JP2008515999A - CRIg polypeptide for prevention and treatment of complement related disorders - Google Patents

Abstract

The present invention includes the recently discovered macrophage specific receptor CRIg and ocular conditions involving complements such as age-related macular degeneration (AMD) and chronic choroidal neovascularization (CNV), It relates to its use in the prevention and treatment of disorders involving complement. In one aspect, the present invention relates to a method for the prevention or treatment of eye diseases involving complement. This includes administering to a subject in need thereof a prophylactically effective amount or a therapeutically effective amount of a complement inhibitor (eg, an alternative complement pathway inhibitor (eg, a CRIg polypeptide or an agonist thereof)). included.

Description

(Field of Invention)
The present invention includes the recently discovered macrophage specific receptor CRIg (formerly called STIgMA) and complements such as age-related macular degeneration (AMD) and chronic choroidal neovascularization (CNV). It relates to its use in the prevention and treatment of disorders involving complement, including related ocular symptoms.

(Background of the Invention)
The complement system is a complex enzyme cascade composed of a series of serum glycoproteins, usually present in the form of inactive proenzymes. Three major pathways (classical complement pathway and alternative complement pathway) can activate complement, which are integrated at the C3 level, here by two similar C3 convertases, C3 is cut into C3a and C3b. A mannose-binding lectin (MBL) pathway, an additional pathway for complement activation, has also been described.

  The components of the classical complement pathway are named with C and numbers (eg, C1, C3). Because of the order in which they were identified, the first four components are numbered C1, C4, C2, and C3. Alternative complement pathway components are marked with letters (eg, B, P, D). Cleaved fragments are indicated with a lowercase letter after the complement designation (eg, C3a and C3b are fragments of C3). Inactive C3b is designated iC3b. The polypeptide chain of the complement protein is shown with a Greek letter after the component (eg, C3α and C3β are the C3 α and β chains). The cell membrane receptor for C3 is abbreviated as CR1, CR2, CR3, and CR4.

  The classical complement pathway of the complement system is a major effector of the humor branch of the human immune response. The factor for activation of the classical complement pathway and the MBL pathway is either an antigen on the target cell or an IgG or IgM antibody bound to a lectin. Binding of the antibody to the antigen exposes one site on the antibody, which is the binding site for C1, the first complement component. C1 binds to the exposed region of the antibody with at least two antigens bound. As a result, its C1r and C1s subunits are activated. Activated C1s is responsible for cleavage involving the following two complement components C4 and C2. C4 is cleaved into two fragments. Among them, larger C4b molecules attach to the immediate target membrane, while smaller C4a molecules are released. The exposed sites on the precipitated C4b can be used to interact with the next complement component, C2. As seen in the previous step, activated C1s cleaves the C2 molecule into two fragments, of which C2a remains and a small C2b fragment is released. C4b2a is also known as C3 convertase, which 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 an IgG antibody. Factor B then binds to C3b bound to the cell to form C3bB. C3bB is then divided into Bb and Ba by factor B to form C3bBb, which is the alternative complement pathway C3 convertase. Properdin (serum protein) then binds to C3bBb to form C3bBbP, which functions as a C3 convertase. As a result, the C3 molecule is cleaved into C3a and C3b by the enzyme. At this point, another complement system tract is activated. Some of C3b binds to C3bBb to form C3bBb3b. This can cleave the C5 molecule into C5a and C5b.

  An alternative complement pathway is the self-amplification pathway, which is important for clearance and recognition of bacteria and other pathogens in the absence of antibodies. This alternative complement pathway can also amplify complement activation after initial complement activation by either the lectin and / or classical complement pathway. The rate-limiting step in the activation of the human alternative complement pathway is the enzymatic action of factor D which cleaves factor B to form C3bBb, the C3 convertase of the alternative complement pathway. (Non-Patent Document 1). There is strong evidence for the role of complement activation and accumulation in adjuvant-induced arthritis (AIA), and collagen-induced arthritis (CIA), and various other diseases and conditions.

  Recently, deficiencies in the control of alternative complement pathways have been implicated in the onset of kidney and eye diseases, including hemolytic uremic syndrome (HUS) and AMD (online in www.sciencedirect.com). Available above). C3 has been shown to be essential for the development of CNV in mice (Non-patent Document 3).

The role of the complement system in inflammatory symptoms and related tissue damage, autoimmune diseases, and diseases involving complement is also well known. Local and distant tissue injury following blood reperfusion (Stahl et al., Supra); adult respiratory distress syndrome (ARDS, NPL 5); complement activation during cardiopulmonary bypass surgery (NPL 6) Dermatomyositis (Non-Patent Document 7); and pemphigus (Non-Patent Document 8) have been suggested to play an important role. Alternative complement pathways also include autoimmune diseases (eg, lupus nephritis, and resulting glomerulonephritis, and vasculitis (see, eg, Non-Patent Document 9); and rheumatoid arthritis, eg, juvenile It is also related to rheumatoid arthritis (Non-Patent Document 10 and Non-Patent Document 11).

  Local increase in complement accumulation and activation correlates with disease severity (Non-Patent Document 12). C5a receptor antagonists (eg, peptides and small organic molecules) have been tested for the treatment of arthritis (Non-Patent Document 13) and various other immunoinflammatory diseases (Non-Patent Document 14; Non-Patent Document 15; And several companies (eg, Promics (Australia)) are conducting human clinical trials to test the effectiveness of C5a antagonists in similar indications, such as dermatomyositis and pemphigus. (Non-patent document 7) Anti-C5a monoclonal antibody reduces cardiopulmonary bypass and coronary endothelial dysfunction induced by cardiac paralysis (non-patent document 16), prevents collagen-induced arthritis, And it is shown that an established disease is relieved (nonpatent literature 17).

  Opsonophagocytosis is the process of accumulation of complement fragments on the surface of particles followed by uptake by phagocytic cells, which circulates including immune complexes, apoptotic cells or cell debris, and pathogens This is important for the clearance of the particles (Non-patent Document 18). Tissue-resident macrophages are known to play an important role in particle clearance from circulation mediated by complement. Kupfer cells, over 90% of which are composed of tissue-resident macrophages, are exposed to blood from the hepatic portal vein without interruption, opsonized viruses, tumor cells, bacteria, fungi, parasites, And strategically placed in hepatic sinusoidal capillaries for efficient clearance of harmful substances from the gastrointestinal tract. This clearance process largely depends on the presence of complement C3 as opsonin (Non-patent Document 19). Upon binding to the bacterial surface via the spacer, C3 is cleaved, amplifying the alternative complement pathway. This response leads to further accumulation of C3 fragments that can act as ligands for complement receptors on macrophages. The importance of this pathway is shown by the very susceptibility of humans lacking C3 to bacterial and viral infections (references).

  Complement receptors CR1, 3 and 4 characterized so far internalize C3b and phagocytic C3 opsonized particles only after PKC activation or Fc receptor stimulation (Non-Patent Literature). 20; Non-patent document 21; Non-patent document 22). Furthermore, CR1 is not expressed on the surface of mouse Kupffer cells (Non-patent Document 23). No complement receptor has been described so far that helps KC in the constitutive clearance of circulating particles.

Anti-C3b (i) antibodies have been reported to promote complement activation, C3b (i) accumulation, and rituximab-induced CD20 ⁺ cell death (Non-patent Document 24).
Stahl et al., American Journal of Pathology, 2003, 162, p. 449-455 Zipfel et al., Mol. Immunol. 2006, Vol. 43, p. 97-106 Bora et al. Immunol. 2005, 174, No. 1, p. 491-7 Molnes et al., Trends in Immunology, 2002, Vol. 23, p. 61-64 Schein et al., Chest, 1987, 91, p. 850-854 Fung et al., J Thorac Cardiovas Surg, 2001, vol. 122, p. 113-122 Kissel, JT et al., NEJM, 1986, 314, p. 329-334 Honguchi et al., J Invest Dermatol, 1989, Vol. 92, p. 588-592 Watanabe et al. Immunol. 2000, 164, p. 786-794 Aggarwal et al., Rheumatology, 2000, 29, p. 189-192 Neumann E.M. Et al., Arthritis Rheum. 2002, volume 4, p. 934-45 Atkinson, J Clin Invest, 2003, 112, p. 1639-1641 Woodruf et al., Arthritis & Rheumatism, 2002, 46, 9, p. 2476-2485 Short et al., Br J Pharmacol, 1999, 126, p. 551-554 Finch et al., J Med Chem, 1999, 42, p. 1965-1074 Tofukuji et al. Thorac. Cardiovasc. Surg. 1998, 116, p. 1060-1069 Wang et al., Proc. Natl. Acad. Sci. USA, 1995, Vol. 92, No. 19, p. 8955-8959 Gasque, P.M. Mol Immunol. 2004, vol. 41, p. 1089-1098 Fujita et al., Immunol. Rev. 2004, 198, p. 185-202 Carpentier et al., Cell Regul, 1991, Volume 2, p. 41-55 Sengelov, Crit. Rev. Immunol. 1995, Vol. 15, p. 107-131 Sengelov et al. Immunol. 1994, 153, p. 804-810 Fang et al. Immunol. 1998, 160, p. 5273-5279 Kennedy et al., Blood, 2003, 101, No. 3, p. 1071-1079

  In view of the known involvement of the complement cascade in various diseases, the identification and development of new agents for the prevention and / or treatment of diseases involving complement is necessary.

