Provided are methods of treatment for skin disorders. In particular, treatment, the skin disorders are generally inflammatory skin disorders, including improper wound healing. Provided are methods of using of a cytokine molecule.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention is based, in part, upon the discovery that an IL-23 fusion protein, e.g., a fusion protein comprising the p19 subunit linked to the p40 subunit, enhanced wound healing response in various mouse models.

The invention provides a method of treating or improving healing comprising administering to a subject an effective amount of an agonist or antagonist of IL-23. Also provided is the above method, wherein the agonist or antagonist comprises a polypeptide of IL-23, or a derivative or variant thereof, a binding composition derived from an antibody that specifically binds to IL-23 or to IL-23R; or a nucleic acid encoding a polypeptide of IL-23, or a derivative or variant thereof. In addition, the invention provides the above method wherein the derivative or variant comprises an IL-23 hyperkine; wherein the agonist comprises a complex of a mature sequence of SEQ ID NO: 10; and a mature sequence of SEQ ID NO: 12; or the above method wherein the nucleic acid further comprises an expression vector.

In another aspect, the invention provides a method of treating or improving healing comprising administering to a subject an effective amount of an agonist or antagonist of IL-23, wherein the healing is of a skin or cutaneous wound; of an ulcer or graft; or is improper healing. Also provided is the above method wherein the treating or improving increases a pressure required to break a healed or healing wound; a stiffness of a healed or healing wound; a rate of healing of a wound; a granulation layer thickness of a healed or healing wound; recruitment of a cell to or towards a wound; or antimicrobial activity. In yet another aspect, the invention provides the above method wherein the cell is a CD11b.sup.+, MHC Class II.sup.+ cell; a monocyte/macrophage; a CD31.sup.+ endothelial cell; or an immune cell. Also provided is the above method wherein the recruitment is in or towards a granulation tissue; wherein the increased wound breaking pressure is about a 15% or about a 20% increase in wound breaking pressure; or the increased stiffness is about a 15% or about a 20% increase in stiffness. In another embodiment, the present invention provides the above method wherein the treating or improving comprises increased angiogenesis; or immune surveillance; or the above method wherein the increased angiogenesis is mediated by ICAM-1 or -2; or the increased immune surveillance is mediated by dendritic cells.

Yet another aspect of the above invention provides a method of treating or improving healing comprising administering to a subject an effective amount of an agonist or antagonist of IL-23, wherein the treating or improving comprises increased expression of a nucleic acid or protein of a cytokine in addition to IL-23; a signaling molecule; an anti-microbial molecule; a protease or protease inhibitor; or a molecule of the extracellular matrix; or the above method wherein the cytokine nucleic acid or protein is IL-17, IL-6, IL-19, GRO-alpha, or GM-CSF; or wherein the nucleic acid or protein is lactoferrin; DEC-205; CD50; nitric oxide synthase; or secretory leukoprotease inhibitor; or CD40L.

Also provided is the above method, wherein the antagonist comprises a nucleic acid; a blocking antibody to IL-23 or to IL-23R; or a soluble receptor derived from an extracellular part of IL-23R; the above method wherein the nucleic acid comprises an anti-sense nucleic acid; or interference RNA.

Yet another aspect of the present invention provides an agonist of IL-23 derived from the binding site of an antibody that specifically binds to an IL-23 receptor; the above agonist that is a polyclonal antibody; a monoclonal antibody; an Fab, Fv, or F(ab').sub.2 fragment; humanized; a peptide mimetic; or detectably labeled. In another embodiment, the present invention provides the above agonist comprising a complex of a polypeptide of the mature sequence of SEQ ID NO:10 and a polypeptide of the mature sequence of SEQ ID NO:12; the above agonist comprising a complex of two polypeptides of the mature sequence of SEQ ID NO:10 and two polypeptides of the mature sequence of SEQ ID NO:12. Moreover the invention provides the above agonist wherein contact of the agonist to a cell expressing hIL-23R and hIL-12beta1 results in an increase in proliferation of the cell. Also provided is a kit comprising the above agonist and a compartment; or instructions for use or disposal. Also provided is a nucleic acid encoding an agonist of IL-23 derived from the binding site of an antibody that specifically binds to an IL-23 receptor.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

All references cited herein are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

As used herein, including the appended claims, the singular forms of words such as "a," "an," and "the," include their corresponding plural references unless the context clearly dictates otherwise.

