WO2013041029A1 - Novel soluble ctla4 variants - Google Patents

Abstract

The present invention provides novel soluble CTLA4 variant compositions useful for intervening T cell mediated activities. The compositions are useful in therapies for immune system disorders such as autoimmune diseases, inflammatory disorders and transplant rejections.

Description

NOVEL SOLUBLE CTLA4 VARIANTS

FIELD OF THE INVENTION

The present invention relates to CTLA4 related compositions and uses in medicine field, particularly in therapeutics for immune system disorders.

BACKGROUND OF THE INVENTION

Cytotoxic T- Lymphocyte Antigen 4 (CTLA4 or CTLA-4; also called CD 152), a member of the immunoglobulin superfamily, is a negative regulator of T cell activities. T cells play a critical role in cell-mediated immune response to antigens. During the course of T cell activation, specific T cell receptor (TCR) interacts with corresponding MHC/peptide complex on an antigen-presenting cell (APC), followed by a costimulatory interaction between receptors on the T cell and their ligands on the APC. Among many costimulatory interactions, the ones between CD28 on T cells and its B7 ligands ~ B7-1 (CD80) and B7-2 (CD86) ~ on APCs are the most critical and best studied. Similar to CD28, CTLA4 is also expressed on T cells and binds to CD80 and CD86 on APCs. It inhibits the immune response in two principal ways: first, it competes binding to CD80/CD86 with CD28, thereby blocking costimulation; and second, it transmits a negative signal to inhibit T cell activation directly.

The CTLA4 protein comprises a V-like extracellular domain (ECD), a transmembrane domain and a cytoplasmic domain. Human and murine CTLA4 proteins show 76% homology. There are two CTLA4 isoforms from alternative splicing: the full length molecule CTLA4-TM has 223 amino acids and is expressed on the surface of T cells, whereas the short, soluble splice variant sCTLA4 has 137 amino acids and is preferentially expressed in nonactivated T cells with a deleted transmembrane and partial cytoplasmic segments.

Because of CTLA4's important role in regulating the costimulatory interaction via binding to CD80 and CD86, CTLA4 agonists have been developed to antagonize CD80/CD86 activities, thereby inhibiting undesirable immune responses in situations such as autoimmune diseases and transplantation.

Abatacept (Orencia®), is a CTLA4- Ig immunoadhesin consisting of the extracellular binding domain of CTLA4 linked to the Fc domain of a human IgG. Bluestone et al., 2006, Immunity 24:233-238. Although abatacept was approved for the treatment of rheumatoid arthritis (RA) and in clinical trials for a number of other autoimmune indications, its binding affinity to B7 ligands, particularly CD86, is not ideal, and the drug is not effective for some indications such as immune tolerance in transplantation. Kirk et al., 1997, Proc Natl Acad Sci USA 94:8789-8794; Levisetti et al, 1997, J Immunol 159:5187-5191. Belatacept, a second generation of abatacept with two substitutions A29Y and L104E in CTLA4 sequence, has recently been approved by the US FDA for prevention of acute rejection in kidney transplant patients. Vincenti et al. (2005), New Engl. J. Med. 353:770-781. Other CTLA-Ig fusion proteins with additional sequence variations have also been described. See, for example, WO

201 1103584.

The present invention provides soluble CTLA4 variants and fusion proteins thereof with improved binding affinities to CD80 and CD86. Also provided are methods of using the novel compositions in treating immune conditions or disorders.

SUMMARY OF THE INVENTION

The present invention is based in part on the generation of novel soluble CTLA4 variants having a surprisingly high binding affinity to CTLA4 ligands CD80 and CD86. Accordingly, the present invention provides soluble CTLA4 variant compositions comprising the extracellular domain (ECD) of a human CTLA4, wherein the binding affinity of the composition to a CTLA4 ligand has a Kd value of less than InM. In one aspect of the invention, the CTLA4 ligand is CD80 (B7-1) or CD86 (B7-2) that is preferably bound on the surface of immune cells such as antigen presenting cells (APCs). In one aspect, the ECD of the human CTLA4 in the soluble CTLA4 variant of the invention has an amino acid sequence as shown below:

KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQV NLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSD (SEQ ID NO:1 )

In one aspect, the soluble CTLA4 variant of the invention is an immunoadhesin comprsing as a first domain the ECD of human CTLA4 and as a second domain the Fc region of an immunoglobulin, wherein the two domains are linked directly or via a linker sequence. In one aspect, the linker is a short peptide sequence having about 1-50 amin acid residues, more preferably 3-30 amino acid residues. The linker sequence can be enriched with glysine (G) residues. The Fc region can be a native Fc region from an immunoglobulin molecule such as an IgG including any and all of its subclasses IgGl- IgG4. In one aspect, the CTLA4-Fc irnmunoadhesin comprises the ECD of human CTLA4 fused via a linker to the Fc region of IgG2. As an example, the soluble CTLA4 variant of the invention is the molecule IGM001 , which has an amino acid sequence according to SEQ ID NO: 2 shown below (linker sequence is underlined):

KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQV NLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDGGGGAGGGGVECPPCPAPPVAGPSVFLFP PKPKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRWSVLTVVHQDWLNGKEYK CKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:2)

The present invention further provides nucleic acid molecules comprising nucleotide sequences encoding the soluble CTLA4 variants as described above. More specifically, the invention provides a DNA molecule having a nucleotide sequence according to SEQ ID NO: 3 below:

ATG GAATG GAG CTGG GTCTTTCTCTTCTTCCTGTCAGTAACG ACTG GTGTCCACTCCAAAG CAATG CACGTGG C

CCAGCCTGCTGTGGTACTGGCCAGCAGCCGAGGCATCGCCAGCTTTGTGTGTGAGTATGCATCTCCAGGCAAA

GCCACTGAGGTCCGGGTGACAGTGCTTCGGCAGGCTGACAGCCAGGTGACTGAAGTCTGTGCGGCAACCTACA

TGATGGGGAATGAGTTGACCTTCCTAGATGATTCCATCTGCACGGGCACCTCCAGTGGAAATCAAGTGAACCTC

ACTATCCAAGGACTGAGGGCCATGGACACGGGACTCTACATCTGCAAGGTGGAGCTCATGTACCCACCGCCATA

CTACCTGGGCATAGGCAACGGAACCCAGATTTATGTAATTGATCCAGAACCGTGCCCAGATTCTGACGGCGGAG

GTGGTGCAGGAGGCGGTGGAGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCC

TCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGT

GAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAG

CCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGA

ACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACC

AAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCA

GCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG

AGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTG

GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACA

CGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAG (SEQ ID NO:3)

Also provided are vectors and host cells comprising the nucleic acid molecules herein. Methods of making the soluble CTLA4 variant of the invention using such vectors and host cells are also provided.

The invention also provides uses of the soluble CTLA4 variants for therapeutic purposes, including treating T cell-associated immune conditions or disorders. Subjects suitable for treatments with the soluble CTLA4 variants include those having autoimmune diseases or those undergoing tissue or organ transplantation in need of suppressing immune rejections.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1B provide sequence informaiton relating to the soluble CTLA4-Fc immunoadhesin IGM001. 1 A showes amino acid sequences for the ECD of human CTLA4 used in IGM001 and the IGM001 itself. Linker sequence in IGM001 is underlined.

Figures 2A-2B illustrate IGM001 (labeled as CWT) binding to cells expressing CD80 (2A) or CD86 (2B). Abatacept was used for comparison.

Figures 3A-3B illustrate IGMOOl 's inhibitory effects on T cell proliferation co- stimulated by CD80 (3A) or CD86 (3B). Abatacept was used for comparison.

Figures 4A-4C illustrate IGM001 's inhibitory effects on T cell production of cytokines (4A: IFN-g; 4B: IL-2; 4C: IL-4). Abatacept was used for comparison.

DETAILED DESCRIPTION OF THE INVENTION

Definitions:

As used herein, a "soluble CTLA4 variant" refers to a molecule comprising an extracellular domain of CTLA4, or a portion thereof that binds at least one CTLA4 ligand such as CD80 or CD86, or both. The soluble CTLA4 variant preferably does not contain any portion of the TM or cytoplasmic domain of CTLA4. One preferred example of the soluble CTLA4 variant has the amino acid sequence of SEQ ID NO:2.

As used herein, a "fusion protein" is defined as one or more amino acid sequences joined together to form one contiguous polypeptide.

An "immunoadhesin," or an "Ig fusion protein," refers to a fusion protein wherein one or more amino acid sequences is operably linked to an Fc region of an immunoglobulin. In general, the fusion partner of the Fc region is a polypeptide capable of binding to a target. For example, the immunoadhesion of the present invention combines the ECD region of CTLA4 that is capable of binding to CD80 and CD86 to an Fc region of an IgG. The Fc region and its fusion partner polypeptide can be linked directly or through a linker sequence. A "linker" as used herein refers to a short amino acid sequence used to link one or more polypeptide in a fusion protein. For example, a linker can be used in an immunoadhesin of the invention to link a soluble CTLA4 polypeptide to an Fc. The linker peptide usually does not exert biological function in itself other than serving as a covalent linker for the two fusion partners in a fusion protein. Linke sequence may predominantly include the following amino acid residues: Gly, Ser, Ala, or Thr. A linker peptide usually has a length that is adequate to link two molecules in such a way that they assume the correct conformation relative to one another so that they retain the desired activity. In one embodiment, the linker is from about 1 to 50 amino acids in length, preferably about 3 to 30 amino acids in length. Useful linkers include glycine- serine polymers, glycine- alanine polymers, alanine-serine polymers, and other flexible linkers.

