HK1112192B - Crig polypeptide for prevention and treatment of complement-associated disorders - Google Patents

Description

CRIg polypeptide for preventing and treating complement related disorder

Technical Field

The present invention concerns the recently discovered macrophage specific receptor, CRIg (formerly STIgMA), and its use in the prevention and/or treatment of complement-associated disorders, including complement-associated ocular conditions such as age-related macular degeneration (AMD) and Choroidal Neovascularization (CNV).

Background

The complement system is a complex enzyme cascade consisting of a series of serum glycoproteins, which are usually present in the inactive proenzyme (pro-enzyme) form. Three major pathways, the classical and alternative pathways, can activate complement, which merge at the level of C3, with two similar C3 convertases cleaving C3 into C3a and C3 b. The mannose-binding lectin (MBL) pathway has also been described as an alternative pathway.

Classical pathway components are labeled with C and values (e.g., C1, C3). The first four components are numbered C1, C4, C2 and C3 in view of their identified order. The bypass pathway components are indicated by letters (e.g., B, P, D). The cut fragments are named in lower case letters following the component name (e.g., C3a and C3b are fragments of C3). The inactive C3b was named iC3 b. The polypeptide chains of complement proteins are designated by the greek letters following this component (e.g., C3 α and C3 β are the α and β chains of C3). Cell membrane receptors for C3 are abbreviated CR1, CR2, CR3 and CR 4.

The classical pathway of the complement system is a major effector of the humoral branch of the human immune response. The trigger to activate the classical and MBL pathways is either IgG or IgM antibodies that bind to the antigen or lectins on the target cell. Binding of the antibody to the antigen exposes a site on the antibody that is the binding site for the first complement component C1. C1 binds to at least two exposed regions of the antibody that bind to the antigen, with the result that its C1r and C1s subunits are activated. Activated C1s is responsible for cleavage of the two complement components C4 and C2 involved below. C4 is cleaved into two fragments, with the larger C4b molecule attaching to the nearby target membrane and the smaller C4a molecule leaving. An exposed site on deposited C4b is available for interaction with the next complement component, C2. As in the previous step, the activated C1s cleaves the C2 molecule into two fragments, with the fragment C2a remaining and the smaller C2b fragment leaving. C4b2a, also known as C3 convertase, remains bound to the membrane. This C3 convertase converts the next complement component, C3, into its active form.

Activation of the alternative complement pathway begins when C3b binds to the cell wall and other cellular components of the pathogen and/or to IgG antibodies. Factor B then associates with cell-bound C3B to form C3 bB. C3bB is then split by factor B into Bb and Ba to form the alternative pathway C3 convertase C3 bBb. Properdin, a serum protein, then binds C3bBb to form C3bBbP that functions as a C3 convertase, enzymatically cleaving the C3 molecule into C3a and C3 b. At this point, the alternative complement pathway is activated. Some C3b bound C3bBb to form C3bBb3b, which is capable of splitting the C5 molecule into C5a and C5 b.

The alternative pathway is a self-amplifying pathway and is important in the clearance and recognition of bacteria and other pathogens in the absence of antibodies. The alternative pathway may also amplify complement activation following initial complement activation by lectins and/or the classical pathway. The rate-limiting step in activation of the alternative pathway in humans is the enzymatic action of factor D on the cleavage of factor B into the alternative pathway C3 converting enzyme C3bBb (Stahl et al, American Journal of Pathology 162: 449-455 (2003)). There is strong evidence for the role of complement activation and deposition in adjuvant-induced arthritis (AIA) and collagen-induced arthritis (CIA) and in a variety of other diseases and conditions.

Recently, defective bypass pathway control has been implicated in the development of renal and ocular diseases, including Hemolytic Uremic Syndrome (HUS) and AMD (Zipfel et al, mol. immunol.43: 97-106(2006), available on-line www.sciencedirect.com). C3 has been found to be critical for CNV formation in mice (Bora et al, J.Immunol.174 (1): 491-7 (2005)).

The role of the complement system in inflammatory conditions and associated tissue damage, autoimmune diseases and complement-associated diseases is also well known.

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Proposed alternative routes to inflammation (Mollnes et al, Trends in Immunology 23: 61-64(2002)), local and distant tissue injury following ischemia and reperfusion (Stahl et al, supra); adult respiratory distress syndrome (ARDS; Schein et al, Chest 91: 850-854 (1987)); complement activation during cardiopulmonary bypass surgery (Fung et al, J Thorac Cardiovasc Surg 122: 113-122 (2001)); dermatomyositis (Kissel, JT et al, NEJM 314: 329-334 (1986)); and pemphigus (Honguchi et al, J Invest Dermatol 92: 588-. The alternative complement pathway has also been implicated in autoimmune diseases such as, for example, lupus nephritis and glomerulonephritis and vasculitis which occur as a result (see, for example, Watanabe et al, J.Immunol.164: 786-.

Local increases in complement deposition and activation have been associated with disease severity (Atkinson, J ClinInvest 112: 1639-1641 (2003)). C5a receptor antagonists, such as peptides and small organic molecules, have been tested for the treatment of Arthritis (Woodruf et al, Arthritis & Rheumatic 46 (9): 2476-10785 (2002)) and a variety of other immunoinflammatory diseases (Short et al, Br J Pharmacol 126: 551-554 (1999); Finch et al, J Med Chem 42: 1965-1074 (1999)); while companies such as chemicals (australia) have been conducting human clinical trials to test the efficacy of C5a antagonists in similar indications. C5a has also been implicated in dermatomyositis and pemphigus (Kissel, JT et al, NEJM 314: 329-334 (1986)). anti-C5 a monoclonal antibodies have also been shown to reduce cardiopulmonary bypass and cardiac arrest-induced coronary endothelial dysfunction (Tofukuji et al, J.Thorac.Cardiovasc.Surg.116: 1060-.

Opsonophagocytosis (Opsonophagocytosis), the process by which complement fragments are deposited on the surface of particles and subsequently taken up by phagocytes, is crucial for the clearance of circulating particles, including immune complexes, apoptotic cells or cell debris, and pathogens (Gasque, p., mol immunol.41: 1089-. Tissue resident macrophages are known to play a major role in complement-mediated clearance of particles from the circulation. Kupffer cells, which account for more than 90% of tissue resident macrophages, are continuously exposed to blood from the portal vein and are strategically located in the antrum of the liver to effectively clear conditioned viruses, tumor cells, bacteria, fungi, parasites and harmful substances from the gastrointestinal tract. This clearance process is largely dependent on the presence of complement C3 as an opsonin (Fujita et al, Immunol. Rev.198: 185-202 (2004)). Upon binding to the bacterial surface via thioesters, C3 is cleaved and amplifies the alternative complement pathway. This reaction results in further deposition of fragment C3, which may act as a ligand for complement receptors on macrophages. The importance of this pathway is represented by the high susceptibility to bacterial and viral infections in humans lacking C3.

Complement receptors CR1, 3 and 4, characterized to date, internalize C3b and phagocytose C3 opsonized particles only upon PKC activation or Fc receptor stimulation (Carpentier et al, Cell Regul 2: 41-55 (1991); Senglov, Crit. Rev. Immunol.15: 107-. In addition, CR1 was not expressed on the surface of murine Kupffer cells (Fang et al, J.Immunol.160: 5273-5279 (1998)). Complement receptors that help KC to constitutively clear circulating granules have not been described to date.

anti-C3 b (i) antibodies have been reported to enhance complement activation, C3b (i) deposition, and rituximab to CD20⁺Cell killing (Kennedy et al, Blood101 (3): 1071-1079 (2003)).

Given that the complement cascade is known to be involved in a variety of diseases, there is a need to identify and develop new drugs for the prevention and/or treatment of complement-associated diseases.

Summary of The Invention

The present invention is based on the identification of a novel member of the complement receptor family and a member of the first immunoglobulin (Ig) superfamily that interacts with the complement system.

In one aspect, the invention concerns a method for preventing or treating a complement-associated eye condition comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of a complement inhibitor, such as an inhibitor of the alternative complement pathway, e.g., a CRIg polypeptide or agonist thereof.

Complement-associated ocular conditions can be, for example, age-related macular degeneration (AMD), Choroidal Neovascularization (CNV), uveitis, diabetic and other ischemia-related retinopathies, endophthalmitis, diabetic macular edema, pathologic myopia, von Hippel-Lindau disease, histoplasmosis of the eye, Central Retinal Vein Occlusion (CRVO), corneal neovascularization, and retinal neovascularization. Preferably, the complement-associated eye condition is AMD or CNV, including all stages of these conditions.

In another aspect, the invention concerns a method for preventing the formation or development of AMD, comprising administering to a subject at risk of developing AMD or diagnosed with AMD, an effective amount of a complement inhibitor, such as an inhibitor of the alternative complement pathway, e.g. a CRIg polypeptide or agonist thereof.

In yet another aspect, the invention concerns a method for treating dry AMD comprising administering to a subject in need thereof a therapeutically effective amount of a complement inhibitor, such as an inhibitor of the alternative pathway, e.g., a CRIg polypeptide or agonist thereof.

In all embodiments, the CRIg polypeptide can be selected, for example, from SEQ ID NOs: 2, 4, 6 and 8 and the extracellular domain (ECD) of said polypeptide. The CRIg polypeptides, including full-length polypeptides and their ECDs, can be fused to immunoglobulin sequences, such as immunoglobulin heavy chain constant region sequences, e.g., Fc regions, and the resulting immunoadhesins can be used as CRIg agonists in the prophylactic and therapeutic methods of the invention. The immunoglobulin is preferably an IgG, such as IgG-1, IgG-2, IgG-3, or IgG-4, more preferably IgG-1 or IgG-3. The IgG1 heavy chain constant region sequence may comprise at least the hinge, CH2, and CH3 regions, or, for example, the CH1, hinge, CH2, and CH3 regions.

In yet another aspect, the invention concerns a method for preventing or treating a complement-associated disease or condition comprising treating a subject in need thereof with a prophylactically or therapeutically effective amount of a complement inhibitor, such as an inhibitor of the alternative pathway, e.g., a CRIg polypeptide or an agonist thereof.

In another aspect, the invention concerns a method for inhibiting the production of a C3b complement fragment in a mammal, comprising administering to the mammal an effective amount of a complement inhibitor, such as an inhibitor of the alternative pathway, e.g., a CRIg polypeptide or agonist thereof.

In yet another aspect, the invention concerns a method for selectively inhibiting the alternative complement pathway in a mammal comprising administering to said mammal an effective amount of a CRIg polypeptide or agonist thereof.

In all aspects, the CRIg polypeptide can be selected, for example, from SEQ ID NOs: 2, 4, 6, 8 and an extracellular region of said polypeptide. The agonist is preferably a CRIg-Ig fusion protein (immunoadhesin) as described above. The immunoglobulin sequence may be, for example, an immunoglobulin constant region sequence, such as the constant region sequence of an immunoglobulin heavy chain. In another embodiment, the immunoglobulin heavy chain constant region sequence is identical to SEQ ID NO: 2, 4, 6 or 8 is fused to an extracellular region of the CRIg polypeptide. In yet another embodiment, the immunoglobulin heavy chain constant region sequence is an IgG, such as that of IgG-1 or IgG-3, wherein the IgG-1 heavy chain constant region sequence may, for example, comprise at least the hinge, CH2, and CH3 regions, or the hinge, CH1, CH2, and CH3 regions.

The complement-associated disease can be, for example, an inflammatory disease or an autoimmune disease.

In a specific embodiment, the complement-associated disease is selected from the group consisting of: rheumatoid Arthritis (RA), Adult Respiratory Distress Syndrome (ARDS), remote tissue injury after ischemia and reperfusion, complement activation during cardiopulmonary bypass surgery, dermatomyositis, pemphigus, lupus nephritis and glomerulonephritis and vasculitis arising therefrom, cardiopulmonary bypass, coronary endothelial dysfunction induced by cardiac arrest (coronary endothelial dysfunction), type II membranoproliferative glomerulonephritis, IgA nephropathy, acute renal failure, cryoglobulinemia, antiphospholipid syndrome, age-related macular degeneration, uveitis, diabetic retinopathy, allograft, hyperacute rejection, hemodialysis, chronic obstructive pulmonary stress syndrome (chronic obstructive pulmonary stress syndrome), asthma, Alzheimer's disease, atherosclerosis, hereditary angioedema, paroxysmal nocturnal hemoglobinuria and aspiration pneumonia.

In another specific embodiment, the complement-associated disease is selected from the group consisting of: inflammatory Bowel Disease (IBD), systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic inflammatory myopathy (dermatomyositis, polymyositis), Sjogren's disease (Sj) gren) syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis (Graves 'disease), Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis, diabetes, immune-mediated nephropathy (glomerulonephritis, tubulointerstitial nephritis), demyelinating diseases of the central and peripheral nervous system such as multiple sclerosis, idiopathic polyneuropathy, hepatobiliary diseases such as infectious hepatitis (a, b, c, d, e and other non-hepadnaviral hepatitis), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous cirrhosis, and the likeInflammatory and fibrotic lung diseases (e.g. cystic fibrosis), gluten-sensitive bowel disease, Whipple's (Whipple) disease, autoimmune or immune-mediated skin diseases including bullous skin disease, erythema multiforme and contact dermatitis, psoriasis, allergic diseases of the lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, transplantation related diseases including graft rejection and graft-versus-host disease.

In yet another specific embodiment, the complement-associated disease is Rheumatoid Arthritis (RA), psoriasis, or asthma.

In all embodiments, the subject may be a mammal, such as a human patient.

In yet another aspect, the invention concerns a method for preventing or treating age-related macular degeneration (AMD) or Choroidal Neovascularization (CNV) in a subject comprising administering to the subject an effective amount of a complement inhibitor, such as an inhibitor of the alternative pathway, e.g., a CRIg polypeptide or agonist thereof.

Brief Description of Drawings

FIGS. 1A-1B show the nucleotide and amino acid sequences of a 321 amino acid human CRIg polypeptide (SEQ ID NOS: 1 and 2, respectively).

FIGS. 2A-2B show the nucleotide and amino acid sequences of a full length, elongated native human CRIg (huCRIg or huCRIg-long) of 399 amino acids (SEQ ID NOS: 3 and 4, respectively).

FIGS. 3A-3B show the nucleotide and amino acid sequences of a 305 amino acid short version of native human CRIg (huCRIg-short) (SEQ ID NOS: 5 and 6, respectively).

FIGS. 4A-4C show the nucleotide and amino acid sequences of a native murine CRIg (mucIg) of 280 amino acids (SEQ ID NOS: 7 and 8, respectively).

FIG. 5 shows the amino acid sequences of full-length huCRIg (SEQ ID NO: 4) and huCRIg-short (SEQ ID NO: 6) in comparison to mucRIG (SEQ ID NO: 8). The hydrophobic signal sequence, IgV, IgC and the transmembrane region are shown. MuCRIg has a predicted single N-linked glycosylation site (NGTG) at position 170. The Ig domain boundaries deduced from exon-intron boundaries of the human CRIg gene are indicated.

Figure 6 shows in situ hybridization of CRIg in frozen sections of mouse liver.

Figure 7 shows in situ hybridization of CRIg in human liver cryosections.

FIG. 8 shows in situ hybridization of CRIg in activated colon and adrenal macrophages, Kupffer cells, and placental Hofbauer cells.

FIG. 9 shows in situ hybridization of CRIg mRNA in RA synovial cells.

Figure 10 shows in situ hybridization of CRIg mRNA in brain microglia.

Figure 11 shows in situ hybridization of CRIg mRNA in cells from human asthmatic tissue.

FIG. 12 shows in situ hybridization of CRIg mRNA in cells from human chronic hepatitis tissue.

Figure 13 shows immunohistochemical analysis of CRIg in adrenal macrophages.

Figure 14 shows immunohistochemical analysis of CRIg in liver Kupffer cells.

Figure 15 shows immunohistochemical analysis of CRIg in brain microglia.

Figure 16 shows immunohistochemical analysis of CRIg in placental Hofbauer cells.

Fig. 17. Northern blot analysis showing huCRIg expression in various tissues. Two transcripts of 1.5 and 1.8kb were present in CRIg-expressing human tissues.

Fig. 18. (A) huCRIg was shown to be present in the bone marrow monocyte lines HL60 and THP-1 and in differentiated macrophagesIncreased expression in TAQMAN ^TMAnd (4) PCR analysis. Low levels of expression were found in Jurkat T cells, MOLT3, MOLT4 and RAMOS B cell lines. (B) hucrig mrna was expressed elevated during monocyte differentiation in vitro. Monocytes isolated from human peripheral blood differentiated by adhesion to plastic for 7 days. Total RNA was extracted at different time points during differentiation. (C) huCRIg protein is expressed in elevated levels during the differentiation of monocytes into macrophages. Monocytes were treated as described in (B), whole cell lysates were run on gel electrophoresis and transferred to nitrocellulose membranes, incubated with polyclonal antibody to huCRIg (4F 7). The polyclonal antibody recognized 48 and 38kDa bands that might represent long and short huCRIg.

Fig. 19. Molecular characterization of huCRIg protein in cell lines. (A) huCRIg-gd was transiently expressed in 293E cells, immunoprecipitated with anti-gd, and blots were incubated with polyclonal antibodies against gd or CRIg extracellular domain (ECD). (B) huCRIg expressed in 293 cells was a monomeric N-glycosylated protein. CRIg tyrosine phosphorylated on sodium perborate treatment of HEK293 cells but did not recruit Syk kinase. Phosphorylated CRIg migrates at slightly higher molecular weight compared to non-glycosylated CRIg.

Fig. 20. Selective expression of huCRIg on human monocyte-derived macrophages. Antibodies specific for B, T, NK cells, monocytes for peripheral blood mononuclear cells and ALEXA^TMMonoclonal antibody (3C9) staining of a 488-conjugated CRIg. There was no expression in all peripheral blood leukocytes and in monocyte-derived dendritic cells, but in vitro differentiated macrophages.

FIG. 21. CRIg mRNA and protein expression was increased by IL-10 and dexamethasone. (A) Real-time PCR showed that CRIg mRNA expression was elevated after treatment with IL-10, TGF β and highly induced by dexamethasone, but down-regulated by LPS, IFN γ and TNF α treatment. (B) Ficoll-isolated peripheral blood mononuclear cells were treated with various cytokines and dexamethasone for 5 days and double stained with anti-CD 14 and anti-CRIg. Flow analysis showed a significant increase in CRIg expression on the surface of monocytes treated with dexamethasone and after treatment with IL-10 and LPS.

FIG. 22. Subcellular localization of CRIg in monocyte-derived macrophages. Monocytes were cultured in macrophage differentiation medium for 7 days, fixed in acetone, and stained with polyclonal anti-CRIg antibody 6F1 or CD63 and secondary goat anti-rabbit FITC. Cells were studied in a confocal microscope. CRIg is found in the cytoplasm where it co-localizes with the lysosomal membrane protein CD 63. CRIg is also expressed in a pattern similar to F-actin at the leading and trailing edges of macrophages. Scale bar 10 μm.

FIG. 23. Localization of CRIg mRNA in chronic inflammatory diseases. In situ hybridization revealed the presence of crig mrna in alveolar macrophages obtained from tissues from patients with pneumonia (a, B) or chronic asthma (C, D). CRIg mRNA was also expressed in liver Kupffer cells in tissues obtained from liver biopsies of chronic hepatitis patients (E, F).

FIG. 24. CRIg mRNA expression is elevated in inflamed synovium. CRIg mRNA is low or absent in synovium of joints obtained from knee replacement in patients without joint inflammation (a, C), but is highly expressed in cells in pannus, possibly synovial cells or synovial macrophages, in patients with osteoarthritis (B, D).

FIG. 25. The CRIg protein was detected in synovial lining cells of patients with degenerative joint disease (A, B, C) using polyclonal antibody 6F 1. No immunohistochemical detection of CRIg was found in the control synovium (D).

FIG. 26. CRIg protein is expressed in tissue-resident macrophage subtypes, and its expression is elevated in chronic inflammatory diseases. (A) CRIg is expressed on the membrane of CHO cells stably expressing CRIg. High expression of CRIg protein was found in alveolar macrophages (B) in tissues obtained from chronic asthma patients. (C) Expression of CRIg in tissue cells of the human small intestine. The sections are obtained from surgically excised tissue and may contain tumors. (D) Expression of CRIg protein in Hofbauer cells in human prenatal placenta. High expression of CRIg protein in macrophages is present in adrenal (E) and Kupffer cells of human liver (F). Staining was performed on acetone-fixed sections 5 μm thick using DAB as chromogen. Images were taken at 20X and 40X magnification.

FIG. 27. Immunohistochemical staining of CD68 and CRIg on vascular plaques obtained from atherosclerotic patients. Serial sections were fixed and stained with monoclonal antibodies (a, B) to human CD68 and polyclonal antibody 6F1(C, D) prepared against human CRIg. CRIg appears in a population of macrophages and foam cells present in atherosclerotic plaques as judged by staining of serial sections, and overlaps with CD68 positive macrophages. Magnification: 10X (A, C) and 20X (B, D).

FIG. 28. Co-staining of CRIg and CD68 on cardiac interstitial macrophages. 5 μm sections were obtained from human hearts (autopsy) and stained with CRIg monoclonal antibody (3C9) and a secondary anti-mouse FITC labeled antibody. CD68 was detected by staining with PE-labeled monoclonal antibodies to CD 68. Magnification: 20X.

FIG. 29. CRIg mRNA levels are significantly elevated in colon tissue obtained from patients with ulcerative colitis, Crohn's (Crohn's) disease, Chronic Obstructive Pulmonary Disease (COPD), and asthma. Real-time PCR was performed on total RNA extracted from multiple tissues. The mRNA of CRIg is significantly elevated in tissues obtained from patients with ulcerative colitis, Crohn's (Crohn) disease, and COPD. Statistical analysis was performed using the Mann-Whitney U test.

FIG. 30. Cells expressing human CRIg show increased adhesion to human endothelial cells. (A) CRIg is stably expressed in the human Jurkat T cell line. (B) Cells were pre-loaded with the fluorescent dye BCECF (Molecular Probes, Oregon) and added to 96-well plates coated with monolayer Human Umbilical Vein Endothelial Cells (HUVECs) with or without treatment with 10ng/ml TNF α. After 3 washes, fluorescence was counted in a spectrofluorimeter, which indicated the number of cells that remained adherent to HUVEC cells. The figure represents 4 independent experiments.

FIG. 31. Inhibition of the development of a mouse model of collagen-induced arthritis (CIA) by muCRIg IgG-Fc fusion protein. One group (CIA) of mice (n ═ 7) was given 100 μ g muCRIg IgG-Fc fusion protein (square), while a CIA mouse control group (n ═ 8) received 100 μ g murine IgG1 (round), 3 times per week for 6 weeks. Mice were tested daily for signs of inflammation and scored according to a standard of 0-16 (see example 25 for details), with results plotted (mean ± SD, Student's T-test p-value ═ 0.0004 control IgG1 versus test mucig protein).

FIG. 32 is the nucleotide sequence (SEQ ID NO: 9) of DNA42257 (consensus sequence).

Figure 33 shows the reduction of joint swelling in mice treated with CRIg-Fc.

Figure 34 shows that murig inhibits joint inflammation.

FIG. 35 shows the maintenance of cortical bone volume in the joints of mice treated with muCRIg-Fc.

Figure 36 shows that CRIg-Fc treatment did not alter the number or morphology of tissue resident macrophages.

Figure 37 shows that muCRIg treatment did not affect serum anti-collagen antibody titers.

Figure 38 shows that muCRIg does not alter T cell independent B cell responses in vivo.

FIG. 39 shows macrophage infiltration in joints following antibody-induced arthritis (AIA), resulting from staining with F4/80 in undecalcified frozen joints.

FIG. 40 shows that mucIg-Fc prevents joint swelling following antibody-induced arthritis in balb/c mice.

FIG. 41 shows that murig inhibits joint infiltration in antibody-induced arthritis.

FIG. 42 shows the generation of muCRIg knockout mice.

FIG. 43 shows that murine CRIg-Fc fusion protein binds to C3-opsonized sheep red blood cells (E-IgM).

FIG. 44 shows that the binding of human CRIg-Fc to E-IgM is dependent on C3.

Figure 45 shows binding of serum conditioned particles to CHO cells expressing CRIg.

Figure 46 shows that murine CRIg-Fc binds complements C3b and iC3b, but not C2, C4, and C3 d.

Figure 47 shows that murine and human CRIg-Fc bind complements C3b, C3bi, and C3C, but not C1, C2, C4, C3a, and C3 d.

Figure 48A shows that murine and human CRIg-Fc inhibit C3 deposition of zymosan.

Figure 48 shows that murine CRIg inhibits alternative pathway induced hemolysis, but does not affect the classical hemolysis pathway.

Figure 49 shows that murine CRIg inhibits alternative pathway induced hemolysis, but does not affect the classical hemolysis pathway.

FIG. 50. CRIg is selectively expressed on tissue resident macrophage subpopulations. (A) CRIg is a member of the single transmembrane immunoglobulin superfamily, consisting of one (human CRIg short (hucrig (s)) and murine CRIg (mucrg)) or two (huCRIgL) immunoglobulin domains. The scale at the top of the left panel indicates the size in amino acids. The right panel shows the distant relationship of hu and mucrigg to the joint adhesion molecule-A (JAM-A) and A33 antigens. The scale at the top of the right panel indicates the percent amino acid similarity.

(B) CRIg is expressed in macrophages but not monocytes. huCRIg staining was analyzed by flow cytometry on human CD14+ monocytes and CD14+ monocytes cultured for 7 days in 10% autologous serum and 20% fetal bovine serum using an anti-human CRIg MAb (3C 9). MuCRIg staining was analyzed using anti-MuCRIg MAb (14G6) on mouse CD11b + and F4/80+ liver Kupffer cells.

(C) Western blot analysis of human and mouse macrophages. Lysates from human CD14+ monocytes or mouse peritoneal macrophages cultured for the indicated times were boiled in reducing SDS buffer, loaded onto 4-10% Tris-glycine gels, and incubated with either polyclonal anti-CRIg antibody (6F1, left panel) or anti-muCRIg monoclonal antibody (14C6, right panel). Pre-immune IgG (left panel) and rat IgG2b (right panel) were used as isotype controls. The arrows in the left panel indicate the positions of the 57 and 50kDa bands that may represent huCRIg (L) and (S).

(D) Co-localization of CRIg with CD68 on liver Kupffer cells. Sections obtained from human and mouse liver were immunostained using monoclonal anti-CRIg (3C9 human and 14G6 mouse) and monoclonal anti-CD 68 antibody.

FIG. 51. Flow cytometry analysis of CRIg expression on peripheral blood leukocytes and analysis of binding of C3 fragment and C3 conditioned particles to CHO cells expressing CRIg.

(A) Flow cytometry analysis of CRIg expression on human and mouse peripheral blood leukocytes.

(B) Soluble C3 fragment or complement opsonized pathogens bind to CHO cells expressing murine CRIg, but do not bind to CHO cells expressing JAM-2. The suspension cells were incubated with the a 488-labeled complement-conditioned particles for 30 minutes at room temperature under continuous rotation. Cells were washed 3 times and particle binding was monitored by flow cytometry analysis. Results are representative of 3 independent experiments.

FIG. 52 is a schematic view. Soluble and cell surface expressed CRIg binds to the C3 fragment in solution or deposited on the cell surface.

(A) CRIg (L) transfected Jurkat cells (Jurkat-CRIg), but not empty vector transfected Jurkat cells (Jurkat-control) formed rosettes with C3 and IgM conditioned sheep red blood cells (E-IgM). The histogram (left panel) shows CRIg expression on human CRIg (l) stably transfected Jurkat cells. E-IgM conditioned with C3-deficient (C3-) or C3-replete (C3+) serum was mixed with CRIg or Jurkat transfected with control vector for 1 hour. This experiment represents 3 independent experiments.

(B) Binding of CIg (L) -Fc to IgM-opsonized sheep erythrocytes (E-IgM) is dependent on the presence of C3 in the serum. E-IgM was opsonized with C3 depleted human serum, to which increasing concentrations of purified human C3 were added. E-IgM was subsequently incubated with huCRIg (L) -Fc fusion protein, followed by detection by flow cytometry using anti-human Fc polyclonal antibodies. This experiment represents 3 independent experiments.

(C) ELISAs showing that CRIg (L) -and CRIg (S) -Fc bind to C3b and iC3 b. Increasing concentrations of huCRIg (L) -and huCRIg (S) -Fc fusion proteins were added to maxisorb plates coated with purified C3b and iC3 b. Binding was detected using an anti-huFc antibody coupled to HRPO. The results shown represent 4 independent experiments using different batches of fusion protein and purified complement components.

(D) Kinetic binding data showing that soluble C3b dimer binds to hucrig (l) -Fc. The affinity of C3b for CRIg fusion proteins was determined using surface plasmon resonance. CRIg protein was captured on CM5 sensor chip via amine coupling of antibodies against Fc fusion tags. Dimer C3b was then injected for a sufficient time to saturate. Kd was calculated from binding curves plotted against concentration showing the response at equilibrium. The calculated affinity for the C3b dimer for hucrig(s) was 44nM, and the affinity for hucrig (l) was 131 nM.

(E) CRIg expressed on cell surface bound to A488-labeled C3b dimer ((C3b)₂) But not native C3. The left panel shows the expression levels of huCRIg (L) on transfected THP-1 cells as analyzed by flow cytometry. (C3b)₂Shows saturable binding to CRIg transfected THP-1 cells. (C3b)₂Combinations of THP-1CRIg are useful (C3b)₂The extracellular domains of C3b and CRIg (CRIg-ECD) compete out, but C3 does not. The results shown are representative of 3 independent experiments.

FIG. 53 is a schematic view. Generation and characterization of CRIg knockout mice.

(A) Generation of targeting vectors for homologous recombination in ES cells.

(B) Southern blot validation of homologous recombination of SRIg alleles in heterozygous female offspring generated by breeding chimeric and wild-type mice.

(C) Comparison of the number of leukocytes in peripheral blood of wild-type and knockout male and female mice.

(D) FACS analysis showing absence of CR1, CR2 and CD11c expression in KC.

(E) FACS analysis of C3-A488 and C3C-A488 bound wild-type and knock-out KC.

FIG. 54 is a schematic view. CRIg expression on Kupffer cells is required for the binding of C3b and iC3 b. (A) There is no CRIg protein on macrophages obtained from CRIg knockout mice. Peritoneal macrophages obtained from CRIg wild-type, heterozygous or knockout mice were incubated with anti-muCRIg mAb (14G6, left panel). Kupffer Cells (KC) obtained from CRIg wild-type and knockout mice were incubated with antibody 14G6 and analyzed by flow cytometry.

(B) Expression levels of CD11b and CD18, the alpha and beta chains of complement receptor 3, and Crry were similar on Kupffer cells obtained from CRIg wild-type mice and CRIg knockout mice. Kupffer cells isolated from CRIg wild-type or knockout mice were incubated with antibodies to CD11b, CD18, and Crry and analyzed by flow cytometry.

(C) Kupffer cells isolated from CRIg wild-type or knockout mice were incubated with activated mouse serum (activated by incubation at 37 ℃ for 30 minutes), C3b, (C3b)₂Incubated with iC3 b. Binding of purified complement components to the cell surface was detected using polyclonal antibodies recognizing various C3-derived fragments. The results shown are representative of 4 experiments.

(D) KC isolated from CRIg knockout mice showed reduced rosetting with IgM-coated sheep erythrocytes (E-IgM) opsonized in C3 replete mouse serum. KC isolated from the liver of CRIg wild-type and knockout mice was incubated with complement C3 conditioned E-IgM for 30 minutes in the presence of control IgG or anti-CR 3 blocking antibody (M1/70). Cells were fixed, and the number of KCs forming rosettes with E-IgM was counted and expressed as a percentage of the total number of KCs.^*＝p<0.05. The results shown are representative of 2 independent experiments.

FIG. 55. CRIg on Kupffer cell cycle.

(A) From C3 wild type (panels 1, 3, 4 and 6) or C3 knock-out miniKupffer Cells (KC) from mice (FIG. 2, 5) were incubated with A488-labeled anti-CRIg antibodies (14G6) and (C3b)₂Incubate at 4 ℃ for 1 hour (panels 1-3) or at 37 ℃ for 10 minutes (panels 4-6). Cells were then transferred to 4 ℃ and incubated with (red histogram) or without (black histogram) anti-a 488 antibody to distinguish between cytoplasmic and cell surface expressed anti-CRIg or C3 b.

(B) Internalization and co-localization of CRIg and C3b in CRIg wild-type KC but not CRIg knockout KC. KC isolated from the livers of CRIg wild-type and knockout mice were cultured on trough slides for 2 days and incubated with a 455-conjugated anti-CRIg antibody and a 488-conjugated C3b for 30 minutes at 37 ℃ to prepare specimens (mount) and photographed.

(C) Antibodies to CRIg but not to Lampl circulate to the cell surface. Kupffer cells were loaded with A488-conjugated anti-mucIg or anti-muLampl antibody for 10 minutes at 37 ℃, washed, and then incubated in the presence of anti-A488-quenching antibody at 37 ℃ for the indicated time. The results shown are representative of 3 independent experiments.

FIG. 56. CRIg is expressed on circulating endosomes recruited to the site of particle uptake.

(A) Cell surface expressed CRIg maps to F-actin positive ruga (membrane ruffles). Monocyte-derived macrophages cultured for 7 days were incubated with a 488-conjugated anti-CRIg a488mAb3C9 (green channel in a1 and A3) and Alexa 546-phalloidin (red channel in a2 and A3) at 4 ℃. Arrows indicate the rugae in which both CRIg and actin stain more strongly than the remaining cell surface (yellow in the merged image in a 3). The scale bar is 20 μm.

(B) CRIg and C3b co-localize with transferrin in circulating endosomes. Macrophages were incubated with CRIg-a488(B1, green channel in B4) or C30a488(B2, red channel in B4) on ice for 1 hour and then captured in the presence of a 647-transferrin (blue channel in B3, B4) for 10 minutes at 37 ℃. The scale bar is 20 μm.

(C) CRIg recruited to phagocytic cups (phagocytic cups) and to the phagosomal membranes. Macrophages were incubated with IgM-coated erythrocytes conditioned with C3 serum replete for 10 minutes (C1-4) or 2 hours (C5-8) at 37 ℃ in the presence of A647-labeled transferrin (blue channel in C2, 6 and C4, 8). Cells were then fixed, permeabilized, and stained with anti-CRIg polyclonal antibodies (green channel in C1, 2 and C4, 5) and a 555-conjugated LAMP-1 antibody (red channel in C3, 7 and C4, 8).

FIG. 57. Transport of CRIg in human monocyte-derived macrophages.

(A) FACS plots showing saturable binding of C3b-a488 to CRIg on day 7 MDM.

(B) MDM was pulsed (pulse) with anti-CRIg antibody and C3b-A488 for 10 min at 37 ℃ in the presence of a 10-fold molar excess of huCRIg (L) -ECD. The binding and uptake of anti-CRIg antibody was specific for CRIg as it could be eliminated by co-incubation of the antibody with a 10-fold molar excess of CRIg-ECD (panel 1), while leaving transferrin uptake unaffected (panel 2).

(C) MDM was incubated for 20 hours at 37 ℃ in the presence of lysosomal protease inhibitors, then cells were washed, fixed with 1% PFA, and the ingested antibody was detected with Cy 3-labeled anti-mouse IgG (red channel in C panels 1 and 3). Cells were co-stained in 10 μ g/ml rabbit anti-CRIg 6F1 followed by FITC-anti-rabbit to detect the overall CRIg distribution (green channel in C panel 2 and C panel 3). The antibody uptake almost completely overlapped the endogenous CRIg signal (yellow in the merged image in panel C3), indicating that antibody uptake did not affect CRIg transport. The scale bar is 20 μm and the 4x magnified inset of the boxed area shown to the right below each channel is 5 μm. C panel 4 human macrophages were incubated in C3 depleted serum for 13 hours, then fixed and labeled with rabbit anti-CRIg F1 and FITC-anti-rabbit. The CRIg distribution was essentially the same as in C3 abundant serum, both of which almost completely overlapped with the circulating endosomal marker transferrin (data not shown). The scale bar is 20 μm.

(D) MDM was incubated with 1. mu.g/ml anti-CRIg-A488 (panel 1), transferrin-A647 (panel 2) for 10 min at 37 ℃, fixed in 4% PFA, permeabilized with saponin buffer, and incubated with mouse-anti-human Lamp-1-A555 (panel 3). Arrows indicate co-localization of CRIg and transferrin in the recycling compartment.

(E) MDM was incubated with 1. mu.g/ml anti-CRIg-A488 (panel 1, green channel in panel 4), transferrin-A647 (panel 2, blue channel in panel 4) for 30 minutes at 37 ℃, washed, and incubated with PKH-stained, complement C3-conditioned sheep erythrocytes (SRBC, panel 3, red channel in panel 4) at a 1:10 macrophage: SRBC ratio.

FIG. 58. Mice lacking CRIg are susceptible to Listeria Monocytogenes (LM) infection.

(A) Survival curves of female CRIg wild-type and CRIg knockout mice infected with LM at the indicated doses by injection into the lateral tail vein, n-5-7 per group. Statistical analysis (Wilcoxon): wild type knock-out, 2X10⁴Colony Forming Unit (CFU) is p<0.005，5x10⁴And 2x10⁵CFU is p<0.0001。

(B) Bacterial count analysis in heart, liver, blood and spleen 10 minutes after LM infection (2X 10)⁷CFU, n 5 per group). Statistical analysis (paired t-test):^**p<0.01，^*p<0.05。

(C) the concentration of cytokines and chemokines in serum of CRIg knockout mice increased 1 day after LM infection. Statistical analysis (unpaired t-test):^***p<0.001。

(D) decreased uptake of LM-A488 in CRIg knock-out mouse KC. 2x10 for mouse⁷LM-A488 infection. After 1 hour, the liver was perfused, incubated with antibody to F4/80, and analyzed by flow cytometry. F4/80 positive KC was then sorted by FACS and collected on poly L-lysine coated slides for observation by fluorescence microscopy. The number of internalized LM-A488 was counted in a confocal microscope and the phagocytic index was calculated. Results are representative of at least two experiments.

(E) CRIg mice have reduced LM slaveAnd (4) clearing in circulation. CRIg and C3 double or single knockout mice were injected intravenously with 2x10⁷And a CFU LM. CFU in blood was counted 10 min after infection. CRIg knockout mice have significantly reduced LM clearance from circulation in the presence of C3 (p)<0.001). There was no significant difference in LM clearance in CRIg wild-type or knockout mice in the absence of C3.

FIG. 59 shows the nucleotide sequence of a human CRIg (short) -IgG fusion (SEQ ID NO: 20).

FIG. 60 shows the nucleotide sequence of a human CRIg (Long) -IgG fusion (SEQ ID NO: 21).

FIG. 61 shows the CRIg (STIGMA) -Fc ligation in two different constructs, both inserted into the ClaI-XbaI site of pRK5 vector.

FIG. 62 shows that mucIg-Fc fusion protein (but not a control Fc fusion protein) inhibits LM clearance from circulation in wild-type but not CRIg knockout cells. CRIg wild-type and knockout mice were injected intravenously with 2x10⁷CFU LM was treated 24 hours and 16 hours prior to treatment with 2 injections of 12 mg/kgmucIg-Fc or control-Fc fusion protein. CFU in blood was counted 10 min after infection. There was a significant reduction in LM clearance from circulation in CRIg wild-type mice treated with mucIg-Fc compared to wild-type mice treated with control-Fc (p) <0.001, unpaired Student's t-test). In CRIg knockout mice, MuCRIg-Fc treatment had no effect on LM clearance.

FIG. 63. The huCRIg molecule was used to inhibit complement-mediated immune hemolysis. A. hCRIg-short and-long fusion proteins were used to inhibit hemolysis in cynomolgus monkey serum RRBC. B. hCRIg-long ECD was used to inhibit hemolysis in cynomolgus monkey serum RRBC.

FIG. 64 is a schematic view. hCRIg-peptide was used to inhibit hemolysis of human serum in two different experiments.

FIG. 65. hCRIg-short-Fc and CRIg-long-Fc fusion proteins were used to inhibit hemolysis in human serum.

FIG. 66. hCRig-Long-ECD and hCRig-short-ECD were used to inhibit hemolysis of human serum, respectively.

FIG. 67 shows the nucleic acid sequence (SEQ ID NO: 25) encoding huCRIg-Long-Fc ("stem-free" construct).