(Summary of the Invention)
The present invention is based on the identification of novel members of the complement receptor family that interact with the complement system and members of the first immunoglobulin (Ig) superfamily.

  In one aspect, the present invention relates to a method for the prevention or treatment of eye diseases involving complement. This includes administering to a subject in need thereof a prophylactically effective amount or a therapeutically effective amount of a complement inhibitor (eg, an alternative complement pathway inhibitor (eg, a CRIg polypeptide or an agonist thereof)). included.

  Complement-related ocular symptoms include, for example, age-related macular degeneration (AMD), chronic choroidal neovascularization (CNV), uveitis, diabetes, and other ischemia-related retinopathy, intraocular It can be inflammation, diabetic macular edema, pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, central retinal vein occlusion (CRVO), corneal neovascularization, retinal neovascularization. Preferably, the ocular condition to which complement is related is AMD or CNV, which includes all stages of these symptoms.

  In another aspect, the invention relates to a method for the prevention of the development or progression of AMD. This involves the risk of developing AMD in at least one eye or in a subject diagnosed with AMD in at least one eye in an effective amount of a complement inhibitor (eg, an alternative complement pathway inhibitor). (E.g., a CRIg polypeptide or an agonist thereof).

  In yet another aspect, the invention relates to a method for the treatment of wet AMD (dry AMD). This includes administering to a subject in need thereof a therapeutically effective amount of a complement inhibitor (eg, an alternative complement pathway inhibitor (eg, a CRIg polypeptide or agonist thereof)).

  In all embodiments, the CRIg polypeptide can be selected from, for example, the CRIg polypeptides of SEQ ID NOs: 2, 4, 6, and 8, and the extracellular domain (ECD) of such polypeptides. CRIg polypeptides (including full-length polypeptides and their ECDs) can be fused to immunoglobulin sequences (eg, immunoglobulin heavy chain constant region sequences (eg, Fc region)) and the resulting immunoadhesins Can be used as CRIg agonists in the prevention and treatment methods of the present invention. Preferably, the immunoglobulin is IgG (eg, IgG-1 or IgG-2 or IgG-3 or IgG4), more preferably IgG-1 or IgG-3. An IgG1 heavy chain constant sequence can include, for example, at least a hinge, CH1, CH2, and CH3 region, or a hinge, CH2, and CH3 region.

  In a further aspect, the present invention relates to a method for the prevention or treatment of a disease or condition in which complement is implicated. This includes administering to a subject in need thereof a prophylactically effective amount or a therapeutically effective amount of a complement inhibitor (eg, an alternative complement pathway inhibitor (eg, a CRIg polypeptide or an agonist thereof)). included.

  In another aspect, the present invention relates to a method for inhibition of production of C3b complement fragments in a mammal. This includes administering to the mammal an effective amount of a complement inhibitor (eg, an alternative complement pathway inhibitor (eg, a CRIg polypeptide or agonist thereof)).

  In yet another aspect, the invention relates to a method for selective inhibition of the mammalian alternative complement pathway. This includes administering to the mammal an effective amount of a CRIg polypeptide or agonist thereof.

  In all embodiments, the CRIg polypeptide can be selected from the group consisting of, for example, the CRIg polypeptides of SEQ ID NOs: 2, 4, 6, 8, and the extracellular region of such polypeptides. The agonist is preferably a CRIg-Ig fusion protein (immunoadhesive) as described herein above. The immunoglobulin sequence can be, for example, an immunoglobulin constant region sequence (eg, a constant region sequence of an immunoglobulin heavy chain). In another embodiment, the immunoglobulin heavy chain constant region sequence is fused to the extracellular region of the CRIg polypeptide of SEQ ID NO: 2, 4, 6, or 8. In further embodiments, the immunoglobulin heavy chain constant region sequence is that of an IgG (eg, IgG-1 or IgG-3). In this case, the IgG-1 heavy chain constant region sequence can include, for example, at least the hinge, CH2, and CH3 regions, or the hinge, CH1, CH2, and CH3 regions.

  The disease in which complement is involved can be, for example, an inflammatory disease or an autoimmune disease.

  In one specific embodiment, complement-related diseases are rheumatoid arthritis (RA), adult respiratory distress syndrome (ARDS), remote tissue injury after ischemia-reperfusion, cardiopulmonary bypass surgery Interstitial activation, dermatomyositis, pemphigus, lupus nephritis and resulting glomerulonephritis and vasculitis, cardiopulmonary bypass surgery, coronary artery endothelial dysfunction induced by heart failure, type II membranoproliferative glomeruli Nephritis, IgA nephropathy, acute renal failure, cryoglobulemia, antiphospholipid syndrome, age-related macular degeneration, uveitis, diabetic retinopathy, allograft, hyperacute rejection, hemodialysis, chronic obstruction From the group consisting of systemic lung disease (COPD), asthma, Alzheimer's disease, atherosclerosis, hereditary angioedema, paroxysmal nocturnal hemoglobinuria, and aspiration pneumonia Be-option.

  In another specific embodiment, the disease associated with complement is inflammatory bowel disease (IBD), systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathy, systemic sclerosis (strong) Dermatosis), idiopathic inflammatory myopathy (dermatomyositis, polymyositis), Sjogren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria) Autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura, immune thrombocytopenia), thyroiditis (Graves disease, Hashimoto's disease thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, Immune kidney disease (glomerulonephritis, tubulointerstitial nephritis), demyelinating disease of central and peripheral nervous system (eg, multiple sclerosis, idiopathic multiple neuropathy), hepatobiliary disease (eg , Infectious hepatitis ( , B, C, D, hepatitis E, and other non-liver tropic viruses)), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, inflammatory and Fibrous lung disease (eg, cystic fibrosis), gluten irritable bowel disease, Whipple disease, autoimmune or immune skin disease (includes vesicular skin disease, erythema multiforme, and contact dermatitis), Selected from the group consisting of psoriasis, allergic diseases of the lung (eg, eosinophilic pneumonia, idiopathic pulmonary fibrosis, and hypersensitivity pneumonia), transplant-related diseases (including transplant rejection and graft-versus-host disease) Is done.

  In yet another specific embodiment, the disease associated with complement is rheumatoid arthritis (RA), psoriasis, or asthma.

  In all embodiments, the subject can be a mammal, eg, a human patient.

  In a further aspect, the present invention relates to a method for the prevention or treatment of age-related macular degeneration (AMD) or chronic choroidal neovascularization (CNV) in a subject. This includes administering to the subject an effective amount of a complement inhibitor (eg, an alternative complement pathway inhibitor (eg, a CRIg polypeptide or agonist thereof)).

Detailed Description of Preferred Embodiments
(I. Definition)
The terms “PRO362”, “JAM4”, “STIgMA”, and “CRIg” are used interchangeably and refer to naturally occurring sequences and variant CRIg polypeptides.

