Compounds, compositions, multimeric vaccines, and methods for treating dermatitis

Abstract

The description provides for polypeptide chains including antigens, for example a mutant IL-31 antigen; a CH2 domain; and a CH3 domain. Polypeptide chains optionally further include a CH1 domain, a hinge region, a CH4 domain, and / or a tailpiece. The disclosure further provides for multimeric and hexameric proteins capable of treatment of itch-related disorders, for example atopic dermatitis, in animals. Use of multimeric and hexameric proteins described herein as vaccines is further described.

Description

COMPOUNDS, COMPOSITIONS, MULTIMERIC VACCINES, AND METHODS FOR TREATING DERMATITIS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 566,601, filed March 18, 2024, U.S. Provisional Application No.63 / 740,064, filed December 30, 2024, U.S. Provisional Application No.63 / 740,046, filed December 30, 2024, and U.S. Provisional Application No.63 / 761,601, filed February 21, 2025, the entire content of each of these applications are herein incorporated herein by reference in their entireties. SEQUENCE LISTING

[0002] A Sequence Listing in XML format, submitted under 37 C.F.R. §§ 1.831-1.835, entitled 2920951-519977 Sequence Listing_4.xml, 73,934 bytes in size, generated on March 17, 2025, and filed via EFS-Web, is provided in lieu of a paper copy. This Sequence Listing is incorporated by reference into the specification for its disclosures. BACKGROUND

[0003] Atopic dermatitis (AD) is the most common allergic skin disease in dogs. The most common clinical sign of AD is pruritus (scratching behavior). Such scratching may cause infection and loss of fur.

[0004] Interleukin-31 (IL-31) is a recently identified cytokine that has been implicated in pruritic behaviors associated with AD. When first characterized in transgenic mice, overexpression of IL-31 was associated with development of inflammatory cell infiltrates in the skin, severe pruritus, alopecia, and other skin lesions consistent with AD. IL-31 has been shown to be elevated preferentially in pruritic versus non-pruritic human skin conditions, and elevated serum levels have been found in the majority of dogs suffering from AD. When canine IL-31 is injected intra-dermally, intravenously, or subcutaneously into dogs, the dogs will respond with pruritic (scratching) behavior. Certain monoclonal antibodies directed against IL-31 have been shown to significantly reduce scratching behavior in AD dogs and in dogs challenged with IL-31.

[0005] Monoclonal antibody treatment targeting IL-31 has been used in the treatment of canine AD. Such treatment, however, requires frequent administration of the antibody. As such, there is a need in the art a long-acting treatment that decreases the frequency and / or severity of AD flares.

[0006] Another approach to treating AD has been vaccination by way of vaccination using an IL-31 antigen. The vaccine induces production of anti-IL-31 antibodies that sequester and neutralize the activity of endogenous IL-31 and thus prevent itch.

[0007] Vaccination allows for a more sustained antibody response. Vaccine therapy also typically has a lower cost than monoclonal antibody therapy. However, the introduction of a vaccine antigen alone is often ineffective in creating a potent, long-lasting immune response. Antigens are thus often coupled with an adjuvant in order to increase immunogenicity.

[0008] The induction of therapeutic antibodies is often challenged by the ability of the antigen to induce an immune response in an animal from which antibody generation is sought. An immunized animal will often not recognize a vaccinated antigen or be tolerant of an antigen that resembles a self-antigen. An animal that is tolerant to the immunogen will not select B cells that can produce antibodies with the most desirable binding properties.

[0009] The conjugation of antigens with virus-like particles (VLPs) has also been used to increase immunogenicity. The success of this approach may be antigen specific, and the process of chemical conjugation can result in modifications to the structure / function of the antigen. These modifications can negatively impact the potency and / or duration of the induced immune response. In other embodiments, these VLP fusions result in shielding of critical epitopes, similarly reducing the efficacy of the induced response.

[0010] Induction of immunity requires the processing of antigen (Ag) by the innate immune system and presentation of the Ag to the adaptive immune systems. Cells of the innate immune system engulf Ag. These are antigen presenting cells (APCs). Dendritic cells (DCs), which are professional APCs present Ag to T cells in in secondary lymphoid organs such as lymph nodes. Ag that is engulfed by APCs is processed through the endosomal / lysosomal pathway. Processed peptides are bound by MHC class II molecules in the endomembrane system and are then translocated to the cell surface where they are presented to a large and diverse population of naïve T cells. This T cell population is composed of individual cells with T cell receptors that are selective for peptide sequences.Those CD4+ T cells with T cell receptors that have the appropriate affinity to the antigenic peptide respond to such peptides and are provided co-stimulatory cytokines by the DC. CD4+ T cells then present the Ag-CD4 receptor complex to quiescent B cells. Those B cells bearing an membrane localized immunoglobulin that binds the peptide are activated. The activated CD4+ T cell induce class switching of the immunoglobulin from IgM to other isotypes of immunoglobulins including IgG.

[0011] The use of multimeric fusion proteins derived from human antibodies to deliver antigens for use in human vaccination is described, for example, in Mekhaeil et al., Nature Scientific Reports, 1:124 (2011), and Webster et al., Plant Biotechnology Journal, 16:1983– 1996 (2018).

[0012] Vaccines using multimeric fusion proteins have been designed to capitalize upon the ability of the fusion proteins to bind Fcγ receptors in the target subject in order to generate an immune response. Hexameric fusion proteins derived from human antibodies, however, do not bind canine Fcγ receptors. However, the inventors have unexpectedly found that such fusion proteins nevertheless generate a robust immune response which efficiently mitigates itch in response to IL31 signaling. SUMMARY OF THE DISCLOSURE

[0013] In aspects, the disclosure provides for fusion proteins comprising polypeptide chains, wherein each polypeptide chain comprises: (a) an antigen polypeptide sequence comprising IL-31 or a fragment or variant thereof that binds to an IL-31 receptor; (b) an Fc region protein sequence comprising an immunoglobulin heavy chain constant region connected to the antigen sequence; and (c) a tailpiece protein sequence connected to the C-terminal to the immunoglobulin heavy chain constant region; wherein the polypeptide chains form an oligomer.

[0014] In other aspects, the disclosure provides for fusion proteins comprising polypeptide chains, wherein each polypeptide chain comprises: (a) an antigen polypeptide sequence comprising IL-31 or a fragment or variant thereof that binds to an IL-31 receptor;(b) a linker connected to the antigen polypeptide sequence; (c) optionally a CH1 polypeptide sequence connected to the linker; (d) a hinge polypeptide sequence; (e) a CH2 polypeptide sequence; (f) a CH3 polypeptide sequence; and (g) a tailpiece polypeptide sequence; wherein the polypeptide chains form an oligomer, and wherein the fusion protein increases an immune response in an animal relative to antigen alone.

[0015] In an aspect, the disclosures provides for a polypeptide chain comprising: an antigen; a CH2 domain; and a CH3 domain.

[0016] In certain embodiments, the antigen comprises IL-31 or a fragment or variant thereof that is capable of binding to an IL-31 receptor. In certain such embodiments, the antigen comprises mature canine IL-31 or a fragment or variant thereof that is capable of binding to canine IL-31 receptor.

[0017] In certain embodiments, the antigen comprises a fragment or variant of IL-31 that is capable of binding to an IL-31 receptor but has reduced signaling as compared with wild- type IL-31. In certain such embodiments, the antigen comprises a fragment or variant of IL- 31 that is capable of binding to an IL-31 receptor but is not capable of inducing an itch effect. In certain such embodiments, the antigen comprises a fragment or variant of mature canine IL-31 that is capable of binding to canine IL-31 receptor but is not capable of inducing an itch effect.

[0018] In certain embodiments, the antigen comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 8 or is a conservatively-substituted variant thereof, provided that the antigen is capable of binding to an IL-31 receptor and comprises an amino acid substitution at the residue position corresponding to residue position 104 of SEQ ID NO: 8. In an aspect, the amino acid substitution comprises a glutamic acid at residue position 104 of SEQ ID NO: 8.

[0019] In certain embodiments, the CH2 domain is from a human or canine antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH2 domain is a CH3 domain from a human antibody or is a functional fragment or variant thereof.

[0020] In certain embodiments, the CH2 domain is from an isotype of IgG, IgA, IgD, IgE, or IgM antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH2 domain is from a canine IgG-A, IgG-B, IgG-C, or IgG-D antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH2 domain is from a human IgG1, IgG2, IgG3, or IgG4 antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH2 domain is from a canine IgG-B, human IgG1 or human IgG4 antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH2 domain is from a human IgG1 antibody or is a functional fragment or variant thereof.

[0021] In certain embodiments, the CH2 domain is a functional fragment or variant of a CH2 domain from human IgG1 antibody and comprises a mutation as compared with the CH2 domain from wild-type human IgG1 antibody such that it is capable of forming a cross- link with another polypeptide chain. In certain such embodiments, the mutation is the presence of an additional cysteine as compared with the CH2 domain from wild-type human IgG1 antibody.

[0022] In certain embodiments, the CH2 domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 10 or is a conservatively-substituted variant thereof.

[0023] In certain embodiments, the CH2 domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 10 or is a conservatively-substituted variant thereof, provided that the polypeptide chaincomprises a cysteine at the residue position corresponding to residue position 309 of SEQ ID NO: 2.

[0024] In certain embodiments, the CH3 domain is from a human or canine antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH3 domain is a CH3 domain from a human antibody or is a functional fragment or variant thereof.

[0025] In certain embodiments, the CH3 domain is from an IgG-B, IgA, IgD, IgE, IgG, or IgM antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH2 domain is from an IgG-B, IgA, or IgG antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH3 domain is from an IgG1, IgG2, IgG3, or IgG4 antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH3 domain is from an IgG1 or IgG4 antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH3 domain is from an IgG1 antibody or is a functional fragment or variant thereof.

[0026] In certain embodiments, the CH3 domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 15 or is a conservatively-substituted variant thereof.

[0027] In certain embodiments, the polypeptide chain further comprises a CH4 domain.

[0028] In certain embodiments, the CH4 domain is from a human or canine antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH4 domain is a CH4 domain from a human antibody or is a functional fragment or variant thereof.

[0029] In certain embodiments, the CH4 domain is from an IgE or IgM antibody or is a functional fragment or variant thereof.

[0030] In certain embodiments, the polypeptide chain further comprises a tailpiece.

[0031] In certain embodiments, the tailpiece is from a human or canine antibody or is a functional fragment or variant thereof. In certain such embodiments, the tailpiece is a tailpiece from a human antibody or is a functional fragment or variant thereof.

[0032] In certain embodiments, the tailpiece is from an IgA or IgM antibody or is a functional fragment or variant thereof. In certain such embodiments, the tailpiece is from an IgM antibody or is a functional fragment or variant thereof.

[0033] In certain embodiments, the tailpiece comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 21 or is a conservatively-substituted variant thereof.

[0034] In certain embodiments, the polypeptide chain comprises, in N-terminus to C- terminus order: (a) an antigen comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of a mature canine IL-31, or a conservatively-substituted variant thereof, that is capable of binding to an IL-31 receptor; (b) a CH1 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the CH1 domain from a human IgG1 antibody, or is a conservatively-substituted variant thereof; In some embodiments, the CH1 domain includes a fragment of the CH1 domain. (c) a hinge region comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of thehinge region from a human IgG1 antibody, or is a conservatively-substituted variant thereof; (d) a CH2 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the CH2 domain from a human IgG1 antibody, or is a conservatively-substituted variant thereof; (e) a CH3 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the CH3 domain from a human IgG1 antibody, or is a conservatively-substituted variant thereof; and (f) a tailpiece comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the tailpiece from a human IgM antibody, or is a conservatively-substituted variant thereof.

[0035] In certain embodiments, the polypeptide chain comprises, in N-terminus to C- terminus order: (a) an antigen comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 7, or is a conservatively-substituted variant thereof, that is capable of binding to a IL-31 receptor;(b) a CH1 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 22, or is a conservatively-substituted variant thereof; (c) a hinge region comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 26, or is a conservatively-substituted variant thereof; (d) a CH2 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 10, or is a conservatively-substituted variant thereof; (e) a CH3 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 15, or is a conservatively-substituted variant thereof; and (f) a tailpiece comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 21, or is a conservatively-substituted variant thereof.

[0036] In certain embodiments, the polypeptide chain comprises, in N-terminus to C- terminus order: (a) an antigen comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 8, provided that the antigen is capable of binding to an IL-31 receptor and comprises a glutamic acid at the residue position corresponding to residue position 104 of SEQ ID NO: 8; (b) a CH1 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 22, or is a conservatively-substituted variant thereof; (c) a hinge region comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 26, provided that the hinge region comprises a serine at the residue position corresponding to residue position 220 of SEQ ID NO: 26; (d) a CH2 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 10, or is a conservatively-substituted variant thereof, provided that the hinge region comprises a cysteine at the residue position corresponding to residue position(e) a CH3 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 15, or is a conservatively-substituted variant thereof; and (f) a tailpiece comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 21, or is a conservatively-substituted variant thereof.

[0037] The present disclosure also relates in part to a nucleic acid encoding the polypeptide chain of the present disclosure.

[0038] In certain embodiments, the nucleic acid has at least 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 31, or is a codon degenerate variant thereof.

[0039] In certain embodiments, the nucleic acid has at least 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 32, or is a codon degenerate variant thereof.

[0040] The present disclosure further relates in part to a vector comprising the nucleic acid of the present disclosure.

[0041] A yet further aspect of the present disclosure is a monomer comprising two polypeptide chains of the present disclosure. In certain embodiments, the polypeptide chains are identical.

[0042] In certain embodiments, the polypeptide chains are cross-linked with each other.

[0043] In certain embodiments, the monomer units are identical.

[0044] In certain embodiments, the multimeric fusion protein comprises 2 to 12 monomer units of the present disclosure . In certain embodiments, the multimeric fusion protein comprise 6 monomer units. In certain embodiments, the multimeric fusion protein is in the form of a cyclic hexamer. In other aspects, the description provides for a hexamer of dimers.

[0045] In certain embodiments, the monomer units are cross-linked with each other.

[0046] In certain embodiments, a disulfide bond is formed between a cysteine present on one polypeptide chain of a first monomer and a cysteine present on one polypeptide chain of a second monomer. In certain such embodiments, a second disulfide bond is formed between a cysteine present on the other polypeptide chain of the first monomer and a cysteine present on one polypeptide of a third monomer.

[0047] In certain embodiments, a cysteine present in the CH2 domain of a polypeptide chain of the first monomer forms a disulfide bond with a cysteine present in the CH2 domain of a polypeptide chain of the second monomer and a cysteine present in the CH2 domain of the other polypeptide chain of the first monomer forms a disulfide bond with a cysteine present in the CH2 domain of a polypeptide chain of the third monomer. In certain such embodiments, the cysteines are present at residue position 309 in each CH2 domain.

[0048] In certain embodiments, a cysteine present in the tailpiece of a polypeptide chain of the first monomer forms a disulfide bond with a cysteine present in the tailpiece of a polypeptide chain of the second monomer and a cysteine present in the tailpiece of the other polypeptide chain of the first monomer forms a disulfide bond with a cysteine present in the tailpiece of a polypeptide chain of the third monomer.

[0049] The present disclosure also relates in part to an antigen comprising a fragment or variant of IL-31 that is capable of binding to an IL-31 receptor. In certain embodiments, the antigen comprises a fragment or variant of mature canine IL-31. In certain suchembodiments, the antigen comprises a fragment or variant of mature canine IL-31 that is capable of binding to canine IL-31 receptor.

[0050] In certain embodiments, the antigen comprises a fragment or variant of IL-31 that is capable of binding to an IL-31 receptor but has reduced signaling as compared with wild- type IL-31. In certain embodiments, the antigen comprises a fragment or variant of IL-31 that is capable of binding to an IL-31 receptor but is not capable of inducing an itch effect. In certain such embodiments, the antigen comprises a fragment or variant of mature canine IL- 31 that is capable of binding to canine IL-31 receptor but is not capable of inducing an itch effect.

[0051] In certain embodiments, the antigen comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 8 or is a conservatively-substituted variant thereof, provided that the antigen is capable of binding to an IL-31 receptor and comprises a glutamic acid at the residue position corresponding to residue position 104 of SEQ ID NO: 8.

[0052] The present disclosure further relates to a method for producing the antigen or polypeptide chain of certain embodiments, comprising introducing a nucleic acid encoding the antigen or polypeptide chain to a cell.

[0053] In certain embodiments, the method further comprises growing the cell to express the antigen or polypeptide chain. In certain such embodiments, the method further comprises isolating the antigen or polypeptide chain.

[0054] The present disclosure further relates to a composition comprising an immunogenically-effective amount of the antigen or multimeric fusion protein of the present disclosure.

[0055] In certain such embodiments, the composition comprises a multimeric fusion protein or hexameric form. In certain such embodiments, at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% of the multimeric fusion proteins present therein are in hexameric form.

[0056] In certain embodiments, the composition is for use in producing an immunogenic effect in a subject.

[0057] A yet further aspect of the present disclosure is a method for inducing an immunological response in a subject in need thereof, the method comprising administering the antigen or multimeric fusion protein of the present disclosure. In certain embodiments, the subject is canine. In an aspect, the compounds described herein, for example, hexameric structures described herein are administered as hexameric structures.

[0058] In certain embodiments, the antigen is administered in an amount of from about 50 µg to 1000 µg / dose, from about 100 µg to 500 µg / dose, from about 100 µg to 300 µg / dose, about 100 µg / dose, about 200 µg / dose, about 300 µg / dose, about 500 µg / dose, or more than 600 µg / dose.

[0059] In certain embodiments, the multimeric fusion protein is administered in an amount of from about 50 µg to 1000 µg / dose, from about 100 µg to 500 µg / dose, from about 100 µg to 300 µg / dose, about 100 µg / dose, about 200 µg / dose, about 300 µg / dose, about 500 µg / dose, or more than 600 µg / dose.

[0060] In aspects, fusion proteins comprising polypeptide chains are in the form of hexameric proteins, for example, those in FIG.1, wherein the hexameric protein comprises six monomer units, and wherein each monomer unit comprises a set of two polypeptide chains. In aspects, each single polypeptide unit comprises a sequence from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0061] The present disclosure also relates to a method for treating a disease or disorder in a subject in need thereof, the method comprising administering the antigen or multimeric fusion protein of the present disclosure. In certain embodiments, the subject is canine.

[0062] In certain embodiments, the disease or disorder is a disease or disorder for which an immunogenic effect produced from exposure to the antigen would have a therapeutic effect. In certain such embodiments, the disease or disorder is an itch-related disorder. In certain such embodiments, the disease or disorder is atopic dermatitis.

[0063] In certain embodiments, the antigen is administered in an amount of from about 50 µg to 1000 µg / dose, from about 100 µg to 500 µg / dose, from about 100 µg to 300µg / dose, about 100 µg / dose, about 200 µg / dose, about 300 µg / dose, about 500 µg / dose, or more than 600 µg / dose.

[0064] In certain embodiments, the multimeric fusion protein is administered in an amount of from about 50 µg to 1000 µg / dose, from about 100 µg to 500 µg / dose, from about 100 µg to 300 µg / dose, about 100 µg / dose, about 200 µg / dose, about 300 µg / dose, about 500 µg / dose, or more than 600 µg / dose.

[0065] The disclosure provides for compositions comprising fusion proteins, oligomers, multimers, and hexamers described herein. In aspects, compositions described herein further comprise at least one adjuvant or combination of adjuvants.

[0066] In other aspects, fusion proteins and compositions described here are used for therapeutic vaccination of companion animals, for example, cats and dogs. In other aspects, hexameric proteins and compositions thereof described herein are used for therapeutic vaccination of companion animals, for example, cats and dogs.

[0067] In an aspect, one or more adjuvants for use with fusion proteins, oligomers, multimers, hexamers, or compositions described herein include, for example, Freund's Complete Adjuvant and Freund's Incomplete Adjuvant, MF59, AS03,aluminum hydroxide, aluminum phosphate, QS-21, Quil A®, AS01, AS04, MPLA, CpG ODN 1018, CpG ODN 7909, IC31, Imiquimod (R837), Resiquimod, 3M-052, 2’,3’-cGAMP, 3’,3’-cGAMP, c-di- GMP, c-di-AMP, CARBOPOL, Carbopol® 971, Carbopol® 934, Carbopol® 941 NF, Mn2+, MnJ, MnARK, and / or nanoMn.

[0068] In aspects, one or more adjuvants for use with fusion proteins, oligomers, multimers, hexamers, or compositions described herein include, for example, Quil A®, CARBOPOL, Carbopol® 971, Carbopol® 934, and / or Carbopol® 941 NF.

[0069] In aspects, the disclosure provides for polypeptide chains comprising (Formula I): A (Antigen) – B (CH1) – C (hinge) – D (CH2) – E (Optional Interdomain Linker) – F (CH3) – G (tailpiece) wherein, A comprises a IL-31 antigen;B comprises an amino acid sequence at least 90% identity to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24; C comprises an amino acid sequence at least 90% identity to SEQ ID NO: 26; D comprises an amino acid sequence at least 90% identity to SEQ ID NO:61; E is optional and if present comprises an amino acid sequence at least 90% Identity to SEQ ID NO: 62; F comprises an amino acid sequence at least 90% identity to SEQ ID NO: 63; and G comprises an amino acid sequence at least 90% identity to SEQ ID NO: 21.

[0070] In further aspects, polypeptide chains A – G of Formula I comprise: A comprises a IL-31 antigen; B comprises an amino acid sequence at least 95% identity to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24; C comprises an amino acid sequence at least 95% identity to SEQ ID NO: 26; D comprises an amino acid sequence at least 95% identity to SEQ ID NO:61; E is optional and if present comprises an amino acid sequence at least 95% Identity to SEQ ID NO: 62; F comprises an amino acid sequence at least 95% identity to SEQ ID NO: 63; and G comprises an amino acid sequence at least 95% identity to SEQ ID NO: 21.

[0071] In further aspects, polypeptide chains A – G of Formula I comprise: A comprises a IL-31 antigen; B comprises an amino acid sequence 100% identical to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24; C comprises an amino acid sequence 100% identical to SEQ ID NO: 26; D comprises an amino acid sequence 100% identical to SEQ ID NO:61; E is optional and if present comprises an amino acid sequence 100% identical to SEQ ID NO: 62; F comprises an amino acid sequence 100% identical to SEQ ID NO: 63; and G comprises an amino acid sequence 100% identical to SEQ ID NO: 21.

[0072] In aspects, compositions described herein may include an adjuvant in an amount of from about 50 µg to 1000 µg / dose, from about 50 µg to 150 µg / dose, from about 25 µg to 100 µg / dose, from about 100 µg to 500 µg / dose, from about 100 µg to 300 µg / dose, about 100 µg / dose, about 200 µg / dose, about 300 µg / dose, about 500 µg / dose, or more than 600 µg / dose.

[0073] In other aspects, compositions described herein may include fusion proteins, oligomers, multimers, and hexamers described herein in an amount of from about 50 µg to 1000 µg / dose, from about 100 µg to 500 µg / dose, from about 100 µg to 300 µg / dose, about 100 µg / dose, about 200 µg / dose, about 300 µg / dose, about 500 µg / dose, or more than 600 µg / dose.

[0074] In further aspects, compositions described herein may include an adjuvant in an amount of from about 50 µg to 1000 µg / dose, 50 µg to 150 µg / dose from about 100 µg to 500 µg / dose, from about 100 µg to 300 µg / dose, about 100 µg / dose, about 200 µg / dose, about 300 µg / dose, about 500 µg / dose, or more than 600 µg / dose and fusion protein oligomers, multimers, and hexamers described herein in an amount of from about 50 µg to 1000 µg / dose, from about 100 µg to 500 µg / dose, from about 100 µg to 300 µg / dose, from about 100 µg to 400 µg / dose, about 100 µg / dose, about 200 µg / dose, about 300 µg / dose, about 500 µg / dose, or more than 600 µg / dose.

[0075] In aspects, fusion proteins, oligomers, multimers, hexamers, or compositions described herein increase a B cell response in an animal relative to an antigen (single antigen; non-hexameric form) alone about 1 fold or more, about 1.5 fold or more, about 2 fold or more, about 5 fold or more, about 10 fold or more, about .2 fold to about 3 fold, about .5 fold to about 5 fold, about 1 fold to about 10 fold, about 2 fold to about 10 fold, about 5 fold to about 50 fold, or about 50 fold to about 500 fold. In an aspect, the animal is a canine or feline.

[0076] In aspects, fusion proteins, oligomers, multimers, hexamers, or compositions described herein increase a B cell response in an animal relative to an antibody such as Lokivetmab (SEQ ID NO: 64 and SEQ ID NO: 65) about 0.2 fold or more, about 0.5 fold or more, about 1 fold or more, about 1.5 fold or more, about 2 fold or more, about 5 fold or more, about 10 fold or more, about .2 fold to about 3 fold, about .5 fold to about 5 fold, about1 fold to about 10 fold, about 2 fold to about 10 fold, about 5 fold to about 50 fold, or about 50 fold to about 500 fold. In an aspect, the animal is a canine or feline.

[0077] In further aspects, a hexameric protein comprising 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2 increases a B cell response in an animal relative to an antibody such as Lokivetmab (SEQ ID NO: 64 and SEQ ID NO: 65) about 0.2 fold or more, about 0.5 fold or more, about 1 fold or more, about 1.5 fold or more, about 2 fold or more, about 5 fold or more, about 10 fold or more, about .2 fold to about 3 fold, about .5 fold to about 5 fold, about 1 fold to about 10 fold, about 2 fold to about 10 fold, about 5 fold to about 50 fold, or about 50 fold to about 500 fold.

[0078] In aspects, fusion proteins, oligomers, multimers, hexamers, or compositions described herein are administered in an amount of from about 50 µg to 1000 µg / dose, from about 100 µg to 500 µg / dose, from about 100 µg to 300 µg / dose, about 100 µg / dose, about 200 µg / dose, about 300 µg / dose, about 500 µg / dose, or more than 600 µg / dose.

[0079] In aspects, compositions described herein exhibit\ stability for period of time of at least 24 weeks or at least 52 weeks at 2-8°C.

