WO2009124056A2

WO2009124056A2 - Alpha-fetoprotein for treating disease

Info

Publication number: WO2009124056A2
Application number: PCT/US2009/038964
Authority: WO
Inventors: Robert Mulroy; Edward J. Stewart; James Murray
Original assignee: Merrimack Pharmaceuticals, Inc.
Priority date: 2008-04-01
Filing date: 2009-03-31
Publication date: 2009-10-08
Also published as: WO2009124056A3

Abstract

Disclosed are methods of treating psoriatic arthritis, IgA nephropathy, Hashimoto's disease, Sjogren's syndrome, ankylosing spondylitis, and uveitis in a mammal, involving administering to the mammal a therapeutically effective amount of an alpha-fetoprotein (AFP) or a biologically active fragment, derivative, or analog thereof. Compositions and kits containing an AFP or biologically active fragment, derivative, or analog thereof are also disclosed.

Description

ALPHA-FETOPROTEIN FOR TREATING DISEASE

Field of the Invention

This invention relates to methods for using AFP to treat disease, e.g., psoriatic arthritis, IgA nephropathy, Hashimoto's disease, Sjogren's syndrome, ankylosing spondylitis, and uveitis (e.g., autoimmune and idiopathic uveitis).

Background of the Invention

Immunoinflammatory disorders are conditions which result in the destruction of healthy tissue by an inflammatory process, deregulation of the immune system, or an unwanted proliferation of immune cells. Examples of immunoinflammatory disorders include autoimmune diseases, in which the body mounts an immune response against itself. The initiation of autoimmune diseases is not well understood, but may involve genetic predisposition or environmental factors. Immunoinflammatory disorders are usually classified clinically in a variety of ways, including family history, characterization of immune cells and antibodies, and responsiveness of the disorder to immunosuppressive pharmaceuticals. Alpha-fetoprotein (AFP) is a 70-kDa glycoprotein produced by the yolk sac and fetal liver. AFP is present in fetal serum at milligram levels, and, at birth, declines to the nanogram levels normally found in adult serum: increased levels of AFP in adult serum are indicative of a yolk sac tumor, a hepatoma, or of liver regeneration. The role of AFP during fetal development is not known, although it has been suggested that AFP protects a gestating fetus from a maternal immune attack or from the effects of maternal estrogen. In vitro and in vivo experiments have shown that AFP has both cell growth-stimulatory and -inhibitory activities, depending upon the target cell, the relative concentration of AFP, and the presence of other cytokines and growth factors. For example, AFP can inhibit the growth of many types of tumor cells, and, in particular, inhibits estrogen-stimulated cell growth. Conversely, AFP stimulates the growth of normal embryonal fibroblasts. AFP has also been shown to have both immunosuppressive and immunoproliferative effects. Summary of the Invention

Alpha-fetoprotein (AFP) and biologically active fragments, derivatives, or analogs thereof are useful for modulating inflammation that is mediated by, e.g., immune and other cells, in a mammal. Accordingly, the invention provides compositions and methods for treating, preventing, or inhibiting disease (e.g., psoriatic arthritis, IgA nephropathy, Hashimoto's disease, Sjogren's syndrome, ankylosing spondylitis, and uveitis) in a mammal (e.g., a human) or for treating, preventing, or reducing one or more symptoms (e.g., 1, 2, 3, 4, or 5) of disease by administering to the mammal a therapeutically effective amount of AFP or a biologically active fragment, derivative, or analog thereof. One or more symptoms of such diseases include, e.g., stiffness, pain, swelling, tenderness of the joints and surrounding soft tissue, fatigue, nail pitting, depression, sensitivity to cold, weight increase or weight decrease, muscle weakness, coarsening of the skin, dry or brittle hair, constipation, muscle cramps, increased menstral flow, goiter, dry eyes, eye irritation, eye burning, eyelids sticking together, dry mouth, hoarse or weak voice, difficulty swalling food, painful salivary glands, back pain and stiffness, fever, night sweats, light sensitivity, blurring or loss of vision, eye pain, and redness of eye.

In a first aspect, the present invention provides a method of treating a patient with psoriatic arthritis, IgA nephropathy, Hashimoto's disease, Sjogren's syndrome, ankylosing spondylitis, and uveitis by administering AFP (or biologically active fragment, derivative, or analog thereof). In an embodiment, the AFP is administered to the patient in a therapeutically effective amount.

In another embodiment, uveitis is an autoimmune uveitis (e.g., birdshot retinochoroidopathy uveitis, sarcoid uveitis, or HLA-B27 uveitis) or an idiopathic uveitis (e.g., Behcet's disease or Vogt-Koyanagi-Harada (VKH) syndrome).

Additional diseases that may be treated using the method of the invention include: Wegener's granulomatosis, inflammatory bowel disease, polymyositis, dermatomyositis, multiple endocrine failure, Schmidt's syndrome, adrenalitis, thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, presenile dementia, demyelinating diseases, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressier' s syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, pemphigoid, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), adult onset diabetes mellitus (Type II diabetes), male and female autoimmune infertility, mixed connective tissue disease, polyarteritis nedosa, systemic necrotizing vasculitis, juvenile onset rheumatoid arthritis, glomerulonephritis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent abortion, anti -phospholipid syndrome, farmer's lung, erythema multiforme, post cardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird-fancier's lung, allergic disease, allergic encephalomyelitis, toxic epidermal necrolysis, alopecia, Alport's syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, leprosy, malaria, leishmaniasis, trypanosomiasis, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema, lymphomatoid granulomatosis, Caplan's syndrome, Kawasaki's disease, dengue, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuch's cyclitis, Henoch-Schonlein purpura, glomerulonephritis, graft versus host disease, transplantation rejection, human immunodeficiency virus infection, echo virus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post vaccination syndromes, congenital rubella infection, Hodgkin's and Non-Hodgkin's lymphoma, renal cell carcinoma, multiple myeloma, Eaton-Lambert syndrome, relapsing polychondritis, malignant melanoma, cryoglobulinemia, Waldenstrom's macroglobulemia, Epstein-Barr virus infection, mumps, Evan's syndrome, and autoimmune gonadal failure.

In other embodiments, the method further involves administering to the mammal AFP (or a biologically active fragment, derivative, or analog thereof) in combination with one or more secondary agents (e.g., a steroid, a non-steroid anti- inflammatory drug (NSAID), a disease-modifying anti-rheumatic drug (DMARD), an integrin antagonist, an inflammation signaling inhibitor (ISI), a tolerizing agent, an immunosuppressive agent, and an immunomodulatory agent), according to methods well known in the art. In another embodiment, the secondary agent is administered in a therapeutically effective amount. In another embodiment, one or more of the secondary agents is administered at a dosage that is lower than the standard dose for the secondary agent when it is administered as the sole or primary treatment.

In other embodiments of the invention, the steroid is a corticosteroid, such as prednisolone (e.g., DELTASONE^® and ORASONE^®); the NSAID is naproxen sodium, diclofenac sodium, diclofenac potassium, aspirin, sulindac, diflunisal, piroxicam, indomethacin, ibuprofen, nabumetone, choline magnesium trisalicylate, sodium salicylate, salicylsalicylic acid, fenoprofen, flurbiprofen, ketoprofen, meclofenamate sodium, meloxicam, oxaprozin, sulindac, tolmetin, or COX-2 inhibitors, such as rofecoxib, celecoxib, valdecoxib, or lumiracoxib; or the DMARD is auranofin, aurothioglucose, azathioprine, chlorambucil, cyclophosphamide, cyclosporine, D-penicillamine, gold sodium thiomalate (injectable gold), hydroxychloroquine, lefiunomide, methotrexate, minocycline, mycophenolate mofetil, or sulfasalazine. In other embodiments of the invention, the integrin antagonist is natalizumab (Elan/Biogen Idee), oMEPUPA-V (Biogen), alefacept, CDP-323

(Celltech); firategrast (SB-68399; GlaxoSmithKline); TR-9109 (Pfizer); ISIS-107248 (Antisense Therapeutics); R- 1295 (Roche); or TBC-4746 (Schering-Plough); the inflammatory signaling inhibitor is a soluble TNF receptor, such as etanercept or lenercept; a soluble pro-inflammatory cell surface signaling molecule, such as soluble CTLA-4 (abatacept); an antibody directed against a pro-inflammatory cytokine or a pro-inflammatory cell surface signaling molecule, such as adalimumab, certolizumab, inflixamab, golimumab, and rituxan; a dominant-negative pro-inflammatory cytokine variant, such as XENP345, XPRO™1595, anakinra, and variants disclosed in U.S. Patent Application Publication Nos. 20030166559 and 20050265962; an inhibitor of the signaling pathways downstream of pro-inflammatory cytokine or proinflammatory cell surface signaling molecules, such as DE 096, 5-amino-2- carbonylthiopene derivatives (as described in WO2004089929), ARRY-797, BIRB 796 B S, ( 1 -5 -tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3 - [4-2(morpholin-4-yl-ethoxy)- naphtalen-l-yl]-urea, CHR-3620, CNI-1493, FR-167653 (Fujisawa Pharmaceutical, Osaka, Japan), ISIS 101757 (Isis Pharmaceuticals), ML3404, NPC31145, PD169316, PHZI l 12, RJW67657, 4-(4-(4-fluorophenyl)-l-(3-phenylpropyl)-5-(4-pyridinyl)-lH- imidazol-2-yl)-3-butyn-l-ol, SCIO-469, SB202190, SB203580, (4-(4-fluorophenyl)- 2-(4-methylsulfinylphenyl)-5-(4-pyridyl)lH-imidazole), SB239063, trans-l -(4- hydroxycyclohexyl)-4-(4-fluorophenyl-methoxypyridimidin-4-yl)imidazole, SB242235, SD-282, SKF-86002, TAK 715, VX702, and VX745; or an inhibitor of TNF alpha converting enzyme (TACE), such as BB-1101, BB-3103, BMS-561392, butynyloxyphenyl β-sulfone piperidine hydroxomates, CH4474, DPC333, DPH- 067517, GM6001, GW3333, Ro 32-7315, TAPI-I, TAPI-2, and TMI 005); or the tolerizing agent is an anti-idiotypic agent, such as monoclonal antibodies, LJP 394 (abetimus, RIQUENT^®, La Jolla Pharmaceuticals).

In other embodiments of the invention, the immunosuppressive agent is a cyclosporine (e.g., cyclosporin A, such as NEORAL^®, SANDIMMUNE^®, and SANGC YA^®), an azathioprine (e.g. IMURAN^®), FK-506, 15-deoxyspergualin, or an antibody (e.g., a monoclonal antibody, such as basiliximab, daclizumab, and muromonab-CD3, or a globulin, such as anti-lymphocyte globulin, anti-thymocyte globulin, and the like).

In other embodiments, the immunomodulatory agent is an interferon (e.g., an interferon-b (REBIF^® (IFN-β- 1 a), AVONEX^® (IFN- β- 1 a), and BETASERON^® (IFN- β-lb)), an interferon-t (TAUFERON™), an interferon-α (e.g., ROFERON-A^® (IFN- α-2a), INTRON-A^® (IFN-α-2b), REBETRON^® (IFN- α-2b), ALFERON-N^® (IFN-α- n3), PEG-INTRON^® (IFN-α-2b covalently conjugated with monomethoxy polyethylene glycol), INFERGEN^® (a non-naturally occurring type 1 interferon with 88% homology to IFN-α-2b), or PEGASYS^® (pegylated IFN-α-la)), or ACTIMMUNE^® (IFN-γ-lb)).

Different administration schedules can be followed in the above method. For instance, the AFP (or biologically active fragment, derivative, or analog thereof) or the secondary agent can be administered one or more times (e.g., 1, 2, 3, 4, 5, or 10 times or more) hourly, daily, weekly, biweekly, or monthly. In addition, the dosage of the AFP (or biologically active fragment, derivative, or analog thereof) per administration may be the same or different.

In other embodiments of the above method, the AFP (or biologically active fragment, derivative, or analog thereof) and the secondary agent are administered coextensively or separately. Many variations in the administration schemes are possible, for example, both the AFP (or biologically active fragment, derivative, or analog thereof) and the secondary agent may be administered to the patient during the first treatment phase. Subsequently, the administration of one (e.g., the AFP or the secondary agent) may be terminated or the dosage amount may be modified (e.g., increased or decreased) while administration of the other is continued (e.g., at the same dosage level or at a modified level (e.g., increased or decreased)). Alternatively, both the AFP (or biologically active fragment, derivative, or analog thereof) and the secondary agent may be administered initially at their maximal or minimal dosages with subsequent dosages of both being reduced or increased, respectively, during the treatment regimen. In addition, the AFP (or biologically active fragment, derivative, or analog thereof) may be administered prior to or following administration of the secondary agent. In an additional embodiment of the method, the AFP (or biologically active fragment, derivative, or analog thereof) is administered at a dosage in the range of 0.1 to 400 mg. When the AFP is administered in combination with one or more secondary agents, the one or more secondary agents can be administered in the same dosage form or in separate dosage forms; the secondary agent is administered in the range of 0.1 to 3,000 mg.

In another embodiment of the method, the AFP (or a biologically active fragment, derivative, or analog thereof) and the one or more secondary agent are administered via the same route of administration or via two different routes of administration. In another embodiment, the method includes diagnosing the patient with one or more of the diseases disclosed herein prior to treatment. In another embodiment, the method includes treating a mammal predisposed to develop or previously diagnosed with one or more of the diseases disclosed herein.

In a second aspect, the invention features a composition formulated for ophthalmic administration that includes an AFP (or a biologically active fragment, derivative, or analog thereof) in an amount (e.g., a therapeutically effective amount) to treat, prevent, or inhibit uveitis, or to treat, prevent, inhibit, reduce, or ameliorate one or more symptoms (e.g., 1, 2, 3, 4, or 5) of uveitis in a patient in need thereof. In a related embodiment, the ophthalmic composition further includes an amount (e.g., a therapeutically effective amount) of one or more secondary agents

(e.g., a steroid, a non-steroid anti-inflammatory drug (NSAID), a disease-modifying anti-rheumatic drug (DMARD), an integrin antagonist, an inflammation signaling inhibitor (ISI), a tolerizing agent, an immunosuppressive agent, and an immunomodulatory agent).

A third aspect of the invention provides a kit that includes 1) an AFP (or a biologically active fragment, derivative, or analog thereof) formulated for ophthalmic administration in an amount (e.g., a therapeutically effective amount) to treat, prevent, or inhibit uveitis, or to treat prevent, inhibit, or reduce one or more of the symptoms of or the progression of uveitis in a patient in need thereof, and 2) instructions for the administration of the AFP to the patient.

In additional embodiments, the kit further includes one or more secondary agents (e.g. a steroid, a non-steroid anti-inflammatory drug (NSAID), a disease- modifying anti-rheumatic drug (DMAPvD), an integrin antagonist, an inflammation signaling inhibitor (ISI), a tolerizing agent, an immunosuppressive agent, and an immunomodulatory agent) and instructions for the administration of the secondary agent to the patient. In different embodiments of the kit, the AFP (or a biologically active fragment, derivative, or analog thereof) and the one or more secondary agents are present in the same composition or are present in the kit in separate compositions. In other embodiments, the AFP (or biologically active fragment, derivative, or analog thereof) and the secondary agent are formulated for the same route of administration or for two different routes of administration. In several embodiments of all the aspects of the invention, the AFP (or biologically active fragment, derivative, or analog thereof) is naturally-occurring, synthetic, or recombinant human AFP having an amino acid sequence that is substantially identical (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or even 100% identical) to SEQ ID NO: 1. In other embodiments of all aspects of the invention, the AFP (or biologically active fragment, derivative, or analog thereof) is non-glycosylated or glycosylated. In additional embodiments of all aspects of the invention, the biologically active AFP fragment is, e.g., Domain I (SEQ ID NO: 5), Domain II (SEQ ID NO: 6), Domain III (SEQ ID NO: 7), Domain I + II (SEQ ID NO: 8), Domain II + III (SEQ ID NO: 9), or Fragment I (SEQ ID NO: 10). In yet other embodiments of all aspects of the invention, the AFP may be formulated at a dosage in the range of 0.1 mg to 400 mg. The AFP (or biologically active fragment, derivative, or analog thereof) and/or one or more secondary agents may be formulated for or administered by one or more of a variety of routes of administration, including, but not limited to, intravenous, intramuscular, oral, by inhalation, parenteral, intraperitoneal, intaarterial, transdermal, sublingual, nasal, through use of suppositories, transbuccal, liposomal, adiposal, ophthalmic, intraocular, subcutaneous, intrathecal, topical, or local administration. In other embodiments of all aspects of the invention, the AFP (or biologically active fragment, derivative, or analog thereof) and/or the one or more secondary agents is formulated in an ophthalmic solution. In additional embodiments of all aspects of the invention, the coadministration of the AFP (or a biologically active fragment, derivative, or analog thereof) and the one or more secondary agents exhibits a therapeutic effect that is greater than that observed when the AFP (or biologically active fragment, derivative, or analog thereof) or the one or more secondary agents are administered alone. In additional embodiments of all aspects of the invention, the AFP, the one or more secondary agents, or both can be administered at a lower dosage than that normally required for achieving a therapeutic effect when either are administered alone (e.g., the AFP or the secondary agent can be administered at a dosage that is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, or 90% lower). In other embodiments of all aspects of the invention, co-administration of an AFP (or biologically active fragment, derivative, or analog thereof) and the secondary agent reduces the toxicity of the secondary agent (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, or 70% or more) relative to the toxicity of the secondary agent when administered at the same concentration in the absence of the AFP. In yet other embodiments of all aspects of the invention, the secondary agent can be administered in combination with the AFP at a dosage that is higher (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95%, or 100% or more) than the normal dosage of the secondary agent, when administered for treating a disease (e.g., psoriatic arthritis, IgA nephropathy, Hashimoto's disease, Sjogren's syndrome, ankylosing spondylitis, and uveitis), without the toxicity normally expected or observed at the increased dose of the secondary agent when it is administered alone. As used herein, the term "alpha-fetoprotein" or "AFP" refers to a polypeptide having an amino acid sequence that is substantially identical to the mature human AFP (SEQ ID NO: 1) or to a polypeptide that is encoded by a nucleic acid sequence that is substantially identical to the nucleic acid sequence that encodes human AFP (NCBI Accession No. NM 001134; SEQ ID NO: 2). Mature human AFP is a protein of 591 amino acids (see, SEQ ID NO: 1), resulting from cleavage of a precursor of 609 amino acids (GenBank Accession No. NP_001125) to remove an 18-amino acid signal sequence. An AFP of this invention has an amino acid sequence that is substantially identical to SEQ ID NO: 1. An AFP of the invention may include all or part of the 18-amino acid signal sequence (SEQ ID NO: 22).

An AFP of the invention can be naturally-occurring, synthetic, or recombinant (e.g., produced in a transgenic cell or a transgenic animal, e.g., a mammal, such as a goat, sheep, camel, cow, pig, rabbit, horse, or llama). Preferably, an AFP of the invention includes any recombinant human AFP (whether or not having the same post-translational modifications as naturally-occurring AFP, e.g., a non-glycosylated form of AFP; see, e.g., U.S. Patent No. 7,208,576, incorporated by reference herein) and other biologically active variants of human AFP.