  A “naturally occurring sequence” CRIg is a polypeptide having the same amino acid sequence as a CRIg polypeptide derived from nature, regardless of its mode of preparation. Thus, CRIg of naturally occurring sequences can be isolated from nature and can also be produced by recombinant and / or synthetic means. The term “naturally occurring sequence CRIg” specifically includes a naturally occurring shortened CRIg or a secreted CRIg (eg, an extracellular domain sequence), a naturally occurring modification. Included are body forms (eg, alternatively spliced forms), as well as naturally occurring allelic variants of CRIg. Naturally occurring sequences of CRIg polypeptides specifically include the 321 amino acid long human CRIg polypeptide of SEQ ID NO: 2 (shown in FIG. 1), which includes an N-terminal With or without a signal sequence, with or without the initiation methionine at position 1, and transmembrane at amino acid positions about 277 to 307 of SEQ ID NO: 2. Some or all of the domain may or may not be included. Naturally occurring sequences of CRIg polypeptides further include the full-length 399 amino acid long human CRIg polypeptide of seq. ID No. 4 (huCRIg, or huCRIg-long, shown in FIGS. 2 and 5), This may or may not include the N-terminal signal sequence, may or may not include the first methionine at position 1, and may be from about positions 277 to 307 of SEQ ID NO: 4. Some or all of the transmembrane domain at the amino acid position may or may not be included. In yet a further embodiment, the naturally occurring sequence CRIg polypeptide is a 305 amino acid short form of human CRIg (huCRIg-short, SEQ ID NO: 6, shown in FIG. 3), which includes: A membrane located at about positions 183 to 213 of SEQ ID NO: 6 with or without an N-terminal signal sequence, with or without the initiation methionine at position 1. Some or all of the penetrating domain may or may not be included. In a different embodiment, the naturally occurring sequence CRIg polypeptide is the 280 amino acid long full length mouse CRIg polypeptide of SEQ ID NO: 8 (muCRIg, shown in FIGS. 4 and 5), which includes , With or without the N-terminal signal sequence, with or without the starting methionine at position 1, and at amino acid positions from about position 181 to position 211 of SEQ ID NO: 8. Some or all of a transmembrane domain may or may not be included. Specifically included in this definition are CRIg polypeptides of animals other than humans, including higher primates and mammals.

  “CRIg variant” means an active CRIg polypeptide as defined below having at least about 80% amino acid sequence identity to a CRIg polypeptide of a naturally occurring sequence. . This includes the C-terminal truncated 321 amino acids huCRIg (SEQ ID NO: 2), full length huCRIg (SEQ ID NO: 4), huCRIg-short (SEQ ID NO: 6), and muCRIg (SEQ ID NO: 8). It is not limited to. Each of these includes all or part of the transmembrane domain, with or without an N-terminal initiation methionine, with or without an N-terminal signal sequence. It may or may not be included, and may or may not include an intracellular domain. In certain embodiments, the CRIg variant has at least 80% amino acid sequence homology with the mature full-length polypeptide of the sequence of SEQ ID NO: 2. In another embodiment, the CRIg variant has at least about 80% amino acid sequence homology with the mature full-length polypeptide of SEQ ID NO: 4. In yet another embodiment, the CRIg variant has at least about 80% amino acid sequence homology with the mature full-length polypeptide of the sequence of SEQ ID NO: 6. In still further embodiments, the CRIg variant has at least about 80% amino acid sequence homology with the mature full-length polypeptide of the sequence of SEQ ID NO: 8. Such CRIg polypeptide variants include, for example, the N-terminus or C-terminus, wherein one or more amino acid residues are inserted at the N-terminus or C-terminus of the sequence of SEQ ID NO: 2, 4, 6, or 8. CRIg polypeptides in which one or more amino acid residues are replaced and / or one or more amino acid residues at the N-terminus or C-terminus are deleted. Other variants include one or more amino acids inserted in the transmembrane region of the indicated polypeptide sequence, one or more amino acids substituted in the transmembrane region, and / or the membrane One or more amino acids are deleted in the penetrating region.

  Typically, a CRIg variant has at least about 80% amino acid sequence identity, or at least about 85% amino acid sequence identity, or at least about 90% with the mature amino acid sequence of SEQ ID NO: 2, 4, 6, or 8. It will have 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, maximum sequence identity occurs in the extracellular domain (ECD) (amino acids from position 1 or from about position 21 to X of SEQ ID NO: 2 or 4, wherein X is any amino acid from positions 271 to 281) Alternatively, an amino acid from position 1 or about position 21 to X of SEQ ID NO: 6, wherein X is any amino acid residue from positions 178 to 186; Amino acid at position 1 or about position 21 to X, where X is any amino acid residue from position 176 to position 184).

  CRIg (PRO362) "Extracellular domain" or "ECD" refers to one form of CRIg polypeptide. This excludes in principle the transmembrane and cytoplasmic domains of the respective full-length molecules. Typically, CRIg ECD contains less than 1% of such transmembrane and / or cytoplasmic domains, and preferably contains less than 0.5% of such domains. As discussed above, the CRIg ECD optionally includes an amino acid residue from position 1 or from about position 21 to X of SEQ ID NO: 2, 4, 6, or 8. Here, X is any amino acid from about positions 271 to 281 of SEQ ID NO: 2 or 4, any amino acid from about positions 178 to 186 of SEQ ID NO: 6, and about positions 176 to 184 of SEQ ID NO: 8. Any amino acid up to.

  “Percent amino acid sequence identity” is for the CRIg (PRO362) sequences identified herein to obtain sequence alignment and, where necessary, percent sequence identity (%). As a percentage of amino acid residues in the candidate sequence that are identical to amino acid residues in the CRIg sequence, without considering any conservative substitutions as part of sequence identity after the introduction of the gap of Each is defined. Alignment for the purpose of determining percent amino acid sequence identity can be done in various ways within the ability of those skilled in the art, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software, etc. Can be done using publicly available computer programs. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to perform maximal alignment over the full length of the sequences being compared. The sequence identity is then calculated relative to the longer sequence. That is, the overall sequence identity may be less than 100% even when the shorter sequence shows 100% sequence identity with a portion of the longer sequence.

  “% Nucleic Acid Sequence Identity” refers to sequence alignment and, if necessary, maximum sequence identity with respect to the sequence encoding CRIg (PRO362) identified herein (eg, DNA45416). Are defined as the percentage of nucleotides in the candidate sequence that are identical to the nucleotides in the sequence encoding CRIg, respectively, after the introduction of a gap to obtain a percentage of. Alignments for the purpose of determining percent nucleic acid sequence identity can be in a variety of ways that are within the ability of those skilled in the art, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Can be done using publicly available computer programs. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to perform maximal alignment over the full length of the sequences being compared. The sequence identity is then calculated relative to the longer sequence. That is, the overall sequence identity may be less than 100% even when the shorter sequence shows 100% sequence identity with a portion of the longer sequence.

  An “isolated” nucleic acid molecule is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule that normally accompanies the natural source of the nucleic acid. An isolated nucleic acid molecule is in a state other than the form or environment as found in nature. Thus, an isolated nucleic acid molecule is different from a nucleic acid molecule that is present in a cell that exists in nature. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the encoded polypeptide. In this case, for example, the nucleic acid molecule is at a chromosomal location different from that of cells existing in nature.

  A nucleic acid molecule encoding an “isolated” CRIg polypeptide has been identified and separated from at least one contaminating nucleic acid molecule normally associated with a natural source of nucleic acid encoding CRIg. A nucleic acid molecule. A nucleic acid molecule encoding an isolated CRIg polypeptide is in a state other than the form or environment found in nature. Thus, a nucleic acid molecule that encodes an isolated CRIg polypeptide is different from the encoding nucleic acid molecule (s) present in a cell that exists in nature. However, an isolated nucleic acid molecule encoding CRIg includes a nucleic acid molecule encoding CRIg contained in cells that normally express CRIg. In this case, for example, the nucleic acid molecule is at a chromosomal location different from that of cells existing in nature.

  The term “complement-related disease” is used herein in the broadest sense, and its onset is associated with abnormal activation of the complement system, eg, complement deficiency. All diseases and pathological symptoms that are present are included. The term specifically includes diseases and pathological conditions where inhibition of C3 convertase is effective. The term further includes diseases and pathological conditions in which inhibition of the alternative complement pathway (including selective inhibition) is efficacious. Complement-related diseases include inflammatory and autoimmune diseases such as rheumatoid arthritis (RA), acute respiratory distress syndrome (ARDS), remote tissue injury after ischemia reperfusion, cardiopulmonary bypass surgery Complement activation, dermatomyositis, pemphigus, lupus nephritis and resulting glomerulonephritis and vasculitis, cardiopulmonary bypass surgery, coronary artery endothelial dysfunction induced by heart failure, type II membranoproliferative thread Globe nephritis, IgA nephropathy, acute renal failure, cryoglobulinemia, antiphospholipid syndrome, degenerative diseases of the skin and other ocular symptoms related to complement, such as age-related macular degeneration (AMD), Chronic choroidal neovascularization (CNV), uveitis, diabetic retinopathy and other ischemia-related retinopathy, endophthalmitis, and other intraocular neovascular disorders such as diabetic macular edema, pathological Near , Von Hippel-Lindau disease, ocular histoplasmosis, central retinal vein occlusion (CRVO), corneal neovascularization, retinal neovascularization, and allograft, hyperacute rejection, hemodialysis, chronic obstructive pulmonary disease (COPD) , Asthma, and aspiration pneumonia, but are not limited to these.

