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WO2025199054A1 - Composés, compositions, vaccins multimères et procédés de traitement de la dermatite - Google Patents

Composés, compositions, vaccins multimères et procédés de traitement de la dermatite

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Publication number
WO2025199054A1
WO2025199054A1 PCT/US2025/020279 US2025020279W WO2025199054A1 WO 2025199054 A1 WO2025199054 A1 WO 2025199054A1 US 2025020279 W US2025020279 W US 2025020279W WO 2025199054 A1 WO2025199054 A1 WO 2025199054A1
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WIPO (PCT)
Prior art keywords
amino acid
acid sequence
seq
dose
sequence
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PCT/US2025/020279
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English (en)
Inventor
Allyn SPEAR
Moiz Kitabwalla
Wahala WAHALA
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Elanco US Inc
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Elanco US Inc
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Publication of WO2025199054A1 publication Critical patent/WO2025199054A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • 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.
  • Interleukin-31 (IL-31) is a recently identified cytokine that has been implicated in pruritic behaviors associated with AD.
  • IL-31 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.
  • 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.
  • 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.
  • 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.
  • Vaccination allows for a more sustained antibody response.
  • Vaccine therapy also typically has a lower cost than monoclonal antibody therapy.
  • 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.
  • 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.
  • VLPs virus-like particles
  • the conjugation of antigens with virus-like particles 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.
  • these VLP fusions result in shielding of critical epitopes, similarly reducing the efficacy of the induced response.
  • 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 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 na ⁇ ve 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.
  • DCs Dendritic cells
  • 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.
  • 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 (2016).
  • Vaccines using multimeric fusion proteins have been designed to capitalize upon the ability of the fusion proteins to bind Fc ⁇ receptors in the target subject in order to generate an immune response.
  • Hexameric fusion proteins derived from human antibodies do not bind canine Fc ⁇ receptors.
  • the inventors have unexpectedly found that such fusion proteins nevertheless generate a robust immune response which efficiently mitigates itch in response to IL31 signaling.
  • 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.
  • 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.
  • the disclosures provides for a polypeptide chain comprising: an antigen; a CH2 domain; and a CH3 domain.
  • the antigen comprises IL-31 or a fragment or variant thereof that is capable of binding to an IL-31 receptor.
  • the antigen comprises mature canine IL-31 or a fragment or variant thereof that is capable of binding to canine IL-31 receptor.
  • 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.
  • 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.
  • 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.
  • the amino acid substitution comprises a glutamic acid at residue position 104 of SEQ ID NO: 8.
  • 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.
  • 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.
  • 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.
  • the mutation is the presence of an additional cysteine as compared with the CH2 domain from wild-type human IgG1 antibody.
  • 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.
  • 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 chain comprises a cysteine at the residue position corresponding to residue position 309 of SEQ ID NO: 2.
  • 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.
  • 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.
  • 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.
  • the polypeptide chain further comprises a CH4 domain.
  • the CH4 domain is from a human or canine antibody or is a functional fragment or variant thereof.
  • the CH4 domain is a CH4 domain from a human antibody or is a functional fragment or variant thereof.
  • the CH4 domain is from an IgE or IgM antibody or is a functional fragment or variant thereof.
  • the polypeptide chain further comprises a tailpiece.
  • the tailpiece is from a human or canine antibody or is a functional fragment or variant thereof.
  • the tailpiece is a tailpiece from a human antibody or is a functional fragment or variant thereof.
  • the tailpiece is from an IgA or IgM antibody or is a functional fragment or variant thereof.
  • the tailpiece is from an IgM antibody or is a functional fragment or variant thereof.
  • 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.
  • 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
  • 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,
  • 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 9
  • 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
  • the present disclosure also relates in part to a nucleic acid encoding the polypeptide chain of the present disclosure.
  • 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.
  • 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.
  • the present disclosure further relates in part to a vector comprising the nucleic acid of the present disclosure.
  • a yet further aspect of the present disclosure is a monomer comprising two polypeptide chains of the present disclosure.
  • 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.
  • 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.
  • 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.
  • 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.
  • the cysteines are present at residue position 309 in each CH2 domain.
  • 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.
  • 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.
  • the antigen comprises a fragment or variant of mature canine IL-31.
  • the antigen comprises a fragment or variant of mature canine IL-31 that is capable of binding to canine IL-31 receptor.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the composition is for use in producing an immunogenic effect in a subject.
  • 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.
  • the subject is canine.
  • the compounds described herein, for example, hexameric structures described herein are administered as hexameric structures.
  • the antigen is administered in an amount of from about 50 ⁇ g to 1000 ⁇ g/dose, from about 100 ⁇ g to 500 ⁇ g/dose, from about 100 ⁇ g to 300 ⁇ g/dose, about 100 ⁇ g/dose, about 200 ⁇ g/dose, about 300 ⁇ g/dose, about 500 ⁇ g/dose, or more than 600 ⁇ g/dose.
  • the multimeric fusion protein is administered in an amount of from about 50 ⁇ g to 1000 ⁇ g/dose, from about 100 ⁇ g to 500 ⁇ g/dose, from about 100 ⁇ g to 300 ⁇ g/dose, about 100 ⁇ g/dose, about 200 ⁇ g/dose, about 300 ⁇ g/dose, about 500 ⁇ g/dose, or more than 600 ⁇ g/dose.
  • 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.
  • 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.
  • 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.
  • the subject is canine.
  • 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.
  • the disease or disorder is an itch-related disorder.
  • the disease or disorder is atopic dermatitis.
