The present invention relates to a novel member of the TNF-Ligand superfamily. More specifically, isolated nucleic acid molecules are provided encoding a human Apoptosis Inducing Molecule II (AIM II). AIM II polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of AIM II activity. Also provided are therapeutic methods for treating lymphadenopathy, aberrant bone development, autoimmune and other immune system diseases, graft versus host disease, rheumatoid arthritis, osteoarthritis and to inhibit neoplasia, such as tumor cell growth.

Description

79 parts

›CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application Ser. No. 60/360,234, filed Mar. 1, 2002; and is a continuation-in-part and claims benefit under 35 U.S.C. §120 of copending U.S. application Ser. No. 09/523,323, filed Mar. 10, 2000; said Ser. No. 09/523,323 claims benefit under35 U.S.C. §119(e) of U.S. Provisional Application Ser. No. 60/168,380, filed Dec. 2, 1999; U.S. Provisional Application Ser. No. 60/148,326, filed Aug. 11, 1999; U.S. Provisional Application Ser. No. 60/142,657, filed Jul. 6, 1999; U.S. Provisional Application Ser. No. 60/137,457, filed Jun. 4, 1999; and U.S. Provisional Application Ser. No. 60/124,041, filed Mar. 11, 1999, and is a continuation-in-part and claims benefit under 35 U.S.C. §120 of U.S. application Ser. No. 09/252,656, filed Feb. 19, 1999 (now U.S. Pat. No. 6,495,520), each of which is incorporated herein by reference; said Ser. No. 09/252,656 claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application Ser. No. 60/075,409, filed Feb. 20, 1998, and is a continuation-in-part and claims benefit under 35 U.S.C. §120 of U.S. application Ser. No. 09/027,287, filed on Feb. 20, 1998 (now U.S. Pat. No. 6,479,254), each of which is herein incorporated by reference; said Ser. No. 09/027,287 is a continuation-in-part and claims benefit under 35 U.S.C. §120 of U.S. application Ser. No. 09/003,886, filed Jan. 7, 1998 (now abandoned), which is herein incorporated by reference; said Ser. No. 09/003,886 is a continuation-in-part and claims benefit under 35 U.S.C. §120 of U.S. application Ser. No. 08/822,953, filed Mar. 21, 1997 (now abandoned.), which is herein incorporated by reference; said Ser. No. 08/822,953 claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application Ser. No. 60/013,923, filed Mar. 22, 1996 and U.S. Provisional Application Ser. No. 60/030,157, filed Oct. 31, 1996, each of which is incorporated herein by reference.

›BACKGROUND OF THE INVENTION

1. Field Of The Invention

2. Related Art

Human tumor necrosis factors α (TNF-α) and β (TNF-β, or lymphotoxin) are related members of a broad class of polypeptide mediators, which includes the interferons, interleukins and growth factors, collectively called cytokines (Beutler, B. and Cerami, A., Annu. Ret. Immunol. 7:625-655 (1989)).

Tumor necrosis factor (TNF-α and TNF-β) was originally discovered as a result of its anti-tumor activity, however, now it is recognized as a pleiotropic cytokine capable of numerous biological activities including apoptosis of some transformed cell lines, mediation of cell activation and proliferation and also as playing important roles in immune regulation and inflammation.

To date, known members of the TNF-ligand superfamily include TNF-α, TNF-β (lymphotoxin-α), LT-β, OX40L, Fas ligand, CD30L, CD27L, CD40L and 4-IBBL. The ligands of the TNF ligand superfamily are acidic, TNF-like molecules with approximately 20% sequence homology in the extracellular domains (range, 12%-36%) and exist mainly as membrane-bound forms with the biologically active form being a trimeric/multimeric complex. Soluble forms of the TNF ligand superfamily have only been identified so far for TNF, LTα, and Fas ligand (for a general review, see Gruss, H. and Dower, S. K., Blood, 85(12):3378-3404 (1995)), which is hereby incorporated by reference in its entirety.

These proteins are involved in regulation of cell proliferation, activation, and differentiation, including control of cell survival or death by apoptosis or cytotoxicity (Armitage, R. J., Curr. Opin. Immunol. 6:407 (1994) and Smith, C. A., Cell 75:959 (1994)).

Mammalian development is dependent on both the proliferation and differentiation of cells as well as programmed cell death which occurs through apoptosis (Walker et al., Methods Achiev. Exp. Pathol. 13:18 (1988). Apoptosis plays a critical role in the destruction of immune thymocytes that recognize self antigens. Failure of this normal elimination process may play a role in autoimmune diseases (Gammon et al., Immunology Today 12:193 (1991)).

Itoh et al. ( Cell 66:233 (1991)) described a cell surface antigen, Fas/CD95 that mediates apoptosis and is involved in clonal deletion of T-cells. Fas is expressed in activated T-cells, B-cells, neutrophils and in thymus, liver, heart and lung and ovary in adult mice (Watanabe-Fukunaga et al., J. Immunology. 148:1274 (1992)). In experiments where a monoclonal Ab to Fas is cross-linked to Fas, apoptosis is induced (Yonehara et al., J. Exp. Med. 169:1747 (1989); Trauth et al., Science 245:301 (1989)). In addition, there is an example where binding of a monoclonal Ab to Fas may stimulate T-cells under certain conditions (Alderson et al, J. Exp. Med. 178:2231 (1993)).

Fas antigen is a cell surface protein of relative MW of 45 Kd. Both human and murine genes for Fas have been cloned by Watanabe-Fukunaga et al., ( J. Immunol. 148:1274 (1992)) and Itoh et al. ( Cell 66:233 (1991)). The proteins encoded by these genes are both transmembrane proteins with structural homology to the Nerve Growth Factor/Tumor Necrosis Factor receptor superfamily, which includes two TNF receptors, the low affinity Nerve Growth Factor receptor and the LT β receptor CD40, CD27, CD30, and OX40.

Recently the Fas ligand has been described (Suda et al., Cell 75:1169 (1993)). The amino acid sequence indicates that Fas ligand is a type II transmembrane protein belonging to the TNF family. Fas ligand is expressed in splenocytes and thymocytes. The purified Fas ligand has a MW of 40 kd.

Recently, it has been demonstrated that Fas/Fas ligand interactions are required for apoptosis following the activation of T-cells (Ju et al., Nature 373:444 (1995); Brunner et al., Nature 373:441 (1995)). Activation of T-cells induces both proteins on the cell surface. Subsequent interaction between the ligand and receptor results in apoptosis of the cells. This supports the possible regulatory role for apoptosis induced by Fas/Fas ligand interaction during normal immune responses.

The polypeptide of the present invention has been identified as a novel member of the TNF ligand super-family based on structural and biological similarities.

Clearly, there is a need for factors that regulate activation, and differentiation of normal and abnormal cells. There is a need, therefore, for identification and characterization of such factors that modulate activation and differentiation of cells, both normally and in disease states. In particular, there is a need to isolate and characterize additional Fas ligands that control apoptosis for the treatment of autoimmune disease, graft versus host disease, rheumatoid arthritis and lymphadenopathy.

›SUMMARY OF THE INVENTION · 1 of 6

The present invention provides isolated nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide encoding the AIM II polypeptide having the amino acid sequence shown in FIGS. 1A and 1B (SEQ ID NO:2) or the amino acid sequence encoded by the cDNA deposited as ATCC Deposit Number 97689 on Aug. 22,1996. The present invention also provides isolated nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide encoding the AIM II polypeptide having the amino acid sequence shown in FIGS. 1C and 1D (SEQ ID NO:39) or the amino acid sequence encoded by the cDNA deposited as ATCC Deposit Number 97483 on Mar. 15, 1996.

The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of AIM II polypeptides or peptides by recombinant techniques.

The invention further provides an isolated AIM II polypeptide having an amino acid sequence encoded by a polynucleotide described herein.

As used herein the term “AIM II” polypeptide includes membrane-bound proteins (comprising, or alternatively consisting of, a cytoplasmic domain, a transmembrane domain, and an extracellular domain) as well as truncated proteins that retain the AIM II polypeptide activity. In one embodiment, soluble AIM II polypeptides comprise, or alternatively consist of, all or part of the extracellular domain of an AIM II protein, but lack the transmembrane region that would cause retention of the polypeptide on a cell membrane. Soluble AIM II may also include part of the transmembrane region or part of the cytoplasmic domain or other sequences, provided that the soluble AIM II protein is capable of being secreted. A heterologous signal peptide can be fused to the N-terminus of the soluble AIM II polypeptide such that the soluble AIM II polypeptide is secreted upon expression.

The invention also provides for AIM II polypeptides, particularly human AIM II polypeptides, which may be employed to treat afflictions such as lymphadenopathy, rheumatoid arthritis, autoimmune disease (e.g., inflammatory autoimmune diseases, myasthenia gravis), graft versus host disease, IgE-mediated allergic reactions, anaphylaxis, adult respiratory distress syndrome, Crohn's disease, allergic asthma, acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), and Graves' disease. These polypeptides of the invention may also be used to stimulate peripheral tolerance, destroy some transformed cell lines, mediate cell activation and proliferation and are functionally linked as primary mediators of immune regulation and inflammatory response.

The invention further provides compositions comprising, or alternatively consisting of, an AIM II polynucleotide or an AIM II polypeptide for administration to cells in vitro, to cells ex vivo and to cells in vivo, or to a multicellular organism. In certain particularly preferred embodiments of this aspect of the invention, the compositions comprise, or alternatively consist of, an AIM II polynucleotide for expression of an AIM II polypeptide in a host organism for treatment of disease. Particularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with aberrant endogenous activity of an AIM II.

The present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by AIM II, which involves contacting cells which express AIM II with the candidate compound, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made in absence of the candidate compound; whereby, an increased cellular response over the standard indicates that the compound is an agonist and a decreased cellular response over the standard indicates that the compound is an antagonist.

In another aspect, a screening assay for AIM II agonists and antagonists is provided. The antagonists may be employed to treat, prevent, diagnose, and/or prognose septic shock, inflammation, cerebral malaria, activation of the HIV virus, graft-host rejection, immunodeficiency, bone resorption, and cachexia (wasting or malnutrition). In a preferred embodiment, the AIM II antagonists of the invention (e.g., anti-AIM II antibodies) are used to treat, prevent, diagnose, and/or prognose graft versus host disease.

In a further aspect of the invention, AIM II may be used to treat rheumatoid arthritis (RA) by inhibiting the increase in angiogenesis or increase in endothelial cell proliferation required to sustain an invading pannus in bone and cartilage as is often observed in RA.

The present invention encompasses methods for using the polynucleotides, polypeptides or antibodies of the invention to treat, prevent, diagnose and/or prognose a disease or disorder of the immune system. In preferred embodiments, the present invention encompasses methods for using the polynucleotides, polypeptides or antibodies of the invention to treat, prevent, diagnose and/or prognose a disease or disorder of the cellular immune system.

In other preferred embodiments, the invention encompasses methods for using the polynucleotides, polypeptides or antibodies of the invention to treat, prevent, diagnose and/or prognose a disease or disorder associated with aberrant T cell activation. In specific embodiments, the invention encompasses methods for using the polynucleotides, polypeptides or antibodies of the present invention to treat, prevent, diagnose and/or prognose a disease or disorder associated with aberrant CD8 + T cell activation. In further specific embodiments, the invention encompasses methods for using the polynucleotides, polypeptides or antibodies of the present invention to treat, prevent, diagnose and/or prognose a disease or disorder associated with aberrant Th1 cell differentiation.

›SUMMARY OF THE INVENTION · 2 of 6

The present invention encompasses AIM II antagonists, including antibodies, polypeptides, polynucleotides (including RNA, DNA, and synthetic polynucleotide derivatives) and small molecules useful in treating, preventing, diagnosing and/or prognosing a disease or disorder of the immune system. In preferred embodiments, the present invention encompasses AIM II antagonists, including antibodies, polypeptides, peptides, polynucleotides (including RNA, DNA, and synthetic polynucleotide derivatives) and small molecules useful in treating, preventing, diagnosing and/or prognosing a disease or disorder of the cellular immune system.

In other preferred embodiments, the present invention encompasses AIM II antagonists including antibodies, polypeptides, polynucleotides (including RNA, DNA, and synthetic polynucleotide derivatives) and small molecules useful in treating, preventing, diagnosing and/or prognosing a disease or disorder associated with aberrant T cell activation. In specific embodiments, the present invention encompasses AIM II antagonists including antibodies, polypeptides, polynucleotides (including RNA, DNA, and synthetic polynucleotide derivatives) and small molecules useful in treating, preventing, diagnosing, and/or prognosing a disease or disorder associated with aberrant CD8 + T cell activation. In further specific embodiments, the present invention encompasses AIM II antagonists including antibodies, polypeptides, polynucleotides (including RNA, DNA, and synthetic polynucleotide derivatives) and small molecules useful in treating, preventing, diagnosing, and/or prognosing a disease or disorder associated with aberrant Th1 cell differentiation.

The present invention encompasses AIM II agonists, including antibodies, polypeptides, polynucleotides (including RNA, DNA, and synthetic polynucleotide derivatives) and small molecules useful in treating, preventing, diagnosing and/or prognosing a disease or disorder of the immune system. In preferred embodiments, the present invention encompasses AIM II agonists, including antibodies, polypeptides, peptides, polynucleotides (including RNA, DNA, and synthetic polynucleotide derivatives) and small molecules useful in treating, preventing, diagnosing and/or prognosing a disease or disorder of the cellular immune system.

In other preferred embodiments, the present invention encompasses AIM II agonists including antibodies, polypeptides, polynucleotides (including RNA, DNA, and synthetic polynucleotide derivatives) and small molecules useful in treating, preventing, diagnosing and/or prognosing a disease or disorder associated with aberrant T cell activation. In specific embodiments, the present invention encompasses AIM II agonists including antibodies, polypeptides, polynucleotides (including RNA, DNA, and synthetic polynucleotide derivatives) and small molecules useful in treating, preventing, diagnosing, and/or prognosing a disease or disorder associated with aberrant CD8 + T cell activation. In further specific embodiments, the present invention encompasses AIM II agonists including antibodies, polypeptides, polynucleotides (including RNA, DNA, and synthetic polynucleotide derivatives) and small molecules useful in treating, preventing, diagnosing, and/or prognosing a disease or disorder associated with aberrant Th1 cell differentiation.

The invention provides a method of inhibiting, blocking, and/or reducing T cell activation comprising contacting a T cell with an effective amount of one or more AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, wherein said effective amount inhibits, blocks, and/or reduces T cell activation. In specific embodiments, this method is performed in vitro. In other specific embodiments, this method is performed in vivo.

The invention also provides a method of inhibiting, blocking, and/or reducing CD8 + T cell activation comprising contacting a CD8 + T cells with an effective amount of one or more AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, wherein said effective amount inhibits, blocks, and/or reduces CD8 + T cell activation. In specific embodiments, this method is performed in vitro. In other specific embodiments, this method is performed in vivo.

In other embodiments, the invention provides a method of inhibiting, blocking, and/or reducing T cell proliferation comprising contacting a T cell with an effective amount of one or more AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, wherein said effective amount inhibits, blocks, and/or reduces T cell proliferation. In specific embodiments, this method is performed in vitro. In other specific embodiments, this method is performed in vivo.

In other embodiments, the invention provides a method of inhibiting, blocking, and/or reducing CD8 + T cell proliferation comprising contacting a CD8 + T cell with an effective amount of one or more AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, wherein said effective amount inhibits, blocks, and/or reduces CD8 + T cell proliferation. In specific embodiments, this method is performed in vitro. In other specific embodiments, this method is performed in vivo.

In other embodiments, the invention provides a method of inhibiting, blocking, and/or reducing T cell secretion of IFN-γ comprising contacting a T cell with an effective amount of one or more AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, wherein said effective amount inhibits, blocks, and/or reduces T cell secretion of IFN-γ. In specific embodiments, this method is performed in vitro. In other specific embodiments, this method is performed in vivo.

In other embodiments, the invention provides a method of inhibiting, blocking, and/or reducing CD8 + T cell secretion of IFN-γ comprising contacting a CD8 + T cell with an effective amount of one or more AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, wherein said effective amount inhibits, blocks, and/or reduces CD8 + T cell secretion of IFN-γ. In specific embodiments, this method is performed in vitro. In other specific embodiments, this method is performed in vivo.

›SUMMARY OF THE INVENTION · 3 of 6

In other embodiments, the invention provides a method of inhibiting, blocking, and/or reducing a Th1 response comprising contacting a T cell with an effective amount of one or more AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, wherein said effective amount inhibits, blocks, and/or reduces a Th1 response. In specific embodiments, this method is performed in vitro. In other specific embodiments, this method is performed in vivo.

The invention provides a method of stimulating, enhancing, and/or promoting T cell activation comprising contacting a T cell with an effective amount of one or more AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, wherein said effective amount, stimulates, enhances, and/or promotes T cell activation. In specific embodiments, this method is performed in vitro. In other specific embodiments, this method is performed in vivo.

The invention also provides a method of stimulating, enhancing, and/or promoting CD8 + T cell activation comprising contacting a CD8 + T cell with an effective amount of one or more AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, wherein said effective amount stimulates, enhances, and/or promotes CD8 + T cell activation. In specific embodiments, this method is performed in vitro. In other specific embodiments, this method is performed in vivo.

In other embodiments, the invention provides a method of stimulating, enhancing, and/or promoting T cell proliferation comprising contacting a T cell with an effective amount of one or more AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, wherein said effective amount stimulates, enhances, and/or promotes T cell proliferation. In specific embodiments, this method is performed in vitro. In other specific embodiments, this method is performed in vivo.

In other embodiments, the invention provides a method of stimulating, enhancing, and/or promoting CD8 + T cell proliferation comprising contacting a CD8 + T cell with an effective amount of one or more AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, wherein said effective amount stimulates, enhances, and/or promotes CD8 + T cell proliferation. In specific embodiments, this method is performed in vitro. In other specific embodiments, this method is performed in vivo.

In other embodiments, the invention provides a method of stimulating, enhancing, and/or promoting T cell secretion of IFN-γ comprising contacting a T cell with an effective amount of one or more AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, wherein said effective amount stimulates, enhances, and/or promotes T cell secretion of IFN-γ. In specific embodiments, this method is performed in vitro. In other specific embodiments, this method is performed in vivo.

In other embodiments, the invention provides a method of stimulating, enhancing, and/or promoting CD8 + T cell secretion of IFN-γ comprising contacting a CD8 + T cell with an effective amount of one or more AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, wherein said effective amount stimulates, enhances, and/or promotes CD8 + T cell secretion of IFN-γ. In specific embodiments, this method is performed in vitro. In other specific embodiments, this method is performed in vivo.

In other embodiments, the invention provides a method of stimulating, enhancing, and/or promoting a Th1 response comprising contacting a T cell with an effective amount of one or more AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, wherein said effective amount stimulates, enhances, and/or promotes a Th1 response. In specific embodiments, this method is performed in vitro. In other specific embodiments, this method is performed in vivo.

The invention provides for pharmaceutical compositions comprising AIM II polynucleotides, polypeptides, antibodies, and agonists and/or antagonists, which may be employed, for instance, to treat, prevent, prognose and/or diagnose tumor and tumor metastasis, infections by bacteria, viruses and other parasites, immunodeficiencies, inflammatory diseases or disorders, lymphadenopathy, autoimmune diseases or disorders, graft versus host disease, stimulate peripheral tolerance, destroy some transformed cell lines, mediate cell activation, survival and proliferation, mediate immune regulation and inflammatory responses, and to enhance or inhibit immune responses.

The invention further provides compositions, comprising AIM II polynucleotides, polypeptides, antibodies, and agonists and/or antagonists, for administration to cells in vitro, to cells ex vivo, and to cells in vivo, or to a multicellular organism. In preferred embodiments, the compositions of the invention comprise an AIM II polynucleotide for expression of an AIM II polypeptide in a host organism for treatment of disease. In a specific preferred embodiment, the compositions of the invention comprise an AIM II polynucleotide for expression of an AIM II polypeptide in a host organism for treatment of an immunodeficiency and/or conditions associated with an immunodeficiency. In another specific preferred embodiment, the compositions of the invention comprise an AIM II polynucleotide for expression of an AIM II polypeptide in a host organism for treatment of an autoimmune disease and/or conditions associated with an autoimmune disease.

Particularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with aberrant endogenous activity of AIM II (e.g., expression to enhance T-cell function by enhancing T cell activation, expanding T-cell numbers or increasing T cell IFN-γ secretion).

The present invention further encompasses methods and compositions for preventing, treating and/or ameliorating diseases or disorders associated with aberrant or inappropriate AIM II expression or function in an animal, preferably a mammal, and most preferably a human, comprising, or alternatively consisting of, administering to an animal in which such treatment, prevention or amelioration is desired one or more AIM II polypeptides of the invention in an amount effective to treat prevent or ameliorate the disease or disorder.

›SUMMARY OF THE INVENTION · 4 of 6

The present invention further encompasses methods and compositions for killing cells of hematopoietic origin, comprising contacting AIM II polypeptide with cells of hematopoietic origin. In preferred embodiments, the cells of hematopoietic origin are T cells. In further preferred embodiments, the cells of hematopoietic origin are CD8 + T cells.

The present invention further encompasses methods and compositions for killing cells of hematopoictic origin, comprising administering to an animal in which such killing is desired, an AIM II polypeptide (e.g., a radiolabelled AIM II polypeptide) in an amount effective to kill cells of hematopoictic origin. In preferred embodiments, the cells of hematopoietic origin are T cells. In further preferred embodiments, the cells of hematopoietic origin are CD8 + T cells.

In preferred embodiments, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are administered to an animal, preferably a human, to treat, prevent, diagnose and/or prognose inflammation and/or an inflammatory disease or disorder.

In other preferred embodiments, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are administered to an animal, preferably a human, to treat, prevent, diagnose and/or prognose an intestinal disease or disorder. In a specific preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are administered to an animal, preferably a human, to treat, prevent, diagnose and/or prognose Inflammatory Bowel Disease (IBD). In another specific preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are administered to an animal, preferably a human, to treat, prevent, diagnose and/or prognose ulcerative colitis. In another specific preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are administered to an animal, preferably a human, to treat, prevent, diagnose and/or prognose Crohn's disease.

In additional embodiments, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are administered to an animal, preferably a human, to treat, prevent, diagnose and/or prognose an autoimmune disease. In specific embodiments, the autoimmune disease treated, prevented, diagnosed and/or prognosed according to the methods of the invention is a member selected from the group consisting of Graft versus host disease (GVHD), Multiple Sclerosis, type 1 diabetes, rheumatoid arthritis, primary biliary cirrhosis, aplastic anemia, myelodysplasia, systemic lupus erhythematosus, idiopathic thrombocytopenia purpura, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (e.g, IgA nephropathy), an immune-based rheumatologic disease (e.g., SLE, rheumatoid arthritis, CREST syndrome (a variant of scleroderma characterized by calcinosis, Raynaud's phenomenon, esophageal motility disorders, sclerodactyly, and telangiectasia.), Seronegative spondyloarthropathy (SpA), Polymyositis/dermatomyositis, Microscopic polyanguitis, Hepatitis C-associated arthritis, Takayasu's arteritis, and undifferentiated connective tissue disorder), Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura), Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis, Goodpasture's syndrome, Pemphigus, Receptor autoimmunities such as, for example, (a) Graves' Disease, (b) Myasthenia Gravis, and (c) insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis/dermatomyositis, pernicious anemia, idiopathic Addison's disease, infertility, glomerulonephritis such as primary glomerulonephritis and IgA nephropathy, bullous pemphigoid, Sjogren's syndrome, diabetes mellitus, and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis), chronic active hepatitis, primary biliary cirrhosis, other endocrine gland failure, vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria, atopic dermatitis, asthma, inflammatory myopathies, and other inflammatory, granulamatous, degenerative, atrophic disorders, or a condition associated with an autoimmune disease.

In a preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are administered to an animal, preferably a human, to treat, prevent, diagnose and/or prognose graft versus host disease (GVHD). In a specific preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are administered to an animal, preferably a human, to treat, prevent, diagnose and/or prognose tissue rejection after allograft transplantation. In another specific preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are administered to an animal, preferably a human, to treat, prevent, diagnose and/or prognose tissue rejection after isograft transplantation. In another specific preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are administered to an animal, preferably a human, to treat, prevent, diagnose and/or prognose tissue rejection after xenograft transplantation.

In another preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are administered to an animal, preferably a human, to treat, prevent, diagnose and/or prognose multiple sclerosis.

›SUMMARY OF THE INVENTION · 5 of 6

In additional embodiments, AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, are administered to an animal, preferably a human, to treat, prevent, diagnose and/or prognose an immunodeficiency. In specific embodiments, the immunodeficiency treated, prevented, diagnosed and/or prognosed according to the methods of the invention is a member selected from the group consisting of DiGeorge anomaly, ataxia telangiectasia, severe combined immunodeficiency (SCID)-X linked, SCID-autosomal, Wiskott-Aldrich Syndrome (WAS), chronic mucocutaneous candidiasis, natural killer cell deficiency, CD4+T-lymphocytopenia, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton's disease, congenital agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, transient hypogammaglobulinemia of infancy, unspecified hypogammaglobulinemia, agammaglobulinemia, common variable immunodeficiency (CVID) (acquired), X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), antibody deficiency with normal or elevated Igs, immunodeficiency with thymoma, Ig heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), selective IgM immunodeficiency, recessive agammaglobulinemia (Swiss type), reticular dysgenesis, neonatal neutropenia, severe-congenital leukopenia, thymic alymphoplasia-aplasia or dysplasia with immunodeficiency, short limbed dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezelof syndrome-combined immunodeficiency with Igs, purine nucleoside phosphorylase deficiency (PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severe combined immunodeficiency and conditions associated with an immunodeficiency.

In a preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, are administered to an animal, preferably a human, to treat, prevent, diagnose and/or prognose ataxia telangiectasia.

In another preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, are administered to an animal, preferably a human, to treat, prevent, diagnose and/or prognose DiGeorge anomaly.

›SUMMARY OF THE INVENTION · 6 of 6

In an additional aspect of the invention, AIM II may be used to inhibit or activate a cellular response mediated by a cellular receptor (e.g., LT-β-R, TR2, CD27, and TRANK) by either inhibiting the binding of a ligand to the receptor or by binding to the receptor and activating a receptor mediated cellular response.

An additional aspect of the invention is related to a method for treating an individual in need of an increased level of AIM II activity in the body comprising administering to such an individual a composition comprising, or alternatively consisting of, a therapeutically effective amount of an isolated AIM II polypeptide of the invention or an agonist thereof.

A still further aspect of the invention is related to a method for treating an individual in need of a decreased level of AIM II activity in the body comprising, administering to such an individual a composition comprising, or alternatively consisting of, a therapeutically effective amount of an AIM II antagonist.

›BRIEF DESCRIPTION OF THE FIGURES · 1 of 3

FIGS. 1A and 1B show the nucleotide (SEQ ID NO:1) and deduced amino acid (SEQ ID NO:2) sequences of AIM II. The protein has a deduced molecular weight of about 26.4 kDa. The predicted Transmembrane Domain of the AIM II protein is underlined.

FIGS. 1C and 1D show the nucleotide (SEQ ID NO:38) and deduced amino acid (SEQ ID NO:39) sequences of a partial AIM II cDNA that was also obtained.

FIGS. 2A-2F show the regions of similarity between the amino acid sequences of the AIM II protein and human TNF-α (SEQ ID NO: 3), human TNF-β (SEQ ID NO:4), human lymphotoxin (SEQ ID NO:5) and human Fas Ligand (SEQ ID NO:6).

FIGS. 3A-3F show an analysis of the AIM II amino acid sequence. Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown. In the “Antigenic Index—Jameson-Wolf” graph, about amino acid residues 13-20, 23-36, 69-79, 85-94, 167-178, 184-196, 221-233 in FIGS. 1A and 1B (SEQ ID NO:2) correspond to the shown highly antigenic regions of the AIM II protein.

FIGS. 4A and 4B show the effect of AIM II on the in vitro proliferation of MDA-MB-231 human breast cancer cells. 5,000 MDA-MB-231/WT (circle), MDA-MB-231/Neo (triangle) or MDA-MB-231/AIM II (square) cells were plated in triplicate in 24-well plates with IMEM in the presence of either 10% FBS (filled circle, square or triangle) or 1% FBS (open circle, square or triangle). The number of live cells was determined by trypan blue exclusion method at day 3, day 5 or day 7. Cells were fed with fresh medium every two days during this time course. FIG. 4B shows colony formation of MDA-MB-231/WT, MDA-MB-231/Neo and MDA-MB-231/AIM II cells in 0.33% agarose.

FIGS. 5A-5C show increased apoptotic cells in MDA-MB-231/AIM II ( FIG. 5C ) in 0.5% serum compared with that of the MDA-MB-231/WT ( FIG. 5A ) or MDA-MB-231/Neo ( FIG. 5B ) cells with Annexin-V FACS analysis as described in Example 5 Material and Methods.

FIGS. 6A-B . FIG. 6A shows an evaluation of the effects of AIM II on growth of xenograft human breast carcinoma MDA-231 in nude mice. Female athymic nude mice were injected s.c. with 10 6 cells of parental MDA-231 (MDA-231-WT), or MDA-231 stably transfected with AIM II, or vector control neo (n=10). Mice were then ear tagged and randomized. Tumor growth was assessed twice weekly with a caliper in the blinded fashion. This panel represents three experiments each with ten mice per group. FIG. 6B shows the effect of AIM II transduction on inhibition of growth of MC-38 murine colon cancer in syngeneic C57BL/6 mice. Female C57BL/6 mice were injected s.c. with 10 6 cells of parental MC-38 (MC-38 -WT), or MC-38 stably transfected with AIM II, or vector control neo (n=10). Mice were then ear tagged and randomized. Tumor growth was assessed twice weekly with a caliper in a coded, blinded fashion. This panel represents four experiments each with ten mice per group.

FIGS. 7A-B . FIG. 7 shows the pFlag-AIM II plasmid construct ( FIG. 7A ) and cytotoxicity of a recombinant soluble form of AIM II (sAIM II) in MDA-MB-231 cells in the presence or absence of IFN-γ ( FIG. 7B ) or with IFN-γ alone ( FIG. 7C ). Experiments were carried out as described in Example 5 Materials and Methods.

FIGS. 8A-8M . Cell surface expression of the LTβR or TR2 by the FACS analyses using LTβR ( FIGS. 8A-8D ) or TR2 ( FIGS. 8E-8H ) mAb. MDA-MB-231 ( FIGS. 8A and 8E ), HT-29 ( FIGS. 8B and 8F ), MC-3 ( FIGS. 8C and 8G ), U93T ( FIGS. 8D and 8H ). FACS binding analyses of soluble AIM II protein alone ( FIG. 8I ) and blocking of a soluble AIM II protein binding by preincubation with the LTβR-Fc fusion protein ( FIG. 8J ) or TR2-Fc fusion protein ( FIG. 8K ) in MDA-MB-231 cells. FIG. 8L summarizes the surface expression of LTβR and TR2 in various cell lines. Effects of LTβR-Fc or TR2-Fc fusion protein to block the sAIM II-mediated cytotoxicity in HT-29 cells ( FIG. 8M ). Cells were plated into 96-well plates and sAIM II (10 ng/ml) was added in the presence of 5 U/ml of IFNγ with various amounts of sLTβR-Fc (open circle with LTβR-Fc alone, filled circle LTβR-Fc, and IFN.γ) or TR2-Fc fusion protein (open triangle with TR-2Fc alone, filled triangle TR2-Fc with sLTγ and IFNγ). Cells were incubated for five days and the viability of cells was determined by XTT assays.

FIG. 9 shows secretion of IFN-γ by sAIM II treated human PBL cells. Human PBLs (5×10 5 cells per well in the 96 well plate) were treated with or without anti-CD3 mAb and IL-2 (20 U/ml) in the presence or absence of sAIM II for 5 days. The supernatants were then collected from the following groups of cells: PBLs in the presence (filled circle) or absence (open circle) of sAIM II, or the resting PBLs with (filled triangle) or without (open triangle) sAIM II. Human IFNγ concentrations were determined by ELISA.

FIG. 10 shows a schematic representation of the pHE4-5 expression vector (SEQ ID NO: 50) and the subcloned AIM II cDNA coding sequence. The locations of the kanamycin resistance marker gene, the AIM II coding sequence, the oriC sequence, and the lacIq coding sequence are indicated.

FIG. 11 shows the nucleotide sequence of the regulatory elements of the pHE promoter (SEQ ID NO:51). The two lac operator sequences, the Shine-Delgamo sequence (S/D), and the terminal HindIII and NdeI restriction sites (italicized) are indicated.

FIG. 12 shows a sensorgram of specificity of binding of MCA-38 AIM II conditioned media to LTβR-Fc versus MCIF-Fc immobilized on BIAcore chip. Conditioned media was analyzed on a BIAcore instrument flowcell derivatized with lymphotoxin beta receptor Fc fusion protein. The conditioned media (100 μl) was flown over the chip at 5 μl/min and washed with HBS buffer also at 5 μl/min. The shown data represents the net bound (off-rate) region of the plot after binding of AIM II to immobilized receptor and is measured in relative mass units (RU) versus time. The binding conditions were performed at high receptor chip densities under diffusion-limited conditions. Legend: LTβR-Fc and MCIF-Fc refer to binding data from LTβR-Fc or MCIF-Fc immobilized BIAcore chip surfaces, respectively.

›BRIEF DESCRIPTION OF THE FIGURES · 2 of 3

FIG. 13 shows the determination of the LT R binding by AIM II eluted from LTβR-Fc column. Binding conditions were as described in FIG. 11 . Legend: LTβR and MCIF refer to binding data from LTβR-Fc or MCIF-Fc immobilized BIAcore chip surfaces, respectively. Undiluted conditioned media from MCA38 cells was analyzed before (pre) and after passage through MCIF-Fc (post-MCIF) and LTβR-Fc (post-LTβR) affinity columns. Fractions (1 ml) eluted from the LTβR (E4-6) and MCIF-Fc (E1-3) affinity columns were diluted 3-fold and tested for binding to LTβR BIAcore chip.

FIGS. 14A-14B show the sequence of TR6 and aligned amino acid sequence of cysteine-rich motif. (A) Shows a deduced amino acid sequence of TR6 (SEQ ID NO:52). The signal sequence is underlined. The potential N-glycosylation site (NCT, amino acid residues 173-175) is underlined with shadow. The N-terminal amino acid sequence of recombinant TR6-(His) reads as VAETPT - - - , which indicates that the first 29 amino acids constitute a signal sequence. (B) Shows an aligned amino acid sequence of cysteine-rich motif of TR6 with other TNF receptor family members (SEQ ID NO:53-58). The amino acid sequence of TR6 (SEQ ID NOs:52 and 59) was aligned with those of TNFR-I, TNFR-II, 4-1BB, TR2 (HVEM), LTβR and TR1 (OPG) on the basis of sequence homology and conserved cysteines.