[0080] The present disclosure further relates to the use of the antigen or multimeric fusion protein of the present disclosure, in the manufacture of a medicament for the treatment of a disease or disorder in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] FIG.1 depicts the structure of a cyclic hexameric fusion protein comprising mutant mature canine IL-31 antigen and an Fc regions derived from a human IgG1 antibody.

[0082] FIG.2 depicts a chromatogram for an anti-CD20 monoclonal antibody standard.

[0083] FIG.3 depicts a chromatogram for multimeric fusion protein comprising wild type mature canine IL-31 antigen and an Fc regions derived from a human IgG1 antibody.

[0084] FIG.4 depicts a chromatogram for multimeric fusion protein comprising mutant mature canine IL-31 antigen and an Fc regions derived from a human IgG1 antibody.

[0085] FIG.5 depicts a chromatogram for multimeric fusion protein comprising wild type mature canine IL-31 antigen and an Fc regions derived from a human IgG4 antibody.

[0086] FIG.6 depicts a chromatogram for multimeric fusion protein comprising mutant mature canine IL-31 antigen and an Fc regions derived from a human IgG4 antibody.

[0087] FIG.7 depicts a chromatogram for multimeric fusion protein comprising wild type mature canine IL-31 antigen and an Fc regions derived from a human IgG-B antibody.

[0088] FIG.8 depicts a chromatogram for multimeric fusion protein comprising mutant mature canine IL-31 antigen and an Fc regions derived from a human IgG-B antibody.

[0089] FIG.9 is an image of a 4–12% Bis-Tris gel. The lanes contain the following samples: (1) Bio-Rad Precision PlusTMprotein unstained standards; (2) wild-type IL31-IgG1 Fc fusion protein; (3) mutant IL31-IgG1 Fc fusion protein; (4) wild type IL31-IgG4 Fc fusion protein; (5) mutant IL-31-IgG4 Fc fusion protein; (6) wild type IL31-IgG-B Fc fusion protein; and (7) mutant IL-31-IgG-B Fc fusion protein.

[0090] FIG.10 is an image of a 4–12% Bis-Tris gel. The lanes contain the following samples: (1) Bio-Rad Precision PlusTMprotein unstained standards; (2) wild-type IL31-IgG1 Fc fusion protein; (3) mutant IL31-IgG1 Fc fusion protein; (4) wild type IL31-IgG4 Fc fusion protein; (5) mutant IL-31-IgG4 Fc fusion protein; (6) wild type IL31-IgG4 Fc fusion protein; and (7) mutant IL-31-IgG4 Fc fusion protein; (8) wild type IL31-LinkCys; (9) mutant IL-31- LinkCys; and (10) IL-31-Mime4H-LinkCys.

[0091] FIG.11 depicts the results of an IL-31 bioassay.

[0092] FIG.12 depicts the mean titer for IL-31 antigen in each of Groups 1, 2, and 6.

[0093] FIG.13 depicts minutes of scratching behavior observed within the first 2 hours following challenge pruritus scores for dogs in Groups 1, 2, and 6 following IL-31 challenges.

[0094] FIG.14 depicts the number of dogs in each of Groups 1, 2, and 6 with at least 50% reduction in pruritus score within each of Groups 1, 2, and 6.

[0095] FIGs.15A and 15B demonstrate the binding of recombinant IL31Ra to wild-type and mutant antigens via using an octet biolayer interferometry (BLI) system. FIG.15A shows the binding of recombinant IL31Ra to wild-type IL31-Fc-Hexamer antigens. FIG.15B shows the binding of recombinant IL31Ra to IL31-Fc-Hexamer antigens.

[0096] FIG.16A depicts the time course of IL-challenge experiments in dogs vaccinated with IL31-Fc-Hexamer antigens, or controls. Dogs were vaccinated with IL31-Fc-Hexamer antigens (SEQ ID NO: 2) on days 0 and 14. Dogs were subsequently challenged with IL-31 at time 14 days after second vaccination, six months, and 12 months, and then scored for pruritus.

[0097] FIG.16B depicts a time course of the efficacy of IL31-Fc-Hexamer antigens for reducing pruritus in dogs challenged with IL-31. Dogs were immunized with IL31-Fc- Hexamer antigens (SEQ ID NO: 2) administered with or without the adjuvant Quil-A®(Invivogen) or a control wherein the dogs were immunized with phosphate buffered saline (PBS). A booster immunization with IL31-Fc-Hexamer antigens (SEQ ID NO: 2) plus Quil- A®further reduced pruritus scores after 12 months.

[0098] FIG.16C depicts the swelling associated with vaccinating animals with PBS, IL31-Fc-Hexamer antigens with QuilA adjuvant or IL31-Fc-Hexamer antigens without adjuvant. Measurements are in milimeters.

[0099] FIG.16D depicts a time course of the efficacy of IL31-Fc-Hexamer antigens for reducing pruritus in dogs challenged with IL-31. Dogs were immunized with IL31-Fc- Hexamer antigens (SEQ ID NO: 2) antigens administered with five different adjuvant formulations (QuilA 100 μg, Carbopol® 971 Low, Carbopol® 971 High, Carbopol® 971 Low + CpG Oligonucleotides, Ethylene Maleic Anhydride / Neocryl®A640 (Covestro), or a control (PBS) wherein the dogs were immunized with phosphate buffered saline (PBS).

[0100] FIG.16E depicts the distribution of pruritic scores for the six groups treated with the formulations from FIG.16D. The data represents the pruritic scores from the day 105 measurement.

[0101] FIG.17A depicts an SDS-PAGE gel analysis of hexameric and monomeric IL31- Fc fusion proteins. In some lanes proteins were separated with or without reducing reagent. In some lanes proteins were separated after being deglycoslyated.

[0102] FIG.17B depicts reverse phase HPLC separation analysis of hexameric and monomeric IL31-Fc fusion proteins. The analysis depicts the various protein species in a preparation of these proteins.

[0103] FIG.17C depicts size exclusion chromatography separation analysis of hexameric and monomeric IL31-Fc fusion proteins. The analysis depicts the various protein species in a preparation of these proteins.

[0104] FIG.18A depicts the stability of IL31-Fc-Hexamer antigens (SEQ ID NO: 2) in excipients at 5ºC, 25ºC, and 40ºC as determined by Tonset, and Tmusing differential scanning fluorimetry.

[0105] FIG.18B depicts the stability of IL31-Fc-Hexamer antigens (SEQ ID NO: 2) in excipients at 5ºC, 25ºC, and 40ºC as determined by SEC as measured by the integration of the hexamer, fragments, and aggregate peaks.

[0106] FIG.18C depicts the stability of IL31-Fc-Hexamer antigens (SEQ ID NO: 2) in excipients at 5ºC, 25ºC, and 40ºC as determined by RP-HPLC as measured by the integration of the hexamer, monomer, and sub-hexamer peaks comprising IgG-Fc-IL31 polypeptides and fragments that are in an oligomerization state of less than six.

[0107] FIG.18D depicts the stability of IL31-Fc-Hexamer antigens (SEQ ID NO: 2) in Buffer1 (20 mM phosphate, 50 mM arginine HCl, 150 mM sucrose, 10 mM Methionine, 0.02% Polaxamer 188, pH 7.5) at 5ºC, 25ºC, and 40ºC as determined by DSF assay melting temperature (Tm).

[0108] FIG.18E depicts the stability of IL31-Fc-Hexamer antigens (SEQ ID NO: 2) in Buffer1 (20 mM phosphate, 50 mM arginine HCl, 150 mM sucrose, 10 mM Methionine, 0.02% Polaxamer 188, pH 7.5) at 5ºC, 25ºC, and 40ºC as determined by the SEC assay as measured by the integration of the hexamer and aggregate peaks as a percent of the total area.

[0109] FIG.18F depicts the stability of IL31-Fc-Hexamer antigens (SEQ ID NO: 2) in Buffer1 (20 mM phosphate, 50 mM arginine HCl, 150 mM sucrose, 10 mM Methionine, 0.02% Polaxamer 188, pH 7.5) at 5ºC, 25ºC, and 40ºC as determined by the RP-HPLC method as measured by the integration of the hexamer, monomer, and sub-hexamer peaks as a percent of the total area.

[0110] FIG.18G depicts the stability of IL31-Fc-Hexamer antigens (SEQ ID NO: 2) in Buffer1 (20 mM phosphate, 50 mM arginine HCl, 150 mM sucrose, 10 mM Methionine, 0.02% Polaxamer 188, pH 7.5) or phosphate pH 7.5 over the course of 24 weeks, asdetermined by the formation of aggregates measured by the integration of the corresponding peak as a percent of the total area in the SEC assay.

[0111] FIG.18H depicts the stability of IL31-Fc-Hexamer antigens (SEQ ID NO: 2) in Buffer1 (20 mM phosphate, 50 mM arginine HCl, 150 mM sucrose, 10 mM Methionine, 0.02% Polaxamer 188, pH 7.5), citrate-phosphate buffer pH 7.5, or phosphate pH 7.5 over the course of 24 weeks, as determined by the integration of the hexamer peak as a percent of the total area in the SEC assay.

[0112] FIG.18I depicts the stability of IL31-Fc-Hexamer antigens (SEQ ID NO: 2) in phosphate buffer pH 7.5, Buffer1(20 mM phosphate, 50 mM arginine HCl, 150 mM sucrose, 10 mM Methionine, 0.02% Polaxamer 188, pH 7.5), citrate-phosphate buffer pH 7.5, phosphate pH 6.0, or citrate-phosphate pH 6.0 over the course of 26 weeks, as determined by Tm in the DSF assay.

[0113] FIG.18J depicts the stability of IL31-Fc-Hexamer antigens (SEQ ID NO: 2) in citrate buffer pH 6.0, citrate-phosphate buffer pH 6.0, citrate-phosphate buffer pH 7.5, Buffer1 (20 mM phosphate, 50 mM arginine HCl, 150 mM sucrose, 10 mM Methionine, 0.02% Polaxamer 188, pH 7.5), or phosphate pH 7.5 over the course of 24 to 26 weeks, as determined by the integration of the hexamer peak as a percent of the total area in the SEC assay or the integration of the hexamer peak as a percent of the total area in the RP-HPLC assay.

[0114] FIG.19A depicts the enzyme linked immunosorbent assay (ELISA) used to determine potency.

[0115] FIG.19B depicts an assay to optimize the proper concentration of the biotinylated S18-A antibody for detection in the ELISA potency assay.

[0116] FIG.19C depicts an assay to optimize the proper concentration of the streptavidin conjugated peroxidase for detection in the ELISA potency assay.

[0117] FIG.19D-FIG.19F depict an assay to determine the dynamic range of the assay the antigen in the ELISA potency assay.

[0118] FIG.19G depicts an ELISA potency assay to determine the potency of batches of IL31-Fc-Hexamer antigens (SEQ ID NO: 2), monomeric IL31-Fc-Hexamer, and batches of highly purified hexamer species.

[0119] FIG.20A and FIG.20B depict an ELISA potency assay on formulations of IL31- Fc-Hexamer antigens (SEQ ID NO: 2) in excipient formulations.

[0120] FIG.21 depicts a comparison of the efficacy of the IL31-Fc-Hexamer antigens (SEQ ID NO: 2) with the standard of care treatment using Lokivetmab.

[0121] FIG.22 depicts the efficacy of L31-Fc-Hexamer antigens (SEQ ID NO: 2) treatment wherein the amount of QuilA adjuvant is tested at 25, 50, 75 or 100 μg per dose as compared to a PBS alone (placebo treatment). DETAILED DESCRIPTION OF THE DISCLOSURE

[0122] The following description and examples illustrate embodiments of the present disclosure in detail. It is to be understood that the present disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are variations and modifications of the present disclosure, which are encompassed within the scope of the present disclosure.

[0123] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0124] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0125] Although various features of the disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment. Representative Terms

[0126] The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0127] In this application, the use of the singular includes the plural unless specifically stated otherwise. As used in the specification, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0128] In this application, the use of “or” means “and / or” unless stated otherwise. The terms “and / or” and “any combination thereof” and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof” can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.” The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

[0129] Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0130] Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures.

[0131] As used in this specification and the claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[0132] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. In another example, the amount “about 10” includes 10 and any amounts from 9 to 11. In yet another example, the term “about” in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value. Alternatively, particularly with respect to biological systems or processes, the term “about” can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0133] A “therapeutically-effective amount” or “therapeutically-effective dose” refers to an amount or dose effective, for periods of time necessary, to achieve a desired therapeutic result. The amount can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the inventive nucleic acid sequences to elicit a desired response in the individual.

[0134] “Polynucleotide” or “oligonucleotide” refers to a polymeric form of nucleotides or nucleic acids of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double and single stranded deoxyribonucleic acid (DNA), triplex DNA, as well as double and single stranded ribonucleic acid (RNA). It also includes modified, for example, by methylation and / or by capping, and unmodified forms of the polynucleotide. The term is also meant to include molecules that include non-naturally occurring or synthetic nucleotides as well as nucleotide analogs.

[0135] Unless otherwise stated, nucleic acid sequences in the text of this specification are given, when read from left to right, in the 5′ to 3′ direction.

[0136] The terms “transfection,” “transformation,” “nucleofection,” or “transduction” as used herein refer to the introduction of one or more exogenous polynucleotides into a host cell or organism by using physical, chemical, and / or electrical methods. The nucleic acidsequences and vectors disclosed herein can be introduced into a cell or organism by any such methods, including, for example, by electroporation, calcium phosphate co-precipitation, strontium phosphate DNA co-precipitation, liposome mediated-transfection, DEAE dextran mediated-transfection, polycationic mediated-transfection, tungsten particle-facilitated microparticle bombardment, viral, and / or non-viral mediated transfection. In some cases, the method of introducing nucleic acids into the cell or organism involves the use of viral, retroviral, lentiviral, or transposon, or transposable element-mediated vectors.

[0137] “Polypeptide,” “peptide,” and their grammatical equivalents as used herein refer to a polymer of amino acid residues. The polypeptide can optionally include glycosylation or other modifications typical for a given protein in a given cellular environment. Polypeptides and proteins disclosed herein (including functional fragments and functional variants thereof) can comprise synthetic amino acids in place of one or more naturally-occurring amino acids. Such synthetic amino acids are known in the art, and include, for example, aminocyclohexane carboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl- cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2- carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N’-benzyl-N’-methyl-lysine, N’,N’-dibenzyl-lysine, 6- hydroxylysine, ornithine, α-aminocyclopentane carboxylic acid, α-aminocyclohexane carboxylic acid, α-aminocycloheptane carboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert- butylglycine. The present disclosure further contemplates that expression of polypeptides or proteins described herein in an engineered cell can be associated with post-translational modifications of one or more amino acids of the polypeptide or protein. Non-limiting examples of post-translational modifications include phosphorylation, acylation including acetylation and formylation, glycosylation (including N-linked and O-linked), amidation, hydroxylation, alkylation including methylation and ethylation, ubiquitylation, addition of pyrrolidone carboxylic acid, formation of disulfide bridges, sulfation, myristoylation, palmitoylation, isoprenylation, farnesylation, geranylation, glypiation, lipoylation and iodination.

[0138] The term “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and Schirmer, R. H., Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids can be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz, G. E. and Schirmer, R. H., supra). Examples of conservative mutations include amino acid substitutions of amino acids within the sub-groups below, for example, lysine for arginine and vice versa such that a positive charge can be maintained; glutamic acid for aspartic acid and vice versa such that a negative charge can be maintained; serine for threonine such that a free –OH can be maintained; and glutamine for asparagine such that a free –NH2 can be maintained. Exemplary conservative amino acid substitutions are shown in the following chart: Type of Amino Acid Substitutable Amino AcidsAn amino acid sequence that differs from a reference amino acid sequence by only conservative amino acid substitutions will be referred to herein as a “conservatively- substituted variant” of the reference sequence.

[0139] In some embodiments, the functional variants can comprise the amino acid sequence of the reference protein with at least one non-conservative amino acid substitution. The term “non-conservative mutations” involves amino acid substitutions between differentgroups, for example, lysine for tryptophan, or phenylalanine for serine, etc. In this case, it is preferable for the non-conservative amino acid substitution to not interfere with, or inhibit the biological activity of, the functional variant. The non-conservative amino acid substitution can enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the homologous parent protein. Amino acid substitutability is discussed in more detail, for example, in L. Y. Yampolsky and A. Stoltzfus, “The Exchangeability of Amino acids in Proteins,” Genetics 2005 Aug.; 170(4):1459-1472.

[0140] The terms “identical” and its grammatical equivalents as used herein or “sequence identity” in the context of two nucleic acid sequences or amino acid sequences of polypeptides refer to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. A “comparison window,” as used herein, refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are aligned optimally. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981); by the alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48:443 (1970); by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci U.S.A., 85:2444 (1988); by computerized implementations of these algorithms (including, but not limited to CLUSTAL in the PC / Gene program by Intelligentics, Mountain View Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., U.S.A.); the CLUSTAL program is well described by Higgins and Sharp, Gene, 73:237-244 (1988) and Higgins and Sharp, CABIOS, 5:151-153 (1989); Corpet et al., Nucleic Acids Res., 16:10881-10890 (1988); Huang et al., Computer Applications in the Biosciences, 8:155-165 (1992); and Pearson et al., Methods in Molecular Biology, 24:307-331 (1994). Alignment is also often performed by inspection and manual alignment. In one class of embodiments, the polypeptides herein are at least 80%, 85%, 90%, 98% 99% or 100% identical to a reference polypeptide (i.e., the full length thereof), or a fragment thereof, e.g., as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters. Similarly, nucleic acids can also be described with reference to a starting nucleic acid, e.g., they can be 50%, 60%, 70%, 75%,80%, 85%, 90%, 98%, 99% or 100% identical to a reference nucleic acid (i.e., the full length thereof) or a fragment thereof, e.g., as measured by BLASTN (or CLUSTAL, or any other available alignment software) using default parameters. When one molecule is said to have certain percentage of sequence identity with a larger molecule, it means that when the two molecules are optimally aligned, the percentage of residues in the smaller molecule finds a match residue in the larger molecule in accordance with the order by which the two molecules are optimally aligned.

[0141] For purposes of this specification and the claims, it is understood that the phrase “having at least 50% sequence identity with” a reference sequence, or referencing any range therein (e.g., “at least 80% sequence identity with”) encompasses the reference sequence itself. Thus, for example, a claim reciting “a nucleic acid having at least 80% sequence identity with SEQ ID NO: 0” encompasses SEQ ID NO: 0 itself.

[0142] The term “substantially identical” and its grammatical equivalents as applied to nucleic acid or amino acid sequences mean that a nucleic acid or amino acid sequence comprises a sequence that has at least 95% sequence identity with a reference sequence using the programs described above, e.g., BLAST, using standard parameters.

[0143] “Homology” is generally inferred from sequence identity between two or more nucleic acids or proteins (or sequences thereof). The precise percentage of identity between sequences that is useful in establishing homology varies with the nucleic acid and protein at issue, but as little as 25% sequence identity is routinely used to establish homology. Higher levels of sequence identity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more can also be used to establish homology. Methods for determining sequence identity percentages (e.g., BLASTP and BLASTN using default parameters) are described herein and are generally available. Nucleic acids and / or nucleic acid sequences are “homologous” when they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Proteins and / or protein sequences are “homologous” when their encoding DNAs are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. The homologous molecules can be termed “homologs.” For example, any naturally occurring proteins can be modified by any available mutagenesis method. When expressed, this mutagenized nucleic acid encodes a polypeptide that is homologous to the protein encoded by the original nucleic acid.

[0144] Also contemplated and included herein are nucleic acid molecules that hybridize to the disclosed sequences. Hybridization conditions may be mild, moderate, or stringent, as is warranted.

[0145] Appropriate stringency conditions which promote DNA hybridization, for example, 6× sodium chloride / sodium citrate (SSC) at about 45° C, followed by a wash of 2×SSC at 50° C, are known or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. “Stringent hybridization conditions” are those that include a salt concentration of 1.0 M NaCl in 50% formamide, at a temperature of 37oC for 4 to 12 hours, followed by a wash in 0.1X SSC at 60-65oC.

[0146] As will be appreciated by the skilled practitioner, slight changes in nucleic acid sequence do not necessarily alter the amino acid sequence of the encoded polypeptide. This disclosure embraces the degeneracy of codon usage as would be understood by one of ordinary skill in the art. For example, as known in the art, different codons will code for the same amino acid as illustrated in the following chart. Amino Acid CodonsThr / T ACT, ACC, ACA, ACG Trp / W TGG As use a nucleicacid sequence means a nucleic acid sequence that differs from the referenced sequence, but that encodes a polypeptide having the same amino acid sequence as that encoded by the referenced sequence.

[0147] Additionally, it will be appreciated by persons skilled in the art that partial sequences often work as effectively as full-length versions. The ways in which the nucleotide sequence can be varied or shortened are well known to persons skilled in the art, as are ways of testing the suitability or effectiveness of the altered genes. In certain embodiments, suitability and / or effectiveness of the altered gene may easily be tested by, for example, conventional gas chromatography. All such variations of the genes are therefore included as part of the present disclosure.

[0148] In the present application, when an amino acid residue in an Fc region or domain thereof (e.g., a CH2, CH3, or CH4 domain), a CH1 domain, a hinge region, or a tailpiece is referred to by its position, the position number given is, in accordance with the conventional practice in the art, the position number of the residue in the heavy chain of a naturally occurring antibody. For example, a C220S mutation in an IgG1 hinge region refers to a cysteine to serine substitution occurring at position 220 in the heavy chain of an IgG1 antibody and a L309C mutation in an IgG1 CH2 region refers to a leucine to cysteine substitution occurring at position 309 of the heavy chain.

[0149] The term “isolated” and its grammatical equivalents as used herein refer to the removal of a nucleic acid from its natural environment. It is to be understood, however, that nucleic acids and proteins can be formulated with diluents or adjuvants and still for practical purposes be isolated.

[0150] The term “purified” and its grammatical equivalents as used herein refer to a molecule or composition, whether removed from nature (including genomic DNA and mRNA) or synthesized (including cDNA) and / or amplified under laboratory conditions, that has been increased in purity, wherein “purity” is a relative term, not “absolute purity.” For example, nucleic acids typically are mixed with an acceptable carrier or diluent when used for introduction into cells. The term “substantially purified” and its grammatical equivalents as used herein refer to a nucleic acid sequence, polypeptide, protein or other compound that is essentially free, i.e., is more than about 50% free of, more than about 70% free of, more than about 90% free of, the polynucleotides, proteins, polypeptides and other molecules that the nucleic acid, polypeptide, protein or other compound is naturally associated with.

[0151] “Transposon,” “transposable element” or “TE” refers to a DNA sequence that can change its position within the genome, sometimes creating or reversing mutations and altering the cell’s genome size. Transposition often results in duplication of the transposon. Class I transposons are copied in two stages: first, they are transcribed from DNA to RNA, and the RNA produced is then reverse transcribed to DNA. This copied DNA is then inserted at a new position into the genome. The reverse transcription step is catalyzed by a reverse transcriptase, which can be encoded by the transposon itself. The characteristics of retrotransposons are similar to retroviruses, such as HIV. The cut-and-paste transposition mechanism of class II transposons does not involve an RNA intermediate. The transpositions are catalyzed by several transposase enzymes. Some transposases non-specifically bind to any target site in DNA, whereas others bind to specific DNA sequence targets. The transposase makes a staggered cut at the target site resulting in single-strand 5’ or 3’ DNA overhangs (sticky ends). This step cuts out the DNA transposon, which is then ligated into a new target site; this process involves activity of a DNA polymerase that fills in gaps and of a DNA ligase that closes the sugar-phosphate backbone. This results in duplication of the target site. The insertion sites of DNA transposons can be identified by short direct repeats which can be created by the staggered cut in the target DNA and filling in by DNA polymerase, followed by a series of inverted repeats important for the transposon excision by transposase. Cut-and-paste transposons can be duplicated if their transposition takes place during S phase of the cell cycle when a donor site has already been replicated, but a target site has not yet been replicated. Transposition can be classified as either “autonomous” or “non-autonomous” in both Class I and Class II transposons. Autonomous transposons can move by themselves while non-autonomous transposons require the presence of another transposon to move. Thisis often because non-autonomous transposons lack transposase (for class II) or reverse transcriptase (for class I).

[0152] “Transposase” refers an enzyme that binds to the end of a transposon and catalyzes the movement of the transposon to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism. In some embodiments, the transposase’s catalytic activity can be utilized to move gene(s) from a vector to the genome.

[0153] An “expression vector” or “vector” is any genetic element, e.g., a plasmid, a mini- circle, a nanoplasmid, chromosome, virus, transposon, behaving either as an autonomous unit of polynucleotide replication within a cell. (i.e. capable of replication under its own control) or being rendered capable of replication by insertion into a host cell chromosome, having attached to it another polynucleotide segment, so as to bring about the replication and / or expression of the attached segment. Suitable vectors include, but are not limited to, plasmids, transposons, bacteriophages and cosmids. Vectors can contain polynucleotide sequences that are necessary to effect ligation or insertion of the vector into a desired host cell and to effect the expression of the attached segment. Such sequences differ depending on the host organism; they include promoter sequences to effect transcription, enhancer sequences to increase transcription, ribosomal binding site sequences and transcription and translation termination sequences. Alternatively, expression vectors can be capable of directly expressing nucleic acid sequence products encoded therein without ligation or integration of the vector into host cell DNA sequences. In some embodiments, the vector is an “episomal expression vector” or “episome,” which is able to replicate in a host cell, and persists as an extrachromosomal segment of DNA within the host cell in the presence of appropriate selective pressure (see, e.g., Conese et al., Gene Therapy, 11:1735-1742 (2004)). Representative commercially-available episomal expression vectors include, but are not limited to, episomal plasmids that utilize Epstein Barr Nuclear Antigen 1 (EBNA1) and the Epstein Barr Virus (EBV) origin of replication (oriP). The vectors pREP4, pCEP4, pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, Calif.) and pBK-CMV from Stratagene (La Jolla, Calif.) represent non-limiting examples of an episomal vector that uses T-antigen and the SV40 origin of replication in lieu of EBNA1 and oriP. A vector also can comprise a selectable marker gene. In certain embodiments where nano plasmids are utilized, strains such as R6K that utilizes an antisense RNA selection marker (e.g. sucrose tolerance) can be used.

