WO2013164435A2 - Protéine de fusion de la protéine lymphopoiétine stromale thymique canine avec la région fc d'igg - Google Patents

Protéine de fusion de la protéine lymphopoiétine stromale thymique canine avec la région fc d'igg Download PDF

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WO2013164435A2
WO2013164435A2 PCT/EP2013/059212 EP2013059212W WO2013164435A2 WO 2013164435 A2 WO2013164435 A2 WO 2013164435A2 EP 2013059212 W EP2013059212 W EP 2013059212W WO 2013164435 A2 WO2013164435 A2 WO 2013164435A2
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canine
tslp
amino acid
fusion protein
seq
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PCT/EP2013/059212
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WO2013164435A3 (fr
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Mohamad Morsey
Jeanine D. Mattson
Dan M. GORMAN
Rene De Waal Malefyt
Leonard G. Presta
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Intervet International B.V.
Intervet Inc.
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Publication of WO2013164435A2 publication Critical patent/WO2013164435A2/fr
Publication of WO2013164435A3 publication Critical patent/WO2013164435A3/fr

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    • 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]
    • C07K14/5418—IL-7
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/14—Decongestants or antiallergics
    • 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

  • the present invention relates to fusion proteins of the thymic stromal lymphopoietin protein ("TSLP") with the Fc region of IgG, nucleic acid molecules encoding the fusion proteins, vectors and host cells comprising the fusion proteins, and methods of making and using the fusion proteins.
  • TSLP thymic stromal lymphopoietin protein
  • the immediate hypersensitivity observed in atopic diseases results from exposure to specific allergens, such as the house dust mite (Dermatophagoides pteronyssinus), pollens, molds, and danders.
  • individuals having an atopic disease are more likely to suffer from asthma, atopic dermatitis, as well as other disorders related to endogenous IgE release.
  • Atopic diseases such as allergic dermatitis, asthma, and the like, also occur in the canine species, including in domestic dogs. Such dogs generally begin to show signs of atopy between one and three years of age. Due to the hereditary nature of the disease, several breeds, including golden retrievers, most terriers, Irish setters, Lhasa apsos, dalmatians, bulldogs, and Old English sheep dogs have a greater tendency to be atopic, though other types of dogs, including mixed breeds, also are known to suffer from this condition. The incidence of at least one particular type of atopy, atopic dermatitis, is increasing significantly in both humans and canines alike.
  • Atopic canines will usually rub, lick, chew, bite or scratch at their feet, muzzle, ears, armpits or groin area, resulting in hair loss, reddening, and thickening of the skin.
  • several skin conditions combine to cause an animal to itch when a single allergy alone would not have resulted in such itching.
  • These aggravating problems can be due to air borne-allergens (pollens, etc.), allergens in food, and allergens from parasites (fleas, etc.).
  • Bacterial and/or yeast infections of the skin also can augment the itching sensation.
  • veterinary practitioners have treated canine atopic dermatitis by administering oral antihistamines, oral or topical corticosteroid anti-inflammatory agents, other immune system suppressants, such as cyclosporine or tacrolimus, fatty acid supplements, and allergen specific immunotherapy (which requires injection of the identified antigen).
  • oral antihistamines oral or topical corticosteroid anti-inflammatory agents
  • other immune system suppressants such as cyclosporine or tacrolimus
  • fatty acid supplements such as cyclosporine or tacrolimus
  • allergen specific immunotherapy which requires injection of the identified antigen
  • the mammalian immune response is based on a series of complex cellular interactions, called the "immune network". Much of the immune response revolves around the network-like interactions of lymphocytes, macrophages, granulocytes, and other cells, with soluble proteins called cytokines playing a critical role in mediating/controlling/regulating these cellular interactions. Thus, cytokines and immune cells serve to mediate specific physiological mechanisms or pathways leading to the various inflammatory disorders.
  • Allergic inflammation is the result of a complex immunological cascade which leads T cells to produce dysregulated TH2-derived cytokines such as IL-4, IL-5, and IL-13. These cytokines, in turn, trigger bronchial hyperreactvity, IgE production,
  • Thymic Stromal Lymphopoietin protein is an IL-7-like cytokine that was initially identified in mice as a factor that supported: (i) the in vitro development of surface lgM + B cells, and (ii) B and T cell proliferation [Friend et al., Exp Hematology 22/321 -328 (1994), see also, Levin et al., J. Immunol. 162: 677-683 (1999), and Liu et al., Annu. Rev. Immunol. 25:193-219 (2007)].
  • TSLP Thymic Stromal Lymphopoietin protein
  • TSLP is now known to bind a cellular receptor comprising IL-7R-alpha subunit and a unique receptor subunit called TSLP-R [see, e.g., co-owned U.S. Patent Nos. 6,890,734; 7,071 ,308; and 7,569,224; and Reche et al., The Journal of Immunology 167 ':336-343 (2001 )].
  • This interaction triggers signal transduction via STAT activation or Thymus and
  • Activation-Regulated Chemokine (TARC) expression in a hematopoietic cell e.g., a myeloid lineage cell such as a monocyte, or a dendritic cell.
  • TSLP has been identified in a number of species including humans [Reche et al., J. Immunol. 336- 343 (2001 ); U.S. 6,555,520], chimpanzees [WO 2008/066444], mice [Sims et al, J. exp. Med., 192:671 -680 (2000)], horses [Klukowska-Rotzler et al., Vet. Immunol. Immunopathol. 135(3-4) 346-349 (2010)], and canines [U.S. Patent Nos.
  • TSLP also may play a significant role in mice in the pathogenesis of allergic diseases such as atopic dermatitis and asthma.
  • transgenic mice in which the expression of TSLP gene was specifically induced in the skin show immunological and clinical features of atopic dermatitis such as eczematous lesions containing inflammatory dermal cellular infiltrates, a dramatic increase in Th 2 CD4 + T cells expressing skin homing receptors, and elevated serum levels of IgE.
  • mice expressing a lung-specific TSLP transgene show immunological and clinical features of asthma including massive infiltration of leuckocytes, goblet cell hyperplasia, sub-epithelial fibrosis, an increase in T helper type 2 cytokines, and increased levels of IgE.
  • IgG antibodies are predominantly involved in the secondary immune response with the presence of IgG antibodies generally corresponding to the maturation of the antibody response.
  • IgG antibodies possess a high affinity for the Fc receptors on phagocytic cells that is dependent on the class of the antibody (i.e., lgG1 , lgG2a, lgG3, or lgG4) as well as to the species of origin of that antibody.
  • Fc regions of IgG antibodies comprise the CH2 and CH3 domains of the IgG heavy chain and the hinge region.
  • Fc regions of IgG have been combined with the functional domains of receptors or ligands for example, in fusion proteins called immunoadhesins, and research tools for constructing such immunoadhesins are commercially available from Life Technologies/lnvitrogen, in Carlsbad, California. Though the Fc regions of the four classes of IgG have about 95% similarity, the structure of the hinge regions differ significantly. Indeed, it is the hinge region of the IgG antibody that provides each of the four IgG classes their unique biological profile.