FIG. 68 shows a nucleic acid sequence encoding huCRIg-Long-Fc with a "stem" inserted between the transmembrane domain and Fc portion of CRIg (SEQ ID NO: 26).

FIG. 69 shows the nucleic acid sequence (SEQ ID NO: 27) encoding huCRIg-short-Fc ("stemless" construct).

FIG. 70 shows the nucleic acid sequence (SEQ ID NO: 28) encoding huCRIg-short-Fc with a "stem" inserted between the transmembrane domain and Fc portion of CRIg.

Fig. 71A and B show the results of the mouse CNV study described in example 23.

Detailed Description

I. Definition of

The terms "PRO 362", "JAM 4", "STIgMA", and "CRIg" are used interchangeably to refer to the native sequence and to a variant CRIg polypeptide.

"native sequence" CRIg refers to a polypeptide having the same amino acid sequence as a CRIg polypeptide derived from nature, regardless of its mode of preparation. Thus, the native sequence CRIg may be isolated from nature or may be generated by recombinant and/or synthetic means. The term "native sequence CRIg" specifically encompasses naturally occurring truncated or secreted forms of CRIg (e.g. extracellular domain sequences), naturally occurring variant forms of CRIg (e.g. alternatively spliced forms) and naturally occurring allelic variants. The native sequence CRIg polypeptide specifically includes the amino acid sequence of SEQ ID NO: 2 (see figure 1), with or without an N-terminal signal sequence, with or without an initiating methionine at position 1, and with or without a sequence located in seq id NO: 2 at about amino acids 277-307. Native sequence CRIg polypeptide also includes SEQ ID NO: 4 (huCRIg or huCRIg-long, see figures 2 and 5), with or without an N-terminal signal sequence, with or without an initiating methionine at position 1, and with or without a peptide sequence located in SEQ ID NO: 4 at about amino acids 277-307. In yet another embodiment, the native sequence CRIg polypeptide is a short version of 305 amino acids human CRIg (huCRIg-short, SEQ ID NO: 6, see FIG. 3), with or without an N-terminal signal sequence, with or without an initiating methionine at position 1, and with or without a sequence located in SEQ ID NO: 6 at about position 183-213. In a different embodiment, the native sequence CRIg polypeptide is seq id NO: 8 (muclg, see fig. 4 and 5), with or without an N-terminal signal sequence, with or without an initiating methionine at position 1, and with or without a full-length murine CRIg polypeptide of SEQ ID NO: any or all of the transmembrane domain at about amino acids 181-211 of 8. CRIg polypeptides from other non-human animals, including higher primates and mammals, are expressly included within this definition.

"CRIg variant" refers to an active CRIg polypeptide having at least about 80% amino acid sequence identity to a native sequence CRIg polypeptide, as defined below, including but not limited to a C-terminally truncated 321 amino acid huCRIg (SEQ ID NO: 2), full-length huCRIg (SEQ ID NO: 4), huCRIg-short (SEQ ID NO: 6), and mucIg (SEQ ID NO: 8), each with or without an N-terminal initiating methionine, with or without an N-terminal signal sequence, with or without all or part of a transmembrane domain, and with or without an intracellular domain. In a specific embodiment, the CRIg variant binds to a polypeptide derived from the sequence of SEQ ID NO: 2 has at least about 80% amino acid sequence homology. In another embodiment, the CRIg variant binds to a peptide from SEQ ID NO: 4 has at least about 80% amino acid sequence homology. In yet another embodiment, the CRIg variant binds to a polypeptide from SEQ ID NO: 6 has at least about 80% amino acid sequence homology. In yet another embodiment, the CRIg variant binds to a peptide from SEQ ID NO: 8, and at least about 80% amino acid sequence homology to the mature, full-length polypeptide within the sequence of seq id No. 8. Such CRIg polypeptide variants include, for example, those in which the amino acid sequence set forth in SEQ ID NO: 2, 4, 6 or 8 having one or more amino acid residue insertions, substitutions and/or deletions at the N-or C-terminus of the sequence. Other variants have one or more amino acid insertions, substitutions and/or deletions within the transmembrane region of the indicated polypeptide sequence.

Typically, a CRIg variant will hybridize to a sequence derived from SEQ ID NO: 2, 4, 6, or 8, or at least about 80% amino acid sequence identity, or at least about 85% amino acid sequence identity, or at least about 90% amino acid sequence identity, or at least about 95% amino acid sequence identity, or at least about 98% amino acid sequence identity, or at least about 99% amino acid sequence identity. Preferably, the highest degree of sequence identity exists within the extracellular domain (ECD) (amino acid 1 or about 21 to X of SEQ ID NO: 2 or 4, wherein X is any amino acid residue at position 271 and 281; or amino acid 1 or about 21 to X of SEQ ID NO: 6, wherein X is any amino acid residue at position 178 and 186; or amino acid 1 or about 21 to X of SEQ ID NO: 8, wherein X is any amino acid residue at position 176 and 184).

CRIg (PRO362) "extracellular domain" or "ECD" refers to a form of CRIg polypeptide that is substantially free of transmembrane and cytoplasmic domains of the respective full-length molecule. Typically, the CRIg ECD will have less than 1% of such transmembrane and/or cytoplasmic domains, and preferably, will have less than 0.5% of such domains. As described above, optionally, the CRIg ECD will comprise the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8 or about 21 to X, wherein X is SEQ ID NO: 2 or 4, any amino acid of about 271 to 281 of SEQ ID NO: 6, and any amino acid from about 178 to 186 of SEQ ID NO: 8 from about 176 to 184.

"percent (%) amino acid sequence identity" with respect to a CRIg (PRO362) sequence identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in the CRIg sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, respectively, and not considering any conservative substitutions as part of the sequence identity. Sequence alignments for the purposes of determining percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. The sequence identity is then calculated relative to the longer sequence, i.e. even if the shorter sequence shows 100% sequence identity to a part of the longer sequence, the overall sequence identity will be less than 100%.

"percent (%) nucleic acid sequence identity" with respect to a CRIg (PRO362) coding sequence (e.g., DNA45416) identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical to the nucleotides in the CRIg coding sequence, after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percent sequence identity, respectively. Sequence alignments for the purposes of determining percent nucleic acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. The sequence identity is then calculated relative to the longer sequence, i.e. even if the shorter sequence shows 100% sequence identity to a part of the longer sequence, the overall sequence identity will be less than 100%.

An "isolated" nucleic acid molecule refers to a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule with which it is ordinarily associated in its natural source. An isolated nucleic acid molecule is distinguished from the form or context in which it is found in nature. An isolated nucleic acid molecule is thus distinguished from a nucleic acid molecule that is present in a natural cell. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in a cell that normally expresses the encoded polypeptide, for example, when the chromosomal location of the nucleic acid molecule in the cell is different from its chromosomal location in a native cell.

An "isolated" CRIg polypeptide-encoding nucleic acid molecule refers to a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule with which it is normally associated in its natural source. An isolated CRIg polypeptide-encoding nucleic acid molecule that is different from the form or context in which it is found in nature. An isolated CRIg polypeptide-encoding nucleic acid molecule is therefore distinguished from encoding nucleic acid molecules present in natural cells. However, an isolated CRIg-encoding nucleic acid molecule includes a CRIg-encoding nucleic acid molecule contained in a cell that normally expresses CRIg, for example when the chromosomal location of the nucleic acid molecule in the cell is different from its chromosomal location in a native cell.

The term "complement-associated disease" is used herein in the broadest sense and includes all diseases and pathological conditions whose pathogenesis involves abnormal activation of the complement system, such as, for example, complement deficiency. The term specifically includes diseases and pathological conditions that benefit from inhibition of the C3 convertase. The term further includes diseases and pathological conditions that benefit from inhibition of the alternative complement pathway, including selective inhibition. Complement-associated diseases include, but are not limited to, inflammatory and autoimmune diseases, such as, for example, Rheumatoid Arthritis (RA), Acute Respiratory Distress Syndrome (ARDS), distal tissue damage following ischemia and reperfusion, complement activation during cardiopulmonary bypass surgery, dermatomyositis, pemphigus, lupus nephritis and glomerulonephritis and vasculitis occurring therefrom, cardiopulmonary bypass, cardiac arrest-induced coronary endothelial dysfunction, type II membranoproliferative glomerulonephritis, IgA nephropathy, acute renal failure, cryoglobulinemia, antiphospholipid syndrome, macular degeneration and other complement-associated eye conditions, such as age-related macular degeneration (AMD), Choroidal Neovascularization (CNV), uveitis, diabetic and other ischemia-related retinopathies, endophthalmitis, and other intraocular neovascular diseases, such as diabetic macular edema, Pathological myopia, von hippel-Lindau disease, histoplasmosis of the eye, Central Retinal Vein Occlusion (CRVO), corneal neovascularization, retinal neovascularization, and allograft, hyperacute rejection, hemodialysis, chronic obstructive pulmonary stress syndrome (COPD), asthma, and aspiration pneumonia.

The term "complement-associated eye condition" is used herein in the broadest sense, including all eye conditions and diseases whose pathology involves complement, including the classical and alternative pathways of complement, in particular the alternative pathway. This group specifically includes all ocular conditions and diseases associated with the alternative pathway whose occurrence, formation, or development can be controlled by inhibition of the alternative pathway. Complement-associated ocular conditions include, but are not limited to, macular degeneration, such as age-related macular degeneration (AMD), including both dry and wet (non-exudative and exudative) forms, Choroidal Neovascularization (CNV), uveitis, diabetic and other ischemia-related retinopathies, endophthalmitis, and other intraocular neovascular diseases, such as diabetic macular edema, pathologic myopia, von Hippel-Lindau disease, histoplasmosis of the eye, Central Retinal Vein Occlusion (CRVO), corneal neovascularization, and retinal neovascularization at all stages. A preferred group of complement-associated eye conditions includes age-related macular degeneration (AMD), including non-exudative (wet) and exudative (dry or atrophic) AMD, Choroidal Neovascularization (CNV), Diabetic Retinopathy (DR), and endophthalmitis.

The terms "inflammatory disease" and "inflammatory disorder" are used interchangeably to refer to a disease or disorder in which a component of the mammalian immune system causes, mediates, or otherwise contributes to an inflammatory response, contributing to the pathogenesis of the mammal. Also included are diseases in which a reduction in immune response has an ameliorating effect on disease progression. The term includes immune-mediated inflammatory diseases, including autoimmune diseases.

The term "T cell mediated" disease refers to a disease in which T cells directly or indirectly mediate or otherwise contribute to the pathogenesis of a mammal. T cell mediated diseases may be associated with cell mediated effects, lymphokine mediated effects, and the like, and even B cell-related effects, if stimulated, for example by lymphokines secreted by T cells.

Examples of immune-related and inflammatory diseases, someIs T cell mediated, including but not limited to Inflammatory Bowel Disease (IBD), systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), Sjogren's disease (Sj)gren) syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis (Graves 'disease), Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis, diabetes, immune-mediated nephropathy (glomerulonephritis, tubulointerstitial nephritis), demyelinating diseases of the central and peripheral nervous system such as multiple sclerosis, idiopathic polyneuropathy, hepatobiliary diseases such as infectious hepatitis (a, b, c, d, e and other non-hepadnaviral hepatitis), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, And sclerosing cholangitis, inflammatory and fibrotic pulmonary diseases (e.g. cystic fibrosis), gluten-sensitive bowel disease, Whipple's (Whipple) disease, autoimmune or immune-mediated skin diseases including bullous skin disease, erythema multiforme and contact dermatitis, psoriasis, allergic diseases of the lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, transplant-related diseases including graft rejection, graft-versus-host disease, Alzheimer's (Alzheimer's) disease, and atherosclerosis.

"tumor" as used herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous (pre-cancerous) cells and tissues.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular cancer, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, liver cancer (liver cancer), vulval cancer, thyroid cancer, liver cancer (hepatoma), and various types of head and neck cancer.

"treatment" and "treating" refer to interventions intended to prevent the development of a disorder or to modify the pathology of a disorder. Thus, "treatment" and "treatment" refer to both therapeutic treatment and preventative or prophylactic measures. Subjects in need of treatment include subjects who have already suffered from the disorder as well as subjects in whom the disorder is to be prevented. In the treatment of immune related diseases, therapeutic agents may directly alter the magnitude of the immune response component response or make the disease more susceptible to treatment with other therapeutic agents, such as antibiotics, antifungal agents, anti-inflammatory agents, chemotherapeutic agents, and the like. In the treatment of complement-associated diseases, the treatment may, for example, prevent or slow the progression of the disease. Thus, treatment of a complement-associated ocular condition specifically includes preventing, inhibiting, or slowing the formation of the condition or progressing from one stage of the condition to another, higher stage or to a more severe associated condition.

"pathology" of diseases such as complement-associated diseases includes all phenomena, including patient comfort (well-beng). This includes, but is not limited to, abnormal or uncontrolled cell growth (neutrophils, eosinophils, monocytes, lymphocytes), antibody production, autoantibody production, complement production, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or exacerbation of any inflammatory or immunological response, infiltration of inflammatory cells (neutrophils, eosinophils, monocytes, lymphocytes) into the cellular space (cellular space), drusen formation, vision loss, and the like.

The term "mammal" as used herein refers to any animal classified as a mammal, including, but not limited to, humans, non-human primates, domestic and livestock animals, and zoo, sports, or pet animals, such as horses, pigs, cows, dogs, cats, and ferrets, among others. In a preferred embodiment of the invention, the mammal is a human or a non-human primate, most preferably a human.

Administration "in combination with" one or more other therapeutic agents includes simultaneous (concurrent) administration and sequential administration in any order.

The term "cytokine" is a generic term for proteins released by one cell population that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; (ii) a relaxin; a prorelaxin; glycoprotein hormones such as Follicle Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH), and Luteinizing Hormone (LH); a liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and-beta; mullerian (Mullerian) inhibitory substances; mouse gonadotropin-related peptides; a statin; an activin; vascular endothelial growth factor; an integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet-derived growth factor; transforming Growth Factors (TGF), such as TGF-alpha and TGF-beta; insulin-like growth factors-I and-II; erythropoietin (EPO); an osteoinductive factor; interferons such as interferon- α, - β, and- γ; colony Stimulating Factors (CSFs), such as macrophage CSFs (M-CSF), granulocyte-macrophage CSFs (GM-CSF), and granulocyte CSFs (G-CSF); interleukins (IL), such as IL-1, IL-1 α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; tumor necrosis factors such as TNF- α or TNF- β; and other polypeptide factors, including LIF and Kit Ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

By "therapeutically effective amount" is meant an amount of active CRIg, CRIg agonist and antagonist required to achieve a measurable improvement in a target disease or condition, such as, for example, a complement-associated (ocular) disease or condition or a state of cancer, e.g., a pathology.

The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. For example, control sequences suitable for prokaryotes include a promoter, an optional operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

A nucleic acid is "operably linked" if it is in a functional relationship with another nucleic acid sequence. For example, a DNA of a presequence (sequence) or secretion leader (secretory leader) is operably linked to a DNA of a polypeptide if it is expressed as a preprotein (preprotein) involved in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of that sequence; alternatively, if the ribosome binding site is positioned to facilitate translation, it is operably linked to a coding sequence. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading order. However, enhancers need not be contiguous. Ligation may be achieved by ligation reactions at convenient restriction sites. If there are no such sites, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

The "stringency" of hybridization reactions can be readily determined by one of ordinary skill in the art, and is generally calculated empirically based on probe length, washing temperature, and salt concentration. Generally, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, it was concluded that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures are less stringent. For additional details and explanation of the stringency of hybridization reactions, see Ausubel et al, Current protocols in Molecular Biology, Wiley Interscience Publishers, 1995.

"stringent conditions" or "high stringency conditions", as defined herein, can be identified as follows: (1) washing with low ionic strength and high temperature, e.g., 0.015M sodium chloride/0.0015M sodium citrate/0.1% sodium lauryl sulfate, 50 ℃; (2) denaturing agents such as formamide, e.g., 50% (v/v) formamide and 0.1% bovine serum albumin/0.1% FicolI/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer pH6.5 with 750mM sodium chloride, 75mM sodium citrate, 42 ℃; or (3) washing with 50% formamide, 5 XSSC (0.75M NaCl, 0.075M sodium citrate), 50mM sodium phosphate (pH6.8), 0.1% sodium pyrophosphate, 5 XDenhardt's solution, sonicated salmon sperm DNA (50. mu.g/ml), 0.1% SDS, and 10% dextran sulfate, 42 ℃, and 42 ℃ in 0.2XSSC (sodium chloride/sodium citrate) and 50% formamide, followed by high stringency washing in 0.1 XSSC with EDTA at 55 ℃.

"moderately stringent conditions" can be as defined in Sambrook et al, Molecular Cloning: a laboratory Manual, New York, Cold Spring Harbor Press, 1989, including the use of less stringent wash solutions and hybridization conditions (e.g., temperature, ionic strength and% SDS) than those described above. An example of moderately stringent conditions is conditions comprising: 20% formamide, 5 XSSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5 XDenhardt's solution, 10% dextran sulfate, and 20mg/ml denatured sheared salmon sperm DNA solution in temperature overnight, followed by 1 XSSC at about 37-50 ℃ washing filter membrane. The skilled person will recognize how to adjust the temperature, ionic strength, etc. as required to accommodate factors such as probe length.

The term "epitope-tagged" as used herein refers to a chimeric polypeptide comprising a polypeptide of the present invention fused to a "tag polypeptide". The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, but is short enough so that it does not interfere with the activity of the polypeptide to which it is fused. The tag polypeptide is also preferably quite unique such that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides typically have at least 6 amino acid residues and typically between about 8 and about 50 amino acid residues (preferably between about 10 and about 20 amino acid residues).

"active" or "activity" in the context of a CRIg polypeptide variant of the invention refers to a form of such polypeptide that retains the biological and/or immunological activity of the native or naturally occurring polypeptide of the invention. One preferred biological activity is the ability to bind C3b and/or to affect complement or complement activation, in particular to inhibit the alternative complement pathway and/or the C3 convertase. Inhibition of C3 convertase can be measured, for example, by measuring inhibition of C3 turnover (turnover) in normal serum during collagen or antibody induced arthritis or inhibition of C3 deposition in arthritic joints.

"biological activity" in the context of antibodies, polypeptides, or other molecules (e.g., organic or inorganic small molecules, peptides, etc.) that mimic CRIg biological activity and can be identified by the screening assays disclosed herein, refers in part to the ability of such molecules to bind C3b and/or to affect complement or complement activation, particularly to inhibit the alternative complement pathway and/or the C3 convertase.

The term CRIg "agonist" is used in the broadest sense and includes any molecule that qualitatively mimics the biological activity (as defined above) of a native sequence CRIg polypeptide. Such CRIg agonists expressly include CRIg-Ig, such as CRIg-Fc fusion polypeptides (immunoadhesins), and also include small molecules that mimic at least one CRIg biological activity. Preferably, the biological activity refers to blocking the complement pathway, particularly the alternative complement pathway.

The term "antagonist" is used in the broadest sense and includes any molecule that partially or completely qualitatively blocks, inhibits, or neutralizes a biological activity of a native polypeptide, such as a native sequence CRIg polypeptide.

Suitable agonist or antagonist molecules specifically include agonistic or antagonistic antibodies or antibody fragments, fusions or amino acid sequence variants of the native polypeptides of the invention, peptides, small molecules, including organic small molecules, and the like.

A "small molecule" is defined herein as having a molecular weight of less than about 600, preferably less than about 1000 daltons.

The term "antibody" is used in the broadest sense and specifically covers, but is not limited to, single anti-CRIg monoclonal antibodies (including agonistic, antagonistic, and neutralizing antibodies) and anti-CRIg antibody compositions with polyepitopic specificity. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical, except for possible naturally occurring mutations that may be present in minor amounts.

"antibody" (Ab) and "immunoglobulin" (Ig) refer to glycoproteins having the same structural features. While antibodies exhibit binding specificity to a particular antigen, immunoglobulins include both antibodies and other antibody-like molecules that lack antigen specificity. The latter class of polypeptides is produced, for example, at low levels by the lymphatic system and at elevated levels by myeloma. The term "antibody" is used in the broadest sense and specifically covers, but is not limited to, intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity.

"native antibodies" and "native immunoglobulins" are typically heterotetrameric glycoproteins of about 150,000 daltons composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies among heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (V) at one end_H) Followed by a plurality of constant regions (C)_H). Each light chain has a variable region (V) at one end_L) To do soThe other end is a constant region (C)_L). The constant region of the light chain is aligned with the first constant region of the heavy chain, and the variable region of the light chain is aligned with the variable region of the heavy chain. It is believed that particular amino acid residues form an interface between the light and heavy chain variable regions.

The term "variable" refers to the fact that certain portions of the variable regions differ widely in antibody sequence and are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in both the light and heavy chain variable regions. The more highly conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FRs, which largely adopt a β -sheet conformation, connected by three CDRs which form loops and, in some cases, form part of the β -sheet structure. The CDRs in each chain are held together in close proximity by the FRs and, together with the CDRs of the other chain, contribute to the formation of the antigen binding site of the antibody (see Kabat et al, NIH Publ. No.91-3242, Vol. I, pages647-669 (1991)). The constant regions are not directly involved in binding of the antibody to the antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; a diabody; linear antibodies (Zapata et al, Protein Eng.8 (10): 1057-1062[1995 ]]) (ii) a A single chain antibody molecule; and multispecific antibodies formed from antibody fragments. Specifically, examples of antibody fragments encompassed by this definition include: (i) a Fab fragment having VL, CL, VH and CH1 domains; (ii) a Fab' fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CH1 domain; (iii) an Fd fragment having VH and CH1 domains; (iv) an Fd' fragment having the VH and CH1 domains and one or more cysteine residues C-terminal to the CH1 domain; (v) fv fragments having the VL and VH structure of a one-armed antibodyA domain; (vi) dAb fragments (Ward et al, Nature 341: 544-546(1989)) which are composed of VH domains; (vii) an isolated CDR region; (viii) f (ab')₂A fragment comprising a bivalent fragment of two Fab' fragments linked by a disulfide bond in the hinge region; (ix) single chain antibody molecules (e.g., single chain Fv; scFv) (Bird et al, Science 242: 423-; (x) "diabodies" having two antigen-binding sites comprising a heavy chain variable region (VH) and a light chain variable region (VL) joined in the same polypeptide chain (see, e.g., EP404, 097; WO 93/11161; Hollinger et al, Proc. Natl. Acad. Sci. USA 90: 6444-; (xi) "Linear antibodies" comprise a pair of Fd segments (VH-CH1-VH-CH1) in tandem, together with a complementary light chain polypeptide, to form a pair of antigen binding regions (Zapata et al, Protein Eng.8 (10): 1057-1062 (1995); U.S. Pat. No. 5,641,870).

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each having an antigen-binding site, and a residual "Fc" fragment. The designation "Fc" reflects the ability to crystallize readily. Pepsin treatment produced an F (ab')₂A fragment which has two antigen binding sites and is still capable of cross-linking antigens.

"Fv" is the smallest antibody fragment that contains the entire antigen recognition and antigen binding site. This region consists of a dimer of one heavy chain variable region and one light chain variable region in tight, non-covalent association. It is in this configuration that the three CDRs of each variable region interact with each other at V_H-V_LThe surface of the dimer defines an antigen binding site. The six CDRs collectively confer antigen binding specificity to the antibody. However, even a single variable region (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

The Fab fragment also contains the constant region of the light chain and the first constant region of the heavy chain (C)_H1). Fab' fragments are generated by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domainAnd, unlike Fab fragments, include one or more cysteines from the antibody hinge region. Fab '-SH is the designation herein for Fab' in which the cysteine residues of the constant region carry a free thiol group. F (ab') ₂Antibody fragments were originally produced as pairs of Fab 'fragments with hinge cysteines between the Fab' fragments. Other chemical couplings of antibody fragments are also known.

Antibody (immunoglobulin) light chains from any vertebrate species can be classified into one of two distinct types, called kappa (κ) and lambda (λ), based on their constant region amino acid sequences.

Immunoglobulins can be assigned to different classes depending on their heavy chain constant region amino acid sequences. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2. The heavy chain constant regions corresponding to different immunoglobulin classes are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical, except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In addition, unlike conventional (polyclonal) antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by a monoclonal antibody originally produced by Kohler et al, Nature 256: 495(1975), or can be prepared by recombinant DNA methods (see, e.g., U.S. patent No. 4,816,567). "monoclonal antibodies" can also be used, for example, as described in Clackson et al, Nature 352: 624-: the technique described in 581-597(1991) was isolated from phage antibody libraries. See also U.S. Pat. nos. 5,750,373, 5,571,698, 5,403,484 and 5,223,409, which describe the use of phagemids and phage vectors for the preparation of antibodies.

Monoclonal antibodies specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remaining portion of the chain is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al, Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)).

"humanized" forms of non-human (e.g., murine) antibodies refer to chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab ', F (ab')₂Or other antigen binding subsequences of antibodies). For the most part, humanized antibodies refer to an immunoglobulin in which several or all residues of the Complementarity Determining Regions (CDRs) in a human immunoglobulin (the acceptor antibody) are replaced with CDR residues from a non-human species (the donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv Framework Region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may comprise residues that are not found in either the recipient antibody or the import CDR or framework sequences. These modifications are made in order to further improve and maximize the performance of the antibody. Typically, a humanized antibody will comprise substantially all of at least one, and typically two, variable regions in which all or substantially all of the CDRs correspond to CDRs of a non-human immunoglobulin and all or substantially all of the FRs are FRs of a human immunoglobulin sequence. Preferably, the humanized antibody will also comprise A small portion of an immunoglobulin constant region (Fc), typically of a human immunoglobulin. See Jones et al, Nature 321: 522-525 (1986); riechmann et al, Nature 332: 323-329 (1988); presta, curr, op, struct, biol.2: 593-596(1992). Humanized antibodies include "primatized" antibodies in which the antigen binding region of the antibody is derived from an antibody generated by immunization of a cynomolgus monkey (macaque monkey) with the antigen of interest. Antibodies comprising residues from old world monkeys (O1dWorld monkeys) are also possible within the invention. See, e.g., U.S. Pat. nos. 5,658,570; 5,693,780, respectively; 5,681,722, respectively; 5,750,105, respectively; and 5,756,096.

"Single chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide is at V_HAnd V_LPolypeptide linkers are also included between the domains to enable sFv formation of the desired structure for antigen binding. For a review of sFv see Pl ü ckthun, The Pharmacology of Monoclonal Antibodies, vol.113, eds Rosenburg and Moore, Springer-Verlag, New York, pp.269-315, 1994.

The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments are in the same polypeptide chain (V) _H-V_L) In (b) comprises a linked heavy chain variable region (V)_H) And light chain variable region (V)_L). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of the other chain and two antigen binding sites are created. Diabodies are described more fully in, for example, EP404, 097; WO 93/11161; hollinger et al, proc.natl.acad.sci.usa90: 6444-6448(1993).

The "CH 2 domain" (also referred to as the "Cg 2" domain) of the human IgG Fc region typically extends from about amino acid residue 231 to about amino acid residue 340. The CH2 domain is unique in that it is not closely paired with another domain. Instead, two N-linked branched carbohydrate chains intervene between the two CH2 domains of the intact native IgG molecule. It is speculated that carbohydrates may provide an alternative to domain-domain pairings and help stabilize the CH2 domain. Burton, molecular. immunol.22: 161-206(1985). The CH2 domain may be a native sequence CH2 domain or a variant CH2 domain herein.

The "CH 3 domain" comprises a stretch of residues from the C-terminus of the CH2 domain in the Fc region (i.e., from about amino acid residue 341 to about amino acid residue 447 of IgG). The CH3 region may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain with an introduced "bulge" (protoberance) in one strand and a corresponding introduced "cavity" (cavity) in the other strand-see U.S. Pat. No. 5,821,333, expressly incorporated herein by reference). Such variant CH3 domains can be used to generate multispecific (e.g., bispecific) antibodies as described herein.

A "hinge region" is generally defined as the segment from about Glu216 or about Cys226 to about Pro230 of human IgG1 (Burton, molecular. Immunol.22: 161-206 (1985)). The hinge region of other IgG isotypes can be aligned to the IgG1 sequence by placing the first and last cysteine residues that form the S-S bond between heavy chains at the same position. The hinge region may be a native sequence hinge region or a variant hinge region herein. The two polypeptide chains of the variant hinge region typically retain at least one cysteine residue per polypeptide chain, such that the two polypeptide chains of the variant hinge region can form a disulfide bond between the two chains. Preferred hinge regions herein are native sequence human hinge regions, for example native sequence human IgG1 hinge regions.

A "functional Fc region" has at least one "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; complement Dependent Cytotoxicity (CDC); fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors; BCR); and the like. Such effector functions typically require the Fc region to be combined with a binding domain (e.g., an antibody variable region), and can be assessed using a variety of assays known in the art for assessing effector functions of such antibodies.

A "native sequence Fc region" comprises an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature.

A "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region due to at least one amino acid modification. Preferably, the variant Fc region has at least one amino acid substitution, such as about one to about ten amino acid substitutions, preferably about one to about five amino acid substitutions, in the native sequence Fc region or in the Fc region of the parent polypeptide as compared to the native sequence Fc region or to the Fc region of the parent polypeptide. A variant Fc region will generally have, for example, at least about 80% sequence identity, or at least about 90% sequence identity, or at least about 95% or greater sequence identity with a native sequence Fc region and/or with an Fc region of a parent polypeptide herein.

An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more of its CDRs that result in an improvement in the affinity of the antibody for the antigen as compared to the parent antibody without those alterations. Preferred affinity matured antibodies will have nanomolar or even picomolar levels of affinity for the target antigen. Affinity matured antibodies can be generated by procedures known in the art. Marks et al, Bio/Technology 10: 779-783(1992) describes affinity maturation by VH and VL domain shuffling. The following documents describe random mutagenesis of CDR and/or framework residues: barbas et al, proc.nat.acad.sci.usa 91: 3809-3813 (1994); schier et al, Gene 169: 147-; yelton et al, j.immunol.155: 1994-2004 (1995); jackson et al, j.immunol.154 (7): 3310-9 (1995); and Hawkins et al, j.mol.biol.226: 889-896(1992).

"Flexible linker" refers herein to a peptide comprising two or more amino acid residues linked by peptide bonds and providing more rotational freedom to two polypeptides (such as two Fd regions) linked by it. The rotational freedom allows for more efficient access to the target antigen by each of the two or more antigen binding sites connected by the flexible linker. Examples of suitable flexible linker peptide sequences include gly-ser, gly-ser-gly-ser, ala-ser, and gly-gly-gly-ser.

"Single chain Fv" or "sFv" antibody fragments comprise the V of an antibody_HAnd V_LDomains, wherein the domains are present on a single polypeptide chain. Typically, the Fv polypeptide is at V_HAnd V_LPolypeptide linkers are also included between the domains to enable sFv formation of the desired structure for antigen binding. For a review of sFv see Pl ü ckthun, The Pharmacology of monoclonal Antibodies, vol.113, Rosenburg and Moore eds, Springer-Verlag, New York, pp.269-315, 1994.

An "isolated" polypeptide such as an antibody refers to a polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment refer to substances that would interfere with diagnostic or therapeutic uses of the antibody and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the polypeptide, including antibodies, are purified (1) to greater than 95% by weight, most preferably greater than 99% by weight, of the antibody as determined by the Lowry method, (2) to an extent sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues by use of a rotor sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using coomassie blue or, preferably, silver staining. An isolated compound, such as an antibody or other polypeptide, includes a compound in situ within a recombinant cell, since at least one component of the compound's natural environment will not be present. However, the isolated compound will generally be prepared by at least one purification step.

The word "label" as used herein refers to a detectable compound or composition that is conjugated, directly or indirectly, to a compound, such as an antibody or polypeptide, to produce a "labeled" compound. The label may be detectable by itself (e.g., a radioisotope label or a fluorescent label), or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.

"solid phase" refers to a non-aqueous matrix to which the compounds of the present invention can adhere. Examples of solid phases contemplated herein include those made partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamide, polystyrene, polyvinyl alcohol, and polysiloxanes (silicones). In certain embodiments, depending on the context, the solid phase may comprise the wells of an assay plate; in other embodiments, it refers to a purification column (e.g., an affinity chromatography column). This term also includes discontinuous solid phases of discrete particles, such as described in U.S. Pat. No. 4,275,149.

"liposomes" refers to vesicles composed of various types of lipids, phospholipids and/or surfactants that can be used to deliver drugs (such as the anti-ErbB 2 antibodies and optional chemotherapeutic agents disclosed herein) to mammals. The components of liposomes are generally arranged in bilayer form, similar to the lipid arrangement of biological membranes.

As used herein, the term "immunoadhesin" refers to antibody-like molecules that combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of an immunoglobulin constant region. Structurally, immunoadhesins comprise fusions of amino acid sequences and immunoglobulin constant region sequences that differ from the antigen recognition and binding site of the antibody (i.e., are "heterologous"), with the desired binding specificity. The adhesin part of an immunoadhesin molecule is typically a contiguous (contiguous) amino acid sequence comprising at least the binding site for a receptor or ligand. The immunoglobulin constant region sequences in immunoadhesins can be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3 or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM.

"angiogenic factor or agent" refers to a growth factor that stimulates vascular development, e.g., promotes angiogenesis, endothelial cell growth, vascular stability, and/or angiogenesis (vasculogenesis). For example, angiogenic factors include, but are not limited to, e.g., VEGF and members of the VEGF family, PlGF, the PDGF family, the fibroblast growth factor family (FGF), TIE ligands (angiogenin), ephrins, ANGPTL3, ANGPTL4, and the like. It will also include factors that accelerate wound healing such as growth hormone, insulin-like growth factor-I (IGF-I), VIGF, Epidermal Growth Factor (EGF), CTGF and members of its family, and TGF-alpha and TGF-beta. See, e.g., klags brun and D' Amore, annu. rev. physiol.53: 217-39 (1991); streit and Detmar, Oncogene 22: 3172 3179 (2003); ferrara & Alitalo, Nature Medicine5 (12): 1359-; tonini et al, Oncogene 22: 6549-6556(2003) (e.g., Table 1 listing angiogenesis factors); sato int.j.clin.oncol.8: 200-206(2003).

An "anti-angiogenic agent" or "angiogenesis inhibitor" refers to a small molecular weight substance, polynucleotide, polypeptide, isolated protein, recombinant protein, antibody, or conjugate or fusion protein thereof that inhibits, either directly or indirectly, angiogenesis (vasculogenesis), or unwanted vascular permeability. For example, the anti-angiogenic agent is an antibody or other antagonist of an angiogenic agent as defined above, e.g., an antibody to VEGF receptor, a small molecule that blocks VEGF receptor signaling (e.g., PTK787/ZK2284, SU 6668). Anti-angiogenic agents also include natural angiogenesis inhibitors such as angiostatin (angiostatin), endostatin (endostatin), and the like. See, e.g., klags brund D' Amore, annu. rev. physiol.53: 217-39 (1991); streit and Detmar, Oncogene 22: 3172-3179(2003).

The term "effective amount" refers to an amount of a drug effective to treat (including prevent) a disease or disorder in a mammal. Thus, in the case of age-related macular degeneration (AMD) or Choroidal Neovascularization (CNV), an effective amount of the drug may reduce or prevent vision loss. For AMD therapy, in vivo efficacy may be measured, for example, by one or more of: evaluating the mean change in Best Corrected Visual Acuity (BCVA) from baseline to expected time; assessing the proportion of subjects who have vision loss of less than 15 letters compared to baseline at the expected time; assessing the proportion of subjects who gained vision in excess of or equal to 15 letters at the expected time compared to baseline; assessing the proportion of subjects whose Snellen vision corresponds to 20/2000 or worse at the expected time; evaluate the NEI Visual function Questionnaire (Visual functional Questionaire); assessing the size of the CNV at the expected time and the amount of CNV leakage, assessed by fluorescein angiography; and the like. If the indication is the prevention of dry AMD from wet AMD or the prevention of AMD from CMV, then the effective amount of the drug may inhibit, slow, or partially or completely block the progression. In this case, the determination of the effective amount involves grading of the disease, monitoring the time course of disease progression, and adjusting the dose as necessary to achieve the desired result.

Detailed description of the invention

The present invention concerns the use of complement receptor (CRIg) polypeptides identified as macrophage-associated single-transmembrane Ig superfamily members (stigmas) or immunoglobulin families, derived from fetal lung library clones, novel macrophage-associated receptors having homology to the a33 antigen and JAM 1. Native human CRIg is expressed as two splice variants, one comprising an N-terminal IgV-like domain and a C-terminal IgC 2-like domain, and one splice form lacking the C-terminal domain (SEQ ID NOS: 4 and 6, respectively). Both receptors have a single transmembrane and cytoplasmic domain containing tyrosine residues that are constitutively phosphorylated in macrophages in vitro. The mouse homolog was found to have 67% sequence homology to human CRIg (SEQ ID NO: 8). The full-length human CRIg polypeptide also has a shorter version, lacking the N-terminal segment (SEQ ID NO: 2).

As shown in the examples below, CRIg binds complement C3b and inhibits C3 convertase. CRIg is selectively expressed on tissue resident macrophages, its expression is upregulated by dexamethasone and IL-10, downregulated by LPS and IFN- γ, and inhibits collagen and antibody induced arthritis independent of B or T cell responses.

In addition, CRIg was found to be highly expressed on Kupffer cells, binds C3b and iC3b opsonins, and is required for rapid clearance of circulating pathogens. Structurally, CRIg differs from known complement receptors in that it lacks the combined C3b and C4b binding short consensus repeats in CR1 and CR2, and the integrin-like domains present in C3 and CR 4. Although complement receptor CR1-4 is expressed on a wide variety of cell types, CRIg expression is limited to tissue-resident macrophages, including liver Kupffer cells.

Subtractive studies established the role of Kupffer cells in the C3-dependent rapid clearance of Listeria early in infection (Kaufmann, Annu Rev. Immunol.11: 129-315 (1993); Gregory et al, J. Immunol.168: 308-315(2002)), but the receptors involved in this process have not been identified to date. The studies presented in the examples below demonstrate that CRIg expressed by macrophages bind to C3b and iC3b deposited on the surface of pathogens. Due to this dual binding activity to C3b and iC3b, CRIg is required for efficient clearance of Listeria Monocytogenes (LM) conditioned by these two C3-degrading components.

The importance of CRIg in the rapid clearance of C3-conditioned particles by the liver is further supported by the failure of CRIg knockout (ko) mice to effectively clear C3-conditioned LM from the circulation, resulting in increased pathogen load and mortality in various organs. In the absence of C3, CRIg knockout wild type (wt) mice cleared Listeria as well, indicating that CRIg function is dependent on the presence of C3.

The role of complement receptor CR1-4 in clearing LMs from liver Kupffer cells has not been clearly established. CRl and CR2 are not present on tissue-resident macrophages and are expressed predominantly on follicular dendritic cells and B cells, where they play a role in regulating T and B cell responses (Krych-Goldber and dAtkinson, Immunol. Rev.180: 112-122 (2001); Molina et al, J.Exp.Med.175: 121-129 (1992); and examples). CR3 is expressed at low levels on KC, but CD18, lacking both CR3 and CR4, shares the beta chain, resulting in knockout mice of non-functional receptors showing reduced, but not increased, susceptibility to infection (Wu et al, infection. immun.71: 5986-5993 (2003)). Thus, CRIg represents a rapid clearance of the major component of the reticuloendothelial phagocyte system in C3 opsonized particles.

In addition to its expression on liver Kupffer cells, CRIg is present on macrophage subsets in a variety of tissues, including peritoneum, heart, lung, adrenal gland and intestine. These macrophages are known to play an important role in the phagocytosis of dead cells and cell debris (Almeida et al, Ann.N.Y.Acad.Sci.1019: 135-140 (2004); Castellucci and Zaccheo, prog.Clin.biol.Res.296: 443-451 (1989); Taylor et al, Annu.Rev.Immunol.23: 901-944 (2005)). Expression of CRIg on these resident macrophages can mediate complement dependent opsonophagocytosis of a variety of particles. This is supported by the finding that CRIg knockout mice exhibit a decrease in LM in their heart and liver tissues despite an increase in circulating LM burden. Thus, CRIg represents a novel receptor expressed in tissue macrophages and serves as a portal for rapid clearance of complement opsonizing pathogens.

The results presented in the examples below further demonstrate that CRIg is expressed on the intracellular pool of circulating vesicles, thereby ensuring a continuous supply of CRIg on the cell surface for binding to C3 conditioned particles. In addition, endosomes expressing CRIg rapidly recruit to the particle contact site where they may contribute to the delivery of membranes to the formation of phagocytes. The importance of CRIg in phagocytosis of C3-opsonized particles was shown that KC lacking CRIg is unable to bind C3b and iC3b, resulting in decreased phagocytosis of C3-opsonized listeria monocytogenes (see examples).