An AFP of this invention may contain modifications of the amino acid sequence of SEQ ID NO: 1, including substitution (e.g., conservative substitution), deletion, or addition of one or more amino acid residues. For instance, a recombinant human AFP is described in U.S. Patent No. 7,208,576, incorporated herein by reference, which contains an asparagine to glutamine substitution at position 233 of SEQ ID NO: 1. Thus, the term "alpha-fetoprotein" encompasses derivatives or analogs of AFP, such as those described herein, as well as an AFP having the same or different post-translational modifications as naturally-occurring AFP (e.g., an AFP of the invention may include additional, non-naturally-occurring post-translational modifications).

An AFP of the invention is not limited to the full-length sequence; it also includes biologically active fragments of AFP. A biologically active fragment of AFP, for use in the compositions and methods of the invention, can be identified using one or more assays described herein (e.g., AMLR assays, AFP-binding to monocyte assays, experiments using the EAE mouse model, and splenocyte assays; see below). A typical biologically active AFP fragment contains at least 5 contiguous amino acids of SEQ ID NO: 1, or at least 8 contiguous amino acids, preferably at least 10, 20, or 50 contiguous amino acids, more preferably at least 100 contiguous amino acids, and most preferably at least 200, 300, 400, or more contiguous amino acids in length. For instance, U.S. Patent No. 6,818,741 (herein incorporated by reference) discloses an 8 -amino acid fragment of human AFP (amino acids 471-478;

EMTPVNPG; SEQ ID NO: 3), as well as other related AFP fragments. An active AFP fragment of this invention may further contain amino acid substitutions, deletions, or additions at a limited number of positions, so long as the AFP fragment has at least 90% identity (e.g., at least 95%, 99%, or 100% sequence identity) to its corresponding sequence within SEQ ID NO: 1. For sequence comparison purposes in this application, the corresponding sequence of SEQ ID NO: 1 is deemed to have the same number of amino acids as a given AFP fragment. For instance, a 34-mer AFP peptide corresponding to the 446-479 segment of SEQ ID NO: 1 (LSEDKLLACGEGAADIIIGHLCIRHEMTPVNPGV; SEQ ID NO: 4) may contain up to 3 amino acids altered from the 446-479 segment of SEQ ID NO: 1. One such example of sequence deviation in biologically active AFP fragments is found in, e.g., U.S. Patent No. 5,707,963 (herein incorporated by reference), which discloses a 34- amino acid fragment of human AFP (SEQ ID NO: 4) with flexibility at two amino acid residues (amino acid 9 and 22 of SEQ ID NO: 4). Some other examples of AFP fragments include, e.g., Domain I (amino acids 2-198 of mature human AFP; SEQ ID NO: 5), Domain II (amino acids 199-390 of mature human AFP; SEQ ID NO: 6), Domain III (amino acids 391-591 of mature human AFP; SEQ ID NO: 7), Domain I+II (amino acids 2-390 of mature human AFP; SEQ ID NO: 8), Domain II+III (amino acids 199-591 of mature human AFP; SEQ ID NO: 9), and human AFP Fragment I (amino acids 267-591 of mature human AFP; SEQ ID NO: 10).

An AFP of this invention exhibits one or more of the biological activities of naturally-occurring human AFP, including, for example, the ability to bind to human leukocytes, the ability to suppress autoimmune reactions, and the ability to reduce the production of inflammatory cytokines. The leukocyte binding assay used for testing AFP activity is described herein and in, e.g., Parker et al., Protein Express.

Purification 38:177-183, 2004. The autoimmune suppression activity for an AFP of this invention can be demonstrated by, e.g., assaying the ability of the AFP to suppress human autologous mixed lymphocyte reactions (AMLR), by assaying the ability of the AFP to suppress experimental autoimmune encephalomyelitis (EAE) in a mouse model using the methods described herein, or by assaying the ability of AFP to reduce production of inflammatory cytokines. A functional AFP of the invention demonstrates at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% of the biological activity of naturally-occurring human AFP.

In this application, the term "amino acid" refers to naturally occurring, non- naturally occurring, and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ- carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones (e.g., peptide mimetics, such as an AFP peptoid), but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics are chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that are capable of functioning in a manner that is similar to a naturally occurring amino acid. An AFP of the invention can include naturally occurring or synthetic amino acids or amino acid mimetics. (See, e.g., peptoids generally described in U.S. Patent No. 5,811,387 and 7,030,216, herein incorporated by reference).

As to amino acid sequences, one of skill will recognize that individual substitutions, deletions, or additions to a polypeptide sequence that alter, add, or delete a single amino acid or a small percentage of amino acids in the sequence constitute a "conservatively modified variant," when the alterations result in the substitution of one or more amino acids with other, chemically similar amino acids. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. As used herein, "autoimmune diseases" are diseases that are primarily caused by activation of the immune cells of the immune system in response to self-antigens and tissues. The term autoimmune diseases, as used herein, also encompasses diseases that do not appear to be primarily autoimmune but have immune manifestations involving immunoglobulins, antigen-specific B cell surface receptors, or antigen-specific T cell receptors. An autoantigen is any protein, or portion of a protein specifically recognized by and bound by an autoantibody. An antibody is any immunoglobulin, antigen-specific B cell surface receptor (surface immunoglobulin), or antigen-specific T cell receptor directed against an antigen. Examples of autoimmune diseases include psoriatic arthritis, IgA nephropathy, Hashimoto's disease, Sjogren's syndrome, ankylosing spondylitis, and uveitis.

The term "disease modifying anti-rheumatic drug" or "DMARD" refers to a therapeutic agent that is used for the treatment of an inflammatory disease. A DMARD can be used to treat, prevent, inhibit, reduce, or ameliorate one or more inflammatory diseases or the symptoms or progression thereof in a patient when administered to the patient in a therapeutically effective amount. Examples of DMARDs known in the art include, e.g., auranofin, aurothioglucose, azathioprine, chlorambucil, cyclophosphamide, D-penicillamine, gold sodium thiomalate (injectable gold), hydroxychloroquine, leflunomide, methotrexate, minocycline, mycophenolate mofetil, and sulfasalazine.

By "human alpha-fetoprotein precursor" is meant a polypeptide having substantially the same amino acid sequence as amino acids 1-609 set forth in Genbank Accession No. V01514 (SEQ ID NO: 20) and encoded by nucleotides 45-1874 of the cDNA sequence set forth in Genbank Accession No. VOl 514 (SEQ ID NO: 21). By "HuAFP secretory signal" or "HuAFP signal peptide" or "HuAFP leader" or "HuAFP signal sequence" is meant a polypeptide having substantially the same amino acid sequence as amino acids 1-18 set forth in Genbank Accession No. VO 1514 (encoded by nucleotides 45-98; see also nucleotides 45-98 of SEQ ID NO: 21 or amino acids 1-18 of SEQ ID NO: 20). The protein secretory signal is cleaved from rHuAFP during protein maturation and extracellular secretion.

By "idiopathic uveitis" is meant uveitis arising spontaneously or from an obscure or unknown cause. Examples of idiopathic uveitis include, without limitation, Behcet's disease and Vogt-Koyanagi-Harada (VKH) syndrome, as well as uveitis associated with another disease or infection, e.g., sympathetic ophthalmia, toxoplasmosis, acute multifocal choroiditis, multiple sclerosis, tuberculosis, syphilis, vasculitides due to Wegener granulomatosis, systemic lupus erythematosus, Whipple disease, ankylosing spondylitis, Crohn disease, relapsing polychondritis, acute multifocal placoid pigment epitheliopathy, CNS lymphoma, brucellosis, herpes simplex, herpes zoster, inflammatory bowel disease, juvenile rheumatoid arthritis, Kawasaki's disease, leptospirosis, Lyme disease, presumed ocular histoplasmosis syndrome, psoriatic arthritis, Reiter's syndrome, syphilis, and toxocariasis.

By "immune cell antiproliferative" is meant a composition capable of inhibiting the growth of an undesirable immune cell (e.g., an autoreactive T cell as measured using the assays described herein).

As used here, "immunomodulatory agent" refers to (1) an interferon or a peptide or protein that has an amino acid sequence substantially identical (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100% identical) to all or a portion of the sequence of an interferon (e.g., a human interferon), such as, e.g., IFN-α (e.g., IFN-α-la; see U.S. Patent Application No. 20070274950, incorporated herein by reference in its entirety), IFN-α- Ib (SEQ ID NO: 11), IFN-α- 2a (see PCT Application No. WO 07/044083, herein incorporated by reference in its entirety), and IFN-α-2b (SEQ ID NO: 12)), IFN-β (e.g., described in U.S. Patent No. 7,238,344, incorporated by reference in its entirety; IFN-β-la (AVONEX^® and

REBIF^®), as described in U.S. Patent No. 6,962,978, incorporated by reference in its entirety, and IFN-β-lb (BETASERON^®, as described in U.S. Patent Nos. 4,588,585; 4,959,314; 4,737,462; and 4,450,103; incorporated by reference in their entirety), IFN-γ (e.g., SEQ ID NO: 13), and IFN-τ (as described in U.S. Patent No. 5,738,845 and U.S. Patent Application Publication Nos. 20040247565 and 20070243163; incorporated by reference in their entirety); (2) a small molecule (e.g., BGl 2 (fumarate), fingolimod (FTY-720), laquinimod, teriflunomide, or atorvastatin, or a molecule that demonstrates the same or substantially the same biological activity as an interferon (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the activity of a human IFN-α, a human IFN-β, a human IFN-γ, or a human IFN-τ in the ability to suppress EAE in a mouse model)); (3) an antibody (e.g., all or part of a monoclonal antibody (e.g., an IL-2 receptor-binding antibody, such as daclizumab; a CD20-binding antibody, such as rituximab; an IL- 12 binding antibody, such asABT- 874; and a CD52-binding antibody, such as alemtuzumab), a polyclonal antibody, or an antibody fusion protein); (4) a peptide (e.g., MBP-8289, NBI-5788, T cell receptor peptide (NEUROV AX^®), and glatiramer acetate (COPAXONE^®)); or (5) a DNA vaccine (e.g., BNT-3009-01). As used herein, an "immunosuppressive agent" is an agent that decreases or inhibits the activity or proliferation of a T cell. Non-limiting examples of immunosuppressive agents include, a cyclosporine (e.g., cyclosporin A, such as NEORAL^®, SANDIMMUNE^®, and SANGCYA^®), an azathioprine (e.g. IMURAN^®), FK-506, 15-deoxyspergualin, and an antibody (e.g., a monoclonal antibody, such as basiliximab, daclizumab, and muromonab-CD3, or a globulin, such as anti- lymphocyte globulin, anti-thymocyte globulin, and the like).

As used herein, an "inflammatory signaling inhibitor" or "ISI" is an agent that decreases the binding between a pro-inflammatory cytokine (e.g., TNF-alpha, TNF- beta, or IL-I) and its receptor (e.g., TNF receptor 1 or 2, or IL-I receptor, respectively); decreases the binding of activating molecules to pro-inflammatory cell surface signaling molecules (e.g., CD-20, CTLA-4, CD80/CD86, or CD28); or decreases the downstream activation of, or activity of, intracellular signaling molecules that are activated following the binding of pro-inflammatory cytokines to their receptors or the binding of activating molecules to pro-inflammatory cell surface signaling molecules (e.g., an agent that decreases the activation of, or activity of, signaling molecules in the p38 MAPK signaling pathway). Preferably, the decrease in binding between a pro-inflammatory cytokine and its receptor, the decrease in binding of an activating molecule to a pro-inflammatory cell surface signaling molecule, or the decrease in intracellular signaling which occurs following the binding of pro- inflammatory cytokines to their receptors or activating molecules to pro-inflammatory cell surface signaling molecules is by at least about 10%, preferably by 20%, 30%, 40%, or 50%, more preferably by 60%, 70%, 80%, 90% or more (up to 100%). An ISI may act by reducing the amount of pro-inflammatory cytokine (e.g., TNF-alpha, TNF-beta, or IL-I) freely available to bind the receptor. For example, an ISI may be a soluble pro-inflammatory cytokine receptor protein (e.g., a soluble TNF receptor fusion protein such as etanercept (ENBREL^®) or lenercept), or a soluble proinflammatory cell surface signaling molecule (e.g., a soluble CTLA-4 (abatacept)), or an antibody directed against a pro-inflammatory cytokine or a pro-inflammatory cell surface signaling molecule (e.g., an anti-TNF antibody, such as adalimumab, certolizumab, inflixamab, or golimumab; an anti-CD20 antibody, such as rituximab; or TRU-015 (TRUBION^®)). In addition, an ISI may act by disrupting the ability of the endogenous wild-type pro-inflammatory cytokine or the pro-inflammatory cell surface signaling molecule to bind to its receptor (e.g., TNF receptor 1 or 2, IL-I receptor, or CDl Ia (e.g., efalizumab (RAPTIV A^®, Genentech))). Examples of dominant-negative TNF-alpha variants are XENP345 (a pegylated version of TNF variant A145R/I97T) and Xpro™1595, and further variants disclosed in U.S. Patent Application Publication Nos. 20030166559 and 20050265962, herein incorporated by reference. An example of a dominant negative IL-I variant is anakinra (KINERET^®), which is a soluble form of IL-I that binds to the IL-I receptor without activating intracellular signaling pathways.

Inflammatory signaling inhibitors, which can be used in the present invention, are also small molecules which inhibit or reduce the signaling pathways downstream of pro-inflammatory cytokine or pro-inflammatory cell surface signaling molecules (e.g., DE 096). Examples of ISIs of this kind include inhibitors of p38 MAP kinase, e.g., 5-amino-2-carbonylthiopene derivatives (as described in WO 04/089929, herein incorporated); ARRY-797; BIRB 796 BS, (l-5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)- 3-[4-2(morpholin-4-yl-ethoxy)-naphtalen-l-yl]-urea); CHR-3620; CNI-1493; FR- 167653 (Fujisawa Pharmaceutical, Osaka, Japan); ISIS 101757 (Isis

Pharmaceuticals); ML3404; NPC31145; PD169316; PHZl 112; RJW67657, (4-(4-(4- fluorophenyl)- 1 -(3 -phenylpropyl)-5 -(4-pyridinyl)- 1 H-imidazol-2-yl)-3 -butyn- 1 -ol; SCIO-469; SB202190; SB203580, (4-(4-fluorophenyl)-2-(4-methylsulfmylphenyl)-5- (4-pyridyl)lH-imidazole); SB239063, rrøH.s-l-(4-hydroxycyclohexyl)-4-(4- fluorophenyl-methoxypyridimidin-4-yl)imidazole; SB242235; SD-282; SKF-86002; TAK 715; VX702; and VX745. Furthermore, an ISI may interfere with the processing of a pro-inflammatory cytokine (e.g., TNF-alpha and TNF-beta) from its membrane bound form to its soluble form. Inhibitors of TACE are ISIs of this class. Examples of inhibitors of TACE include BB-1101, BB-3103, BMS-561392, butynyloxyphenyl β-sulfone piperidine hydroxomates, CH4474, DPC333, DPH-

067517, GM6001, GW3333, Ro 32-7315, TAPI-I, TAPI-2, and TMI 005. Additional examples of ISIs include short peptides derived from the E. coli heat shock proteins engineered for disease-specific immunomodulatory activity (e.g., dnaJPl). An ISI may be directly administered or delivered by means of gene therapy using, e.g., a viral vector (such as an adeno-associated viral vector) that expresses a soluble TNF-receptor immunoglobulin Fc fusion protein, as described in U.S. Patent Application Publication Nos. 20030113295 and 20030103942, incorporated by reference.

As used here, "integrin antagonist" refers to an agent that suppresses or inhibits the biological activity of an integrin molecule, such as the α4 subunit of an integrin molecule. The agent may act directly or indirectly on the α4 integrin subunit (NCBI Accession No. P13612; SEQ ID NO: 14; Takada et al., EMBO J. 8:1361-1368, 1989; or SEQ ID NO: 15) by inhibiting the activity or expression of the α4 integrin subunit, or may act on the target to which the intact integrin containing an <x4 subunit binds. For example, an antibody or blocking peptide that binds to vascular cell adhesion molecule- 1 (VCAM-I), thus preventing the binding of α4βl integrin to VCAM-I is considered an integrin antagonist for purposes of the present invention. Non-limiting exemplary integrin antagonists suitable for use with the present invention may include proteins, blocking peptides, antibodies, such as natalizumab (TYSABRI^®), small molecule inhibitors, and nucleic acid inhibitors. Examples of nucleic acid inhibitors used as integrin antagonists are those that include a sequence which is complimentary to a sequence substantially identical to all or part of the mRNA sequence of human α4 integrin (NCBI Accession No. NM 000885; SEQ ID NO: 16; Takada et al. supra; or SEQ ID NO: 17). For example, a sequence that is complementary to nucleotides 1-25 of SEQ ID NO: 16 or SEQ ID NO: 17. Examples of α4 integrin antagonists include, but are not limited to, natalizumab (Elan/Biogen Idee; see, e.g., U.S. Patent Nos. 5,840,299; 6,033,665; 6,602,503; 5,168,062; 5,385,839; and 5,730,978; incorporated by reference herein), oMEPUPA-V (Biogen; U.S. Patent No. 6,495,525; incorporated by reference herein), alefacept, CDP-323 (Celltech); firategrast (SB-68399; GlaxoSmithKline); TR-9109 (Pfizer); ISIS- 107248 (Antisense Therapeutics); R- 1295 (Roche); and TBC-4746 (Schering-Plough). Additional non-limiting examples of α4 integrin antagonists include the small molecules described in U.S. Patent Nos. 5,821,231; 5,869,448; 5,936,065; 6,265,572; 6,288,267; 6,365,619; 6,423,728; 6,426,348; 6,458,844; 6.479,666; 6,482,849; 6,596,752; 6,667,331; 6,668,527; 6,685,617; 6,903,128; and 7,015,216 (each herein incorporated by reference); in U.S. Patent Application Publication Nos. 2002/0049236; 2003/0004196; 2003/0018016; 2003/0078249; 2003/0083267; 2003/0100585; 2004/0039040; 2004/0053907; 2004/0087574; 2004/0102496; 2004/0132809; 2004/0229858; 2006/0014966; 2006/0030553; 2006/0166866; 2006/0166961; 2006/0241132; 2007/0054909; and 2007/0232601 (each herein incorporated by reference); in European Patent Nos. EP 0842943; EP 0842944; EP 0842945; EP 0903353; and EP 0918059; and in PCT Publication Nos. WO 95/15973; WO 96/06108; WO 96/40781; WO 98/04247; WO 98/04913; WO 98/42656; WO 98/53814; WO 98/53817; WO 98/53818; WO 98/54207; WO 98/58902; WO 99/06390; WO 99/06431; WO 99/06432; WO 99/06433; WO 99/06434; WO 99/06435; WO 99/06436; WO 99/06437; WO 99/10312; WO 99/10313; WO 99/20272; WO 99/23063; WO 99/24398; WO 99/25685; WO 99/26615; WO 99/26921; WO 99/26922; WO 99/26923; WO 99/35163; WO 99/36393; WO 99/37605; WO 99/37618; WO 99/43642; WO 01/42215; and WO 02/28830; all of which are incorporated by reference herein.