  • the present invention provides new and more practical treatments for atopic disorders, including atopic dermatitis and its associated clinical manifestations.
  • the present invention provides novel fusion proteins that comprise canine thymic stromal lymphopoietin protein (and/or antigenic fragments of the protein) and the Fc region of IgG from a non-canine mammalian species (i.e., an Fc region of a non-canine mammalian IgG antibody) to form an Fc-TSLP.
  • the Fc-TSLP comprises a canineTSLP (or an antigenic fragment of the canine TSLP) together with a murine Fc region of the IgG.
  • the Fc-TSLP comprises a canineTSLP (or an antigenic fragment of the canine TSLP) together with a human Fc region of the IgG.
  • the non-canine mammalian Fc region of the IgG of an Fc-TSLP of the present invention is from a murine IgG antibody.
  • the Fc region of the murine IgG in the Fc-TSLP fusion protein comprises the amino acid sequence of SEQ ID NO: 10.
  • the Fc region of the murine IgG in the Fc-TSLP fusion protein is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 9.
  • the Fc region of the murine IgG in the Fc-TSLP fusion protein comprises the amino acid sequence of SEQ ID
  • the Fc region of the murine IgG in the Fc-TSLP fusion protein is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 109.
  • the non-canine mammalian Fc region of the IgG is from a human antibody.
  • the Fc region of the human IgG in the Fc-TSLP fusion protein comprises the amino acid sequence of SEQ ID NO: 108.
  • the Fc region of the human IgG in the Fc-TSLP fusion protein is encoded by a nucleic acid
  • non-canine mammalian Fc region of the IgG is from an equine antibody. In yet other embodiments the non-canine mammalian Fc region of the IgG is from a porcine antibody. In still other embodiments the non-canine mammalian Fc region of the IgG is from a bovine antibody.
  • the IgG antibody is an lgG2a. In other embodiments the IgG antibody is an lgG1 . In still other embodiments the IgG antibody is an lgG3. In yet other embodiments the IgG antibody is an lgG4.
  • Antigenic fragments of a canine TSLP include those that comprise one or more epitopes individually defined by the amino acid sequences of SEQ ID NOs: 1 1 -106 [see also, U.S. 7,718,772 B2, the contents of which are hereby incorporated by reference in its entirety]. Without listing each combination separately, it should be understood that an Fc-TSLP fusion protein of the present invention can comprise a canine TSLP protein and/or one or more antigenic canine TSLP fragments together with an Fc region of any non-canine mammalian IgG.
  • such an antigenic fragment of canine TSLP comprises one or more epitopes that comprise an amino acid sequence of SEQ ID NOs: 32, 33, 34, 35, and/or 36.
  • the antigenic fragment can have an amino acid sequence contained within the overlap of the amino acid sequences of SEQ ID NOs: 32, 33, 34, 35, and/or 36, i.e., NPPDCLARIERLTLHRIRGCAS (SEQ ID NO: 105).
  • an antigenic fragment of the canine TSLP protein is capable of binding an anti-human TSLP monoclonal antibody.
  • Antigenic fragments of the amino acid sequence of NPPDCLARIERLTLHRIRGCAS (SEQ ID NO: 105) can range in size from about 5 to about 21 amino acid residues.
  • the antigenic fragment has the amino acid sequence of SEQ ID NOs
  • the present invention provides an Fc-TSLP comprising an amino acid sequence for the TSLP portion of the fusion protein that has 80% or greater identity to the amino acid sequence of SEQ ID NO: 6.
  • Fc-TSLP comprising an amino acid sequence for the TSLP portion of the fusion protein that has 80% or greater identity to the amino acid sequence of SEQ ID NO: 6.
  • antibodies that bind the canine TSLP protein comprising the amino acid sequence of SEQ ID NO: 6 are detectable in the resulting canine sera obtained from the vaccinated canine subject.
  • the Fc-TSLP comprises an amino acid sequence for the TSLP portion of the fusion protein that has 80% or greater identity to the amino acid sequence of SEQ ID NO: 6; and is cross reactive with an antibody raised against the canine TSLP comprising the amino acid of SEQ ID NO: 6.
  • the present invention further provides an Fc-TSLP comprising an amino acid sequence that has 80% or greater identity to the amino acid sequence of SEQ ID NO: 6 for the TSLP portion of the fusion protein, and which binds to an epitope- specific canine TSLP monoclonal antibody.
  • Fc-TSLP comprises an amino acid sequence that has 90% or greater identity to the amino acid sequence of SEQ ID NO: 6 for the TSLP portion of the fusion protein. In still another embodiment, that Fc-TSLP comprises an amino acid sequence that has 95% or greater identity to the amino acid sequence of SEQ ID NO: 6 for the TSLP portion of the fusion protein.
  • the Fc-TSLP comprises the amino acid sequence of SEQ ID NO: 2 for the TSLP portion of the fusion protein.
  • the Fc-TSLP comprises the mature canine TSLP protein that comprises the amino acid sequence of SEQ ID NO: 6 for the TSLP portion of the fusion protein.
  • the Fc-TSLP comprises a canine TSLP together with a non- canine mammalian lgG2a.
  • the Fc-TSLP comprises a canine TSLP together with a murine lgG2a.
  • that Fc- TSLP comprises an amino acid sequence that has 90% or greater identity to the amino acid sequence of SEQ ID NO: 8.
  • Fc-TSLP comprises an amino acid sequence that has 95% or greater identity to the amino acid sequence of SEQ ID NO: 8. In yet other embodiments, that Fc-TSLP comprises an amino acid sequence that has 98% or greater identity to the amino acid sequence of SEQ ID NO: 8. In more particular embodiments of this type the Fc-TSLP comprises the amino acid sequence of SEQ ID NO: 8.
  • an Fc-TSLP of the present invention comprises an amino acid sequence that has 90% or greater identity to the amino acid sequence of SEQ ID NO: 4.
  • that Fc-TSLP comprises an amino acid sequence that has 95% or greater identity to the amino acid sequence of SEQ ID NO: 4.
  • that Fc-TSLP comprises an amino acid sequence that has 98% or greater identity to the amino acid sequence of SEQ ID NO: 4.
  • the Fc-TSLP comprises the amino acid sequence of SEQ ID NO: 4.
  • the present invention further provides nucleic acids that encode any and/or all of the Fc-TSLPs of the present invention. Accordingly, individual nucleic acids are provided that encode each respective Fc-TSLP of the present invention.
  • the nucleic acids encode an Fc-TSLP comprising the amino acid sequence of SEQ ID NO: 2 for the TSLP portion of the fusion protein. In other embodiments, the nucleic acids encode an Fc-TSLP comprising the amino acid sequence of SEQ ID NO: 6 for the TSLP portion of the fusion protein. In particular embodiments of this type, the nucleic acids comprise the nucleotide sequence of SEQ ID NO: 1 for the TSLP portion of the fusion protein. In other embodiments of this type, the nucleic acids comprise the nucleotide sequence of SEQ ID NO: 5 for the TSLP portion of the fusion protein.
  • the nucleic acids encode an Fc-TSLP comprising the amino acid sequence of SEQ ID NO: 4.
  • the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 3.