FIGS. 15A-15C show the identification of the membrane-bound TR6 ligand. HEK293 EBNA cells were transfected with pCEP4 control vector (shaded area) or with pCEP4/encoding full-length AIM II (LIGHT) cDNA (solid line). Cells were incubated with (A) HVEM/TR2-Fc (0.34 (g), (B) LTβR-Fc (0.34 (g), (C) TR6-(His) (0.34 (g) or buffer control (same as vector). Cells were stained with anti-hIgG-FITC for detecting HVEM/TR2 and LTβR binding. For detecting TR6 binding, cells were stained with anti-poly(His) and anti-mIgG-FITC. They were analyzed for binding by FACS.

FIGS. 16A and 16B show TR6 inhibits AIM II (LIGHT)-induced cell death in HT29 cells. (A) HT29 cells were incubated in 96-well plates with control medium, 10 U/ml IFN-γ alone, purified sAIM II (sLIGHT) protein (10 ng/ml) in the absence or presence of IFN-γ (10 U/ml), purified sLTβR-Fc (200 ng/ml) or TR6-(His) (200 ng/ml) in the presence of IFN-γ (10 U/ml) and sAIM II (sLIGHT) (10 ng/ml). (B) Cells were incubated with various dose of TR6-(His) and IFN-γ (10 U/ml) with (open circle) or without (filled circle) sAIM II (sLIGHT) (10 ng/ml). In all assays, cells were cultured for 4 days, and proliferation was detected during the last 6 h of culture by the addition of 1 μCi of [ 3 H]thymidine. Cells were harvested, and thymidine incorporation was determined using a liquid scintillation counter.

FIGS. 17A-B . FIG. 17 shows the amino acid sequence and expression of mouse AIM II (LIGHT). The putative amino acid sequence of mouse AIM II deduced from the cDNA sequence was aligned with that of human AIM II ( FIG. 17A ). To obtain the optimal alignment, several gaps (-) were introduced with the ClustalW program using Macvector software (version 6.5). Homology regions are boxed and identical residues shaded. The transmembrane region (TM) is underlined. The asterisk (*) indicates the position of the predicted N-glycosylation site. The amino acid numbers are shown on the sides of the alignment. The data shown in FIG. 17B was generated using human 293 cells transfected with the pmAIM II or pcDNA3 and stained with LTβR-Ig or the anti-AIM II antibody, followed by FITC-conjugated goat anti-human IgG or anti-rabbit IgG, respectively (solid line). Staining with human IgGI or rabbit IgG were used as controls (shaded area). ( FIG. 17B .)

FIGS. 18A-C . FIG. 18 shows the costimulatory activity of mouse AIM II. Purified T cells (1×10 6 cells/ml) were stimulated with either 5×10 4 cells/ml of irradiated COS cells, which were transfected with pcDNA3 or pmAIM II plasmid for 72 hours ( FIG. 18A ), or indicated doses of immobilized AIM II.flag fusion proteins ( FIG. 18B ) in 96-well flat-bottomed microplates in the presence or absence of immobilized anti-CD3. The concentration of anti-CD3 mAb was 2 μg/ml in FIG. 18A and 0.2μg/ml in FIG. 18B . Purified T cells (1×10 6 cells/ml) of either CD28 −/− or CD28 +/+ splenocytes were stimulated with immobilized AIM II.flag fusion protein (4 μg/ml) or soluble anti-CD28 (1μg/ml) in the presence of immobilized anti-CD3 (0.2 μg/ml) ( FIG. 18C ). In all assays, the cells were incubated for 72 hr and the proliferative responses of the T cells were monitored by 3 H-TdR incorporation during the last 15 hr.

FIG. 19 shows that AIM II DNA injection induces enhanced cytolytic T cell (CTL) responses and regression of established P815 tumor with memory tumor immunity. To generate the data shown in FIG. 19A , DBA/2 mice were inoculated with 2×10 5 cells of P815 cells at day 0 and then intratumorally injected with medium, pcDNA3 or pmAIM II mixed with liposome at day 7, 9 and 11. Seven days after last injection, spleen cells were harvested and re-stimulated with irradiated P815 cells for 4 days. The CTL activity was assessed by a standard 4 hr 51 Cr release assay against P815 and L1210 cells in indicated effector:target (E/T) ratios. The results are expressed as the means +SD of triplicate wells. Similar results were obtained in three independent experiments.

To generate the data shown in FIG. 19B , DBA/2 mice were inoculated s.c. with 2×10 5 cells of P815 cells at day 0. Seven days later the mice with palpable tumor nodules were intratumorally injected with either medium, pcDNA3 or pmAIM II mixed with liposome. The injections were repeated on day 10, 14 and 17. Tumor sizes were assessed by measuring diameters, and the tumor with more than 2 mm in the longest diameter was considered as a palpable tumor. The results are expressed as percentage of mice with palpable tumor. Similar results were obtained in three independent experiments.

To generate the data shown in FIG. 19C , the mice which had regressed P815 tumor after pmAIM II treatment were s.c. challenged with 2×10 5 P815 cells at the right back and the same number of L1210 cells at the left back 40 days after primary tumor inoculation. Naive DBA/2.mice receiving both P815 and L1210 challenges were used as controls. Tumor sizes were assessed by measuring perpendicular diameters. The results are expressed as average +SD of 5 mice in each group.

›BRIEF DESCRIPTION OF THE FIGURES · 3 of 3

FIGS. 20A-E . FIG. 20 shows the inhibition of GVHD by blockage of AIM II. ( FIGS. 20A and 20B ) Sub-lethally (4 Gy) irradiated BDFI mice were injected i.v. with 7×10 7 cells of B6 splenocytes on day 0. The recipient mice were administered i.v. with either LTβR-Ig or control human IgG1 at 100 μg per mouse on day—1, 2, 5, 8, 11, 14 and 17 (control Ig were administered until day 11 because all mice died in day 12). The survival ( FIG. 20A ) and body weight ( FIG. 20B ) of the recipients were monitored daily. The results in FIG. 20B are expressed as average grams +SEM of five mice in each group.

Non-irradiated BDFI mice were injected i.v. with 7×10 7 cells of either B6 (B6-F1) or BDF 1 (F1-F1) splenocytes on day 0. The recipient mice were administered i.v. with either LTβR-Ig or control human IgG1 at 100 μg/mouse on day —1, 1, 3, 5, 7, 9. At day 11, the splenocytes were prepared from the recipient mice and assayed for their CTL activity against P815 (H-2 d ) and EL-4 (H-2 b ) in a standard 51 Cr release assay without further stimulation in vitro. ( FIG. 20C .)

Non-irradiated BDF1 mice were injected i.v. with 7×10 7 cells of B6 LTα −/− or LTα +/+ splenocytes on day 0. BDF1 mice injected with BDF1 splenocytes (7×10 7 cells) were used as controls (F1-F1). On day 11, CTL activity of recipient splenocytes were assessed as described above. ( FIG. 20D .)

Purified T cells (1×10 6 cells/ml) of B6 LTα −/− splenocytes were cultured with irradiated BALB/c splenocytes (1×10 6 cells/ml) in the presence of 25 μg of LTβR-Ig or control human IgG1. After 5 days, the CTL activity against C26 (H-2 d ) and EL-4 (H-2 b ) was examined in a standard 51 Cr release assay. ( FIG. 20E .) The results are expressed as the means +SD of triplicate wells ( FIGS. 20C , 20 D, and 20 E).

FIGS. 21A-B . FIG. 21 shows cytokine production by modulation of AIM II costimulatory pathway. Purified T cells (1×10 6 cells/ml) were stimulated with immobilized AIM II.flag fusion protein (3.2 μg/ml) in 96-well flat-bottomed microplates in the presence or absence of immobilized anti-CD3 (0.5 μg/ml). After 48 hours, the culture supernatants were collected and the cytokine production was assessed by sandwich ELISA. ( FIG. 21A .) The data shown in FIG. 21B was generated using T cells (1×10 6 cells/ml) purified from LTα −/− splenocytes cultured with irradiated BALB/c splenocytes (1×10 6 cells/ml) in the presence of 25 μg/ml of LTβR-Ig fusion protein or control human IgG1. After 72 hours, the culture supernatants were harvested and the cytokine production was assessed by sandwich ELISA. IL-10 was not detectable (n.d.) (<0.3 ng/ml) in all wells. ( FIG. 21B .).

FIGS. 22A-F shows that AIM II depletion from culture medium, using an anti-AIM II antibody (αTL-5 Ab), results in decreased activation of CD8 + T-cells as measured by γ-IFN secretion, compared to the anti-DNP antibody isotype negative control (αDNP Ab). CTLA-4.Fc fusion protein, a known stimulator of IFN-γ secretion, is included as a positive control. The data shown were obtained when naïve T cells (CD45RA + Tc; FIGS. 22A , 22 E, and 22 F), effector—memory T cells (CD45RO + Tc; FIG. 22B ), CD4 + (CD4 + Tc; FIG. 22D ), and CD8 + T-cells (CD8 + Tc; FIG. 22C ) were activated in vitro by allogeneic mature (mDC—( FIGS. 22E and 22F )) and immature Dendritic cells (iDC—( FIGS. 22A-D )). The data indicate that AIM II depletion significantly reduced γ-IFN secretion by naïve and CD8 + T-cells, thereby suggesting a specific role for AIM II in the activation and maturation of CD8 + T-cells. See e.g., Example 25.

FIG. 23 shows that AIM II depletion from culture medium, using an anti-AIM II antibody, results in decreased proliferation of T-cells as measured by [ 3 H]-thymidine incorporation. Human T-cells were separated by negative selection on magnetic beads and grown on anti-CD3 and anti-CD28 coated plates without treatment (untx) or in the presence of anti-DNP (PKI6.3), anti-AIM II (α-AIM II), or anti-LTβR (α-LTβR) antibodies at 1 μg/ml. The cells were seeded at 100,000 /well and grown for 4 days. The data indicate that AIM II depletion significantly reduced T-cell proliferation, thereby suggesting a specific role for AIM II in the activation and maturation of T-cells. See e.g., Example 26.

FIG. 24 shows that AIM II depletion from culture medium, using an anti-AIM II antibody, results in decreased γ-IFN secretion by T-cells as measured by ELISA. Human T-cells were separated by negative selection on magnetic beads and grown on anti-CD3 and anti-CD28 coated plates without treatment (untx) or in the presence of anti-DNP (PKI6.3), anti-AIM II (α-AIM II), or anti-LTβR (α-LTβR) antibodies at 1 μg/ml. The cells were seeded at 100,000 /well and grown for 4 days. The data indicate that AIM II depletion significantly reduced T-cell production of γ-IFN more profoundly than the positive control α-LTβR antibody, thereby suggesting a specific role for AIM II in the activation and maturation of T-cells. See e.g., Example 26.

›DETAILED DESCRIPTION · 1 of 68

The present invention provides isolated nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide encoding an AIM II polypeptide having the amino acid sequence shown in FIGS. 1A and 1B (SEQ ID NO:2), which was determined by sequencing a cDNA. The AIM II protein of the present invention shares sequence homology with human TNF-α (SEQ ID NO:3), human TNF-β (SEQ ID NO:4), human lymphotoxin (SEQ ID NO:5) and human Fas Ligand (SEQ ID NO:6) ( FIGS. 2A-2F ). The nucleotide sequence shown in FIGS. 1A and 1B (SEQ ID NO:1) were obtained by sequencing the cDNA, which was deposited on Aug. 22, 1996, at the American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110-2209, USA, and given accession number 97689. The deposited cDNA is contained in the pBluescript SK(-) plasmid (Stratagene, La Jolla, Calif.). The nucleotide sequence shown in FIGS. 1C and 1D was obtained by sequencing the cDNA, which was deposited on Mar. 15, 1996, at the American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110-2209, USA, and given accession number 97483.

Nucleic Acid Molecules

Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.

Using the information provided herein, such as the nucleotide sequence in FIGS. 1A and 1B , a nucleic acid molecule of the present invention encoding an AIM II polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material. Illustrative of the invention, the nucleic acid molecule described in FIGS. 1A and 1B (SEQ ID NO:1) was discovered in a cDNA library derived from human macrophage ox LDL (HMCCB64). The gene was also identified in cDNA libraries from activated T-cells (HT4CC72). The determined nucleotide sequence of the AIM II cDNA of FIGS. 1A and 1B (SEQ ID NO:1) contains an open reading frame encoding a protein of 240 amino acid residues, with an initiation codon at positions 49-51 of the nucleotide sequence in FIGS. 1A and 1B (SEQ ID NO:1), an extracellular domain comprising, or alternatively consisting of, amino acid residues from about 60 to about 240 in FIGS. 1A and 1B (SEQ ID NO:2), a transmembrane domain comprising, or alternatively consisting of, amino acid residues from about 37 to about 59 in FIGS. 1A and 1B (SEQ ID NO:2), a intracellular domain comprising, or alternatively consisting of, amino acid residues from about 1 to about 36 in FIGS. 1A and 1B (SEQ ID NO:2) and a deduced molecular weight of about 26.4 kDa. The AIM II protein shown in FIGS. 1A and 1B (SEQ ID NO:2) is about 27% identical and about 51% similar to the amino acid sequence of human Fas Ligand ( FIGS. 2A-2F ) and is about 26% identical and about 47% similar to the amino acid sequence of human TNF-α ( FIGS. 2A-2F ). TNF-ligand like molecules function as dimers, given that AIM II is homologous to TNF-ligand like molecules, it is likely that it also functions as a homodimer.

As one of ordinary skill would appreciate, due to the possibilities of sequencing errors discussed above, the predicted AIM II polypeptide encoded by the deposited cDNA comprises about 240 amino acids, but maybe anywhere in the range of 230-250 amino acids. It will further be appreciated that, depending on the criteria used, concerning the exact “address” of, the extracellular, intracellular and transmembrane domains of the AIM II polypeptide differ slightly. For example, the exact location of the AIM II extracellular domain in FIGS. 1A and 1B (SEQ ID NO:2) may vary slightly (e.g., the address may “shift” by about 1 to 5 residues) depending on the criteria used to define the domain.

As indicated, nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically. The DNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.

By “isolated” nucleic acid molecule(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.

›DETAILED DESCRIPTION · 2 of 68

However, a nucleic acid contained in a clone that is a member of a library (e.g., a genomic or cDNA library) that has not been isolated from other members of the library (e.g., in the form of a homogeneous solution containing the clone and other members of the library) or a chromosome isolated or removed from a cell or a cell lysate (e.g., a “chromosome spread,” as in a karyotype), is not “isolated” for the purposes of the invention. As discussed further herein, isolated nucleic acid molecules according to the present invention may be produced naturally, recombinantly, or synthetically.

Isolated nucleic acid molecules of the present invention include DNA molecules comprising, or alternatively consisting of, an open reading frame (ORF) shown in FIGS. 1A and 1B (SEQ ID NO:1) or FIGS. 1C and 1D (SEQ ID NO:38); DNA molecules comprising, or alternatively consisting of, the coding sequence for the AIM II protein shown in FIGS. 1A and 1B (SEQ ID NO:2) or FIGS. 1C and 1D (SEQ ID NO:39); and DNA molecules which comprise, or alternatively consist of, a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the AIM II protein. Of course, the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate such degenerate variants.

Nucleic acid molecules according to the present invention further include those encoding the full-length AIM II polypeptide lacking the N-terminal methionine.

In addition, the invention provides a nucleic acid molecule having a nucleotide sequence related to a portion of SEQ ID NO:1 which has been determined from the following related cDNA: HT4CC72R (SEQ ID NO:20).

In another aspect, the invention provides isolated nucleic acid molecules encoding the AIM II polypeptide having an amino acid sequence encoded by the cDNA contained in the plasmid deposited as ATCC Deposit No. 97689 on August 22, 1996, or by the cDNA contained in the plasmid deposited as ATCC Deposit No. 97483 on Mar. 15, 1996. Preferably, this nucleic acid molecule will encode the polypeptide encoded by one of the above-described deposited cDNAs. The invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in FIGS. 1A and 1B (SEQ ID NO:1) or FIGS. 1C and 1D (SEQ ID NO:38) or the nucleotide sequence of one of the AIM II cDNAs contained in the above-described deposited plasmids, or a nucleic acid molecule having a sequence complementary to one of the above sequences. Such isolated molecules, particularly DNA molecules, are useful, for example, as probes for gene mapping, by in situ hybridization with chromosomes, and for detecting expression of the AIM II gene in human tissue, for instance, by Northern blot analysis.

Further embodiments of the invention include isolated nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide having a nucleotide sequence at least 80% identical, and more preferably at least 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to (a) a nucleotide sequence encoding the AIM II polypeptide having the complete amino acid sequence in FIGS. 1A and 1B (SEQ ID NO:2) or FIGS. 1C and 1D (SEQ ID NO:39); (b) a nucleotide sequence encoding the AIM II polypeptide having the amino acid sequence in FIGS. 1A and 1B (SEQ ID NO:2), but lacking the N-terminal methionine; (c) a nucleotide sequence encoding the AIM II polypeptide having the complete amino acid sequence encoded by the cDNA contained in ATCC Deposit No.97689 or ATCC Deposit 97483; (d) a nucleotide sequence encoding the AIM II polypeptide extracellular domain; (e) a nucleotide sequence encoding the AIM II polypeptide transmembrane domain; (f) a nucleotide sequence encoding the AIM II polypeptide intracellular domain; (g) a nucleotide sequence encoding a soluble AIM II polypeptide having the extracellular and intracellular domains but lacking the transmembrane domain; and (h) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f) or (g) above.

Additional embodiments of the invention include isolated nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide having a nucleotide sequence at least 80% identical, and more preferably at least 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to (a) a nucleotide sequence encoding the AIM II polypeptide having the sequence of amino acids about 1 to about 208 in FIGS. 1C and 1D (SEQ ID NO:39); (b) a nucleotide sequence encoding the AIM II polypeptide having the sequence of amino acids about 7 to about 208 in FIGS. 1C and 1D (SEQ ID NO:39); (c) a nucleotide sequence encoding the AIM II polypeptide having the sequence of amino acids about 34 to about 208 in FIGS. 1C and 1D (SEQ ID NO:39); and (d) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), or (c) above.

As a practical matter, whether any particular nucleic acid molecule is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the nucleotide sequence shown in FIGS. 1A and 1B (SEQ ID NO:1) or FIGS. 1C and 1D (SEQ ID NO: 38) or to the nucleotide sequence of the deposited cDNA can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (198 1), to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.

›DETAILED DESCRIPTION · 3 of 68

By a polynucleotide having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the AIM II polypeptide. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. The query sequence may be an entire sequence shown in FIGS. 1A and 1B or FIGS. 1C and 1D , the ORF (open reading frame), or any fragment specified as described herein.

As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the presence invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. ( Comp. App. Biosci. 6:237-245 (1990)). In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present invention. Only bases outside the 5′ and 3′ bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.

For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5′ end of the subject sequence and therefore, the FASTDB alignment does not show a match/alignment of the first 10 bases at the 5′ end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5′ and 3′ ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5′ or 3′ of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5′ and 3′ of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to made for the purposes of the present invention.

The present application is directed to nucleic acid molecules at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in FIGS. 1A and 1B (SEQ ID NO:1) or FIGS. 1C and 1D (SEQ ID NO:38) or to the nucleic acid sequence of the deposited cDNAs, irrespective of whether they encode a polypeptide having AIM II activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having AIM II activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer. Uses ofthe nucleic acid molecules ofthe present invention that do not encode a polypeptide having AIM II activity include, inter alia, (1) isolating the AIM II gene or allelic variants thereof in a cDNA library; (2) in situ hybridization (e.g., “FISH”) to metaphase chromosomal spreads to provide precise chromosomal location of the AIM II gene, as described in Verma et al., Human Chromosomes: A Manual of Basic Techniques , Pergamon Press, New York (1988); and (3) Northern Blot analysis for detecting AIM II mRNA expression in specific tissues.

Preferred, however, are nucleic acid molecules having sequences at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in FIGS. 1A and 1B (SEQ ID NO:1) or FIGS. 1C and 1D (SEQ ID NO:38) or to the nucleic acid sequence of one of the deposited cDNAs which do, in fact, encode a polypeptide having AIM II protein activity. By “a polypeptide having AIM II activity” is intended polypeptides exhibiting activity similar, but not necessarily identical, to an activity of the AIM II protein of the invention, as measured in a particular biological assay. For example, AIM II protein cytotoxic activity can be measured using propidium iodide staining to demonstrate apoptosis as described by Zarres et al., Cell 70: 31-46 (1992). Alternatively, AIM II induced apoptosis can also be measured using TUNEL staining as described by Gavierli et al., J. Cell. Biol. 119: 493-501 (1992). Further included within the scope of the invention are polypeptides encoded by these polynucleotides.

›DETAILED DESCRIPTION · 4 of 68

Briefly, the propidium iodide staining is performed as follows. Cells either from tissue or culture are fixed in formaldehyde, cut into frozen sections and stained with propidium iodide. The cell nuclei are visualized by propidium iodide using confocal fluorescent microscopy. Cell death is indicated by pyknotic nuclei (chromosome clumping, shrinking and/or fragmentation of nuclei).

Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of one of the deposited cDNAs or the nucleic acid sequence shown in FIGS. 1 A and 1 B (SEQ ID NO:1) or FIGS. 1C and 1D (SEQ ID NO:38) will encode a polypeptide “having AIM II protein activity.” In fact, since degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having AIM II protein activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid).

For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, J. U. et al., “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990), wherein the authors indicate that proteins are surprisingly tolerant of amino acid substitutions.

Preferably, the polynucleotides and polynucleotide fragments of the invention encode a polypeptide which demonstrates an AIM II functional activity. By a polypeptide demonstrating an AIM II “functional activity” is meant, a polypeptide capable of displaying one or more known functional activities associated with a full-length and/or secreted AIM II polypeptide. Such functional activities include, but are not limited to, biological activity, antigenicity, ability to bind (or compete with a polypeptide for binding) to an anti-AIM II antibody, immunogenicity (ability to generate antibody which binds to a polypeptide), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide (e.g., TR2 (International Publication No. WO 96/34095), LT-β receptor, TR6 (International Publication No. WO 98/30694), and CD27)).

The functional activity of AIM II polypeptides, and fragments, variants derivatives, and analogs thereof, can be assayed by various methods.

For example, in one embodiment where one is assaying for the ability to bind or compete with full-length polypeptide for binding to anti-AIM II antibody, various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.

In another embodiment, where a ligand is identified (e.g., DR5 (See, International Publication No. WO 98/41629), TRIO (See, International Publication No. WO 98/54202), 312C2 (See, International Publication No. WO 98/06842), and TR11, TR11SV1, and TR11SV2 (See, U.S. application Ser. No. 09/176,200)), or the ability of a polypeptide fragment, variant or derivative of the invention to multimerize is being evaluated, binding can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky, E. et al., 1995, Microbiol. Rev. 59:94-123. In another embodiment, physiological correlates of binding to its substrates (signal transduction) can be assayed.

Other methods will be known to the skilled artisan and are within the scope of the invention.

The present invention is further directed to polynucleotides comprising, or alternatively consisting of, fragments of the isolated nucleic acid molecules described herein. By a fragment of an isolated nucleic acid molecule having the nucleotide sequence of one of the deposited cDNAs or the nucleotide sequence shown in FIGS. 1A and 1B (SEQ ID NO:1) or FIGS. 1C and 1D (SEQ ID NO:38) is intended fragments at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein. In this context “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) nucleotides. Of course, larger fragments 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1125 or 1150 nt in length are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of one of the deposited cDNAs or as shown in FIGS. 1A and 1B (SEQ ID NO:1) or FIGS. 1C and ID (SEQ ID NO:38). By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of one of the deposited cDNAs or the nucleotide sequence as shown in FIGS. 1A and 1B (SEQ ID NO: 1) or FIGS. 1C and 1D (SEQ ID NO:38).

›DETAILED DESCRIPTION · 5 of 68

The present invention is further directed to polynucleotides comprising, or alternatively consisting of, fragments of isolated nucleic acid molecules which encode subportions of AIM II domains. In particular, the invention provides polynucleotides comprising, or alternatively consisting of, the nucleotide sequences of a member selected from the group consisting of nucleotides 49-108, 59-118, 69-128, 79-138, 89-148, 99-156, 109-156, 109-168, 119-178, 129-188, 139-198, 149-208, 157-216, 157-225, 168-227, 178-237, 188-247, 198-257, 208-267, 218-277, 226-285, 236-295, 246-305, 256-315, 266-325, 276-335, 286-345, 296-355, 306-365, 316-375, 326-385, 336-395, 346-405, 356-415, 366-425, 376-435, 386-445, 396-455, 409-469, 456-515, 476-536, 496-556, 516-575, 536-595, 576-635, 596-655, 636-695, 656-715, 696-755, 706-765, and 716-768 of SEQ ID NO:1.

The present invention is further directed to polynucleotides comprising, or alternatively consisting of, isolated nucleic acid molecules which encode domains of AIM II. In one aspect, the invention provides polynucleotides comprising, or alternatively consisting of, nucleic acid molecules which encode beta-sheet regions of AIM II set out in Table 2. Representative examples of such polynucleotides comprise, or alternatively consist of, nucleic acid molecules which encode a polypeptide having an amino acid sequence selected from the group consisting of amino acid residues from about 7 to about 14, amino acid residues from about 18 to about 23, amino acid residues from about 17 to about 25, amino acid residues from about 33 to about 46, amino acid residues from about 35 to about 39, amino acid residues from about 57 to about 60, amino acid residues from about 67 to about 72, amino acid residues from about 102 to about 107, amino acid residues from about 121 to about 126, amino acid residues from about 131 to about 166, amino acid residues from about 141 to about 152, amino acid residues from about 158 to about 169, amino acid residues from about 213 to about 221, and amino acid residues from about 232 to about 240 of SEQ ID NO:2. The invention is further directed to isolated polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of amino acid residues from about 7 to about 14, amino acid residues from about 18 to about 23, amino acid residues from about 17 to about 25, amino acid residues from about 33 to about 46, amino acid residues from about 35 to about 39, amino acid residues from about 57 to about 60, amino acid residues from about 67 to about 72, amino acid residues from about 102 to about 107, amino acid residues from about 121 to about 126, amino acid residues from about 131 to about 166, amino acid residues from about 141 to about 152, amino acid residues from about 158 to about 169, amino acid residues from about 213 to about 221, and amino acid residues from about 232 to about 240 of SEQ ID NO:2. In this context “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) amino acids.

Nucleic acid fragments of the present invention include nucleic acid molecules encoding beta-sheet regions of the AIM II protein, as well as isolated nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide having a nucleotide sequence at least 80% identical, and more preferably at least 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to nucleic acid molecules encoding beta-sheet regions of the AIM II protein. Polynucleotides encoding polypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to beta-sheet regions are also with the scope of the invention, as are the polypeptides encoded by these polynucleotides.

The present invention is also directed to polynucleotides comprising, or alternatively consisting of, isolated nucleic acid molecules which encode a polypeptide having an amino acid sequence selected from the group consisting of amino acid residues from about 94 to about 100, amino acid residues from about 121 to about 124, amino acid residues from about 127 to about 135, amino acid residues from about 139 to about 149, amino acid residues from about 160 to about 168, amino acid residues from about 175 to about 185, amino acid residues from about 197 to about 209, amino acid residues from about 213 to about 220, and amino acid residues from about 232 to about 240 of SEQ ID NO:2. The invention is further directed to isolated polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of amino acid residues from about 94 to about 100, amino acid residues from about 121 to abdut 124, amino acid residues from about 127 to about 135, amino acid residues from about 139 to about 149, amino acid residues from about 160 to about 168, amino acid residues from about 175 to about 185, amino acid residues from about 197 to about 209, amino acid residues from about 213 to about 220, and amino acid residues from about 232 to about 240 of SEQ ID NO:2. In this context “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) amino acids.

Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding epitope-bearing portions of the AIM II protein. In particular, such nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising, or alternatively consisting of, one, two, three, four, five, or more amino acid sequences selected from amino acid residues from about 13 to about 20 in FIGS. 1A and 1B (SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 23 to about 36 in FIG. 1 (SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 69 to about 79 in FIGS. 1A and 1B (SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 85 to about 94 in FIGS. 1A and 1B (SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 167 to about 178 in FIGS. 1A and 1B (SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 184 to about 196 in FIGS. 1A and 1B (SEQ ID NO: 2); and a polypeptide comprising, or alternatively consisting of, amino acid residues from about 221 to about 233 in FIGS. 1A and 1B (SEQ ID NO:2). In this context “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) amino acids. The inventors have determined that the above polypeptide fragments are antigenic regions of the AIM II protein. Methods for determining other such epitope-bearing portions of the AIM II protein are described in detail below. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

›DETAILED DESCRIPTION · 6 of 68

As is discussed be low in more detail, AIM II polynucleotides may be used in accordance with the present invention for a variety of applications, particularly those that make use of the chemical and biological properties of the AIM II. Among these applications is autoimmune disease, immunodeficiency and aberrant cellular proliferation. Additional applications relate to diagnosis and to treatment of disorders of cells, tissues and organisms.

In another aspect, the invention provides an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, the complement of a polynucleotide fragment described herein, or the cDNA plasmid contained in ATCC Deposit 97689 or ATCC Deposit 97483. By “stringent hybridization conditions” is intended overnight incubation at 42° C. in a solution comprising, or alternatively consisting of: 50% fornmamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65 C.

By a polynucleotide which hybridizes to a “portion” of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 nt of the reference polynucleotide. In this context “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) nucleotides. These are useful as diagnostic probes and primers as discussed above and in more detail below.

By a portion of a polynucleotide of “at least 20 nt in length,” for example, is intended 20 or more contiguous nucleotides from the nucleotide sequence of the reference polynucleotide (e.g., one of the deposited cDNAs or the nucleotide sequence as shown in FIGS. 1A and 1B (SEQ ID NO:1) or FIGS. 1C and 1D (SEQ ID NO:38)).

Of course, a polynucleotide which hybridizes only to a poly A sequence (such as the 3 terminal poly(A) tract of the AIM II cDNA shown in FIGS. 1A and 1B (SEQ ID NO: 1) or FIGS. 1C and 1D (SEQ ID NO:38)), or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA generated from an oligo-dT primed cDNA library).

As indicated, nucleic acid molecules of the present invention which encode an AIM II polypeptide may include, but are not limited to those encoding the amino acid sequence of the polypeptide, by itself; the coding sequence for the polypeptide and additional sequences, such as those encoding a leader or secretory sequence, such as a pre-, or pro- or pre-pro-protein sequence; the coding sequence of the polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5′ and 3′ sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example—ribosome binding and stability of mRNA; an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities. Thus, the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. The “HA” tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al., Cell 37:-767 (1984). As discussed below, other such fusion proteins include the AIM II fused to Fc at the N- or C-terminus.

The present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of the AIM II protein. Variants may occur naturally, such as a natural allelic variant. By an “allelic variant” is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II Lewin, B., ed., John Wiley & Sons, New York (1985).

Non-naturally occurring variants may be produced using art-known mutagenesis techniques, which include, but are not limited to: oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site-directed mutagenesis (see, e.g., Carter et al., Nucl. Acids Res. 13:4331 (1986); and Zoller et al., Nucl. Acids Res. 10:6487 (1982)), cassette mutagenesis (see, e.g., Wells et al., Gene 34:315 (1985)), restriction selection mutagenesis (see, e.g., Wells et al., Philos. Trans. R. Soc. London Ser.A 317:415 (1986)).

Such variants include those produced by nucleotide substitutions, deletions or additions which may involve one or more nucleotides. The variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the AIM II protein or portions thereof. Also especially preferred in this regard are conservative substitutions.

›DETAILED DESCRIPTION · 7 of 68

Vectors and Host Cells

The present invention also relates to vectors which include the isolated DNA molecules of the present invention, host cells which are genetically engineered with the recombinant vectors, and the production of AIM II polypeptides or fragments thereof by recombinant techniques.

The polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome-binding site for translation. The coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.

In addition to the use of expression vectors in the practice of the present invention, the present invention further includes novel expression vectors comprising operator and promoter elements operatively linked to nucleotide sequences encoding a protein of interest. One example of such a vector is pHE4-5 which is described in detail below.

As summarized in FIGS. 10 and 11 , components of the pHE4-5 vector (SEQ ID NO:50) include: 1) a neomycin-phosphotransferase gene as a selection marker, 2) an E. coli origin of replication, 3) a T5 phage promoter sequence, 4) two lac operator sequences, 5) a Shine-Delgamo sequence, 6) the lactose operon repressor gene (lacIq). The origin of replication (oriC) is derived from pUC19 (LTI, Gaithersburg, Md.). The promoter sequence and operator sequences were made synthetically. Synthetic production of nucleic acid sequences is well known in the art. C LONTECH 95/96 Catalog, pages 215-216, C LONTECH, 1020 East Meadow Circle, Palo Alto, Calif. 94303. A nucleotide sequence encoding AIM II (SEQ ID NO:1), is operatively linked to the promoter and operator by inserting the nucleotide sequence between the NdeI and Asp7 18 sites of the pHE4-5 vector.

As noted above, the pHE4-5 vector contains a lacIq gene. LacIq is an allele of the lacI gene which confers tight regulation of the lac operator. Amann, E. et al., Gene 69:301-315 (1988); Stark, M., Gene 51:255-267 (1987). The lacIq gene encodes a repressor protein which binds to lac operator sequences and blocks transcription of down-stream (i.e., 3′) sequences. However, the lacIq gene product dissociates from the lac operator in the presence of either lactose or certain lactose analogs, e.g., isopropyl B-D-thiogalactopyranoside (IPTG). AIM II thus is not produced in appreciable quantities in uninduced host cells containing the pHE4-5 vector. Induction of these host cells by the addition of an agent such as IPTG, however, results in the expression of the AIM II coding sequence.

The promoter/operator sequences of the pHE4-5 vector (SEQ ID NO:51) comprise a T5 phage promoter and two lac operator sequences. One operator is located 5′ to the transcriptional start site and the other is located 3′ to the same site. These operators, when present in combination with the lacIq gene product, confer tight repression of down-stream sequences in the absence of a lac operon inducer, e.g., IPTG. Expression of operatively linked sequences located down-stream from the lac operators may be induced by the addition of a lac operon inducer, such as IPTG. Binding of a lac inducer to the lacIq proteins results in their release from the lac operator sequences and the initiation of transcription of operatively linked sequences. Lac operon regulation of gene expression is reviewed in Devlin, T., T EXTBOOK OF B IOCHEMISTRY WITH C LINICAL C ORRELATIONS, 4th Edition (1997), pages 802-807.

The pHE4 series of vectors contain all of the components of the pHE4-5 vector except for the AIM II coding sequence. Features of the pHE4 vectors include optimized synthetic T5 phage promoter, lac operator, and Shine-Delgamo sequences. Further, these sequences are also optimally spaced so that expression of an inserted gene may be tightly regulated and high level of expression occurs upon induction.

