Methods of Using IL-1 Antagonists to Treat Autoinflammatory Disease

Abstract

Methods of treating, inhibiting, or ameliorating an autoinflammatory disorder, disease, or condition in a subject in need thereof, comprising administering to a subject in need a therapeutic amount of an interleukin 1 (IL-1) antagonist, wherein the autoinflammatory disorder, disease, or condition is treated, inhibited, or ameliorated. The IL-1 antagonist is an IL-1 trap, preferably comprising a sequence selected from the group consisting of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or a substantially identical having at least 95% identity to the sequence shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and capable of binding and inhibiting IL-1. The therapeutic methods are useful for treating a human adult or child suffering from Neonatal Onset Multisystem Inflammatory Disorder (NOMID/CINCA), Muckle-Wells Syndrome (MWS), Familial Cold Autoinflammatory Syndrome (FCAS), familial mediterranean fever (FMF), or systemic onset juvenile rheumatoid arthritis (Still's Disease).

Description

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 11/144,987, filed 3 Jun. 2005, now U.S. Pat. No. 0,000,000, which claims the benefit under 35 USC 119(e) of U.S. Ser. No. 60/577,023 filed 4 Jun. 2004, which applications are incorporated by reference in their entirety.

BACKGROUND

1. Field of the Invention

The invention relates to methods of using interleukin-1 (IL-1) antagonists to treat autoinflammatory diseases, including familial mediterranean fever (FMF), cryopyrin mutation-associated disorders,

2. Description of Related Art

One important group of autoinflammatory disorders encompasses autosomal dominant conditions associated with mutations in CIAS-1, a gene that encodes a pyrin-related protein called “cryopyrin” (Feldmann et al. (2002) Am. J. Hum. Genet. 71:198-203; Hoffman et al. (2001) Nat. Genet. 29:301-305). These disorders include Neonatal Onset Multisystem Inflammatory Disorder (NOMID/CINCA), Muckle-Wells Syndrome (MWS), and Familial Cold Autoinflammatory Syndrome (FCAS). These disorders present as a spectrum of clinical manifestations ranging from FCAS being the mildest to the seriously disabling disease of NOMID/CINCA. An urticaria like skin rash is common to the entire spectrum of CMSI associated diseases. In patients with FCAS, this rash is inducible by cold exposure while most patients with MWS or NOMID present with daily rashes that are consistently provoked by a number of different stimuli. Conjunctivitis is present in all forms of disease expression, however, hearing loss, aseptic meningitis and arthritis are mainly seen in patients with MWS and NOMID/CINCA. The disfiguring and disabling body overgrowth at the epiphyses and patellae is only seen in patients with NOMID/CINCA.

FMF is a recessively inherited condition characterized by episodes of fever and serositis or synovitis; some subjects also develop systemic amyloidosis (Balow et al. (1997) Genomics 44:280-291). The FMF gene encodes a novel protein called pyrin that is the prototype of a family of molecules involved in the regulation of apoptosis (cell-death) and inflammation. The precise biochemical mechanism by which these proteins function, and by which mutations cause disease, is still unknown.

Still's Disease (systemic onset juvenile rheumatoid arthritis), is manifest by spiking fevers, evanescent salmon color rash, arthritis, arthralgia, and hepatosplenomegaly (Masson et al. (1995) Rev. Rhum. Engl. Ed. 62:748-757; Spiegel et al. (2000) Arthritis Rheum. 43:2402-2409). There are as yet no definitive genetic associations with Still's Disease and the pathogenesis is poorly understood. Interestingly, many of the signs and symptoms of Still's disease are similar to those with autoinflammatory disease. Still's Disease typically first occurs during childhood, but can also have its onset in adulthood.

Similarly, Kawasaki disease is disease affecting children that is accompanied by fevers, swelling and arthritic joints, and rash, as well as vascular inflammation that can cause permanent coronary damage in approximately 15-25% of affected children. The etiology of this disease is very similar to autoinflammatory disease.