[0154] The term “selectable marker gene” refers to a nucleic acid sequence that allows cells expressing the nucleic acid sequence to be specifically selected for or against, in the presence of a corresponding selective agent. Suitable selectable marker genes are known in the art and described in, e.g., International Patent Application Publications WO 1992 / 08796 and WO 1994 / 28143; Wigler et al., Proc. Natl. Acad. Sci. USA, 77: 3567 (1980); O’Hare et al., Proc. Natl. Acad. Sci. USA, 78: 1527 (1981); Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78: 2072 (1981); Colberre-Garapin et al., J. Mol. Biol., 150:1 (1981); Santerre et al., Gene, 30: 147 (1984); Kent et al., Science, 237: 901-903 (1987); Wigler et al., Cell, 11: 223 (1977); Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48: 2026 (1962); Lowy et al., Cell, 22: 817 (1980); and U.S. Pat. Nos.5,122,464 and 5,770,359.

[0155] The term “coding sequence” refers to a segment of a polynucleotide that encodes for protein or polypeptide. The region or sequence is bounded nearer the 5’ end by a start codon and nearer the 3’ end with a stop codon. Coding sequences can also be referred to as open reading frames.

[0156] The term “operably linked” refers to refers to the physical and / or functional linkage of a DNA segment to another DNA segment in such a way as to allow the segments to function in their intended manners. A DNA sequence encoding a gene product is operably linked to a regulatory sequence when it is linked to the regulatory sequence, such as, for example, promoters, enhancers and / or silencers, in a manner, that allows modulation of transcription of the DNA sequence, directly or indirectly. For example, a DNA sequence is operably linked to a promoter when it is ligated to the promoter downstream with respect to the transcription initiation site of the promoter and in the correct reading frame with respect to the transcription initiation site so as to allow transcription elongation to proceed through the DNA sequence. An enhancer or silencer is operably linked to a DNA sequence coding for a gene product when it is ligated to the DNA sequence in such a manner as to, respectively, increase or decrease the transcription of the DNA sequence. Enhancers and silencers can be located upstream or downstream of or embedded within the coding regions of the DNA sequence. A DNA for a signal sequence is operably linked to DNA coding for a polypeptide if the signal sequence is expressed as a pre-protein that participates in the secretion of the polypeptide. Linkage of DNA sequences to regulatory sequences is typically accomplished by ligation at suitable restriction sites or via adapters or linkers inserted in the sequence using restriction endonucleases known to one of skill in the art.

[0157] The terms “induce,” “induction” and their grammatical equivalents as used herein refer to an increase in nucleic acid sequence transcription, promoter activity and / or expression brought about by a transcriptional regulator, relative to some basal level of transcription.

[0158] The term “transcriptional regulator” refers to a biochemical element that acts to prevent or inhibit the transcription of a promoter-driven DNA sequence under certain environmental conditions (e.g., a repressor or nuclear inhibitory protein), or to permit or stimulate the transcription of the promoter-driven DNA sequence under certain environmental conditions (e.g., an inducer or an enhancer).

[0159] The term “enhancer,” as used herein, refers to a DNA sequence that increases transcription of, for example, a nucleic acid sequence to which it is operably linked. Enhancers can be located many kilobases away from the coding region of the nucleic acid sequence and can mediate the binding of regulatory factors, patterns of DNA methylation, or changes in DNA structure. A large number of enhancers from a variety of different sources are well known in the art and are available as or within cloned polynucleotides (from, e.g., depositories such as the ATCC as well as other commercial or individual sources). A number of polynucleotides comprising promoters (such as the commonly-used CMV promoter) also comprise enhancer sequences. Enhancers can be located upstream or downstream of coding sequences or within coding sequences. The term “Ig enhancers” refers to enhancer elements derived from enhancer regions mapped within the immunoglobulin (Ig) locus (such enhancers include for example, the heavy chain (mu) 5’ enhancers, light chain (kappa) 5’ enhancers, kappa and mu intronic enhancers, and 3’ enhancers (see generally Paul W. E. (ed), Fundamental Immunology, 3rd Edition, Raven Press, New York (1993), pages 353-363; and U.S. Pat. No.5,885,827).

[0160] The term “promoter” refers to a region of a polynucleotide that initiates transcription of a coding sequence. Promoters are located near the transcription start sites of genes, on the same strand and upstream on the DNA (towards the 5’ region of the sense strand). Some promoters are constitutive as they are active in all circumstances in the cell, while others are regulated becoming active in response to specific stimuli, e.g., an inducible promoter. The term “promoter activity” and its grammatical equivalents as used herein refer to the extent of expression of nucleotide sequence that is operably linked to the promoter whose activity is being measured. Promoter activity can be measured directly by determiningthe amount of RNA transcript produced, for example by Northern blot analysis or indirectly by determining the amount of product coded for by the linked nucleic acid sequence, such as a reporter nucleic acid sequence linked to the promoter.

[0161] “Inducible promoter” refers to a promoter, that is induced into activity by the presence or absence of transcriptional regulators, e.g., biotic or abiotic factors. Inducible promoters are useful because the expression of genes operably linked to them can be turned on or off at certain stages of development of an organism or in a particular tissue. Non- limiting examples of inducible promoters include alcohol-regulated promoters, tetracycline- regulated promoters, steroid-regulated promoters, metal-regulated promoters, pathogenesis- regulated promoters, temperature-regulated promoters and light-regulated promoters. The inducible promoter can be part of a gene switch or genetic switch.

[0162] As used herein, the phrase “functional fragment” when used with reference to a polypeptide refers to a fragment of such polypeptide that possesses the primary function of the referenced polypeptide. For example, a functional fragment of a polypeptide that serves as a transmembrane domain is a fragment of that polypeptide that also serves as a transmembrane domain. In certain embodiments, the functional fragment of a polypeptide is shorter than the referenced polypeptide by at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues at the N- and / or C-terminus. When used with reference to a nucleic acid, the phrase “functional fragment” refers to a fragment of the referenced nucleic acid that encodes a polypeptide having the same primary function as the polypeptide encoded by the referenced nucleic acid.

[0163] As used herein, the phrase “functional variant” when used with reference to a polypeptide refers to a polypeptide that differs from the referenced polypeptide but possesses the primary function of the referenced polypeptide. For example, a functional variant of a polypeptide that serves as a transmembrane domain is a fragment of that polypeptide that also serves as a transmembrane domain. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the referenced amino acid sequence and / or is a conservatively-substituted variant of the referenced sequence. When used with reference to a nucleic acid, the phrase “functional variant” refers to a nucleic acid that differs from the referenced nucleic acid but encodes a polypeptide having the same primary function as the polypeptide encoded by the referenced nucleic acid. In certain embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% sequence identity with the referenced nucleic acid sequence, hybridizes under stringent hybridization conditions with the complement of the referenced nucleic acid sequence, or is a codon degenerate variant of the nucleic acid sequence.

[0164] The term “antibody,” also known as immunoglobulin (Ig), as used herein can refers to a monoclonal or polyclonal antibody. The term “monoclonal antibodies,” as used herein, refers to antibodies that are produced by a single clone of B-cells and bind to the same epitope. In contrast, “polyclonal antibodies” refer to a population of antibodies that is produced by different B-cells and bind to different epitopes of the same antigen. The antibodies can be from any animal origin. An antibody may, for example, be an IgG-B, IgG (including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, IgM, or IgY antibody. In some embodiments, the antibody can a single-chain whole antibody.

[0165] An antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (VH) region and three or four C-terminal constant regions and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The constant regions of a heavy chain are, in N-terminal to C-terminal order, the CH1, CH2, and CH3 domains and, where it exists, the CH4 domain. Each heavy chain may further comprise a hinge region that connects the CH1 domain with the CH2 domain. Each heavy chain may also further comprise a tailpiece that is C-terminal to the constant regions.

[0166] The “Fab region” of the antibody refers to the portion consisting of the VL, VH, CL, and CH1 domains from both the heavy and light chains. The “Fc region” comprises the CH2 and CH3 domains and, where applicable, the CH4 domain and / or tailpiece of both heavy chains of an antibody. In certain antibodies, the Fab region and the Fc region are connected by way of a hinge region that may be present in the heavy chains.

[0167] As used herein, the terms “CH1 domain,” “CH2 domain,” “CH3 domain,” “CH4 domain,” “hinge region,” and “tailpiece” refer respectively to a CH1 domain, CH2 domain, CH3 domain, CH4 domain, hinge region, or tailpiece from an antibody (e.g., an IgG-B, IgA, IgD, IgE, IgG, IgM, or IgY antibody) or a functional fragment or variant thereof.

[0168] The term “naturally-occurring,” as used to describe an antibody or a portion thereof (e.g., a CH1 domain, a CH2 domain, a CH3 domain, a CH4 domain, a hinge region, atailpiece, or an Fc region) refers to a naturally-occurring antibody or a portion of a naturally- occurring antibody. The antibody may be from any species, for example human or canine.

[0169] The terms “non-naturally occurring,” “non-natural,” “synthetic,” and “artificial,” as used to describe a polypeptide or a portion thereof refers to a polypeptide or portion thereof comprising an amino acid sequence that does not occur in nature. In certain embodiments, the non-naturally occurring polypepide or portion thereof is a functional variant of a naturally-occurring antibody or portion thereof (e.g., a CH1 domain, a CH2 domain, a CH3 domain, a CH4 domain, a hinge region, a tailpiece, or an Fc region). The antibody may be from any species, for example human or canine.

[0170] “Patient” or “subject” refers to a mammalian subject diagnosed with or suspected of having or developing an itch-related disorder such as atopic dermatitis. In some embodiments, the term “patient” refers to a mammalian subject with a higher than average likelihood of developing such a disorder. Exemplary patients can be humans, apes, dogs, pigs, cattle, cats, horses, goats, sheep, rodents and other mammalians that can benefit from the therapies disclosed herein. Exemplary human patients can be male and / or female. “Patient in need thereof” or “subject in need thereof” means a patient diagnosed with or suspected of having a disease or disorder, for instance, but not restricted to itch-related disorders.

[0171] “Administering” refers to herein as providing one or more compositions described herein to a patient or a subject. By way of example and not limitation, composition administration, e.g., injection, can be performed by intravenous (i.v.) injection, sub-cutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, or intramuscular (i.m.) injection. One or more such routes can be employed. Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time. Alternatively, or concurrently, administration can be by the oral route. Additionally, administration can also be by surgical deposition of a bolus or pellet of cells, or positioning of a medical device.

[0172] As used herein, the terms “treatment,” “treating,” and their grammatical equivalents refer to obtaining a desired pharmacologic and / or physiologic effect. In some embodiments, the effect is therapeutic, i.e., the effect partially or completely cures a disease and / or adverse symptom attributable to the disease. In some embodiments, the term “treating” can include “preventing” a disease or a condition.

[0173] As used herein, a “treatment interval” refers to a treatment cycle, for example, a course of administration of a therapeutic agent that can be repeated, e.g., on a regular schedule. In some embodiments, a dosage regimen can have one or more periods of no administration of the therapeutic agent in between treatment intervals.

[0174] The terms “administered in combination,” “co-administration,” “co- administering,” and “co-providing” as used herein, mean that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as "simultaneous” or “concurrent delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

[0175] In some embodiments, the first treatment and second treatment can be administered simultaneously (e.g., at the same time), in the same or in separate compositions, or sequentially. Sequential administration refers to administration of one treatment before (e.g., immediately before, less than 5, 10, 15, 30, 45, 60 minutes; 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24, 48, 72, 96 or more hours; 4, 5, 6, 7, 8, 9 or more days; 1, 2, 3, 4, 5, 6, 7, 8 or more weeks before) administration of an additional, e.g., secondary, treatment. The order of administration of the first and secondary treatment can also be reversed.

[0176] The terms “therapeutically effective amount,” therapeutic amount,” “immunogenically effective amount,” and their grammatical equivalents refer to an amounteffective, at dosages and for periods of time necessary, to achieve a desired therapeutic or immunogenic result. The effective amount can vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of a composition described herein to elicit a desired response in one or more subjects. The precise amount of the compositions of the present disclosure to be administered can be determined by a physician or veterinarian with consideration of the age, weight, and condition of the patient (subject).

[0177] As used herein, terms used in the identification of biological moieties may include, or may not include, a dash “ – ” within the term. The presence or absence of a dash does not change the intended meaning or identification of the biological moiety. II. The Polypeptide Chain

[0178] The present disclosure relates in part to a polypeptide chain comprising: an antigen; a CH2 domain; and a CH3 domain. In certain embodiments, the polypeptide chain further comprises a CH1 domain; a hinge region; a CH4 domain; and / or a tailpiece.

[0179] In certain embodiments, the polypeptide chain comprises, in order from its N- terminus to its C-terminus: the antigen; the CH2 domain; and the CH3 domain. In certain embodiments, the polypeptide chain further comprises a CH1 domain that is between the antigen and the CH2 domain. In certain embodiments, the polypeptide chain further comprises a hinge region between the antigen and the CH2 domain or between the CH1 domain and the CH2 domain. In certain embodiments, the polypeptide chain further comprises a CH4 domain that is C-terminal to the CH3 domain. In certain embodiments, the polypeptide chain further comprises a tailpiece that is C-terminal to the CH3 domain or the CH4 domain. In certain embodiments, the polypeptide chain comprises, in order from its N- terminus to its C-terminus: the antigen; the CH1 domain; the hinge region; the CH2 domain; the CH3 domain; and the tailpiece.

[0180] It is understood that each of the aforementioned elements of the polypeptide chain (the antigen, the CH1 domain, the hinge region, the CH2 domain, the CH3 domain, the CH4 domain, and the tailpiece) may individually be linked to one other directly by way of a covalent bond between such elements. For example, the antigen and the CH1 domain may be linked together by way of a covalent bond between the C-terminal residue of the antigen and the N-terminal residue of the CH1 domain, the CH1 domain and the hinge region may be linked together by way of a covalent bond between the C-terminal residue of the CH1 domainand the N-terminal residue of the hinge region, the hinge region and the CH2 domain may be linked together by way of a covalent bond between the C-terminal residue of the hinge region and the N-terminal residue of the CH2 domain, the CH2 domain and the CH3 domain may be linked together by way of a covalent bond between the C-terminal residue of the CH2 domain and the N-terminal residue of the CH3 domain, the CH3 domain and the CH4 domain may be linked together by way of a covalent bond between the C-terminal residue of the CH3 domain and the N-terminal residue of the CH4 domain, and the CH4 domain and the tailpiece may be linked together by way of a covalent bond between the C-terminal residue of the CH4 domain and the N-terminal residue of the tailpiece.

[0181] Alternatively, any element of the polypeptide chain may be linked to another element by way of a linker peptide. Any linker peptide known in the art for linking two polypeptides may be used

[0182] It is understood that each element of the polypeptide (the antigen, the CH1 domain, the hinge region, the CH2 domain, the CH3 domain, the CH4 domain, and the tailpiece) may individually be naturally-occurring or non-natural. It is also understood that, when any two elements of the polypeptide chain are naturally-occurring, they may each be from a different antibody or antibody subclass. For example, a polypeptide chain may comprise a naturally-occurring CH1 domain from an IgA1 antibody, a naturally-occurring CH2 domain from an IgG1 antibody, and a non-natural CH3 domain that has 90% sequence identity with an IgG4 antibody.

[0183] In certain embodiments, the polypeptide chain comprises, in N-terminus to C- terminus order: (a) an antigen comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of a mature canine IL-31, or a conservatively-substituted variant thereof, that is capable of binding to an IL-31 receptor; (b) a CH1 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the CH1 domain from a human IgG1 antibody, or is a conservatively-substituted variant thereof; (c) a hinge region comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the hinge region from a human IgG1 antibody, or is a conservatively-substituted variant thereof; (d) a CH2 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the CH2 domain from a human IgG1 antibody, or is a conservatively-substituted variant thereof; (e) a CH3 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the CH3 domain from a human IgG1 antibody, or is a conservatively-substituted variant thereof; and (f) a tailpiece comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the tailpiecefrom a human IgM antibody, or is a conservatively-substituted variant thereof.

[0184] In certain such embodiments, the polypeptide comprises, in N-terminus to C- terminus order: (a) an antigen comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 7, or is a conservatively-substituted variant thereof, that is capable of binding to a IL-31 receptor; (b) a CH1 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 22, or is a conservatively-substituted variant thereof; (c) a hinge region comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 26, or is a conservatively-substituted variant thereof; (d) a CH2 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 10, or is a conservatively-substituted variant thereof; (e) a CH3 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 15, or is a conservatively-substituted variant thereof; and (f) a tailpiece comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 21, or is a conservatively-substituted variant thereof.

[0185] In certain such embodiments, the polypeptide chain comprises, in N-terminus to C-terminus order: (a) an antigen comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 8, or is a conservatively-substituted variant thereof, that is capable of binding to a IL-31 receptor; (b) a CH1 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 22, or is a conservatively-substituted variant thereof; (c) a hinge region comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 26, or is a conservatively-substituted variant thereof;(d) a CH2 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 10, or is a conservatively-substituted variant thereof; (e) a CH3 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 15, or is a conservatively-substituted variant thereof; and (f) a tailpiece comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 21, or is a conservatively-substituted variant thereof.

[0186] In certain embodiments, the polypeptide chain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 1 or is a conservatively-substituted variant thereof.

[0187] In certain embodiments, the polypeptide chain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 2 or is a conservatively-substituted variant thereof. In certain such embodiments, the polypeptide chain comprises a glutamic acid at the residue position corresponding to residueposition 104 of SEQ ID NO: 2, a serine at the residue position corresponding to residue position 220 of SEQ ID NO: 2, and cysteine at the residue position corresponding to residue position 309 of SEQ ID NO: 2.

[0188] In certain embodiments, the polypeptide chain comprises, in N-terminus to C- terminus order: (a) an antigen comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of a mature canine IL-31, or a conservatively-substituted variant thereof, that is capable of binding to an IL-31 receptor; (b) a CH1 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the CH1 domain from a human IgG4 antibody, or is a conservatively-substituted variant thereof; (c) a hinge region comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the hinge region from a human IgG4 antibody, or is a conservatively-substituted variant thereof; (d) a CH2 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of theCH2 domain from a human IgG4 antibody, or is a conservatively-substituted variant thereof; (e) a CH3 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the CH3 domain from a human IgG4 antibody, or is a conservatively-substituted variant thereof; and (f) a tailpiece comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the tailpiece from a human IgM antibody, or is a conservatively-substituted variant thereof.

[0189] In certain such embodiments, the polypeptide chain comprises, in N-terminus to C-terminus order: (a) an antigen comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 7, or is a conservatively-substituted variant thereof, that is capable of binding to a IL-31 receptor; (b) a CH1 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 23, or is a conservatively-substituted variant thereof;(c) a hinge region comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 28, or is a conservatively-substituted variant thereof; (d) a CH2 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 12, or is a conservatively-substituted variant thereof; (e) a CH3 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 18, or is a conservatively-substituted variant thereof; and (f) a tailpiece comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 21, or is a conservatively-substituted variant thereof.

[0190] In certain such embodiments, the polypeptide chain comprises, in N-terminus to C-terminus order: (a) an antigen comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 8,or is a conservatively-substituted variant thereof, that is capable of binding to a IL-31 receptor; (b) a CH1 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 23, or is a conservatively-substituted variant thereof; (c) a hinge region comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 28, or is a conservatively-substituted variant thereof; (d) a CH2 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 12, or is a conservatively-substituted variant thereof; (e) a CH3 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 18, or is a conservatively-substituted variant thereof; and (f) a tailpiece comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 21,or is a conservatively-substituted variant thereof.

[0191] In certain embodiments, the polypeptide chain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 3 or is a conservatively-substituted variant thereof.

[0192] In certain embodiments, the polypeptide chain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 4 or is a conservatively-substituted variant thereof. In certain such embodiments, the polypeptide chain comprises a proline at the residue position corresponding to residue position 228 of SEQ ID NO: 4, a glutamic acid at the residue position corresponding to residue position 235 of SEQ ID NO: 4, cysteine at the residue position corresponding to residue position 309 of SEQ ID NO: 4, and glycine at the residue position corresponding to residue position 329 of SEQ ID NO: 4.

[0193] In certain embodiments, the polypeptide chain comprises, in N-terminus to C- terminus order: (a) an antigen comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of a mature canine IL-31, or a conservatively-substituted variant thereof, that is capable of binding to an IL-31 receptor; (b) a CH1 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%sequence identity, or 100% sequence identity with the amino acid sequence of the CH1 domain from a canine IgG-B antibody, or is a conservatively-substituted variant thereof; (c) a hinge region comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the hinge region from a canine IgG-B antibody, or is a conservatively-substituted variant thereof; (d) a CH2 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the CH2 domain from a canine IgG-B antibody, or is a conservatively-substituted variant thereof; (e) a CH3 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the CH3 domain from a canine IgG-B antibody, or is a conservatively-substituted variant thereof; and (f) a tailpiece comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the tailpiece from a human IgM antibody, or is a conservatively-substituted variant thereof.

[0194] In certain such embodiments, the polypeptide chain comprises, in N-terminus to C-terminus order: (a) an antigen comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 7, or is a conservatively-substituted variant thereof, that is capable of binding to a IL-31 receptor; (b) a CH1 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 24, or is a conservatively-substituted variant thereof; (c) a hinge region comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 30, or is a conservatively-substituted variant thereof; (d) a CH2 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 14, or is a conservatively-substituted variant thereof; (e) a CH3 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 20, or is a conservatively-substituted variant thereof; and (f) a tailpiece comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 21, or is a conservatively-substituted variant thereof.

[0195] In certain such embodiments, the polypeptide chain comprises, in N-terminus to C-terminus order: (a) an antigen comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 8, or is a conservatively-substituted variant thereof, that is capable of binding to a IL-31 receptor; (b) a CH1 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 24, or is a conservatively-substituted variant thereof; (c) a hinge region comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 30, or is a conservatively-substituted variant thereof; (d) a CH2 domain comprising an amino acid sequence having at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 14, or is a conservatively-substituted variant thereof; (e) a CH3 domain comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 20, or is a conservatively-substituted variant thereof; and (f) a tailpiece comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 21, or is a conservatively-substituted variant thereof.

[0196] In certain embodiments, the polypeptide chain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 5 or is a conservatively-substituted variant thereof.

[0197] In certain embodiments, the polypeptide chain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 6 or is a conservatively-substituted variant thereof. In certain such embodiments, the polypeptide chain comprises a glutamic acid at the residue position corresponding to residue position 104 of SEQ ID NO: 6.

[0198] In aspects, the disclosure provides for polypeptide chains comprising: A (Antigen) – B (CH1) – C (hinge) – D (CH2) – E (Optional Interdomain Linker) – F (CH3) –G (tailpiece)Formula I

[0199] In aspects, Formula I may cover the following representative combinations of Table A. In further aspects, proteins of Formula I and Table A form the basis of monomer units described herein (for example, two polypeptide chains of Formula I and Table A), which in turn may be combined to form a multimeric fusion protein described herein. In aspects, the fusion protein comprises 2 to 12 monomer units of the description. In certain embodiments, the multimeric fusion protein comprise 6 monomer units. In certain embodiments, the multimeric fusion protein is in the form of a cyclic hexamer. In other aspects, the description provides for a hexamer of dimers. In aspects, polypeptide chains of Formula I and Table A form the basis of monomer units which are combined to form a cyclic hexamer such as, for example, in FIG.1. In further aspects, polypeptide chains of Formula I and Table A are utilized in methods described herein, for example, in methods of vaccination of animals, such as canines or felines.)DeIDIce Q ot 1o1ipli %EQ0Sy2ty2ot%E5S tiot :ttnOit :eN nO eN)D D o ,2,31QEIQEQIQt 4E y2 2 2 QDI HS Q SES Q S tit :O:O:OES QC %0oESro %5oESro n %5oE(B9ttsyat ,i2,349tt yt ,2,e34diN N NDet2 2 2 sIDD 9tStyt ,2ln: : : ai etn 2:2:2:%I Isi 0QQQaetn 2:teadiOOOlN N NteadiOOO N N N01ESESEl SteadiO N DIQE)Sneogtityntn n nitA( egegengeAitnit itdian1an31 a1%5-3L-3-9tIL s 8nILnI anel :t O A A AaNot 1ot 1ot 1ot 1ot 1ot 1n n n n, ,eN N N N o n N N N o ndedededl%e l%eiDI iDI iDiD an 59 diDD D an 59 di%00Q%I IQ%Q%Qoit,%I I IQQQoit,%1E0S01E0S01E0S01ES p% O00901ESESES p% O00901%0%0%0% % % 00 00 0 01 1 10 0 0r r r1 1 1o o oror r,8,8,8,7 o,8 o 8%:%:%:%:%: ,%:8O8O8O8O8O8O 9,N 9 N 9 N 9 N 9 N 9 N%5DI ,%5DI ,%5DI ,%DI ,%DI ,%DI9,Q%E9,QE9,Q5E9,Q5E9 Q5E9 QE0S9 ot%0S9 ot%0S9 ot%0S,9ot%0S,9ot%0S9 ottsyatit y t y t y t y t yetslnatitsatits tits tits titeelneelnaeelnaeelnaelnt d t d t d t d ted tea i a i a i a i a i adiot 1ot 1ot 1ot 1S,2S,r%0%N, , ,D%0%ND %0%N %0%N2:o,O349t0s01I90Qts01I90Qts0D1I90 DIQts01 QN 2:2: ae, E ae, E a , E a , EDIOO l % N Nta8 Sl % 9otta8 S el % 9otta8 S el % 9otta8 S9ot%0%0% % 000001r1 101o,8r%:o,8r:or,8:o,8:8O%8O%O%O 9,N 9 N89 N89 N%5DI ,%5DI ,%5DI ,%5DI9,Q%E9 Q0S, E9 Q 9 QS, ES, ES9 ot%09 ot%09 ot%09 ottsyatitsytitsytitsyti et aet a t a tlnln eln elnted ted tetea i a i adi adiot 1ot 1ot 1ot 1ot 1ot 1 1ee09t0s0DI 0900DI 090 DI 090 DI 090 DI 090 DIN N Na1 Qt1 Qt 01 Qt 01 Qt 01 Qt 01 Q DIDIDIe,lEsae, Esae, Esae, Esae, Esa , Et%a8 Sl % 9otta8 Sl % 9otta8 Sl %Sl %S el %SQ 9otta89otta89otta89otEQ SEQ SES%0% % % % %0000 0 0 01 1010 0 0r r r1r1 1o o o oror,8: ,8: ,8,8,8 o,8%O%O%:O%:%:O%:89,N89 N89 N8O 9N89 N8O 9N%5DI ,%DI ,%DI ,%DI ,DI ,DIn9,Q5%E9 Q5E9 Q5% %E9 Q5E9 Q59 Qegit0S,o% S,o% S,o% S, Eo% S, Eno % Soa9t 0tsy9t 0 t 0 t 0 t 0 tatit y9t y9y9y9y 13ets tits titts titts titts tit -lnaLeelnaeelnaeelnaelnaelnItaditaditaditeaditeaditeadi nAonis enn o1o ni nroe, vid snhtevno taioi iywtvcratuwl i ltoa aivtu,e e esotnitDsI 16: no retsitsn er n i bQohtocmausESOitNpeonnoobcusroe, v sido tai nhoti evidi nwtvcra ituwyltc oitD6 la av atu,e e esonitsI2: no riesotnitsn er n i bQohtocmausESOtNpeonnooicmbaus,en onro en oois , , niyllmnaaoi 2t234o:2:2 yllmnaoitneoeireviuth ttitOO:Oan eviutitptoravsbN N N oi ta su DDDtp v buho rti,e s I I Io re sosndiQQQht sndtc aESES S i iwwo c Ewoccaenoyllanoitpdoichati owni8:maOeNvit naoiD IvtrutQes itnsE obS cusA. The Antigen

[0200] The antigen may comprise IL-31, or a fragment or variant thereof that is capable of binding to an IL-31 receptor. The IL-31 may be from any species. In certain embodiments, the IL-31 may be canine.