  • the nucleic acids encode an Fc-TSLP comprising the amino acid sequence of SEQ ID NO: 8.
  • the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 7.
  • the present invention also provides vectors that can individually comprise any and/or all of the nucleic acids that encode one or more Fc-TSLP of the present invention.
  • the vectors are expression vectors that comprise and express a nucleic acid of the present invention.
  • the expression vector is a recombinant viral vector encoding an Fc-TSLP of the present invention.
  • an expression vector comprises a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 7.
  • the expression vector comprises a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3.
  • the present invention provides host cells that comprise such expression vectors.
  • the host cell is optionally a prokaryote or a eukaryote host cell.
  • the prokaryote host cell is an Escherichia coli.
  • the host cell is E. coli BL21 (DE3)/pLysS that contains the T7 RNA polymerase gene under the control of the isopropyl ⁇ -D- thiogalactopyranoside (IPTG)-inducible lacUV5 promoter.
  • the present invention also provides vaccines and/or pharmacines and/or
  • the present invention further provides vaccines and/or pharmacines and/or immunogenic compositions that can comprise an effective amount of an expression vector of the present invention that can express an Fc- TSLP of the present invention.
  • Such vectors can be used, e.g., in pharmacines that are suitable for administration into a canine having atopic dermatitis, see below.
  • the expression vector is a recombinant viral vector.
  • the recombinant viral vector is a canine adenovirus type 2 vector.
  • the expression vector is a naked DNA vector.
  • the expression vectors of the present invention can be used, for example, e.g., in pharmacines that are suitable for administration into a canine having atopic dermatitis.
  • the vaccines and/or pharmacines and/or immunogenic compositions of the present invention can further comprise a pharmaceutically acceptable carrier and/or an adjuvant.
  • a vaccine and/or pharmacine and/or immunogenic composition of the present invention can further comprise an effective amount of a non-TSLP
  • a vaccine and/or pharmacine and/or immunogenic composition of the present invention may be employed in a method of inducing anti-TSLP antibodies.
  • One such method comprises immunizing an animal subject with an effective amount of the vaccine and/or pharmacine and/or immunogenic composition of the present invention.
  • This method optionally includes a method of downregulating TSLP activity in the animal subject and/or a method of treating or preventing allergic symptoms in an atopic animal subject that comprises immunizing the animal subject with an effective amount of the vaccine and/or pharmacine and/or immunogenic
  • the allergic symptoms ameliorated can include allergic dermatitis, and/or asthma, and the like.
  • the animal subject is a canine.
  • the animal subject is a feline.
  • a vaccine and/or pharmacine and/or immunogenic composition of the present invention may be administered by many routes including: intramuscular injection, subcutaneous injection, intravenous injection, intradermal injection, oral
  • the present invention also provides methods of producing an Fc-TSLP protein of the present invention.
  • One such method comprises culturing a host cell of the present invention in a suitable culture medium. This method can further include the step of isolating and/or purifying the Fc-TSLP protein from the cultured host cell or the culture medium. The resulting isolated and/or purified Fc-TSLP protein is also part of the present invention.
  • the present invention additionally provides diagnostic methods employing the inventive Fc-TSLP proteins.
  • the present invention provides a method of diagnosing atopic dermatitis in an animal subject comprising obtaining an epidermal sample from the animal subject and determining the presence of TSLP in the epidermal sample with an antibody raised against a canine Fc-TSLP.
  • composition of the present invention also can be used to elicit antiserum for screening and/or identifying a TSLP, e.g., as an aid in a test kit for identifing animals that overexpress TSLP.
  • a composition of the present invention also can be used to elicit antiserum for screening and/or identifying a TSLP, e.g., as an aid in a test kit for identifing animals that overexpress TSLP.
  • the animal subject is a canine. In another embodiment, the animal subject is a feline.
  • the present invention further provides isolated and/or recombinant canine thymic stromal lymphopoietin protein receptor (TSLPR) proteins and canine interleukin-7Ra (IL-7Ra) proteins, isolated and/or recombinant nucleic acids that encode canine TSLPR and/or canine IL-7Ra, along with recombinant vectors that comprise and express the canine TSLPR and canine IL-7Ra proteins either individually or together.
  • TSLPR canine thymic stromal lymphopoietin protein receptor
  • IL-7Ra canine interleukin-7Ra
  • isolated and/or recombinant nucleic acids that encode canine TSLPR and/or canine IL-7Ra along with recombinant vectors that comprise and express the canine TSLPR and canine IL-7Ra proteins either individually or together.
  • the present invention provides cells that have have been modified to express canine TSLPR and/or canine IL-7R
  • the cell is a BaF3 cell.
  • the isolated and/or recombinant canine TSLPR of the present invention comprises an amino acid sequence that has 95% or greater identity to the amino acid sequence of SEQ ID NO: 1 14. In still other embodiments, the isolated and/or recombinant canine TSLPR comprises an amino acid sequence that has 98% or greater identity to the amino acid sequence of SEQ ID NO: 1 14. In more particular embodiments of this type the isolated and/or recombinant canine TSLPR comprises the amino acid sequence of SEQ ID NO: 1 14. In a specific embodiment of this type, the isolated and/or recombinant canine TSLPR comprises the amino acid sequence of SEQ ID NO: 1 12.
  • the present invention provides isolated and/or recombinant nucleic acids encoding a canine TSLPR that comprises an amino acid sequence that has 95% or greater identity to the amino acid sequence of SEQ ID NO: 1 14.
  • the isolated and/or recombinant nucleic acid encodes a canine TSLPR that comprises an amino acid sequence that has 98% or greater identity to the amino acid sequence of SEQ ID NO: 1 14.
  • the isolated and/or recombinant nucleic acid encodes a canine TSLPR that comprises the amino acid sequence of SEQ ID NO: 1 14.
  • the isolated and/or recombinant nucleic acid comprises the nucleotide sequence of SEQ ID NO: 1 13.
  • the isolated and/or recombinant nucleic acid encodes a canine TSLPR that comprises the amino acid sequence of SEQ ID NO: 1 12.
  • the isolated and/or recombinant nucleic acid comprises the nucleotide sequence of SEQ ID NO: 1 1 1 .
  • the isolated and/or recombinant canine IL-7Ra of the present invention comprises an amino acid sequence that has 95% or greater identity to the amino acid sequence of SEQ ID NO: 1 18. In still other embodiments, the isolated and/or recombinant canine IL-7Ra comprises an amino acid sequence that has 98% or greater identity to the amino acid sequence of SEQ ID NO: 1 18. In more particular embodiments of this type the isolated and/or recombinant canine IL-7Ra comprises the amino acid sequence of SEQ ID NO: 1 18. In a specific embodiment of this type, the isolated and/or recombinant canine IL-7Ra comprises the amino acid sequence of SEQ ID NO: 1 16.
  • the present invention provides an isolated and/or
  • the isolated and/or recombinant nucleic acid encoding a canine IL-7Ra that comprises an amino acid sequence that has 95% or greater identity to the amino acid sequence of SEQ ID NO: 1 18.
  • the isolated and/or recombinant nucleic acid encodes a canine IL-7Ra that comprises an amino acid sequence that has 98% or greater identity to the amino acid sequence of SEQ ID NO: 1 18.