Additional examples of α4 integrin antagonists include the phenylalanine derivatives described in: U.S. Patent Nos. 6,197,794; 6,229,011; 6,329,372; 6,388,084; 6,348,463; 6,362,204; 6,380,387; 6,445,550; 6,806,365; 6,835,738; 6,855,706; 6,872,719; 6,878,718; 6,911,451; 6,916,933; 7,105,520; 7,153,963; 7,160,874; 7,193,108; 7,250,516; and 7,291,645 (each herein incorporated by reference). Additional amino acid derivatives that are α.4 integrin antagonists include those described in, e.g., U.S. Patent Application Publication Nos. 2004/0229859 and 2006/021 1630 (herein incorporated by reference), and PCT Publication Nos. WO 01/36376; WO 01/47868; and WO 01/70670; all of which are incorporated by reference herein.

Other examples of α4 integrin antagonists include the peptides, and the peptide and semi-peptide compounds described in, e.g., PCT Publication Nos. WO 94/15958; WO 95/15973; WO 96/00581; WO 96/06108; WO 96/22966 (Leu-Asp-Val tripeptide; Biogen, Inc.); WO 97/02289; WO 97/03094; and WO 97/49731. An additional example of an α4 integrin antagonist is the pegylated molecule described in

U.S. Patent Application Publication No. 2007/066533 (herein incorporated by reference). Examples of antibodies that are α.4 integrin antagonists include those described in, e.g., PCT Publication Nos. WO 93/13798; WO 93/15764; WO 94/16094; and WO 95/19790. Additional examples of α4 integrin antagonists are described herein. By "non-glycosylated human alpha-fetoprotein" is meant a polypeptide having substantially the same amino acid sequence as the mature human alpha-fetoprotein (SEQ ID NO: 1), except including a mutation at amino acid position 233 (e.g., substitution of the naturally occurring asparagine residue with another amino acid, e.g., a glutamine residue (as set forth in SEQ ID NOS: 18 and 19), which eliminates the single N-linked glycosylation site). The asparagine at position 233 can be replaced with, e.g., any other naturally-occurring amino acid, such as glycine, alanine, or leucine, that does not promote glycosylation at position 233.

By "non-steroidal anti-inflammatory drug" or "NSAID" is meant a nonsteroidal agent that prevents or diminishes inflammation. NSAIDs include, e.g., naproxen sodium, diclofenac sodium, diclofenac potassium, aspirin, sulindac, diflunisal, piroxicam, indomethacin, ibuprofen, nabumetone, choline magnesium trisalicylate, sodium salicylate, salicylsalicylic acid, fenoprofen, flurbiprofen, ketoprofen, meclofenamate sodium, meloxicam, oxaprozin, sulindac, tolmetin, and COX-2 inhibitors, such as rofecoxib, celecoxib, valdecoxib, and lumiracoxib. By "purified" or "substantially pure" is meant that the compound or agent

(e.g., an AFP or a secondary agent) is partially or completely separated from other components (e.g., proteins, lipids, other compounds, and water), thus increasing the effective concentration of the compound or agent relative to an unpurified compound or agent. A compound or agent of the present invention can be at least 80%, 85%, 90%, 95%, 99%, or even 100% pure.

By "recombinant human alpha-fetoprotein mutant N233Q" or "rHuAFP N233Q" is meant a polypeptide having substantially the same amino acid sequence as the mature human alpha-fetoprotein described above, except including a mutation at amino acid position 233 of SEQ ID NO: 1 from an asparagine residue to a glutamine residue (as set forth in SEQ ID NO: 18), which eliminates the single glycosylation site of HuAFP. The nucleic acid sequence of the precursor non-glycosylated human alpha-fetoprotein includes nucleotides 45 through 1874 of the sequence set forth in SEQ ID NO: 19. By "steroid" is meant any naturally occurring or synthetic compound characterized by a hydrogenated cyclopentanoperhydrophenanthrene ring system. Naturally occurring steroids are generally produced by the adrenal cortex. Synthetic steroids may be halogenated. Exemplary steroids are algestone, 6-alpha- fluoroprednisolone, 6-alpha-methylprednisolone, 6-alpha-methylprednisolone 21- acetate, 6-alpha-methylprednisolone 21-hemisuccinate sodium salt, 6-alpha-9-alpha- difluoroprednisolone 21 -acetate 17-butyrate, amcinafal, beclomethasone, beclomethasone dipropionate, beclomethasone dipropionate monohydrate, 6-beta- hydroxycortisol, betamethasone, betamethasone- 17-valerate, budesonide, clobetasol, clobetasol propionate, clobetasone, clocortolone, clocortolone pivalate, cortisone, cortisone acetate, cortodoxone, deflazacort, 21-deoxycortisol, deprodone, descinolone, desonide, desoximethasone, dexamethasone, dexamethasone-21 -acetate, dichlorisone, diflorasone, diflorasone diacetate, diflucortolone, doxibetasol, fludrocortisone, flumethasone, flumethasone pivalate, flumoxonide, flunisolide, fluocinonide, fluocinolone acetonide, 9-fluorocortisone, fluorohydroxyandrostenedione, fluorometholone, fluorometholone acetate, fluoxymesterone, flupredidene, fluprednisolone, flurandrenolide, formocortal, halcinonide, halometasone, halopredone, hyrcanoside, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone cypionate, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, hydrocortisone probutate, hydrocortisone valerate, 6-hydroxydexamethasone, isoflupredone, isoflupredone acetate, isoprednidene, meclorisone, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, paramethasone, paramethasone acetate, prednisolone, prednisolone acetate, prednisolone metasulphobenzoate, prednisolone sodium phosphate, prednisolone tebutate, prednisolone-21 -hemisuccinate free acid, prednisolone-21 -acetate, prednisolone-21 (beta-D-glucuronide), prednisone, prednylidene, procinonide, tralonide, triamcinolone, triamcinolone acetonide, triamcinolone acetonide 21-palmitate, triamcinolone diacetate, triamcinolone hexacetonide, and wortmannin. Particularly desirable corticosteroids are prednisolone, cortisone, dexamethasone, hydrocortisone, methylprednisolone, fluticasone, prednisone, triamcinolone, and diflorasone.

A "therapeutically effective amount" of a therapeutic agent (e.g., an AFP or a secondary agent) is an amount of the agent that is sufficient to treat or reduce one or more symptoms (e.g., 1, 2, 3, 4, or 5) of disease (e.g., psoriatic arthritis, IgA nephropathy, Hashimoto's disease, Sjogren's syndrome, ankylosing spondylitis, and uveitis) or the severity of one or more symptoms of disease (e.g., a reduction of at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% in the severity of one or more symptoms relative to an untreated control patient). These symptoms include, e.g., stiffness, pain, swelling, tenderness of the joints and surrounding soft tissue, fatigue, nail pitting, depression, sensitivity to cold, weight increase or weight decrease, muscle weakness, coarsening of the skin, dry or brittle hair, constipation, muscle cramps, increased menstral flow, goiter, dry eyes, eye irritation, eye burning, eyelids sticking together, dry mouth, hoarse or weak voice, difficulty swalling food, painful salivary glands, back pain and stiffness, fever, night sweats, light sensitivity, blurring or loss of vision, eye pain, and redness of eye. The treatment or reduction in one or more of the symptoms of disease (or their severity) can be determined by using one of several methods known in the art and the methods described herein. The amount may vary depending on the effect to be achieved. For instance, a "therapeutically effective amount" of a secondary agent for treating one or more of the diseases recited herein when used in combination with AFP (or a biologically fragment, derivative, or analog thereof) may be different from the "therapeutically effective amount" of the secondary agent when used alone or as the primary agent for treating disease. A "therapeutically effective amount" of an AFP and/or one or more secondary agents may also be used for the prophylactic treatment of one or more of the diseases described herein (e.g., uveitis).

By "treating" is meant a reduction (e.g., by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or even 100%) in the progression, severity, or frequency of one or more symptoms (e.g., 1, 2, 3, 4, or 5 of the symptoms) of one or more of the diseases recited herein in a human patient. By "treating" is also meant the prophylactic treatment of one of the diseases described herein (e.g., uveitis).

By "tolerizing agent" is meant an agent that "vetos" activation of T or B cells involved in the pathogenesis of an immunoinflammatory disease (e.g., an autoimmune uveitis or idiopathic uveitis). Examples of tolerizing agents include anti-idiotypic agents, such as monoclonal antibodies, LJP 394 (abetimus, RIQUENT^®, La Jolla Pharmaceuticals), and the tolerizing agents disclosed in, e.g., U.S. Patent No. 6,428,782 and PCT Application No. WO 07/112410, herein incorporated by reference.

By "uveitis" is an inflammatory disease of the uveal tract. The uveal tract consists of the pigmented tissue of the eye, including the iris, ciliary body, and choroid. There are two different classes of uveitis: autoimmune uveitis (e.g., birdshot retinochoroidopathy uveitis, sarcoid uveitis, and HLA-B27 uveitis) and idiopathic uveitis (e.g., Behcet's disease and Vogt-Koyanagi-Harada (VKH) syndrome). Uveitis can also be classified by which part of the uvea it affects. Anterior uveitis refers to inflammation of the iris alone (called iritis) or the iris and the ciliary body. Intermediate uveitis refers to inflammation of the ciliary body. Posterior uveitis is inflammation of the choroid. Diffuse uveitis is inflammation of all areas of the uvea. Uveitis may also be acute or chronic in nature.

Uveitis may occur alone or it may be associated with another disease or infection, e.g., sympathetic ophthalmia, toxoplasmosis, acute multifocal choroiditis, multiple sclerosis, tuberculosis, syphilis, vasculitides due to Wegener granulomatosis, systemic lupus erythematosus, Whipple disease, ankylosing spondylitis, Crohn disease, relapsing polychondritis, acute multifocal placoid pigment epitheliopathy, CNS lymphoma, brucellosis, herpes simplex, herpes zoster, inflammatory bowel disease, juvenile rheumatoid arthritis, Kawasaki's disease, leptospirosis, Lyme disease, presumed ocular histoplasmosis syndrome, psoriatic arthritis, Reiter's syndrome, syphilis, and toxocariasis.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Detailed Description

As summarized above, the invention provides methods, kits, and compositions for treating, preventing, or inhibiting disease (e.g., psoriatic arthritis, IgA nephropathy, Hashimoto's disease, Sjogren's syndrome, ankylosing spondylitis, and uveitis), or for treating, preventing, or reducing one or more of the symptoms of one or more of these disease(s) by administering an AFP, or a biologically active fragment, derivative, or analog thereof, alone or in combination with one or more secondary agents. Methods of Treating Disease by Administering a Composition of the Present Invention

The present invention features methods of treating disease (e.g., psoriatic arthritis, IgA nephropathy, Hashimoto's disease, Sjogren's syndrome, ankylosing spondylitis, and uveitis), in a patient by administering a therapeutically effective amount of an AFP (or biologically active fragment, derivative, or analog thereof) to the patient. The compositions of the invention may, but need not, also include additional secondary agents, such as those described below. The compositions of the invention can be administered to a patient to treat or inhibit disease in a human patient or to treat, prevent, ameliorate, inhibit the progression of, or reduce the severity of one or more symptoms of disease in a human patient. The AFP (or biologically active fragment, derivative, or analog thereof) and secondary agent may be administered coextensively or separately, in a single dose or in multiple doses. The AFP (or biologically active fragment, derivative, or analog thereof) and secondary agent may be formulated for the same route of administration or formulated for different routes of administration.

A physician may adjust the dose (e.g., increase or decrease the dose) of the AFP or of the secondary agent administered to the patient based on the severity of, occurrence of, or progression of the disease (e.g., psoriatic arthritis, IgA nephropathy, Hashimoto's disease, Sjogren's syndrome, ankylosing spondylitis, and uveitis) in the patient. For example, a physician can increase the dose of the AFP or of the secondary agent if necessary to alleviate one or more symptoms of the disease (e.g., psoriatic arthritis, IgA nephropathy, Hashimoto's disease, Sjogren's syndrome, ankylosing spondylitis, and uveitis) in a patient. Alternatively, a physician can decrease the dose of the AFP or of the secondary agent based on an improvement in one or more symptoms of the disease (e.g., psoriatic arthritis, IgA nephropathy, Hashimoto's disease, Sjogren's syndrome, ankylosing spondylitis, and uveitis) in the patient or to avoid toxicity associated with, e.g., the administration of the secondary agent. Particular diseases that benefit from the methods and compositions disclosed herein are described below. Diseases

As is discussed above, autoimmune diseases are characterized by a loss of tolerance to self antigens, causing cells of the immune systems, e.g., T or B cells (or both), to react against self tissue antigens. Autoimmune diseases may involve any organ system, although some are affected more commonly than others. Examples of autoimmune and inflammatory diseases that can be treated with the compositions and methods of the invention include, e.g., psoriatic arthritis, IgA nephropathy, Hashimoto's disease, Sjogren's syndrome, ankylosing spondylitis, and uveitis.

The art provides a wide variety of experimental animal systems, transgenic and non-transgenic, for testing therapies for human illness involving autoimmune diseases (see, e.g., Paul, W.E., Fundamental Immunology, 2nd ed., Raven Press, New York, 1989; and Kandel et al., Principles of Neural Science, 3rd ed., Appleton and Lange, Norwalk, CT, 1991; and Current Protocols In Immunology, Coligan, J.E., Kruisbeek, A.M., Margulies, D.H., Shevach, E.M., and Strober, eds., Green Publishing Associates (John Wiley & Sons), New York, 1992).

In addition to an AFP (or a biologically active fragment, derivative, or analog thereof), one or more secondary agents (e.g., an NSAID, a corticosteroid, and an immunosuppressive agent) can be administered for use in the treatment of psoriatic arthritis, IgA nephropathy, Hashimoto's disease, Sjogren's syndrome, ankylosing spondylitis, or uveitis. For individuals with severe complications, the administration of one or more secondary agents (e.g., corticosteroids or immunosuppressive drugs) may be particularly desired.

Psoriatic Arthritis Psoriatic arthritis is a specific type of arthritis that has been diagnosed in approximately 23 percent of people who have psoriasis. It commonly affects the ends of the fingers and toes, but can also affect the spine. The disease can be difficult to diagnose, particularly in its milder forms and earlier stages. Early diagnosis is important for preventing long-term damage to joints and tissue. Most people with psoriatic arthritis also have psoriasis. Rarely, a person can have psoriatic arthritis without having psoriasis. The symptoms of psoriatic arthritis include: stiffness, pain, swelling and tenderness of the joints and surrounding soft tissue, reduced range of motion in the joints, morning stiffness and tiredness, nail changes, including pitting (small indentations in the nail) or lifting of the nail (found in 80 percent of people with psoriatic arthritis), and redness and pain of the eye (similar to conjunctivitis). Psoriatic arthritis can develop at any time and appears, on average, about 10 years after the first signs of psoriasis. For most people it appears between the ages of 30 and 50. It affects men and women equally. The diagnosis and monitoring of a patient having psoriatic arthritis may be performed by measuring the number or severity of the above symptoms in a patient.

In about one of seven people with psoriatic arthritis, arthritis symptoms occur before any skin lesions. Like rheumatoid arthritis, psoriatic arthritis is thought to be caused by a malfunctioning immune system. Psoriatic arthritis is usually milder than rheumatoid arthritis, but some patients with psoriatic arthritis have as severe a disease as patients with rheumatoid arthritis. Psoriatic arthritis can start slowly with mild symptoms, or it can develop quickly.

Traditional treatment options for psoriatic arthritis include skin care, light treatment (UVB or PUVA), corrective cosmetics, medications, e.g., glucocorticoids, non-steroid anti-inflammatory drugs (NSAIDs), disease-modifying anti-rheumatic drugs (DMAPvDs; such as methotrexate, sulfasalazine, gold, cyclosporine), biologic response modulators (BRJVIs; such as adalimumab, etanercept, infliximab, rituximab, anakinra, and abatacept), exercise, rest, heat and cold, splints, and surgery, although rarely is this necessary.

The present invention provides methods and compositions for treating psoriatic arthritis by administering an AFP (or a biologically active fragment, derivative, or analog thereof) to a patient in need thereof, alone or in combination with one or more secondary agents. The effectiveness of an AFP (or a biologically active fragment, derivative, or analog thereof) on the treatment of psoriatic arthritis can be evaluated using a mouse model of psoriatic arthritis, which is described in, e.g., U.S. Patent No. 5,945,576, incorporated herein by reference. To evaluate the effectiveness of an AFP (or a biologically active fragment, derivative, or analog thereof) for the treatment of psoriatic arthritis, i.e., the ability to prevent or ameliorate psoriatic arthritis, an AFP is administered to a severe combined immune deficiency ("SCID") mouse, a CD-3 deficient mouse, a TCRαβ deficient mouse, or a T cell signaling defective mouse according to standard methods, e.g., intravenously, subcutaneously, or intraperitoneally, at an appropriate dosage on a daily basis. Generally, administration is initiated prior to the onset of psoriatic arthritis and/or after the clinical appearance of psoriatic arthritis. Control animals receive a placebo, e.g., human serum albumin, similarly administered as for glycosylated or non-glycosylated rHuAFP or related molecules. The effect of the test molecule on psoriatic arthritis is monitored according to standard methods. For example, analysis of the cellular component(s) of a synovial joint are monitored on a daily basis. If desired, histological inspection (e.g., by using any standard histochemical or immunohistochemical procedure, see e.g., Ausubel et al., supra; Bancroft and Stevens, supra) of the synovial joint is performed and tissue samples examined microscopically for evidence of psoriatic arthritis, e.g., evidence of erosion of collagen and cartilage matrix in a joint. Comparative studies between treated and control animals are used to determine the relative efficacy of an AFP (or a biologically active fragment, derivative, or analog thereof) in preventing or ameliorating psoriatic arthritis. In addition to an AFP (or a biologically active fragment, derivative, or analog thereof), one or more secondary agents (e.g., an NSAID, a corticosteroid, and an immunosuppressive agent) can be administered for use in the treatment of psoriatic arthritis. For individuals with severe complications, the administration of one or more secondary agents (e.g., corticosteroids or immunosuppressive drugs) may be particularly desired.

IgA Nephropathy

IgA nephropathy is a kidney disorder caused by deposits of the protein immunoglobulin A (IgA) inside the glomeruli (filters) within the kidney. The glomeruli normally filter wastes and excess water from the blood and send them to the bladder as urine. The IgA protein prevents this filtering process, leading to blood and protein in the urine and swelling in the hands and feet. This chronic kidney disease may progress over a period of 10 to 20 years and, if left untreated, can result in end- stage renal disease, which requires that the patient go on dialysis or receive a kidney transplant.

Diagnosis and monitoring of IgA nephopathy in a patient is performed by the administration and analysis of the following renal function tests: urinalysis (red blood cells in the urine or albumin), serum creatinin, and blood urea nitrogen (BUN) level. The IgA protein, an antibody, is a normal part of the body's immune system, the system that protects against disease. Genetic factors probably contribute to the disease. Traditional treatment focuses on slowing the progression of the disease and preventing complications. Treatment includes limiting the intake of protein in the diet to reduce the buildup of waste in the blood, reducing cholesterol levels through diet, medication, or both, and the administration of ACE inhibitors, corticosteroids, fish oil supplements containing omega 3 fatty acids, and immunosuppressive agents, e.g., mycophenolate mofetil (MMF). The present invention features the administration of an AFP and biologically active fragments, derivatives, or analogs thereof, either alone or in combination with one or more secondary agents, for the treatment of IgA nephropathy.