[0201] In certain embodiments, the antigen comprises a canine IL-31, or a fragment or variant thereof that is capable of binding to canine IL-31 receptor. In certain such embodiments, the antigen comprises a mature canine IL-31, or a fragment or variant thereof that is capable of binding to canine IL-31 receptor.

[0202] In certain embodiments, the antigen comprises a fragment or variant of IL-31 that is capable of binding to an IL-31 receptor but has reduced signaling as compared with wild- type IL-31. In certain such embodiments, the antigen comprises a fragment or variant of canine IL-31 that is capable of binding to canine IL-31 receptor but has reduced signaling as compared with wild-type canine IL-31. In certain such embodiments, the antigen comprises a fragment or variant of mature canine IL-31 that is capable of binding to canine IL-31 receptor but has reduced signaling as compared with wild-type canine IL-31.

[0203] In certain embodiments, the antigen comprises a fragment or variant of IL-31 that is capable of binding to an IL-31 receptor but is not capable of inducing an itch effect. In certain such embodiments, the antigen comprises a fragment or variant of canine IL-31 that is capable of binding to canine IL-31 receptor but is not capable of inducing an itch effect. In certain such embodiments, the antigen comprises a fragment or variant of mature canine IL- 31 that is capable of binding to canine IL-31 receptor but is not capable of inducing an itch effect.

[0204] In certain embodiments, the antigen comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 7 or is a conservatively-substituted variant thereof, provided that the antigen is capable of binding to an IL-31 receptor.

[0205] In certain embodiments, the antigen comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 8 or is a conservatively-substituted variant thereof, provided that the antigen is capable of binding to an IL-31 receptor and comprises a glutamic acid at the residue position corresponding to residue position 104 of SEQ ID NO: 8. B. The CH2 Domain

[0206] The polypeptide chain comprises a CH2 domain. The CH2 domain may be naturally-occurring or non-natural. The CH2 domain may from an antibody from any species, for example human or canine, or is a functional fragment or variant of a CH2 domain from such an antibody. In certain embodiments, the CH2 domain is from a human antibody, or is a functional fragment or variant of a CH2 domain from a human antibody.

[0207] In certain embodiments, the CH2 domain is from an IgG-B, IgA, IgD, IgE, IgG, or IgM antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH2 domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the CH2 domain of an IgG-B, IgA, IgD, IgE, IgG, or IgM antibody, or is a conservatively-substituted variant thereof.

[0208] In certain such embodiments, the CH2 domain is from an IgG-B, IgA, or IgG antibody or is a functional fragment or variant thereof.

[0209] In certain embodiments, the CH2 domain is from a canine IgG-B antibody or is a functional fragment or variant thereof.

[0210] In certain embodiments, the CH2 domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 13 or is a conservatively-substituted variant thereof.

[0211] In certain embodiments, the CH2 domain is from an IgA antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH2 domain is from an IgA1 or IgA2 antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH2 domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the CH2 domain from an IgA1 or IgA2 antibody, or is a conservatively-substituted variant thereof.

[0212] In certain embodiments, the CH2 domain is from an IgG antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH2 domain is from an IgG1, IgG2, IgG3, or IgG4 antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH2 domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the CH2 domain from an IgG1, IgG2, IgG3, or IgG4 antibody, or is a conservatively-substituted variant thereof.

[0213] In certain such embodiments, the CH2 domain is from an IgG1 or IgG4 antibody or is a functional fragment or variant thereof.

[0214] In certain such embodiments, the CH2 domain is from an IgG1 antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH2 domain is from a human IgG1 antibody or is a functional fragment or variant thereof.

[0215] In certain embodiments, the CH2 domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 9 or is a conservatively-substituted variant thereof.

[0216] In certain such embodiments, the CH2 domain is from an IgG4 antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH2 domain is from a human IgG4 antibody or is a functional fragment or variant thereof.

[0217] In certain embodiments, the CH2 domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 11 or is a conservatively-substituted variant thereof.

[0218] In certain embodiments, the CH2 domain is a functional fragment or variant of a naturally-occurring CH2 domain that comprises a mutation such that it is capable of forming additional or fewer cross-links, for example disulfide bonds, with another polypeptide chain (an inter-chain cross-link) or with another portion of the same polypeptide chain (an intra- chain cross-link). For example, the CH2 domain may comprise additional or fewer cysteine residues as compared with the corresponding naturally-occurring CH2 domain. The cysteine residue serves as a position where a disulfide bond may form with other another cysteine in the same polypeptide chain or in a different polypeptide chain.

[0219] In certain embodiments, the CH2 domain is a functional fragment or variant of a CH2 domain from a human IgG1 or IgG4 antibody or a canine IgG-B antibody and comprises a mutation as compared with the CH2 domain from, respectively, wild-type human IgG1 or IgG4 antibody or wild-type canine IgG-B antibody such that it is capable of forming a cross-link, for example a disulfide bond, with another polypeptide chain. In certain such embodiments, the mutation is the presence of an additional cysteine as compared with the CH2 domain from wild-type human IgG1 or IgG4 antibody or wild-type canine IgG-B antibody.

[0220] In certain embodiments, the CH2 domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 10 or is a conservatively-substituted variant thereof, provided that the antigen comprises a cysteine at the residue position corresponding to residue position 309 of SEQ ID NO: 10.

[0221] In certain embodiments, the CH2 domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 12 or is a conservatively-substituted variant thereof, provided that the antigen comprises a cysteine at the residue position corresponding to residue position 309 of SEQ ID NO: 12.

[0222] In certain embodiments, the CH2 domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 14 or is a conservatively-substituted variant thereof, provided that the antigen comprises a cysteine at the residue position corresponding to residue position 309 of SEQ ID NO: 14.

[0223] In certain embodiments, the CH2 domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 61 or is a conservatively-substituted variant thereof, provided that the antigen comprises a cysteine at the residue position corresponding to residue position 309 of SEQ ID NO: 61. C. The CH3 Domain

[0224] The polypeptide chain comprises a CH3 domain. The CH3 domain may be naturally-occurring or non-natural. The CH3 domain may from an antibody from any species, for example human or canine, or is a functional fragment or variant of a CH3 domain from such an antibody. In certain embodiments, the CH3 domain is from a human antibody, or is a functional fragment or variant of a CH3 domain from a human antibody.

[0225] In certain embodiments, the CH3 domain is from an IgG-B, IgA, IgD, IgE, IgG, or IgM antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH3 domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the CH3 domain of an IgG-B, IgA, IgD, IgE, IgG, or IgM antibody, or is a conservatively-substituted variant thereof.

[0226] In certain such embodiments, the CH3 domain is from an IgG-B, IgA, or IgG antibody or is a functional fragment or variant thereof.

[0227] In certain embodiments, the CH3 domain is from a canine IgG-B antibody or is a functional fragment or variant thereof.

[0228] In certain embodiments, the CH3 domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 20 or is a conservatively-substituted variant thereof.

[0229] In certain embodiments, the CH3 domain is from an IgA antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH3 domain is from an IgA1 or IgA2 antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH3 domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the CH3 domain from an IgA1 or IgA2 antibody, or is a conservatively-substituted variant thereof.

[0230] In certain embodiments, the CH3 domain is from an IgG antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH3 domain is from an IgG1, IgG2, IgG3, or IgG4 antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH3 domain comprises an amino acid sequence having atleast about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the CH3 domain from an IgG1, IgG2, IgG3, or IgG4 antibody, or is a conservatively-substituted variant thereof.

[0231] In certain such embodiments, the CH3 domain is from an IgG1 or IgG4 antibody or is a functional fragment or variant thereof.

[0232] In certain embodiments, the CH3 domain is from an IgG1 antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH3 domain is from a human IgG1 antibody or is a functional fragment or variant thereof.

[0233] In certain embodiments, the CH3 domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 15 or is a conservatively-substituted variant thereof. In certain such embodiments, the CH3 domain retains an arginine at the residue position corresponding to residue position 355 of SEQ ID NO: 15.

[0234] In certain embodiments, the CH3 domain is from an IgG4 antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH3 domain is from a human IgG4 antibody or is a functional fragment or variant thereof.

[0235] In certain embodiments, the CH3 domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 17 or is a conservatively-substituted variant thereof.

[0236] In certain embodiments, the CH3 domain is a functional fragment or variant of a naturally-occurring CH3 domain that comprises a mutation such that it is capable of formingadditional or fewer cross-links, for example disulfide bonds, with another polypeptide chain (an inter-chain cross-link) or with another portion of the same polypeptide chain (an intra- chain cross-link). For example, the CH3 domain may comprise additional or fewer cysteine residues as compared with the corresponding naturally-occurring CH3 domain. The cysteine residue serves as a position where a disulfide bond may form with other another cysteine in the same polypeptide chain or in a different polypeptide chain.

[0237] In certain embodiments, the CH3 domain is a functional fragment or variant of a CH3 domain from a human IgG1 antibody and comprises a mutation as compared with the CH3 domain from wild-type human IgG1 antibody such that it is capable of forming a cross- link, for example a disulfide bond, with another polypeptide chain. In certain such embodiments, the mutation is the presence of an additional cysteine as compared with the CH3 domain from wild-type human IgG1 antibody.

[0238] In certain embodiments, the CH3 domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 16 or is a conservatively-substituted variant thereof, provided that the antigen comprises a cysteine at the residue position corresponding to residue position 355 of SEQ ID NO: 16.

[0239] In certain embodiments, the CH3 domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 63 or is a conservatively-substituted variant thereof. D. The CH4 Domain

[0240] The polypeptide chain may further comprise a CH4 domain. The CH4 domain may be naturally-occurring or non-natural. The CH4 domain may from an antibody from any species, for example human or canine, or is a functional fragment or variant of a CH4 domain from such an antibody. In certain embodiments, the CH4 domain is from a human antibody, or is a functional fragment or variant of a CH4 domain from a human antibody.

[0241] In certain embodiments, the CH4 domain is from an IgE or IgM antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH3 domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the CH4 domain of an IgE or IgM antibody, or is a conservatively-substituted variant thereof.

[0242] In certain embodiments, the CH4 domain is a functional fragment or variant of a naturally-occurring tailpiece that comprises a mutation such that it is capable of forming additional or fewer cross-links, for example disulfide bonds, with another polypeptide chain (an inter-chain cross-link) or with another portion of the same polypeptide chain (an intra- chain cross-link). For example, the CH4 domain may comprise additional or fewer cysteine residues as compared with the corresponding naturally-occurring CH4 domain. The cysteine residue serves as a position where a disulfide bond may form with other another cysteine in the same polypeptide chain or in a different polypeptide chain. E. The Tailpiece

[0243] The polypeptide chain may further comprise a tailpiece. The tailpiece is the region of the polypeptide chain that causes the monomers to assemble into a multimeric fusion protein. For example, in certain embodiments, the tailpiece is capable of forming a cross-link with a polypeptide chain in another monomer, thus causing such monomers to assemble into a multimeric fusion protein. In certain such embodiments, both polypeptide chains of a monomer form cross-links with different monomers, thus resulting in a fusion protein comprising at least three monomers.

[0244] The tailpiece may be naturally-occurring or non-natural. The tailpiece may from an antibody from any species, for example human or canine, or is a functional fragment or variant of a tailpiece domain from such an antibody. In certain embodiments, the tailpiece is from a human antibody, or is a functional fragment or variant of a tailpiece from a human antibody.

[0245] In certain embodiments, the tailpiece is from an IgA or IgM antibody or is a functional fragment or variant thereof. In certain such embodiments, the tailpiece domaincomprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the tailpiece of an IgA or IgM antibody, or is a conservatively-substituted variant thereof.

[0246] In certain embodiments, the tailpiece is from an IgM antibody or is a functional fragment or variant thereof. In certain such embodiments, the tailpiece comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the tailpiece from an IgM antibody, or is a conservatively-substituted variant thereof.

[0247] In certain embodiments, the tailpiece comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 21 or is a conservatively-substituted variant thereof.

[0248] In certain embodiments, the tailpiece is a functional fragment or variant of a naturally-occurring tailpiece that comprises a mutation such that it is capable of forming additional or fewer cross-links, for example disulfide bonds, with another polypeptide chain (an inter-chain cross-link) or with another portion of the same polypeptide chain (an intra- chain cross-link). For example, the tailpiece may comprise additional or fewer cysteine residues as compared with the corresponding naturally-occurring tailpiece. The cysteine residue serves as a position where a disulfide bond may form with other another cysteine in the same polypeptide chain or in a different polypeptide chain.

[0249] In certain embodiments, the Fc region of each polypeptide chain further comprises a tailpiece of an antibody. The tailpiece is the region of the polypeptide chain that causes the monomers to assemble into a multimeric fusion protein. For example, in certainembodiments, the tailpiece is capable of forming a linkage with a polypeptide chain in another monomer, thus causing such monomers to assemble into a multimeric fusion protein. In certain such embodiments, both polypeptide chains of a monomer form linkages with different monomers, thus resulting it a fusion protein comprising at least three monomers.

[0250] In certain embodiments, tailpiece is non-natural and is designed to have increased length and / or flexibility as compared to naturally-occurring tailpieces. F. The CH1 Domain

[0251] The polypeptide chain may further comprise a CH1 domain. The CH1 domain may be naturally-occurring or non-natural. The CH1 domain may from an antibody from any species, for example human or canine, or is a functional fragment or variant of a CH1 domain from such an antibody. In certain embodiments, the CH1 domain is from a human antibody, or is a functional fragment or variant of a CH1 domain from a human antibody. In aspects, the CH1 domain may be modified without influencing the function of the fusion protein, hexameric protein, or vaccine thereof.

[0252] In certain embodiments, the CH1 domain is from an IgG-B, IgA, IgD, IgE, IgG, or IgM antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH1 domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the CH1 domain of an IgG-B, IgA, IgD, IgE, IgG, or IgM antibody, or is a conservatively-substituted variant thereof.

[0253] In certain embodiments, the CH1 domain is from a canine IgG-B antibody or is a functional fragment or variant thereof.

[0254] In certain such embodiments, the CH1 domain is from an IgA or IgG antibody or is a functional fragment or variant thereof.

[0255] In certain embodiments, the CH1 domain is from an IgA antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH1 domain is from an IgA1 or IgA2 antibody or is a functional fragment or variant thereof. In certain suchembodiments, the CH1 domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the CH1 domain from an IgA1 or IgA2 antibody, or is a conservatively-substituted variant thereof.

[0256] In certain embodiments, the CH1 domain is from an IgG antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH1 domain is from an IgG1, IgG2, IgG3, or IgG4 antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH1 domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the CH1 domain from an IgG1, IgG2, IgG3, or IgG4 antibody, or is a conservatively-substituted variant thereof.

[0257] In certain such embodiments, the CH1 domain is from an IgG1 or IgG4 antibody or is a functional fragment or variant thereof.

[0258] In certain embodiments, the CH1 domain is from an IgG1 antibody or is a functional fragment or variant thereof. In certain such embodiments, the CH1 domain is from a human IgG1 antibody or is a functional fragment or variant thereof.

[0259] In certain embodiments, the CH1 domain is a functional fragment of the CH1 domain from antibody, for example an IgG-B, IgA, IgD, IgE, IgG, or IgM antibody or functional fragment or variant thereof.

[0260] In certain embodiments, the CH1 domain is a functional fragment of the CH1 domain from a canine IgG-B antibody. In certain such embodiments, the CH1 domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identitywith the amino acid sequence of SEQ ID NO: 24 or is a conservatively-substituted variant thereof.

[0261] In certain embodiments, the CH1 domain is a functional fragment of the CH1 domain from a human IgG1 antibody. In certain such embodiments, the CH1 domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 22 or is a conservatively-substituted variant thereof.

[0262] In certain embodiments, the CH1 domain is a functional fragment of the CH1 domain from a human IgG4 antibody. In certain such embodiments, the CH1 domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 23 or is a conservatively-substituted variant thereof.

[0263] In certain embodiments, the CH1 domain is a functional fragment or variant of a naturally-occurring CH1 domain that comprises a mutation such that it is capable of forming additional or fewer cross-links, for example disulfide bonds, with another polypeptide chain (an inter-chain cross-link) or with another portion of the same polypeptide chain (an intra- chain cross-link). For example, the CH1 domain may comprise additional or fewer cysteine residues as compared with the corresponding naturally-occurring CH1 domain. The cysteine residue serves as a position where a disulfide bond may form with other another cysteine in the same polypeptide chain or in a different polypeptide chain. G. The Hinge Region

[0264] The polypeptide chain may further comprise a hinge region. The hinge region may be naturally-occurring or non-natural. The hinge region may from an antibody from any species, for example human or canine, or is a functional fragment or variant of a hinge regionfrom such an antibody. In certain embodiments, the hinge region is from a human antibody, or is a functional fragment or variant of a hinge region from a human antibody.

[0265] Examples of suitable hinge regions include those disclosed in US 2007 / 0081406, the contents of which are incorporated by reference in their entirety.

[0266] In certain embodiments, the hinge region is from an IgG-B, IgA, IgD, IgE, IgG, or IgM antibody or is a functional fragment or variant thereof. In certain such embodiments, the hinge region comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the hinge region of an IgG-B, IgA, IgD, IgE, IgG, or IgM antibody, or is a conservatively-substituted variant thereof.

[0267] In certain embodiments, the hinge region is from an IgG-B antibody or is a functional fragment or variant thereof.

[0268] In certain embodiments, the hinge region comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 29 or is a conservatively-substituted variant thereof.

[0269] In certain such embodiments, the hinge region is from an IgA or IgG antibody or is a functional fragment or variant thereof.

[0270] In certain embodiments, the hinge region is from an IgA antibody or is a functional fragment or variant thereof. In certain such embodiments, the hinge region is from an IgA1 or IgA2 antibody or is a functional fragment or variant thereof. In certain such embodiments, the hinge region comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or100% sequence identity with the amino acid sequence of the hinge region from an IgA1 or IgA2 antibody, or is a conservatively-substituted variant thereof.

[0271] In certain embodiments, the hinge region is from an IgG antibody or is a functional fragment or variant thereof. In certain such embodiments, the hinge region is from an IgG1, IgG2, IgG3, or IgG4 antibody or is a functional fragment or variant thereof. In certain such embodiments, the hinge region comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the hinge region from an IgG1, IgG2, IgG3, or IgG4 antibody, or is a conservatively-substituted variant thereof.

[0272] In certain such embodiments, the hinge region is from an IgG1 or IgG4 antibody or is a functional fragment or variant thereof.

[0273] In certain embodiments, the hinge region is from an IgG1 antibody or is a functional fragment or variant thereof. In certain such embodiments, the hinge region is from a human IgG1 antibody or is a functional fragment or variant thereof.

[0274] In certain embodiments, the hinge region comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 25 or is a conservatively-substituted variant thereof.

[0275] In certain embodiments, the hinge region is from an IgG4 antibody or is a functional fragment or variant thereof. In certain such embodiments, the hinge region is from a human IgG4 antibody or is a functional fragment or variant thereof.

[0276] In certain embodiments, the hinge region comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 27 or is a conservatively-substituted variant thereof.

[0277] In certain embodiments, the hinge region is a functional fragment or variant of a naturally-occurring hinge region that comprises a mutation such that it is capable of forming additional or fewer cross-links, for example disulfide bonds, with another polypeptide chain (an inter-chain cross-link) or with another portion of the same polypeptide chain (an intra- chain cross-link). For example, the hinge region may comprise additional or fewer cysteine residues as compared with the corresponding naturally-occurring hinge region. The cysteine residue serves as a position where a disulfide bond may form with other another cysteine in the same polypeptide chain or in a different polypeptide chain.

[0278] In certain embodiments, the hinge region is a functional fragment or variant of a hinge region from a human IgG1 antibody and comprises a mutation as compared with the hinge region from wild-type human IgG1 antibody such that it is capable of forming at least one fewer cross-link, for example a disulfide bond, with another polypeptide chain or another portion of the same polypeptide chain. In certain such embodiments, the mutation is the presence of one fewer cysteine as compared with the hinge region from wild-type human IgG1 antibody.

[0279] In certain embodiments, the hinge region comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 26 or is a conservatively-substituted variant thereof, provided that the antigen comprises an arginine at the residue position corresponding to residue position 220 of SEQ ID NO: 26. III. Nucleic Acids

[0280] The present disclosure also relates to nucleic acids encoding the polypeptide chain of the present disclosure and nucleic acids encoding elements thereof: the antigen, the CH1 domain, the hinge region, the CH2 domain, the CH3 domain, the CH4 domain, and the tailpiece.

[0281] In an embodiment, the nucleic acid encodes an antigen of the present disclosure. In certain such embodiments, the nucleic acid has at least 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 37 or 38, or is a codon degenerate variant thereof.

[0282] In an embodiment, the nucleic acid encodes a CH2 domain of the present disclosure. In certain such embodiments, the nucleic acid has at least 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 39–44 or is a codon degenerate variant thereof.

[0283] In an embodiment, the nucleic acid encodes a CH3 domain of the present disclosure. In certain such embodiments, the nucleic acid has at least 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 45–50, or is a codon degenerate variant thereof.

[0284] In an embodiment, the nucleic acid encodes a tailpiece of the present disclosure. In certain such embodiments, the nucleic acid has at least 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 51, or is a codon degenerate variant thereof.

[0285] In an embodiment, the nucleic acid encodes a CH1 domain of the present disclosure. In certain such embodiments, the nucleic acid has at least 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 52–54, or is a codon degenerate variant thereof.

[0286] In an embodiment, the nucleic acid encodes a hinge region of the present disclosure. In certain such embodiments, the nucleic acid has at least 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the nucleotide sequence of any one of SEQ ID NO: 55–60, or is a codon degenerate variant thereof.

[0287] In an embodiment, the nucleic acid encodes a polypeptide chain of the present disclosure.

[0288] In certain such embodiments, the nucleic acid has at least 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 1 or 2, or a codon degenerate variant thereof.

[0289] In certain such embodiments, the nucleic acid has at least 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 3 or 4, or a codon degenerate variant thereof.

[0290] In certain such embodiments, the nucleic acid has at least 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 5 or 6, or a codon degenerate variant thereof.IV. Vectors and Delivery Systems

[0291] A nucleic acid of the present disclosure (e.g., one encoding an antigen or a polypeptide chain of the present disclosure) can be delivered to cells on a long oligonucleotide, which is then inserted into a specific genome location. In certain embodiments, the nucleic acid can be integrated into a cell’s genome through gene editing systems that utilize CRISPR, TALEN or Zinc-Finger nucleases.

[0292] The nucleic acid can be delivered to a target cell by any suitable delivery system, including non-viral and viral delivery systems. The present disclosure thus also relates in part to a vector comprising the nucleic acid encoding the polypeptide chain of the present disclosure.

[0293] Any vector known in the art for use in delivering nucleic acids may be used in the practice of the present disclosure. In certain embodiments, the vector is a plasmid, a mini- circle DNA, a nanoplasmid, a viral vector, an episomal vector, or a non-viral vector. Examples of viral vectors for use in the present disclosure include lentiviral vectors and retroviral vectors. Examples of non-viral vectors for use in the present disclosure include transposons. In certain embodiments, the vector may include sequences for serine recombinase mediated integration. Where the vector is a plasmid, mini-circle DNA, or a nanoplasmid, the plasmid, mini-circle DNA or nanoplasmid can further include a bacterial origin of replication.

[0294] An example of a non-viral vector for use in delivering a nucleic acid of the present disclosure is a lipid formulation. Any lipid formulation known in the art for delivering such nucleic acids may be used in the practice of the present disclosure. In certain embodiments, the polynucleotide can be associated with a lipid. For example, the polynucleotide may encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.

[0295] Another example of a non-viral vector is a transposon. Any transposon known in the art for delivering a nucleic acid may be used in the practice of the present disclosure.When a transposon is used to deliver a nucleic acid, a transposase or a nucleic acid encoding the same is typically also delivered to the cell. A transposase is an enzyme that binds to a transposon and catalyzes its integration into the genome of a cell. An example of a transposase for use in the present disclosure is the piggyBac transposase.

[0296] Any viral vector known in the art for delivering nucleic acids may be used in the practice of the present disclosure. Examples of such vectors include, but are not limited to, adenoviral vectors (e.g., the adenovirus-based Per.C6 system available from Crucell, Inc. (Leiden, The Netherlands)), adeno-associated virus based vectors, lentivirus-based vectors (e.g., the lentiviral-based pLPI from Life Technologies (Carlsbad, Calif.)), retroviral vectors (e.g., the pFB-ERV plus pCFB-EGSH), and herpes virus-based vectors.

[0297] In an embodiment, the viral vector is an adenoviral vector.

