WO2014018931A1 - Methods and compositions for treating autoimmune disease - Google Patents

Abstract

Methods of treating a patient having or at risk of developing an autoimmune disease (e.g., type 1 diabetes) are provided. The method comprise administering to the patient a composition containing a CD21 agonist. The CD21 agonist may be a polypeptide, an antibody or an antigen-binding fragment thereof, a nucleic acid molecule, or a small molecule. The CD21 agonist induces expression of TNF-a and/or intracellular signaling through NF-KB upon binding to CD21 receptor in a cell of the patient. Compositions and kits are also disclosed.

Description

METHODS AND COMPOSITIONS FOR TREATING AUTOIMMUNE DISEASE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No. 61/675,885, filed on July 26, 2012, which is hereby incorporated by reference.

FIELD OF THE INVENTION

In general, the present invention relates to the treatment of autoimmune disorders, such as, type 1 diabetes.

BACKGROUND OF THE INVENTION

Autoimmune diseases are believed to involve immune responses to the body's own components that are not observed under normal conditions, which result in a pathological state that causes various tissue disorders and/or functional disorders. Autoimmune diseases are broadly classified into systemic autoimmune diseases and organ-specific autoimmune diseases according to their characteristics.

Examples of autoimmune diseases include insulin-dependent diabetes (also known as type 1 diabetes), systemic lupus erythematosus, chronic rheumatoid arthritis, Hashimoto's disease, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, ulcerative colitis, psoriatic arthritis, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barre, hypothyroidism, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, juvenile arthritis, lichen planus, lupus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, Stiff-Man syndrome, Devic's disease, Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis.

In particular, type 1 diabetes is a severe, childhood, autoimmune disease, characterized by insulin deficiency that prevents normal regulation of blood glucose levels. Insulin is a peptide hormone produced by β cells within the islets of Langerhans of the pancreas. Insulin promotes glucose utilization, protein synthesis, formation and storage of neutral lipids, and is the primary source of energy for brain and muscle tissue. Type 1 diabetes is caused by an autoimmune reaction that results in complete destruction of the β cells of the pancreas, which eliminates insulin production and eventually results in hyperglycemia and ketoacidosis. Insulin injection therapy has been useful in preventing severe hyperglycemia and ketoacidosis, but fails to completely normalize blood glucose levels. Although insulin injection therapy has been quite successful, it does not prevent the premature vascular deterioration that is the leading cause of morbidity among diabetics today. Diabetes-related vascular deterioration, which includes both microvascular deterioration and acceleration of atherosclerosis, can eventually cause renal failure, retinal deterioration, angina pectoris, myocardial infarction, peripheral neuropathy, and atherosclerosis.

Some of the investigated therapies against autoimmune diseases include anti-tumor necrosis factor-alpha (anti-TNF-a) therapies (e.g., Adalimumab, Infliximab, and Etanercept) and anti-cluster of differentiation 3 receptor (anti-CD3) therapies (e.g., Otelixizumab, Teplizumab, and Visilizumab). The anti-TNF-α therapies provide only moderate benefit in select autoimmune diseases, while clinical trials for type 1 diabetes treatment using the anti-CD3 therapies produced disappointing results (e.g., Sherry et al., Lancet 378:487-497, 2011 ; GlaxoSmithKline, March 1 1 , 201 1 ,

http://www.gsk.com/media/pressreleases/2011/2-11_pressrelease_10039.htm).

Tumor necrosis factor-alpha (TNF-a) is a naturally occurring cytokine that was described in 1975 as the serum factor induced after Bacillus Calmette-Guerin (BCG) injection as a means to fight tumors (Carswell et al., Proc. Natl. Acad. Sci. U.S.A. 72:3666-3670, 1975). The cloning of TNF-a and its two receptors uncovered sequence homology to the genomes of microbial pathogens (e.g., Loetscher et al., Cell 62:351 , 1990). This surprising sequence overlap represents a system of intricate microbial responses to modulate host TNF-a secretion and the activity of its receptors (Rahman et al., PloS Pathogens 2:66, 2006).

TNF-a expression is induced by diverse bacteria, parasites, and viruses as a host first line defense to infections. Viruses, such as the Epstein-Barr virus, encode receptors and proteins that even augment TNF-a and TNF-a signaling (Liebowitz, New Engl. J. Med. 338:1461-1463, 1998; Guasparri et al., Blood 1 11 :3813-3821 , 2008; Wang et al., Cell 43:831 -840, 1985). Alternatively, a variety of viruses have been shown to express proteins that repress TNF-a signaling activity and function in the host (Rahman et al., PloS Pathogens 2:66, 2006). Some evidence suggests that viral infections (e.g., Epstein- Barr virus infections) may cause autoimmune disease (Sairenji et al., Diabetologia 34:33-39, 1991 ).

There remains a need for methods and compositions for treating autoimmune diseases.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of treating a mammal (e.g., a human) having an autoimmune disease including administering to the mammal (e.g., the human) a composition containing a CD21 agonist. The CD21 agonist may act by inducing expression of tumor necrosis factor-alpha (TNF-a) in the mammal upon binding of the CD21 agonist to CD21 receptor. In certain embodiments of the invention, the CD21 agonist is an agent selected from the group consisting of a polypeptide, an antibody or antigen-binding fragment thereof, a nucleic acid molecule, and a small molecule.

In some embodiments of the methods of the invention, the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, an Fab, a humanized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a monovalent antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a single-chain Fv molecule, a bispecific single chain Fv ((scFv')₂) molecule, a domain antibody, a diabody, a triabody, an affibody, a domain antibody, a SMIP, a nanobody, a Fv fragment, a Fab fragment, a F(ab')₂ molecule, and a tandem scFv (taFv) fragment. In some embodiments of the methods of the invention, the CD21 agonist polypeptide is selected from the group consisting of a receptor, a ligand, and an aptamer. In certain embodiments of the methods of the invention, a CD21 agonist is a polypeptide, the sequence of which has at least 90% sequence identity to all or a portion of the sequence of SEQ ID NO: 1 or 2. In certain embodiments of the methods of the invention, the CD21 agonist is a polypeptide, the sequence of which has at least 90% sequence identity to the sequence of any one of SEQ ID NO: 3 to 10. In certain embodiments of the methods of the invention, the CD21 agonist is a polypeptide, the sequence of which has one or more of amino acid sequences having at least 90% sequence identity to the sequence of any one of SEQ ID NO: 3 to 6. In certain embodiments of the methods of the invention, the CD21 agonist is a polypeptide containing the combination of amino acid sequences of two or more of SEQ ID NOs: 3 to 6. In certain embodiments of the methods of the invention, the CD21 agonist is a polypeptide containing the combination of amino acid sequences having at least 90% sequence identity to the sequences of SEQ ID NOs: 3 to 5. In certain embodiments of the methods of the invention, the CD21 agonist is a polypeptide containing the combination of amino acid sequences having at least 90% sequence identity to the sequences of SEQ ID NOs: 3 and 4. In certain embodiments of the methods of the invention, the CD21 agonist is a polypeptide containing the combination of amino acid sequences having at least 90% sequence identity to the sequences of SEQ ID NOs: 4 to 6. In certain embodiments of the methods of the invention, the CD21 agonist is a polypeptide containing the combination of amino acid sequences having at least 90% sequence identity to the sequences of SEQ ID NOs: 5 and 6. In certain embodiments of the methods of the invention, the CD21 agonist is a polypeptide containing the combination of amino acid sequences having at least 90% sequence identity to the sequences of SEQ ID NOs: 3, 4, and 6. In certain embodiments of the methods of the invention, the CD21 agonist is a polypeptide containing the combination of amino acid sequences having at least 90% sequence identity to the sequences of SEQ ID NOs: 3, 5, and 6. In certain embodiments of the methods of the invention, the CD21 agonist is a polypeptide containing the combination of amino acid sequences having at least 90% sequence identity to the sequences of SEQ ID NOs: 3 and 6. In certain embodiments of the methods of the invention, the CD21 agonist is a polypeptide containing the combination of amino acid sequences having at least 90% sequence identity to the sequences of SEQ ID NOs: 4 and 6. In certain embodiments of the methods of the invention, the CD21 agonist contains a polypeptide containing the combination of amino acid sequences having at least 90% sequence identity to the sequences of SEQ ID NOs: 4 and 5.

In some embodiments of the methods of the invention, binding of the CD21 agonist to CD21 is inhibited by monoclonal antibody 72A1 or Fab fragment thereof.

In different embodiments of the methods of the invention, the CD21 agonist is selected from the group consisting of an IFNa, IFNp, CD23, and C3d protein and CD21-binding fragments thereof. In some embodiments of the methods of the invention, the CD21 agonist is selected from the group consisting of an IFNa, IFN , and C3d protein. In some embodiments of the methods of the invention, the CD21 agonist is IFNa or IFN . In some embodiments of the methods of the invention, the CD21 agonist is IFNa. In some embodiments of the methods of the invention, the CD21 agonist is IFN . In certain embodiments of the methods of the invention, the CD21 agonist is not C3d protein.

In different embodiments of the methods of the invention, the CD21 agonist is an agent (e.g., a polypeptide, an antibody or antigen-binding fragment thereof, or a small molecule) that binds to the first and/or second consensus repeat (SCR) domains of the CD21 receptor. In different embodiments of the methods of the invention, the CD21 agonist is an agent that binds to an epitope of CD21 defined by one or more of amino acid residues Arg13, Ser15, Arg28, Lys41 , Arg36, Arg83, Lys57, and Lys67, or a conservative substitution thereof (numbering relative to SEQ ID NO: 1 1 ). In some embodiments of the methods of the invention, the epitope of CD21 may further contain an Arg residue at position 13 and a Ser at position 15. In some embodiments of the methods of the invention, the epitope of CD21 may further contain an Arg at position 28 and a Lys at position 41 . In some embodiments of the methods of the invention, the epitope of CD21 may further contain an Arg at position 36 and an Arg at position 83. In some embodiments of the methods of the invention, the epitope of CD21 may further contain a Lys at position 57 and a Lys at position 67. In different embodiments of the methods of the invention, the CD21 agonist binds to an epitope of CD21 defined by one or more of amino acid residues I9, N1 1 , R13, Y16, A22, R28, Y29, C31 , S32, G33, T34, R36, K41 , S42, K48, K50, D56, K57, Y64, K67, Y68, S70, R83, G84, R89, H90, D92, S93, A97, T100, N101 , S109, and S128. In some embodiments of the methods of the invention, the CD21 agonist binds to an epitope of CD21 defined by one or more of amino acid residues N1 1 , R13, Y16, A22, R28, S32, R36, K41 , D42, K50, K57, Y64, K67, Y68, R83, G84, and R89. In some embodiments of the methods of the invention, the CD21 agonist binds to an epitope of CD21 defined by one or more of amino acid residues R13, S15, R28, R36, K41 , K56, K67, R83, and R89. In some embodiments of the methods of the invention, the CD21 agonist binds to an epitope of CD21 defined by one or more of amino acid residues R13, S15, R28, R36, K41 , K50, K56, K57, K67, R83, and R89. In some embodiments of the methods of the invention, the CD21 agonist binds to an epitope of

CD21 defined by one or more of amino acid residues R13, Y16, R28, S42, K48, K50, Y68, R83, G84, and R89. In some embodiments of the methods of the invention, the CD21 agonist binds to an epitope of CD21 defined by one or more of amino acid residues R13, R28, Y68, R83, G84, and R89. In some embodiments of the methods of the invention, the CD21 agonist binds to an epitope of CD21 defined by one or more of amino acid residues R13 and R28. In some embodiments of the methods of the invention, the CD21 agonist binds to an epitope of CD21 defined by one or more of amino acid residues R13, S 5, and R28. In some embodiments of the methods of the invention, the CD21 agonist binds to an epitope of CD21 defined by one or more of amino acid residues R13, R28, and K41. In some embodiments of the methods of the invention, the CD21 agonist binds to an epitope of CD21 defined by one or more of amino acid residues R13, S15, R28, and K41. In some embodiments of the methods of the invention, the CD21 agonist binds to an epitope of CD21 defined by one or more of amino acid residues R28 and K41 . In some embodiments of the methods of the invention, the CD21 agonist binds to an epitope of CD21 defined by one or more of amino acid residues S15, R28, and K41. In some embodiments of the methods of the invention, the CD21 agonist binds to an epitope of CD21 defined by one or more of amino acid residues R13, S15, R28, R36, and K41 . In some embodiments of the methods of the invention, the CD21 agonist binds to an epitope of CD21 defined by one or more of amino acid residues R 3, R28, R36, and K41 . In some embodiments of the methods of the invention, the CD21 agonist binds to an epitope of CD21 defined by one or more of amino acid residues R28, R36, and 41 . In some embodiments of the methods of the invention, the CD21 agonist binds to an epitope of CD21 defined by one or more of amino acid residues R13, S15, R28, R36, K41 , and R83. In some embodiments of the methods of the invention, the CD21 agonist binds to an epitope of CD21 defined by one or more of amino acid residues R13, R28, R36, K41 , and R83. In some embodiments of the methods of the invention, the CD21 agonist binds to an epitope of CD21 defined by one or more of amino acid residues R28, R36, K41 , and R83. In some embodiments of the methods of the invention, the CD21 agonist binds to an epitope of CD21 defined by one or more of amino acid residues R36, K41 , and R83. In some embodiments of the methods of the invention, the CD21 agonist binds to an epitope of CD21 defined by one or more of amino acid residues R36, K41 , and R83. In some embodiments of the methods of the invention, the CD21 agonist binds to an epitope of CD21 defined by one or more of amino acid residues R28, K41 , and R83. In some embodiments of the methods of the invention, the CD21 agonist binds to an epitope of CD21 defined by one or more of amino acid residues R13, S15, R28, K41 and R83. In preferred embodiments, CD21 contains a polypeptide having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 1 .

In many embodiments of the methods of the invention, the CD21 agonist induces (either directly or indirectly) activation of the NF-κΒ pathway in an autoreactive immune cell of a mammal (e.g. a human) upon binding to the CD21 receptor.

In some embodiments of the methods of the invention, administration of the composition containing a CD21 agonist (e.g., a polypeptide, an antibody or antigen-binding fragment thereof, a nucleic acid molecule, or a small molecule) to a mammal (e.g., a human) induces at least a 1 % increase in autoreactive immune cell death in the mammal relative to the level of autoreactive immune cell death observed in the mammal prior to the treatment. In some embodiments, the autoreactive immune cell may be an autoreactive T cell. In certain embodiments, the autoreactive T cell may be an autoreactive CD8⁺ T cell.