  • the antigen is administered in an amount of from about 50 ⁇ g to 1000 ⁇ g/dose, from about 100 ⁇ g to 500 ⁇ g/dose, from about 100 ⁇ g to 300 ⁇ g/dose, about 100 ⁇ g/dose, about 200 ⁇ g/dose, about 300 ⁇ g/dose, about 500 ⁇ g/dose, or more than 600 ⁇ g/dose.
  • the multimeric fusion protein is administered in an amount of from about 50 ⁇ g to 1000 ⁇ g/dose, from about 100 ⁇ g to 500 ⁇ g/dose, from about 100 ⁇ g to 300 ⁇ g/dose, about 100 ⁇ g/dose, about 200 ⁇ g/dose, about 300 ⁇ g/dose, about 500 ⁇ g/dose, or more than 600 ⁇ g/dose.
  • the disclosure provides for compositions comprising fusion proteins, oligomers, multimers, and hexamers described herein.
  • compositions described herein further comprise at least one adjuvant or combination of adjuvants.
  • fusion proteins and compositions described here are used for therapeutic vaccination of companion animals, for example, cats and dogs.
  • hexameric proteins and compositions thereof described herein are used for therapeutic vaccination of companion animals, for example, cats and dogs.
  • 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.
  • Freund's Complete Adjuvant and Freund's Incomplete Adjuvant include, for example, Freund's Complete Adjuvant and Freund
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions described herein may include an adjuvant in an amount of from about 50 ⁇ g to 1000 ⁇ g/dose, from about 50 ⁇ g to 150 ⁇ g/dose, from about 25 ⁇ g to 100 ⁇ g/dose, from about 100 ⁇ g to 500 ⁇ g/dose, from about 100 ⁇ g to 300 ⁇ g/dose, about 100 ⁇ g/dose, about 200 ⁇ g/dose, about 300 ⁇ g/dose, about 500 ⁇ g/dose, or more than 600 ⁇ g/dose.
  • compositions described herein may include fusion proteins, oligomers, multimers, and hexamers described herein in an amount of from about 50 ⁇ g to 1000 ⁇ g/dose, from about 100 ⁇ g to 500 ⁇ g/dose, from about 100 ⁇ g to 300 ⁇ g/dose, about 100 ⁇ g/dose, about 200 ⁇ g/dose, about 300 ⁇ g/dose, about 500 ⁇ g/dose, or more than 600 ⁇ g/dose.
  • compositions described herein may include an adjuvant in an amount of from about 50 ⁇ g to 1000 ⁇ g/dose, 50 ⁇ g to 150 ⁇ g/dose from about 100 ⁇ g to 500 ⁇ g/dose, from about 100 ⁇ g to 300 ⁇ g/dose, about 100 ⁇ g/dose, about 200 ⁇ g/dose, about 300 ⁇ g/dose, about 500 ⁇ g/dose, or more than 600 ⁇ g/dose and fusion protein oligomers, multimers, and hexamers described herein in an amount of from about 50 ⁇ g to 1000 ⁇ g/dose, from about 100 ⁇ g to 500 ⁇ g/dose, from about 100 ⁇ g to 300 ⁇ g/dose, from about 100 ⁇ g to 400 ⁇ g/dose, about 100 ⁇ g/dose, about 200 ⁇ g/dose, about 300 ⁇ g/dose, about 500 ⁇ g/dose, or more than 600 ⁇ g/dose.
  • 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.
  • the animal is a canine or feline.
  • 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, 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.
  • the animal is a canine or feline.
  • 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.
  • 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
  • fusion proteins, oligomers, multimers, hexamers, or compositions described herein are administered in an amount of from about 50 ⁇ g to 1000 ⁇ g/dose, from about 100 ⁇ g to 500 ⁇ g/dose, from about 100 ⁇ g to 300 ⁇ g/dose, about 100 ⁇ g/dose, about 200 ⁇ g/dose, about 300 ⁇ g/dose, about 500 ⁇ g/dose, or more than 600 ⁇ g/dose.
  • compositions described herein exhibit ⁇ stability for period of time of at least 24 weeks or at least 52 weeks at 2-8°C.
  • 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.
  • FIG.2 depicts a chromatogram for an anti-CD20 monoclonal antibody standard.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG.9 is an image of a 4–12% Bis-Tris gel.
  • the lanes contain the following samples: (1) Bio-Rad Precision Plus TM protein 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.
  • FIG.10 is an image of a 4–12% Bis-Tris gel.
  • the lanes contain the following samples: (1) Bio-Rad Precision Plus TM protein 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.
  • FIG.11 depicts the results of an IL-31 bioassay.
  • FIG.12 depicts the mean titer for IL-31 antigen in each of Groups 1, 2, and 6.
  • 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.
  • 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.
  • 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.
  • FIG.16A depicts the time course of IL-challenge experiments in dogs vaccinated with IL31-Fc-Hexamer antigens, or controls.
  • FIG.16B depicts a time course of the efficacy of IL31-Fc-Hexamer antigens for reducing pruritus in dogs challenged with IL-31.
  • 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.
  • 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 ⁇ g, 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).
  • PBS phosphate buffered saline
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG.18A depicts the stability of IL31-Fc-Hexamer antigens (SEQ ID NO: 2) in excipients at 5oC, 25oC, and 40oC as determined by T onset , and T m using differential scanning fluorimetry.
  • 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.
  • 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.
  • 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).
  • Buffer1 (20 mM phosphate, 50 mM arginine HCl, 150 mM sucrose, 10 mM Methionine, 0.02% Polaxamer 188, pH 7.5
  • 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.
  • Buffer1 (20 mM phosphate, 50 mM arginine HCl, 150 mM sucrose, 10 mM Methionine, 0.02% Polaxamer 188, pH 7.5
  • 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.