[0298] In an embodiment, the viral vector is a lentiviral vector. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

[0299] In order to assess the expression of the polypeptide chain described herein, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors or non-viral vectors. In other aspects, the selectable marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes can be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neomycin resistance gene (neo) and ampicillin resistance gene and the like.

[0300] Reporter genes can be used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after thenucleic acid has been introduced into the recipient cells. Suitable reporter genes include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., FEBS Letters 479: 79- 82 (2000)). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions can be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription. V. Multimeric Fusion Proteins

[0301] Two of the polypeptide chains of the present disclosure may be cross-linked with each other by way of at least one disulfide bond, thus forming a two-chain monomer unit. Such a disulfide bond typically forms between cysteine residues present in the hinge region of each polypeptide chain. Such monomer units are capable of self-assembly in a cell.

[0302] The polymer chains within a monomer unit may be identical to each other or different. In certain embodiments, a monomer unit comprises two identical polypeptide chains. In aspects, representative polypeptide chains are those described in SEQ ID NO: 1 – 6, Formula I, and Table A.

[0303] The monomer units, in turn, are capable of cross-linking with each other to form multimeric fusion proteins. In certain embodiments, the multimeric fusion protein comprises two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve monomer units. Such multimeric fusion proteins are capable of self-assembly in a cell.

[0304] In certain embodiments, the multimeric fusion protein comprises six monomer units (a “hexamer”). In certain such embodiments, the multimeric fusion protein is in the form of a cyclic hexamer.

[0305] The monomer units in a multimeric fusion protein may each be identical or different from one another. In certain embodiments, each monomer unit in a multimeric fusion protein is identical to each other.

[0306] In certain embodiments, the multimeric fusion protein comprises six identical monomers and is in the form of a cyclic hexamer.

[0307] In certain embodiments, two monomer units are connected to one another by way of at least one disulfide bond formed between a cysteine present on one polypeptide chain of the first monomer and a cysteine present on one polypeptide chain of a second monomer. In such an embodiment, a cysteine present on the other polypeptide chain of the first monomer may form a disulfide bond with a cysteine present on one polypeptide of a third monomer.

[0308] In certain such embodiments, the cysteine is present in the CH2 domain of the polypeptide chain of the first monomer and a disulfide bond is formed between such cysteine and a cysteine present in the CH2 domain of a polypeptide chain of the second monomer. In such an embodiment, a cysteine present in the CH2 domain of the other polypeptide chain of the first monomer may form a disulfide bond with a cysteine present in the CH2 domain on one polypeptide chain of a third monomer.

[0309] In certain such embodiments, the monomers are identical to one another and each comprise two polypeptide chains that are identical to one another. In such an embodiment, the cysteine present at position 309 in the CH2 domain of one polypeptide chain of the first monomer forms a disulfide bond with the cysteine present at position 309 in the CH2 domain of one polypeptide chain of the second monomer and a cysteine present at position 309 in the CH2 domain of the other polypeptide chain of the first monomer forms a disulfide bond with a cysteine present at position 309 in the CH2 domain on one polypeptide of a third monomer.

[0310] In certain such embodiments, the cysteine is present in the tailpiece of the polypeptide chain of the first monomer and a disulfide bond is formed between such cysteine and a cysteine present in the tailpiece of a polypeptide chain of the second monomer. In such an embodiment, a cysteine present in the tailpiece of the other polypeptide chain of the first monomer may form a disulfide bond with a cysteine present in the tailpiece on one polypeptide of a third monomer.

[0311] In certain such embodiments, the monomers are identical to one another and each comprises two polypeptide chains that are identical to one another. In such an embodiment, the cysteine present in the tailpiece of one polypeptide chain of the first monomer forms a disulfide bond with a cysteine present in the tailpiece of one polypeptide chain of the second monomer and a cysteine present in the tailpiece of the other polypeptide chain of the first monomer forms a disulfide bond with a cysteine present in the tailpiece in the tailpiece of one polypeptide of a third monomer.VI. Antigens

[0312] The present disclosure relates in part to an antigen that is a fragment or variant of IL-31 that is capable of binding to an IL-31 receptor. The IL-31 may be from any species. In certain embodiments, the Il-31 may be canine.

[0313] In certain embodiments, the antigen comprises a fragment or variant of canine IL- 31 that is capable of binding to canine IL-31 receptor. In certain such embodiments, the antigen comprises a fragment of variant of mature canine IL-31 that is capable of binding to canine IL-31 receptor.

[0314] In certain embodiments, the antigen comprises a fragment or variant of IL-31 that is capable of binding to an IL-31 receptor but has reduced signaling as compared with wild- type IL-31. In certain such embodiments, the antigen comprises a fragment or variant of canine IL-31 that is capable of binding to canine IL-31 receptor but has reduced signaling as compared with wild-type canine IL-31. In certain such embodiments, the antigen comprises a fragment or variant of mature canine IL-31 that is capable of binding to canine IL-31 receptor but has reduced signaling as compared with wild-type canine IL-31.

[0315] In certain embodiments, the antigen comprises a fragment or variant of IL-31 that is capable of binding to an IL-31 receptor but is not capable of inducing an itch effect. In certain such embodiments, the antigen comprises a fragment or variant of canine IL-31 that is capable of binding to canine IL-31 receptor but is not capable of inducing an itch effect. In certain such embodiments, the antigen comprises a fragment or variant of mature canine IL- 31 that is capable of binding to canine IL-31 receptor but is not capable of inducing an itch effect.

[0316] In certain embodiments, the antigen comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 8 or is a conservatively-substituted variant thereof, provided that the antigen is capable of binding to an IL-31 receptor and comprises a glutamic acid at the residue position corresponding to residue position 104 of SEQ ID NO: 8.VII. Methods for Producing the Antigen and Multimeric Fusion Proteins

[0317] The present disclosure also relates in part to a method for producing the antigen of the present disclosure comprising introducing a nucleic acid encoding the antigen to a cell. The polypeptide chains of the present disclosure are capable of assembling in the cells to form monomers which, in turn, are capable of assembling in the cells to form multimeric fusion proteins.

[0318] The present disclosure also relates in part to a method for producing the multimeric fusion protein of the present disclosure comprising introducing a nucleic acid encoding the polypeptide chain of the present disclosure to a cell.

[0319] In certain embodiments of the above methods, the method comprises transfecting a cell with a nucleic acid of the present disclosure. In certain such methods, the method comprises transfecting a cell with a vector comprising the nucleic acid.

[0320] In certain embodiments, the nucleic acid may comprise a transposon. In embodiments wherein a transposon is used, a transposase or a functional fragment or variant thereof, or a nucleic acid encoding the same, may be introduced to the cell. In certain embodiments wherein the cell is transfected with a transposon, the method further comprises transfecting the cell with a vector encoding a transposase.

[0321] In certain embodiments of the above methods, the cell is transduced with a nucleic acid of the present disclosure. The cells may be transduced with a viral vector comprising such nucleic acid.

[0322] Methods of introducing into a cell and expressing genes in a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method known in the art. For example, the vector can be transferred into a cell by physical, chemical, or biological means.

[0323] Physical methods for introducing a nucleic acid into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are known in the art. See, for example, Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (2001)). In some embodiments, amethod for the introduction of a nucleic acid into a host cell is calcium phosphate transfection or polyethylenimine (PEI) Transfection. In some embodiments, a method for introduction of a polynucleotide into a host cell is electroporation.

[0324] Chemical methods for introducing a nucleic acid into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0325] As a biological method, a nucleic acid of the present disclosure may, for example, be introduced to the cell using viral-based delivery systems. Representative viral expression vectors include, but are not limited to, the adenovirus-based vectors (e.g., the adenovirus- based Per.C6 system available from Crucell, Inc. (Leiden, The Netherlands)), adeno- associated virus based vectors, lentivirus-based vectors (e.g., the lentiviral-based pLPI from Life Technologies (Carlsbad, Calif.)), retroviral vectors (e.g., the pFB-ERV plus pCFB- EGSH), and herpes virus-based vectors. In an embodiment, the viral vector is a lentivirus vector. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity. In general, and in embodiments, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No.6,326,193).

[0326] Regardless of the method used to introduce exogenous nucleic acids into a host cell, in order to confirm the presence of the recombinant nucleic acid sequence in the host cell, a variety of assays can be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR and “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots).

[0327] The method of the present disclosure may further include growing the cell containing the nucleic acid encoding the antigen or the polypeptide chain to express the antigen or the polypeptide chain. As discussed, the polypeptide chains of the present disclosure are capable of assembling in the cells to form monomers which, in turn, are capable of assembling in the cells to form multimeric fusion proteins.

[0328] The method may further include isolating the antigen, the polypeptide chain, or a multimeric fusion protein comprising the polypeptide chain. VIII. Compositions

[0329] The present disclosure relates in part to a composition comprising an immunogenically-effective amount of the antigen of the present disclosure.

[0330] The present disclosure also relates in part to a composition comprising an immunogenically-effective amount of the multimeric fusion protein of the present disclosure.

[0331] In certain embodiments, the composition comprises a heterologous mixture of multimeric fusion proteins having differing amounts of monomers. For example, the composition may comprise multimeric fusion proteins having six monomers and multimeric fusion proteins comprising twelve monomers.

[0332] In certain embodiments, at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% of the multimeric fusion proteins present in such a composition are in hexameric form.

[0333] The present disclosure also relates in part to a composition as described above for use in producing an immunogenic effect in a subject. In certain embodiments, the subject is canine.

[0334] In certain embodiments, the composition further comprises a carrier, a diluents, and / or an excipient. Any carrier, diluent, or excipient known in the art for use with a polypeptide is contemplated for use in the practice of the present disclosure. For example, compositions of the present disclosure may comprise: buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose, dextrans, or mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0335] In certain embodiments, the kit comprises a carrier, package, label, or container. Suitable containers include, for example, bottles, vials, syringes, and test tubes. Adjuvants

[0336] Fusion protein chains, polypeptide chains, multimers, oligomers, or hexamers described herein can further be mixed with one or more adjuvants. In aspects, the adjuvants may be mixed into a composition or added separately from the fusion protein chains, oligomers, or hexamers described herein.

[0337] Representative adjuvants include oil-based adjuvants, such as Freund's Complete Adjuvant and Freund's Incomplete Adjuvant, Amphigen, and glycerine. Other adjuvants such as emulsion adjuvants: MF59, AS03, MetaStim® (squalene, Pluronic), Montanide ISA (ISA 61), Montanide IMS (IMS 3012 VG) are provided herein. Acrylic copolymers such as Neocryl®A640 (Covestro) are provided. Aluminum adjuvants including aluminum hydroxide and aluminum phosphate are also provided. Particulate adjuvants extracted from the bark of Quillaja Saponaria: containing the active ingredient QS-21 (Cambridge Biotech Inc., Cambridge Mass.), and Quil-A®(which is the saponin adjuvant), chitosan; Q-VET-S (Q-Vant Biosciences, Forestdale Mass.). Immunostimulating complexes (ISCOMs): classic and ISCOM-Matrix, AbISCO-200® AbISCO-300® are also provided. Other particulate adjuvants: AS01. TLR4 agonists AS04 and MPLA. Synthetic DNA TLR9 agonists: CpG ODN 1018, CpG ODN 7909, 1668-PTO, 2006-PTO, and IC31 are further provided. Imidazoquinolines that stimulate TLR 7 / 8: Imiquimod (R837), Resiquimod, and 3M-052. Cyclic dinucleotides that can stimulate the cGAS-STING pathway: 2’,3’-cGAMP, 3’,3’- cGAMP, c-di-GMP, c-di-AMP. Mucoadhesive polyacrylic acid polymers: CARBOPOL®934 and CARBOPOL®941 NF, Carbopol® 971, Montanide ™ PET Gel A and vaccine grade Poly (I:C) (VacciGrade; Invivogen) are also provided.

[0338] Mucoadhesive polyacrylic acid (PAA) polymers polyacrylates include linear polyacrylic acid of 100 and 450 kDa (PAA 100 and 450), with allyl pentaerythritol, allyl sucrose and divinyl glycol cross-linked PAAs (PAA AP, PAA AS / AP, PAA DG) as well as hydrophobically modified cross-linked PAAs (PAA C10-30). Crosspolymers of acrylates / C10-30 alkyl acrylate with low (PAA C10-30low) and medium (PAA C10-30medium) degree of C10-30 alkylation exhibit high mucoadhesive properties with the additional benefit of emulsifying properties.

[0339] Additional adjuvants for use may include, for example, poly(di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus Research Institute, USA); derivatives of lipopolysaccharides such as monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton, Mont.), muramyl dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland); and Leishmania elongation factor (a purified Leishmania protein; Corixa Corporation, Seattle, Wash.). Manganese and derivatives that stimulate the cGAS-STING pathway Mn2+, nanoscale manganese jelly (MnJ), MnARK, and nanoMn. Some adjuvants include surface active substances (e.g. lysolecithin, pluronic polyols, polyanions, peptides, nanoemulsions, keyhole limpet hemocyanin, and dinitrophenol).

[0340] In aspects, adjuvants may be added to a composition comprising a nanoemulsion adjuvant and an immunogen, or, the adjuvant may be formulated with carriers, for example liposomes, or metallic salts (e.g., aluminium salts) prior to combining with or co- administration with a composition comprising a nanoemulsion adjuvant and an immunogen. In some embodiments, an immunogenic composition comprising a nanoemulsion adjuvant and an immunogen comprises a single additional immunostimulatory compound / molecule and / or adjuvant. In other embodiments, an immunogenic composition comprising a nanoemulsion adjuvant and an immunogen comprises two or more additional immunostimulatory compounds / molecules and / or adjuvants. Additional immunogenic compositions with mucoadhesives include: Cross-linked derivatives of poly(acrylic acid) (e.g., Carbopol and polycarbophil); Carbopol® (Lubrizol), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides (e.g., alginate and chitosan), hydroxypropyl methylcellulose, lectins, fimbrial proteins, and carboxymethylcellulose. Additional representative adjuvants may be found in U.S.11,173,207, the content of which is incorporated by reference in its entirety.

[0341] In some embodiments, adjuvants such as cytokines (e.g., interleukins (e.g., IL-2, IFN-γ, IL-4, etc.), macrophage colony stimulating factor, tumor necrosis factor, etc.), detoxified mutants of a bacterial ADP-ribosylating toxin such as a cholera toxin (CT), a pertussis toxin (PT), or an^E. Coli^heat-labile toxin (LT), particularly LT-K63 (where lysine is substituted for the wild-type amino acid at position 63) LT-R72 (where arginine is substituted for the wild-type amino acid at position 72), CT-S109 (where serine is substituted for thewild-type amino acid at position 109), and PT-K9 / G129 (where lysine is substituted for the wild-type amino acid at position 9 and glycine substituted at position 129) (See, e.g., WO93 / 13202 and WO92 / 19265, the contents which are incorporated by reference in their entireties), and / or other immunogenic compounds / substances (e.g., that stimulate an immune response) are used in or in combination with an immunogenic composition comprising an adjuvant formulation comprising an emulsion in combination with one or more immunostimulatory compounds of the present disclosure.

[0342] In some embodiments, the amount of Quil-A®present is about 5 μg, about 10 μg, about 25μg, about 50μg, about 75μg, about 100μg, or about 200 μg in compositions, fusion protein compositions, or IL-31 hexamer compositions described herein. In other embodiments, the amount of Quil-A®is present is about from 5 μg – 200 μg, from 5 μg – 100 μg, about 10 μg to about 100 μg, about 10 μg to about 80 μg, about 20 μg to about 60 μg, about 25 μg to about 100 μg, about 30 μg to about 50 μg, about 25 μg, about 50 μg, about 75 μg, about 100 μg in compositions, fusion protein compositions, or IL-31 hexamer compositions described herein.

[0343] In some embodiments, the concentration of Carbopol, for example, Carbopol®971, is about 0.005%, about 0.01%, about 0.02%, about 0.05%, about 0.075%, about 0.1%, about 0.125%, about 0.15% or about 0.2% in compositions, fusion protein compositions, or IL-31 hexamer compositions described herein. In another embodiments, the concentration of Carbopol, for example, Carbopol®971, is about 0.005% to about 0.2%, about 0.01% to about 0.1%, 0.02%, to about 0.1%, or about 0.1 to about 0.05% in compositions, fusion protein compositions, or IL-31 hexamer compositions described herein.

[0344] In some embodiments, compositions comprising immunizing IL-31 hexamer proteins including SEQ ID NOs: 1-6 described herein and adjuvants including Quil A®, Carbopol®971, EMA / Neocryl®A640, 1668-PTO, and 2006-PTO described herein exhibit an increase of antibodies that block canine IL-31 from binding to the IL-31 receptor in dogs relative to IL-31 hexamer proteins alone (without adjuvants) in the amount of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 40%, at least about 60%, at least about 80%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, or at least about 500% or more.

[0345] In some embodiments, compositions comprising one of SEQ ID NO: 1 – 6 with adjuvants selected from Quil A®, Carbopol®971, EMA / Neocryl®A640, 1668-PTO, or 2006- PTO exhibit an increase of canine IL-31 antibodies which block canine IL-31 from binding the IL-31 receptor proteins in dogs relative to compositions comprising one of SEQ ID NO: 1 – 6 alone (without adjuvants) in the amount of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 40%, at least about 60%, at least about 80%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, or at least about 500% or more.

[0346] In some embodiments, compositions comprising SEQ ID NO: 2 with an adjuvant from Quil A®, Carbopol®971, EMA / Neocryl®A640, 1668-PTO, or 2006-PTO exhibit an increase of canine IL-31 antibodies which block canine IL-31 from binding the IL-31 receptor proteins in dogs relative to compositions comprising SEQ ID NO: 2 alone (without adjuvants) in the amount of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 40%, at least about 60%, at least about 80%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, or at least about 500% or more.

[0347] In aspects, fusion proteins or hexameric proteins comprising units of SEQ ID NO: 2 with adjuvant Quil A®, Carbopol®971, EMA / Neocryl®A640, 1668-PTO, or 2006-PTO exhibit an increase of canine IL-31 inactivating antibodies in dogs relative to compositions comprising SEQ ID NO: 2 alone (without adjuvants) in the amount of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 40%, at least about 60%, at least about 80%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, or at least about 500% or more.

[0348] In other aspects, compositions described herein may include fusion proteins, polypeptide chains, oligomers, multimers, and hexamers described herein in an amount of from about 50 µg to about 1000 µg / dose, from about 100 µg to about 500 µg / dose, from about 100 µg to about 300 µg / dose, about 100 µg / dose, about 200 µg / dose, about 300 µg / dose, about 500 µg / dose, or more than 600 µg / dose.

[0349] In further aspects, compositions described herein may include an adjuvant in an amount of from about 50 µg to 1000 µg / dose, 50 µg to 150 µg / dose from about 100 µg to 500 µg / dose, from about 100 µg to 300 µg / dose, about 100 µg / dose, about 200 µg / dose, about300 µg / dose, about 500 µg / dose, or more than 600 µg / dose and fusion proteins, oligomers, multimers, and hexamers described herein in an amount of from about 50 µg to 1000 µg / dose, from about 100 µg to 500 µg / dose, from about 100 µg to 300 µg / dose, from about 100 µg to 400 µg / dose, about 100 µg / dose, about 200 µg / dose, about 300 µg / dose, about 500 µg / dose, or more than 600 µg / dose. IX. Stability of Compositions

[0350] In some embodiments, compositions described herein exhibit stability over a period for a period of time of at least 1 day, at least about 2 days, at least about 5 days, at least about 10 days, at least about 25 days, at least about 50 days, at least about 100 days, at least about 150 days, at least about 200 days, at least about 250 days, at least about 300 days, at least about 365 days, at least about 500 days, or at least about 1000 days.

[0351] In some embodiments, compositions described herein and an adjuvant described herein, such as Quil A®, Carbopol®971, EMA / Neocryl®A640, 1668-PTO, and / or 2006- PTO, exhibit stability such that the compositions can be used as an effective pharmaceutical vaccine from 1ºC to 30 ºC. In some embodiments, the composition of IL-31 IgG hexamers and adjuvant is stable at a temperature from 1 ºC to 2 ºC, 1 ºC to 4 ºC, 1 ºC to 8 ºC, 1 ºC to 16 ºC, 1 ºC to 2 ºC, 1 ºC to 25 ºC, 1 ºC to 30 ºC, 2 ºC to 4 ºC, 2 ºC to 8 ºC, 2 ºC to 16 ºC, 2 ºC to 25 ºC, 2 ºC to 30 ºC, 4 ºC to 8 ºC, 4 ºC to 16 ºC, 4 ºC to 2 ºC, 4 ºC to 25 ºC, 4 ºC to 30 ºC, 8 ºC to 16 ºC, 8 ºC to 25 ºC, 8 ºC to 30 ºC, 16 ºC to 25 ºC, 16 ºC to 30 ºC, 2 ºC to 25 ºC, 2 ºC to 30 ºC, or 25 ºC to 30 ºC. In some embodiments, the composition of IL-31 IgG hexamers and adjuvant is stable at 1 ºC, 2 ºC, 4 ºC, 8 ºC, 16 ºC, 2 ºC, 25 ºC, or 30 ºC.

[0352] In some embodiments, compositions described are stable at 5°C for a period of time of at least 1 day, at least about 2 days, at least about 5 days, at least about 10 days, at least about 25 days, at least about 50 days, at least about 100 days, at least about 150 days, at least about 200 days, at least about 250 days, at least about 300 days, at least about 365 days, at least about 500 days, or at least about 1000 days.

[0353] In some embodiments, compositions described are stable at 5°C for a period of time of at least 1 day, at least about 2 days, at least about 5 days, at least about 10 days, at least about 25 days, at least about 50 days, at least about 100 days, at least about 150 days, at least about 200 days, at least about 250 days, at least about 300 days, at least about 365 days, at least about 500 days, or at least about 1000 days.

[0354] In some embodiments, compositions described are stable at 40°C for a period of time of at least 1 day, at least about 2 days, at least about 5 days, at least about 10 days, at least about 25 days, at least about 50 days, at least about 100 days, at least about 150 days, at least about 200 days, at least about 250 days, at least about 300 days, at least about 365 days, at least about 500 days, or at least about 1000 days.

[0355] In some embodiments, compositions described herein are stable at 5° C from 1 to 365 days in a buffer selected from Buffer1 (20 mM phosphate, 50 mM arginine HCl, 150 mM sucrose, 10 mM Methionine, 0.02% Polaxamer 188, pH 7.5), buffer1 plus excipients or adjuvants, phosphate (pH 7.5), citrate-phosphate (pH7.5), phosphate (pH 6.0), and citrate- phosphate (pH 6.0).

[0356] In other embodiments, compositions described herein comprise the following combination of compounds and associated properties: (a) a composition comprising one of SEQ ID NO: 1, 2, 3, 4, 5, or 6; and (b) stability at a temperature between 2°C and 8°C for a period of time of at least 1 day, at least about 2 days, at least about 5 days, at least about 10 days, at least about 4 weeks, at least about 50 days, at least about 100 days, at least about 150 days, at least 24 weeks, at least 26 weeks, at least about 200 days, at least about 250 days, at least about 300 days, at least about 365 days, at least about 500 days, or at least about 1000 days.

[0357] In other embodiments, compositions described herein comprise the following combination of compounds and associated properties: (a) a composition comprising SEQ ID NO: 2; and (b) stability at a temperature between 2°C and 8°C for a period of time of at least 1 day, at least about 2 days, at least about 5 days, at least about 10 days, at least about 4 weeks, at least about 50 days, at least about 100 days, at least about 150 days, at least 24 weeks, at least 26 weeks, at least about 200 days, at least about 250 days, at least about 300 days, at least about 365 days, at least about 500 days, or at least about 1000 days.

[0358] In other embodiments, compositions described herein comprise the following combination of compounds and associated properties: (a) polypeptide chains comprising SEQ ID NO: 2, wherein the polypeptide chains are in the form of a hexameric protein (for example, see Fig.1); and(b) stability at a temperature between 2°C and 8°C for a period of time of at least 1 day, at least about 2 days, at least about 5 days, at least about 10 days, at least about 4 weeks, at least about 50 days, at least about 100 days, at least about 150 days, at least 24 weeks, at least 26 weeks, at least about 200 days, at least about 250 days, at least about 300 days, at least about 365 days, at least about 500 days, or at least about 1000 days.

[0359] In other embodiments, compositions described herein comprise the following combination of compounds and associated properties: (a) a hexameric protein comprising SEQ ID NO: 2; (b) adjuvants selected from Quil A®, Carbopol®971, EMA / Neocryl®A640, 1668-PTO, or 2006-PTO; and (c) stability at a temperature between 2°C and 8°C for a period of time of at least 1 day, at least about 2 days, at least about 5 days, at least about 10 days, at least about 4 weeks, at least about 50 days, at least about 100 days, at least about 150 days, at least 24 weeks, at least 26 weeks, at least about 200 days, at least about 250 days, at least about 300 days, at least about 365 days, at least about 500 days, or at least about 1000 days. X. Methods of Treatment

[0360] The present disclosure also relates to a method of inducing an immunological response in a subject in need thereof, the method comprising administering the aforementioned antigen, multimeric fusion protein, or a composition comprising the antigen or multimeric fusion protein to the subject. Administration may be in a immunogenically- effective amount. In certain embodiments, the subject is a mammal, for example a canine.

[0361] The present disclosure also relates to a method of treating a disease or disorder in a subject in need thereof, the method comprising administering the aforementioned antigen, multimeric fusion protein, or a composition comprising the antigen or multimeric fusion protein to the subject. Administration may be in a therapeutically-effective amount. In certain embodiments, the subject is a mammal, for example a canine. In an aspect, the antigen, multimeric fusion protein, or composition are administered to an animal in need thereof, for example, a canine, from about 50 µg to 1000 µg / dose, from about 100 µg to 500 µg / dose, from about 100 µg to 300 µg / dose, about 100 µg / dose, about 200 µg / dose, about 300 µg / dose, about 500 µg / dose, or more than 600 µg / dose.