In some embodiments of the methods of the invention, administration of the composition containing a CD21 agonist (e.g., a polypeptide, an antibody or antigen-binding fragment thereof, a nucleic acid molecule, or a small molecule) to a mammal (e.g., a human) induces at least a 1 % increase in the number of regulatory T cells in the mammal relative to the number of regulator T cells present in the mammal prior to the treatment.

In some embodiments of the methods of the invention, administering the composition containing a CD21 agonist (e.g., a polypeptide, an antibody or antigen-binding fragment thereof, a nucleic acid molecule, or a small molecule) to a mammal (e.g., a human) results in at least a 1 % increase in C-peptide levels in the mammal relative to the C-peptide levels in the mammal prior to the treatment.

In any of the above embodiments of the methods of the invention, the autoimmune disease is selected from type I diabetes, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, systemic lupus erythmatosous, ulcerative colitis, psoriatic arthritis, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, hypothyroidism, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, juvenile arthritis, lichen planus, lupus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, Stiff-Man syndrome, Devic's disease, Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis. In particular, the autoimmune disease is selected from type 1 diabetes, celiac sprue-dermatitis, Crohn's disease, Graves' disease, hypothyroidism, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis, sarcoidosis, Sjogren's syndrome, and ulcerative colitis. In some embodiments of the methods of the invention, the autoimmune disease is type 1 diabetes. In certain embodiments of the methods of the invention, a mammal (e.g., a human) is a long-term insulin dependent diabetic. In some embodiments, the invention provides a method of treating a mammal (e.g., a human) having an autoimmune disease including administering to the mammal a composition containing a CD21 agonist that induces expression of tumor necrosis factor-alpha (TNF-a) in the mammal upon binding to CD21 , where the CD21 agonist is C3d and the autoimmune disease is selected from type I diabetes, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, systemic lupus erythmatosous, ulcerative colitis, psoriatic arthritis, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, hypothyroidism, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, juvenile arthritis, lichen planus, lupus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, Stiff-Man syndrome, Devic's disease, Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis.

In some embodiments of the methods of the invention, the method further includes administering an agent selected from the group consisting of TNF-a, a TNF-a inducing substance, and a TNF-a receptor 2 agonist to a mammal (e.g., a human). In certain embodiments of the methods of the invention, the TNF-a inducing substance is selected from the group consisting of Bacillus Calmette-Guerin (BCG), complete Freund's adjuvant, tissue plasminogen factor, lipopolysaccharide (LPS), interleukin-1 , interleukin-2, lymphotoxin, and cachectin. In specific embodiments of the methods of the invention, Bacillus Calmette-Guerin (BCG) is administered intraderma!ly. In certain embodiments of the methods of the invention, the TNF-a receptor 2 agonist is selected from the group consisting of a TNF-a mutein, an anti-TNF-a receptor 2 antibody, and a small molecule that binds a TNF-a receptor 2.

In some embodiments of the methods of the invention, the composition containing a CD21 agonist is administered to a mammal (e.g., a human) prior to the development of one or more symptoms of the autoimmune disease. In other embodiments of the methods of the invention, the composition is administered to a mammal (e.g., a human) after the development of one or more symptoms of the autoimmune disease. In some embodiments of the methods of the invention, treating a mammal (e.g., a human) results in a decrease in one or more symptoms of the autoimmune disease relative to, e.g., an untreated mammal.

In any of the above embodiments of the methods of the invention, the symptoms of an

autoimmune disease are selected from the group consisting of increased levels of autoantibodies, increased levels of autoreactive T cells, loss of targeted cells, hyperglycemia, hypoglycemia, fatigue, depression, sensitivity to cold, weight gain, muscle weakness, constipation, insomnia, irritability, ketoacidosis, weight loss, bulging eyes, blurred eyesight, muscle tremors, skin rashes, painful or swollen joints, sensitivity to the sun, loss of coordination, frequent urination, and paralysis. The methods of the invention may improve one or more of these symptoms. In some embodiments of the methods of the invention, the autoimmune disease is type I diabetes and the one or more symptoms of the autoimmune disease are selected from the group consisting of increased levels of autoreactive T cells, hyperglycemia, hypoglycemia, fatigue, ketoacidosis, weight loss, skin rashes, blurred eyesight, and frequent urination. Preferably, the CD21 agonist is administered intravenously. In particular, the CD21 agonist is an antibody or antigen-binding fragment thereof that is administered intravenously.

In any of the above embodiments of the methods of the invention, the composition containing a

CD21 agonist is administered intravenously, parenterally, topically, intra-arterially, intracranially, intradermally, subcutaneously, intramuscularly, intraorbital^, intraventricularly, intraspinally,

intraperitoneally, intranasally, or orally.

In any of the above embodiments of the methods of the invention, the composition containing a CD21 agonist (e.g., a polypeptide, an antibody or antigen-binding fragment thereof, a nucleic acid molecule, or a small molecule as defined above and herein) is administered one or more times daily, weekly, monthly, or yearly. In some embodiments of the methods of the invention the composition containing a CD21 agonist is administered twice daily, biweekly, bi-annually, tri-annually, or quarterly.

In any of the embodiments of the methods of the invention, the method further includes

performing a diagnostic step prior to the administering step in order to ascertain the presence of autoimmune disease in a subject. For example, the method may include performing a diagnostic step to determine the presence type 1 diabetes in a subject prior to administering a CD21 agonist-containing composition.

A second aspect of the invention features CD21 agonist agent-containing compositions. For example, the composition may be a pharmaceutical composition that contains a CD21 agonist (e.g., a polypeptide, an antibody or antigen-binding fragment thereof, a nucleic acid molecule, or a small molecule as defined above and herein) and a pharmaceutically acceptable carrier or excipient for use in treating an autoimmune disease in a mammal (e.g., a human). In an embodiment, the pharmaceutical composition further contains, or is formulated for administration with, an additional agent, such as, e.g., TNF-a, a TNF-a inducing substance (e.g., BCG), or a TNF-a receptor 2 agonist (e.g., a TNF-a receptor 2 agonist antibody), as is described above.

In any of the embodiments of the invention, the mammal is a human.

A third aspect of the invention features a kit. The kit of the invention contains a pharmaceutical composition containing a CD21 agonist (e.g., a polypeptide, an antibody or antigen-binding fragment thereof, a nucleic acid molecule, or a small molecule as defined above and herein), a pharmaceutically-acceptable carrier or excipient, and, optionally, an additional agent (e.g., TNF-a, a TNF-a inducing substance, or a TNF-a receptor 2 agonist) as described above. In some embodiments of the invention, the kit contains a therapeutically effective amount of the pharmaceutical composition (described above) for treating autoimmune disease. The kits may further include instructions for administering the pharmaceutical composition contained therein. Furthermore, the kits may also include one or more additional pharmaceutical compositions containing an additional agent (e.g., TNF-a, a TNF-a inducing substance, or a TNF-a receptor 2 agonist) as described above, instructions or administration schedules for a patient suffering from an autoimmune disease, and, optionally, a device for administering the pharmaceutical composition(s) (e.g., a syringe).

Definitions

The term "about" is used herein to mean a value that is ± 0% of the recited value.

The term "antibody," as used herein, includes whole antibodies or immunoglobulins and any antigen- binding fragment or single chains thereof. Antibodies, as used herein, can be mammalian (e.g., human or mouse), humanized, chimeric, recombinant, synthetically produced, or naturally isolated. In most mammals, including humans, whole antibodies have at least two heavy (H) chains and two light (L) chains connected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as V_H) and a heavy chain constant region. The heavy chain constant region consists of three domains, C_H , C_H2, and C_H3 and a hinge region between C_H1 and C_H2. Each light chain consists of a light chain variable region

(abbreviated herein as V_L) and a light chain constant region. The light chain constant region consists of one domain, C_L. The V_H and V_L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each V_H and V_L is composed of three CDRs and four FRs, arranged from amino- terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Antibodies of the present invention include all known forms of antibodies and other protein scaffolds with antibody-like properties. For example, the antibody can be a monoclonal antibody, a polyclonal antibody, human antibody, a humanized antibody, a bispecific antibody, a monovalent antibody, a chimeric antibody, or a protein scaffold with antibody-like properties, such as fibronectin or ankyrin repeats. The antibody can have any of the following isotypes: IgG (e.g., lgG1 , lgG2, lgG3, and lgG4), IgM, IgA (e.g., lgA1 , lgA2, and IgAsec), IgD, or IgE.

The term "antigen-binding fragment," as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to a specific antigen (e.g., CD21 receptor). The antigen- binding function of an antibody can be performed by fragments of a full-length antibody. The antibody fragments can be a Fab, Fab'2, scFv, SMIP, diabody, a triabody, an affibody, a nanobody, an aptamer, or a domain antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the V_L, V_H, C_L, and C_H1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two

Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V_H and C_H1 domains; (iv) a Fv fragment consisting of the V_u and V_H domains of a single arm of an antibody, (v) a dAb including V_H and V_L domains; (vi) a dAb fragment (Ward et al.. Nature 341 :544-546, 1989), which consists of a V_H domain; (vii) a dAb which consists of a VH or a V_L domain; (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, V_L and V_H, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V_L and V_H regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al., Science 242: 423-426, 1988, and Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883, 1988). These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

The term "aptamer," as used herein, refers to any peptide that binds to a specific target molecule, preferably a receptor, and more preferably, a CD21 receptor. Preferably, an aptamer that binds CD21 is an agonist that induces TNF-a expression.

The terms "CD21 receptor," "CD21 ," "complement receptor 2," and "CR2" are used

interchangably herein. A CD21 receptor is described in detail in U.S. Pat. No. 6,820,01 1 and WO 201 1 /143637, which are hereby incorporated by reference in their entirety. CD21 receptor is a 145 kiloDalton ("kDa") transmembrane protein. The full-length human wild-type CD21 receptor is a protein containing the amino acid sequence represented by SEQ ID NO: 1 1 and containing 15 or 16 short consensus repeat (SCR) extracellular domains, a 28 amino acid single pass transmembrane domain, and a short 34 amino acid intracellular domain (Fingeroth, et al., J. Virol. 62:1442-1447, 1988; Fujisaku, et al., J. Biol. Chem. 264:21 18-2125, 1989; Moore, et al., Proc. Nat. Acad. Sci. USA 84:9194-9198, 1987; Weis, et al., Proc. Nat. Acad. Sci. USA 83:5639-5643, 1986; Weis, et al., J. Exp. Med. 167:1047-1066, 1988). Each of the extracellular SCRs contains approximately 60-70 amino acid residues and is connected by linker regions of three to eight amino acid residues. All SCRs contain a number of conserved amino acid residues including four cysteine residues, which form disulfide bridges between first and third Cys residues and second and fourth Cys residues. CD21 is primarily present on B cells, where it is found in complex with other membrane proteins that promote normal humoral and cellular immune responses (Ahearn, et al., Adv. Immunol. 46:183-219, 1989; Cooper, et al., Ann. Rev. Immunol. 6:85-1 13, 1988; Holers, et al., Mosby, 363-391 , 1995; Tolnay, et al., Clin. Immunol. Immunopathol. 88: 123-132, 1998). Using the amino-terminal SCR domains, i.e., SCR1 and SCR2, CD21 binds four classes of ligands - complement component 3 (C3) proteolytic fragments iC3b, C3dg, and C3d (lida, et al., J. Exp. Med. 158:1021 -1033, 1983; Weis, et al., Proc. Nat. Acad. Sci. USA 81 :881 -885, 1984), the Epstein-Barr virus (EBV) glycoprotein gp350/220 (Fingeroth, et al., Proc. Nat. Acad. Sci. USA 81 :4510-4514, 1984;

Nemerow, et al ., Cell 56:369-377, 1989; Nemerow, et al., J. Virol. 55:347-351 , 1985), the low affinity IgE receptor CD23 (Aubry, et al., J. Immunol. 152:5806-5813, 1994; Aubry, et al., Nature 358:505-507, 1992), and the cytokine interferon alpha (IFN-a) (Asokan, et al., J. Immunol. 1 77:383-394, 2006; Delcayre, et al., J. Virol. 67:2918-2921 , 2003; Delcayre, et al., EMBO J. 10:919-926, 1991 ). SCR1 is located within the human CD21 amino acid sequence between Cys23 and Cys82 (i.e., between the first Cys and the fourth Cys residues) of SEQ ID NO: 1 1 . SCR2 is located within the human CD21 amino acid sequence between Cys91 and Cys146 (i.e., between the fifth Cys and the eighth Cys residues) of SEQ ID NO: 11 . The sequence of first and second short consensus repeat (SCR) domains of CD21 is provided in SEQ ID NO: 12.

The term "CD21 agonist," as used herein, refers to an agent (e.g., a polypeptide, an antibody or an antigen-binding fragment thereof, a nucleic acid molecule, or a small molecule) that binds to CD21 receptor and triggers a physiological reaction (e.g., an increase in expression of TNF-a, NF-κΒ signaling, killing of autoreactive immune cells (e.g., autoreactive T cells), an increase in serum levels of C-peptide, and/or a decrease in, or amelioration of one or more symptoms of an autoimmune disease (e.g., type 1 diabetes)).

The term "chimeric antibody" refers to an immunoglobulin or antibody whose variable regions derive from a first species and whose constant regions derive from a second species. Chimeric antibodies can be constructed, for example, by genetic engineering, from immunoglobulin gene segments belonging to different species (e.g., from a mouse and a human).

The terms "conservatively substituted" and "conservative substitution," as used herein, refer to a substitution of one amino acid residue within a polypeptide for another amino acid residue, both amino acid residues in question belonging to the same group. For purposes of classifying amino acids substitutions as conservative or nonconservative, amino acids are grouped as follows: Group I (hydrophobic sidechains): norleucine, Met, Ala, Val, Leu, lie; Group II (neutral hydrophilic side chains): Cys, Ser, Thr; Group III (acidic side chains): Asp, Glu; Group IV (basic side chains): Asn, Gin, His, Lys, Arg; Group V (residues influencing chain orientation): Gly, Pro; and Group VI (aromatic side chains): Trp, Tyr, Phe. Conservative substitutions involve substitutions between amino acids within the same group. Non-conservative substitutions involve substitutions between amino acids within different groups.