  • the isolated and/or recombinant nucleic acid encodes a canine IL-7Ra that comprises the amino acid sequence of SEQ ID NO: 1 18.
  • the isolated and/or recombinant nucleic acid comprises the nucleotide sequence of SEQ ID NO: 1 17.
  • the isolated and/or recombinant nucleic acid encodes a canine IL-7Ra that comprises the amino acid sequence of SEQ ID NO: 1 16.
  • the isolated and/or recombinant nucleic acid comprises the nucleotide sequence of SEQ ID NO: 1 15.
  • the figures depict the change in Optical Density measured between 570-600 nm (OD 570-600nm) of modified BaF3 cells as a function of the log dilution of purified IgG obtained from the sera collected from pairs of individual dogs either prior to the vaccination (pre-vaccinated) or after the vaccination (post-vaccinated) as described in Example 2 below.
  • FIGURES 1 A and 1 B Assay for neutralization of canine TSLP activity by
  • FIGURE 1 A without pre-incubation with sera (0);
  • FIGURE 1 B without pre-incubation with sera (0); pre-incubation with sera from pre- vaccinated dogs, UEK ( ⁇ ) and WKO (— ); pre-incubation with sera from
  • FIGURES 2A and 2B Assay for neutralization of canine TSLP activity by
  • FIGURE 2A without pre-incubation with sera (0); pre-incubation with sera from pre-vaccinated dogs, ZCO ( ⁇ ) and YGO (— );
  • FIGURE 2B without pre-incubation with sera (0); pre-incubation with sera from pre-vaccinated dogs, YDO ( ⁇ ) and WLO (— ); pre-incubation with sera from post-vaccinated dogs, YDO (A) and dog WLO ( ⁇ ).
  • FIGURES 3A and 3B Assay for neutralization of canine TSLP activity by
  • FIGURE 3A without pre-incubation with sera (0); pre-incubation with sera from pre-vaccinated dogs, ISO ( ⁇ ) and FVK (— );
  • FIGURE 3B without pre-incubation with sera (0); pre-incubation with sera from pre-vaccinated dogs, FCK ( ⁇ ) and URK (— ); pre-incubation with sera from post-vaccinated dogs, FCK (A) and URK ( ⁇ ).
  • FIGURES 4A and 4B Assay for neutralization of canine TSLP activity by
  • FIGURE 4A without pre-incubation with sera (0); pre-incubation with sera from pre-vaccinated dogs, WWO ( ⁇ ) and ZSO (— ); pre-incubation with sera from post-vaccinated dogs, WWO (A) and dog ZSO ( ⁇ ).
  • FIGURE 4B without pre-incubation with sera (0); pre-incubation with sera from pre-vaccinated dogs, WZO ( ⁇ ) and ZFO (— ); pre-incubation with sera from post-vaccinated dogs, WZO (A) and ZFO ( ⁇ ).
  • Atopic dermatitis (“AD”) is a Th2 mediated allergic inflammatory disease. This disease manifests itself with many similar clinical features in human and canine patients.
  • the immunopathogenesis of atopic dermatitis in dogs appears to be comparable to atopic dermatitis in humans with respect to cell types and cytokines involved in the skin lesions.
  • the binding of the TARC ligand (CCL22) to the CC chemokine receptor 4 (CCR4) which is selectively expressed on Th2 lymphocytes, induces selective migration of these cells to allergic lesions. It has been reported that TARC and its receptor CCR4 are upregulated in lesions of canine AD skin.
  • TSLP is a strong inducer of TARC in humans, and TSLP was found in the lesions of canine AD [U.S. 7,718,772 B2, the contents of which are hereby incorporated by reference in their entirety]. Accordingly, the present invention provides fusion proteins constructed from canine TSLP or fragments thereof combined with Fc IgG from another mammalian source. Surprisingly, these canine Fc-TSLP fusion proteins were found to be superior to the other canine TSLP fusion proteins tested with respect to inducing antibodies in dogs that could neutralize canineTSLP activities (see, Example 2 below).
  • an Fc-TSLP fusion protein of the present invention can comprise a canine TSLP protein, (with or without its natural signal peptide), and/or one or more antigenic canine TSLP fragment, together with an Fc region of any non-canine mammalian IgG.
  • nucleic acids of the present invention can encode all such fusion proteins, and vectors of the present invention, including expression vectors, can comprise the respective nucleic acids.
  • canine Fc-TSLP proteins of the present invention can be used as an immunogen for eliciting anti-TSLP antibodies for use as a research and/or a diagnostic reagent in domestic dogs, or in other mammalian species, such as felines.
  • a canine Fc-TSLP protein and/or a nucleic acid that encodes a canine Fc-TSLP protein of the present invention, and/or a
  • recombinant vector that comprises such a nucleic acid and can effectuate the expression of the canine Fc-TSLP in a host cell may serve to upregulate elements of the immune system of immune-impaired mammals e.g., via STAT activation, or
  • TARC expression e.g., in hematopoietic cells.
  • nucleic acids encoding canine TSLPR and/or canine IL-7Ra of the present invention can be inserted into a cell line to express the heterodimer TSLP receptor, which inter alia, can be use detect the presence of free canine TSLP in the cell as exemplified below.
  • the canine TSLPR and canine IL-7Ra proteins of the present invention can be used to generate antibodies to allow detection of their expression in such cell lines.
  • Such antibodies include monoclonal and caninized antibodies.
  • a monoclonal antibody elicited in a non-canine species can be optionally engineered to be caninized, so as to be minimally antigenic when injected into a canine subject.
  • Isolated antibodies generated through the use of the canine TSLPR and/or canine IL-7Ra proteins, and which are specific for the canine TSLPR and canine IL-7Ra proteins respectively, and/or for the canine TSLPR - IL-7Ra heterodimer are also part of the present invention.
  • the antibodies of the present invention can be used to block or moderate the action of canine TSLP in an animal and thereby, moderate, eliminate, and/or prevent one or more atopic symptoms, such as those arising in asthma and/or atopic dermatitis, in the animal subject.
  • polypeptide includes reference to one or more of such polypeptides.
  • approximately is used interchangeably with the term
  • “comprising” and the like is intended to be read in an inclusive and open sense, i.e., as specifying the presence of the features explicitly stated, but not precluding the presence of additional features, e.g., in more specific embodiments of the invention.
  • the term “comprises” can further include all possible combinations of the specific features provided in the present Specification that is conceivable for the invention, even if such a combination of features is not explicitly recited together.
  • “comprises” encompasses related, but more narrow terminology such as “consists of, “consisting of, “consisting essentially of, etc., and therefore, such terms can be substituted for "comprises” for example, when it is desired to define more specific embodiments.
  • binding composition refers to molecules that bind with specificity to a TSLP, e.g., in an antibody-antigen interaction.
  • the specificity may be more or less inclusive, e.g., specific to a particular embodiment, or to groups of related
  • a canine Fc-TSLP e.g., a canine Fc-TSLP.
  • canine includes all domestic dogs, Canis lupus familiaris or Canis familiaris, unless otherwise indicated.
