 206 µg, 207 µg, 208 µg, 209 µg, 210 µg, 211 µg, 212 µg, 213 µg, 214 µg, 215 µg, 216 µg, 217 µg, 218 µg, 219 µg, 220 µg, 221 µg, 222 µg, 223 µg, 224 µg, 225 µg, 226 µg, 227 µg, 228 µg, 229 µg, 230 µg, 231 µg, 232 µg, 233 µg, 234 µg, 235 µg, 236 µg, 237 µg, 238 µg, 239 µg, 240 µg, 241 µg, 242 µg, 243 µg, 244 µg, 245 µg, 246 µg, 247 µg, 248 µg, 249 µg, 250 µg, 255 µg, 260 µg, 265 µg, 270 µg, 275 µg, 280 µg, 285 µg, 290 µg, 295 µg, or 300 µg.

[0029] In an embodiment, the fusion protein further comprises an ER import signal sequence onthe N-terminal.

[0030] In an embodiment, the fusion protein further comprises an affinity tag on the C-terminal.The affinity tag can be a hexa-histidine-tag.

[0031] In an embodiment, a composition can comprise a fusion protein described herein. Thecomposition can be a pharmaceutical composition. The pharmaceutical composition can further comprise a pharmaceutical excipient, carrier, diluent, adjuvant, or a combination thereof. The composition can be formulated for intravenous, subcutaneous, infusion, oral, intrathecal, intraperitoneal, parenteral administration, or a combination thereof. The composition can further comprise an anti-IL-31 antibody, anti-IL-5 antibody, anti-IL-22 antibody, or a combination thereof.

[0032] In an embodiment, the effective amount of a fusion protein described herein in acomposition can be between about 0.1 and 10 mg / kg. The effective amount can be between about 1 and 500 μg, optionally between about 150 and 250 μg. The effective amount can be about 150 µg, 151 µg, 152 µg, 153 µg, 154 µg, 155 µg, 156 µg, 157 µg, 158 µg, 159 µg, 160 µg, 161 µg, 162 µg, 163 µg, 164 µg, 165 µg, 166 µg, 167 µg, 168 µg, 169 µg, 170 µg, 171 µg, 172 µg, 173 µg, 174 µg, 175 µg, 176 µg, 177 µg, 178 µg, 179 µg, 180 µg, 181 µg, 182 µg, 183 µg, 184 µg, 185 µg, 186 µg, 187 µg, 188 µg, 189 µg, 190 µg, 191 µg, 192 µg, 193 µg, 194 µg, 195 µg, 196 µg, 197 µg, 198 µg, 199 µg, 200 µg, 201 µg, 202 µg, 203 µg, 204 µg, 205 µg, 206 µg, 207 µg, 208 µg, 209 µg, 210 µg, 211 µg, 212 µg, 213 µg, 214 µg, 215 µg, 216 µg, 217 µg, 218 µg, 219 µg, 220 µg, 221 µg, 222 µg, 223 µg, 224 µg, 225 µg, 226 µg, 227 µg, 228 µg, 229 µg, 230 µg, 231 µg, 232 µg, 233 µg, 234 µg, 235 µg, 236 µg, 237 µg, 238 µg, 239 µg, 240 µg, 241 µg, 242 µg, 243 µg, 244 µg, 245 µg, 246 µg, 247 µg, 248 µg, 249 µg, or 250 µg.

[0033] In an embodiment, a composition can comprise a fusion protein described herein for thetreatment of inflammation. The inflammation can be associated with atopic dermatitis. The inflammation can be associated with allergic dermatitis.

[0034] In an embodiment, a composition can comprise a fusion protein described herein for thetreatment of atopic dermatitis.

[0035] In an embodiment, a composition can comprise a fusion protein described herein for thetreatment of allergic dermatitis.

[0036] In an embodiment, a nucleotide sequence can encode a fusion protein described herein.

[0037] In an embodiment, an expression vector can comprise a nucleotide sequence encoding afusion protein described herein.

[0038] In an embodiment, a recombinant host cell can comprise a nucleotide sequence encodinga fusion protein described herein.

[0039] In an embodiment, a recombinant host cell can comprise an expression vector comprisingthe nucleotide sequence encoding a fusion protein described herein.

[0040] In an embodiment, a method of treating or preventing inflammation comprisingadministering to a mammal in need thereof an effective amount of a fusion protein described herein.

[0041] In an embodiment, a method of treating or preventing inflammation comprisingadministering to a mammal in need thereof an effective amount of the composition comprising an effective amount of a fusion protein described herein.

[0042] In an embodiment, the dermatological condition comprises at least one skin disorderselected from psoriasis, atopic dermatitis, skin rash, skin irritation, skin sensitization, allergic reactions, pruritus, and combinations thereof.

[0043] In an embodiment, the inflammation is associated with atopic dermatitis.

[0044] In an embodiment, the inflammation is associated with allergic dermatitis.

[0045] In an embodiment, the mammal is a non-human mammal.

[0046] In an embodiment, the mammal is a canine.

[0047] In an embodiment, the mammal is a dog. The dog can be at least 9 months of age. Thedog can be at least 12 months of age.

[0048] In an embodiment, the mammal is a cat.

[0049] In an embodiment, the administering is performed daily.

[0050] In an embodiment, the administering is performed twice daily.

[0051] In an embodiment, the administering is performed weekly.

[0052] In an embodiment, the administering is performed monthly.

[0053] In an embodiment, the administering is performed twice, once a day 0 and a second timeat day 28.

[0054] In an embodiment, the effective amount is between about 0.1 and 10 mg / kg. Theeffective amount can be between about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg. The effective amount can be between about 0.1 and 1 mg / kg, 0.5 and 2 mg / kg, 0.75 and 5 mg / kg, or 1 and 10 mg / kg.

[0055] Use of a fusion protein described herein for the manufacture of a medicament for thetreatment of inflammation in a mammal. The inflammation can be associated with atopic dermatitis or allergic dermatitis.

[0056] BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The patent or application file contains at least one drawing executed in color. Copies ofthis patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee

[0058] FIG. 1 depicts a IL-13 / IL-4 / Flagellin construct. The IL-13 / IL-4 / Flagellin constructdescribed herein can comprise the amino acid sequence of SEQ. ID. NO: 1.

[0059] FIG. 2 depicts the bulk antibody titers, anti-cIL-4 (A) and anti-cIL-13 (B) afterimmunization with the IL-13 / IL-4 / Flagellin construct. The inventors observed no / low titers in primary immunizations and high titers after booster shots. The booster shots achieved endpoint titers exceeding 1:100,000 in the case of cIL-4, and > 1:10,000 in the case of cIL-13.FIG. 3 depicts the suppression of cytokine responses, anti-cIL-4 (A) and anti-cIL-13 (B) after immunization with the IL-13 / IL-4 / Flagellin construct. The inventors observed in the case of cIL- 4 neutralizing antibodies already in the primary immunization phase at days 21 and 28. These titers were greatly increased after booster shots, reaching full cIL-4 neutralization titers of ≥ 1:40,000 at day 35 - day 56, with a subsequent gradual decrease to 1:20,000 (day 64), 1:5000 (day 70) and 1:4000 (day 84). In the case of cIL-13, neutralizing antibodies were not detected in the primary immunization phase, but were strongly present after booster shots. These titers reached full cIL-13 neutralization at ≥ 1:1280 at day 35, ≥ 1:640 at day 42 - day 49, ≥ 1:160 at day 56 - day 70 and 1:80 at day 84

[0060] FIG. 4 depicts the bulk antibody titers, anti-cIL-4 (A) and anti-cIL-13 (B) afterimmunization with the IL-13 / IL-4 / Flagellin construct. The inventors observed no / low titers in primary immunizations and high titers after booster shots. The booster shots achieved endpoint titers exceeding 1:40,000 in the case of both cIL-4 and cIL-13.

[0061] FIG. 5 depicts the suppression of cytokine responses, anti-cIL-4 (A) and anti-cIL-13 (B)after immunization with the IL-13 / IL-4 / Flagellin construct. The inventors observed in the case of cIL-4 neutralizing antibodies already in the primary immunization phase at day 28. These titers were greatly increased after booster shots, reaching full cIL-4 neutralization titers of ≥ 1:40,000 at day 35 - day 42, with a subsequent gradual decrease to 1:20,000 (day 49 – day 56), 1:5000 (day 64), 1:2500 (day 70) and 1:1000 at day 84. In the case of cIL-13, neutralizing antibodies were not detected in the primary immunization phase, but were strongly present after boostershots. These titers reached full cIL-13 neutralization at ≥ 1:1280 at day 35 – day 42, ≥ 1:640 at day 49, ≥ 1:320 at day 56, ≥ 1:160 at day 64 - day 70 and 1:80 at day 84

[0062] FIG. 6 depicts the bulk antibody titers, anti-cIL-4 (A) and anti-cIL-13 (B) afterimmunization with the IL-13 / IL-4 / Flagellin construct. The inventors observed no / low titers in primary immunizations and high titers after booster shots. The booster shots achieved endpoint titers exceeding 1:100,000 in the case of both cIL-4 and cIL-13.

[0063] FIG. 7 depicts the suppression of cytokine responses, anti-cIL-4 (A) and anti-cIL-13 (B)after immunization with the IL-13 / IL-4 / Flagellin construct. The inventors observed in the case of cIl-4 neutralizing antibodies already in the primary immunization phase at days 21 and 28. These titers were greatly increased after booster shots, reaching full cIL-4 neutralization titers of ≥ 1:40,000 at day 35 , ≥ 1:20,000 at day 42, with a subsequent gradual decrease to 1:10,000 (day 49 – day 56), 1:5000 (day 63 – day 70) and 1:2500 at day 84. In the case of cIL-13, neutralizing antibodies were not detected in the primary immunization phase, but were strongly present after booster shots. These titers reached full cIL-13 neutralization at ≥ 1:5000 at day 35, ≥ 1:1280 at day 42 – day 49, ≥ 1:640 at day 56, ≥ 1:320 at day 64 - day 70 and 1:160 at day 84. Taken together, immunization of dogs with cIL-13-cIL-4-poly-BsFliC (SEQ ID NO: 1), after the booster shots, leads to the development of high titers of specific antibodies against both cIL-4 and cIL-13, and serum form these immunized animals effectively suppresses cIl-4 and cIL-13 activity in a relevant dog macrophage readout system in all three immunized dogs.