As used herein, an "autoimmune disease" refers to a disease or disorder arising from and/or directed against an individual's own tissues or organs, or a co-segregate or manifestation thereof, or resulting condition therefrom. Typically, various clinical and laboratory markers of autoimmune diseases may exist including, but not limited to, hypergammaglobulinemia, high levels of autoantibodies, antigen-antibody complex deposits in tissues, clinical benefit from corticosteroid or immunosuppressive treatments, and lymphoid cell aggregates in affected tissues.

An autoimmune disease can be an organ-specific disease (i.e., the immune response is specifically directed against an organ system such as the endocrine system, the hematopoietic system, the skin, the cardiopulmonary system, the gastrointestinal and liver systems, the renal system, the thyroid, the ears, the neuromuscular system, the central nervous system, etc.) or a systemic disease which can affect multiple organ systems (for example, systemic lupus erythematosus (SLE), rheumatoid arthritis, polymyositis, etc.). Exemplary diseases include autoimmune rheumatologic disorders (such as, for example, rheumatoid arthritis, Sjogren's syndrome, scleroderma, lupus such as SLE and lupus nephritis, polymyositis/dermatomyositis, cryoglobulinemia, anti-phospholipid antibody syndrome, and psoriatic arthritis), autoimmune gastrointestinal and liver disorders (such as, for example, inflammatory bowel diseases (e.g., ulcerative colitis and Crohn's disease), autoimmune gastritis and pernicious anemia, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, and celiac disease), vasculitis (such as, for example, ANCA-negative vasculitis and ANCA-associated vasculitis, including Churg-Strauss vasculitis, Wegener's granulomatosis, and microscopic polyangiitis), autoimmune neurological disorders (such as, for example, multiple sclerosis, opsoclonus myoclonus syndrome, myasthenia gravis, neuromyelitis optica, Parkinson's disease, Alzheimer's disease, and autoimmune polyneuropathies), renal disorders (such as, for example, glomerulonephritis, Goodpasture's syndrome, and Berger's disease), autoimmune dermatologic disorders (such as, for example, psoriasis, urticaria, hives, pemphigus vulgaris, bullous pemphigoid, and cutaneous lupus erythematosus), hematologic disorders (such as, for example, thrombocytopenic purpura, thrombotic thrombocytopenic purpura, post-transfusion purpura, and autoimmune hemolytic anemia), atherosclerosis, uveitis, autoimmune hearing diseases (such as, for example, inner ear disease and hearing loss), Behcet's disease, Raynaud's syndrome, organ transplant, and autoimmune endocrine disorders (such as, for example, diabetic-related autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM), Addison's disease, and autoimmune thyroid disease (e.g., Graves' disease and thyroiditis)).

Specific examples of autoimmune diseases as defined herein, which in some cases encompass those listed above, include, but are not limited to, arthritis (acute and chronic, rheumatoid arthritis including juvenile-onset rheumatoid arthritis and stages such as rheumatoid synovitis, gout or gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, menopausal arthritis, estrogen-depletion arthritis, and ankylosing spondylitis/rheumatoid spondylitis), autoimmune lymphoproliferative disease, inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails, atopy including atopic diseases such as hay fever and Job's syndrome, dermatitis including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, hives, dermatitis herpetiformis, nummular dermatitis, seborrheic dermatitis, non-specific dermatitis, primary irritant contact dermatitis, and atopic dermatitis, x- linked hyper IgM syndrome, allergic intraocular inflammatory diseases, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, myositis, polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis such as systemic sclerosis, multiple sclerosis (MS) such as spino-optical MS, primary progressive MS (PPMS), and relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, ataxic sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease (IBD) (for example, Crohn's disease, autoimmune-mediated gastrointestinal diseases, gastrointestinal inflammation, colitis such as ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, and transmural colitis, and autoimmune inflammatory bowel disease), bowel inflammation, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndrome, including adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, an autoimmune hematological disorder, graft-versus-host disease, angioedema such as hereditary angioedema, cranial nerve damage as in meningitis, herpes gestationis, pemphigoid gestationis, pruritis scroti, autoimmune premature ovarian failure, sudden hearing loss due to an autoimmune condition, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis,

encephalitis such as Rasmussen's encephalitis and limbic and/or brainstem encephalitis, uveitis, such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis, glomerulonephritis (GN) with and without nephrotic syndrome such as chronic or acute glomerulonephritis such as primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN

(MPGN), including Type I and Type II, and rapidly progressive GN (RPGN), proliferative nephritis, autoimmune polyglandular endocrine failure, balanitis including balanitis

circumscripta plasmacellularis, balanoposthitis, erythema annulare centrifugum, erythema dyschromicum perstans, eythema multiform, granuloma annulare, lichen nitidus, lichen sclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus, lichen planus, lamellar ichthyosis, epidermolytic hyperkeratosis, premalignant keratosis, pyoderma gangrenosum, allergic conditions and responses, food allergies, drug allergies, insect allergies, rare allergic disorders such as mastocytosis, allergic reaction, eczema including allergic or atopic eczema, asteatotic eczema, dyshidrotic eczema, and vesicular palmoplanar eczema, asthma such as asthma bronchiale, bronchial asthma, and auto-immune asthma, conditions involving infiltration of T cells and chronic inflammatory responses, immune reactions against foreign antigens such as fetal A-B-0 blood groups during pregnancy, chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte adhesion deficiency, lupus, including lupus nephritis, lupus cerebritis, pediatric lupus, non-renal lupus, extra-renal lupus, discoid lupus and discoid lupus erythematosus, alopecia lupus, SLE, such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and lupus erythematosus disseminatus, juvenile onset (Type I) diabetes mellitus, including pediatric IDDM, adult onset diabetes mellitus (Type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, diabetic retinopathy, diabetic nephropathy, diabetic colitis, diabetic large-artery disorder, immune responses associated with acute and delayed

hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis,

granulomatosis including lymphomatoid granulomatosis, agranulocytosis, vasculitides (including large-vessel vasculitis such as polymyalgia rheumatica and giant-cell (Takayasu's) arteritis, medium-vessel vasculitis such as Kawasaki's disease and polyarteritis nodosa/periarteritis nodosa, immunovasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis such as fibrinoid necrotizing vasculitis and systemic necrotizing vasculitis, ANCA- negative vasculitis, and ANCA-associated vasculitis such as Churg-Strauss syndrome (CSS), Wegener's granulomatosis, and microscopic polyangiitis), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia perniciosa), Addison's disease, pure red cell anemia or aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia(s), cytopenias such as pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, Alzheimer's disease, Parkinson's disease, multiple organ injury syndrome such as those secondary to septicemia, trauma or hemorrhage, antigen-antibody complex- mediated diseases, anti- glomerular basement membrane disease, anti-phospholipid antibody syndrome, motoneuritis, allergic neuritis, Behcet's disease/syndrome, Castleman's syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid or

pemphigus such as pemphigoid bullous, cicatricial (mucous membrane) pemphigoid, skin pemphigoid, pemphigus vulgaris, paraneoplastic pemphigus, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, and pemphigus erythematosus, epidermolysis bullosa acquisita, ocular inflammation, including allergic ocular inflammation such as allergic conjunctivis, linear IgA bullous disease, autoimmune- induced conjunctival inflammation, autoimmune

polyendocrinopathies, Reiter's disease or syndrome, thermal injury due to an autoimmune condition, preeclampsia, an immune complex disorder such as immune complex nephritis, antibody-mediated nephritis, neuroinflammatory disorders, polyneuropathies, chronic neuropathy such as IgM polyneuropathies or IgM-mediated neuropathy, thrombocytopenia (as developed by myocardial infarction patients, for example), including thrombotic

thrombocytopenic purpura (TTP), post-transfusion purpura (PTP), heparin-induced

thrombocytopenia, and autoimmune or immune-mediated thrombocytopenia including, for example, idiopathic thrombocytopenic purpura (ITP) including chronic or acute ITP, scleritis such as idiopathic cerato-scleritis, episcleritis, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases including thyroiditis such as autoimmune thyroiditis,

Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave's disease, Grave's eye disease (ophthalmopathy or thyroid-associated ophthalmopathy), polyglandular syndromes such as autoimmune polyglandular syndromes, for example, type I (or polyglandular endocrinopathy syndromes), paraneoplastic syndromes, including neurologic paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton- Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis such as allergic encephalomyelitis or encephalomyelitis allergica and experimental allergic encephalomyelitis (EAE), myasthenia gravis such as thymoma- associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant-cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, pneumonitis such as lymphoid interstitial pneumonitis (LIP), bronchiolitis obliterans (non- transplant) vs. NSIP, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient acantholytic dermatosis, cirrhosis such as primary biliary cirrhosis and pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac or Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia such as mixed cryoglobulinemia, amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease such as autoimmune inner ear disease (AIED), autoimmune hearing loss, polychondritis such as refractory or relapsed or relapsing polychondritis, pulmonary alveolar proteinosis, keratitis such as Cogan's syndrome/nonsyphilitic interstitial keratitis, Bell's palsy, Sweet's disease/syndrome, rosacea autoimmune, zoster-associated pain, amyloidosis, a non-cancerous lymphocytosis, a primary lymphocytosis, which includes monoclonal B cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and channelopathies of the CNS, autism, inflammatory myopathy, focal or segmental or focal segmental

glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis, chorioretinitis,

autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, Dressler's syndrome, alopecia areata, alopecia totalis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), male and female autoimmune infertility, e.g., due to anti-spermatozoan antibodies, mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, parasitic diseases such as leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, fibrosing mediastinitis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, flariasis, cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), or Fuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, SCID, acquired immune deficiency syndrome (AIDS), echovirus infection, sepsis (systemic inflammatory response syndrome (SIRS)), endotoxemia, pancreatitis, thyroxicosis, parvovirus infection, rubella virus infection, post-vaccination syndromes, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, giant-cell polymyalgia, chronic hypersensitivity pneumonitis, conjunctivitis, such as vernal catarrh, keratoconjunctivitis sicca, and epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, transplant organ reperfusion, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway/pulmonary disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders (cerebral vascular insufficiency) such as arteriosclerotic encephalopathy and

arteriosclerotic retinopathy, aspermiogenese, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica, enteritis allergica, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman- Rich's disease, sensoneural hearing loss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leucopenia, mononucleosis infectiosa, traverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica (sympathetic ophthalmitis), neonatal ophthalmitis, optic neuritis, orchitis granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma gangrenosum, Quervain's thyreoiditis, acquired spenic atrophy, non-malignant thymoma, lymphofollicular thymitis, vitiligo, toxic-shock syndrome, food poisoning, conditions involving infiltration of T cells, leukocyte-adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T- lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome, antigen- antibody complex-mediated diseases, antiglomerular basement membrane disease, autoimmune polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, autoimmune polyglandular syndromes, including polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), cardiomyopathy such as dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, allergic sinusitis, an eosinophil-related disorder such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or granulomas containing eosinophils, anaphylaxis, spondyloarthropathies, seronegative spondyloarthritides, polyendocrine

autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome, angiectasis, autoimmune disorders associated with collagen disease, rheumatism such as chronic arthrorheumatism, lymphadenitis, reduction in blood pressure response, vascular dysfunction, tissue injury, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and disease accompanying vascularization, allergic hypersensitivity disorders, glomerulonephritides, reperfusion injury, ischemic re-perfusion disorder, reperfusion injury of myocardial or other tissues, lymphomatous tracheobronchitis, inflammatory dermatoses, dermatoses with acute inflammatory components, multiple organ failure, bullous diseases, renal cortical necrosis, acute purulent meningitis or other central nervous system inflammatory disorders, ocular and orbital inflammatory disorders, granulocyte transfusion-associated syndromes, cytokine-induced toxicity, narcolepsy, acute serious inflammation, chronic intractable inflammation, pyelitis, endarterial hyperplasia, peptic ulcer, valvulitis, and endometriosis.

"Rheumatoid arthritis" (RA) refers to a chronic systemic autoimmune inflammatory disease that mainly involves the synovial membrane of multiple joints with resultant injury to the articular cartilage, resulting in joint destruction. The main presenting symptoms in RA are pain, stiffness, swelling, and/or loss of function of one or more joints.

A "subject" herein is typically a human. In certain embodiments, a subject is a non- human mammal. Exemplary non-human mammals include laboratory, domestic, pet, sport, and stock animals, e.g., mice, cats, dogs, horses, and cows. Typically, the subject is eligible for treatment, e.g., treatment of an autoimmune disorder, treatment related to a tissue transplant, or the like.

As used herein, "treatment" refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, methods and compositions of the invention are useful in attempts to delay development of a disease or disorder.

An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A "therapeutically effective amount" of a therapeutic agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the therapeutic agent are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

The term "immunosuppressive agent" as used herein for therapy refers to substances that act to suppress or mask the immune system of the mammal being treated herein. This would include substances that suppress cytokine production, down-regulate or suppress self-antigen expression, or mask the MHC antigens. Examples of such agents include 2-amino-6-aryl-5- substituted pyrimidines (see U.S. Pat. No. 4,665,077); nonsteroidal antiinflammatory drugs (NSAIDs); ganciclovir, tacrolimus, glucocorticoids such as Cortisol or aldosterone, antiinflammatory agents such as a cyclooxygenase inhibitor, a 5 -lipoxygenase inhibitor, or a leukotriene receptor antagonist; purine antagonists such as azathioprine or mycophenolate mofetil (MMF); alkylating agents such as cyclophosphamide; bromocryptine; danazol; dapsone; glutaraldehyde (which masks the MHC antigens, as described in U.S. Pat. No. 4,120,649); anti- idiotypic antibodies for MHC antigens and MHC fragments; cyclosporin A; steroids such as corticosteroids or glucocorticosteroids or glucocorticoid analogs, e.g., prednisone,