The pathogenesis of autoinflammatory disease is not completely understood. There is a growing body of evidence that interleukin-1 (IL-1) plays a role in a number of these conditions and that targeting of this cytokine can provide important benefits (Hoffman et al. (2004) Arthritis. Rheum. 50:345-349). There is clearly a need to develop improved therapeutic treatment of these autoinflammatory diseases

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the invention features a method of treating, inhibiting, or ameliorating an autoinflammatory disorder, comprising administering to a subject in need an interleukin 1 (IL-1) antagonist. An IL-1 antagonist is a compound capable of blocking or inhibiting the biological action of IL-1, including fusion proteins capable of trapping IL-1, such as an IL-1 trap. In a preferred embodiment, the IL-1 trap is an IL-1-specific fusion protein comprising two IL-1 receptor components and a multimerizing component, for example, an IL-1 trap described in U.S. patent publication No. 2003/0143697, published 31 Jul. 2003, herein specifically incorporated by reference in its entirety. In a specific embodiment, the IL-1 trap is the fusion protein shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26. A preferred IL-1 trap is shown in SEQ ID NO:10. The invention encompasses the use of an IL-1 trap substantially identical to the protein of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, that is, a protein having at least 95% identity, at least 97% identity, at least 98% identity to the protein of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and capable of binding and inhibiting IL-1. Further, in specific embodiments, the IL-1 antagonist is a modified IL-1 trap comprising one or more receptor components and one or more immunoglobulin-derived components specific for IL-1 and/or an IL-1 receptor. In another embodiment, the IL-1 antagonist is a modified IL-1 trap comprising one or more immunoglobulin-derived components specific for IL-1 and/or an IL-1 receptor.

The subject being treated is most preferably a human diagnosed as suffering from an autoinflammatory disorder. More specifically, the subject is a human adult or child diagnosed with an autoinflammatory disorder associated with mutations in CIAS-1, such as Neonatal Onset Multisystem Inflammatory Disorder (NOMID/CINCA), Muckle-Wells Syndrome (MWS), and Familial Cold Autoinflammatory Syndrome (FCAS); familial mediterranean fever (FMF); or systemic onset juvenile rheumatoid arthritis (Still's Disease) or Kawasaki Disease.

The method of the invention includes administration of the IL-1 antagonist by any means known to the art, for example, subcutaneous, intramuscular, intranasal, intraarterial, intravenous, topical, transvaginal, transdermal, transanal administration or oral routes of administration.

In a second aspect, the invention features a method of treating, inhibiting, or ameliorating Neonatal Onset Multisystem Inflammatory Disorder (NOMID/CINCA), Muckle-Wells Syndrome (MWS), and Familial Cold Autoinflammatory Syndrome (FCAS); familial Mediterranean fever (FMF); or systemic onset juvenile rheumatoid arthritis (Still's Disease), the method comprising administering to a subject in need an interleukin 1 (IL-1) antagonist. In a preferred embodiment, the IL-1 antagonist is a fusion protein capable of trapping IL-1 (IL-1 trap). In a specific embodiment, the IL-1 trap is the fusion protein shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or a substantially identical protein capable of binding and inhibiting IL-1. A preferred IL-1 trap is shown in SEQ ID NO:10. Preferably, the subject treated is a child or adult human diagnosed with a disease or condition selected from the group consisting of NOMID/CINCA, MWS, FCAS, FMF, Still's Disease and Kawasaki Disease.

In a third aspect, the invention features a method of treating, inhibiting, or ameliorating Neonatal Onset Multisystem Inflammatory Disorder (NOMID/CINCA), comprising administering to a subject in need an interleukin 1 (IL-1) antagonist. In a preferred embodiment, the IL-1 antagonist is a fusion protein capable of trapping IL-1 (IL-1 trap). In a specific embodiment, the IL-1 trap is the fusion protein shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or a substantially identical protein capable of binding and inhibiting IL-1. A preferred IL-1 trap is shown in SEQ ID NO:10.

In a fourth aspect, the invention features a method of treating, inhibiting, or ameliorating Muckle-Wells Syndrome (MWS), the method comprising administering to a subject in need an interleukin 1 (IL-1) antagonist. In a preferred embodiment, the IL-1 antagonist is a fusion protein capable of trapping IL-1 (IL-1 trap). In a specific embodiment, the IL-1 trap is the fusion protein shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or a substantially identical protein capable of binding and inhibiting IL-1. A preferred IL-1 trap is shown in SEQ ID NO:10.