  The term “complement-related ocular condition” is used herein in its broadest sense, and includes the classical and alternative complement pathways in its development, in particular alternatives All ocular conditions and diseases that involve complement of the complement pathway are included. Specifically, this group includes all ophthalmic conditions and diseases associated with the alternative complement pathway whose development, development, or progression can be controlled by inhibition of the alternative complement pathway. . Ocular symptoms involving complement include degenerative diseases of the eye, such as all stages of age-related macular degeneration (AMD) (including dry and wet (non-exuded and exuded forms)), chronic choroid Neovascularization (CNV), uveitis, diabetic retinopathy and other ischemia-related retinopathy, endophthalmitis, and other intraocular neovascular disorders such as diabetic macular edema, pathological myopia, Examples include, but are not limited to, von Hippel-Lindau disease, ocular histoplasmosis, central retinal vein occlusion (CRVO), corneal neovascularization, and retinal neovascularization. Preferred groups of ocular conditions involving complement include age-related macular degeneration (AMD) (including wet (wet) AMD and non-wet (dry or atrophic) AMD), chronic choroidal neovascularization (CNV), diabetic retinopathy (DR), and endophthalmitis.

  The terms “inflammatory disease” and “inflammatory disorder” are used interchangeably and a component of the mammal's immune system causes, mediates, or otherwise causes an inflammatory response that contributes to the morbidity of the mammal. Means a disease or disorder related in this way. Also included are diseases that have a palliative effect on the progression of the disease by reducing the inflammatory response. The term includes immune inflammatory diseases, including autoimmune diseases.

  The term “T cell mediated” disease refers to a disease in which T cells directly or indirectly mediate morbidity in mammals, or where T cells are otherwise related. Diseases mediated by T cells may be related to cell mediated effects, lymphokine mediated effects, etc., and B cells are stimulated by, for example, lymphokines secreted by T cells. May be related to effects related to B cells.

  Examples of immune and inflammatory diseases, some of which are mediated by T cells, include inflammatory bowel disease (BD), systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathy, systemic Systemic sclerosis (scleroderma), idiopathic inflammatory myopathy (dermatomyositis, polymyositis), Sjogren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, seizures) Nocturnal hemoglobinuria), autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura, immune thrombocytopenia), thyroiditis (Graves disease, Hashimoto's disease thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroid) Inflammation), diabetes mellitus, immune kidney disease (glomerulonephritis, tubulointerstitial nephritis), demyelinating diseases of the central and peripheral nervous system (eg, multiple sclerosis, idiopathic multiple neuropathy), Hepatobiliary Disease (eg, infectious hepatitis (A, B, C, D, hepatitis E, and other non-liver tropic viruses)), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, And sclerosing cholangitis, inflammatory and fibrotic lung disease (eg cystic fibrosis), gluten-sensitive bowel disease, Whipple disease, autoimmune or immune skin disease (vesicular skin disease, erythema multiforme, And contact dermatitis), psoriasis, pulmonary allergic diseases (eg eosinophilic pneumonia, idiopathic pulmonary fibrosis, and hypersensitivity pneumonia), transplant-related diseases (transplant rejection and graft-versus-host disease) ), Alzheimer's disease, and atherosclerosis.

  “Tumor” as used herein refers to all neoplastic cells, whether malignant or benign, and whether all are precancerous cells or tissues. Means growth and proliferation.

  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, liver cancer, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, liver cancer, female vulva cancer, thyroid cancer, liver cancer, and various Types of head and neck cancer.

  “Treatment” is an intervention performed to prevent the onset of a disorder or to change the pathology of a disorder. Thus, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those that require treatment include those already with the disorder as well as those who wish to prevent the disorder. In the treatment of diseases related to immunity, the therapeutic agent may directly alter the magnitude of the response of the components of the immune response, and may also cause other therapeutic agents (eg, antibiotics, antifungals, In some cases, the sensitivity of the disease may be further enhanced for treatment with inflammatory drugs, chemotherapeutic drugs, and the like. In the treatment of diseases involving complement, the treatment may, for example, prevent disease progression or slow disease progression. Therefore, the treatment of ocular symptoms involving complement is specifically more advanced in preventing, inhibiting, or delaying the onset of symptoms, or from one stage of symptoms to another. Includes prevention, inhibition, or delay of progression to a stage or to a more severely related symptom.

  A “condition” of a disease (eg, a disease involving complement) includes all phenomena that endanger a patient's satisfactory life. This includes abnormal or uncontrollable cell proliferation (neutrophils, eosinophils, monocytes, lymphocyte cells), antibody production, autoantibody production, complement production, normality of neighboring cells Interference with function, abnormal level release of cytokines or other secreted products, suppression or aggravation of any inflammatory or immunological response, inflammatory cells (neutrophils, eosinophils, monocytes) into the cell space Lymphocytes) infiltration, drusen formation, blindness, and the like.

  The term “mammal”, as used herein, means any animal classified as a mammal. This includes humans, non-human primates, livestock animals, and zoo animals, sport animals, or pet animals such as horses, pigs, cows, dogs, cats, and ferrets. There is no limitation. In preferred embodiments of the present invention, the mammal is a human or non-human primate, most preferably a human.

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

  The term “cytokine” is a general term for proteins released by one cell population that act on another cell as an intercellular mediator. Examples of such cytokines are lymphokines, monokines, and conventional polypeptide hormones. Among cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, glycoprotein hormones such as follicle stimulation Hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH), liver growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-α and tumor necrosis factor-β, Muellerian tube inhibition Substance, mouse gonadotropin related peptide, inhibin, activin, vascular endothelial growth factor, integrin, thrombopoietin (TPO), nerve growth factor (eg NGF-β platelet growth factor), transforming growth factor (TGF) (eg , TGF-α and TGF-β), insulin-like growth factor-I and insulin-like growth factor-II, erythropoietin (EPO), osteoinductive factors, interferons (eg, interferon-α, interferon-β and interferon-γ) Colony stimulating factor (CSF) (eg, macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF)), interleukin (IL) (eg IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12), Tumor necrosis factor (eg, TNF-α or TNF-β) and other polypeptides including LIF and kit ligand (KL) It includes factors. As used herein, the term cytokine refers to a biological activity of a protein from a natural source, or from a recombinant cell culture, as well as a naturally occurring sequence of cytokines. Equivalents are included.

  A “therapeutically effective amount” is necessary to obtain a measurable improvement in a condition, eg, a disease state or symptom of a target disease (eg, a disease or symptom that is associated with complement, or cancer). The amount of active CRIg, CRIg agonist and antagonist.

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

  A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, a presequence or secretory leader DNA is operably linked to a polypeptide DNA if it is expressed as a 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 the sequence. Alternatively, a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. In general, “operably linked” means that the linked DNA sequences are contiguous and, in the case of a secretory leader, are contiguous and within the reading frame. Means that. However, enhancers are not necessarily continuous. Ligation is performed by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to conventional practice.

  The “stringency” of a hybridization reaction can be readily determined by one skilled in the art and is usually calculated empirically as a function of probe length, wash temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, and shorter probes require lower temperatures. Hybridization typically takes advantage of the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting point. 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, of course, the higher the relative temperature, the more likely the reaction conditions tend to be more stringent, while the lower the temperature, the less such a tendency. For further 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 used herein, can be identified as follows: (1) Cleaning involves low ionic strength and high temperature. For example, at 50 ° C., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate; (2) a denaturant (eg, formamide) during hybridization. Used, for example, at 42 ° C., containing 750 mM sodium chloride, 75 mM sodium citrate, 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM phosphoric acid 50% (v / v) formamide containing sodium buffer (pH 6.5); or (3) 50% ethanol at 42 ° C. Muamide, 5 × SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 × Denhardt's solution, sonicated Use salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate, 0.2 × SSC (sodium chloride / sodium citrate) at 42 ° C., and 50 ° C. at 55 ° C. Wash with% formamide followed by high stringency at 55 ° C. with 0.1 × SSC containing EDTA.

  “Moderate stringency conditions” can be identified as described in Sambrook et al., Molecular Cloning A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, which is described above. Use of wash solutions and hybridization conditions (eg, temperature, ionic strength, and% SDS) that are less stringent than the others. An example of moderately stringent conditions is 20% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% Incubation at 37 ° C. overnight in a solution containing dextran sulfate and 20 mg / mL denatured sheared salmon sperm DNA, followed by washing the filter at about 37-50 ° C. with 1 × SSC. Those skilled in the art will recognize methods for adjusting temperature, ionic strength, etc. as needed to accommodate factors such as probe length.