I. General.

Interleukin-23 (IL-23) is a heterodimeric cytokine composed of a novel p19 subunit (a.k.a. IL-B30) and the p40 subunit of IL-12 (Oppmann, et al., supra). The p19 subunit was identified during a computational search for members of the IL-6 helical cytokine family characterized by their unique four .alpha.-helix bundle. Genetic analysis of the family, of which oncostatin-M, IL-11, cardiotrophin-1, and leukaemia inhibitory factor are members, reveals the closest evolutionary neighbor of p19 to be the p35 subunit of IL-12. Like p35, p19 requires co-expression of p40 for biological activity (Wiekowski, et al. (2001) J. Immunol. 166:7563-7570). The IL-23 receptor (IL-23R) comprises a novel receptor subunit (IL-23R), that binds p19, and IL-12R.beta.1, that binds p40 (Parham, et al. (2002) J. Immunol. 168:5699-5708). These two receptor subunits form the functional signaling complex and are expressed on CD4.sup.+ CD45Rb.sup.lo memory T cells as well as interferon-gamma (IFNgamma) activated bone marrow macrophages (Parham, et al., supra).

Preliminary characterization of IL-23 suggests that it has potent effects on memory T cells from both humans and mice, as measured by proliferation and IFNgamma production. Consistent with the immunostimulatory properties of IL-23, mice in which haematopoetic cells constitutively express transgenic p19 have widespread multi-organ inflammation that results in premature death (Wiekowski, et al., supra). The inflammatory disease is characterized by intense macrophage infiltration, neutrophilia, and elevated levels of proinflammatory monokines such as IL-1 and TNF, suggesting that IL-23 may also act on myeloid cells.

Recent studies analyzing the necessity of IL-12 in resistance to infectious diseases have yielded divergent results, depending on whether p35.sup.-/- or p40.sup.-/- mice are used. The former, which specifically lack IL-12 but express IL-23, are resistant to infection, whereas the latter, unable to express both IL-12 and IL-23, are more susceptible.

Transgenic mice deficient for the p19 subunit of IL-23 (IL-23p19) were resistant to EAE, a CNS autoimmune disease mediated by TH1 cells and inflammatory macrophages, while wild-type and heterozygous p19 control mice were highly susceptible. Mice deficient in the p40 subunit of IL-12 (IL-12p40 deficient mice) were also resistant to EAE, while mice deficient in the p35 subunit of IL-12 (IL-12p35 deficient mice) were highly susceptible to EAE. This is indicative of a role of IL-23 in the induction of EAE.

p19 deficient mice had a notable altered wound healing response following subcutaneous injection of an oil emulsion in mice. The p19 deficient mice were also defective in a variety of mouse disease models that required monocyte/macrophage activation. Monocytes/macrophages are known to stimulate wound repair, see, e.g., Schaffer and Nanney (1996) Intl. Rev. Cytol 169:151-181. In particular, it is shown below that delivery of IL-23 polypeptide into mouse skin could attract CD11b.sup.+/Class II.sup.+ activated monocyte/macrophage populations.

III. Agonists and Antagonists.

The present invention provides methods of using IL-23 agonists including the full length cytokine protein (SEQ ID NO: 2 or 4). Also provided is a fusion protein, also known as "IL-23 hyperkine" (SEQ ID NO: 6 or 8), comprising p19 linked to p40 with a FLAG sequence as described for IL-6 in, e.g., Oppmann, et al., supra; Fischer, et al. (1997) Nature Biotechnol. 15:142-145; Rakemann, et al. (1999) J. Biol. Chem. 274:1257-1266; and Peters, et al. (1998) J. Immunol. 161:3575-3581, thereof. The invention also provides agonistic anti-IL-23R antibodies that are agonistic to the IL-23 receptor, e.g., antibodies that stimulate the IL-23 receptor in the absence or presence of IL-23.

Peptides of those sequences, or variants thereof, will be used to induce receptor signaling. Also contemplated are small molecules which also induce receptor signaling. Agonists of the present invention will be useful in the treatment of various inflammatory skin disorders, including but not limited to wound healing, skin disorders associated with impaired recruitment of myeloid/monocyte cells.

The invention provides IL-23 antagonists, e.g., a blocking antibody that binds to IL-23, a blocking antibody that binds to IL-23R, a soluble receptor based on the extracellular portion of IL-23R, and nucleic acids. The IL-23 antagonists of the present invention encompass nucleic acids that are anti-sense nucleic acids and RNA interference nucleic acids (see, e.g., Arenz and Schepers (2003) Naturwissenschaften 90:345-359; Sazani and Kole (2003) J. Clin. Invest. 112:481-486; Pirollo, et al. (2003) Pharmacol. Therapeutics 99:55-77; Wang, et al. (2003) Antisense Nucl. Acid Drug Devel. 13:169-189).