[0064] FIG. 8 depicts the ex vivo blood stimulation assays, immunized animals, cIL-4 or cIL-13stimulation, cTARC mRNA induction (qPCR) at Day 0 (A), Day 28 (B), Day 42 (C), Day 56 (D), Day 72 (E), and Day 84 (F). At day 0 (-5), blood of the three dogs to be vaccinated with cIL-13-cIL-4-poly-BsFliC and the conteol dog responded well to ex vivo stimulation with cIL-4 and cIL-13 with respect to cTARC mRNA induction (90-400-fold for cIL-4 and 30-100-fold for cIL-13, Fig. 8A). The primary immunization phase probed at day 28 led to a decrease in cIL-4 induced cTARC mRNA production in all three immunized dogs by 98-100% relative to the control dog. Upon cIL-13 stimulation, blood of 2 dogs showed 90% and 100% cTARC mRNA reduction, while one dog was at 50% reduction (Fig. 8B). Two weeks into the booster immunization, at day 42, decreases of cTARC mRNA were 98-100% for cIL-4 stimulated blood and 90-100% for cIL-13 stimulated blood, in all three dogs relative to the non-vaccinated control dog (Fig.8C). At day 56 and day 84, the cTARC mRNA decreases were 96-100% and 99.5-100% for cIL-4, respectively, and 100% and 99-100%, respectively (Fig.8D and 8F). At day 72, the measured cTARC suppression dropped to 80-100% for cIL-4 stimulation, and 30-100% for cIL-13 stimulation. This day 72 measurement was however burdened with very low signals overall.

[0065] Taken together, immunization of dogs with cIL-13-cIL-4-poly-BsFliC (SEQ ID NO:1)effectively suppresses both cIL-4 and cIL-13 function in the animals’ blood at least up to day 84. This suppression is already visible in primary immunization phase (at least day 28). Definitions

[0066] Unless otherwise defined, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0067] “Non-human mammals,” as used herein, refers broadly to non-human mammaliananimals, including but not limited to dogs, cats, mice, rats, guinea pigs, rabbits, ferrets, cows, horses, sheep, goats, and pigs. Non-human mammals can be mammalian pets or companion animals, including but not limited to dogs and cats and also mice, rats, guinea pigs, ferrets, and rabbits. The non-human mammal can be a dog or a cat.

[0068] “Treatment” refers broadly to both therapeutic treatment and prophylactic or preventativemeasures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. As used herein, the term “treating,” refers broadly to treating a disease, arresting, or reducing the development of the disease or its clinical symptoms, and / or relieving the disease, causing regression of the disease or its clinical symptoms. Therapy encompasses prophylaxis, treatment, remedy, reduction, alleviation, and / or providing relief from a disease, signs, and / or symptoms of a disease. Therapy encompasses an alleviation of signs and / or symptoms in patients with ongoing disease signs and / or symptoms. Therapy also encompasses “prophylaxis”. The term “reduced”, for purpose of therapy, refers broadly to the clinical significant reduction in signs and / or symptoms. Therapy includes treating relapses orrecurrent signs and / or symptoms. Therapy encompasses but is not limited to precluding the appearance of signs and / or symptoms anytime as well as reducing existing signs and / or symptoms and eliminating existing signs and / or symptoms. Therapy includes treating chronic disease (“maintenance”) and acute disease. For example, treatment includes treating or preventing relapses or the recurrence of signs and / or symptoms.

[0069] “Host cell,” as used herein refers broadly to the particular subject cell transfected with anucleic acid molecule and the progeny or potential progeny of such a cell. Progeny may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.

[0070] “Effective amount,” as used herein, refers broadly to an amount of construct to breakself-tolerance to a normally self-expressed antigen and result in a self immune response against said self-expressed antigen. Effective amount may be an amount that results in an immune response to the self antigen at a level sufficient to affect the function of the self antigen. Effective amount may be an amount that results in an immune response to the self antigen at a level to interfere with the binding of a self agonist with the self antigen. For example, the effective amount may be an amount sufficient to result in an immune response the interferes with binding of myostatin to an activin receptor. The term “effective amount” is synonymous with “therapeutically effective amount” for purposes described herein.

[0071] “Mammal,” as used herein, refers broadly to any and all warm-blooded vertebrateanimals of the class Mammalia, characterized by a covering of hair on the skin and, in the female, milk-producing mammary glands for nourishing the young. Mammals include, but are not limited to, humans, domestic and farm animals, and zoo, sports, or pet animals. Mammal also includes any and all those listed on the Mammal Species of the World maintained by the National Museum of Natural History, Smithsonian Institution in Washington D.C. Similarly, the term “subject” or “patient” includes both human and veterinary subjects and / or patients.

[0072] “Fusion protein,” a used herein, refers broadly to the in frame genetic linkage of at leasttwo heterologous polypeptides. Upon transcription / translation, a single protein is made. In this way, multiple proteins, or fragments thereof can be incorporated into a single polypeptide. “Operably linked” refers to the functional linkage between two or more elements. For example, an operable linkage between two polypeptides fuses both polypeptides together in frame toproduce a single polypeptide fusion protein. In an aspect, the fusion protein can further comprise a third polypeptide which can comprise a linker sequence.

[0073] “Variant,” as used herein refers broadly to a polypeptide that possesses a similar oridentical function as the IL-4 or IL-13 but does not necessarily comprise a similar or identical amino acid sequence of the IL-4 or IL-13, or possesses a similar or identical structure of the IL-4 or IL-13. A variant having a similar amino acid identity refers to a polypeptide that satisfies at least one of the following: (a) a polypeptide comprising, or alternatively consisting of, an amino acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence of the IL-4 or IL-13; (b) a polypeptide encoded by a nucleotide sequence, the complementary sequence of which hybridizes under stringent conditions to a nucleotide sequence encoding the IL-4 or IL-13, of at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, or at least 150 amino acid residues; and (c) a polypeptide encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99%, identical to the nucleotide sequence encoding the IL-4 or IL-13. A polypeptide with similar structure to an IL-4 or IL-13 described herein refers to a polypeptide that has a similar secondary, tertiary or quaternary structure of an IL-4 or IL-13 as described herein. The structure of a polypeptide can be determined by methods known to those skilled in the art, including but not limited to, X-ray crystallography, nuclear magnetic resonance, and crystallographic electron microscopy. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding positionin the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by thesequences (e.g., % identity = number of identical overlapping positions / total number of positionsx 100%). In one embodiment, the two sequences are the same length.

[0074] The determination of percent identity between two sequences can be accomplished usinga mathematical algorithm known to those of skill in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993). The BLASTn and BLASTx programs of Altschul, et al. J. Mol. Biol. 215:403-410(1990) have incorporated such an algorithm. BLAST nucleotide searches can be performed with the BLASTn program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid molecules described herein. BLAST protein searches can be performed with the BLASTx program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to a protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. Nucleic Acids Res.25:3389-3402 (1997). Alternatively, PSI-BLAST can be used to perform an iterated search that detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used.

[0075] Another example of a mathematical algorithm utilized for the comparison of sequences isthe algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0), which is part of the GCG sequence alignment software package, has incorporated such an algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM asdescribed in Torellis and Robotti Comput. Appl. Biosci., 10 :3-5(1994); and FASTA described inPearson and Lipman Proc. Natl. Acad. Sci. 85:2444-8(1988). Within FASTA, ktup is a controloption that sets the sensitivity and speed of the search.

[0076] “Conservative” amino acid substitutions are those substitutions that do not substantiallyaffect or decrease the affinity of a protein, such as the IL-4 antibody or IL-13 antibody against IL-4 or IL-13, respectively. For example, the IL-4 antibody or IL-13 antibody binds IL-14 or IL- 13, respectively, can include at most about 1, at most about 2, at most about 5, at most about 10, or at most about 15 conservative substitutions and bind IL-4 or IL-13. The term “conservativevariant” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that the IL-4 antibody or IL-13 antibody binds IL-4 or IL-13, respectively. Non-conservative substitutions are those that reduce binding to IL-4 or IL-13.

[0077] Conservative amino acid substitution tables providing functionally similar amino acidsare well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0078] The term “derivative” as used herein, refers to a variant polypeptide described herein thatcomprises, or alternatively consists of, an amino acid sequence of an antibody described herein that binds to IL-4 or IL-13, which has been altered by the introduction of amino acid residue substitutions, deletions or additions. The term “derivative” as used herein also refers to a protein that binds IL-4 or IL-13 that has been modified, e.g., by the covalent attachment of any type of molecule to the polypeptide. For example, but not by way of limitation, an antibody or protein that binds to IL-4 or IL-13 can be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. A derivative of the antibody or protein that binds to IL-4 or IL-13 can be modified by chemical modifications using techniques known to those of skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Further, a derivative of an antibody or protein that binds to IL-4 or IL-13 can contain one or more non-classical amino acids. A polypeptide derivative possesses a similar or identical function as an antibody or protein that binds to IL-4 or IL-13 described herein.

[0079] “Host cell,” as used herein refers broadly to a cell transfected with a nucleic acidmolecule and the progeny or potential progeny of such a cell. Progeny may not be identical to theparent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome. IL-4 / IL-13 / FLAGELLIN SELF-VACCINE

[0080] IL-4 and IL-13 are Th2 cytokines that are potent mediators of type 2-associatedinflammation, such as those found in atopic dermatitis and allergic dermatitis in canines. IL-13 and IL-4 are potent mediators of type 2–associated inflammation such as those found in atopic dermatitis. IL-4 shares overlapping biological functions with IL-13, a finding that is mainly explained by their ability to signal via the type 2 IL-4 receptor (R), which is composed of IL-4Rα in association with IL-13Rα1.

[0081] The IL-4 / IL-13 / Flagellin self vaccine described herein breaks self tolerance in the animalto allow for the production of anti-IL-4 and anti-IL13 antibodies which reduce the amount and activity of IL-4 and / or IL-13. A reduction in the amount and activity of IL-4 and / or IL-13 can provide relief from atopic dermatitis and allergic dermatitis.