  The term “epitope tagged” as used herein means a chimeric polypeptide comprising a polypeptide of the invention fused to a “tag polypeptide”. The tag polypeptide is sufficient residues to provide an epitope against which antibodies can be generated, which is still short enough so as not to interfere with the activity of the fused polypeptide. . The tag polypeptide is preferably highly specific so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least 6 amino acid residues, usually between about 8 and 50 amino acid residues (preferably about 10 to 20 amino acids). Between amino acid residues).

  “Active” or “activity” retains the biological and / or immunological activity of a polypeptide of the invention, natural or naturally occurring, for a variant of the CRIg polypeptide of the invention. Means the form (s) of such polypeptides. A preferred biological activity is the ability to bind to C3b and / or affect complement or complement activation, specifically inhibiting alternative complement pathways and / or C3 convertase Is ability. Inhibition of C3 convertase can be measured, for example, by measuring inhibition of C3 turnover in normal serum during collagen-induced or antibody-induced arthritis, or inhibition of C3 accumulation is It is an arthritic joint.

  “Biological activity” refers to the ability to bind C3b for an antibody, polypeptide, or another molecule that mimics the biological activity of CRIg and can be identified by the screening assays disclosed herein. And / or part of the ability of such molecules to affect complement or complement activation, specifically such molecules that inhibit alternative complement pathways and / or C3 convertase Means ability.

  The term CRIg “agonist” is used in the broadest sense and includes a qualitative biological activity of a CRIg polypeptide of a naturally occurring sequence (as defined herein above). Any molecule that mimics is included. This CRIg agonist specifically includes CRIg-Ig (eg, CRIg-Fc fusion polypeptide (immunoadhesive)), but also includes small molecules that mimic the biological activity of at least one CRIg. . Preferably, the biological activity is a block of the complement pathway (particularly the alternative complement pathway).

  The term “antagonist” is used in the broadest sense and includes, in part, a qualitative biological activity of a naturally occurring polypeptide (eg, a CRIg polypeptide of a naturally occurring sequence). Or any molecule that completely blocks, inhibits or neutralizes.

  Suitable agonist or antagonist molecules specifically include the naturally occurring polypeptides, peptides, small molecule (including small organic molecules) agonist antibodies or agonist antibodies, or antibody fragments, fragments, Fusions, amino acid sequence variants and the like are included.

  “Small molecule” is defined herein as having a molecular weight of less than about 600 Daltons, preferably less than about 1000 Daltons.

  The term “antibody” is used in the broadest sense and specifically includes one anti-CRIg monoclonal antibody (including agonists, antagonists, and neutralizing antibodies), and anti-CRIg having multi-epitope specificity. Antibody compositions are included, but are not limited to these. The term “monoclonal antibody” as used herein refers to a substantially homogeneous population of antibodies (ie, the individual antibodies that are included in the population can be present in nature in minor amounts). Is identical except for the possibility of mutations).

  “Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity for a particular antigen, immunoglobulins include both antibodies and antibody-like molecules that lack antigen specificity. The latter type of polypeptide is produced, for example, at low levels by the lymphatic system and at high levels by myeloma. The term “antibody” is used in the broadest sense and specifically includes complete monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two complete antibodies (eg, bispecific antibodies). , And antibody fragments include, but are not limited to, where they exhibit the desired biological activity.

“Naturally occurring antibodies” and “naturally occurring immunoglobulins” usually consist of two identical light (L) chains and two identical heavy (H) chains, about 150 It is a heterotetrameric glycoprotein of 1,000 daltons. Each light chain is linked to one heavy chain by one disulfide covalent bond, but the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable domain (V _H ) followed by a number of constant domains. Each light chain has a variable domain (V _L ) at one end and a constant domain at the other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Certain amino acid residues are thought to form the interface between the light chain variable domain and the heavy chain variable domain.

  The term “variable” refers to the fact that a particular portion of a variable domain varies widely in sequence between antibodies and is used for the binding and specificity of an individual particular antibody to that particular antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions, both present in the light chain variable domain and the heavy chain variable domain. The more highly conserved portions of variable domains are called the framework (FR). Each naturally occurring heavy and light chain variable domain has three CDRs (which are predominantly β-sheet structures. These form a loop bond, and some are β-sheet structures. 4 FR regions joined together by the same). The CDRs of each chain are kept in close proximity to each other by the FR region, and together with CDRs derived from other chains, contribute to the formation of the antigen binding site of the antibody (Kabat et al., NIH Public. 91-3342, Vol.I, pages 647-669 (1991)). Constant domains are not directly involved in antibody binding to antigen, but exhibit a variety of effector functions (eg, antibody involvement in antibody-dependent cytotoxicity).

“Antibody fragments” include a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab ′, F (ab ′) ₂ , and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8 (10): 1057-1062 (1995)); Chain antibody molecules; as well as multispecific antibodies formed from antibody fragments. Specifically, examples of antibody fragments included within the definition of the present invention include: (i) Fab fragments having VL, CL, VH, and CH1 domains; (ii) C of the CH1 domain A Fab ′ fragment that is a Fab fragment having one or more cysteine residues at the terminal; (iii) an Fd fragment having VH and CH1 domains; (iv) a VH and CH1 domain, and a CH1 domain An Fd ′ fragment having one or more cysteine residues at the C-terminus; (v) an Fv fragment having the VL and VH domains of one arm of the antibody; (vi) a dAb consisting of the VH domain Fragment (Ward et al., Nature 341, 544-546 (1989)); (vii) isolated CDR region; (viii) F (ab ′) 2 fragment, disulfide at the hinge region A bivalent fragment comprising two Fab ′ fragments linked by a linking bond; (ix) a single chain antibody molecule (eg, single chain Fv; scFv) (Bird et al., Science 242: 423-426 (1988); Huston et al., PNAS (USA) 85: 5879-5883 (1988)); (x) heavy chain variable having two antigen binding sites and linked to a light chain variable domain (VL) in the same polypeptide chain. “Diabodies” comprising a domain (VH) (eg, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993)); (xi) tandem "Linear antibody" (VH-CH1-VH-CH1) containing a pair of Fd segments (this is complementary A form a pair of antigen binding regions with the light chain polypeptide) (Zapata et al, Protein Eng.8 (10): 1057-1062 (1995); and U.S. Pat. No. 5,641,870).

Digestion of the antibody with papain results in two identical antigen-binding fragments called “Fab” fragments (each having one antigen-binding site) and the remaining “Fc” fragments. The name “Fc” reflects the ability to crystallize easily. Treatment with pepsin yields an F (ab ′) ₂ fragment that has two antigen binding sites and is capable of cross-linking antigen.

“Fv” is the minimum antibody fragment which contains a complete antigen recognition and binding site. This region is composed of a dimer in which one heavy chain variable domain and one light chain variable domain are tightly and non-covalently associated. In this conformation, the three CDRs of individual variable domains interact to delineate the antigen binding site on the surface of the V _H -V _L dimer. In summary, the six CDRs confer antigen binding specificity to the antibody. However, one variable domain (or half of an Fv that contains only three CDRs specific to the antigen) still has the ability to recognize and bind antigens, although with lower affinity than the complete binding site. Yes.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab ′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines derived from the antibody hinge region. Fab′-SH is the name herein for Fab ′ in which the cysteine residue (s) of the constant domain have a free thiol group. F (ab ′) ₂ antibody fragments were originally produced as pairs of Fab ′ fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

  The “light chains” of antibodies (immunoglobulins) from any vertebrate species are based on the amino acid sequence of their constant domains and are of two distinct types (called kappa (κ) and lambda (λ)). It can be divided into one.

  Based on the amino acid sequences of their heavy chain constant domains, immunoglobulins can be divided into various classes. There are five major classes for immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Some of these can be further divided into subclasses (isoforms) (eg, IgG1, IgG2, IgG3, IgG4, IgA, and IgA2). The heavy chain constant domains that correspond to the different classes of immunoglobulins are called γ, μ, δ, α, and ε, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known.

  The term “monoclonal antibody” as used herein refers to a substantially homogeneous population of antibodies (ie, the individual antibodies that are included in the population can be present in nature in minor amounts). Is identical except for the possibility of mutations). Monoclonal antibodies are highly specific and are directed against one antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations (which usually include various antibodies directed against various determinants (epitopes)), each monoclonal antibody is one of those in the antigen. Oriented to one determinant. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma cultures that are not contaminated with other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a population of substantially homogeneous 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 can be made by the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), and can be produced by recombinant DNA methods (eg, US Pat. No. 4,816,567). “Monoclonal antibodies” are also described, for example, in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al. Mol. Biol. 222: 581-597 (1991) can also be used to isolate from a phage antibody library. See also U.S. Pat. Nos. 5,750,373, 5,571,698, 5,403,484, and 5,223,409. These describe the preparation of antibodies using phagemids and phage vectors.