Among known bacterial promoters suitable for use in the production of proteins of the present invention include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous Sarcoma Virus (RSV), and metallothionein promoters, such as the mouse metallothionein-I promoter.

The pHE4-5 vector also contains a Shine-Delgarno sequence 5′ to the AUG initiation codon. Shine-Delgamo sequences are short sequences generally located about 10 nucleotides up-stream (i.e., 5′) from the AUG initiation codon. These sequences essentially direct prokaryotic ribosomes to the AUG initiation codon.

›DETAILED DESCRIPTION · 8 of 68

Thus, the present invention is also directed to expression vector useful for the production of the proteins of the present invention. This aspect of the invention is exemplified by the pHE4-5 vector (SEQ ID NO:50).

Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.

Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986).

The polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.

A preferred fusion protein comprises a heterologous region from immunoglobulin that is useful to solubilize proteins. For example, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232 262). On the other hand, for some uses it would be desirable to be able to delete the Fe part after the fusion protein has been expressed, detected and purified in the advantageous manner described. This is the case when Fe portion proves to be a hindrance to use in therapy and diagnosis, for example when the fusion protein is to be used as antigen for immunizations. In drug discovery, for example, human proteins, such as, hIL-5-receptor has been fused with Fe portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. See, D. Bennett et al., Journal of Molecular Recognition, Vol. 8:52-58 (1995) and K. Johanson et al., The Journal of Biological Chemistry , Vol. 270, No. 16:9459-9471 (1995).

The AIM II protein can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification. Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.

In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., AIM II coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with AIM II polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous AIM II polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous AIM II polynucleotide sequences via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; WO 96/29411, published Sep. 26, 1996; WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).

AIM II Polypeptides and Fragments

The invention further provides an isolated AIM II polypeptide having the amino acid sequence encoded by one of the deposited cDNAs, or the amino acid sequence in FIGS. 1A and 1B (SEQ ID NO:2) or FIGS. 1C and 1D (SEQ ID NO:39), or a peptide or polypeptide comprising, or alternatively consisting of, a portion of the above polypeptides.

The polypeptides of the present invention include the polypeptide encoded by one of the deposited cDNAs, the polypeptide of FIGS. 1A and 1B (SEQ ID NO:2) or FIGS. 1C and 1D (SEQ ID NO:39), the polypeptide of FIGS. 1A and 1B (SEQ ID NO:2) lacking the N-terminal methionine, the extracellular domain, the transmembrane domain, the intracellular domain, soluble polypeptides comprising, or alternatively consisting of, all or part of the extracellular and intracellular domains but lacking the transmembrane domain, as well as polypeptides which are at least 80% identical, more preferably at least 85%, 90%, 92% or 95% identical, still more preferably at least 96%, 97%, 98% or 99% identical to the polypeptide encoded by one of the deposited cDNAs, to the polypeptide of FIGS. 1A and 1B (SEQ ID NO:2) or FIGS. 1C and 1D (SEQ ID NO:39), and also include portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids.

›DETAILED DESCRIPTION · 9 of 68

The polypeptides of the present invention further include (a) the AIM II polypeptide having the sequence of amino acids about 1 to about 208 in FIGS. 1C and 1D (SEQ ID NO:39); (b) the AIM II polypeptide having the sequence of amino acids about 7 to about 208 in FIGS. 1C and 1D (SEQ ID NO:39); and (c) the AIM II polypeptide having the sequence of amino acids about 34 to about 208 in FIGS. 1C and 1D (SEQ ID NO:39), as well as polypeptides; which are at least 80% identical, more preferably at least 85%, 90%, 92% or 95% identical, still more preferably at least 96%, 97%, 98% or 99% identical to the polypeptide encoded by one of the deposited cDNAs, to the polypeptide of FIGS. 1A and 1B (SEQ ID NO:2) or FIGS. 1C and 1D (SEQ ID NO:39), and also include portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids.

By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a reference amino acid sequence of an AIM II polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of the AIM II polypeptide. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence shown in FIGS. 1A and 1B (SEQ ID NO:2) or FIGS. 1C and 1D (SEQ ID NO:39) or to the amino acid sequence encoded by one of the deposited cDNAs can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.

By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, (indels) or substituted with another amino acid. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequences shown in Table 1 or to the amino acid sequence encoded by the deposited DNA can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. ( Comp. App. Biosci. 6:237-245 (1990)). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues of the query (reference) sequence that extend past the N- or C-termini of the subject sequence are considered for the purposes of manually adjusting the percent identity score. That is, only residues which are not matched/aligned with the N- or C-termini of the query sequence are counted when manually adjusting the percent identity score.

›DETAILED DESCRIPTION · 10 of 68

For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a match/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to be made for the purposes of the present invention.

The natural processed form of AIM II that was affinity purified on an LT-β receptor column from conditioned media of MCA-38 cells transformed with full length AIM II cDNA is Leu-83 to Val-240 in SEQ ID NO:2. (See, Example 10). However, it appears that AIM II is processed differently in COS cells, producing an AIM II that is cleaved between Glu-67 and Met-68 to yield a polypeptide having amino acids 68-240 in SEQ ID NO: 2. In addition, COS cells also cleave the AIM II between Met-68 and Val-69, resulting a polypeptide having amino acids 69-240 in SEQ ID NO:2.

The polypeptides of the present invention are preferably provided in an isolated form. By “isolated polypeptide” is intended a polypeptide removed from its native environment. Thus, a polypeptide produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention. Also intended as an “isolated polypeptide” are polypeptides that have been purified, partially or substantially, from a recombinant host. For example, a recombinantly produced version of the AIM II polypeptide can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988).

The AIM II polypeptides of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomers and multimers of the AIM II polypeptides of the invention, their preparation, and compositions (preferably, pharmaceutical compositions) containing them. In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In additional embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.

Multimers encompassed by the invention may be homomers or heteromers. As used herein, the term homomer, refers to a multimer containing only AIM II polypeptides of the invention (including AIM II fragments, variants, splice variants, and fusion proteins, as described herein). These homomers may contain AIM II polypeptides having identical or different amino acid sequences. In a specific embodiment, a homomer of the invention is a multimer containing only AIM II polypeptides having an identical amino acid sequence. In another specific embodiment, a homomer of the invention is a multimer containing AIM II polypeptides having different amino acid sequences. In specific embodiments, the multimer of the invention is a homodimer (e.g., containing AIM II polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing AIM II polypeptides having identical and/or different amino acid sequences). In additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., polypeptides of different proteins) in addition to the AIM II and AIM II polypeptides of the invention. In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.

Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation. Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution. In another embodiment, heteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution. In other embodiments, multimers of the invention are formed by covalent associations with and/or between the AIM II polypeptides of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in SEQ ID NO:2 or SEQ ID NO:39, or contained in the polypeptide encoded by a cDNA of the deposits designated as ATCC Accession 97689 or 97483). In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide. In another instance, the covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in an AIM II fusion protein. In one example, covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., U.S. Pat. No. 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in an AIM II-Fc fusion protein of the invention (as described herein). In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another TNF family ligand/receptor member that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g., WO 98/49305, the contents of which are herein incorporated by reference in its entirety).

›DETAILED DESCRIPTION · 11 of 68

The multimers of the invention may be generated using chemical techniques known in the art. For example, polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Further, polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art. In one embodiment, polypeptides contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In a specific embodiment, polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain (or hydrophobic or signal peptide) and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).

The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions. For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fe region, or portion thereof. The antibody portion fused to a polypeptide of the present invention may comprise the hinge region, CH1 domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof. The polypeptides of the present invention may be fused or conjugated to the above antibody portions to increase the in vivo half-life of the polyp eptides or for use in immunoassays using methods known in the art. The polypeptides may also be fused or conjugated to the above antibody portions to form multimers. For example, Fe portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions. Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,112,946; EP 0 307 434, EP 0 367 166; WO 96/04388, WO 91/06570; Ashkenazi, A. et al. PNAS 88:10535-10539 (1991); Zheng, X. X. et al. J. Immunol. 154:5590-5600 (1995); and Vil, H. et al. PNAS 89:11337-11341 (1992) (said references incorporated by reference in their entireties).

As used herein the term “AIM II” polypeptide includes membrane-bound proteins (comprising, or alternatively consisting of, a cytoplasmic domain, a transmembrane domain, and an extracellular domain) as well as truncated proteins that retain the AIM II functional activity. In one embodiment, soluble AIM II polypeptides comprise, or alternatively consisting of, all or part of the extracellular domain of an AIM II protein, but lack the transmembrane region that would cause retention of the polypeptide on a cell membrane. Soluble AIM II may also include part of the transmembrane region or part of the cytoplasmic domain or other sequences, provided that the soluble AIM II protein is capable of being secreted. A heterologous signal peptide can be fused to the N-terminus of the soluble AIM II polypeptide such that the soluble AIM II polypeptide is secreted upon expression.

The polypeptide of the present invention have uses that include, but are not limited to, functioning as a molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art.

It will be recognized in the art that some amino acid sequences of the AIM II polypeptide can be varied without significant effect of the structure or function of the protein. If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the protein which determine activity.

Thus, the invention further includes variations of the AIM II polypeptide which show substantial AIM II polypeptide activity or which include regions of AIM II protein such as the protein portions discussed below. Such mutants include deletions, insertions, inversions, repeats, and type substitutions. As indicated above, guidance concerning which amino acid changes are likely to be phenotypically silent can be found in Bowie, J. U., et al., “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990). Polynucleotides encoding these fragments, derivatives or analogs are also encompassed by the invention.

›DETAILED DESCRIPTION · 12 of 68

Thus, the fragment, derivative or analog of the polypeptide of FIGS. 1A and 1B (SEQ ID NO:2) or FIGS. 1C and 1D (SEQ ID NO:39), or that encoded by one of the deposited cDNAs, maybe (i) one in which one or more of the amino acid residues (e.g., 3, 5, 8, 10, 15 or 20) are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or,(ii) one in which one or more of the amino acid residues includes a substituent group (e.g., 3, 5, 8, 10, 15 or 20), or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein. Polynucleotides encoding these fragments, derivatives or analogs are also encompassed by the invention.

Of particular interest are substitutions of charged amino acids with another charged amino acid and with neutral or negatively charged amino acids. The latter results in proteins with reduced positive charge to improve the characteristics of the AIM II protein. The prevention of aggregation is highly desirable. Aggregation of proteins not only results in a loss of activity but can also be problematic when preparing pharmaceutical formulations, because they can be immunogenic. (Pinckard et al., Clin Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993)).

The replacement of amino acids can also change the selectivity of binding to cell surface receptors. Ostade et al., Nature 361:266-268 (1993) describes certain mutations resulting in selective binding of TNF-α to only one of the two known types of TNF receptors. Thus, the AIM II receptor of the present invention may include one or more (e.g., 3, 5, 8, 10, 15 or 20) amino acid substitutions, deletions or additions, either from natural mutations or human manipulation.

As indicated, changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table 1).

Of course, the number of amino acid substitutions a skilled artisan would make depends on many factors, including those described above. Generally speaking, the number of substitutions for any given AIM II polypeptide will not be more than 50, 40, 30, 25, 20, 15, 10, 5 or 3.

Amino acids in the AIM II protein of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as receptor binding or in vitro, or in vitro proliferative activity. Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)).

Also included in the present invention are amino terminal deletion mutants. Such mutants include those comprising the amino acid sequence shown in SEQ ID NO:2 having a deletion of at least first N-terminal amino acid but not more than the first 114 N-terminal amino acid residues of SEQ ID NO:2. Alternatively, the deletion will include at least the first 35 N-terminal amino acid residues but not more than the first 114 N-terminal amino acid residues of SEQ ID NO:2. Alternatively, the deletion will include at least the first 59 N-terminal amino acid residues but not more than the first 114 N-terminal amino acid residues of SEQ ID NO:2. Alternatively, the deletion will include at least the first 67 N-terminal amino acid residues but not more than the first 114 N-terminal amino acid residues of SEQ ID NO:2. Alternatively, the deletion will include at least the first 68 N-terminal amino acid residues but not more than the first 114 N-terminal amino acid residues of SEQ ID NO:2. Alternatively, the deletion will include at least the first 73 N-terminal amino acid residues but not more than the first 114 N-terminal amino acid residues of SEQ ID NO:2. Alternatively, the deletion will include at least the first 82 N-terminal amino acid residues but not more than the first 114 N-terminal amino acid residues of SEQ ID NO:2. Alternatively, the deletion will include at least the first 100 N-terminal amino acid residues but not more than the first 114 N-terminal amino acid residues of SEQ ID NO:2. Polynucleotides encoding these deletion mutants are also encompassed by the invention, as are polynucleotides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to polynucleotides encoding the deletion mutants described above and polynucleotides encoding polypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to these deletion mutants. The present invention also encompasses the above polynucleotides fused to a heterologous polynucleotides and polypeptide expression products of these polynucleotides.

In addition to the ranges of N-terminal deletion mutants described above, the present invention is also directed to all combinations of the above described ranges. For example, the deletions of at least the first 59 N-terminal amino acid residues but not more than the first 67 N-terminal amino acid residues of SEQ ID NO:2; deletions of at least the first 59 N-terminal amino acid residues but not more than the first 68 N-terminal amino acid residues of SEQ ID NO:2; deletions of at least the first 59 N-terminal amino acid residues but not more than the first 73 N-terminal amino acid residues of SEQ ID NO:2; deletions of at least the first 59 N-terminal amino acid residues but not more than the first 82 N-terminal amino acid residues of SEQ ID NO:2; deletions of at least the first 59 N-terminal amino acid residues but not more than the first 100 N-terminal amino acid residues of SEQ ID NO:2; deletions of at least the first 67 N-terminal amino acid residues but not more than the first 73 N-terminal amino acid residues of SEQ ID NO:2; deletions of at least the first 67 N-terminal amino acid residues but not more than the first 82 N-terminal amino acid residues of SEQ ID NO: 2; deletions of at least the first 67 N-terminal amino acid residues but not more than the first 100 N-terminal amino acid residues of SEQ ID NO:2; deletions of at least the first 68 N-terminal amino acid residues but not more than the first 73 N-terminal amino acid residues of SEQ ID NO:2; deletions of at least the first 68 N-terminal amino acid residues but not more than the first 82 N-terminal amino acid residues of SEQ ID NO:2; deletions of at least the first 68 N-terminal amino acid residues but not more than the first 100 N-terminal amino acid residues of SEQ ID NO:2; deletions of at least the first 73 N-terminal amino acid residues but not more than the first 82 N-terminal amino acid residues of SEQ ID NO:2; deletions of at least the first 73 N-terminal amino acid residues but not more than the first 100 N-terminal amino acid residues of SEQ ID NO:2; deletions of at least the first 82 N-terminal amino acid residues but not more than the first 100 N-terminal amino acid residues of SEQ ID NO:2; etc. etc. etc. . . . Polynucleotides encoding these deletion mutants are also encompassed by the invention, as are polynucleotides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to polynucleotides encoding the deletion mutants described above and polynucleotides encoding polypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to these deletion mutants. The present invention also encompasses the above polynucleotides fused to a heterologous polynucleotides and polypeptide expression products of these polynucleotides.

›DETAILED DESCRIPTION · 13 of 68

Preferred AIM II polypeptides are those having one or more of the sequences shown below (numbering starts with the first amino acid in the protein (Met)): Gln(residue 60) to Val(residue 240); Leu(61) to Val(240); His(62) to Val(240); Trp(63) to Val(240); Arg(64) to Val(240); Leu(65) to Val(240); Gly(66) to Val(240); Glu(67) to Val(240); Met(68) to Val(240); Val(69) to Val(240); Thr(70) to Val(240); Arg(71) to Val(240); Leu(72) to Val(240); Pro(73) to Val(240); Asp(74) to Val(240); Gly(75) to Val(240); Pro(76) to Val(240); Ala(77) to Val(240); Gly(78) to Val(240); Ser(79) to Val(240); Trp(80) to Val(240); Glu(81) to Val(240); Gln(82) to Val(240); Leu(83) to Val(240); Ile(84) to Val(240); Gln(85) to Val(240); Glu(86) to Val(240); Arg(87) to Val(240); Arg(88) to Val(240); Ser(89) to Val(240); His(90) to Val(240); Glu(91) to Val(240); Val(92) to Val(240); Asn(93) to Val(240); Pro(94) to Val(240); Ala(95) to Val(240); Ala(96) to Val(240); His(97) to Val(240); Leu(98) to Val(240); Thr(99) to Val(240); Gly(100) to Val(240); Ala(101) to Val(240); Asn(102) to Val(240); Ser(103) to Val(240); Ser(104) to Val(240); Leu(105) to Val(240); Thr(106) to Val(240); Gly(107) to Val(240); Ser(108) to Val(240); Gly(109) to Val(240); Gly(110) to Val(240); Pro(111) to Val(240); Leu(112) to Val(240); Leu(113) to Val(240); Trp(114) to Val(240)

Particularly preferred embodiments include polypeptides comprising, or alternatively consisting of, one or more of the AIM II N-terminal deletions Gln-60 to Val-240 (AIM II (aa 60-240)), Met-68 to Val-240 (AIM II (aa 68-240)), Val-69 to Val-240 (AIM II (aa 69-240)), Asp-74,to Val-240 (AIM II (aa 74-240)), Leu-83 to Val-240 (AIM II (aa 83-240)), and Ala-101 to Val-240 (AIM II (aa 101-240)). Polynucleotides encoding these polypeptides are also encompassed by the invention, as are polynucleotides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to polynucleotides encoding the polypeptides described above and polynucleotides encoding polypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides. The present invention also encompasses the above polynucleotides fused to a heterologous polynucleotides and polypeptide expression products of these polynucleotides.

Even if deletion of one or more amino acids from the N-terminus of a protein results in modification or loss of one or more biological functions of the protein, other biological activities may still be retained. Thus, the ability of shortened AIM II muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that an AIM II mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six AIM II amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the AIM II amino acid sequence shown in FIGS. 1A and 1B (SEQ ID NO:2 up to the phenylalanine residue at position number 235, and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n-314 of FIGS. 1A and 1B (SEQ ID NO:2), where n is an integer in the range of 2 to 235, and 236 is the position of the first residue from the N-terminus of the complete AIM II polypeptide believed to be required for at least immunogenic activity of the AIM 11 polypeptide. Polynucleotides encoding these polypeptides are also encompassed by the invention.

More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of residues of E-2 to V-240; E-3 to V-240; S-4 to V-240; V-5 to V-240; V-6 to V-240; R-7 to V-240; P-8 to V-240; S-9 to V-240; V-10 to V-240; F-11 to V-240; V-12 to V-240; V-13 to V-240; D-14 to V-240; G-15 to V-240; Q-16 to V-240; T-17 to V-240; D-18 to V-240; I-19 to V-240; P-20 to V-240; F-21 to V-240; T-22 to V-240; R-23 to V-240; L-24 to V-240; G-25 to V-240; R-26 to V-240; S-27 to V-240; H-28 to V-240; R-29 to V-240; R-30 to V-240; Q-31 to V-240; S-32 to V-240; C-33 to V-240; S-34 to V-240; V-35 to V-240; A-36 to V-240; R-37 to V-240; V-38 to V-240; G-39 to V-240; L-40 to V-240; G-41 to V-240; L-42 to V-240; L-43 to V-240; L-44 to V-240; L-45 to V-240; L-46 to V-240; M-47 to V-240; G-48 to V-240; A-49 to V-240; G-50 to V-240; L-51 to V-240; A-52 to V-240; V-53 to V-240; Q-54 to V-240; G-55 to V-240; W-56 to V-240; F-57 to V-240; L-58 to V-240; L-59 to V-240; Q-60 to V-240; L-61 to V-240; H-62 to V-240; W-63 to V-250; R-64 to V-240; L-65 to V-240; G-66 to V-240; E-67 to V-240; M-68 to V-240; V-69 to V-240; T-70 to V-240; R-71 to V-240; L-72 to V-240; P-73 to V-240; D-74 to V-240; G-75 to V-240; P-76 to V-240; A-77 to V-240; G-78 to V-240; S-79 to V-240; W-80 to V-240; E-81 to V-240; Q-82 to V-240; L-83 to V-240; 1-84 to V-240; Q-85 to V-240; E-86 to V-240; R-87 to V-240; R-88 to V-240; S-89 to V-240; H-90 to V-240; E-91 to V-240; V-92 to V-240; N-93 to V-240; P-94 to V-240; A-95 to V-240; A-96 to V-240; H-97 to V-240; L-98 to V-240; T-99 to V-240; G-100 to V-240; A-101 to V-240; N-102 to V-240; S-103 to V-240; S-104 to V-240; L-105 to V-240; T-106 to V-240; G-107 to V-240; S-108 to V-240; G-109 to V-240; G-110 to V-240; P-111 to V-240; L-112 to V-240; L-113 to V-240; W-114 to V-240; E-115 to V-240; T-116 to V-240; Q-117 to V-240; L-118 to V-240; G-119 to V-240; L-120 to V-240; A-121 to V-240; F-122 to V-240; L-123 to V-240; R-124 to V-240; G-125 to V-240; L-126 to V-240; S-127 to V-240; Y-128 to V-240; H-129 to V-240; D-130 to V-240; G-131 to V-240; A-132 to V-240; L-133 to V-240; V-134 to V-240; V-135 to V-240; T-136 to V-240; K-137 to V-240; A-138 to V-240; G-139 to V-240; Y-140 to V-240; Y-141 to V-240; Y-142 to V-240; I-143 to V-240; Y-144 to V-240; S-145 to V-240; K-146 to V-240; V-147 to V-240; Q-148to V-240; L-149 to V-240; G-150 to V-240; G-151 to V-240; V-152 to V-240; G-153 to V-240; C-154 to V-240; P-155 to V-240; L-156 to V-240; G-157 to V-240; L-158 to V-240; A-159 to V-240; S-160 to V-240; T-161 to V-240; I-162 to V-240; T-163 to V-240; H-164 to V-240; G-165 to V-240; L-166 to V-240; Y-167 to V-240; K-168 to V-240; R-169 to V-240; T-170 to V-240; P-171 to V-240; R-172 to V-240; Y-173 to V-240; P-174 to V-240; E-175 to V-240; E-176 to V-240; L-177 to V-240; E-178 to V-240; L-179 to V-240; L-180 to V-240; V-181 to V-240; S-182 to V-240; Q-183 to V-240; Q-184 to V-240; S-185 to V-240; P-186 to V-240; C-187 to V-240; G-188 to V-240; R-189 to V-240; A-190 to V-240; T-191 to V-240; S-192 to V-240; S-193 to V-240; S-194 to V-240; R-195 to V-240; V-196 to V-240; W-197 to V-240; W-198 to V-240; D-199 to V-240; S-200 to V-240; S-201 to V-240; F-202 to V-240; L-203 to V-240; G-204 to V-240; G-205 to V-240; V-206 to V-240; V-207 to V-240; H-208 to V-240; L-209 to V-240; E-210 to V-240; A-211 to V-240; G-212 to V-240; E-213 to V-240; E-214 to V-240; V-215 to V-240; V-216 to V-240; V-217 to V-240; R-218 to V-240; V-219 to V-240; L-220 to V-240; D-221 to V-240; E-222 to V-240; R-233 to V-240; L-224 to V-240; V-225 to V-240; R-226 to V-240; L-227 to V-240; R-228 to V-240; D-229 to V-240; G-230 to V-240; T-231 to V-240; R-232 to V-240; S-233 to V-240; Y-234 to V-240; and F-235 to V-240 of the AIM II sequence shown in SEQ ID NO:2 (which is identical to the sequence shown as FIGS. 1A and 1B , with the exception that the amino acid residues in SEQ ID NO:2 are numbered consecutively from 1 through 240 from the N-terminus to the C-terminus). The present invention is also directed to nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequences encoding the polypeptides described above. The present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotide sequences are also encompassed by the invention.

›DETAILED DESCRIPTION · 14 of 68

As mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more biological functions of the protein, other biological activities may still be retained. Thus, the ability of the shortened AIM II mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that an AIM II mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six AIM II amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the AIM II polypeptide shown in FIGS. 1A and 1B (SEQ ID NO:2), up to the Valine residue at position number 6, and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues 1-m of FIGS. 1A and 1B (i.e., SEQ ID NO:2), where m is an integer in the range of 6 to 239, and 6 is the position of the first residue from the C-terminus of the complete AIM II polypeptide believed to be required for at least immunogenic activity of the AIM II polypeptide. Polynucleotides encoding these polypeptides are also encompassed by the invention.

More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of residues M-1 to M-239; M-1 to F-238; M-1 to A-237; M-1 to G-236; M-1 to F-235; M-1 to Y-234; M-1 to S-233; M-1 to R-232; M-1 to T-231; M-1 to G-230; M-1 to D-229; M-1 to R-228; M-1 to L-227; M-1 to R-226; M-1 to V-225; M-1 to L-224; M-1 to R-223; M-1 to E-222; M-1 to D-221; M-1 to L-220; M-1 to V-219; M-1 to R-218; M-1 to V-217; M-1 to V-216; M-1 to V-215; M-1 to E-214; M-1 to E-213; M-1 to G-212; M-1 to A-211; M-1 to E-210; M-1 to L-209; M-1 to H-208; M-1 to V-207; M-1 to V-206; M-1 to G-205; M-1 to G-204; M-1 to L-203; M-1 to F-202; M-1 to S-201; M-1 to S-200; M-1 to D-199; M-1 to W-198; M-1 to W-197; M-1 to V-196; M-1 to R-195; M-1 to S-194; M-1 to S-193; M-1 to S-192; M-1 to T-191; M-1 to A-190; M-1 to R-189; M-1 to G-188; M-1 to C-187; M-1 to P-186; M-1 to S-185; M-1 to Q-184; M-1 to Q-183; M-1 to S-182; M-1 to V-181; M-1 to L-180; M-1 to L-179; M-1 to E-178; M-1 to L-177; M-1 to E-176; M-1 to E-175; M-1 to P-174; M-1 to Y-173; M-1 to R-172; M-1 to P-171; M-1 to T-170; M-1 to R-169; M-1 to K-168; M-1 to Y-167; M-1 to L-166; M-1 to G-165; M-1 to H-164; M-1 to T-163; M-1 to I-162; M-1 to T-161; M-1 to S-160; M-1 to A-159; M-1 to L-158; M-1 to G-157; M-1 to L-156; M-1 to P-155; M-1 to C-154; M-1 to G-153; M-1 to V-152; M-1 to G-151; M-1 to G-150; M-1 to L-149; M-1 to Q-148; M-1 to V-147; M-1 to K-146; M-1 to S-145; M-1 to Y-144; M-1 to I-143; M-1 to Y-142; M-1 to Y-141; M-1 to Y-140; M-1 to G-139; M-1 to A-138; M-1 to K-137; M-1 to T-136; M-1 to V-135; M-1 to V-134; M-1 to L-133; M-1 to A-132; M-1 to G-131; M-1 to D-130; M-1 to H-129; M-1 to Y-128; M-1 to S-127; M-1 to L-126; M-1 to G-125; M-1 to R-124; M-1 to L-123; M-1 to F-122; M-1 to A-121; M-1 to L-120; M-1 to G-119; M-1 to L-118; M-1 to Q-117; M-1 to T-116; M-1 to E-115; M-1 to W-114; M-1 to L-113; M-1 to L-112; M-1 to P-111; M-1 to G-110; M-1 to G-109; M-1 to S-108; M-1 to G-107; M-1 to T-106; M-1 to L-105; M-1 to S-104; M-1 to S-103;M-1 to N-102; M-1 to A-101;M-1 to G-100;M-1 to T-99; M-1 to L-98; M-1 to H-97; M-1 to A-96; M-1 to A-95; M-1 to P-94; M-1 to N-93; M-1 to V-92; M-1 to E-91; M-1 to H-90; M-1 to S-89; M-1 to R-88; M-1 to R-87; M-1 to E-86; M-1 to Q-85; M-1 to I-84; M-1 to L-83; M-1 to Q-82; M-1 to E-81; M-1 to W-80; M-1 to S-79; M-1 to G-78; M-1 to A-77; M-1 to P-76; M-1 to G-75; M-1 to D-74; M-1 to P-73; M-1 to L-72; M-1 to R-71; M-1 to T-70; M-1 to V-69; M-1 to M-68; M-1 to E-67; M-1 to G-66; M-1 to L-65; M-1 to R-64; M-1 to W-63; M-1 to H-62; M-1 to L-61; M-1 to Q-60; M-1 to L-59; M-1 to L-58; M-1 to F-57; M-1 to W-56; M-1 to G-55; M-1 to Q-54; M-1 to V-53; M-1 to A-52; M-1 to L-51; M-1 to G-50; M-1 to A-49; M-1 to G-48; M-1 to M-47; M-1 to L-46; M-1 to L-45; M-1 to L-44; M-1 to L-43; M-1 to L-42; M-1 to G-41; M-1 to L-40; M-1 to G-39; M-1 to V-38; M-1 to R-37; M-1 to A-36; M-1 to V-35; M-1 to S-34; M-1 to C-33; M-1 to S-32; M-1 to Q-31; M-1 to R-30; M-1 to R-29; M-1 to H-28; M-1 to S-27; M-1 to R-26; M-1 to G-25; M-1 to L-24; M-1 to R-23; M-1 to T-22; M-1 to F-21; M-1 to P-20; M-1 to 1-19; M-1 to D-18; M-1 to T-17; M-1 to Q-16; M-1 to G-15; M-1 to D-14; M-1 to V-13; M-1 to V-12; M-1 to F-11; M-1 to V-10; M-1 to S-9; M-1 to P-8; M-1 to R-7; M-1 to V-6 of the sequence of the AIM II sequence shown in FIGS. 1A and 1B (which is identical to the sequence shown as SEQ ID NO: 2, with the exception that the amino acid residues in SEQ ID NO:2 are numbered consecutively from 1 through 240 from the N-terminus to the C-terminus). The present invention is also directed to nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequences encoding the polypeptides described above. The present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotide sequences are also encompassed by the invention.

The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of an AIM II polypeptide, which may be described generally as having residues n-m of FIGS. 1A and 1B (i. e., SEQ ID NO:2), where n and m are integers as described above.

›DETAILED DESCRIPTION · 15 of 68

In additional embodiments, the present invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues 83-m 1 of FIGS. 1A and 1B (i.e., SEQ ID NO:2), where m 1 is an integer from 89 to 239, corresponding to the position of the amino acid residue in FIGS. 1A and 1B (SEQ ID NO: 2). For example, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of residues L-83 to M-239; L-83 to F-238; L-83 to A-237; L-83 to G-236; L-83 to F-235; L-83 to Y-234; L-83 to S-233; L-83 to R-232; L-83 to T-231; L-83 to G-230; L-83 to D-229; L-83 to R-228; L-83 to L-227; L-83 to R-226; L-83 to V-225; L-83 to L-224; L-83 to R-223; L-83 to E-222; L-83 to D-221; L-83 to L-220; L-83 to V-219; L-83 to R-218; L-83 to V-217; L-83 to V-216; L-83 to V-215; L-83 to E-214; L-83 to E-213; L-83 to G-212; L-83 to A-211; L-83 to E-210; L-83 to L-209; L-83 to H-208; L-83 to V-207; L-83 to V-206; L-83 to G-205; L-83 to G-204; L-83 to L-203; L-83 to F-202; L-83 to S-201; L-83 to S-200; L-83 to D-199; L-83 to W-198; L-83 to W-197; L-83 to V-196; L-83 to R-195; L-83 to S-194; L-83 to S-193; L-83 to S-192; L-83 to T-191; L-83 to A-190; L-83 to R-189; L-83 to G-188; L-83 to C-187; L-83 to P-186; L-83 to S-185; L-83 to Q-184; L-83 to Q-183; L-83 to S-182; L-83 to V-181; L-83 to L-180; L-83 to L-179; L-83 to E-178; L-83 to L-177; L-83 to E-176; L-83 to E-175; L-83 to P-174; L-83 to Y-173; L-83 to R-172; L-83 to P-171; L-83 to T-170; L-83 to R-169; L-83 to K-168; L-83 to Y-167; L-83 to L-166; L-83 to G-165; L-83 to H-164; L-83 to T-163; L-83 to I-162; L-83 to T-161; L-83 to S-160; L-83 to A-159; L-83 to L-158; L-83 to G-157; L-83 to L-156; L-83 to P-155; L-83 to C-154; L-83 to G-153; L-83 to V-152; L-83 to G-151; L-83 to G-150; L-83 to L-149; L-83 to Q-148; L-83 to V-147; L-83 to K-146; L-83 to S-145; L-83 to Y-144; L-83 to I-143; L-83 to Y-142; L-83 to Y-141; L-83 to Y-140; L-83 to G-139; L-83 to A-138; L-83 to K-137; L-83 to T-136; L-83 to V-135; L-83 to V-134; L-83 to L-133; L-83 to A-132; L-83 to G-131; L-83 to D-130; L-83 to H-129; L-83 to Y-128; L-83 to S-127; L-83 to L-126; L-83 to G-125; L-83 to R-124; L-83 to L-123; L-83 to F-122; L-83 to A-121; L-83 to L-120; L-83 to G-119; L-83 to L-118; L-83 to Q-117; L-83 to T-116; L-83 to E-115; L-83 to W-114; L-83 to L-113; L-83 to L-112; L-83 to P-111; L-83 to G-110; L-83 to G-109; L-83 to S-108; L-83 to G-107; L-83 to T-106; L-83 to L-105; L-83 to S-104; L-83 to S-103; L-83 to N-102; L-83 to A-101; L-83 to G-100; L-83 to T-99; L-83 to L-98; L-83 to H-97; L-83 to A-96; L-83 to A-95; L-83 to P-94; L-83 to N-93; L-83 to V-92; L-83 to E-91; L-83 to H-90; and L-83to S-89; of the sequence of the AIM II sequence shown in FIGS. 1A and 1B (SEQ ID NO:2). The present application is also directed to nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding the AIM II polypeptides described above. The present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotide sequences are also encompassed by the invention.

In additional embodiments, the polynucleotides ofthe invention encode functional attributes of AIM II. Preferred embodiments of the invention in this regard include fragments that comprise, or alternatively consist of, one, two, three, four or more of one or more of the following functional domains: alpha-helix and alpha-helix forming regions (“alpha-regions”), beta-sheet and beta-sheet forming regions (“beta-regions”), turn and turn-forming regions (“turn-regions”), coil and coil-forming regions (“coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of AIM II.

The data representing the structural or functional attributes of AIM II set forth in FIGS. 3A-3F and/or Table 2 was generated using the various identified modules and algorithms of the DNA* STAR set on default parameters. In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table 2 can be used to determine regions of AIM II which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or IV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.

Certain preferred regions in these regards are set out in FIGS. 3A-3F , but may, as shown in Table 2, be represented or identified in tabular form. The DNA*STAR computer algorithm used to generate FIGS. 3A-3F (set on the original default parameters) was used to present the data in FIGS. 3A-3F in a tabular format (See Table 2). The tabular format of the data in FIG. 3A-3F may be used to easily determine specific boundaries of a preferred region.