[0362] The present disclosure also relates to the use of antigens or multimeric fusion proteins of the present disclosure, or a nucleic acid encoding the antigen or a polypeptide chain of the present disclosure, in the manufacture of a medicament for the treatment of a disease or disorder in a subject. In an aspect, the antigen or multimeric fusion proteins described herein are administered to an animal in need thereof, for example, a canine, from about 50 µg to 1000 µg / dose, from about 100 µg to 500 µg / dose, from about 100 µg to 300 µg / dose, about 100 µg / dose, about 200 µg / dose, about 300 µg / dose, about 500 µg / dose, or more than 600 µg / dose.

[0363] In certain embodiments, the disease or disorder is a disease or disorder for which an immunogenic effect produced from exposure to the multimeric fusion protein would have a therapeutic effect.

[0364] In certain such embodiments, the disease or disorder is an itch-related disorder, for example atopic dermatitis.

[0365] In certain embodiments, the method involves the administration of a composition described herein. Such methods may be carried out in any manner known in the art, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The composition described herein can be administered to a subject subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.

[0366] In certain embodiments, the composition is administered to a particular site on a body, for example the interscapular region.

[0367] The dosage of the above treatments to be administered to a subject will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for canine administration can be performed according to art-accepted practices.

[0368] In certain embodiments, the immunological response lasts for at least two weeks, one month, at least two months, at least three months, at least 6 months, at least one year, or at least 2 years or more.

[0369] It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that, for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

[0370] In some embodiments, the compositions described herein can be administered as a combination therapy with an additional therapeutic agent. Examples of such agents include biologic agents and small molecules. XI. Immune Response to IL-31 and Reduction In Pruritic Response In Dogs.

[0371] In some embodiments, administration of IL-31 hexamers results in the breaking of self-tolerance to canine IL-31 in dogs. In some embodiments, administration of hexamers results in autoimmunity and the formation of antibodies to canine IL-31 in dogs. In some embodiments, administration of hexamers results in the breaking of self-tolerance to canine IL-31 in dogs. In some embodiments, the administration of IL-31 hexamers results in autoimmunity and the formation of antibodies to canine IL-31 in dogs wherein administration of canine IL-31 does not result in autoimmunity and the formation of antibodies to canine IL- 31 in dogs. In some embodiments, the administration of IL-31 hexamers results in the breaking of self-tolerance in to canine IL-31 in dogs wherein administration of canine IL-31 does not result in the breaking of self-tolerance to IL-31 in dogs.

[0372] In some embodiments, the canine IL-31 hexamer that result in autoimmunity and the formation of antibodies to IL-31 comprises peptide chains of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In some embodiments, the canine IL-31 hexamer that result in autoimmunity and the formation of antibodies to IL-31 is SEQ ID NO: 2. In some embodiments, the canine IL-31 is selected from SEQ ID NO: 7 or SEQ ID NO: 8.

[0373] In some embodiments, administration of canine IL-31 hexamers to dogs results in autoimmunity to canine IL-31 sufficient to inactivate physiological levels of canine IL-31 from binding to the IL-31 receptor lasting a period selected from at least 1 day, at least 2 days, at least 7 days, at least 14 days, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 9 months, at least 1 year, at least 2 years, at least 5 years, or at least 10years. In some embodiments, administration of canine IL-31 hexamers results in a reduction in pruritic responses in dogs lasting a period selected from at least 1 day, at least 2 days, at least 7 days, at least 14 days, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 9 months, at least 1 year, at least 2 years, 5 years, or at least 10 years.

[0374] In some embodiments, additional doses or boosters of IL-31 hexamers results in higher anti IL-31 antibody titer relative to the initial course of immunization. In some embodiments, additional doses or boosters of IL-31 hexamers described herein results in a longer lasting reduction in pruritic response in dogs relative to the initial course of immunization. In some embodiments the number of boosters administered are selected from one, two, three, four, and five booster doses of IL-31 hexamers. In some embodiments, the administration of a booster dose of IL-31 hexamers described herein results in an additional immunity to canine IL-31 relative to dogs receiving no booster selected from at least 1 month, at least two months, at least 3 months, at least 6 months, at least 9 months, at least 1 year, at least 2 years, at least 5 years and at least 10 years. In some embodiments, the administration of a booster dose of IL-31 hexamers described herein results in a longer lasting reduction in pruritic response of at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.5 times, at least 1.8 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, and at least 20 times as long as Lokivetmab (SEQ ID NO: 64, heavy chain; SEQ ID NO: 65: light chain).

[0375] In some embodiments, the administration of IL-31 hexamers described herein results in an antibody titer to canine IL-31 relative to Lokivetmab selected from at least 0.5 fold, at least 0.75 fold, at least equal to, at least 1.1 fold, at least 1.25 fold, at least 1.5 fold, at least 1.75 fold, at least 2 fold, at least 4 fold, at least 8 fold, or at least 10 fold. In some embodiments, the administration of IL-31 hexamers described herein results in an antibody titer to canine IL-31 relative to Lokivetmab at 64 days after administration selected from 0.5 fold, 0.75 fold, 1.1 fold, 1.25 fold, 1.5 fold, 1.75 fold, 2 fold, 4 fold, 8 fold, or 10 fold. In some embodiments, the administration of IL-31 hexamers results in an antibody titer to canine IL-31 relative to Lokivetmab at 85 days after administration selected from at least 0.5 fold, at least 0.75 fold, at least 1.1 fold, at least 1.25 fold, at least 1.5 fold, at least 1.75 fold, at least 2 fold, at least 4 fold, at least 8 fold, or at least 10 fold. In some embodiments, the administration of IL-31 hexamers described herein results in an antibody titer to canine IL-31 relative to Lokivetmab at 134 days after administration selected from at least 0.5 fold, at least0.75 fold, at least 1.1 fold, at least 1.25 fold, at least 1.5 fold, at least 1.75 fold, at least 2 fold, at least 4 fold, at least 8 fold, or at least 10 fold. In some embodiments, the administration of IL-31 hexamers described herein results in an antibody titer to canine IL-31 relative to Lokivetmab at 183 days after administration selected from at least 0.5 fold, at least 0.75 fold, at least 1.1 fold, at least 1.25 fold, at least 1.5 fold, at least 1.75 fold, at least 2 fold, at least 4 fold, 8 fold, or at least 10 fold. In some embodiments, the administration of IL-31 hexamers described herein results in an antibody titer to canine IL-31 relative to Lokivetmab at 204 days after administration selected from at least 0.5 fold, at least 0.75 fold, at least 1.1 fold, at least 1.25 fold, at least 1.5 fold, at least 1.75 fold, at least 2 fold, at least 4 fold, at least 8 fold, or at least 10 fold.

[0376] In some embodiments, the administration of IL-31 hexamers described herein results in an antibody titer to canine IL-31 relative to administering canine IL-31 selected from 0.5 fold, 0.75 fold, 1.1 fold, 1.25 fold, 1.5 fold, 1.75 fold, 2 fold, 4 fold, 8 fold, 10 fold, 20 fold, 50 fold, or 100 fold. In some embodiments, the administration of IL-31 hexamers described herein results in an antibody titer to canine IL-31 relative to administering canine IL-31 at 64 days after administration selected from of 0.5 fold, 0.75 fold, 1.1 fold, 1.25 fold, 1.5 fold, 1.75 fold, 2 fold, 4 fold, 8 fold, 10 fold, 20 fold, 50 fold, or 100 fold. In some embodiments, the administration of IL-31 hexamers described herein results in an antibody titer to canine IL-31 relative to administering canine IL-31 at 85 days after administration selected from 0.5 fold, 0.75 fold, 1.1 fold, 1.25 fold, 1.5 fold, 1.75 fold, 2 fold, 4 fold, 8 fold, 10 fold, 20 fold, 50 fold, or 100 fold. In some embodiments, the administration of IL-31 hexamers described herein results in an antibody titer to canine IL-31 relative to administering canine IL-31 at 134 days after administration selected from 0.5 fold, 0.75 fold, 1.1 fold, 1.25 fold, 1.5 fold, 1.75 fold, 2 fold, 4 fold, 8 fold, 10 fold, 20 fold, 50 fold ,and 100 fold. In some embodiments, the administration of IL-31 hexamers described herein results in an antibody titer to canine IL-31 relative to administering canine IL-31 at 183 days after administration selected from 0.5 fold, 0.75 fold, 1.1 fold, 1.25 fold, 1.5 fold, 1.75 fold, 2 fold, 4 fold, 8 fold, 10 fold, 20 fold, 50 fold, or 100 fold. In some embodiments, the administration of IL-31 hexamers described herein results in an antibody titer to canine IL-31 relative to administering canine IL-31 at 204 days after administration selected from 0.5 fold, 0.75 fold, 1.1 fold, 1.25 fold, 1.5 fold, 1.75 fold, 2 fold, 4 fold, 8 fold, 10 fold, 20 fold, 50 fold, or 100 fold.

[0377] In some embodiments administration of canine IL-31 hexamers described herein results in the production of IL-31 antibodies that block IL-31 from binding to the IL31 receptor. In some embodiments, administration of canine IL-31 hexamers results in the production of IL-31 antibodies that block IL-31 from binding to an IL-31 receptor alpha (IL- 31RA). In some embodiments, administration of canine IL-31 hexamers described herein results in the production of IL-31 inactivating antibodies that block IL-31 from binding to an oncostatin M receptor beta (OSMR). In some embodiments, administration of canine IL-31 hexamers described herein results in the production of IL-31 antibodies that block IL-31 from binding to both IL-31RA and OSMR.

[0378] In some embodiments, the antibodies elicited by administration of IL-31 hexamers described herein results in inactivation of physiological canine IL-31 for binding to IL-31RA or OSMR or the complex of IL-31RA and OSMR at a percentage selected from at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, and at least 99%.

[0379] In some embodiments, the administration of IL-31 hexamers described herein results in a reduction in a pruritic response in dogs as a result of IL-31 challenge. In some embodiments, the administration of IL-31 hexamers described herein results in a reduction in a pruritic response in dogs as a result of immune sensitivity. In some embodiments, the administration of IL-31 hexamers described herein results in a reduction in a pruritic response in dogs as a result of an allergic response. In some embodiments, the administration of IL-31 hexamers described herein results in a reduction in a pruritic response in dogs as a result of an allergic response to an allergen. In some embodiments, the administration of IL-31 hexamers described herein results in a reduction of a pruritic response in dogs as a result of IL-31 challenge wherein the reduction in the pruritic response relative to untreated dogs is selected from at least a 5% reduction, at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, a 40% reduction, a 45% reduction, a 50% reduction, a 55% reduction, a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least an 80% reduction, at least an 85% reduction, at least a 90% reduction, or at least a 99% reduction.

[0380] In some embodiments, the white blood cell count will be a number selected from 4 k per μl, 4.5 k per μl, 4.9 k per μl, 5 k per μl, 6 k per μl, 7 k per μl, 8 k per μl, 9 k per μl, 10k per μl, 11 k per μl, 12k per μl, 13k per μl, 14k per μl, 15k per μl,16 k per μl, 17k per μl, 17.9k per μl, 18k per μl, 19k per μl, or 20k per μl. In some embodiments, the number of white blood cells in dogs treated with IL-31 hexamers are higher than dogs not treated with IL-31 hexamers. In some embodiments, the number of white blood cells in dogs treated with IL-31 hexamers plus adjuvants are higher than dogs not treated with IL-31 hexamers.

[0381] In some embodiments, the administration of IL-31 hexamers described herein results in efficacy of reducing the pruritic effect and stable immunity selected from 50% efficacy and 12 months of duration, 60% efficacy and 12 months of duration, 70% efficacy and 12 months of duration, 80% efficacy and 12 months of duration, 90% efficacy and 12 months of duration, 95% efficacy and 12 months of duration, 95% efficacy and 8 months of duration, 95% efficacy and 9 months of duration, 95% efficacy and 10 months of duration, 95% efficacy and 11 months of duration, 95% efficacy and 13 months of duration, 95% efficacy and 14 months of duration, 95% efficacy and 15 months of duration, 95% efficacy and 16 months of duration, 90% efficacy and 8 months of duration, 90% efficacy and 9 months of duration, 90% efficacy and 10 months of duration, 90% efficacy and 11 months of duration, 90% efficacy and 13 months of duration, 90% efficacy and 14 months of duration, 90% efficacy and 15 months of duration, 90% efficacy and 16 months of duration, 80% efficacy and 8 months of duration, 80% efficacy and 9 months of duration, 80% efficacy and 10 months of duration, 80% efficacy and 11 months of duration, 80% efficacy and 13 months of duration, 80% efficacy and 14 months of duration, 80% efficacy and 15 months of duration, 80% efficacy and 16 months of duration, 70% efficacy and 8 months of duration, 70% efficacy and 9 months of duration, 70% efficacy and 10 months of duration, 70% efficacy and 11 months of duration, 70% efficacy and 13 months of duration, 70% efficacy and 14 months of duration, 70% efficacy and 15 months of duration, 70% efficacy and 16 months of duration, 60% efficacy and 8 months of duration, 60% efficacy and 9 months of duration, 60% efficacy and 10 months of duration, 60% efficacy and 11 months of duration, 60% efficacy and 13 months of duration, 60% efficacy and 14 months of duration, 60% efficacy and 15 months of duration, 60% efficacy and 16 months of duration, 50% efficacy and 8 months of duration, 50% efficacy and 9 months of duration, 50% efficacy and 10 months of duration, 50% efficacy and 11 months of duration, 50% efficacy and 13 months ofduration, 50% efficacy and 14 months of duration, 50% efficacy and 15 months of duration, and 50% efficacy and 16 months of duration.

[0382] In some embodiments, the administration of IL-31 hexamers described herein results in an IL-31 inhibiting titer and stable immunity selected from 50% IL-31 inhibiting titer and 12 months of duration, 60% IL-31 inhibiting titer and 12 months of duration, 70% IL-31 inhibiting titer and 12 months of duration, 80% IL-31 inhibiting titer and 12 months of duration, 90% IL-31 inhibiting titer and 12 months of duration, 95% IL-31 inhibiting titer and 12 months of duration, 95% IL-31 inhibiting titer and 8 months of duration, 95% IL-31 inhibiting titer and 9 months of duration, 95% IL-31 inhibiting titer and 10 months of duration, 95% IL-31 inhibiting titer and 11 months of duration, 95% IL-31 inhibiting titer and 13 months of duration, 95% IL-31 inhibiting titer and 14 months of duration, 95% IL-31 inhibiting titer and 15 months of duration, 95% IL-31 inhibiting titer and 16 months of duration, 90% IL-31 inhibiting titer and 8 months of duration, 90% IL-31 inhibiting titer and 9 months of duration, 90% IL-31 inhibiting titer and 10 months of duration, 90% IL-31 inhibiting titer and 11 months of duration, 90% IL-31 inhibiting titer and 13 months of duration, 90% IL-31 inhibiting titer and 14 months of duration, 90% IL-31 inhibiting titer and 15 months of duration, 90% IL-31 inhibiting titer and 16 months of duration, 80% IL-31 inhibiting titer and 8 months of duration, 80% IL-31 inhibiting titer and 9 months of duration, 80% IL-31 inhibiting titer and 10 months of duration, 80% IL-31 inhibiting titer and 11 months of duration, 80% IL-31 inhibiting titer and 13 months of duration, 80% IL-31 inhibiting titer and 14 months of duration, 80% IL-31 inhibiting titer and 15 months of duration, 80% IL-31 inhibiting titer and 16 months of duration, 70% IL-31 inhibiting titer and 8 months of duration, 70% IL-31 inhibiting titer and 9 months of duration, 70% IL-31 inhibiting titer and 10 months of duration, 70% IL-31 inhibiting titer and 11 months of duration, 70% IL-31 inhibiting titer and 13 months of duration, 70% IL-31 inhibiting titer and 14 months of duration, 70% IL-31 inhibiting titer and 15 months of duration, 70% IL-31 inhibiting titer and 16 months of duration, 60% IL-31 inhibiting titer and 8 months of duration, 60% IL-31 inhibiting titer and 9 months of duration, 60% IL-31 inhibiting titer and 10 months of duration, 60% IL-31 inhibiting titer and 11 months of duration, 60% IL-31 inhibiting titer and 13 months of duration, 60% IL-31 inhibiting titer and 14 months of duration, 60% IL-31 inhibiting titer and 15 months of duration, 60% IL-31 inhibiting titer and 16 months of duration, 50% IL-31 inhibiting titer and 8 months of duration, 50% IL-31 inhibiting titer and 9 months of duration, 50% IL-31 inhibiting titer and 10 months ofduration, 50% IL-31 inhibiting titer and 11 months of duration, 50% IL-31 inhibiting titer and 13 months of duration, 50% IL-31 inhibiting titer and 14 months of duration, 50% IL-31 inhibiting titer and 15 months of duration, and 50% IL-31 inhibiting titer and 16 months of duration.

[0383] In other embodiments, compositions described herein comprise: (a) a hexameric protein comprising SEQ ID NO: 2, adjuvants selected from Quil A®, 0.1% Carbopol®971, 0.02% Carbopol®971,EMA / Neocryl®A640, 1668-PTO, or 2006-PTO; (b) stability at 2-8°C for a period of time of at least 1 day, at least about 2 days, at least about 5 days, at least about 10 days, at least about 25 days, at least about 50 days, at least about 100 days, at least about 150 days, at least about 200 days, at least about 250 days, at least about 300 days, at least about 365 days, at least about 500 days, or at least about 1000 days; (c) reduction in pruritic response of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, 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%, or a at least 99% reduction; and / or (d) reduction in pruritic response of at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.5 times, at least 1.8 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, and at least 20 times as long as Lokivetmab (SEQ ID NO: 64, heavy chain; SEQ ID NO: 65: light chain).

[0384] In other embodiments, compositions described herein comprise: (a) a hexameric protein comprising SEQ ID NO: 2 and adjuvant100 ug Quil®A; (b) stability at 2-8°C for a period of time of at least 24 weeks or at least 52 weeks; (c) reduction in pruritic score of at least 50% relative to PBS / placebo treatment; and / or (d) reduction in pruritic score of at least as long as Lokivetmab (SEQ ID NO: 64, heavy chain; SEQ ID NO: 65: light chain).

[0385] In other embodiments, compositions described herein comprise: (a) a hexameric protein comprising SEQ ID NO: 2, and adjuvant 0.1% Carbopol®; (b) stability at 2-8°C for a period of time of at least 24 weeks or at least 52 weeks; (c) reduction in pruritic score of 50% relative to PBS / placebo treatment; and / or(d) reduction in pruritic score of at least as long as Lokivetmab (SEQ ID NO: 64, heavy chain; SEQ ID NO: 65: light chain).

[0386] In other embodiments, compositions described herein comprise: (a) a hexameric protein comprising SEQ ID NO: 2, and adjuvant 0.02% Carbopol®; (b) stability at 2-8°C for a period of time of at least 24 weeks or at least 52 weeks; (c) reduction in pruritic score of 50% relative to PBS / placebo treatment; and / or (d) reduction in pruritic score of at least as long as Lokivetmab (SEQ ID NO: 64, heavy chain; SEQ ID NO: 65: light chain).

[0387] In other embodiments, compositions described herein comprise: (a) a hexameric protein comprising SEQ ID NO: 2, and adjuvant 0.02% Carbopol®plus 1668-PTO, and 2006-PTO; (b) stability at 2-8°C for a period of time of at least 24 weeks or at least 52 weeks; (c) reduction in pruritic score of 50% relative to PBS / placebo treatment; and / or (d) reduction in pruritic score of at least as long as Lokivetmab (SEQ ID NO: 64, heavy chain; SEQ ID NO: 65: light chain).

[0388] In other embodiments, compositions described herein comprise: (a) a hexameric protein comprising SEQ ID NO: 2, and adjuvant EMA / Neocryl®A640; (b) stability at 2-8°C for a period of time of at least 24 weeks or at least 52 weeks; (c) reduction in pruritic score of 50% relative to PBS / placebo treatment; and / or (d) reduction in pruritic score of at least as long as Lokivetmab (SEQ ID NO: 64, heavy chain; SEQ ID NO: 65: light chain).

[0389] In other embodiments, compositions described herein comprise: (a) a hexameric protein comprising SEQ ID NO: 2 and 0.1% Carbopol®; (b) stability at 2-8°C for a period of time of at least 26 weeks, at least about 200 days, at least about 250 days, at least about 300 days, at least about 365 days, at least about 500 days, or at least about 1000 days; (c) reduction in pruritic response of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, or a 99% reduction; and / or (d) a reduction in pruritic response of at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.5 times, at least 1.8 times, at least 2 times, at least 3 times, at least 4times, at least 5 times, at least 10 times, and at least 20 times as long as Lokivetmab (SEQ ID NO: 64, heavy chain; SEQ ID NO: 65: light chain). EXAMPLES

[0390] These Examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.

[0391] The following abbreviations and special terms apply to the Examples and Drawings only. These abbreviations and special terms are not otherwise limiting, and neither replace nor narrow the broader definitions set forth above, which shall continue to apply to the claims. IL31[WT]–IgG1 Fc fusion – refers to a fusion protein of SEQ ID NO: 1 IL31–IgG1 Fc fusion (Variant 1) – refers to a fusion protein of SEQ ID NO: 2 IL31[WT]–IgG4 Fc fusion – refers to a fusion protein of SEQ ID NO: 3 IL31–IgG4 Fc fusion (Variant 2)– refers to a fusion protein of SEQ ID NO: 4 IL31[WT]–IgG-B Fc fusion – refers to a fusion protein of SEQ ID NO: 5 IL31–IgG-B Fc fusion (Variant 3) – refers to a fusion protein of SEQ ID NO: 6 Example 1. Expression and Purification of IL-31[WT]-IgG1 Fc Hexameric Fusion Protein

[0392] A codon-optimized polynucleotide (SEQ ID NO: 31) encoding a polypeptide chain of the present disclosure (SEQ ID NO: 1) was cloned into an expression plasmid. Plasmids were transfected into CHO suspension cells and stably integrated into a chromosome using piggyBac transposase. Cultures of the cells were grown in media.

[0393] The cells were then removed by centrifugation. Multimeric fusion proteins were purified from the supernatants by protein A chromatography using MabSelectTMSuReTMresin (61 ml) with step elution using a 20 mM citrate pH 3.0 buffer and a 1x PBS, pH 7.4 washing buffer and 1 M potassium phosphate dibasic neutralization buffer. Example 2. Expression and Purification of IL-31-IgG1 Fc (Variant 1) Hexameric FusionProtein

[0394] A codon-optimized polynucleotide (SEQ ID NO: 32) encoding a polypeptide chain of the present disclosure (SEQ ID NO: 2) was cloned into an expression plasmid. Expression and purification of the fusion protein was performed using the procedures described in Example 1. Example 3. Expression and Purification of IL-31[WT]-IgG4 Fc Hexameric Fusion Protein

[0395] A codon-optimized polynucleotide (SEQ ID NO: 33) encoding a polypeptide chain of the present disclosure (SEQ ID NO: 3) was cloned into an expression plasmid. Expression and purification of the fusion protein was performed using the procedures described in Example 1. Example 4. Expression and Purification of IL-31-IgG4 Fc (Variant 2) Hexameric Fusion Protein

[0396] A codon-optimized polynucleotide (SEQ ID NO: 34) encoding a polypeptide chain of the present disclosure (SEQ ID NO: 4) was cloned into an expression plasmid. Expression and purification of the fusion protein was performed using the procedures described in Example 1. Example 5. Expression and Purification of IL-31[WT]-IgG-B Fc Hexameric Fusion Protein

[0397] A codon-optimized polynucleotide (SEQ ID NO: 35) encoding a polypeptide chain of the present disclosure (SEQ ID NO: 5) was cloned into an expression plasmid. Expression and purification of the fusion protein was performed using the procedures described in Example 1. Example 6. Expression and Purification of IL-31-IgG-B Fc (Variant 3) Hexameric Fusion Protein

[0398] A codon-optimized polynucleotide (SEQ ID NO: 36) encoding a polypeptide chain of the present disclosure (SEQ ID NO: 6) was cloned into an expression plasmid.Expression and purification of the fusion protein was performed using the procedures described in Example 1. Example 7. Confirmation of Multimer Production

[0399] Samples containing the fusion protein were analyzed to confirm production of multimers.

[0400] Analytical size exclusion high performance liquid chromatography (HPLC) was performed using a DionexTMUltimate 3000 HPLC (Thermo Fisher); solvent degasser, high pressure RS pump, thermostat auto-sampler, column compartment and a photo diode array detector (capable of monitoring 280nm absorbance). The system was controlled and the chromatograms processed by Chromeleon 6.8 software. The mobile phase was isocratic at a flow rate of 0.3 mL / min and a single injection was monitored for 8 minutes. Column temperature was controlled at 25 °C and the absorbance was monitored at 280 nm. The mAb control and sample injections were set to 20 ul. The final salt peak eluted at approximately 5.5 minutes, monomer IgG peak at approximately 4 minutes, IgG dimer peak at 3.6 minutes and IgG fragment peak eluted on the tail of the monomer peak at approximately 4.8 minutes.