The subcellular localization and intracellular trafficking of CRIg is distinct from the known complement C3 receptor. Although CRIg is localized to an endosome in a constitutive cycle, CR1, CR3 and CR4 are localized to secretory vesicles which fuse with the plasma membrane upon cytokine stimulation of the Cell (Sengelov et al, J.Immunol.153: 804-. As a result, CRIg expression on the cell surface was down-regulated upon stimulation of the cells, whereas CR1 and CR3 cell surface expression was elevated upon stimulation. This elevation serves as an important step in binding and phagocytosis, and like CRIg, CR3 concentrates in the phagocytic cups and phagosomes surrounding C3 opsonized particles (Aderem and underhill, annu. rev. immunol. 17: 593-623 (1999)). The constitutive circulation and endocytosis of ligand by CRIg in resting macrophages is consistent with the role of particles that play a role in complement-bound opsonization (e.g. recruitment of activated phagocytes) during the initial stages of bacterial infection prior to inflammatory response, and during clearance of particles from the circulation under non-inflammatory conditions.

Complement plays a key role in body defense and, together with other components of the immune system, protects individuals from pathogen invasion into the body. However, complement can also cause damage to host tissues if not properly activated or controlled. Inappropriate activation of complement has been implicated in the pathogenesis of a variety of diseases known as complement-associated diseases or disorders, such as immune complexes and autoimmune diseases, and a variety of inflammatory conditions, including complement-mediated inflammatory tissue damage. The pathology of complement-associated diseases varies, and may involve a longer or shorter period of complement activation, a complete cascade, activation of only one of the cascades (e.g., the classical or alternative pathway), activation of only some components of the cascade, etc. In some diseases, the complement biological activity of the complement fragment results in tissue damage and disease. Therefore, inhibitors of complement have high therapeutic potential. Selective inhibitors of the alternative pathway would be particularly useful because removal of pathogens and other organisms from the blood by the classical pathway would remain intact.

C3b is known to covalently opsonize the surface of microorganisms invading the body and act as ligands for complement receptors present on phagocytic cells, ultimately leading to phagocytosis of pathogens. In many pathological conditions, such as those listed above, complement will be activated on the cell surface, including the vessel wall, cartilage in joints, globules (glomerili) in the liver, or cells lacking intrinsic complement inhibitors. Complement activation leads to inflammation caused by the chemoattractant properties of the anaphylatoxins C3a and C5a, and can cause damage to self cells by the formation of a membrane attack complex. Without being bound to any particular theory, by binding to C3b, CRIg is believed to inhibit C3 convertase, thereby preventing or alleviating complement-mediated diseases, examples of which have been listed above.

Compounds of the invention

1. Native sequence and variant CRIg polypeptides

The preparation of native CRIg molecules and their nucleic acid and polypeptide sequences has been discussed above. Example 1 shows SEQ ID NO: 4 cloning of full-length huCRIg. CRIg polypeptides can be produced by culturing cells transformed or transfected with a vector comprising a CRIg nucleic acid. Of course, alternative methods are envisaged which are well known in the art and which can be used to prepare CRIg. For example, CRIg sequences or portions thereof can be generated by direct peptide synthesis using Solid Phase techniques (see, e.g., Stewart et al, Solid-Phase peptide Synthesis, W.H.Freeman Co., San Francisco, Calif. (1969); Merrifield, J.am. chem. Soc.85: 2149-. In vitro protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be accomplished, for example, using an Applied Biosystems peptide synthesizer (Foster City, Calif.) using the manufacturer's instructions. The various moieties of CRIg can be chemically synthesized separately and combined using chemical or enzymatic methods to generate full-length CRIg.

CRIg variants can be prepared by introducing appropriate nucleotide changes into DNA encoding the native sequence CRIg polypeptide, or by synthesizing the desired CRIg polypeptide. Those skilled in the art will appreciate that amino acid changes may alter post-translational processing of CRIg, such as changing the number or position of glycosylation sites or altering membrane anchoring characteristics.

Variations in the native sequence CRIg polypeptide described herein can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations, such as those set forth in U.S. patent No. 5,364,934. The variation can be a substitution, deletion or insertion of one or more codons encoding the native sequence or a variant CRIg, resulting in a change in its amino acid sequence compared to the corresponding native sequence or variant CRIg. Optionally, the variation is by replacing at least one amino acid in one or more domains of the native sequence CRIg polypeptide with any other amino acid. Guidelines for determining which amino acid residues can be inserted, substituted or deleted without adversely affecting the desired activity can be found by comparing CRIg to the sequence of a homologous known protein molecule and minimizing the number of amino acid sequence changes made in regions of high homology.

Amino acid substitutions may be the result of replacing one amino acid with another having similar structural and/or chemical properties, such as replacing leucine with serine, i.e., conservative amino acid substitutions. Insertions or deletions may optionally be in the range of 1-5 amino acids. The allowable variation can be determined by systematically making amino acid insertions, deletions, or substitutions in the sequence and testing the resulting variants for activity in vitro assays described in the examples below.

Mutagenesis can be performed using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter et al, Nucl. acids Res.13: 4331 (1986); Zoller et al, Nucl. acids Res.10: 6487(1987)), cassette mutagenesis (Wells et al, Gene 34: 315(1985)), restriction selection mutagenesis (Wells et al, Philos. Trans. R.Soc. London SerA 317: 415(1986)), or other known techniques can be performed on the cloned DNA to generate CRIg variant DNA.

Scanning amino acid analysis can also be employed to identify one or more amino acids that can vary along a contiguous sequence. Preferred scanning amino acids are relatively small neutral amino acids. Such amino acids include alanine, glycine, serine and cysteine. Alanine is generally the preferred scanning amino acid in this class because it eliminates the beta-carbon side chain and is less likely to alter the backbone conformation of the variant. Alanine is also generally preferred because it is the most common amino acid. Furthermore, it is often found in both hidden and exposed locations (Creighton, The Proteins, W.H.Freeman & Co., N.Y.; Chothia, J.mol.biol.150: 1 (1976)). If alanine substitution does not result in sufficient amounts of variant, an isostructural (isoteric) amino acid may be used.

It was found that elimination or inactivation of all or part of the transmembrane and/or cytoplasmic domain does not impair the biological activity of CRIg. Thus, CRIg variants with deletion/inactivation of the transmembrane and/or cytoplasmic regions are clearly within the scope of the invention. Similarly, the presence of biologically active natural short huCRIg and murine homologues demonstrates that the IgC2 region can be eliminated without compromising biological activity.

Covalent modifications of native sequences and variant CRIg polypeptides are included within the scope of the invention. One type of covalent modification involves reacting targeted amino acid residues of CRIg with an organic derivatizing agent capable of reacting with selected side chains or N-or C-terminal residues of the CRIg polypeptide. Derivatization with bifunctional agents is useful, for example, for crosslinking CRIg to a water-insoluble support matrix or surface, e.g., in methods for purifying anti-CRIg antibodies. Commonly used cross-linking agents include, for example, 1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, e.g., esters with 4-azidosalicylic acid, homobifunctional imidoesters including disuccinimidyl esters such as 3, 3' -dithiobis (succinimidyl propionate), bifunctional maleimides such as bis-N-maleimide-1, 8-octane, and reagents such as methyl 3- [ (p-azidophenyl) dithio ] propionimidate.

Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amino groups of the lysine, arginine and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., SanFrancisco, pp.79-86(1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification to a CRIg polypeptide included within the scope of the invention includes altering the native glycosylation pattern of the polypeptide. By "altering the native glycosylation pattern" is herein meant deleting one or more carbohydrate modules (moieities) found in the native sequence CRIg, and/or adding one or more glycosylation sites not present in the native sequence CRIg, and/or altering the proportion and/or composition of sugar residues attached to the glycosylation sites. The predicted native glycosylation site was found at position 170 of murine CRIg, with the sequence NGTG.

Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatic attachment of a carbohydrate module to an asparagine side chain. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine may also be involved in O-linked glycosylation. Native sequence CRIg has insignificant N-glycosylation. The addition of glycosylation sites to CRIg polypeptides can be achieved by altering the amino acid sequence. The alteration can be made, for example, by adding or replacing one or more serine or threonine residues to the native sequence CRIg (for O-linked glycosylation sites), or by adding a recognition sequence for N-linked glycosylation. The CRIg amino acid sequence can optionally be altered by changes at the DNA level, particularly by mutating the DNA encoding the CRIg polypeptide at preselected bases, to generate codons that will be translated into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on a CRIg polypeptide is by chemically or enzymatically coupling a glycoside to the polypeptide. Such methods are described in the art, for example, WO87/05330, published on 9/11 1987; and Aplin and Wriston, CRC crit. rev. biochem, pp.259-306 (1981).

Elimination of the carbohydrate module present on the CRIg polypeptide can be achieved by chemical or enzymatic methods, or by mutational substitution of codons encoding amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and are described, for example, in Hakimuddin et al, arch, biochem, biophysis, 259: 52(1987) and Edge et al, anal. biochem.118: 131(1981). Enzymatic cleavage of carbohydrate modules on polypeptides can be achieved by using a variety of endo-and exoglycosidases, such as Thotakura et al, meth.enzvmol.138: 350 (1987).

Another type of covalent modification to CRIg involves linking the CRIg polypeptide to one of a variety of non-proteinaceous polymers, such as polyethylene glycol, polypropylene glycol or polyoxyalkylene (polyoxakylene), for example as described in U.S. Pat. nos. 4,640,835; 4,496,689, respectively; 4,301,144, respectively; 4,670,417, respectively; 4,791,192 or 4,179,337.

The native sequence and variant CRIg of the invention can also be modified in such a way that a chimeric molecule comprising the CRIg, including a fragment of the CRIg, fused to another heterologous polypeptide or amino acid sequence is formed. In one embodiment, such chimeric molecules comprise a fusion of CRIg with a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind. Epitope tags are typically located at the amino or carboxy terminus of the CRIg polypeptide. The presence of such epitope tagged forms of CRIg polypeptide can be detected using antibodies directed against the tag polypeptide. Furthermore, the provision of an epitope tag makes the CRIg polypeptide easy to purify by affinity purification using anti-tag antibodies or other types of affinity matrices that bind the epitope tag. A variety of tag polypeptides and their corresponding antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; influenza HA tag polypeptide and its antibody 12CA5(Field et al, mol.cell.biol.8: 2159-2165 (1988)); the C-myc tag and its antibodies 8F9, 3C7, 6E10, G4, B7 and 9E10(Evan et al, Molecular and cellular biology 5: 3610-3616 (1985)); and the herpes simplex virus glycoprotein D (gD) tag and its antibodies (Paborsky et al, Protein Engineering3 (6): 547-553 (1990)). Other tag polypeptides include the Flag peptide (Hopp et al, Biotechnology 6: 1204-1210 (1988)); KT3 epitope peptide (Martin et al, Science 255: 192-; alpha-tubulin epitope peptide (Skinner et al, J.biol.chem.266: 15163-15166 (1991)); and the T7 gene 10 protein peptide tag (Lutz-Freyrmuth et al, Proc. Natl. Acad. Sci. USA 87: 6393-.

In another embodiment, the chimeric molecule can comprise a fusion of the CRIg polypeptide or fragment thereof with an immunoglobulin or a specific region of an immunoglobulin. For a bivalent form of the chimeric molecule, such a fusion may be to the Fc region of an IgG molecule. These fusion polypeptides are antibody-like molecules that combine the binding specificity of a heterologous protein ("adhesin") with the effector functions of immunoglobulin constant regions, often referred to as immunoadhesins. Structurally, immunoadhesins comprise fusions of amino acid sequences and immunoglobulin constant region sequences that differ from the antigen recognition and binding site of the antibody (i.e., are "heterologous"), with the desired binding specificity. The adhesin part of an immunoadhesin molecule is typically a contiguous (contiguous) amino acid sequence comprising at least the binding site for a receptor or ligand. The immunoglobulin constant region sequences in immunoadhesins can be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3 or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM.

Chimeras (immunoadhesins) constructed from receptor sequences linked to appropriate immunoglobulin constant region sequences are known in the art. Immunoadhesins reported in the literature include the T cell receptor (Gascoigne et al, Proc. Natl. Acad. Sci. USA 84: 2936-2940 (1987)); CD4(Capon et al, Nature 337: 525-; l-selectin (homing receptor) (Watson et al, J.cell.biol.110: 2221-2229 (1990); Watson et al, Nature 349: 164-167 (1991)); CD44(Aruffo et al, Cell 61: 1303-; CD28 and B7(Linsley et al, J.Exp.Med.173: 721-; CTLA-4(Lisley et al, J.Exp.Med.174: 561-; CD22(Stamenkovic et al, Cell 66: 1133-11144 (1991)); TNF receptors (Ashkenazi et al, Proc. Natl. Acad. Sci. USA 88: 10535-; NP receptors (Bennett et al, J.biol.chem.266: 23060-23067 (1991)); and IgE receptor alpha (Ridgway et al, J.cell.biol.115: abstr.1448 (1991)).

The simplest and most straightforward immunoadhesin design combines the binding domains of the "adhesin" protein with the hinge and Fc regions of immunoglobulin heavy chains. Typically, in making the CRIg-immunoglobulin chimeras of the invention, a nucleic acid encoding the CRIg polypeptide or CRIg polypeptide extracellular domain (ECD) will be fused C-terminally to a nucleic acid encoding the N-terminus of an immunoglobulin constant region sequence, although N-terminal fusions are also possible.

Typically, in such fusions, the encoded chimeric polypeptide will retain at least the functionally active immunoglobulin heavy chain constant region hinge and CH2 and CH3 domains. The C-terminus of the Fc portion of the constant region is also fused, or directly to the N-terminus of heavy chain CH1 or the corresponding region of the light chain.

The precise site at which the fusion is performed is not critical; specific sites are well known and can be selected for the purpose of optimizing the biological activity, secretion or binding characteristics of CRIg-immunoglobulin chimeras.

In some embodiments, CRIg-immunoglobulin chimeras assemble into monomers or homo-or homomultimers, particularly dimers or tetramers, substantially as described in WO 91/08298.

In a preferred embodiment, a sequence of a native sequence human CRIg polypeptide, such as, for example, huCRIg (Long) (SEQ ID NO: 4) or huCRIg (short) (SEQ ID NO: 6), or CRIg extracellular domain sequences (including the ECD of huCRIg (Long) and huCRIg (short)) is fused N-terminally to a C-terminal portion (particularly an Fc domain) of an antibody comprising an immunoglobulin, for example, immunoglobulin G1(IgGl) effector function. It is possible to fuse the entire heavy chain constant region to CRIg or CRIg extracellular domain sequences. More preferably, however, a sequence starting immediately upstream of the papain cleavage site (which chemically defines an IgG Fc; residue 216, 114 as the first residue of the heavy chain constant region or other immunoglobulin analogue site) in the hinge region is used in the fusion. In a particularly preferred embodiment, the CRIg amino acid sequence is fused to the hinge region and CH2 and CH3 of the IgGl, IgG2 or IgG3 heavy chain, or to the CH1, hinge, CH2 and CH3 domains. The precise site at which the fusion is performed is not critical and the optimal site can be determined by routine experimentation. Specific CRIg immunoadhesin structures are illustrated in FIGS. 59-61.

In some embodiments, the CRIg-immunoglobulin chimeras assemble into multimers, particularly homodimers or tetramers. Typically, these assembled immunoglobulins will have a known subunit structure. The basic four-chain building block is the form in which IgG, IgD and IgE are present. The four-chain unit repeats in higher molecular weight immunoglobulins; IgM typically exists as a pentamer of elementary four-chain units held together by disulfide bonds; IgA globulin and occasionally IgG globulin are also present in the serum in multimeric form. In the case of multimers, each four-chain unit may be the same or different.

Alternatively, a CRIg or CRIg extracellular domain sequence can be inserted between immunoglobulin heavy and light chain sequences, resulting in an immunoglobulin comprising a chimeric heavy chain. In this embodiment, the CRIg sequence is fused to the 3' end of the immunoglobulin heavy chain in each arm of the immunoglobulin, either between the hinge and the CH2 domain or between the CH2 and CH3 domains. Hoogenboom et al, mol.immunol.28: 1027-1037(1991) reported similar constructs.

Although the presence of an immunoglobulin light chain is not required in the immunoadhesins of the present invention, an immunoglobulin light chain can be present, either covalently linked to the CRIg-immunoglobulin heavy chain fusion polypeptide or directly fused to the CRIg extracellular domain. In the former case, the DNA encoding the immunoglobulin light chain is typically co-expressed with the DNA encoding the CRIg-immunoglobulin heavy chain fusion protein. Upon secretion, the hybrid heavy and light chains will be covalently linked to provide an immunoglobulin-like structure comprising two pairs of disulfide-linked immunoglobulin heavy-light chains. Suitable methods for preparing such structures are disclosed, for example, in U.S. Pat. No. 4,816,567, issued on 3/28 1989.

Nucleotide sequences encoding certain CRIg-Ig fusion proteins of the invention are shown in figures 59, 60 and 67-70. For example, as shown in figures 67-70, a fusion protein can comprise a linker between CRIg and an immunoglobulin sequence, such as, for example, a short peptide sequence, e.g., DKTHT. In addition, in some constructs, the sequence between the CRIg Transmembrane (TM) region and the immunoglobulin (Fc) region (referred to herein as the "stem" sequence) may be deleted. The amino acid positions at which the linker begins in the various CRIg constructs shown in figures 67-70 are as follows: huCRIg-long-Fc + stem: 267 th position; huCRIg-long-Fc-stem: 233 th bit; huCRIg-short-Fc + stem: position 173; huCRIg-short-Fc-stem: 140 th bit.

2. Preparation of native sequence and variant CRIg Polypeptides

DNA encoding a CRIg polypeptide can be obtained from a cDNA library prepared from tissues thought to have CRIg mRNA and express it at detectable levels. Thus, human CRIg DNA can be conveniently obtained from cDNA libraries prepared from human tissues, such as described in example 1. CRIg encoding genes can also be obtained from genomic libraries or by oligonucleotide synthesis.

Libraries can be screened with probes designed to identify the gene of interest or the protein encoded thereby, such as antibodies to CRIg or oligonucleotides of at least about 20-80 bases. Screening of cDNA or genomic libraries with selected probes can be performed using standard procedures, such as Sambrook et al, Molecular Cloning: a Laboratory Manual, New York, Cold Spring Harbor Laboratory Press, 1989. An alternative method for isolating the gene encoding CRIg is to use PCR methodology (Sambrook et al, supra; Dieffenbach et al, PCR Primer: A Laboratory Manual, Cold spring harbor Laboratory Press, 1995).

Example 1 describes techniques for screening cDNA libraries. The oligonucleotide sequence chosen as a probe should be long enough and unambiguous enough to minimize false positives. The oligonucleotide is preferably labeled so that it is detectable upon hybridization to the DNA in the library being screened. The marking method isAs is well known in the art, including the use of radiolabels, like³²P-labeled ATP, biotinylated or enzyme-labeled. Hybridization conditions, including medium and high stringency, are described in Sambrook et al, supra.

Sequences identified in such library screening methods can be compared and aligned with other known sequences available in deposited and public databases such as GenBank or other private sequence databases. Sequence identity within defined regions of a molecule or across the full-length sequence (either at the amino acid level or at the nucleotide level) can be determined by sequence alignment using computer software programs that employ various algorithms to measure homology, such as BLAST, BLAST-2, ALIGN, DNAstar, and INHERIT.

Nucleic acids having protein coding sequences can be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequences disclosed for the first time herein, and, if necessary, using Sambrook et al, supra, for routine primer extension procedures to detect precursors and processing intermediates of mRNA that may not be reverse transcribed into cDNA.

Host cells are transfected or transformed with the expression or cloning vectors described herein for CRIg production and cultured in conventional nutrient media appropriately adjusted for the induction of promoters, selection of transformants, or amplification of genes encoding the desired sequences. Culture conditions, such as medium, temperature, pH, etc., can be selected by one of skill in the art without undue experimentation. In general, principles, protocols and practical techniques for maximizing Cell culture productivity can be found in Mammalian Cell Biotechnology: aPractical Approach, M.Butler, ed., IRL Press, 1991 and Sambrook et al, supra.

Methods of transfection are known to the skilled worker, e.g.CaPO₄And electroporation. Depending on the host cell used, transformation is carried out using standard techniques appropriate for such cells. Calcium treatment with calcium chloride, as described in Sambrook et al, supra, or electroporation is commonly used for prokaryotes or with a robust cell wall barrierOther cells. Infection with Agrobacterium tumefaciens (Agrobacterium tumefaciens) is used for transformation of certain plant cells, as described by Shaw et al, Gene 23: 315(1983) and WO89/05859, published 1989 on 29.6. For mammalian cells without such cell walls, Graham and van der Eb, Virology 52: 456-457 (1978). See U.S. Pat. No. 4,399,216 for a general case of mammalian cell host system transfection. Transformation into yeast is generally performed according to Van Solingen et al, j.bact.130: 946(1977) and Hsiao et al, proc.natl.acad.sci. (USA) 76: 3829 (1979). However, other methods for introducing DNA into cells may be used, such as nuclear microinjection, electroporation, fusion of bacterial protoplasts with intact cells, or polycations such as polybrene, polyornithine. For various techniques for transforming mammalian cells see Keown et al, Methods in Enzvmology 185: 527- & 537(1990) and mansource et al, Nature 336: 348-352(1988).

Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryotes, yeast, or higher eukaryotes. Suitable prokaryotes include, but are not limited to, eubacteria, such as gram-negative or gram-positive organisms, for example enterobacteriaceae, such as e. Various E.coli strains are publicly available, such as E.coli K12 strain MM294(ATCC31,446); escherichia coli X1776(ATCC31,537); escherichia coli strains W3110(ATCC27,325) and K5772(ATCC53,635).

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for CRIg-encoding vectors. Saccharomyces cerevisiae (Saccharomyces cerevisiae) is a commonly used lower eukaryotic host microorganism.

Suitable host cells for expressing glycosylated CRIg are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, and plant cells. Examples of useful mammalian host cell lines include Chinese Hamster Ovary (CHO) and COS cells. More specific examples include monkey kidney CVl cells transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cells (293 or 293 cells subcloned for growth in suspension culture, Graham et al, J.Gen Virol.36: 59 (1977)); chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA77: 4216 (1980)); mouse Sertoli (sertoli) cells (TM4, Mather, biol. reprod.23: 243-251 (1980)); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); and mouse breast tumor cells (MMT060562, ATCC CCL 51). The selection of suitable host cells is considered to be within the skill of the art.

Nucleic acid encoding CRIg (e.g. cDNA or genomic DNA) can be inserted into a replicative vector for cloning (DNA amplification) or expression. A variety of vectors are publicly available. The vector may be in the form of, for example, a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence can be inserted into the vector by a variety of procedures. Typically, the DNA is inserted into an appropriate restriction endonuclease site using techniques known in the art. Carrier members typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors comprising one or more of these building blocks employs standard ligation techniques known to the skilled artisan.

CRIg polypeptides can be produced recombinantly not only directly, but also as fusion polypeptides with heterologous polypeptides, which can be signal sequences or other polypeptides having specific cleavage sites at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be part of the CRIg DNA inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected from, for example, alkaline phosphatase, penicillinase, 1pp, or a heat-stable enterotoxin II leader sequence. For yeast secretion, the signal sequence may be, for example, a yeast invertase leader, an alpha factor leader (including the sugar yeast and Kluyveromyces alpha-factor leader, see U.S. Pat. No. 5,010,182), or an acid phosphatase leader, a Candida albicans glucoamylase leader (EP 362,179 published 4/1990), or the signal described in WO90/13646 published 11/15/1990. In mammalian cell expression, mammalian signal sequences can be used to direct secretion of proteins, such as signal sequences for secreted polypeptides from the same or related species, and viral secretion leader sequences.

Both expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from plasmid pBR322 is suitable for most gram-negative bacteria, the 2. mu. plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV, or BPV) can be used for cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene, also referred to as a selectable marker. Typical selection genes encode the following proteins: (a) conferring resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline; (b) supplementing the nutritional deficiency; or (c) provide key nutrients not available from complex media, such as a gene encoding a bacillus D-alanine racemase.

An example of a suitable selectable marker for mammalian cells is one that can identify cells having the ability to take up a CRIg nucleic acid, such as DHFR or thymidine kinase. When wild-type DHFR is used, a suitable host cell is a CHO cell line deficient in DHFR activity, prepared and propagated as described in Urlaubet al, proc.natl.acad.sci.usa77: 4216 (1980). A suitable selection Gene for yeast is the trp1 Gene present in the yeast plasmid YRp7 (Stinchcomb et al, Nature 282: 39 (1979); Kingsman et al, Gene 7: 141 (1979); Tschemper et al, Gene 10: 157 (1980)). the trp1 gene provides a selectable marker for yeast mutants lacking the ability to grow in tryptophan, such as ATCC No.44076 or PEP4-1 (Jones, Genetics 85: 12 (1977)).

Expression and cloning vectors typically contain a promoter operably linked to a CRIg nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use in prokaryotic hosts include the beta-lactamase and lactose promoter systems (Chang et al, Nature 275: 615 (1978); Goeddel et al, Nature 281: 544(1979)), alkaline phosphatase, tryptophan (trp) promoter systems (Goeddel, Nucleic acids Res.8: 4057 (1980); EP36,776), and hybrid promoters such as the tac promoter (deBoer et al, Proc. Natl. Acad. Sci. USA 80: 21-25 (1983)). Promoters for bacterial systems will also comprise a Shine-Dalgarno (s.d.) sequence operably linked to DNA encoding CRIg.

Examples of promoter sequences suitable for use in yeast hosts include the promoters of 3-phosphoglycerate kinase (Hitzeman et al, J.biol. chem.255: 2073(1980)) or other glycolytic enzymes (Hess et al, J.adv. enzyme Reg.7: 149 (1968); Holland, Biochemistry 17: 4900(1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters which are inducible promoters having the additional advantage of transcription controlled by growth conditions are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Vectors and promoters suitable for yeast expression are further described in EP73,657.

Transcription of CRIg from vectors in mammalian host cells is controlled, for example, by promoters derived from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published on 7/5 1989), adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis b virus and simian virus 40(SV40), from heterologous mammalian promoters such as the actin promoter or immunoglobulin promoter, and from heat shock promoters, provided such promoters are compatible with the host cell system.

Transcription of DNA encoding CRIg polypeptides by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300bp, that act on a promoter to increase its transcription. Many enhancer sequences are known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). However, typically an enhancer from a eukaryotic cell virus is used. Examples include the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Enhancers can be spliced into the vector at positions 5 ' or 3 ' to the CRIg coding sequence, but are preferably located at sites 5 ' to the promoter.

Expression vectors for eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are typically available from the 5 'and occasionally 3' ends of untranslated regions of eukaryotic or viral DNA or cDNA. These regions comprise nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding CRIg.

Other methods, vectors and host cells suitable for the synthesis of CRIg in recombinant vertebrate cell culture after modification are described in Gething et al, Nature 293: 620-625 (1981); mantei et al, Nature 281: 40-46 (1979); EP117, 060; and EP117, 058.

Amplification and/or expression of the gene can be measured directly in the sample, for example by conventional Southern blotting, Northern blotting for quantification of mRNA transcription (Thomas, Proc. Natl. Acad. Sci. USA 77: 5201. 5205(1980)), dot blotting (DNA analysis) or in situ hybridization using a suitable labelled probe according to the sequences provided herein. Alternatively, antibodies that recognize specific duplexes including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes may be used. The antibody may then be labeled and an assay may be performed in which the duplex is bound to a surface such that the presence of antibody bound to the duplex is detectable when the duplex is formed on the surface.

Alternatively, for direct quantification of gene product expression, gene expression can be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assays of cell cultures or body fluids. Antibodies useful for immunohistochemical staining and/or sample fluid assays may be monoclonal or polyclonal and may be prepared in any mammal. Conveniently, antibodies can be made against a native sequence CRIg polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against a foreign sequence fused to CRIg DNA and encoding a particular antibody epitope.

Various forms of CRIg can be recovered from the culture broth or from host cell lysates. If membrane bound, it may be released from the membrane using a suitable detergent solution (e.g.Triton-X100) or by enzymatic cleavage. The cells employed in CRIg expression can be disrupted by a variety of physical or chemical means, such as freeze-thaw cycles, sonication, mechanical disruption, or a lytic agent.

It may be desirable to purify CRIg from recombinant cellular proteins or polypeptides. The following scheme is illustrative of a suitable purification scheme: fractionation on an ion exchange column; ethanol precipitation; reversed phase HPLC; chromatography on silica or cation exchange resins such as DEAE; carrying out chromatographic focusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein a Sepharose column to remove contaminants such as IgG; and a metal chelating column that binds to an epitope-tagged form of the CRIg polypeptide. A variety of protein purification Methods can be employed, such Methods are known in the art and are described, for example, in Deutscher, Methods in enzymology 182 (1990); scopes, Protein Purification: principles and Practice, Springer-Verlag, New York (1982). The purification step chosen will depend on, for example, the nature of the generation process used and the particular CRIg produced.

Agonists of CRIg Polypeptides

Agonists of CRIg polypeptides will mimic the qualitative biological activity of the native sequence CRIg polypeptide. Preferably, the biological activity is the ability to bind C3b and/or to affect complement or complement activation, in particular to inhibit the alternative complement pathway and/or the C3 convertase. Agonists include, for example, immunoadhesins, peptidomimetics, and non-peptide organic small molecules that mimic the biological activity of the native CRIg.

CRIg-Ig immunoadhesins have been discussed above.

Another group of CRIg agonists are peptide mimetics of the native sequence CRIg polypeptide. Peptidomimetics include, for example, peptides comprising non-naturally occurring amino acids, provided that the compound retains the biological activity of CRIg as described herein. Similarly, peptidomimetics and analogs can include structures that mimic important structural elements of the CRIg polypeptides of the invention and chemical structures of non-amino acids that retain CRIg biological activity. The term "peptide" as used herein refers to an amino acid sequence of less than about 50 amino acid residues, preferably less than about 40 amino acid residues, constrained (i.e. having some structural elements like e.g. the presence of amino acids that initiate β -turns or β -sheet folding, or cyclized e.g. due to the presence of disulfide bond Cys residues) or unconstrained (e.g. linear), including or multimers such as dimers between them. For peptides of less than about 40 amino acid residues, peptides of between about 10 and about 30 amino acid residues are preferred, especially peptides of about 20 amino acid residues. However, upon reading this disclosure, the skilled artisan will recognize that a peptide is not distinguished by the length of the particular peptide, but rather by its ability to bind C3b and inhibit the C3 convertase, particularly the alternative complement pathway C3 convertase.

Peptides can be conveniently prepared using solid phase peptide synthesis (Merrifield, J.Am.chem.Soc.85: 2149 (1964); Houghten, Proc.Natl.Acad.Sci.USA 82: 5132 (1985)). Solid phase synthesis begins with the carboxy terminus of the putative peptide and couples the protected amino acid to an inert solid support. The inert solid support may be any macromolecule capable of serving as a C-terminal anchor for the initial amino acid. Typically, the macromolecular support is a crosslinked polymer resin (e.g., a polyamide or polystyrene resin), such as that shown in FIGS. 1-1 and 1-2, pages 2 and 4, supra, of Stewart and Young. In one embodiment, the C-terminal amino acid is coupled to a polystyrene resin to form a benzyl ester. The macromolecular support is selected such that the peptide anchor is stable under conditions used for alpha-amino deprotection of the blocked amino acid in peptide synthesis. If a base-labile α -protecting group is used, it is desirable to use an acid-labile linkage between the peptide and the solid support. For example, acid-labile ether resins are effective for Fmoc amino acid peptide synthesis that is base-labile, as described by Stewart and Young, supra, page 16. Alternatively, peptide anchor linkages and α -protecting groups that are differentially unstable to acidolysis may be used. For example, aminomethyl resins such as phenylacetamide methyl (Pam) resins work well in conjunction with Boc-amino acid peptide synthesis, as described by Stewart and Young, supra, pages 11-12.

After coupling of the starting amino acid to the inert solid support, the α -amino protecting group of the starting amino acid is removed, for example with trifluoroacetic acid (TFA) in dichloromethane and neutralized, for example with Triethylamine (TEA). After the alpha-amino group of the starting amino acid is deprotected, the next alpha-amino group and side chain protected amino acid in the synthesis is added. The remaining alpha-amino groups and optionally side chain protected amino acids are then coupled in the desired order by condensation to obtain the intermediate compound attached to the solid support. Alternatively, some amino acids may be coupled to each other to form fragments of the desired peptide, followed by addition of peptide fragments to the growing solid phase peptide chain.

The condensation reaction between two amino acids or an amino acid and a peptide or a peptide and a peptide may be carried out according to a commonly used condensation method such as an acid method, a mixed anhydride method, a DCC (N, N '-dicyclohexylcarbodiimide) or DIC (N, N' -diisopropylcarbodiimide) method, an active ester method, a p-nitrophenyl ester method, a BOP (benzotriazole-1-yl-oxy-tris [ dimethylamino ] phosphine hexafluorophosphate) method, an N-hydroxysuccinate imino ester method, and the like, and a Woodward reagent K method.

Common in the chemical synthesis of peptides is the protection of any reactive side chain group of an amino acid with a suitable protecting group. Finally, these protecting groups are removed after sequential assembly of the desired polypeptide chain. Also common is the protection of the alpha-amino group on an amino acid or peptide fragment when the C-terminal carboxyl group of the amino acid or peptide fragment reacts with the free N-terminal amino group of the growing solid phase polypeptide chain, followed by selective deprotection of the alpha-amino group to allow addition of the next amino acid or peptide fragment to the solid phase polypeptide chain. Thus, common in polypeptide synthesis is the generation of intermediate compounds comprising each amino acid residue positioned in the desired sequence in the peptide chain, wherein the individual residues still carry side chain protecting groups. These protecting groups can be removed substantially simultaneously after cleavage from the solid phase to yield the desired polypeptide product.

The α -and ε -amino side chains may be protected with the following groups: benzyloxycarbonyl (abbreviated as Z), Isonicotinoxycarbonyl (iNOC), o-chlorobenzyloxycarbonyl [ Z (2Cl) ], p-nitrobenzyloxycarbonyl [ Z (NO2) ], p-methoxybenzyloxycarbonyl [ Z (OMe)) ], t-butoxycarbonyl (Boc), t-pentyloxycarbonyl (Aoc), isobornyloxycarbonyl, adamantyloxycarbonyl, 2- (4-biphenyl) -2-propoxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl (Fmoc), methanesulfonylethoxycarbonyl (Msc), trifluoroacetyl, phthaloyl, formyl, 2-Nitrobenzenesulfonyl (NPS), diphenylphosphinothio (Ppt) and dimethylphosphinothio (Mpt), and the like.

Protecting groups for carboxyl functions are, for example, benzyl ester (OBzl), cyclohexyl ester (Chx), 4-nitrobenzyl ester (ONb), tert-butyl ester (Obut), 4-pyridylmethyl ester (OPic), etc. It is often desirable that certain amino acids having functional groups other than amino and carboxyl groups, such as arginine, cysteine, and serine, be protected by suitable protecting groups. For example, the guanidino group of arginine may be protected with nitro group, p-toluenesulfonyl group, benzyloxycarbonyl group, adamantyloxycarbonyl group, p-methoxybenzenesulfonyl group, 4-methoxy-2, 6-dimethylbenzenesulfonyl group (Nds), 1, 3, 5-trimethylbenzenesulfonyl group (Mts), or the like. The thiol group of cysteine may be protected with p-methoxybenzyl, trityl, etc.

Many of the blocked amino acids described above are commercially available, such as Novabiochem (San Diego, Calif.), Bachem CA (Torrence, Calif.), or Penninsula Labs (Belmont, Calif.).

Stewart and Young, supra, provides detailed information regarding the peptide preparation scheme. Alpha-amino protection is described on pages 14-18 and side chain blocking is described on pages 18-28. The amine, hydroxyl and thiol functional protecting groups are listed at page 149-151.

After completion of the desired amino acid sequence, the peptide can be cleaved from the solid support, recovered and purified. Cleaving the peptide from the solid support by means of a reagent capable of breaking the peptide-solid phase linkage, and optionally deprotecting the blocked functional group on the peptide. In one embodiment, the peptide is cleaved from the solid phase by acidolysis with liquid hydrofluoric acid (HF), which also removes any remaining side chain protecting groups. Preferably, to avoid alkylation of residues in the peptide (e.g., alkylation of methionine, cysteine and tyrosine residues), the acidolysis reaction mixture contains thiocresol and cresol scavengers. After HF cleavage, the resin was washed with ether and the free peptide was extracted from the solid phase with successive washes of acetic acid solution. The combined wash solutions were lyophilized and the peptide was purified.

Antagonists of CRIg polypeptide

Antagonists of the native sequence CRIg polypeptide are useful in the treatment of conditions benefiting from excessive complement activation, including tumors.

A preferred group of antagonists includes antibodies that specifically bind native CRIg. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies.

Methods for preparing polyclonal antibodies are known to the skilled worker. Polyclonal antibodies can be produced in animals, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected into the animal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include the CRIg polypeptide of the present invention or a fragment or fusion protein thereof. It may be useful to couple an immunizing agent to a protein known to be immunogenic in the mammal being immunized. This is achieved byExamples of immunoiogenic-like proteins include, but are not limited to, keyhole limpetHemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants that can be used include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). Immunization protocols can be selected by those skilled in the art without undue experimentation.

Alternatively, the antibody that recognizes and binds the polypeptide of the present invention or functions as an antagonist thereof may be a monoclonal antibody. Monoclonal antibodies can be prepared using hybridoma methods, such as Kohler and Milstein, Nature 256: 495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes may be immunized in vitro.

The immunizing agent will typically include a CRIg polypeptide, antigenic fragment thereof or fusion protein of the invention. Typically, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103). Immortalized cell lines are generally transformed mammalian cells, in particular myeloma cells of rodent, bovine and human origin. Typically, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable medium, preferably containing one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will contain hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent HGPRT-deficient cells from growing.

Preferred immortalized cell lines are those that fuse efficiently, support stable high levels of antibody expression by the selected antibody-producing cells, and are sensitive to media such as HAT media. More preferred immortalized Cell lines are murine myeloma lines, as obtainable, for example, from the SalkInstitute Cell Distribution Center (SalkInstitute Cell Distribution Center, San Diege, California, USA) and the American Type Culture Collection (American Type Culture Collection, Rockville, Maryland, USA). Human myeloma and mouse-human heteromyeloma cell lines for the Production of human monoclonal antibodies have also been described (Kozbor, J.Immunol.133: 3001 (1984); Brodeur et al, monoclonal antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987) pp.51-63).

The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against or having similar activity as the polypeptide of the invention. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of monoclonal antibodies can be determined, for example, by Munson and pollard, anal. biochem. 107: 220(1980) by Scatchard analysis.

After identification of the desired hybridoma cells, the clones can be subcloned by limiting dilution procedures and cultured by standard methods (Goding, supra). Suitable media for this purpose include, for example, Dulbecco's modified Eagle's Medium or RPMI-1640 medium. Alternatively, the hybridoma cells can be cultured in vivo in a mammal as ascites fluid.

Monoclonal antibodies secreted by the subclones can be separated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures, such as, for example, protein a-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

Monoclonal antibodies can also be generated by recombinant DNA techniques, such as described in U.S. patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention are a preferred source of such DNA. Once isolated, the DNA may be placed into an expression vector, which is then transfected into a host cell that does not otherwise produce immunoglobulin proteins, such as a simian COS cell, a Chinese Hamster Ovary (CHO) cell, or a myeloma cell, to obtain synthesis of monoclonal antibodies in the recombinant host cell. The DNA may also be modified, for example, by substitution, i.e., replacement of the homologous murine sequences with the coding sequences for the human heavy and light chain constant regions (U.S. Pat. No. 4,816,567; Morrison et al, supra), or by covalent conjugation of the immunoglobulin coding sequence with all or part of the coding sequence for a non-immunoglobulin polypeptide. Such non-immunoglobulin polypeptides may be substituted for the constant regions of an antibody of the invention, or it may be substituted for the variable regions of one antigen-binding site of an antibody of the invention to produce a chimeric bivalent antibody.