The above-mentioned preferred regions set out in FIGS. 3A-3F and in Table 2 include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in FIGS. 1A and 1B . As set out in FIGS. 3A-3F and in Table 2, such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions (columns I, III, V, and VII in Table 2), Chou-Fasman alpha-regions, beta-regions, and turn-regions (columns II, IV, and VI in Table 2), Kyle-Doolittle hydrophilic regions (column VIII in Table 2), Hopp-Woods hydrophobic regions (column IX in Table 2), Eisenberg alpha- and beta-amphipathic regions (columns X and XI in Table 2), Karplus-Schulz flexible regions (column XII in Table 2), Jameson-Wolf regions of high antigenic index (column XIII in Table 2), and Emini surface-forming regions (column XIV in Table 2).

›DETAILED DESCRIPTION · 16 of 68

Among highly preferred fragments in this regard are those that comprise regions of AIM II that combine several structural features, such as several of the features set out above in Table 2.

The present invention is further directed to isolated polypeptides comprising, or alternatively consisting of, fragments of AIM II. In particular, the invention provides isolated polypeptides comprising, or alternatively consisting of, the amino acid sequences of a member selected from the group consisting of amino acids 1-60, 11-70,21-80, 31-90,41 -100, 51-110, 61-120, 71-130, 81-140, 91-150, 101-160, 111-170, 121-180, 131-190, 141-200, 151-210, 161-220, 171-230, and 181-240 of SEQ ID NO:2, as well as isolated polynucleotides which encode these polypeptides.

The present invention is also directed to isolated polypeptides comprising, or alternatively consisting of, domains of AIM II. In particular, the invention provides polypeptides comprising, or alternatively consisting of, beta-sheet regions of AIM II set out in Table 2. These polypeptides include polypeptides comprising, or alternatively consisting of, amino acid sequences of a member selected from the group consisting of amino acid residues from about 7 to about 14, amino acid residues from about 18 to about 23, amino acid residues from about 17 to about 25, amino acid residues from about 33 to about 46, amino acid residues from about 35 to about 39, amino acid residues from about 57 to about 60, amino acid residues from about 67 to about 72, amino acid residues from about 102 to about 107, amino acid residues from about 121 to about 126, amino acid residues from about 131 to about 166, amino acid residues from about 141 to about 152, amino acid residues from about 158 to about 169, amino acid residues from about 213 to about 221, and amino acid residues from about 232 to about 240 of SEQ ID NO:2. The invention is further directed to isolated polynucleotides comprising, or alternatively consisting of, nucleic acid molecules which encode the beta-sheet regions set out in Table 2, and isolated polypeptides comprising, or alternatively consisting of, amino acid sequences at least 80% identical, and more preferably at least 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to nucleic acid molecules encoding beta-sheet regions of the AIM II protein.

The invention is also directed to isolated polypeptides comprising, or alternatively consisting of, fragments of AIM II which have one or more functional activity associated with AIM II polypeptides having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:39, as well as isolated polynucleotides encoding these polypeptides.

As to the selection of peptides or polypeptides bearing an antigenic epitope (i.e., that contain a region of a protein molecule to which an antibody can bind), it is well known in that art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner, R. A. (1983) Antibodies that react with predetermined sites on proteins. Science 219:660-666. Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals.

Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies that bind specifically to a polypeptide of the invention. See, for instance, Wilson et al. Cell 37:767-778 (1984) at 777.

The present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:39, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in deposited clone identified as ATCC Accession No.97689 or 97483 or encoded by a polynucleotide that hybridizes to the complement of the sequence of SEQ ID NO:1 or SEQ ID NO:38 or contained in deposited clone identified as ATCC Accession No. 97689 or 97483 under stringent hybridization conditions or lower stringency hybridization conditions as defined supra. The present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO:1 or SEQ ID NO:38), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined supra.

The term “epitopes,” as used herein, refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human. In a preferred embodiment, the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide. An “immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,” as used herein, is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.

Fragments that function as epitopes may be produced by any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985), further described in U.S. Pat. No. 4,631,211).

›DETAILED DESCRIPTION · 17 of 68

In the present invention, antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at-least 8, at least 9, at least 10, at least 15, at least 20, at least 25, and, most preferably, between about 15 to about 30 amino acids. Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies that specifically bind the epitope. Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).

Similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). A preferred immunogenic epitope includes the secreted protein. The polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as, for example, rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).

Non-limiting examples of antigenic polypeptides or peptides that can be used to generate AIM II-specific antibodies include: a polypeptide comprising, or alternatively consisting of, amino acid residues from about 13 to about 20 in FIGS. 1A and 1B (SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 23 to about 36 in FIGS. 1A and 1B (SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 69 to about 79 in FIGS. 1A and 1B (SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 85 to about 94 in FIGS. 1A and 1B (SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 167 to about 178 in FIGS. 1A and 1B (SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 184 to about 196 in FIGS. 1A and 1B (SEQ ID NO:2); and a polypeptide comprising, or alternatively consisting of, amino acid residues from about 221 to about 233 in FIGS. 1A and 1B (SEQ ID NO:2). In this context, the term “about” includes the particular recited ranges and ranges larger or smaller by several (5, 4, 3, 2, or 1) amino acids at either terminus or at both termini. As indicated above, the inventors have determined that the above polypeptide fragments are antigenic regions of the AIM II protein. Polynucleotides encoding these polypeptides are also encompassed by the invention.

The epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means. Houghten, R. A. General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985). This “Simultaneous Multiple Peptide Synthesis (SMPS)” process is further described in U.S. Pat. No. 4,631,211 to Houghten et al. (1986).

Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). If in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde. Animals such as, for example, rabbits, rats, and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 micrograms of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody that can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.

As one of skill in the art will appreciate, and as discussed above, the polypeptides of the present invention comprising an immunogenic or antigenic epitope can be fused to other polypeptide sequences. For example, the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination thereof and portions thereof) resulting in chimeric polypeptides. Such fusion proteins may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). IgG Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion disulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin (“HA”) tag or flag tag) to aid in detection and purification of the expressed polypeptide. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix-binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni 2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.

›DETAILED DESCRIPTION · 18 of 68

Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308-313 (1998) (each of these patents and publications are hereby incorporated by reference in its entirety). In one embodiment, alteration of polynucleotides corresponding to SEQ ID NO:1 and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence. In another embodiment, polynucleotides of the invention, or the encoded polypeptides, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide coding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

A list of exemplified amino acid sequences comprising immunogenic epitopes are shown in Table 2. It is pointed out that Table 2 only lists amino acid residues comprising epitopes predicted to have the highest degree of antigenicity using the algorithm of Jameson and Wolf, Comp. Appl. Biosci. 4:181-186 (1988) (said references incorporated by reference in their entireties). The Jameson-Wolf antigenic analysis was performed using the computer program PROTEAN, using default parameters (Version 3.11 for the Power MacIntosh, DNASTAR, Inc., 1228 South Park Street Madison, Wis.). Table 2 and portions of polypeptides not listed in Table 2 are not considered non-immunogenic. The immunogenic epitopes of Table 2 is an exemplified list, not an exhaustive list, because other immunogenic epitopes are merely not recognized as such by the particular algorithm used. Amino acid residues comprising other immunogenic epitopes may be routinely determined using algorithms similar to the Jameson-Wolf analysis or by in vivo testing for an antigenic response using methods known in the art. See, e.g., Geysen et al., supra; U.S. Pat. Nos 4,708,781; 5,194,392; 4,433,092; and 5,480,971 (said references incorporated by reference in their entireties).

It is particularly pointed out that the amino acid sequences of Table 2 comprise immunogenic epitopes. Table 2 lists only the critical residues of immunogenic epitopes determined by the Jameson-Wolf analysis. Thus, additional flanking residues on either the N-terminal, C-terminal, or both N- and C-terminal ends may be added to the sequences of Table 2 to generate an epitope-bearing polypeptide of the present invention. Therefore, the immunogenic epitopes of Table 2 may include additional N-terminal or C-terminal amino acid residues. The additional flanking amino acid residues may be contiguous flanking N-terminal and/or C-terminal sequences from the -polypeptides of the present invention, heterologous polypeptide sequences, or may include both contiguous flanking sequences from the polypeptides of the present invention and heterologous polypeptide sequences.

The immunogenic and antigenic epitope-bearing fragments may be specified by either the number of contiguous amino acid residues, as described above, or further specified by N-terminal and C-terminal positions of these fragments on the amino acid sequence of SEQ ID NO:2. Every combination of a N-terminal and C-terminal position that a fragment of, for example, at least 7 or at least 15 contiguous amino acid residues in length could occupy on the amino acid sequence of SEQ ID NO:2 is included in the invention. Again, “at least 7 contiguous amino acid residues in length” means 7 amino acid residues in length or any integer between 7 amino acids and the number of amino acid residues of the full-length polypeptide of the present invention. Specifically, each and every integer between 7 and the number of amino acid residues of the full-length polypeptide are included in the present invention.

Immunogenic and antigenic epitope-bearing polypeptides of the invention are useful, for example, to make antibodies which specifically bind the polypeptides of the invention, and in immunoassays to detect the polypeptides of the present invention. The antibodies are useful, for example, in affinity purification of the polypeptides of the present invention. The antibodies may also routinely be used in a variety of qualitative or quantitative immunoassays, specifically for the polypeptides of the present invention using methods known in the art. See, e.g., Harlow et al., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press; 2nd Ed. 1988).

The epitope-bearing polypeptides of the present invention may be produced by any conventional means for making polypeptides including synthetic and recombinant methods known in the art. For instance, epitope-bearing peptides may be synthesized using known methods of chemical synthesis. For instance, Houghten has described a simple method for the synthesis of large numbers of peptides, such as 10-20 mgs of 248 individual and distinct 13 residue peptides representing single amino acid variants of a segment of the HA1 polypeptide, all of which were prepared and characterized (by ELISA-type binding studies) in less than four weeks (Houghten et al., Proc. Natl. Acad. Sci. U.S.A. 82:5131-5135 (1985)). This “Simultaneous Multiple Peptide Synthesis (SMPS)” process is further described in U.S. Pat. No. 4,631,211 to Houghten and coworkers (1986). In this procedure the individual resins for the solid-phase synthesis of various peptides are contained in separate solvent-permeable packets, enabling the optimal use of the many identical repetitive steps involved in solid-phase methods. A completely manual procedure allows 500-1000 or more syntheses to be conducted simultaneously (Houghten et al., Proc. Natl. Acad. Sci. U.S.A. 82:5131-5135 (1985) at 5134).

›DETAILED DESCRIPTION · 19 of 68

Epitope-bearing polypeptides of the present invention are used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). If in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer maybe boosted by coupling of the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 μg of peptide or carrier protein and Freund's adjuvant. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.

As one of skill in the art will appreciate, and discussed above, the polypeptides of the present invention comprising an immunogenic or antigenic epitope can be fused to heterologous polypeptide sequences. For example, the polypeptides of the present invention maybe fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, any combination thereof including both entire domains and portions thereof) resulting in chimeric polypeptides. These fusion proteins facilitate purification, and show an increased half-life in vivo. This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EPA 0,394,827; Traunecker et al., Nature 331:84-86 (1988). Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion can also be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem. 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag to aid in detection and purification of the expressed polypeptide.

In addition, proteins of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller, M. et al., Nature 310:105-111 (1984)). For example, a peptide corresponding to a fragment of the AIM II polypeptides of the invention can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the AIM II polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, alpha-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, alpha-Abu, alpha-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, omithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, alpha-alanine, fluoro-amino acids, designer amino acids such as alpha-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

Non-naturally occurring variants may be produced using art-known mutagenesis techniques, which include, but are not limited to oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see, e.g., Carter et al., Nucl. Acids Res. 13:4331 (1986); and Zoller et al., Nucl. Acids Res. 10:6487 (1982)), cassette mutagenesis (see, e.g., Wells et al., Gene 34:315 (1985)), restriction selection mutagenesis (see, e.g., Wells et al., Philos. Trans. R. Soc. London SerA 317:415 (1986)).

The invention additionally, encompasses AIM II polypeptides which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications maybe carried out by known techniques, including but not limited to, specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH 4 , acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin; etc.

Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression. The polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.

Also provided by the invention are chemically modified derivatives of AIM II which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemical moieties for derivation may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.

›DETAILED DESCRIPTION · 20 of 68

The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog). For example, the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

As noted above, the polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in U.S. Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosures of each of which are incorporated herein by reference.

The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the protein. There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues, glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.

As suggested above, polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues. For example, polyethylene glycol can be linked to a protein via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the protein or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the protein.

One may specifically desire proteins chemically modified at the N-terminus. Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.

As indicated above, pegylation of the proteins of the invention may be accomplished by any number of means. For example, polyethylene glycol maybe attached to the protein either directly or by an intervening linker. Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern. J of Hematol. 68:1-18 (1998); U.S. Pat. Nos. 4,002,531; 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of which are incorporated herein by reference.

One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride (ClSO 2 CH 2 CF 3 ). Upon reaction of protein with tresylated MPEG, polyethylene glycol is directly attached to amine groups of the protein. Thus, the invention includes protein-polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.

Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, U.S. Pat. No.5,612,460, the entire disclosure of which is incorporated herein by reference, discloses urethane linkers for connecting polyethylene glycol to proteins. Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. A number additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in WO 98/32466, the entire disclosure of which is incorporated herein by reference. Pegylated protein products produced using the reaction chemistries set out herein are included within the scope of the invention.

›DETAILED DESCRIPTION · 21 of 68

The number of polyethylene glycol moieties attached to each protein of the invention (i.e., the degree of substitution) may also vary. For example, the pegylated proteins of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules. Similarly, the average degree of substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per protein molecule. Methods for determining the degree of substitution are discussed, for example, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

Antibodies

The present invention further relates to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, preferably an epitope, of the present invention (as determined by immunoassays well known in the art for assaying specific antibody-antigen binding). Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. The term “antibody,” as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

Most preferably the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodies of the invention may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine, donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).

Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention that they recognize or specifically bind. The epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or listed in the Tables and Figures. Antibodies that specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. Further included in the present invention are antibodies that bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein). Antibodies of the present invention may also be described or specified in terms oftheir binding affinity to a polypeptide of the invention. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10 −2 M, 10 −2 M, 5×10 −3 M, 10 −3 M, 5×10 −4 M, 10 −4 M, 5×10 −5 M, 10 −5 M, 5×10 −6 M, 10 −6 M, 5×10 −7 M, 10 −7 M, 5×10 −8 M, 10 −8 M, 5×10 −9 M, 10 −9 M, 5×10 −10 M, 10 −10 M, 5×10 −11 M, 10 −11 M, 5×10 −12 M, 10 −12 M, 5×10 −13 M, 10 −13 M, 5×10 −14 M, 10 −14 M, 5×10 −15 M, and 10 −15 M.

›DETAILED DESCRIPTION · 22 of 68

The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonists of the polypeptides ofthe present invention. For example, the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully. The invention features both receptor-specific antibodies and ligand-specific antibodies. The invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra). In specific embodiments, antibodies are provided that inhibit ligand or receptor activity by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody.

The invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand. Likewise, included in the invention are neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor. Further included in the invention are antibodies which activate the receptor. These antibodies may act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation. The antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein. Thus, the invention further relates to antibodies which act as agonists or antagonists of the polypeptides of the present invention. The above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6): 1981 -1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4):1786-1794(1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al., J. Cell Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995); Mulleretal., Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996) (which are all incorporated by reference herein in their entireties).

Antibodies of the present invention may be used, for example, but not limited to, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference herein in its entirety).

As discussed in more detail below, the antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions. For example, antibodies of the present invention maybe recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.

The antibodies of the invention include derivatives that are modified, i.e, by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.

The antibodies of the present invention may be generated by any suitable method known in the art. Polyclonal antibodies to an antigen of interest can be produced by various procedures well known in the art. For example, a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum . Such adjuvants are also well known in the art.

›DETAILED DESCRIPTION · 23 of 68

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Thus, the term “monoclonal antibody” is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma and recombinant and phage display technology.

Methods for producing and screening for specific antibodies using hybridoma technology are routine and well-known in the art and are discussed in detail in Example 23. Briefly, mice can be immunized with a polypeptide of the invention or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.

Accordingly, the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.

Antibody fragments that recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab′)2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.

For example, the antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular, such phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phages used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187:9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties).

Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999(1993); and Skerra et al, Science 240:1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entireties. Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementacety determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).

›DETAILED DESCRIPTION · 24 of 68

Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 96/34096; WO 96/33735; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598, which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, Calif.) and GenPharm (San Jose, Calif.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Bio/technology 12:899-903 (1988)).

Further, antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that “mimic” polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J 7(5):437-444 (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies which bind to and competitively inhibit polyp eptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that “mimic” the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand. For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligands/receptors, and thereby block its biological activity.

A. Polynucleotides Encoding Antibodies.

The invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO:2.

The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody maybe assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.

›DETAILED DESCRIPTION · 25 of 68

Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA techniques, one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278:457-479 (1998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention. Preferably, as discussed supra, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.

In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No.4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., 1988, Science 242:1038-1041).

B. Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, e.g., a heavy or light chain of an antibody of the invention, requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.

›DETAILED DESCRIPTION · 26 of 68

A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis ) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia ) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably, bacterial cells such as Escherichia coli , and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., 1986, Gene 45:101; Cockett et al., 1990, Bio/Technology 8:2).

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J.2:1791), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest maybe ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts. (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:51-544).

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.

›DETAILED DESCRIPTION · 27 of 68

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.

A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1, which are incorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).

The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.

C. Antibody Conjugates

The present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20 or 50 amino acids of the polypeptide) of the present invention to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. The antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20 or 50 amino acids of the polypeptide) of the present invention. For example, antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors. Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452 (1991), which are incorporated by reference in their entireties.

The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions. For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof. The antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CH1 domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof. The polypeptides may also be fused or conjugated to the above antibody portions to form multimers. For example, Fe portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions. Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-11341(1992) (said references incorporated by reference in their entireties).

›DETAILED DESCRIPTION · 28 of 68

As discussed, supra, the polypeptides of the present invention may be fused or conjugated to the above antibody portions to increase the in vivo half-life of the polypeptides or for use in immunoassays using methods known in the art. Further, the polypeptides of the present invention may be fused or conjugated to the above antibody portions to facilitate purification. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86 (1988). The polypeptides of the present invention fused or conjugated to an antibody having disulfide-linked dimeric structures (due to the IgG) may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. (EP A 232,262). Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion-protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5 receptor, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).

Moreover, the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate their purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the “flag” tag.

The present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. See, for example, U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 125 I, 131 I, 111 In or 99 Tc.

Further, an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.

›DETAILED DESCRIPTION · 29 of 68

Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.

D. Assays for Antibody Binding

The antibodies of the invention may be assayed for immunospecific binding by any method known in the art. The immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).

Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trayslol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C., adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C., washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32 P or 125 I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.

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The binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3 H or 125 I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest is conjugated to a labeled compound (e.g., 3 H or 125 I) in the presence of increasing amounts of an unlabeled second antibody.

E. Antibody Based Therapies

The present invention is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disorders or conditions described herein. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof as described herein.

The present invention encompasses antagonistic AIM II antibodies useful in treating, preventing, diagnosing, and/or prognosing a disease or disorder of the immune system. The present invention also encompasses antagonistic AIM II antibodies useful in treating, preventing, diagnosing, and/or prognosing a disease or disorder of the cellular immune system.

The present invention also encompasses antagonistic AIM II antibodies useful in treating, preventing, diagnosing, and/or prognosing a disease or disorder associated with aberrant T cell activation.

The present invention also encompasses antagonistic AIM II antibodies useful in treating, preventing, diagnosing, and/or prognosing a disease or disorder associated with aberrant CD8 + T cell activation.

The present invention encompasses agonistic AIM II antibodies, useful in treating, preventing, diagnosing, and/or prognosing a disease or disorder of the immune system. The present invention also encompasses agonistic AIM II antibodies useful in treating, preventing, diagnosing, and/or prognosing a disease or disorder of the cellular immune system.

The present invention also encompasses agonistic AIM II antibodies useful in treating, preventing, diagnosing, and/or prognosing a disease or disorder associated with aberrant T cell activation.

The present invention also encompasses agonistic AIM II antibodies useful in treating, preventing, diagnosing, and/or prognosing a disease or disorder associated with aberrant CD8 + T cell activation.

The present invention encompasses a method for using the AIM II antibodies of the present invention to treat, prevent, diagnose and/or prognose a disease or disorder of the immune system. The present invention also encompasses a method for using the AIM II antibodies of the present invention to treat, prevent, diagnose and/or prognose a disease or disorder of the cellular immune system.

The present invention also encompasses a method for using the AIM II antibodies of the present invention to treat, prevent, diagnose and/or prognose a disease or disorder associated with aberrant T cell activation.

The invention provides a method of inhibiting, blocking, and/or reducing T cell activation comprising contacting a T cell with an effective amount of one or more AIM II antibodies of the invention, wherein said effective amount inhibits, blocks, and/or reduces T cell activation. In specific embodiments, this method is performed in vitro. In specific embodiments, this method is performed in vivo.

The invention also provides a method of inhibiting, blocking, and/or reducing CD8 + T cell activation comprising contacting a CD8 + T cell with an effective amount of one or more AIM II antibodies of the invention, wherein said effective amount inhibits, blocks, and/or reduces CD8 + T cell activation. In specific embodiments, this method is performed in vitro. In specific embodiments, this method is performed in vivo.

In other embodiments, the invention provides a method of inhibiting, blocking, and/or reducing T cell proliferation comprising contacting a T cell with an effective amount of one or more AIM II antibodies of the invention, wherein said effective amount inhibits, blocks, and/or reduces T cell proliferation. In specific embodiments, this method is performed in vitro. In specific embodiments, this method is performed in vivo.

In other embodiments, the invention provides a method of inhibiting, blocking, and/or reducing CD8 + T cell proliferation comprising contacting a CD8 + T cell with an effective amount of one or more AIM II antibodies of the invention, wherein said effective amount inhibits, blocks, and/or reduces CD8 + T cell proliferation. In specific embodiments, this method is performed in vitro. In specific embodiments, this method is performed in vivo.

In other embodiments, the invention provides a method of inhibiting, blocking, and/or reducing T cell secretion of IFN-γ comprising contacting a T cell with an effective amount of one or more AIM II antibodies of the invention, wherein said effective amount inhibits, blocks, and/or reduces T cell secretion of IFN-γ. In specific embodiments, this method is performed in vitro. In specific embodiments, this method is performed in vivo.

In other embodiments, the invention provides a method of inhibiting, blocking, and/or reducing CD8 + T cell secretion of IFN-γ comprising contacting a CD8 + T cell with an effective amount of one or more AIM II antibodies of the invention, wherein said effective amount inhibits, blocks, and/or reduces CD8 + T cell secretion of IFN-γ. In specific embodiments, this method is performed in vitro. In specific embodiments, this method is performed in vivo.

›DETAILED DESCRIPTION · 31 of 68

The invention provides a method of stimulating, enhancing, and/or promoting T cell activation comprising contacting a T cell with an effective amount of one or more AIM II antibodies of the invention, wherein said effective amount stimulates, enhances, and/or promotes T cell activation. In specific embodiments, this method is performed in vitro. In specific embodiments, this method is performed in vivo.

The invention also provides a method of stimulating, enhancing, and/or promoting CD8 + T cell activation comprising contacting a CD8 + T cell with an effective amount of one or more AIM II antibodies of the invention, wherein said effective amount stimulates, enhances, and/or promotes CD8 + T cell activation. In specific embodiments, this method is performed in vitro. In specific embodiments, this method is performed in vivo.

In other embodiments, the invention provides a method of stimulating, enhancing, and/or promoting T cell proliferation comprising contacting a T cell with an effective amount of one or more AIM II antibodies of the invention, wherein said effective amount stimulates, enhances, and/or promotes T cell proliferation. In specific embodiments, this method is performed in vitro. In specific embodiments, this method is performed in vivo.

In other embodiments, the invention provides a method of stimulating, enhancing, and/or promoting CD8+ T cell proliferation comprising contacting a CD8 + T cell with an effective amount of one or more AIM II antibodies of the invention, wherein said effective amount stimulates, enhances, and/or promotes CD8 + T cell proliferation. In specific embodiments, this method is performed in vitro. In specific embodiments, this method is performed in vivo.

In other embodiments, the invention provides a method of stimulating, enhancing, and/or promoting T cell secretion of IFN-γ comprising contacting a T cell with an effective amount of one or more AIM II antibodies of the invention, wherein said effective amount stimulates, enhances, and/or promotes T cell secretion of IFN-γ. In specific embodiments, this method is performed in vitro. In specific embodiments, this method is performed in vivo.

In other embodiments, the invention provides a method of stimulating, enhancing, and/or promoting CD8 + T cell secretion of IFN-γ comprising contacting a CD8 + T cell with an effective amount of one or more AIM II antibodies of the invention, wherein said effective amount stimulates, enhances, and/or promotes CD8 + T cell secretion of IFN-γ. In specific embodiments, this method is performed in vitro. In specific embodiments, this method is performed in vivo.

The invention provides a method of regulating, modulating, and/or controlling T cell activation comprising contacting a T cell with an effective amount of one or more AIM II antibodies of the invention, wherein said effective amount regulates, modulates, and/or controls T cell activation. In specific embodiments, this method is performed in vitro. In specific embodiments, this method is performed in vivo.

The invention also provides a method of regulating, modulating, and/or controlling CD8 + T cell activation comprising contacting a CD8 + T cell with an effective amount of one or more AIM II antibodies of the invention, wherein said effective amount regulates, modulates, and/or controls CD8 + T cell activation. In specific embodiments, this method is performed in vitro. In specific embodiments, this method is performed in vivo.

In other embodiments, the invention provides a method of regulating, modulating, and/or controlling T cell proliferation comprising contacting a T cell with an effective amount of one or more AIM II antibodies of the invention, wherein said effective amount regulates, modulates, and/or controls T cell proliferation. In specific embodiments, this method is performed in vitro. In specific embodiments, this method is performed in vivo.

In other embodiments, the invention provides a method of regulating, modulating, and/or controlling CD8 + T cell proliferation comprising contacting a CD8 + T cell with an effective amount of one or more AIM II antibodies of the invention, wherein said effective amount regulates, modulates, and/or controls CD8 + T cell proliferation. In specific embodiments, this method is performed in vitro. In specific embodiments, this method is performed in vivo.

In other embodiments, the invention provides a method of regulating, modulating, and/or controlling T cell secretion of IFN-γ comprising contacting a T cell with an effective amount of one or more AIM II antibodies of the invention, wherein said effective amount regulates, modulates, and/or controls T cell secretion of IFN-γ. In specific embodiments, this method is performed in vitro. In specific embodiments, this method is performed in vivo.

In other embodiments, the invention provides a method of regulating, modulating, and/or controlling CD8 + T cell secretion of IFN-γ comprising contacting a CD8 + T cell with an effective amount of one or more AIM II antibodies of the invention, wherein said effective amount regulates, modulates, and/or controls CD8 + T cell secretion of IFN-γ. In specific embodiments, this method is performed in vitro. In specific embodiments, this method is performed in vivo.

The invention further provides compositions, comprising AIM II polynucleotides, polypeptides, antibodies, and agonists and/or antagonists, for administration to cells in vitro, to cells ex vivo, and to cells in vivo, or to a multicellular organism. In preferred embodiments, the compositions of the invention comprise an AIM II polynucleotide for expression of an AIM II polypeptide in a host organism for treatment of disease. In a most preferred embodiment, the compositions of the invention comprise an AIM II polynucleotide for expression of an AIM II polypeptide in a host organism for treatment of an immunodeficiency and/or conditions associated with an immunodeficiency. In another most preferred embodiment, the compositions of the invention comprise an AIM II polynucleotide for expression of an AIM II polypeptide in a host organism for treatment of an autoimmune disease and/or conditions associated with an autoimmune disease. Particularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with aberrant endogenous activity of AIM II (e.g., expression to enhance T-cell function by enhancing T cell activation, expanding T-cell numbers or increasing T cell IFN-γ secretion).

›DETAILED DESCRIPTION · 32 of 68

The present invention further encompasses methods and compositions for killing cells of hematopoietic origin, comprising administering to an animal in which such killing is desired, an AIM II polypeptide (e.g., a radiolabelled AIM II polypeptide) in an amount effective to kill cells of hematopoietic origin. In preferred embodiments, the cells of hematopoietic origin are T cells. In further preferred embodiments, the cells of hematopoietic origin are CD8 + T cells.

The antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein such as, for example, graft versus host disease and autoimmune diseases, disorders, or conditions associated with such diseases or disorders (including, but not limited to, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (e.g., IgA nephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura), Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis, systemic lupus erhythematosus, Goodpasture's syndrome, Pemphigus, Receptor autoimmunities such as, for example, (a) Graves' Disease, (b) Myasthenia Gravis, and (c) insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoid arthritis, scleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis/dermatomyositis, pernicious anemia, idiopathic Addison's disease, infertility, glomerulonephritis such as primary glomerulonephritis and IgA nephropathy, bullous pemphigoid, Sjogren's syndrome, diabetes mellitus, and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis), chronic active hepatitis, primary biliary cirrhosis, other endocrine gland failure, vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria, atopic dermatitis, asthma, inflammatory myopathies, and other inflammatory, granulamatous, degenerative, and atrophic disorders), and immunodeficiencies or conditions associated with such diseases or disorders (including, but not limited to, severe combined immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton's disease, congenital agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, transient hypogammaglobulinemia of infancy, unspecified hypogammaglobulinemia, agammaglobulinemia, common variable immunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS), X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), antibody deficiency with normal or elevated Igs, immunodeficiency with thymoma, Ig heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), selective IgM immunodeficiency, recessive agammaglobulinemia (Swiss type), reticular dysgenesis, neonatal neutropenia, severe congenital leukopenia, thymic alymphoplasia-aplasia or dysplasia with immunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezelof syndrome-combined immunodeficiency with Igs, purine nucleoside phosphorylase deficiency (PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome), severe combined immunodeficiency, DiGeorge anomaly, thymic hypoplasia, chronic mucocutaneous candidiasis, natural killer cell deficiency, idiopathic CD4+ T-lymphocytopenia, and immunodeficiency with predominant T-cell defect.

›DETAILED DESCRIPTION · 33 of 68

In additional embodiments, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are administered to an animal, preferably a human, to treat, prevent, diagnose and/or prognose an autoimmune disease. In specific embodiments, the autoimmune disease treated, prevented, diagnosed and/or prognosed according to the methods of the invention is a member selected from the group consisting of Graft versus host disease (GVHD), Multiple Sclerosis, type 1 diabetes, rheumatoid arthritis, primary biliary cirrhosis, aplastic anemia, myelodysplasia, systemic lupus erhythematosus, idiopathic thrombocytopenia purpura, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (e.g, IgA nephropathy), an immune-based rheumatologic disease (e.g., SLE, rheumatoid arthritis, CREST syndrome (a variant of scleroderma characterized by c alcinosis, R aynaud's phenomenon, e sophageal motility disorders, s clerodactyly, and t elangiectasia.), Seronegative spondyloarthropathy (SpA), Polymyositis/dermatomyositis, Microscopic polyangiitis, Hepatitis C-asociated arthritis, Takayasu's arteritis, and undifferentiated connective tissue disorder), Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura), Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis, Goodpasture's syndrome, Pemphigus, Receptor autoimmunities such as, for example, (a) Graves' Disease, (b) Myasthenia Gravis, and (c) insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis/dermatomyositis, pernicious anemia, idiopathic Addison's disease, infertility, glomerulonephiltis such as primary glomerulonephritis and IgA nephropathy, bullous pemphigoid, Sjogren's syndrome, diabetes mellitus, and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis), chronic active hepatitis, primary biliary cirrhosis, other endocrine gland failure, vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria, atopic dermatitis, asthma, inflammatory myopathies, and other inflammatory, granulamatous, degenerative, atrophic disorders, or a condition associated with an autoimmune disease.

›DETAILED DESCRIPTION · 34 of 68

In additional embodiments, AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, are administered to an animal, preferably a human, to treat, prevent, diagnose and/or prognose an immunodeficiency. In specific embodiments, the immunodeficiency treated, prevented, diagnosed and/or prognosed according to the methods of the invention is a member selected from the group consisting of DiGeorge anomaly, ataxia telangiectasia, severe combined immunodeficiency (SCID)-X linked, SCID-autosomal, Wiskott-Aldrich Syndrome (WAS), chronic mucocutaneous candidiasis, natural killer cell deficiency, CD4+ T-lymphocytopenia, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton's disease, congenital agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, transient hypogammaglobulinemia of infancy, unspecified hypogammaglobulinemia, agammaglobulinemia, common variable immunodeficiency (CVID) (acquired), X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), antibody deficiency with normal or elevated Igs, immunodeficiency with thymoma, Ig heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), selective IgM immunodeficiency, recessive agammaglobulinemia (Swiss type), reticular dysgenesis, neonatal neutropenia, severe congenital leukopenia, thymic alymphoplasia-aplasia or dysplasia with immunodeficiency, short limbed dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezelof syndrome-combined immunodeficiency with Igs, purine nucleoside phosphorylase deficiency (PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severe combined immunodeficiency and conditions associated with an immunodeficiency.

›DETAILED DESCRIPTION · 35 of 68

In a specific embodiment, antibodies of the invention are be used to treat, inhibit, prognose, diagnose or prevent graft versus host disease and autoimmune disease.

In a specific embodiment, antibodies of the invention are be used to treat, inhibit, prognose, diagnose or prevent rheumatoid arthritis.

In another specific embodiment, antibodies of the invention are used to treat, inhibit, prognose, diagnose or prevent systemic lupus erythematosis. The treatment and/or prevention of diseases and disorders associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases and disorders. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.

A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g., as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred. Thus, in a preferred embodiment, human antibodies, fragments derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides, including fragments thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10 −6 M, 10 −6 M, 5×10 −7 M, 10 −7 M, 5×10 −8 M, 10 −8 M, 5×10 −9 M, 10 −9 M, 5×10 −10 M, 10 −10 M, 5×10 −11 M, 10 −11 M, 5×10 −12 M, 10 −12 M, 5×10 −13 M, 10 −13 M, 5×10 −14 M, 10 −14 M, 5×10 −15 M, and 10 −15 M.

Identification of AIM II Agonists and Antagonists

The invention also provides a method of screening compounds to identify those which enhance or block the action of AIM II on cells, such as its interaction with AIM II-binding molecules such as receptor molecules. An agonist is a compound which increases the natural biological functions of AIM II or which functions in a manner similar to AIM II, while antagonists decrease or eliminate such functions.