In a fifth aspect, the invention features a method of treating, inhibiting, or ameliorating Familial Cold Autoinflammatory Syndrome (FCAS) the method comprising administering to a subject in need an interleukin 1 (IL-1) antagonist. In a preferred embodiment, the IL-1 antagonist is a fusion protein capable of trapping IL-1 (IL-1 trap). In a specific embodiment, the IL-1 trap is the fusion protein shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or a substantially identical protein capable of binding and inhibiting IL-1. A preferred IL-1 trap is shown in SEQ ID NO:10.

In a sixth aspect, the invention features a method of treating, inhibiting, or ameliorating familial mediterranean fever (FMF), the method comprising administering to a subject in need an interleukin 1 (IL-1) antagonist. In a preferred embodiment, the IL-1 antagonist is a fusion protein capable of trapping IL-1 (IL-1 trap). In a specific embodiment, the IL-1 trap is the fusion protein shown in SEQ ID NO:4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or a substantially identical protein capable of binding and inhibiting IL-1. A preferred IL-1 trap is shown in SEQ ID NO:10.

In a seventh aspect, the invention features a method of treating, inhibiting, or ameliorating systemic onset juvenile rheumatoid arthritis (Still's Disease), the method comprising administering to a subject in need an interleukin 1 (IL-1) antagonist. In a preferred embodiment, the IL-1 antagonist is a fusion protein capable of trapping IL-1 (IL-1 trap). In a specific embodiment, the IL-1 trap is the fusion protein shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or a substantially identical protein capable of binding and inhibiting IL-1. A preferred IL-1 trap is shown in SEQ ID NO:10.

In specific embodiments of the therapeutic method of the invention, the subject is treated with a combination of an IL-1 trap and a second therapeutic agent. The second therapeutic agent may be a second IL-1 antagonist, such as, for example, anakinra (Kineret®, Amgen), a recombinant, nonglycosylated form of the human IL-1 receptor antagonist (IL1Ra), or an anti-IL-18 drug such as IL-18BP or a derivative, an IL-18 Trap, anti-IL-18, anti-IL-18R1, or anti-IL-18Racp. Other co-therapies include low dose colchine for FMF, aspirin or other NSAIDs, steroids such as prednisolone, methotrexate, low dose cyclosporine A, TNF inhibitors such as Enbrel®, or Humira®, other inflammatory inhibitors such as inhibitors of caspase-1, p38, IKK1/2, CTLA-4Ig, anti-IL-6 or anti-IL6Ra, etc.

In an eighth aspect, the invention features a therapeutic method of treating an autoinflammatory disease or condition, comprising administering a pharmaceutical composition comprising an IL-1 trap and a pharmaceutically acceptable carrier, in a dose range of 50-100 mg/kg on a weekly basis for a treatment period of between 1 week to one year or more.

Other objects and advantages will become apparent from a review of the ensuing detailed description.

DETAILED DESCRIPTION

Before the present methods are described, it is to be understood that this invention is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only the appended claims.

As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus for example, a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety.

General Description

In vitro experiments have demonstrated that cryopyrin can up-regulate IL-1 production through a pathway involving PYRIN domain interactions between cryopyrin and, ASC, and then homotypic caspase recruitment domain interactions between ASC and caspase-1 (interleukin 1 converting enzyme). IL-1 protein levels was found to be constitutively elevated on Western blot analysis from monocyte/macrophage lysates from two subjects with NOMID/CINCA (Aksentiejevich et al. (2002) Arthritis. Rheum. 46:3340-3348).