  • non-canine mammalian IgG antibody is a mammalian IgG antibody or fragment thereof other than a canine IgG antibody or fragment.
  • feline refers to any member of the Felidae family.
  • Members of this family include wild, zoo, and domestic members, such as any member of the subfamilies Felinae, Panterinae or Acinonychinae.
  • Nonlimiting examples of species included within the Felidae family are cats, lions, tigers, pumas, jaguars, leopards, snow leopards, panthers, North American mountain lions, cheetahs, lynx, bobcats, caracals or any cross breeds thereof.
  • Cats also include domestic cats, pure-bred and/or mongrel companion cats, show cats, laboratory cats, cloned cats and wild or feral cats.
  • parenteral administration includes subcutaneous injections, submucosal injections, intravenous injections, intramuscular injections, intradermal injections, and infusion.
  • polypeptide is used interchangeably with the terms “protein” and “peptide” and denotes a polymer comprising two or more amino acids connected by peptide bonds.
  • polypeptide as used herein includes a significant fragment or segment, and encompasses a stretch of amino acid residues of at least about 8 amino acids, generally at least about 12 amino acids, typically at least about 16 amino acids, preferably at least about 20 amino acids, and, in particularly preferred embodiments, at least about 30 or more amino acids, e.g., 35, 40, 45, 50, etc.
  • Such fragments may have ends which begin and/or end at virtually all positions, e.g., beginning at residues 1 , 2, 3, etc., and ending at, e.g., 155, 154, 153, etc., in all practical combinations.
  • a polypeptide may lack certain amino acid residues that are encoded by a gene or by an mRNA.
  • a gene or mRNA molecule may encode a sequence of amino acid residues on the N-terminus of a polypeptide (i.e., a signal sequence) that is cleaved from, and therefore, may not be part of the final protein.
  • TSLP and "TSLP protein” are used interchangeably as an abbreviation for the protein known as thymic stromal lymphopoietin protein.
  • cTSLP is an abbreviation for canine TSLP.
  • the use of the term "excluding the 28 amino acid residue signal sequence” with regard to a percentage of identity to a particular amino acid sequence indicates that the % identity determination should be made in comparison to the amino acid sequence minus the 28 amino acid residue signal sequence, (e.g., that of amino acid residues 29-155 of SEQ ID NO: 2).
  • an Fc region of an IgG antibody comprises the CH2 and CH3 domains of the IgG heavy chain along with the hinge region.
  • Fc-TSLP and "Fc-TSLP protein” and “TSLP-Fc” and “TSLP-Fc protein” are used interchangeably as an abbreviation for the fusion protein
  • An "Fc-TSLP" of the present invention preferably is constructed with the TSLP portion being towards the N-Terminus of the fusion protein with respect to the Fc portion [see, Example 1 below].
  • mFc lgG2a is murine Fc lgG2a.
  • an antigenic fragment in regard to a particular protein is a fragment of that protein (including large fragments that are missing as little as a single amino acid from the full-length protein) that is antigenic, i.e., capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor.
  • an antigenic fragment of a TSLP e.g., a canine TSLP, is a fragment of the TSLP that is antigenic.
  • Such a fragment need not be itself immunogenic, i.e., capable of eliciting an immune response without a carrier, so long as it can be used to generate an antibody to the TSLP protein after conjugating the fragment to a carrier molecule for immunization.
  • an antigenic fragment of the present invention is immunodominant for antibody and/or T cell receptor recognition.
  • a "canine Fc-TSLP” is an Fc-TSLP in which the portion of the fusion protein from a TSLP is from a canine TSLP, regardless of which species the corresponding IgG originated from. This convention is used herein for all
  • a "human Fc-TSLP” is an Fc-TSLP in which the portion of the fusion protein from a TSLP is from a human TSLP, regardless of which species the corresponding IgG originated from.
  • an amino acid sequence is 100% "homologous" to a second amino acid sequence if the two amino acid sequences are identical, and/or differ only by neutral or conservative substitutions as defined below. Accordingly, an amino acid sequence is about 80% "homologous" to a second amino acid sequence if about 80% of the two amino acid sequences are identical, and/or differ only by neutral or conservative substitutions. Functionally equivalent amino acid residues often can be substituted for residues within the sequence resulting in a conservative amino acid substitution. Such alterations define the term "a conservative substitution" as used herein. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration.
  • substitutions for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • Amino acids containing aromatic ring structures are phenylalanine, tryptophan, and tyrosine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such alterations will not be expected to affect apparent molecular weight as determined by polyacrylamide gel electrophoresis, or isoelectric point.
  • Particularly preferred conservative substitutions are: Lys for Arg and vice versa such that a positive charge may be maintained; Glu for Asp and vice versa such that a negative charge may be maintained; Ser for Thr such that a free --OH can be maintained; and Gin for Asn such that a free NH 2 can be maintained.
  • amino acids also can be placed in the following similarity groups: (1 ) proline, alanine, glycine, serine, and threonine; (2) glutamine, asparagine, glutamic acid, and aspartic acid; (3) histidine, lysine, and arginine; (4) cysteine; (5) valine, leucine, isoleucine, methionine; and (6) phenylalanine, tyrosine, and tryptophan.
  • similarity groups (1 ) proline, alanine, glycine, serine, and threonine; (2) glutamine, asparagine, glutamic acid, and aspartic acid; (3) histidine, lysine, and arginine; (4) cysteine; (5) valine, leucine, isoleucine, methionine; and (6) phenylalanine, tyrosine, and tryptophan.
  • two highly homologous DNA sequences can be identified by their own homology, or the homology of the amino acids they encode. Such comparison of the sequences can be performed using standard software available in sequence data banks.
  • two highly homologous DNA sequences encode amino acid sequences having about 80% identity, more preferably about 90% identity and even more preferably about 95% identity. More particularly, two highly homologous amino acid sequences have about 80% identity, preferably about 90% identity, more preferably about 95% identity and even more preferably about 98% identity.
  • protein and DNA sequence percent identity can be determined using software such as MacVector v9, commercially available from Accelrys (Burlington, Massachusetts) and the Clustal W algorithm with the alignment default parameters, and default parameters for identity. See, e.g., Thompson, et al. [Nucleic Acids Res. 22:4673-4680 (1994)].
  • ClustalW is freely downloadable for Dos, Macintosh and Unix platforms from, e.g., EMBLI, the European Bioinformatics Institute. The present download link is found at http://www.ebi.ac.uk/clustalw/.
  • nucleic acid or a "nucleic acid” or a “nucleic acid molecule” are used interchangeably and denote a molecule comprising nucleotides including, but is not limited to, RNA, cDNA, genomic DNA and even synthetic DNA sequences. The terms are also contemplated to encompass nucleic acid molecules that include any of the art-known base analogs of DNA and RNA.
  • a nucleic acid "coding sequence” or a "sequence encoding" a particular protein or peptide is a nucleotide sequence which is transcribed and translated into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory elements.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic ⁇ e.g., mammalian) DNA, and even synthetic DNA sequences.
  • a transcription termination sequence will usually be located 3' to the coding sequence.
  • operably linked refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
  • control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence.