[0082] The IL-13 / IL-4 / Flagellin fusion protein constructs described herein can comprise an IL-13 polypeptide linked to an IL-4 polypeptide, with the IL-4-linker-IL-13 portion repeated, linked to a flagellin protein. The flagellin protein can be a B. subtilis protein. The IL-4 and / or IL-13 can be canine IL-4 and / or IL-13. The IL-4 and IL-13 can be linked by a linker comprising, for example, a polyglycine peptide, e.g., GGGGG (SEQ ID NO: 8), GGGGGGGG (SEQ ID NO: 9), or a glycine-serine sequence, e.g., GGGGSGGGGSGGGGG (SEQ ID NO: 10), GGGSG (SEQ ID NO: 11), GSGGS (SEQ ID NO: 12). For example, the IL-13 / IL-4 / Flagellin fusion protein constructs described herein can comprise the following formulas: Flagellin–linker–IL-4–linker–IL-13–linker–IL-4–linker–IL-13 IL-4–linker–IL-13–linker–IL-4–linker–IL-13–linker–Flagellin Flagellin–linker–IL-13–linker–IL-4–linker–IL-13–linker–IL-4 IL-13–linker–IL-4–linker–IL-13–linker–IL-4–linker–Flagellin Flagellin–linker–IL-4–linker–IL-13IL-13–linker–IL-4–linker–flagellin

[0083] The IL-13 / IL-4 / Flagellin fusion protein constructs described herein can further comprisean artificial optimized ER import signal sequence, e.g., MGWSCIILFLVATATGVHS (SEQ ID NO: 13), at the N-terminus. The IL-13 / IL-4 / Flagellin fusion protein constructs described herein can further comprise a polyhistidine tag, e.g., a stretch of 5-8 histidines, e.g., SEQ ID NO: 14. The IL-13 / IL-4 / Flagellin fusion protein constructs described herein can further comprise Tetanus toxin T cell epitopes, e.g., Tetanus toxin P2, Tetanus toxin P30, or a combination thereof. Tetanus toxin P2 can comprise the amino acid sequence of SEQ ID NO: 6. The Tetanus toxin P30 can comprise the amino acid sequence of SEQ ID NO: 7. The Tetanus toxin T cell epitopes may be interspersed in the construct between the flagellin, IL-4, and IL-13 polypeptides. Further, the conjugation of the flagellin to the T-cell epitopes and IL-4 and IL-13 polypeptides via afusion protein construct is different from conjugation by chemical linkage. For example, the IL-13 / IL-4 / Flagellin fusion protein constructs described herein can comprise the following formulas: ER import signal sequence-Flagellin-linker-IL-4-linker-Tetanus toxin epitope-linker-IL-13- linker- Tetanus toxin epitope-linker-IL-4-linker- Tetanus toxin epitope-linker-IL-13 Flagellin -linker- Tetanus toxin epitope-linker-IL-4-linker- Tetanus toxin epitope-linker-IL-13- linker- Tetanus toxin epitope-linker- IL-4-linker- Tetanus toxin epitope-linker-IL-13- polyhistidine tag ER import signal sequence- Flagellin -linker-IL-4-linker- Tetanus toxin epitope-linker-IL-13- linker- Tetanus toxin epitope-linker-IL-4-linker- Tetanus toxin epitope-linker-IL-13- polyhistidine tag ER import signal sequence–IL-4-linker- Tetanus toxin epitope-linker-IL-13-linker- Tetanus toxin epitope-linker-IL-4-linker- Tetanus toxin epitope-linker-IL-13-linker–Flagellin IL-4-linker- Tetanus toxin epitope-linker-IL-13-linker- Tetanus toxin epitope-linker- IL-4-linker- Tetanus toxin epitope-linker-IL-13–linker–flagellin–polyhistidine tagER import signal sequence-IL-4-linker- Tetanus toxin epitope-linker-IL-13-linker- Tetanus toxin epitope-linker- IL-4-linker- Tetanus toxin epitope-linker-IL-13–linker–flagellin–polyhistidine tag ER import signal sequence-Flagellin-linker-IL-13-linker- Tetanus toxin epitope-linker-IL-4- linker- Tetanus toxin epitope-linker- IL-13-linker- Tetanus toxin epitope-linker-IL-4-linker Flagellin -linker-IL-13-linker- Tetanus toxin epitope-linker-IL-4-linker- Tetanus toxin epitope- linker- IL-13-linker- Tetanus toxin epitope-linker-IL-4-polyhistidine tag ER import signal sequence- Flagellin -linker-IL-13-linker- Tetanus toxin epitope-linker-IL-4- linker- IL-13-linker- Tetanus toxin epitope-linker-IL-4-polyhistidine tag ER import signal sequence–IL-13-linker- Tetanus toxin epitope-linker-IL-4-linker- Tetanus toxin epitope-linker- IL-13-linker- Tetanus toxin epitope-linker-IL-4-linker–Flagellin IL-13-linker- Tetanus toxin epitope-linker-IL-4-linker- Tetanus toxin epitope-linker- IL-13- linker- Tetanus toxin epitope-linker-IL-4–linker–flagellin–polyhistidine tag ER import signal sequence-IL-13-linker- Tetanus toxin epitope-linker-IL-4-linker- Tetanus toxin epitope-linker- IL-13-linker- Tetanus toxin epitope-linker-IL-4–linker–flagellin–polyhistidine tag

[0084] Immunization of dogs with cIL-13-cIL-4-poly-BsFliC (SEQ ID NO: 1) leads tosuppression of cIL-4 and cIL-13 induction of TARC mRNA in blood samples already at day 28 (primary immunization phase) and becoming highly efficient (95% - 100%) at day 42, 56, 72 and 86). Immunization of dogs with cIL-13-cIL-4-poly-BsFliC breaks self-tolerance in dogs against both cIL-4 and cIL-13 leading to functional suppression of these cytokines in vivo. The IL-13 / IL- 4 / Flagellin fusion protein construct can comprise the amino acid sequence of SEQ ID NO: 1 IL-13-IL-4-polyh-BsFliC protein construct (888 amino acids) [SEQ ID NO : 1] MGWSCIILFLVATATGVHSSPSPVTPSPTLKELIEELVNITQNQASLCNGSMVWSVNLTA GMYCAALESLINVSDCSAIQRTQRMLKALCSQKPAAGQISSERSRDTKIEVIQLVKNLLT YVRGVYRHGNFRGGGGGQYIKANSKFIGITELGGGSGHNFNITIKEIIKMLNILTARNDSC MELTVKDVFTAPKNTSDKEIFCRAATVLRQIYTHNCSNRYLRGLYRNLSSMANKTCSM NEIKKSTLKDFLERLKVIMQKKYYRHGGGSGFNNFTVSFWLRVPKVSASHLEGSGGSSPSPVTPSPTLKELIEELVNITQNQASLCNGSMVWSVNLTAGMYCAALESLINVSDCSAIQR TQRMLKALCSQKPAAGQISSERSRDTKIEVIQLVKNLLTYVRGVYRHGNFRGGGGGHNF NITIKEIIKMLNILTARNDSCMELTVKDVFTAPKNTSDKEIFCRAATVLRQIYTHNCSNRY LRGLYRNLSSMANKTCSMNEIKKSTLKDFLERLKVIMQKKYYRHGGGSGFNNFTVSFW LRVPKVSASHLEGGGGGQYIKANSKFIGITELGGGGGGGGGGMRINHNIAALNTLNRLS SNNSASQKNMEKLSSGLRINRAGDDAAGLAISEKMRGQIRGLEMASKNSQDGISLIQTA EGALTETHAILQRVRELVVQAGNTGTQDKATDLQSIQDEISALTDEIDGISNRTEFNGKK LLDGTYKVDTATPANQKNLVFQIGANATQQISVNIEDMGADALGIKEADGSIAALHSVN DLDVTKFADNAADTADIGFDAQLKVVDEAINQVSSQRAKLGAVQNRLEHTINNLSASG ENLTAAESRIRDVDMAKEMSEFTKNNILSQASQAMLAQANQQPQNVLQLLRHHHHH

[0085] Flagellin fusion proteins can be used in fusion protein constructs to break self-tolerancefor the induction of neutralizing antibodies. Flagellin is a TLR-5 agonist. Toll-like receptor 5 (TLR5), expressed on B-cells, recognizes flagellin from both Gram-positive and Gram- negative bacteria. Activation of the receptor stimulates the production of proinflammatory cytokines, such as TNF-α, through signaling via the adaptor protein MyD88. The inventors surprisingly discovered that the flagellin fusion protein was highly expressed in a mammalian cell line. This was unexpected because the mammalian expression system requires the nascent protein to pass through the endoplasmic reticulum (ER), this leads to oxidation and protein folding. The flagellin protein does not comprise any cysteines, which may lead to better expression in the mammalian expression system. Without wishing to be bound to a particular theory, the inventor theorized that the flagellin fusion protein may break self-tolerance by activating endogenous quiescent B-cells that are specific against the IL-4 or IL-13 polypeptides. With the addition of the T-cell epitopes the flagellin fusion protein activates both cellular and humoral immune response against the self proteins, IL-4 and IL-13. Polypeptide Variants

[0086] The receptor trap construct proteins described herein can have sequence identitypolypeptides of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of to the sequences. In further embodiments, the polypeptides have at least 60%, 65%, 70%,71%, 72%, 73%, 74%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of identity in their amino acid sequences with other polypeptides to which they are compared.Nucleic Acids, Vectors, and Host Cells

[0087] Described herein are isolated nucleic acid molecules encoding the IL-13 / IL-4 / Flagellinfusion protein constructs described herein (including molecules comprising, or alternatively consisting of, fragments or variants thereof). The nucleic acids can be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid can be isolated by purification away from other cellular components or other contaminants (e.g., other cellular nucleic acids or proteins) by standard techniques, including alkaline / SDS treatment, CsCI banding, column chromatography, agarose gel electrophoresis and others well known in the art. See, e.g., Ausubel, et al. (2011) Current Protocols in Molecular Biology John Wiley & Sons, Inc. A nucleic acid described herein can be, for example, DNA or RNA and can or cannot contain intronic sequences. The nucleic acid can be a cDNA molecule. Nucleic acids described herein can be obtained using standard molecular biology techniques. Specifically, degenerate codon substitutions can be achieved by generating, e.g., sequences in which the third position of one or more selected codons is substituted with mixed-base and / or deoxyinosine residues. Batzer et al., Nucleic Acid Res., 19:5081, 1991; Ohtsuka et al., J. Biol. Chem., 260: 2605-8, 1985; Rossolini et al., Mol. Cell. Probes, 8:91-8, 1994.

[0088] Described herein are methods for recombinantly producing IL-13 / IL-4 / Flagellin fusionprotein constructs described herein. Methods of producing the recombinant proteins are well known to those of ordinary skill in the art. The IL-13 / IL-4 / Flagellin fusion protein constructs described herein can also be produced by constructing, using conventional techniques well known to those of ordinary skill in the art, an expression vector containing an operon and a DNA sequence encoding the IL-13 / IL-4 / Flagellin fusion protein constructs described herein (e.g., vectors, especially plasmids, cosmids, viruses, bacteriophages and other vectors common in genetic engineering, which contain the above-mentioned nucleic acid molecules). The nucleic acid molecules contained in the vectors can be linked to regulatory elements that ensure the transcription in prokaryotic and eukaryotic cells.

[0089] Vectors contain elements that facilitate manipulation for the expression of a foreignprotein within the target host cell. Manipulation of sequences and production of DNA for transformation can be, for example, first performed in a bacterial host (e.g., E. coli) and usually vectors include sequences to facilitate such manipulations, including a bacterial origin of replication and appropriate bacterial selection marker. Selection markers encode proteinsnecessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene do not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics or other toxins, complement auxotrophic deficiencies, or supply critical nutrients not available from complex media. Exemplary vectors and methods for transformation of yeast are described in the art. See, e.g., Burke et al., Methods in Yeast Genetics Cold Spring Harbor Laboratory Press, 2000.

[0090] The polynucleotide coding for the IL-13 / IL-4 / Flagellin fusion protein constructsdescribed herein can be operably linked to transcriptional and translational regulatory sequencesthat provide for expression of the polypeptide in yeast cells. These vector components caninclude, but are not limited to, one or more of the following: an enhancer element, a promoter, and a transcription termination sequence. Sequences for the secretion of the polypeptide can also be included (e.g., a signal sequence).