[0401] A standard of Anti-CD20 mAb (L# SO-DP-738-16-06202016) was used to calibrate the system and for control monitoring purposes. The results are summarized in Table 1 and the chromatogram is shown in FIG.2. Table 1 No. Peak Name Retention Area Height Relative Relative %)n.a. BSA 66 kDa n.a. n.a. n.a. n.a. n.a.of multimers. The results for HPLC performed for Example 1 (IL-31[WT]–IgG1 Fc fusion protein) are summarized in Table 2 and the related chromatogram shown in FIG.3. The chromatographs and results show the production of multimers by showing the estimates of size. Monomer single segments will have a retention time similar to the IgG standard. Multimers will have shorter retention times. Table 2 No. Peak Name RetentionArea Height Relative Relative time (min)mAU*min mAU Area % Height

[0403] The results for HPLC performed for Example 2 ((IL-31–IgG1 Fc (Variant 1) fusion protein)) are summarized in Table 3 and the related chromatogram shown in Fig.4. Table 3 Retention Area Height Relative Relative No Peak Name )IL-31 IgG1 1 Hexamer 3.073 45.801 282.409 86.69 93.23 (Variant 1)protein) are summarized in Table 4 and the related chromatogram shown in Fig.5. The unassigned peak corresponds to an component that has not been determined. Table 4 No. Peak Name RetentionArea Height Relative Relative time (min)(mAU*min) (mAU) Area (%) Height

[0405] The results for HPLC performed for Example 4 ((IL-31–IgG4 Fc (Variant 2) fusion protein)) are summarized in Table 5 and the related chromatogram shown in Fig.6. Table 5 No Peak Name RetentionArea Height Relative Relative Height

[0406] The results for HPLC performed for Example 5 (IL-31[WT]–IgG-B Fc fusion protein) are summarized in Table 6 and the related chromatogram shown in Fig.7. Table 6No. Peak NameRetentionArea Height Relative Relative time (min)mAU*min mAU Area % Height Wild-t IL-31

[0407] The results for HPLC performed for Example 6 ((IL-31–IgG-B Fc (Variant 3) fusion protein)) are summarized in Table 7 and the related chromatogram shown in Fig.8. Table 7 No. Peak NameRetentionArea Height Relative Relative time (min)mAU*min mAU Area % Height

[0408] Reducing and non-reducing gel analysis was also performed. Endotoxin was tested using the limulus amoebocyte lysate (LAL) assay and samples used in assays were <1 EU / mg. It was found that about 85% of the IL-31[WT]–IgG1 Fc fusion proteins, about 85% of the IL-31–IgG1 Fc (Variant 1) fusion proteins, about 53% of the IL-31[WT]–IgG4 Fc fusion proteins, about 53% of the IL-31–IgG4 (Variant 2) Fc fusion proteins, about 83% of the IL-31[WT]–IgG-B Fc fusion proteins, and about 83% of the IL-31–IgG-B (Variant 3) Fc fusion proteins produced were in hexameric form.Table 8 Name Concentration (mg / ml) Volume (ml) Total protein (mg)Table 9 Name Concentration (Cedex; Volume (ml)Example 8. Expression and Purification of the IL-31 Variant Antigens

[0409] An expression plasmid was generated containing a codon optimized polynucleotide encoding the IL-31 antigen containing an F104E mutation. The expression plasmid was transfected into CHO suspension cells and integrated into a chromosome using piggyBac transposase.

[0410] The antigen was expressed in a 14-day fed-batch culture and secreted into media. The culture media was clarified by sterile filtration and the vaccine antigen purified from clarified media using Protein A chromatography. The purified antigen was buffer exchanged into 1X PBS as enabling formulation. Example 9. Binding of the IL-31 Variant Antigens to IL31 Receptor

[0411] The extracellular domain of IL-31 receptor alpha (IL31Ra), which contains the IL- 31 binding domain, was expressed recombinantly in HEK293 cells.

[0412] An octet biolayer interferometry (BLI) system was used to demonstrate binding of recombinant IL31Ra to wild-type IL31-Fc-Hexamer antigens and IL31-Fc-Hexamer antigens containing the F104E mutation. See FIGS.15A and 15B.

[0413] Comparable binding of wild-type and F104E versions of the hexamer to the IL31Ra indicate that the F104E mutant has maintained native secondary and tertiary structure. Example 10. In vivo Studies

[0414] A head-to-head vaccination study with HexIgG-IL31 conducted with normal beagle dogs weighing between 6 and 15 kg of both genders at Ridglan Farms revealed promising anti-pruritic 7 month efficacy for HexIgG-IL31.

[0415] The age of the dogs was ≥6 months at the start of vaccination. The dogs were housed in a BSL2 facility, in rooms where video monitoring of the cages was available. All dogs received a physical examination to ensure they were healthy enough to participate in the study. Any dogs with a recent history of steroid or other immunomodulatory / immunosuppressive drug use were excluded.

[0416] All dogs had a baseline IL31 challenge prior to enrollment. Dogs were ranked according to pruritus score within gender. To qualify for randomization, dogs must have had had a minimum pruritus score of 35 in a two hour monitoring period. The dogs were randomized to treatment stratified by sex with a block size of 6. The first six dogs on each list were randomly assigned, one to each treatment. The next six dogs were assigned to the 6 treatment groups, and so on, thus ensuring a roughly even distribution of gender and baseline pruritus score amongst treatment groups. A total of six treatment groups, each contained 8 dogs, were formulated by this method.

[0417] Among the six treatment groups, three of them were treated with placebo, positive control, and the Hexameric IgG-Fc-IL31 vaccine, respectively. The conditions of these three groups are described as follows: Group (1): Placebo (vaccine buffer solution: 5 mM Na phosphate, 2 mM EDTA, pH 7.5 ), 100 μg saponin adjuvant; Group (2): Lokivetmab (positive control) dosed according to label instructions (2 mg / kg subcutaneously); Group (6): Hexameric IgG-Fc-IL31 vaccine, 300 μg total dose, 100 μg saponin adjuvant.

[0418] Lokivetmab, is also referred to a Cytopoint®, see, for example, https: / / drugs.ncats.io / drug / 3D6091GOHH

[0419] The Hexameric IgG-Fc-IL31 vaccine included 300 μg of the multimeric fusion protein described herein wherein the fusion protein includes 6 monomer units formed by a polypeptide chain including the amino acid sequence of SEQ ID NO: 2.

[0420] Dogs in Groups 1 and 6 were dosed subcutaneously on Days 0, 21, 42, and 63.4 dogs in Group 6 were given a booster on Day 189 as antibody titers waned during the monitoring period. Group 2 dogs were dosed at varying intervals (6, 4, 2 weeks) prior to IL- 31 challenges. The first dose was in a previously shaved site on the right side of the interscapular region. Subsequent vaccinations alternated sides. Group 1 (placebo) dogs were dosed in the same volume as the vaccinated dogs.

[0421] All dogs in Groups 1, 2, and 6 were monitored for vaccine-associated reactions. Prior to vaccination, dogs were monitored for general attitude and behavior, as well as examined at the site of the proposed injection. Post-vaccination, dogs were observed continuously for 30–45 minutes for any signs of reaction. To determine if the vaccine antigen was pruritogenic, dogs were monitored via video camera between 3 and 4 hours post- vaccination and pruritus evaluated on a 0–10 scale, with 0 being no pruritus and 10 being continual scratching behavior. Injection sites and behavior were evaluated and recorded at 4 hours post vaccination. Particular attention was paid to the recording of any pruritic behavior in conjunction with vaccination. Injection site reactions continued to be monitored at 24, 48, and 72 hours post-injection. If injection site reactions were still present at 72 hours post- injection, monitoring continued until they are resolved.

[0422] Temperatures were monitored before, and at 4, 24, 48, and 72 hours post- vaccination. If temperatures were elevated at 72 hours, they were monitored until returned to normal. If temperatures were normal at 4 and 24 hours post-vaccination, then this was discontinued for 48 and 72 hours. If temperatures were normal during two consecutive vaccinations, then this was discontinued for subsequent vaccinations. Because excited dogs can have an elevated temperature, the upper limit of normal was defined as 103.5° F.

[0423] Serum was collected before, and 3 weeks after, each vaccination to monitor vaccine titers. Titers were also checked prior to IL31 challenge at 19, 26, 29, 39, and 52 weeks. Five to ten mL of blood was collected from an easily accessible peripheral vein using an appropriate needle and a coagulation or serum separation tube. Blood was allowed to coagulate, and then spun down. Serum was separated into two labeled aliquots and frozen at approximately -60° C until shipped to the lab on dry ice. Serum for antibody titers was sent to Elanco labs in Greenfield, Indiana. Anti-IL-31 antibody titers is depicted in FIG.12.

[0424] Whole blood and serum was collected in 5 mL tubes on Days 0, 84, 133, and 182. These samples were sent to the diagnostic lab for CBC and chemistry panel, in order to further monitor preliminary tolerability of the vaccine.

[0425] After week 29, the study was ended for Group 1.

[0426] IL-31 challenges occurred prior to vaccination (baseline), and during Weeks 12, 19, 26, 39, and 52 after the first vaccination. Individual body weights (kg) were be obtained prior to each challenge. Dose of IL-31 was 0.85 μg / kg delivered intravenously. Followingadministration of the IL-31, the dogs were allowed to “rest and reacclimate” for one hour and then recording and monitoring of pruritus commenced. For each discrete 1 minute period during the observation period, the observer answered the question, “Did the dog display any pruritic behavior? Yes / No.” Pruritic behavior is defined as: scratching, licking, chewing, head shaking, rubbing, and / or scooting. Pruritus scores are depicted in FIG.13. The number of dogs with at least a 50% decline in pruritus scores is depicted in FIG.14. Example 11. Formulation, Adjuvant, and Analysis

[0427] A Hexameric IgG-Fc-IL31 (“HexIgG-IL31”) protein (SEQ ID NO: 2) was either formulated in 20 mM phosphate, 50 mM arginine HCl, 150 mM sucrose, 10 mM Methionine, 0.02% Polaxamer 188, pH 7.5 (“Buffer1”) or lyophilized. The HexIgG-IL31 protein was mixed with one of the following adjuvant formulations: a) Formulated Hexameric IgG-Fc-IL31 plus Ethylene / maleic anhydride (EMA) and Neocryl®A640 (Covestro) Ingredient Volume (µl) Final Concentrationb) Formulated Hexameric IgG-Fc-IL31 plus Carbopol® 971 (Lubrizol) at Lower Concentration Ingredient Volume (µl) Final t tiFinal Injection 1000 Volumec) Formulated Hexameric IgG-Fc-IL31 + Carbopol® 971 at Higher Concentration Ingredient Volume (µl) Final Concentrationd) Formulated Hexameric IgG-Fc-IL31 plus 10% Carbopol® 971 and CpG oligonucleotides 1668-PTO and 2006-PTO (Invivogen) Ingredient Volume (µl) Final Concentratione) Formulated Hexameric IgG-Fc-IL31 plus Quil-A® (Invivogen) Ingredient Volume (µl) FinalIL-31 – hIgG1 (SEQ 500 300 ID NO: 2) (600 / ml)f) Hexameric IgG-Fc-IL31 plus EMA / Neocryl®RTU (Covestro) Ingredient Concentration Amount (%)g) Hexameric IgG-Fc-IL31 plus Carbopol® 971 (ready for injection formulation) Ingredient Concentration Amount (%)h) Lyophilized HexIgG-IL31 plus Admix1Carbopol® 971 Ingredient Concentration Amount (%) IL 31 hI G1 SE N Ai) Lyophilized Hexameric IgG-Fc-IL31 + Admix1EMA / Neocryl®A640 Ingredient Concentration Amount (%)Table 10: Protocol and analysis of experiments conducted in FIG.16B 1 Admix means that the lyophilized vaccine is reconstituted with the adjuvant.Study Week (below numbers in “weeks”) es o es es

[0428] The formulations were administered to dog groups, thereby breaking tolerance to canine IL-13 (SEQ ID NO: 8) and building IL31 immunity in the dogs. An experiment wherein the Hexameric IgG-Fc-IL31 was administered without adjuvant or with the adjuvant Quil-A®on days 0 and 14 (FIG.16A) and then challenged with IL31 after two weeks, 6 months, and 12 months. FIG.16B demonstrates that the protection from IL31 challenge by Hexameric IgG-Fc-IL31 administered with Quil-A®provided reduced pruritic symptoms from IL-31 challenge at 2 weeks, 6 months, and persisted for at least 12 months as compared to control immunization with PBS. A booster with Hexameric IgG-Fc-IL31 with Quil-A®after 12 months demonstrates increased protection from the IL-31 challenge (FIG.16D). In FIG.16C swelling as a result of the immunization is shown. Swelling was mild to moderate and transient. Swelling was never associated with pain as determined by palpitation.

[0429] To test the efficacy of different adjuvant formulations dogs were immunized with Hexameric IgG-Fc-IL31 (SEQ ID NO: 2) plus formulations with one of the following: No adjuvant 100 ug Quil A® Carbopol® 971 (0.02%) Carbopol® 971 (0.1%)Carbopol® 971 (0.02%) + CpG oligonucleotides: 1668-PTO, 2006-PTO Ethylene / Maleic Anhydride / Neocryl®A640.

[0430] For comparison, a control group of dogs received PBS instead of Hexameric IgG- Fc-IL31. The dogs were challenged with IL-31 two and 15 weeks after the second vaccination to determine the efficacy of the immunization. The results are shown in FIG. 16C. From the data shown in FIG.16D, the Hexameric IgG-Fc-IL31 (SEQ ID NO: 2) plus adjuvants Carbopol® 971 (0.02%), Carbopol® 971 (0.1%), and EMA / Neocryl®A640 provided relief from the IL31 challenge two weeks after the administration. The negative control is PBS, and the positive control is IL31 hexamers (SEQ ID NO: 2) plus 100 μg Quil A®. After 15 weeks all study groups containing Hexameric IgG-Fc-IL31 and adjuvant demonstrated relief from the IL31 challenge, as measured by the pruritic score (Table 11, FIG.16E). Table 11 Carbopol® 971 l® l® Low Dose and EMA / ® 1

[0431] The result of a tolerance assessment conducted on the dogs treated in FIG.16D is shown in Table 12A and Table 12B. Table 12A Local Tolerance Assessment, Day 0 Day 0 Day 1 (24 hrs) Day 2 (48 hrs)Group Swelling Induration Pain Swelling Induration Pain Swelling IndurationTable 12B Local Tolerance Assessment, Day 14 Day 15 Day 14 Day 16 (48 hrs) onµSlight erythema for the dog with induration.Carbopol 0.1% 1(1) 0 0 0 1 (2) 1(1) 0 0 Same as diffExample 12. Analysis of Hexamers Using SDS-PAGE and Chromatographic Techniques

[0432] To determine the state of hexamerization an SDS-PAGE separation was performed (FIG.17A). Hexameric IgG-Fc-IL31, and monomeric IgG-Fc-IL31 were separated with and without a reducing agent such as mercapthoethanol or dithiothreitol. Hexameric IgG-Fc-IL31, and monomeric IgG-Fc-IL31 were separated with and without treatment to deglycosylate the protein.

[0433] Reverse phase chromatography was used to separate preparations of Hexameric IgG-Fc-IL31 into components including hexameric protein complexes of IL-31-Fc, IL31-Fc monomer, and other subspecies (FIG.17B).

[0434] Size exclusion chromatography was used to separate preparations of Hexameric IgG-Fc-IL31 into components including hexameric protein complexes of IL-31-Fc, Sub- hexamer comprising IgG-Fc-IL31 polypeptides and fragments that are in an oligomerization state of less than six, and IL31-Fc monomer (FIG.17C). Example 13. IL-31 IgG Hexamer Stability In Storage

[0435] Hexameric IgG-Fc-IL31 protein (SEQ ID NO: 2) stability was determined by differential scanning fluorimetry (DSF), size exclusion chromatography, and reverse phase chromatography. Samples were assayed as a time course of stability with or without an excipient at storage temperatures of 5° C, 25° C, and 40° C. Indicators in the DSF assay for stability were melting temperature (Tm), temperature of unfolding onset (Tonset) (FIG.18A).

[0436] A size exclusion chromatography assay (SEC) indicators for Hexameric IgG-Fc- IL31 (SEQ ID NO: 2) stability were individually, the integration of the aggregate peak, hexamer peak, and fragment peak, as a percent of the total area of all peaks. These percentages were assayed weekly for four weeks, then plotted (FIG.18B). At all temperatures the Hexameric IgG-Fc-IL31 protein in Buffer1 (20 mM phosphate, 50 mM arginine HCl, 150 mM sucrose, 10 mM Methionine, 0.02% Polaxamer 188, pH 7.5) and Buffer1 with excipients demonstrated similar stability behavior in the SEC assay.

[0437] A reverse phase high performance liquid chromatography (RP-HPLC) assay was used to separate the hexamer, monomer, and sub-hexamer species of Hexameric IgG-Fc-IL31 (SEQ ID NO: 2) to determine stability of the protein at 5° C, 25° C, and 40° C in Buffer1 or Buffer1 containing excipients (FIG.18C). The result demonstrates that the protein demonstrates similar stability at 5° C and 25° C over the course of one to four weeks. The result demonstrates that the protein demonstrates similar stability with or without excipients.

[0438] Using the DSF melting temperature assay, the stability of the Hexameric IgG-Fc- IL31 (SEQ ID NO: 2) in Buffer1 was determined at 5° C, 25° C, and 40° C wherein the Tm was assayed over the course of four weeks (FIG.18D). The result demonstrates that the protein demonstrates similar stability at 5° C and 25° C.

[0439] Using the SEC assay the percent hexamer and percent aggregates were used to determine the stability of Hexameric IgG-Fc-IL31 (SEQ ID NO: 2) in Buffer1 over the course of 4 weeks of storage at 5° C, 25° C, and 40° C (FIG.18E). The result demonstrates that the protein demonstrates similar stability at 5° C and 25° C.

[0440] Using the RP-HPLC assay the percent hexamer, percent monomer, and percent aggregates were used to determine the stability of Hexameric IgG-Fc-IL31 (SEQ ID NO: 2) in Buffer1 over the course of 4 weeks of storage (FIG.18F).

[0441] Using the SEC assay the percent aggregates was used to determine the stability of Hexameric IgG-Fc-IL31 (SEQ ID NO: 2) in Buffer1 or phosphate, pH 7.5 over the course of 24 weeks of storage at temperatures ranging from 2°C to 8°C (FIG.18G). The result demonstrates that in both buffers the protein demonstrates similar stability.

[0442] Using the SEC assay the percent hexamer was used to determine the stability of Hexameric IgG-Fc-IL31 (SEQ ID NO: 2) in Buffer1, phosphate pH 7.5, or citrate-phosphatebuffer pH7.5 over the course of 24 weeks of storage at temperatures ranging from 2°C to 8°C (FIG.18H). The result demonstrates that in the three buffers the protein demonstrates similar stability.

[0443] Using the DSF melting temperature assay, the stability of the Hexameric IgG-Fc- IL31 (SEQ ID NO: 2) in storage at temperatures ranging from 2°C to 8°C in Buffer1, phosphate pH 7.5, citrate-phosphate pH 7.5, phosphate pH 6.0, and citrate-phosphate pH 6.0 were determined wherein the Tm was assayed over the course of 26 weeks (FIG.18I). The result demonstrates that the protein demonstrates similar stability in Buffer1, phosphate pH 7.5, citrate-phosphate pH 7.5.

[0444] Using the SEC assay for percent hexamer and RP-HPLC assay for percent hexamer the stability of Hexameric IgG-Fc-IL31 (SEQ ID NO: 2) in citrate pH 6.0, citrate- phosphate pH 6.0, citrate-phosphate pH 7.5, Buffer1, and phosphate pH 7.5 was determined over the course of 24 to 26 weeks of storage at temperatures ranging from 2°C to 8°C (FIG. 18J). The result demonstrates that in all six buffers the protein demonstrates similar stability. Example 14. Hexamer Potency Assay

[0445] To determine the stability of Hexameric IgG-Fc-IL31 (SEQ ID NO: 2) by potency, an ELISA assay was employed (FIG.19A). The capture antibody was an S18-A monoclonal antibody with affinity to IL-31 was dissolved in 50mM NaCO3 / NaHCO3pH 9.7 and was used to coat the wells of an assay plate. The plate was blocked in 1.15% non-fat dry milk. The wells were washed in 10 mM PBS containing 0.03% Tween. The detection antibody was a biotinylated S18-A antibody. The detection complex was a peroxidase conjugated with streptavidin. The detection substrate for the peroxidase was 3,3',5,5'- Tetramethylbenzidine (TMB).

[0446] To determine the proper concentration of the biotinylated S18-A antibody for detection, the antibody was diluted in a range of 1:100 to 1:107. The capture antibody was used at 1:800. The peroxidase conjugated with streptavidin was diluted 1:500. The antigen (Hexameric IgG-Fc-IL31, SEQ ID NO: 2) was diluted to 300 ng / ml. The ELISA was developed for 30 minutes before reading. A comparison between the Ref and OPV with a negative control as shown in FIG.19B. From this it was concluded that the optimal dilution for the detection antibody was 1:10000.

[0447] To determine the proper concentration of the streptavidin conjugated peroxidase for detection, the conjugate was diluted in a range of 1:102to 1:106. This was assayed with the S18-A antibody capture antibody at 1:6000, the detection antibody at 1:105, and the antigen (Hexameric IgG-Fc-IL31, SEQ ID NO: 2) at 300 ng / ml. The results were compared to those of Ref and OPV with a negative control as shown in FIG.19C. From this assay it was determined that the best dilution for the peroxidase streptavidin conjugate was 1:1000.

[0448] To determine the dynamic range of the assay the antigen (Hexameric IgG-Fc- IL31, SEQ ID NO: 2) was diluted in a range of 103to 108fold from the stock of 0.3 mg / ml (300 ng / ml to 3 pg / ml). This was assayed with the S18-A antibody capture antibody at 1:6000, and the biotinylated S18-A detection Ab at 1:10000. The streptavidin conjugated peroxidase was diluted 1:1000 (FIG.19D, FIG.19E, FIG.19F, Table 13. Table 13 Expected Observed Prototype # Fraction Concentration RP Concentration Percent A r

[0449] The potency assay was used to compare various batches of IL31 IgG Fc fusion proteins (SEQ ID NO: 2) (FIG.19G).Example 15: Potency Assay to Determine Stability of Hexameric IgG-Fc-IL31 in Formulations

[0450] To determine the stability of Hexameric IgG-Fc-IL31 (SEQ ID NO: 2) in formulations of Ref at 0.3 mg / ml, Buffer1 (20 mM phosphate, 50 mM arginine HCl, 150 mM sucrose, 10 mM Methionine, 0.02% Polaxamer 188, pH 7.5), Buffer1 containing Quil-A®, Buffer1 containing Carbopol® 971 (0.02%) (FIG.20A), Carbopol® 971 (0.1%), Carbopol® 971 plus CpG containing oligonucleotides, or ethylene / maleic anhydride (FIG.20B). Example 16: Efficacy and Tolerability of IL-31 immunization in Dogs

[0451] Dogs were placed in three treatment groups and treated as follows (see table 9): 1. Placebo, 100 µg QuilA (n=8) (negative control) 2. Lokivetmab 2mg / kg (n=8) (positive control) 3. Hex-IgG 300 µg and 100 µg QuilA (n=8) The dosing regimen was Groups 1 and 3 received 4 subcutaneous immunizations of placebo (Group 1) or Hexameric IgG-Fc-IL31, (SEQ ID NO: 2) 21 days apart (week 0, week 3, week 6, week 9), where the injection was 1.5 mL per injection. Group 3 (4 dogs) received a booster on week 28. Group 2: received Lokivetmab 2mg / kg, 6weeks before 1st IL31 challenge, 4weeks before 2nd IL31 challenge, and 2 weeks before the other IL31 challenges. IL-31 challenges were conducted Prior to treatment, on week 12, week 19, week 26, week 39, and week 52. Table 14 Week 5 2 xComplete Blood Count3, Blood Ch i t T t x x

[0452] The safety and tolerability was evaluated by swelling and pain at the injection site (Tables 15 and 16). Table 15: Number of dogs exhibiting swelling at the injection site. 1st Vax 2nd Vax 3rd Vax 4th VaxTable 16: Number of dogs exhibiting transient pain upon palpitation, induration, increased heat at the injection site, or erythema at the injection site. 1st Vax 2nd Vax 3rd Vax 4th Vax3CBC measures: red blood cell count, white blood cell count, platelets, hemoglobin, hematocrit & mean corpuscular volume. Blood chemistry measures sodium, potassium, chloride, bicarbonate, blood urea nitrogen, creatine, and glucose.

[0453] In FIG. 21A the IL31 hexamer vaccination was shown to have efficacy from 12 weeks post vaccination and continuing for at least 52 weeks. The IL31 hexamer vaccination at least as efficacious as Lokivetmab. Example 17: Titration of QuilA dosing for protection from Pruritus

[0454] To determine the best concentration of QuilA for providing protection in an IL31 challenge, five groups of six dogs were treated as follows: 1. Negative Control (PBS / Mock immunization)2. 300 μg Hexameric IgG-Fc-IL31 protein + 25 μg QuilA3. 300 μg Hexameric IgG-Fc-IL31 protein + 50 μg QuilA4. 300 μg Hexameric IgG-Fc-IL31 protein + 75 μg QuilA5. Positive Control (300 μg Hexameric IgG-Fc-IL31 protein +100 μg QuilA)

[0455] The dogs received 2 subcutaneous injections two weeks apart at the beginning of the study and were evaluated for local tolerance and efficacy against IL-31 challenge. In FIG. 22 the data demonstrates that the adjuvant amounts of 50, 75, and 100 μg of QuilA have efficacy as compared to the PBS control at reducing the pruritic response in dogs in response to an IL31 challenge.

[0456] Dogs were observed for tolerability of the injection (Table 17A, Table 17B). There were more reactions after the second injection. No pain was recorded after any injection. The efficacy at providing protection is shown in FIG. 22. This result shows that the injection is sufficient for protection with Quil A®adjuvant as low as 50 μg per injection.5yglanailmrDle ow N S 4g 2nix0llero2xro2x1x1x xw N03N0 0 0 05S 2 1 2A71el , g , gbaatmniamniTneeh hc ehc1mtt h tyr artyary moecsrecsa cD g4x2x5x3x3 2 5 4nill0e20x2030x40x x x502040w0x0x0x x x x3xS 6 2 90406020608t,nag ,age mneinimh htcm teh hc0myr arttyr aryoecCs ecsaD gn3 4 3 3ilxl0x5x0x0e2wx3x3x3xS02060404go1- 1 234 2- - - -1-2-3- 4- DDI 4 4 4 4 5 5 5 5gpμgg) μ )μuo5Al 5Al0)0Alr2G(i 72uQ(i 14uQ(i5uQyaD91ya ewezLxm3x3x3x2x3x3xDSiSWm(060503060308ybhtegn )zi em SLym(4 g1niylbltHxa ehtd higi LxDwSWeH W o 1 1 2 3 4 3 DgDI-2-3-3-3-3-4gp) gm uo Am)5Ar2li05liG(2 uQ(3 uQa mr1 22x 12x x 0x 25 5 2x 0 xx x5 1 50x x x x101050103013x3 2 3 3 3 3 3 30x0x0x x x x x x2 3 503030 0 0 0x x x x x2x3x3 30 0 0 0 0 0 0x0x4 7 4 7 8 6 7 6032x20x40x20x4054- 1 2 5 6 712 44-4-4-5-5-5-5-5-5g gm)m57 A( l0)i01 A(li4 uQ5 uQExample 18 Clinical Pathology Analysis of Blood

[0457] Dogs received two subcutaneous injections on SD0 and SD14 of either placebo [T01; sterile PBS], adjuvanted hexameric IL-31 [T02; 150 µg hex hIgG1-Fc-cIL-31 (SEQ ID NO: 2) plus 100 µg Quil-A®, or non-adjuvanted hexameric IL-31 [T03; 150 µg hex hIgG1- Fc-cIL-31 (SEQ ID NO: 2 plus PBS]. The T03 group completed the study at six months (Study Day 196) due to a lack of demonstrable pruritus reduction.