IL-1 is synthesized in pro-form and is activated via cleavage by the enzyme caspase-1. Caspase-1 can be activated by a protein called ASC, a protein usually under negative regulation by pyrin, the gene mutated in FMF. These mutations result in the inability of pyrin to inhibit ASC and thereby lead to the activation of caspase-1 and, secondarily, IL-1 (Srinivasula et al. (2002) J. Biol. Chem. 277:21119-21122). Studies of mice with targeted disruption of the pyrin gene show evidence of caspase 1 activation and increased IL-1 release. Studies of subjects with FMF have shown elevated levels of IL-1 gene expression. Colchicine has been found to often be effective in preventing FMF disease exacerbations (Dinarello et al. (1974) N. Engl. J. Med. 291:934-937); however, approximately 10% of subjects with FMF are refractory to treatment with this agent. Furthermore, persistent IL-1 over-expression and elevated levels of acute phase reactants have been observed in patients with FMF between flares, even when treated with colchicine (Notarnicola et. al. (2002) Genes Immun. 3:43-45).

IL-1 is generally recognized as an important regulator of inflammatory processes and yet there is currently only targeted therapy available that specifically works via inhibition of this cytokine, anakinra (Kineret®, Amgen), a recombinant, nonglycosylated form of the human IL-1 receptor antagonist, IL-1Ra. This agent has a relatively short half-life and must be administered as a once-daily subcutaneous injection. While generally well tolerated, some subjects exhibit significant injection site reactions. The combination of once-daily administration and injection site reactions can be undesirable for many subjects. There is need for additional therapies with improved pharmacological characteristics that are targeted against IL-1.

DEFINITIONS

By the term “blocker”, “inhibitor”, or “antagonist” is meant a substance that retards or prevents a chemical or physiological reaction or response. Common blockers or inhibitors include but are not limited to antisense molecules, antibodies, antagonists and their derivatives. More specifically, an example of an IL-1 blocker or inhibitor is an IL-1 antagonist including, but not limited to, IL-1 trap, which binds and inhibits IL-1.

By the term “therapeutically effective dose” is meant a dose that produces the desired effect for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

By the term “substantially identical” is meant a protein sequence having at least 95% identity to an amino acid sequence selected from the group consisting of the amino acid sequences SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, and capable of binding IL-1 and inhibiting the biological activity of IL-1.

The term “identity” or “homology” is construed to mean the percentage of amino acid residues in the candidate sequence that are identical with the residue of a corresponding sequence to which it is compared, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent identity for the entire sequence, and not considering any conservative substitutions as part of the sequence identity. Neither N- or C-terminal extensions nor insertions will be construed as reducing identity or homology. Methods and computer programs for the alignment are well known in the art. Sequence identity may be measured using sequence analysis software (e.g., Sequence Analysis Software Package, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Ave., Madison, Wis. 53705). This software matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.

IL-1 Trap Antagonists

Interleukin-1 (IL-1) traps are multimers of fusion proteins containing IL-1 receptor components and a multimerizing component capable of interacting with the multimerizing component present in another fusion protein to form a higher order structure, such as a dimer. Cytokine traps are a novel extension of the receptor-Fc fusion concept in that they include two distinct receptor components that bind a single cytokine, resulting in the generation of antagonists with dramatically increased affinity over that offered by single component reagents. In fact, the cytokine traps that are described herein are among the most potent cytokine blockers ever described. Briefly, the cytokine traps called IL-1 traps are comprised of the extracellular domain of human IL-1R Type I (IL-1RI) or Type II (IL-1RII) followed by the extracellular domain of human IL-1 Accessory protein (IL-1AcP), followed by a multimerizing component. In a preferred embodiment, the multimerizing component is an immunoglobulin-derived domain, such as, for example, the Fc region of human IgG, including part of the hinge region, the CH2 and CH3 domains. An immunoglobulin-derived domain may be selected from any of the major classes of immunoglobulins, including IgA, IgD, IgE, IgG and IgM, and any subclass or isotype, e.g. IgG1, IgG2, IgG3 and IgG4; IgA-1 and IgA-2. Alternatively, the IL-1 traps are comprised of the extracellular domain of human IL-1AcP, followed by the extracellular domain of human IL-1RI or IL-1RII, followed by a multimerizing component. For a more detailed description of the IL-1 traps, see WO 00/18932, which publication is herein specifically incorporated by reference in its entirety. Preferred IL-1 traps have the amino acid sequence shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, or a substantially identical protein at least 95% identity to a sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26, and capable of binding and inhibiting IL1.