[0091] Nucleic acids are “operably linked” when placed into a functional relationship withanother nucleic acid sequence. For example, DNA for a signal sequence is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. Generally, “operably linked” refers broadly to contiguous linked DNA sequences, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous.

[0092] Promoters are untranslated sequences located upstream (5’) to the start codon of astructural gene (generally within about 100 to 1000 bp) that control the transcription and translation of particular nucleic acid sequences to which they are operably linked. Such promoters fall into several classes: inducible, constitutive, and repressible promoters (e.g., that increase levels of transcription in response to absence of a repressor). Inducible promoters can initiate increased levels of transcription from DNA under their control in response to some change in culture conditions (e.g., the presence or absence of a nutrient or a change in temperature).

[0093] The expression vectors are transfected into a host cell by convention techniques wellknown to those of ordinary skill in the art to produce a transfected host cell, said transfected hostcell cultured by conventional techniques well known to those of ordinary skill in the art to produce the IL-13 / IL-4 / Flagellin fusion protein constructs described herein.

[0094] The host cells used to express the IL-13 / IL-4 / Flagellin fusion protein constructsdescribed herein can be either a bacterial cell such as E. coli, yeast (e.g., S. cerevisiae), or a eukaryotic cell (e.g., a mammalian cell line). A mammalian cell of a well-defined type for this purpose, such as a myeloma cell, 3T3, HeLa, C6A2780, Vero, MOCK II, a Chinese hamster ovary (CHO), Sf9, Sf21, COS, NS0, or HEK293 cell line can be used.

[0095] The general methods by which the vectors can be constructed, transfection methodsrequired to produce the host cell and culturing methods required to produce the antibodies, and fragments thereof, from said host cells all include conventional techniques. Although preferably the cell line used to produce the IL-13 / IL-4 / Flagellin fusion protein constructs described herein is a mammalian cell line, any other suitable cell line, such as a bacterial cell line such as an E. coli-derived bacterial strain, or a yeast cell line, can be used.

[0096] Similarly, once produced IL-13 / IL-4 / Flagellin fusion protein constructs described hereincan be purified according to standard procedures in the art, such as for example cross-flow filtration, ammonium sulphate precipitation, and affinity column chromatography. Compositions

[0097] The compositions can further comprise a pharmaceutical excipient, carrier, diluent,adjuvant, or a combination thereof. The compositions can be a pharmaceutical composition.

[0098] The pharmaceutical compositions described herein may further comprise at least one ofany suitable auxiliaries including, but not limited to, diluents, binders, stabilizers, buffers, thickeners, antioxidants, salts, lipophilic solvents, surfactants, preservatives, adjuvants, or combinations thereof. For example, the adjuvant can be POLYGEN (terminally-sterilized, low molecular weight, copolymer adjuvant). The compositions can further comprise immunostimulatory oligonucleotides (ODN), for example ODN 1668, 2006-PTO, or a combination thereof. ODN1668 is a CpG oligodeoxynucleotide (ODN) and a TLR-9 agonist. 2006-PTO is a CpG oligodeoxynucleotide (ODN) and a TLR-9 agonist. Examples and methods of preparing such sterile solutions are well known in the art and can be found in well-known texts such as, but not limited to, REMINGTON’S PHARMACEUTICAL SCIENCES (Gennaro, Ed., 18th Edition, Mack Publishing Co. (1990)). Pharmaceutically acceptable carriers can be routinely selected that are suitable for the mode of administration, solubility and / or stability ofthe IL-13 / IL-4 / Flagellin fusion protein constructs described herein. “Pharmaceutically acceptable carrier,” as used herein, refers broadly to any and all solvents, dispersion media, coatings, antibacterial and antifungal agent, isotonic and absorption delaying agents for pharmaceutical active substances as are well known in the art. Except insofar as any conventional media or agent is incompatible with the compound, its use in the therapeutic compositions is contemplated. Supplementary compounds can also be incorporated into the compositions.

[0099] The pharmaceutical composition may comprise a surfactant. Suitable surfactants areamphiphilic compounds. Mono-, di-, or tri-esters of sorbitan with fatty acids, polyoxyethylated compounds, such as polyoxyethylene sorbitan fatty acid esters, polyoxyethlyene castor oil derivatives, and poloxamers, may be used as surfactants. Polyoxyethylated compounds, also referred to as polyethoxylated compounds, are prepared for example by reaction with ethylene oxide. They have one or more concatenated units of the formula —[O—CH2—CH2]—. Polyoxyethylated compounds which may be mentioned in particular are: nonionic amphiphilic polyoxyethylated compounds such as - poloxamers, preferably with molar masses of from 100 to 5000 g / mol, particularly preferably with molar masses of from 1000 to 3500 g / mol. Poloxamer is the international non-proprietary name for block copolymers of ethylene oxide and methyloxirane, - polyoxyethylene fatty acid glycerides, also called non-ionic emulsifiers, preferably for example glycerol polyethylene glycol ricinoleate, - polyoxyethylene sorbitan fatty acid esters, preferably for example polyoxyethylene 20 sorbitan monooleate, - polyoxyethylene fatty acids such as macrogol 15 hydroxystearate (=Solutol HS15, obtainable by reacting 15 mol of ethylene oxide and 1 mol of 12-hydroxystearic acid) - polyoxyethylene fatty alcohols such as hydroxypolyethoxydodecane.

[0100] Fatty acid or fatty alcohol stands in particular for the corresponding compounds having atleast 6 carbon atoms and normally not more than 30 carbon atoms.

[0101] The pharmaceutical composition can comprise a thickener. The thickener may be in anamount of from 0.1% to 10% (w / w), from 0.1% to 8% (w / w), from 0.5% to 5% (w / w), or from 0.5% to 2.5% (w / w). The pharmaceutical composition can comprise thickener is in an amount of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%.9%, or 10% (w / w). The pharmaceutical composition can comprise a thickener is in an amount of about 1.0% (w / w).

[0102] Suitable thickeners include but are not limited to cellulose derivatives, for example,methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, microcrystalline cellulose; bentonites, kaolin, pectin, starches, modified starch, waxes, agar, paraffins, gelatin, alginates, polyvinylpyrrolidone, crospovidone, cetyl alcohol, stearates such as, for example, magnesium stearate, zinc stearate or glyceryl stearate, saturated or unsaturated long-chain fatty acids (C8-C24, high molecular weight polyethylene glycols (e.g., polyethylene glycol 2000), glycerol ester, and combinations thereof.

[0103] The thickener can be a glycerol ester and is preferably a glycerol ester with C12-C24 fattyacids and / or is a monoester, a diester, a triester, or a mixture thereof. The thickener may be glycerol dibehenate. Glycerol dibehenate is also referred to as glyceryl dibehenate or glycerin dibehenate.

[0104] The pharmaceutical composition may comprise an antioxidant. The antioxidant can be inan amount of from 0.01% to 2% (w / w), from 0.01% to 1.5% (w / w), 0.5 to 2 wt.%, from 0.01% to 1.5% (w / w), from 0.001% to 1% (w / w), or from 0.01% to 0.3% (w / w). The pharmaceutical compositions described herein can comprise an antioxidant in an amount of about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, or 2% (w / w).

[0105] Suitable antioxidants include but are not limited to ascorbyl palmitate,butylhydroxytoluene, butylhydroxyanisole, lecithins, sulfites (Na sulfite, Na metabisulfite), organic sulfides (cystine, cysteine, cysteamine, methionine, thioglycerol, thioglycolic acid, thiolactic acid), phenols (tocopherols, as well as vitamin E and vitamin E DPGS (d-alpha- tocopheryl polyethylene glycol 1000 succinate)), butylated hydroxyanisole, butylated hydroxytoluene, gallic acid (propyl, octyl, propyl gallate, and dodecyl gallate), organic acids (ascorbic acid, citric acid, tartaric acid, lactic acid) and salts and esters thereof may be mentioned. Preferably, antioxidants may be selected from the group consisting of ascorbyl palmitate, butylhydroxytoluene, butylhydroxyanisole, citric acid, lecithins, propyl gallate, and tocopherol.

[0106] The pharmaceutical composition can comprise an antioxidant selected from the groupconsisting of ascorbyl palmitate, butylhydroxytoluene, butylhydroxyanisole, citric acid, lecithins, propyl gallate, tocopherol, or a combination thereof.

[0107] The pharmaceutical compositions described herein can comprise a preservative. Suitablepreservatives include but are not limited to carboxylic acids (sorbic acid, propionic acid, benzoic acid, lactic acid), phenols (cresols, p-hydroxybenzoic esters such as methylparaben, propylparaben), aliphatic alcohols (benzyl alcohol, ethanol, butanol), quaternary ammonium compounds (benzalkonium chloride, cetylpyridinium chloride). Preferably, preservatives may be ethanol, propylene glycol, butanol, chlorobutanol, benzoic acid, sorbic acid, and para- hydroxybenzoic esters. Methyl 4-hydroxybenzoate, ethyl 4-hydroxybenzoate, and propyl 4- hydroxybenzoate are also known as preferred para-hydroxybenzoic esters.

[0108] The pharmaceutical composition can comprise a preservative selected from the groupconsisting of ethanol, propylene glycol, butanol, chlorobutanol, benzoic acid, sorbic acid, para- hydroxybenzoic esters, and combinations thereof.

[0109] Pharmaceutical excipients and additives useful in the present invention can also include,but are not limited to, proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, terra-, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination in ranges of 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein. Representative amino acid components, which can also function in a buffering capacity, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and combinations thereof.

[0110] Carbohydrate excipients suitable for use in the present invention includemonosaccharides e.g., fructose, maltose, galactose, glucose, D-mannose, sorbose; disaccharides, e.g., lactose, sucrose, trehalose, cellobiose; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches; and alditols, e.g., mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), myoinositol and combinations thereof.

[0111] The pharmaceutical compositions described herein may further comprise coloring agents,emulsifying agents, surfactants, thickening agents, suspending agents, ethanol, chelators (e.g., EDTA), buffers (e.g., citrate buffer), flavoring, water, or combinations thereof.

[0112] Chelators such as EDTA and EGTA can optionally be added to the pharmaceuticalcompositions to reduce aggregation. These additives are particularly useful if a pump or plastic container is used to administer the pharmaceutical composition. The presence of pharmaceutically acceptable surfactant mitigates the propensity for the composition to aggregate.

[0113] The pharmaceutical compositions described herein may comprise an emulsifier,including, but are not limited to ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0114] Additionally, the pharmaceutical compositions described herein can comprise polymericexcipients / additives such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as hydroxypropyl-β-cyclodextrin), polyethylene glycols, flavoring agents, anti-microbial agents, sweeteners, antioxidants, anti-static agents, surfactants (e.g., polysorbates such as “Tween® 20” and “Tween® 80”), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA or EGTA). These and additional known pharmaceutical excipients and / or additives suitable for use in the pharmaceutical compositions described herein and are known in the art, e.g., as listed in REMINGTON: THE SCIENCE & PRACTICE OF PHARMACY (19th ed., Williams & Williams (1995)) and PHYSICIAN’S DESK REFERENCE (52nd ed., Medical Economics (1998)).