The etiology of human IgA nephropathy has been studied using animal models that resemble the essential characteristics of the human disease. To evaluate the effectiveness of an AFP (or biologically active fragments, derivatives, or analogs thereof) in treating IgA nephropathy, an appropriate laboratory animal, e.g., a mouse (e.g., the ddY mouse, see, e.g., Imai et al., Kidney Int. 27:756-761, 1985, the uteroglobulin deficient mouse, see, e.g., Pouria et al., Gut 46:452-253, 2000), or a primate (e.g., a marmoset, see, e.g., Schroeder et al., Nephrol. Dial. Transplant. 14:1875-1880, 1999) can be utilized. An AFP (or a biologically active fragment, derivative, or analog thereof) is administered to an appropriate laboratory animal according to standard methods, e.g., intravenously, subcutaneously, or intraperitoneally, at an appropriate dosage on a daily basis. Control animals receive a placebo, e.g., human serum albumin, similarly administered as for glycosylated or non-glycosylated rHuAFP or related molecules. The effect of the test molecule on IgA nephopathy is monitored according to standard methods. For example, effectiveness can be determined by measuring a decrease in persistent proteinuria and improvement in renal function (e.g., a decrease in serum creatinine levels). If desired, histological inspection (e.g., by using any standard histochemical or immunohistochemical procedure, see e.g., Ausubel et al., supra; Bancroft and Stevens, supra) of the kidney is performed and tissue samples examined microscopically for evidence of a decrease in inflammation of the kidney. Comparative studies between treated and control animals are used to determine the relative efficacy of an AFP (or a biologically active fragment, derivative, or analog thereof) in preventing or ameliorating IgA nephropathy.

In addition to an AFP (or a biologically active fragment, derivative, or analog thereof), one or more secondary agents (e.g., an NSAID, a corticosteroid, and an immunosuppressive agent) can be administered for use in the treatment of IgA nephropathy. For individuals with severe complications, the administration of one or more secondary agents (e.g., corticosteroids or immunosuppressive drugs) may be particularly desired.

Hashimoto 's Thyroiditis Hashimoto's thyroiditis is a type of autoimmune thyroid disease in which the immune system attacks and destroys the thyroid gland. The thyroid helps set the rate of metabolism - the rate at which the body uses energy. Hashimoto's thyroiditis prevents the gland from producing enough thyroid hormones for the body to work correctly. It is the most common form of hypothyroidism (underactive thyroid). Some patients with Hashimoto's thyroiditis may have no symptoms. The common symptoms are fatigue, depression, and sensitivity to cold, weight gain, muscle weakness, coarsening of the skin, dry or brittle hair, constipation, muscle cramps, increased menstrual flow, and goiter (enlargement of the thyroid gland). Hashimoto's thyroiditis can usually be treated with thyroid hormone replacement. Diagnosis and monitoring of Hashimoto's thyroiditis in a patient is performed by measuring the number and/or severity of the above symptoms in a patient or assessed by measuring the thyroid stimulating hormone (TSH), throxine (T4), triiodothyronine (T3), and anti-thyroid peroxidase antibody serum levels.

The present invention features the administration of an AFP (or biologically active fragments, derivatives, or analogs thereof), either alone or in combination with thyroid hormone replacement therapy, or one or more secondary agents for the treatment of Hashimoto's thyroiditis.

Examples of animal models that spontaneously develop Hashimoto's thyroiditis resembling the human disease include the nonobese diabetic (NOD) mouse (Boechat et al., Brazilian J. Med. Biol. Res. 35:289-295, 2002) and the obese strain

(OS) of chickens (Kaiser et al., J Immunol. 168:4216-4220, 2002). Hashimoto's thyroiditis can also be experimentally induced in NOD mice by administration of an overload of iodine after a goiter-inducing phase (see, e.g., Many et al., J. Endocrinology 147:311-320, 1995, and Rose et al., Crit. Rev. Immunol. 17:511-517, 1997). NOD mice develop thyroid infiltrates similar to those observed in human Hashimoto's thyroiditis and also express autoantibodies directed at thyroid cell membrane antigens. NOD.H.2h4, a mouse strain derived from NOD, is also prone to the development of thyroid infiltrates that are clearly related to the amount of iodine intake (see, e.g., Rasooly et al., Clin. Immunol. Immunopathol. 81:287-292, 1996). There is also a model of experimental autoimmune thyroiditis in transgenic mice producing AFP (see, Matsuura et al., Tumour Biol. 20(3): 162-171, 1999).

Therefore, the art provides a variety of animal models resembling human Hashimoto's thyroiditis, which can be used to examine and assess approaches for the prevention or amelioration of symptoms by administering an AFP (or a biologically active fragment, derivative, or analog thereof). To evaluate the immunosuppressive effect of an AFP (or a biologically active fragment, derivative, or analog thereof) on the development of thyroiditis, i.e., the ability of an AFP to treat or prevent thyroiditis, the test compound is administered to an appropriate test animal, e.g, a NOD mouse, according to standard methods, e.g., intravenously, subcutaneously, or intraperitoneally, at an appropriate dosage on a daily basis. Generally, administration is initiated prior to the onset of thyroiditis and/or after the clinical appearance of thyroiditis characteristics. Control animals receive a placebo, e.g., human serum albumin, similarly administered as the AFP or related molecules. The effect of test molecules on thyroiditis is monitored according to standard methods. For example, thyroid stimulating hormone and thyroid hormone levels are monitored on a daily basis. If desired, histological inspection (e.g., by using any standard histochemical or immunohistochemical procedure, see e.g., Ausubel et al., supra; Bancroft and Stevens, supra) of thyroid cells is performed and tissue samples examined microscopically for evidence of thyroiditis or thyroid autoantibodies. Comparative studies between treated and control animals are used to determine the relative efficacy of the test molecules in preventing or ameliorating the autoimmune thyroid disease. In addition to an AFP (or a biologically active fragment, derivative, or analog thereof), one or more secondary agents (e.g., an NSAID, a corticosteroid, and an immunosuppressive agent) can be administered for use in the treatment of Hashimoto's thyroiditis. For individuals with severe complications, the administration of one or more secondary agents (e.g., corticosteroids or immunosuppressive drugs) may be particularly desired.

Sjogren 's Syndrome Sjogren's syndrome is a chronic, incurable, autoimmune disorder in which the body's immune system reacts against itself; resulting in the destruction of the exocrine (mucous-secreting) glands of the body. There are two types of Sjogren's syndrome: primary and secondary. Primary Sjogren's syndrome is localized and affects mainly the eyes and mouth, which become infiltrated and dysfunctional. Secondary Sjogren's syndrome is systemic and is accompanied by a disease that affects the body's connective tissue, such as lupus or rheumatoid arthritis. Approximately 50% of people with Sjogren's syndrome are described as having secondary Sjogren's syndrome.

Some of the symptoms of Sjogren's syndrome are dry eyes, eye irritation, eye burning, eyelids sticking together, dry mouth, hoarse or weak voice, difficulty in swallowing food, and enlarged, painful salivary glands. There are many tests available to diagnose and monitor Sjogren's syndrome including, e.g., examination of the eyes and measurement of tear production (the Schirmer test); measurement of saliva production; x-ray examination of certain salivary glands (sialography); blood tests to determine the presence of antibodies to two identifiable antigens (Ro and La); and a lip gland biopsy.

Some of the medical complications of Sjogren's syndrome include skin, nose, and vaginal dryness; and damage to the kidneys, blood vessels, lungs, liver, pancreas, and brain. Sjogren's symptoms may level off without becoming better or worse, they may worsen, or they may go into remission. Some people may only experience the mild symptoms of dry eyes and mouth, while others go through cycles of good health followed by severe disease. Children born to women with Sjogren's syndrome have an increased chance of having serious heart defects.

Animal models such as the NOD3 (or, simply, NOD) mouse, which is prone to Sjogren's syndrome, can be used to evaluate the effectiveness of an AFP (or a biologically active fragments, derivatives, or analogs thereof) in treating or ameliorating the symptoms of Sjogren's syndrome. Generally, administration of an AFP (or a biologically active fragment, derivative, or analog thereof) is initiated prior to the development of Sjogren's syndrome in the animal model and/or after the clinical appearance of Sjogren's syndrome. Control animals receive a placebo, e.g., human serum albumin, similarly administered as for the AFP or related molecules. The effect of the test molecules on Sjogren's syndrome is monitored according to standard methods. Comparative studies between treated and control animals are used to determine the relative efficacy of the test molecules in preventing or ameliorating Sjogren's syndrome.

In addition to an AFP (or a biologically active fragment, derivative, or analog thereof), one or more secondary agents (e.g., an NSAID, a corticosteroid, and an immunosuppressive agent) can be administered for use in the treatment of Sjogren's syndrome. For individuals with severe complications, the administration of one or more secondary agents (e.g., corticosteroids or immunosuppressive drugs) may be particularly desired.

Ankylosing Spondylitis

Ankylosing spondylitis is a painful, progressive, rheumatic disease. It mainly affects the spine, but can also affect other joints, e.g., tendons and ligaments. Other areas, such as the eyes, lungs, bowel, and heart can also be involved. Inflammation occurs at the site where certain ligaments or tendons attach to bone (enthesis). This is followed by some erosion of bone at the site of the attachment (enthesopathy). As the inflammation subsides, a healing process takes place and new bone develops. Movement becomes restricted where bone replaces the elastic tissue of ligaments or tendons. Repetition of this inflammatory process leads to further bone formation and the individual bones which make up your backbone, the vertebrae, can fuse together. The pelvis is commonly affected first, with the lower back, chest wall, and neck becoming involved at different times.

Ankylosing spondylitis is more common in men, with nearly three times as many men affected with the disorder than women, and typically strikes during the late teens and twenties, with the average age being 24. Symptoms of ankylosing spondylitis include slow or gradual onset of back pain and stiffness over weeks or months, rather than hours or days, early-morning stiffness and pain, wearing off or reducing during the day with exercise, persistence for more than three months (as opposed to coming on in short attacks), feeling better after exercise and feeling worse after rest, weight loss, especially in the early stages, fatigue, feeling feverish, and experiencing night sweats. Diagnosis and monitoring of ankylosing spondylitis in a patient is performed by measuring the number and/or severity of the above symptoms in a patient or assessed by measuring the erythrocyte sedimentation rate (ESR). Ankylosing spondylitis also affects other joints, causing aching, pain and swelling in the hips, knees, and ankles. In most cases the pain and swelling will settle down after treatment. Ankylosing spondylitis can also affect the eyes, heart, and lungs. These effects are not life-threatening and they can be treated with relative ease. Ankylosing spondylitis can cause inflammation of the iris and its attachment to the outer wall of the eye, the uvea. Forty percent of people with ankylosing spondylitis will develop iritis or uveitis on one or more occasions. Usually the first symptom is a slight blurring of vision in one eye but the main symptom is a sharp pain together with a dramatically bloodshot eye.

Ankylosing spondylitis is typically treated using NSAIDs to reduce inflammation and relieve pain and stiffness. Simple pain killers, such as paracetamol, can also be administered. For others, especially those who suffer from inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis) or peripheral joint arthritis, a DMARD like sulphasalazine may be required.

An animal model of ankylosing spondylitis, such as the ank/ank mouse (Mahowald et al., J. Rheumatol. 16:60-66, 1989) or the HLA-B27 transgenic rat (see, e.g., Taurog et al., Clin. Rheumatol. Suppl. 1 :22-27, 1996), in which ankylosing spondylitis occurs with symptoms similar to that of the human disease, can be used to evaluate the effectiveness of an AFP (or a biologically active fragment, derivative, or analog thereof) in treating or ameliorating the symptoms of ankylosing spondylitis. Generally, administration of an AFP (or a biologically active fragment, derivative, or analog thereof) is initiated prior to the development of ankylosing spondylitis in the animal model and/or after the clinical appearance of ankylosing spondylitis. Control animals receive a placebo, e.g., human serum albumin, similarly administered as for the AFP or related molecules. The effect of the test molecules on ankylosing spondylitis is monitored according to standard methods, including, for example, the monitoring of the inflammation and swelling of the joints. Comparative studies between treated and control animals are used to determine the relative efficacy of the test molecules in preventing or ameliorating ankylosing spondylitis. In addition to an AFP (or a biologically active fragment, derivative, or analog thereof), one or more secondary agents (e.g., an NSAID, a corticosteroid, a DMARD, and an immunosuppressive agent) can be administered for use in the treatment of ankylosing spondylitis. For individuals with severe complications, the administration of one or more secondary agents (e.g., corticosteroids or immunosuppressive drugs) may be particularly desired.

Uveitis

Uveitis is inflammation inside the eye, specifically affecting one or more of the three parts of the eye that make up the uvea: the iris (the colored part of the eye), the ciliary body (behind the iris, responsible for manufacturing the fluid inside the eye), and the choroid (the vascular lining tissue underneath the retina). Uveitis is the third leading cause of blindness in the United States, after diabetes and macular degeneration. Three different forms of autoimmune inflammatory uveitis are sarcoid uveitis, birdshot retinochoroidopathy uveitis, and HLA-B27 uveitis. There are other forms of uveitis that are idiopathic: e.g., Behcet's disease and Vogt-Koyanagi-Harada (VKH) syndrome. The term uveitis is often used to describe inflammation that affects both the uvea and other ocular tissues such as the retina. This serious inflammation threatens vision and can progress to blindness. Uveitis is often classified by which part of the uvea it affects. Anterior uveitis refers to inflammation of the iris alone (called iritis) or the iris and the ciliary body. Intermediate uveitis refers to inflammation of the ciliary body. Posterior uveitis is inflammation of the choroid. Diffuse uveitis is inflammation of all areas of the uvea. Uveitis may also be classified as acute or chronic. Uveitis may be associated with another disease or infection, e.g., sympathetic ophthalmia, toxoplasmosis, acute multifocal choroiditis, multiple sclerosis, tuberculosis, syphilis, vasculitides due to Wegener granulomatosis, systemic lupus erythematosus, Whipple disease, ankylosing spondylitis, Crohn disease, relapsing polychondritis, acute multifocal placoid pigment epitheliopathy, CNS lymphoma, brucellosis, herpes simplex, herpes zoster, inflammatory bowel disease, juvenile rheumatoid arthritis, Kawasaki's disease, leptospirosis, Lyme disease, presumed ocular histoplasmosis syndrome, psoriatic arthritis, Reiter's syndrome, syphilis, and toxocariasis. Diagnosis and monitoring of uveitis in a patient is performed by measuring the number and/or severity of the symptoms (e.g., light sensitivity, blurring or loss of vision, pain, and redness of eye) in a patient.

About 80% of all uveitis cases are believed to be of autoimmune origin. Abnormal immunological reactions in these patients, which target proteins in the eye, result in a progressive destruction of the retina. Current therapy is limited to symptomatic treatment of the inflammation and a general suppression of immune responses. The mainstay of treatment involves systemic corticosteroids, often in combination with immunosuppressive agents, such as cyclosporin A or azathioprine. High dose steroids, which can cause potentially serious side effects in the eye, are often required to control the disease. TNF therapies, such as infliximab, etanercept, and adalimumab are also used to treat autoimmune uveitis.

Experimental autoimmune uveitis (EAU) is a disease of the neural retina induced by immunization with retinal antigens, such as interphotoreceptor retinoid- binding protein (IRBP) and arrestin (retinal soluble antigen, S-Ag; see e.g., Pennesi et al., J Clin. Invest. 111 :1171-1180, 2003; Willbanks et al., Hum. Immunol. 53:188- 194, 1997; Takeuchi et a\., Jpn J. Ophthalmol. 45:463-469, 2001 ; and Nussenblatt, Int. Rev. Immunol. 21 :273-289, 2002). EAU serves as a model for human autoimmune uveitic diseases associated with major histocompatibility complex (HLA) genes, in which patients exhibit immunological responses to retinal antigens. The effectiveness of an AFP (or biologically active fragment, derivative, or analog thereof) on the treatment of uveitis can be evaluated using a mouse model of experimentally-induced uveitis (e.g., an HLA transgenic mouse, such as an HLA-DR3 TG mouse, an HLA-DR4 TG mouse, an HLA-DQ6 TG mouse, and an HLA-DQ8 TG mouse). To evaluate an AFP (or biologically active fragment, derivative, or analog thereof) on uveitis, i.e., the ability to prevent or ameliorate uveitis, an AFP (or biologically active fragment, derivative, or analog thereof) is administered to a mouse in which uveitis has been experimentally induced, according to standard methods, e.g., intravenously, subcutaneously, intraperitoneally, or by ophthalmic administration at an appropriate dosage on a daily basis. Generally, administration is initiated prior to the onset of uveitis and/or after the clinical appearance of uveitis. Control animals receive a placebo, e.g., human serum albumin, similarly administered as for AFP or related molecules. The effect of the test molecule on uveitis is monitored according to Standard methods. For example, analysis of the extent of inflammatory cell infiltration of the ciliary body, choroid, vitreous, or retina, and the presence of discrete lesions of the tissue (e.g., vasculitis, granuloma formation, retinal folding and/or detachment, and photoreceptor damage). If desired, histological inspection (e.g., by using any standard histochemical or immunohistochemical procedure, see e.g., Ausubel et al., supra; Bancroft and Stevens, supra) of the eyes is performed and tissue samples examined microscopically for evidence of uveitis. Comparative studies between treated and control animals are used to determine the relative efficacy of an AFP (or a biologically active fragment, derivative, or analog thereof) in preventing or ameliorating uveitis or the conditions associated with uveitis.

Preferably, the AFP (or a biologically active fragment, derivative, or analog thereof) prevents or ameliorates (decreases, suppresses, relieves, or promotes remission of) the symptoms of uveitis. In one embodiment, the AFP or biologically active fragment, analog, or derivative thereof is administered (e.g., topically or through injection) to an eye in an ophthalmic solution. In another embodiment, the AFP molecule (or a biologically active fragment, derivative, or analog thereof) is administered directly to the eye at a dose that is substantially different (e.g., increased or decreased) from the dose administered systemically. An AFP can also be administered to a patient systemically for the treatment of uveitis. The effectiveness of an AFP (or a biologically active fragment, derivative, or analog thereof) for the treatment of uveitis in humans can be evaluated directly in a medical study. One such study to test the effectiveness of a rHuAFP to treat inflammatory uveitis in humans is discussed in the Examples.

In addition to an AFP (or a biologically active fragment, derivative, or analog thereof), one or more secondary agents (e.g., an NSAID, a corticosteroid, a DMARD, and an immunosuppressive agent) can be administered for use in the treatment of uveitis. For individuals with severe complications, the administration of one or more secondary agents (e.g., corticosteroids or immunosuppressive drugs) may be particularly desired.