III. Antibodies and Related Reagents.

Antibodies and binding compositions derived from an antigen-binding site of an antibody are provided. These include humanized antibodies, monoclonal antibodies, polyclonal antibodies, and binding fragments, such as Fab, F(ab).sub.2, and Fv fragments, and engineered versions thereof. The antibody or binding composition may be agonistic or antagonistic. Antibodies that simultaneously bind to a ligand and receptor are contemplated. Monoclonal antibodies will usually bind with at least a K.sub.D of about 1 mM, more usually at least about 300 .mu.M, typically at least about 100 .mu.M, more typically at least about 30 .mu.M, preferably at least about 10 .mu.M, and more preferably at least about 3 .mu.M or better.

Monoclonal, polyclonal, and humanized antibodies can be prepared. See, e.g., Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ. Press, New York, N.Y.; Kontermann and Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 139-243; Carpenter, et al. (2000) J. Immunol. 165:6205; He, et al. (1998) J. Immunol. 160:1029; Tang, et al. (1999) J. Biol. Chem. 274:27371-27378).

Single chain antibodies, single domain antibodies, and bispecific antibodies are described, see, e.g., Malecki, et al. (2002) Proc. Natl. Acad. Sci. USA 99:213-218; Conrath, et al. (2001) J. Biol. Chem. 276:7346-7350; Desmyter, et al. (2001) J. Biol. Chem. 276:26285-26290, Kostelney, et al. (1992) J. Immunol. 148:1547-1553; U.S. Pat. Nos. 5,932,448; 5,532,210; 6,129,914; 6,133,426; 4,946,778.

The invention also encompasses deamidated binding compositions, e.g., antibodies, and methods of using deamidated binding compositions (see, e.g., Zhang and Czupryn (2003) J. Pharm. Biomed. Anal. 30:1479-1490; Perkins, et al. (2000) Pharm. Res. 17:1110-1117; Lehrman, et al. (1992) J. Protein Chem. 11:657-663).

Antigen fragments may be joined to other materials, such as fused or covalently joined polypeptides, to be used as immunogens. An antigen and its fragments may be fused or covalently linked to a variety of immunogens, such as keyhole limpet hemocyanin, bovine serum albumin, or ovalbumin (Coligan, et al. (1994) Current Protocols in Immunol., Vol. 2, 9.3-9.4, John Wiley and Sons, New York, N.Y.). Peptides of suitable antigenicity can be selected from the polypeptide target, using an algorithm, such as those of Parker, et al. (1986) Biochemistry 25:5425-5432; Welling, et al. (1985) FEBS Lett. 188:215-218; Jameson and Wolf (1988) Cabios 4:181-186; or Hopp and Woods (1983) Mol. Immunol. 20:483-489.

Purification of antigen is not necessary for the generation of antibodies. Immunization can be performed by DNA vector immunization. See, e.g., Wang, et al. (1997) Virology 228:278-284. Alternatively, animals can be immunized with cells bearing the antigen of interest. Splenocytes can then be isolated from the immunized animals, and the splenocytes can fused with a myeloma cell line to produce a hybridoma. Resultant hybridomas can be screened for production of the desired antibody by functional assays or biological assays, that is, assays not dependent on possession of the purified antigen. Immunization with cells may prove superior for antibody generation than immunization with purified antigen (Meyaard, et al. (1997) Immunity 7:283-290; Wright, et al. (2000) Immunity 13:233-242; Preston, et al. (1997) Eur. J. Immunol. 27:1911-1918; Kaithamana, et al. (1999) J. Immunol. 163:5157-5164).

Antibody affinity, i.e., antibody to antigen binding properties can be measured, e.g., by surface plasmon resonance or enzyme linked immunosorbent assay (ELISA) (see, e.g., Maynard and Georgiou (2000) Annu. Rev. Biomed. Eng. 2:339-376; Karlsson, et al. (1991) J. Immunol. Methods 145:229-240; Neri, et al. (1997) Nat. Biotechnol. 15:1271-1275; Jonsson, et al. (1991) Biotechniques 11:620-627; Friguet, et al. (1985) J. Immunol. Methods 77:305-319; Hubble (1997) Immunol Today 18:305-306).

Antibodies of the present invention will usually bind with at least a K.sub.D of about 10.sup.-3 M, more usually at least 10.sup.-6 M, typically at least 10.sup.-7 M, more typically at least 10.sup.-8 M, preferably at least about 10.sup.-9 M, and more preferably at least 10.sup.-10 M, and most preferably at least 10.sup.-11 M (see, e.g., Presta, et al. (2001) Thromb. Haemost. 85:379-389; Yang, et al. (2001) Crit. Rev. Oncol. Hematol. 38:17-23; Carnahan, et al. (2003) Clin. Cancer Res. (Suppl.) 9:3982s-3990s; Wilchek, et al. (1984) Meth. Enzymol. 104:3-55).