In specific embodiments, the IL-1 antagonist comprises an antibody fragment capable of binding IL-1α, IL-1β, IL-1R1 and/or IL-1 RAcp, or a fragment thereof. The preferred embodiment would an antagonist of IL-1β. One embodiment of an IL-1 antagonist comprising one or more antibody fragments, for example, single chain Fv (scFv), is described in U.S. Pat. No. 6,472,179, which publication is herein specifically incorporated by reference in its entirety. In all of the IL-1 antagonist embodiments comprising one or more antibody-derived components specific for IL-1 or an IL-1 receptor, the components may be arranged in a variety of configurations, e.g., a IL-1 receptor component(s)-scFv(s)-multimerizing component; IL-1 receptor component(s)-multimerizing component-scFv(s); scFv(s)-IL-1 receptor component(s)-multimerizing component, ScFv-ScFv-Fc, etc., so long as the molecule or multimer is capable of inhibiting the biological activity of IL-1

Anti-IL-1 Human Antibodies and Antibody Fragments

In another embodiment of the IL-1 antagonist useful in the method of the invention, examples of anti-IL-1 antibodies are disclosed in U.S. Pat. No. 4,935,343; U.S. Pat. No. 5,681,933; WO 95/01997; EP 0267611, U.S. Pat. No. 6,419,944; WO 02/16436 and WO 01/53353. The IL-1 antagonist of the invention may include an antibody or antibody fragment specific for an IL-1 ligand (e.g., IL-1α or IL-1β) and/or an IL-1 receptor (e.g., IL-1R1 and/or IL-1RAcp). Antibody fragments include any fragment having the required target specificity, e.g. antibody fragments either produced by the modification of whole antibodies (e.g. enzymatic digestion), or those synthesized de novo using recombinant DNA methodologies (scFv, single domain antibodies or dabs, single variable domain antibodies) or those identified using human phase display libraries (see, for example, McCafferty et al. (1990) Nature 348:552-554). Alternatively, antibodies can be isolated from mice producing human or human-mouse chimeric antibodies using standard immunization and antibody isolation methods, including but not limited to making hybridomas, or using B cell screening technologies, such as SLAM. Immunoglobulin binding domains also include, but are not limited to, the variable regions of the heavy (V_H) or the light (V_L) chains of immunoglobulins. Or by immunizing people and isolating antigen positive B cells and cloning the cDNAs encoding the heavy and light chain and coexpressing them in a cell, such as CHO.

The term “antibody” as used herein refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant regions, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. Within each IgG class, there are different isotypes (eg. IgG₁, IgG₂, IgG₃, IgG₄). Typically, the antigen-binding region of an antibody will be the most critical in determining specificity and affinity of binding.

An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one light chain (about 25 kD) and one heavy chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The terms “variable light chain” (V_L) and variable heavy chain (V_H) refer to these light and heavy chains respectively.

Antibodies exist as intact immunoglobulins, or as a number of well-characterized fragments produced by digestion with various peptidases. For example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)′₂, a dimer of Fab which itself is a light chain joined to V_H-C_H1 by a disulfide bond. The F(ab)′₂ may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)′₂ dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab with part of the hinge region. While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology.

Methods for preparing antibodies are known to the art. See, for example, Kohler & Milstein (1975) Nature 256:495-497; Harlow & Lane (1988) Antibodies: a Laboratory Manual, Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.). The genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell, e.g., the genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody. Monoclonal antibodies can be humanized using standard cloning of the CDR regions into a human scaffold. Gene libraries encoding human heavy and light chains of monoclonal antibodies can also be made from hybridoma or plasma cells. Random combinations of the heavy and light chain gene products generate a large pool of antibodies with different antigenic specificity. Techniques for the production of single chain antibodies or recombinant antibodies (U.S. Pat. No. 4,946,778; U.S. Pat. No. 4,816,567) can be adapted to produce antibodies used in the fusion proteins and methods of the instant invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express human, human-mouse chimeric, or humanized antibodies. Alternatively, phage display technology can be used to identify human antibodies and heteromeric Fab fragments that specifically bind to selected antigens.