  Monoclonal antibodies herein specifically include heavy and / or light chain portions corresponding to sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass. Antibodies that are the same or homologous, but the remainder of the chain (s) is from another species or belong to another antibody class or subclass, as well as the corresponding in fragments of such antibodies “Chimeric” antibodies (immunoglobulins) that are the same as or homologous to the sequence are included as long as they exhibit the desired biological activity (US Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. (USA), 81: 6851-6855 (1984)).

“Humanized” forms of non-human (eg, murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof that contain minimal sequence derived from non-human immunoglobulin (eg, Fv, Fab, Fab ′, F (ab ′) ₂ , or other antigen binding subsequence of the antibody). For the most part, humanized antibodies are human immunoglobulins (recipient antibodies), and some or all of the residues from the recipient's complementarity determining regions (CDRs) have the desired specificity, affinity, and It has been replaced with residues from the CDRs of a competent non-human species (donor antibody) (eg mouse, rat or rabbit). In some instances, certain Fv framework region (FR) residues of the human immunoglobulin can also be replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance. Generally, a humanized antibody will comprise substantially the entire at least one, usually two variable domains. In this case, all or substantially all of the CDR region corresponds to that of a non-human immunoglobulin, and all or substantially all of the FR region is of a human immunoglobulin sequence. A humanized antibody preferably also includes at least a portion of an immunoglobulin constant region (Fc), and typically includes at least a portion of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525 (1986); Reichmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992). Humanized antibodies include “primatized” antibodies, where the antigen-binding region of the body is derived from antibodies produced by immunizing macaque monkeys with the antigen of interest. Antibodies that include residues from Old World monkeys are also probably included in the present invention. See, for example, US Pat. Nos. 5,658,570; 5,693,780; 5,681,722; 5,750,105; and 5,756,096. That.

“Single-chain Fv” or “sFv” antibody fragments comprise the V _H and V _L domains of antibody. These domains are present in one polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the _VH and _VL domains that allows the sFv to form the desired structure for antigen binding. For an overview of sFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, edited by Rosenburg and Moore. , Springer-Verlag, New York, pages 269-315 (1994).

The term “diabody” refers to a small antibody fragment having two antigen binding sites. This fragment includes a heavy chain variable domain (V _H ) linked to a light chain variable domain (V _L ) in the same polypeptide chain (V _H -V _L ). By using linkers that are too short to allow pairing between two domains on the same strand, these domains are directed to pair with the complementary domains of another strand, Two antigen binding sites are generated. Diabodies are described, for example, in EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

  The “CH2 domain” (also referred to as “Cg2” domain) of a human IgG Fc region typically extends from about amino acid residue about position 231 to about 340 amino acid residue. The CH2 domain is unique in that it does not pair closely with another domain. Rather, two N-linked branched carbohydrate chains are placed between the two CH2 domains of a completely natural IgG molecule. Carbohydrates can provide an alternative to domain-domain pairings and have been speculated to help stabilize the CH2 domain. Burton, Molec. Immunol. 22: 161-206 (1985). The CH2 domain herein may be a CH2 domain or a variant CH2 domain of a naturally occurring sequence.

  “CH3 domain” includes a stretch of residues C-terminal to the CH2 domain in the Fc region (ie, from about amino acid residue 341 to about 447 amino acid residue of IgG). Where the CH3 region herein is a CH3 domain of a naturally occurring sequence, a variant CH3 domain (for example, “protroberrance” is introduced in one chain thereof). And a CH3 domain in which the other chain has a corresponding “cavity” introduced; see US Pat. No. 5,821,333, incorporated herein by reference) Can be. Such variant CH3 domains can be used to make multispecific (eg, bispecific) antibodies as described herein.

  A “hinge region” is generally defined as extending from about Glu216 or about Cys226 of human IgG1 to about Pro230 (Burton, Molec. Immunol. 22: 161-206 (1985)). The hinge region of the other IgG isoform is aligned with the IgG1 sequence by determining the position of the first and last cysteine residues that form an intra-heavy chain SS bond at the same position Can do. The hinge region herein may be a naturally occurring sequence hinge region or a variant hinge region. The two polypeptide chains of the variant hinge region generally retain at least one cysteine residue for one polypeptide chain, so that the two polypeptide chains of the variant hinge region are 2 Disulfide bonds can be formed between the two chains. A preferred hinge region herein is a human hinge region of a naturally occurring sequence, for example, a human IgG1 hinge region of a naturally occurring sequence.

  A “functional Fc region” has at least one “effector function” of an Fc region of a naturally occurring sequence. Exemplary “effector functions” include C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody dependent cellular mediated cytotoxicity (ADCC); phagocytosis; For example, down regulation of B cell receptor (BCR) and the like can be mentioned. Such effector functions usually require an Fc region that binds to the binding domain (eg, the variable domain of an antibody), and various known in the art for assessing the effector function of such antibodies. Can be assessed using these assays.

  The “naturally occurring sequence Fc region” includes the same amino acid sequence as the amino acid sequence of the Fc region found in nature.

  A “variant Fc region” includes an amino acid sequence that differs from the amino acid sequence of an Fc region of a naturally occurring sequence due to modification of at least one amino acid. Preferably, the variant Fc region comprises at least one amino acid substitution relative to the Fc region of the naturally occurring sequence or the Fc region of the original polypeptide (eg, Fc of the naturally occurring sequence). From about 1 to about 10 amino acid substitutions, and preferably from about 1 to about 5 amino acid substitutions) in the region or in the Fc region of the original polypeptide. A variant Fc region herein is typically, for example, at least about 80% sequence identity with the Fc region of a naturally occurring sequence and / or the Fc region of the original polypeptide, or with them. Have at least about 90% sequence identity, or at least about 95% or more sequence identity therewith.

  An “affinity matured” antibody has one or more of its CDRs that result in an improvement in the affinity of the antibody for the antigen compared to the original antibody that does not have those change (s). An antibody having one or more changes. Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Antibodies with mature affinity are produced by procedures known in the art. Marks et al., Bio / Technology 10: 779-783 (1992) describes affinity maturation by shuffling VH and VL domains. Random mutagenesis of CDR and / or framework residues is described by Barbes et al., Proc. Natl. Acad. Sci. USA, 91: 3809-3813 (1994); Schier et al., Gene 169: 147-155 (1995); Yelton et al., J. Biol. Immunol. 155: 1994-2004 (1995); Jackson et al., J Immunol. 154 (7): 3310-9 (1995); and Hawkins et al., J. Biol. Mol. Biol. 226: 889-896 (1992).

  A “flexible linker” as used herein is linked by peptide bond (s) and provides additional rotational freedom for two linked polypeptides (eg, two Fd regions). Means a peptide comprising two or more amino acid residues. Such rotational degrees of freedom allow two or more antigen binding sites to be linked by a flexible linker, each allowing more efficient access to the target antigen (s). Examples of suitable flexible linker peptide sequences include gly-ser, gly-ser-gly-ser, ala-ser, and gly-gly-gly-ser.

“Single-chain Fc” or “sFv” antibody fragments comprise the V _H and V _L domains of antibody. These domains are present in one polypeptide chain. In general, it includes a polypeptide linker between the further V _H and V _L domains in the Fv polypeptide, thereby, possible that the sFv to form the desired structure for antigen binding Become. For an overview of sFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, edited by Rosenburg and Moore. , Springer-Verlag, New York, pages 269-315 (1994).

  An “isolated” polypeptide (eg, an antibody) is one that has been identified, separated, and / or recovered from a component of its natural environment. Its natural environmental contaminants are substances that interfere with the diagnostic or therapeutic use of antibodies, including enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Can be. In a preferred embodiment, the polypeptide (including antibodies) is (1) more than 95% by weight antibody, most preferably more than 99% by weight antibody as determined by the Lowry method, (2) To the extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) Coomassie blue staining, or preferably silver staining Used to purify until homogenous by SDS-PAGE under reducing or non-reducing conditions. Isolated compound (eg, antibody or other polypeptide) includes a compound in situ in recombinant cells. This is because there is no at least one component of the compound's natural environment. Ordinarily, however, isolated compound will be prepared by at least one purification step.

  The term “label”, as used herein, is a detectable that binds directly or indirectly to a compound (eg, an antibody or polypeptide), resulting in a “labeled” compound. A compound or composition is meant. The label may be detectable per se (eg, a radioisotope label or fluorescent label), or in the case of an enzyme label, may catalyze a chemical change in the detectable substrate compound or composition. .