For example, a cellular compartment, such as a membrane preparation, may be prepared from a cell that expresses a molecule that binds AIM II, such as a molecule of a signaling or regulatory pathway modulated by AIM II. The preparation is incubated with labeled AIM II in the absence or the presence of a candidate molecule which may be an AIM II agonist or antagonist. The ability of the candidate molecule to bind the binding molecule or AIM II itself is reflected in decreased binding of the labeled ligand. Molecules which bind gratuitously, i.e., without inducing the effects of AIM II when bound to the AIM II binding molecule, are most likely to be good antagonists. Molecules that bind well and elicit effects that are the same as or closely related to AIM II, are good agonists.

AIM II-like effects of potential agonists and antagonists may by measured, for instance, by determining activity of a second messenger system following interaction of the candidate molecule with a cell or appropriate cell preparation, and comparing the effect with that of AIM II or molecules that elicit the same effects as AIM II. Second messenger systems that may be useful in this regard include but are not limited to AMP guanylate cyclase, ion channel or phosphoinositide hydrolysis second messenger systems.

Another example of an assay for AIM II antagonists is a competitive assay that combines AIM II and a potential antagonist with membrane-bound AIM II receptor molecules or recombinant AIM II receptor molecules under appropriate conditions for a competitive inhibition assay. AIM II can be labeled, such as by radioactivity, such that the number of AIM II molecules bound to a receptor molecule can be determined accurately to assess the effectiveness of the potential antagonist.

Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to a polypeptide of the invention, and thereby inhibit or extinguish its activity. Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a receptor molecule, without inducing AIM II-induced activities, thereby preventing the action of AIM II by excluding AIM II from binding. Antagonists of the invention include fragments of the AIM II polypeptide having the amino acid sequence shown in SEQ ID NO:2.

Other potential antagonists include antisense molecules. Antisense technology can be used to control gene expression through antisense DNA or RNA or through triple-helix formation. Antisense techniques are discussed, for example, in Okano, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Triple helix formation is discussed in, for instance, Lee et al., Nucleic Acids Research 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991). The methods are based on binding of a polynucleotide to a complementary DNA or RNA. For example, the 5′ coding portion of a polynucleotide that encodes the mature polypeptide of the present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription thereby preventing transcription and the production of AIM II. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into AIM II polypeptide. The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of AIM II.

›DETAILED DESCRIPTION · 36 of 68

Therapeutic Uses of AIM II, AIM II Agonists and AIM II Antagonists

The Tumor Necrosis Factor (TNF) family ligands are known to be among the most pleiotropic cytokines, inducing a large number of cellular responses, including cytotoxicity, anti-viral activity, immunoregulatory activities, and the transcriptional regulation of several genes (Goeddel, D. V. et al., “Tumor Necrosis Factors: Gene Structure and Biological Activities,” Symp. Quant. Biol. 51:597-609 (1986), Cold Spring Harbor; Beutler, B., and Cerami, A., Annu. Rev. Biochem. 57:505-518 (1988); Old, L. J., Sci. Am. 258:59-75 (1988); Fiers, W., FEBS Lett. 285:199-224 (1991)). The TNF-family ligands induce such various cellular responses by binding to TNF-family receptors.

›DETAILED DESCRIPTION · 37 of 68

In other embodiments, the invention provides a method-of stimulating, enhancing, and/or promoting CD8 + T cell secretion of IFN-γ comprising contacting a CD8 + T cell with an effective amount of one or more AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, wherein said effective amount stimulates, enhances, and/or promotes CD8 + T cell secretion of IFN-γ. In specific embodiments, this method is performed in vitro. In other specific embodiments, this method is performed in vivo.

›DETAILED DESCRIPTION · 38 of 68

In other embodiments, the invention provides a method of regulating, modulating, and/or controlling CD8 + T cell secretion of IFN-γ comprising contacting a CD8 + T cell with an effective amount of one or more AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, wherein said effective amount regulates, modulates, and/or controls CD8 + T cell secretion of IFN-γ. In specific embodiments, this method is performed in vitro. In other specific embodiments, this method is performed in vivo.

In other embodiments, the invention provides a method of regulating, modulating, and/or controlling a Th1 response comprising contacting a T cell with an effective amount of one or more AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, wherein said effective amount regulates, modulates, and/or controls a Th1 response. In specific embodiments, this method is performed in vitro. In other specific embodiments, this method is performed in vivo.

AIM II polynucleotides, polypeptides, agonists or antagonists of the invention may be used in developing treatments for any disorder mediated (directly or indirectly) by defective, or insufficient amounts of AIM II. AIM II polypeptides, agonists or antagonists may be administered to a patient (e.g., mammal, preferably human) afflicted with such a disorder. Alternatively, a gene therapy approach may be applied to treat such disorders. Disclosure herein of AIM II nucleotide sequences permits the detection of defective AIM II genes, and the replacement thereof with normal AIM II-encoding genes. Defective genes may be detected in in vitro diagnostic assays, and by comparison of the AIM II nucleotide sequence disclosed herein with that of a AIM II gene derived from a patient suspected of harboring a defect in this gene.

The AIM II polypeptide of the present invention may be employed to treat lymphoproliferative disease which results in lymphadenopathy, the AIM II mediates apoptosis by stimulating clonal deletion of T-cells and may therefore, be employed to treat autoimmune disease, to stimulate peripheral tolerance and cytotoxic T-cell mediated apoptosis. The AIM II may also be employed as a research tool in elucidating the biology of autoimmune disorders including systemic lupus erythematosus (SLE), Graves' disease, immunoproliferative disease lymphadenopathy (IPL), angioimmunoproliferative lymphadenopathy (AIL), immunoblastive lymphadenopathy (IBL), rheumatoid arthritis, diabetes, and multiple sclerosis, allergies and to treat graft versus host disease.

The AIM II polynucleotides, polypeptides and/or agonists or antagonists of the invention may also be used to treat, prevent, diagnose and/or prognose diseases which include, but are not limited to, autoimmune disorders, immunodeficiency disorders, and graft versus host disease.

The invention further provides compositions, comprising AIM II polynucleotides, polypeptides, antibodies, and agonists and/or antagonists, for administration to cells in vitro, to cells ex vivo, and to cells in vivo, or to a multicellular organism. In preferred embodiments, the compositions of the invention comprise an AIM II polynucleotide for expression of an AIM II polypeptide in a host organism for treatment of disease. In a most preferred embodiment, the compositions of the invention comprise an AIM II polynucleotide for expression of an AIM II polypeptide in a host organism for treatment of an immunodeficiency and/or conditions associated with an immunodeficiency. In another most preferred embodiment, the compositions of the invention comprise an AIM II polynucleotide for expression of an AIM II polypeptide in a host organism for treatment of an autoimmune disease and/or conditions associated with an autoimmune disease. Particularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with aberrant endogenous activity of AIM II (e.g., expression to enhance T-cell function by enhancing T cell activation, expanding T-cell numbers or increasing T cell IFN-γ secretion).

›DETAILED DESCRIPTION · 39 of 68

The present invention further encompasses methods and compositions for killing cells of hematopoictic origin, comprising contacting AIM II polypeptide with cells of hematopoietic origin. In preferred embodiments, the cells of hematopoietic origin are T cells. In further preferred embodiments, the cells of hematopoietic origin are CD8 + T cells.

In additional embodiments, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are administered to an animal, preferably a human, to treat, prevent, diagnose and/or prognose an autoimmune disease. In specific embodiments, the autoimmune disease treated, prevented, diagnosed and/or prognosed according to the methods of the invention is a member selected from the group consisting of Graft versus host disease (GVHD), Multiple Sclerosis, type 1 diabetes, rheumatoid arthritis, primary biliary cirrhosis, aplastic anemia, myelodysplasia, systemic lupus erhythematosus, idiopathic thrombocytopenia purpura, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (e.g, IgA nephropathy), an immune-based rheumatologic disease (e.g., SLE, rheumatoid arthritis, CREST syndrome (a variant of scleroderma characterized by c alcinosis, R aynaud's phenomenon, e sophageal motility disorders, s clerodactyly, and t elangiectasia.), Seronegative spondyloarthropathy (SpA), Polymyositis/dermatomyositis, Microscopic polyanguitis, Hepatitis C-asociated arthritis, Takayasu's arteritis, and undifferentiated connective tissue disorder), Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura), Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis, Goodpasture's syndrome, Pemphigus, Receptor autoimmunities such as, for example, (a) Graves' Disease, (b) Myasthenia Gravis, and (c) insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis/dermatomyositis, pernicious anemia, idiopathic Addison's disease, infertility, glomerulonephritis such as primary glomerulonephritis and IgA nephropathy, bullous pemphigoid, Sjogren's syndrome, diabetes mellitus, and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis), chronic active hepatitis, primary biliary cirrhosis, other endocrine gland failure, vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria, atopic dermatitis, asthma, inflammatory myopathies, and other inflammatory, granulamatous, degenerative, atrophic disorders, or a condition associated with an autoimmune disease.

›DETAILED DESCRIPTION · 40 of 68

In another preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are administered to an animal, preferably a human, to treat-prevent, diagnose and/or prognose autoimmune thyroiditis.

In additional embodiments, AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, are administered to an animal, preferably a human, to treat, prevent, diagnose and/or prognose an immunodeficiency. In specific embodiments, the immunodeficiency treated, prevented, diagnosed and/or prognosed according to the methods of the invention is a member selected from the group consisting of DiGeorge anomaly, ataxia telangiectasia, severe combined immunodeficiency (SCID)-X linked, SCID-autosomal, Wiskott-Aldrich Syndrome (WAS), chronic mucocutaneous candidiasis, natural killer cell deficiency, CD4+ T-lymphocytopenia, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton's disease, congenital agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, transient hypogammaglobulinemia of infancy, unspecified hypogammaglobulinemia, agammaglobulinemia, common variable immunodeficiency (CVID) (acquired), X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), antibody deficiency with normal or elevated Igs, immunodeficiency with thymoma, Ig heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), selective IgM immunodeficiency, recessive agammaglobulinemia (Swiss type), reticular dysgenesis, neonatal neutropenia, severe congenital leukopenia, thymic alymphoplasia-aplasia or dysplasia with immunodeficiency, short limbed dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezelof syndrome-combined immunodeficiency with Igs, purine nucleoside phosphorylase deficiency (PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severe combined immunodeficiency and conditions associated with an immunodeficiency.

›DETAILED DESCRIPTION · 41 of 68

The AIM II polypeptide of the present invention may also be employed to inhibit neoplasia, such as tumor cell growth. The AIM II polypeptide may be responsible for tumor destruction through apoptosis and cytotoxicity to certain cells. AIM II may also be employed to treat diseases which require growth promotion activity, for example, restenosis, since AIM II has proliferation effects on cells of endothelial origin. AIM II may, therefore, also be employed to regulate hematopoiesis in endothelial cell development.

Diseases associated with increased cell survival, or the inhibition of apoptosis, that may be treated, prevented, diagnosed and/or prognosed with the AIM II polynucleotides, polypeptides and/or agonists or antagonists of the invention include, but are not limited to, cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Grave's disease, Hashimoto's thyroiditis, autoimmune diabetes, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis, autoimmune gastritis, autoimmune thrombocytopenic purpura, and rheumatoid arthritis) and viral infections (such as herpes viruses, pox viruses and adenoviruses), inflammation, graft vs. host disease (acute and/or chronic), acute graft rejection, and chronic graft rejection. In preferred embodiments, AIM II polynucleotides, polypeptides, agonists, or antagonists of the invention are used to inhibit growth, progression, and/or metastasis of cancers, in particular those listed above or in the paragraph that follows.

Additional diseases or conditions associated with increased cell survival, that may be treated, prevented, diagnosed and/or prognosed with the AIM II polynucleotides, polypeptides and/or agonists or antagonists of the invention include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myclocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibro sarcoma, myxo sarcoma, liposarcoma, chondro sarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma.

›DETAILED DESCRIPTION · 42 of 68

Diseases associated with increased apoptosis, that may be treated, prevented, diagnosed and/or prognosed with the AIM II polynucleotides, polypeptides and/or agonists or antagonists of the invention include, but are not limited to, AIDS; neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and brain tumor or prior associated disease); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Grave's disease Hashimoto's thyroiditis, autoimmune diabetes, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus, immune-related glomerulonephritis, autoimmune gastritis, thrombocytopenic purpura, and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia), graft vs. host disease (acute and/or chronic), ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury or disease (e.g., hepatitis related liver injury, cirrhosis, ischemia/reperfusion injury, cholestosis (bile duct injury) and liver cancer); toxin-induced liver disease (such as that caused by alcohol), septic shock, ulcerative colitis, cachexia and anorexia. In preferred embodiments, AIM II polynucleotides, polypeptides, agonists, and/or antagonists are used to treat the diseases and disorders listed above.

Many of the pathologies associated with HIV are mediated by apoptosis, including HIV-induced nephropathy and HIV encephalitis. Thus, in additional preferred embodiments, AIM II polynucleotides, polypeptides, agonists or antagonists of the invention are used to treat AIDS and pathologies associated with AIDS.

Another embodiment of the present invention is directed to the use of AIM II polynucleotides, polypeptides, or antagonists to reduce AIM II-mediated death of T cells in HIV-infected patients. The role of T cell apoptosis in the development of AIDS has been the subject of a number-of studies (see, for example, Meyaard et al., Science 257:217-219 (1992); Groux et al., J. Exp. Med., 175:331 (1992); and Oyaizu et al., in Cell Activation and Apoptosis in HIV Infection , Andrieu and Lu, Eds., Plenum Press, New York, pp. 101-114 (1995)). Fas-mediated apoptosis has been implicated in the loss of T cells in HIV individuals (Katsikis et al., J. Exp. Med. 181:2029-2036 (1995). It is also likely that T cell apoptosis occurs through multiple mechanisms. For example, at least some of the T cell death seen in HIV patients is likely to be mediated by AIM II.

Activated human T cells are induced to undergo programmed cell death (apoptosis) upon triggering through the CD3/T cell receptor complex, a process termed activated-induced cell death (AICD). AICD of CD4 T cells isolated from HIV-infected asymptomatic individuals has been reported (Groux et al., supra). Thus, AICD may play a role in the depletion of CD4+ T cells and the progression to AIDS in HIV-infected individuals. Thus, the present invention provides a method of inhibiting AIM II-mediated T cell death in HIV patients, comprising administering AIM II polynucleotides, polypeptides, or antagonists of the invention to the patients. In one embodiment, the patient is asymptomatic when treatment with AIM II polynucleotides, polypeptides, or antagonists commences. If desired, prior to treatment, peripheral blood T cells may be extracted from an HIV patient, and tested for susceptibility to AIM II-mediated cell death by procedures known in the art. In one embodiment, a patient's blood or plasma is contacted with AIM II antagonists (e.g., anti-AIM II antibodies) of the invention ex vivo. The AIM II antagonists may be bound to a suitable chromatography matrix by procedures known in the art. The patient's blood or plasma flows through a chromatography column containing AIM II antagonist bound to the matrix, before being returned to the patient. The immobilized AIM II antagonist binds AIM II, thus removing AIM II protein from the patient's blood.

In additional embodiments an AIM II polynucleotide, polypeptide, or antagonist of the invention is administered in combination with other inhibitors of T cell apoptosis. For example, as discussed above, Fas-mediated apoptosis also has been implicated in loss of T cells in HIV individuals (Katsikis et al., J. Exp. Med. 181:2029-2036 (1995)). Thus, a patient susceptible to both Fas ligand mediated and AIM II-mediated T cell death may be treated with both an agent that blocks AIM II/AIM II receptor interactions and an agent that blocks Fas-ligand/Fas interactions. Suitable agents for blocking binding of Fas-ligand to Fas include, but are not limited to, soluble Fas polypeptides; multimeric forms of soluble Fas polypeptides (e.g., dimers of sFas/Fc); anti-Fas antibodies that bind Fas without transducing the biological signal that results in apoptosis; anti-Fas-ligand antibodies that block binding of Fas-ligand to Fas; and muteins of Fas-ligand that bind Fas but do not transduce the biological signal that results in apoptosis. Preferably, the antibodies employed according to this method are monoclonal antibodies. Examples of suitable agents for blocking Fas-ligand/Fas interactions, including blocking anti-Fas monoclonal antibodies, are described in WO 95/10540, hereby incorporated by reference.

In another example, agents which block binding of TRAIL to a TRAIL receptor are administered with the AIM II polynucleotides, polypeptides, or antagonists of the invention. Such agents include, but are not limited to, soluble TRAIL receptor polypeptides (e.g., a soluble form of OPG, DR4 (WO 98/32856); TR5 (WO 98/30693); DR5 (WO 98/41629); and TR10 (WO 98/54202)); multimeric forms of soluble TRAIL receptor polypeptides; and TRAIL receptor antibodies that bind the TRAIL receptor without transducing the biological signal that results in apoptosis, anti-TRAIL antibodies that block binding of TRAIL to one or more TRAIL receptors, and muteins of TRAIL that bind TRAIL receptors but do not transduce the biological signal that results in apoptosis. Preferably, the antibodies employed according to this method are monoclonal antibodies.

›DETAILED DESCRIPTION · 43 of 68

AIM II polypeptides of the invention may also be employed to regulate hematopoiesis and, in particular, erythropoiesis. Hematopoiesis is a multi-step cell proliferation and differentiation process which begins with a pool of multipotent stem cells. These cells can proliferate and differentiate into hematopoietic progenitors in reply to different stimuli. The AIM II polypeptides of the invention, as well as agonists and antagonists thereof, may be used to either stimulate or inhibit development of hematopoietic cells and, in particular, erythropoietic precursor cells.

The AIM II polypeptides used to regulate development of hematopoietic cells may be contacted-with hematopoietic target cells in a number of forms, e.g., as full-length or mature polypeptides, AIM II polypeptide fragments, or as hybrid fusion proteins composed of AIM II and a non-AIM II polypeptide (e.g., colony stimulating factors, erythropoietin, interferons, interleukins, etc.). Methods for producing such fusion proteins are described, for example, in Mele et al., U.S. Pat. No. 5,916,773.

As used herein, the phrase “hematopoietic cells” refers cells of hematopoietic origin such as erythrocytes, thrombocytes, lymphocytes, eosinophils, basophils, macrophages, and monocytes. Further, the phrase “erythropoietic precursor cells” refers cells of erythropoietic origin, such as erythrocytes.

As discussed below in Examples 6, 9 and 22, AIM II polypeptides (e.g., a polypeptide comprising, or alternatively consisting of amino acid residues 69 to 240 or 83 to 240 of SEQ ID NO:2), as well as agonists thereof (e.g., anti-AIM II antibodies, soluble forms of AIM II having amino acids sequences contained in the extracellular domain of AIM II) can be used to stimulate the production of cytokines (e.g., GM-CSF, G-CSF, IL-2, IL-3, IL-5, IL-6, IL-7, IL-12, IL-13, IL-15, anti-CD40, CD40L, IFN-γ, TNF-α, vascular endothelial growth factor (VEGF)) by various cell types (e.g., T cells).

As discussed below in Examples 25 and 26, AIM II functions in the activation of T cells and most noticeably in the activation of CD8 + T cells in response to certain stimuli.

AIM II polypeptides, as well as agonists thereof, can be used to modulate T cell immune responses (e.g., to inhibit antigen induced T cell proliferation).

AIM II polypeptides, as well as agonists thereof, can also be used to modulate cell-mediated immune responses (e.g., activate cytolytic T cells).

AIM II polypeptides, as well as agonists thereof, can be used to modulate apoptosis.

Further, AIM II polypeptides, as well as agonists thereof, can be used to induce cytolytic T cell responses and facilitate the formation of antigen-specific memory.

AIM II polynucleotides or polypeptides of the invention, or agonists or antagonists thereof, may also find application as the following:

A vaccine adjuvant that enhances immune responsiveness to specific antigen.

An adjuvant to enhance tumor-specific immune responses.

An adjuvant to enhance anti-viral immune responses. Anti-viral immune responses that may be enhanced using the compositions of the invention as an adjuvant, include virus and virus associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: AIDS, meningitis, Dengue, EBV, and hepatitis (e.g., Hepatitis B). In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: HIV/AIDS, Respiratory syncytial virus, Dengue, Rotavirus, Japanese B encephalitis, Influenza A and B, Parainfluenza, Measles, Cytomegalovirus, Rabies, Junin, Chikungunya, Rift Valley fever, Herpes simplex, and yellow fever.

An adjuvant to enhance anti-bacterial or anti-fungal immune responses. Anti-bacterial or anti-fungal immune responses that may be enhanced using the compositions of the invention as an adjuvant, include bacteria or fungus and bacteria or fungus associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: tetanus, Diphtheria, botulism, and meningitis type B. In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella typhi, Salmonella paratyphi, Meisseria meningitidis, Streptococcus pneumoniae , Group B streptococcus, Shigella spp., Enterotoxigenic Escherichia coli , Enterohemorrhagic E. coli, Borrelia burgdorferi , and Plasmodium (malaria).

An adjuvant to enhance anti-parasitic immune responses. Anti-parasitic immune responses that may be enhanced using the compositions of the invention as an adjuvant, include parasite and parasite associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a parasite. In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to Plasmodium (malaria).

As an agent to boost immunoresponsiveness among B cell and/or T cell immunodeficient individuals, such as, for example, an individual who has undergone a partial or complete splenectomy. B cell immunodeficiencies that may be ameliorated or treated by administering the AIM II polypeptides or polynucleotides of the invention, or agonists thereof, include, but are not limited to, severe combined immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton's disease, congenital agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, transient hypogammaglobulinemia of infancy, unspecified hypogammaglobulinemia, agammaglobulinemia, common variable immunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS), X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), antibody deficiency with normal or elevated Igs, immunodeficiency with thymoma, Ig heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), selective IgM immunodeficiency, recessive agammaglobulinemia (Swiss type), reticular dysgenesis, neonatal neutropenia, severe congenital leukopenia, thymic alymphoplasia-aplasia or dysplasia with immunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezelof syndrome-combined immunodeficiency with Igs, purine nucleoside phosphorylase deficiency (PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severe combined immunodeficiency. T cell immunodeficiencies that may be ameliorated or treated by administering the AIM II polypeptides or polynucleotides of the invention, or agonists thereof, include, but are not limited to, DiGeorge anomaly, thymic hypoplasia, chronic mucocutaneous candidiasis, natural killer cell deficiency, idiopathic CD4+ T-lymphocytopenia, and immunodeficiency with predominant T-cell defect.

›DETAILED DESCRIPTION · 44 of 68

As an agent to boost immunoresponsiveness among individuals having an acquired loss of B cell function. Conditions resulting in an acquired loss of B or T cell function that may be ameliorated or treated by administering the AIM II polypeptides or polynucleotides of the invention, or agonists thereof, include, but are not limited to, HIV Infection, AIDS, bone marrow transplant, and B cell chronic lymphocytic leukemia (CLL).

As an agent to boost immunoresponsiveness among individuals having a temporary immune deficiency. Conditions resulting in a temporary immune deficiency that may be ameliorated or treated by administering the AIM II polypeptides or polynucleotides of the invention, or agonists thereof, include, but are not limited to, recovery from viral infections (e.g., influenza), conditions associated with malnutrition, recovery from infectious mononucleosis, or conditions associated with stress, recovery from measles, recovery from blood transfusion, recovery from surgery.

AIM II polynucleotides or polypeptides of the invention, or agonists or antagonists thereof, may be used to diagnose, prognose, treat or prevent one or more of the following diseases or disorders, or conditions associated therewith: primary immuodeficiencies, immune-mediated thrombocytopenia, Kawasaki syndrome, bone marrow transplant (e.g., recent bone marrow transplant in adults or children), chronic B-cell lymphocytic leukemia, HIV infection (e.g., adult or pediatric HIV infection), chronic inflammatory demyelinating polyneuropathy, and post-transfusion purpura.

Additionally, AIM II polynucleotides or polypeptides of the invention, or agonists or antagonists thereof, may be used to diagnose, prognose, treat or prevent one or more of the following diseases, disorders, or conditions associated therewith, Guillain-Barre syndrome, anemia (e.g., anemia associated with parvovirus B19, patients with stable multiple myeloma who are at high risk for infection (e.g., recurrent infection), autoimmune hemolytic anemia (e.g., warm-type autoimmune hemolytic anemia), thrombocytopenia (e.g., neonatal thrombocytopenia), and immune-mediated neutropenia), transplantation (e.g., cytomegalovirus (CMV)-negative recipients of CMV-positive organs), hypogammaglobulinemia (e.g., hypogammaglobulinemic neonates with risk factor for infection or morbidity), epilepsy (e.g., intractable epilepsy), systemic vasculitic syndromes, myasthenia gravis (e.g., decompensation in myasthenia gravis), dermatomyositis, and polymyositis.

Autoimmune disorders and conditions associated with these disorders that maybe treated, prevented, and/or diagnosed with the AIM II polynucleotides, polypeptides, and/or antagonist of the invention (e.g., anti-AIM II antibodies), include, but are not limited to, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (e.g., IgA nephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura), Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye disease.

Additional autoimmune disorders (that are highly probable) that may be treated, prevented, and/or diagnosed with the compositions of the invention include, but are not limited to, autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis) (often characterized, e.g., by cell-mediated and humoral thyroid cytotoxicity), systemic lupus erhythematosus (often characterized, e.g., by circulating and locally generated immune complexes), Goodpasture's syndrome (often characterized, e.g., by anti-basement membrane antibodies), Pemphigus (often characterized, e.g., by epidermal acantholytic antibodies), receptor autoimmunities such as, for example, (a) Graves' Disease (often characterized, e.g., by TSH receptor antibodies), (b) Myasthenia Gravis (often characterized, e.g., by acetylcholine receptor antibodies), and (c) insulin resistance (often characterized, e.g., by insulin receptor antibodies), autoimmune hemolytic anemia (often characterized, e.g., by phagocytosis of antibody-sensitized RBCs), autoimmune thrombocytopenic purpura (often characterized, e.g., by phagocytosis of antibody-sensitized platelets.

Additional autoimmune disorders (that are probable) that may be treated, prevented, and/or diagnosed with the compositions of the invention include, but are not limited to, rheumatoid arthritis (often characterized, e.g., by immune complexes in joints), scleroderma with anti-collagen antibodies (often characterized, e.g., by nucleolar and other nuclear antibodies), mixed connective tissue disease (often characterized, e.g., by antibodies to extractable nuclear antigens (e.g., ribonucleoprotein)), polymyositis/dermatomyositis (often characterized, e.g., by nonhistone ANA), pernicious anemia (often characterized, e.g., by antiparietal cell, microsomes, and intrinsic factor antibodies), idiopathic Addison's disease (often characterized, e.g., by humoral and cell-mediated adrenal cytotoxicity, infertility (often characterized, e.g., by antispermatozoal antibodies), glomerulonephritis (often characterized, e.g., by glomerular basement membrane antibodies or immune complexes) such as primary glomerulonephritis and IgA nephropathy, bullous pemphigoid (often characterized, e.g., by IgG and complement in basement membrane), Sjogren's syndrome (often characterized, e.g., by multiple tissue antibodies, and/or a specific nonhistone ANA (SS-B)), diabetes mellitus (often characterized, e.g., by cell-mediated and humoral islet cell antibodies), and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis) (often characterized, e.g., by beta-adrenergic receptor antibodies).

›DETAILED DESCRIPTION · 45 of 68

Additional autoimmune disorders (that are possible) that may be treated, prevented, and/or diagnosed with the compositions of the invention include, but are not limited to, chronic active hepatitis (often characterized, e.g., by smooth muscle antibodies), primary biliary cirrhosis (often characterized, e.g., by mitchondrial antibodies), other endocrine gland failure (often characterized, e.g., by specific tissue antibodies in some cases), vitiligo (often characterized, e.g., by melanocyte antibodies), vasculitis (often characterized, e.g., by Ig and complement in vessel walls and/or low serum complement), post-MI (often characterized, e.g., by myocardial antibodies), cardiotomy syndrome (often characterized, e.g., by myocardial antibodies), urticaria (often characterized, e.g., by IgG and IgM antibodies to IgE), atopic dermatitis (often characterized, e.g., by IgG and IgM antibodies to IgE), asthma (often characterized, e.g., by IgG and IgM antibodies to IgE), inflammatory myopathies, and many other inflammatory, granulamatous, degenerative, and atrophic disorders.

In a preferred embodiment, the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are treated, prevented, and/or diagnosed using anti-AIM II antibodies.

In a specific preferred embodiment, rheumatoid arthritis is treated, prevented, and/or diagnosed using anti-AIM II antibodies and/or other antagonist of the invention.

In a specific preferred embodiment, lupus is treated, prevented, and/or diagnosed using anti-AIM II antibodies and/or other antagonist of the invention.

In a specific preferred embodiment, nephritis associated with lupus is treated, prevented, and/or diagnosed using anti-AIM II antibodies and/or other antagonist of the invention.

In a specific embodiment, AIM II polynucleotides or polypeptides, or antagonists thereof (e.g., anti-AIM II antibodies) are used to treat or prevent systemic lupus erythematosus and/or diseases, disorders or conditions associated therewith. Lupus-associated diseases, disorders, or conditions that may be treated or prevented with AIM II polynucleotides or polypeptides, or antagonists of the invention, include, but are not limited to, hematologic. disorders (e.g., hemolytic anemia, leukopenia, lymphopenia, and thrombocytopenia), immunologic disorders (e.g., anti-DNA antibodies, and anti-Sm antibodies), rashes, photosensitivity, oral ulcers, arthritis, fever, fatigue, weight loss, serositis (e.g., pleuritus (pleuricy)), renal disorders (e.g. nephritis), neurological disorders (e.g., seizures, peripheral neuropathy, CNS related disorders), gastroinstestinal disorders, Raynaud phenomenon, and pericarditis. In a preferred embodiment, the AIM II polynucleotides or polypeptides, or antagonists thereof (e.g., anti-AIM II antibodies) are used to treat or prevent renal disorders associated with systemic lupus erythematosus. In a most preferred embodiment, AIM II polynucleotides or polypeptides, or antagonists thereof(e.g., anti-AIM II antibodies) are used to treat or prevent nephritis associated with systemic lupus erythematosus.

In certain embodiments, soluble AIM II polypeptides of the invention (e.g., a polypeptide comprising, or alternatively consisting of, amino acids 69 to 240 or 83 to 240 of SEQ ID NO:2), or agonists thereof, are administered, to treat, prevent, prognose and/or diagnose an immunodeficiency (e.g., severe combined immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton's disease, congenital agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, transient hypogammaglobulinemia of infancy, unspecified hypogammaglobulinemia, agammaglobulinemia, common variable immunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS), X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), antibody deficiency with normal or elevated Igs, immunodeficiency with thymoma, Ig heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), selective IgM immunodeficiency, recessive agammaglobulinemia (Swiss type), reticular dysgenesis, neonatal neutropenia, severe congenital leukopenia, thymic alymphoplasia-aplasia or dysplasia with immunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezelof syndrome-combined immunodeficiency with Igs, purine nucleoside phosphorylase deficiency (PNP), MHC Class IT deficiency (Bare Lymphocyte Syndrome) and severe combined immunodeficiency, DiGeorge anomaly, thymic hypoplasia, chronic mucocutaneous candidiasis, natural killer cell deficiency, idiopathic CD4+ T-lymphocytopenia, and immunodeficiency with predominant T-cell defect ) or conditions associated with an immunodeficiency.

A number of soluble forms of the polypeptides of the present invention can be used to treat, prevent, prognose and/or diagnose disease states. These soluble forms will generally lack the AIM II transmembrane domain. Examples of soluble AIM II polypeptides include those where the extracellular and intracellular domains are present, but the transmembrane domain has been deleted. Further examples comprising, or alternatively consisting of, amino acids 60 to 240, 69 to 240, or 83 to 240 of SEQ ID NO:2.

In a specific embodiment, AIM II polypeptides or polynucleotides of the invention, or agonists thereof, is administered to treat, prevent, prognose and/or diagnose common variable immunodeficiency.

In a specific embodiment, AIM II polypeptides or polynucleotides of the invention, or agonists thereof, is administered to treat, prevent, prognose and/or diagnose X-linked agammaglobulinemia.

In another specific embodiment, AIM II polypeptides or polynucleotides of the invention, or agonists thereof, is administered to treat, prevent, prognose and/or diagnose severe combined immunodeficiency (SCID).

›DETAILED DESCRIPTION · 46 of 68

In another specific embodiment, AIM II polypeptides or polynucleotides of the invention, or agonists thereof, is administered to treat, prevent, prognose and/or diagnose Wiskott-Aldrich syndrome.

In another specific embodiment, AIM II polypeptides or polynucleotides of the invention, or agonists thereof, is administered to treat, prevent, prognose and/or diagnose X-linked Ig deficiency with hyper IgM.

In another specific embodiment, AIM II polypeptides or polynucleotides of the invention, or agonists thereof, is administered to treat, prevent, prognose and/or diagnose; DiGeorge anomaly.

AIM II antagonists also may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described.

The antagonists may further be employed for instance to treat cachexia which is a lipid clearing defect resulting from a systemic deficiency of lipoprotein lipase, which is believed to be suppressed by AIM II. The AIM II antagonists may also be employed to treat cerebral malaria in which AIM II may play a pathogenic role.

The AIM II antagonists may also be employed to prevent graft-host rejection (e.g., by preventing the stimulation of the immune system in the presence of a graft).

The AIM II antagonists may also be employed to prevent graft-host disease. In a preferred embodiment, anti-AIM II antibody is employed to prevent graft-host disease.

The AIM II antagonists may also be employed to inhibit bone resorption and, therefore, to treat and/or prevent osteoporosis.

The antagonists may also be employed as anti-inflammatory agents, and to treat endotoxic shock. This critical condition results from an exaggerated response to bacterial and other types of infection.

AIM II antagonists may also find application as the following:

As inhibitors of the production of one or more cytokines.

As inhibitors of cytolytic T cell responses and the formation of antigen-specific memory.

As modulators of T cell immune responses (e.g., inhibitors of antigen induced T cell proliferation).

As modulators of cell mediated immune responses (e.g., inhibitors of cytolytic T cell activation).

In a specific embodiment, nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention, by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.

For general reviews of the methods of gene therapy, see Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; and Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY.

In a preferred aspect, the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host. In particular, such nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody nucleic acids (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438). In specific embodiments, the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, of the antibody.

Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180 dated Apr. 16, 1992 (Wu et al.); WO 92/22635 dated Dec. 23, 1992 (Wilson et al.); WO 92/20316 dated Nov. 26, 1992 (Findeis et al.); WO93/14188 dated Jul. 22, 1993 (Clarke et al.), WO 93/20221 dated Oct. 14, 1993 (Young)). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).

›DETAILED DESCRIPTION · 47 of 68

In a specific embodiment, viral vectors that contain nucleic acid sequences encoding an antibody of the invention are used. For example, a retroviral vector can be used (see Miller et al., 1993, Meth. Enzymol. 217:581-599). These retroviral vectors have been to delete retroviral sequences that are not necessary for packaging of the viral genome and integration into host cell DNA. The nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitate delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen et al., 1994, Biotherapy 6:291-302, which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., 1994, J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114.

Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3:499-503 present a review of adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy 5:3-10 demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., 1991, Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155; Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234; PCT Publication WO94/12649; and Wang et al., 1995, Gene Therapy 2:775-783. In a preferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300; U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther. 29:69-92) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.

In a preferred embodiment, the cell used for gene therapy is autologous to the patient.

In an embodiment in which recombinant cells are used in gene therapy, nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see, e.g., PCT Publication WO 94/08598, dated Apr. 28, 1994; Stemple and Anderson, 1992, Cell 71:973-985; Rheinwald, 1980, Meth. Cell Bio. 21A:229; and Pittelkow and Scott, 1986, Mayo Clinic Proc. 61:771).

In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription.

As noted above, antagonists and agonists of the invention include AIM II polypeptides. These polypeptides can modulate their effect by, for example, binding to cellular proteins such as receptors. Methods for identifying peptides which interact with a specific protein are known in the art. For example, Phizicky and Fields, “Protein-protein interactions: methods for detection and analysis” Microbiol. Rev. 59:94-123 (1995), describe methods for screening peptides to identify those having binding affinity for a second polypeptide. Phizicky and Fields discuss methods such as protein affinity chromatography, affinity blotting, co-immunoprecipitation, and cross-linking. Additional molecular biological methods suitable for use with the present invention include protein probing of expression libraries, the two-hybrid system, cell panning, and phage display.

›DETAILED DESCRIPTION · 48 of 68

Another method for identifying AIM II polypeptides of the invention which bind to a cell surface receptor involves transfecting eukaryotic cells with DNA encoding the receptor, such that the cells expresses the receptor on their surfaces, followed by contacting the cells with a labeled (e.g., radioactive label, biotin, etc.) AIM II polypeptide. The amount of labeled AIM II polypeptide bound to the cells is measured and compared to that bound to control cells. The control cells will generally be cells which do not express the surface receptor. The detection of an increased amount of label bound to the cells which express the receptor as compared to the control cells indicates that the cells which expresses the receptor bind the AIM II polypeptide.

Further, as one skilled in the art would recognize, cells which express and retain AIM II polypeptides can be used to identify AIM II ligands. In one such an embodiment, cells which express AIM II would be contacted with potential ligands which have been detectably labeled. Further, such ligands may be polypeptides which are expressed as part of a library of sequences on the surface of a phage (e.g., a phage display library).

Once an AIM II polypeptide has been identified which binds to the cell surface receptor of interest, assays can be performed to determine whether the AIM II polypeptide induces or inhibits a receptor-mediated cellular response normally elicited by the particular receptor. Whether an AIM II polypeptide activates a receptor-mediated cellular response may be determined by measuring a cellular response known to be elicited by the receptor in the presence of the AIM II polypeptide or another ligand. Further, whether an AIM II polypeptide inhibits a receptor-mediated cellular response may be determined by measuring a cellular response known to be elicited by the receptor in the presence of both a molecule which is known to induce the cellular response and the AIM II polypeptide.

Soluble forms of the polypeptides of the present invention (e.g., an AIM polypeptide comprising, or alternatively consisting of, amino acid 69 to 240 or 83-240 of SEQ ID NO:2), for example, may be utilized in the ligand binding and receptor activation/inhibition assay described above.

AIM II has also been shown to inhibit the differentiation and survival of photoreceptor cells. Further, AIM II has been shown to inhibit the production of rhodopsin by retinal cells. Thus, AIM II polypeptides and AIM II agonists are useful for inhibiting the differentiation and survival of photoreceptor cells (e.g., rods and cones) and inhibiting rhodopsin production by retinal cells (e.g., rods).

AIM II polypeptides, agonists or antagonists of the invention may be used to treat cardiovascular disorders, including peripheral artery disease, such as limb ischemia.

Cardiovascular disorders include cardiovascular abnormalities, such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia and Scimitar Syndrome. Congenital heart defects include aortic coarctation, cor triatriatum, coronary vessel anomalies, crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition of great vessels, double outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septal defects, such as aortopulmonary septal defect, endocardial cushion defects, Lutembacher's Syndrome, trilogy of Fallot, and ventricular heart septal defects.

Cardiovascular disorders also include heart disease, such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestive caardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture, ventricular septal rupture, heart valve diseases, myocardial diseases, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, and ventricular fibrillation. Tachycardias include paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

Heart valve disease include aortic valve insufficiency, aortic valve stenosis, heart murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis.

Myocardial diseases include alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and myocarditis.

Myocardial ischemias include coronary disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.

›DETAILED DESCRIPTION · 49 of 68

Cardiovascular diseases also include vascular diseases such as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior vena cava syndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis, and venous insufficiency.

Aneurysms include dissecting aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, and iliac aneurysms.

Arterial occlusive diseases include arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya disease, renal artery obstruction, retinal artery occlusion, and thromboanguitis obliterans.

Cerebrovascular disorders include carotid artery diseases, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery diseases, cerebral embolism and thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subarachnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), subdlavian steal syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine, and vertebrobasilar insufficiency.

Embolisms include air embolisms, amniotic fluid embolisms, cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and thromboembolisms. Thrombosis include coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, and thrombophlebitis.

Ischemia includes cerebral ischemia, ischemic colitis, compartment syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion injuries, and peripheral limb ischemia. Vasculitis includes aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and Wegener's granulomatosis.

In one embodiment, an AIM II polynucleotide, polypeptide, agonist, or antagonist of the invention is used to treat thrombotic microangiopathies. One such disorder is thrombotic thrombocytopenic purpura (TTP) (Kwaan, H. C., Semin. Hematol. 24:71 (1987); Thompson et al., Blood 80:1890 (1992)). Increasing TTP-associated mortality rates have been reported by the U.S. Centers for Disease Control (Torok et al., Am. J Hematol. 50:84 (1995)). Plasma from patients afflicted with TTP (including HIV+ and HIV− patients) induces apoptosis of human endothelial cells of dermal microvascular origin, but not large vessel origin (Laurence et al., Blood 87:3245 (1996)). Plasma of TTP patients thus is thought to contain one or more factors that directly or indirectly induce apoptosis. Another thrombotic microangiopathy is hemolytic-uremic syndrome (HUS) (Moake, J. L., Lancet 343:393 (1994); Melnyk et al., Arch. Intern. Med. 155:2077 (1995); Thompson et al., supra). Thus, in one embodiment, the invention is directed to use of AIM II to treat the condition that is often referred to as “adult HUS” (even though it can strike children as well). A disorder known as childhood/diarrhea-associated HUS differs in etiology from adult HUS. In another embodiment, conditions characterized by clotting of small blood vessels may be treated using AIM II. Such conditions include, but are not limited to, those described herein. For example, cardiac problems seen in about 5-10% of pediatric AIDS patients are believed to involve clotting of small blood vessels. Breakdown of the microvasculature in the heart has been reported in multiple sclerosis patients. As a further example, treatment of systemic lupus erythematosus (SLE) is contemplated. In one embodiment, a patient's blood or plasma is contacted with AIM II polypeptides of the invention ex vivo. The AIM II polypeptides of the invention may be bound to a suitable chromatography matrix by procedures known in the art. According to this embodiment, the patient's blood or plasma flows through a chromatography column containing AIM II polynucleotides and/or polypeptides of the invention bound to the matrix, before being returned to the patient. The immobilized AIM II binds AIM II, thus removing AIM II protein from the patient's blood. Alternatively, AIM II polynucleotides, polypeptides, agonists or antagonists of the invention may be administered in vivo to a patient afflicted with a thrombotic microangiopathy. In one embodiment, a soluble form of AIM II polypeptide of the invention is administered to the patient. Thus, the present invention provides a method for treating a thrombotic microangiopathy, involving use of an effective amount of AIM II polynucleotide, polypeptide, agonist or antagonist. An AIM II polypeptide may be employed in in vivo or ex vivo procedures, to inhibit AIM II-mediated damage to (e.g., apoptosis of) microvascular endothelial cells.

AIM II polynucleotides, polypeptides, agonists or antagonists of the invention may be employed in combination with other agents useful in treating a particular disorder. For example, in an in vitro study reported by Laurence et al., Blood 87:3245 (1996), some reduction of TTP plasma-mediated apoptosis of microvascular endothelial cells was achieved by using an anti-Fas blocking antibody, aurintricarboxylic acid, or normal plasma depleted of cryoprecipitate. Thus, a patient may be treated with a polynucleotide and/or polypeptide of the invention in combination with an agent that inhibits Fas-ligand-mediated apoptosis of endothelial cells, such as, for example, an agent described above. In one embodiment, an AIM II polynucleotide, polypeptide, agonist or antagonist, and an anti-FAS blocking antibody are both administered to a patient afflicted with a disorder characterized by thrombotic microangiopathy, such as TTP or HUS. Examples of blocking monoclonal antibodies directed against Fas antigen (CD95) are described in WO 95/10540, hereby incorporated by reference.

›DETAILED DESCRIPTION · 50 of 68

The naturally occurring balance between endogenous stimulators and inhibitors of angiogenesis is one in which inhibitory influences predominate. Rastinejad et al., Cell 56:345-355 (1989). In those rare instances in which neovascularization occurs under normal physiological conditions, such as wound healing, organ regeneration, embryonic development, and female reproductive processes, angiogenesis is stringently regulated and spatially and temporally delimited. Under conditions of pathological angiogenesis such as, that characterizing solid tumor growth, these regulatory controls fail. Unregulated angiogenesis becomes pathologic and sustains progression of many neoplastic and non-neoplastic diseases. A number of serious diseases are dominated by abnormal neovascularization including solid tumor growth and metastases, arthritis, some types of eye disorders, and psoriasis. See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkman etal., N. Engl. J. Med. 333:1757-1763 (1995); Auerbach et al., J. Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research, Klein and Weinhouse, eds., Academic Press, New York, pp. 175-203 (1985); Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science 221:719-725 (1983). In a number of pathological conditions, the process of angiogenesis contributes to the disease state. For example, significant data have accumulated which suggest that the growth of solid tumors is dependent on angiogenesis. Folkman and Klagsbrun, Science 235:442-447 (1987).

The present invention provides for treatment of diseases or disorders associated with neovascularization by administration of the AIM II polynucleotides and/or polypeptides of the invention (including AIM II agonists and/or antagonists). Malignant and metastatic conditions which can be treated with the polynucleotides and polypeptides of the invention include, but are not limited to those malignancies, solid tumors, and cancers described herein and otherwise known in the art (for a review of such disorders, see Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)).

Additionally, ocular disorders associated with neovascularization which can be treated with the AIM II polynucleotides and polypeptides of the present invention (including AIM II agonists and AIM II antagonists) include, but are not limited to: neovascular glaucoma, diabetic retinopathy, retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of prematurity, macular degeneration, corneal graft neovascularization, as well as other eye inflammatory diseases, ocular tumors and diseases associated with choroidal or iris neovascularization. See, e.g., reviews by Waltman et al., Am. J. Ophthal. 85:704-710 (1978) and Gartner et al., Surv. Ophthal. 22:291-312 (1978).

Additionally, disorders which can be treated with the AIM II polynucleotides and polypeptides of the present invention (including AIM II agonists and AIM II antagonists) include, but are not limited to, hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing, granulations, hemophilic joints, hypertrophic scars, nonunion fractures, Osler-Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.

Polynucleotides and/or polypeptides of the invention, and/or agonists and/or antagonists thereof, are useful in the diagnosis and treatment or prevention of a wide range of diseases and/or conditions. Such diseases and conditions include, but are not limited to, cancer (e.g., immune cell related cancers, breast cancer, prostate cancer, ovarian cancer, follicular lymphoma, cancer associated with mutation or alteration of p53, brain tumor, bladder cancer, uterocervical cancer, colon cancer, colorectal cancer, non-small cell carcinoma of the lung, small cell carcinoma of the lung, stomach cancer, etc.), lymphoproliferative disorders (e.g., lymphadenopathy), microbial (e.g., viral, bacterial, etc.) infection (e.g., HIV-1 infection, HIV-2 infection, herpesvirus infection (including, but not limited to, HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-7, EBV), adenovirus infection, poxvirus infection, human papilloma virus infection, hepatitis infection (e.g., HAV, HBV, HCV, etc.), Helicobacter pylori infection, invasive Staphylococcia, etc.), parasitic infection, nephritis, bone disease (e.g., osteoporosis), atherosclerosis, pain, cardiovascular disorders (e.g., neovascularization, hypovascularization or reduced circulation (e.g., ischemic disease (e.g., myocardial infarction, stroke, etc.)), AIDS, allergy, inflammation, neurodegenerative disease (e.g., Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, pigmentary retinitis, cerebellar degeneration, etc.), graft rejection (acute and chronic), graft vs. host disease, diseases due to osteomyelodysplasia (e.g., aplastic anemia, etc.), joint tissue destruction in rheumatism, liver disease (e.g., acute and chronic hepatitis, liver injury, and cirrhosis), autoimmune disease (e.g., multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, immune complex glomerulonephritis, autoimmune diabetes, autoimmune thrombocytopenic purpura, Grave's disease, Hashimoto's thyroiditis, etc.), cardiomyopathy (e.g., dilated cardiomyopathy), diabetes, diabetic complications (e.g., diabetic nephropathy, diabetic neuropathy, diabetic retinopathy), influenza, asthma, psoriasis, glomerulonephritis, septic shock, and ulcerative colitis.

Polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are useful in promoting angiogenesis, wound healing (e.g., wounds, bums, and bone fractures).

Polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are also useful as an adjuvant to enhance immune responsiveness to specific antigen and/or anti-viral immune responses.

More generally, polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are useful in regulating (i.e., elevating or reducing) immune response. For example, polynucleotides and/or polypeptides of the invention maybe useful in preparation or recovery from surgery, trauma, radiation therapy, chemotherapy, and transplantation, or may be used to boost immune response and/or, recovery in the elderly and immunocompromised individuals. Alternatively, polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are useful as immunosuppressive agents, for example in the treatment or prevention of autoimmune disorders. In specific embodiments, polynucleotides and/or polypeptides of the invention are used to treat or prevent chronic inflammatory, allergic or autoimmune conditions, such as those described herein or are otherwise known in the art.

›DETAILED DESCRIPTION · 51 of 68

The uses of the AIM II polypeptides, particularly human AIM II polypeptides, include but are not limited to, the treatment or prevention of viral hepatitis, Herpes viral infections, allergic reactions, adult respiratory distress syndrome, neoplasia, anaphylaxis, allergic asthma, allergen rhinitis, drug allergies (e.g., to penicillin, cephalosporins), primary central nervous system lymphoma (PCNSL), glioblastoma, chronic lymphocytic leukemia (CLL), lymphadenopathy, autoimmune disease, graft versus host disease, rheumatoid arthritis, osteoarthritis, Graves' disease, acute lymphoblastic leukemia (ALL), lymphomas (Hodgkin's disease and non-Hodgkin's lymphoma (NHL)), ophthalmopathy, uveoretinitis, the autoimmune phase of Type 1 diabetes, myasthenia gravis, glomerulonephritis, autoimmune hepatological disorder, autoimmune inflammatory bowel disease, and Crohn's disease. In addition, the AIM II polypeptide of the present invention may be employed to inhibit neoplasia, such as tumor cell growth. The combination of AIM II protein with immunotherapeutic agent such as IL-2 or IL-12 may result in synergistic or additive effects that would be useful for the treatment of established cancers. The AIM II polypeptide may also be useful for tumor therapy. AIM II may further be employed to treat diseases which require growth promotion activity, for example, restenosis, since AIM II has proliferative effects on cells of endothelial origin. AIM II may, therefore, also be employed to regulate hematopoiesis in endothelial cell development.

The AIM II polypeptides of the invention may also be employed to inhibit the differentiation and proliferation of T cells and B cells. AIM II induced inhibition of T and B cell activation, differentiation and/or proliferation may be employed to treat a number of immunological based diseases, several of which are referred to above. Further, depending on the particular AIM II polypeptide employed, the AIM II polypeptides of the invention may also be employed to stimulate activation, differentiation and/or proliferation of T cells and B cells.

AIM II may act as a cytokine adjuvant or costimulatory molecule. The following experiments are performed to assess the in vivo AIM II-protein on the host immune system.

Tumor or non-tumor bearing mice are treated with AIM II protein at three different doses (0.1 mg/kg, 1 mg/kg and 10 mg/kg, i.p., QD, 10-14 days, N=5 per group) before or after immunization with tumor antigen or superantigen, the mice are sacrificed weekly post treatment after blood collection. The spleens or the lymph nodes are used for the following in vitro analyses well known to those skilled in the art:

FACS analyses: Expression of surface markers for T cells, B cells, NK cells, Monocytes, Dendritic cells, costimulatory and adhesion molecules. Cytokine production assays T cell proliferation or cytotoxicity assay

AIM II protein and tumor antigen may result in the induction of protective immunity, which could lead to protecting mice from subsequent tumor challenge. In order to examine this possibility the following experiment can be performed using syngeneic C57BL/6 mice to test the effect of AIM II on induction of tumor or Ag-specific protective immunity.

MC-38 tumor-free mice treated with AIM II protein will be challenged with MC-38 or irrelevant murine sarcoma MCA-102 using techniques well known to those skilled in the art.

Three possible results could be observed:

If generation of tumor-specific protective immunity upon AIM II treatment is demonstrated, the following depletion experiments are performed to identify which leukocyte sub-population is responsible for the tumor rejection. The mice will be treated with various mAb which recognize either CD4+ or CD8+ T cells, NK cells, granulocyte (Gr1+), or specific cytokine such as IFNγ using techniques well known to those skilled in the art. Tumor growth in these antibody-treated mice is measured.

AIM II may also be used to treat rheumatoid arthritis (RA) by inhibiting the increase in angiogenesis or the increase in endothelial cell proliferation required to sustain an invading pannus in bone and cartilage as is often observed in RA. Endothelial cell proliferation is increased in the synovia of RA patients as compared to patients with osteoarthritis (OA) or unaffected individuals. Neovascularization is needed to sustain the increased mass of the invading pannus into bone and cartilage. Inhibition of angiogenesis is associated with a significant decrease in the severity of both early and chronic arthritis in animal models.

The AIM II polypeptide is believed to possess binding activities for a number of proteins, including several human cellular receptors. These receptors include the lymphotoxin-β-receptor (LT-β-R), TR2 (also referred to as the Herpes virus entry mediator (HVEM) and ATAR), CD27, TR6 (also referred to as DcR3), TR9 (DR6) and TRANK (also referred to as receptor activator of nuclear factor-kappa B (RANK)).

Each of the receptors listed immediately above is involved in various physiological processes which may be modulated by the AIM II polypeptides of the invention. More specifically, the polypeptides of the invention can be used to stimulate or block the action of ligands which bind cellular receptors having AIM II binding activity (e.g., LT-β-R, TR2, CD27, TR6, TR9 and TRANK).

LT-β, which binds to the LT-β-R, has been implicated in the development of secondary lymphoid tissues and the maintenance of organized lymphoid tissues in adults. LT-β-R may, in some instances, function in conjunction with TR2 to mediate cellular responses and has been shown to be expressed in a number of tissues in the lung including a subpopulation of T-lymphocytes. LT-β-R has also been implicated in the formation of germinal centers and thus appears to be involved in humoral immune responses. Rennert et al., Int. Immunol. 9:1627-1639 (1997).

The AIM II polypeptides of the invention may be employed to inhibit the formation of germinal centers and LT-β-R mediated humoral responses by blocking access of cellular ligands to LT-β-R. Further, polypeptides of the invention may stimulate the formation of germinal centers and LT-β-R mediated humoral responses by activating LT-β-R.

›DETAILED DESCRIPTION · 52 of 68

One skilled in the art would recognize that different portions of the AIM II polypeptide may have different effects on LT-β-R. One skilled in the art would also recognize that the effect that the AIM II polypeptides of the invention would have on LT-β-R would vary with the individual peptide and the effect it has when bound to LT-β-R. Methods for screening molecules having agonistic and antagonistic activities of cellular receptor are described above.

The core protein of Hepatitis C virus (HCV) has also been shown to associate with LT-β-R and enhance signaling mediated by this receptor. Chen et al., J. Virol. 71:9417-9426 (1997). Further, the interaction of this protein with LT-β-R may contribute to the chronically activated, persistent state of HCV-infected cells. The AIM II polypeptides of the invention may be employed to block HVC stimulation of LT-β-R and the pathology associated with this virus.

TR2 is a member of the tumor necrosis factor (TNF) receptor family which is expressed in a number of human tissues and cell lines. This protein is expressed constitutively and in relatively high levels in peripheral blood T cells, B cells, and monocytes. Kwon et al., J. Biol. Chem. 272:14272-14276 (1997). TR2 serves a number of functions in vivo, including the mediation of Herpes viral entry into cells during infection. Further, a TR2-Fc fusion protein has been demonstrated to inhibit mixed lymphocyte reaction-mediated proliferation. These data suggest that the TR2 and its ligand play a role in T cell stimulation. It has been shown along these lines that overexpression of TR2 activates NF-κB and AP-1. This activation appears to occur through a TNF receptor-associated factor (TRAF)-mediated mechanism.

The AIM II polypeptides of the invention may be employed to inhibit T cell activation, and thus T cell mediated immune responses, by blocking access to TR2 by cellular ligands which activate this receptor. Similarly, polypeptides of the invention may stimulate T cell activation by activating TR2. As noted above for LT-β-R, one skilled in the art would recognize that different portions of the AIM II polypeptide may either inhibit or stimulate TR2 mediated cellular responses.

The AIM II polypeptides of the invention may also be employed to prevent or treat Herpes viral infections.

Expression of CD27, as well as its ligand CD70, is predominantly confined to lymphocytes. Further, CD27 has been shown to interact with CD70 and to be involved in the induction of IgE synthesis in B cells. Nagumo et al., J. Immunol. 161:6496-6502 (1998). In addition, activation of CD27 may enhance IgE synthesis. Inhibition of the interaction between CD27 and CD70 thus may inhibit IgE production and allergic responses.

The AIM II polypeptides of the invention may be used for modulating immune responses. For example, AIM II polypeptides may be used to regulate the function of B cells by inhibiting the interaction between CD27 and CD70. AIM II polypeptides may thus bind to CD27 and inhibit B cell differentiation and proliferation, as well as the secretion of proteins (e.g., IgE) by these cells. Therefore, AIM II polypeptides may be employed to suppress IgE antibody formation in the treatment of IgE-induced immediate hypersensitivity reactions, such as allergic rhinitis (also know as hay fever), bronchial asthma, allergic asthma, anaphylaxis, atopic dermatitis and gastrointestinal food allergy.

CD27 is also believed to be the receptor for a pro-apoptotic protein commonly known as Siva. Pandanilametal., Kidney Int. 54:1967-1975 (1998). AIM II polypeptides of the invention may be employed to inhibit the interaction between Siva and CD27 and thus prevent Siva/CD27 mediated induction of apoptosis. Diseases associated with decreased cell survival, or increased apoptosis, include AIDS; neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration); myelodysplastic syndromes (such as aplastic anemia), ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.

AIM II polypeptides of the invention may also be employed to enhance activation of the Siva/CD27 apoptotic pathway and thus facilitate the induction of apoptosis. Diseases associated with increased cell survival, or the inhibition of apoptosis, include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors), autoimmune disorders (such as systemic lupus erythematosus, immune-related glomerulonephritis, and rheumatoid arthritis) and viral infections (such as herpes viruses, pox viruses and adenoviruses), information graft v. host disease, acute graft rejection, and chronic graft rejection.

While CD27 may be membrane-bound, a soluble form of CD27 is produced in the course of the immune response. Soluble CD27 (sCD27) is found in a number of body fluids and may be measured to monitor local and systemic immune activation. Further, CD27 is expressed on human malignant B cells and high levels of sCD27 are present in the sera of patients with various B-cell malignancies. Kersten et al., Blood 87:1985-1989 (1996). These elevated levels of sCD27 have been shown to strongly correlate with tumor load.

sCD27 has also been shown to be elevated in patients with a variety lymphoid malignancies and solid tumors of the central nervous system. These afflictions include primary central nervous system lymphoma (PCNSL) and lymphoid malignancies located in the meninges (e.g., acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL)).

Soluble CD27 has also been found to be elevated in patients with a number of non-hyperproliferative diseases. For example, sCD27 has been shown to be elevated in patients with untreated Graves' hyperthyroidism. Kallio et al., J. Lab. Clin Med. 132:478-482 (1998). Further, increases in sCD27 serum levels have been found in patients with systemic lupus erythematosus (SLE) and this increase has been shown to be associated with the activity of the disease. Font et al., Clin. Immunol. Immunopathol. 81:239-243 (1996); Swaak et al., Clin. Rheumatol. 14:293-300 (1995). Also, B cells from most patients with chronic lymphocytic leukemia (CLL) have been shown to co-express both membrane-bound and soluble CD27, as well as CD70. Ranheim et al., Blood 85:3556-3565 (1995).

›DETAILED DESCRIPTION · 53 of 68

It has been postulated that sCD27 may prevent leukemic B cells from stimulating T cells via CD70, and thus may impair the ability of B cells to function as antigen-presenting cells. Ranheim et al., Blood 85:3556-3565 (1995). Polypeptides of the invention may be employed to inhibit interactions between sCD27 and CD70, and, thus, to enhance the ability of B cells to act as antigen-presenting cells.

AIM II polypeptides of the invention may also be employed to treat diseases and afflictions associated with increased levels of sCD27. While not wishing to be bound to a mechanistic theory, AIM II polypeptides may be useful in treatment regimens for these conditions since AIM II binds sCD27 and prevents it from interacting with cellular ligands.

AIM II polypeptides bind to TR6. (See, Example 13). TR6 (DcR3) is a novel soluble member of the TNFR superfamily, which is constitutively expressed in lung tissue, tumor cells and in endothelial cells. TR6 binds specifically to AIM II and FasL and inhibits their activities. Like DcR1, DcR2 and another soluble member of TNFR superfamily, OPG, TR6 may act as an inhibitor of signaling through TNF family members, FasL and AIM II. TR6 appears to have important roles in the inhibition of apoptosis and tumor modulation. TR6 may act as an inhibitor of AIM II interaction and may also act in distinct roles, depending on cell types. TR6 (DcR3) may act as a decoy receptor and contribute to immune evasion both in slow and rapid tumor cell death, that are mediated by AIM II or FasL mediated apoptosis pathway respectively. Indeed, TR6 (DcR3) has been shown to bind to FasL and might contribute to immune evasion by certain tumors (Pitti, R. M., et al., Nature 396:699-703 (1998)). AIM II and FasL are known to be expressed in activated T cells. Therefore, it is believed that TR6 and its ligands are important for interactions between activated T lymphocytes and endothelium. TR6 may be involved in activated T cell trafficking as well as endothelial cell survival. Another possibility is that TR6 may act as a cytokine to trigger membrane-bound FasL or AIM II and directly transduce signals through FasL or AIM II. Recently, the Desbarats and Suzuki groups reported that FasL could itself transduce signals, leading to cell-cycle arrest and cell death in CD4 + T cells but cell proliferation in CD8 + T cells (Desbarats, J., et al., Nature Medicine 4:1377-1382 (1998); Suzuki, I., and Fink, P. J., J. Exp. Med. 187:123-128 (1998)). Therefore, TR6 may act as a cytokine for signaling through FasL and AIM II.

Because TR6 appears to have important roles in the inhibition of apoptosis and tumor modulation, AIM II polypeptides of the invention may also be employed to treat diseases and afflictions associated with increased levels of TR6, or conditions where increased apoptosis would be desirable. While not wishing to be bound to a mechanistic theory, AIM II polypeptides may be useful in treatment regimens for these conditions since AIM II binds TR6 and prevents TR6 from interacting with cellular ligands to block apoptosis.

AIM II polypeptides are also believed to bind to RANK. (See Anderson et al., Nature 390:175-179 (1997).) RANK is a protein which has been implicated in osteoclast differentiation and regulation of interactions between T cells and dendritic cells. RANK apparently mediates its cellular effects via interaction with RANKL (also referred to as osteoprotegerin ligand (OPGL), TRANCE and ODF).

Mice having a disrupted RANKL gene show severe osteoporosis, exhibit defective tooth eruption, and lack osteoclasts. These mice also exhibit defects in T and B lymphocyte differentiation. Additionally, RANKL-deficient mice lack lymph nodes but exhibit normal splenic structure and Peyer's patches. These data indicate that RANKL mediated pathways regulate lymph node organogenesis, lymphocyte development, and osteoclast differentiation and proliferation.

There are two main classes of bone cells: cells which make bone, osteoblasts, and cells which resorb bone, osteoclasts. These cells each have very precise functions and the balance between their activities is critical to the maintenance of the skeletal system. For example, in human adults, between 10 to 15% of trabecular bone surfaces are covered with osteoid (new unmineralized bone made by osteoblasts) while about 4% have active resorptive surfaces. The dynamic nature of the continuing flux of bone cell activity is illustrated by the fact that approximately 18% of total skeletal calcium is often removed and deposited over a period of one year.

The AIM II polypeptides of the invention may be employed to regulate osteoclast differentiation and proliferation (bone formation), as well as bone development and degradation. Polypeptides of the invention may, for example, be employed to inhibit osteoclast differentiation and proliferation and, thus, may be employed to decease the rate of bone degradation. Inhibition of osteoclast differentiation and proliferation and bone degradation may be useful in the treatment of conditions such as osteoporosis, skeletal and dental abnormalities, bone cancers, osteoarthritis, osteogenesis imperfecta, and Hurler and Marfan syndromes. Polypeptides of the invention may also be employed in processes for reshaping bone and teeth and in periodontal reconstructions where lost bone replacement or bone augmentation is required, such as in a jaw bone and supplementing alveolar bone loss resulting from periodontal disease to delay or prevent tooth loss (see, e.g., Sigurdsson et al., J. Periodontol. 66:511-21 (1995)).

The AIM II polypeptides of the invention may further be used to regulate T and B lymphocyte differentiation and proliferation. AIM II polypeptides may thus bind to RANK and inhibit the differentiation and proliferation of T and B lymphocyte, as well as the secretion of proteins (e.g., immunoglobulins) from these cells. AIM II polypeptides may therefore be employed to suppress lymphocyte-mediated immune responses, for example, to prevent graft rejection. AIM II polypeptides may also be used to inhibit osteoclast differentiation and proliferation. AIM II polypeptides may thus be employed to treat diseases such as bone cancers.

›DETAILED DESCRIPTION · 54 of 68

The present invention also provides AIM II polypeptides which mimic one or more of the natural ligands of RANK and stimulate RANK-mediated cellular responses. These cellular responses include the activation of T and B lymphocyte differentiation and proliferation and induction of osteoclast differentiation. AIM II polypeptides may thus be employed to treat diseases such as infections (e.g., bacterial, viral, and protozoal infections). AIM II polypeptides may also be employed to enhance immune responses (e.g., in the treatment of AIDS and AIDS related complexes) and to increase bone degradation rates.

AIM II polypeptide also binds TR9. TR9 (also referred to as DR6) is a novel member of the tumor necrosis factor family of receptors which possesses a cytoplasmic death domain. (See, WO 98/56892). TR9 induces apoptosis in mammalian cells and is capable of engaging the NF-κB and JNK pathways. TR9 is believed to play a role in inflammatory responses and immune regulation.

The AIM II polypeptides may be used to modulate inflammatory responses and immune regulation via TR9. AIM II polypeptides may also be used to induce apoptosis via TR9 via the NF-κB and JNK pathways. AIM II polypeptides may thus be employed to treat diseases such as infections. AIM II polypeptides may also be employed to enhance immune responses.

The AIM II polypeptide may be cleaved in vivo to form a soluble form of the molecule. As noted in Example 10, a cleavage site appears to be located between amino acid residues 82 and 83 of the sequence shown in SEQ ID NO:2. Cleavage of the AIM II polypeptide at this location is believed to result in the production of a soluble form of the molecule which comprises, or alternatively consisting of, amino acids 83-240 in SEQ ID NO:2. Soluble forms of AIM II are especially useful for the treatment of diseases where systemic administration of these peptides is preferred. Further, soluble forms of AIM II are also useful for topical administration. The complete and mature AIM II polypeptides of the invention, as well as subfragments of these polypeptides, may be employed to treat afflictions associated with receptors and other ligands to which these molecules bind.

In one embodiment, the invention provides a method of delivering compositions containing the polypeptides of the invention (e.g., compositions containing AIM II polypeptides or anti-AIM II antibodies associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs) to targeted cells, expressing the membrane-bound form of AIM II on their surface, or alternatively, an AIM II receptor (e.g., TR2, LTβ receptor, and CD27) on their surface. AIM TI polypeptides or anti-AIM II antibodies of the invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions.

In one embodiment, the invention provides a method for the specific delivery of compositions of the invention to cells by administering polypeptides of the invention (e.g., AIM II or anti-AIM II antibodies) that are associated with heterologous polypeptides or nucleic acids. In one example, the invention provides a method for delivering a therapeutic protein into the targeted cell. In another example, the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.

In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention (e.g., AIM II polypeptides or anti-AIM II antibodies) in association with toxins or cytotoxic prodrugs.

In a specific embodiment, the invention provides a method for the specific destruction of cells expressing TR2, LTβ receptor, and/or CD27 on their surface (e.g., activated T cells, and/or T cell and/or B cell related leukemias or lymphomas) by administering AIM II polypeptides in association with toxins or cytotoxic prodrugs.

The present invention further encompasses methods and compositions for killing cells of hematopoietic origin, comprising contacting AIM II polypeptide with cells of hematopoietic origin. In preferred embodiments, the cells of hematopoietic origin are T cells. In yet further preferred embodiments, the cells of hematopoietic origin are CD8 + T cells.

The present invention further encompasses methods and compositions for killing cells of hematopoietic origin, comprising administering to an animal in which such killing is desired, an AIM II polypeptide (e.g., a radiolabelled AIM II polypeptide) in an amount effective to kill cells of hematopoietic origin. In preferred embodiments, the cells of hematopoietic origin are T cells. In yet further preferred embodiments, the cells of hematopoietic origin are CD8 + T cells.

In another specific embodiment, the invention provides a method for the specific destruction of cells expressing the membrane-bound form of AIM II on their surface (e.g., spleen, bone marrow, kidney and PBLs) by administering anti-AIM II antibodies in association with toxins or cytotoxic prodrugs.

By “toxin” is meant compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, cytotoxins (cytotoxic agents), or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. “Toxin” also includes a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213 Bi, or other radioisotopes such as, for example, 103 Pd, 133Xe, 131 I, 68 Ge, 57 Co, 65 Zn, 85 Sr, 32 P, 35 S, 90 Y, 153 Sm, 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, 113 Sn, 90 Yttrium, 117 Tin, 186 Rhenium, 166 Holmium, an 188 Rhenium; luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.

›DETAILED DESCRIPTION · 55 of 68

Techniques known in the art may be applied to label proteins (including antibodies) of the invention. Such techniques include, but are not limited to, the use of bifunctional conjugating agents (see, e.g., U.S. Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contents of each of which are hereby incorporated by reference in its entirety). A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

By “cytotoxic prodrug” is meant a non-toxic compound that is converted by an enzyme, normally present in the cell, into a cytotoxic compound. Cytotoxic prodrugs that may be used according to the methods of the invention include, but are not limited to, glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubisin, and phenoxyacetamide derivatives of doxorubicin.