  • Buffer1 (20 mM phosphate, 50 mM arginine HCl, 150 mM sucrose, 10 mM Methionine, 0.02% Polaxamer 188, pH 7.5
  • 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, as determined by the formation of aggregates measured by the integration of the corresponding peak as a percent of the total area in the SEC assay.
  • Buffer1 (20 mM phosphate, 50 mM arginine HCl, 150 mM sucrose, 10 mM Methionine, 0.02% Polaxamer 188, pH 7.5
  • phosphate pH 7.5 over the course of 24 weeks
  • 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.
  • 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 citrate-phosphate buffer pH 7.5
  • phosphate pH 7.5 over the course of 24 weeks
  • 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.
  • 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.
  • FIG.19A depicts the enzyme linked immunosorbent assay (ELISA) used to determine potency.
  • FIG.19B depicts an assay to optimize the proper concentration of the biotinylated S18-A antibody for detection in the ELISA potency assay.
  • FIG.19C depicts an assay to optimize the proper concentration of the streptavidin conjugated peroxidase for detection in the ELISA potency assay.
  • FIG.19D-FIG.19F depict an assay to determine the dynamic range of the assay the antigen in the ELISA potency assay.
  • 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.
  • FIG.20A and FIG.20B depict an ELISA potency assay on formulations of IL31- Fc-Hexamer antigens (SEQ ID NO: 2) in excipient formulations.
  • 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.
  • 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 ⁇ g per dose as compared to a PBS alone (placebo treatment).
  • SEQ ID NO: 2 L31-Fc-Hexamer antigens
  • 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.
  • 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.
  • 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.
  • the term “about” can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.
  • 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.
  • 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).
  • DNA double and single stranded deoxyribonucleic acid
  • RNA double and single stranded ribonucleic acid
  • nucleic acid sequences in the text of this specification are given, when read from left to right, in the 5′ to 3′ direction.
  • transfection refers to the introduction of one or more exogenous polynucleotides into a host cell or organism by using physical, chemical, and/or electrical methods.
  • nucleic acid sequences 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.
  • 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.
  • 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 can comprise synthetic amino acids in place of one or more naturally-occurring amino acids.
  • Such synthetic amino acids include, for example, aminocyclohexane carboxylic acid, norleucine, ⁇ -amino n-decanoic acid, homoserine, S-acetylaminomethyl- cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, ⁇ -phenylserine ⁇ -hydroxyphenylalanine, phenylglycine, ⁇ -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-
  • 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.
  • 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.
  • 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).
  • 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 Acids An 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.
  • the functional variants can comprise the amino acid sequence of the reference protein with at least one non-conservative amino acid substitution.
  • non-conservative mutations involves amino acid substitutions between different groups, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • BLASTN or CLUSTAL, or any other available alignment software
  • nucleic acid having at least 80% sequence identity with SEQ ID NO: 0 encompasses SEQ ID NO: 0 itself.
  • 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.
  • “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.
  • nucleic acid molecules that hybridize to the disclosed sequences.
  • Hybridization conditions may be mild, moderate, or stringent, as is warranted.
  • 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 37 o C for 4 to 12 hours, followed by a wash in 0.1X SSC at 60-65 o C.
  • nucleic acid sequence 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 Codons Thr/T ACT, ACC, ACA, ACG Trp/W TGG As use a nucleic acid 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.
  • 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.
  • 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.
  • 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.
  • 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
  • a L309C mutation in an IgG1 CH2 region refers to a leucine to cysteine substitution occurring at position 309 of the heavy chain.
  • 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.
  • 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.”
  • nucleic acids typically are mixed with an acceptable carrier or diluent when used for introduction into cells.
  • 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.
  • 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. This is often because non-autonomous transposons lack transposase (for class II) or reverse transcriptase (for class I).
  • 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.
  • the transposase’s catalytic activity can be utilized to move gene(s) from a vector to the genome.
  • 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.
  • 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.
  • 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.
  • 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).
  • EBNA1 Epstein Barr Nuclear Antigen 1
  • EMV Epstein Barr Virus
  • 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.
  • strains such as R6K that utilizes an antisense RNA selection marker (e.g. sucrose tolerance) can be used.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • a promoter-driven DNA sequence under certain environmental conditions
  • an inducer or an enhancer e.g., an inducer or an enhancer.
  • 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.
  • 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 also comprise enhancer sequences. Enhancers can be located upstream or downstream of coding sequences or within coding sequences.
  • 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).
  • 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.
  • 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 determining the 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.
  • “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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% 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.
  • antibody also known as immunoglobulin (Ig)
  • Ig immunoglobulin
  • 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.
  • the antibody can a single-chain whole antibody.
  • 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 (V L ) 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.
  • the “Fab region” of the antibody refers to the portion consisting of the V L , V H , 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.
  • the Fab region and the Fc region are connected by way of a hinge region that may be present in the heavy chains.
  • 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.
  • Naturally-occurring 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.
  • non-naturally occurring refers to a polypeptide or portion thereof comprising an amino acid sequence that does not occur in nature.
  • 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.
  • “Patient” or “subject” refers to a mammalian subject diagnosed with or suspected of having or developing an itch-related disorder such as atopic dermatitis.
  • 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.
  • “Administering” refers to herein as providing one or more compositions described herein to a patient or a subject.
  • composition administration e.g., injection
  • i.v. intravenous
  • s.c. sub-cutaneous
  • i.d. intradermal
  • i.p. intraperitoneal
  • intramuscular injection i.m.
  • 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.
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect is therapeutic, i.e., the effect partially or completely cures a disease and/or adverse symptom attributable to the disease.
  • the term “treating” can include “preventing” a disease or a condition.
  • 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.
  • a dosage regimen can have one or more periods of no administration of the therapeutic agent in between treatment intervals.