The term "diabetic," as used herein, refers to a subject or a patient to be treated according to the methods described herein and may be one who has been diagnosed by a medical practitioner as having type 1 diabetes or is at risk of developing type 1 diabetes. Diagnosis may be performed by any technique and method known in the art. One skilled in the art will understand that a subject to be treated according to the present invention may have been subjected to standard tests or may have been identified, without examination, as one at high risk due to the presence of one or more risk factors (e.g., fasting plasma glucose levels of at least 6.1 mmol/L, plasma glucose levels of at least 1 1.1 mmol/L two hours after oral glucose load of 75 g, decreased serum levels of C-peptide, presence of autoreactive immune cells (e.g., autoreactive T cells, preferably autoreactive CD8⁺ T cells)).

The terms "effective amount" or "amount effective to" or "therapeutically effective amount" means an amount of a composition containing a CD21 agonist (e.g., a polypeptide, an antibody or antigen-binding fragment thereof, a nucleic acid molecule, or a small molecule) sufficient to produce a desired result, for example, one or more of an increase in expression of TNF-a, NF-κΒ signaling, autoreactive immune cells (e.g., autoreactive T cells) killing, an increase in serum levels of C-peptide, and/or a decrease in, or amelioration of symptoms of, an autoimmune disease (e.g., type 1 diabetes) in a subject upon administration of the composition. Preferably, an effective amount of a composition containing a CD21 agonist is an amount that reduces, inhibits, or prevents the occurrence of one or more symptoms of, e.g., autoimmune disease (e.g., type 1 diabetes) or is an amount that reduces the severity of, or the length of time during which a subject suffers from, one or more symptoms of the autoimmune disease (e.g., by at least 10%, 20%, or 30%, more preferably by at least 50%, 60%, or 70%, and most preferably by at least 80%, 90%, 95%, 99%, or more, relative to a control subject that is not treated with a CD21 agonist of the invention). An effective amount of a composition containing a CD21 agonist used to practice the methods described herein may vary depending upon the manner of administration and the age, body weight, and general health of the subject being treated. A physician or researcher can decide the appropriate amount and dosage regimen.

The term "gp350/220," as used herein, refers to Epstein-Barr virus envelope glycoprotein having a sequence of SEQ ID NO: 1 or 2, i.e., gp350 or gp220, both of which originate from the same gene and constitute splice variants.

The term "gp350," as used herein, referes to a ca. 350 kDa protein having a sequence of SEQ ID

NO: 1 .

The term "gp220," as used herein, referes to a ca. 220 kDa protein having a sequence of SEQ ID

NO: 2, which is a splice variant of gp350.

The term "human antibody," as used herein, is intended to include antibodies, or fragments thereof, having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences as described, for example, by Kabat et al (Sequences of proteins of

Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 991 ). Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site- specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e., a humanized antibody or antibody fragment).

The term "humanized antibody" refers to any antibody or antibody fragment that includes at least one immunoglobulin domain having a variable region that includes a variable framework region substantially derived from a human immunoglobulin or antibody and complementarity determining regions (e.g., at least one CDR) substantially derived from a non-human immunoglobulin or antibody.

The term "immune cell," as used herein, refers to any cell that is involved in the generation, regulation, or effect of the acquired or innate immune system. Immune cells include, e.g., T cells (e.g., CD4⁺ cells or CD8⁺ cells), B cells, natural killer (NK) cells, macrophages, monocytes and dendritic cells, and neutrophils.

The term "ligand," as used herein, refers to any molecule that binds specifically and reversibly to another chemical entity to form a larger complex and acts as an agonist or an antagonist of the chemical entity, to which the ligand binds. Preferably, a ligand, as used herein, refers to a molecule that binds to a CD21 receptor as an agonist. Preferably, the ligand is a polypeptide.

The term "mutein," as used herein, refers to a polypeptide that differs in its amino acid sequence by at least one or more amino acids. For example, a mutein may have an amino acid sequence with greater than 90% but less than 100% sequence identity relative to the amino acid sequence of a

reference polypeptide.

The terms "patient" or "subject," as used interchangeably herein, refer to any animal, e.g., a mammal (e.g., a human). A subject to be treated according to the methods described herein (e.g., a subject diagnosed with an autoimmune disease (e.g., type 1 diabetes)) may be one who has been diagnosed by a medical practitioner as having such a condition or one at risk for developing the condition (e.g., an autoimmune disease (e.g., type 1 diabetes)). Diagnosis may be performed by any techniques and methods known in the art. One skilled in the art will understand that a subject to be treated according to the present invention may have been subjected to standard tests or may have been identified, without examination, as one at high risk due to the presence of one or more risk factors (e.g., fasting plasma glucose levels of at least 6.1 mmol/L, plasma glucose levels of at least 1 1 .1 mmol/L two hours after oral glucose load of 75 g, decreased serum levels of C-peptide, presence of autoreactive immune cells (e.g., autoreactive T cells, preferably autoreactive CD⁺ T cells)).

The terms "peptide," "polypeptide," and "protein," as used herein (and collectively referred to as a "polypeptide"), refers to any chain of more than two amino acids, regardless of post-translational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally occurring polypeptide, or constituting a non-naturally occurring polypeptide (e.g., a synthetic polypeptide).

Preferably, the polypeptide is a receptor, a ligand, or an aptamer. A polypeptide may be said to be "isolated" or "substantially pure" when physical, mechanical, or chemical methods have been employed to remove the polypeptide from cellular constituents. An "isolated polypeptide," means a polypeptide that is removed from cellular constituents and is at least 60% by weight free from the proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the polypeptide is at least 75%, more preferably at least 90%, and most preferably at least 99% by weight pure. An isolated polypeptide may be obtained by standard techniques, for example, by extraction from a natural source (e.g., cell lines), by expression of a recombinant nucleic acid encoding the polypeptide, or by chemically synthesizing the polypeptide or peptide. Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

The terms "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient," as used interchangeably herein, refer to any ingredient other than the compounds described herein (for example, a vehicle capable of suspending or dissolving the active compound) and having the properties of being nontoxic and non-inflammatory in a patient. Carriers and excipients may include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, or waters of hydration. Exemplary carriers and excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethyicellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine,

methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, xylitol, water, phosphate- buffered saline (PBS), acetate-buffered saline (ABS), Ringer's solution, dextrose, glycerol, ethanol, or the like and combinations thereof. DETAILED DESCRIPTION

I have discovered methods of treating autoimmune disease by activating CD21 using a CD21 agonist agent, such as a polypeptide, antibody or antigen-binding fragment thereof, a nucleic acid molecule, or a small molecule. CD21 agonist agents promote an increase in the expression of tumor necrosis factor-alpha (TNF-a), which results in intracellular signaling through transcription nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κΒ) that leads to the death of autoreactive immune cells, induction of beneficial regulatory T cells, regeneration of cellular damage caused by autoreactive immune cells (.e.g, autoreactive T cells), and/or restoration of biological activities (e.g., a beneficial, transient restoration of insulin secretion in type 1 diabetes patients, as evidenced by increased levels of C-peptide). The induction of TNF-a can be achieved using CD21 agonists, such as Epstein-Barr virus (EBV) glycoprotein gp350/220 and fragments and muteins thereof, CD21 agonist antibodies and antigen- binding fragments thereof, interferon-alpha (IFN-a), interferon-beta (IFN-β), and complement C3d protein. These CD21 agonists can be used to treat autoimmune disease in a patient diagnosed with or at risk of developing an autoimmune disease.

Without being bound by any theories, it is believed that binding of the CD21 agonist to a CD21 receptor that is naturally expressed on cells in the body may induce expression of TNF-a, which is then released into the bloodstream (e.g., resulting in an increase in serum levels of TNF-a). Circulating TNF-a then binds TNF-a receptors (e.g., TNF-a receptor 2) on autoreactive immune cells, which activates intracellular NF-κΒ signaling. Intracellular NF-κΒ signaling in autoreactive immune cells is believed to induce apoptosis of the autoreactive immune cells, thereby treating autoimmune disease in those patients administered the CD21 agonist. Alternatively, it is believed that the CD21 agonist may bind to a CD21 receptor on the surface of an autoreactive immune cell (e.g., an autoreactive T cell, such as an autoreactive CD8⁺ T cell), which upregulates TNF-a expression in the autoreactive immune cell, thereby inducing intracellular NF-κΒ signaling that leads to apoptosis of the autoreactive immune cell.

The methods and compositions of the invention are described below.

Methods of Treating Autoimmune Disease

The invention features methods of treating a mammal (e.g., a human) having or at risk of developing an autoimmune disease involving administering to the mammal (e.g., the human) a composition containing a CD21 agonist that induces expression of tumor necrosis factor-alpha (TNF-a) in the mammal (e.g., the human) upon binding to CD21. The increase in the expression of TNF-a is believed to induce activation of the NF-κΒ pathway in an autoreactive immune cell (e.g., an autoreactive T cell, such as an autoreactive CD8⁺ T cell) directly or indirectly in the mammal (e.g., the human) following binding of the CD21 agonist to CD21 receptor, which results in apoptosis of the autoreactive immune cell and an improvement in one or more symptoms of autoimmune disease in the mammal (e.g., the human).

Autoimmune diseases that can be treated by administering a CD21 agonist include one or more of the following: type I diabetes, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, systemic lupus erythmatosous, ulcerative colitis, psoriatic arthritis, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, hypothyroidism, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, juvenile arthritis, lichen planus, lupus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, Stiff-Man syndrome, Devic's disease, Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis. Preferably, the autoimmune diseases that can be treated according to the methods of the invention include type 1 diabetes, celiac sprue-dermatitis, Crohn's disease, Graves' disease, hypothyroidism, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis, sarcoidosis, Sjogren's syndrome, and ulcerative colitis. More preferably, the autoimmune disease is type 1 diabetes. Most preferably, the mammal (e.g., the human) that is treated according to the methods of the invention is a long-term insulin dependent diabetic.

The methods of the invention involve the administration of a CD21 agonist that may be a polypeptide (e.g., a receptor, a ligand, or an aptamer), an antibody or antigen-binding fragment thereof (e.g., a CD21 agonist antibody), a nucleic acid molecule (e.g., a gp350/220-encoding or CD21 -binding nucleic acid molecule or a nucleic acid molecule that binds to CD21 or that encodes a protein that binds CD21 ), or a small molecule.

For example, the methods of the invention include administering to a mammal having or being at risk of developing an autoimmune disease a composition containing a CD21 agonist that is a polypeptide having at least 90% (e.g., 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to all or a portion of the sequence of SEQ ID NO: 1 or 2, where the portion of the sequence contains at least one polypeptide having at least 90% (e.g., 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a sequence selected from any one or more of SEQ ID NO: 3 to 10. Some compositions include a CD21 agonist such as Epstein-Barr virus (EBV) envelope glycoproteins, gp350 (SEQ ID NO: 1 ) or gp220 (SEQ ID NO: 2), or fragments thereof (SEQ ID NO: 3-10) for administering to a mammal having or at risk of developing an autoimmune disease. Some methods of the invention include administering compositions containing a CD21 agonist, such as IFN-a, IFN-β, CD23, or a C3d protein or fragment thereof. The methods of the invention may also exclude the administration of a C3d protein or fragment thereof as a CD21 agonist.

The methods of the invention also include administering a CD21 agonist that is an antibody or antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof may be a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, an Fab, a humanized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen- binding fragment thereof, a monovalent antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a single-chain Fv molecule, a bispecific single chain Fv ((scFv')₂) molecule, a domain antibody, a diabody, a triabody, an affibody, a domain antibody, a SM!P, a nanobody, a Fv fragment, a Fab fragment, a F(ab')₂ molecule, or a tandem scFv (taFv) fragment. The antibody or antigen-binding fragment thereof of the invention may bind one or more epitopes within the first and/or second short consensus repeat (SCR) domains of CD21 and induce expression of TNF-a upon binding. For example, the antibody or antigen-binding fragment thereof may bind to one or more epitopes described in the following paragraph.

The CD21 agonist of the invention may be an agent (e.g., a polypeptide, an antibody or antigen- binding fragment thereof, or a small molecule) that binds one or more epitopes within the first and/or second short consensus repeat (SCR) domains of CD21 . The one or more epitopes within SCR1 and SCR2 of CD21 receptor to which a CD21 agonist of the invention may bind may contain one or more of the following amino acid residues: I9, N11 , R13, Y16, A22, R28, Y29, C31 , S32, G33, T34, R36, K41 ,

S42, K48, K50, D56, K57, Y64, K67, Y68, S70, R83, G84, R89, H90, D92, S93, A97, T100, N101 , S109, and S128. The one or more epitopes within SCR1 of CD21 receptor to which a CD21 agonist of the invention may bind may contain one or more of the following amino acid residues: R28, Y29, C31 , S32, G33, T34, R36, K41 , S42, K48, K50, D56, K57, Y64, K67, Y68, and S70. The one or more epitopes within SCR2 of CD21 receptor to which a CD21 agonist of the invention may bind may contain one or more of the following amino acid residues: D92, S93, A97, T100, N101 , S109, and S128. The one or more epitopes within SCR1 and SCR2 domains of CD21 to which a CD21 agonist of the invention may bind may contain one or more of the following amino acid residues: R13, R28, and K41. The one or more epitopes within the SCR1 and SCR2 domains of CD21 bound by a CD21 agonist may further contain R36 and R83. The one or more epitopes within SCR1 and SCR2 domains of CD21 bound by a CD21 agonist may further contain K67 and R89. The one or more epitopes within SCR1 and SCR2 domains of CD21 bound by a CD21 agonist may further contain one or more of the following amino acid residues: N1 1 , Y16, A22, S32, D42, K50, K57, Y64, Y68, and G84. The one or more epitopes within SCR1 and SCR2 domains of CD21 bound by a CD21 agonist may further contain one or more of the following amino acid residues: I9, Y29, C31 , G33, T34, D56, S70, H90, D92, S93, A97, T100, N101 , S109, and S128. In some embodiments, the one or more epitopes of CD21 to which a CD21 agonist binds do not include one or more of the following amino acid residues: I9, Y29, C31 , G33, T34, D56, S70, H90, D92, S93, A97, T100, N101 , S109, and S128.

According to some methods of the invention, binding of a CD21 agonist to CD21 receptor is inhibited by monoclonal antibody 72A1 or Fab fragment thereof (clone 72A1 | mAb 10219; EMD Millipore, Billerica, MA), which is known to competitively inhibit binding of gp350 to CD21. Accordingly, CD21 agonists can be identified by binding to monoclonal antibody 72A1 or Fab fragment thereof .