Antibody Screening and Selection

Screening and selection of preferred antibodies can be conducted by a variety of methods known to the art. Initial screening for the presence of monoclonal antibodies specific to a target antigen may be conducted through the use of ELISA-based methods, for example. A secondary screen is preferably conducted to identify and select a desired monoclonal antibody for use in construction of the multi-specific fusion proteins of the invention. Secondary screening may be conducted with any suitable method known to the art. One preferred method, termed “Biosensor Modification-Assisted Profiling” (“BiaMAP”) is described in co-pending U.S. Ser. No. 60/423,017 filed 1 Nov. 2002, herein specifically incorporated by reference in its entirety. BiaMAP allows rapid identification of hybridoma clones producing monoclonal antibodies with desired characteristics. More specifically, monoclonal antibodies are sorted into distinct epitope-related groups based on evaluation of antibody:antigen interactions. Antibodies capable of blocking either a ligand or a receptor may be identified by a cell based assay, such as a luciferase assay utilizing a luciferase gene under the control of an NFKB driven promoter. Stimulation of the IL-1 receptors by IL-1 ligands leads to a signal through NFKB thus increasing luciferase levels in the cell. Blocking antibodies are identified as those antibodies that blocked IL-1 induction of luciferase activity.

Treatment Population

The therapeutic methods of the invention are useful for treating individuals affected with CIAS-1 mutation disorders (NOMID, MWS, FCAS), FMF, or Still's Disease. Commonly accepted diagnostic criteria for CIAS-1 mutation associated disease (NOMID, MWS, FCAS), Familial Mediterranean Fever, or Still's Disease (adult- or juvenile-onset) are know to those skilled in the art. In the case of patients diagnosed with FMF, the therapeutic method of the invention may be particularly useful for those with disease refractory to therapy with colchicine.

Methods of Administration

The invention provides methods of treatment comprising administering to a subject an effective amount of an agent of the invention. In a preferred aspect, the agent is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects).

Various delivery systems are known and can be used to administer an agent of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction can be enteral or parenteral and include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved, for example, and not by way of limitation, by local infusion during surgery, topical application, e.g., by injection, by means of a catheter, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, fibers, commercial skin substitutes or angioplasty balloons or stents.

In another embodiment, the active agent can be delivered in a vesicle, in particular a liposome (see Langer (1990) Science 249:1527-1533). In yet another embodiment, the active agent can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer (1990) supra). In another embodiment, polymeric materials can be used (see Howard et al. (1989) J. Neurosurg. 71:105). In another embodiment where the active agent of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see, for example, U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.

Combination Therapies

In numerous embodiments, the IL-1 antagonists of the present invention may be administered in combination with one or more additional compounds or therapies. Combination therapy may be simultaneous or sequential. The IL-1 traps of the invention may be combined with, for example, TNF-inhibiting agents such as etanercept (Enbrel®, Amgen), infliximab (Remicade®, Centocor), Humira® (Abbott), thalidomide, steroids, anakinra (Kinaret®, Amgen), or colchicine. Colchicine is a mainstay of therapy for subjects with FMF; in this study, subjects will not be removed from treatment with this medication. For Still's Disease (and classical autoinflammatory diseases), compounds such as methotrexate, cyclosporine, chlorambucil, cyclophosphamide (DMARDs) have been used as monotherapy or in combination with no consistent response. Some subjects respond to high doses of steroids. DMARDs, and more recently anti-TNF agents have been used with variable success. The IL-1 traps of the invention may also be combined with anti-IL-18 drugs, such as for example, IL-18BP or a derivative, an IL-18 Trap, anti-IL-18, anti-IL-18R1, or anti-IL-18Racp. Other co-therapies include low dose colchine for FMF, aspirin or other NSAIDs, steroids such as prednisolone, methotrexate, low dose cyclosporine A, TNF inhibitors such as Enbrel®, or Humira®, other inflammatory inhibitors such as inhibitors of caspase-1, p38, IKK1/2, CTLA-4Ig, anti-IL-6 or anti-IL6Ra, etc.

Pharmaceutical Compositions

The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of an active agent, and a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.