Additional Diseases

Additional diseases that may be treated using the methods of the invention include: Wegener's granulomatosis, inflammatory bowel disease, polymyositis, dermatomyositis, multiple endocrine failure, Schmidt's syndrome, adrenalitis, thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, presenile dementia, demyelinating diseases, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressier' s syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, pemphigoid, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), adult onset diabetes mellitus (Type II diabetes), male and female autoimmune infertility, mixed connective tissue disease, polyarteritis nedosa, systemic necrotizing vasculitis, juvenile onset rheumatoid arthritis, glomerulonephritis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent abortion, anti-phospholipid syndrome, farmer's lung, erythema multiforme, post cardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird-fancier's lung, allergic disease, allergic encephalomyelitis, toxic epidermal necrolysis, alopecia, Alport's syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, leprosy, malaria, leishmaniasis, trypanosomiasis, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema, lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome, Kawasaki's disease, dengue, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuch's cyclitis, Henoch-Schonlein purpura, glomerulonephritis, graft versus host disease, transplantation rejection, human immunodeficiency virus infection, echo virus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post vaccination syndromes, congenital rubella infection, Hodgkin's and Non-Hodgkin's lymphoma, renal cell carcinoma, multiple myeloma, Eaton-Lambert syndrome, relapsing polychondritis, malignant melanoma, cryoglobulinemia, Waldenstrom's macroglobulemia, Epstein- Barr virus infection, mumps, Evan's syndrome, and autoimmune gonadal failure. Compositions of the Present Invention

The present invention provides compositions including an AFP (or a biologically active fragment, derivative, or analog thereof) for the treatment of a disease (e.g., psoriatic arthritis, IgA nephropathy, Hashimoto's disease, Sjogren's syndrome, ankylosing spondylitis, and uveitis). The compositions of the invention may be formulated for any route of administration (e.g., the formulations described herein, preferably for ophthalmic administration) and may be administered in a single dose or in multiple doses to a subject in need thereof. The compositions of the invention may also include one or more secondary agents, e.g., a steroid, a non-steroid anti-inflammatory drug (NSAID), a disease-modifying anti-rheumatic drug

(DMARD), an integrin antagonist, an inflammation signaling inhibitor (ISI), a tolerizing agent, an immunosuppressive agent, and an immunomodulatory agent.

AFPs for Use in the Compositions and Methods of the Invention

Alpha- fetoprotein for use in the compositions, kits, and methods of the present invention is described below. For the purposes of the present invention, both naturally-occurring human AFP, synthetically-produced AFP, and recombinantly- produced (e.g., produced in a transgenic cell or a transgenic animal, e.g., a mammal, such as a goat, sheep, camel, cow, pig, rabbit, horse, or llama) AFP polypeptides or biologically active fragments thereof can be used. Naturally occurring human AFP can be obtained by, e.g., purification from umbilical cord blood or umbilical cord serum. Recombinant AFP polypeptide or biologically active fragment thereof can be obtained, e.g., by using a prokaryotic or eukaryotic expression system, such as those described in, e.g., U.S. Patent Nos. 5,384,250; 6,331,611; and 7,208,576; and U.S. Patent Application Publication No. 2005/0026815 (each of which is herein incorporated by reference). These methods include the purification of AFP from a biological fluid of transgenic mammal that has been engineered to express AFP into the biological fluid, as well as other methods known in the art. These AFPs can be used in the present invention notwithstanding the fact that the use of these different expression systems (e.g., production in a prokaryotic host cell, a eukaryotic host cell, or a transgenic animal or plant) may result in a recombinant AFP or fragment thereof having different post-translational modifications than that in the wild-type AFP (e.g., a different number of attached sugar residues (e.g., at least 1, 2, 3, 4, 5, 6, 7, or 10 sugar molecules), a different type of glycosylation (e.g., O-linked glycosylation or N- linked glycosylation, or the lack thereof), a different type of sugar residues (e.g., mannose, galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, glucuronate, sialic acid, or xylose, or different combinations thereof), or different amino acids glycosylated). For instance, naturally occurring human AFP is a variably glycosylated protein (e.g., glycosylation at asparagine 233 of SEQ ID NO: 1). In contrast, the recombinant AFP or fragment may be unglycosylated when produced by a prokaryotic host cell or may be somewhat differently glycosylated when produced by a eukaryotic host cell. Alternatively, a recombinant AFP can be genetically modified to eliminate glycosylation (e.g., by removing a glycosylation site, for instance replacing asparagine 233 of SEQ ID NO: 1 with any amino acid other than asparagine), regardless of the expression system in which it is produced. Human AFP is available through various commercial suppliers, including Fitzgerald Industries International (Concord, MA), Cell Sciences (Canton, MA), and Biodesign International (Saco, ME).

Furthermore, it is possible to employ well-known chemical synthesis methods to synthesize an AFP polypeptide or fragment, particularly when the AFP fragment is a peptide of a relatively short length, e.g., with less than 50 or 100 amino acids, although larger fragments can be synthetically produced.

Any AFP polypeptide or fragment thereof, regardless of its origin or status of post-translational modification, can be used in the present invention if the polypeptide or fragment has the same or substantially the same biological activity (e.g., at least about 40%, desirably at least about 50%, 60%, 70%, and more desirably at least about 80%, 90%, 100%, or 100% or more of the biological activity) of native human AFP

(e.g., as determined based on the ability of the AFP to bind to human leukocytes, to suppress human autologous mixed lymphocyte reactions (AMLR), to suppress EAE in a mouse model, or to inhibit release of inflammatory cytokines in a splenocyte assay). Similarly, fragments of the human AFP can also be used in the compositions and methods of the present invention, so long as the fragments retain the same or substantially the same biological activity of naturally-occurring human AFP (as determined using one or more of the assays for AFP biological activity described below). Fragments of human AFP can be generated by methods known to those skilled in the art, e.g., proteolytic cleavage or recombinant expression, or may result from normal protein processing (e.g., removal from a nascent polypeptide of amino acids). Fragments of human AFP can also be produced recombinantly using the techniques described above. Chemical methods can also be useful for synthesizing active AFP fragments. Specific examples of fragments of AFP are described in detail below and in U.S. Patent No. 5,707,963 and U.S. Patent No. 6,818,741, herein incorporated by reference.

Different methods for the production of an AFP are described below.

Production of Recombinant Human Alpha-Fetoprotein in E. coli

Methods for producing recombinant human AFP in a prokaryotic cell are described in U.S. Patent No. 5,384,250; 5,965,528; 6,288,034; 6,331,611; and 6,744,108, each of which is hereby incorporated by reference.

Production of Recombinant AFP in Baculovirus (BrAFP)

Methods for producing recombinant human AFP in a baculovirus expression system are described in U.S. Patent Application Publication No. 2005/0026815, hereby incorporated by reference.

Production of Alpha-Fetoprotein in Eukaryotes

Recombinant alpha-fetoprotein can be expressed in mammalian cells and transgenic animals. Methods for producing recombinant human AFP in mammalian cells and transgenic animals (e.g., e.g., a mammal, such as a goat, sheep, camel, cow, pig, rabbit, horse, or llama) are described in PCT Application Publication Nos.

WO 00/40693 and WO 00/53759, U.S. Patent No. 7,208,576, and U.S. Patent Application Publication No. 2007/0169211, each herein incorporated by reference in their entirety.

Mammalian cells (for example, CHO, COS, and myeloma cells) can also be used as hosts for the expression of alpha-fetoprotein cDNAs and fragments or analogs thereof to produce the corresponding proteins and peptides (see, e.g., WO 00/053759, herein incorporated by reference). For expression of constructs leading to direct expression of rHuAFP, active COS or CHO cell expression systems are preferred. The rHuAFP cDNAs can be introduced to plasmids and allowed to integrate into chromosomal DNA especially for CHO cells, or allowed to replicate to very high copy number especially in COS cells. The plasmids generally require a selectable marker for maintenance in transfected hosts, an efficient eukaryotic promoter to allow a high level of transcription from the cDNAs, convenient restriction enzyme sites for cloning and polyadenylation, and transcription termination signals for message stability. Several such vectors, which are suitable for expression of rHuAFP or biologically active fragments thereof, have been described in the literature (see, e.g, Bebbington et al., BioTechnology, 10:169-175, 1992; and Wright, Methods, 2:125- 135, 1991) and are commercially available (such as pRc/CMV, Invitrogen Corp.).

Fragments, Derivatives, and Analogs

In general, fragments, derivatives, or analogs of AFP are produced according to the techniques of polypeptide expression and purification described in U.S. Patent No. 5,384,250, U.S. Patent No. 6,331,611, and U.S. Patent No. 7,208,576, each or which is incorporated herein by reference. For example, suitable fragments, derivatives, or analogs of rHuAFP can be produced by transformation of a suitable host bacterial or eukaryotic cell with part of a HuAFP-encoding cDNA fragment (e.g., the cDNA described above) in a suitable expression vehicle. Alternatively, such fragments, derivatives, or analogs can be generated by standard techniques of PCR and cloned into appropriate expression vectors (Bebbington, supra, and Wright, supra). Accordingly, once a fragment, derivative, or analog of rHuAFP is expressed, it may be isolated by various chromatographic and/or immunological methods known in the art. Lysis and fractionation of rHuAFP-containing cells prior to affinity chromatography may be performed by standard methods. Once isolated, the recombinant protein can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques In Biochemistry And Molecular Biology, Work and Burdon, eds., Elsevier, 1980).

Fragments of rHuAFP molecules can be generated by methods known to those skilled in the art, e.g., chemical synthesis or proteolytic cleavage of naturally- occurring or recombinantly expressed AFP.

Recombinant HuAFP fragments of interest include, but are not limited to, Domain I (amino acids 1 (Arg) - 198 (Ser), SEQ ID NO: 5), Domain II (amino acids 199 (Ser) - 390 (Ser), SEQ ID NO: 6), Domain III (amino acids 391 (GIn) - 591 (VaI), SEQ ID NO: 7), Domain I+II (amino acids 1 (Arg) - 390 (Ser), SEQ ID NO: 8), Domain II+III (amino acids 199 (Ser) - 591 (VaI), SEQ ID NO: 9), and rHuAFP Fragment I (amino acids 267 (Met) - 591 (VaI), SEQ ID NO: 10). Activity of a fragment is evaluated experimentally using conventional techniques and assays, e.g., the assays described herein.

The invention further includes functional derivatives or analogs of full-length rHuAFP or fragments thereof. Analogs can differ from AFP by amino acid sequence differences (e.g., additions, deletions, conservative or non-conservative substitutions), or by modifications (e.g., post-translational modifications) that do not affect sequence, or by both. The derivatives/analogs of the invention will generally exhibit at least 90%, more preferably 95%, or even 99% amino acid identity with all or part of native human AFP amino acid sequence (SEQ ID NO: 1).

An AFP fragment, derivative, or analog may differ from a naturally occurring human AFP due to post-translational modifications (which do not normally alter primary sequence), which include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, or pegylation; such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes. Also included are cyclized peptide molecules and analogs that contain residues other than L-amino acids, e.g., D-amino acids, non-naturally occurring, or synthetic amino acids, e.g., β- or γ-amino acids, or L-amino acids with non-naturaly side chains (see, e.g., Noren et al., Science 244:182, 1989). Methods for site-specific incorporation of non-natural amino acids into the protein backbone of proteins is described, e.g., in Ellman et al., Science 255:197, 1992. Also included are chemically synthesized polypeptides or peptides with modified peptide bonds (e.g., non-peptide bonds as described in U.S. Patent No. 4,897,445 and U.S. Patent No. 5,059,653; herein incorporated by reference) or modified side chains to obtain the desired pharmacological properties as described herein. Useful AFP, AFP fragments, AFP derivatives, of AFP analogs having the same or substantially the same biological activity (e.g., at least about 40%, preferably at least about 50%, 60%, 70%, and more preferably at least about 80%, 90%, 100%, or 100% or more of the biological activity) of wild-type AFP can be identified using art-recognized methods, such as those described below). Some preferred functional AFP derivatives contain one or more conservative substitutions, in which certain amino acid residues are substituted by other residues having similar chemical structures (e.g., alanine for glycine, arginine for lysine, etc.). The derivatives/analogs mentioned above may include allelic variants, inter-species variants, and genetic variants, both natural and induced (for example, resulting from random mutagenesis by, e.g., site-specific mutagenesis according to methods described in scientific literature such as Sambrook et ah, Molecular Cloning, A Laboratory Manual, 3rd ed., 2001; Kriegler, Gene Transfer and Expression: A Laboratory Manual, 1990; and Ausubel et al., eds., Current Protocols in Molecular Biology, 1994.

AFP Activity Assays

As stated above, AFP polypeptides or AFP fragments suitable for use in the compositions and methods of the present invention may include various derivatives, analogs, or fragments of the naturally occurring human AFP, so long as the polypeptides or fragments retain the same or substantially the same biological activity of mature human AFP. The biological activity of an AFP of the present invention can be demonstrated by using, e.g., one or more of the following assays.

A first assay for testing a candidate AFP for biological activity entails measuring the ability of the AFP to specifically bind to human leukocytes (e.g., peripheral monocytes). A binding assay suitable for this purpose is described in, e.g., Parker et al., Protein Express. Purification 38:177-183, 2004. Briefly, a competitive assay format is used to test a candidate AFP for its ability to specifically bind to U937 cells, a human monocytic cell line. The cells are maintained in RPMI media with 10% fetal bovine serum. Prior to the binding assay, the cells are washed twice with serum-free media and adjusted to 2.5 x 10⁶ cells/ml in phosphate-buffered saline (PBS). Native human AFP (SEQ ID NO: 1) or non-glycosylated human AFP (see, e.g., SEQ ID NO: 18, where, e.g., residue 233 is glutamine) is labeled with a detectable label, e.g., fluorescein, in a proper reaction followed by removal of the unattached labeling material, for instance, by gel filtration. In the case of labeling human AFP with fluorescein, the protein is mixed with a solution of fluorescein-5- isothiocyanate in dimethyl sulfoxide for 1 hour in the dark, followed by gel filtration to remove unbound dye. Labeled human AFP is stored in 20% glycerol at -20 ⁰C until use.

For the binding assay, a certain number of U937 cells (e.g., 40 μl of cell suspension at 2.5 x 10⁶ cells/ml concentration) are mixed with a pre-determined amount of labeled human AFP (e.g., at a final concentration of 0.5 μM) and with unlabeled human AFP or an unlabeled candidate AFP, each at a set of final concentrations (e.g., 20, 10, 5, 2.5, 1.25, and 0.625 μM) to determine the IC₅₀ values for both human AFP and the candidate AFP. At the conclusion of the binding process, the cells are then washed with PBS and suspended in fresh PBS so that the labeled AFP remaining on U937 cells can be measured, e.g., by flow cytometry. A second assay for testing a candidate AFP for biological activity entails measuring the ability of the AFP to suppress autoimmune reactions, either in AMLR or in a mouse model of EAE. Methods are known in the art for testing AMLR and its inhibition. For instance, U.S. Patent Nos. 5,965,528 and 6,288,034, and U.S. Patent Application Publication No. 2005/0026815 (each of which is herein incorporated by reference) describe the AMLR system as follows: isolation of human peripheral blood mononuclear cells (PBMC), their fractionation into non-T-cell populations, and the AMLR, performed according to standard procedures. Briefly, responder T-cells are isolated by passing 1.5 x 10⁸ PMBC over a commercial anti-Ig affinity column (US Biotek Laboratories, Seattle, WA) and 2 x 10⁵ responder cells are subsequently cultured with 2 x 10⁵ autologous ¹³⁷Cs-irradiated (2500 rads) non-T stimulator cells from a single donor. The medium employed consists of RPMI- 1640 supplemented with 20 mM HEPES (Invitrogen), 5 x 10^"5 M 2-mercaptoethanol (BDH, Montreal, QC), 4 mM L-glutamine (Invitrogen), 100 U/ml penicillin (Invitrogen), and 100 μg/ml streptomycin sulfate, with the addition of 10% fresh human serum autologous to the responder T-cell donor for the AMLR. Varying concentrations of purified recombinant human AFP, human serum albumin, anti-human AFP monoclonal antibody clone #164 (125 μg/ml final concentration in culture) (Leinco Technologies, St. Louis, MO) are added at the initiation of cultures. AMLR cultures are incubated for 4 to 7 days, at 37 ⁰C in 95% air and 5% CO₂. At the indicated intervals, DNA synthesis is assayed by a 6 hour pulse with 1 μCi of ³H-thymidine (specific activity 56 to 80 Ci/mmole; ICN Radioisotopes, Cambridge, MA). The cultures are harvested on a multiple sample harvester (Skatron, Sterling, VA), and the incorporation Of³H-TdR is measured in a Packard 2500 TR liquid scintillation counter. Results are expressed as mean cpm ± the standard error of the mean of triplicate or quadruplicate cultures. The immunosuppressive activity of a candidate AFP within the scope of the present invention can be assessed by its ability to suppress human autologous mixed lymphocyte reactions (AMLR). Generally, the candidate AFP is tested for its ability to inhibit the proliferative response of autoreactive lymphocytes stimulated by autologous non-T-cells, by measuring lymphocyte autoproliferation throughout a time course of 4 to 7 days. Suppression of AMLR in a dose-dependent manner is demonstrated by results from dose-response studies performed at the peak of T-cell autoproliferation where an AFP is added at the initiation of cultures. Furthermore, parallel viability studies can be used to establish that the inhibitory activity of an AFP polypeptide or fragment on human autoreactive T-cells is not due to non-specific cytotoxic effects.

A third assay for testing a candidate AFP for biological activity involves the use of a myelin oligodendrocyte glycoprotein (MOG) mouse model of experimental autoimmune encephalomyelitis (EAE) (see, e.g., Fritz et al., J Immunol. 130:1024, 1983; Naiki et al., Int. J. Immunopharmacol. 13:235, 1991; and Goverman, Lab. Anim. Sci., 46:482, 1996). In this in vivo assay, genetically susceptible strains of mice are subcutaneously immunized with MOG emulsified in Complete Freund's Adjuvant (CFA), which leads to the development of EAE in the animals. A candidate AFP is administered to a selected group of mice on a daily basis, beginning prior to, at the same time, or subsequent to the start of the administration of MOG to the animals. The symptoms of EAE in these animals are monitored and compared to those in a control group (e.g., those receiving only saline injections) over a certain time period, e.g., 30 days. Severity of EAE in each animal is given a score between 1-5 based on defined clinical symptoms; the average score of animals in a group indicates the disease state of the group. A biologically active AFP will reduce the severity of EAE in animals receiving MOG compared to controls (e.g., at least a 50% reduction in the severity of disease, e.g., after 2, 5, 10, 15, 20, 25, or 30 days or more of treatment). A fourth assay that can be used to test a candidate AFP for biological activity examines the ability of the candidate AFP to inhibit or reduce the release of inflammatory cytokines from mitogen-stimulated in vitro splenocyte cultures obtained from naϊve mice (e.g., as described in Hooper and Evans, J. Reprod. Immunol.16: 83- 961,1989; and Kruisbeek, in Current Protocols in Immunology, Vol. I₅ Section 3.1.1- 3.1.5, 2000). Splenocytes are stimulated with phytohemagglutinin (PHA), concavalin A (ConA), or lipopolysaccharide (LPS) in the presence of increasing concentrations of an AFP for 24 hours. Human serum albumin is used as a negative control for the assays. A lO point dose response study has shown that biologically active AFP inhibits or substantially inhibits the secretion of PHA induced IFN-γ in a reproducible manner.

Supplemental Secondary Agents for Use in the Methods, Compositions, and Kits of the Invention

In addition to an AFP (or a biologically active fragment, derivative, or analog thereof), the therapies of the invention can also include the administration of one or more secondary agents (e.g. a steroid, a non-steroid anti-inflammatory drug (NSAID), a disease-modifying anti-rheumatic drug (DMARD), an integrin antagonist, an inflammation signaling inhibitor (ISI), a tolerizing agent, an immunosuppressive agent, and an immunomodulatory agent). Non-limiting examples of secondary agents for use in the invention are listed in Table 1 and discussed in detail below.