Administering a composition containing a CD21 agonist of the invention activates NF-κΒ signaling in a cell of a mammal (e.g., a human) upon binding of the CD21 agonist to the CD21 receptor directly or indirectly via expression of TNF-a in an autoreactive immune cell of the mammal (e.g., the human). Activation of the NF-κΒ pathway promotes death (e.g., apoptosis) of the autoreactive immune cell.

Treatment of an autoimmune disease according to the methods of the invention may result in at least about a 1 % decrease (e.g., at least a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, or even 100% decrease) in the severity of, or alleviation in, one or more (e.g., 1 , 2, 3, 4, or 5) symptoms of an autoimmune disease in a mammal relative to the severity of the one or more symptoms in a control mammal (e.g., a mammal with an autoimmune disease not receiving the treatment) as assessed using, e.g., a control sample (e.g., a biological sample taken from a mammal prior to the start of treatment). Treatment may result in the regeneration of targeted cells or tissue damaged by the autoimmune disease, or in a reduction in hyperglycemia, hypoglycemia, fatigue, depression, sensitivity to cold, weight gain, muscle weakness, constipation, insomnia, irritability, ketoacidosis, weight loss, weight gain, bulging eyes, blurred eyesight, muscle tremors, skin rashes, painful or swollen joints, sensitivity to the sun, loss of coordination, frequent urination, and paralysis, relative to a control mammal (e.g., a mammal with an autoimmune disease not receiving the treatment or a mammal with an autoimmune disease before the treatment with the composition containing a CD21 agonist of the invention). The efficacy of treatment of an autoimmune disease according to the present methods may also be shown by observation of at least about a 1 % increase (e.g., at least a 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,

15%, 20%, 25%, 30%, 35%, 40%, or 50% increase) in autoreactive T cell (e.g., autoreactive CD8⁺ T) cell death (e.g., apoptosis) in a mammal (e.g., a human) relative to a control mammal (e.g., a mammal with an autoimmune disease not receiving the treatment) as assessed using, e.g., a control sample (e.g., a biological sample taken from a mammal prior to the start of treatment). Treatment of an autoimmune disease according to the present methods may also result in at least about a 10% decrease (e.g., at least a 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100% decrease) in the autoantibody or autoreactive immune cell levels in a mammal (e.g., a human) relative to the autoantibody or autoreactive immune cell levels in a control mammal (e.g., a mammal with an autoimmune disease not receiving the treatment) as assessed using, e.g., a control sample (e.g., a biological sample taken from a mammal prior to the start of treatment). Treatment of an autoimmune disease may also result in at least about a 0.1 % increase (e.g., at least a 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 %, 2%, 3%, 5%, 10%, 20%, 30%, 40%, or 50% increase) in the number of regulatory T cells in a mammal (e.g., a human) relative to a control mammal (e.g., a mammal with an autoimmune disease not receiving the treatment) as assessed using, e.g., a control sample (e.g., a biological sample taken from a mammal prior to the start of treatment). In addition, the successful treatment of an autoimmune disease may provide for the regeneration of the targeted cells or tissues, or an increase (e.g., at least about a 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, or even 100% increase) in one or more (e.g., 1 , 2, 3, 4, or 5) biological activities of the targeted cells (e.g., insulin production by β islet cells in a type 1 diabetic following treatment according to the present methods) or tissue within a mammal relative to a control mammal (e.g., a mammal with an autoimmune disease not receiving the treatment) as assessed using, e.g., a control sample (e.g., a biological sample taken from a mammal prior to the start of treatment). Preferably, the mammal is a human.

A number of different methods for assessing treatment efficacy in type 1 diabetics are also known in the art. For example, successful treatment of type 1 diabetes can be assessed by observing at least about a 1 % decrease (e.g., at least a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%,

90%, or even 100% decrease) in one or more (e.g., 1 , 2, 3, 4, or 5) symptoms of type 1 diabetes, such as hyperglycemia, hypoglycemia, thirst, frequent urination, extreme hunger, weight loss, fatigue, blurred vision, and ketoacidosis (DKA). In addition, treatment of insulin-dependent diabetes desirably results in at least a 1 % increase in pancreatic β-cell number (e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, or even 100%) increase in the pancreatic β-cell number relative to a control mammal not receiving the treatment, at least about a 5% increase (e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, or even 100% increase) in insulin production, at least about a 1 % increase (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, or even 100% increase) in C-peptide levels, at least a 1 % decrease in glycated hemoglobin A1 c levels (e.g., at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or even 100%), at least about a 1 % increase (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% increase) in autoreactive CD8⁺ T cell (e.g., autoreactive insulin CD8⁺ T cell) death (e.g., apoptosis), or at least about a 0.1 % increase in the number of regulatory T cells in the mammal (at least a 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 %, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 10%, 20%, 30%, 40%, or 50% increase) relative to a control mammal (e.g., a mammal having type 1 diabetes and not receiving the treatment) as assessed using, e.g., a control sample (e.g., a biological sample taken prior to the start of treatment from a mammal having type 1 diabetes). Treatment of a mammal having type 1 diabetes also desirably results in at least about a 5% decrease (e.g., at least a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100% decrease) in the levels of anti- glutamic acid dehydrogenase (anti-GAD) auto-antibodies or the levels of anti-pancreatic beta cell-specific zinc transporter (anti-ZnT8A) auto-antibodies relative to a control mammal (e.g., a mammal having type 1 diabetes and not receiving the treatment) as assessed using, e.g., a control sample (e.g., a biological sample taken prior to the start of treatment from a mammal having type 1 diabetes). Preferably, the mammal is a human.

According to some methods of the invention, a composition containing CD21 agonist of the invention may be formulated using methods and techniques known in the art for administration by any route (e.g., a route selected from among intravenous, intradermal, parenteral, intra-arterial,

subcutaneous, intramuscular, intraorbital, topical, intraventricular, intraspinal, intraperitoneal, intranasal, intracranial, or oral routes of administration).

The methods of treatment described herein may also include the administration of one or more additional agents that are known in the art for treating autoimmune disease. These agents may include TNF-a, a TNF-a inducing substance, or a TNF-a receptor 2 agonist. For example, the administration of a composition containing a CD21 agonist of the invention, as described herein, may be administered to a mammal in conjunction with one or more additional substances/therapies that increase TNF-a concentration, expression, or activity. Non-limiting examples of TNF-a inducing substances or therapies include Bacillus Calmette-Guerin (BCG), complete Freund's Adjuvant, tissue plasminogen activator (TPA), lipopolysaccharide (LPS), interleukin-1 , interleukin-2, UV light, lymphotoxin, and cachectin.

Preferably, the additional agent is BCG. Non-limiting examples of TNF-a receptor agonists include small molecules (e.g., the small molecule agonists described in Hymowitz et al (Nature Chem. Biol. :353-354, 2005)) and antibodies that bind to TNF-a receptor 2 (e.g., clones MR2-1 , 80M2 (Cell Sciences HM2022) from Cell Sciences, Sigma T1815 (clone 22221 .31 1 ), and anti-TNF-a receptor 2 agonist antibodies described in U.S. Pat. No. 7,582,313, U.S. Pat. No. 8,017,392, and U.S. Pat. No. 8,173,129, which are incorporated herein by reference in their entirety). One or more additional agents can be formulated using methods and techniques known in the art for administration by any route (e.g., intravenous, intradermal, parenteral, intra-arterial, subcutaneous, intramuscular, intraorbital, topical, intraventricular, intraspinal, intraperitoneal, intranasal, intracranial, or oral routes of administration). The route of administration of one or more additional agent is not constrained by the preferred route of administration of the composition containing a CD21 agonist of the invention. Preferably, the additional agent is BCG formulated for intradermal administration.

A composition containing a CD21 agonist of the invention can be administered to a mammal (e.g., a human) prior to the development of one or more symptoms of the autoimmune disease.

Alternatively, a composition containing a CD21 agonist of the invention can be administered to a mammal (e.g., a human) after the development of one or more symptoms of the autoimmune disease. Optionally, one or more additional agent(s) described supra are administered to the mammal (e.g., a human). The symptoms of autoimmune diseases include: hyperglycemia, hypoglycemia, fatigue, depression, sensitivity to cold, weight gain, muscle weakness, constipation, insomnia, irritability, ketoacidosis, weight loss, weight gain, bulging eyes, blurred eyesight, muscle tremors, skin rashes, painful or swollen joints, sensitivity to the sun, loss of coordination, frequent urination, and paralysis. Preferably, the autoimmune disease is type 1 diabetes. The symptoms of type 1 diabetes are one or more of hyperglycemia, hypoglycemia, thirst, frequent urination, extreme hunger, weight loss, fatigue, blurred vision, and ketoacidosis (DKA).

A composition containing a CD21 agonist of the invention can be administered to a mammal (e.g., a human) one or more times daily, weekly, monthly, or yearly. For example, the CD21 agonist of the invention can be administered to a mammal (e.g., a human) twice daily, biweekly, bi-annually, tri- annually, or quarterly. Optionally, one or more additional agents described supra may also be administered to the mammal (e.g., a human) at the same or a different frequency, and/or in the same or a different composition.

CD21 Agonists for Use in Treating Autoimmune Disease

A CD21 agonist of the invention may be selected from a polypeptide, an antibody or antigen- binding fragment thereof, a nucleic acid molecule, and a small molecule. The CD21 agonist is one that binds to CD21 receptor and induces TNF-a expression (e.g., at least 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%», 90%, 100%, 150%, 200%, 300%, 400%, 500%, 700%, 1000%), 5000%, 10000%, or higher increase in the expression of TNF-a protein in a cell treated with CD21 agonist relative to the expression of TNF-a protein in an untreated cell).

Examples of CD21 agonists of the invention include natural CD21 ligands, such as EBV envelope gp350/220 protein, C3d, CD23 receptor, IFN-a, and IFN-β. Other CD21 agonists include agents that are capable of binding to one or more epitopes within CD21 that are known to be bound by natural CD21 agonist ligands (e.g., ligands that bind one or more epitopes in SCR1 and/or SCR2). Investigations of CD21-gp350/220 and CD21-IFN-a binding have identified a number of specific amino acid residues that are contained within one or more epitopes of CD21 involved in CD21 agonism. These residues within CD21 (using the numbering in SEQ ID NO: 11 ) include one or more of N11 , R13, Y16, A22, R28, S32, R36, K41 , D42, K50_: K57, Y64, K67, Y68, R83, G84, and R89. Amino acid residues of CD21 that are present in epitopes that are bound by multiple ligands (e.g., gp350/220 and IFN-a) include one or more of R13, R28, Y68, R83, G84, and R89. One or more of the following amino acid residues of CD21 are present in epitopes that are bound by gp350/220: R13, S15, R28, R36, K41 , 56, K67, R83, R89, and, to a lesser extent, K50 and K57 (see, e.g., Young et al. J. Biol C em. 282:36614-36625, 2007). One or more of the amino acid residues that define epitope(s) within CD21 that are involved in IFN-a binding to CD21 are: R13, Y16, R28, S42, K48, K50, Y68, R83, G84, and R89. One or more of the amino acid residues within CD21 that are contained within one or more epitopes of CD21 receptor binding by C3d are: I9, R13, Y16, A22, R28, Y29, C31 , S32, G33, T34, K48, D56, K57, Y68, S70, R83, G84, R89, H90, D92, S93, A97, T100, N101 , S109, and S128. CD21 agonists of the invention also include agents that do not bind to epitopes within CD21 defined by one or more of the following amino acid residues within CD21 that are not involved in CD21 binding by either gp350/220 or IFN-a are: I9, Y29, C31 , G33, T34, D56, S70, H90, D92, S93, A97, T100, N101 , S109, and S128. Thus, CD21 agonists of the invention also include agents that are capable of binding to one or more epitopes within CD21 (e.g., within SCR1 and/or SCR2 of CD21 ) that are defined by one or more of the amino acid residues discussed above.

CD21 agonist polypeptides

The CD21 agonist may be a polypeptide (e.g., a receptor, a ligand, or an aptamer). The polypeptide (e.g., a ligand) may originate from a natural polypeptide of a DNA or RNA virus (e.g., EBV, such as EBV gp350/220 or muteins or fragments thereof) and may mediate (either directly or indirectly) an increase in the expression of TNF-a, e.g., at least 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 700%, 1000%, 5000%, 10000%, or higher increase in the expression of TNF-a protein or an mRNA encoding TNF-a protein in a cell treated with CD21 agonist relative to the expression of TNF-a protein or an mRNA encoding TNF-a protein in a cell untreated with CD21 agonist) or the activity of TNF-a (e.g., an increase in the activation of a TNF-a receptor (e.g., TNF-a receptor 2), a TNF-a signaling cascade, or an NF-KB signaling cascade, relative to a cell untreated with CD21 agonist) in a mammalian cell (in vitro or in vivo). Preferably, the virus is Epstein-Barr virus (EBV). Preferably, the ligand is EBV envelope gp350/220 glycoprotein or a mutein or fragment thereof.

CD21 agonist polypeptides of the invention exhibit at least 90% or more (e.g., 91 %, 92%, 93%,

94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a naturally-occurring EBV gp350/220 polypeptide having an amino acid sequence of SEQ ID NO: 1 or 2, an EBV gp350/220 polypeptide fragment having an amino acid sequence of any one of SEQ ID NOs: 3 to 10, or a polypeptide that includes combinations of the sequences of SEQ ID NOs: 3 to 10), and/or may differ from a naturally- occurring EBV gp350/220 polypeptide or a fragment thereof by having one or more conservative amino acid substitutions within the sequences defined by SEQ ID NOs: 3 to 10 or one or more conservative or non-conservative amino acid substitutions, deletions, or additions in regions of an EBV gp350/220 polypeptide outside the sequences defined by SEQ ID NOs: 3 to 10. EBV EBV gp350/220 polypeptide or fragment thereof that contains one or more mutations (e.g., amino acid substitutions, deletions, or additions) retains the ability to bind a CD21 receptor and induce TNF-a expression, although other biological activities of the gp350/220 protein or fragment threof may be absent or reduced. CD21 agonist polypeptides of the invention may also include amino acid substitutions, deletions, or additions relative to the naturally-occurring polypeptide that further stabilize or increase the half-life of the CD21 agonist polypeptide (e.g., the addition of an Fc domain to the polypeptide (e.g., at the N- or C-terminus)).