In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The active agents of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

The amount of the active agent of the invention which will be effective in the treatment of delayed-type hypersensitivity can be determined by standard clinical techniques based on the present description. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the condition, and should be decided according to the judgment of the practitioner and each subject's circumstances. However, suitable dosage ranges for intravenous administration are generally about 20 micrograms to 2 grams of active compound per kilogram body weight. Suitable dosage ranges for intra-nasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

For systemic administration, a therapeutically effective dose can be estimated initially from in vitro assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC₅₀ as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data.

Dosage amount and interval may be adjusted individually to provide plasma levels of the compounds that are sufficient to maintain therapeutic effect. In cases of local administration or selective uptake, the effective local concentration of the compounds may not be related to plasma concentration. One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation.

The amount of compound administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration, and the judgment of the prescribing physician. The therapy may be repeated intermittently while symptoms are detectable or even when they are not detectable. The therapy may be provided alone or in combination with other drugs.

Kits

The invention also provides an article of manufacturing comprising packaging material and a pharmaceutical agent contained within the packaging material, wherein the pharmaceutical agent comprises at least one IL-1-specific fusion protein of the invention and wherein the packaging material comprises a label or package insert which indicates that the IL-1-specific fusion protein can be used for treating an autoinflammatory disease or condition.

Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments which are given for illustration of the invention and are not intended to be limiting thereof.

EXAMPLES

The following example is put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Example 1

Effect of IL-1 Trap on Human Autoinflammatory Disease

An initial study is conducted with 10 adult subjects suffering from autoinflammatory disease or Still's Disease. Subjects are screened for eligibility, clinical symptoms determined, and baseline blood is drawn on 3 occasions one week apart to determine baseline levels of inflammation.

All subjects receive IL-1 trap with a dosing regimen of 100 mg once a day for 3 consecutive days, a regimen expected to provide 2-4 weeks of significant IL-1 inhibitory activity. The primary outcomes are measured during this period and include drug safety, clinical efficacy analysis, and the change in biomarkers of inflammation (e.g., acute phase reactants such as CRP, serum amyloid A, and ESR) at Day 10 following initiation of treatment with IL-1 trap. Patients receive IL-1 trap for 3 days, and are observed for up to 8 weeks (with no additional treatment) following their first dose of IL-1 trap with weekly assessment of acute phase reactants, cytokine levels, and safety lab measurements

History and Physical Exam. A careful, complete standardized history and physical exam is performed, appropriate for the disease under study to assure uniform data collection on every patient. Vital signs and weight is obtained at each visit. The clinical data is based on a detailed questionnaire including all the reported clinical manifestations. The following evaluation procedures pertain specifically to CIAS-1 mutation associated disorders and are performed as clinically indicated: dermatological evaluation; opthalmologic evaluation; ear/nose/throat evaluation; neurology evaluation; lumbar puncture; head MRI; radiographs, joint MRI; and pharmacokinetic profiling.

Example 2

Treatment of CAPS with an IL-1 Antagonist

An open label pilot study of IL-1 trap (SEQ ID NO:10) for the treatment of three autoinflammatory diseases was conducted. Diseases under study include CAPS, familial Mediterranean fever (FMF), and adult Still's disease.

CAPS Study. Four subjects with CAPS were initially enrolled. The dosing strategy includes an initial “loading regimen” of 300 mg administered subcutaneously (administered as 100 mg once daily on three consecutive days). The rationale for this regimen was to enable rapid attainment of drug levels compatible with steady state dosing of 100 mg IL-1 trap protein per week, a regimen previously shown to offer promise for the treatment of rheumatoid arthritis. After initial dosing, subjects were observed to ascertain response of signs and symptoms of autoinflammatory disease. If a favorable response was observed, subjects are observed (with no further treatment) until return of signs and symptoms (flare). Upon flare, subjects are eligible for entry into an extension phase that entails re-treatment with the loading regimen and then once-weekly dosing with 100 mg.