methylprednisolone, and dexamethasone; dihydrofolate reductase inhibitors such as methotrexate (oral or subcutaneous); hydroxycloroquine; sulfasalazine; leflunomide; cytokine or cytokine receptor antibodies including anti-interferon-alpha, -beta, or -gamma antibodies, anti-tumor necrosis factor-alpha antibodies (golimumab, certolizumab pegol, infliximab or adalimumab), anti-TNF-alpha immunoadhesin (etanercept), anti-tumor necrosis factor-beta antibodies, anti- interleukin-2 antibodies and anti-IL-2 receptor antibodies; anti- LFA-I antibodies, including anti- CD1 la and anti-CD 18 antibodies; heterologous anti- lymphocyte globulin; pan-T antibodies, including anti-CD3; soluble peptide containing a LFA-3 binding domain; streptokinase; TGF- beta; streptodornase; RNA or DNA from the host; FK506; RS-61443; deoxyspergualin;

rapamycin; T-cell receptor; T-cell-receptor fragments; and T-cell-receptor antibodies such as T10B9. By "greater affinity" or "improved affinity" or "enhanced affinity" or "better affinity" than a parent polypeptide, as used herein is meant that a variant binds to a ligand or receptor with a significantly higher equilibrium constant of association (KA or Ka) or lower equilibrium constant of dissociation (KD or Kd) than the parent polypeptide done under the same conditions, for example, when the amounts of variant and parent polypeptide in the binding assay are essentially the same. For example, a CTLA4 variant with improved CD80 binding affinity may display from about 1.5, 2, 5, 10, 20, 50, 75, 100 fold or more , improvement in CD80 binding affinity compared to the parent CTLA4 polypeptide, where CD80 binding affinity is determined, for example, by the binding methods disclosed herein, including but not limited to Biacore™, by one skilled in the art. CTLA4 variants may improve binding selectively to CD80 relative to CD86. Alternatively, variants may improve binding selectively to CD86 relative to CD80.

The term "glycosylation" means the attachment of oligosaccharides (carbohydrates containing two or more simple sugars linked together e.g. from two to about twelve simple sugars linked together) to a glycoprotein. The oligosaccharide side chains are typically linked to the backbone of the glycoprotein through either N- or O-linkages. The oligosaccharides of immunoadhesins disclosed herein occur generally are attached to a CH2 domain of an Fc region as N-linked oligosaccharides. "N-linked glycosylation" refers to the attachment of the carbohydrate moiety to an asparagine residue in a glycoprotein chain. The skilled artisan will recognize that, for example, each of murine lgGl , lgG2a, lgG2b and lgG3 as well as human lgGl , lgG2, lgG3, lgG4, IgA and IgD CH2 domains have a single site for N-linked

glycosylation at residue 297.

Recombination Production of the Compositions of the Invention

The compositions of the invention, such as a fusion polypeptide comprising a soluble CTLA4 variant and a Fc region of an immunoglobulin, can be produced recombinantly, using techniques and materials readily obtainable. For recombinant production of a polypeptide of the invention, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression in a host cell. DNA encoding the polypeptide of the invention is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the protein). Many vectors are available. The vector components generally include, but are not limited to, one or more of the following components: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Suitable DNA sequences for each of these components are known and widely used in the field. For example, promoters suitable for use with prokaryotic hosts include the phoA promoter, β- lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are suitable. Promoters for use in bacterial systems also will contain a Shine- Dalgarno (S.D.) sequence operably linked to the DNA encoding the polypeptide of the invention. Transcription of compositions of the invention from vectors in mammalian host cells is controlled by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and typically Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems.

Suitable host cells for cloning or expressing DNA encoding the compositions of the invention in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described herein. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g. , Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis , Pseudomonas such as P. aeruginosa, and Streptomyces. Typically, the E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli BL21(DE3), E. coli XI 776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptide of the invention-encoding vectors.

Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.

wicker amii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K . thermotolerans, and ^. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Penicilliiim, Tolypocladium, and Aspergillus hosts such as ^i. nidulans and niger.

Suitable host cells for the expression of compositions of the invention can be derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-l variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the invention, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.

Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Examples of useful mammalian host cell lines are monkey kidney CVl line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al, J. Gen Virol. 36:59 (1977)) ; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cellsADHFR (CHO, Urlaub et al, Proc. Natl. Acad. Sci. USA 77:4216 (1980)) ; mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980) ); monkey kidney cells (CVl ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al, Annals N. Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

Host cells are transformed with the above-described expression or cloning vectors for polypeptide of the invention production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

Polypeptide Purification

A polypeptide or protein of the invention may be recovered from a subject. When using recombinant techniques, a polypeptide of the invention can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. Compositions of the invention may be recovered from culture medium or from host cell lysates. If membrane -bound, it can be released from the membrane using a suitable detergent solution (e.g. Triton-X 100) or by enzymatic cleavage. Cells employed in expression of a polypeptide of the invention can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.

The following procedures are exemplary of suitable protein purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC;

chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column, DEAE, etc.);

chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG; and metal chelating columns to bind epitope-tagged forms of compositions of the invention. Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer- Verlag, New York (1982). The purification step(s) selected will depend, for example, on the nature of the production process used and the particular polypeptide of the invention produced.

Pharmaceutical Formulations

Pharmaceutical formulations of the claimed composition as described herein are prepared by mixing such composition having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX^®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized formulations are described in US Patent No. 6,267,958. Aqueous formulations include those described in US Patent No. 6,171,586 and WO2006/044908, the latter formulations including a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano- particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the drug, which matrices are in the form of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

Combination Therapies

The soluble CTLA4 variants disclosed herein may be administered concomitantly with one or more other therapeutic regimens or agents. The additional therapeutic regimes or agents may be used to improve the efficacy or safety of the soluble CTLA4 variant. For example, a soluble CTLA4 variants may be administered to the patient along with another

immuosuppressive agent chemotherapy, radiation therapy, or both chemotherapy and radiation therapy.

The terms "in combination with" and "co-administration" are not limited to the administration of said prophylactic or therapeutic agents at exactly the same time. Instead, it is meant that the composition disclosed herein and the other agent or agents are administered in a sequence and within a time interval such that they may act together to provide a benefit that is increased versus treatment with only either the composition disclosed herein or the other agent or agents. In some embodiments, soluble CTLA4 variants disclosed herein and the other agent or agents act additively, and sometimes synergistically. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. The skilled medical practitioner can determine empirically, or by considering the pharmacokinetics and modes of action of the agents, the appropriate dose or doses of each therapeutic agent, as well as the appropriate timings and methods of administration.

A pharmaceutical composition of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

Compositions of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical

practitioners. The composition need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of leading composition present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of a composition of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of composition, the severity and course of the disease, whether the composition is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the composition, and the discretion of the attending physician. The composition is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg- lOmg/kg) of composition can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the composition would be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the composition). An initial higher loading dose, followed by one or more lower doses may be administered. The progress of this therapy is easily monitored by conventional techniques and assays.

Articles of Manufacture

In another embodiment of the invention, an article of manufacture containing materials useful for the treatment of the disorders described above is provided. The article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agent in the composition is the claimed poplypeptide. The label on, or associated with, the container indicates that the composition is used for treating the condition of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate -buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

Whereas particular embodiments of the invention have been described above for purposes of illustration, it will be appreciated by those skilled in the art that numerous variations of the details may be made without departing from the invention as described.

Example 1. Making of a Soluble CTLA4 variant IGM001 and Its Binding Affinity to CTLA4 Ligands

This example describes the recombinant generation of one soluble CTLA4 variant IGM001 (also labeled as CWT in some figures herein), which is a immunoadhesin comprising the ECD of CTLA4 fused via a linker to the Fc region of IgG2.

DNAs encoding a signal peptide, the ECD of CTLA4 and a linker sequence were synthesized using commercial DNA synthersizer, and subcloned into a mammalian expression vector containing the Fc region of the human IgG2. The resulting vectors were transfected into CHO host cells and grown in mammalian cell culture. After sufficient time for growth and protein expression, expressed fusion proteins were purified from the culture by protein A affinity. The fusioin protein products were subject to sequence analysis to confirm the fidelity of the sequences.

The resulting soluble CTLA4-Fc immunoadhesin, IGMOO 1 , has the amino acid sequence shown in Figure 1 and as SEQ ID NO:2.

Purified IGMOO 1 was analyzed for binding to CD80 and CD86 using a Biacore assay. The commercially available CDLA4-Ig product abatacept was used as comparision in the Biacore binding assay.

The results of Biacore assay showing fitted kinetic rate constants and binding affinities are summarized in Table 1. IGMOO 1 has much higher affinities to both CTLA4 ligands CD80 and CD86 when comparied to those of abatacept (4-6 fold higher), and the slower dissociation rates (Koff) seem to be the main attribute to the stronger binding.

Table 1. Fitted kinetic rate constants and binding affinities of CTLA4-Fc constructs to CTLA4 ligands.