Mutations in the polypeptide may be made in specific domains of the CD21 agonist polypeptide or may be made at amino acid positions that are conserved or at amino acid positions that are not conserved in the CD21 agonist polypeptide sequence.

In certain embodiments, the CD21 agonist polypeptide contains an amino acid sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to all or a portion of the sequence of SEQ ID NO: 1 or 2.

In certain embodiments, the polypeptide of the invention contains an amino acid sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NO: 3 to 10 (or to a peptide that includes combinations of one or more of these sequences).

Investigations studying the binding of gp350/220 protein to CD21 receptor have been used previously to uncover key regions within gp350/220 that are responsible for the binding and physiological responses (Beisel et a!., J. Virol. 54:665-674, 1985; Nemerow et al., J. Virol. 61 :1416-1420, 1987;

Nemerow et al. Cell 56:369-377, 1989; Urquiza et al., J. Biol. Chem. 280:35598-35605, 2005; all of these documents are hereby incorporated by reference). Beisel et al. disclose peptides having a sequence of SEQ ID NO: 1 or 2, where the former sequence corresponds to that of gp350 and the latter corresponds to that of gp220. Proteins gp350 and gp220 are encoded by the same gene and constitute splicing variants, each of which acts as a CD21 agonist. The peptide having a sequence of SEQ ID NO: 3 (EDPGFFNVE) binds to purified CD21 and to CD21 ⁺ but not to CD21 ^" B- and T-lymphoblastoid cell lines (SEQ ID NO: 3 corresponds to the sequence between residues 21 and 29 of SEQ ID NO: 1 or 2). The peptide having a sequence of SEQ ID NO: 3 coupled to bovine serum albumin (BSA) inhibits CD21 binding to EBV and gp350/220 (SEQ ID NO: 1 or 2). Other peptides, such as those having the sequence of SEQ ID NOs: 4, 5, or 6, bind to CD21 , inhibit CD21 binding to EBV, bind to peripheral blood lymphocytes (CD21 ⁺ cells) to induce synthesis of interleukin-6 (IL-6) in these cells, and inhibit binding of mAb 72A1 to EBV (SEQ ID NO: 4 corresponds to the sequence between residues 142 and 161 of SEQ ID NO: 1 or 2; SEQ ID NO: 5 corresponds to the sequence between residues 282 and 301 ; SEQ ID NO: 6 corresponds to the sequence between residues 822 and 841 ). As IL-6 is upregulated through activation of NF-KB signaling, increased synthesis of IL-6 in a cell is indicative of NF-κΒ signaling activation. Thus, a polypeptide that includes the sequence of any one or more of SEQ ID NOs: 3 to 6 (e.g., a polypeptide that includes combinations of SEQ ID NOs: 3 to 6, such as a polypeptide that includes each of SEQ ID NOs: 3 to 6, SEQ ID NOs: 3 to 5, SEQ ID NOs: 3 and 4, SEQ ID NOs: 4 to 6, SEQ ID NOs: 5 and 6, SEQ ID NOs: 3, 4, and 6, SEQ ID NOs: 3, 5, and 6, SEQ ID NOs: 3 and 6, SEQ ID NOs: 4 and 6, SEQ ID NOs: 4 and 5) or a polypeptide having at least 90% or more (e.g., 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity thereto can be used as a CD21 agonist according to the methods of the invention.

CD21 agonist antibodies or antigen-binding fragments thereof

The CD21 agonist may be an antibody or antigen-binding fragment thereof. According to the present invention, an antibody or antigen-binding fragment thereof may bind to one or more epitopes within the first and/or second short consensus repeat (SCR) domains of CD21 . The one or more epitopes within the first and/or second short consensus repeat (SCR) domains of CD21 that may be bound by the antibody or antigen-binding fragment thereof of the invention are those defined by the amino acid residues described previously (e.g., one or more of 19, N1 1 , R13, Y16, A22, R28, Y29, C31 , S32, G33, T34, R36, K41 , S42, K48, K50, D56, K57, Y64, K67, Y68, S70, R83, G84, R89, H90, D92, S93, A97, T100, N101 , S109, and S128 (using the numbering of SEQ ID NO: 1 1 )).

The antibody or antigen-binding fragment thereof of the invention may be a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, an Fab, a humanized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a monovalent antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a single-chain Fv molecule, a bispecific single chain Fv ((scFv')₂) molecule, a domain antibody, a diabody, a triabody, an affibody, a domain antibody, a SMIP, a nanobody, a Fv fragment, a Fab fragment, a F(ab')₂ molecule, or a tandem scFv (taFv) fragment.

According to the present invention, a CD21 agonist antibody or antigen-binding fragment thereof is one that binds to CD21 (e.g., the CD21 antibody may bind to one or more of epitopes within CD21 defined as discussed above), thereby promoting the activation of NF- Β signaling in an autoreactive immune cell (e.g., by the induction of TNF-a expression). The CD21 agonist antibodies of the invention or antigen-binding fragments thereof exhibit a specific binding affinity for CD21 in the range between 10⁶- 10¹³ M^"1 (e.g., 10⁷-10¹³ M^~1, 10⁸-10¹³ M^" , 10⁹-10^{13 "1}, or 10¹⁰-10¹³ M^"1). The CD21 agonist antibodies of the invention or antigen-binding fragments thereof exhibit a specific binding affinity for CD21 of at least 10⁶, 10⁷, 10^s, 10⁹, or 10¹⁰ M^" or greater (e.g., up to, e.g., 10^{13 ~1} ). Examples of known anti-CD21 antibodies include: anti-CD21 2G9 antibody (clone 2G9, Novus Biologicals, Littleton, CO), anti-CD21 LT21 antibody (clone LT21 , BioLegend, Inc., San Diego, CA), and anti-CD21 BU33 antibody (clone

BU33, Ancell Corporation, Bayport, MN).

Many of the CD21 agonist antibodies or antigen-binding fragments thereof described herein can undergo non-critical amino-acid substitutions, additions or deletions in both the variable and constant regions without loss of CD21 binding specificity or effector functions, or without intolerable reduction of specific CD21 binding affinity (e.g., above about 10^s, 10⁷, 10⁸, 10⁹, or 10¹⁰ M^"1). Usually, a CD21 agonist antibody or antigen-binding fragment thereof incorporating such alterations exhibits substantial sequence identity to a reference CD21 agonist antibody or antigen-binding fragment thereof from which it is derived. Occasionally, a mutated CD21 agonist antibody or antibody antigen-binding fragment thereof can be selected having the same specificity and increased affinity compared with a reference antibody or antibody fragment from which it was derived. Phage-display technology offers powerful techniques for selecting such antibodies. See, e.g., Dower et al., WO 91/17271 ; McCafferty et al., WO 92/01047; and Huse, WO 92/06204, which are incorporated herein by reference in their entirety.

CD21 agonist nucleic acid molecules

The CD21 agonist of the invention may also be a nucleic acid molecule. For example, the CD21 agonist nucleic acid molecule may bind to CD21 receptor or encode a polypeptide of the invention (e.g., a polypeptide having the amino acid sequence of any one or more of SEQ ID NOs: 1 to 10 or a polypeptide having a sequence that is at least 90% (e.g., 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identical thereto), thereby inducing the expression of TNF-a.

CD21 agonist nucleic acid molecule(s), either directly or indirectly (e.g., by encoding a CD21 agonist polypeptide) exhibit the ability to increase TNF-a synthesis in cells, to activate NF-κΒ signaling, to increase autoreactive immune cell (e.g., autoreactive T cell, such as autoreactive CD8⁺ T cell) death in a tissue relative to the level of autoreactive immune cell death in a tissue that is not treated with the CD21 agonist nucleic molecule(s), or to increase the number of regulatory T cells relative to the number of regulatory T cells in a tissue that is not treated with the CD21 agonist nucleic molecule(s).

CD21 agonist nucleic acid molecule(s) directly binding CD21 or encoding one or more CD21 agonist polypeptide(s) of the invention may be cloned into a vector that, optionally, is operably linked to a control sequence which is capable of providing for the expression of the encoded polypeptide by the host cell, e g., an expression vector. The term "operably linked" refers to a juxtaposition wherein the

components described are in a relationship permitting them to function in their intended manner. A regulatory sequence, such as a promoter, "operably linked" to a coding sequence is positioned is such a way that the expression of the coding sequence is achieved under conditions compatible with the regulatory sequence.

CD21 agonist small molecules

The invention also features small molecule CD21 agonists that bind to CD21 and activate NF-KB signaling in an autoreactive immune cell (e.g., by inducing synthesis of TNF-a). A CD21 agonist small molecule of the invention may be identified according to methods of high throughput screening (HTS) of candidate small molecule agents for their ability to bind CD21 , particularly, one or more epitope(s) within SCR1 and/or SCR2 of CD21 that is defined by one or more of the amino acid residues discussed above. Candidate CD21 agonist small molecules can also be screened for their ability to increase TNF-a synthesis in cells, to activate NF-κΒ signaling, to increase autoreactive immune cell (e.g., autoreactive T cell, such as autoreactive CD8⁺ T cell) death in a tissue relative to the level of autoreactive immune cell death in a tissue that is not treated with the small molecule, or to increase the number of regulatory T cells relative to the number of regulatory T cells in a tissue that is not treated with the small molecule. In general, candidate CD21 agonist small molecules should bind target sequences within CD21 with a specific binding affinity in excess of 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M^" or greater (e.g., up to, e.g., 10¹³ M^"1 ), or falling within the range between 10⁶-10¹³ M^"1 (e.g., 10⁷-10¹³ M^"1, 10⁸-10¹³ M^"1, 10⁹-10¹³ M^"1, or 10¹⁰-10¹³ M^"1).

Peptides, polypeptides, phages, or fusion molecules, or libraries thereof, encoding at least a fragment containing the SCR1 and/or SCR2 domains of CD21 , may be used in HTS binding assays and methods to identify CD21 agonist small molecules of the invention. In general, fluorescence and luminescence based assays (e.g., ELISA, colorimetric assays) are used to measure binding affinities of candidate small molecules contacted against single or multiple target compounds that contain at least the SCR1 and/or SCR2 domains of CD21 . Upon the identification of a candidate small molecule from a first screening process, it may be useful to further scrutinize the binding affinity and ability of the candidate by means of a second, different HTS assay. This could be accomplished, for example, by contacting the promising candidate small molecule with variants of a fragment containing the SCR1 and/or SCR2 domains of CD21. A discussion of HTS methodologies is found in Verkman (Am. J. Physiol. Cell Physiol. 286, C465-C474, 2004) and Dove (Nat Biotechnol 21 :859-864, 2003). Examples of HTS screening methods for the discovery of useful small molecule agents are found in, e.g., U.S. Patent Nos. 7,279,286 and 7,276,346, and are incorporated by reference herein in their entirety.

Candidate small molecules that have undergone HTS screening may be further modified to empirically improve binding affinities to CD21 or other properties directed to increased TNF-a expression according to the design considerations discussed below.

CD21 agonist small molecules of the invention can also be generated according to the principles of rational design. Computer modeling technology allows visualization of the three-dimensional atomic structure of a selected molecule and the design of new compounds that can interact with CD21 receptor via one or more of the epitope(s) found in the SCR1 and SCR2 domains of CD21 receptor or other epitopes unique to CD21 receptor (that promote TNF-a expression upon binding by a candidate small molecule). The three-dimensional construct typically depends on data from x-ray crystallographic analyses or NMR imaging of the selected molecule or epitope. A computer graphics system enables prediction of how a candidate small molecule compound can bind to the target CD21 receptor or epitope therein and allows experimental manipulation of the structures of the small molecule and target protein to perfect binding specificity. A prediction of what the molecule-protein interaction will be when small changes are made in one or both can be determined by using molecular mechanics software and computationally intensive computers. An example of a molecular modeling system described generally above includes the CHARMm and QUANTA programs (Polygen Corporation, Waltham, Mass.).

CHARMm performs the energy minimization and molecular dynamics functions, while QUANTA performs the construction, graphic modeling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules that intact with each other. Another molecular modeling program that can be used to identify small molecules for use in the methods of the invention is DOCK (Kuntz Laboratory, UCSF).

The conformation and structural properties of CD21 receptor are known to those skilled in the art (see, e.g., US 6,820,011 , WO 2011/143637, and Young et al, J. Biol. Chem. 282:36614-36625, 2007). This knowledge can be used to design small molecules capable of binding to, and promoting activation of, a CD21 receptor.

CD21 agonist small molecules of the invention can be organic or inorganic compounds, and even nucleic acids. Specific binding to a CD21 receptor or epitope therein can be achieved by including chemical groups having the correct spatial location and charge in the small molecule. In a preferred embodiment, compounds are designed with hydrogen bond donor and acceptor sites arranged to be complementary to the CD21 receptor epitope(s) known to be involved in the induction of TNF-a expression. An agent is formed with chemical side groups ordered to yield the correct spatial arrangement of hydrogen bond acceptors and donors when the agent is in a specific conformation induced and stabilized by binding to the CD21 epitope(s). Additional binding forces such as ionic bonds and Van der Waals interactions can also be considered when synthesizing a small molecule of the invention. The likelihood of forming the desired conformation can be refined and/or optimized using molecular computational programs.

Organic compounds can be designed to be rigid, or to present hydrogen bonding groups on edge or plane, which can interact with complementary sites. Rebek (Science 235, 1478-1484, 1987) and Rebek er a/. (J Am Chem Soc 109, 2426-2431 , 1987) have summarized these approaches and the mechanisms involved in binding of compounds to regions of proteins.

Synthetic methods can be used by one skilled in the art to make small molecules that interact with functional groups found in the SCR1 and/or SCR2 domains of CD21 . Methods of preparing CD21 agonists: polypeptides and

antibodies or antigen-binding fragments thereof

Methods for the expression and purification of recombinant proteins, such as polypeptides having at least 90% or more sequence identity to a naturally-occurring polypeptide (e.g., EBV gp350/220 glycoprotein, IFN-a, IFN-β, or CD23 receptor) or a fragment thereof, are known in the art. For example, expression of the recombinant protein may be performed in competent bacterial or yeast strains or in mammalian cells (e.g., Chinese hamster ovary cells), or expression may be performed in transgenic mammals (e.g., cows or goats) with the recombinant protein expressed in, e.g., the serum or milk of the mammal. Purification of the recombinant proteins may be performed using standard techniques known in the art, including, but not limited to precipitation, size exclusion, and/or column chromatography methods, and may also include a step of affinity chromatography when the recombinant protein has been designed to contain an affinity moiety (e.g., a His₆ tag or streptavidin tag). Desirably, the one or more (e.g., 1 , 2, 3, 4, or 5 or more) polypeptides or the nucleic acid encoding one or more (e.g., 1 , 2, 3, 4, or 5 or more) polypeptides is/are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100% pure from other cellular components (e.g., other cellular proteins or nucleic acids).