[0115] The present disclosure provides stable pharmaceutical compositions as well as preservedsolutions and formulations containing a preservative, as well as multi-use preserved formulations suitable for pharmaceutical or veterinary use, comprising IL-13 / IL-4 / Flagellin fusion protein constructs described herein in a pharmaceutically acceptable formulation.

[0116] The pharmaceutical compositions described herein can be presented in unit-dose ormulti-dose containers, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, water for injections, immediately prior to use. Extemporaneous suspensions can be prepared from sterile powders, granules and tablets.

[0117] Acceptable liquid carriers for use in the pharmaceutical compositions, include, but are notlimited to, vegetable oils, e.g., peanut oil, cotton seed oil, sesame oil or combinations thereof. The compositions may further comprise a buffer, including but not limited to phosphate buffer saline (PBS), Ringer’s solution, lactated Ringer’s solution, Krebs Ringer Buffer solution, HEPES buffer, PIPES buffer, MES buffer, Tris buffer, TES buffer, TAPS buffer, Bicine buffer, Tricine buffer, TAPSO buffer, PIPES buffer, cacodylate buffer, or a mixture thereof. The composition may comprise phosphate buffer solution (PBS). The pH of the composition may be about pH 7.4.

[0118] The IL-13 / IL-4 / Flagellin fusion protein constructs described herein can be administeredin combination with additional therapeutics. In an embodiment, the IL-13 / IL-4 / Flagellin fusion protein constructs described herein can be administered in combination with one or more steroids. For example, prednisone, prednisolone, triamcinolone, betamethasone, dexamethasone, flumethasone, fludrocortisone, hydrocortisone, methylprednisolone, and combinations thereof. In another embodiment, the present receptor traps can be administered in combination with one or more antihistamines. For example, hydroxyzine, chlorpheniramine, chlorphenamine, dimetindene, diphenhydramine, loratadine, cetirizine, clemastine, and combinations thereof. In yet another embodiment, the present receptor traps can be administered in combination one or more with calcineurin inhibitors. For example, cyclosporine, tacrolimus, and a combination thereof. In yet another embodiment, the present receptor traps can be administered in combination with one or more JAK inhibitors. For example, oclacitinib. In another embodiment, the IL-13 / IL-4 / Flagellin fusion protein constructs described herein can be administered in combination with one or more neutralizing monoclonal antibodies. For example, anti-IL-4 antibodies, anti-IL-5 antibodies, anti-IL-13 antibodies, anti-IL-17A antibodies, anti-IL-17C antibodies, anti-IL-22 antibodies, anti-IL-31 antibodies, anti-IL-33 antibodies, and combinations thereof. In another embodiment, the IL-13 / IL-4 / Flagellin fusion protein constructs described herein can be administered in combination with one or more nonsteroidal anti-inflammatory drugs (NSAID). For example, meloxicam. The IL-13 / IL-4 / Flagellin fusion protein constructs described herein can be administered concurrently or sequentially with the additional therapeutics using the same or a different route of administration. For example, the IL-13 / IL- 4 / Flagellin fusion protein constructs described herein can be administered concurrently or sequentially with anti-IL-31 antibodies, anti-IL-5 antibodies, anti-IL-22 antibodies, or a combination thereof.Effective Amount

[0119] The effective amount of the IL-13 / IL-4 / Flagellin fusion protein constructs describedherein in a composition in an amount sufficient to treat a dermatological condition. The dermatological condition comprises at least one skin disorder selected from psoriasis, atopic dermatitis, skin rash, skin irritation, skin sensitization, allergic reactions, pruritus, and combinations thereof.

[0120] The effective amount of the IL-13 / IL-4 / Flagellin fusion protein constructs describedherein can range from about 1 nanogram (ng) to 1 gram (g).

[0121] The effective amount of the IL-13 / IL-4 / Flagellin fusion protein constructs describedherein can be about 1 ng to 1,000 ng.

[0122] The effective amount may be between about 1 ng and 100 ng, 10 ng and 500 ng, 200 ngand 800 ng, or 250 ng and 750 ng.

[0123] The effective amount of the IL-13 / IL-4 / Flagellin fusion protein constructs describedherein can be about 1 ng, 2 ng, 3 ng, 4 ng, 5 ng, 6 ng, 7 ng, 8 ng, 9 ng, 10 ng, 11 ng, 12 ng, 13 ng, 14 ng, 15 ng, 16 ng, 17 ng, 18 ng, 19 ng, 20 ng, 21 ng, 22 ng, 23 ng, 24 ng, 25 ng, 26 ng, 27 ng, 28 ng, 29 ng, 30 ng, 31 ng, 32 ng, 33 ng, 34 ng, 35 ng, 36 ng, 37 ng, 38 ng, 39 ng, 40 ng, 41 ng, 42 ng, 43 ng, 44 ng, 45 ng, 46 ng, 47 ng, 48 ng, 49 ng, 50 ng, 51 ng, 52 ng, 53 ng, 54 ng, 55 ng, 56 ng, 57 ng, 58 ng, 59 ng, 60 ng, 61 ng, 62 ng, 63 ng, 64 ng, 65 ng, 66 ng, 67 ng, 68 ng, 69 ng, 70 ng, 71 ng, 72 ng, 73 ng, 74 ng, 75 ng, 76 ng, 77 ng, 78 ng, 79 ng, 80 ng, 81 ng, 82 ng, 83 ng, 84 ng, 85 ng, 86 ng, 87 ng, 88 ng, 89 ng, 90 ng, 91 ng, 92 ng, 93 ng, 94 ng, 95 ng, 96 ng, 97 ng, 98 ng, 99 ng, 100 ng, 110 ng, 120 ng, 130 ng, 140 ng, 150 ng, 160 ng, 170 ng, 180 ng, 190 ng, 200 ng, 210 ng, 220 ng, 230 ng, 240 ng, 250 ng, 260 ng, 270 ng, 280 ng, 290 ng, 300 ng, 310 ng, 320 ng, 330 ng, 340 ng, 350 ng, 360 ng, 370 ng, 380 ng, 390 ng, 400 ng, 410 ng, 420 ng, 430 ng, 440 ng, 450 ng, 460 ng, 470 ng, 480 ng, 490 ng, 500 ng, 525 ng, 550 ng, 575 ng, 600 ng, 625 ng, 650 ng, 675 ng, 700 ng, 725 ng, 750 ng, 775 ng, 800 ng, 825 ng, 850 ng, 875 ng, 900 ng, 950 ng, or 975 ng.

[0124] The effective amount of the IL-13 / IL-4 / Flagellin fusion protein constructs describedherein can be about 1 µg to 1,000 µg.

[0125] The effective amount can be between about 1 µg and 100 µg, 10 µg and 500 µg, 150 µgand 250 µg, 175 µg and 225 µg, 180 µg and 240 µg, 150 µg and 300 µg, 200 µg and 800 µg, or 250 µg and 750 µg. The effective amount can be about 200 µg.

[0126] The effective amount of the IL-13 / IL-4 / Flagellin fusion protein constructs describedherein can be about 1 µg, 2 µg, 3 µg, 4 µg, 5 µg, 6 µg, 7 µg, 8 µg, 9 µg, 10 µg, 11 µg, 12 µg, 13 µg, 14 µg, 15 µg, 16 µg, 17 µg, 18 µg, 19 µg, 20 µg, 21 µg, 22 µg, 23 µg, 24 µg, 25 µg, 26 µg, 27 µg, 28 µg, 29 µg, 30 µg, 31 µg, 32 µg, 33 µg, 34 µg, 35 µg, 36 µg, 37 µg, 38 µg, 39 µg, 40 µg, 41 µg, 42 µg, 43 µg, 44 µg, 45 µg, 46 µg, 47 µg, 48 µg, 49 µg, 50 µg, 51 µg, 52 µg, 53 µg, 54 µg, 55 µg, 56 µg, 57 µg, 58 µg, 59 µg, 60 µg, 61 µg, 62 µg, 63 µg, 64 µg, 65 µg, 66 µg, 67 µg, 68 µg, 69 µg, 70 µg, 71 µg, 72 µg, 73 µg, 74 µg, 75 µg, 76 µg, 77 µg, 78 µg, 79 µg, 80 µg, 81 µg, 82 µg, 83 µg, 84 µg, 85 µg, 86 µg, 87 µg, 88 µg, 89 µg, 90 µg, 91 µg, 92 µg, 93 µg, 94 µg, 95 µg, 96 µg, 97 µg, 98 µg, 99 µg, 100 µg, 110 µg, 1203µg, 130 µg, 140 µg, 150 µg, 160 µg, 170 µg, 180 µg, 190 µg, 191 µg, 192 µg, 193 µg, 194 µg, 195 µg, 196 µg, 197 µg, 198 µg, 199 µg, 200 µg, 201 µg, 202 µg, 203 µg, 204 µg, 205 µg, 206 µg, 207 µg, 208 µg, 209 µg, 210 µg, 220 µg, 230 µg, 240 µg, 250 µg, 260 µg, 270 µg, 280 µg, 290 µg, 300 µg, 310 µg, 320 µg, 330 µg, 340 µg, 350 µg, 360 µg, 370 µg, 380 µg, 390 µg, 400 µg, 410 µg, 420 µg, 430 µg, 440 µg, 450 µg, 460 µg, 470 µg, 480 µg, 490 µg, 500 µg, 525 µg, 550 µg, 575 µg, 600 µg, 625 µg, 650 µg, 675 µg, 700 µg, 725 µg, 750 µg, 775 µg, 800 µg, 825 µg, 850 µg, 875 µg, 900 µg, 950 µg, or 975 µg.

[0127] The effective amount of the IL-13 / IL-4 / Flagellin fusion protein constructs describedherein can be about 1 mg to 1,000 mg.

[0128] The effective amount can be between about 1 mg and 100 mg, 10 mg and 500 mg, 200mg and 800 mg, or 250 mg and 750 mg.

[0129] The effective amount of the IL-13 / IL-4 / Flagellin fusion protein constructs describedherein can be about 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 61 mg, 62 mg, 63 mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg, 69 mg, 70 mg, 71 mg, 72 mg, 73 mg, 74 mg, 75 mg, 76 mg, 77 mg, 78 mg, 79 mg, 80 mg, 81 mg, 82 mg, 83 mg, 84 mg, 85 mg, 86 mg, 87 mg, 88 mg, 89 mg, 90 mg, 91 mg, 92 mg, 93 mg, 94 mg, 95 mg, 96 mg, 97 mg, 98 mg, 99 mg, 100 mg, 110 mg, 1203mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg, 430 mg, 440 mg, 450 mg, 460 mg, 470 mg, 480 mg, 490 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 950 mg, or 975 mg.