[0458] ALP (Alkaline Phosphatase) levels remained within the normal range (5-160 U / L) across all treatment groups throughout the study, suggesting no discernible effect of the vaccine on ALP levels overall.

[0459] AST (Aspartate Aminotransferase): AST levels were generally within the normal range (16-55 U / L) for study days 28-288 post 2nd vaccination. At baseline [Study Day (SD) -42], dogs 1 and 2, both in the Treatment group 2 (T02): (hex IgG1-Fc-IL-31 (SEQ ID NO: 2) (150 µg) + Quil-A®(100 µg) / 1.5mL Dose) treatment group, had values below the normal range (15 U / L and 14 U / L, respectively). Dog 3 (T02) had one low out-of-range value (15 U / L) at SD 380. At SD 478, dogs 1 and 2, both in group T02, had AST values just above the normal range (56 U / L and 58 U / L respectively). The remaining dogs in the T01, T02, and T03 treatment groups had AST values that fell within the normal range for all study days AST Levels Below / Above the Normal Reference Interval (16-55 U / L) in Vaccinated (T02) Dogs Dog ID Treatment SD-42 SD 380 SD 478 Grou * ID (Baseline)

[0460] ALT (Alanine Aminotransferase): ALT levels remained predominantly within the normal range (18-121 U / L) throughout the study. The T02 treatment group had no excursions outside the normal range. In the T01 treatment group, dog 1 had a high ALT value(261 U / L) at Study Day (SD) 112, above the normal range. Dog 1 in the T03 treatment group had two low ALT values: one at SD 0 (16 U / L) and another at SD 56 (16 U / L), both below the normal range. ALT Levels Below / Above the Normal Reference Interval (18-121 U / L) in Control (T01) and Vaccinated (T03) Dogs Dog ID Treatment SD 0 (Prior to SD 56 SD 112 Group* ID Vaccination) s

[0461] Gamma-glutamyl Transferase (GGT): GGT levels were within the normal reference range (0-13 U / L) in all study dogs and consistent across the treatment groups to indicate no detectable cholestatic liver injury or issues with bile flow.

[0462] Total Bilirubin: Excursions were observed only in dogs from the T02 group. While values generally remained within or close to the normal range (0 - 0.3 mg / dL), five excursions occurred in four dogs. Three of these excursions were seen at or near baseline: two dogs (4 of and 2 of T02) had values of 0.4 mg / dL at Study Day (SD) -42 (baseline), and one dog (5 of T02) had a value of 0.4 mg / dL on Study Day (SD) 0 (prior to vaccination).5 and 4 of T02 also had excursions at SD 1, both at 0.4 mg / dL. Of the two remaining excursions, 2 of T02, who had a baseline value of 0.4 mg / dL, maintained the same value on SD 196, while dog 1 of T02 showed a notable increase to 0.8 mg / dL on SD 28 but transient in nature. The slightly elevated bilirubin levels observed in some dogs at or near baseline may indicate pre-existing hyperbilirubinemia. Total Bilirubin Levels Above the Normal Reference Interval (0.0.3 mg / DL) in Vaccinated (T02) Dogs Dog Treatment Group ID* SD -42 SD 0 SD 1 SD 28 SD 196* T02= hex IgG1-Fc-IL-31 (150 µg) plus Quil-A®(100 µg) / 1.5mL Dose; -**= Total bilirubin levels fell within the normal range. SD is study day.

[0463] Albumin Levels: Remained largely within the normal range (2.7-3.9 g / dL). Three excursions below the lower limit of the normal range were noted in treatment group T03: one dog on Study Day (SD) 0, one dog on Study Day 01 and another dog on Day 196), all at 2.6 g / dL. No excursions below, or above, the limits of the normal range were observed in treatment group T02 dogs. Albumin Values Below the Normal Reference Interval (2.7-3.9 g / DL) in Vaccinated Dogs Dog ID Treatment Group ID* SD 0 SD 1 SD 196 2 T03 2.6 2.6 -*. .

[0464] Globulin: Largely remained within the normal range (2.4-4.0 g / dL). Isolated excursions occurred in one dog (1) in group T03 at baseline (SD -42, 2.1 g / dL), and in another (dog4) from the T02 group on Study Day (SD) 0, prior to vaccination (4.1 g / dL) and SD 196 (2.3 g / dL). Single animals in the T02 and T03 groups also exhibited excursions on SD 56: 2 (T02, 2.3 g / dL) and 1 (T03, 2.3 g / dL). One dog in group T02 (1) showed excursions below the normal range on SD 288 (2.3 g / dL), SD 380 (2.3 g / dL), and SD 478 (2.3 g / dL). One dog in T01 (dog 4) also exhibited an excursion below the normal range on SD 380 (2.3 g / dL) and SD 478 (2.3 g / dL). One excursion in the control group T03 also showed an excursion below normal at SD 196 (dog 1, 2.3 g / dL). All excursions, apart from dog 4 in group T02 on SD 0 (prior to vaccination), fell below the lower limit of the normal range. All excursions remained close to the reference interval. Globulin Values Below / Above the Normal Reference Interval (2.7-3.9 g / DL) in Control (T01) & Vaccinated (T02 and T03) Dogs Dog ID Treatment SD -42 SD 0 SD 56 SD 196 SD 288 SD 380 SD 4781 T02 -** -** -** -** 2.3 2.3 2.3 2 T02 -** -** 2.3 -** -** -** -** ** ** ** ** ** **e normal range. SD is study day.

[0465] ALB / GLOB Ratio: There were no excursions from the normal reference interval (0.7-1.5) reported, indicating no inflammatory process during the in-life phase of the study.

[0466] Total Protein: In groups T02 (hex-hIgG1-Fc-Il-31 Vaccine and Quil-A®adjuvant), total protein excursions below the normal reference range (5.5-7.5 g / dL) were observed in three dogs, dog 2, T02 exhibited a value of 5.4 g / dL at baseline on Study Day (SD)-42, dog 5, T02 recorded 5.4 g / dL at SD56 and dog 7 T02 presented with 5.3 g / dL at SD112. In group T03 (hex-hIgG1-Fc-Il-31 Vaccine without any adjuvant), four dogs had total protein levels below the normal range. At Day 0 (prior to vaccination), dog 4 and dog 2 showed respective values of 5.2 g / dL, while dog 1 presented with 4.9 g / dL. Dog 2 had another excursion at SD56 (5.4 g / dL), and both PZY1 and dog 1 had low readings at SD112 (5.4 g / dL each). Dog 1 also measured low at SD196 (5.4 g / dL). All observed excursions were close to the lower end of the normal range. Total Protein Levels Below the Normal Reference Interval (5.5-7.5 g / dL) in Vaccinated (T02 & T03) Dogs Dog ID Treatment Baseline SD0 SD56 SD112 SD196 SD288 G * SD 42*T02= hex IgG1-Fc-IL-31 (150 µg) + Quil-A®(100 µg) / 1.5mL Dose; *T03= hex IgG1-Fc- IL-31 (150 µg) / 1.5mL Dose; -**= Total protein levels fell within the normal range. SD is study day.

[0467] While all three (Albumin, Globulin and Albumin / Globulin Ratio) parameters showed some level of fluctuation, globulin displayed a greater number of excursions and exhibited values outside of normal reference range, primarily below the lower normal limit. This variability may stem from a combination of factors: a sensitivity of globulin's lower normal limit to fluctuations, and individual dog responses.

[0468] Efficacy [Immunogenicity]: A robust anti-IL-31 antibody response was observed in group T02 (adjuvanted vaccine) after the second vaccination. In this group, one dog showed elevated titers as early as SD 14. By SD 28, all dogs in T02 group exhibited high serum antibody titers, maintained until SD56. Titers subsequently declined, with undetectable levels in four dogs by SD 112 and low titers in the remaining four. Antibody analysis was discontinued after SD 112. No antibody response was observed in the placebo group (T01) (except for the dog that was apparently mis-dosed on D14 and had an increase of antibodies on D112) or the non-adjuvanted vaccine group (T03). Neutralizing antibody assessment was not conclusive and will not be further developed.

[0469] Efficacy [Onset of Immunity (OOI) and Duration of Immunity (DOI)]: All enrolled dogs demonstrated a strong pruritus response to the cIL-31 challenge at baseline (mean scores of approximately 65 and 70) on SD -42. Three groups were evaluated: T01 (placebo control), T02 (Quil-A®-adjuvanted hex hIgG1-Fc-cIL-31vaccine), and T03 (non- adjuvanted hex hIgG1-Fc-cIL-31vaccine). T03 group showed median pruritus scores above the threshold (>35 over the first 2 hours), indicating a lack of efficacy, and was not further continued after six months (SD 196). Subsequent analyses focused on T01 and T02. Two weeks after the second vaccination (SD 28, onset of immunity), T02 group demonstrated significantly lower median pruritus score of 10 compared to T01 group score of 49.5. This difference persisted at six months (SD 196), with median scores of 70 for T01 and 19.5 for T02 respectively, and at 12 months (SD 380), with median scores of 56 for T01 and 19 for T02 respectively, demonstrating sustained efficacy.

[0470] This demonstrates that the hexameric hIgG1-Fc-cIL-31 vaccine formulated with Quil-A®adjuvant (T02 group) effectively reduced cIL-31-induced pruritus in healthy beaglesfor up to one year following a two-dose vaccination regimen in an cIL-31 challenge model. Onset of immunity was observed two weeks after the second vaccination (SD 28), with sustained efficacy observed for up to 12 months (SD 380). No antibody response or pruritus reduction was observed in dogs receiving the non-adjuvanted formulation (T03 group), highlighting the crucial role of the adjuvant. The vaccine demonstrated an acceptable systemic safety profile over 15 months post-vaccination. While effective in stimulating an immune response, the Quil-A®adjuvant induced transient injection site swelling, underscoring the need for a safer alternative. Isolated and sporadic fluctuations in some clinical pathology parameters were noted but deemed not clinically significant and unrelated to vaccination. Similarly, hematological parameters, such as WBC and RBC indices, and platelet counts, showed transient and isolated fluctuations within the normal range, supporting the overall systemic safety profile. No adverse effects on renal or hepatic function were observed.SEQUENCE LISTING SEQ ID NO: Description Sequence D P V V A I K D S P V V A I K D A VL235E, QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA L309C, VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS D S A V S D P V G TI P D ScIgG-B ACLESVFKSLNSGPQPASKTKVDKPVPKRENGRVPRP (Variant 3) PDCPPCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCV G TI P D D S E EAmino acid LFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY sequence for VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWL Y L V H V H A S A Swith R355C mutation K K A S V Khuman IgG1 CH1 domainwith S228P mutation G G A G A G C A C G G C CCTCCACCTACCGCGTCGTGTCCGTGTTGACCGTGTG CCATCAAGACTGGCTGAACGGAAAGGAGTACAAGT G G A T T G G A G A G C G G G C TAACGCCAAGACCAAGCCGAGAGAAGAACAGTACA ACTCCACCTACCGCGTCGTGTCCGTGTTGACCGTGT G A G G A T T G G A G A G C A G G C C TGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGA CCAAGCCTCGCGAGGAACAGTTCAACAGCACCTAT C C A G C T A A C C G G A G A G C G G C C CGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAG ACCAAGCCTCGCGAGGAACAGTTCAACAGCACCTA A T C A A C C G G C G G A G A G C A C G C A G A ACCCCGAAGTGCAGATTTCATGGTTCGTGGACGGAA AGCAGATGCAGACCGCCAAAACTCAGCCTCGGGAG C A A G A A A G G A G A G C G G G G T A AAGTGACCTGTGTGGTGGTCGACCTCGATCCGGAGG ACCCCGAAGTGCAGATTTCATGGTTCGTGGACGGA A A C A A G A A A G A T C G C A G G Amature CTCCTGGAGGATTACCAGAAGAAGGAAACCGGGGT canine IL-31 CCCTGAGTCGAATCGCACCCTGCTGTTGTGTCTCAC G C G A A T G A A A T G ANucleic acid CTGGGTGGTCCCAGCGTGTTCCTGTTCCCGCCGAAA sequence for CCTAAGGACACGCTGATGATCTCGAGAACCCCGGA A T A C A A A T A C A A A G C A Acanine IgG-B GTGACCTGTGTGGTGGTCGACCTCGATCCGGAGGA CH2 domain CCCCGAAGTGCAGATTTCATGGTTCGTGGACGGAA G C T C T C A C T C A T GGAGAACAATTACAAGACGACTCCGCCCGTGCTGGA TAGCGACGGATCCTTCTTCCTCTACTCCCGCCTTAC T C A T G T C A T G T C A A ACTCGATGAGGACGGCTCGTACTTCCTGTACTCCAAG CTGTCCGTGGACAAGAGCAGATGGCAACGGGGGGA A C T Thinge region with C220S C C Einterdomain linker S D C L E R A T G H F NKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSST EYLSHELYSCEITHKSLPSTLIKSFQRSEC

Claims

CLAIMS 1. A fusion protein comprising polypeptide chains, wherein each polypeptide chain comprises: (a) an antigen polypeptide sequence comprising IL-31 or a fragment or variant thereof that binds to an IL-31 receptor; (b) an Fc region protein sequence comprising an immunoglobulin heavy chain constant region connected to the antigen sequence; and (c) a tailpiece protein sequence connected to the C-terminal to the immunoglobulin heavy chain constant region; wherein the polypeptide chains form an oligomer.

2. A fusion protein comprising polypeptide chains, wherein each polypeptide chain comprises: (a) an antigen polypeptide sequence comprising IL-31 or a fragment or variant thereof that binds to an IL-31 receptor; (b) a linker connected to the antigen polypeptide sequence; (c) optionally a CH1 polypeptide sequence connected to the linker; (d) a hinge polypeptide sequence; (e) a CH2 polypeptide sequence; (f) a CH3 polypeptide sequence; and (g) a tailpiece polypeptide sequence; wherein the polypeptide chains form an oligomer, and wherein the fusion protein increases an immune response in an animal relative to antigen alone.

3. The fusion protein of claim 1 or 2, wherein the antigen comprises mature canine IL-31 or a fragment or variant thereof that is capable of binding to canine IL-31 receptor.

4. The fusion protein of any one of claims 1–3, wherein the antigen comprises a fragment or variant of IL-31 that is capable of binding to an IL-31 receptor but has reduced signaling as compared with wild-type IL-31.

5. The fusion protein of any one of claims 1–4, wherein the antigen comprises a fragment or variant of IL-31 that is capable of binding to an IL-31 receptor but is not capable of inducing an itch effect.

6. The fusion protein of any one of claims 1 – 5, wherein two individual polypeptide chains are cross-linked with each other and form a monomer subunit.

7. The fusion protein of claim 6, wherein multiple monomer subunits form a multimeric protein structure, 8. The fusion protein of claim 6, wherein the multimeric protein structure is a hexameric protein structure.

9. The fusion protein of claim 8, wherein the hexameric protein comprises identical monomeric subunits.

10. The fusion protein of claim 8, wherein the hexameric protein comprises monomeric subunits that are not identical.

11. The fusion protein of any one of claims 6 – 10 comprising 2 to 12 monomer units.

12. The fusion protein of claim 11, wherein the monomer units are identical.

13. The fusion protein of anyone of claims 6 – 11, comprising 6 monomer units.

14. The fusion protein of any one of claims 6 – 13, wherein the fusion protein is in the form of a cyclic hexamer.

15. The fusion protein of claim 1, wherein the immunoglobulin heavy chain constant region comprises a human IgG1 immunoglobulin Fc, or optionally a human IgG2 Fc, a human IgG3 Fc, a human IgG4 Fc, a human IgA Fc, a human IgD Fc, a human IgE Fc, a human IgM Fc, a canine IgG-a Fc, a canine IgG-b Fc, a canine IgG-c Fc, a canine IgG-d Fc, a murine IgG1 Fc, a murine IgG2 Fc, a murine IgG3 Fc, a murine IgM, or a functional variant thereof.

16. The fusion protein of any one of claims 1–15, wherein the antigen comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO:

8.

17. The fusion protein of claim 2, wherein the CH2 domain is a functional fragment or variant of a CH2 domain from human IgG1 antibody and comprises a mutation as compared with the CH2 domain from wild-type human IgG1 antibody such that it is capable of forming a cross-link with another polypeptide chain.

18. The fusion protein of any one of claims 2 or 17, wherein the CH2 domain comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO:

10.

19. The fusion protein of claim 18, wherein the CH2 domain comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 10, provided that the polypeptide chain comprises a cysteine at the residue position corresponding to residue position 309 of SEQ ID NO:

2.

20. The fusion protein of any one of claims 2 and 17 -19, wherein the CH3 domain comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO:

15.

21. The fusion protein of any one of claims 1 – 20, wherein the tailpiece comprises an amino acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 21.

2. The fusion protein of any one of claims 1 – 15, comprising, in N-terminus to C-terminus order: (a) an antigen comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of a mature canine IL-31, or a conservatively-substituted variant thereof, that is capable of binding to an IL-31 receptor; (b) a CH1 domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the CH1 domain from a human IgG1 antibody, or is a conservatively-substituted variant thereof; (c) a hinge region comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the hinge region from a human IgG1 antibody, or is a conservatively-substituted variant thereof; (d) a CH2 domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the CH2 domain from a human IgG1 antibody, or is a conservatively-substituted variant thereof; (e) a CH3 domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of the CH3 domain from a human IgG1 antibody, or is a conservatively-substituted variant thereof; and (f) a tailpiece comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequenceidentity with the amino acid sequence of the tailpiece from a human IgM antibody, or is a conservatively-substituted variant thereof.

23. The fusion protein of any one of claims 1 – 15, comprising, in N-terminus to C-terminus order: (a) an antigen comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 8 or is a conservatively- substituted variant thereof, that is capable of binding to a IL-31 receptor; (b) a CH1 domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 22, or is a conservatively- substituted variant thereof; (c) a hinge region comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 26, or is a conservatively- substituted variant thereof; (d) a CH2 domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 10, or is a conservatively- substituted variant thereof; (e) a CH3 domain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 15, or is a conservatively- substituted variant thereof; and(f) a tailpiece comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 21, or is a conservatively- substituted variant thereof.

24. A nucleic acid encoding the polypeptide chain of any one of claims 1–23.

25. The nucleic acid of claim 24, wherein the nucleic acid at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 31, or is a codon degenerate variant thereof.

26. The nucleic acid of claim 24, wherein the nucleic acid has at least 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 32, or is a codon degenerate variant thereof.

27. A vector comprising the nucleic acid of any one of claims 24–26.

28. A method for producing the antigen of any one of claims 24 – 26, comprising introducing a nucleic acid encoding the antigen to a cell.

29. The method of claim 28, further comprising growing the cell to express the antigen.

30. The method of claim 29, further comprising isolating the antigen.

31. A method for producing the polypeptide chain of any one of claims 1–23, comprising introducing a nucleic acid encoding the polypeptide chain to a cell.

32. A composition comprising an immunogenically-effective amount of the fusion protein of any one of claims 1 – 23.

33. The composition of claim 32, comprising a multimeric fusion protein hexameric form.

34. The composition of claim 32 or 33, wherein at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% of the multimeric fusion proteins present therein are in hexameric form.

35. The composition of any one of claims 32 – 34 for use in producing an immunogenic effect in a subject.

36. A method for inducing an immunological response in a subject in need thereof, the method comprising administering the fusion protein of any one of claims 1 – 23 to a subject.

37. The method of claim 36, wherein the subject is canine.

38. The method of claim 36 or 37, wherein the antigen is administered in an amount of from about 50 µg to 1000 µg / dose, from about 100 µg to 500 µg / dose, from about 100 µg to 300 µg / dose, about 100 µg / dose, about 200 µg / dose, about 300 µg / dose, about 500 µg / dose, or more than 600 µg / dose..

39. A method for inducing an immunological response in a subject in need thereof, the method comprising administering the fusion protein of any one of claims 1 – 23 to a subject.

40. The method of claim 39, wherein the subject is canine.

41. A method for treating a disease or disorder in a subject in need thereof, the method comprising administering the fusion protein of any one of claims 1 – 23 to a subject.

42. The method of claim 41, wherein the subject is canine.

43. The method of claim 41 or 42, wherein the disease or disorder is a disease or disorder for which an immunogenic effect produced from exposure to the antigen would have a therapeutic effect.

44. The method of any one of claims 41–43, wherein the disease or disorder is an itch-related disorder.

45. The method of any one of claims 41–43, wherein the disease or disorder is atopic dermatitis.

46. The method of any one of claims 41 – 45, wherein the antigen is administered in an amount of from about 50 µg to 1000 µg / dose, from about 100 µg to 500 µg / dose, from about 100 µg to 300 µg / dose, about 100 µg / dose, about 200 µg / dose, about 300 µg / dose, about 500 µg / dose, or more than 600 µg / dose.

47. A polypeptide chain comprising an amino acid sequence at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, or SEQ ID NO:

6.

48. The polypeptide chain of claim 47, wherein the polypeptide chain comprises a cystine at position 309.

49. A composition of comprising the fusion protein of any one of claims 1 – 23 or the protein of any one of claims 47 or 48.

50. The composition of claim 49, wherein said composition is formulated as a vaccine composition.

51. The composition of any one of claims 49 or 50 wherein the composition further comprises one or more adjuvants.

52. The composition of claim 51, wherein the one or more adjuvants is selected from the group of Quil A®, Carbopol®971, CARBOPOL® 934, and / or CARBOPOL® 941 NF.

53. The composition of claim 52, wherein one or more adjuvants is present in the composition in an amount of from about 50 µg to 1000 µg / dose, 50 µg to 150 µg / dose from about 100 µg to 500 µg / dose, from about 100 µg to 300 µg / dose, about 100 µg / dose, about 200 µg / dose, about 300 µg / dose, about 500 µg / dose, or more than 600 µg / dose.

54. The composition of any one of claims 49 – 53, wherein the fusion protein is present in the composition in an amount of from about 50 µg to 1000 µg / dose, from about 100 µg to 500 µg / dose, from about 100 µg to 300 µg / dose, about 100 µg / dose, about 200 µg / dose, about 300 µg / dose, about 500 µg / dose, or more than 600 µg / dose.

55. The composition of any one of claims 49 – 54, wherein the composition comprises an adjuvant in an amount of from about 50 µg to 1000 µg / dose, 50 µg to 150 µg / dose from about 100 µg to 500 µg / dose, from about 100 µg to 300 µg / dose, about 100 µg / dose, about 200 µg / dose, about 300 µg / dose, about 500 µg / dose, or more than 600 µg / dose and a fusion protein in an amount of from about 50 µg to 1000 µg / dose, from about 100 µg to 500 µg / dose, from about 100 µg to 300 µg / dose, from about 100 µg to 400 µg / dose, about 100 µg / dose, about 200 µg / dose, about 300 µg / dose, about 500 µg / dose, or more than 600 µg / dose.

56. A polypeptide chain comprising the below: A (Antigen) – B (CH1) – C (hinge) – D (CH2) – E (Optional Interdomain Linker) – F (CH3) – G (tailpiece) wherein, A comprises an IL-31 antigen; B comprises an amino acid sequence at least 90% identity to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24; C comprises an amino acid sequence at least 90% identity to SEQ ID NO: 26; D comprises an amino acid sequence at least 90% identity to SEQ ID NO:61; E is optional and if present comprises an amino acid sequence at least 90% Identity to SEQ ID NO: 62; F comprises an amino acid sequence at least 90% identity to SEQ ID NO: 63; and G comprises an amino acid sequence at least 90% identity to SEQ ID NO:

21.

57. The polypeptide chain of claim 56, wherein: A comprises an IL-31 antigen; B comprises an amino acid sequence at least 95% identity to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24;C comprises an amino acid sequence at least 95% identity to SEQ ID NO: 26; D comprises an amino acid sequence at least 95% identity to SEQ ID NO:61; E is optional and if present comprises an amino acid sequence at least 95% Identity to SEQ ID NO: 62; F comprises an amino acid sequence at least 95% identity to SEQ ID NO: 63; and G comprises an amino acid sequence at least 95% identity to SEQ ID NO:

21.

58. The polypeptide chain of claim 56, wherein: A comprises an IL-31 antigen; B comprises an amino acid sequence 100% identical to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24; C comprises an amino acid sequence 100% identical to SEQ ID NO: 26; D comprises an amino acid sequence 100% identical to SEQ ID NO:61; E is optional and if present comprises an amino acid sequence 100% identical to SEQ ID NO: 62; F comprises an amino acid sequence 100% identical to SEQ ID NO: 63; and G comprises an amino acid sequence 100% identical to SEQ ID NO:

21.

59. A fusion protein comprising a polypeptide chain of any one of claims 56 – 58.

60. The fusion protein of claim 59, wherein the polypeptide chains form an oligomer, and wherein the fusion protein increases an immune response in an animal relative to antigen alone.

61. The fusion protein of any one of claims 59 and 60, wherein the fusion protein is in the form of a cyclic hexamer.

62. The fusion protein of any one of claims 59 – 61, wherein the antigen comprises mature canine IL-31 or a fragment or variant thereof that is capable of binding to canine IL-31 receptor.

63. A composition comprising a fusion protein of any one of claims 59 – 62, wherein said composition comprises one or more adjuvants.

64. The composition of claim 63, wherein the one or more adjuvants is selected from the group of Quil A®, Carbopol®971, CARBOPOL® 934, and / or CARBOPOL® 941 NF.

65. The composition of any one of claims 32 – 35, 49 – 55, or 63 – 64, wherein the composition exhibits stability for period of time of at least 24 weeks or at least 52 weeks at 2-8°C.

66. A method of eliciting an immune response in a canine by administering the composition of any one of claims 49 – 55 or 63 – 65 to a canine.

67. Use of the fusion protein of any one of claims 1 – 23 or 59 – 62, the polypeptide chain of any one of claims 47 – 48 or 56 – 58, or the composition of any one of claims 49 – 55 or 63 – 65 as a vaccine.

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