The production of non-human monoclonal antibodies, e.g., murine, lagomorpha, and equine, is well known and can be accomplished by, for example, immunizing an animal with a preparation containing cells bearing a CD21 receptor or a fragment thereof (e.g., a fragment containing a portion or all of the CD21 SCR1 and/or SCR2 domains or a fragment that includes one or more of the epitopes within these domains as noted above) or an isolated CD21 receptor or a fragment thereof, such as an extracellular domain (e.g., the SCR1 and/or SCR2 domains). Antibody-producing cells obtained from the immunized animals are immortalized and screened, or screened first for the production of antibody which binds to a CD21 receptor and induces TNF-ct expression, and then immortalized. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Pubs., N.Y., 1988. Alternatively, substantially monospecific antibody populations can be produced by chromatographic purification of polyclonal sera. The production of human and humanized antibodies or antigen-binding fragments thereof can be achieved as follows. Suitable human antibody sequences are identified by alignments of the amino acid sequences of the murine (e.g., lagomorpha, equine) variable regions with the sequences of known human antibodies. The comparison may be performed separately for heavy and light chains but the principles are similar for each. Methods of preparing chimeric and humanized antibodies and antibody fragments are described in, e.g., U.S. Pat. Nos. 4,816,567,

5,530,101 , 5,622,701 , 5,800,815, 5,874,540, 5,914,1 10, 5,928,904, 6,210,670, 6,677,436, and 7,067,313 and U.S. Pat. Application Nos. 2002/0031508, 2004/026531 1 , and 2005/0226876. Preparation of an antibody or antigen-binding fragments thereof is further described in, e.g., U.S. Pat. Nos. 6,331 ,415, 6,818,216, and 7,067,313.

Pharmaceutical Compositions of the Invention

The compositions of the invention (e.g., compositions containing a CD21 agonist or compositions that include a CD21 agonist and an additional agent, such as TNF-a, a TNF-a inducing substance, or a TNF-a receptor 2 agonist) may be formulated using any method known in the art. For eample, the composition may be formulated for intravenous, intradermal, parenteral, intra-arterial, subcutaneous, intramuscular, intraorbital, intraventricular, topical, intraspinal, intraperitoneal, intranasal, intracranial, or oral administration. The compositions of the invention may include one or more (e.g., 1 , 2, 3, 4, or 5 or more) CD21 agonists of the invention.

Compositions of the invention that contain a CD21 agonist (e.g., a polypeptide, an antibody or antigen-binding fragment thereof, a nucleic acid molecule, or a small molecule), as described above, may also contain TNF-a, one or more (e.g., 1 , 2, 3, 4, or 5 or more) TNF-a receptor 2 agonists, and/or one or more (e.g., 1 , 2, 3, 4, or 5 or more) TNF-a inducing substances. A TNF-a receptor 2 agonist is a molecule (e.g., a small molecule, a polypeptide, or an antibody) that binds and/or activates TNF-a receptor 2. Non-limiting examples of TNF-a receptor agonists include small molecules (e.g., the small molecule agonists described in Hymowitz et al (Nature Chem. Biol. 1 :353-354, 2005)) and antibodies that bind to TNF-a receptor 2 (e.g., clones MR2-1 , 80M2 (Cell Sciences H 2022) from Cell Sciences, Sigma T 815 (clone 22221 .31 1 ), and anti-TNF-a receptor 2 agonist antibodies described in U.S. Pat. No.

7,582,313, U.S. Pat. No. 8,017,392, and U.S. Pat. No. 8,173,129, which are incorporated herein by reference in their entirety). Non-limiting examples of TNF-a inducing substances include BCG, complete Freund's adjuvant, tissue plasminogen factor, lipopolysaccharide (LPS), interleukin-1 , interleukin-2, lymphotoxin, and cachectin.

The compositions may be administered to a mammal (e.g., a human) prior to the development of symptoms of the autoimmune disease (i.e., an asymptomatic mammal) or the compositions may be administered to the patient after diagnosis with an autoimmune disease or after presentation with one or more (e.g., 1 , 2, 3, 4, or 5) symptoms of an autoimmune disease (i.e., in a patient with short-term disease (new-onset or recent diagnosis of autoimmune disease), e.g., diagnosis or development of one or more symptoms of an autoimmune disease within 2 weeks, 1 month, two months, three months, four months, six months, one year, or two years; or long-term or established autoimmune disease, e.g., a mammal diagnosed with or having one or more symptoms of an autoimmune disease over two years, five years, ten years, fifteen years, twenty years, twenty-five years, thirty years, thirty-five years, or forty years).

The compositions may be administered to a mammal (e.g., a human) in one or more doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more doses). If more than one dose is administered, the doses may be administered via the same mode of administration (e.g., intravenous or intradermal administration) or by different modes of administration (e.g., intravenous and intramuscular administration). The mammal may also be administered different doses at different times. For example, the mammal may be administered a higher initial dose and lower subsequent doses over the course of treatment or vice versa.

A dose of the composition may be administered daily, weekly, monthly, or yearly. For example, a dose of the composition may be administered twice daily, biweekly, bi-annually, tri-annually, or quarterly. The dose of the composition may be determined by a skilled physician upon consideration of a subject's clinical symptoms and/or physical condition (e.g., weight, sex, height, and severity of the autoimmune disease). The composition may be administered by intravenous, intradermal, parenteral, intra-arterial, subcutaneous, intramuscular, intraorbital, topical, intraventricular, intraspinal, intraperitoneal, intranasal, intracranial, or oral administration.

The compositions of the invention may be prepared in a pharmaceutically acceptable carrier or excipient. Such suitable carriers or exipients may be selected from, for example, water, phosphate- buffered saline (PBS), acetate-buffered saline (ABS), Ringer's solution, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, a composition for administration to a mammal can contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, or pH buffering agents that enhance the effectiveness of the composition. The compositions of the invention may also be prepared in any acceptable salt formulation.

Nucleic acid molecule(s) encoding a polypeptide of the invention may be administered by means of specialized delivery vectors (e.g., using gene therapy). Gene therapy methods are discussed in Verme et al. (Nature 389:239-242, 1997). Both viral and non-viral vector systems can be used. The vectors may be, for example, plasmids, artificial chromosomes (e.g., bacterial, mammalian, or yeast artificial chromosomes), virus or phage vectors provided with a origin of replication, and optionally, a promoter for the expression of the nucleic acid encoding the polypeptide and optionally, a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example, an ampicillin or kanamycin resistance gene in the case of a bacterial plasmid or a resistance gene for a fungal vector. Vectors may be used in in vitro, for example, for the production of DNA, RNA, or the polypeptide, or may be used to transfect or transform a host cell, for example, a mammalian host cell, e.g., for the production of the polypeptide encoded by the vector. The vectors may also be adapted to be used in vivo, for example, in a method of vaccination or gene therapy.

Examples of suitable viral vectors include, retroviral, lentiviral, adenoviral, adeno-associated viral, herpes viral, including herpes simplex viral, alpha-viral, pox viral, such as Canarypox and vaccinia-viral based systems. Gene transfer techniques using these viruses are known in the art. Retrovirus vectors, for example, may be used to stably integrate the nucleic acids of the invention into the host genome. Replication-defective adenovirus vectors by contrast remain episomal and therefore allow transient expression. Vectors capable of driving expression in insect cells (e.g., baculovirus vectors), in human cells, yeast, or in bacteria may be employed in order to produce quantities of the polypeptide(s) encoded by the nucleic acids of the invention, for example, for use in subunit vaccines or in immunoassays.

A replication-deficient simian adenovirus vector may be used as a live vector. These viruses contain an E1 deletion and can be grown on cell lines that are transformed with an E1 gene. Examples of these replication-deficient simian adenovirus vectors are described in U.S. Patent No. 6,083,716 and WO 03/046124 (each of which is herein incorporated by reference). These vectors can be manipulated to insert a nucleic acid of the invention, such that the encoded polypeptide(s) may be expressed.

Promoters and other expression regulatory signals may be selected to be compatible with the host cell for which expression is designed. For example, mammalian promoters include the

metallothionein promoter, which can be induced in response to heavy metals, such as cadmium, and the β-actin promoter. Viral promoters, such as the SV40 large T antigen promoter, human cytomegalovirus (CMV), immediate early (1 E) promoter, rous sarcoma virus LTR promoter, adenovirus promoter, or a HPV promoter, particularly the HPV upstream regulatory region (URR) may also be used. All these promoters, as well as additional promoters, are well-described in the art.

Diagnosis of Autoimmune Disease

A subject may be diagnosed with an autoimmune disease by a physician using methods known in the art (e.g., molecular diagnostic tests and/or examination for the clinical symptoms of an autoimmune disease; see, e.g., U.S. Pat. Publication No. 2004/0229785). The clinical symptoms of an autoimmune disease depend on the specific tissue targeted by the autoreactive immune cells in the subject (e.g., β- islet cells in type 1 diabetes, thymocytes in Hashimoto's disease, and mucosal epithelial cells in Crohn's disease). Non-limiting examples of symptoms of autoimmune diseases include increased levels of autoantibodies, increased levels of autoreactive immune cells (autoreactive CD8⁺ T cells), loss of targeted cells or targeted tissue damage, hyperglycemia, hypoglycemia, fatigue, depression, sensitivity to cold, weight gain, muscle weakness, constipation, insomnia, irritability, weight loss, weight gain, bulging eyes, muscle tremors, skin rashes, painful or swollen joints, sensitivity to the sun, loss of coordination, and paralysis.

Specific examples of symptoms of type 1 diabetes include decreased pancreatic β-cell number, decreased insulin production, decreased C-peptide levels, increased glycated hemoglobin levels (i.e., hemoglobin A1 c levels or HbA1c levels), increased thirst, frequent urination, extreme hunger, weight loss, fatigue, blurred vision, diabetic ketoacidosis (DKA), increased insulin-specific autoreactive immune cells (autoreactive CD8⁺ T cells), decreased T regulatory cells, skin rashes, hyperglycemia, hypoglycemia, and increased levels of autoantibodies (e.g., increased levels of anti-glutamic acid dehydrogenase and anti- pancreatic beta cell-specific zinc transporter antibodies).

A subject to be treated using the methods of the invention may be identified as being at risk for the development of an autoimmune disease (e.g., having at least a 5%, 10%, 15%, 20%, 25%, 30%, 40%, or 50% increased chance of developing an autoimmune disease) using molecular genetic methods known in the art to identify genetic defects in the subject's CD8⁺ T cells or by analysis of the medical history of the subject's family.

Animal Models and Biological In Vitro Assays

for Testing the Compositions of the Invention

The efficacy or activity of the CD21 agonist-containing compositions may be tested using in vitro, in vivo, and/or animal model assays, or combinations thereof. In vitro assays may be performed to determine whether the compositions of the invention stimulate TNF-a expression (e.g., an increase in TNF-a protein or TNF-a mRNA), activate TNF-a signaling pathways, agonize TNF-a receptor 2, or activate NF- Β signaling pathways in a mammalian cell (e.g., a human cell, such as a human

autoreactive immune cell). The compositions of the invention may also be tested in vitro for their ability to induce autoreactive CD8⁺ T cell death (see, e.g., the methods described in Ban et al., Proc. Natl. Acad. Sci. 105:13644-13649, 2008). Also, the compositions of the invention can be tested in vitro for the ability to augment (e.g., increase) the number of T_reg cells, their function, or their phenotype.

The CD21 agonist-containing compositions of the invention may also be tested in vivo for their ability to induce autoreactive immune cell (autoreactive CD8⁺ T cell) death (e.g., apoptosis), to increase the number of T regulatory cells, to reduce the production of autoantibodies, and to increase the number (and/or biological activity) of the cells or tissue in a mammal targeted by the autoimmune disease or a biological activity mediated by the cells or tissue. For example, there are several animal models of autoimmune diseases available in the art, e.g., for type 1 diabetes (human CD21 transgenic mice treated with streptozotocin, see, e.g., Kulik et al (Mol. Immunol. 48:883-894, 2011 ), and NOD mouse (non-obese diabetic mouse, the Jackson Laboratory, Sacramento, CA)), for autoimmune thyroid disease and type 1 diabetes (BB rats, BioBreeding Laboratories), for lupus ((NZB x NZW)F1 , MRL/lpr mice, and NOD mice), and rheumatoid arthritis (collagen-induced arthritis in DBA 1 mice). These animal models may be used to assess the therapeutic efficacy of a CD21 agonist-containing composition of the invention to treat an autoimmune disease. As described above, the treated animals may be observed for a reduction in the severity or the alleviation of one or more symptoms of an autoimmune disease. In addition, the level of induced autoreactive immune cell death (autoreactive CD8⁺ T cell apoptosis), the level of T regulatory cells, and the levels of autoantibodies, as well as the number and biological activity of the targeted cells and tissues may be assessed in the treated animals, and compared to the values observed in control animals (e.g., control animals that do not receive treatment or using a biological sample obtained from an animal prior to treatment).

Kits

The present invention further provides kits that include a pharmaceutical composition containing a CD21 agonist of the invention, a pharmaceutically-acceptable carrier or excipient, and, optionally, an additional agent (e.g., TNF-a, a TNF-a inducing substance, or a TNF-a receptor 2 agonist) as described above; the pharmaceutical composition present in a therapeutically effective amount for treating autoimmune disease. The kits may include instructions explaining how a practitioner (e.g.. a physician, nurse, or patient) may administer the composition contained therein. Furthermore, the kits may also include additional components, such as one or more additional pharmaceutical compositions containing an additional agent (e.g., TNF-a, a TNF-a inducing substance, or a TNF-a receptor 2 agonist) as described above, instructions or administration schedules for a patient suffering from an autoimmune disease, and, optionally, a device(s) for administering the pharmaceutical composition(s) (e.g., a syringe).

The following examples are meant to illustrate the invention. They are not meant to limit the invention in any way.

EXAMPLES

Example 1. Treatment of Type I Diabetes in a Human

The efficacy of a composition containing a CD21 agonist of the invention for the treatment of type 1 diabetes can be ascertained by following the procedure described below.