  • the control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between a promoter and the coding sequence and the promoter can still be considered “operably linked" to the coding sequence.
  • heterologous nucleotide sequence is a nucleotide sequence that is added to a nucleotide sequence of the present invention by recombinant methods to form a nucleic acid that is not naturally formed in nature.
  • Such nucleic acids can encode fusion ⁇ e.g., chimeric) proteins.
  • the heterologous nucleotide sequence can encode peptides and/or proteins that contain regulatory and/or structural properties.
  • the heterologous nucleotide sequence can encode a protein or peptide that functions as a means of detecting the protein or peptide encoded by the nucleotide sequence of the present invention after the recombinant nucleic acid is expressed.
  • heterologous nucleotide sequence can function as a means of detecting a nucleotide sequence of the present invention.
  • a heterologous nucleotide sequence can comprise non- coding sequences including restriction sites, regulatory sites, promoters and the like.
  • a "vector” or “replication vector” is a replicon, such as a plasmid, naked DNA vector, virus, phage, or cosmid, to which another nucleic acid segment may be attached or incorporated so as to bring about the replication of the attached segment.
  • the term also includes a replicon that includes the incorporated or attached nucleic acid segment of interest.
  • Vectors that can be used in this invention include microbial plasmids, naked DNA vectors, viruses (in particular animal viruses), bacteriophage, integratable DNA fragments, and other vehicles that may facilitate integration of the nucleic acids into the genome of the host. Plasmids are the most commonly used vector, but all other vectors that serve an equivalent function and that are or become known in the art are suitable for use herein. [See, e.g., Pouwels et ai, Cloning Vectors: A Laboratory Manual, 1985 and Supplements, Elsevier, N.Y., and Rodriguez et al. (eds.), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, 1988, Buttersworth, Boston, MA.]
  • Insertion of a nucleic acid encoding an inventive Fc-TSLP of the present invention into a vector is easily accomplished when the termini of both the nucleic acid and the vector comprise compatible restriction sites. If this cannot be done, it may be necessary to modify the termini of the nucleotide sequence and/or vector by digesting back single-stranded nucleic acid overhangs ⁇ e.g., DNA overhangs) generated by restriction endonuclease cleavage to produce blunt ends, or to achieve the same result by filling in the single-stranded termini with an appropriate
  • desired sites may be produced, e.g., by ligating nucleotide sequences (linkers) onto the termini.
  • linkers may comprise specific oligonucleotide sequences that define desired restriction sites. Restriction sites can also be generated through the use of the polymerase chain reaction (PCR). [See, e.g., Saiki et al., Science 239:487-491 (1988)].
  • PCR polymerase chain reaction
  • Recombinant expression vectors used in this invention are typically self-replicating DNA or RNA constructs comprising nucleic acids encoding an Fc-TSLP of the present invention which is usually operably linked to suitable genetic control elements that are capable of regulating expression of the nucleic acids in compatible host cells.
  • Genetic control elements may include a prokaryotic promoter system or a eukaryotic promoter expression control system, and typically include a transcriptional promoter, an optional operator to control the onset of transcription, transcription enhancers to elevate the level of mRNA expression, a sequence that encodes a suitable ribosome binding site, and sequences that terminate transcription and translation.
  • Expression vectors may also contain an origin of replication that allows the vector to replicate independently of the host cell.
  • FC-TSLP expression of nucleic acids encoding the inventive FC-TSLP can be carried out by conventional methods in either prokaryotic or eukaryotic cells.
  • a “host cell” is a cell that contains, or is capable of containing, and expressing, an exogenous nucleic acid molecule, either transiently or permanently.
  • a cell has been "transformed” by exogenous DNA when such exogenous DNA has been introduced inside the cell membrane.
  • Exogenous DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the exogenous DNA may be maintained on an episomal element, such as a plasmid.
  • a stably transformed cell is one in which the exogenous DNA has become integrated into the
  • chromosome so that it is inherited by daughter cells through chromosome
  • Prokaryotes include both gram negative and positive organisms, e.g., E. coli and B. subtilis.
  • Eukaryotes include established tissue culture cell lines from animal cells, both of non-mammalian origin, e.g., insect cells, and birds, and mammalian origin, e.g., human, primates, and rodents.
  • Prokaryotic host-vector systems include a wide variety of vectors for many different species.
  • Vectors for amplifying DNA include pBR322 or many of its derivatives, or the pET42b(+) expression vector (Novagen).
  • Prokaryotic expression control sequences typically used include promoters, including those derived from the ⁇ -lactamase and lactose promoter systems [Chang et al., Nature, 798:1056 (1977)], e.g., pUC-series, the tryptophan (trp) promoter system
  • Yeast as well as higher eukaryotic tissue culture cells are also contemplated as hosts for the recombinant production of an inventive Fc-TSLP.
  • any higher eukaryotic tissue culture cell line might be used, including insect baculovirus expression systems, mammalian cells are preferred. Transformation or transfection and propagation of such cells have become a routine procedure. Examples of useful cell lines include HeLa cells, Chinese hamster ovary (CHO) cell lines, baby rat kidney (BRK) cell lines, insect cell lines (e.g. SF9), bird cell lines (e.g.
  • Expression vectors for such cell lines usually include, for example, an origin of replication, a promoter, a translation initiation site, RNA splice sites (if genomic DNA is used), a polyadenylation site, and a transcription termination site. These vectors also usually contain a selection gene or amplification gene.
  • Suitable expression vectors may be plasmids, naked DNA vectors, viruses (such as canine adenovirus type 2), or retroviruses carrying indogenous and/or exogenous promoters derived, e.g., from such sources as adenovirus, SV40, parvoviruses, vaccinia virus, or cytomegalovirus.
  • viruses such as canine adenovirus type 2
  • retroviruses carrying indogenous and/or exogenous promoters derived, e.g., from such sources as adenovirus, SV40, parvoviruses, vaccinia virus, or cytomegalovirus.
  • suitable expression vectors include pCR®3.1 , pCDNAI , pCD [Okayama et al., Mol. Cell Biol.
  • the inventive Fc-TSLP can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, R. Scopes, PROTEIN
  • compositions of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more
  • homogeneity are most preferred for pharmaceutical uses. Purification can be partial, or to homogeneity as desired. If the Fc-TSLP is to be used therapeutically, the protein should be substantially free of endotoxin. Selective purification of expressed Fc-TSLP on a bound anti-TSLP antibody column, or on a bound TSLP-receptor column are available strategies for obtaining highly purified canine TSLP protein.
  • nucleic acids can be purified by precipitation, chromatography, ultracentrifugation and other means.
  • Proteins and polypeptides, as well as peptides can be purified by various methods including, without limitation, preparative disc-gel electrophoresis, isoelectric focusing, HPLC, reversed-phase HPLC, gel filtration, ion exchange and partition
  • polypeptide in a recombinant system in which the protein contains an additional sequence tag that facilitates purification, such as, but not limited to, a polyhistidine sequence or a sequence that specifically binds to an antibody, such as FLAG® and GST.
  • the polypeptide can then be purified from a crude lysate of the host cell by chromatography on an appropriate solid-phase matrix.