  • administered in combination means 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.
  • 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.”
  • the delivery of one treatment ends before the delivery of the other treatment begins.
  • the treatment is more effective because of combined administration.
  • 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.
  • 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.
  • 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.
  • the terms “therapeutically effective amount,” therapeutic amount,” “immunogenically effective amount,” and their grammatical equivalents refer to an amount effective, 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).
  • terms used in the identification of biological moieties may include, or may not include, a dash “ – ” within the term.
  • the present disclosure relates in part to a polypeptide chain comprising: an antigen; a CH2 domain; and a CH3 domain.
  • the polypeptide chain further comprises a CH1 domain; a hinge region; a CH4 domain; and/or a tailpiece.
  • the polypeptide chain comprises, in order from its N- terminus to its C-terminus: the antigen; the CH2 domain; and the CH3 domain.
  • the polypeptide chain further comprises a CH1 domain that is between the antigen and the CH2 domain.
  • 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.
  • each of the aforementioned elements of the polypeptide chain may individually be linked to one other directly by way of a covalent bond between such elements.
  • 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 domain and 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
  • the CH4 domain and the tailpiece may be linked together by way of a covalent bond between the C-termin
  • 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 [00182] 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.
  • 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.
  • 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
  • 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 9
  • 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
  • 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.
  • 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.
  • the polypeptide chain comprises a glutamic acid at the residue position corresponding to residue position 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.
  • 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
  • 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
  • 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
  • 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.
  • 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.
  • 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.
  • 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
  • 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
  • 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
  • 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.
  • 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.
  • the polypeptide chain comprises a glutamic acid at the residue position corresponding to residue position 104 of SEQ ID NO: 6.
  • polypeptide chains comprising: A (Antigen) – B (CH1) – C (hinge) – D (CH2) – E (Optional Interdomain Linker) – F (CH3) – G (tailpiece)
  • Formula I may cover the following representative combinations of Table A.
  • 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.
  • the fusion protein comprises 2 to 12 monomer units of the description.
  • the multimeric fusion protein comprise 6 monomer units.
  • the multimeric fusion protein is in the form of a cyclic hexamer.
  • the description provides for a hexamer of dimers.
  • 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.
  • 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.
  • 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.
  • the IL-31 may be canine.
  • the antigen comprises a canine IL-31, or a fragment or variant thereof that is capable of binding to canine IL-31 receptor.
  • the antigen comprises a mature canine IL-31, or a fragment or variant thereof that is capable of binding to canine IL-31 receptor.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • the CH2 domain is from a human antibody, or is a functional fragment or variant of a CH2 domain from a human antibody.
  • the CH2 domain is from an IgG-B, IgA, IgD, IgE, IgG, or IgM antibody or is a functional fragment or variant thereof.
  • 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.
  • the CH2 domain is from an IgG-B, IgA, or IgG antibody or is a functional fragment or variant thereof. [00209] In certain embodiments, the CH2 domain is from a canine IgG-B antibody or is a functional fragment or variant thereof.
  • 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: 13 or is a conservatively-substituted variant thereof.
  • the CH2 domain is from an IgA antibody or is a functional fragment or variant thereof.
  • the CH2 domain is from an IgA1 or IgA2 antibody or is a functional fragment or variant thereof.
  • 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.
  • 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.
  • 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.
  • the CH2 domain is from an IgG1 or IgG4 antibody or is a functional fragment or variant thereof.
  • 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. [00215] 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: 9 or is a conservatively-substituted variant thereof.
  • 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. [00217] 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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 antigen comprises a cysteine at the residue position corresponding to residue position 309 of SEQ ID NO: 10.
  • 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.
  • 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.
  • 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.
  • the CH3 Domain [00224] 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.
  • the CH3 domain is from a human antibody, or is a functional fragment or variant of a CH3 domain from a human antibody.
  • the CH3 domain is from an IgG-B, IgA, IgD, IgE, IgG, or IgM antibody or is a functional fragment or variant thereof.
  • 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.
  • the CH3 domain is from an IgG-B, IgA, or IgG antibody or is a functional fragment or variant thereof.
  • the CH3 domain is from a canine IgG-B antibody or is a functional fragment or variant thereof.
  • 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.
  • the CH3 domain is from an IgA antibody or is a functional fragment or variant thereof.
  • the CH3 domain is from an IgA1 or IgA2 antibody or is a functional fragment or variant thereof.
  • 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.
  • 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.
  • 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 IgG1, IgG2, IgG3, or IgG4 antibody, or is a conservatively-substituted variant thereof.
  • the CH3 domain is from an IgG1 or IgG4 antibody or is a functional fragment or variant thereof.
  • 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. [00233] 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.
  • the CH3 domain retains an arginine at the residue position corresponding to residue position 355 of SEQ ID NO: 15.
  • 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.
  • 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.
  • 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 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).
  • 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.
  • 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.
  • the mutation is the presence of an additional cysteine as compared with the CH3 domain from wild-type human IgG1 antibody.
  • 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.
  • 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.
  • the CH4 Domain [00240]
  • 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.
  • the CH4 domain is from a human antibody, or is a functional fragment or variant of a CH4 domain from a human antibody.
  • the CH4 domain is from an IgE or IgM antibody or is a functional fragment or variant thereof.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • both polypeptide chains of a monomer form cross-links with different monomers, thus resulting in a fusion protein comprising at least three monomers.
  • 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.
  • the tailpiece is from a human antibody, or is a functional fragment or variant of a tailpiece from a human antibody.
  • the tailpiece is from an IgA or IgM antibody or is a functional fragment or variant thereof.
  • the tailpiece 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 tailpiece of an IgA or IgM antibody, or is a conservatively-substituted variant thereof.