Antibodies to IL-23R, where the anti-IL-23R antibody has substantially the same nucleic acid and amino acid sequence as those recited herein, but possessing substitutions that do not substantially affect the functional aspects of the nucleic acid or amino acid sequence, are within the definition of the contemplated invention. Variants with truncations, deletions, additions, and substitutions of regions which do not substantially change the biological functions of these nucleic acids and polypeptides are also within the definition of the contemplated invention.

A humanized antibody encompasses a human antibody, antibody fragment, single chain antibody, and the like, that has one or more amino acid residues introduced into it from a source which is non-human (import antibody). The amino acids used for grafting may comprise the entire variable domain of the source, one or more of the complementary determining regions (CDRs) of the source, or all six of the CDRs of the source antibody. With grafting of the import amino acids or polypeptide regions on to the host antibody, the corresponding amino acids or regions of the host antibody are generally removed. A humanized antibody will comprise substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab').sub.2, Fabc, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The framework regions and CDRs are highly conserved in sequence and conformation and can be accurately predicted, e.g., for use in grafting CDRs into an acceptor human antibody framework. CDR regions can be grafted into a naturally occurring human acceptor framework, or in a consensus framework derived from many human antibodies. A number of human variable light (V.sub.L) and variable heavy (V.sub.H) consensus sequences have been identified. For humanization, a chain of the mouse antibody can be compared with the available human framework chains, where the human chain of closest homology is chosen for grafting (see, e.g., Maynard and Georgiou, supra; Li, et al. (2002) Immunol. Revs. 190:53-68; Co, et al. (1991) Proc. Natl. Acad. Sci. USA 88:2869-2873; Sims, et al. (1993) J. Immunol. 151:2296-2308; Sato, et al. (1994) Mol. Immunol. 31:371-381; Morea, et al. (2000) Methods 20:267-279; Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5.sup.th ed., 4 vol., U.S. Department of Health Human Services, NIH, USA; U.S. Pat. No. 6,538,111, issued to Koike, et al.; U.S. Pat. No. 6,329,511, issued to Vasquez, et al.).

The humanized antibody of the present invention also encompasses substitutions, deletions, and/or insertions, using standard techniques of site-directed mutagenesis, e.g., those used for alanine scanning, see, e.g., Jin and Wells (1994) Protein Sci. 3:2351-2357; Cunningham and Wells (1997) Curr. Opin. Struct. Biol. 7:457-462; Jones, et al. (1998) J. Biol. Chem. 273:11667-11674; U.S. Pat. No. 4,816,567 issued to Cabilly, et al.

Embodiments of the present invention encompass fusion proteins, purification tags, and epitope tag, at an N-terminus, C-terminus, or positions within the polypeptide, e.g., FLAG tag and GSH-S transferase fusion protein. Amino acid changes can alter, add, or eliminate post-translational processes of the agonist anti-IL-23R antibody, e.g., sites for O- and N-glycosylation, and positions of cysteine residues used for disulfide formation, see, e.g., Wright and Morrison (1997) Trends Biotechnol. 15:26-32; Kunkel, et al. (2000) Biotechnol. Prog. 16:462-470.

Binding properties of the humanized antibody can be improved by the following procedure, e.g., involving site-directed mutagenesis. Computer modeling allows visualization of which mouse framework amino acid residues are likely to interact with mouse CDRs. These "contacting" mouse framework amino acids are then superimposed on the homologous human framework. Where the superimposition indicates that the mouse "contacting" framework amino acid is different from the corresponding human framework amino acid, human amino acid is changed to the corresponding mouse framework amino acid. "Contact" means interchain contact between a light chain and heavy chain, where, e.g., the amino acids are predicted to be within about 3 Angstroms of each other.

Site-directed mutagenesis can also be desirable where the amino acid of the human framework is rare for that position and the corresponding amino acid in the mouse immunoglobin is common for that position in human immunoglobin sequences. Here, the human framework amino acid can be mutated to the corresponding donor framework amino acid, see, e.g., U.S. Pat. No. 6,407,213, issued to Carter et al.; U.S. Pat. No. 6,180,370, issued to Queen, et al., Jung, et al. (2001) J. Mol. Biol. 309:701-716.

The humanized antibody can comprise at least a portion of an immunoglobulin constant region (Fc), e.g., of a human immunoglobulin. The antibody can optionally include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG1, IgG2, IgG3 and IgG4. Standard methods can be used to improve, or remove, effector function. Effector function includes binding to FcRn, FcgammaR, and complement. Half-life can be improved, e.g., by using human IgG2 or IgG4 subclasses or by altering residues in the hinge region (see, e.g., Clark (2000) Immunol Today 21:397-402; Presta, et al. (2002) Biochem. Soc. Trans. 30:487-490; Morea, et al. (2000) Methods 20:267-279).