  • antibodies, or binding fragments thereof, produced against the polypeptide can be used as purification reagents.
  • the solvent and electrolytes will usually be a biologically compatible buffer, of a type used for preservation of biological activities, and will usually approximate a physiological aqueous solvent.
  • the solvent will have a neutral pH, typically between about 5 and 10, and preferably about 7.5.
  • one or more detergents will be added, typically a mild non-denaturing one, e.g., CHS
  • cholesteryl hemisuccinate or CHAPS (3-[3 cholamidopropyl) dimethylammoni- o]-1 - propane sulfonate), or a low enough concentration as to avoid significant disruption of structural or physiological properties of the protein.
  • a harsh detergent may be used to effect significant denaturation.
  • heterologous proteins from E. coli or other bacteria can be isolated from inclusion bodies by means of solubilization using strong denaturants, and subsequent refolding.
  • Denaturants include, simply by way of example, urea, potassium thiocyanate, guanadine HCI ("GuHCI”), potassium iodate, and/or sodium iodide and combinations of these.
  • GuHCI is employed as a reducing agent, e.g., from about 6 to about 8 M in concentration, under alkaline conditions, e.g., about pH 8.
  • another reducing agent, dithiothreitol (“DTT”) is employed, either alone or in combination with GuHCI.
  • the concentration ranges, simply by way of example, from about 50 mM to about 0.5 mM DTT.
  • a reducing agent must be present to separate or denature the disulfide bonds.
  • One exemplary reducing buffer is: 0.1 M Tris pH 8.0, 6 M guanidine, 2 mM EDTA, and 0.3 M DTE (dithioerythritol). Renaturation is typically accomplished by dilution ⁇ e.g., 100-fold) of the denatured and reduced protein into a refolding buffer, in the presence of an oxidizing agent.
  • oxidizing agent Any suitable art-known oxidizing agent can be employed, provided that it allows for correct refolding in good yields.
  • oxidation and refolding can be provided by low molecular weight thiol reagents in reduced and oxidized form, as described in Saxena, et al., [Biochemistry 9: 5015-5021 (1970), incorporated by reference herein, and especially as described by Buchner, et al., supra.]
  • Renaturation is typically accomplished by dilution ⁇ e.g., 100-fold) of the denatured and reduced protein into a refolding buffer.
  • a refolding buffer is: Tris HCI 100 mM, pH 10.0, 25 mM EDTA, NaCI 0.1 M, GSSG 551 mg/L, 0.5 M Arginine.
  • GSSG is the oxidized form of glutathione.
  • the size and structure of the polypeptide should generally be in a substantially stable state, and usually not in a denatured state.
  • the polypeptide may be
  • polypeptides in a quaternary structure e.g., to confer solubility, or associated with lipids or detergents.
  • Substantially pure typically means that the protein is free from other contaminating proteins, nucleic acids, or other biologicals derived from the original source organism. Purity may be assayed by standard methods, typically by weight, and will ordinarily be at least about 40% pure, generally at least about 50% pure, often at least about 60% pure, typically at least about 80% pure, preferably at least about 90% pure, and in most preferred embodiments, at least about 95% pure. Carriers or excipients will often be added. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay and other methods known in the art. From a functional aspect, an isolated canine Fc-TSLP according to the invention is one sufficiently separated from other materials, so as to be capable of eliciting an immune response that is specific for the TSLP protein.
  • the present invention also provides Fc-TSLPs that comprise an antigenic fragment of a TSLP.
  • an antigenic fragment of the TSLP contains between 5 and 150 amino acid residues. In one particular embodiment the antigenic fragment of the TSLP contains greater than 120 amino acid residues. In another embodiment an antigenic fragment of the TSLP contains between 10 and 120 amino acid residues. In still another embodiment an antigenic fragment of the TSLP contains between 20 and 100 amino acid residues. In yet another embodiment an antigenic fragment of the TSLP contains between 25 and 75 amino acid residues.
  • Appropriate TSLP peptides useful as antigenic fragments for inclusion in specific TSLP fusion proteins of the present invention can be selected from those provided in U.S. 7,718,772 B2, the contents of which are hereby incorporated by reference in its entirety, and to variants thereof. A table listing the amino acid sequences of such peptides, essentially reproduced from U.S. 7,718,772 B2, is provided below.
  • Epitopes from such peptides and variants thereof can also be selected based on their reactivity with polyclonal or monoclonal antibodies that react with native TSLP proteins and especially those antibodies that are capable of neutralizing TSLP bioactivity.
  • Fusion proteins of the present invention including such antigenic fragments of TSLP can be obtained from a recombinant source and can be obtained through recombinant expression.
  • the present invention further provides vaccines and/or immunogenic compositions and/or pharmacines that include an effective amount of an Fc-TSLP of the present invention.
  • vaccines can be used to treat allergic diseases such as atopic dermatitis or other diseases in which TSLP plays a role in the disease pathogenesis.
  • an immunogenic composition can be employed for inducing
  • endogenous anti-TSLP antibodies in an animal subject in need thereof, e.g., in order to treat clinical signs of a disease or disorder that is responsive to the
  • Vaccines, immunogenic compositions, and pharmacines are well known to the art and can, but do not necessarily include, for example, physiologically compatible buffers and saline and the like, as well as pharmaceutically acceptable adjuvants.
  • Pharmaceutically acceptable adjuvants of the present invention may be obtained from any of a number of sources including from natural sources, recombinant sources, and/or be chemically synthesized, etc. Examples of chemical compounds used as adjuvants include, but are not limited to aluminum compounds,
  • metabolizable and non-metabolizable oils block polymers, ISCOM's (immune stimulating complexes), vitamins and minerals (including but not limited to: vitamin E, vitamin A, selenium, and vitamin B12), and Quil A (saponins), Freund's complete adjuvant, polymers of acrylic acid cross-linked with polyalkenyl ethers or divinyl glycol, as sold under the trademark CARBOPOL® (e.g., CARBOPOL® 941 ).
  • CARBOPOL® e.g., CARBOPOL® 941
  • Additional adjuvants include aluminum phosphate such as REHYDRAPHOSTM, aluminum hydroxide, such as REHYDROGELTM, and/or an oil-in-water emulsions such as EMULSIGEN®, a uniformly dispersed micron size oil droplets-in-water emulsion.
  • adjuvants that sometimes have been referred to specifically as immune stimulants, include, bacterial and fungal cell wall components (e.g., lipopolysaccarides, lipoproteins, glycoproteins, muramylpeptides, £>efa-1 ,3/1 ,6- glucans), various complex carbohydrates derived from plants (e.g., glycans, acemannan), various proteins and peptides derived from animals (e.g., hormones, cytokines, co-stimulatory factors), and novel nucleic acids derived from viruses and other sources (e.g., double stranded RNA, CpG).
  • bacterial and fungal cell wall components e.g., lipopolysaccarides, lipoproteins, glycoproteins, muramylpeptides, £>efa-1 ,3/1 ,6- glucans
  • various complex carbohydrates derived from plants e.g., glycans, acemannan
  • any number of combinations of the aforementioned substances may provide an adjuvant effect, and therefore, can form an adjuvant of the present invention.