  • the tailpiece is from an IgM antibody or is a functional fragment or variant thereof.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • both polypeptide chains of a monomer form linkages with different monomers, thus resulting it a fusion protein comprising at least three monomers.
  • tailpiece is non-natural and is designed to have increased length and/or flexibility as compared to naturally-occurring tailpieces.
  • 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.
  • the CH1 domain is from a human antibody, or is a functional fragment or variant of a CH1 domain from a human antibody.
  • the CH1 domain may be modified without influencing the function of the fusion protein, hexameric protein, or vaccine thereof.
  • the CH1 domain is from an IgG-B, IgA, IgD, IgE, IgG, or IgM antibody or is a functional fragment or variant thereof.
  • 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.
  • the CH1 domain is from a canine IgG-B antibody or is a functional fragment or variant thereof.
  • the CH1 domain is from an IgA or IgG antibody or is a functional fragment or variant thereof.
  • the CH1 domain is from an IgA antibody or is a functional fragment or variant thereof.
  • the CH1 domain is from an IgA1 or IgA2 antibody or is a functional fragment or variant thereof.
  • 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.
  • the CH1 domain is from an IgG antibody or is a functional fragment or variant thereof.
  • the CH1 domain is from an IgG1, IgG2, IgG3, or IgG4 antibody or is a functional fragment or variant thereof.
  • 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.
  • the CH1 domain is from an IgG1 or IgG4 antibody or is a functional fragment or variant thereof.
  • the CH1 domain is from an IgG1 antibody or is a functional fragment or variant thereof.
  • the CH1 domain is from a human IgG1 antibody or is a functional fragment or variant thereof.
  • 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.
  • the CH1 domain is a functional fragment of the CH1 domain from a canine IgG-B antibody.
  • 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: 24 or is a conservatively-substituted variant thereof.
  • the CH1 domain is a functional fragment of the CH1 domain from a human IgG1 antibody.
  • 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.
  • the CH1 domain is a functional fragment of the CH1 domain from a human IgG4 antibody.
  • 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.
  • 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).
  • 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.
  • 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 region from such an antibody.
  • the hinge region is from a human antibody, or is a functional fragment or variant of a hinge region from a human antibody.
  • suitable hinge regions include those disclosed in US 2007/0081406, the contents of which are incorporated by reference in their entirety.
  • the hinge region is from an IgG-B, IgA, IgD, IgE, IgG, or IgM antibody or is a functional fragment or variant thereof.
  • 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.
  • the hinge region is from an IgG-B antibody or is a functional fragment or variant thereof.
  • 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.
  • the hinge region is from an IgA or IgG antibody or is a functional fragment or variant thereof. [00270] 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.
  • 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 IgA1 or IgA2 antibody, or is a conservatively-substituted variant thereof.
  • the hinge region is from an IgG antibody or is a functional fragment or variant thereof.
  • the hinge region is from an IgG1, IgG2, IgG3, or IgG4 antibody or is a functional fragment or variant thereof.
  • 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.
  • the hinge region is from an IgG1 or IgG4 antibody or is a functional fragment or variant thereof. [00273] 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.
  • 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.
  • 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.
  • 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: 27 or is a conservatively-substituted variant thereof.
  • 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).
  • 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.
  • 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.
  • the mutation is the presence of one fewer cysteine as compared with the hinge region from wild-type human IgG1 antibody.
  • 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 [00280] 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. [00281] In an embodiment, the nucleic acid encodes an antigen of the present disclosure.
  • 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.
  • the nucleic acid encodes a CH2 domain of the present disclosure.
  • 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.
  • the nucleic acid encodes a CH3 domain of the present disclosure.
  • 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.
  • the nucleic acid encodes a tailpiece of the present disclosure.
  • 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.
  • the nucleic acid encodes a CH1 domain of the present disclosure.
  • 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: 52–54, or is a codon degenerate variant thereof.
  • the nucleic acid encodes a hinge region of the present disclosure.
  • 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.
  • the nucleic acid encodes a polypeptide chain of the present disclosure.
  • 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.
  • 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.
  • 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.
  • 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.
  • the nucleic acid can be integrated into a cell’s genome through gene editing systems that utilize CRISPR, TALEN or Zinc-Finger nucleases.
  • 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.
  • the vector is a plasmid, a mini- circle DNA, a nanoplasmid, a viral vector, an episomal vector, or a non-viral vector.
  • viral vectors for use in the present disclosure include lentiviral vectors and retroviral vectors.
  • non-viral vectors for use in the present disclosure include transposons.
  • the vector may include sequences for serine recombinase mediated integration.
  • 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.
  • 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.
  • the polynucleotide can be associated with a lipid.
  • 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.
  • a non-viral vector is a transposon.
  • transposase Any transposon known in the art for delivering a nucleic acid may be used in the practice of the present disclosure.
  • 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.
  • Any viral vector known in the art for delivering nucleic acids may be used in the practice of the present disclosure.
  • adenoviral vectors e.g., the adenovirus-based Per.C6 system available from Crucell, Inc. (Leiden, The Netherlands)
  • adeno-associated virus based vectors e.g., the lentiviral-based pLPI from Life Technologies (Carlsbad, Calif.)
  • retroviral vectors e.g., the pFB-ERV plus pCFB-EGSH
  • herpes virus-based vectors e.g., the viral vector is an adenoviral vector.
  • 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.
  • 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.
  • 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.
  • Reporter genes can be used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
  • 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 the nucleic 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.
  • 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.
  • 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.
  • 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.
  • the polymer chains within a monomer unit may be identical to each other or different.
  • a monomer unit comprises two identical polypeptide chains.