The subjects can be studied using blood monitoring of the number of autoreactive T cells and/or the number of T regulatory cells, and the pancreas can also be monitored for signs of regeneration.

Although almost all immunomodulatory trials in type 1 diabetes are conducted in very-recent-onset diabetics with the aim of slowing the inevitable decline in the pancreas function with immunosuppression, the study may include subjects with longstanding type 1 diabetes (i.e., long term diabetics), on average 15 years duration, having no clinically- detectable pancreas function.

A study, using type 1 diabetics, can be performed to investigate whether administration of the composition of the invention would result in an immunomodulatory or pancreas inductive effect. In the study, each composition containing a CD21 agonist of the invention (e.g., gp350 or a fragment thereof or a CD21 agonist antibody) can be administered to a separate group of diabetics and saline can be administered to another group. The CD21 agonist composition can be administered once weekly (or at longer intervals, such as once quarterly, bi-annually, or annually) intravenously in a dosage containing 0.5-100 nmol of the CD21 agonist per kg weight of each subject. Blood can be drawn from normal control volunteers that are studied simultaneously with each diabetic sample. In both samples the T cell response can be monitored. All patients can undergo monitoring as described below.

Immune Monitoring

1 . T Cell Assays

CD4⁺ and CD8⁺ T cells can be isolated from fresh human blood within 1 .5 h of venipuncture using Dynai CD4 positive isolation kit and Dynal CD8 positive isolation kit (Invitrogen, Carlsbad, CA). This method is unique in yielding cells that are both free of magnetic particles and free of the positive selection with the antibody.

2. Detection of Autoreactive CD8⁺ T Cells in Type 1 Diabetes

Highly purified, viable and high yield CD8⁺ T cells can be utilized for tetramer staining, as previously described (Verginis et al., Proc. Natl. Acad. Sci. U.S.A. 105:3479-3484, 2008). Tetramers are diagnostic reagents that are composed of the binding region of specific HLA class I proteins with loaded peptides in the exterior binding groves. The tetramers are then made fluorescent and act as diagnostic reagents that can bind to T cells with specific reactivity to the presented peptide fragment. For detection of autoreactive T cells to insulin, tetramers to HLA *0210 insulin beta 10-18 with a fragment of

HLVEALYLV (Beckman Coulter #T02001 ) can be used. For negative control tetramers, the following tetramer reagents can be used: HLA *0201 Her-2/neu with a sequence to KIFGSLAFL (Beckman Coulter #T02001 ), a breast cancer peptide, HLA *0201 null without a non-specific peptide fragment (Beckman Coulter #T01010) and/or a tetramer to the CMV virus HLA-A *0201 CMGPP65 with a sequence of NLVPMVATV (Beckman Coulter #T01009).

Tetramer reagent staining can be conducted both after 12 h of culture at 26°C followed by 6 h at 37 °C and/or after 1 h rest at 26 °C followed by 12 h at 37 °C. The cells can be then stained with Sytox- green (MBL International Co., Woburn, MA) and/or CD8 antibodies (BD Biosciences, San Jose, CA). All cells can be stained at 4 °C in the dark for 30 minutes and then can be washed twice in Hank's buffer with 2% heat-inactivated bovine serum . On average, 100,000 highly pure CD8⁺ T cells can be analyzed to ensure clear data points and to allow for the detection of the rare autoreactive T cells. All cells can be fresh to prevent fixation artifacts and to allow for the quantification of dead versus viable cells. Cell viability can be quantified by either of two stains that fluorescently label dead cells, Sytox (MBL

International Co., Woburn, MA) or propidium iodide (PI).

3. Detection of T_REG CD4⁺ Cells in Type 1 Diabetics

Two different methods can be used for the detection of T_REG cells. T_REG cells can be detected using CD4, CD25^bright, and Foxp3 staining or with CD4, CD25^bri9ht, and CD127^low antibody staining.

Briefly, isolated CD4 positive cells can be incubated with CD4-PE-Cy5 (clone RPA-T4) and CD25-PE (clone BC96) antibodies for 20 minutes at room temperature. After washing, cells can be fixed with Foxp3 Fix/Perm solution (Biolegend) for 20 minutes at room temperature. Cells can be washed and permeabilized with Biolegend's Foxp3 Perm Buffer for 1 5 minutes at room temperature. Cells can be then stained with Foxp3 Alexa Fluor477 antibody (clone 259D) for 30 minutes. Isotype controls can be done for each sample prior to flow cytometric analysis. Alternatively, for detection of T regulatory cells, staining can also be performed with a CD4 antibody (clone RPA-T4, BD Biosciences, San Jose, CA) and an anti-human CD127 antibody (clone hll_-7R-M21 , BD Biosciences). Other methods for detecting T_REG cells are described in U.S. Serial No. 61/763,217, which is incorporated herein by reference in its entirety. Treatment with a CD21 agonist composition

1 . Effect of the CD21 agonist on Insulin Autoreactive T cells

Autoreactive T cells in diabetics show cytotoxicity against self-peptides correctly presented through HLA class I alleles. For example, peptide-specific autoreactive T cells against the insulin B chain 10-18, detected in vitro with the insulin tetramer reagent, have been identified in rare islet allograft recipients and in about 20-35% of long-term diabetic patients, when improved blood isolation methods are utilized. Insulin autoreactive T cells detected in blood are rare and found at levels of 0.22-1.5% of the CD8⁺ T cells in long-term diabetic patients (Verginis et al., Proc. Natl. Acad. Sci. U.S.A. 05:3479-3484, 2008). In culture, these insulin autoreactive cells selectively die with short exposures to added TNF. In this study, the effect of endogenous TNF elevations secondary to administration of the CD21 agonist on in vivo autoreactive T cells can be determined.

As a first step, the presence of detectable CD8⁺ T cells with reactivity to the insulin peptide fragment in the HLA-A2 (*0201 ) allele can be measured in each diabetic.

Generally, detectable numbers of insulin-specific autoreactive T cells would be expected to appear in the circulation of diabetics after administration of the CD21 agonist. The placebo-treated diabetic patients would be expected to show little if any appearance of insulin-autoreactive T cells or fluctuations in the numbers of insulin-autoreactive T cells with sequential blood samples compared to the control samples.

All insulin autoreactive T cells can be serially studied for detection in peripheral blood and can also be studied for cell viability at each monitoring point. Increased levels of circulating and released autoreactive CD8⁺ T cells might be predicted to represent dying cells from the boost in TNF levels from administration of the CD21 agonist. Overall, the augmented and detectable insulin autoreactive T cells in the circulation would be expected to be predominantly dead insulin autoreactive T cells. Thus, at least 1 % increase in the number of dead insulin autoreactive T cells would be indicative of successful treatment of the type 1 diabetic using the CD21 agonist.

2. Effect of the CD21 agonist on Induction of T_REG Cells

The levels of T_REG CD4⁺ T cells can be measured in fresh blood specimens from a diabetic and compared to a non-diabetic control sample. Two different T_REG analysis methods can be used to quantify the possible change in T_REG cells with the CD21 agonist-exposed diabetics compared to the placebo- treated diabetics, i.e., the numbers of CD4⁺, CD25⁺, and Foxp3⁺ T cells or the numbers of CD4⁺, CD25⁺, and CD127^low T cells. Using this analysis method, at least one CD21 agonist-treated diabetic would be expected to demonstrate statistically significant elevations (at least 0.1 %) in the relative numbers of T_REG cells. The placebo-treated diabetics are expected to exhibit no change in the numbers of T_REG cells measured by Foxp3 staining.

3. Effect of the CD21 agonist on Insulin Secretion

The secretion of pancreatic insulin is associated with the co-secretion of the pro-insulin fragment, i.e., the C-peptide fragment. Diabetics can be monitored for changes in fasting C-peptide levels, a marker of restored endogenous pancreatic islet activity. Diabetics would be expected to exhibit a statistically significant (at least 1 %) change upward in C-peptide levels following administration of the CD21 agonist as compared to the placebo. Example 2. In Vitro Assays of CD8⁺ T Cell Death

Assays to determine the ability of the compositions of the invention to mediate autoreactive CD8⁺ T cell death may be performed using the in vitro assay described in Ban et al. (Proc. Natl. Acad. Sci. U.S.A. 105:13644-13649, 2008). In an example of this assay, viable subpopulations of CD4⁺ and CD8⁺ T cells may be isolated from an autoimmune patient (e.g., a type 1 diabetic subject) and incubated with the CD21 agonist of the invention. The ability of the composition to elicit cell death in the CD4⁺ and CD8⁺ populations may be measured using two different cell death assays: lactate dehydrogenase (LDH) assay (necrotic cell death) and the caspase 3/7 assay (a luminescent assay of apoptosis).

Similar experiments to determine the effect of a composition of the invention on cell death in autoreactive diabetic CD8⁺ T cells to an insulin fragment may also be performed. These assays are performed by first incubating purified CD8⁺ T cells with fluorescently-labeled tetramers to HLA 0210 with an insulin beta 10-18 fragment (HLVEALYLV), washing the cells twice in Hank's buffer with 2% heat inactivated bovine serum, and isolating the labeled autoreactive CD8⁺ T cells using a flow cytometer. The isolated autoreactive CD8⁺ T cells are then treated with compositions of the invention and the amount of cell death (necrotic and/or apoptotic cell death) is measured using either a LDH assay or a caspase 3/7 assay (described supra). Compositions useful for treatment can result in an increase in necrotic or apoptotic autoreactive CD8⁺ T cell death. Suitable controls for these experiments include CD8⁺ T cells from a subject that does not have type 1 diabetes, samples of untreated autoreactive CD8⁺ T cells, or samples of autoreactive CD8⁺ T cells treated with a control polypeptide (e.g., albumin). Example 3. Mouse Model of Insulin-Dependent Diabetes

To assess the therapeutic effect of a CD21 agonist of the invention, a transgenic mouse expressing human CD21 (Kulik et al., Moi. Immunol. 48:883-894, 2011 ) may be used. Some CD21 agonists of the invention, e.g., gp350/220, are known to bind CD21 from a human but not from a mouse (see, e.g., Prota et al (Proc. Nat. Acad. Sci. USA 99:10641 -10646, 2002). The mice would be administered a dose of streptozotocin to elicit the symptoms of type 1 diabetes. The mice can be administered a composition containing gp350, gp220, a polypeptide including a sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1 to 10 that induce an elevation of at least 1 % in the level of TNF-a (e.g., the concentration of TNF-a found in the blood), IFN-a, IFN-β, CD23, or C3d. The mice can subsequently be assessed for a change in the symptoms of insulin-dependent diabetes: hyperglycemia, hypoglycemia, thirst, frequent urination, extreme hunger, weight loss, fatigue, blurred vision, and ketoacidosis (DKA). In addition, the mice can be monitored for an increase in pancreatic β-cell number, an increase in insulin production, an increase in C-peptide levels, a decrease in glycated hemoglobin A1 c levels, an increase in autoreactive CD8⁺ T cell death (e.g., apoptosis), or increase in the number of regulatory T cells. The mice can also be monitored for a decrease in the levels of autoantibodies, such as anti-glutamic acid dehydrogenase (anti-GAD) auto-antibodies and anti-pancreatic beta cell-specific zinc transporter (anti- ZnT8A) auto-antibodies. The experimental values for the treated mice can be compared relative to control mice (e.g., a transgenic mouse expressing human CD21 treated with streptozotocin receiving the composition or receiving a placebo, or a biological sample from the transgenic mouse expressing human CD21 treated with streptozotocin prior to treatment with the composition). A composition that may be used to treat a subject having type 1 diabetes would desirably result in a decrease or alleviation of one or more symptoms of insulin-dependent diabetes, an increase in pancreatic β-cell number, an increase in insulin production, an increase in C-peptide levels, a decrease in glycated hemoglobin A1c levels, an increase in autoreactive CD8⁺ T cell death (e.g., apoptosis), or an increase in the number of regulatory T cells in the treated mice compared to the control mice.

Example 4. Combination Therapy

Combination therapy can be studied following the procedure described in Example 1 , except, in addition to the CD21 agonist of the invention, an autoimmune patient, such as a diabetic, can be administered BCG intradermally, while the control group can be administered intradermal injections of saline.

1 . Effect of the Combination of the CD21 Agonist of the Invention

and BCG on Autoreactive T cells

Autoreactive T cells in autoimmune disease patients receiving treatment with the combination therapy can be studied according to the procedure described in Example 1. Thus, at least 1 % increase in the number of dead insulin autoreactive T cells (exceeding the increase observed with the composition of the invention alone) would be indicative of successful treatment of the autoimmune disease patient using the combination therapy.

2. Effect of the Combination of the CD21 Agonist of the Invention

and BCG on Induction of T_REG Cells

The levels of TREG CD4 T cells in autoimmune disease patients receiving the combination therapy can be analyzed as described in Example 1 . Using this analysis method, at least one autoimmune disease patient receiving the combination therapy is expected to demonstrate statistically significant elevations (at least 0.1 % relative to control) in the relative numbers of T_REG cells (exceeding the elevations observed in the case of the composition of the invention alone).

3. Effect of the Combination of the CD21 Agonist of the Invention

and BCG on Insulin Secretion

If the autoimmune disease patient is a diabetic, the combination therapy would be expected to produce a statistically significant change upward (at least 1 % relative to control) in C-peptide levels following administration of the composition of the invention and BCG as compared to the CD21 agonist of the invention alone.