The CDR and framework regions of the humanized antibody need not correspond precisely to the import or host sequences, e.g., these sequences can be mutagenized by substitution, insertion or deletion of at least one residue so that the residue at that site does not correspond to either the consensus or the import antibody. Such mutations, however, will not be extensive. Usually, at least 75% of the humanized antibody residues will correspond to those of the parental FR and CDR sequences, more often 90%, and most preferably greater than 95%.

Ordinarily, amino acid sequence variants of the humanized anti-IL-23R antibody will have an amino acid sequence having at least 75% amino acid sequence identity with the original humanized antibody amino acid sequences of either the heavy or the light chain (e.g. as in SEQ ID NOs:2 and 4), more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%. Identity or homology with respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the humanized anti-IL-23R residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. None of N-terminal, C-terminal, or internal extensions, deletions, or insertions into the antibody sequence shall be construed as affecting sequence identity or homology.

An alternative to humanization is to use human antibody libraries displayed on phage or human antibody libraries contained in transgenic mice (see, e.g., Vaughan, et al. (1996) Nat. Biotechnol. 14:309-314; Barbas (1995) Nature Med. 1:837-839; de Haard, et al. (1999) J. Biol. Chem. 274:18218-18230; McCafferty et al. (1990) Nature 348:552-554; Clackson et al. (1991) Nature 352:624-628; Marks et al. (1991) J. Mol. Biol. 222:581-597; Mendez, et al. (1997) Nature Genet. 15:146-156; Hoogenboom and Chames (2000) Immunol Today 21:371-377; Barbas, et al. (2001) Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Kay, et al. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, San Diego, Calif.; de Bruin, et al. (1999) Nat. Biotechnol. 17:397-399).

IV. Nucleic Acids, Vectors, and Protein Purification.

An "expression vector" is a nucleic acid construct, generated recombinantly or synthetically, with one or more predetermined nucleic acid elements that permit transcription of a particular nucleic acid. Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.

The light chain and heavy chain of the agonistic anti-IL-23R antibody can be encoded by one nucleic acid, where expression of the light chain is operably linked to a first promoter, and where expression of the heavy chain is operably linked to a second promoter. Alternatively, both light and heavy chains can be encoded by one nucleic acid, where expression of both chains is operably linked to one promoter. The nucleic acid or nucleic acids encoding the light chain and the heavy chain can be provided as one or as two vectors. The methods of the present invention encompass incorporation of the one or two vectors into the genome of a host cell (see, e.g., Chadd and Chamow (2001) Curr. Opin. Biotechnol. 12:188-194; Houdebine (2000) Transgenic Res. 9:305-320; Stoger, et al. (2002) Curr. Opin. Biotechnol. 13:161-166).

The nucleic acid encoding the light chain can further comprise a first vector, while the nucleic acid encoding the heavy chain can further comprise a second vector. Alternatively, one vector may comprise the nucleic acids encoding the light chain and the heavy chain.

For long-term or scaled-up expression of the agonistic anti-IL-23R antibody, one vector, containing the nucleic acids encoding both the light chain and the heavy chain, can be incorporated into the host genome, e.g., where incorporation is at one point or a plurality of points in the host genome. Coexpression of the light chain and heavy chain in a host cell produces a soluble antibody. The host cell can be, e.g., a mammalian, transformed or immortalized, insect, plant, yeast, or bacterial cell. The host cell may further comprise a transgenic animal. Combinations of the above embodiments are contemplated, e.g., where the light chain is simultaneously expressed by a vector that is incorporated in the host cell's genome and by a vector that is not incorporated in the genome.

Purification of an antibody, or fragments thereof, can involve ion exchange chromatography, immunoprecipitation, epitope tags, affinity chromatography, high pressure liquid chromatography, and use of stabilizing agents, detergents or emulsifiers (Dennison and Lovrien (1997) Protein Expression Purif. 11: 149-161; Murby, et al. (1996) Protein Expression Purif. 7:129-136; Ausubel, et al. (2001) Curr. Protocols Mol. Biol., Vol. 3, John Wiley and Sons, New York, N.Y., pp. 17.0.1-17.23.8; Rajan, et al. (1998) Protein Expression Purif. 13:67-72; Amersham-Pharmacia (2001) Catalogue, Amersham-Pharmacia Biotech, Inc., pp. 543-567, 605-654; Gooding and Regnier (2002) HPLC of Biological Molecules, 2.sup.nd ed., Marcel Dekker, NY).