  By “solid phase” is meant a non-aqueous matrix to which a compound of the invention can be attached. Examples of solid phases include, herein, parts from glass (eg, glass with controlled pores), polysaccharides (eg, agarose), polyacrylamide, polystyrene, polyvinyl alcohol, and silicon. Or the whole is formed. In certain embodiments, depending on the circumstances, the solid phase can include the wells of an assay plate. In other embodiments, the solid phase is a purification column (eg, an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in US Pat. No. 4,275,149.

  “Liposomes” are small vesicles composed of various types of lipids, phospholipids, and / or surfactants, which are drugs to mammals (eg, anti-ErbB2 antibodies disclosed herein) , And optionally chemotherapeutic drugs). The components of liposomes are generally arranged in a bilayer form, similar to the lipid arrangement of biological membranes.

  The term “immunoadhesive”, as used herein, refers to an antibody-like molecule that combines the binding specificity of a heterologous protein (“adhesive”) with the effector function of an immunoglobulin constant domain. ing. Structurally, immunoadhesins include an amino acid sequence having a desired binding specificity other than the antigen recognition and binding site of an antibody (ie, it is “heterologous”) and an immunoglobulin constant domain. A fusion with a sequence is included. The adhesion part of an immunoadhesive molecule is usually a contiguous amino acid sequence comprising at least a receptor or ligand binding site. The immunoglobulin constant domain sequence of the immunoadhesive can be any immunoglobulin (eg, IgG-1, IgG-2, IgG-3, or IgG-4 subtype, IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM).

  An “antigenic factor or antigenic substance” is a growth factor that stimulates blood vessel development (eg, promotes angiogenesis, endothelial cell proliferation, vascular stability, and / or angiogenesis, etc.). For example, examples of the angiogenic factor include VEGF and a member of the VEGF family, PIGF, PDGF family, fibroblast growth factor family (FGF), TIE ligand (angiopoietin), ephrin, ANGPTL3, and ANGPTL4. However, it is not limited to these. This includes factors that promote healing, such as growth hormone, insulin-like growth factor-I (IGF-I), VIGF, epidermal growth factor (EGF), members of CTGF and its family, and TGF-α and TGF- β is also included. For example, Klagsbrun and D'Amore, Annu. Rev. Physiol. 53: 217-39 (1991); Strite and Detmar, Oncogene, 22: 3172-3179 (2003); Ferrara & Alitaro, Nature Medicine 5 (12): 1359-1364 (1999); Tonini et al., Oncogene, 22: 6549-6556 (2003) (eg, Table 1 listing angiogenic factors); and Sato Int. , J .; Clin. Oncol. 8: 200-206 (2003).

  An “anti-angiogenic agent” or “angiogenesis inhibitor” is a low molecular weight substance, polynucleotide, polypeptide, isolated that directly or indirectly inhibits angiogenesis, angiogenesis, or unwanted vascular permeability. Protein, recombinant protein, antibody, or a conjugate or fusion protein thereof. For example, an anti-angiogenic agent is an antibody or other antagonist to an angiogenic agent as defined above, eg, an antibody to VEGF, an antibody to VEGF receptor, a small molecule that blocks signal transduction by VEGF receptor (eg, , PTK787 / ZK2284, SU6668). Anti-angiogenic substances also include naturally occurring angiogenesis inhibitors (eg, angiostatin, endostatin, etc.). For example, Klagsbrun and D'Amore, Annu. Rev. Physiol. 53: 217-39 (1991); Street and Detmar, Oncogene, 22: 3172-3179 (2003).

  The term “effective amount” means an amount of an agent effective to treat (including prevent) a mammalian disease or disorder. Therefore, in the case of age-related macular degeneration (AMD) or chronic choroidal neovascularization (CNV), blinding can be reduced or prevented with an effective amount of drug. For AMD treatment, in vivo efficacy can be measured, for example, by one or more of the following: assessing the mean change in best corrected visual acuity (BCVA) from baseline to the desired time, baseline Assessing the percentage of subjects who lost less than 15 characters in visual acuity at a desired time compared to the percentage of subjects who acquired 15 characters or more in visual acuity at a desired time compared to baseline Assessing the proportion of subjects having a visual-acoustic Snellen equivalent of 20/2000 or worse at the desired time point, assessing NEI Visual Functioning Questionnaire, fluorescent fundus vessels CNV magnitude and CNV at the desired time point as assessed by contrast enhancement To evaluate the leakage amount. If the indication is progression from dry AMD to wet AMD, or prevention of progression from AMD to CMV, an effective amount of the drug inhibits, delays, or partially or completely blocks such progression be able to. In this case, determining an effective amount includes staging the disease, monitoring the progression of the disease over time, and adjusting the dosage necessary to obtain the desired result.

(II. Detailed description)
The present invention relates to the use of a novel macrophage-related receptor having homology to the A33 antigen and JAM1. It is cloned from a fetal lung library and is a single transmembrane Ig superfamily member associated macrophage associated (STigMA) polypeptide, or complement receptor of the immunoglobulin family (Complement Receptor of the Immunoglobulin family) (CRIg) polypeptide. Naturally occurring human CRIg is expressed as two types of spliced variants, one containing an N-terminal IgV-like domain and a C-terminal IgC2-like domain, the spliced form being a C-terminal domain Are deleted (SEQ ID NOs: 4 and 6, respectively). Both receptors have one transmembrane domain and a cytoplasmic domain, which contains tyrosine residues that are constitutively phosphorylated in macrophages in vitro. The mouse homologue was found to have 67% sequence homology to human CRIg (SEQ ID NO: 8). The full-length human CRIg polypeptide also has a short version in which the N-terminal segment is missing (SEQ ID NO: 2).

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

  In addition, CRIg is highly expressed in Kupffer cells, binds to C3b and iC3b opsonins, and has been shown to be required for rapid clearance of circulating pathogens. Structurally, CRIg lacks a short consensus repeat of bound C3b- and C4b binding in CR1 and CR2 and an integrin-line domain present in C3 and CR4. In that it differs from known complement receptors. Complement receptors CR1-4 are expressed in a wide variety of cell types, but CRIg expression is limited to tissue resident macrophages including liver Kupffer cells.

  Depletion experiments have established the role of Kupffer cells in the rapid C3-dependent clearance of Listeria during the early stages of infection (Kaufmann, Annu Rev. Immunol. 11: 129-163 (1993); Gregory et al. J. Immunol.168: 308-315 (2002)), but the receptors involved in this process have not been identified so far. The experiments shown in the examples below demonstrate that CRIg expressed by macrophages binds to C3b and iC3b accumulated on the surface of pathogens. Due to this double bond activity against C3b and iC3b, CRIg is required for efficient clearance of Listeria monocytogenes (LM) opsonized with both C3 degradation components.

  The importance of CRIg in the rapid hepatic clearance of C3 opsonized particles is further supported by the failure of CRIg knockout (ko) mice to efficiently clear C3-opsonized LM from the circulation. This results in increased pathogen load and increased mortality in various organs. In the absence of C3, CRIg ko wild type (wt) mice are equally well cleared of Listeria, indicating a dependence of CRIg function on the presence of C3.

  The role of complement receptors C1-4 in the clearance of LM by liver Kupffer cells has not been well established. CR1 and CR2 are not present in tissue-resident macrophages and are predominantly expressed on follicular dendritic cells and B cells, where they play a role in regulating T and B cell responses (Krych-Goldber and Atkinson, Immunol. Rev. 180: 112-122 (2001); Molina et al., J. Exp. Med. 175: 121-129 (1992), and Examples). Although CR3 is expressed at low levels in KC, the ko mouse that lacks the CD18 β chain common to both CR3 and CR4, resulting in a non-functional receptor, It has been shown not to be susceptible to infection but to be difficult to infect (Wu et al., Infect. Immun. 71: 5986-5993 (2003)). Thus, CRIg represents a major component of the reticuloendothelial phagocytic system in the rapid clearance of C3-opsonized particles.

  In addition to its expression in liver Kupffer cells, CRIg is present in macrophage subpopulations in various tissues, including peritoneum, heart, lung, adrenal gland, and intestine. These macrophages are known to play a central role in the phagocytosis of dead cells and cell debris (Almeida et al., Ann. NY 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)). The expression of CRIg in these resident macrophages can mediate the complement-dependent opsonophagocytosis of various particles. This is supported by the finding that CRIg ko mice show a decrease in LM in their heart and liver tissues despite an increase in circulating ML loading. Thus, CRIg presents a novel receptor expressed in tissue macrophages, which plays a role as a keep portal for the rapid clearance of pathogens opsonized with complement.