The antibody is preferably a monovalent antibody. Methods for making monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chains and modified heavy chains. The heavy chain is typically truncated at any point in the Fc region, thereby preventing heavy chain cross-linking. Alternatively, the relevant cysteine residue is replaced with another amino acid residue or deleted, thereby preventing cross-linking.

In vitro methods are also suitable for the production of monovalent antibodies. Digestion of antibodies to generate fragments thereof, particularly Fab fragments, can be accomplished using conventional techniques known in the art.

The antibodies of the invention may also include humanized or human antibodies. A humanized form of a non-human (e.g., murine) antibody refers to a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof that minimally comprises sequences derived from a non-human immunoglobulin(s) ((Such as Fv, Fab ', F (ab')₂Or other antigen binding subsequences of antibodies). Humanized antibodies include those in which Complementarity Determining Region (CDR) residues in a human immunoglobulin (acceptor antibody) are replaced with CDR residues from a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced with corresponding non-human residues. Humanized antibodies may also comprise residues that are not found in both the acceptor antibody and the import CDR or framework sequences. Typically, the humanized antibody will comprise substantially all of at least one, and typically two, variable regions in which all or substantially all of the CDRs correspond to CDRs of a non-human immunoglobulin and all or substantially all of the FRs are FRs of a human immunoglobulin consensus sequence. The humanized antibody will optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al, Nature 321: 522-525 (1986); Riechmann et al, Nature 332: 323-329 (1988); Presta, curr. Op. Structure. biol. 2: 593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art. Typically, humanized antibodies have one or more amino acid residues introduced into them from a source other than human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable region. Humanization can be carried out essentially following the method of Winter and co-workers (Jones et al, Nature 321: 522-525 (1986); Riechmann et al, Nature 332: 323-327 (1988); Verhoeyen et al, Science 239: 1534-1536(1988)), using rodent CDRs or CDR sequences in place of the corresponding sequences of a human antibody. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) in which substantially less than the entire human variable region is replaced with the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced with residues from analogous sites in rodent antibodies.

Human antibodies can also be generated using a variety of techniques known in the art, including phage display libraries and Winter (J.mol.biol.227: 381 (1991); Marks et al, J.mol.biol.222: 581 (1991)). The techniques of Cole et al and Boerner et al can also be used to prepare human Monoclonal Antibodies (Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, p.77 (1985); Boerner et al, J.Immunol.147 (1): 86-95 (1991); U.S.5,750, 373). Similarly, human antibodies can be generated by introducing human immunoglobulin loci into transgenic animals, such as mice, in which endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production was observed, very similar in all respects to that seen in humans, including gene rearrangement, assembly, and antibody repertoire. Such a process is described, for example, in us patent 5,545,807; 5,545,806; 5,569,825; 5,625,126, respectively; 5,633,425, respectively; 5,661,016, and the following scientific publications: marks et al, Bio/Technology 10: 779 783 (1992); lonberg et al, Nature 368: 856-859 (1994); morrison, Nature 368: 812-13 (1994); fisherworld et al, Nature Biotechnology 14: 845-51 (1996); neuberger, Nature Biotechnology 14: 826 (1996); lonberg and huskzar, lntern. rev. immunol.13: 65-93(1995).

Bispecific antibodies refer to monoclonal, preferably human or humanized antibodies having binding specificity for at least two different antigens. In this case, one binding specificity may be for the polypeptide of the invention, the other for any other antigen, preferably for a cell surface protein or receptor subunit.

Methods for generating bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies has been based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two heavy chains have different specificities (Millstein and Cuello, Nature 305: 537-539 (1983)). Due to the random assignment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is usually accomplished by an affinity chromatography step. WO93/08829 and Trunecker et al, EMBO J.10, published on 5/13 1993: 3655-3659(1991) a similar procedure is disclosed.

Antibody variable regions with the desired binding specificity (antibody-antigen binding site) can be fused to immunoglobulin constant region sequences. Preferably, the fusion uses an immunoglobulin heavy chain constant region comprising at least part of the hinge, CH2, and CH3 regions. Preferably, a first heavy chain constant region (CH1) is present in at least one of the fusions that comprises the site necessary for light chain binding. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain is inserted into separate expression vectors and co-transfected into a suitable host organism. For more details on the generation of bispecific antibodies see, e.g., Suresh et al, Methods in Enzymology 121: 210(1986).

Heteroconjugate antibodies consist of two covalently joined antibodies. Such antibodies are suggested, for example, for targeting immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for the treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089). It is contemplated that antibodies can be prepared in vitro using methods known in synthetic protein chemistry, including methods involving cross-linking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of reagents suitable for this purpose include iminothiolate and methyl 4-mercaptobutylimidate and are disclosed, for example, in U.S. Pat. No. 4,676,980.

It may be desirable to modify the antibodies of the invention with respect to effector function, e.g., to enhance the efficacy of the antibodies in treating immune related diseases. For example, cysteine residues may be introduced in the Fc region to allow interchain disulfide bonds to form in this region. The homodimeric antibody so produced may have improved internalization capacity and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al, j.exp.med.176: 1191-1195(1992) and shop, B., J.Immunol.148: 2918-2922(1992). Homodimeric antibodies with enhanced anti-tumor activity can also be used as described in Wolff et al, Cancer Research 53: 2560, 2565 (1993). Alternatively, the antibody may be engineered to have dual Fc regions, and thus may have enhanced complement lysis and ADCC capabilities. See Stevenson et al, Anti-Cancer Drug Design 3: 219-230(1989).

The invention also relates to immunoconjugates comprising an antibody conjugated to a cytotoxic agent, such as a chemotherapeutic agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin or a fragment thereof), or a radioisotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that may be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii toxalbumin, Dianthin toxalbumin, Phytolacca Americana gericaa toxalbumin (PAPI, PAPII and PAP-S), Momordica charantia (momordia) inhibitor, curcin (curcin), crotin (crotin), Saponaria officinalis (saparonia officinalis) inhibitor, gelonin (gelonin), mitomycin (restrictocin), restrictocin (restrictocin), triphenols (thymol), trichothecin (theomycin) and neomycin (neomycin). There are a variety of radioisotopes available for the production of radioconjugated antibodies. Examples include ²¹²Bi、¹³¹I、¹³¹In、⁹⁰Y, and¹⁸⁶Re。

conjugates of the antibody and cytotoxic agent may be generated using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), Iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate hcl), reactive esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis (p-diazoniumbenzoyl) ethylenediamine), diisocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene). For example, the methods can be described as Vitetta et al, Science 238: 1098(1987) ricin immunotoxin was prepared as described in (1098). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotides to antibodies. See WO 94/11026.

In another embodiment, the antibody may be conjugated to a "receptor" (such as streptavidin) for use in tissue pre-localization, wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from circulation using a clearing agent, followed by administration of a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radionucleotide).

5. Target disease

5.1 complement-associated diseases and conditions

The CRIg polypeptide and its agonist, especially CRIg-Ig immunoadhesin, can be used for preventing and/or treating complement related diseases and pathological conditions. Such diseases and conditions include, but are not limited to, inflammatory and autoimmune diseases.

Specific examples of complement-associated diseases include, but are not limited to, Rheumatoid Arthritis (RA), Acute Respiratory Distress Syndrome (ARDS), distal tissue injury following ischemia and reperfusion, complement activation during cardiopulmonary bypass surgery, dermatomyositis, pemphigus, lupus nephritis and glomerulonephritis and vasculitis arising therefrom, cardiopulmonary bypass, cardiac arrest-induced coronary endothelial dysfunction, type II membranoproliferative glomerulonephritis, IgA nephropathy, acute renal failure, cryoglobulinemia, antiphospholipid syndrome, macular degeneration, and other complement-associated eye conditions such as age-related macular degeneration (AMD), Choroidal Neovascularization (CNV), uveitis, diabetic and other ischemia-associated retinopathies, endophthalmitis, and other intraocular neovascular diseases such as diabetic edema, pathologic myopia, von hippel-Lindau disease, Histoplasmosis of the eye, Central Retinal Vein Occlusion (CRVO), corneal neovascularization, retinal neovascularization, and allograft, hyperacute rejection, hemodialysis, chronic obstructive pulmonary stress syndrome (COPD), asthma, and aspiration pneumonia.

5.2 complement-associated eye conditions

CRIg polypeptides and agonists thereof, in particular CRIg-Ig immunoadhesins, are particularly useful for the prevention and treatment of complement-associated eye conditions (all pathologies involving complement, including complement of the classical and alternative pathways, in particular the alternative pathway, complement eye conditions and diseases), such as, for example, macular degenerative diseases such as all stages of age-related macular degeneration (AMD), including both dry and wet (non-exudative and exudative) forms, Choroidal Neovascularization (CNV), uveitis, diabetic and other ischemia-related retinopathies, endophthalmitis, and other intraocular neovascular diseases such as diabetic macular edema, pathologic myopia, von Hippel-Lindau disease, histoplasmosis of the eye, Central Retinal Vein Occlusion (CRVO), corneal neovascularization, and retinal neovascularization. A preferred group of complement-associated eye conditions include age-related macular degeneration (AMD), including non-exudative (wet) and exudative (dry or atrophic) AMD, Choroidal Neovascularization (CNV), Diabetic Retinopathy (DR), and endophthalmitis.

AMD refers to age-related macular degeneration, the leading cause of irreversible visual dysfunction in individuals over the age of 60. There are two types of AMD, non-exudative (dry) and exudative (wet). The dry or non-exudative form involves atrophy and hypertrophic changes of the Retinal Pigment Epithelium (RPE) beneath the central retina (macula), and deposits (drusen) on the RPE. Patients with non-exudative AMD can develop wet or exudative forms of AMD, in which abnormal blood vessels called choroidal neovascular membranes (CNVMs) develop under the retina, exude fluid and blood, eventually causing discoid scarring in the retina and under the retina that blinds the person. Non-exudative AMD, which is usually the precursor of exudative AMD, is more common. Non-exudative AMD behaves differently; hard drusen, soft drusen, RPE geographic atrophy (geogenic atrophy), and pigment aggregation may be present. Complement components are deposited on the RPE early in AMD and are the major component of drusen.

The present invention is specifically concerned with the treatment of high risk AMD, including class 3 and class 4 AMD. Class 3 AMD is characterized by the absence of advanced AMD in both eyes, 20/32 or better vision in at least one eye and at least one large wart (e.g., 125 μm), extensive (as measured by drusen area) drusen, or Geographic Atrophy (GA) not involving the center of the macula, or any combination thereof. Class 3 AMD (still considered "dry" AMD) has a high risk of conversion to Choroidal Neovascularization (CNV).

Class 4 high risk AMD (classified as "wet" AMD) is characterized by 20/32 or better vision in the index eye (index eye) and by the absence of advanced AMD (a feature involving GA in the center of the macula or choroidal neovascularization). The contralateral eye (fellow eye) is characterized by advanced AMD, or vision below 20/32, attributable to AMD macular degeneration. Generally, high risk AMD, if untreated, rapidly progresses to Choroidal Neovascularization (CNV) at a rate that is about 10-30 times greater than the rate of development of class 1 or 2 (less at risk) AMD.

CRIg and its agonists, such as CRIg immunoadhesins, are particularly useful for preventing the development of AMD (particularly class 3 or class 4 AMD) to CNV, and/or for preventing the formation/development of AMD or CNV in the unaffected or less affected contralateral eye. In this context, the term "preventing" is used in the broadest sense and includes completely or partially blocking and slowing the progression of the disease, as well as delaying the onset of more severe forms of the disease. Patients at high risk of developing or developing high risk (class 4) AMD or CMV particularly benefit from this aspect of the invention.

Complement factor h (cfh) polymorphisms are known to be associated with an individual's risk of developing AMD and/or CNV. Mutations in CFH can activate complement, which in turn can lead to AMD/CNV. Recently, Complement Factor H (CFH) polymorphisms have been reported to account for 50% of the risk of AMD (Klein et al, Science 308: 385-9 (2005)). A common haplotype of CFH (HF1/CFH) has been found to predispose individuals to age-related macular degeneration (Hageman et al, Proc. Natl. Acad. Sci. USA102 (2): 7227-. AMD has been isolated as a frequently-stained dominant trait, with the disease locus mapped to chromosome 1q25-q31, with a maximum lod score (lod score) of about 3.20(Klein et al, Arch Opthalmol.116 (8): 1082-9(1998), Majewski et al, am.J.Hum.Genet.73 (3): 540-50(2003), Seddon et al, am.J.Hum.Genet.73 (4): 780-90(2003), Weeks et al, am.J.Ophthalmol.132 (5): 682-92 (92), (2001), Iyengar et al, am.J.Hum.Genet.74 (1-20-39 (2004)); chromosome 2q3/2q32, with a maximum lod score of 2.32/2.03(Seddon et al, supra) between markers D12S1391 and D2S 1384; 3p13, maximum lod score 2.19 between markers D12S1300 and D12S1763 (Majewski et al, supra; Schick et al, am.J.hum.Genet.72 (6): 1412-24 (2003)); 6q14, maximum lod score 3.59/3.17(Kniazeva et al, am. J. Ophthlmol.130 (2): 197 202(2000)) between markers D6S1056 and DS 249; 9q33, max lod score 2.06 at marker D9S934 (Mejwski et al, see above); 10q26, maximum lod score 3.06 at marker D10S1230 (Majewski et al, supra; Iyengaret al, supra; Kenealy et al, mol. Vis.10: 57-61 (2004)); 17q25, max lod score 3.16 at marker D17S928 (Weeks et al, supra); and 22q12, max lod score 2.0 at marker D22S1045 (Seddon et al, supra). Thus, genetic screening is an important part of identifying who is a prophylactic treatment in patients, including particularly good candidates to prevent the disease from developing into more severe forms, such as progression from AMD to CNV.

In addition, given the solid evidence of the link between complement activation and age-related macular degeneration (AMD), the present invention provides novel methods for the prevention and treatment of CNV and AMD through complement inhibition, particularly inhibition of the alternative pathway. In addition to CRIg, inhibitors of the alternative pathway include fusion proteins (e.g., immunoadhesins), agonistic anti-CRIg antibodies, and peptidic and non-peptidic small molecules.

5.3 inflammatory conditions and autoimmune diseases

A more extensive list of inflammatory conditions as examples of complement-associated diseases includes, for example, Inflammatory Bowel Disease (IBD), systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), Sjogren's disease (Sj)gren) syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis (Graves 'disease), Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes, immune-mediated nephropathy (glomerulonephritis, tubulointerstitial nephritis), demyelinating diseases of the central and peripheral nervous system such as multiple sclerosis, idiopathic polyneuropathy, hepatobiliary diseases such as infectious hepatitis (a, b, c, d, e and other non-hepadnaviral hepatitis), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, And sclerosing cholangitis, inflammatory and fibrotic pulmonary diseases (e.g. cystic fibrosis), gluten-sensitive bowel disease, Whipple's (whippple) disease, autoimmune or immune-mediated skin diseases including bullous skin disease, erythema multiforme and contact dermatitis, psoriasis, allergic diseases of the lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, graft-related diseases including graft rejection and graft-versus-host disease.

In systemic lupus erythematosus, the main mediators of the disease are the production of autoreactive antibodies to self-proteins/tissues and the subsequent production of immune-mediated inflammation. The antibody mediates tissue damage either directly or indirectly. Although T lymphocytes have not been shown to be directly involved in tissue damage, T lymphocytes are required for the formation of autoreactive antibodies. The development of the disease is therefore dependent on T lymphocytes. A variety of organs and systems are affected clinically, including the kidney, lung, musculoskeletal system, mucocutaneous, eye, central nervous system, cardiovascular system, gastrointestinal tract, bone marrow, and blood.

Rheumatoid Arthritis (RA) is a chronic systemic autoimmune inflammatory disease that involves mainly the synovium of multiple joints, thereby causing damage to articular cartilage. The pathogenesis is dependent on T lymphocytes and is associated with the production of rheumatoid factors, autoantibodies against self IgG, thereby leading to the formation of immune complexes, which reach high levels in the synovial fluid and blood. These complexes in the joints can induce significant lymphocyte and monocyte infiltration into the synovium and subsequent significant synovial changes; joint cavities/fluids are infiltrated by similar cells and many neutrophils are added. The affected tissue is primarily the joint, often in a symmetrical pattern. However, there are two major forms of extra-articular disease. One form is the formation of extra-articular lesions with ongoing progressive arthropathy and the typical lesions of pulmonary fibrosis, vasculitis and skin ulcers. The second form of extra-articular disease is the so-called Felty syndrome, which occurs late in the course of RA, sometimes after the arthropathy has subsided, and is implicated in the occurrence of neutropenia, thrombocytopenia and splenomegaly. This can be accompanied by vasculitis and infarction, skin ulceration and gangrene formation in multiple organs. Patients also often develop rheumatoid nodules in the subcutaneous tissue above the affected joint; the late nodules have necrotic centers surrounded by mixed inflammatory cell infiltrates. Other manifestations that may occur in RA include: pericarditis, pleuritis, coronary arteritis, interstitial pneumonia with pulmonary fibrosis, keratoconjunctivitis sicca, and rheumatoid nodules.

Juvenile chronic arthritis is a chronic idiopathic inflammatory disease, often beginning before the age of 16. Its phenotype is somewhat similar to RA; some patients who are positive for rheumatoid factor are classified as juvenile rheumatoid arthritis. Diseases are subdivided into three main categories: a few joints, multiple joints and systemic. Arthritis can be severe, often destructive, and results in joint stiffness and growth retardation. Other manifestations may include chronic anterior uveitis and systemic amyloidosis.

Spondyloarthropathies are a group of disorders with some common clinical features and are commonly associated with the expression of the HLA-B27 gene product. These disorders include: ankylosing spondylitis, Reiter's syndrome (reactive arthritis), arthritis associated with inflammatory bowel disease, spondylitis associated with psoriasis, juvenile spondyloarthropathy and undifferentiated spondyloarthropathy. Distinctive features include sacroiliac arthritis with or without spondylitis; inflammatory asymmetric arthritis; is associated with HLA-B27 (alleles of the serologically defined MHC class I HLA-B locus); ocular inflammation; and the lack of autoantibodies associated with other rheumatoid diseases. The most relevant cells critical for the induction of disease are CD8+ T lymphocytes, cells that target antigens presented by MHC class I molecules. CD8+ T cells react with the MHC class I allele HLA-B27 as if it were an exogenous peptide expressed by an MHC class I molecule. It has been hypothesized that epitopes of HLA-B27 may mimic antigenic epitopes of bacteria or other microorganisms, thereby inducing a CD8+ T cell response.

The etiology of systemic sclerosis (scleroderma) is unknown. The disease is characterized by skin sclerosis; this may be induced by active inflammatory processes. Scleroderma may be local or systemic; vascular damage is common and endothelial cell damage in the microvasculature is an early and important event in the development of systemic sclerosis; vascular damage may be mediated by immunity. The presence of mononuclear cellular infiltrates in skin lesions and antinuclear antibodies in many patients suggests an immunological basis. ICAM-1 is often upregulated on the cell surface of fibroblasts in skin lesions, suggesting that T cell interactions with these cells may play a role in the pathogenesis of the disease. Other organs involved include: gastrointestinal tract: smooth muscle atrophy and fibrosis, leading to abnormal peristalsis/movement; kidney: concentric subendothelial hyperplasia affecting the small segmental and interlobal arteries, thereby reducing renal cortical blood flow, resulting in proteinuria, azotemia, and hypertension; skeletal muscle: atrophy, interstitial fibrosis, inflammation; lung: interstitial pneumonia and interstitial fibrosis; and a core: shrinkage band necrosis, scarring/fibrosis.

Idiopathic inflammatory myopathies, including dermatomyositis, polymyositis, and others, are chronic inflammatory disorders of muscle of unknown etiology that result in muscle weakness. Muscle damage/inflammation is often symmetrical and progressive. Autoantibodies are associated with most forms. These myositis-specific autoantibodies target and inhibit the function of components, proteins and RNAs involved in protein synthesis.

Sjogren's disease) The syndrome is caused by immune-mediated inflammation and subsequent disruption of lacrimal and salivary gland function. The disease may be associated with or accompanied by inflammatory connective tissue disease. The disease is associated with autoantibody production against the Ro and La antigens, both of which are small RNA-protein complexes. Damage leads to keratoconjunctivitis sicca, xerostomia, and other manifestations or associations, including biliary cirrhosis, peripheral or sensory neuropathy, and palpable purpura.

Systemic vasculitis includes diseases where the primary injury is inflammation and subsequent damage to the blood vessels results in ischemia/necrosis/degeneration of the tissue supplied by the affected vessels and, in some cases, ultimately end-organ dysfunction. Vasculitic conditions may also occur as secondary injury or sequelae to other immune-inflammatory mediated diseases such as rheumatoid arthritis, systemic sclerosis, and the like, particularly in diseases that are also associated with immune complex formation. The diseases in the group of primary systemic vasculitis included: systemic necrotizing vasculitis: polyarteritis nodosa, allergic vasculitis, granulomatosis, and polyangiitis; wegener's granulomatosis; lymphomatoid granulomatosis; and giant cell arteritis. Various vasculitic conditions include: mucosal and cutaneous lymph node syndrome (MLNS or Kawasaki) disease), isolated CNS vasculitis, Behet's (Behet) disease, thromboangiitis obliterans (Buerger's) disease, and cutaneous necrotizing venulitis. The pathogenesis of most of the types of vasculitis listed is thought to be primarily due to the deposition of immunoglobulin complexes in the vessel wall and subsequent induction of an inflammatory response via ADCC, complement activation, or both.

Sarcoidosis is a condition of unknown etiology characterized by the presence of epithelioid granulomas in almost any tissue of the body; the lung is most commonly involved. Pathogenesis involves the persistent presence of activated macrophages and lymphoid cells at the site of disease and the subsequent chronic sequelae caused by the release of local and systemic active products by these cell types.

Autoimmune hemolytic anemia, including autoimmune hemolytic anemia, immune pancytopenia, and paroxysmal nocturnal hemoglobinuria, is a result of the production of antibodies that react with antigens expressed on the surface of red blood cells (and in some cases other blood cells, including platelets) as a reflection of the removal of those antibody-coated cells via complement-mediated lysis and/or ADCC/Fc receptor-mediated mechanisms.

In autoimmune thrombocytopenia, including thrombocytopenic purpura, and other immune-mediated thrombocytopenias in clinical settings, platelet destruction/removal occurs due to antibody or complement attachment to the platelets and subsequent removal via complement lysis, ADCC, or Fc receptor-mediated mechanisms.

Thyroiditis, including Graves 'disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis and atrophic thyroiditis, is due to an autoimmune response against thyroid antigens and the production of antibodies that react with proteins present in the thyroid gland and which are usually specific for the thyroid gland. Existing experimental models include spontaneous models: rats (BUF and BB rats) and chickens (obese chicken strain); inducible models: animals were immunized with thyroglobulin, thyroid microsomal antigen (thyroid peroxidase).

Type I diabetes or insulin-dependent diabetes is the autoimmune destruction of islet beta cells; this destruction is mediated by autoantibodies and autoreactive T cells. Antibodies to insulin or insulin receptors may also produce insulin-unresponsive phenotypes.

Immune-mediated renal diseases, including glomerulonephritis and tubulointerstitial nephritis, are due to antibody or T lymphocyte-mediated damage to renal tissue, either directly due to the production of autoreactive antibodies or T cells against renal antigens, or indirectly due to the deposition in the kidney of antibodies and/or immune complexes that are reactive against other, non-renal antigens. Thus, other immune-mediated diseases that result in immune complex formation may also induce immune-mediated nephropathy as an indirect sequelae. Both direct and indirect immune mechanisms result in an inflammatory response that produces/induces lesion formation in renal tissue, resulting in impaired organ function and in some cases progression to renal failure. Both humoral and cellular immune mechanisms may be involved in the pathogenesis of injury.

Demyelinating diseases of the central and peripheral nervous system, including multiple sclerosis; idiopathic demyelinating polyneuropathy or Guillain-barre syndrome (Guillain-barre); and chronic inflammatory demyelinating polyneuropathy, which are considered to be autoimmune-based and result in demyelination of nerves due to direct damage to oligodendrocytes or to myelin. In MS, there is evidence that the induction and development of the disease is dependent on T lymphocytes. Multiple sclerosis is a T-lymphocyte dependent inflammatory demyelinating disease and has a relapsing-remitting course or a lengthy progressive course. The etiology is unknown; however, viral infections, genetic cachexia, environmental and autoimmune can all play a role. The lesions contain infiltrates, microglia and infiltrated macrophages mediated primarily by T lymphocytes; CD4+ T lymphocytes are the predominant cell type at the site of injury. The mechanism of oligodendrocyte cell death and subsequent demyelination is unknown, but may be driven by T lymphocytes.

Inflammatory and fibrotic pulmonary diseases including eosinophilic pneumonia; idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, may involve an unregulated immune-inflammatory response. Inhibiting this response would have therapeutic benefit.

Autoimmune or immune-mediated skin diseases, including bullous skin disease, erythema multiforme, and contact dermatitis, are mediated by autoantibodies, which develop T-dependent lymphocytes.

Psoriasis is a T lymphocyte-mediated inflammatory disease. The lesions contain infiltrates of T lymphocytes, macrophages and antigen processing cells, and some neutrophils.

Allergic diseases including asthma; allergic rhinitis; atopic dermatitis; food hypersensitivity; and urticaria, which is dependent on T lymphocytes. These diseases are mediated primarily by T lymphocyte-induced inflammation, IgE-mediated inflammation, or a combination of both.

Transplantation-related diseases including transplant rejection and Graft Versus Host Disease (GVHD), T-dependent lymphocytes; inhibiting T lymphocyte function has improving effect.

6. Method of treatment

For the prevention, treatment, or reduction of the severity of complement-associated (immune-related) diseases, the appropriate dosage of a compound of the invention will depend on the type of disease to be treated, the severity and course of the disease, whether the agent is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the compound, and the judgment of the attending physician, as defined above. The compound is suitably administered to the patient at once or over a series of treatments. Preferably, it is desirable to determine the dose-response curve of the pharmaceutical composition of the invention first in vitro and then in a useful animal model prior to testing in humans.

For example, depending on the type and severity of the disease, an initial candidate dose for administration to a patient is about 1. mu.g/kg to 15mg/kg (e.g., 0.1-20mg/kg) of the polypeptide, e.g., either by one or more separate administrations or by continuous infusion. Depending on the factors mentioned above, typical daily dosages may range from about 1. mu.g/kg to 100mg/kg or more. For repeated administrations over several days or longer, depending on the condition, the treatment is continued until the desired suppression of disease symptoms occurs. However, other dosage regimens may also be useful. The progress of such therapy is readily monitored by conventional techniques and assays.

The compounds of the present invention are typically administered by ocular, intraocular, and/or intravitreal injection when used to prevent or treat ocular diseases or conditions. Other methods of administration that may also be used include, but are not limited to, topical, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal, and intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.

Formulations for ocular, intraocular, or intravitreal administration can be prepared using methods and ingredients known in the art. One of the major requirements for effective treatment is proper penetration through the eye. Unlike diseases of the anterior segment of the eye, where drugs can be delivered locally, retinal diseases require more site-specific approaches. Eye drops and ointments rarely penetrate the back of the eye and the blood-eye barrier prevents systemically administered drugs from penetrating into the ocular tissues. Thus, the method of choice for drug delivery to treat retinal diseases, such as AMD and CNV, is typically direct intravitreal injection. Intravitreal injections are usually repeated at intervals, depending on the condition of the patient and the nature and half-life of the drug being delivered. Smaller molecules are generally preferred for intraocular (e.g., intravitreal) penetration. In the case of CRIg, all forms, including ECD of huCRIg short and long forms and its ig (fc) fusions, full length huCRIg long and short forms and its ig (fc) fusions, are suitable for intraocular (including intravitreal) delivery.

The efficacy of treatment of complement-associated ocular conditions, such as AMD or CNV, can be measured by assessing multiple endpoints commonly used in intraocular diseases. For example, vision loss may be assessed. Loss of vision can be assessed by, but is not limited to, for example: measuring the mean change from baseline to Best Corrected Vision (BCVA) at the expected time point (e.g., in BCVA based on an Early Treatment Diabetic Retinopathy Study (ETDRS) vision test table and evaluation at a 4 meter test distance); measuring the proportion of subjects who have vision loss of less than 15 letters compared to baseline at the expected time point; measuring the proportion of subjects who gained vision in excess of or equal to 15 letters compared to baseline at the expected time point; measuring the proportion of subjects whose Snellen vision corresponds to 20/2000 or worse at the expected time point; measuring the NEI visual function Questionnaire (VisualFunctioning Questionaire); measuring the size of the CNV and the amount of CNV leakage at the expected time points, e.g. by fluorescein angiography; and the like. Ocular evaluations may be performed, including for example, but not limited to, performing an ocular examination, measuring intraocular pressure, evaluating vision, measuring slit lamp pressure (slit lamp pressure), evaluating intraocular inflammation, and the like.

CRIg antagonists, such as antibodies to CRIg, are useful in immune-assisted therapy for the treatment of tumors (cancers). It is now well established that T cells recognize human tumor specific antigens. A group of tumor antigens encoded by the MAGE, BAGE and GAGE gene families are silent in all adult normal tissues, but are expressed in significant amounts in tumors, such as melanoma, lung, head and neck, and bladder cancers. DeSmet, c.etal, proc.natl.acad.sci.usa93: 7149(1996). Co-stimulation of T cells has been shown to induce tumor regression and anti-tumor responses both in vitro and in vivo. Melero, I.et al, Nature Medicine 3: 682 (1997); kwon, e.d.et al, proc.natl.acad.sci.usa94: 8099 (1997); lynch, d.h.et al, Nature Medicine 3: 625 (1997); finn, o.j.and Lotze, m.t., j.immunol.21: 114(1998). The CRIg antagonists of the present invention can be administered alone or in combination with growth regulators, cytotoxic agents or chemotherapeutic agents as adjuvants to stimulate T cell proliferation/activation and anti-tumor responses to tumor antigens. The growth-regulating agent, cytotoxic agent or chemotherapeutic agent may be administered in conventional amounts using known administration protocols. The immunostimulatory activity of the CRIg antagonists of the invention allows for a reduction in the amount of growth regulators, cytotoxic agents or chemotherapeutic agents, thereby potentially reducing toxicity to the patient.

Although some macrophages are implicated in tumor eradication, many solid tumors are known to contain macrophages that support tumor growth (double et al, J Pathol 196: 254-. These macrophages may contain CRIg on their surface. Antibodies that block CRIg's ability to inhibit complement activation can be used to activate complement on tumor cells and help eradicate tumors via complement-mediated lysis. This method would be particularly useful in tumors containing CRIg-positive macrophages.

In the treatment methods of the present invention, the compositions herein may be combined with one or more other therapeutic modalities for preventing or treating the target disease or condition. Thus, for example, if the goal is to prevent or treat a complement-associated ocular condition, administration of CRIg (including all forms and their EC regions and/or Ig fusions) may be combined with or supplemented with the anti-VEGF-A antibody ranibizumab (Lucenitis)^TMGenentech, Inc.), which is in clinical development for the treatment of AMD. In a recently concluded phase III clinical trial, except that the primary efficacy endpoint of the study was reached in patients with wet AMD, vision was maintained, as measured by the diabetic retinopathy early stage treatment (ETDRS) eye chart, with 25% (59/238) using 0.3mg Lucentis ^TMTreated patients and 34% (81/240) with 0.5mg Lucentis^TMThe treated patients had improved vision by 15 letters or more, compared to about 5% of the patients in the control group (11/238). Almost 40% (188/478) by Lucentis^TMThe treated patients achieved a vision score of 20/40 or better at 12 months compared to 11% in the control group (26/238). At 12 months, Lucentis was used compared to visual acuity at study addition^TMTreated patients received an average of 7 letters, while control patients lost an average of 10.5 letters.

Administration of CRIg (including all forms) can be used in combination with other therapies for such indications if the goal is to treat complement-associated inflammatory or autoimmune diseases. Thus, for example, if Rheumatoid Arthritis (RA) is the target, other arthritic drugs such as salicylates (e.g., aspirin), traditional non-steroidal anti-inflammatory molecules (NSAIDs) (such as, for example, Asaid, austemper, cathelam, Ibuprofen (Ibuprofen), Naproxen (Naproxen), etc.), COX-2 inhibitors (e.g., Celebrex, Vioxx) may be combined. In this context, "combination" means simultaneous administration or sequential administration in any order, and may be in any dosage form, either the same or different delivery routes.

7. Screening assays and animal models

Ig fusions of CRIg and CRIg agonists, including CRIg and CRIg ECDs, can be evaluated in a variety of cell-based assays and animal models of complement-associated diseases or disorders.

Thus, for example, efficacy in the prevention and/or treatment of arthritis can be assessed in a collagen-induced arthritis model (Terato et al. Brit. J. Rheum.35: 828-838(1966)) as shown in example 7 below. Potential arthritis prophylactic/therapeutic agents can also be screened in antibody-mediated arthritis models induced by intravenous injection of a mixture of four monoclonal antibodies, as described by Terato et al, j.immunol.148: 2103-8 (1992); terato et al, Autoimmiturity 22: 137-47 (1995); and example 8 below. Candidates for the prevention and/or treatment of arthritis may also be studied in transgenic animal models, such as, for example, TNF-alpha transgenic mice (Taconic). These animals express human tumor necrosis factor (TNF- α), a cytokine that has been implicated in the pathogenesis of rheumatoid arthritis in humans. TNF- α expression in these mice results in severe chronic arthritis of the anterior and posterior paws, providing a simple mouse model of inflammatory arthritis.

In recent years, animal models of psoriasis have also been developed. Thus, sebum-free (Asebia) (ab), thin and flaky skin (fsn), and chronic proliferative dermatitis (cpd) are spontaneous mouse mutations with psoriatic skin changes. Transgenic mice whose skin overexpresses cytokines such as interferon-gamma, interleukin-1 alpha, keratinocyte growth factor, transforming growth factor-alpha, interferon-6, vascular endothelial growth factor, or bone morphogenic protein-6 may also be used to study psoriasis and identify therapeutic agents for treating psoriasis in vivo. Psoriatic lesions are also described in β 2-integrin suballent and β 1-integrin transgenic mice backcrossed to the PL/J strain, scid/scid mice reconstituted with CD4+/CD45RBhi T lymphocytes, and HLA-B27/h β 2m transgenic rats. A xenograft model using human skin transplanted onto immunodeficient mice is also known. Thus, the compounds of the invention may be described in Schon, m.p. eal, nat. med.3: 183(1997) in the scid/scid mouse model, where mice display histopathological skin lesions similar to psoriasis. Another suitable model is, for example, Nickoloff, b.j.et., am.j.path.146: 580(1995) human skin/scid mouse chimera prepared as described. See, e.g., Schon, m.p, J Invest Dermatology 112: 405-410(1999).

Recombinant (transgenic) animal models can be engineered by introducing the coding portion of the gene of interest into the genome of the animal of interest using standard techniques for generating transgenic animals. Animals that can serve as subjects for transgenic manipulation include, but are not limited to, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, such as baboons, chimpanzees, and other monkey species. Techniques known in the art for introducing transgenes into such animals include prokaryotic microinjection (Hoppe and warger, U.S. Pat. No. 4,873,191); retroviral-mediated gene transfer into the germline (e.g., Van der Putten et al, Proc. Natl. Acad. Sci. USA 82: 6148-; gene targeting in embryonic stem cells (Thompson et al, Cell 56: 313-321 (1989)); embryo electroporation (Lo, mol.cell.biol.3: 1803-1814 (1983)); sperm-mediated gene transfer (Lavitrano et al, Cell 57: 717-73 (1989)). For a review see, for example, U.S. Pat. No. 4,736,866.

For the purposes of the present invention, transgenic animals include those which carry the transgene only in a portion of their cells ("mosaics"). The transgenes may be integrated as a single transgene or as a tandem, e.g., a head-to-head or head-to-tail tandem. It is also possible to selectively introduce transgenes into specific cell types, for example Lasko et al, proc.natl.acad.sci.usa89 as follows: 623, and 636 (1992).

Expression of the transgene in the transgenic animal can be monitored by standard techniques. For example, Southern blot analysis or PCR amplification can be used to verify the integration of the transgene. The mRNA expression level can then be analyzed using techniques such as in situ hybridization, Northern blot analysis, PCR, or immunocytochemistry.

The animal may be further tested for signs of immunopathology, for example by histological examination to determine whether immune cells have infiltrated a particular tissue. Blocking experiments can also be performed in which transgenic animals are treated with CRIg or candidate agonists to determine the extent of effect on complement and complement activation, including the classical and alternative pathways, or T cell proliferation. In these experiments, blocking antibodies that bind to the polypeptides of the invention are administered to animals and the biological effect of interest is monitored.

Alternatively, a "knock-out" animal can be constructed having a defective or altered gene encoding CRIg as a result of homologous recombination between the endogenous gene encoding the CRIg polypeptide and altered genomic DNA encoding the same introduced into an animal's embryonic cells. For example, a cDNA encoding CRIg can be used to clone genomic DNA encoding CRIg according to established techniques. A portion of the genomic DNA encoding CRIg may be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5 'and 3' ends) are included in the vector (for a description of homologous recombination vectors see, e.g., Thomas and Capecchi, Cell 51: 503 (1987)). The vector is introduced into an embryonic stem Cell line (e.g., by electroporation), and cells in which the introduced DNA undergoes homologous recombination with endogenous DNA are selected (see, e.g., Li et al, Cell 69: 915 (1992)). The selected cells are then injected into blastocysts of an animal (e.g., mouse or rat) to form aggregated chimeras (see, e.g., Bradley, in Teratoccinomas and Embryonic Stem Cell: A practical apparatus, E.J.Robertson, ed. (IRL, Oxford, 1987), pp.113-152). The chimeric embryo can then be implanted into a suitable pseudopregnant female surrogate mother and the embryo produced at term to produce a "knockout" animal. Progeny comprising the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal comprise the homologously recombined DNA. Knockout animals can be characterized by, for example, the ability to defend against certain pathological conditions and develop pathological conditions due to the lack of a CRIg polypeptide.

Thus, the biological activity of CRIg or a potential agonist thereof can be further studied in murine CRIg knockout mice, as described in example 7 below.

An asthma model has been described in which antigen-induced airway hyperresponsiveness, pulmonary eosinophilia and inflammation are induced by sensitizing animals with ovalbumin and then challenging the animals with the same protein delivered by aerosol. Several animal models (guinea pig, rat, non-human primate) showed similar symptoms to atopic asthma in humans after challenge with aerosol antigens. Murine models have many of the characteristics of human asthma. Suitable procedures for testing the activity and efficacy of CRIg and CRIg agonists in the treatment of asthma are described in Wolyniec, w.w.et al, am.j.respir.cell mol.biol.18: 777(1998) and references cited therein.

Contact hypersensitivity is a simple in vivo assay of cell-mediated immune function. In this procedure, epidermal cells are exposed to exogenous haptens, which elicit a delayed hypersensitivity reaction, which is measured and quantified. Contact hypersensitivity involves an initial sensitization phase followed by a priming phase. The priming phase occurs when epidermal cells encounter antigens they have previously been exposed to. Swelling and inflammation occur, making it an excellent model of allergic contact dermatitis in humans. One suitable procedure is described in detail in CurrentProtocols in Immunology, eds. j.e.cologan, a.m.kruisbeek, d.h.margulies, e.m.shevach and w.strober, John Wiley & Sons, inc., 1994, unit 4.2. See also Grabbe, s.and Schwarz, t., immun.today19 (1): 37-44(1998).

Graft versus host disease occurs when immunocompetent cells are transplanted into an immunosuppressed or tolerated patient. The donor cells recognize and respond to host antigens. The response can vary from severe inflammation, which is life threatening, to mild cases of diarrhea and weight loss. The graft versus host disease model provides a means to assess T cell reactivity to MHC antigens and secondary transplantation antigens. One suitable protocol is described in detail in Current Protocols in Immunology, supra, Unit 4.3.

An animal model of skin allograft rejection is a means of testing the ability of T cells to mediate tissue destruction in vivo, an indication and measure of their role in antiviral and tumor immunity. The most common and accepted model uses rat tail skin grafts. Repeated experiments showed that skin allograft rejection is mediated by T cells, helper T cells and killer-effector T cells rather than antibodies. Auchincloss, H.Jr.and Sachs, D.H., Fundamental Immunology, 2nd ed., W.E.Paul ed., Raven Press, NY, 1989, 889-. One suitable protocol is described in detail in Current protocols Immunology, supra, Unit 4.4. Other transplant rejection models that can be used to test CRIg and CRIg agonists are Tanabe, m.et al, Transplantation 58: 23(1994) and Tinubu, s.a.et., j.immunol.4330-4338 (1994).