K_on = on rate; K_off = off rate; K_D = equilibrium dissociation constant; Fold change is the K_D comparison between abatacept and IGMOO 1.

IGMOO 1 was also tested for its binding to cells expressing CD80 or CD86 using FACS analysis. CHO cells transfected with CD80+ or CD86+ were incubated with increasing concentrations of IGMOO 1 or abatacept. Bound fusion proteins were detected using fluorescein isothiocyanate-conjugated goat anti-human IgG, and mean fluorescence intensity (MFI) was calculated from data histograms. As shown in Figures 2 A and 2B, binding of IGMOOl to CD80+ cells is comparable to that of abatacept; whereas binding of IGMOOl to CD86+ cells seems stronger than that of abatacept at higher protein concentrations.

Example 2. Inhibitory Functions of the Soluble CTLA4 Variant IGMOOl

The high affinity soluble CTLA4 variant IGMOOl was subject to a series of functional assays to illustrate its biological activities in comparison to the commercially available abatacept.

First, IGMOOl was tested for its ability to inhibit ligand-induced T-cell proliferation using a [3H]thymidine incorporation assay. Human CD4-positive T cells were isolated and cultured in the presence of CD80-positive or CD86-positive CHO cells and various

concentrations of inhibitor (either IGMOOl or abatacept) or a negative control. Proliferative responses were measured by the addition of [3H]thymidine during the final phase of culturing.

The results are shown in Figures 4A and 4B. IGMOOl is more effective than abatacept in inhibiting T cell proliferations stimulated by either CD80 or CD86. Therefore, IGMOOl is a more potent inhibitor of T cell costimulation.

Next, IGMOOl 's inhibitory effects on T cell costimulation were further confirmed by assays for cytokine production in T cells. Duplicate secondary allostimulation plates were set up. After several days, culture media was assayed for cytokine expressions using ELISA kits. As shown in Figures 5A-C, IGMOOl is more potent than abatacept at blocking T cell IFN-g, IL-2 and IL-4 cytokine production following a secondary allogeneic stimulus.

All cited references are herein expressly incorporated by reference in their entirety.

Whereas particular embodiments of the invention have been described above for purposes of illustration, it will be appreciated by those skilled in the art that numerous variations of the details may be made without departing from the invention as described in the appended claims.

Claims

CLAIMS We claim:

1. A soluble CTLA4 variant composition comprising the extracellular domain (ECD) of a human CTLA4, wherein the binding affinity of the composition to a CTLA4 ligand has a Kd value of less than InM.

2. The composition of claim 1 , wherein the CTLA4 ligand is CD80 (B7- 1 ) or CD86 (B7-2).

3. The composition of claim 2, wherein the CD80 or CD86 is bound on the surface of an immune cell.

4. The composition of claim 3, wherein the immune cell is an antigen presenting cell.

5. The composition of claim 1, wherein the ECD of the human CTLA4 has an amino acid sequence according to SEQ ID NO: 1.

6. The composition of claim 1, wherein the soluble CTLA4 variant is an immunoadhesin having the ECD of human CTLA4 fused to the Fc region of an immunoglobulin.

7. The composition of claim 6, wherein the ECD of human CTLA4 is fused to the Fc region of an immunoglobulin through a linker.

8. The composition of claim 7, wherein the linker is a peptide sequence with 3-30 amino acid residues.

9. The composition of claim 8, wherein the linker is enriched with Glysine.

10. The composition of claim 6, wherein the immunoglobulin is an IgGl or IgG2.

11. The composition of claim 6, wherein the Fc region of the immunoglobulin is modified to remove or reduce its effector function.

12. The composition of claim 6, wherein the immunoadhesin comprises an amino acid sequence according to SEQ ID NO:2.

13. A nucleic acid molecule comprising a nucleotide sequence encoding the soluble CTLA4 variant according to any one of claims 1 -12.

14. The nucleic acid molecule of claim 13, having a sequence according to SEQ ID NO:3.

15. A vector comprsing the nucleotide sequence of claim 14.

16. A host cell comprising a vector of claim 15.

17. A method of treating a T cell-associated immune condition or disorder in a subject, comprising administering to the subject an effective amount of the soluble CTLA4 variant of claim 1.

18. The method of claim 17, wherein the T cell-associated immune disorder is an autoimmune disease.

19. The method of claim 18, wherein the autoimmune disease is rheumatoid arthritis (RA).

20. The method of claim 17, wherein the T cell-associated immune condition is transplant rejection.

21. Use of the soluble CTLA4 variant of claim 1 in the manufacture of a medicament for treating a T cell-associated immune condition or disorder.

22. A method of making a CTLA4 immunoadhesin, comprising culturing the host cell of claim 16 under conditions suitable for the production of CTLA4 immunoadhesin.