Table 1

Steroids

For example, the compositions and kits of the invention may include, or the methods of the invention may involve the administration of, a steroid as a supplemental secondary agent. A steroid is a naturally occurring or synthetic compound characterized by a hydrogenated cyclopentanoperhydrophenanthrene ring system. Non-limiting exemplary steroids that may be used in the invention include algestone, 6-alpha-fluoroprednisolone, 6-alpha-methylprednisolone, 6-alpha- methylprednisolone 21 -acetate, 6-alpha-methylprednisolone 21-hemisuccinate sodium salt, 6-alpha-9-alpha-difluoroprednisolone 21 -acetate 17-butyrate, amcinafal, beclomethasone, beclomethasone dipropionate, beclomethasone dipropionate monohydrate, 6-beta-hydroxycortisol, betamethasone, betamethasone- 17-valerate, budesonide, clobetasol, clobetasol propionate, clobetasone, clocortolone, clocortolone pivalate, cortisone, cortisone acetate, cortodoxone, deflazacort, 21-deoxycortisol, deprodone, descinolone, desonide, desoximethasone, dexamethasone, dexamethasone- 21 -acetate, dichlorisone, diflorasone, diflorasone diacetate, diflucortolone, doxibetasol, fludrocortisone, flumethasone, flumethasone pivalate, flumoxonide, flunisolide, fluocinonide, fluocinolone acetonide, 9-fluorocortisone, fluorohydroxyandrostenedione, fluorometholone, fluorometholone acetate, fluoxymesterone, flupredidene, fluprednisolone, flurandrenolide, formocortal, halcinonide, halometasone, halopredone, hyrcanoside, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone cypionate, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, hydrocortisone probutate, hydrocortisone valerate, 6-hydroxydexamethasone, isoflupredone, isoflupredone acetate, isoprednidene, meclorisone, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, paramethasone, paramethasone acetate, prednisolone, prednisolone acetate, prednisolone metasulphobenzoate, prednisolone sodium phosphate, prednisolone tebutate, prednisolone-21-hemisuccinate free acid, prednisolone-21 -acetate, prednisolone-21 (beta-D-glucuronide), prednisone, prednylidene, procinonide, tralonide, triamcinolone, triamcinolone acetonide, triamcinolone acetonide 21-palmitate, triamcinolone diacetate, triamcinolone hexacetonide, and wortmannin. Particularly desirable corticosteroids are prednisolone, cortisone, dexamethasone, hydrocortisone, methylprednisolone, fluticasone, prednisone, triamcinolone, and diflorasone.

NSAIDs

The compositions and kits of the invention may also include, and the methods of the invention may also involve the administration of, an non-steroidal antiinflammatory drug (NSAID). An NSAID is a non-steroidal agent that prevents or diminishes inflammation. Non-limiting examples of NSAIDs that may be used in the present invention include naproxen sodium, diclofenac sodium, diclofenac potassium, aspirin, sulindac, diflunisal, piroxicam, indomethacin, ibuprofen, nabumetone, choline magnesium trisalicylate, sodium salicylate, salicylsalicylic acid, fenoprofen, flurbiprofen, ketoprofen, meclofenamate sodium, meloxicam, oxaprozin, sulindac, tolmetin, and COX-2 inhibitors such as rofecoxib, celecoxib, valdecoxib, and lumiracoxib.

DMARDs

In addition, compositions and kits of the invention may also include, and the methods of the invention may also involve the administration of, a disease-modifying anti-rheumatic drug (DMARD). A DMARD can be used treat, prevent, or reduce one or more of the symptoms of or the progression of an inflammatory disease in a patient. Non-limiting examples of DMARDs that may be used in the present invention include auranofin, aurothioglucose, azathioprine, chlorambucil, cyclophosphamide, cyclosporine, D-penicillamine, gold sodium thiomalate (injectable gold), hydroxychloroquine, leflunomide, methotrexate, minocycline, mycophenolate mofetil, and sulfasalazine.

Integrin Antagonists

In another example, the compositions, kits, and methods of the invention may include an integrin antagonist. An integrin antagonist suppresses or inhibits the biological activity of an integrin molecule, such as the α4 subunit of an integrin molecule. An integrin antagonist may include proteins, blocking peptides, antibodies, such as natalizumab, small molecule inhibitors, and nucleic acid inhibitors. Examples of nucleic acid inhibitors used as integrin antagonists are those that include a sequence which is complimentary to a sequence substantially identical to all or part of the mRNA sequence of human α4 integrin (NCBI Accession No. NM 000885; SEQ ID NO: 16; Takada et al. supra; or SEQ ID NO: 17). For example, a sequence that is complementary to nucleotides 1-25 of SEQ ID NO: 16 or SEQ ID NO: 17. Examples of α4 integrin antagonists include, but are not limited to, natalizumab (Elan/Biogen Idee; see, e.g., U.S. Patent Nos. 5,840,299; 6,033,665; 6,602,503; 5,168,062; 5,385,839; and 5,730,978), oMEPUPA-V (Biogen; U.S. Patent No. 6,495,525; incorporated by reference herein), CDP-323 (Celltech); firategrast (SB-68399; GlaxoSmithKline); TR-9109 (Pfizer); ISIS-107248 (Antisense Therapeutics); R-1295 (Roche); and TBC-4746 (Schering-Plough). Additional non-limiting examples of α4 integrin antagonists include the small molecules described in U.S. Patent Nos. 5,821,231; 5,869,448; 5,936,065; 6,265,572;

6,288,267; 6,365,619; 6,423,728; 6,426,348; 6,458,844; 6.479,666; 6,482,849;

6,596,752; 6,667,331; 6,668,527; 6,685,617; 6,903,128; and 7,015,216 (each herein incorporated by reference); in U.S. Patent Application Publication Nos.

2002/0049236; 2003/0004196; 2003/0018016; 2003/0078249; 2003/0083267;

2003/0100585; 2004/0039040; 2004/0053907; 2004/0087574; 2004/0102496;

2004/0132809; 2004/0229858; 2006/0014966; 2006/0030553; 2006/0166866;

2006/0166961; 2006/0241132; 2007/0054909; and 2007/0232601 (each herein incorporated by reference); in European Patent Nos. EP 0842943; EP 0842944; EP

0842945; EP 0903353; and EP 0918059; and in PCT Publication Nos. WO 95/15973;

WO 96/06108; WO 96/40781; WO 98/04247; WO 98/04913; WO 98/42656; WO

98/53814; WO 98/53817; WO 98/53818; WO 98/54207; WO 98/58902; WO

99/06390; WO 99/06431; WO 99/06432; WO 99/06433; WO 99/06434; WO 99/06435; WO 99/06436; WO 99/06437; WO 99/10312; WO 99/10313; WO

99/20272; WO 99/23063; WO 99/24398; WO 99/25685; WO 99/26615;

WO 99/26921; WO 99/26922; WO 99/26923; WO 99/35163; WO 99/36393; WO

99/37605; WO 99/37618; WO 99/43642; WO 01/42215; and WO 02/28830; all of which are incorporated by reference herein. Additional examples of α4 integrin antagonists include the phenylalanine derivatives described in: U.S. Patent Nos. 6,197,794; 6,229,011; 6,329,372; 6,388,084; 6,348,463; 6,362,204; 6,380,387; 6,445,550; 6,806,365; 6,835,738; 6,855,706; 6,872,719; 6,878,718; 6,911,451; 6,916,933; 7,105,520; 7,153,963; 7,160,874; 7,193,108; 7,250,516; and 7,291,645 (each herein incorporated by reference). Additional amino acid derivatives that are α.4 integrin antagonists include those described in, e.g., U.S. Patent Application Publication Nos. 2004/0229859 and 2006/0211630 (herein incorporated by reference), and PCT Publication Nos. WO 01/36376; WO 01/47868; and WO 01/70670; all of which are incorporated by reference herein. Other examples of oc4 integrin antagonists include the peptides, and the peptide and semi-peptide compounds described in, e.g., PCT Publication Nos. WO 94/15958; WO 95/15973; WO 96/00581; WO 96/06108; WO 96/22966 (Leu-Asp-Val tripeptide; Biogen, Inc.); WO 97/02289; WO 97/03094; and WO 97/49731. An additional example of an α4 integrin antagonist is the pegylated molecule described in U.S. Patent Application Publication No. 2007/066533 (herein incorporated by reference).

Examples of antibodies that are α.4 integrin antagonists include those described in, e.g., PCT Publication Nos. WO 93/13798; WO 93/15764; WO 94/16094; and WO 95/19790.

Inflammatory Signaling Inhibitors (ISIs)

An inflammatory signaling inhibitor (ISI) may also be used in conjuction with the compositions, kits, and methods of the present invention. An inflammatory signaling inhibitor is an agent that decreases the binding between a pro-inflammatory cytokine (e.g., TNF-alpha, TNF-beta, or IL-I) and its receptor (e.g., TNF receptor 1 or 2, or IL-I receptor, respectively); decreases the binding of activating molecules to pro-inflammatory cell surface signaling molecules (e.g., CD-20, CTLA-4, CD80/CD86, or CD28); or decreases the downstream activation of, or activity of, intracellular signaling molecules that are activated following the binding of pro- inflammatory cytokines to their receptors or the binding of activating molecules to pro-inflammatory cell surface signaling molecules (e.g., an agent that decreases the activation of, or activity of, signaling molecules in the p38 MAPK signaling pathway). An ISI may act by reducing the amount of pro-inflammatory cytokine (e.g., TNF-alpha, TNF-beta, or IL-I) freely available to bind the receptor.

Non-limiting examples of inflammatory signaling inhibitors include a soluble TNF receptor fusion protein such as etanercept (ENBREL " ) or lenercept, a soluble pro-inflammatory cell surface signaling molecule (e.g., a soluble CTLA-4 (Abatacept)), and an antibody directed against a pro-inflammatory cytokine or a pro- inflammatory cell surface signaling molecule (e.g., an anti-TNF antibody, such as adalimumab, certolizumab, inflixamab, or golimumab; or an anti-CD20 antibody, such as rituximab, or TRU-Ol 5 (TRUBION^®)). In addition, an ISI may act by disrupting the ability of the endogenous wild-type pro-inflammatory cytokine or the pro-inflammatory cell surface signaling molecule to bind to its receptor (e.g., TNF receptor 1 or 2, or IL-I receptor). Examples of dominant-negative TNF-alpha variants are XENP345 (a pegylated version of TNF variant A145R/I97T) and XPRO™1595, and further variants disclosed in U.S. Patent application publication Nos. 20030166559 and 20050265962, herein incorporated by reference. An example of a dominant negative IL-I variant is anakinra (KINERET^®), which is a soluble form of IL-I that binds to the IL-I receptor without activating intracellular signaling pathways.

Inflammatory signaling inhibitors, which can be used in the methods, kits, and compositions of the invention, are also small molecules which inhibit or reduce the signaling pathways downstream of pro-inflammatory cytokine or pro-inflammatory cell surface signaling molecules (e.g., DE 096). Examples of ISIs of this kind include inhibitors of p38 MAP kinase, e.g., 5-amino-2-carbonylthiopene derivatives (as described in WO2004089929, herein incorporated); ARRY-797; BIRB 796 BS, (1-5- tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-2(moφholin-4-yl-ethoxy)-naphtalen-l-yl]- urea); CHR-3620; CNI-1493; FR-167653 (Fujisawa Pharmaceutical, Osaka, Japan); ISIS 101757 (Isis Pharmaceuticals); ML3404; NPC31145; PD169316; PHZl 112; RJW67657, (4-(4-(4-fluorophenyl)-l-(3-phenylpropyl)-5-(4-pyridinyl)-lH-imidazol- 2-yl)-3-butyn-l-ol; SCIO-469; SB202190; SB203580, (4-(4-fluorophenyl)-2-(4- methylsulfmylphenyl)-5-(4-pyridyl)lH-imidazole); SB239063, frαrø-l-(4- hydroxycyclohexyl)-4-(4-fluorophenyl-methoxypyridimidin-4-yl)imidazole; SB242235; SD-282; SKF-86002; TAK 715; VX702; and VX745. Furthermore, an ISI may interfere with the processing of a pro-inflammatory cytokine (e.g., TNF-alpha and TNF-beta) from its membrane bound form to its soluble form. Inhibitors of TACE are ISIs of this class. Examples of inhibitors of TACE include BB-1101, BB- 3103, BMS-561392, butynyloxyphenyl β-sulfone piperidine hydroxomates, CH4474, DPC333, DPH-067517, GM6001, GW3333, Ro 32-7315, TAPI-I, TAPI-2, and TMI 005. Additional examples of ISIs include short peptides derived from the E. coli heat shock proteins engineered for disease-specific immunomodulatory activity(e.g., dnaJPl).

An ISI may be directly administered or delivered by means of gene therapy, e.g., an adeno-associated viral vector that expresses a soluble TNF-receptor immunoglobulin Fc fusion protein, as described in U.S. Patent Application

Publication Nos. 20030113295 and 20030103942, incorporated by reference. Tolerizing Agents

Additionally, the compositions, kits, and methods of the invention may include a tolerizing agent. A tolerizing agent energizes or "vetos" T cells by preventing development of normal T cell-dependent responses. Non-limiting examples of tolerizing agents include anti-idiotypic agents, such as monoclonal antibodies, LJP 394 (abetimus, Riquent, La Jolla Pharmaceuticals), and the tolerizing agents disclosed in US Patent No. 6,428,782 and PCT Application No. WO 07/112410, herein incorporated by reference.

Immunosuppressive Agents

In another example, the compositions, kits, and methods of the invention may include an immunosuppressive agent. An immunosuppressive agent decreases or inhibits the activity or proliferation of a T cell. Non-limiting examples of immunosuppressive agents include a cyclosporine (e.g., cyclosporin A, such as NEORAL^®, SANDIMMUNE^®, and SANDCYA^®), an azathioprine (e.g. IMURAN^®), FK-506, 15-deoxyspergualin, and an antibody (e.g., a monoclonal antibody, such as basiliximab, daclizumab, and muromonab-CD3, or a globulin, such as anti- lymphocyte globulin, anti-thymocyte globulin, and the like).

Immunomodulatory Agents

The compositions, kits, and methods of the invention may also include an immunomodulatory agent. An immunomodulatory agent is: (1) an interferon or a peptide or protein that has an amino acid sequence substantially identical (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100% identical) to all or a portion of the sequence of an interferon (e.g., a human interferon), such as

IFN-α (e.g., IFN-α-la; see U.S. Patent Application No. 20070274950, incorporated herein by reference in its entirety), IFN-α-lb (SEQ ID NO: 11), IFN-α-2a (see PCT Application No. WO 07/044083, herein incorporated by reference in its entirety), and IFN-α-2b (SEQ ID NO: 12)), IFN-β (e.g., described in U.S. Patent No. 7,238,344, incorporated by reference in its entirety; IFN-β- Ia as described in U.S. Patent No.

6,962,978, incorporated by reference in its entirety; and IFN-β- Ib (as described in U.S. Patent Nos. 4,588,585; 4,959,314; 4,737,462; and 4,450,103; incorporated by reference in their entirety)), IFN-γ (e.g., SEQ ID NO: 13), and IFN-τ (as described in U.S. Patent No. 5,738,845 and U.S. Patent Application Publication Nos. 20040247565 and 20070243163; incorporated by reference in their entirety); (2) a small molecule (e.g., BG12 (fumarate), fϊngolimod (FTY-720), laquinimod, teriflunomide, or atorvastatin, or a molecule that demonstrates the same or substantially the same biological activity to an interferon (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the activity of a human IFN-α, a human IFN-β, a human IFN-γ or a human IFN-τ in the ability to suppress EAE in a mouse model)); (3) an antibody (e.g., all or part of a monoclonal antibody (e.g., an IL-2 receptor-binding antibody, such as daclizumab; a CD20-binding antibody, such as rituximab; an IL- 12 binding antibody, such as ABT-874; and a CD52-binding antibody, such as alemtuzumab), a polyclonal antibody, or an antibody fusion protein); (4) a peptide (e.g., MBP-8289, NBI-5788, T cell receptor peptide (NEUROV AX^®), or glatiramer acetate (COPAXONE^®)); or (5) a DNA vaccine (e.g., BNT-3009-01).

Pharmaceutical Compositions

The present invention also relates to a pharmaceutical composition for the treatment of one or more of the diseases disclosed herein which contains a therapeutically effective amount of an AFP. When treating uveitis, the pharmaceutical composition containing an AFP is preferably formulated for ophthalmic administration. The compositions of the invention may also include one or more secondary agents. The AFP and the one or more secondary agent may be present in the same pharmaceutical composition (a single dosage form) or separate pharmaceutical compositions (separate dosage forms) to be administered coextensively or separately. In addition, the composition can include one or more different AFPs or secondary agents. The compositions can be formulated for use in a variety of drug delivery systems. One or more physiologically acceptable excipients or carriers can also be included in the compositions for proper formulation. Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sceinces, Mack Publishing Company, Philadelphia, PA, 17th ed., 1985. See, e.g., Langer, Science 49:1527-1533, 1990, for a brief review of methods for drug delivery.

The AFP and/or secondary agent can be formulated for parenteral, intranasal, topical, oral, ophthalmic, or local administration, such as by a transdermal means, for therapeutic treatment. Commonly, the compositions are administered parenterally (e.g., by intra-arterial, intravenous, intramuscular, or subcutaneous injection), by oral ingestion, or by topical application at areas affected by the disease. Thus, the invention features compositions for parenteral administration that include an AFP and/or a secondary agent dissolved or suspended in an acceptable carrier, preferably an aqueous carrier, e.g., water, buffered water, saline, PBS, and the like. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents, and the like. In a preferred embodiment, the both the AFP and/or the secondary agent are formulated in an ophthalmic solution for administration to the eye (i.e., ophthalmic administration). The invention also features compositions formulated for oral delivery, which may contain inert ingredients such as binders or fillers for the formulation of a tablet, a capsule, and the like. Furthermore, this invention features compositions for local administration, which may contain inert ingredients such as solvents or emulsifiers for the formulation of a cream, an ointment, and the like. In different embodiments of the invention, the AFP and the secondary agent may be administered in the same or separate compositions for administration via the same or two different routes of administration. Compositions of the invention may be sterilized by conventional sterilization techniques or they may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 11 , more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of an AFP and/or a secondary agent, such as in a sealed package of tablets or capsules (e.g., a blister pack). The composition in liquid or gel form can also be packaged in a container for a flexible quantity, such as in a squeezable tube or an eye dropper designed for a topically applicable cream, ointment, or eye drops.