[0130] The effective amount of the IL-13 / IL-4 / Flagellin fusion protein constructs describedherein can be about 0.1 mg / kg to 10 mg / kg.

[0131] The effective amount can be between about 0.1 mg / kg and 5 mg / kg, 1 mg / kg and 5mg / kg, 0.2 mg / kg and 8 mg / kg, or 0.25 mg / kg and 10 mg / kg.

[0132] The effective amount of the IL-13 / IL-4 / Flagellin fusion protein constructs describedherein can be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg. The effective amount can be between about 0.1 and 1 mg / kg, 0.5 and 2 mg / kg, 0.75 and 5 mg / kg, or 1 and 10 mg / kg. Therapeutic Methods

[0100] The IL-13 / IL-4 / Flagellin fusion protein constructs described herein and / or compositioncomprising an effective amount thereof, can used to treat a dermatological condition. The dermatological condition can comprise at least one skin disorder selected from psoriasis, atopic dermatitis, skin rash, skin irritation, skin sensitization, allergic reactions, pruritus, and combinations thereof.

[0101] The route of administration of the IL-13 / IL-4 / Flagellin fusion protein constructsdescribed herein and compositions comprising the same can be by known routes, e.g. injection or infusion by intravenous, intraperitoneal, intracerebral, subcutaneous, intramuscular, intraocular, inhaled, optionally intranasal, intrapulmonary, intraarterial, intracerebrospinal, or intralesional routes, or by sustained release systems. Preferably The IL-13 / IL-4 / Flagellin fusion protein constructs described herein are given systemically.

[0102] Therapeutic and pharmaceutical compositions comprising the IL-13 / IL-4 / Flagellin fusionprotein constructs described herein can be used to treat dermatological conditions. The dermatological condition can comprise at least one skin disorder selected from psoriasis, atopic dermatitis, skin rash, skin irritation, skin sensitization, allergic reactions, pruritus, and combinations thereof.

[0103] As a general proposition, the initial pharmaceutically effective amount of the IL-13 / IL-4 / Flagellin fusion protein constructs described herein administered parenterally will be in therange of about 0.1 to 50 mg / kg of patient body weight per day, with the typical initial range of the IL-13 / IL-4 / Flagellin fusion protein constructs described herein used being 0.3 to 20 mg / kg / day, more preferably 0.3 to 15 mg / kg / day. The desired dosage can be delivered by a single bolus administration, by multiple bolus administrations, or by continuous infusion administration of the IL-13 / IL-4 / Flagellin fusion protein constructs described herein, depending on the pattern of pharmacokinetic decay that the practitioner wishes to achieve.

[0104] Further, the effective amount of the IL-13 / IL-4 / Flagellin fusion protein constructsdescribed herein administered (optionally parenterally) can be in the range of about 0.1 to 50 mg / kg of patient body weight. The IL-13 / IL-4 / Flagellin fusion protein constructs described herein can be administered every 1, 2, 3, or 4 weeks. The IL-13 / IL-4 / Flagellin fusion protein constructs described herein can be administered every 1, 2, 3, 4, 5, or 6 months. For example, the IL-13 / IL-4 / Flagellin fusion protein constructs described herein can be administered every 2-3 months. The IL-13 / IL-4 / Flagellin fusion protein constructs described herein can be administered every 2-3 weeks.

[0105] The IL-13 / IL-4 / Flagellin fusion protein constructs described herein administered(optionally parenterally) and administered as a booster, about 25 days to 35 days later, e.g., about 28 days. Kits

[0106] A pharmaceutical pack or kit may comprise one or more containers filled with one ormore of the ingredients of the pharmaceutical compositions comprising the IL-13 / IL-4 / Flagellin fusion protein constructs described herein. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for veterinary uses.

[0107] Kits that can be used in the methods described herein. A kit may comprise the IL-13 / IL-4 / Flagellin fusion protein constructs described herein or compositions comprising the same, in one or more containers. Routes of administration

[0108] The IL-13 / IL-4 / Flagellin fusion protein constructs described herein can be administeredparentally by injection or by gradual infusion over time. Although the tissue to be treated can typically be accessed in the body by systemic administration and therefore most oftentreated by intravenous administration of therapeutic compositions, other tissues and delivery means are contemplated where there is a likelihood that the tissue targeted contains the target molecule. Thus, the IL-13 / IL-4 / Flagellin fusion protein constructs described herein may be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracaviatary, intravesically, transdermally, topically, intraocually, orally, intranasally, or a combination thereof. Systemic, optionally intreperionally or intravenously, are preferred routes of administration.

[0109] Further details described herein can be found in the following example, which furtherdefines the scope described herein. All references cited throughout the specification, and the references cited therein, are hereby expressly incorporated by reference in their entirety. EXAMPLES EXAMPLE 1 cIL13-cIL-4-polyBsFlicC VACCINE CONSTRUCT

[0110] A consecutive arrangement of cIL-13 and cIL-4 mature polypeptides was designed, wherethe cytokines in the first cIL-13 / cIL-4 module were separated by Tetanus toxin T cell epitope p2 encased by pentapeptide spacers formed by glycine / serine (G / S). This was followed by Tetanus toxin T cell epitope p30. In the following second cIL-13 / cIL-4 module attached to this first arrangement; the two cytokine sequences were only separated by a G / S spacer. This was then followed by two copies of Tetanus toxin T cell epitope p30 and one Tetanus toxin T cell epitope p2, both encased in G / S spacers. The Tetanus toxin T cell epitopes are added here to aid in the breakage of self-tolerance. This poly-cIL-13 / cIL-4 protein sequence arrangement was then followed, via an octaglycine linker, by the protein sequence of Bacillus subtilis flagellin. N-terminally, a signal sequence for import into the endoplasmic reticulum and the secretory pathway was attached, and C- terminally a tag (His6) for straightforward purification was added. This construct is shown in SEQ ID NO: 1.

[0111] Based on the cIL-13-cIL-4-poly-BsFliC polypeptide sequence, an encoding DNA wasdesigned and synthesized (SEQ. ID. NO: 2), including a Kozak sequence upstream the start ATG (and the stop codon TGA) to improve expression in mammalian cells, and flanking unique restriction enzyme sites (EcoRI and HindIII) for subcloning.

[0112] The complete cIL13-cIL4-poly-BsFliC DNA sequence was sub-cloned into thepcDNA3.4 mammalian expression vector. Large amounts of transfection grade plasmid was prepared for Expi293F cell expression. Cell Culture and Transient Transfection

[0113] Expi293F cells were grown in serum-free Expi293 expression Medium (Thermo FisherScientific). The cells were maintained in Erlenmeyer Flasks (Corning Inc.) at 37°C with 8% CO2 on an orbital shaker (VWR Scientific). One day before transfection, the cells were seeded at an appropriate density in Corning Erlenmeyer Flasks. On the day of transfection, DNA and transfection reagent were mixed at an optimal ratio and then added into the flask with cells ready for transfection. The recombinant plasmids encoding target protein was transiently co-transfected into suspension Expi293F cell cultures. The cell culture supernatants collected on day 6 were used for purification. Purification and Analysis

[0114] Cell culture broth was centrifuged. Cell culture supernatant was loaded onto an Ni2+-NTA affinity purification column at an appropriate flowrate. After washing and elution with appropriate buffers, the eluted fractions were pooled and buffer exchanged to the final formulation buffer. The purified protein was analyzed by SDS-PAGE, Western blot analysis to determine the molecular weight and purity. The concentration was determined by Bradford assay with BSA as a standard. SDS-PAGE Analysis

[0115] Buffer: Reducing Loading buffer: 300 mM Tris-HCl, 10% SDS, 30% Glycerol, 0.5%bromophenol blue, 250 mM DTT, pH 6.8. Non-Reducing Loading buffer: 300 mM Tris- HCl,10% SDS, 30% glycerol, 0.5% bromophenol blue, pH 6.8. Reducing and non-reducing loading buffer were added to protein sample respectively and the final concentration of protein was close to 0.5 mg / ml. After mixing the sample, heating at 100oC for 5-10 minutes was performed (only for reducing condition). The protein samples were centrifuged at 10000 rpm for 1 minutes, and then loaded onto a precast gel (Genscript, Cat.No. M42012) in a gel chamber and the appropriate running buffer. Electrophoresis was performed at 140V for approximately 60min. Western- (Immuno-)blotting

[0116] Western blotting was performed using standard methods using a mouse-anti-His6 tagmonoclonal antibody (mAb) (GenScript, Cat.No. A00186) as well as a protein Marker(GenScript, Cat. No. M00521) and a multiple-tag (GenScript, Cat.No. M0101) marker as positive control. Detection of bound anti His6-mAb was performed by incubation with a peroxidase-labeled anti mouse IgG and enhanced chemoluminescence (ECL) detection.

[0117] The Coomassie Blue stained gel showed a dominant band at ~ 150 kDa under reductiveconditions. Omission of the reducing agent showed the same band, but in addition higher molecular weight material that is likely aggregated cIL13-cIL4-poly-BsFliC protein. This is confirmed by Western blot analysis where the same bands are detected by the anti-His6 monoclonal antibody.

[0118] By the above production and purification procedure, approximately 15.4 mg of soluble(in phosphate-buffered saline, PBS) cIL13-cIL4-poly-BsFliC were obtained from 100 ml crude cell culture supernatant. The dominant band in lane R of the SDS-PAGE / Coomassie Blue (Fig. 3) is much larger than expected from the protein sequence (94848.87 Da, calculated from the mature sequence using the ProtParam tool. It is likely that the difference is accounted for by extensive N-glycosylation, since the protein sequence contains 27 N-glycosylation sites, of which 23, based on NetNGlyc analysis, are likely to be modified. The NetNGlyc analysis of cIL13-cIL4-poly-BsFliC are shown in the table below:

[0119] Table 1: NetNGlyc analysis of cIL13-cIL4-poly-BsFliC(Threshold=0.5) ---------------------------------------------------------------------- SeqName Position Potential Jury N-Glyc agreement result ---------------------------------------------------------------------- Sequence 20 NITQ 0.7781 (9 / 9) +++ Sequence 30 NGSM 0.5974 (7 / 9) + Sequence 38 NLTA 0.6330 (6 / 9) + Sequence 53 NVSD 0.6737 (9 / 9) ++ Sequence 142 NITI 0.7051 (8 / 9) + Sequence 159 NDSC 0.5311 (6 / 9) + Sequence 176 NTSD 0.5308 (4 / 9) + Sequence 197 NCSN 0.5911 (7 / 9) + Sequence 209 NLSS 0.7038 (9 / 9) ++ Sequence 215 NKTC 0.6126 (9 / 9) ++ Sequence 254 NFTV 0.7187 (9 / 9) ++ Sequence 297 NITQ 0.7461 (9 / 9) ++ Sequence 307 NGSM 0.5423 (7 / 9) + Sequence 315 NLTA 0.5859 (6 / 9) + Sequence 330 NVSD 0.6208 (9 / 9) ++ Sequence 399 NITI 0.6792 (8 / 9) + Sequence 416 NDSC 0.4811 (3 / 9) - Sequence 433 NTSD 0.4772 (7 / 9) - Sequence 454 NCSN 0.5368 (6 / 9) + Sequence 466 NLSS 0.6581 (8 / 9) +Sequence 472 NKTC 0.5579 (6 / 9) + Sequence 511 NFTV 0.6782 (9 / 9) ++ Sequence 578 NNSA 0.3946 (7 / 9) - Sequence 687 NRTE 0.6778 (9 / 9) ++ Sequence 721 NATQ 0.5472 (7 / 9) + Sequence 807 NLSA 0.4429 (6 / 9) - Sequence 814 NLTA 0.5183 (6 / 9) + ---------------------------------------------------------------------- EXAMPLE 2 INDIRECT ELISA FOR IGG ANTIBODIES DIRECTED AGAINST CYTOKINES cIL4 AND cIL13 IN CANINE SERUM