Animal models of delayed hypersensitivity also provide assays for cell-mediated immune function. Delayed hypersensitivity responses are T cell-mediated immune responses in vivo characterized by inflammation that peaks only after a period of time has elapsed following antigen challenge. These responses also occur in tissue-specific autoimmune diseases, such as Multiple Sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE, a model of MS). One suitable protocol is described in detail in Current Protocols in Immunology, supra, Unit 4.5.

EAE is a T cell mediated autoimmune disease characterized by T cell and monocyte inflammation and subsequent demyelination of axons in the central nervous system. EAE is generally considered a relevant animal model for human MS. Bolton, c., Multiple Sclerosis 1: 143(1995). Both acute and relapsing-remitting models have been developed. CRIg and its agonists and antagonists can be tested for T cell stimulatory or inhibitory activity against immune mediated demyelinating diseases using Current Protocols in Immunology, see Protocols described above in units15.1 and 15.2. See also the model for myelin diseases in which oligodendrocytes or Schwann (Schwann) cells are transplanted into the central nervous system as described by Duncan, I.D. et al, Molec.Med.Today554-561 (1997).

An animal model of age-related macular degeneration (AMD) consists of mice with a null mutation in the Cc1-2 or Ccr-2 gene. The main features of AMD developed in these mice include accumulation of lipofuscin in the Retinal Pigment Epithelium (RPE) and drusen underneath, photoreceptor atrophy and Choroidal Neovascularization (CNV). These features develop later than 6 months of age. Drusen formation, photoreceptor atrophy and choroidal neovascularization can be tested for CRIg and CRIg agonists.

CNVs can be tested in a variety of laser-induced choroidal neovascularization models. Thus, for example, CNV can be induced in rats and macaques by intense laser photocoagulation leading to choroidal neovascularization. The development and treatment of this condition can be assessed, for example, by fluorescein angiography, histological and immunohistochemical assessment, and pharmacokinetic, hemolytic, antibody screening and complement activation assays of sera collected from animals at various time intervals before and after treatment. The efficacy of prophylactic administration can be monitored by similar methods, including monitoring vascular leakage by fluorescein angiography, inhibition of complement deposition at the site of laser burn, eye examination, ophthalmography, collection of vitreous and retinal tissue, and the like. More details are provided in the examples below.

The model of myocardial ischemia-reperfusion can be performed in mice or rats. Animals were tracheotomized and ventilated with a small animal ventilator. Polyethylene catheters were placed in the internal and external carotid arteries for measurement of mean arterial blood pressure. Myocardial ischemia reperfusion was initiated by ligation of the left anterior descending artery (LAD) with 6-O suture. Ischemia is created by tightening a reversible ligation around the LAD to completely occlude the vessel. After 30 minutes the ligation was removed and perfused cardiovascularly for 4 hours. The efficacy of CRIg and CRIg agonists can be tested by measuring cardiac infarct size, myocardial ammonia kinase activity, myeloperoxidase activity, and immunohistochemistry using anti-C3 antibody.

One model of diabetic retinopathy involves treating mice or rats with streptozocin (streptozocin). CRIg and CRIg agonists can be tested for their effect on retinal and vitreous cavity veno-dilation, intraretinal microvascular abnormalities and neovascularization.

A model for membranoproliferative glomerulonephritis can be established as follows: female mice were i.p. immunized with 0.5mg of control rabbit IgG in CFA (day-7). Seven days later (day 0), 1mg of rabbit anti-mouse Glomerular Basement Membrane (GBM) antibody was injected i.v. via tail vein. The rise of anti-rabbit IgG antibodies in serum was measured by ELISA. A 24 hour urine sample was collected from mice in metabolic cages and mouse renal function was assessed by measuring urine protein in addition to blood urea nitrogen.

Pharmaceutical composition

The active molecules of the invention, including polypeptides and agonists thereof, as well as other molecules identified by the screening assays disclosed above, may be administered in the form of a pharmaceutical composition for the treatment of inflammatory diseases.

Therapeutic formulations of active molecules of the invention, preferably CRIg polypeptides or CRIg agonists, are prepared for storage by mixing the active molecule of the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers in lyophilized form or as an aqueous solution (Remington's pharmaceutical Sciences, 16th edition, Osol, a.ed., 1980). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexane diamine chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol 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 counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants, such as TWEEN^TM、PLURONICS^TMOr polyethylene glycol (PEG).

Compounds identified by the screening assays of the invention can be formulated in a similar manner using standard techniques well known in the art.

Lipofection or liposomes can also be used to deliver the polypeptide, antibody or antibody fragment into the cell. When antibody fragments are used, the smallest fragment that specifically binds to the binding domain of the target protein is preferred. For example, based on the variable region sequences of antibodies, peptide molecules can be designed that retain the ability to bind to a target protein sequence. Such peptides may be chemically synthesized, and/or generated by recombinant DNA techniques (see, e.g., Marasco et al, Proc. Natl. Acad. Sci. USA 90: 7889-7893 (1993)).

The formulations herein may also contain more than one active compound necessary for the particular indication being treated, preferably with activities complementary to each other and without adverse effects. Alternatively, or in addition, the composition may contain a cytotoxic agent, cytokine or growth inhibitory agent. Suitably, such molecules are combined in amounts effective for the intended purpose.

The active molecule may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's pharmaceutical sciences, 16th edition, Osol, a.ed., (1980).

Formulations for in vivo administration must be sterile. This can be easily achieved by filtration using sterile filtration membranes.

Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and L-glutamic acid gamma ethyl ester, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRONDEPOT ^TM(injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D- (-) -3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid are capable of releasing molecules for over 100 days, certain hydrogels release proteins for shorter periods of time. When encapsulated antibodies are maintained in vivo for extended periods of time, they may denature or aggregate by exposure to a humid environment at 37 ℃, resulting in a loss of biological activity and possible changes in immunogenicity. Rational stabilization strategies can be devised based on the relevant mechanisms. For example, if the aggregation mechanism is found to be intermolecular S — S bond formation via thiol-disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling humidity, employing appropriate additives, and developing specific polymer matrix compositions.

For intraocular administration, injection formulations are generally used, usually administered about six weeks apart. The eye was anesthetized prior to each injection.

However, it is also possible to use implants with sustained release formulations of CRIg or agonists, such as CRIg-Ig or CRIg ECD-Ig fusions, for intravitreal release.

The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

All patents and references cited in this specification are expressly incorporated herein by reference in their entirety.

Examples

Unless otherwise indicated, commercial reagents mentioned in the examples were used according to the manufacturer's instructions. The source of those cells identified by ATCC number in the examples and throughout the specification which follow is the American Type Culture Collection (American Type Culture Collection, 10801 university boulevard, Manassas, VA 20110-.

Example 1: isolation of a cDNA clone encoding human CRIg (PRO362)

The extracellular domain (ECD) sequences of about 950 known secreted proteins (including secretion signals, if any) in the Swiss-Prot public protein database were used to search the Expressed Sequence Tag (EST) database. EST databases include public EST databases (e.g., GenBank) and private ESTDNA databasesIncyte Pharmaceuticals, Palo Alto, Calif.). Searches the results of the 6-reading frame translation of the ECD protein sequence and the EST sequence were compared using the computer programs BLAST or BLAST-2 (e.g., Altsull et al, Methods in Enzymology 266: 460-. BLAST scores were clustered into 70 (or in some cases 90) or higher comparison programs "phrap" (Phil Green, University of Washington, Seattle, Washington) that do not encode known proteins and assembled into consensus DNA sequences.

Consensus DNA sequences were assembled using phrap relative to other EST sequences. This consensus sequence is referred to herein as DNA42257(SEQ ID NO: 9) (see FIG. 32). Based on the consensus sequence of DNA42257(SEQ ID NO: 9) shown in FIG. 32, oligonucleotides were synthesized: 1) to identify a cDNA library containing the sequence of interest by PCR, and 2) as a probe for isolating clones of the full-length coding sequence of CRIg. The forward and reverse PCR primers typically range from 20-30 nucleotides and are often designed to produce PCR products of approximately 100 and 1000bp in length. The probe sequences are typically 40-55bp in length. In some instances, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5 kbp. To screen several libraries for full-length clones, DNA from the libraries was screened by PCR amplification with PCR primer pairs according to Ausubel et al, Current Protocols in Molecular Biology. The positive library is then used to isolate clones encoding the gene of interest using one of the probe oligonucleotides and primer pairs.

Synthesis of PCR primers (forward and reverse):

forward PCR primer 1(42257.f1)

5’-TATCCCTCCAATTGAGCACCCTGG-3’(SEQ ID NO：10)；

Forward PCR primer 2(42257.f2)

5’-GTCGGAAGACATCCCAACAAG-3’(SEQ ID NO：11)；

Reverse PCR primer 1(42257.r1)

5’-CTTCACAATGTCGCTGTGCTGCTC-3’(SEQ ID NO：12)；

Reverse PCR primer 2(42257.r2)

5’-AGCCAAATCCAGCAGCTGGCTTAC-3’(SEQ ID NO：13)。

In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was constructed based on the DNA42257 consensus sequence:

Hybridization Probe (42257.p1)

5’-TGGATGACCGGAGCCACTACACGTGTGAAGTCACCTGGCAGACTCC

TGAT-3’(SEQ ID NO：14)。

To screen several libraries for the origin of full-length clones, DNA from the libraries was screened by PCR amplification using the PCR primer pairs described above. The positive library was then used to isolate clones encoding the CRIg gene using one of the probe oligonucleotides and PCR primers.

RNA was isolated from human fetal brain tissue (LIB153) for construction of cDNA libraries. The cDNA library used to isolate the cDNA clones was constructed by standard methods using commercially available reagents such as those purchased from Invitrogen, san diego, CA. The cDNA is primed with an oligo dT containing a NotI site, ligated blunt-ended with a SalI semi-phosphorylated (hemiphosphorylated) adaptor, cleaved with NotI, appropriately separated by gel electrophoresis, and cloned in a defined orientation between the unique XhoI and NotI sites of a suitable cloning vector such as pRKB or pRKD; pRK5B is a pRK5D precursor without SfiI sites; see Holmes et al, Science 253: 1278-once 1280 (1991).

DNA sequencing of clones isolated as described above resulted in the DNA sequence of the isolated CRIg polypeptide (referred to herein as UNQ317(DNA45416-1251) (SEQ ID NO: 1)).

FIG. 1(SEQ ID NO: 1) shows the complete nucleotide sequence of UNQ317(DNA 45416-1251). The clone UNQ367(DNA45416-1251) (SEQ ID NO: 1) contained a single open reading frame, and the apparent translation initiation site was located at nucleotide 1082-1084 (FIG. 1, SEQ ID NO: 1). The predicted polypeptide precursor is 321 amino acids long (FIG. 1, SEQ ID NO: 2). The CRIg protein shown in figure 1 has an estimated molecular weight of about 35,544 daltons, and a pI of about 8.51. Analysis of the 321 amino acid CRIg polypeptide shown in FIG. 1(SEQ ID NO:2) demonstrated the presence of a glycosaminoglycan attachment site from about amino acid 149 to about amino acid 152, and a transmembrane domain from about amino acid 276 to about amino acid 306. Clone UNQ317(DNA45416-1251) has been deposited under ATCC accession number 209620.

Like JAM family members, CRIg (PRO362), recently called CRIg, is a type 1 transmembrane molecule, a member of the immunoglobulin superfamily. The extracellular domain of long human CRIg (huCRIg (L))) encodes both V and C2 type terminal Ig domains (Smith and Xue, J.mol.biol.274: 530-545(1997)), while the short form (huCRIg (S))) encodes only a single V type Ig, similar to murine CRIg (muCRIg) (FIG. 50A). The C-terminal cytoplasmic domain of both human and murine CRIg contained a consensus AP-2 internalization motif (YARL and DSQALI, Bonafacino & Trub, Ann Rev Biochem 72: 395(2003)), respectively. HuCRIg and mucIg share 67% overall sequence homology, and 83% homology exists in the IgV domain. Among the JAM family members, huCRIg is most closely related to JAM-A. Sequence similarity is limited to a conserved stretch of residues that form the Ig domain fold (fig. 50A). Both human and murine CRIg are located on the X chromosome Xq12, with a syntenic (syntenic) position on the chromosome flanked by a membrane iron transport accessory protein (hephaestin) and a moesin protein.

Example 2: preparation and purification of proteins

The extracellular domains of hu and muCRIg were cloned into a modified pRK5 expression vector encoding a human or murine IgG1 Fc region downstream of the CRIg sequence. The Fc portion of murine IgG1 contained a double mutation (D265A, N297A) that prevented Fc receptor binding (Gong et al, J.Immunol.174: 817-826(2005)) for controlling Fc receptor modulation. Human IREM-1 and mouse CLM-1 Fc fusion proteins or murine anti-gp 120 IgG antibodies were used as controls. LFH-tagged CRIg was prepared by fusing the ECD of CRIg with yeast leucine zipper, Flag and C-terminal (6) histidine. Proteins were overexpressed in CHO cells by transient transfection. Cells were cultured in a fully automated bioreactor using a medium based on F-12/Dulbecco's modified Eagle's medium supplemented with Ultra-Low IgG serum (Invitrogen) and Primatone HS (Sigma). Cultures were maintained for 7-12 days until harvest. The Fc fusion protein was purified by protein A affinity chromatography followed by Sephacryl S-300 gel filtration. LFH fusion proteins were purified on nickel columns. Human CRIg-ECD protein was affinity purified on Millipore Glyceryl-CPG (173700404) column adsorbed with monoclonal antibody 3C 9. The protein eluted at pH 3.0. hu and mucIg-HIS were prepared as follows: the CRIg ECD was cloned into a baculovirus expression vector containing a C-terminal (6) histidine. Plasmid DNA was transfected into Sf9 cells, H5 cells were infected with the supernatant, and the protein was purified on a nickel column. Identification of all purified proteins confirmed by N-terminal sequence analysis that all human or murine CRIg preparations had lipopolysaccharide concentrations < 5 Eu/mg.

Example 3: preparation of antibodies

Polyclonal antibodies were prepared as follows: new Zealand rabbits were immunized with 200. mu.g of huCRIg (L) -His in perfluoronated adjuvant and boosted 6 weeks after the first immunization. monoclonal antibodies to muCRIg and huCRIg were prepared as follows: wistar rats and Balb/c mice were immunized with 50. mu.g of his-labeled CRIg fusion protein by paw pad injection. Clones were selected for reactivity with human and murine CRIg-ECD by ELISA, FACS, Western blot and immunohistochemistry. The antibodies obtained were used for subsequent testing, unless otherwise indicated.

Example 4: infiltration of inflammatory cells into guinea pig skin

The following examples show that huCRIg (PRO362) is PRO-inflammatory, i.e. it stimulates infiltration of inflammatory cells (i.e. neutrophils, eosinophils, monocytes or lymphocytes) into guinea pig skin. The assay described herein monitors the ability of this protein to induce inflammatory cell infiltration into guinea pig skin. Compounds that stimulate inflammatory infiltration are useful in therapies that enhance the beneficial inflammatory response. Compounds that inhibit lymphocyte proliferation are useful in therapies where inhibition of an inflammatory response is beneficial. The therapeutic agent can take the form of, for example, a murine-human chimeric, humanized or human antibody directed against CRIg, a small molecule, peptide, etc., that mimics the biological activity of CRIg, a CRIg fusion protein, a CRIg extracellular region, and the like.

Hairless guinea pigs weighing 350 g or more (Charles River Labs) were anesthetized by intramuscular injection of ketamine (75-80mg/kg body weight) and xylazine (5mg/kg body weight). Protein samples of huCRIg and control proteins were injected subcutaneously into the back of each animal in 100 μ l volumes per injection site. About 16-24 injection points per animal. 1mL of Evans blue (Evans blue) dye (1% in physiological buffered saline) was injected intracardially. Animals were euthanized after 6 hours and examined for viable tissue at each skin injection site and fixed in formalin. The skin was treated for histopathological evaluation. The spots were evaluated for inflammatory cell infiltration into the skin. Positive was scored as having visible inflammatory cells. Samples that induced inflammatory cell infiltration were scored as proinflammatory substances. CRIg tested positive in this assay, indicating anti-inflammatory activity.

Example 5: CRIg (PRO362) mRNA and polypeptide expression

A. In situ hybridization and immunohistochemistry

CRIg mRNA expression in various types of tissues was assessed by in situ hybridization, immunohistochemistry and RT-PCR.

For in situ hybridization, tissues were fixed (4% formalin), paraffin embedded, sectioned (3-5 μm thick), deparaffinized, deproteinized with proteinase K (20 μ g/ml) (15 min, 37 ℃) and processed for in situ hybridization. Probes for the polypeptides of the invention were prepared by PCR. The primer contains a T7 or T3RNA polymerase initiation site, allowing transcription of the sense or antisense probe from the amplification product in vitro. ³³The P-UTP labeled sense and antisense probes were hybridized overnight (55 deg.C), washed (0.1 XSSC, 55 deg.C, 2 hours), immersed in NBT2 nuclear channel emulsion (Eastman Kodak, Rochester, N.Y.), exposed (4 deg.C, 4-6 weeks), developed, and counterstained with hematoxylin and eosin. Typically, representative pairs of bright and dark images appear.

Immunohistochemical staining was performed on 5mm thick frozen sections using a DAKO automated staining machine. Endogenous peroxidase activity was blocked with Kirkegaard and Perry blocking solution (1:10, 4 min, 20 ℃). TBS/10% NGS in 0.05% Tween-20(DAKO) was used for dilution and blocking. MAb4F7.22.2 anti-CRIg (anti-PRO 362) or mouse IgG was used at 0.13 mg/ml. Biotinylated goat anti-mouse IgG (Vector Labs, Burlingame, Calif.) was used at 1:200 and detected using the Vector Labs Standard ABC Elite kit (Vector Labs, Burlingame, Calif.). Slides were developed with Pierce metal-enhanced diaminobenzidine (Pierce Chemicals, Rockford, IL). Double immunohistochemistry for CRIg (PRO362) and CD68 expression was performed on frozen sections to visualize the localization of CRIg expression to macrophages. Detection was by phycoerythrin and FITC markers using mAb4F7.22.2 anti-CRIg and anti-CD 68mAb KP-1 from DAKO, respectively.

Expression has been examined in a wide variety of tissues and cell types in humans and other animals.

a. Normal tissue

Normal adult tissues examined included tonsil, lymph node, spleen, kidney, bladder, lung, heart, aorta, coronary artery, liver, gall bladder, prostate, stomach, small intestine, colon, pancreas, thyroid, skin, adrenal gland, placenta, uterus, ovary, testis, retina and brain (cerebellum, brainstem, cerebral cortex). Normal human fetal tissues including E12-E16 week old brain, spleen, intestine and thyroid were also examined. In addition, expression in murine liver was investigated.

b. Inflamed tissue

The inflamed tissues examined by in situ hybridization include tissues with chronic inflammatory diseases such as lungs with chronic asthma, chronic bronchopneumonia, chronic bronchitis/chronic obstructive pulmonary disease, kidneys with chronic lymphocytic interstitial nephritis, and liver with chronic inflammation and cirrhosis due to chronic hepatitis c infection, autoimmune hepatitis or alcoholic cirrhosis.

c. Primary tumor (Primary Neoplasms)

Primary human tumors examined for PRO362 expression by in situ hybridization include breast, lung squamous cell, lung adenocarcinoma, prostate adenocarcinoma, and colon adenocarcinoma.

2. Results

CRIg (PRO362) was found to be expressed in mouse liver frozen sections (fig. 6), human liver frozen sections (fig. 7), and various tissue macrophage-like cells including colon macrophages (fig. 8A), Kupffer cells (fig. 8B), adrenal macrophages (fig. 8C), Hofbauer cells (fig. 8D), synovial cells (fig. 9), blastocyst (alvelar) macrophages, resident macrophages in the intestinal lamina propria, and interstitial macrophages in various tissues. CRIg was also significantly expressed in brain microglia (figure 10). CRIg expression in these tissues was significantly elevated when activated in the presence of tumors or inflammatory diseases including rheumatoid arthritis (figure 9), inflammatory bowel disease, chronic hepatitis (figure 12), pneumonia, chronic asthma (figure 11), glioma and bronchitis.

To further examine CRIg expression, immunohistochemical staining was performed on multiple tissue types. Double immunohistochemical staining of CRIg and CD68 was performed on tissue macrophages, including adrenal macrophages, liver Kupffer cells, brain microglia cells, and placenta Hofbauer cells to determine whether CRIg and CD68 were expressed in the same tissue.

CRIg was found to be co-expressed with CD68 in adrenal macrophages (fig. 13), liver Kupffer cells (fig. 14), brain microglia cells (fig. 15), and placental Hofbauer cells (fig. 16).

Example 6: CRIg (PRO362) is implicated in chronic inflammation

As described in example 1 and below, a novel macrophage-associated receptor with homology to the a33 antigen and JAM1 was cloned and identified as a single transmembrane Ig superfamily member macrophage-associated polypeptide (CRIg or PRO 362).

CRIg is expressed as two splice variants. A variant is a 399 amino acid polypeptide comprising an N-terminal IgV-like domain and a C-terminal IgC 2-like domain, designated huCRIg or huCRIg-long (SEQ ID NO: 4). The 355 amino acid long spliced form lacking the C-terminal domain is called huCRIg-short (SEQ ID NO: 6). Both receptors have a single transmembrane domain and a cytoplasmic domain containing tyrosine residues that are constitutively phosphorylated in macrophages in vitro.

This study demonstrates that CRIg is selectively expressed in a subset of macrophages residing in the tissue and is associated with chronic inflammation.

Materials and methods

Cells

After informed consent, blood was obtained from a vein puncture of healthy adult volunteers and isolated using Ficoll-PaquePLUS (Amersham Pharmacia Biotech) according to the manufacturer's instructions. PBMCs were harvested from the interface, washed with cold PBS, lysed with 0.2% NaCl for 30 seconds, and neutralized with 1.6% NaCl. Cells were counted and stored on ice until use. To isolate the peripheral blood subsets, an unsealed MACS kit (Miltenyi Biotech, Auburn, CA) was used according to the manufacturer's instructions. Differentiation into macrophage phenotype was induced by culturing CD14+ monocytes in HG-DMEM medium containing 10% (v/v) autologous human serum, 20% fetal bovine serum, and 10mM 1-glutamine, penicillin, and streptomycin for up to 2 weeks. The medium was changed on day 5. For flow cytometry analysis, cells were dissociated from culture dishes using ice-cold cell dissociation fluid (Sigma). Lysates for Western blot analysis were prepared by adding 0.5ml lysis buffer directly to the wells. The lysate was mixed with sample buffer containing SDS and beta-mercaptoethanol, electrophoresed on Tris-glycine gel, and transferred to nitrocellulose membrane. Cell viability was assessed by trypan blue exclusion assay.

Flow cytometry

Cells for flow cytometry analysis were blocked with PBS containing 2% fetal bovine serum and 5. mu.g/ml human IgG (Calbiochem, San Diego, Calif.) for 30 min at 4 ℃. Next, the cells were incubated with the anti-CRIg (anti-PRO 362) monoclonal antibody 3C 9. After washing with PBS, cells were stained with CD1lb, CD14, CD163, CD15, CD68 antibody (Pharmingen) conjugated with Phycoerythrin (PE).

Cell-cell adhesion study

Expression of pRK expression vector containing full-length CRIg was stably expressed in human Jurkat T cell line using neomycin selection and Autoclone (Autoclone) protocol as described elsewhereAnd (4) selecting. Cells were preloaded with the fluorescent dye BCECF (Molecular Probes, Oregon) and added to 96-well Maxisorb plates (CORNING) coated with a monolayer of Human Umbilical Vein Endothelial Cells (HUVEC) with or without 10ng/ml TNF α treatment^TM) In (1). Cells were washed gently as follows: incubation buffer (HBSS containing 10mM CaCI, 10mM magnesium, and 1.5mM NaCl) was added to the wells, and the plates were then inverted onto a piece of absorbent paper. After 3 washes, fluorescence counts were performed in a fluorescence spectrometer. The fluorescence readings represent the number of cells still adhering to the HUVEC cells.

Northern blot analysis

Random priming of full-length CRIg cDNA Using Ambion kit following manufacturer's recommendations ³²The P-labeled probe probes multiple tissue Northern blots. The blot was exposed to a phosphorus imaging screen for 4 hours at 22 ℃. Blots were excised and re-probed with a commercial probe for human or mouse beta-actin (Clontech) to assess RNA load and quantity in each lane and with StormPhosphorus imager (molecular dynamics, Sunnyvale, Calif.) analysis.

Real-time Rtpcr analysis

For quantitative PCR analysis (TAQMAN)^TM) Total mRNA (100ng) from human tissue or primary cells (PerkinElmer Life Sciences) and primers based on the CRIg coding sequence were recommended.

Preparation of Fc and His fusion proteins

Human CRIg was cloned into baculovirus expression vector pHIF (Pharmingen). The HIS-tagged CRIg fusion protein is formed by fusing CRIg extracellular domain and 8 histidines. His-tagged fusion proteins were purified from the supernatant of suspension-cultured baculovirus-infected insect cells using nickel affinity resin.

Preparation of monoclonal and polyclonal antibodies

For the present experiment, female BALBc was immunized and boosted with 10 μ g crig-His8 by paw pad injection as previously described by Ghilardi et al, j.biol.chem.277: 1683l-16836 (2002). Individual clones were screened for CRIg-His by ELISA. Selected clones were tested against JAM family members and human IgG Fc. Clones were titrated to single cell density and screened again. Clone 3C9(IgG1) was found to be selectively reactive to CRIg. Clones were used for ascites production and purified on protein g (amersham Pharmacia biotech); protein concentration was determined using Pierce BCA reagent (Pierce, Rockford, IL).

Polyclonal antibodies were prepared by injecting New Zealand rabbits with 150 μ g CRIg-His. Serum titers were determined by ELISA. Serum was collected at peak circulating IgG levels and purified on a protein a column.

In situ hybridization

PCR primers (at top 5 '-TCTCTGTCTCCAAGCCCACAG (SEQ ID NO: 18) and at bottom 5' -CTTTGAGGAGTCTTTGACC (SEQ ID NO: 19)) were designed to amplify a 700bp huJAM4 fragment. The primer contains a T7 or T3RNA polymerase initiation site, allowing transcription of the sense or antisense probe from the amplification product, respectively, in vitro. Normal adult tissues include tonsils, lymph nodes, spleen, kidney, lung and heart. Tissues with chronic inflammatory diseases include lungs with chronic asthma, chronic bronchitis, liver with chronic inflammation and cirrhosis due to chronic hepatitis c infection. Tissues were fixed in 4% formalin, paraffin embedded, sectioned (3-5 μm thick), deparaffinized, deproteinized with 20 μ g/ml proteinase K (15 min, 37 ℃) and processed for in situ hybridization as described elsewhere.

Immunohistochemistry

Human livers were obtained from ardis corporation, Lexington, MA. Immunohistochemical staining was performed on frozen liver sections 5-6 μm thick using a DAKO automated stainer. Endogenous peroxidase activity was blocked with Kirkegaard and Perry blocking solution (1:10, 4 min, 20 ℃). TBS/10% Normal Goat Serum (NGS) in 0.05% Tween-20 was used for dilution and blocking. Mab3C9 was used at 1. mu.g/ml. Slides were developed with Pierce metal enhanced diaminobenzidine (Pierce Chemicals).

For immunofluorescent staining of sections, sections were blocked with PBS/10% NGS and incubated with mAb3C9 for 1 hour at 20 ℃. A rabbit anti-mouse FITC-labeled secondary antibody conjugated to FITS was used as a detection agent. For the double staining procedure, sections were subsequently stained with PE-conjugated monoclonal antibody to human CD 68.

Results

huCRIg was cloned from a human fetal cDNA library using degenerate primers recognizing the conserved Ig domain of human JAM1 as described in example 1. Sequencing of several clones revealed an open reading frame of 321 amino acids (FIG. 1, SEQ ID NO: 2). Blast search confirmed similarity to the type 1 transmembrane protein Z39Ig (Langnaese et al, Biochim Biophys Acta 1492: 522-525 (2000)). This 321 amino acid protein was later found to have lost some of the C-terminal amino acid residues. The full-length huSIgMA protein was determined to have 399 amino acid residues as shown in FIG. 2 (SEQ ID NO: 4). The extracellular region of CRIg consists of 2 Ig-like domains, including one N-terminal group V domain and one C-terminal group C2 domain. Cloning using 3 'and 5' primers yielded a splice variant of CRIg which lacks the membrane-proximal IgC domain, CRIg-short (305 amino acids, FIG. 3, SEQ ID NO: 6).

Cloning and sequence comparison of murine CRIg

The murine Expressed Sequence Tag (EST) database was searched using huCRIg's complete open reading frame and tblastn algorithm. Sequencing the DNA of 3 clones yielded the same 280 amino acids complete open reading frame. Primers for 3 priming regions were used to clone full-length transcripts from a mouse spleen library. Murine clones resemble the spliced form of huCRIg, i.e., lack a C-terminal Ig-like domain. The extracellular IgV domain is well conserved between human and murine receptors, with 93% identity. The murine cytoplasmic domain is poorly conserved, 20 amino acids shorter than its human counterpart, with 40% identity. Nucleic acid encoding murine crig (mucrig) and the deduced amino acid sequences are shown in figure 4 as SEQ ID NOS: 7 and 8.

CRIg is expressed on resident macrophage subsets in various tissues and its expression is elevated in inflammation

Northern blot analysis of huCRIg showed that both transcripts of 1.5 and 1.8kb (FIG. 17) were expressed most in adrenal glands, lung, heart and placenta, and less in other organs such as spinal cord, thyroid, mammary gland and lymph nodes. In all tissues, the 1.8kb transcript is the most abundantly expressed transcript, probably encoding the long CRIg. A single transcript of approximately 1.4kb was detected in the mouse liver and heart.

TAQMAN^TMReal-time PCR analysis

To identify specific cell lines expressing CRIg, real-time quantitative PCR and primers/probes specific for the N-terminal Ig domain were used. Low but detectable mRNA expression was found in PMA-treated myeloid cell line HL-60 and monocyte cell line THP-1. Expression was not seen in B and T cell lines (fig. 18A).

CRIg expression on differentiated monocytes.

To determine the details of when CRIg is expressed in differentiating monocytes/macrophages, we measured CRIg mRNA levels in non-adherent and adherent monocytes induced to differentiate in the presence of human autologous serum. CRIg mRNA levels gradually increased over time to reach a maximum level at day 7 post-plating (figure 18B). At this stage of differentiation, mRNA levels were 100-fold higher than in undifferentiated monocytes.

Western blot analysis of monocyte/macrophage lysates showed a parallel increase in CRIg protein expression with an increase in CRIg mRNA expression (figure 18C), indicating that CRIg is expressed when monocytes differentiate to form macrophages. A48 kDa band and a 40kDa band appeared on the blot, possibly representing long and short forms of human CRIg.

Molecular characterization of CRIg

CRIg migrated similarly under reducing and non-reducing conditions, indicating that it was expressed in monomeric form (figure 19A). When CRIg was deglycosylated with PNGase F, the migration pattern was only slightly altered, indicating that N-glycosylation was not significant. CRIg was phosphorylated when cells overexpressing CRIg were treated with peroxyvanadate (figure 19B). Migration of phosphorylated CRIg appeared as a slightly higher Mw protein (55 kDa). In human HEK293 cells, tyrosine phosphorylated CRIg cytoplasmic domain did not recruit Syk kinase (results not shown).

Flow cytometry analysis of CRIg expression on peripheral blood mononuclear cells

To determine the expression pattern of CRIg in circulating leukocytes, flow cytometry analysis was performed on lymphocytes isolated from blood of healthy donors using monoclonal anti-human CRIg antibody 3C 9. Antibodies were prepared by immunizing Balb/C mice with an octameric His-tagged human CRIg extracellular domain. The antibody is a non-blocking antibody, and is directly related to ALEXA^TMA488 conjugate, which can be used to detect native proteins in acetone-fixed cryosections. Counterstaining was performed with PE-conjugated antibodies to several immune cell surface antigens. CRIg was not found on the surface of all leukocytes, including B-, T-, NK cells, monocytes and granulocytes (fig. 20). However, CRIg expression was found on monocytes cultured for 7 days in macrophage differentiation medium.

Modulation of CRIg expression in monocytes

To investigate the modulation of CRIg expression, macrophages were cultured for 7 days in the presence of various pro-and anti-inflammatory cytokines and CRIg expression levels were determined by real-time PCR or flow analysis. After 2 days of macrophage treatment with IL-10 and TGF- β, CRIg mRNA expression was elevated, while IL-4, IL13 and LPS down-regulated expression (FIG. 21A). Dexamethasone treatment increased expression by 5-fold compared to control untreated macrophages. To determine the regulation of CRIg expressed on the cell surface, flow cytometry was performed on peripheral blood mononuclear cells treated for 5 days with various cytokines and dexamethasone. CRIg was detected using monoclonal antibody clone 3C9 coupled to ALEXATMA 488. Cells were co-stained with anti-CD 14 antibody. After 5 days of monocyte treatment with IL-10 and LPS, the surface expression of CRIg was found to be elevated (FIG. 21B). After treatment with dexamethasone, a significant increase in surface CRIg expression was found.

Subcellular distribution of CRIg

To study the subcellular distribution of CRIg, Monocyte Derived Macrophages (MDM) were cultured for 15 days, then fixed, stained with monoclonal antibody (clone 3C9) or polyclonal rabbit antibody 4F7, followed by staining with FITC-conjugated secondary antibody and PE-labeled anti-CD 63 antibody. Confocal microscopy showed that CRIg is highly expressed in the perinuclear cytoplasm, overlapping with the expression of the lysosomal membrane protein CD63 (fig. 22). CRIg is also expressed on the anterior and posterior edges of macrophages where its staining pattern does not overlap with CD 63.

CRIg expression in Normal and diseased tissues

The CRIg expression in tissue resident macrophages and its changes in expression in tissues with chronic inflammatory diseases were studied. Crig mrna expression was determined using in situ hybridization on a panel of paraformaldehyde-fixed human tissues. High expression levels were found in alveolar macrophages obtained from lung autopsies of patients with pneumonia or chronic asthma (fig. 23, a, B, C and D). High mRNA expression was found in liver Kupffer cells of chronic hepatitis patients (fig. 23, E and F).

High expression of CRIGmRNA was found in synovial membranes of rheumatoid arthritis patients in previous studies (Walker, Biochimica et Biophysica Acta 1574: 387 390(2002)) and electronic library screening. Therefore, the expression pattern of CRIg in synovium obtained from patients with rheumatoid arthritis, osteoarthritis and degenerative (degenerative) bone disease was investigated. High expression of CRIg mRNA was found in synovial cells obtained from osteoarthritic patients (figure 24, B). Synovial cells in the superficial layer had the highest CRIg expression (figure 24, D). In addition, CRIg expression in frozen sections of human synovium obtained from rheumatoid arthritis patients was studied using the polyclonal antibody 6F 1. CRIg is expressed in a fraction (20-40%) of synovium in synovium and tissue phagocytic macrophages (fig. 25, a, B, C). These cells are most likely type a macrophage-like synovial cells. No staining was seen in the control synovium (fig. 25, D).

Expression of CRIg protein is also found on macrophages in many different tissues. Frozen sections prepared from CHO cells stably expressing CRIg showed membrane localization of CRIg (fig. 26A). CRIg protein was found in alveolar macrophages (fig. 26, B), histiocytes in the lamina propria of the small intestine (fig. 26, C), Hofbauer cells in the placenta (fig. 26, D), macrophages in the adrenal gland (fig. 26, E) and Kupffer cells in the liver (fig. 26, F).

Atherosclerotic plaques contain a large number of macrophages or macrophage-foam cells that adhere tightly to the aortic lumen wall. The expression of CRIg in atherosclerotic plaques was studied, taking into account its role in macrophage-endothelial adhesion. Alternate sections of the plaques were stained with either anti-CD 63 (fig. 27, a and B) or anti-CRIg (fig. 27, C and D). Overlapping staining patterns against CD63 and CRIg were found in foam cells lining the vessel wall, suggesting a role for CRIg in atherosclerosis.

To determine whether CRIg is selectively expressed on macrophages, double staining immunofluorescence was performed on cardiac stromal macrophages (fig. 28). As shown in the overlay (fig. 28, third panel), CRIg-positive stromal macrophages were also mostly CD 68-positive. Not all CD 68-positive macrophages were CRIg-positive, indicating that the latter are specific for a subset of tissue-resident macrophages.

To quantify mRNA expression levels in Inflammatory Bowel Disease (IBD) syndrome, mRNA was extracted from colon tissue obtained from patients with ulcerative colitis, Crohn's disease (Crohn) or from patients without IBD manifestations. Relative expression levels were measured by real-time PCR using primers specific for CRIg. Expression levels were 16-fold higher in patients with ulcerative colitis and 5-fold higher in patients with crohn's disease compared to control tissues (fig. 29, a). Similarly, relative RNA equivalents in lung tissue were determined and found to be highest in tissues of patients with chronic obstructive pulmonary disease (COPD: 14-fold higher than normal), with no significant difference from normal in asthmatic patients (FIG. 29, B).

It is well known that molecules of the Ig superfamily mediate cell surface recognition and cell-cell adhesion. Since CRIg expression is higher in interstitial macrophages lining blood vessels, the involvement of CRIg in macrophage-endothelial cell adhesion was studied. Jurkat cell lines stably transfected with full length CRIg-Long (FIG. 30A) were loaded with the fluorescent dye BCECF and cells were added to wells of a 96-well maxisorb plate that had been cultured with a monolayer of HUVEC cells. Adhesion was measured by the amount of fluorescence remaining after 3 gentle washes. Jurkat cells expressing CRIg adhered both the control and TNF α -stimulated endothelium more than Jurkat cells stably transfected with the control plasmid (fig. 30B).

Discussion of the related Art

This study described for the first time the tissue distribution, regulation of expression and molecular characterization of the novel Ig superfamily member, CRIg/Z39Ig, confirming its selective expression in tissue resident macrophages.

CRIg expression was found on resident macrophages with a fully differentiated phenotype. Its expression is elevated in tissues of chronic inflammatory-like, rheumatoid arthritis and inflammatory bowel disease. Elevated CRIg expression in these diseases, which are generally characterized by Th 2-type diseases, may be consistent with the modulation of expression by Th2 cytokines in vitro. Whether this increased expression is due to increased presence of CRIg-positive macrophages or increased expression on inflammatory macrophages remains to be determined.

CRIg mediates one of the effector functions of human macrophages, including bacterial recognition, phagocytosis, antigen presentation, and cytokine release. These results indicate a role for CRIg in adhesion and possible migration of macrophages towards the vascular endothelial cell wall.

CRIg expression is elevated in non-microbial inflammatory diseases like ulcerative colitis and Chronic Obstructive Pulmonary Disease (COPD), but is down-regulated in isolated macrophages when treated with LPS or other bacterial cell wall components like lipoteichoic acid and bacterial lipoproteins. Prolonged treatment with LPS for more than 2 days resulted in increased CRIg expression. This may be due to the autocrine effect of IL-10 secreted by macrophages stimulated by LPS. Both mRNA and protein levels of CRIg were found to be significantly upregulated when monocytes or macrophages were treated with dexamethasone. It was found that very little monocyte/macrophage surface receptor expression was elevated upon treatment with dexamethasone. One example is CD163, but its induction by dexamethasone is very insignificant. It is of interest to upregulate CRIg by the anti-inflammatory cytokines IL10 and TGF β, suggesting that CRIg may mediate the anti-inflammatory effects of glucocorticosteroids.

As described herein, CRIg is expressed on a portion of CD 68-positive macrophages, which may be activated macrophages. Blocking and activating antibodies using CRIg and CRIg-Fc fusion proteins, their role in macrophage effector function, adhesion and migration and their role in chronic inflammatory diseases have been investigated and are described in example 7.

Only few cell surface markers are specifically expressed on differentiated macrophages, such as CD68 and CD 163. Although CD68 was clearly expressed on all human macrophage populations, the antigen was also detected on other myeloid cells as well as on some non-myeloid cells. Thus, CRIg is the first cell surface antigen selectively expressed on a portion of stromal mature macrophages.

Example 7: CRIg fusion protein in collagen-induced arthritis (CIA) in DBA-1J mice

The aim of this experiment was to compare CRIg fusion protein with control murine IgG1 in disease development and CIA (collagen-induced arthritis, an experimental animal model system for rheumatoid arthritis) development.