V. Kits.

This invention contemplates an agonistic anti-IL23R antibody, fragments thereof, nucleic acids encoding an agonistic anti-IL-23R antibody, or fragments thereof, in a diagnostic kit. Encompassed is the use of binding compositions, including antibodies or antibody fragments, for the detection of IL-23R and metabolites and breakdown products thereof, and for the detection of IL-23R-dependent activities, e.g., biochemical or cellular activity. Conjugated antibodies are useful for diagnostic or kit purposes, and include antibodies coupled with a label or polypeptide, e.g., a dye, isotopes, enzyme, or metal, see, e.g., Le Doussal, et al. (1991) J. Immunol. 146:169-175; Gibellini, et al. (1998) J. Immunol. 160:3891-3898; Hsing and Bishop (1999) J. Immunol. 162:2804-2811; Everts, et al. (2002) J. Immunol. 168:883-889.

The invention provides a kit, where the kit comprises a compartment containing an agonistic anti-IL-23R antibody, an antigenic fragment thereof, or a nucleic acid encoding an agonistic anti-IL-23R antibody, or a fragment thereof. In another embodiment the kit has a compartment, a nucleic acid, e.g., a probe, primer, or molecular beacon, see, e.g., Zammatteo, et al. (2002) Biotech. Annu. Rev. 8:85-101; Klein (2002) Trends Mol. Med. 8:257-260.

The kit may comprise, e.g., a reagent and a compartment, a reagent and instructions for use, or a reagent with both a compartment and instructions for use. A kit for determining the binding of a test compound, e.g., acquired from a biological sample or from a chemical library, can comprise a control compound, a labeled compound, and a method for separating free labeled compound from bound labeled compound. Diagnostic assays can be used with biological matrices such as live cells, cell extracts and lysates, fixed cells, cell cultures, bodily fluids, or forensic samples. Various assay formats exist, such as radioimmunoassays (RIA), ELISA, and lab on a chip (U.S. Pat. Nos. 6,176,962 and 6,517,234).

The method can further comprise contacting a sample from a control subject, normal subject, or normal tissue or fluid from the test subject, with the binding composition. Moreover, the method can additionally comprise comparing the specific binding of the composition to the test subject with the specific binding of the composition to the normal subject, control subject, or normal tissue or fluid from the test subject. Expression or activity of a test sample or test subject can be compared with that from a control sample or control subject. A control sample can comprise, e.g., a sample of non-affected or non-inflamed tissue in a patient suffering from an immune disorder. Expression or activity from a control subject or control sample can be provided as a predetermined value, e.g., acquired from a statistically appropriate group of control subjects.

VI. Diagnostic Uses; Therapeutic Compositions; Methods.

The present invention provides methods for the treatment and diagnosis of healing, improper healing, wound healing, and improper wound healing, e.g., of the skin. Provided are methods of improving normal wound healing, e.g., by improving the rate of healing, and of treating improper wound healing, e.g., wounds characterized by ulcers or excess fibrosis. Moreover, the invention provides methods of treating and preventing wound-related infections.

Gene therapy of skin disorders may be performed using a variety of methods. Delivery vehicles are well described in the art, see, e.g., Boulikas (1998) Gene Therapy and Molecular Biology, Vol. 1, Gene Therapy Press, Palo Alto, Calif.; Jolly, et al. (1994) Cancer Gene Therapy 1:51-64; Kimura, et al. (1994) Human Gene Therapy 5:845-852; and Kaplitt, et al. (1994) Nat. Genetics 6:148-153.

To prepare pharmaceutical or sterile compositions including a cytokine or a small molecule agonist, the entity is admixed with a pharmaceutically acceptable carrier or excipient which is preferably inert. Preparation of such pharmaceutical compositions is known in the art, see, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984).

Cytokines are normally administered parentally, preferably intravenously. Since such proteins or peptides may be immunogenic they are preferably administered slowly, either by a conventional IV administration set or from a subcutaneous depot, e.g. as taught by Tomasi, et al, U.S. Pat. No. 4,732,863. Means to minimize immunological reactions may be applied. Small molecule entities may be orally active. For treatment of skin disorders, the present invention may also be administered topically, see, e.g., Gilman, et al. (eds.) (1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed. (1990), Mack Publishing Co., Easton, Pa.