[0120] Coating of polystyrene ELISA plates (384 microtiter plate: BRAND™ 781740, orequivalent) with 10µL / well of 1 µg / ml of cIL4 (HEK cell expression, GenScript U1119GH110- 3, 0,84 mg / ml) and 1 µg / ml of cIL13 (HEK cell expression, Genscript U5991GJ120-4, 0,19 mg / ml) in coating buffer PBS (ThermoFisher Phosphate-Buffered Saline - pH 7.2, Catalogue No. 20012-019), or equivalent). Incubation overnight (O / N) at 4°C or 1 hour (h) at room temperature (RT) with closed lid. Removal of coating solution, washings 3 x with PBS, 0.05% (v / v) Tween 20, 35 µl / well. Blockage of nonspecific binding sites with 35 µl / well of PBS, 0.05% (v / v) Tween 20, 5% (w / v) non-fat dry milk powder (MP) (Roth T145.2) (= blocking solution). Incubation at RT for > 1 hour with closed lid. Performance of the sample (canine serum) dilution in a new nonabsorptive 96 well microtiter plate (Sarstedt 82.1583.001, or equivalent). Starting at 1:20 and then carrying out 11 dilution levels with a serial dilution factor of 2 (1:2 steps). Removal of blocking solution (discard) and addition of 20 µl / well of the diluted samples. Incubation at RT for > 1 hour with closed lid. Removal of the diluted samples, washings 3 x with PBS 0.05% (v / v) Tween 20, 35 µl / well. Addition of 20 µl / well of (diluted in blocking solution):1:2000 Goat IgG anti-Dog IgG (Fc specific)-Alkaline Phosphatase (Sigma: SAB3700103, Lot: RI6082, 0,61 mg / mL) (generally aim at: 100-400 ng / ml conjugate). Incubation at room temperature (RT, about 25oC) with closed lid for > 1 hour. Removal of conjugate solution, washing 2 x with PBS 0.05% (v / v) Tween 20, 35 µl / well. Washing 1 x with AP buffer (50 mM NaHCO3 / Na2CO3, 2 mM MgCl2, pH 9.6), 50 µl / well or 35 µl / well. Development of ELISA by adding 90 µl / well of 5 mM 4-nitrophenyl phosphate disodium salt hexahydrate (pNPP, Applichem) in 50 mM NaHCO3 / Na2CO3, 2 mM MgCl2, pH 9.6 buffer. Kinetic monitoring of the optical density (OD) at 405 nm in an ELISA reader (mOD / min) at RT. Determination of the curve slope in a linearrange. Slopes > 200 mOD / min are considered problematic. Measurement with a photometer from BioTek, Software: Gen5; Vmax star: Vmax determination over data points 2-5; Vmax variable: Vmax determination via 4 data points with maximal slope within the measurement intervals 1-7; Single point measurement (OD 405nm): different timepoints after adding substrate (e.g., after 30, 60, 120 min). See FIGs. 2, 4, and 6. EXAMPLE 3 DETECTION OF NEUTRALIZING ANTIBODIES IN CANINE SERUM SAMPLES VIA A cIL-4 / cIL-13 CELL CULTURE STIMULATION SYSTEM

[0121] DH82-STAT6-SEAP cells were made by transfecting the dog monocyte cell line DH82(ATCC CRL-3590) by a STAT6-SEAP (secreted embryonic alkaline phosphatase). DH82- STAT6-SEAP cells were grown in DMEM (Thermo Fisher, 616965-026), 10% iFCS, at 370C, 5% CO2. For selection purposes, culture medium was supplemented with 0.5 mg / ml geneticin (Invivogen). Dilution of canine sera were performed in 40 μl full growth medium in a 384 well cell culture plate supplemented with 1 ng / ml cIL-4 or 10 ng / ml cIL-13 and incubated for 1 hour. DH82-STAT6-SEAP cells were harvested, adjusted to 2 x105cells / ml in full growth medium, and 40 μl cell suspension were added to each of the canine sera dilutions. The cells were incubated at 370C, 5% (v / v) CO2 for 96 hours. Cell culture supernatant samples (2 – 10 ml) were used for measurement of alkaline phosphatase (SEAP) activity by adding them to 5 mM para- nitrophenyl phosphate (pNPP) in AP buffer (50 mM NaHCO3 / Na2CO3, 2 mM MgCl2, pH 9.6), 90 µl / well for 384 well plates.

[0122] Measurement of the optical density (OD) at 405 nm at RT in an ELISA reader in a kineticmode (mOD / min) by determining the curve slope in a linear area (= mOD405nm / min), or in an endpoint mode (OD) by determining the optical density at a fixed time point. Graphic representation of OD405nm or OD405nm / min versus canine serum dilutions. See, e.g., FIG.3, 5, and 7. Detection of neutralizing antibodies in canine blood samples via direct stimulation of blood cells. EDTA-stabilized dog blood samples (from day 0 and from strategic sampling times throughout the immunization scheme, e.g., day 28, day 42, day 56, day 72, day 84). At each time point, 495 µL dog blood were supplemented with 5 µL cytokine (100 x solutions of the respective cytokine, leading up to 1 – 10 ng / ml final concentrations of the cytokines) in a 24-well culture plate. Incubation for 6 h at 35°C, 5%CO2 and 96% relative humidity. Blood lysis and RNA stabilization was performed with lysis buffer from Sarstedt S-MONOVETTE® RNAEXACT (01.2048.001). RNA isolation was performed with the Macherey-Nagel: NucleoSpin RNA Blood, Mini kit for RNA from blood (REF 740200.50). Isolated RNA was analysed and quantified using an Implen NanoPhotometer®, type NP80. Quantitative RT- PCR (qPCR) was performed with the TaqMan® Assay Cf02622128_m1 (ThermoFisher) with probe and primers for canine CCL-17 (TARC) and the QuantiNova Probe RT-PCR Kit (Qiagen 208354), with the primers, reaction mixtures and conditions outlined by the manufacturers. A canine α-actin TaqMan probe and PCR primers were used as a housekeeping gene control. Typically 25 ng total RNA was used as template. The qPCR was performed using a CFX96 Real-Time System (BioRad). FIG.8A-8F. EXAMPLE 4 EX VIVO BLOOD STIMULATION ASSAYS, IMMUNIZED ANIMALS, cIL-4 OR cIL-13 STIMULATION, CTARC MRNA INDUCTION (QPCR)

[0123] The inventors immunized dogs with cIL-13-cIL-4-flagellin fusion construct describedherein. Dogs were immunized with a primary immunization comprising a subcutaneous immunization of a composition comprising 200 μg cIL-13-cIL-4-fusion construct described herein, 100 μg 1668-PTO, 100 μg 2006-PTO, and 10% polygen / PBS at Day 0.28 days later, a subcutaneous booster immunization was administered comprising a composition comprising 200 μg cIL-13-cIL-4-flagellin fusion construct, 100 μg 1668-PTO, 100 μg 2006-PTO, and 10% polygen / PBS. 1668-PTO is a phosphodiester CpG oligonucleotides used as an adjuvant.2006- PTO is B-class CpG ODN with a preference for TLR9 that is used an adjuvant.

[0124] The inventors surprisingly discovered that immunization of dogs with cIL-13-cIL-4-poly-BsFliC leads to suppression of cIL-4 and cIL-13 induction of TARC mRNA in blood samples already at day 28 (primary immunization phase) and becoming highly efficient (95% - 100%) at day 42, 56, 72 and 86). Immunization of dogs with cIL-13-cIL-4-poly-BsFliC breaks self- tolerance in dogs against both cIL-4 and cIL-13 leading to functional suppression of these cytokines in vivo. The flagellin fusion proteins constructs described herein can be used to break self-tolerance for the induction of neutralizing antibodies against IL-4 and IL-13. See FIG.8A-F.

Claims

What is claimed:

1. A fusion protein comprising:an IL-13 polypeptide; an IL-4 polypeptide; and a flagellin polypeptide.

2. The fusion protein of claim 1, wherein the N-terminal of the flagellin polypeptide islinked to the C-terminal of the IL-4 polypeptide.

3. The fusion protein of claim 1 or 2, wherein the N-terminal of the IL-4 polypeptide islinked to the C-terminal of IL-13 polypeptide.

4. The fusion protein of any one of claims 1-3, wherein the fusion protein comprises:a flagellin polypeptide linked to an IL-4 polypeptide linked to an IL-13 polypeptide linked to an IL-4 polypeptide linked to an IL-13 polypeptide.

5. The fusion protein of any one of claims 1-3, wherein the fusion protein comprises:a flagellin polypeptide linked to an IL-13 polypeptide linked to an IL-4 polypeptide linked to an IL-13 polypeptide linked to an IL-4 polypeptide.

6. The fusion protein of any one of claims 1-3, wherein the fusion protein comprises:IL-4 polypeptide linked to an IL-13 polypeptide linked to an IL-4 polypeptide linked to an IL-13 polypeptide linked to a flagellin polypeptide.

7. The fusion protein of any one of claims 1-3, wherein the fusion protein comprises:an IL-13 polypeptide linked to an IL-4 polypeptide linked to an IL-13 polypeptide linked to an IL-4 polypeptide linked to a flagellin polypeptide.

8. The fusion protein of any one of claims 1-3, wherein the fusion protein comprises:a flagellin polypeptide linked to an IL-4 polypeptide linked to an IL-13 polypeptide.

9. The fusion protein of any one of claims 1-3, wherein the fusion protein comprises:IL-13 polypeptide linked to an IL-4 polypeptide linked to a flagellin polypeptide.

10. The fusion protein of any one of claims 1-3, wherein the fusion protein comprises:an IL-4 polypeptide linked to an IL-13 polypeptide to a flagellin polypeptide.