As discussed in example 4, CRIg is specifically highly expressed in a fraction of macrophages and elevated in chronic inflammatory tissues. Murine CRIg is highly expressed in macrophages and synovial cells in inflamed joints of collagen-induced arthritic mice. In vitro studies have shown that CRIg is involved in adhesion of macrophages to endothelium. CRIg-Fc fusion proteins affect the course of autoimmune disease, in this case collagen-induced arthritis in mice, either by affecting the properties of tissue macrophages or by affecting the immune response of other cells (e.g., T cells, B cells, epithelial cells, endothelial cells). This can lead to inflammation, swelling and mitigation of long-term bone erosion in the joints.

The muCRIg-Fc fusion protein was prepared as follows: the hinge, CH2 and CH3 domains of murine IgG1 were fused to the extracellular domain (amino acids 1-200) of murine CRIg. Fusions containing double mutations that prevent Fc receptor binding are used to control Fc receptor regulation. The nucleotide sequence of the muCRIg-Fc fusion protein is shown as SEQ ID NO: shown at 17. (similar huCRIg-Ig and huCRIg-short-Ig coding sequences are shown in SEQ ID NOS: 15 and 16, respectively.) proteins were produced by transient transfection of CHO cells with plasmid DNA. The fusion protein was purified as follows: the cell supernatant was run through a protein a column followed by ion exchange chromatography to eliminate aggregates. Serum half-life was assessed as follows: C57B6 mice were injected with a dose of 4mg/kg CRIg-Fc and sera were collected from the mice at the indicated time intervals. Serum levels of murine CRIg-Fc were determined by sandwich ELISA using anti-CRIg mab recognizing different epitopes on the CRIg extracellular domain.

Animal model species: mouse

And (2) breeding: DBA-1J

The supplier: JACKSON

Age range: 7-8 weeks old

Mice were selected as species to study collagen-induced arthritis (CIA), as CIA is an inflammatory polyarthritis with similar clinical and pathological features as human Rheumatoid Arthritis (RA). This animal model has been used by several laboratories, and the histopathology of CIA resembles that seen in RA with synovial hyperplasia and its progression to pannus formation, cartilage degeneration/destruction and marginal bone erosion and subsequent joint deformity. In addition, mice are the lowest phylogenetically mammals. Furthermore, no in vitro model has been able to mimic the complex multifactorial pathogenesis of RA.

Design of experiments

Test groups:

1) 6mg/kg mIgG1 isotype was injected Subcutaneously (SC) in 200 μ l saline 3 times per week for 7 weeks (n ═ 8).

2) 4mg/kg mucig was injected Subcutaneously (SC) in 100 μ l saline 3 times per week for 7 weeks (n ═ 8).

Mice were immunized intradermally with bovine CII (100 μ g, Sigma, St Louis) emulsified in cfs (difco). After 21 days, mice were challenged again with CII emulsified in ifa (difco). Starting on day 24, one group of mice (n ═ 7) was given 100 μ g muCRIg (PRO362) Fc 3 times a week for 6 weeks, and the second group (n ═ 8) received 100 μ g murine IgG1 as a control. Mice were examined daily for signs of joint inflammation and scored as follows: 0, normal; erythema and slight swelling, limited to the ankle; erythema and mild swelling, spreading from the ankle to the metatarsal and metacarpal joints; erythema and moderate swelling, spreading from the ankle to the metatarsal or metacarpal joints; erythema and severe swelling, spreading from the ankle to the paw toe. The maximal arthritis score was 4 per paw and 16 per mouse (fig. 31).

On day 0, all mice were immunized with 100 μ g bovine type II collagen emulsified in 100 μ l Freund's complete adjuvant (CFA). Type II collagen in CFA was injected intradermally into the right side of the tail root. On day 21, a second immunization, 100 μ g of bovine type II collagen in 100 μ l Freund's incomplete adjuvant, was given intradermally to the left of the tail. Investigators examined animals (M-F) daily. Nestlet was used as an enrichment device (enrichmentdevice) to provide additional packing for the animals. If necessary, wet food was placed on the bottom of the cage. The weakened animals were sacrificed after consulting the veterinarian. Terminal faxitron X-rays and microcts were collected at the end of the study to assess joint damage/erosion. In addition, animals were weighed before treatment and at termination.

On day 35 and at study termination, group 1-8 mice were bled for determination of serum pK and anti-type II collagen antibody titers (100 μ l orbital bleeds).

On day 70, all mice were finally bled intracardiacally under 3% isoflurane for terminal blood image (terminalomogram), white blood cell differential count and serum pK (G3) assessment.

On day 70 after induction of arthritis, mice were euthanized. All limbs were collected for radiographic, 5CT and histopathology.

Results

Systemic injection of CRIg fusion protein muCRIg-Fc into collagen-induced arthritis mice (animal model of rheumatoid arthritis) showed a significant reduction in CIA development in the experimental group of mice (boxes) receiving CRIg fusion protein compared to the control group of mice (circles) receiving IgG1 (see figure 31: p value ═ 0.0004). Collagen-induced arthritis was induced by injection of bovine type II collagen emulsified in freund's complete adjuvant. The booster immunization was given 21 days after the first immunization. Animals were treated 3 times a week with either murine CRIg-Fc fusion protein or with anti-gp 120IgG 1. The dose was 4mg/kg, in 100. mu.l PBS, injected subcutaneously. Treatment started on day 21 and continued through day 70. Mice were observed daily for hind paw swelling as a sign of arthritis. The severity of arthritis is graded as 1-16 as follows: 0 ═ absence of erythema and swelling, 1 ═ erythema and slight swelling limited to the middle paw (mid-foot) (tarsal bone) or ankle, 2 ═ erythema and slight swelling, spread from ankle to middle paw, 3 ═ erythema and moderate swelling, spread from ankle to metatarsal joint, 4 ═ erythema and severe swelling, covering ankle, paw and toe.

Repeat the test

The above protocol was adapted, and the results of previous trials in a collagen-induced arthritis (CIA) model were repeated and validated. The adapted protocol included investigation of the potential effect of radioactive irradiation on disease development and progression based on the results of in vivo micct imaging.

70 DBA-1J mice (7-8 weeks old, Jackson Laboratories) were divided into 5 treatment groups, two groups (G1 and G3) of 15 mice each, two groups (G4 and G5) of 10 mice each, and one group (G2) of 20 mice.

Treatment group:

g1: mu.l saline was injected subcutaneously with 4mg/kg of MuIgG1 isotype 3 times per week for 7 weeks (n-15).

G2: mu.l saline was injected subcutaneously with 4mg/kg MuCRIg-IgG1 3 times per week for 7 weeks (n ═ 20).

G3: mu.l saline was injected subcutaneously with 4mg/kg muttnfrii-IgG 1 isotype 3 times per week for 7 weeks (n-15).

G4: 4mg/kg of MuIgG1 isotype was injected subcutaneously in 100. mu.l saline 3 times per week for 7 weeks and anesthetized with in vivo microCT (n 10).

G5: mu.l saline was injected subcutaneously with 1.0mg/kg MuTNFRII-IgG1 3 times per week for 7 weeks and anesthetized with in vivo microCT (n 10).

TNF is a cytokine secreted by mononuclear phagocytes, Ag-stimulated T cells, NK cells and mast cells. It is involved in normal inflammatory and immune responses. TNF-alpha plays an important role in the pathogenesis of Rheumatoid Arthritis (RA). Elevated levels of TNF are found in synovial fluid of RA patients. In this protocol, the interaction between TNF and its cell surface receptor was blocked using mTNFRII-Fc as a positive control.

On day 0, all mice from G1 to G5 were immunized with 100 μ G bovine type II collagen emulsified in 100 μ l Freund's complete adjuvant (CFA). Type II collagen in CFA was injected intradermally into the right side of the tail root. On day 21, the tail was given a second immunization intradermally, i.e., 100 μ g of bovine type II collagen in 100 μ l of Freund's incomplete adjuvant.

Animals were examined daily. Mice from group G4-5 were anesthetized with isoflurane each week and subjected to in vivo microCt. Terminal faxitron X-rays and microcts were collected at the end of the study to assess joint damage/erosion.

On day 35 and at study termination, mice from group G1-5 were bled for serum pK and anti-type II collagen antibody titers (100 μ l orbital bleeds). On day 70, all mice were finally bled intracardiacally under 3% isoflurane for terminal hemograms, differential white blood count and serum pK (G3).

At day 70 after induction of arthritis, mice were euthanized. All limbs were collected for radiographic imaging, micct and histopathology.

Figure 33 shows a significant reduction in joint swelling in CRIg-Fc treated mice.

Immunohistochemistry on formalin-fixed, paraffin-embedded tissues (H & E staining) obtained from muCRIg-treated animals at day 70 showed that joint inflammation was inhibited by treatment. FIG. 34 shows a section of H & E stained metatarsal joints of DBA1/J mice 70 days after immunization with type II collagen. A. A large inflammatory cellular infiltrate was found in the peritendinous and periarticular cavity areas; B.A detail view; crig-Fc treatment of low-grade inflammatory infiltrates in the joints of mice. Few inflammatory cells were found in the area around the tendon sheaths and joint cavities; D.B detailed description.

FIG. 35 shows that cortical bone volume is preserved in the joints of muCRIg-Fc treated mice. Mice in the control IgG-treated group and CRIg-Fc-treated group were sacrificed 70 days after collagen injection and the joints were scanned by μ CT. Bone erosion and loss of bone density in the joints of mice representing the CRIg-Fc group and the control IgG group are shown in the left panel compared to muIgG1 treated animals. maintenance of cortical bone volume was significantly greater in the muCRIg-Fc treated animals. The image is a three-dimensional surface rendering made from μ CT data using Analyze image analysis software.

Figure 36 shows that CRIg-Fc neither changes the number of macrophages resident in the tissue nor its morphology. Liver and lung from anti-gp 120IgG1 (left panel) or CRIg-Fc (right panel) treated mice were dissected, fixed in formalin and then embedded in paraffin. The 7 micron sections were stained with antibody F4/80. A close examination of the sections showed an equal number of F4/80 positive macrophages in both test groups. In addition, no difference was observed in the morphology of macrophages.

FIG. 37 shows that treatment with mucIg-Fc did not affect serum anti-collagen antibody titers. Serum titers of anti-collagen antibodies were determined 70 days after immunization. No differences were found in the serum titers of the IgG1, IgG2a and IgM antibody subtypes between animals treated with CRIg-Fc and animals treated with anti-gp 120. This means that CRIg-Fc does not affect antibody responses in mice immunized with type II collagen. FIG. 38 shows that mucIg-Fc reduces the number of circulating inflammatory macrophages. Peripheral blood was collected from CRIg-Fc and anti-gp 120 treated animals 70 days after immunization and analyzed by flow cytometry using markers for inflammatory and non-inflammatory monocytes. Animals treated with CRIg-Fc showed a significant increase in inflammatory monocyte numbers and a decrease in non-inflammatory monocyte numbers compared to the anti-gp 120 treated group.

In summary, the results of the experiments described in this example demonstrate that muCRIg-Fc fusion protein inhibits collagen-induced arthritis. Specifically, the results show that CRIg-Fc inhibits joint swelling, inhibits inflammation, maintains cortical joint bone volume, and reduces circulating inflammatory macrophage numbers.

Other experiments have shown that CRIg-Fc does not affect B cell or T cell responses in vivo.

Example 8: CRIg fusion protein in antibody-mediated CIA in mice

Antibody-mediated arthritis differs from collagen-induced arthritis in that no antigen (bovine type II collagen) is injected, but instead an antibody that recognizes type II collagen is injected. Thus, adaptive B and T cell responses are bypassed and effector functions on macrophages and neutrophils are directly induced via Fc receptor and complement mediated activation.

Can be administered by Arthrogen-CIA by intravenous injectionAntibody-mediated CIA was induced by a combination of four different monoclonal antibodies produced by the mouse B hybridoma cell line (Terato et al, j. immunol.148: 2103-8 (1992)). 3 of said monoclonal antibodies recognizeThe fourth monoclonal antibody reacted with LyC1 at a fragment of 84 amino acid residues, a clustered autoantigenic epitope within LyC2 of CB11 (a minimal arthritic fragment of collagen type II). All four antibodies recognize conserved epitopes common to a variety of types of type II collagen and cross-react with both homologous and heterologous types II collagen (Terato et al, supra; Terato et al, Autoimmiturity 22: 137-47 (1995)). Arthrogen-CIA Arthritis-inducing monoclonal antibody mixtures are commercially available (Chemicon International, Inc., Temecula, Calif., catalog No. 90035).

Scheme(s)

10 BALB-c mice (CR/Hollister) aged 4-5 weeks were divided into two groups of 5 mice each.

Starting the day before injection of the antibody mixture (day-1), animals were treated daily with 100 μ g mucrig-Fc or 100 μ g control-Fc (anti-gp 120IgG1) for up to day 14. On day 14. Animals were examined at least twice daily and written records of observations were kept. Disease severity was scored by visual observation.

Visual scoring system:

0 ═ absence of erythema and swelling

1-erythema and slight swelling, restricted to the middle paw

2-erythema and slight swelling, spreading from the ankle to the middle paw

Erythema and moderate swelling, spreading from the ankle to the metatarsal joints

Erythema and severe swelling, covering the ankle, paw and paw toe

Nestlet was used as an enrichment device (enrichment device) to provide additional packing for the animals.

All animals were sacrificed on day 14 and joints were harvested for immunohistochemical staining or hematoxylin-eosin staining. Blood samples were collected for hydrographic analysis.

Results

FIG. 39 shows macrophage infiltration in joints after antibody-induced arthritis (AIA) and staining with F4/80 in non-decalcified frozen joints. Female Balb/C mice were injected intravenously with 2mg of anti-collagen antibody (arthrogen) followed by intraperitoneal injection of 25. mu.g of LPS 3 days later. 14 days after antibody injection, mice were euthanized and the paws were collected and embedded in polyvinyl alcohol. Sections of 7 μm thickness were excised from the frozen joints and stained with murine CRIg and an antibody to the macrophage specific marker F4/80.

FIG. 40 shows that muCRIg prevents joint swelling after antibody-induced arthritis in Balb/c mice. Arthritis was induced by the method of Terato and coworkers (Terato et al, 1992, supra; Terato et al, 1995, supra) using a cocktail of 4 monoclonal antibodies (Chemicon) that recognize conserved epitopes on type II collagen. Female Balb/C mice, 6 weeks old, were injected intravenously with 2mg of anti-CII antibody followed by intraperitoneal injection of 25. mu.g LPS 3 days later. Animals were treated daily with either murine CRIg-Fc fusion protein or with a control-Fc fusion protein. The dose was 4mg/kg, in 100. mu.l PBS, injected subcutaneously. Treatment began the day before the injection of anti-collagen antibodies and continued until day 14 mice were euthanized. Animals were observed daily for hind paw swelling following LPS injection as a sign of arthritis. The severity of arthritis is graded as 1-16 as follows: 0 ═ erythema and swelling, 1 ═ erythema and slight swelling, limited to the middle paw (tarsal bone) or ankle, 2 ═ erythema and slight swelling, spreading from ankle to middle paw, 3 ═ erythema and moderate swelling, spreading from ankle to metatarsal joint, 4 ═ erythema and severe swelling, encompassing ankle, paw and paw toe.

Therapeutic treatment was performed similarly to prophylactic treatment, except treatment was initiated on day 4, not day-1. MuCRIg-Fc treatment reduced inflammatory cytokine levels in the paw of AIA mice. The concentration of cytokines C3a and C5a in the arthritic hindpaw was measured according to Kagari et al, j.immunol.169: 1459-66 (2002). Briefly, at the indicated time points after antibody-induced arthritis, the paws were collected and frozen in liquid nitrogen. Thereafter, the paw was ground on a liquid nitrogen cooled metal plate and dispersed in ice cold PBS containing 0.1% pmsf (sigma). The sample was homogenized on ice using a vitatron (nl) homogenizer, the insoluble fraction was removed by centrifugation at 14000g for 10 minutes, and the supernatant was collected. Cytokine in the supernatant was measured using cytokine ELISA purchased from BDPharmingen.

MuCRIg-Fc treatment inhibited the deposition of complement C3 on cartilage in AIA, but did not inhibit the deposition of IgG2 a. Female Balb/C mice were injected intravenously with 2mg of anti-collagen antibody (arthrogen) followed by intraperitoneal injection of 25. mu.g of LPS 3 days later. 14 days after antibody injection, mice were euthanized, the paws were collected, embedded in polyvinyl alcohol, and then frozen in isopentane chilled on dry ice. Sections of 7 μm thickness were excised from the cryo-articular and stained with FITC-conjugated polyclonal antibody of murine C3 (Calbiochem) and A594-conjugated polyclonal antibody of murine IgG2a (Jackson Immunoresearch). The sections were photographed under a Leitz fluorescence microscope.

The results of immunohistochemistry with H & E staining are shown in figure 41. Control treated mice (muIgG1) had moderate to severe arthritis (left panel), while muclg treated mice had little to no arthritis (right panel). This result shows that the muCRIg inhibitory antibody induces joint inflammation in arthritis.

In summary, animals treated with murine CRIg-Fc had significantly reduced clinical scores compared to animals treated with anti-gp 120 IgG 1. CRIg shows both prophylactic and therapeutic efficacy in this animal model. A reduction in the severity of arthritis is also manifested by a reduction in inflammatory cells, especially neutrophils, in the joints. An increase in the number of neutrophils in the circulation may reflect a decrease in the number of neutrophils migrating into the joint. In parallel with the clinical manifestations of RA, muCRIg-Fc inhibits local IL-1 β and IL-6 production. Muclg treatment did not affect immune complex deposition, but inhibited the deposition of complement C3 on cartilage. Effector function was found to be independent of Fc receptor binding. huCRIg-short-Fc also showed significant prophylactic activity.

Example 9: murine CRIg-Fc binds C3 opsonized sheep red blood cells (E-IgM)

SRBC (MP Biomedicals, ICN/Cappel) were coated with rat IgM (E-IgM) (Forssmann Ag, Pharmingen). E-IgM was opsonized with normal mouse serum or serum from C3 knockout mice. Conditioned E-IgM was incubated with different concentrations of murine CRIg-Fc. Binding of the fusion protein to E-IgM was monitored by flow cytometry using FITC-labeled antibodies to the Fc portion of the fusion protein.

As shown in figure 43, murine CRIg bound E-IgM conditioned with normal mouse serum in a dose-dependent manner, but not with C3-deficient serum, indicating that CRIg selectively binds to murine C3 or C3 fragments.

Example 10: binding of human CRIg-Fc to E-IgM is dependent on C3

SRBC (MP Biomedicals, ICN/Cappel) were coated with rat IgM (E-IgM) (Forssmann Ag, Pharmingen). E-IgM was opsonized with human serum deficient in C3 or C5. The conditioned E-IgM was incubated with different concentrations of human CRIg-Fc. Binding of the fusion protein to E-IgM was monitored by flow cytometry using FITC-labeled antibodies to the Fc portion of the fusion protein.

As shown in figure 44, human CRIg bound E-IgM conditioned with C5-deficient serum in a dose-dependent manner, but not with C3-deficient serum, indicating that CRIg selectively binds to human C3 or C3 fragments. Similar results were obtained using the human CRIg ECD.

Example 11: binding of serum conditioning particles to CHO cells expressing CRIg

Mu.l of fresh C57B6 female serum + 20. mu.g/ml mCRig-mFc (PUR5270-B) or mPIGR-mFc (4699) were mixed together. At 37 ℃ in PBS/0.2% gelatin/0.18% glucose/1 mM MgCl₂(PBSgg + +) to which A488 particles from Molecular Probes, zymosan, Staphylococcus aureus or Escherichia coli were added for 60 minutes. Conditioned particles were washed twice with PBS and then added to CHO cells expressing either murine CRIg (clone 5C10) or human JAM2 at 37 ℃ for 30 minutes in the presence or absence of CRIg-Fc or control-Fc protein. Cells were washed twice with PBS and analyzed for particle binding to the cell surface in a FACS caliper.

As shown in figure 45, particles conditioned with C3-deficient serum bound CHO cells expressing CRIg, but not CHO cells expressing JAM 2. Binding was abolished in the presence of CRIg-Fc fusion protein, but not in the presence of control-Fc fusion protein, indicating that the binding site for CRIg to C3b is located in the extracellular domain.

Example 12: MuCRIg Fc binds C3b

Using BiacoreThe surface plasmon resonance assay was monitored in real time using a 2000 instrument and the data was analyzed using BiaEvaluation 3.0 software (Biacore AB, Uppsala, Sweden). Carboxylated dextran chips (sensor chip CM5, research grade, from Biacore AB) were used in all assays. The flow-through of the CM5 chip was used for a standard amine coupling protocol, or prepared for direct enzymatic coupling of C3b, using a standard activation-deactivation protocol without any protein addition between steps. The activation step was performed with a fresh solution containing N-hydroxysuccinimide and N-ethyl-N' - (dimethylaminopropyl) -carbodiimide (Biacore AB, injected at a flow rate of 5. mu.l/min for 7-15 minutes) followed by deactivation with ethanolamine-HCl (1.0M, pH8.5) (Biacore AB, injected for 7-15 minutes). Hepes buffered saline (biagarde, Biacore AB) or VBS was used as the whole flow buffer. After these initial steps, VBS or VBS was used as a continuous flow buffer at a flow rate of 5. mu.l/min, using only degassed buffer.

Coupling protein amines to Biacoreon-chip-C3 b, iC3b, C3C and C3d were coupled to CM5 chips using standard amine coupling protocols recommended by the manufacturer. The protein to be coupled is dialyzed against 10mM acetate buffer (pH 5.0-5.7) to obtain a negative net charge for amine coupling. Briefly, the chip surface was activated with N-ethyl-N' - (dimethylaminopropyl) -carbodiimide (injection 7-15 minutes, 5. mu.l/min) and then purified C3b (50. mu.g/ml, 20. mu.l), C3C (70. mu.g/ml, 30. mu.l) or C3d (130. mu.g/ml, 20. mu.l) was injected until a coupling level suitable for the binding assay was reached, i.e., 1,000 and 5,000 Resonance Units (RU). The flow cell is then deactivated as described above. Prior to the assay, the flow cell was thoroughly washed with VBS in 10mM acetate buffer, pH4.6, and 3M NaCl.

Using BiacoreBinding assay of (2) -we tested the binding of CRIg-Fc to amine-coupled C3b, C3C and C3 d. To BiacoreInjection, dialysis of the reagents against VBS, dilution with VBS, and filtration (0.20 μm Minisart)Sartorius corp., Edgewood, NY) or concentrated (10 min, 14,000 xg). The protein concentration of the post-dialysis reagent was measured using the BCA protein assay (Pierce). The fusion protein was injected separately through a control flow cell (flow cell activated and deactivated but without any coupled protein, "blank channel") and through a protein coupled flow cell at a flow rate of 5. mu.l/min and 22 ℃. All binding assays were performed at least in duplicate using independently prepared sensor chips.

As shown in fig. 46, murine CRIg-Fc showed specific binding of C3b to the sensor chip, with a calculated Kd of 250 nM.

Example 13: mouse and human CRIg-Fc binding complement C3b

Maxisorb plates were coated overnight with 3. mu.g/ml C1, C3a, b, C, d, C4, C6 in PBS. Plates were blocked with PBS + 4% BSA for 2 hours and incubated with various concentrations of murine or human CRIg-Fc fusion protein in PBS + 4% BSA + 0.1% Tween for 1 hour at room temperature. The plates were washed and incubated with peroxidase-conjugated goat anti-mouse or goat anti-human Fc antibody. After washing, the plates were incubated with TNB substrate and OD values were read on a plate reader.

The results shown in figure 47 indicate that murine and human CRIg binding to C3b, C3C, and C3bi increased in a concentration-dependent manner with no binding to C1, C2, C4, C3a, and C3 d.

Example 14: mouse and human CRIg-Fc inhibit the deposition of C3 on zymosan

Inhibition of the alternative pathway was investigated using a flow cytometry analysis using C3 deposition on zymosan A particles (Sigma) (Quigg et al, J.Immunol.160: 4553-4560 (1998)). Briefly, 50mg zymosan particles in 10ml 0.15M NaCl were first activated as follows: boiled for 60 minutes and then washed 2 times with PBS. In each alternative pathway assay condition, the final concentration of 10mM EGTA and 5mM MgCl was added ₂2x10 was added to the reaction tube⁷And (4) granules. Samples containing 10mM EDTA (negative control) or increasing amounts of murine CRIg-Fc as described herein were then added. Mu.l BALB/c serum was added as a complement source and all samples were supplemented to 100. mu.l with PBS. The samples were incubated at 37 ℃ for 20 minutes and the reaction was stopped by the addition of 10mM EDTA. The particles were centrifuged and the supernatant was taken and frozen for subsequent analysis. The particles were then washed twice with cold PBS, 1% BSA, and then incubated on ice for 1 hour with FITC-conjugated goat anti-mouse C3(Cappel, Durham, NC). The samples were then washed twice with cold PBS, 1% BSA and weighedSuspended in PBS and then analyzed by flow cytometry using an EPICS cytometer (Coulter, Hialeah, FL). Percent inhibition was calculated using the following formula: [1- [ sample mean channel fluorescence-background (10mM EDTA condition)/Positive control mean channel fluorescence (No Crry-Ig) -background]]x100。

Supernatants from reactions were also analyzed by Western blot to determine the extent of C3 cleavage. In this analysis, 5. mu.l of the supernatant was mixed with an equal volume of SDS-PAGE loading buffer containing 10% 2-ME. Samples were subjected to SDS-PAGE on 7.5% acrylamide gels and transferred overnight to Hybond Enhanced Chemiluminescence (ECL) paper (Amersham, Arlington Heights, IL) in 0.19M Tris, 0.025M glycine, 20% methanol buffer. Thereafter, the membranes were blocked in 10% milk in PBS, 0.1% Tween for 1 hour. The anti-C3 mAb RmC11H9(Quigg et al, supra), which had been pre-titrated, was then added to the blot in the same buffer containing 1% BSA. After washing, horseradish peroxidase-conjugated goat anti-rat IgG (Southern Biotechnology, Birmingham, AL) (pre-adsorbed against mouse IgG) was added for 1 hour, and then the blot was washed and developed with an Enhanced Chemiluminescence (ECL) system (Amersham).

Inhibition of complement activation by CRIg-Fc on zymosan particles was analyzed by flow cytometry to detect surface-bound C3 (fig. 48A), or by Western blot analysis of aliquots of zymosan reaction supernatants and detection using anti-C3 mAb (fig. 48B). The positions of the complete C3 and C3' strands in B are shown by the right arrow. Lanes 10mM EDTA represent the negative control, with increasing doses of CRIg-Fc shown at the top of lanes 2-7.

Example 15: CRIg inhibits alternative pathway hemolysis of SRBC

For the alternative route, rabbit erythrocytes (RRBC) were washed with veronal buffer (Bio whitetracker) containing 0.1% gelatin at 1 × 10⁹Cells/ml were resuspended in GVB. Mu.l of the cell suspension was added to 10. mu.l of inhibitor-containing C1q depleted serum. The mixture was incubated in a greenhouse at 37 ℃ for 35 min with shakingA clock. 200 μ l GVB containing 10mM EDTA was added, the cells were centrifuged at 2500rpm for 5 minutes and a 100 μ l aliquot was read at 412 nm.

For the classical pathway, sheep red blood cells conditioned with IgM (E-IgM) were incubated in fB deficient sera. The methodology is similar to the bypass pathway measurement.

The results shown in figure 49 show that murine CRIg inhibits alternative pathway induced hemolysis but does not affect classical pathway hemolysis. Similar results were obtained with human CRIg.

Example 16: CRIg selectively inhibits the alternative complement pathway

Hemolytic assay using whole serum

According to Kostavasili et al, j.immunol.158: 1763-71(1997), rabbit erythrocytes (Er) were used to assess the alternative complement pathway. Briefly, Er (Colorado Serum, Denver, CO) was washed 3 times with GVB and resuspended to 1X10⁹And/ml. Add 10. mu.l Er to 10. mu.l GVB/EGTA (0.1M EGTA/0.1M MgCl)₂) Inhibitor, 10. mu. l C1q depleted human serum, adjusted to 100. mu.l volume with GVB, and incubated at 37 ℃ for 30 minutes. The reaction was stopped by adding 250. mu.l GVB/10mM EDTA and centrifuged at 500Xg for 5 minutes. Hemolysis was determined by the absorbance at 412nm of 200. mu.g of supernatant. The percent dissolution was normalized by setting the dissolution that occurred in the absence of inhibitor to 100% dissolution.

To determine the effect of CRIg on the classical pathway of complement, a similar procedure was followed except that Er was replaced with E-IgM and the assay was performed in fB deficient human serum in GVB + +.

Measurement of C3 cleavage mediated by C3 convertase

To examine the effect of CRIg on liquid phase C3 cleavage of C3 convertase (C3b. bb) (from Kostavasili et al, supra), 0.4 μ M purified C3 was incubated with huCRIg-long, huCRIg-short, muCRIg or factor H in GVB (20 μ l volume) for 15 minutes at 37 ℃. Thereafter, 0.4. mu.M factor B and 0.04. mu.M factor D were added to a total volume of 30. mu.l in the presence of 50mM MgEGTA to activate the pathway. After incubation at 37 ℃ for 30 min, the reaction mixture was quenched with 30. mu.l Laemmli's sample buffer containing 2-ME (BioRad), boiled for 3 min, and run on an 8% SDS-PAGE gel (Invitrogen). Proteins were visualized by staining with SimplyBlue dye (Invitrogen, Carlsbad, CA). The gel was scanned for densitometry analysis and the percentage of C3 that was cleaved was calculated. Controls were incubated in GVBE (GVB with 10mM EDTA) to inhibit cleavage.

Microtiter plate assays for the alternative pathway DAA were performed as previously described (Krych-Goldberg et al, J.biol.chem.274: 31160-8 (1999)). Microtiter plates were coated overnight with 5. mu.g/ml C3b (Advanced Research Technologies) phosphate buffered saline. The plates were blocked with phosphate buffered saline containing 1% bovine serum albumin and 0.1% Tween20 for 2 hours at 37 ℃ with 10ng factor B, 1ng factor D and 0.8mM NiCl in 2.5mM Flopana buffer pH7.4 containing 71mM NaCl and 0.05% Tween20₂Incubate together at 37 ℃ for 15 minutes. Using the same buffer, sequential incubations were performed for 1 hour with 0.01-1. mu.g CRIg-Fc, 0.129. mu.g goat anti-human factor B antibody, and 100. mu.g 1:15,000 diluted anti-goat antibody conjugated to horseradish peroxidase (Jackson Immunoresearch laboratories, West Grove, Pa.). Developing with o-phenylenediamine. In this assay, DAF and factor H act as mediators of decay-promoting activity as expected, and C3a release is detected using the Amersham pharmacia Biotech des-Arg RIA kit.

C5 convertase assay

Deposition of C3b on zymosan, i.e. 1X10¹⁰Each zymosan pellet was resuspended in 0.2ml10mg/ml C3, 5. mu.g trypsin was added and then incubated at 22 ℃ for 10 minutes. Trypsin-induced deposition of C3b was repeated and cells were washed 6 times with 5ml GVB. Yeast glycan pellets were resuspended in 100. mu.l GVB, 50. mu.l GVB with factor B (35. mu.g) and factor D (0.5. mu.g) and 50. mu.l 10mM NiCl ₂And (4) mixing. After 5 minutes incubation at 22 ℃ 5. mu.l of 0.2M EDTA was added. Cells were exposed to 22 ℃ by adding 50. mu. l C3 (500. mu.g) to amplify bound C3bIncubate for 30 minutes. The C3 b-bearing zymosan particles were washed and the scaling up procedure was repeated until the desired number of C3 b/zymosan was obtained.

Since the time required for the formation of the C5 convertase is less than 1 minute, the enzyme is formed in the same reaction mixture in which the assay is performed. Enzyme velocity (velocity) was determined in 0.5 ml siliconized microcentrifuge tubes at saturating concentrations of factor B, factor D and C6 as previously described. The assay mixtures contained varying concentrations of C5 (preincubated at 37 ℃ for 20 min to eliminate the background C5B, 6-like activity due to freeze/thaw), factor B (1.2. mu.g, 516nM), factor D (0.1. mu.g, 167nM), C6 (2.5. mu.g, 833nM) and 0.5mM TiCl₂. The reaction was started by the addition of ZymC3b, ESC3b or ERC3 b. Based on the density of C3b per cell, cell concentrations were adjusted to give 9-35ng of bound C3b in a final volume of 25. mu.g GVB, resulting in enzyme concentrations of 2-8 nM. After incubation at 37 ℃ for 15 min, further cleavage of C5 was prevented by transferring the assay tube to an ice bath and adding ice-cold GVBE. The assay mixture, diluted appropriately, was immediately titrated for C5b, 6 formation by a hemolytic assay using EC. C5b, 6 was quantified using a standard curve prepared with purified C5b, 6. Controls were established with cold temperatures and dilutions sufficient to reduce C5 cleavage to undetectable levels in subsequent steps. Using EC lysis as an endpoint, lysis of rabbit red blood cells (ER) or sheep red blood cells (ES) was shown to contribute < 2% of C5b, 6 titers.

The sensitivity of EC to hemolytic lysis by human C5b-9 was used to measure C5b, 6 by hemolysis. To an aliquot (25. mu.l) of diluted sample from the C5 convertase assay was added 1.2X10 to a final volume of 225. mu.l GVBE⁷A mixture of individual ECs and 5. mu.l pooled Normal Human Serum (NHS) served as the source of complement proteins C7-C9. The reaction mixture was incubated at 37 ℃ for 10 minutes, and then the undissolved cells were removed by centrifugation at 10,000Xg for 1 minute. The amount of released hemoglobin was quantified spectrophotometrically at 414 nm. EC dissolution in 2% Nonidet P-40 was measured as 100% dissolution. Controls containing C5 and C6 but no C5 convertase were subtracted as background. Controls containing the C5 convertase but no purified C5 or C6 demonstrated that NHS used as a source of C7-9 did not form significant amounts from EC solubilizationAmount C5b, 6.

Results

The results are shown in FIGS. 49(A) - (E) (not provided).

Figure 49(a) (not provided) shows that CRIg inhibits hemolysis of rabbit erythrocytes in Clq-deficient sera (alternative pathway), but does not inhibit hemolysis of IgM-conditioned sheep erythrocytes in fB-deficient sera (classical pathway), suggesting that hatCRIg selectively inhibits the alternative complement pathway.

As shown in figure 49(B) (not provided), CRIg inhibited liquid phase C3 convertase activity. The gel shows inhibition of cleavage of the C3115 kDa alpha chain with increasing concentrations of human CRIg-ECD (10-100 nM).

Figures 49(C) and (D) (not provided) show that CRIg neither acts as a cofactor for factor I mediated C3 cleavage nor as a promoter of C3 convertase decay.

The data presented in figure 49(E) (not provided) shows that CRIg inhibits the alternative pathway C5 convertase formed on zymosan particles.

Example 17: CRIg is expressed on a fraction of tissue macrophages

Monoclonal antibodies specific for human and mouse CRIg were prepared for determining CRIg expression as described in example 3. Although there was no CRIg on peripheral blood C14+ monocytes, CRIg was readily detected by flow cytometry on monocyte-derived macrophages (figure 50B). huCRIg was absent from peripheral blood CD4+ and CD8+ T cells, CD19+ B cells, CD56+ NK cells, CD15+ granulocytes (fig. 51A). Similar to huCRIg, there was no muCRIg on peripheral blood and splenic leukocytes, including CD11B + bone marrow cells, but muCRIg was detected on liver Kupffer cells (KC, fig. 50B). The expression of hucrig (l) and (S) proteins was confirmed by 55 and 48K Mr proteins upon differentiation of monocytes into macrophages (fig. 50C). Similarly, mouse CRIg was detected in Peritoneal Macrophages (PM) by 48K Mr glycoprotein. MuCRIg has a predicted N-linked glycosylation site and is glycosylated, exhibiting a mobility change of about 5kDa on the gel (results not shown).

CRIg expression in the liver was further analyzed by immunohistochemistry due to the high levels of CRIg mRNA detected in the liver. CRIg was expressed on CD68+ KC in human and mouse liver, but CRIg was also detected on macrophages in adrenal, placenta, synovium, intestine and peritoneum (data not shown). Human spleen macrophages, Langerhans (Langerhan) cells, microglia and bone marrow derived macrophages, as well as various human and mouse macrophage cell lines (THP-1, RAW275, PU1.1, j 774; results not shown) do not have CRIg. Taken together, these results indicate that CRIg is highly expressed on resident macrophage populations in a variety of tissues.

Example 18: CRIg binds C3b and iC3b

Materials and methods

Complement proteins

According to Hammer et al, j.biol.chem.256 (8): 3995-4006(1981) isolated human and mouse C3 and additionally removed contaminating IgG using a protein A column. For the acquisition of hC3b, hC3 was combined with CVF, hfB and hfD in a molar ratio of 10:10:1 in the presence of 10mM MgCl₂Incubated at 37 ℃ for 1 hour. The hC3b fragment was subsequently isolated by strong anion exchange of monoQ5/50(Amersham Biosciences, Piscataway, N.J.) and Superdex S-20010/300GL gel filtration column (Amersham Biosciences, Piscataway, N.J.), purity according to Coomassie blue stained gel >95 percent. To prepare the C3b dimer, C3b prepared above was reacted with bismaleimide hexane in methanol (Pierce) at a 2.2:1 molar ratio in PBS pH7.0 at 4 ℃ for 3 days. Crosslinking occurs through free thiol groups by breaking thioester bonds. With this procedure, the yield was higher than 50%. The dimer was purified using Superdex S-20010/300GL gel filtration column (Amersham Biosciences, Piscataway, NJ). According to coomassie blue stained gel, dimer purity was 95%. Hydrolyzed C3 was prepared by adding 2M methylamine at pH7.0 to C3 in PBS containing 10mM EDTA, resulting in a final concentration in the reaction volumeThe concentration was 50 mM. The reaction was carried out at 37 ℃ for 4 hours, then purified on a Superdex S-20010/300GL gel filtration column (Amersham biosciences, Piscataway, N.J.) and iC3b and C3C (Advanced research technologies) were purified on a Superdex S-20010/300GL gel filtration column to separate the monomers from the dimer. C3D, factor B, factor D and factor P, complement component C1-9, antibody sensitized sheep red blood cells and cobra venom factor were obtained from Advanced Research technologies (San Diego, Calif.).

Results

Expression of CRIg on a highly phagocytic population prompted us to explore whether CRIg is involved in post-opsonic particle binding. Complement and Fc receptors have been shown to mediate phagocytosis. (reviewed in Aderem and Underhill, Annu. Rev. Immunol.17: 593-623 (1999); Underhill and Ozinsky, Annu. Rev. Immunol.20: 825-852 (2002)). To determine whether CRIg bound complement C3, sheep red blood cells coated with rabbit IgG (E-IgG) or mouse IgM (E-IgM) were analyzed for their ability to form rosettes with the Jurkat T cell line expressing CRIg in the presence of C3 or C5 deficient human serum. Jurkat cells expressing CRIg (L) in the presence of C3 (C3+) but not control Jurkat cells rosetted with E-IgM but not in the absence of C3 (C3-) (FIG. 52A). CRIg does not appear to be involved in Fc receptor mediated binding, as Es opsonized with IgG do not form rosettes with Jurkat CRIg cells (results not shown).

To test whether CRIg can bind directly to complement components on the cell surface, a soluble form of human CRIg was prepared in which the ECD of CRIg was fused to the Fc portion of human IgG 1. The fusion protein huCRIg-long-Fc but not control-Fc bound to conditioned E-IgM in the presence of C3, but not in the absence of C3 (fig. 52B). Binding was again restored when C3 deficient sera were reconstituted with purified human C3. Type V Ig domains were sufficient for binding because huCRIg (S) -Fc and mucIg-Fc were both capable of binding E-IgM (results not shown).

As a result of complement activation inducing an enzymatic cascade, C3 cleaves into its multiple breakdown products C3b, iC3b, C3C, C3dg and C3d, each of which can act as a binding partner for CRIg. Using a plate-bound ELISA, huCRIg (L) and huCRIg(s) -Fc but not control Fc showed saturable binding to C3b and iC3b (fig. 52C), but not to C3, C3a, C3C or C3d (results not shown). Similar results were observed for huCRIGL-ECD and mucIg-Fc without the Fc portion, with greater binding to iC3b than to C3b (results not shown). In contrast, soluble C3b also bound to and competed with hucrig (l) -Fc coated on plates (results not shown). Thus, CRIg can bind C3b and iC3b in solution, or when C3b and iC3b are bound to a substrate. Since C3b exists in multimeric form when deposited on the cell surface, the binding of CRIg to the artificially assembled C3b dimer (C3b2) was further evaluated. C3b2 bound hucrig (l) with a Kd of 131nM (fig. 52D) and 44nM (fig. 52D) as measured by surface plasmon resonance.