Diagnosis and Imaging

The invention further relates to the use of the AIM II polynucleotides to detect complementary polynucleotides such as, for example, as a diagnostic reagent. Detection of a mutated form of an AIM II associated with a dysfunction will provide a diagnostic tool that can add or define a diagnosis of a disease or susceptibility to disease which results from under-expression, over-expression or altered expression of AIM II, such as, for example, autoimmune diseases, malignancies and/or immunodeficiencies. The polynucleotide encoding the AIM II may also be employed as a diagnostic marker for expression of the polypeptide of the present invention.

The present inventors have discovered that AIM II is expressed in spleen, thymus and bone marrow tissue. For a number of disorders, such as septic shock, inflammation, cerebral malaria, activation of the HIV virus, graft-host rejection, bone resorption, rheumatoid arthritis and cachexia, it is believed that significantly higher or lower levels of AIM II gene expression can be detected in certain tissues (e.g., spleen, thymus and bone marrow tissue) or bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a “standard” AIM II gene expression level, i.e., the AIM II expression level in tissue or bodily fluids from an individual not having the disorder. Thus, the invention provides a diagnostic method useful during diagnosis of a disorder, which involves: (a) assaying AIM II gene expression level in cells or body fluid of an individual; (b) comparing the AIM II gene expression level with a standard AIM II gene expression level, whereby an increase or decrease in the assayed AIM II gene expression level compared to the standard expression level is indicative of a disorder.

It is also believed that certain tissues in mammals with cancer express significantly reduced levels of the AIM II protein and mRNA encoding the AIM II protein when compared to a corresponding “standard” mammal, i.e., a mammal of the same species not having the cancer. Further, it is believed that reduced levels of the AIM II protein can be detected in certain body fluids (e.g., sera, plasma, urine, and spinal fluid) from mammals with cancer when compared to sera from mammals of the same species not having the cancer. Thus, the invention provides a diagnostic method useful during tumor diagnosis, which involves assaying the expression level of the gene encoding the AIM II protein in mammalian cells or body fluid and comparing the gene expression level with a standard AIM II gene expression level, whereby an decrease in the gene expression level over the standard is indicative of certain tumors.

Where a tumor diagnosis has already been made according to conventional methods, the present invention is useful as a prognostic indicator, whereby patients exhibiting enhanced AIM II gene expression may experience a better clinical outcome relative to patients expressing the gene at a lower level.

By “assaying the expression level of the gene encoding the AIM II protein” is intended qualitatively or quantitatively measuring or estimating the level of the AIM II protein or the level of the mRNA encoding the AIM II protein in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the AIM II protein level or mRNA level in a second biological sample).

Preferably, the AIM II protein level or mRNA level in the first biological sample is measured or estimated and compared to a standard AIM II protein level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the cancer. As will be appreciated in the art, once a standard AIM II protein level or mRNA level is known, it can be used repeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained from an individual, cell line, tissue culture, or other source which contains AIM II protein or mRNA. Biological samples include mammalian body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) which contain secreted mature AIM II protein, and ovarian, prostate, heart, placenta, pancreas liver, spleen, lung, breast and umbilical tissue.

›DETAILED DESCRIPTION · 56 of 68

The present invention is useful for detecting cancer in mammals. In particular the invention is useful during diagnosis of the following types of cancers in mammals: breast, ovarian, prostate, bone, liver, lung, pancreatic and spleen. Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Particularly preferred are humans.

Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic purposes to detect, diagnose, or monitor diseases and/or disorders associated with the aberrant expression and/or activity of a polypeptide of the invention. The invention provides for the detection of aberrant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of aberrant expression.

The invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.

The diagnostic assays of the invention can be used for the diagnosis and prognosis of any disease related to the altered expression or production of AIM II. These assays are believed to be particularly useful for the diagnosis and prognosis of graft versus host disease, autoimmune disease and immunodeficiencies.

In preferred embodiments, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are used to treat, prevent, diagnose and/or prognose inflammation and/or an inflammatory disease or disorder.

In other preferred embodiments, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are used to treat, prevent, diagnose and/or prognose an intestinal disease or disorder. In a specific preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are used to treat, prevent, diagnose and/or prognose Inflammatory Bowel Disease (IBD). In another specific preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are used to treat, prevent, diagnose and/or prognose ulcerative colitis. In another specific preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are used to treat, prevent, diagnose and/or prognose Crohn's disease.

In additional embodiments, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are used to treat, prevent, diagnose and/or prognose an autoimmune disease. In specific embodiments, the autoimmune disease or disorder treated, prevented, diagnosed and/or prognosed according to the methods of the invention is a member selected from the group consisting of Graft versus host disease (GVHD), Multiple Sclerosis, type 1 diabetes, rheumatoid arthritis, primary biliary cirrhosis, aplastic anemia, myelodysplasia, systemic lupus erhythematosus, idiopathic thrombocytopenia purpura, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (e.g, IgA nephropathy), an immune-based rheumatologic disease (e.g., SLE, rheumatoid arthritis, CREST syndrome (a variant of scleroderma characterized by c alcinosis, R aynaud's phenomenon, e sophageal motility disorders, s clerodactyly, and t elangiectasia.), Seronegative spondyloarthropathy (SpA), Polymyositis/dermatomyositis, Microscopic polyanglitis, Hepatitis C-asociated arthritis, Takayasu's arteritis, and undifferentiated connective tissue disorder), Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura), Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis, Goodpasture's syndrome, Pemphigus, Receptor autoimmunities such as, for example, (a) Graves' Disease, (b) Myasthenia Gravis, and (c) insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis/dermatomyositis, pernicious anemia, idiopathic Addison's disease, infertility, glomerulonephritis such as primary glomerulonephritis and IgA nephropathy, bullous pemphigoid, Sjogren's syndrome, diabetes mellitus, and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis), chronic active hepatitis, primary biliary cirrhosis, other endocrine gland failure, vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria, atopic dermatitis, asthma, inflammatory myopathies, and other inflammatory, granulamatous, degenerative, atrophic disorders, or a condition associated with an autoimmune disease.

›DETAILED DESCRIPTION · 57 of 68

In a preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are used to treat, prevent, diagnose and/or prognose graft versus host disease (GVHD). In a specific preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are used to treat, prevent, diagnose and/or prognose tissue rejection after allograft transplantation. In another specific preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are used to treat, prevent, diagnose and/or prognose tissue rejection after isograft transplantation. In another specific preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are used to treat, prevent, diagnose and/or prognose tissue rejection after xenograft transplantation.

In a preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are used to treat, prevent, diagnose and/or prognose rheumatoid arthritis.

In another preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are used to treat, prevent, diagnose and/or prognose multiple sclerosis.

In another preferred embodiment, AIM II polynucleotides, polypeptides, antibodies and/or antagonists of the invention, are used to treat, prevent, diagnose and/or prognose type 1 (immune-mediated) diabetes.

In another preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are used to treat, prevent, diagnose and/or prognose Graves' disease.

In another preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are used to treat, prevent, diagnose and/or prognose autoimmune thyroiditis.

In another preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are used to treat, prevent, diagnose and/or prognose Hashimoto's disease.

In another preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are used to treat, prevent, diagnose and/or prognose aplastic anemia.

In another preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are used to treat, prevent, diagnose and/or prognose myelodysplasia.

In another preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are used to treat, prevent, diagnose and/or prognose vitiligo.

In another preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or antagonists of the invention, are used to treat, prevent, diagnose and/or prognose vasculitis.

In additional embodiments, AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, are used to treat, prevent, diagnose and/or prognose an immunodeficiency. In specific embodiments, the immunodeficiency treated, prevented, diagnosed and/or prognosed according to the methods of the invention is a member selected from the group consisting of DiGeorge anomaly, ataxia telangiectasia, severe combined immunodeficiency (SCID)-X linked, SCID-autosomal, Wiskott-Aldrich Syndrome (WAS), chronic mucocutaneous candidiasis, natural killer cell deficiency, CD4+ T-lymphocytopenia, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton's, disease, congenital agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, transient hypogammaglobulinemia of infancy, unspecified hypogammaglobulinemia, agammaglobulinemia, common variable immunodeficiency (CVID) (acquired), X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), antibody deficiency with normal or elevated Igs, immunodeficiency with thymoma, Ig heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), selective IgM immunodeficiency, recessive agammaglobulinemia (Swiss type), reticular dysgenesis, neonatal neutropenia, severe congenital leukopenia, thymic alymphoplasia-aplasia or dysplasia with immunodeficiency, short limbed dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezelof syndrome-combined immunodeficiency with Igs, purine nucleoside phosphorylase deficiency (PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severe combined immunodeficiency and conditions associated with an immunodeficiency.

In a preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, are used to treat, prevent, diagnose and/or prognose ataxia telangiectasia.

In another preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, are used to treat, prevent, diagnose and/or prognose DiGeorge anomaly.

In another preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, are used to treat, prevent, diagnose and/or prognose severe combined immunodeficiency (SCID).

In another preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, are used to treat, prevent, diagnose and/or prognose X-linked severe combined immunodeficiency (SCID).

In another preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, are used to treat, prevent, diagnose and/or prognose autosomal recessive severe combined immunodeficiency (SCID).

In another preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, are used to treat, prevent, diagnose and/or prognose Wiskott-Aldrich syndrome.

In another preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, are used to treat, prevent, diagnose and/or prognose adenosine deaminase deficiency (ADA deficiency).

›DETAILED DESCRIPTION · 58 of 68

In another preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, are used to treat, prevent, diagnose and/or prognose reticular dysgenesis.

In another preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, are used to treat, prevent, diagnose and/or prognose thymic alymphoplasia.

In another preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, are used to treat, prevent, diagnose and/or prognose short limbed dwarfism.

In another preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, are used to treat, prevent, diagnose and/or prognose Nezelof syndrome.

In another preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, are used to treat, prevent, diagnose and/or prognose purine nucleoside phosphorylase (PNP) deficiency.

In another preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, are used to treat, prevent, diagnose and/or prognose MHC Class II deficiency (Bare Lymphocyte Syndrome).

In another preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, are used to treat, prevent, diagnose and/or prognose chronic mucocutaneous candidiasis.

In another preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, are used to treat, prevent, diagnose and/or prognose natural killer cell deficiency.

In another preferred embodiment, AIM II polynucleotides, polypeptides, antibodies, and/or agonists of the invention, are used to treat, prevent, diagnose and/or prognose idiopathic CD4+ T-lymphocytopenia.

Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, M. et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M. et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 112 In), and technetium ( 99 Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a disease or disorder associated with aberrant expression of a polypeptide of interest in an animal, preferably a mammal and most preferably a human. In one embodiment, diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest. Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.

It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99 mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et al, “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.

In a specific embodiment, the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).

›DETAILED DESCRIPTION · 59 of 68

Total cellular RNA can be isolated from a biological sample using the single-step guanidinium-thiocyanate-phenol-chloroform method described in Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels of mRNA encoding the AIM II protein are then assayed using any appropriate method. These include Northern blot analysis (Harada et al., Cell 63:303-312 (1990)), S1 nuclease mapping (Fujita et al., Cell 49:357-367 (1987)), the polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR) (Makino et al., Technique 2:295-301 (1990)), and reverse transcription in combination with the ligase chain reaction (RT-LCR).

Assaying AIM II protein levels in a biological sample can occur using antibody-based techniques. For example, AIM II protein expression in tissues can be studied with classical immunohistological methods (Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096 (1987)).

Other antibody-based methods useful for detecting AIM II protein expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).

Suitable labels are known in the art and include enzyme labels, such as, Glucose oxidase, and radioisotopes, such as iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 C), tritium ( 3 H), indium ( 112 In), and technetium ( 99m Tc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.

AIM II “Knock-Outs” and Homologous Recombination

Endogenous gene expression can also be reduced by inactivating or “knocking out” the gene and/or its promoter using targeted homologous recombination. (e.g., see Smithies et al., Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell 5:313-321 (1989); each of which is incorporated by reference herein in its entirety). For example, a mutant, non-functional polynucleotide of the invention (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous polynucleotide sequence (either the coding regions or regulatory regions of the gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express polypeptides of the invention in vivo. In another embodiment, techniques known in the art are used to generate knockouts in cells that contain, but do not express the gene of interest. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the targeted gene. Such approaches are particularly suited in research and agricultural fields where modifications to embryonic stem cells can be used to generate animal offspring with an inactive targeted gene (see, e.g., Thomas & Capecchi 1987 and Thompson 1989, supra). However this approach can be routinely adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors that will be apparent to those of skill in the art. The contents of each of the documents recited in this paragraph are herein incorporated by reference in its entirety.

In further embodiments of the invention, cells that are genetically engineered to express the polypeptides of the invention, or alternatively, that are genetically engineered not to express the polypeptides of the invention (e.g., knockouts) are administered to a patient in vivo. Such cells maybe obtained from the patient (i.e., animal, including human) or an MHC compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypeptides of the invention, e.g., by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. The coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention. The engineered cells which express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically, e.g., in the circulation, or intraperitoneally. Alternatively, the cells can be incorporated into a matrix and implanted in the body, e.g., genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft. (See, e.g., Anderson et al U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959, each of which is incorporated by reference herein in its entirety).

When the cells to be administered are non-autologous or non-MHC compatible cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells. For example, the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.

Transgenic Non-Human Animals

The polypeptides of the invention can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals. In a specific embodiment, techniques described herein or otherwise known in the art are used to express polypeptides of the invention in humans, as part of a gene therapy protocol.

›DETAILED DESCRIPTION · 60 of 68

Any technique known in the art may be used to introduce the transgene (i.e., polynucleotides of the invention) into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology ( NY ) 11:1263-1270 (1993); Wright et al., Biotechnology ( NY ) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der. Putten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; gene targeting in embryonic stem cells (Thompson etal., Cell 56:313-321 (1989)); electroporation of cells or embryos (Lo, Mol. Cell. Biol. 3:1803-1814 (1983)); introduction of the polynucleotides of the invention using a gene gun (see, e.g., Ulmer et al., Science 259:1745 (1993); introducing nucleic acid constructs into embryonic pluripotent stem cells and transferring the stem cells back into the blastocyst; and sperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989); etc. For a review of such techniques, see Gordon, “Transgenic Animals,” Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by reference herein in its entirety. Further, the contents of each of the documents recited in this paragraph are herein incorporated by reference in its entirety. See also, U.S. Pat. No. 5,464,764 (Capecchi et al., Positive-Negative Selection Methods and Vectors); U.S. Pat. No. 5,631,153 (Capecchi et al., Cells and Non-Human Organisms Containing Predetermined Genomic Modifications and Positive-Negative Selection Methods and Vectors for Making Same); U.S. Pat. No. 4,736,866 (Leder et al., Transgenic Non-Human Animals); and U.S. Pat. No. 4,873,191 (Wagner et al., Genetic Transformation of Zygotes); each of which is hereby incorporated by reference in its entirety.

Any technique known in the art may be used to produce transgenic clones containing polynucleotides of the invention, for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campbell et al., Nature 380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)), each of which is herein incorporated by reference in its entirety).

The present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals or chimeric. The transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. When it is desired that the polynucleotide transgene be integrated into the chromosomal site of the endogenous gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. The contents of each of the documents recited in this paragraph are herein incorporated by reference in its entirety.

Once transgenic animals have been generated, the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenic gene-expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.

Once the founder animals are produced, they may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal. Examples of such breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene on a distinct background that is appropriate for an experimental model of interest.

Transgenic and “knock-out” animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of AIM II polypeptides, studying conditions and/or disorders associated with aberrant AIM II expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.

›DETAILED DESCRIPTION · 61 of 68

Other Uses

The present invention also relates to methods for separating cells into subpopulations based on whether these cells bind either the AIM II polypeptides of the invention or antibodies having specificity for these polypeptides. These separation methods will generally be based on the principle that cells which either express a surface receptor which binds AIM II polypeptides or have an AIM II polypeptide on their surface can be identified using labeled AIM II polypeptides or AIM II specific antibodies. Such cells can then be separated from other cells in a population which do not bind these polypeptides or antibodies. Methods for separating cells, commonly known as “cell sorting”, are known in the art and are discussed in Crane, U.S. Pat. No. 5,489,506.

Thus, in one aspect, the invention provides methods for separating cells which bind either AIM II polypeptides or antibodies having specificity for AIM II polypeptides comprising contacting a population of cells with either an AIM II polypeptide or an antibody having specificity for the AIM II polypeptide, wherein the AIM II polypeptide or antibody is labeled with a detectable label and separating cells which bind either the AIM II polypeptide or anti-AIM II polypeptide antibody from cells which do not bind these molecules. Cells which bind AIM II polypeptides are believed to include those which express the lymphotoxin-β-receptor (LT-β-R), TR2, CD27, and TRANK.

In another embodiment, the polypeptides of the present invention are used as a research tool for studying the biological effects that result from inhibiting LTβ receptor/AIM II, TR6/AIM II, and/or TRANK/AIM II interactions on different cell types. AIM II polypeptides also may be employed in in vitro assays for detecting LTβ receptor, TR6, and/or TRANK or AIM II or the interactions thereof.

This invention also provides a method for identification of molecules, such as receptor molecules, that bind AIM II. Genes encoding proteins that bind AIM II, such as receptor proteins, can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting. Such methods are described in many laboratory manuals such as, for instance, Coligan et al., Current Protocols in Immunology, 1(2):Chapter 5 (1991).

For instance, expression cloning may be employed for this purpose. To this end polyadenylated RNA is prepared from a cell responsive to AIM II, a cDNA library is created from this RNA, the library is divided into pools and the pools are transfected individually into cells that are not responsive to AIM II. The transfected cells then are exposed to labeled AIM II. (AIM II can be labeled by a variety of well-known techniques including standard methods of radio-iodination or inclusion of a recognition site for a site-specific protein kinase.) Following exposure, the cells are fixed and binding of AIM IT is determined. These procedures conveniently are carried out on glass slides.

Pools are identified of cDNA that produced AIM II-binding cells. Sub-pools are prepared from these positives, transfected into host cells and screened as described above. Using an iterative sub-pooling and re-screening process, one or more single clones that encode the putative binding molecule, such as a receptor molecule, can be isolated.

Alternatively, a labeled ligand can be photo affinity linked to a cell extract, such as a membrane or a membrane extract, and prepared from cells that express a molecule that it binds, such as a receptor molecule. Cross-linked material is resolved by polyacrylamide gel electrophoresis (“PAGE”) and exposed to X-ray film. The labeled complex containing the ligand-receptor can be excised, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing can be used to design unique or degenerate oligonucleotide probes to screen cDNA libraries to identify genes encoding the putative receptor molecule.

Polypeptides of the invention also can be used to assess AIM II binding capacity of AIM II binding molecules, such as receptor molecules, in cells or in cell-free preparations.

The nucleic acid molecules of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.

In certain preferred embodiments in this regard, one of the cDNAs herein disclosed is used to clone genomic DNA of an AIM II protein gene. This can be accomplished using a variety of well-known techniques and libraries, which generally are available commercially. The genomic DNA then is used for in situ chromosome mapping using well-known techniques for this purpose.

In addition, in some cases, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3 untranslated region of the gene is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromomes.

Fluorescence in situ hybridization (“FISH”) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with probes from the cDNA as short as 50 or 60 bp. For a review of this technique, see Verma et al., Human Chromosomes: A Manual Of Basic Techniques , Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance In Man , available on-line through Johns Hopkins University, Welch Medical Library. The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).

›DETAILED DESCRIPTION · 62 of 68

Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.

Demonstration of Therapeutic or Prophylactic Activity

The compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample. The effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays. In accordance with the invention, in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.

Kits

The present invention provides kits that can be used in the above methods. In one embodiment, a kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In a specific embodiment, the kits of the present invention contain a substantially isolated polypeptide comprising an epitope which is specifically immunoreactive with an antibody included in the kit. Preferably, the kits of the present invention further comprise a control antibody which does not react with the polypeptide of interest. In another specific embodiment, the kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate).

In another specific embodiment of the present invention, the kit is a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides. Such a kit may include a control antibody that does not react with the polypeptide of interest. Such a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one anti-polypeptide antigen antibody. Further, such a kit includes means for detecting the binding of said antibody to the antigen (e.g., the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry). In specific embodiments, the kit may include a recombinantly produced or chemically synthesized polypeptide antigen. The polypeptide antigen of the kit may also be attached to a solid support.

In a more specific embodiment the detecting means of the above-described kit includes a solid support to which said polypeptide antigen is attached. Such a kit may also include a non-attached reporter-labeled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the said reporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the invention. The diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support. In a specific embodiment, the antibody may be a monoclonal antibody. The detecting means of the kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention. After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support. The reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying out this diagnostic method. The kit generally includes a support with surface-bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.

Therapeutic/Prophylactic Administration and Compositions

It will be appreciated that conditions, such as those discussed above, can be treated by administration of AIM II protein. As a result, the invention further provides a method of treating an individual in need of an increased level of AIM II activity comprising administering to such an individual a pharmaceutical composition comprising, or alternatively consisting of, an effective amount of an isolated AIM II polypeptide of the invention, effective to increase the AIM II activity level in such an individual. Thus, the invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention. In a preferred aspect, the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.

›DETAILED DESCRIPTION · 63 of 68

Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; additional appropriate formulations and routes of administration can be selected from among those described herein-below.

Various delivery systems are known and can be used to administer a compound 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 include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions 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. Thus, pharmaceutical compositions containing the AIM II of the invention may be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray.

The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrastemal, subcutaneous and intra-articular injection and infusion.

Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or 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 compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the invention, care must be taken to use materials to which the protein does not absorb.

In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)

In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng.14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, N.Y. (1984); Ranger and Peppas, J., 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J.Neurosurg. 71:105). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer (1990, Science 249:1527-1533).

In a specific embodiment where the compound 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 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.

In the treatment of rheumatoid arthritis, particularly preferred modes of administration of AIM II polypeptides of the present invention include, intradermal, subcutaneous and intra-articular injection and infusion. Preferably, AIM II polypeptide administered intra-articularly or intra-dermally will be in the range of about 0.1 to about 1.0 mg/kg of patient body weight per dose.

The compositions of the invention may be administered alone or in combination with other therapeutic agents (e.g., a costimulatory molecule). Therapeutic agents that may be administered in combination with the compositions of the invention include, but are not limited to, other members of the TNF family, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti-inflammatories, conventional immunotherapeutic agents, cytokines and/or growth factors. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second. Thus, in effect, the therapeutic agents may be administered to individuals either at the same time or at different times. In most instances when the therapeutic agents are administered to individuals at different times, they will generally be administered in a manner such that the therapeutic effects of these agents overlap for a period of time.

›DETAILED DESCRIPTION · 64 of 68

The present invention encompasses administration of compositions of the invention in combination with other therapeutic agents to treat, prevent, diagnose and/or prognose a disease or disorder of the immune system. In preferred embodiments, the present invention encompasses administration of compositions of the invention in combination with other therapeutic agents to treat, prevent, diagnose and/or prognose a disease or disorder of the cellular immune system.

In preferred embodiments the invention encompasses administration of compositions of the invention in combination with immunosuppressant agents. In specific embodiments, compositions of the invention maybe administered in combination with one or more immunosuppressant agents selected from the group consisting of steroids, cyclosporine, cyclosporine analogs, cyclophosphamide, methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin, ORTHOCLONE™ (OKT3), SANDIMMUNE™/NEORAL™/SANGDYA™ (cyclosporin), PROGRAF™ (tacrolimus), CELLCEPT™ (mycophenolate), Azathioprine, glucorticosteroids, RAPAMUNE™ (sirolimus) and other immunosuppressive agents that act by suppressing the function of responding T cells.

In a specific preferred embodiment compositions of the invention are administered in combination with immunosuppressant steroids.

In another specific preferred embodiment compositions of the invention are administered in combination with cyclosporine.

In another specific preferred embodiment compositions of the invention are administered in combination with cyclosporine analogs including, for example, SANDIMMUNE™/NEORAL™/SANGDYA™.

In another specific preferred embodiment compositions of the invention are administered in combination with cyclophosphamide.

In another specific preferred embodiment compositions of the invention are administered in combination with methylprednisone.

In another specific preferred embodiment compositions of the invention are administered in combination with prednisone.

In another specific preferred embodiment compositions of the invention are administered in combination with azathioprine.

In another specific preferred embodiment compositions of the invention are administered in combination with FK-506.

In another specific preferred embodiment compositions of the invention are administered in combination with 15-deoxyspergualin.

In another specific preferred embodiment compositions of the invention are administered in combination with ORTHOCLONE™ (OKT3).

In another specific preferred embodiment compositions of the invention are administered in combination with PROGRAF™ (tacrolimus).

In another specific preferred embodiment compositions of the invention are administered in combination with CELLCEPT™ (mycophenolate).

In another specific preferred embodiment compositions of the invention are administered in combination with RAPAMUNE™ (sirolimus).

In preferred embodiments the invention encompasses administration of compositions of the invention in combination with compounds which stimulate secretion of inflammatory cytokines. In a specific embodiment, compositions of the invention may be administered in combination with CTLA-4.Fc. In another specific preferred embodiment, compositions of the invention may be administered in combination with an anti-CTLA-4 antibody.

In preferred embodiments, compositions of the invention may be administered in combination with polynucleotides, polypeptides, antibodies, agonists and/or antagonists of TNF family re.ceptors and/or TNF family ligands. In specific embodiments, compositions of the invention may be administered in combination with one or more TNF family members selected from the group consisting of TNF-alpha, lymphotoxin-alpha (LT-α, also known as TNF-β), LT-β (found in complex heterotrimer LT-α2-β), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-γ (International Publication No. WO 96/14328), AIM-I (International Publication No. WO 97/33899), APRIL (J. Exp. Med. 188(6): 1185-1190), endokine-α (International Publication No. WO 98/07880), TR6 (International Publication No. WO 98/30694), OPG, and neutrokine-α (International Publication No. WO 98/18921, OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3 (International Publication No. WO 97/33904), DR4 (International Publication No. WO 98/32856), TR5 (International Publication No. WO 98/30693), TR6 (International Publication No. WO 98/30694), TR7 (International Publication No. WO 98/41629), TRANK, TR9 (International Publication No. WO 98/56892), TR10 (International Publication No. WO 98/54202), 312C2 (International Publication No. WO 98/06842), TR12, and soluble forms of CD154, CD70 and CD153.

In a specific preferred embodiment, compositions of the invention are administered in combination with ENBREL™.

In another specific preferred embodiment, compositions of the invention are administered in combination with AVEREND™.

In preferred embodiments, compositions of the invention may be administered in combination with one or more anti-inflammatory agents. Anti-inflammatory agents that may be administered with the compositions of the invention include, but are not limited to, glucocorticoids and the nonsteroidal anti-inflammatories, amino arylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, and tenidap.

In a specific preferred embodiment, compositions of the invention are administered in combination with aminoarylcarboxylic acid derivatives.

In another specific preferred embodiment, compositions of the invention are administered in combination with arylacetic acid derivatives.

›DETAILED DESCRIPTION · 65 of 68

In another specific preferred embodiment, compositions of the invention are administered in combination with arylbutyric acid derivatives.

In another specific preferred embodiment, compositions of the invention are administered in combination with arylcarboxylic acids.

In another specific preferred embodiment, compositions of the invention are administered in combination with arylpropionic acid derivatives.

In another specific preferred embodiment, compositions of the invention are administered in combination with pyrazoles.

In another specific preferred embodiment, compositions of the invention are administered in combination with pyrazolones.

In another specific preferred embodiment, compositions of the invention are administered in combination with salicylic acid derivatives.

In another specific preferred embodiment, compositions of the invention are administered in combination with thiazinecarboxamides.

In another specific preferred embodiment, compositions of the invention are administered in combination with e-acetamidocaproic acid

In another specific preferred embodiment, compositions of the invention are administered in combination with S-adenosylmethionine.

In another specific preferred embodiment, compositions of the invention are administered in combination with 3-amino-4-hydroxybutyric acid.

In another specific preferred embodiment, compositions of the invention are administered in combination with amixetrine.

In another specific preferred embodiment, compositions of the invention are administered in combination with bendazac.

In another specific preferred embodiment, compositions of the invention are administered in combination with benzydamine.

In another specific preferred embodiment, compositions of the invention are administered in combination with bucolome.

In another specific preferred embodiment, compositions of the invention are administered in combination with difenpiramide.

In another specific preferred embodiment, compositions of the invention are administered in combination with ditazol.

In another specific preferred embodiment, compositions of the invention are administered in combination with emorfazone.

In another specific preferred embodiment, compositions of the invention are administered in combination with guaiazulene.

In another specific preferred embodiment, compositions of the invention are administered in combination with nabumetone.

In another specific preferred embodiment, compositions of the invention are administered in combination with nimesulide.

In another specific preferred embodiment, compositions of the invention are administered in combination with orgotein.

In another specific preferred embodiment, compositions of the invention are administered in combination with oxaceprol.

In another specific preferred embodiment, compositions of the invention are administered in combination with paranyline.

In another specific preferred embodiment, compositions of the invention are administered in combination with perisoxal.

In another specific preferred embodiment, compositions of the invention are administered in combination with pifoxime.

In another specific preferred embodiment, compositions of the invention are administered in combination with proquazone.

In another specific preferred embodiment, compositions of the invention are administered in combination with proxazole.

In another specific preferred embodiment, compositions of the invention are administered in combination with tenidap.

In one embodiment, the compositions of the invention are administered in combination with other members of the TNF family. TNF, TNF-related or TNF-like molecules that may be administered with the compositions of the invention include, but are not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-α, also known as TNF-β), LT-β (found in complex heterotrimer LT-α2-β), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-γ (International Publication No. WO 96/14328), AIM-I (International Publication No. WO 97/33899), APRIL (J. Exp. Med. 188(6):1185-1190), endokine-α (International Publication No. WO 98/07880), TR6 (International Publication No. WO 98/30694), OPG, and neutrokine-a (International Publication No. WO 98/18921, OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3 (International Publication No. WO 97/33904), DR4 (International Publication No. WO 98/32856), TR5 (International Publication No. WO 98/30693), TR6 (International Publication No. WO 98/30694), TR7 (International Publication No. WO 98/41629), TRANK, TR9 (International Publication No. WO 98/56892), TR10 (International Publication No. WO 98/54202), 312C2 (International Publication No. WO 98/06842), TR12, and soluble forms of CD154, CD70 and CD153.

In a preferred embodiment, the compositions of the invention are administered in combination with CD40 ligand (CD40L), a soluble form of CD40L (e.g., AVREND™), biologically active fragments, variants, or derivatives of CD40L, anti-CD40L antibodies (e.g., agonistic or antagonistic antibodies), and/or anti-CD40 antibodies (e.g., agonistic or antagonistic antibodies).

In a preferred embodiment, the compositions of the invention are administered in combination with one, two, three, four, five, or more of the following compositions: tacrolimus (Fujisawa), thalidomide (e.g., Celgene), anti-Tac(Fv)-PE40 (e.g., Protein Design Labs), inolimomab (Biotest), MAK-195F (Knoll), ASM-981 (Novartis), interleukin-1 receptor (e.g., Immunex), interleukin-4 receptor (e.g., Immunex), ICM3 (ICOS), BMS-188667 (Bristol-Myers Squibb), anti-TNF Ab (e.g., Therapeutic antibodies), CG-1088 (Celgene), anti-B7 Mab (e.g., Innogetics), MEDI-507 (BioTransplant), ABX-CBL (Abgenix).

In certain embodiments, compositions of the invention are administered in combination with antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors. Nucleoside reverse transcriptase inhibitors that may be administered in combination with the compositions of the invention, include, but are not limited to, RETROVIR™ (zidovudine/AZT), VIDEX™ (didanosine/ddI), HIVID™ (zalcitabine/ddC), ZERIT™ (stavudine/d4T), EPIVIR™ (lamivudine/3TC), and COMBIVIR™ (zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitors that may be administered in combination with the compositions of the invention, include, but are not limited to, VERAMU™ (nevirapine), RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitors that may be administered in combination with the compositions of the invention, include, but are not limited to, CRIXIVAN™ (indinavir), NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VIRACEPT™ (nelfinavir). In a specific embodiment, antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be used in any combination with compositions of the invention to treat, prevent, and/or diagnose AIDS and/or to treat, prevent, and/or diagnose HIV infection.

›DETAILED DESCRIPTION · 66 of 68

In other embodiments, compositions of the invention may be administered in combination with anti-opportunistic infection agents. Anti-opportunistic agents that may be administered in combination with the compositions of the invention, include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™, ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™, CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™, FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™, PYRIMETHIAMINE™, LEUCOVORIN™, NEUPOGEN™, (filgrastim/G-CSF), and LEUKINE™ (sargramostim/GM-CSF). In a specific embodiment, compositions of the invention are used in any combination with TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, and/or ATOVAQUONE™ to prophylactically treat, prevent, and/or diagnose an opportunistic Pneumocystis carinii pneumonia infection. In another specific embodiment, compositions of the invention are used in any combination with ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, and/or ETHAMBUTOL™ to prophylactically treat, prevent, and/or diagnose an opportunistic Mycobacterium avium complex infection. In another specific embodiment, compositions of the invention are used in any combination with RIFABUTIN™, CLARITHROMYCIN™, and/or AZITHROMYCIN™ to prophylactically treat, prevent, and/or diagnose an opportunistic Mycobacterium tuberculosis infection. In another specific embodiment, compositions of the invention are used in any combination with GANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ to prophylactically treat, prevent, and/or diagnose an opportunistic cytomegalovirus infection. In another specific embodiment, compositions of the invention are used in any combination with FLUCONAZOLE™, ITRACONAZOLE™, and/or KETOCONAZOLE™ to prophylactically treat, prevent, and/or diagnose an opportunistic fungal infection. In another specific embodiment, compositions of the invention are used in any combination with ACYCLOVIR™ and/or FAMCICOLVIR™ to prophylactically treat, prevent, and/or diagnose an opportunistic herpes simplex virus type I and/or type II infection. In another specific embodiment, compositions of the invention are used in any combination with PYRIMETHIAMINE™ and/or LEUCOVORIN™ to prophylactically treat, prevent, and/or diagnose an opportunistic Toxoplasma gondli infection. In another specific embodiment, compositions of the invention are used in any combination with LEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat, prevent, and/or diagnose an opportunistic bacterial infection.

In a further embodiment, the compositions of the invention are administered in combination with an antiviral agent. Antiviral agents that may be administered with the compositions of the invention include, but are not limited to, acyclovir, ribavirin, amantadine, and remantidine.

In a further embodiment, the compositions of the invention are administered in combination with an antibiotic agent. Antibiotic agents that may be administered with the compositions of the invention include, but are not limited to, amoxicillin, aminoglycosides, beta-lactam (glycopeptide), beta-lactamases, Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin, erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins, quinolones, rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim, trimethoprim-sulfamthoxazole, and vancomycin.