In other preferred routes of administration, the AFP and/or secondary agent can be given to a patient by injection or implantation of a slow release preparation, for example, in a slowly dissociating polymeric or crystalline form; this sort of sustained administration can follow an initial delivery of the drug by more conventional routes (for example, those described above). Alternatively, the AFP can be administered using an infusion pump (e.g., an external or implantable infusion pump), thus allowing a precise degree of control over the rate of drug release. The compositions of the invention containing an effective amount of an AFP and/or one or more secondary agents can be administered for therapeutic treatments. For example, in prophylactic applications, compositions of the invention containing an AFP and/or one or more secondary agents are administered to a patient susceptible to or otherwise at risk of developing one or more of the diseases described herein. In this use, the precise amounts again depend on the patient's state of health, but generally range from about 0.1 mg to about 400 mg of an AFP per dose (e.g., 0.5 mg, 1 mg, 5 mg, 10 mg, 50 mg, 60 mg, 100 mg, 200 mg, 300 mg, or 400 mg per dose) and from about 0.1 mg to about 3,000 mg of a secondary agent per dose (e.g., 1 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1,000 mg, 1,500 mg, 2,000 mg, 2,500 mg, or 3,000 mg per dose). A dose of the AFP and/or secondary agent can be administered to a patient one or more times per hour, day, week, month, or year (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times per hour, day, week, month, or year). More commonly, a single dose per week of an AFP and/or a secondary agent is administered to a patient. In therapeutic applications, compositions of the invention can be administered to a patient already suffering from a disease (e.g., psoriatic arthritis, IgA nephropathy, Hashimoto's disease, Sjogren's syndrome, ankylosing spondylitis, and uveitis) in an amount sufficient to treat or at least partially arrest one or more of the symptoms of the disease (e.g., one or more of the diseases described herein) and one or more of its complications. Amounts effective for this use may depend on the severity of the disease or condition and the general state of the patient, but may range from about 0.1 mg to about 400 mg of an AFP per dose (e.g., 0.5 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 200 mg, 300 mg, or 400 mg per dose) and from about 0.1 mg to about 3,000 mg of a secondary agent per dose (e.g., 1 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 ,000 mg, 1 ,500 mg, 2,000 mg, 2,500 mg, or 3,000 mg per dose). In several embodiments, the patient may receive an AFP (with or without a secondary agent) in the range of about 0.1 to about 400 mg per dose one or more times per week (e.g., 2, 3, 4, 5, 6, or 7 or more times per week), preferably about 5 mg to about 300 mg per dose one or more times per week, and even more preferably about 5 mg to about 200 mg per dose one or more times per week. The patient may also receive a biweekly dose of an AFP in the range of about 50 mg to about 800 mg or a monthly dose of an AFP in the range of about 50 mg to about 1,200 mg.

In other embodiments, an AFP may be administered to a patient in a typical dosage range of about 0.1 mg to about 400 mg per dose per week, about 1.0 mg to about 300 mg per dose per week, about 5 mg to about 200 mg per dose per week, about 10 mg to about 100 mg per dose per week, about 20 mg to about 80 mg per dose per week, about 100 mg to about 300 mg per dose per week, or about 100 mg to about 200 mg per dose per week. An AFP may be administered in the range of about 0.5 mg to about 100 mg per dose every other day, preferably about 5 mg to about 75 mg per dose every other day, more preferably about 10 mg to about 50 mg per dose every other day, and even more preferably 20 mg to about 40 mg per dose every other day. An AFP may also be administered in the range of about 0.5 mg to about 100 mg per dose three times per week, preferably about 5 mg to about 75 mg per dose three times per week, more preferably about 10 mg to about 50 mg per dose three times per week, and even more preferably about 20 mg to about 40 mg per dose three times per week. Preferably the AFP is administered at 20 mg per dose three times per week or 60 mg per dose once a week.

In a preferred embodiment, the AFP (alone or in conjunction with a secondary agent) is administered to a patient in the amount of 60 mg/dose one or more times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per week, for several weeks (e.g., 2, 3, 5, 10, or 15 weeks or more).

Furthermore, the method(s) of the invention can also employ combination therapy in which the AFP is administered either simultaneously or sequentially with a secondary agent such as a general or specific tolerizing agent (e.g., an anti-idiotypic agent, such as a monoclonal antibody), a therapeutic vaccine, an oral agent (e.g., insulin, collagen or myelin basic protein), a cytokine (e.g., U-15), an immunosuppressive agent, a steroid, a DMARD, an NSAID, an ISI, an integrin antagonist, or an immunomodulatory agent (e.g., an interferon, such as α-interferon). Preferably, the secondary agent is administered in an effective dose which is at or lower than the standard dose when the secondary agent is used by itself. Preferred secondary agents include cyclosporines, FK-506, steroids, azathioprines, 15- deoxyspergualin, infliximab, etanercept, and adalimumab. In an additional embodiment, the AFP may be administered in combination with a secondary agent at dosages that allow a synergistic improvement in therapeutic efficacy.

For the above described combination therapies, the immunosuppressive agent is delivered in the range of 0.1 to 3,000 mg per dose one or more times per week (e.g.,

2, 3, 4, 5, 6, or 7 or more times per week), 0.1 to 2,500 mg per dose one or more times per week, 0.1 to 2,000 mg per dose one or more times per week, 0.1 to 1 ,500 mg per dose one or more times per week, 0.1 to 1,000 mg per dose one or more times per week, 0.1 to 800 mg per dose one or more times per week, 0.1 to 600 mg per dose one or more times per week, 0.1 to 500 mg per dose one or more times per week, 0.1 to 400 mg per dose one or more times per week, 0.1 to 300 mg per dose one or more times per week, 0.1 to 250 mg per dose one or more times per week, 0.1 to 200 mg per dose one or more times per week, 0.1 to 150 mg per dose one or more times per week, 0.1 to 100 mg per dose one or more times per week, or 0.1 to 50 mg per dose one or more times per week.

For the above described combination therapies, the tolerizing agent is delivered in the range of 0.1 to 1,500 mg per dose one or more times per week (e.g., 2,

3, 4, 5, 6, or 7 or more times per week), 0.1 to 1,200 mg per dose one or more times per week, 0.1 to 1,000 mg per dose one or more times per week, 0.1 to 800 mg per dose one or more times per week, 0.1 to 600 per dose one or more times per week, 0.1 to 500 mg per dose one or more times per week, 0.1 to 400 mg per dose one or more times per week, 0.1 to 300 mg per dose one or more times per week, 0.1 to 200 mg per dose one or more times per week, 0.1 to 150 mg per dose one or more times per week, 0.1 to 100 mg per dose one or more times per week, 0.1 to 80 mg per dose one or more times per week, 0.1 to 60 mg per dose one or more times per week, 0.1 to 40 mg per dose one or more times per week, 0.1 to 20 mg per dose one or more times per week, or 0.1 to 10 mg per dose one or more times per week.

For the above described combination therapies, the steroid is delivered in the range of 0.1 to 1,500 mg per dose one or more times per week (e.g., 2, 3, 4, 5, 6, or 7 or more times per week), 0.1 to 1,200 mg per dose one or more times per week, 0.1 to 1,000 mg per dose one or more times per week, 0.1 to 800 mg per dose one or more times per week, 0.1 to 600 per dose one or more times per week, 0.1 to 500 mg per dose one or more times per week, 0.1 to 400 mg per dose one or more times per week, 0.1 to 300 mg per dose one or more times per week, 0.1 to 200 mg per dose one or more times per week, 0.1 to 150 mg per dose one or more times per week, 0.1 to 100 mg per dose one or more times per week, 0.1 to 80 mg per dose one or more times per week, 0.1 to 60 mg per dose one or more times per week, 0.1 to 40 mg per dose one or more times per week, 0.1 to 20 mg per dose one or more times per week, or 0.1 to 10 mg per dose one or more times per week. For the above described combination therapy, the DMARD is administered in the range of about 0.1 to 3,000 mg per dose one or more times per week (e.g., 2, 3, 4, 5, 6, or 7 or more times per week), 0.1 to 2,500 mg per dose one or more times per week, 0.1 to 2,000 mg per dose one or more times per week, 0.1 to 1,500 mg per dose one or more times per week, 0.1 to 1 ,000 mg per dose one or more times per week, 0.1 to 800 mg per dose one or more times per week, 0.1 to 600 mg per dose one or more times per week, 0.1 to 500 mg per dose one or more times per week, 0.1 to 400 mg per dose one or more times per week, 0.1 to 300 mg per dose one or more times per week, 0.1 to 250 mg per dose one or more times per week, 0.1 to 200 mg per dose one or more times per week, 0.1 to 150 mg per dose one or more times per week, 0.1 to 100 mg per dose one or more times per week, or 0.1 to 50 mg per dose one or more times per week.

For the above described combination therapy, the NSAID is administered in the range of 0.1 to 1,500 mg per dose one or more times per week (e.g., 2, 3, 4, 5, 6, or 7 or more times per week), 0.1 to 1,200 per dose one or more times per week, 0.1 to 1 ,000 mg per dose one or more times per week, 0.1 to 800 mg per dose one or more times per week, 0.1 to 600 mg per dose one or more times per week, 0.1 to 500 mg per dose one or more times per week, 0.1 to 400 mg per dose one or more times per week, 0.1 to 300 mg per dose one or more times per week, 0.1 to 200 mg per dose one or more times per week, 0.1 to 150 mg per dose one or more times per week, 0.1 to 100 mg per dose one or more times per week, 0.1 to 80 mg per dose one or more times per week, 0.1 to 60 mg per dose one or more times per week, 0.1 to 40 mg per dose one or more times per week, 0.1 to 20 mg per dose one or more times per week, or 0.1 to 10 mg per dose one or more times per week. During the above described combination treatment, a patient may also receive an ISI in the range of about 0.1 to 3,000 mg per dose one or more times per week (e.g., 2, 3, 4, 5, 6, or 7 or more times per week), 0.1 to 2,500 mg per dose one or more times per week, 0.1 to 2,000 mg per dose one or more times per week, 0.1 to 1,500 mg per dose one or more times per week, 0.1 to 1 ,000 mg per dose one or more times per week, 0.1 to 800 mg per dose one or more times per week, 0.1 to 600 mg per dose one or more times per week, 0.1 to 500 mg per dose one or more times per week, 0.1 to 400 mg per dose one or more times per week, 0.1 to 300 mg per dose one or more times per week, 0.1 to 200 mg per dose one or more times per week, 0.1 to 150 mg per dose one or more times per week, 0.1 to 100 mg per dose one or more times per week, or 0.1 to 50 mg per dose one or more times per week. A patient may also receive an ISI in the range of 0.1 to 3,000 mg per dose once every two or three weeks. During the above described combination treatment, a patient may also receive an immunomodulatory agent in the range of about 50 μg to 300 mg per dose one or more times per week (e.g., 2, 3, 4, 5, 6, or 7 or more times per week), 0.1 to 250 mg per dose one or more times per week, 0.1 to 200 mg per dose one or more times per week, 0.1 to 150 mg per dose one or more times per week, 0.1 to 125 mg per dose one or more times per week, 0.1 to 100 mg per dose one or more times per week, 0.1 to 80 mg per dose one or more times per week, 1.0 to 70 mg per dose one or more times per week, 1.0 to 60 mg per dose one or more times per week, 1.0 to 50 mg per dose one or more times per week, 1.0 to 40 mg per dose one or more times per week, 1.0 to 30 mg per dose one or more times per week, 1.0 to 30 mg per dose one or more times per week, or 0.1 to 25 mg per dose one or more times per week. A patient may also receive an ISI in the range of 0.1 to 1,200 mg per dose once every two or three weeks. A dose of the AFP and/or one or more secondary agent can be administered therapeutically to a patient one or more times per hour, day, week, month, or year (e.g., 2, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times per hour, day, week, month, or year). More commonly, a single dose per week of an AFP and/or a secondary agent is administered to a patient. In non-limiting embodiments of the methods of the present invention, an AFP and/or a secondary agent are administered to a patient: continuously for 1, 2, 3, or 4 hours; 1 , 2, 3, or 4 times a day; every other day or every third, fourth, fifth, or sixth day; 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times a week; biweekly; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 times a month; bimonthly; 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times every six months; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times a year; or biannually. The AFP (or biologically active fragment, derivative, or analog thereof) and the secondary agent may be administered at different frequencies during a therapeutic regime (i.e., administered at a higher frequency in the later stages of a disease (e.g., one or more of the diseases described herein) (e.g., administered once a week in the initial stages of the disease and administered three times a week a later stage of the disease) or administered at a higher frequency in the early stages of the disease (e.g., administered three times a week during the initial stages of the disease and administered once a week at a later stage of the disease)). In additional embodiments, the AFP and the secondary agent may be administered to a patient at the same frequency or at a different frequency.

The amount of the AFP and/or secondary agent required to achieve the desired therapeutic effect depends on a number of factors, such as the secondary agents chosen, the mode of administration, and clinical condition of the recipient. A skilled artisan will be able to determine the appropriate dosages of secondary agent and AFP (or biologically active fragment thereof) to achieve the desired results.

The administration of an AFP and a secondary agent according to the methods of this invention refers to the use of the two active ingredients in the same general time period or using the same general administration method. It is not always necessary, however, to administer both at the exact same time. For instance, if an AFP and an secondary agent are administered to a patient suffering from one or more of the diseases described herein in two separate pharmaceutical compositions, the two compositions need not be delivered to the patient during the same time period or even during two partially overlapping time periods. In some cases, the administration of the second agent (e.g., an AFP) may begin shortly after completion of the administration period for the first agent (e.g., a secondary agent), or vice versa. The time gap between the two administration periods may vary from one or more hours, days, weeks, or months. In some cases, one therapeutic agent (e.g., an AFP) may be administered first with the second (e.g., a secondary agent) in a separate time period, and subsequently administered without the second in a following period. A typical schedule of this type may require a higher dosage of the first therapeutic agent in the first, co-administration period, and a lower dosage in the second period, and vice versa. The same applies for the second agent.

Single or multiple administrations of the compositions of the present invention that include an effective amount of an AFP and/or a secondary agent can be carried out with the dose levels and the pattern being selected by the treating physician. The dose and administration schedule may be determined and adjusted based on the severity of the disease in a patient, the manner of administration, the age and body weight of the patient, and mass of the patient. The dose and administration may also be monitored and adjusted throughout the course of treatment according to the methods commonly practiced by clinicians or those described herein.

Treatment is started generally with the diagnosis or suspicion of one or more of the diseases described herein and is generally repeated on a daily basis. Protection or prevention from the development (or progression or exacerbation) of the disease is also achieved by administration of an AFP prior to the onset of the disease. If desired, the efficacy of the treatment or protection regimens is assessed with the methods of monitoring or diagnosing patients for the disease.

The methods of the invention can also be used to treat non-human mammals, for example, domestic pets or livestock.

Kits of the Invention

The invention also provides kits for treating uveitis. The kits typically include a pharmaceutical composition containing an AFP polypeptide (or a biologically active fragment, derivative, or analog thereof) that is formulated for ophthalmic administration and instructions for administering the AFP to a patient having uveitis. Additionally, in different embodiments, the kits may include one or more secondary agents (e.g., a steroid, a non-steroid anti-inflammatory drug (NSAID), a disease- modifying anti-rheumatic drug (DMARD), an integrin antagonist, an inflammation signaling inhibitor (ISI), a tolerizing agent, an immunosuppressive agent, and an immunomodulatory agent) and instructions for administering the one or more secondary agents to a patient having uveitis. In different embodiments, the AFP (or biologically active fragment, derivative, or analog thereof) and one or more secondary agents can be present in a single pharmaceutical composition or separate pharmaceutical compositions.

Preferably, the kits include pharmaceutical compositions (e.g., AFP alone or AFP with one or more secondary agents) formulated for ophthalmic administration (e.g., in an ophthalmic solution). Optionally, instruments or devices necessary for administering the pharmaceutical composition(s) may be included in the kits. For instance, a kit of this invention may provide one or more prefilled syringes or an eye dropper containing an effective amount of an AFP (or biologically active fragment, derivative, or analog thereof). Furthermore, the kits may also include additional components such as instructions or administration schedules for a patient suffering from uveitis to use the pharmaceutical composition(s) containing an AFP (or biologically active fragment, derivative, or analog thereof) and/or one or more secondary agents. The following examples are meant to illustrate the invention and should not be construed as limiting.

Examples

Example 1: Clinical Study of the Effect of AFP on Uveitis Pre-clinical data from rodents and non-human primates demonstrated that rHuAFP is well tolerated and is not associated with significant toxicity. A Phase I study of rHuAFP in healthy volunteers has been completed and demonstrates that rHuAFP is well-tolerated, with an adverse event profile that is similar to placebo. The pharmacokinetic profile of rHuAFP in healthy volunteers demonstrated dose- proportionality of serum levels and a half-life of approximately 5 days, and suggested an optimal target dose of 2-60 mg. In addition, a double-blind, placebo-controlled Phase IB trial in adult patients with active rheumatoid arthritis while on stable doses of methotrexate has been completed. Data from this study suggest that the adverse event profile is similar to that observed in the Phase I healthy volunteer study and that rHuAFP appears to have beneficial biological activity in patients with rheumatoid arthritis. A larger Phase II study to further assess the safety and efficacy of three doses of rHuAFP, compared to placebo, in adult patients with active rheumatoid arthritis despite treatment with stable doses of methotrexate has recently been completed. This study was a multi-center, double-blind, randomized, placebo-controlled study that tested 3 doses of rHuAFP (2.5 mg, 7.5 mg, and 20 mg) versus placebo. A total of 259 patients were enrolled at 39 centers in the United States. Recombinant human AFP was administered weekly by the patient as a subcutaneous injection in their abdominal area for a total of 6 months.

Consistent with previous studies, rHuAFP was safe and well-tolerated across all dosing groups. Adverse event relationship to rHuAFP was similar between all groups and there was no difference in the severity of adverse events between the groups.

Another Phase HA study to assess the safety, tolerability, and preliminary biologic activity of 20 mg of rHuAFP in adult patients with moderate to severe chronic plaque psoriasis has also been recently completed. Data analysis is ongoing for this study, but a preliminary review of the safety data suggests that rHuAFP is safe and well-tolerated.

Uveitis is an inflammatory disease of the uveal tract, which consists of the pigmented tissue of the eye, including the iris, ciliary body, and choroid. The cell- mediated inflammation is characterized by an infiltration of T lymphocytes and monocytes/macrophages. Uveitis produces eye pain, light sensitivity, and diminished vision, including blindness, all of which significantly impact the ability of patients to perform the routine activities of work, household, and social activities, and time away from the job because of the symptoms and because of the need for frequent appointments with the ophthalmologist for monitoring of the inflammation and its response to therapy, sufficient to interfere with the ability to work effectively. Uveitis is associated with a variety of etiologies, including various infections and autoimmune diseases, such as rheumatoid arthritis, autoimmune vasculitis, and sarcoidosis. Two forms of uveitis are of particular interest for this study: sarcoid uveitis and birdshot retinochoroidopathy. Sarcoid Uveitis

Sarcoidosis (also known as Hutchinson disease and Boeck disease) is a multisystem autoimmune disease in which T lymphocytes and macrophages predominate at inflammatory loci (granulomas), especially in the lung, skin, and peripheral lymph nodes. The disease is characterized by the presence of non- caseating granulomas that are similar to granulomas that develop in response to various infections and inflammatory agents such as mycobacteria, beryllium, and fungi. However, no infectious organisms or inflammatory agents have been recovered from the sarcoid granulomas, suggesting that sarcoidosis represents a granulatomatous response to autoantigens. The disease is generally self-limited and patients are frequently followed without specific therapy, although some patients require treatment with steroids. Sarcoidosis is a relatively common disorder, affecting all races and both sexes, from childhood to older age, in all geographic areas. The prevalence of sarcoidosis in the United States is approximately 10-40 per 100,000 individuals. Sarcoid uveitis has an extremely poor long-term outcome (loss of vision) with corticosteroid therapy alone. Approximately 25% of patients with sarcoidosis develop uveitis at some point. The uveitis may be the presenting complaint or may persist after the systemic features of sarcoidosis subside, such that many patients require long-term treatment with steroids and/or immunomodulatory therapy.