To complete these biochemical studies, we evaluated the binding specificity of cell surface CRIg for the C3-derived product. A488-labeled C3b2 dimer formed the surface of THP-1 cells that bound CRIg + but not CRIg- (FIG. 52E). Binding was specific because it was competed by the addition of soluble unlabeled C3b2, C3b monomer, and hucrig (l) -ECD instead of native C3. In addition to binding to soluble complement fragments, muCRIg expressed on the surface of CHO cell lines also bound to various particles conditioned in C3 replete but not defective serum (fig. 51B). Taken together, these studies demonstrated that CRIg expressed on the cell surface, as well as soluble CRIg (CRIg-Fc), are receptors for iC3b and C3 b.

Example 19: CRIg expression on Kupffer cells is required for soluble or particle-bound C3 fragment binding

Materials and methods

Generation of CRIg knockout (ko) mice

All animals were kept under sterile, pathogen-free conditions and animal trials were approved by the institutional animal care and use committee (institutional animal care and use committee) for the Genentech study. CRIg knockout embryonic stem cells were prepared by introducing a linearized targeting vector with exon 1 replaced with a neomycin resistance gene into C2B6 Embryonic Stem (ES) cells by electroporation. Clones resistant to neomycin were selected and homologous recombination was verified by Southern blotting. 7 of the 100 clones screened were positive for homologous recombination. Two targeted clones were injected into C57VL/6 blastocysts, transferred to pseudopregnant surrogate mothers (foster mothers), and the resulting male chimeric mice were mated with C57BL/6 female mice to obtain +/-mice. Germline transmission of the 2ES clone was verified by Southern blot analysis or tail DNA from F1 progeny (fig. 42B). +/-mouse hybridization was performed to generate-/-CRIg mice. The phenotypes of these two clones were identical. For routine genotyping by PCR methods, a 306bp fragment was amplified for the wild type allele and a 406bp fragment for the mutant allele using a common sense primer 5'-CCACTGGTCCCAGAGAAAGT-3' (SEQ ID NO: 22) and wild type specific antisense primer (5'-CACTATTAGGTGGCCCAGGA-3') (SEQ ID NO: 23) and knock-out specific antisense primer (5'-GGGAGGATTGGGAAGACAAT-3') (SEQ ID NO: 24). The generation of C3 knockout mice has been previously described (Naughton et al, Immunol.156: 3051-3056 (1996)). To generate CRIg/C3 dual knockout mice, C3 knockout mice mixed with s129/B6 background (F2) were crossed with CRIg knockout mice. Then, F1 females, heterozygous for both alleles, were crossed to C3 heterozygous males, hemizygous for the CRIg allele. The offspring thus mated were used for the study. C57B6 mice used for CRIg expression analysis by flow cytometry were purchased from jackson laboratories (bar harbor).

Western blotting and deglycosylation

Human and murine macrophages were lysed in PBS containing 1% SDS, 0.1% Triton X-100 and protease inhibitor cocktail (Boehringer). After centrifugation at 10,000g, the soluble fraction was subjected to SDS gel electrophoresis and transferred to nitrocellulose membrane. CRIg protein was visualized using anti-CRIg antibody and HRPO-conjugated secondary antibody, and the bound antibody was then detected for chemiluminescence by ECL (Amersham). To determine the glycosylation state of CRIg, cells expressing CRIg-gD were immunoprecipitated with anti-gD antibody, treated with PNGase, O-glycosylase and neuraminidase according to the manufacturer's instructions (Biolabs, NE), and then subjected to Western blot analysis using biotinylated anti-gD antibody.

Results

To investigate the biological function of CRIg, mice with null mutations in the CRIg gene were generated by homologous recombination as described above and shown in figure 42A. Deletions were verified by Southern blotting (fig. 53B), Western blotting of peritoneal exudate cell lysates (fig. 54A) and flow cytometry (fig. 54B). Mice were born at the expected mendelian ratio, with no gross phenotypic or histopathological abnormalities. The absolute number of immune cells in different lymphoid compartments was similar in blood, spleen and lymph nodes from wild type and knockout animals (fig. 53C). In addition, no difference was observed in F4/80+ KC and cardiac macrophage numbers when analyzed by flow cytometry and immunohistochemistry, respectively (results not shown). The expression levels of other complement-binding proteins, including the alpha and beta chains of CR3 and the complement receptor-associated gene y (crry) on KC, were unchanged (fig. 54C). Similarly, there is a similarity between wild type and knock-out KCs in that expression of CR1, CR2 or CD11c, CR4 β chain is low or undetectable.

Next, CRIg wild-type and CRIg knockout KC were tested for their ability to bind to C3 degradation products. The C3 fragment (C3B, C3B2 and iC3B) was easily deposited on the surface of CRIg wild-type KC (fig. 54B). In contrast, no binding of C3b, C3b2, iC3b or iC3b2 was detected in CRIg wild-type KC. Little or no binding was detected by C3 and C3C to wild-type or knock-out KC (fig. 53E).

To extend the assay from binding of soluble C3 fragment to binding of C3 fragment bound to the cell surface, the effect of CRIg on KC binding to C3-conditioned IgM-coated erythrocytes was examined. CRIg knockout KC exhibited about 60% less rosetting by E-IgM when compared to CRIg wild-type KC (figure 54D). A further reduction (< 20%) in rosette formation was observed upon addition of CR3 blocking antibody, so CR3 contributed little to the overall binding activity. Thus, CRIg expression is required for binding of C3 degradation products and C3 opsonization particles to Kupffer cells.

Example 20: CRIg internalization and expression on recirculating endosomes

Since the binding of C3 opsonizing particles to their receptors can trigger subsequent endocytosis (Fearon et al, J.Exp.Med.153: 1615-. A488-conjugated anti-CRIg mAb was preincubated with KC at 4 ℃. Addition of anti-a 488 antibody at 4 ℃ inhibited the fluorescence of surface-bound anti-CRIg antibody, as shown in panel 1 of figure 47A. When a 488-conjugated anti-CRIg mAb was incubated with KC for 30 minutes at 37 ℃ and then with anti-a 488 antibody, fluorescence was not inhibited (fig. 55A panel 4), indicating that anti-CRIg antibody internalized upon transfer to cells at 4 ℃ -37 ℃ and thus failed to quench anti-a 488 antibody. Similar results were found for C3b (fig. 55A panels 3 and 6). Internalization of anti-CRIg antibodies was independent of the presence of C3, as antibody uptake occurred in KC isolated from C3 knockout mice (fig. 55A, panels 2 and 5) and in the absence of serum (results not shown). Immunohistochemistry further confirmed the presence of anti-CRIg antibodies and C3B in the cytoplasm of KCs from CRIg wild-type but not knockout mice (figure 55B). Over time, KC coated with a 488-conjugated anti-CRIg antibody incubated in the presence of extracellular anti-a 488 antibody, a decrease in fluorescence over time was observed, indicating that anti-CRIg antibody was recycled back to the cell surface (figure 55C). The time course of the recycle was again independent of C3, as the kinetics of quenching were similar in the presence and absence of C3 (results not shown). In contrast, antibodies to the lysosomal protein Lampl remained intracellular and did not diminish over time. These results indicate that CRIg functions as a C3b receptor located on a collection of constitutive recycle films.

To further determine the subcellular compartment in which CRIg was recirculated, deconvolution microscopy was usedHuman Monocyte Derived Macrophages (MDM) were visualized by microscopy using transferrin as a marker for recycled endosomes and Lampl as a marker for lysosomes. MDM cultured for 7 days expressed CRIg on 60% of cells showing saturable C3b binding (fig. 55A), which could be competed with the extracellular domain of hucrig (l) (results not shown). Macrophages coated with anti-CRIg antibody at 4 ℃ exhibited local (focal) CRIg expression in F-actin rich filament protrusions (arrows, figure 56A, panels 1-3). In addition, CRIg antibody co-localized to the cell surface with C3b (results not shown). Cells were transferred from 4 ℃ to 37 ℃ and then incubated at 37 ℃ for 10 minutes (FIG. 56B), resulting in rapid internalization of CRIg antibody and C3B into transferrin located at the cell periphery⁺Endosomal compartment (FIG. 56B, panels 1-4, arrows) proximal to Lamp1⁺Compartment (arrow, fig. 57D, panels 1-4). The tracking time was extended until 24 hours, the CRIg was still localized within the internal body compartment and not degraded in lysosomes (results not shown). Incubation of macrophages with anti-CRIg antibodies did not affect the CRIg distribution because the internalized CRIg antibodies completely overlapped the full pool of CRIg detected with polyclonal antibodies after immobilization (figure 57C, panels 1-3), independent of the presence of C3 in the medium (figure 57C, panel 4). Taken together, these results indicate that CRIg is present on the recycling and early endosomes, and that internalization of CRIg occurs in the absence of ligand or cross-linking antibody.

Since C3b and iC3b are mostly deposited on serum-exposed particles (Brown, curr. opin. immunol.3: 76-82(1991)), we next explored the localization of CRIg-positive endosomes in macrophages during phagocytosis of C3 opsonic particles. Upon encountering iC3 b-opsonized sheep red blood cells (E-IgM), CRIg redistributed rapidly (10 minutes) from transferrin-positive vesicles to the developing phagosomes, appearing as rings around engulfed red blood cells (fig. 56C, panels 1 and 4, arrows). Phagocytes matured 2 hours after incubation of macrophages with C3-conditioned particles, as shown by their translocation into the lysosomal compartment (fig. 56C, panels 5-8). CRIg was highly expressed in the phagosomal membrane surrounding the C3 opsonic particle (FIG. 56C, panels 5 and 8, arrows) and most macrophages were notRe-presence in transferrin⁺The inner body is in the chamber. Although CRIg is still present on a portion of the phagosome in the lysosomal compartment, its expression does not overlap with that of LAMP-1 (fig. 56C, panels 7 and 8, arrows). LAMP-1⁺The absence of CRIg in the membrane is unlikely to be due to degradation of CRIg by lysosomes, as protease inhibitors persist during the incubation period. Some have taken up E-IgM but lack CRIg ⁺In phagocytic macrophages, CRIg⁺With transferrin⁺Compartment colocalization (bold arrows, FIG. 56C5, panels 5 and 8, bold arrows) indicating CRIg following transfer of E-IgM to the lysosomal compartment⁺Back to the recirculation compartment.

Taken together, these results indicate that CRIg is recruited from the endosome to the site of particulate uptake, involved in the initial phase of phagosome formation, but upon phagosome-lysosomal fusion, it escapes from the phagosome back into the endosomal compartment.

Example 21: mice deficient in CRIg are susceptible to Listeria monocytogenes infection

Materials and methods

1. Microorganism, mouse infection and Listeria growth assessment by CFU count

All experiments used virulent Listeria Monocytogenes (LM) (ATCC strain 43251). Bacterial virulence was maintained by serial passage in BALB/c mice. Fresh isolates were obtained from infected spleens and cultured in brain heart infusion (liquid) or brain heart infusion plates (Difco Laboratories, Detroit, MI). The bacteria were washed repeatedly, resuspended in sterile Phosphate Buffered Saline (PBS), and then stored in small aliquots in PBS containing 40% glycerol at-80 ℃. Mice were inoculated intravenously with listeria monocytogenes in the tail vein at different doses. To observe bacterial growth in various organs, we injected mice intravenously with 1x10 ⁴A single Colony Forming Unit (CFU) of Listeria that does not cause any response in either CRIg knockout mice or CRIg wild type miceDeath. Viable bacteria counts in the inoculum, liver and spleen homogenates, and infected cells were determined by plating 10-fold serial dilutions onto brain-heart infusion agar (Difco Laboratories) plates. After 24 hours incubation at 37 ℃, the number of CFUs was counted.

2. Determination of Listeria-A488 uptake in Kupffer cells

Live Listeria monocytogenes were labeled with the A-488 labeling kit according to the manufacturer's instructions (Molecular Probes, Oregon). The number of viable listeria after the labeling procedure was assessed by colony counting. CRIg wild-type mice or CRIg knockout mice were injected intravenously with 1 million CFU LMs. After 1 hour, the liver was perfused and Kupffer cells were isolated according to the method described above. Cells were stained with PE-labeled anti-F4/80 antibody, positive cells were isolated using anti-PE beads (miltenyi), followed by sorting with a MoFlo flow cytometer (DakoCytomation, ft. F4/80 positive cells were collected on coverslips and the number of internalized labeled bacteria was estimated using confocal and optical microscopy. The number of bacteria per cell was counted in 400 cells of 4 different fields per slide. Phagocyte index is calculated as the average number of bacteria per cell multiplied by the percentage of kupffer cells containing at least 1 bacterium. The results show the mean and standard deviation of the phagocyte index obtained from four different animals.

Results

To explore the role of CRIg in phagocytosis of complement opsonic particles in vivo based on its binding to C3b/iC3b opsonic particles, CRIg wild-type and CRIg knockout mice were infected with different doses of Listeria Monocytogenes (LM), a gram-positive facultative bacterium that, when exposed to serum, activated the alternative complement pathway that deposited primarily C3b and iC3b on the surface of the bacterium (Croize et al, in fe. immunol.61: 5134-. CRIg knockout mice were significantly more susceptible to LM infection, showing increased lethality (fig. 58A). In contrast, pretreatment of CRIg-Ig fusion protein increased susceptibility in CRIg wild-type mice but not CRIg knockout mice (figure 62).

Consistent with the role of CRIg in the binding and phagocytosis of complement C3 opsonic particles, CRIg knockout mice decreased in clearing LM from blood, resulting in increased LM loading in the spleen and lungs (fig. 58B). The LM load was also reduced in the liver and heart of infected mice, which probably reflects the presence of CRIg-expressing macrophages in these tissues (figure 58B). The inflammatory response in CRIg knockout mice was elevated, as evidenced by elevated serum levels of IFN-. gamma.TNF-. alpha.and IL-6 (FIG. 58C). Consistent with the requirement for CRIg to clear C3 conditioning particles, CRIg knockout KC showed a significant reduction in binding and phagocytosis by LM compared to CRIg wild-type KC (figure 58D). Finally, the rise in listerial loads detected in the blood of CRIg knockout mice was dependent on C3, as infection of C3 knockout mice abrogated the difference in bacterial titers between CRIg knockout mice and CRIg wild-type mice (figure 58E). Interestingly, circulating levels of bacteria were significantly lower in the C3 knockout mice compared to the C3 replete mice, which likely reflects an increased involvement of C3-independent mechanisms responsible for listeria clearance in the C3 knockout mice. However, rapid clearance in the absence of C3 did not result in long-term effective clearance of pathogens, as C3-deficient mice died within 2 days after gram-positive bacterial infection (Cunnion et al, J.Lab.Clin.Med.143: 358: 365 (2004)). These results strongly suggest that CRIg expressed on liver Kupffer cells plays a crucial role in the rapid clearance of complement C3-opsonized pathogens from the circulation.

Example 22: inhibition of complement-mediated immune hemolysis with huCRIg molecules

It is well understood that rabbit erythrocytes specifically activate the alternative complement pathway, leading to lysis of cells by the C5b-9 complex (Polhill, et al, J.Immunol.121 (1): 363-370 (1978)). Specifically, rabbit erythrocytes initiate the alternative complement cascade, resulting in the formation of MAC causing lysis of these cells. If the test compound is capable of inhibiting the alternative pathway, then the addition of reagents to rabbit red blood cells soaked in serum (in this case macaque or human C1q depleted serum) should be capable of preventing cytolysis. This can be assessed by monitoring the change in absorbance at a wavelength of 412nm caused by hemoglobin released by the lysed erythrocytes. In the macaque serum test, blood was collected from the femoral vein of macaques. No anticoagulant was used. The sample was allowed to coagulate at room temperature. The samples were centrifuged and the serum collected and stored in a refrigerator set to maintain-60 ℃ to-80 ℃. Rabbit Red Blood Cells (RRBC) were washed 3 times with GVB (1 XFlorida buffer (Biowhittaker), 0.1% gelatin), resuspended to 1X10 with GVB⁹And/ml. GVB, huCRIg (short or long, or long ECD) were added followed by 10. mu.l GVB +/EGTA (GVB, 0.1M EGTA, 0.1M MgCl) ₂). Mu.l of macaque serum or C1q subtracted serum (Quidel) was added followed by 10. mu.l of RRBC and the mixture was snap-mixed. After incubation in the greenhouse at 37 ℃ for 45 minutes with shaking, 250. mu.l GVB/10mM EDTA were added and the mixture was centrifuged at 2500rpm for 5 minutes. Using a 250. mu.l aliquot, readings were taken at 412 nm. The results shown in FIGS. 63A and B (cynomolgus monkey serum) and FIGS. 64-66 (human serum) demonstrate that the CRIg compounds tested inhibit complement.

hST-L: human CRIg-Long

hST-S: human CRIg-short

hST-L ECD: human CRIg-Long ECD

hPIGR: human polyimmunoglobulin receptor

fH: complement factor H

Example 23: testing of murine CRIg-Fc fusion proteins in a mouse model of choroidal neovascularization

Choroidal Neovascularization (CNV) can be induced experimentally by laser burning the retina. In this study, 40C 57BL-6 mice (Charles River Laboratory) were divided into two treatment groups. Group 1 (control): on days-1, 3 and 5, 12mg/kg gp120mIgGl was injected intraperitoneally. Group 2: on days-1, 3 and 5, 12mg/kg murine CRIg (mCRIg) was injected intraperitoneally.

Animals of each group were anesthetized by subcutaneous (s.c.) injection of a mixture of ketamine (25mg/g) and xylazine (1.28 mg/g). The pupil was dilated with a drop of 1% tropicamide. The animal is then fixed in a plastic mold. 3 laser irradiation spots were generated around the optic nerve in the eye with a diode laser (100 μm spot diameter) using an OcuLight GL diode laser (532nm), Zeiss30W slit lamp and micromanipulator. The laser was irradiated to the right eye at a crack size of 120mW, 0.1 second and 100 μm. The laser irradiation spot divided by the bubble formed indicated that the Brach's membrane broke.

Laser irradiation spots were evaluated 7 days after the laser irradiation treatment using confocal microscopy. At this time, the animals were anesthetized with isoflurane and 0.5ml PBS containing 50mg/ml fluorescein labeled dextran (Sigma) was perfused through the heart. The eyes were enucleated, fixed with 10% phosphate buffered formalin, the retinas discarded, and the remaining eye cups (eye cups) were placed flat on a glass slide. Histopathological examination included immunohistochemical staining of complement fragments and elastin from choroidal tiled specimens, and analysis of CNV complex size by confocal microscopy monitoring of the FITC-dextran stained vasculature in the eye.

The results are shown in FIGS. 71A and B, where burn wells in the right eye were scored according to a scale of 0-3 and 0-5, respectively.

Example 24: testing of CRIg ECD and CRIg-Fc fusion proteins in cynomolgus monkeys suffering laser-induced retinal injury

24 macaques, all males or all females, or 12 males and 12 females were used in this study. The animals are 2-7 years old and weigh 2-5 kg.

TABLE 2

Group nomenclature and dosage levels

Administration was via intravenous injection via the cephalic vein. Animals were dosed at least once prior to laser treatment, three times per week for the remainder of the study. The dose was determined from the most recently recorded body weight and ranged from 10-15 mg/kg.

On day 4, the macula of each eye of all animals was laser treated by CORL, burned with a 532nm diode green laser (OcuLight GL, IRIDEX Corp Inc, Mountain View, California), using a slit lamp delivery system and Kaufman-wall (Ocular Instruments Inc., Bellevue, Wash) flat bottom contact lenses. The laser and support equipment are supplied by the CORL. Animals were anesthetized with ketamine and xylazine. The 9 regions were symmetrically distributed in the macula of each eye. The laser parameters included a 75 micron spot diameter and a 0.1 second duration. The power used was assessed by the ability to generate blisters and small amounts of bleeding. A second laser spot was placed near the first following the same laser treatment procedure (except for wattage adjustment) unless bleeding was observed at the time of the first laser treatment. For regions not adjacent to the dimple, the primary power was set to 500 mW; if a second spot is provided, the power is 650 mW. For the adjacent concave regions, the power was set at 400mW (primary) and 550mW (secondary). The power setting can be adjusted according to the situation observed during laser irradiation, at the discretion of the ophthalmologist.

Clinical ophthalmic examination

Each animal was subjected to a clinical ophthalmic examination prior to the start of treatment and on days 8, 15, 22 and 29. Animals were anesthetized with ketamine and the eyes (pupils) were dilated with mydriatic agents. The attachments and fronts of both eyes were examined using a slit-lamp biomicroscope. The fundus of both eyes was examined using an indirect ophthalmoscope. Other suitable instruments may be used to examine the eye and take a picture, at the discretion of the ophthalmologist.

Eye photographing

Pictures of the eyes (OP) were taken once each on the day of laser treatment (post-laser treatment), on days 10, 17, 24 and 31 of dosing phase 1, and on day 6 of dosing phase 2 (day of necropsy). When fluorescein angiography is performed simultaneously during the dosing phase 1, OP is performed first.

Animals were anesthetized with ketamine, maintained under anesthesia with isoflurane anesthetic while simultaneously performing fluorescein angiography, and anesthetized with ketamine and xylazine when performed alone (i.e., after laser treatment). The eye (pupil) is dilated with a mydriatic drug. Color photographs were taken of each eye, including a stereophotography of the retina and associated ocular abnormalities, a posterior pole, and a non-stereophotography of two mid-peripheral fields (temporal and nasal).

Fluorescein angiography

All animals were subjected to fluorescein angiography once each on days 10, 17, 24 and 31 (days 6, 13, 20 and 27 after laser irradiation) before the start of treatment and on day 1 of dosing.

Animals were fasted prior to fluorescein angiography. Animals were anesthetized with ketamine, maintained under anesthesia with isoflurane, and eyes (pupils) were dilated with mydriatic drugs. Animals were intubated because of the possibility of vomiting following fluorescein injection. Animals were injected intravenously with fluorescein. Pictures were taken at the beginning and end of the fluorescein injection. After fluorescein injection, serial rapid stereophotographs were taken of the posterior pole of the right eye, followed by stereophotographic pairing of the posterior pole of the left eye 1 minute ago, and then for each eye, at about 1-2 and 5 minutes. Between about 2-5 minutes, a non-stereo photograph of the mid-peripheral field (temporal and nasal) was taken for each eye. If fluorescein leakage was observed at the 5 minute time point, a stereo photographic match was made at approximately 10 minutes.

Fluorescein angiogram evaluations were performed according to the following grading system for evidence of excessive permeability (fluorescein leakage) or any other abnormality.

Grade of damage	Definition of
		I	Without high fluorescence
II	High fluorescence and no leakage
		III	High fluorescence, early or intermediate transition and late leakage
IV	Bright high fluorescence, early or intermediate transition and late leakage across the borders of the treatment area

Material preservation

The following materials have been deposited at the American type culture Collection (ATCC, American type culture Collection, 10801 University Boulevard, Manassas, VA20110-2209, USA):

deposit date of the name ATCC deposit

DNA 45416-12512096201998 year 2, month 5

This deposit is made under the terms of the Budapest treaty on the international recognition of the deposit of microorganisms for patent procedures and the rules thereof. This ensures that the preserved viable cultures are maintained for 30 years from the date of preservation. This deposit is available through the ATCC under the terms of the Budapest treaty and subject to an agreement between Genentech corporation and the ATCC which ensures that the public, at the time of grant of the pertinent United states patent or at the time of publication to the public of any United states or foreign patent application, is permanently and unrestrictedly able to obtain progeny of the deposited culture, and that individuals who are determined by the U.S. patent and trademark office in accordance with 35USC § 122 and in accordance with its regulatory guidelines (including 37CFR § 1.14, specifically 886OG638) will be entitled to obtain progeny.

The assignee of the present application has agreed that if a deposited culture dies, is lost or is damaged when cultured under appropriate conditions, he will be quickly replaced with the same culture upon notification. The availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

The foregoing written description is considered to be sufficient to enable those skilled in the art to practice the invention. The scope of the invention is not limited by the deposited constructs, as the deposited embodiments are intended to be a single illustration of certain aspects of the invention, and any functionally equivalent construct is within the scope of the invention. The deposit of material herein does not constitute an admission that the written description contained herein is not sufficient to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific exemplifications presented.

Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and are within the scope of the appended claims.

Sequence listing

<110> Jiantaike Biotechnology company (GENENTECH, INC)

ASHKENAZI，AVI

HELMY，KARIM YUSSEF

FONG，SHERMAN

GODDARD，AUDREY

GURNEY，AUSTIN L.

KATSCHKE JR.，JAMES KATCHKE

VAN LOOKEREN，MENNO

WOOD，WILLIAM I

<120> CRIg polypeptide for preventing and treating complement-associated disorders

<130>39766-0100CP4

<140> to be specified

<141>2005-10-10

<150>US 11/159,919

<151>2005-06-22

<150>US 10/964,263

<151>2004-10-12

<150>US 10/767,374

<151>2004-01-29

<150>US 10/767,904

<151>2004-01-29

<150>US 09/953,499

<151>2001-09-14

<150>PCT/US03/31207

<151>2003-10-01

<150>US 10/633,008

<151>2003-07-31

<150>US 10/265,542

<151>2002-10-03

<150>US 09/254,465

<151>1999-03-05

<150>US 09/380,138

<151>1999-08-25

<150>PCT/US99/05028

<151>1999-03-08

<150>PCT/US98/24855

<151>1998-11-20

<150>US 60/078,936

<151>1998-03-20

<160>28

<170> FastSEQ for Windows version 4.0

<210>1

<211>2181

<212>DNA

<213> human (Homo sapiens)

<400>1

cccacgcgtc cgcccacgcg tccgcccacg ggtccgccca cgcgtccggg ccaccagaag 60

tttgagcctc tttggtagca ggaggctgga agaaaggaca gaagtagctc tggctgtgat 120

ggggatctta ctgggcctgc tactcctggg gcacctaaca gtggacactt atggccgtcc 180

catcctggaa gtgccagaga gtgtaacagg accttggaaa ggggatgtga atcttccctg 240

cacctatgac cccctgcaag gctacaccca agtcttggtg aagtggctgg tacaacgtgg 300

ctcagaccct gtcaccatct ttctacgtga ctcttctgga gaccatatcc agcaggcaaa 360

gtaccagggc cgcctgcatg tgagccacaa ggttccagga gatgtatccc tccaattgag 420

caccctggag atggatgacc ggagccacta cacgtgtgaa gtcacctggc agactcctga 480

tggcaaccaa gtcgtgagag ataagattac tgagctccgt gtccagaaac tctctgtctc 540

caagcccaca gtgacaactg gcagcggtta tggcttcacg gtgccccagg gaatgaggat 600

tagccttcaa tgccaggctc ggggttctcc tcccatcagt tatatttggt ataagcaaca 660

gactaataac caggaaccca tcaaagtagc aaccctaagt accttactct tcaagcctgc 720

ggtgatagcc gactcaggct cctatttctg cactgccaag ggccaggttg gctctgagca 780

gcacagcgac attgtgaagt ttgtggtcaa agactcctca aagctactca agaccaagac 840

tgaggcacct acaaccatga catacccctt gaaagcaaca tctacagtga agcagtcctg 900

ggactggacc actgacatgg atggctacct tggagagacc agtgctgggc caggaaagag 960

cctgcctgtc tttgccatca tcctcatcat ctccttgtgc tgtatggtgg tttttaccat 1020

ggcctatatc atgctctgtc ggaagacatc ccaacaagag catgtctacg aagcagccag 1080

gtaagaaagt ctctcctctt ccatttttga ccccgtccct gccctcaatt ttgattactg 1140

gcaggaaatg tggaggaagg ggggtgtggc acagacccaa tcctaaggcc ggaggccttc 1200

agggtcagga catagctgcc ttccctctct caggcacctt ctgaggttgt tttggccctc 1260

tgaacacaaa ggataattta gatccatctg ccttctgctt ccagaatccc tgggtggtag 1320

gatcctgata attaattggc aagaattgag gcagaagggt gggaaaccag gaccacagcc 1380

ccaagtccct tcttatgggt ggtgggctct tgggccatag ggcacatgcc agagaggcca 1440

acgactctgg agaaaccatg agggtggcca tcttcgcaag tggctgctcc agtgatgagc 1500

caacttccca gaatctgggc aacaactact ctgatgagcc ctgcatagga caggagtacc 1560

agatcatcgc ccagatcaat ggcaactacg cccgcctgct ggacacagtt cctctggatt 1620

atgagtttct ggccactgag ggcaaaagtg tctgttaaaa atgccccatt aggccaggat 1680

ctgctgacat aattgcctag tcagtccttg ccttctgcat ggccttcttc cctgctacct 1740

ctcttcctgg atagcccaaa gtgtccgcct accaacactg gagccgctgg gagtcactgg 1800

ctttgccctg gaatttgcca gatgcatctc aagtaagcca gctgctggat ttggctctgg 1860

gcccttctag tatctctgcc gggggcttct ggtactcctc tctaaatacc agagggaaga 1920

tgcccatagc actaggactt ggtcatcatg cctacagaca ctattcaact ttggcatctt 1980

gccaccagaa gacccgaggg aggctcagct ctgccagctc agaggaccag ctatatccag 2040

gatcatttct ctttcttcag ggccagacag cttttaattg aaattgttat ttcacaggcc 2100

agggttcagt tctgctcctc cactataagt ctaatgttct gactctctcc tggtgctcaa 2160

taaatatcta atcataacag c 2181

<210>2

<211>321

<212>PRT

<213> human

<400>2

Met Gly Ile Leu Leu Gly Leu Leu Leu Leu Gly His Leu Thr Val Asp

1 5 10 15

Thr Tyr Gly Arg Pro Ile Leu Glu Val Pro Glu Ser Val Thr Gly Pro

20 25 30

Trp Lys Gly Asp Val Asn Leu Pro Cys Thr Tyr Asp Pro Leu Gln Gly

35 40 45

Tyr Thr Gln Val Leu Val Lys Trp Leu Val Gln Arg Gly Ser Asp Pro

50 55 60

Val Thr Ile Phe Leu Arg Asp Ser Ser Gly Asp His Ile Gln Gln Ala

65 70 75 80

Lys Tyr Gln Gly Arg Leu His Val Ser His Lys Val Pro Gly Asp Val

85 90 95

Ser Leu Gln Leu Ser Thr Leu Glu Met Asp Asp Arg Ser His Tyr Thr

100 105 110

Cys Glu Val Thr Trp Gln Thr Pro Asp Gly Asn Gln Val Val Arg Asp

115 120 125

Lys Ile Thr Glu Leu Arg Val Gln Lys Leu Ser Val Ser Lys Pro Thr

130 135 140

Val Thr Thr Gly Ser Gly Tyr Gly Phe Thr Val Pro Gln Gly Met Arg

145 150 155 160

Ile Ser Leu Gln Cys Gln Ala Arg Gly Ser Pro Pro Ile Ser Tyr Ile

165 170 175

Trp Tyr Lys Gln Gln Thr Asn Asn Gln Glu Pro Ile Lys Val Ala Thr

180 185 190

Leu Ser Thr Leu Leu Phe Lys Pro Ala Val Ile Ala Asp Ser Gly Ser

195 200 205

Tyr Phe Cys Thr Ala Lys Gly Gln Val Gly Ser Glu Gln His Ser Asp

210 215 220

Ile Val Lys Phe Val Val Lys Asp Ser Ser Lys Leu Leu Lys Thr Lys

225 230 235 240

Thr Glu Ala Pro Thr Thr Met Thr Tyr Pro Leu Lys Ala Thr Ser Thr

245 250 255

Val Lys Gln Ser Trp Asp Trp Thr Thr Asp Met Asp Gly Tyr Leu Gly

260 265 270

Glu Thr Ser Ala Gly Pro Gly Lys Ser Leu Pro Val Phe Ala Ile Ile

275 280 285

Leu Ile Ile Ser Leu Cys Cys Met Val Val Phe Thr Met Ala Tyr Ile

290 295 300

Met Leu Cys Arg Lys Thr Ser Gln Gln Glu His Val Tyr Glu Ala Ala

305 310 315 320

Arg

<210>3

<211>1372

<212>DNA

<213> human

<400>3

ccaactgcac ctcggttcta tcgataggag gctggaagaa aggacagaag tagctctggc 60

tgtgatgggg atcttactgg gcctgctact cctggggcac ctaacagtgg acacttatgg 120

ccgtcccatc ctggaagtgc cagagagtgt aacaggacct tggaaagggg atgtgaatct 180

tccctgcacc tatgaccccc tgcaaggcta cacccaagtc ttggtgaagt ggctggtaca 240

acgtggctca gaccctgtca ccatctttct acgtgactct tctggagacc atatccagca 300

ggcaaagtac cagggccgcc tgcatgtgag ccacaaggtt ccaggagatg tatccctcca 360

attgagcacc ctggagatgg atgaccggag ccactacacg tgtgaagtca cctggcagac 420

tcctgatggc aaccaagtcg tgagagataa gattactgag ctccgtgtcc agaaactctc 480

tgtctccaag cccacagtga caactggcag cggttatggc ttcacggtgc cccagggaat 540

gaggattagc cttcaatgcc aggctcgggg ttctcctccc atcagttata tttggtataa 600

gcaacagact aataaccagg aacccatcaa agtagcaacc ctaagtacct tactcttcaa 660

gcctgcggtg atagccgact caggctccta tttctgcact gccaagggcc aggttggctc 720

tgagcagcac agcgacattg tgaagtttgt ggtcaaagac tcctcaaagc tactcaagac 780

caagactgag gcacctacaa ccatgacata ccccttgaaa gcaacatcta cagtgaagca 840

gtcctgggac tggaccactg acatggatgg ctaccttgga gagaccagtg ctgggccagg 900

aaagagcctg cctgtctttg ccatcatcct catcatctcc ttgtgctgta tggtggtttt 960

taccatggcc tatatcatgc tctgtcggaa gacatcccaa caagagcatg tctacgaagc 1020

agccagggca catgccagag aggccaacga ctctggagaa accatgaggg tggccatctt 1080

cgcaagtggc tgctccagtg atgagccaac ttcccagaat ctgggcaaca actactctga 1140

tgagccctgc ataggacagg agtaccagat catcgcccag atcaatggca actacgcccg 1200

cctgctggac acagttcctc tggattatga gtttctggcc actgagggca aaagtgtctg 1260

ttaaaaatgc cccattaggc caggatctgc tgacataatc tagagtcgac ctgcagaagc 1320

ttggccgcca tggcccaact tgtttattgc agcttataat ggttacaaat aa 1372

<210>4

<211>399

<212>PRT

<213> human

<400>4

Met Gly Ile Leu Leu Gly Leu Leu Leu Leu Gly His Leu Thr Val Asp

1 5 10 15

Thr Tyr Gly Arg Pro Ile Leu Glu Val Pro Glu Ser Val Thr Gly Pro

20 25 30

Trp Lys Gly Asp Val Asn Leu Pro Cys Thr Tyr Asp Pro Leu Gln Gly

35 40 45

Tyr Thr Gln Val Leu Val Lys Trp Leu Val Gln Arg Gly Ser Asp Pro

50 55 60

Val Thr Ile Phe Leu Arg Asp Ser Ser Gly Asp His Ile Gln Gln Ala

65 70 75 80

Lys Tyr Gln Gly Arg Leu His Val Ser His Lys Val Pro Gly Asp Val

85 90 95

Ser Leu Gln Leu Ser Thr Leu Glu Met Asp Asp Arg Ser His Tyr Thr

100 105 110

Cys Glu Val Thr Trp Gln Thr Pro Asp Gly Asn Gln Val Val Arg Asp

115 120 125

Lys Ile Thr Glu Leu Arg Val Gln Lys Leu Ser Val Ser Lys Pro Thr

130 135 140

Val Thr Thr Gly Ser Gly Tyr Gly Phe Thr Val Pro Gln Gly Met Arg

145 150 155 160

Ile Ser Leu Gln Cys Gln Ala Arg Gly Ser Pro Pro Ile Ser Tyr Ile

165 170 175

Trp Tyr Lys Gln Gln Thr Asn Asn Gln Glu Pro Ile Lys Val Ala Thr

180 185 190

Leu Ser Thr Leu Leu Phe Lys Pro Ala Val Ile Ala Asp Ser Gly Ser

195 200 205

Tyr Phe Cys Thr Ala Lys Gly Gln Val Gly Ser Glu Gln His Ser Asp

210 215 220

Ile Val Lys Phe Val Val Lys Asp Ser Ser Lys Leu Leu Lys Thr Lys

225 230 235 240

Thr Glu Ala Pro Thr Thr Met Thr Tyr Pro Leu Lys Ala Thr Ser Thr

245 250 255

Val Lys Gln Ser Trp Asp Trp Thr Thr Asp Met Asp Gly Tyr Leu Gly

260 265 270

Glu Thr Ser Ala Gly Pro Gly Lys Ser Leu Pro Val Phe Ala Ile Ile

275 280 285

Leu Ile Ile Ser Leu Cys Cys Met Val Val Phe Thr Met Ala Tyr Ile

290 295 300

Met Leu Cys Arg Lys Thr Ser Gln Gln Glu His Val Tyr Glu Ala Ala

305 310 315 320

Arg Ala His Ala Arg Glu Ala Asn Asp Ser Gly Glu Thr Met Arg Val

325 330 335

Ala Ile Phe Ala Ser Gly Cys Ser Ser Asp Glu Pro Thr Ser Gln Asn

340 345 350

Leu Gly Asn Asn Tyr Ser Asp Glu Pro Cys Ile Gly Gln Glu Tyr Gln

355 360 365

Ile Ile Ala Gln Ile Asn Gly Asn Tyr Ala Arg Leu Leu Asp Thr Val

370 375 380

Pro Leu Asp Tyr Glu Phe Leu Ala Thr Glu Gly Lys Ser Val Cys

385 390 395

<210>5

<211>1090

<212>DNA

<213> human

<400>5

gtccaactgc acctcggttc tatcgatagg aggctggaag aaaggacaga agtagctctg 60

gctgtgatgg ggatcttact gggcctgcta ctcctggggc acctaacagt ggacacttat 120

ggccgtccca tcctggaagt gccagagagt gtaacaggac cttggaaagg ggatgtgaat 180

cttccctgca cctatgaccc cctgcaaggc tacacccaag tcttggtgaa gtggctggta 240

caacgtggct cagaccctgt caccatcttt ctacgtgact cttctggaga ccatatccag 300

caggcaaagt accagggccg cctgcatgtg agccacaagg ttccaggaga tgtatccctc 360

caattgagca ccctggagat ggatgaccgg agccactaca cgtgtgaagt cacctggcag 420

actcctgatg gcaaccaagt cgtgagagat aagattactg agctccgtgt ccagaaacac 480

tcctcaaagc tactcaagac caagactgag gcacctacaa ccatgacata ccccttgaaa 540

gcaacatcta cagtgaagca gtcctgggac tggaccactg acatggatgg ctaccttgga 600

gagaccagtg ctgggccagg aaagagcctg cctgtctttg ccatcatcct catcatctcc 660

ttgtgctgta tggtggtttt taccatggcc tatatcatgc tctgtcggaa gacatcccaa 720

caagagcatg tctacgaagc agccagggca catgccagag aggccaacga ctctggagaa 780

accatgaggg tggccatctt cgcaagtggc tgctccagtg atgagccaac ttcccagaat 840

ctgggcaaca actactctga tgagccctgc ataggacagg agtaccagat catcgcccag 900

atcaatggca actacgcccg cctgctggac acagttcctc tggattatga gtttctggcc 960

actgagggca aaagtgtctg ttaaaaatgc cccattaggc caggatctgc tgacataatc 1020

tagagtcgac ctgcagaagc ttggccgcca tggcccaact tgtttattgc agcttataat 1080

ggttacaata 1090

<210>6

<211>305

<212>PRT

<213> human

<400>6

Met Gly Ile Leu Leu Gly Leu Leu Leu Leu Gly His Leu Thr Val Asp

1 5 10 15

Thr Tyr Gly Arg Pro Ile Leu Glu Val Pro Glu Ser Val Thr Gly Pro

20 25 30

Trp Lys Gly Asp Val Asn Leu Pro Cys Thr Tyr Asp Pro Leu Gln Gly

35 40 45

Tyr Thr Gln Val Leu Val Lys Trp Leu Val Gln Arg Gly Ser Asp Pro

50 55 60

Val Thr Ile Phe Leu Arg Asp Ser Ser Gly Asp His Ile Gln Gln Ala

65 70 75 80

Lys Tyr Gln Gly Arg Leu His Val Ser His Lys Val Pro Gly Asp Val

85 90 95

Ser Leu Gln Leu Ser Thr Leu Glu Met Asp Asp Arg Ser His Tyr Thr

100 105 110

Cys Glu Val Thr Trp Gln Thr Pro Asp Gly Asn Gln Val Val Arg Asp

115 120 125

Lys Ile Thr Glu Leu Arg Val Gln Lys His Ser Ser Lys Leu Leu Lys

130 135 140

Thr Lys Thr Glu Ala Pro Thr Thr Met Thr Tyr Pro Leu Lys Ala Thr

145 150 155 160

Ser Thr Val Lys Gln Ser Trp Asp Trp Thr Thr Asp Met Asp Gly Tyr

165 170 175

Leu Gly Glu Thr Ser Ala Gly Pro Gly Lys Ser Leu Pro Val Phe Ala

180 185 190

Ile Ile Leu Ile Ile Ser Leu Cys Cys Met Val Val Phe Thr Met Ala

195 200 205

Tyr Ile Met Leu Cys Arg Lys Thr Ser Gln Gln Glu His Val Tyr Glu

210 215 220

Ala Ala Arg Ala His Ala Arg Glu Ala Asn Asp Ser Gly Glu Thr Met

225 230 235 240

Arg Val Ala Ile Phe Ala Ser Gly Cys Ser Ser Asp Glu Pro Thr Ser

245 250 255

Gln Asn Leu Gly Asn Asn Tyr Ser Asp Glu Pro Cys Ile Gly Gln Glu

260 265 270

Tyr Gln Ile Ile Ala Gln Ile Asn Gly Asn Tyr Ala Arg Leu Leu Asp

275 280 285

Thr Val Pro Leu Asp Tyr Glu Phe Leu Ala Thr Glu Gly Lys Ser Val

290 295 300

Cys

305

<210>7

<211>1590

<212>DNA

<213> little mouse (Mus musculus)