Parenteral therapeutics may be administered in aqueous vehicles such as water, saline, or buffered vehicles with or without various additives and/or diluting agents. Alternatively, a suspension, such as a zinc suspension, can be prepared to include the peptide. Such a suspension can be useful for subcutaneous (SQ) or intramuscular (IM) injection, see, e.g., Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications 2d ed., Dekker, NY; Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Tablets 2d ed., Dekker, NY; Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Disperse Systems Dekker, NY; Fodor, et al. (1991) Science 251:767-773, Coligan (ed.) Current Protocols in Immunology; Hood, et al. Immunology Benjamin/Cummings; Paul (ed.) Fundamental Immunology; Academic Press; Parce, et al. (1989) Science 246:243-247; Owicki, et al. (1990) Proc. Natl. Acad. Sci. USA 87:4007-4011; and Blundell and Johnson (1976) Protein Crystallography, Academic Press, New York.

Selecting an administration regimen for a therapeutic depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells, timing of administration, absorption through epithelial layers, etc. Preferably, an administration regimen maximizes the amount of therapeutic delivered to the patient consistent with an acceptable level of side effects. Accordingly, the amount of biologic delivered depends in part on the particular entity and the severity of the condition being treated. Guidance in selecting appropriate doses of cytokine or small molecules are determined using standard methodologies.

Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms of, e.g., the inflammation or level of inflammatory cytokines produced. Preferably, a biologic that will be used is derived from the same species as the animal targeted for treatment, thereby minimizing a humoral response to the reagent.

Antibodies, antibody fragments, and cytokines can be provided by continuous infusion, or by doses at intervals of, e.g., one day, one week, or 1-7 times per week. Doses may be provided intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, or by inhalation. A preferred dose protocol is one involving the maximal dose or dose frequency that avoids significant undesirable side effects. A total weekly dose is generally at least 0.05 .mu.g/kg body weight, more generally at least 0.2 .mu.g/kg, most generally at least 0.5 .mu.g/kg, typically at least 1 .mu.g/kg, more typically at least 10 .mu.g/kg, most typically at least 100 .mu.g/kg, preferably at least 0.2 mg/kg, more preferably at least 1.0 mg/kg, most preferably at least 2.0 mg/kg, optimally at least 10 mg/kg, more optimally at least 25 mg/kg, and most optimally at least 50 mg/kg, see, e.g., Yang, et al. (2003) New Engl. J. Med. 349:427-434; Herold, et al. (2002) New Engl. J. Med. 346:1692-1698; Liu, et al. (1999) J. Neurol. Neurosurg. Psych. 67:451-456; Portielji, et al. (2003) Cancer Immunol. Immunother. 52:133-144. The desired dose of a small molecule therapeutic, e.g., a peptide mimetic, natural product, or organic chemical, is about the same as for an antibody or polypeptide, on a moles/kg basis.

The present invention also provides for administration of biologics in combination with known therapies, e.g., steroids, particularly glucocorticoids, which alleviate the symptoms, e.g., associated with inflammation, or antibiotics or anti-infectives. Daily dosages for glucocorticoids will range from at least about 1 mg, generally at least about 2 mg, and preferably at least about 5 mg per day. Generally, the dosage will be less than about 100 mg, typically less than about 50 mg, preferably less than about 20 mg, and more preferably at least about 10 mg per day. In general, the ranges will be from at least about 1 mg to about 100 mg, preferably from about 2 mg to 50 mg per day. Suitable dose combinations with antibiotics, anti-infectives, or anti-inflammatories are also known.

The present invention provides agonists and antagonists of IL-23 for modulating genes relating to healing, e.g., wound healing. Also provided are methods of diagnosis of healing, e.g., involving detecting expression or changes in expression of IL-23 modulated genes and gene products. These genes and gene products include, e.g., nitric oxide synthase 2 (NOS2), lactoferrin, IL-19, DEC-205, CD50 (ICAM-2), IL-25, TNFSF7 (CD27L), eosinophilic basic protein, and others.

The invention provides a method to modulate expression of MMP-7, e.g., for the treatment of wound healing. Matrix proteolysis is a hallmark of inflammation. Matrilysin, a metalloprotease, is used in wound repair (see, e.g., Parks, et al. (2001) Chest 120:36S-41S; Wilson, et al. (1999) Science 286:113-117).

The present invention provides methods for modulating activities and proteins relating to neutrophils, such as neutrophil chemoattractants and proteins and metabolites expressed by neutrophils. IL-23 stimulates IL-17 expression which, in turn, stimulates production of chemokines that attract neutrophils. Increased expression or activity of neutrophil response, lactoferrin, IL-17, IL-6, and nitric oxide, are found in a number of inflammatory conditions, and can play a role in modulating wound healing (see, e.g., Tsokos, et al. (2002) Virchows Arch. 441:494-499; Linden (2001) Int. Arch. Allergy Immunol. 126:179-184; Sheppard (2002) Chest 121:21S-25S; Redington (2000) Monaldi Arch. Chest Dis. 55:317-323; Vignola, et al. (2001) Curr. Allergy Asthma Rep. 1:108-115).