Birdshot Retinochoroidopathy (BSRC)

BSRC is a rare, local (ocular) autoimmune disease affecting the choroid and inner retina, with a T lymphocyte predominance in the pathogenesis, with bilateral retinal dysfunction and dependence on immunomodulatory therapy (e.g., a secondary agent) for prevention of blindness. Only about 200 cases have been reported worldwide, but the Massachusetts Eye Research and Surgery Institute (MERSI) is a referral resource for this rare disorder, with approximately 40 patients currently under treatment. BSRC affects almost exclusively Caucasians between the ages of 35 and 70 years of age, with 70% of them being female. It is the most highly HLA-linked of the known autoimmune disorders, with 96% of the patients with BSRC being HLA-A

29 positive. Unmet Medical Need

Systemic therapy, including prednisone, methotrexate (MTX), mycophenolate mofetil, cyclosporine, and even alklyating agents, such as cyclophosphamide or chlorambucil, are often effective in patients with moderate or severe disease. The limiting element of these therapies is the substantial incidence of acute and cumulative adverse effects, including liver toxicity (MTX), kidney toxicity (cyclosporine), hypertension (cyclosporine), and increased susceptibility to infection from generalized immunosuppression (mycophenolate motefil and the alklyating agents). Subjects taking systemic medications require careful and frequent monitoring by their physicians, with frequent hematologic monitoring.

Biological response modifiers, including human monoclonal antibodies and receptor decoys (including Amevive®, Enbrel®, Remicade®, Humira®, and Zenapax®), are a relatively new class of agents used to treat otherwise treatment- resistent uveitis. These agents selectively suppress key elements of T cell activation and mediators they generate that lead to the pathologic events that give rise to uveitis. However, the biologies' route of administration (injection or infusion), the high cost of treatments, and the fact that not all patients respond to a given agent are limiting their use. Despite the availability of these pharmacological interventions and their use in combination, many subjects continue to have active disease. Many others must discontinue therapy due to unacceptable adverse reactions to these agents. Hence, it is important to continue to search for novel therapeutic alternatives that are safe and effective for treatment of uveitis.

The safety and toxicology of rHuAFP has been evaluated in mice, rats, dogs, and non-human primates. In all of the studies, rHuAFP was well tolerated and there were no significant toxicological events. The acute and single dose studies that were performed were not confounded by the production of antibodies. However, in a 28- day multiple dose mouse study, the mice made antibodies to rHuAFP which resulted in low exposure to rHuAFP. The marmosets in a 26-week study also made antibodies to rHuAFP, but the antibodies were generally non-neutralizing and did not affect exposure.

A Phase I safety study with rHuAFP was performed in healthy volunteer subjects in the United Kingdom. This was a placebo-controlled, single ascending dose (SAD) and multiple (repeat) ascending dose (MAD) study to assess the safety, tolerability, pharmacokinetics, and pharmacodynamics (immune function) of rHuAFP when administered by subcutaneous injection. A total of 58 subjects were enrolled; 16 subjects received placebo and 42 received rHuAFP at single doses ranging from 1 mg to 84 mg or multiple injections of rHuAFP at doses of 4 or 21 mg. The results of this study demonstrated that rHuAFP is well tolerated at single doses ranging from 1 mg to 84 mg and at multiple (repeated) doses up to 21 mg, when administered once every 96 hours for 28 days (i.e., 7 doses). The adverse event profile was similar for the placebo and active drug cohorts and no dose-response trends were observed. The most commonly reported adverse events included pharyngolaryngeal pain, headache, flu-like illness, cough, abdominal pain, lethargy, injection site reactions, and procedural site reactions (pain and swelling at the site of DTH skin testing). One serious adverse event was reported of acute gastroenteritis requiring hospitalization for antibiotics and fluid replacement. The event was judged to be unrelated to rHuAFP and probably related to a viral illness or food exposure. One subject was discontinued from the study after four doses (every 96 hours) of 21 mg of rHuAFP, due to elevations of liver function tests. There were transient elevations in ALT (peak level = 171 IU/L) and AST (peak level = 74 IU/L), both of which returned to normal within 30 days of stopping study rHuAFP. The event was judged to be related to a recent viral infection and was unrelated to rHuAFP. The PK profile in the Phase I study demonstrated dose-independent (linear) kinetics with a terminal half-life of approximately 5 days. Repeat doses of 4 and 21 mg resulted in peak serum levels of approximately 700 and 4,000 ng/ml, respectively. None of the patients made an antibody response to rHuAFP. In addition, studies of immune function were performed at baseline and after completion of either single or multiple dose administration of blinded rHuAFP. There were no differences in results between the placebo and active rHuAFP on immune functions, including lymphocyte profiles by flow cytometry, T cell cytokine production, immunoglobulin levels, and cutaneous delayed type hypersensitivity (DTH) testing to Candida and tuberculin antigens. These data indicate that rHuAFP does not have significant negative impact on non-autoreactive immune cell activity. Dose, Route, and/or Schedule of Administration

Patients for treatment include those exhibiting uveitis (whether autoimmune or idiopathic). In particular, though, patients for treatment include those individuals with a history of relapsing sarcoid or BSRC uveitis for whom immunomodulatory monotherapy has been used to control the disease. Serum levels of about 0.3 - 0.5 μg/ml is the target concentration of rHuAFP in clinical studies. PK data from a Phase I study of rHuAFP administered as a single subcutaneous injection at doses ranging from 1 mg to 84 mg and as repeat doses once every 96 hours at doses of 4 and 21 mg to healthy adult volunteers demonstrated dose-proportionality of serum levels and a half-life of approximately 5 days, suggesting an optimal target dose in the range of 2- 60 mg.

Recombinant HuAFP was well tolerated at all doses studied in the Phase I study. A double-blind, placebo-controlled Phase IB study has been completed in adult patients with active RA while on stable doses of methotrexate. These patients received a once- weekly subcutaneous injection of 21 mg of rHuAFP or placebo for 12 weeks. Safety data indicates that the rHuAFP has an adverse event profile similar to that reported in the Phase I study. In addition, in non-human primate toxicology studies, the no adverse effect level (NOAEL) was 10 mg/kg, which is equivalent to a human dose of approximately 100 mg. Thus, a weekly dose of 60 mg can be administered to treat uveitis. Accordingly, rHuAFP formulated at 20 mg/ml would be administered to patients at 3 doses per week to achieve a 60 mg weekly dose. The treatment period can range from, e.g., 25-40 weeks. Treatment can include a monotherapy period (e.g., AFP only treatment) or a taper period, in which lower subsequent doses of a secondary agent are administered to the patient.

AFP Formulation rHuAFP is a non-glycosylated version of human alpha-fetoprotein produced by recombinant DNA technology. Twenty mg of rHuAFP can be supplied in a volume of 1.1 ml in a 2-ml, clear glass septum vial. To achieve a 60 mg dose, patients can inject 1 ml (20 mg) of rHuAFP 3 times/week (a total of 60 mg/week). In a clinical study, the matching placebo for rHuAFP can be phosphate buffered saline (PBS), pH 7.2. A volume of 1.1 ml can be supplied in the same 2-ml, clear glass septum vial used for rHuAFP. To maintain the blind, patients that are assigned to placebo can inject a matching volume (i.e. 1 ml) of placebo 3 times/week. Along with the rHuAFP, the patients can be supplied with an ancillary kit that contains materials required for self-injection (i.e., syringes, band-aids, sharps container, and alcohol swipes). The undispensed rHuAFP can be stored between 2 and 8 ⁰C with protection from light and humidity at the central dispensing site or clinical site until such time as it is given to the patient. No special procedures for the safe handling of rHuAFP are required. However, vigorous agitation of the vials prior to injection should be avoided to prevent foaming of the solution.

rHuAFP Administration

Patients can be trained by clinical staff to self-inject rHuAFP. To achieve a 60 mg weekly dose, patients can be required to inject 20 mg of rHuAFP 3 times/week. To maintain the blind, placebo patients can also be required to inject the same volume of placebo 3 times/week. Patients can be trained to withdraw 1 ml of rHuAFP from each vial with a syringe and inject subcutaneously in the abdominal area. If the patient's abdominal area is inappropriate for injection, alternatives sites such as the thigh or arm may be used at the discretion of the investigator. The injection site location must be recorded on the dosing log and should be constant for the patient throughout the study. Alternatively, ophthalmic administration of rHuAFP can be employed.

The rHuAFP can be administered by the patient at the clinical site, in the presence of the clinical staff at day 0, at 4 weeks, at 8 weeks, and from 12 weeks to 32 weeks. All other weekly injections of rHuAFP can be administered by the patient (or a qualified and trained caregiver) at their home. During the tapering schedule, patients can return to the clinic to receive their adjusted secondary agent dose, but they will still self-inject rHuAFP at home on their regular schedule.

There can be a total of 3 injections of rHuAFP/ week. Recombinant human AFP may be administered on the following weekly schedule: Day 1 : Dose #1 of weekly dose

Day 3: Dose #2 of weekly dose Day 6: Dose #3 of weekly dose For example, in the clinical trial, if a patient's first dose is on a Monday, then their next two doses may be on Wednesday and Saturday for week #1. The first dose for week #2 may be on the following Monday. Patients should adhere to the same weekly schedule throughout the study. In the example above, the patient would therefore dose on Mondays, Wednesdays and Saturdays of each week throughout the remainder of the study.

Injections can be administered by a spouse or caregiver, but they must be present at the clinic during the day 1 visit for injection training, and should be present for each clinic visit so the clinic staff can observe their injection technique. At screening, patients can also be given the option to have rHuAFP administered by a nurse at the clinic. If patients choose this option, they must agree to return to the clinic 3 times/week to receive their dose.

Taper or Washout of Secondary Agents Patients can enter the study on stable doses of secondary agents (e.g., an oral corticosteroid, a DMARD (such as methotrexate), an immunosuppressive agent (e.g., cyclosporine), etc.) that is administered to control their uveitis. Depending on the current therapy used to treat uveitis (e.g., the secondary agent administering prior to treatment with rHuAFP), the background uveitis treatment can be tapered off or washed out. In both cases, patients can be treated with rHuAFP for a total of 36 weeks. The first 8 weeks of rHuAFP treatment is considered the stabilization period. As such, patients that flare during this period will not be considered non-evaluable with respect to efficacy endpoints.

After the patient has been stabilized on rHuAFP for a total of 8 doses, the secondary agent taper can begin. During the rHuAFP stabilization period, patients must not miss more than 2 consecutive doses of rHuAFP before initiating the secondary agent taper. In addition, patients must not miss a dose of rHuAFP in the week prior to the initiation of the secondary agent taper. If this occurs, the patient should extend the stabilization period for one week before initiating the secondary agent taper. The patient can return to the clinic on a weekly basis throughout the secondary agent taper to receive their secondary agent therapy to ensure that the taper is properly calculated and the correct dose is administered. Some therapies that are used to treat uveitis can require a washout period prior to first dose with rHuAFP. For the wash out, patients can stop dosing and allow the designated period of time to expire prior to receiving the first dose of rHuAFP.

Measurements of Efficacy

The following efficacy assessments will be performed to assess the patient's response to therapy: visual acuity; slit lamp examination to grade the degree of inflammation (uveitis); Visual Function Quality of Life Index; repeated fluorescein angiography, electroretinography, and cytokine analysis. The Visual Function Quality of Life Index is a questionnaire consists of 10- items that assess the patient's quality of life during the last 7 days. Each question has four possible responses ranging in score from 0-3: not at all (0); a little (1); a lot or (2); and very much (3). The total score represents the sum of the 10 items, from 0-30 points.

Other Embodiments

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth.

What is claimed is:

Claims

1. A method of treating a mammal having a disease selected from the group consisting of psoriatic arthritis, IgA nephropathy, Hashimoto's disease, Sjogren's syndrome, ankylosing spondylitis, and uveitis, comprising administering to said mammal a therapeutically effective amount of alpha-fetoprotein (AFP) or a biologically active fragment, derivative, or analog thereof.

2. The method of claim 1, wherein said disease is uveitis.

3. The method of claim 2, wherein said uveitis is an autoimmune uveitis.

4. The method of claim 3, wherein said autoimmune uveitis is birdshot retinochoroidopathy uveitis, sarcoid uveitis, or HLA-B27 uveitis.

5. The method of claim 2, wherein said uveitis is an idiopathic uveitis.

6. The method of claim 5, wherein said idiopathic uveitis is Behcet's disease or Vogt-Koyanagi-Harada (VKH) syndrome.

7. The method of claim 1, wherein said AFP or biologically active fragment, derivative, or analog thereof is human recombinant AFP.

8. The method of claim 1, wherein said AFP or biologically active fragment, derivative, or analog thereof is non-glycosylated.

9. The method of claim 1, wherein said AFP or biologically active fragment, derivative, or analog thereof is glycosylated.

10. The method of claim 1, wherein said biologically active fragment is selected from Domain I (SEQ ID NO: 5), Domain II (SEQ ID NO: 6), Domain III (SEQ ID NO: 7), Domain I + II (SEQ ID NO: 8), Domain II + III (SEQ ID NO: 9), or Fragment I (SEQ ID NO: 10).

11. The method of claim 1 , wherein said mammal is human.

12. The method of claim 1, wherein said AFP or biologically active fragment, derivative, or analog thereof is administered daily, weekly, biweekly, or monthly.

13. The method of claim 12, wherein said AFP or biologically active fragment, derivative, or analog thereof is administered daily.

14. The method of claim 1, wherein said AFP or biologically active fragment, derivative, or analog thereof is administered in the range of about 0.1 mg to 400 mg per dose.

15. The method of claim 1, wherein said administering results in a reduction in the severity of one or more symptoms of the disease.

16. The method of claim 15, wherein said one or more symptoms of the disease are selected from the group consisting of stiffness, pain, swelling, tenderness of the joints and surrounding soft tissue, fatigue, nail pitting, depression, sensitivity to cold, weight increase or weight decrease, muscle weakness, coarsening of the skin, dry or brittle hair, constipation, muscle cramps, increased menstral flow, goiter, dry eyes, eye irritation, eye burning, eyelids sticking together, dry mouth, hoarse or weak voice, difficulty swallowing food, painful salivary glands, back pain and stiffness, fever, night sweats, light sensitivity, blurring or loss of vision, eye pain, and redness of eye.

17. The method of claim 1, further comprising administering one or more of a secondary agent selected from the group consisting of a steroid, a non-steroid anti- inflammatory drug (NSAID), a disease-modifying anti-rheumatic drug (DMARD), an integrin antagonist, an inflammation signaling inhibitor (ISI), a tolerizing agent, an immunosuppressive agent, and an immunomodulatory agent.

18. The method of claim 17, wherein said AFP is administered prior to said one or more secondary agent.

19. The method of claim 17, wherein said AFP is administered after said one or more secondary agent.

20. The method of claim 1 or 17, wherein said AFP or said one or more secondary agent is administered intravenously, intramuscularly, orally, by inhalation, parenterally, intraperitoneally, intraarterially, transdermally, sublingually, nasally, in a suppository, transbuccally, liposomally, adiposally, intraocularly, subcutaneously, intrathecally, topically, locally, or ophthalmically.

21. The method of claim 20, wherein said AFP or said one or more secondary agent is administered topically or through ophthalmic administration.

22. The method of claim 17, wherein said AFP and said one or more secondary agent are administered by two different routes of administration.

23. The method of claim 17, wherein said AFP and said one or more secondary agent are administered by the same route of administration.

24. The method of claim 17, wherein said secondary agent is administered in an effective dose which is below the standard dose when the secondary agent is administered alone.

25. A composition for treating a mammal having uveitis comprising a therapeutically effect amount of an alpha- fetoprotein (AFP) or a biologically active fragment, derivative, or analog thereof, wherein said composition is formulated for ophthalmic administration.

26. The composition of claim 25, wherein said AFP or biologically active fragment, derivative, or analog thereof is in an ophthalmic solution.

27. The composition of claim 25, wherein said AFP or biologically active fragment, derivative, or analog thereof is human recombinant AFP.

28. The composition of claim 25, wherein said AFP or biologically active fragment, derivative, or analog thereof is non-glycosylated.

29. The composition of claim 25, wherein said AFP or biologically active fragment, derivative, or analog thereof is glycosylated.

30. The composition of claim 25, wherein said biologically active fragment is selected from Domain I (SEQ ID NO: 5), Domain II (SEQ ID NO: 6), Domain III (SEQ ID NO: 7), Domain I + II (SEQ ID NO: 8), Domain II + III (SEQ ID NO: 9), or Fragment I (SEQ ID NO: 10).

31. The composition of claim 25, further comprising one or more of a secondary agent selected from the group consisting of a steroid, a non-steroid antiinflammatory drug (NSAID), a disease-modifying anti-rheumatic drug (DMARD), an integrin antagonist, an inflammation signaling inhibitor (ISI), a tolerizing agent, an immunosuppressive agent, and an immunomodulatory agent.

32. A kit comprising:

(i) a therapeutically effective amount of an alpha-fetoprotein (AFP) or a biologically active fragment or analog thereof formulated for ophthalmic administration; and

(ii) instructions for administering said AFP or biologically active fragment or analog thereof to a patient having uveitis.

33. The kit of claim 32, wherein said uveitis is an autoimmune uveitis.

34. The kit of claim 33, wherein said autoimmune uveitis is birdshot retinochoroidopathy uveitis or sarcoid uveitis.

35. The kit of claim 32, wherein said uveitis is an idiopathic uveitis.

36. The kit of claim 35, wherein said idiopathic uveitis is Behcet's disease or Vogt-Koyanagi-Harada (VKH) syndrome.

37. The kit of claim 32, wherein said AFP or biologically active fragment, derivative, or analog thereof is human recombinant AFP.

38. The kit of claim 32, wherein said AFP or biologically active fragment, derivative, or analog thereof is non-glycosylated.

39. The kit of claim 32, wherein said AFP or biologically active fragment, derivative, or analog thereof is glycosylated.

40. The kit of claim 32, wherein said biologically active fragment is selected from Domain I (SEQ ID NO: 5), Domain II (SEQ ID NO: 6), Domain III (SEQ ID NO: 7), Domain I + II (SEQ ID NO: 8), Domain II + III (SEQ ID NO: 9), or Fragment I (SEQ ID NO: 10).

41. The kit of claim 32, further comprising one or more of a secondary agent selected from the group selected from the group consisting of a steroid, a non-steroid anti-inflammatory drug (NSAID), a disease-modifying anti-rheumatic drug (DMARD), an integrin antagonist, a inflammation signaling inhibitor (ISI), tolerizing agent, immunosuppressive agent, and an immunomodulatory agent, and instructions for administering said one or more secondary agent to a patient having uveitis

42. The kit of claim 41, wherein said secondary agent is formulated for ophthalmic administration.

43. The kit of claim 32 or 42, wherein said AFP or biologically active fragment, derivative, or analog thereof, or said secondary agent is in an ophthalmic solution.