Preliminary Results. Results indicated that all subjects experienced rapid and extensive improvement in inflammatory signs and symptoms upon treatment with IL-1 trap, including improvement in both patient- and physician-reported disease manifestation. Major declines in inflammatory biomarkers, such as CRP and SAA were also observed. Signs and symptoms returned within a median of 21 days (range 9-26) of initial dosing and then responded promptly to re-treatment. Table 1 provides a summary of the daily diary scores, acute phase reactants and clinical assessments (‡ Performed on 3 patients; * statistically significant difference from previous timepoint at p<0.1 level; ** statistically significant difference from previous timepoint at p<0.05 level).

	TABLE 1
	Baseline	Maximal Efficacy	Flare
	median (range)	median (range)	median (range)
Daily Diary Score	6.06	(2.2-7.56)	1.67	(0-3.3)*	4.5	(2-7.33)
Acute phase reactants
SAA (mg/L)	96	(16.1-468)	8.25	(2-19)	84	(50-236)‡
CRP (mg/dL)	7.28	(2.32-8.65)	0.72	(0.07-1.15)**	2.93	(0.076-6.21)
ESR (mm/hr)	56.67	(22-92)	24	(7-45)**	34	(11-70)*
Blood Count
WBC	15.28	(9.33-19.4)	7.58	(7.21-9.9)**	8.48	(6.34-11.47)
Hgb	12.95	(8.1-14.7)	13.3	(8.2-15.6)*	13.1	(7.9-14.57)
Plt	356.5	(291-445.5)	303.25	(240-377)**	291	(257-359.3)
Questionnaires‡
Physician global VAS (cm)	6.85	(4.1-6.95)	0.2	(0.2-2.6)	3.3	(3.1-3.5)
Patient global VAS (cm)	5.2	(3.95-6.9)	1.1	(0.95-3.05)**	3.6	(3.1-6.45)**
Fatigue VAS (cm)	5.55	(3.25-8)	1.15	(0.5-3.9)	6.6	(3.15-6.9)
Pain VAS (cm)	7.55	(3.6-7.7)	0.95	(0.2-1.05)*	4.1	(0.5-6.55)
SF-36 Physical Health	44.38	(42.5-47.5)	50.63	(33.75-92.5)	41.56	(35-69.4)
SF-36 Mental Health	41.625	(28.5-57.8)	75.88	(55-96)	39.6	(37-57)

Claims

1. A method of treating, inhibiting, or ameliorating an autoinflammatory disorder, disease, or condition in a subject in need thereof, comprising administering to the subject a therapeutic amount of an interleukin 1 (IL-1) fusion protein antagonist once a week, wherein the autoinflammatory disorder, disease, or condition is treated, inhibited, or ameliorated, wherein the IL-1 fusion protein antagonist comprises two IL-1 receptor components and a multimerizing component, wherein the fusion protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:10, wherein the subject is a human adult or child diagnosed with Neonatal Onset Multisystem Inflammatory Disorder (NOMID/CINCA), Muckle-Wells Syndrome (MWS), Familial Cold Autoinflammatory Syndrome (FCAS), familial Mediterranean fever (FMF), tumor necrosis factor receptor-associated periodic fever syndrome (TRAPS), or systemic onset juvenile idiopathic arthritis (Still's Disease).

2. The method of claim 1, wherein the fusion protein comprises a sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:10.

3. The method of claim 1, wherein administration is subcutaneous, intramuscular, or intravenous.

4. The method of claim 1, wherein the therapeutically effective amount is between 1-20 mg/kg.

5. A method of treating, inhibiting, or ameliorating an autoinflammatory disorder associated with mutations in CIAS-1 in a subject in need thereof, comprising administering once a week to the subject a therapeutic amount of an interleukin 1 (IL-1) antagonist, wherein the IL-1 antagonist comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:10, wherein the autoinflammatory disorder is treated, inhibited, or ameliorated, and wherein the autoinflammatory disorder associated with mutations in CIAS-1 is one of Neonatal Onset Multisystem Inflammatory Disorder (NOMID/CINCA), Muckle-Wells Syndrome (MWS), and Familial Cold Autoinflammatory Syndrome (FCAS).

6. The method according to claim 5, wherein the IL-1 antagonist comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:10.

7. The method of claim 5, wherein administration is subcutaneous, intramuscular, or intravenous.

8. The method of claim 5, wherein the therapeutically effective amount is between 1-20 mg/kg.