Provided are methods of modulating expression of lactoferrin, a protein produced by neutrophils (see, e.g., Boyton, et al. (2002) Brit. Medical Bull. 61:1-12; Singh, et al. (2002) Nature 417:552-555; Gomez, et al. (2002) Infect. Immun. 70:7050-7053).

Provided are methods for modulating expression of neutrophil elastase, e.g., for modulating wound healing (see, e.g., Tkalcevic, et al. (2000) Immunity 12:201-210; Aprikyan, et al. (2001) Curr. Opinion Immunol 13:535-538; Tremblay, et al. (2003) Curr. Opin. Investig. Drugs 4:556-565; Lee, et al. (2001) Curr. Opinion Crit. Care 7:1-7; Shapiro (2002) Am. J. Respir. Cell Mol. Biol. 26:266-268).

Also provided are methods to modulate neutrophil attractants for promoting wound healing, e.g., IL-17, nitric oxide, and GRO-alpha. IL-17 modulates neutrophil recruitment (see, e.g., Ye, et al. (2001) J. Exp. Med. 194:519-527; Ye, et al. (2001) Am. J. Respir. Cell Mol. Biol. 25:335-340). Nitric oxide, synthesized by nitric oxide synthase, can promote wound healing, e.g., by attracting monocytes and neutrophils to the wound, see, e.g., Schwentker, et al. (2002) Nitric Oxide 7: 1-10; MacMicking, et al. (1997) Annu. Rev. Immunol 15:323-350. CXCL-1 (a.k.a. GRO-alpha) promotes wound healing, e.g., by attracting neutrophils to wounds and stimulating keratinocyte proliferation and angiogenesis (see, e.g., Gillitzer, et al. (2001) J. Leukoc. Biol. 69:513-521; Li and Thornhill (2000) Cytokine 12:1409-1413).

IL-6 promotes the healing of injuries, e.g., skin wounds, see, e.g., Gallucci, et al. (2001) J. Interferon Cytokine Res. 21:603-609; Sugawara, et al. (2001) Cytokine 15:328-336; Erdag, et al. (2002) Ann. Surg. 235:113-124; Nadeau, et al. (2002) Microbes Infect. 4:1379-1387; Imanishi, et al. (2000) Prog. Retin. Eye Res. 19:113-129; Gregory, et al. (1998) J. Immunol. 160:6056-6061.

Interferon-gamma (IFNgamma) mediates proper wound healing, e.g., by modulating actin and collagen content, contractile capacity, and scar formation, see, e.g., Moulin, et al. (1998) Exp. Cell Res. 238:283-293; Ahdieh, et al. (2001) Am. J. Physiol. Cell Physiol. 281:C2029-C2038; Cornelissen, et al. (2000) J. Dent. Res. 79:1782-1788; Shtrichman, et al. (2001) Curr. Opin. Microbiol. 4:251-259; Ikeda, et al. (2002) Cytokine Growth Factor Rev. 13:95-109; Rottenberg, et al. (2002) Curr. Opin. Immunol 14:444-451).

IFNgamma production is stimulated by CD27 (a.k.a. TNFRSF7). CD27 also stimulates cell proliferation and is implicated in the activation and development of T cells, and in T cell-dependent antibody production, including IgE production, by B cells (see, e.g., Takeda, et al. (2000) J. Immunol. 164:1741-1745; Nagumo, et al. (1998) J. Immunol. 161:6496-6502; Tomiyama, et al. (2002) J. Immunol. 168:5538-5550; Busse and Lemanske (2001) New Engl. J. Med. 344:350-362).

MUC5ac serves a number of biological functions, including wound healing, see, e.g., Dohrman, et al. (1998) Biochim. Biophys. Acta 1406:251-259; Rose, et al. (2000) J. Aerosol. Med. 13:245-261; Rogers (2000) Monaldi Arch. Chest Dis. 55:324-332; Enss, et al. (2000) Inflamm. Res. 49:162-169.
 

Claim 1 of 15 Claims

1. A method of treating a cutaneous ulcer or graft comprising administering to a subject an effective amount of an IL-23 complex comprising: a) a polypeptide comprising residues 352 to 521 of SEQ ID NO: 8 or a conservatively modified variant thereof comprising an individual amino acid substitution; and b) a polypeptide comprising residues 25 to 330 of SEQ ID NO: 8 or a conservatively modified variant thereof comprising an individual amino acid substitution; wherein the IL-23 complex increases the expression of IL-17 at least 2-fold in excisional wounds on the backs of C57BI/6NT mice.

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