Conventional nonspecific immunosuppressive agents, that may be administered in combination with the compositions of the invention include, but are not limited to, steroids, cyclosporine, cyclosporine analogs cyclophosphamide, cyclophosphamide IV, methylprednisolone, prednisolone, azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressive agents that act by suppressing the function of responding T cells.

In specific embodiments, compositions of the invention are administered in combination with immunosuppressants. Immunosuppressants preparations that may be administered with the compositions of the invention include, but are not limited to, ORTHOCLONE™ (OKT3), SANDIMMUNE™/NEORAL™/SANGDYA™ (cyclosporin), PROGRAF™ (tacrolimus), CELLCEPT™ (mycophenolate), Azathioprine, glucorticosteroids, and RAPAMUNE™ (sirolimus). In a specific embodiment, immunosuppressants may be used to prevent rejection of organ or bone marrow transplantation.

In a preferred embodiment, the compositions of the invention are administered in combination with steroid therapy. Steroids that may be administered in combination with the compositions of the invention, include, but are not limited to, oral corticosteroids, prednisone, and methylprednisolone (e.g., IV methylprednisolone). In a specific embodiment, compositions of the invention are administered in combination with prednisone. In a further specific embodiment, the compositions of the invention are administered in combination with prednisone and an immunosuppressive agent. Immunosuppressive agents that may be administered with the compositions of the invention and prednisone are those described herein, and include, but are not limited to, azathioprine, cylophosphamide, and cyclophosphamide IV. In another specific embodiment, compositions of the invention are administered in combination with methylprednisolone. In a further specific embodiment, the compositions of the invention are administered in combination with methylprednisolone and an immunosuppressive agent. Immunosuppressive agents that may be administered with the compositions of the invention and methylprednisolone are those described herein, and include, but are not limited to, azathioprine, cylophosphamide, and cyclophosphamide IV.

In a preferred embodiment, the compositions of the invention are administered in combination with an antimalarial. Antimalarials that may be administered with the compositions of the invention include, but are not limited to, hydroxychloroquine, chloroquine, and/or quinacrine.

In a preferred embodiment, the compositions of the invention are administered in combination with an NSAID.

›DETAILED DESCRIPTION · 67 of 68

In a nonexclusive embodiment, the compositions of the invention are administered in combination with one, two, three, four, five, ten, or more of the following drugs: NRD-101 (Hoechst Marion Roussel), diclofenac (Dimethaid), oxaprozin potassium (Monsanto), mecasermin (Chiron), T-614 (Toyama), pemetrexed disodium (Eli Lilly), atreleuton (Abbott), valdecoxib (Monsanto), eltenac (Byk Gulden), campath, AGM-1470 (Takeda), CDP-571 (Celltech Chiroscience), CM-101 (CarboMed), ML-3000 (Merckle), CB-2431 (KS Biomedix), CBF-BS2 (KS Biomedix), IL-1Ra gene therapy (Valentis), JTE-522 (Japan Tobacco), paclitaxel (Angiotech), DW-166HC (Dong Wha), darbufelone mesylate (Warner-Lambert), soluble TNF receptor 1 (synergen; Amgen), IPR-6001 (Institute for Pharmaceutical Research), trocade (Hoffman-La Roche), EF-5 (Scotia Pharmaceuticals), BIIL-284 (Boehringer Ingelheim), BIIF-1149 (Boehringer Ingelheim), LeukoVax (Inflammatics), MK-663 (Merck), ST-1482 (Sigma-Tau), and butixocort propionate (WarnerLambert).

In a preferred embodiment, the compositions of the invention are administered in combination with one, two, three, four, five or more of the following drugs: methotrexate, sulfasalazine, sodium aurothiomalate, auranofin, cyclosporine, penicillamine, azathioprine, an antimalarial drug (e.g., as described herein), cyclophosphamide, chlorambucil, gold, ENBREL™ (Etanercept), anti-TNF antibody, and prednisolone. In a more preferred embodiment, the compositions of the invention are administered in combination with an antimalarial, methotrexate, anti-TNF antibody, ENBREL™ and/or suflasalazine. In one embodiment, the compositions of the invention are administered in combination with methotrexate. In another embodiment, the compositions of the invention are administered in combination with anti-TNF antibody. In another embodiment, the compositions of the invention are administered in combination with methotrexate and anti-TNF antibody. In another embodiment, the compositions of the invention are administered in combination with suflasalazine. In another specific embodiment, the compositions of the invention are administered in combination with methotrexate, anti-TNF antibody, and suflasalazine. In another embodiment, the compositions of the invention are administered in combination ENBREL™. In another embodiment, the compositions of the invention are administered in combination with ENBREL™ and methotrexate. In another embodiment, the compositions of the invention are administered in combination with ENBREL™, methotrexate and suflasalazine. In another embodiment, the compositions of the invention are administered in combination with ENBREL™, methotrexate and suflasalazine. In other embodiments, one or more antimalarials is combined with one of the above-recited combinations. In a specific embodiment, the compositions of the invention are administered in combination with an antimalarial (e.g., hydroxychloroquine), ENBREL™, methotrexate and suflasalazine. In another specific embodiment, the compositions of the invention are administered in combination with an antimalarial (e.g., hydroxychloroquine), sulfasalazine, anti-TNF antibody, and methotrexate.

Conventional nonspecific immunosuppressive agents, that maybe administered in combination with the compositions of the invention include, but are not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressive agents that act by suppressing the function of responding T cells.

In a further embodiment, the compositions of the invention are administered in combination with an antibiotic agent. Antibiotic agents that may be administered with the compositions of the invention include, but are not limited to, tetracycline, metronidazole, amoxicillin, beta-lactamases, aminoglycosides, macrolides, quinolones, fluoroquinolones, cephalosporins, erythromycin, ciprofloxacin, and streptomycin.

In an additional embodiment, the compositions of the invention are administered alone or in combination with an anti-inflammatory agent. Anti-inflammatory agents that may be administered with the compositions of the invention include, but are not limited to, glucocorticoids and the nonsteroidal anti-inflammatories, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, and tenidap.

In another embodiment, compositions of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered with the compositions of the invention include, but are not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cis-platin, and vincristine sulfate); hormones (e.g., medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and testolactone); nitrogen mustard derivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogen mustard) and thiotepa); steroids and combinations (e.g., bethamethasone sodium phosphate); and others (e.g., dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

›DETAILED DESCRIPTION · 68 of 68

In an additional embodiment, the compositions of the invention are administered in combination with cytokines. Cytokines that may be administered with the compositions of the invention include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, IFN-γ and TNF-α.

In an additional embodiment, the compositions of the invention are administered in combination with angiogenic proteins. Angiogenic proteins that may be administered with the compositions of the invention include, but are not limited to, Glioma Derived Growth Factor (GDGF), as disclosed in European Patent Number EP-399816; Platelet Derived Growth Factor-A (PDGF-A), as disclosed in European Patent Number EP-682110; Platelet Derived Growth Factor-B (PDGF-B), as disclosed in European Patent Number EP-282317; Placental Growth Factor (PIGF), as disclosed in WO 92/06194; Placental Growth Factor-2 (PIGF-2), as disclosed in Hauser et al., Growth Factors 4:259-268 (1993); Vascular Endothelial Growth Factor (VEGF), as disclosed in WO 90/13649; Vascular Endothelial Growth Factor-A (VEGF-A), as disclosed in European Patent Number EP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosed in WO 96/39515; Vascular Endothelial Growth Factor B-186 (VEGF-B186), as disclosed in WO 96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in WO 98/02543; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E), as disclosed in German Patent Number DE19639601. The above mentioned references are incorporated by reference herein.

In an additional embodiment, the compositions of the invention are administered in combination with Fibroblast Growth Factors. Fibroblast Growth Factors that may be administered with the compositions of the

›Tables in the description — 4

TABLE 1 — Conservative Amino Acid Substitutions.

Aromatic	Phenylalanine
	Tryptophan
	Tyrosine
Hydrophobic	Leucine
	Isoleucine
	Valine
Polar	Glutamine
	Asparagine
Basic	Arginine
	Lysine
	Histidine
Acidic	Aspartic Acid
	Glutamic Acid
Small	Alanine
	Serine
	Threonine
	Methionine
	Glycine

TABLE 2

	Res Position	I	II	III	IV	V	VI	VII	VIII	IX	X	XI	XII	XIII	XIV
Met	1	A	.	.	.	.	.	.	0.19	−0.71	.	.	.	0.95	1.49
Glu	2	A	.	.	.	.	.	.	−0.28	−0.50	*	.	.	0.50	0.87
Glu	3	A	.	.	B	.	.	.	0.22	−0.29	*	.	.	0.30	0.50
Ser	4	A	.	.	B	.	.	.	0.40	−0.71	*	.	.	0.60	0.99
Val	5	A	.	.	B	.	.	.	0.49	−0.90	*	*	.	0.60	0.89
Val	6	A	.	.	B	.	.	.	0.23	−0.51	.	.	.	0.60	0.69
Arg	7	.	.	B	.	.	T	.	−0.47	0.13	*	*	.	0.10	0.38
Pro	8	.	.	B	.	.	T	.	−1.32	0.53	*	*	.	−0.20	0.44
Ser	9	.	.	B	.	.	T	.	−1.88	0.53	*	.	.	−0.20	0.44
Val	10	.	.	B	.	.	T	.	−1.02	0.53	*	*	.	−0.20	0.17
Phe	11	.	.	B	.	.	.	.	−0.51	0.53	.	*	.	−0.40	0.18
Val	12	.	.	B	.	.	.	.	−0.62	0.53	.	*	.	−0.12	0.13
Val	13	.	.	B	.	.	T	.	−0.72	0.54	.	*	.	0.36	0.31
Asp	14	.	.	B	.	.	T	.	−0.42	0.39	.	*	F	1.09	0.52
Gly	15	.	.	.	.	T	T	.	−0.46	−0.40	.	*	F	2.52	1.17
Gln	16	.	.	.	.	T	T	.	0.03	−0.36	.	*	F	2.80	1.11
Thr	17	.	.	.	B	.	.	C	0.19	−0.57	.	*	F	2.22	1.03
Asp	18	.	.	B	B	.	.	.	0.73	0.21	.	*	F	0.69	0.90
Ile	19	.	.	B	B	.	.	.	0.84	0.27	.	*	F	0.41	0.75
Pro	20	.	.	B	B	.	.	.	0.38	−0.13	*	*	.	0.73	1.02
Phe	21	.	.	B	B	.	.	.	0.03	0.07	*	.	.	−0.30	0.50
Thr	22	.	.	B	B	.	.	.	0.46	0.50	*	.	.	−0.26	0.71
Arg	23	.	.	B	B	.	.	.	0.16	−0.19	*	.	F	1.13	0.90
Leu	24	.	.	.	B	T	.	.	1.01	−0.23	*	.	F	2.02	1.39
Gly	25	.	.	.	B	T	.	.	1.33	−0.51	*	.	F	2.66	1.31
Arg	26	.	.	.	.	T	T	.	2.14	−1.00	*	.	F	3.40	1.31
Ser	27	.	.	.	.	T	T	.	2.46	−1.00	*	.	F	3.06	3.11
His	28	.	.	.	.	T	T	.	2.04	−1.29	*	.	F	3.03	5.44
Arg	29	.	.	.	.	T	T	.	2.19	−1.33	*	.	F	3.00	3.72
Arg	30	.	.	.	.	T	.	.	2.23	−0.76	*	*	F	2.77	1.49
Gln	31	.	.	.	.	T	T	.	1.27	−0.76	*	.	F	2.94	1.47
Ser	32	.	.	.	.	T	T	.	0.98	−0.61	*	.	F	3.10	0.56
Cys	33	.	.	B	.	.	T	.	1.12	−0.11	*	.	.	1.94	0.29
Ser	34	.	.	B	.	.	T	.	0.16	−0.11	*	.	.	1.63	0.32
Val	35	.	.	B	B	.	.	.	−0.30	0.13	*	*	.	0.32	0.18
Ala	36	.	.	B	B	.	.	.	−1.11	0.17	.	*	.	0.01	0.33
Arg	37	.	.	B	B	.	.	.	−1.16	0.29	*	*	.	−0.30	0.20
Val	38	.	.	B	B	.	.	.	−1.30	0.33	*	*	.	−0.30	0.27
Gly	39	.	.	B	B	.	.	.	−1.81	0.37	*	*	.	−0.30	0.22
Leu	40	.	A	B	.	.	.	.	−1.77	0.56	*	*	.	−0.60	0.09
Gly	41	.	A	B	.	.	.	.	−1.99	1.24	*	*	.	−0.60	0.10
Leu	42	.	A	B	.	.	.	.	−2.91	1.29	*	*	.	−0.60	0.09
Leu	43	.	A	B	.	.	.	.	−2.66	1.54	.	.	.	−0.60	0.09
Leu	44	.	A	B	.	.	.	.	−2.66	1.47	.	.	.	−0.60	0.09
Leu	45	.	A	B	.	.	.	.	−2.43	1.47	.	.	.	−0.60	0.10
Leu	46	.	A	B	.	.	.	.	−2.43	1.29	.	.	.	−0.60	0.13
Met	47	A	A	.	.	.	.	.	−2.43	1.03	.	.	.	−0.60	0.15
Gly	48	A	.	.	.	.	T	.	−2.21	1.03	.	.	.	−0.20	0.15
Ala	49	A	.	.	.	.	T	.	−2.26	0.84	.	.	.	−0.20	0.19
Gly	50	A	.	.	.	.	T	.	−1.44	0.80	.	.	.	−0.20	0.14
Leu	51	A	.	.	.	.	T	.	−0.98	0.59	.	.	.	−0.20	0.25
Ala	52	.	A	B	.	.	.	.	−0.67	0.59	.	*	.	−0.60	0.24
Val	53	A	A	.	.	.	.	.	−1.02	1.00	.	.	.	−0.60	0.26
Gln	54	.	A	B	.	.	.	.	−1.24	1.36	.	*	.	−0.60	0.27
Gly	55	.	A	B	.	.	.	.	−1.71	1.36	.	*	.	−0.60	0.22
Trp	56	A	A	.	.	.	.	.	−0.90	1.54	.	*	.	−0.60	0.24
Phe	57	.	A	B	.	.	.	.	−1.12	1.30	.	*	.	−0.60	0.24
Leu	58	.	A	B	.	.	.	.	−0.30	1.59	.	*	.	−0.60	0.20
Leu	59	.	A	B	.	.	.	.	−0.59	1.66	*	*	.	−0.60	0.26
Gln	60	.	A	B	.	.	.	.	−0.13	1.66	*	*	.	−0.60	0.32
Leu	61	.	A	.	.	.	.	C	−0.66	0.87	*	*	.	−0.40	0.76
His	62	.	A	.	.	.	.	C	−0.30	0.87	*	*	.	−0.40	0.76
Trp	63	.	A	.	.	.	.	C	0.51	0.61	*	*	.	−0.40	0.43
Arg	64	A	A	.	.	.	.	.	0.72	0.21	*	*	.	−0.30	0.91
Leu	65	A	A	.	.	.	.	.	−0.13	0.14	*	*	.	−0.30	0.66
Gly	66	.	A	.	.	T	.	.	0.37	0.29	*	*	.	0.10	0.47
Glu	67	.	A	B	.	.	.	.	0.51	−0.14	*	*	.	0.30	0.34
Met	68	.	A	B	.	.	.	.	−0.01	−0.14	*	*	.	0.30	0.82
Val	69	.	A	B	.	.	.	.	−0.33	−0.14	*	.	.	0.64	0.68
Thr	70	.	A	B	.	.	.	.	0.48	−0.14	*	.	.	0.98	0.61
Arg	71	.	A	B	.	.	.	.	0.48	−0.14	*	*	F	1.62	1.02
Leu	72	.	.	B	.	.	T	.	0.27	−0.33	*	.	F	2.36	1.37
Pro	73	.	.	.	.	T	T	.	0.28	−0.54	*	.	F	3.40	1.46
Asp	74	.	.	.	.	T	T	.	0.79	−0.53	*	.	F	2.91	0.75
Gly	75	.	.	.	.	.	T	C	0.80	−0.10	*	.	F	2.07	0.91
Pro	76	.	.	.	.	.	T	C	0.40	−0.40	*	.	F	1.73	0.78
Ala	77	.	.	.	.	.	T	C	1.21	0.09	.	.	F	0.79	0.49
Gly	78	.	.	.	.	.	T	C	1.42	0.09	.	.	F	0.45	0.86
Ser	79	.	.	.	.	.	T	C	0.61	0.06	*	.	F	0.45	0.97
Trp	80	A	A	.	.	.	.	.	0.07	0.31	*	.	F	−0.15	0.79
Glu	81	A	A	.	.	.	.	.	0.28	0.50	*	.	.	−0.60	0.56
Gln	82	A	A	.	.	.	.	.	0.87	0.47	.	*	.	−0.60	0.72
Leu	83	A	A	.	.	.	.	.	1.32	0.09	.	.	.	−0.15	1.19
Ile	84	A	A	.	.	.	.	.	1.73	−0.83	.	.	.	0.75	1.35
Gln	85	A	A	.	.	.	.	.	1.72	−0.83	.	.	F	0.90	1.52
Glu	86	A	A	.	.	.	.	.	1.69	−0.84	.	.	F	0.90	2.48
Arg	87	A	A	.	.	.	.	.	1.69	−1.03	.	.	F	0.90	4.81
Arg	88	.	A	.	.	T	.	.	1.64	−1.71	.	.	F	1.30	4.81
Ser	89	.	A	.	.	T	.	.	2.53	−1.47	.	.	F	1.30	2.06
His	90	.	A	.	.	.	.	C	2.32	−1.07	.	.	.	0.95	1.69
Glu	91	.	A	.	.	.	.	C	1.73	−0.64	*	.	.	0.95	1.34
Val	92	.	A	.	.	.	.	C	1.03	−0.14	*	.	.	0.65	1.01
Asn	93	.	.	.	.	.	T	C	0.89	−0.03	.	*	.	0.90	0.75
Pro	94	A	.	.	.	.	T	.	0.38	−0.03	.	*	.	0.70	0.59
Ala	95	A	.	.	.	.	T	.	0.10	0.66	.	*	.	−0.20	0.65
Ala	96	A	.	.	.	.	T	.	−0.24	0.50	.	*	.	−0.20	0.59
His	97	A	.	.	.	.	.	.	0.02	0.53	.	*	.	−0.40	0.37
Leu	98	A	.	.	.	.	.	.	0.02	0.60	.	.	.	−0.40	0.37
Thr	99	.	.	B	.	.	.	.	−0.07	0.50	.	.	.	−0.40	0.60
Gly	100	.	.	.	.	.	.	C	0.22	0.39	.	.	F	0.25	0.59
Ala	101	.	.	.	.	.	.	C	0.00	0.27	.	.	F	0.25	0.96
Asn	102	.	.	B	.	.	T	.	−0.28	0.27	.	.	F	0.25	0.55
Ser	103	.	.	B	.	.	T	.	0.19	0.27	.	.	F	0.25	0.80
Ser	104	.	.	B	.	.	T	.	0.20	0.27	.	*	F	0.25	0.78
Leu	105	.	.	B	.	.	T	.	0.20	0.16	.	.	F	0.25	0.65
Thr	106	.	.	B	.	.	.	.	0.44	0.19	.	*	F	0.05	0.48
Gly	107	.	.	.	.	T	T	.	0.23	0.23	.	.	F	0.65	0.35
Ser	108	.	.	.	.	T	T	.	−0.28	0.27	.	.	F	0.65	0.66
Gly	109	.	.	.	.	.	T	C	−0.79	0.27	.	.	F	0.45	0.38
Gly	110	.	.	.	.	.	T	C	−0.27	0.47	.	.	F	0.15	0.32
Pro	111	.	A	.	.	.	.	C	0.04	0.96	.	.	F	−0.25	0.25
Leu	112	.	A	.	.	.	.	C	0.08	0.57	.	.	F	−0.25	0.43
Leu	113	.	A	B	.	.	.	.	0.38	0.63	.	*	.	−0.60	0.63
Trp	114	.	A	B	.	.	.	.	−0.09	0.60	.	.	.	−0.60	0.71
Glu	115	.	A	B	.	.	.	.	−0.09	0.86	.	*	.	−0.60	0.71
Thr	116	A	A	.	.	.	.	.	−0.69	0.60	.	*	F	−0.45	0.85
Gln	117	A	A	.	.	.	.	.	−0.47	0.60	.	*	F	−0.45	0.67
Leu	118	A	A	.	.	.	.	.	−0.36	0.19	.	.	.	−0.30	0.39
Gly	119	A	A	.	.	.	.	.	−0.88	0.97	*	*	.	−0.60	0.23
Leu	120	A	A	.	.	.	.	.	−0.77	1.17	*	*	.	−0.60	0.11
Ala	121	.	A	B	.	.	.	.	−0.80	0.77	*	.	.	−0.60	0.26
Phe	122	.	A	B	.	.	.	.	−1.61	0.51	*	.	.	−0.60	0.26
Leu	123	.	A	B	.	.	.	.	−1.10	0.77	*	.	.	−0.60	0.26
Arg	124	.	A	B	.	.	.	.	−1.00	0.47	*	.	.	−0.60	0.35
Gly	125	.	A	B	.	.	.	.	−0.22	0.73	.	.	.	−0.60	0.63
Leu	126	.	.	B	.	.	.	.	0.37	0.44	*	.	.	−0.25	1.05
Ser	127	.	.	.	.	.	.	C	0.72	−0.24	*	.	.	0.70	0.89
Tyr	128	.	.	.	.	.	.	C	0.94	0.19	*	*	.	0.10	0.89
His	129	.	.	.	.	T	T	.	0.02	0.26	*	.	.	0.65	1.09
Asp	130	.	.	.	.	T	T	.	−0.49	0.26	.	.	.	0.50	0.67
Gly	131	.	.	B	.	.	T	.	−0.53	0.51	.	.	.	−0.20	0.32
Ala	132	.	.	B	.	.	T	.	−0.54	0.40	*	.	.	−0.20	0.17
Leu	133	.	.	B	B	.	.	.	−0.26	0.39	*	.	.	−0.30	0.15
Val	134	.	.	B	B	.	.	.	−0.81	0.39	*	.	.	−0.30	0.30
Val	135	.	.	B	B	.	.	.	−1.16	0.46	*	.	.	−0.60	0.30
Thr	136	.	.	B	B	.	.	.	−1.06	0.39	.	.	.	−0.30	0.36
Lys	137	.	.	B	.	.	T	.	−0.71	0.46	.	.	F	−0.05	0.77
Ala	138	.	.	B	.	.	T	.	−0.14	0.57	.	.	.	−0.05	1.62
Gly	139	.	.	B	.	.	T	.	−0.18	0.69	.	.	.	−0.05	1.76
Tyr	140	.	.	B	.	.	T	.	0.43	0.89	*	.	.	−0.20	0.62
Tyr	141	.	.	B	B	.	.	.	0.44	1.64	.	.	.	−0.60	0.96
Tyr	142	.	.	B	B	.	.	.	0.44	1.53	.	*	.	−0.45	1.30
Ile	143	.	.	B	B	.	.	.	0.18	1.10	.	*	.	−0.45	1.66
Tyr	144	.	.	B	B	.	.	.	0.52	0.99	.	*	.	−0.60	0.78
Ser	145	.	.	B	B	.	.	.	−0.04	0.63	.	*	.	−0.60	0.87
Lys	146	.	.	B	B	.	.	.	−0.14	0.56	.	*	.	−0.45	1.02
Val	147	.	.	B	B	.	.	.	−0.24	0.30	.	*	.	−0.30	0.64
Gln	148	.	.	B	B	.	.	.	−0.21	−0.03	.	*	.	0.30	0.48
Leu	149	.	.	B	B	.	.	.	−0.31	0.23	.	*	.	−0.30	0.18
Gly	150	.	.	B	B	.	.	.	−0.68	0.66	.	*	.	−0.60	0.24
Gly	151	.	.	B	B	.	.	.	−0.93	0.59	.	*	.	−0.60	0.07
Val	152	.	.	B	B	.	.	.	−0.89	0.61	.	.	.	−0.60	0.14
Gly	153	.	.	B	.	.	.	.	−1.23	0.61	.	.	.	−0.40	0.11
Cys	154	.	.	B	.	.	T	.	−1.23	0.61	.	.	.	−0.20	0.11
Pro	155	.	.	B	.	.	T	.	−1.48	0.87	.	.	.	−0.20	0.13
Leu	156	.	.	B	.	.	T	.	−1.43	0.73	.	.	.	−0.20	0.13
Gly	157	.	.	B	.	.	T	.	−0.89	0.69	.	.	.	−0.20	0.32
Leu	158	.	.	B	B	.	.	.	−1.43	0.60	.	.	.	−0.60	0.30
Ala	159	.	.	B	B	.	.	.	−1.08	0.86	.	.	.	−0.60	0.26
Ser	160	.	.	B	B	.	.	.	−0.90	0.66	.	.	.	−0.60	0.37
Thr	161	.	.	B	B	.	.	.	−0.43	0.73	*	.	F	−0.45	0.62
Ile	162	.	.	B	B	.	.	.	−0.90	0.47	*	.	.	−0.60	0.60
Thr	163	.	.	B	B	.	.	.	−0.33	0.66	*	.	.	−0.60	0.37
His	164	.	.	B	B	.	.	.	0.30	1.03	*	.	.	−0.60	0.40
Gly	165	.	.	B	B	.	.	.	0.71	0.54	.	.	.	−0.45	1.15
Leu	166	.	.	B	B	.	.	.	0.71	−0.14	.	.	.	0.75	1.56
Tyr	167	.	.	.	B	T	.	.	1.39	−0.14	*	.	.	1.45	1.66
Lys	168	.	.	.	B	T	.	.	1.81	−0.21	*	.	F	1.90	2.59
Arg	169	.	.	B	.	.	.	.	1.60	−0.64	*	.	F	2.30	6.15
Thr	170	.	.	.	.	.	T	C	1.73	−0.57	*	.	F	3.00	6.15
Pro	171	.	.	.	.	.	T	C	2.54	−0.90	*	.	F	2.70	4.75
Arg	172	.	.	.	.	.	T	C	2.79	−0.90	*	.	F	2.40	4.20
Tyr	173	.	.	.	.	.	T	C	1.93	−0.90	*	*	F	2.10	5.05
Pro	174	.	A	.	.	.	.	C	1.82	−0.70	*	*	F	1.40	2.69
Glu	175	A	A	.	.	.	.	.	1.32	−1.13	*	*	F	0.90	2.38
Glu	176	A	A	.	.	.	.	.	0.72	−0.44	*	*	F	0.60	1.25
Leu	177	A	A	.	.	.	.	.	−0.24	−0.51	*	*	.	0.60	0.67
Glu	178	A	A	.	.	.	.	.	−0.30	−0.30	*	.	.	0.30	0.29
Leu	179	A	A	.	.	.	.	.	−0.09	0.09	.	.	.	−0.30	0.22
Leu	180	A	A	.	.	.	.	.	−0.09	0.49	.	.	.	−0.60	0.47
Val	181	A	A	.	.	.	.	.	−0.39	0.20	.	*	.	−0.30	0.47
Ser	182	A	A	.	.	.	.	.	0.21	0.59	.	.	F	−0.45	0.76
Gln	183	.	.	.	.	T	.	.	−0.46	0.33	.	.	F	0.60	1.42
Gln	184	.	.	B	.	.	.	.	0.01	0.21	.	*	F	0.20	1.02
Ser	185	.	.	.	.	.	T	C	0.93	0.00	*	*	F	0.45	0.76
Pro	186	.	.	.	.	T	T	.	1.20	−0.39	.	*	F	1.25	0.85
Cys	187	.	.	.	.	T	T	.	1.19	−0.29	.	*	F	1.25	0.50
Gly	188	.	.	B	.	.	T	.	0.89	−0.20	.	*	F	1.15	0.54
Arg	189	.	.	B	B	.	.	.	0.59	−0.20	.	*	F	1.05	0.47
Ala	190	.	.	B	B	.	.	.	0.59	−0.24	*	*	F	1.50	1.16
Thr	191	.	.	.	B	.	.	C	0.91	−0.43	.	*	F	2.00	1.58
Ser	192	.	.	.	.	.	T	C	0.72	−0.86	.	*	F	3.00	1.58
Ser	193	.	.	B	.	.	T	.	0.78	−0.21	.	*	F	2.20	1.16
Ser	194	.	.	B	.	.	T	.	0.38	0.20	*	*	F	1.15	0.84
Arg	195	.	.	B	.	.	T	.	0.97	0.63	*	*	F	0.55	0.66
Val	196	.	.	B	B	.	.	.	0.98	0.24	*	*	.	0.00	0.82
Trp	197	.	.	.	B	T	.	.	0.98	0.24	.	*	.	0.10	0.82
Trp	198	.	.	B	B	.	.	.	0.58	0.24	.	*	.	−0.30	0.56
Asp	199	.	.	B	.	.	T	.	0.07	1.03	.	*	F	−0.05	0.66
Ser	200	.	.	.	.	.	T	C	−0.39	1.07	.	*	F	0.15	0.52
Ser	201	.	.	.	.	.	T	C	0.12	0.59	.	.	F	0.15	0.49
Phe	202	.	.	.	.	T	T	.	−0.44	0.10	.	.	F	0.65	0.29
Leu	203	.	.	.	B	.	.	C	−1.01	0.74	.	.	.	−0.40	0.16
Gly	204	.	.	.	B	.	.	C	−1.04	1.00	.	.	.	−0.40	0.09
Gly	205	.	.	.	B	.	.	C	−1.56	1.11	.	.	.	−0.40	0.14
Val	206	.	A	B	.	.	.	.	−1.26	1.01	.	*	.	−0.60	0.14
Val	207	.	A	B	.	.	.	.	−1.14	0.33	.	.	.	−0.30	0.24
His	208	A	A	.	.	.	.	.	−0.68	0.40	.	.	.	−0.60	0.25
Leu	209	A	A	.	.	.	.	.	−0.33	0.40	.	.	.	−0.60	0.33
Glu	210	A	A	.	.	.	.	.	0.01	−0.24	.	*	.	0.30	0.77
Ala	211	A	A	.	.	.	.	.	0.01	−0.89	.	.	F	0.75	0.98
Gly	212	A	A	.	.	.	.	.	0.01	−0.74	.	*	F	0.75	0.88
Glu	213	A	A	.	B	.	.	.	−0.81	−0.79	*	*	F	0.75	0.38
Glu	214	A	A	.	B	.	.	.	0.11	−0.14	*	*	F	0.45	0.28
Val	215	A	A	.	B	.	.	.	−0.74	−0.64	*	*	.	0.60	0.55
Val	216	A	A	.	B	.	.	.	−0.97	−0.43	*	*	.	0.30	0.24
Val	217	A	A	.	B	.	.	.	−0.62	0.26	*	*	.	−0.30	0.11
Arg	218	A	A	.	B	.	.	.	−0.62	0.26	*	*	.	−0.30	0.25
Val	219	A	A	.	B	.	.	.	−0.51	−0.39	*	*	.	0.30	0.59
Leu	220	A	A	.	B	.	.	.	−0.47	−1.03	*	.	.	0.75	1.56
Asp	221	A	A	.	B	.	.	.	−0.47	−0.99	*	.	F	0.75	0.66
Glu	222	A	A	.	.	.	.	.	0.50	−0.34	*	.	F	0.45	0.66
Arg	223	A	A	.	.	.	.	.	−0.42	−0.99	*	*	F	0.90	1.56
Leu	224	A	A	.	.	.	.	.	0.54	−0.99	*	*	.	0.60	0.77
Val	225	.	A	B	.	.	.	.	1.36	−0.99	*	*	.	0.94	0.87
Arg	226	.	A	B	.	.	.	.	1.01	−0.99	.	*	.	1.28	0.74
Leu	227	.	.	B	.	.	T	.	0.70	−0.56	*	*	.	2.02	0.89
Arg	228	.	.	B	.	.	T	.	0.70	−0.76	.	*	F	2.66	1.73
Asp	229	.	.	.	.	T	T	.	1.21	−1.40	*	*	F	3.40	1.73
Gly	230	.	.	.	.	T	T	.	1.82	−1.01	*	*	F	3.06	2.81
Thr	231	.	.	.	.	T	.	.	1.01	−0.94	*	*	F	2.52	2.25
Arg	232	.	.	B	.	.	.	.	1.48	−0.16	*	*	F	1.48	1.17
Ser	233	.	.	B	.	.	T	.	0.78	0.27	*	*	F	0.74	1.17
Tyr	234	.	.	B	.	.	T	.	0.08	0.34	*	.	.	0.10	0.82
Phe	235	.	.	B	.	.	T	.	−0.18	0.64	.	.	.	−0.20	0.36
Gly	236	.	.	B	.	.	T	.	−0.72	1.26	.	.	.	−0.20	0.27
Ala	237	.	A	B	.	.	.	.	−1.22	1.51	.	.	.	−0.60	0.13
Phe	238	.	A	B	.	.	.	.	−1.31	1.19	.	.	.	−0.60	0.19
Met	239	.	A	B	.	.	.	.	−1.46	0.83	.	.	.	−0.60	0.24
Val	240	.	A	B	.	.	.	.	−1.14	0.83	.	.	.	−0.60	0.30

Indication from #1: Evidence of tumor-specific protective immunity Indication from #2: Evidence of non-tumor specific immunity Indication from #3: Lack of protective immunity

Result #1	Result #2	Result #3
MC-38.WT:	tumor (−)	tumor (−)	tumor (+)
MCA-102:	tumor (+)	tumor (−)	tumor (+)

TABLE 3 — N-terminus of AIM2 purified from MCA-38 or CHO-11 clone conditioned media. 1 = Affinity purified AIM II from MCA-38 or CHO-11 conditioned media. 2 = Amino acid sequence at junction of ck-beta-8 and extracellular region of AIM II. Double underlined sequence corresponds to ck beta 8 signal sequence (SQA), and in the case of the GS residues sequence introduced during cloning. AIM II sequence starts at the 6th residue, Q. Values in parenthesis represent percentage of each sequence found in AIM II sample.

N-terminus	———————————————————————————— LIQER...
MCA-38 1
N-terminus	SQAGS......................
CHO11 1 (40%)
N-terminus	______GSQLH................
CHO-11 1 (40%)
ck-beta-8-AIM2	SOAGS QLHWRLGEMVTRLPDGPAGSWEQLIQERN
sequence

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Claims as granted

12 claims

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17 codes

IPC · International Patent Classification

Section A — Human necessities

A61K38/00
A61K48/00
A61K39/00
A61K38/21
A61K39/395
A61K45/00
A61K45/06

Section C — Chemistry; metallurgy

C07K14/00
C07K16/28
C07K14/47
C07K14/705

USPC · US Patent Classification

424/130.1530/387.1424/139.1424/85.4530/351424/85.1

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