  • a purified Fc-TSLP protein of the present invention can be blended with an adjuvant such as EMULSIGEN® or REHYDRAPHOSTM and used to form vaccines that are able to induce neutralizing anti-TSLP antibodies.
  • the vaccines and/or immunogenic compositions and/or pharmacines of the present invention may be administered by any route including by oral administration, by intranasal administration, or by parenteral administration, including intramuscular injection, subcutaneous injection, intravenous injection, intradermal injection, and by any combination thereof.
  • a pharmacine of the present invention can be given in doses ranging from 10 g - 2 mg per dose. As the skilled artisan would readily appreciate, the appropriate dosage will depend on many factors including the species of the mammal, its weight, the type of ailment, the degree of severity of the ailment, and the like. SEQUENCE LISTING TABLES
  • the present invention provides an exemplifing antigen that is composed of a canine TSLP fused to the Fc region from a mouse lgG2a antibody (an
  • Fc-TSLP Fc-TSLP
  • the gene encoding the fusion protein was cloned in an expression plasmid which is transfected into cell lines such as HEK 293 and CHO cells.
  • the transfected cells are selected in the presence of antibiotics and used to produce the recombinant vaccine antigen in a variety of production systems including tissue culture flasks, roller bottles or bioreactors.
  • the vaccine antigen which is secreted into the media of the cultured cells is purified by passage of the culture fluids over affinity columns such as protein A.
  • murine lgG2a Fc Unbolded, unitalicized Amino Acid Sequence (SEQ ID NO: 4)
  • cTSLP-human lgG1 Fc cTSLP-hlgG1 Fc
  • cTSLP-murine lgG2a Fc cTSLP-murlgG2a Fc
  • This neutralized IgG fraction can be used in the TSLP bioassay as described below.
  • a bioassay to measure cTSLP activity was developed using BaF3 cells which had been genetically modified to express the canine heterodimer TSLP receptor,
  • IL-7Ra/TSLPR [see e.g., Reche et al., The Journal of Immunology 167:336-343 (2001 )].
  • synthesized and codon optimized nucleic acids encoding canine TSLPR comprising the nucleotide sequence of SEQ ID NO: 1 1 1 and canine IL-7Ra comprising the nucleotide sequence of SEQ ID NO: 1 15 were subcloned into retroviral vector pMXneo-CD8 Ieader-His6 and pMXpuro respectively [see e.g., EP 1 798 286 B1 ] via Clal(5')/EcoRI(3') and BamHI(5') /Notl(3') restriction sites respectively.
  • Canine TSLPR was introduced into BF3 cells by retrotransduction and drug selection (neomycin), then canine IL-7RA was introduced into bulk selected cells by retrotransduction and puromycin selection.
  • BaF3 cells are an immortalized murine bone marrow-derived pro-B-cell line that depends on the presence of IL-3 for growth and proliferation.
  • Canine TSLP can substitute for IL-3 in these genetically modified BaF3 cells. Accordingly, the addition of either cTSLP or IL-3 allows the genetically modified BaF3 cells to grow and proliferate. Conversely, the genetically modified BaF3 cells undergo apoptosis in the absence of both cTSLP and IL-3. Therefore, the genetically modified BaF3 cells become dependent on cTSLP to grow and proliferate when IL-3 is left out of the incubation well.
  • the growth and proliferation of the cells can be quantified by optical density determinations between 570 and 600 nm with the aid of AlamarBlue® dye.
  • this unique bioassay can be used to determine whether or not the dogs vaccinated with a specific TSLP fusion protein described above, produced neutralizing IgG antibodies against cTSLP. This assay was performed as follows:
  • Figures 1 A-1 B show the results obtained from two pairs of dogs (RFK and ZXO; and UEK and WTO) vaccinated with the cTSLP-GST fusion protein.
  • the incubations with the cTSLP were performed with IgG purified from sera obtained (i) prior to the vaccination, or (ii) after the third vaccination with cTSLP-GST fusion protein (post- vaccination) as detailed above.
  • Canine TSLP incubated with sera from these four dogs induced the genetically modified BaF3 cells to proliferate to the same extent as canine TSLP alone (also shown), which indicates that sera from the dogs vaccinated with the cTSLP-GST fusion protein did not contain IgG antibodies capable of inhibiting the cTSLP activity, i.e., the sera did not contain neutralizing IgG antibodies.
  • Figures 2A-2B show the results obtained from two pairs of dogs (ISO and FVK, and YDO and WLO) vaccinated with the cTSLP-i efa gal fusion protein.
  • the incubations with the cTSLP were performed with IgG purified from sera obtained (i) prior to the vaccination, or (ii) after the third vaccination with cTSLP-i efa gal fusion protein (post-vaccination) as detailed above.
  • Canine TSLP incubated with sera from these four dogs induced the genetically modified BaF3 cells to proliferate to the same extent as canine TSLP alone (also shown), which indicates that sera from the dogs vaccinated with the cTSLP-befa gal fusion protein did not contain IgG antibodies capable of inhibiting the cTSLP activity, i.e., the sera did not contain neutralizing IgG antibodies.
  • Figures 3A-3B show the results obtained from two pairs of dogs (ISO and FVK, and FCK and URK) vaccinated with the cTSLP-human lgG1 Fc fusion protein.
  • the incubations with the cTSLP were performed with IgG purified from sera obtained (i) prior to the vaccination, or (ii) after the third vaccination with cTSLP-human lgG1 Fc fusion protein (post-vaccination) as detailed above.
  • cTSLP incubated with sera from one of the two dogs depicted in each of Figure 3A and Figure 3B, FVK and URK respectively inhibited the genetically modified BaF3 cells from proliferating in a dose-dependent manner, whereas sera from the other two dogs, ISO and FCK respectively, did not.
  • the incubations with the cTSLP were performed with IgG purified from sera obtained (i) prior to the vaccination, or (ii) after the third vaccination with cTSLP-murine lgG2a Fc fusion protein (post-vaccination) as detailed above.
  • cTSLP incubated with sera from each of the dogs as depicted in Figures 4A and 4B, inhibited the genetically modified BaF3 cells from proliferating in a dose-dependent manner.
  • an additional positive control and an additional negative control was performed.
  • the genetically modified BaF3 cells were incubated in the absence of cTSLP, but in presence of IL-3.
  • the genetically modified BaF3 cells cells grew and proliferated, having an OD570-600 nm that was approximately twice that with cTSLP alone.
  • the genetically modified BaF3 cells were incubated in the absence of both IL-3 and cTSLP. As expected, these cells did not grow or proliferate, having an OD570-600 nm that was essentially at the baseline.

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Abstract

La présente invention concerne des protéines de fusion de TSLP canine ou de ses fragments antigéniques, avec la région Fc d'IgG, ainsi que les acides nucléiques qui codent ces protéines de fusion. La présente invention concerne en outre des procédés de fabrication et d'utilisation de ces protéines de fusion et acides nucléiques.
PCT/EP2013/059212 2012-05-04 2013-05-03 Protéine de fusion de la protéine lymphopoiétine stromale thymique canine avec la région fc d'igg WO2013164435A2 (fr)

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