<400>7

gtccaactgc acctcggttc tatcgattcg aattcggcca cactggccgg atcctctaga 60

gatccctcga cctcgaccca cgcgtccgag cagcaagagg atggaaggat gaatagaagt 120

agcttcaaat aggatggaga tctcatcagg cttgctgttc ctgggccacc taatagtgct 180

cacctatggc caccccaccc taaaaacacc tgagagtgtg acagggacct ggaaaggaga 240

tgtgaagatt cagtgcatct atgatcccct gagaggctac aggcaagttt tggtgaaatg 300

gctggtaaga cacggctctg actccgtcac catcttccta cgtgactcca ctggagacca 360

tatccagcag gcaaagtaca gaggccgcct gaaagtgagc cacaaagttc caggagatgt 420

gtccctccaa ataaataccc tgcagatgga tgacaggaat cactatacat gtgaggtcac 480

ctggcagact cctgatggaa accaagtaat aagagataag atcattgagc tccgtgttcg 540

gaaatataat ccacctagaa tcaatactga agcacctaca accctgcact cctctttgga 600

agcaacaact ataatgagtt caacctctga cttgaccact aatgggactg gaaaacttga 660

ggagaccatt gctggttcag ggaggaacct gccaatcttt gccataatct tcatcatctc 720

cctttgctgc atagtagctg tcaccatacc ttatatcttg ttccgctgca ggacattcca 780

acaagagtat gtctatggag tgagcagggt gtttgccagg aagacaagca actctgaaga 840

aaccacaagg gtgactacca tcgcaactga tgaaccagat tcccaggctc tgattagtga 900

ctactctgat gatccttgcc tcagccagga gtaccaaata accatcagat caacaatgtc 960

tattcctgcc tgctgaacac agtttccaga aactaagaag ttcttgctac tgaagaaaat 1020

aacatctgct aaaatgcccc tactaagtca aggtctactg gcgtaattac ctgttactta 1080

tttactactt gccttcaaca tagctttctc cctggcttcc tttcttctta gacaacctaa 1140

agtatctatc tagtctgcca attctggggc cattgagaaa tcctgggttt ggctaagaat 1200

atactacatg cacctcaaga aatctagctt ctgggcttca cccagaacaa ttttcttcct 1260

agggccttca caactcttct ccaaacagca gagaaattcc atagcagtag aggttcttta 1320

tcatgcctcc agacagcgtg agtctcagtc ctacaaactc agacaagcac atgggtctag 1380

gattactcct ctttctctag ggccagatga cttttaattg atattactat tgctacatta 1440

tgaatctaat gcacatgtat tcttttgttg ttaataaatg tttaatcatg acatcaaaaa 1500

aaaaaaaaaa aagggcggcc gcgactctag agtcgacctg cagtagggat aacagggtaa 1560

taagcttggc cgccatggcc caacttgttt 1590

<210>8

<211>280

<212>PRT

<213> mouse

<400>8

Met Glu Ile Ser Ser Gly Leu Leu Phe Leu Gly His Leu Ile Val Leu

1 5 10 15

Thr Tyr Gly His Pro Thr Leu Lys Thr Pro Glu Ser Val Thr Gly Thr

20 25 30

Trp Lys Gly Asp Val Lys Ile Gln Cys Ile Tyr Asp Pro Leu Arg Gly

35 40 45

Tyr Arg Gln Val Leu Val Lys Trp Leu Val Arg His Gly Ser Asp Ser

50 55 60

Val Thr Ile Phe Leu Arg Asp Ser Thr Gly Asp His Ile Gln Gln Ala

65 70 75 80

Lys Tyr Arg Gly Arg Leu Lys Val Ser His Lys Val Pro Gly Asp Val

85 90 95

Ser Leu Gln Ile Asn Thr Leu Gln Met Asp Asp Arg Asn His Tyr Thr

100 105 110

Cys Glu Val Thr Trp Gln Thr Pro Asp Gly Asn Gln Val Ile Arg Asp

115 120 125

Lys Ile Ile Glu Leu Arg Val Arg Lys Tyr Asn Pro Pro Arg Ile Asn

130 135 140

Thr Glu Ala Pro Thr Thr Leu His Ser Ser Leu Glu Ala Thr Thr Ile

145 150 155 160

Met Ser Ser Thr Ser Asp Leu Thr Thr Asn Gly Thr Gly Lys Leu Glu

165 170 175

Glu Thr Ile Ala Gly Ser Gly Arg Asn Leu Pro Ile Phe Ala Ile Ile

180 185 190

Phe Ile Ile Ser Leu Cys Cys Ile Val Ala Val Thr Ile Pro Tyr Ile

195 200 205

Leu Phe Arg Cys Arg Thr Phe Gln Gln Glu Tyr Val Tyr Gly Val Ser

210 215 220

Arg Val Phe Ala Arg Lys Thr Ser Asn Ser Glu Glu Thr Thr Arg Val

225 230 235 240

Thr Thr Ile Ala Thr Asp Glu Pro Asp Ser Gln Ala Leu Ile Ser Asp

245 250 255

Tyr Ser Asp Asp Pro Cys Leu Ser Gln Glu Tyr Gln Ile Thr Ile Arg

260 265 270

Ser Thr Met Ser Ile Pro Ala Cys

275 280

<210>9

<211>1503

<212>DNA

<213> human

<400>9

gcaggcaaag taccagggcc gcctgcatgt gagccacaag gttccaggag atgtatccct 60

ccaattgagc accctggaga tggatgaccg gagccactac acgtgtgaag tcacctggca 120

gactcctgat ggcaaccaag tcgtgagaga taagattact gagctccgtg tccagaaact 180

ctctgtctcc aagcccacag tgacaactgg cagcggttat ggcttcacgg tgccccaggg 240

aatgaggatt agccttcaat gccagggttc ggggttctcc tcccatcagt tatatttggt 300

ataagcaaca gactaataac cagggaaccc atcaaagtag caaccctaag taccttactc 360

ttcaagcctg cggtgatagc cgactcaggc tcctatttct gcactgccaa gggccaggtt 420

ggctctgagc agcacagcga cattgtgaag tttgtggtca aagactcctc aaagctactc 480

aagaccaaga ctgaggcacc tacaaccatg acatacccct tgaaagcaac atctacagtg 540

aagcagtcct gggactggac cactgacatg gatggctacc ttggagagac cagtgctggg 600

ccaggaaaga gcctgcctgt ctttgccatc atcctcatca tctccttgtg ctgtatggtg 660

gtttttacca tggcctatat catgctctgt cggaagacat cccaacaaga gcatgtctac 720

gaagcagcca gggcacatgc cagagaggcc aacgactctg gagaaaccat gagggtggcc 780

atcttcgcaa gtggctgctc cagtgatgag ccaacttccc agaatctggg gcaacaacta 840

ctctgatgag ccctgcatag gacaggagta ccagatcatc gcccagatca atggcaacta 900

cgcccgcctg ctggacacag ttcctctgga ttatgagttt ctggccactg agggcaaaag 960

tgtctgttaa aaatgcccca ttaggccagg atctgctgac ataattgcct agtcagtcct 1020

tgccttctgc atggccttct tccctgctac ctctcttcct ggatagccca aagtgtccgc 1080

ctaccaacac tggagccgct gggagtcact ggctttgccc tggaatttgc cagatgcatc 1140

tcaagtaagc cagctgctgg atttggctct gggcccttct agtatctctg ccgggggctt 1200

ctggtactcc tctctaaata ccagagggaa gatgcccata gcactaggac ttggtcatca 1260

tgcctacaga cactattcaa ctttggcatc ttgccaccag aagacccgag gggaggctca 1320

gctctgccag ctcagaggac cagctatatc caggatcatt tctctttctt cagggccaga 1380

cagcttttaa ttgaaattgt tatttcacag gccagggttc agttctgctc ctccactata 1440

agtctaatgt tctgactctc tcctggtgct caataaatat ctaatcataa cagcaaaaaa 1500

aaa 1503

<210>10

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide Probe

<400>10

tatccctcca attgagcacc ctgg 24

<210>11

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide Probe

<400>11

gtcggaagac atcccaacaa g 21

<210>12

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide Probe

<400>12

cttcacaatg tcgctgtgct gctc 24

<210>13

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide Probe

<400>13

agccaaatcc agcagctggc ttac 24

<210>14

<211>50

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide Probe

<400>14

tggatgaccg gagccactac acgtgtgaag tcacctggca gactcctgat 50

<210>15

<211>7496

<212>DNA

<213> human

<400>15

ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca 60

ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct 120

ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 180

acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac 240

ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 300

aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 360

tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat 420

gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat 480

gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc 540

ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt 600

ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga 660

caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt 720

gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttggccc 780

acccccttgg cttcgttaga acgcggctac aattaataca taaccttatg tatcatacac 840

atacgattta ggtgacacta tagaataaca tccactttgc ctttcacatc cactttgcct 900

ttctctccac aggtgtccac tcccaggtcc aactgcacct cggttctatc gattaaacca 960

ccatggggat cttactgggc ctgctactcc tggggcacct aacagtggac acttatggcc 1020

gtcccatcct ggaagtgcca gagagtgtaa caggaccttg gaaaggggat gtgaatcttc 1080

cctgcaccta tgaccccctg caaggctaca cccaagtctt ggtgaagtgg ctggtacaac 1140

gtggctcaga ccctgtcacc atctttctac gtgactcttc tggagaccat atccagcagg 1200

caaagtacca gggccgcctg catgtgagcc acaaggttcc aggagatgta tccctccaat 1260

tgagcaccct ggagatggat gaccggagcc actacacgtg tgaagtcacc tggcagactc 1320

ctgatggcaa ccaagtcgtg agagataaga ttactgagct ccgtgtccag aaactctctg 1380

tctccaagcc cacagtgaca actggcagcg gttatggctt cacggtgccc cagggaatga 1440

ggattagcct tcaatgccag gctcggggtt ctcctcccat cagttatatt tggtataagc 1500

aacagactaa taaccaggaa cccatcaaag tagcaaccct aagtacctta ctcttcaagc 1560

ctgcggtgat agccgactca ggctcctatt tctgcactgc caagggccag gttggctctg 1620

agcagcacag cgacattgtg aagtttgtgg tcaaagactc ctcaaagcta ctcaagacca 1680

agactgaggc acctacaacc atgacatacc ccttgaaagc aacatctaca gtgaagcagt 1740

cctgggactg gaccactgac atggatggcg ggcgcgccca ggtcaccgac aaagctgcgc 1800

actatactct gtgcccaccg tgcccagcac ctgaactcct ggggggaccg tcagtcttcc 1860

tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag gtcacatgcg 1920

tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac gtggacggcg 1980

tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc acgtaccgtg 2040

tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag tacaagtgca 2100

aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa gccaaagggc 2160

agccccgaga accacaggtg tacaccctgc ccccatcccg ggaagagatg accaagaacc 2220

aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc gtggagtggg 2280

agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg gactccgacg 2340

gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag caggggaacg 2400

tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag aagagcctct 2460

ccctgtctcc gggtaaatga gtgcgacggc cctagagtcg acctgcagaa gcttctagag 2520

tcgacctgca gaagcttggc cgccatggcc caacttgttt attgcagctt ataatggtta 2580

caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag 2640

ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatcgatc gggaattaat 2700

tcggcgcagc accatggcct gaaataacct ctgaaagagg aacttggtta ggtaccttct 2760

gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct 2820

ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa 2880

agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 2940

ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt 3000

ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct 3060

ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc 3120

tgttaattcg aacacgcaga tgcagtcggg gcggcgcggt cccaggtcca cttcgcatat 3180

taaggtgacg cgtgtggcct cgaacaccga gcgaccctgc agcgacccgc ttaacagcgt 3240

caacagcgtg ccgcagatct gatcaagaga caggatgagg atcgtttcgc atgattgaac 3300

aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact 3360

gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc 3420

gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg 3480

cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg 3540

tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt 3600

catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc 3660

atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag 3720

cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg 3780

ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc 3840

tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt 3900

ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg 3960

ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt 4020

acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct 4080

tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg 4140

agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga 4200

cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccgg 4260

gagatggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat 4320

gacggcaata aaaagacaga ataaaacgca cgggtgttgg gtcgtttgtt cataaacgcg 4380

gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat 4440

acgcccgcgt ttcttccttt tccccacccc aacccccaag ttcgggtgaa ggcccagggc 4500

tcgcagccaa cgtcggggcg gcaagcccgc catagccacg ggccccgtgg gttagggacg 4560

gggtccccca tggggaatgg tttatggttc gtgggggtta ttcttttggg cgttgcgtgg 4620

ggtcaggtcc acgactggac tgagcagaca gacccatggt ttttggatgg cctgggcatg 4680

gaccgcatgt actggcgcga cacgaacacc gggcgtctgt ggctgccaaa cacccccgac 4740

ccccaaaaac caccgcgcgg atttctggcg ccgccggacg aactaaacct gactacggca 4800

tctctgcccc ttcttcgctg gtacgaggag cgcttttgtt ttgtattggt caccacggcc 4860

gagtttccgc gggaccccgg ccagggcacc tgtcctacga gttgcatgat aaagaagaca 4920

gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg 4980

aaggctctca agggcatcgg tcgagcggcc gcatcaaagc aaccatagta cgcgccctgt 5040

agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc 5100

agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc 5160

tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg 5220

cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga 5280

tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc 5340

caaactggaa caacactcaa ccctatctcg ggctattctt ttgatttata agggattttg 5400

ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt 5460

aacaaaatat taacgtttac aattttatgg tgcaggcctc gtgatacgcc tatttttata 5520

ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt 5580

gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag 5640

acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca 5700

tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc 5760

agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat 5820

cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc 5880

aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg atgacgccgg 5940

gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc 6000

agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat 6060

aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga 6120

gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc 6180

ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgccag cagcaatggc 6240

aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt 6300

aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc 6360

tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc 6420

agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca 6480

ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca 6540

ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt 6600

ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta 6660

acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 6720

agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 6780

ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 6840

cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa 6900

gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 6960

cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 7020

gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 7080

caccgaactg agatacctac agcgtgagca ttgagaaagc gccacgcttc ccgaagggag 7140

aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 7200

tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 7260

gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagctggca 7320

cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttacct 7380

cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 7440

tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg aattaa 7496

<210>16

<211>7201

<212>DNA

<213> human

<400>16

ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca 60

ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct 120

ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 180

acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac 240

ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 300

aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 360

tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat 420

gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat 480

gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc 540

ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt 600

ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga 660

caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt 720

gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttggccc 780

acccccttgg cttcgttaga acgcggctac aattaataca taaccttatg tatcatacac 840

atacgattta ggtgacacta tagaataaca tccactttgc ctttcacatc cactttgcct 900

ttctctccac aggtgtccac tcccaggtcc aactgcacct cggttctatc gatgctctca 960

ataaaccacc atggggatct tactgggcct gctactcctg gggcacctaa cagtggacac 1020

ttatggccgt cccatcctgg aagtgccaga gagtgtaaca ggaccttgga aaggggatgt 1080

gaatcttccc tgcacctatg accccctgca aggctacacc caagtcttgg tgaagtggct 1140

ggtacaacgt ggctcagacc ctgtcaccat ctttctacgt gactcttctg gagaccatat 1200

ccagcaggca aagtaccagg gccgcctgca tgtgagccac aaggttccag gagatgtatc 1260

cctccaattg agcaccctgg agatggatga ccggagccac tacacgtgtg aagtcacctg 1320

gcagactcct gatggcaacc aagtcgtgag agataagatt actgagctcc gtgtccagaa 1380

acactcctca aagctactca agaccaagac tgaggcacct acaaccatga catacccctt 1440

gaaagcaaca tctacagtga agcagtcctg ggactggacc actgacatgg atggggggcg 1500

cgcccaggtc accgacaaag ctgcgcacta tactctgtgc ccaccgtgcc cagcacctga 1560

actcctgggg ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat 1620

ctcccggacc cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt 1680

caagttcaac tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga 1740

ggagcagtac aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg 1800

gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga 1860

gaaaaccatc tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc 1920

atcccgggaa gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta 1980

tcccagcgac atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac 2040

cacgcctccc gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga 2100

caagagcagg tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca 2160

caaccactac acgcagaaga gcctctccct gtctccgggt aaatgagtgc gacggcccta 2220

gagtcgacct gcagaagctt ctagagtcga cctgcagaag cttggccgcc atggcccaac 2280

ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat 2340

aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat 2400

catgtctgga tcgatcggga attaattcgg cgcagcacca tggcctgaaa taacctctga 2460

aagaggaact tggttaggta ccttctgagg cggaaagaac cagctgtgga atgtgtgtca 2520

gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct 2580

caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca 2640

aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc 2700

cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta 2760

tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt 2820

tggaggccta ggcttttgca aaaattcgaa cacgcagatg cagtcggggc ggcgcggtcc 2880

caggtccact tcgcatatta aggtgacgcg tgtggcctcg aacaccgagc gaccctgcag 2940

cgacccgctt aacagcgtca acagcgtgcc gcagatctga tcaagagaca ggatgaggat 3000

cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga 3060

ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc 3120

ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga 3180

atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg 3240

cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc 3300

cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg 3360

atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga 3420

aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc 3480

tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca 3540

tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg 3600

tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct 3660

atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg 3720

accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc 3780

gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac 3840

gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt 3900

cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga 3960

gttcttcgcc caccccggga gatgggggag gctaactgaa acacggaagg agacaatacc 4020

ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg ggtgttgggt 4080

cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga 4140

gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccaa cccccaagtt 4200

cgggtgaagg cccagggctc gcagccaacg tcggggcggc aagcccgcca tagccacggg 4260

ccccgtgggt tagggacggg gtcccccatg gggaatggtt tatggttcgt gggggttatt 4320

cttttgggcg ttgcgtgggg tcaggtccac gactggactg agcagacaga cccatggttt 4380

ttggatggcc tgggcatgga ccgcatgtac tggcgcgaca cgaacaccgg gcgtctgtgg 4440

ctgccaaaca cccccgaccc ccaaaaacca ccgcgcggat ttctggcgcc gccggacgaa 4500

ctaaacctga ctacggcatc tctgcccctt cttcgctggt acgaggagcg cttttgtttt 4560

gtattggtca ccacggccga gtttccgcgg ggcacctgtc ctacgagttg catgataaag 4620

aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact 4680

gggttgaagg ctctcaaggg catcggtcga gcggccgctc aaagcaacca tagtacgcgc 4740

cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 4800

ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 4860

ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 4920

tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc 4980

cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 5040

tgttccaaac tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga 5100

ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 5160

attttaacaa aatattaacg tttacaattt tatggtgcag gcctcgtgat acgcctattt 5220

ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga 5280

aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc 5340

atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt 5400

caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct 5460

cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt 5520

tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt 5580

tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgatgac 5640

gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac 5700

tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct 5760

gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg 5820

aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg 5880

gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gccagcagca 5940

atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa 6000

caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt 6060

ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc 6120

attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg 6180

agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt 6240

aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt 6300

catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc 6360

ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct 6420

tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 6480

ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc 6540

ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac 6600

ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct 6660

gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat 6720

aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg 6780

acctacaccg aactgagata cctacagcgt gagcattgag aaagcgccac gcttcccgaa 6840

gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg 6900

gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga 6960

cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc 7020

tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt 7080

tacctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt 7140

ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgaatta 7200

a 7201

<210>17

<211>5988

<212>DNA

<213> human

<400>17

tcgagctcgc ccgacattga ttattgacta gttattaata gtaatcaatt acggggtcat 60

tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 120

gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 180

cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact 240

tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta 300

aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt 360

acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg 420

ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg 480

ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc 540

cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt 600

tagtgaaccg tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagac 660

accgggaccg atccagcctc cgcggccggg aacggtgcat tggaacgcgg attccccgtg 720

ccaagagtga cgtaagtacc gcctatagag tctataggcc cacccccttg gcttggccca 780

cccccttggc ttcgttagaa cgcggctaca attaatacat aaccttatgt atcatacaca 840

tacgatttag gtgacactat agaataacat ccactttgcc tttcacatcc actttgcctt 900

tctctccaca ggtgtccact cccaggtcca actgcacctc ggttctatcg attgaattcc 960

acgcgtccga gcagcaagag gatggaagga tgaatagaag tagcttcaaa taggatggag 1020

atctcatcag gcttgctgtt cctgggccac ctaatagtgc tcacctatgg ccaccccacc 1080

ctaaaaacac ctgagagtgt gacagggacc tggaaaggag atgtgaagat tcagtgcatc 1140

tatgatcccc tgagaggcta caggcaagtt ttggtgaaat ggctggtaag acacggctct 1200

gactccgtca ccatcttcct acgtgactcc actggagacc atatccagca ggcaaagtac 1260

agaggccgcc tgaaagtgag ccacaaagtt ccaggagatg tgtccctcca aataaatacc 1320

ctgcagatgg atgacaggaa tcactataca tgtgaggtca cctggcagac tcctgatgga 1380

aaccaagtaa taagagataa gatcattgag ctccgtgttc ggaaatataa tccacctaga 1440

atcaatactg aagcacctac aaccctgcac tcctctttgg aagcaacaac tataatgagt 1500

tcaacctctg acttgaccac taatgggact ggaaaacttg aggagaccat tgctggttca 1560

gggggggtca ccgacaagaa aattgtgccc agggattgtg gttgtaagcc ttgcatatgt 1620

acagtcccag aagtatcatc tgtcttcatc ttccccccaa agcccaagga tgtgctcacc 1680

attactctga ctcctaaggt cacgtgtgtt gtggtagaca tcagcaagga tgatcccgag 1740

gtccagttca gctggtttgt agatgatgtg gaggtgcaca cagctcagac gcaaccccgg 1800

gaggagcagt tcaacagcac tttccgctca gtcagtgaac ttcccatcat gcaccaggac 1860

tggctcaatg gcaaggagtt caaatgcagg gtcaacagtg cagctttccc tgcccccatc 1920

gagaaaacca tctccaaaac caaaggcaga ccgaaggctc cacaggtgta caccattcca 1980

cctcccaagg agcagatggc caaggataaa gtcagtctga cctgcatgat aacagacttc 2040

ttccctgaag acattactgt ggagtggcag tggaatgggc agccagcgga gaactacaag 2100

aacactcagc ccatcatgga cacagatggc tcttacttcg tctacagcaa gctcaatgtg 2160

cagaagagca actgggaggc aggaaatact ttcacctgct ctgtgttaca tgagggcctg 2220

cacaaccacc atactgagaa gagcctctcc cactctcctg gtaaatgagt cgacctgcag 2280

aagcttggcc gccatggccc aacttgttta ttgcagctta taatggttac aaataaagca 2340

atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt 2400

ccaaactcat caatgtatct tatcatgtct ggatcgggaa ttaattcggc gcagcaccat 2460

ggcctgaaat aacctctgaa agaggaactt ggttaggtac cttctgaggc ggaaagaacc 2520

agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa 2580

gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc 2640

cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc 2700

taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct 2760

gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga 2820

agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctgtta acagcttggc 2880

actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg 2940

ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg 3000

cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct 3060

tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt 3120

agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc 3180

agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc 3240

tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg 3300

cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga 3360

tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc 3420

caaactggaa caacactcaa ccctatctcg ggctattctt ttgatttata agggattttg 3480

ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt 3540

aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc 3600

gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt 3660

ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag 3720

aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt 3780

ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga 3840

aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc 3900

atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt 3960

caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct 4020

cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt 4080

tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt 4140

tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac 4200

gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac 4260

tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct 4320

gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg 4380

aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg 4440

gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca 4500

atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa 4560

caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt 4620

ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc 4680

attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg 4740

agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt 4800

aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt 4860

catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc 4920

ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct 4980

tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 5040

ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc 5100

ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac 5160

ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct 5220

gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat 5280

aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg 5340

acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa 5400

gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg 5460

gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga 5520

cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc 5580

aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct 5640

gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct 5700

cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca 5760

atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg 5820

tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat 5880

taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc 5940

ggataacaat ttcacacagg aaacagctat gacatgatta cgaattaa 5988

<210>18

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide Probe

<400>18

tctctgtctc caagcccaca g 21

<210>19

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide Probe

<400>19

ctttgaggag tctttgacc 19

<210>20

<211>1293

<212>DNA

<213> Artificial sequence

<220>

<223> huCRIg-short-IgG fusions

<400>20

acctcggttc tatcgatgct ctcaataaac caccatgggg atcttactgg gcctgctact 60

cctggggcac ctaacagtgg acacttatgg ccgtcccatc ctggaagtgc cagagagtgt 120

aacaggacct tggaaagggg atgtgaatct tccctgcacc tatgaccccc tgcaaggcta 180

cacccaagtc ttggtgaagt ggctggtaca acgtggctca gaccctgtca ccatctttct 240

acgtgactct tctggagacc atatccagca ggcaaagtac cagggccgcc tgcatgtgag 300

ccacaaggtt ccaggagatg tatccctcca attgagcacc ctggagatgg atgaccggag 360

ccactacacg tgtgaagtca cctggcagac tcctgatggc aaccaagtcg tgagagataa 420

gattactgag ctccgtgtcc agaaacactc ctcaaagcta ctcaagacca agactgaggc 480

acctacaacc atgacatacc ccttgaaagc aacatctaca gtgaagcagt cctgggactg 540

gaccactgac atggacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg 600

gggaccgtca gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac 660

ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa 720

ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta 780

caacagcacg taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg 840

caaggagtac aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat 900

ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga 960

agagatgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga 1020

catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc 1080

cgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag 1140

gtggcagcag gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta 1200

cacgcagaag agcctctccc tgtctccggg taaatgagtg cgacggccct agagtcgacc 1260

tgcagaagct tctagagtcg acctgcagaa gct 1293

<210>21

<211>1556

<212>DNA

<213> Artificial sequence

<220>

<223> huCRIg-long-IgG fusions

<400>21

atcgattaaa ccaccatggg gatcttactg ggcctgctac tcctggggca cctaacagtg 60

gacacttatg gccgtcccat cctggaagtg ccagagagtg taacaggacc ttggaaaggg 120

gatgtgaatc ttccctgcac ctatgacccc ctgcaaggct acacccaagt cttggtgaag 180

tggctggtac aacgtggctc agaccctgtc accatctttc tacgtgactc ttctggagac 240

catatccagc aggcaaagta ccagggccgc ctgcatgtga gccacaaggt tccaggagat 300

gtatccctcc aattgagcac cctggagatg gatgaccgga gccactacac gtgtgaagtc 360

acctggcaga ctcctgatgg caaccaagtc gtgagagata agattactga gctccgtgtc 420

cagaaactct ctgtctccaa gcccacagtg acaactggca gcggttatgg cttcacggtg 480

ccccagggaa tgaggattag ccttcaatgc caggctcggg gttctcctcc catcagttat 540

atttggtata agcaacagac taataaccag gaacccatca aagtagcaac cctaagtacc 600

ttactcttca agcctgcggt gatagccgac tcaggctcct atttctgcac tgccaagggc 660

caggttggct ctgagcagca cagcgacatt gtgaagtttg tggtcaaaga ctcctcaaag 720

ctactcaaga ccaagactga ggcacctaca accatgacat accccttgaa agcaacatct 780

acagtgaagc agtcctggga ctggaccact gacatggata aaactcacac atgcccaccg 840

tgcccagcac ctgaactcct ggggggaccg tcagtcttcc tcttcccccc aaaacccaag 900

gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 960

gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 1020

acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 1080

ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 1140

ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 1200

tacaccctgc ccccatcccg ggaagagatg accaagaacc aggtcagcct gacctgcctg 1260

gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 1320

aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc 1380

aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 1440

catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaaatga 1500

gtgcgacggc cctagagtcg acctgcagaa gcttctagag tcgacctgca gaagct 1556

<210>22

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide primers

<400>22

ccactggtcc cagagaaagt 20

<210>23

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide primers

<400>23

cactattagg tggcccagga 20

<210>24

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide primers

<400>24

gggaggattg ggaagacaat 20

<210>25

<211>1454

<212>DNA

<213> Artificial sequence

<220>

<223> huCRIg-long-Fc fusions

<400>25

atcgattaaa ccaccatggg gatcttactg ggcctgctac tcctggggca cctaacagtg 60

gacacttatg gccgtcccat cctggaagtg ccagagagtg taacaggacc ttggaaaggg 120

gatgtgaatc ttccctgcac ctatgacccc ctgcaaggct acacccaagt cttggtgaag 180

tggctggtac aacgtggctc agaccctgtc accatctttc tacgtgactc ttctggagac 240

catatccagc aggcaaagta ccagggccgc ctgcatgtga gccacaaggt tccaggagat 300

gtatccctcc aattgagcac cctggagatg gatgaccgga gccactacac gtgtgaagtc 360

acctggcaga ctcctgatgg caaccaagtc gtgagagata agattactga gctccgtgtc 420

cagaaactct ctgtctccaa gcccacagtg acaactggca gcggttatgg cttcacggtg 480

ccccagggaa tgaggattag ccttcaatgc caggctcggg gttctcctcc catcagttat 540

atttggtata agcaacagac taataaccag gaacccatca aagtagcaac cctaagtacc 600

ttactcttca agcctgcggt gatagccgac tcaggctcct atttctgcac tgccaagggc 660

caggttggct ctgagcagca cagcgacatt gtgaagtttg tggtcaaaga ctccgataaa 720

actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 780

ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 840

gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 900

gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 960

gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 1020

gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 1080

ccccgagaac cacaggtgta caccctgccc ccatcccggg aagagatgac caagaaccag 1140

gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 1200

agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 1260

tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1320

ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1380

ctgtctccgg gtaaatgagt gcgacggccc tagagtcgac ctgcagaagc ttctagagtc 1440

gacctgcaga agct 1454

<210>26

<211>1556

<212>DNA

<213> Artificial sequence

<220>

<223> huCRIg-long-Fc fusions

<400>26

atcgattaaa ccaccatggg gatcttactg ggcctgctac tcctggggca cctaacagtg 60

gacacttatg gccgtcccat cctggaagtg ccagagagtg taacaggacc ttggaaaggg 120

gatgtgaatc ttccctgcac ctatgacccc ctgcaaggct acacccaagt cttggtgaag 180

tggctggtac aacgtggctc agaccctgtc accatctttc tacgtgactc ttctggagac 240

catatccagc aggcaaagta ccagggccgc ctgcatgtga gccacaaggt tccaggagat 300

gtatccctcc aattgagcac cctggagatg gatgaccgga gccactacac gtgtgaagtc 360

acctggcaga ctcctgatgg caaccaagtc gtgagagata agattactga gctccgtgtc 420

cagaaactct ctgtctccaa gcccacagtg acaactggca gcggttatgg cttcacggtg 480

ccccagggaa tgaggattag ccttcaatgc caggctcggg gttctcctcc catcagttat 540

atttggtata agcaacagac taataaccag gaacccatca aagtagcaac cctaagtacc 600

ttactcttca agcctgcggt gatagccgac tcaggctcct atttctgcac tgccaagggc 660

caggttggct ctgagcagca cagcgacatt gtgaagtttg tggtcaaaga ctcctcaaag 720

ctactcaaga ccaagactga ggcacctaca accatgacat accccttgaa agcaacatct 780

acagtgaagc agtcctggga ctggaccact gacatggata aaactcacac atgcccaccg 840

tgcccagcac ctgaactcct ggggggaccg tcagtcttcc tcttcccccc aaaacccaag 900

gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 960

gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 1020

acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 1080

ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 1140

ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 1200

tacaccctgc ccccatcccg ggaagagatg accaagaacc aggtcagcct gacctgcctg 1260

gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 1320

aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc 1380

aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 1440

catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaaatga 1500

gtgcgacggc cctagagtcg acctgcagaa gcttctagag tcgacctgca gaagct 1556

<210>27

<211>1172

<212>DNA

<213> Artificial sequence

<220>

<223> huCRIg-short-Fc fusion

<400>27

atcgattaaa ccaccatggg gatcttactg ggcctgctac tcctggggca cctaacagtg 60

gacacttatg gccgtcccat cctggaagtg ccagagagtg taacaggacc ttggaaaggg 120

gatgtgaatc ttccctgcac ctatgacccc ctgcaaggct acacccaagt cttggtgaag 180

tggctggtac aacgtggctc agaccctgtc accatctttc tacgtgactc ttctggagac 240

catatccagc aggcaaagta ccagggccgc ctgcatgtga gccacaaggt tccaggagat 300

gtatccctcc aattgagcac cctggagatg gatgaccgga gccactacac gtgtgaagtc 360

acctggcaga ctcctgatgg caaccaagtc gtgagagata agattactga gctccgtgtc 420

cagaaacact ccgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 480

ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 540

cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 600

tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 660

aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 720

aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 780

tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggaa 840

gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 900

atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 960

gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1020

tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1080

acgcagaaga gcctctccct gtctccgggt aaatgagtgc gacggcccta gagtcgacct 1140

gcagaagctt ctagagtcga cctgcagaag ct 1172

<210>28

<211>1293

<212>DNA

<213> Artificial sequence

<220>

<223> huCRIg-short-Fc fusion

<400>28

acctcggttc tatcgatgct ctcaataaac caccatgggg atcttactgg gcctgctact 60

cctggggcac ctaacagtgg acacttatgg ccgtcccatc ctggaagtgc cagagagtgt 120

aacaggacct tggaaagggg atgtgaatct tccctgcacc tatgaccccc tgcaaggcta 180

cacccaagtc ttggtgaagt ggctggtaca acgtggctca gaccctgtca ccatctttct 240

acgtgactct tctggagacc atatccagca ggcaaagtac cagggccgcc tgcatgtgag 300

ccacaaggtt ccaggagatg tatccctcca attgagcacc ctggagatgg atgaccggag 360

ccactacacg tgtgaagtca cctggcagac tcctgatggc aaccaagtcg tgagagataa 420

gattactgag ctccgtgtcc agaaacactc ctcaaagcta ctcaagacca agactgaggc 480

acctacaacc atgacatacc ccttgaaagc aacatctaca gtgaagcagt cctgggactg 540

gaccactgac atggacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg 600

gggaccgtca gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac 660

ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa 720

ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta 780

caacagcacg taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg 840

caaggagtac aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat 900

ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga 960

agagatgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga 1020

catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc 1080

cgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag 1140

gtggcagcag gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta 1200

cacgcagaag agcctctccc tgtctccggg taaatgagtg cgacggccct agagtcgacc 1260

tgcagaagct tctagagtcg acctgcagaa gct 1293

Claims (31)

1. Use of a CRIg polypeptide or a CRIg immunoadhesin comprising a CRIg polypeptide fused to an immunoglobulin sequence in the manufacture of a medicament for preventing the formation or progression of age-related macular degeneration (AMD), wherein said preventing comprises administering an effective amount of the CRIg polypeptide or CRIg immunoadhesin to a subject at risk of developing AMD, or diagnosed with AMD, in at least one eye.
2. 2. The use of claim 1 wherein said CRIg polypeptide is SEQ ID NO: ECD of CRIg polypeptide of 2, 4, 6 or 8.
3. 3. The use of claim 1, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 4 or 6.
4. 4. The use of claim 3, wherein the fusion polypeptide is selected from the group consisting of SEQ ID NO: 20, 21, 25, 26, 27 and 28, or a pharmaceutically acceptable salt thereof.
5. 5. The use of any one of claims 1-4, wherein the subject is a human.
6. 6. The use of claim 5, wherein the human subject has been diagnosed with AMD in at least one eye.
7. 7. The use of claim 6, wherein said AMD is type 3 or type 4 dry AMD.
8. 8. The use of claim 7, wherein the subject has been identified as at risk for developing a CNV.
9. 9. The use of claim 8, wherein the subject is genetically at risk of developing a CNV.
10. 10. The use of claim 7, wherein the human subject has been diagnosed with AMD in both eyes.
11. 11. The use of claim 10, wherein the human subject has 3 or 4 types of AMD in both eyes.
12. 12. The use of claim 5, wherein the administration slows the development of AMD.
13. 13. The use of claim 5, wherein the administration delays the progression of AMD to CNV.
14. 14. The use of claim 5, wherein the administration prevents the progression of AMD to CNV.
15. 15. The use of claim 6, wherein the human subject has been diagnosed with AMD in only one eye.
16. 16. The use of claim 15, wherein the administration delays the formation of AMD in the other eye.
17. 17. The use of claim 15, wherein the administration prevents the formation of AMD in the other eye.
18. 18. The use of claim 5, wherein said administering is by intravitreal injection.
19. 19. The use of claim 5, further comprising administering an additional agent to prevent or treat AMD or CNV.
20. 20. The use of claim 19, wherein the additional agent is an anti-VEGF-a antibody.
21. Use of a CRIg polypeptide or a CRIg immunoadhesin comprising a CRIg polypeptide fused to an immunoglobulin sequence for the preparation of a medicament for the prophylactic or therapeutic treatment of dry age-related macular degeneration (AMD), wherein said treatment comprises administering to a subject in need thereof a prophylactically or therapeutically effective amount of a CRIg polypeptide or CRIg immunoadhesin.
22. 22. The use of claim 21 wherein said CRIg polypeptide is selected from the group consisting of: SEQ ID NO: 2, 4, 6, 8 and the extracellular domain (ECD) of said polypeptide.
23. 23. The use of claim 22 wherein said CRIg polypeptide is SEQ ID NO: ECD of CRIg polypeptide of 2, 4, 6 or 8.
24. 24. The use of claim 23 wherein said CRIg polypeptide is SEQ ID NO: ECD of CRIg polypeptide of 4 or 6.
25. 25. The use of claim 21, wherein the immunoglobulin sequence is an immunoglobulin constant region sequence.
26. 26. The use of claim 25, wherein the immunoglobulin constant region sequence is of an immunoglobulin heavy chain.
27. 27. The use of claim 26, wherein the immunoglobulin heavy chain constant region sequence is identical to seq id NO: the extracellular region of the CRIg polypeptide of 2, 4, 6 or 8 is fused to produce a CRIg-Ig fusion protein.
28. 28. The use of claim 27, wherein the immunoglobulin constant region sequence is IgG.
29. 29. The use of claim 28, wherein the IgG is IgG-1 or IgG-3.
30. 30. The use of claim 29, wherein the IgG-1 heavy chain constant region sequence comprises at least the hinge, CH2, and CH3 regions.
31. 31. The use of claim 29, wherein the IgG-1 heavy chain constant region sequence comprises the CH1, hinge, CH2, and CH3 regions.