APPENDIX: SEQUENCE LISTINGS

SEQ ID NO: 1 (amino acid sequence of gp350 protein)

1 meaallvcqy tiqslihltg edpgffnvei pefpfyptcn vctadvnvti nfdvggkkhq 61 ldldfgqltp htkavyqprg afggsenatn lfllellgag elaltmrskk lpinvttgee

121 qqvslesvdv yfqdvfgtmw chhaemqnpv ylipetvpyi kwdncnstni ta vraqgld 181 vtlplslpts aqdsnfsvkt emlgneidie cimedgeisq vlpgdnkfni tcsgyeshvp

241 sggiltstsp vatpipgtgy ayslrltprp vsrflgnnsi lyvfysgngp kasggdyciq

301 snivfsdeip asqdmptntt dityvgdnat ysvpmvtsed anspnvtvta fwa pnntet

361 dfkckwzlts gtpsgcenis gafasnrtfd itvsglgtap ktliitrtat natttthkvi 421 fskapesttt sptlnttgfa dpntttglps sthvptnlta pastgptvst advtsptpag

481 ttsgaspvtp spspwdngte skapdmtsst spvttptpna tsptpavttp tpnatsptpa

541 vttptpnats ptlgktspts avttptpnat sptlgktspt savttptpna tsptlgktsp

601 tsavttptpn atgptvgets pqanatnhtl ggtsptpvvt sqpknatsav ttgqhnitss

661 stssmslrps snpetlspst sdnstshmpl ltsahptgge nitqvtpasi sthhvstssp 721 aprpgttsqa sgpgnsstst kpgevnvtkg tppqnatspq apsgqktavp tvtstggkan

781 sttggkhttg hgartstept tdyggdsttp rprynattyl ppstssklrp rwtftsppvt

841 taqatvpvpp tsqprfsnls mlvlqwasla vltlllllvm adcafrrnls tshtyttppy 901 ddaetyv SEQ ID NO: 2 (amino acid sequence of gp220 protein)

1 meaallvcqy tiqslihltg edpgffnvei pefpfyptcn vctadvnvti nfdvggkkhq

61 ldldfgqltp htkavyqprg afggsenatn lfllellgag elaltmrskk lpinvttgee

121 qqvslesvdv yfqdvfgcmw chhaemqnpv ylipetvpyi kwdncnstni tavvraqgld

181 vtlplslpts aqdsnfsvkt emlgneidie cimedgeisq vlpgdnkfni tcsgyeshvp 241 sggiltstsp vatpipgtgy ayslrltprp vsrflgnnsi lyvfysgngp kasggdyciq

301 snivfsdeip asqdmptntt di yvgdnat ysvpmvtsed anspnvtvta fwawpnntet

361 dfkckwtlts gtpsgcenis gafasnrtfd itvsglgtap ktliitrtat natttthkvi

421 fskapesttt sptlnttgfa dpntttglps sthvptnlta pastgptvst advtsptpag

481 ttsgaspvtp spspwdngte stppqnatsp qapsgqktav ptvtstggka nsttggkhtt 541 ghgartstep ttdyggdstt prprynatty lppstssklr prwtftsppv ttacatvpvp

601 ptsqprfsnl smlvlqwasl avltlllllv madcafrrnl stshtyttpp yddaetyv

SEQ ID NO: 3 (amino acid sequence of gp350/220 fragment)

1 edpgffnve

SEQ ID NO: 4 (amino acid sequence of gp350/220 fragment)

1 hhaemqnpvy lipetvpyik

SEQ ID NO: 5 (amino acid sequence of gp350/220 fragment)

1 yslrltprpv srflgnnsil

SEQ ID NO: 6 (amino acid sequence of gp350/220 fragment)

1 pstssklrpr wtftsppvtt y SEQ ID NO: 7 (amino acid sequence of gp350/220 fragment)

1 fgqltp htkavyqprg afggsenatn lfllellgag elal^~mrskk lpinvttgee

61 qqvslesvdv yfqdvfgtmw chhaemqnpv ylipetvpyi kwdncnstni tavv

SEQ ID NO: 8 (amino acid sequence of gp350/220 fragment) 1 eshvp sggiltstsp vatpipgtgy ayslrltprp vsrflgnnsi lyvfysgngp

61 kasggdyciq snivfsdeip asqdmptntt dityvgd

SEQ ID NO: 9 (amino acid sequence of gp350/220 fragment)

1 meaallvcqy tiqslihltg edpgffnvei pefpfyptcn vctadvnvti nfdvggkkhq

61 Idldfgqltp htkavyqprg afggsenatn lfllellgag elaltrtirskk lpinvttgee

121 qqvslesvdv yfqdvfgtmw chhaemqnpv ylipetvpyi kwdncnstni tavvraqgld

181 vtlplslpts aqdsnfsykt emlgneidie cimedgeisq vlpgdnkfni tcsgyeshvp

241 sggiltstsp vatpipgtgy ayslrltprp vsrflgnnsi lyvfysgngp kasggdyciq 301 snivfsdeip asqdmptntt dityvgdnat ysvpmvtsed anspnvtvta fwawpnntet

361 dfkckwtlts g^~psgcenis gafasnrtfd itvsglgtap ktliitrtat natttthkvi

421 fakapcottt nptlnttgfa dpntttglps sthvptnlta pastgptvst

SEQ ID NO: 10 (amino acid sequence of gp350/220 fragment)

1 pstssklrpr wtftsppvtt y

SEQ ID NO: 1 1 (amino acid sequence of full-length human CD21 )

MGAAGLLGVFLALVAPGVLGISCGSPPPILNGRISYYSTPIAVGTVIRYSCSGTFRTiTGEKSLLCITKDKVDGTWDK PAPKCEYF KYSSCPEPIVPGGYKIRGSTPYRHGDSVTFACKTNFSMNGNKSVWCQANNM GPTRLPTCVSVFPLEC PALP IHNGHHTSENVGSIAPGLSVTYSCESGYLLVGEKIINCLSSGKWSAVPPTCEEARCKSLGRFPNGKVKEPPI LRVGVTANFFCDEGYRLQGPPSSRCVIAGQGVAWTKMPVCEEIFCP3PPPILNGRHIGNSLANVSYGSIVTYTCDPD P GVNF1LIGESTLRCTVDSQKTGT SGPAPRCELSTSAVQCPHPQILRGRMVSGQKDRYTYNDTVIFACMFGFTL KGS QIRCNAQGTWEPSAPVCE ECQAPPNILNGQ EDRHMVRFDPGTSIKYSCNPGYVLVGEESIQCTSEGV TPP VPQCKVAACEATGRQLLT PQHQFVRFDVN3SCGEGYKLSGSVYQECQGTIPWFMEIRLCKEITCPPPPVIYNGAHT GSSLEDFPYGTTVTYTCNPGPERGVEFSLIGESTIRCTSNDQERGTWSGPAPLCKLSLLAVQCSHVHIANGYKISGK EAPYFYNDTVTFKCYSGFTLKGSSQIRCKRDNTWDPEI PVCEKGCQPPPGLHHGRHTGGNTVFFVSGMTVDYTCDPG YLLVGNKSIHCMPSGNWSPSAPRCEETCQHVRQSLQELPAGSRVELVNTSCQDGYQLTGHAYQM3QDAENGIWFK I PLCKVIHCHPPPVIVNGKHTGMMAENFLYGNEVSYECDQGFYLLGEK CSAEVILKAVJILERAFPQCLRSLCPNPEV KHGYKLNKTHSAYSHNDIVYVDCNPGFIMNGSRVIRCHTD T VPGVPTCIK AFIGCPPPPKTPNGNHTGGNIARF SPGMSILYSCDQGYLVVGEPLLLCTHEGTWSQPAPHC EV CSSPADMDGIQKGLEPRKMYQYGAVVTLECEDGYML EGSPQSQCQSDHQWNPPLAVCRSRSLAPVLCGIAAGLILLTFLIVITLYVISKHRERNYYTDTSQKEAFHLEAREVY SVDPYNPAS

SEQ ID NO: 12 (amino acid sequence of short consensus repeat (SCR) domains )

ISCGSPPPILNGRISYYSTPIAVGTVIRYSCSGTFRLIGEKSLLGITKDKVDGTWDKPAPKCEYFNKYSSGPEPIVP GGYKIRGSTPYRHGDSVTFACKTNFS NGNKSVWGQANN WGPTRLPTCVS

Other Embodiments

All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Other embodiments are in the claims.

Claims

Claims

1. A method of treating a mammal having an autoimmune disease comprising administering to said mammal a composition comprising a CD21 agonist that induces expression of tumor necrosis factor- alpha (TNF-a) in said mammal upon binding to CD21.

2. The method of claim 1 , wherein said CD21 agonist is selected from the group consisting of a polypeptide, an antibody or antigen-binding fragment thereof, a nucleic acid molecule, and a small molecule.

3. The method of claim 2, wherein said antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, an Fab, a humanized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a monovalent antibody or antigen- binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a single-chain Fv molecule, a bispecific single chain Fv ((scFv')₂) molecule, a domain antibody, a diabody, a triabody, an affibody, a domain antibody, a SMIP, a nanobody, a Fv fragment, a Fab fragment, a F(ab')₂ molecule, and a tandem scFv (taFv) fragment.

4. The method of claim 2, wherein said polypeptide is selected from the group consisting of a receptor, a ligand, and an aptamer.

5. The method of any one of claims 1 to 4, wherein said CD21 agonist comprises a polypeptide having at least 90% sequence identity to all or a portion of the sequence of SEQ ID NO: 1 or 2.

6. The method of any one of claims 1 to 5, wherein said CD21 agonist comprises a polypeptide having at least 90% sequence identity to the sequence of any one of SEQ ID NO: 3 to 10.

7. The method of any one of claims 1 to 6, wherein said CD21 agonist comprises a polypeptide having at least 90% sequence identity to the sequence of SEQ ID NO: 1 or 2, preferably wherein said CD21 agonist comprises a polypeptide having the sequence of SEQ ID NO: 1 or 2.

8. The method of any one of claims 1 to 7, wherein binding of said CD21 agonist to said CD21 is inhibited by monoclonal antibody 72A1 or Fab fragment thereof.

9. The method of claim 1 or 2, wherein said CD21 agonist is selected from the group consisting of an IFNa, IFNp, and C3d protein.

10. The method of any one of claims 1 to 9, wherein said CD21 agonist binds to the first and second consensus repeat (SCR) domains of said CD21.

11 . The method of claim 10, wherein said CD21 agonist binds to an epitope of said CD21 comprising one or more of amino acid residues Arg13, Ser15, Arg28, Lys41 , Arg36, Arg83, Lys57, and Lys67, or a conservative substitution thereof.

12. The method of claim 0, wherein said CD21 agonist binds to an epitope of said CD21 comprising Arg residue at position 13 and Ser at position 15, preferably wherein said CD21 comprises a polypeptide having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 11.

13. The method of claim 12, wherein said epitope further comprises Arg at position 28 and Lys at position 41.

14. The method of claim 12 or 13, wherein said epitope further comprises Arg at position 36 and Arg at position 83.

15. The method of any one of claims 12 to 14, wherein said epitope further comprises Lys at position 57 and Lys at position 67.

16. The method of any one of claims 1 to 15, wherein said CD21 agonist induces activation of the NF-κΒ pathway in an autoreactive immune cell of said mammal upon binding to said CD21.

17. The method of any one of claims 1 to 16, wherein administration of said composition to said mammal induces at least a 1 % increase in autoreactive immune cell death in said mammal relative to the level of autoreactive immune cell death observed in said mammal prior to said treatment.

18. The method of claim 17, wherein said autoreactive immune cell is an autoreactive T cell.

19. The method of claim 18, wherein said autoreactive T cell is an autoreactive CD8⁺ T cell.

20. The method of any one of claims 1 to 19, wherein administration of said composition to said mammal induces at least a 1 % increase in the number of regulatory T cells in said mammal relative to the number of regulator T cells present in said mammal prior to said treatment.

21. The method of any one of claims 1 to 20, wherein administering said composition to said mammal results in at least a 1 % increase in C-peptide levels in said mammal relative to the C-peptide levels in said mammal prior to said treatment.

22. The method of any one of claims 1 to 21 , wherein said mammal is a human.

23. The method of any one of claims 1 to 22, wherein said autoimmune disease is selected from type I diabetes, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, systemic lupus erythrnatosous, ulcerative colitis, psoriatic arthritis, essential mixed cryoglobulinemia, fibromyalgia- fibromyositis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, hypothyroidism, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, juvenile arthritis, lichen planus, lupus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, Stiff-Man syndrome, Devic's disease, Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis.

24. The method of claim 23, wherein said autoimmune disease is type 1 diabetes.

25. The method of claim 24, wherein said mammal is a long-term insulin dependent diabetic.

26. The method of any one of claims 1 to 25, wherein said method further comprises administering an agent selected from the group consisting of TNF-a, TNF-a inducing substance, and TNF-a receptor 2 agonist to said mammal.

27. The method of claim 26, wherein said TNF-a inducing substance is selected from the group consisting of Bacillus Calmette-Guerin (BCG), complete Freund's adjuvant, tissue plasminogen factor, lipopolysaccharide (LPS), interleukin-1 , interleukin-2, lymphotoxin, and cachectin.

28. The method of claim 27, wherein said Bacillus Calmette-Guerin (BCG) is administered intradermally.

29. The method of claim 26, wherein said TNF-a receptor 2 agonist is selected from the group consisting of a TNF-a mutein, an anti-TNF-a receptor 2 antibody, and a small molecule.

30. The method of any one of claims 1 to 29, wherein said composition is administered to said mammal prior to the development of one or more symptoms of said autoimmune disease.

31 . The method of any one of claims 1 to 29, wherein said composition is administered to said mammal after the development of one or more symptoms of said autoimmune disease.

32. The method of any one of claims 1 to 31 , wherein said treating results in a decrease in one or more symptoms of said autoimmune disease.

33. The method of claim 32, wherein said symptoms of said autoimmune disease are selected from the group consisting of increased levels of autoantibodies, increased levels of autoreactive T cells, loss of targeted cells, hyperglycemia, hypoglycemia, fatigue, depression, sensitivity to cold, weight gain, muscle weakness, constipation, insomnia, irritability, ketoacidosis, weight loss, bulging eyes, blurred eyesight, muscle tremors, skin rashes, painful or swollen joints, sensitivity to the sun, loss of coordination, frequent urination, and paralysis.

34. The method of claim 32, wherein said autoimmune disease is type I diabetes and said one or more symptoms of said autoimmune disease are selected from the group consisting of increased levels of autoreactive T cells, hyperglycemia, hypoglycemia, fatigue, ketoacidosis, weight loss, skin rashes, blurred eyesight, and frequent urination.

35. The method of any one of claims 1 to 34, wherein said composition is administered intravenously, parenterally, topically, intra-arterially, intracranially, intradermal^, subcutaneously, intramuscularly, intraorbitally, intraventricularly, intraspinally, intraperitoneaily, intranasally, or orally.

36. The method of claim 35, wherein said composition is administered one or more times daily, weekly, monthly, or yearly.

37. The method of claim 35, wherein said composition is administered twice daily, biweekly, bi- annually, tri-annually, or quarterly.

38. A pharmaceutical composition comprising a CD21 agonist for use in treating an autoimmune disease in a mammal, wherein said CD21 agonist induces expression of tumor necrosis factor-alpha (TNF-a) in said mammal upon binding to CD21.

39. The pharmaceutical composition of claim 38 further comprising a pharmaceutically acceptable excipient.