  • representative polypeptide chains are those described in SEQ ID NO: 1 – 6, Formula I, and Table A.
  • the monomer units are capable of cross-linking with each other to form multimeric fusion proteins.
  • 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.
  • 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.
  • the monomer units in a multimeric fusion protein may each be identical or different from one another.
  • each monomer unit in a multimeric fusion protein is identical to each other.
  • the multimeric fusion protein comprises six identical monomers and is in the form of a cyclic hexamer.
  • 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.
  • 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.
  • 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.
  • 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.
  • the monomers are identical to one another and each comprise two polypeptide chains that are identical to one another.
  • 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.
  • 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.
  • 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.
  • the monomers are identical to one another and each comprises two polypeptide chains that are identical to one another.
  • 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.
  • 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.
  • the Il-31 may be canine.
  • 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. [00314] 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the method comprises transfecting a cell with a nucleic acid of the present disclosure.
  • the method comprises transfecting a cell with a vector comprising the nucleic acid.
  • the nucleic acid may comprise a transposon.
  • 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.
  • the method further comprises transfecting the cell with a vector encoding a transposase.
  • 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.
  • Methods of introducing into a cell and expressing genes in a cell are known in the art.
  • 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.
  • the vector can be transferred into a cell by physical, chemical, or biological means.
  • 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)).
  • a method for the introduction of a nucleic acid into a host cell is calcium phosphate transfection or polyethylenimine (PEI) Transfection.
  • PEI polyethylenimine
  • a method for introduction of a polynucleotide into a host cell is electroporation.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the method may further include isolating the antigen, the polypeptide chain, or a multimeric fusion protein comprising the polypeptide chain.
  • Compositions [00329] The present disclosure relates in part to a composition comprising an immunogenically-effective amount of the antigen of the present disclosure.
  • the present disclosure also relates in part to a composition comprising an immunogenically-effective amount of the multimeric fusion protein of the present disclosure.
  • the composition comprises a heterologous mixture of multimeric fusion proteins having differing amounts of monomers.
  • the composition may comprise multimeric fusion proteins having six monomers and multimeric fusion proteins comprising twelve monomers.
  • 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.
  • the present disclosure also relates in part to a composition as described above for use in producing an immunogenic effect in a subject.
  • the subject is canine.
  • 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.
  • 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.
  • the kit comprises a carrier, package, label, or container. Suitable containers include, for example, bottles, vials, syringes, and test tubes.
  • Adjuvants [00336] 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. [00337] Representative adjuvants include oil-based adjuvants, such as Freund's Complete Adjuvant and Freund's Incomplete Adjuvant, Amphigen, and glycerine.
  • 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.
  • Immunostimulating complexes 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.
  • 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 TM PET Gel A and vaccine grade Poly (I:C) (VacciGrade; Invivogen) are also provided.
  • 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 C10-30 low ) and medium (PAA C10-30 medium ) degree of C10-30 alkylation exhibit high mucoadhesive properties with the additional benefit of emulsifying properties.
  • 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.).
  • PCPP polymer polymer
  • Virus Research Institute, USA poly(di(carboxylatophenoxy)phosphazene
  • MPL monophosphoryl lipid A
  • MDP muramyl dipeptide
  • t-MDP threonyl-muramy
  • Some adjuvants include surface active substances (e.g. lysolecithin, pluronic polyols, polyanions, peptides, nanoemulsions, keyhole limpet hemocyanin, and dinitrophenol).
  • 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.
  • an immunogenic composition comprising a nanoemulsion adjuvant and an immunogen comprises a single additional immunostimulatory compound/molecule and/or adjuvant.
  • 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.
  • adjuvants such as cytokines (e.g., interleukins (e.g., IL-2, IFN- ⁇ , 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.
  • cytokines e.g., interleukins (e.g., IL-2, IFN- ⁇ , IL-4, etc.), macrophage colony stimulating factor, tumor necrosis factor, etc.
  • a bacterial ADP-ribosylating toxin such as a cholera toxin (CT), a pertussis toxin (PT), or an ⁇ E.
  • Coli ⁇ heat-labile toxin 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 the wild-type amino acid at position 109
  • PT-K9/G129 where lysine is substituted for the wild-type amino acid at position 9 and glycine substituted at position 129)
  • immunogenic compounds/substances e.g., that stimulate an immune response
  • an immunogenic composition comprising an adjuvant formulation comprising an emulsion in combination with one or more immunostimulatory compounds of the present disclosure.
  • the amount of Quil-A ® present is about 5 ⁇ g, about 10 ⁇ g, about 25 ⁇ g, about 50 ⁇ g, about 75 ⁇ g, about 100 ⁇ g, or about 200 ⁇ g in compositions, fusion protein compositions, or IL-31 hexamer compositions described herein.
  • the amount of Quil-A ® is present is about from 5 ⁇ g – 200 ⁇ g, from 5 ⁇ g – 100 ⁇ g, about 10 ⁇ g to about 100 ⁇ g, about 10 ⁇ g to about 80 ⁇ g, about 20 ⁇ g to about 60 ⁇ g, about 25 ⁇ g to about 100 ⁇ g, about 30 ⁇ g to about 50 ⁇ g, about 25 ⁇ g, about 50 ⁇ g, about 75 ⁇ g, about 100 ⁇ g in compositions, fusion protein compositions, or IL-31 hexamer compositions described herein.
  • the concentration of Carbopol 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • compositions described herein may include fusion proteins, polypeptide chains, oligomers, multimers, and hexamers described herein in an amount of from about 50 ⁇ g to about 1000 ⁇ g/dose, from about 100 ⁇ g to about 500 ⁇ g/dose, from about 100 ⁇ g to about 300 ⁇ g/dose, about 100 ⁇ g/dose, about 200 ⁇ g/dose, about 300 ⁇ g/dose, about 500 ⁇ g/dose, or more than 600 ⁇ g/dose.