11. The fusion protein of any one of claims 1-10, wherein the fusion protein comprises twoIL-4 polypeptides.

12. The fusion protein of any one of claims 1-11, wherein the fusion protein comprises twoIL-13 polypeptides.

13. The fusion protein of any one of claims 1-12, wherein the flagellin polypeptide and theIL-4 polypeptide are joined by a linker.

14. The fusion protein of any one of claims 1-13, wherein the IL-4 polypeptide and IL-13polypeptide are joined by a linker.

15. The fusion protein of any one of claims 1-14, wherein the linker is an oligopeptidecomprising about 4 to 20 amino acids.

16. The fusion protein of claim 15, wherein the oligopeptide is a polyglycine oligopeptide.

17. The fusion protein of claim 16, wherein the oligopeptide comprises the amino acidsequence of SEQ ID NO: 8 or 9.

18. The fusion protein of claim 15, wherein the oligopeptide is a polyglycine / serineoligopeptide.

19. The fusion protein of claim 18, wherein the oligopeptide comprises the amino acidsequence of SEQ ID NO: 10, 11, or 12.

20. The fusion protein of any one of claims 1-19, wherein the flagellin polypeptide is a B.subtilis sequence.

21. The fusion protein of any one of claims 1-20, wherein the flagellin polypeptide comprisesan amino acid sequence with at least 75% homology to the amino acid sequence of SEQ ID NO: 5.

22. The fusion protein of any one of claims 1-21, wherein the flagellin polypeptide comprisesan amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 5.

23. The fusion protein of any one of claims 1-22, wherein the IL-4 polypeptide is a canineIL-4 polypeptide.

24. The fusion protein of any one of claims 1-23, wherein the IL-4 comprises an amino acidsequence with at least 75% homology to the amino acid sequence of SEQ ID NO: 4.

25. The fusion protein of any one of claims 1-24, wherein the IL-4 comprises an amino acidsequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 4.

26. The fusion protein of any one of claims 1-25, wherein the IL-13 is a canine IL-13polypeptide.

27. The fusion protein of any one of claims 1-26, wherein the IL-13 comprises an amino acidsequence with at least 75% homology to the amino acid sequence of SEQ ID NO: 3.

28. The fusion protein of any one of claims 1-27, wherein the IL-13 comprises an amino acidsequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 3.

29. The fusion protein of any one of claims 1-28, wherein the fusion protein comprises anamino acid sequence with at least 75% homology to the amino acid sequence of SEQ ID NO: 1.

30. The fusion protein of any one of claims 1-29, wherein the fusion protein comprises anamino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 1.

31. The fusion protein of any one of claims 1-30, wherein the fusion protein furthercomprises a Tetanus toxin T-cell epitope.

32. The fusion protein of claim 31, wherein the Tetanus toxin T-cell epitope is between theFlagellin and IL-14, between the Flagellin and IL-4, between the IL-4 and IL-13, or a combination thereof.

33. The fusion protein of claim 31 or 32, wherein the Tetanus toxin T-cell epitope is aTetanus toxin T cell epitope p2, Tetanus toxin T cell epitope p30, or a combination.

34. The fusion protein of any one of claims 31-33, wherein the Tetanus toxin T-cell epitopecomprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 6 or 7.

35. The fusion protein of any one of claims 1-34, wherein the fusion protein furthercomprises an ER import signal sequence on the N-terminal.

36. The fusion protein of any one of claims 1-35, wherein the fusion protein furthercomprises an affinity tag on the C-terminal.

37. The fusion protein of claim 36, wherein the affinity tag is a hexahistidine-tag.

38. A composition comprising the fusion protein of any one of claims 1-37.

39. The composition of claim 38, wherein the composition is a pharmaceutical composition.

40. The pharmaceutical composition of claim 39, wherein the pharmaceutical compositionfurther comprises a pharmaceutical excipient, carrier, diluent, adjuvant, or a combination thereof.

41. The composition of any one of claims 38-40, wherein the composition is formulated forintravenous, subcutaneous, infusion, oral, intrathecal, intraperitoneal, parenteral administration, or a combination thereof.

42. The composition of any one of claims 38-41, wherein the composition further comprisesan anti-IL-31 antibody, anti-IL-5 antibody, anti-IL-22 antibody, or a combination thereof.

43. A nucleotide sequence encoding the fusion protein of any one of claims 1-37.

44. An expression vector comprising the nucleotide sequence of claim 43.

45. A recombinant host cell comprising the nucleotide sequence of claim 44.

46. A recombinant host cell comprising the expression vector of claim 45.

47. A method of treating or preventing inflammation comprising administering to a mammalin need thereof an effective amount of the fusion protein of any one of claims 1-37.

48. A method of treating or preventing inflammation comprising administering to a mammalin need thereof an effective amount of the composition any one of claims 38-42.

49. The method of claim 47 or 48, wherein the dermatological condition comprises at leastone skin disorder selected from psoriasis, atopic dermatitis, skin rash, skin irritation, skin sensitization, allergic reactions, pruritus, and combinations thereof.

50. The method of any one of claims 47-49, wherein the inflammation is associated withatopic dermatitis.

51. The method of any one of claims 47-50, wherein the inflammation is associated withallergic dermatitis.

52. The method of any one of claims 47-51, wherein the mammal is a non-human mammal.

53. The method of any one of claims 47-52, wherein the mammal is a canine.

54. The method of any one of claims 47-53, wherein the mammal is a dog.

55. The method of claim 54, wherein the dog is at least 9 months of age.

56. The method of claim 54, wherein the dog is at least 12 months of age.

57. The method of any one of claims 47-52, wherein the mammal is a cat.

58. The method of any one of claims 47-57, wherein the administering is performed daily.

59. The method of any one of claims 47-58, wherein the administering is performed twicedaily.

60. The method of any one of claims 47-59, wherein the administering is performed weekly.

61. The method of any one of claims 47-60, wherein the administering is performedmonthly.

62. The method of any one of claims 47-61, wherein the administering is twice.

63. The method of any one of claims 47-62, wherein the administering is performed twice,once a day 0 and a second time at day 28.

64. The method of any one of claims 47-63, wherein the effective amount is between about0.1 and 10 mg / kg.

65. The method of any one of claims 47-64, wherein the effective amount is between about0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg.

66. The method of any one of claims 47-65, wherein the effective amount is between about0.1 and 1 mg / kg, 0.5 and 2 mg / kg, 0.75 and 5 mg / kg, or 1 and 10 mg / kg.

67. The method of any one of claims 47-66, wherein the effective amount is between about100 μg and 300 μg.

68. The method of any one of claims 47-67, wherein the effective amount is between about100 μg and 200 μg, 150 μg and 250 μg, 175 μg and 225 μg, 195 μg and 205 μg, or 150 μg and 300 μg.

69. The method of any one of claims 47-68, wherein the effective amount is about 100 µg,105 µg, 110 µg, 115 µg, 120 µg, 125 µg, 130 µg, 135 µg, 140 µg, 145 µg, 150 µg, 151 µg, 152 µg, 153 µg, 154 µg, 155 µg, 156 µg, 157 µg, 158 µg, 159 µg, 160 µg, 161 µg, 162 µg, 163 µg, 164 µg, 165 µg, 166 µg, 167 µg, 168 µg, 169 µg, 170 µg, 171 µg, 172 µg, 173 µg, 174 µg, 175 µg, 176 µg, 177 µg, 178 µg, 179 µg, 180 µg, 181 µg, 182 µg, 183 µg, 184 µg, 185 µg, 186 µg, 187 µg, 188 µg, 189 µg, 190 µg, 191 µg, 192 µg, 193 µg, 194 µg, 195 µg, 196 µg, 197 µg, 198 µg, 199 µg, 200 µg, 201 µg, 202 µg, 203 µg, 204 µg, 205 µg, 206 µg, 207 µg, 208 µg, 209 µg, 210 µg, 211 µg, 212 µg, 213 µg, 214 µg, 215 µg, 216 µg, 217 µg, 218 µg, 219 µg, 220 µg, 221 µg, 222 µg, 223 µg, 224 µg, 225 µg, 226 µg, 227 µg, 228 µg, 229 µg, 230 µg, 231 µg, 232 µg, 233 µg, 234 µg, 235 µg, 236 µg, 237 µg, 238 µg, 239 µg, 240 µg, 241 µg, 242 µg, 243 µg, 244 µg, 245 µg,246 µg, 247 µg, 248 µg, 249 µg, 250 µg, 255 µg, 260 µg, 265 µg, 270 µg, 275 µg, 280 µg, 285 µg, 290 µg, 295 µg, or 300 µg.

70. A composition for comprising an effective amount of the fusion protein of any one ofclaims 1-37.

71. The composition of claim 70, wherein the composition is a pharmaceutical compositionfurther comprising a pharmaceutically acceptable excipient, carrier, diluent, vehicle, adjuvant, or a combination thereof.

72. The composition of claim 70 or 71, wherein the effective amount is between about 0.1and 10 mg / kg.

73. The composition of any one of claims 70-72, wherein the effective amount is betweenabout 1 and 500 μg, optionally between about 150 and 250 μg.

74. The composition of any one of claims 65-68, wherein the effective amount is about 150µg, 151 µg, 152 µg, 153 µg, 154 µg, 155 µg, 156 µg, 157 µg, 158 µg, 159 µg, 160 µg, 161 µg, 162 µg, 163 µg, 164 µg, 165 µg, 166 µg, 167 µg, 168 µg, 169 µg, 170 µg, 171 µg, 172 µg, 173 µg, 174 µg, 175 µg, 176 µg, 177 µg, 178 µg, 179 µg, 180 µg, 181 µg, 182 µg, 183 µg, 184 µg, 185 µg, 186 µg, 187 µg, 188 µg, 189 µg, 190 µg, 191 µg, 192 µg, 193 µg, 194 µg, 195 µg, 196 µg, 197 µg, 198 µg, 199 µg, 200 µg, 201 µg, 202 µg, 203 µg, 204 µg, 205 µg, 206 µg, 207 µg, 208 µg, 209 µg, 210 µg, 211 µg, 212 µg, 213 µg, 214 µg, 215 µg, 216 µg, 217 µg, 218 µg, 219 µg, 220 µg, 221 µg, 222 µg, 223 µg, 224 µg, 225 µg, 226 µg, 227 µg, 228 µg, 229 µg, 230 µg, 231 µg, 232 µg, 233 µg, 234 µg, 235 µg, 236 µg, 237 µg, 238 µg, 239 µg, 240 µg, 241 µg, 242 µg, 243 µg, 244 µg, 245 µg, 246 µg, 247 µg, 248 µg, 249 µg, or 250 µg.

75. The composition of any one of claims 70-74, wherein the inflammation is associated withatopic dermatitis.

76. The composition of any one of claims 70-75, wherein the inflammation is associated withallergic dermatitis.

77. Use of the fusion protein of any one of claims 1-37 for the manufacture of a medicamentfor the treatment of inflammation in a mammal.

78. The use of claim 77, wherein the inflammation is associated with atopic dermatitis orallergic dermatitis.

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