  • compositions described herein may include an adjuvant in an amount of from about 50 ⁇ g to 1000 ⁇ g/dose, 50 ⁇ g to 150 ⁇ g/dose from about 100 ⁇ g to 500 ⁇ g/dose, from about 100 ⁇ g to 300 ⁇ g/dose, about 100 ⁇ g/dose, about 200 ⁇ g/dose, about 300 ⁇ g/dose, about 500 ⁇ g/dose, or more than 600 ⁇ g/dose and fusion proteins, oligomers, multimers, and hexamers described herein in an amount of from about 50 ⁇ g to 1000 ⁇ g/dose, from about 100 ⁇ g to 500 ⁇ g/dose, from about 100 ⁇ g to 300 ⁇ g/dose, from about 100 ⁇ g to 400 ⁇ g/dose, about 100 ⁇ g/dose, about 200 ⁇ g/dose, about 300 ⁇ g/dose, about 500 ⁇ g/dose, or more than 600 ⁇ g/dose.
  • an adjuvant in an amount of from about 50 ⁇ g to 1000 ⁇ g
  • 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.
  • compositions described herein and an adjuvant described herein exhibit stability such that the compositions can be used as an effective pharmaceutical vaccine from 1oC to 30 oC.
  • 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, 8 oC to 16 oC, 8 oC to 25 oC, 8 oC to 30 o
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the subject is a mammal, for example a canine.
  • 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.
  • the subject is a mammal, for example a canine.
  • the antigen, multimeric fusion protein, or composition are administered to an animal in need thereof, for example, a canine, from about 50 ⁇ g to 1000 ⁇ g/dose, from about 100 ⁇ g to 500 ⁇ g/dose, from about 100 ⁇ g to 300 ⁇ g/dose, about 100 ⁇ g/dose, about 200 ⁇ g/dose, about 300 ⁇ g/dose, about 500 ⁇ g/dose, or more than 600 ⁇ g/dose.
  • 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.
  • the antigen or multimeric fusion proteins described herein are administered to an animal in need thereof, for example, a canine, from about 50 ⁇ g to 1000 ⁇ g/dose, from about 100 ⁇ g to 500 ⁇ g/dose, from about 100 ⁇ g to 300 ⁇ g/dose, about 100 ⁇ g/dose, about 200 ⁇ g/dose, about 300 ⁇ g/dose, about 500 ⁇ g/dose, or more than 600 ⁇ g/dose.
  • 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.
  • the disease or disorder is an itch-related disorder, for example atopic dermatitis.
  • 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.
  • composition described herein can be administered to a subject subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
  • the composition is administered to a particular site on a body, for example the interscapular region.
  • 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.
  • 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.
  • 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.
  • the compositions described herein can be administered as a combination therapy with an additional therapeutic agent.
  • 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.
  • 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.
  • 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.
  • 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.
  • the canine IL-31 hexamer that result in autoimmunity and the formation of antibodies to IL-31 is SEQ ID NO: 2.
  • the canine IL-31 is selected from SEQ ID NO: 7 or SEQ ID NO: 8.
  • 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 10 years.
  • 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.
  • additional doses or boosters of IL-31 hexamers results in higher anti IL-31 antibody titer relative to the initial course of immunization.
  • 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.
  • the number of boosters administered are selected from one, two, three, four, and five booster doses of IL-31 hexamers.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. [00377] 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.
  • 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).
  • 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).
  • 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.
  • 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%.
  • 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.
  • 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.
  • the white blood cell count will be a number selected from 4 k per ⁇ l, 4.5 k per ⁇ l, 4.9 k per ⁇ l, 5 k per ⁇ l, 6 k per ⁇ l, 7 k per ⁇ l, 8 k per ⁇ l, 9 k per ⁇ l, 10k per ⁇ l, 11 k per ⁇ l, 12k per ⁇ l, 13k per ⁇ l, 14k per ⁇ l, 15k per ⁇ l,16 k per ⁇ l, 17k per ⁇ l, 17.9k per ⁇ l, 18k per ⁇ l, 19k per ⁇ l, or 20k per ⁇ l.
  • 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.
  • 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 10 months of duration, 90%
  • 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,
  • 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%
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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 4 times, at least 5 times, at least 10 times, and at least 20 times as long as Lokivetmab (SEQ ID NO:
  • 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.
  • 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. [00393] The cells were then removed by centrifugation.
  • Multimeric fusion proteins were purified from the supernatants by protein A chromatography using MabSelect TM SuRe TM resin (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 Fusion Protein [00394]
  • 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.
  • Example 3 Expression and Purification of IL-31[WT]-IgG4 Fc Hexameric Fusion Protein [00395]
  • 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.
  • Analytical size exclusion high performance liquid chromatography was performed using a Dionex TM Ultimate 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.
  • Example 2 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 N o. Peak Name Retention Area Height Relative Relative t ime (min) mAU*min mAU Area % Height [00403] 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.
  • 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.
  • 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.
  • 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 N o.
  • Example 9 Binding of the IL-31 Variant Antigens to IL31 Receptor [00411] The extracellular domain of IL-31 receptor alpha (IL31Ra), which contains the IL- 31 binding domain, was expressed recombinantly in HEK293 cells.
  • Dogs were ranked according to pruritus score within gender. To qualify for randomization, dogs must have had 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. [00417] Among the six treatment groups, three of them were treated with placebo, positive control, and the Hexameric IgG-Fc-IL31 vaccine, respectively.
  • Group (1) Placebo (vaccine buffer solution: 5 mM Na phosphate, 2 mM EDTA, pH 7.5 ), 100 ⁇ g saponin adjuvant;
  • Group (2) Lokivetmab (positive control) dosed according to label instructions (2 mg/kg subcutaneously);
  • the Hexameric IgG-Fc-IL31 vaccine included 300 ⁇ g 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.
  • 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. [00421] 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.
  • 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 ( ⁇ l) Final Concentration b) Formulated Hexameric IgG-Fc-IL31 plus Carbopol® 971 (Lubrizol) at Lower Concentration Ingredient Volume ( ⁇ l) Final t ti Final Injection 1000 Volume c) Formulated Hexameric IgG-Fc-IL31 + Carbopol® 971 at Higher Concentration Ingredient Volume ( ⁇ l) Final Concentration d) Formulated Hexameric IgG-Fc-IL31 plus 10% Carbopol® 971 and CpG oligonucleotides 1668-PTO and 2006-PTO (Invivogen) Ingredient Volume ( ⁇ l) Final Concentration e) Formulated Hexameric IgG-Fc-IL31 plus Quil-
  • 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).
  • 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.
  • 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.
  • 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
  • the positive control is IL31 hexamers (SEQ ID NO: 2) plus 100 ⁇ g Quil A®.
  • 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 Induration Table 12B Local Tolerance Assessment, Day 14 Day 15 Day 14 Day 16 (48 hrs) on ⁇ Slight erythema for the dog with induration. Carbopol 0.1% 1(1) 0 0 0 1 (2) 1(1) 0 0 Same as diff Example 12. Analysis of Hexamers Using SDS-PAGE and Chromatographic Techniques [00432] 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. [00433] 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).
  • 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 (T m ), temperature of unfolding onset (T onset ) (FIG.18A).
  • T m melting temperature
  • T onset temperature of unfolding onset
  • FIG.18B 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).
  • 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.
  • 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).
  • RP-HPLC reverse phase high performance liquid chromatography
  • the protein demonstrates similar stability at 5° C and 25° C.
  • 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).
  • 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).
  • Hexamer Potency Assay 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 NaCO 3 / NaHCO 3 pH 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).
  • TMB 3,3',5,5'- Tetramethylbenzidine
  • the conjugate was diluted in a range of 1:10 2 to 1:10 6 . This was assayed with the S18-A antibody capture antibody at 1:6000, the detection antibody at 1:10 5 , 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.
  • the antigen (Hexameric IgG-Fc- IL31, SEQ ID NO: 2) was diluted in a range of 10 3 to 10 8 fold 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.
  • Example 15 Potency Assay to Determine Stability of Hexameric IgG-Fc-IL31 in Formulations
  • 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 Buffer1 containing Carbopol® 971 (0.02%)
  • Carbopol® 971 0.1%)
  • Example 16 Efficacy and Tolerability of IL-31 immunization in Dogs
  • SEQ ID NO: 2 Hexameric IgG-Fc-IL31
  • 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 x Complete Blood Count 3 , Blood Ch i t T t x x [00452] 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.
  • 1 st Vax 2nd Vax 3rd Vax 4th Vax Table 16 Number of dogs exhibiting transient pain upon palpitation, induration, increased heat at the injection site, or erythema at the injection site.
  • 1 st Vax 2nd Vax 3rd Vax 4th Vax 3 CBC 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. [00453] 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
  • Negative Control PBS / Mock immunization
  • AST Aspartate Aminotransferase: AST levels were generally within the normal range (16-55 U/L) for study days 28-288 post 2nd vaccination.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • T01 placebo control
  • T02 Quil-A ® -adjuvanted hex hIgG1-Fc-cIL-31vaccine
  • T03 non- adjuvanted hex hIgG1-Fc-cIL-31vaccine
  • T03 did not further continued after six months (SD 196).

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Abstract

La description concerne des chaînes polypeptidiques comprenant des antigènes, par exemple un antigène IL-31 mutant ; un domaine CH2 ; et un domaine CH3. Les chaînes polypeptidiques comprennent éventuellement en outre un domaine CH1, une région charnière, un domaine CH4 et/ou un empennage. L'invention concerne en outre des protéines multimères et hexamères capables de traiter des troubles liés aux démangeaisons, par exemple la dermatite atopique, chez les animaux. L'invention concerne en outre l'utilisation de protéines multimères et hexamères décrites ici en tant que vaccins.
PCT/US2025/020279 2024-03-18 2025-03-17 Composés, compositions, vaccins multimères et procédés de traitement de la dermatite Pending WO2025199054A1 (fr)

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WO2022025643A1 (fr) * 2020-07-29 2022-02-03 (주)메디톡스 Protéine de fusion fc hétérodimère, et composition, utilisation et procédé associés
WO2022212593A1 (fr) * 2021-03-31 2022-10-06 The Regents Of The University Of California Fusions agent de liaison bispécifique-ligand pour la dégradation de protéines cibles
WO2023047129A1 (fr) * 2021-09-23 2023-03-30 St George's Hospital Medical School Peptides de fusion immunogènes comprenant un fragment fc et une sous-unité b non toxique d'une toxine ab5

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022025643A1 (fr) * 2020-07-29 2022-02-03 (주)메디톡스 Protéine de fusion fc hétérodimère, et composition, utilisation et procédé associés
WO2022212593A1 (fr) * 2021-03-31 2022-10-06 The Regents Of The University Of California Fusions agent de liaison bispécifique-ligand pour la dégradation de protéines cibles
WO2023047129A1 (fr) * 2021-09-23 2023-03-30 St George's Hospital Medical School Peptides de fusion immunogènes comprenant un fragment fc et une sous-unité b non toxique d'une toxine ab5

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