  The results shown in the examples below show that CRIg is expressed in the intracellular pool of recycled vesicles, thereby providing a sustained supply of CRIg at the cell surface for binding to C3 opsonized particles. It further shows that is sure. In addition, endosomes expressing CRIg are rapidly recruited to the contact site of the particles, which can help induce the membrane to form phagosomes. The importance of CRIg in the phagocytosis of C3 opsonized particles is that KC lacking CRIg cannot bind to C3b and iC3b, thereby causing C3 opsonized Listeria monocytogenes (LM). Is shown by the occurrence of reduced phagocytosis (see Examples).

  Intracellular localization and intracellular transport of CRIg is different from known complement C3 receptors. CRIg is localized to structurally recycled endosomes, whereas CR1, CR3, and CR4 are present in secretory vesicles, which bind to plasma membranes when cells are stimulated with cytokines (Sengelov et al., J. Immunol. 153: 804-810 (1994); and Sengelov et al., Crit. Rev. Immunol. 15: 107-131 (1995)), and only after receptor crosslinking, The ligand is internalized by the process (Carpentier et al., Cell Regul. 2: 41-55 (1991); Brown et al., Curr. Opin. Immunol. 3: 76-82 (1991)). As a consequence, CRIg expression on the cell surface is down-regulated after cell stimulation, while CR1 and CR3 expression on the cell surface increases after stimulation. This increase plays an important step in binding and phagocytosis, and like CRIg, CR3 collects peripheral C3 opsonized particles in phagocytic cups and phagosomes (Aderem and Underhill, Annu. Rev. Immunol. 17). : 593-623 (1999)). Structural reuse and endocytosis of ligands by CRIg in resting macrophages plays a role in the binding of complement opsonized particles during the early stages of bacterial infection prior to the inflammatory response (eg, activation Phagocytic mobilization), as well as a role in the binding of complement opsonized particles during removal of the particles from the circulation under non-inflammatory conditions.

  Complement plays an important role in defense of the body and, together with other components of the immune system, in protecting individuals from pathogen invasion into the body. However, if not properly activated or controlled, complement can also cause damage to host tissues. Inappropriate activation of complement results in diseases or disorders in which complement is involved (eg, immune complex diseases and autoimmune diseases, as well as various inflammatory diseases (complement-mediated damage to inflammatory tissues). It is related to the onset of various diseases called)). Disease conditions involving complement vary, long-term or short-term complement activation, whole cascade activation, one of the cascades (classical complement pathway or alternative complement pathway) only Activation, activation of only some components of the cascade, and the like. In some diseases, the biological activity of complement of complement fragments results in tissue damage and disease. Thus, complement inhibitors have high therapeutic potential. Alternative inhibitors of alternative complement pathways are particularly useful. This is because the clearance of pathogens and other organisms from the blood by the classical complement pathway remains incomplete.

  It is known that C3b opsonizes the surface of microorganisms that invade the body by covalent bonds, acts as a ligand for a complement receptor present in phagocytic cells, and ultimately leads to phagocytosis of pathogens. In many pathological situations, such as those listed above, complement is the surface of cells including blood vessel walls, joint cartilage, liver glomeruli, and cells that lack native complement inhibitors. It is activated by. Complement activation can cause inflammation caused by the chemotactic properties of anaphylatoxins C3a and C5a, and the membrane attack complex can cause damage to autologous cells. Without being bound by any particular theory, by binding C3b, CRIg inhibits C3 convertase, thereby preventing or reducing complement-mediated diseases, examples of which are listed above. I think that.

(Compound of the present invention)
(1. CRIg polypeptide and variant CRIg polypeptide of natural sequence)
The preparation of naturally occurring CRIg molecules (with their nucleic acid and polypeptide sequences) has been discussed above. Example 1 shows the cloning of the full length huCRIg of SEQ ID NO: 4. CRIg polypeptides can be produced by culturing cells transformed or transfected with a vector containing a CRIg nucleic acid. Of course, it is anticipated that CRIg can be prepared using other methods well known in the art. For example, CRIg sequences or portions thereof can be produced by direct peptide synthesis using solid phase techniques (eg, Stewart et al., Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco, CA). (1969); Merrifield, J. Am. Chem. Soc., 85: 2149-2154 (1963)). In vitro protein synthesis can be performed using manual techniques or automatically. Automatic synthesis can be performed using, for example, Applied Biosystems Peptide Synthesizer (Foster City, CA) according to the manufacturer's instructions. Various portions of CRIg can be chemically synthesized separately and combined using chemical or enzymatic methods to form full length CRIg.

  CRIg variants can be prepared by introducing appropriate nucleotide changes into the DNA encoding the CRIg polypeptide of the naturally occurring sequence, or by synthesis of the desired CRIg polypeptide. Those skilled in the art will appreciate that amino acid changes can alter the post-translational process of CRIg, such as changing the number or position of glycosylation sites, or changing membrane binding properties. It will be.

  For example, variations in the CRIg polypeptides of the naturally occurring sequences described herein are for conservative and non-conservative mutations, eg, as shown in US Pat. No. 5,364,934. Can be created using any of the techniques and guidelines. A variation can be a substitution, deletion or insertion of one or more codons encoding a CRIg of a naturally occurring sequence or a variant CRIg, whereby a CRIg of the corresponding naturally occurring sequence. Or a change occurs in its amino acid sequence compared to the variant CRIg. In some cases, the variation is due to substitution of any other amino acid with at least one amino acid in one or more domains of a CRIg polypeptide of a naturally occurring sequence. Guidance in determining which amino acid residues can be inserted, substituted, or deleted without adversely affecting the desired activity is compared to the sequence of known protein molecules that are homologous by comparing the sequence of CRIg. It can be sought by minimizing the number of amino acid sequence changes made in highly likely regions.

  An amino acid substitution may be the result of the replacement of one amino acid with another amino acid having similar structural and / or chemical properties (eg, replacement of leucine with serine), ie, a conservative amino acid substitution. . Insertions or deletions can optionally range from 1 to 5 amino acids. Acceptable variations are made by systematically creating amino acid insertions, deletions, or substitutions in the sequence, and testing the resulting variants for activity in the in vitro assays described in the Examples below. Can be determined.

  Variations can be made using methods known in the art, such as oligonucleotide-mediated (site-specific) 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 selective mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317: 415 (1986)), or other known techniques can be performed on cloned DNA. To obtain CRIg variant DNA.

  Scan amino acid analysis can also be used to identify one or more amino acids that can be varied along a contiguous sequence. Among these, preferred scan amino acids are relatively small neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is usually the preferred scan amino acid in this group. This is because the side chain exceeding the β-carbon is excluded by this, and the three-dimensional structure of the main chain of the variant is unlikely to change. Alanine is also generally preferred. Because this is the most common amino acid. Furthermore, this is frequently seen in both buried and exposed positions (Creighton, The Proteins, (WH Freeman & Co., NY); Chothia, J. Mol. Biol., 150: 1 (1976)). If a sufficient amount of modification is not obtained by alanine substitution, isomeric amino acids can be used.

  It has been shown that removal or inactivation of all or part of the transmembrane and / or cytoplasmic region does not impair the biological activity of CRIg. Accordingly, CRIg variants in which the transmembrane region and / or cytoplasmic region are deleted / inactivated are specifically included within the scope herein. Similarly, the IgC2 region can be removed without compromising biological fluid activity, as evidenced by the presence of the short biologically active form of huCRIg and the mouse homologue.

  Covalent modifications of naturally occurring sequences of CRIg polypeptides and variant CRIg polypeptides are within the scope of the present invention. One type of covalent modification involves organic derivatization in which the targeted amino acid residues of CRIg can be reacted with selected side chains or N-terminal or C-terminal residues of the CRIg polypeptide. Reacting with the agent. Derivatization with bifunctional substances is useful, for example, for use in methods for purifying anti-CRIg antibodies, such as for cross-linking CRIg to a water-insoluble support matrix or surface. Commonly used crosslinking agents include, for example, 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinic acid ester, such as 4-azidosalicylic acid ester, homobifunctional Functional imide esters (including disuccinimidyl esters such as 3,3′-dithiobis (succinimidyl propionate)), bifunctional maleimides such as bis-N-maleimide-1,8-octane, and methyl-3- Examples include [(p-azidophenyl) dithio] propionic imide.

  Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, hydroxylation of proline and lysine, and phosphorylation of the hydroxyl group of seryl or threonyl residues, respectively. , Lysine, arginine, and histidine side chain α-amino group methylation (TE Creighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86) ), Acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

  Another type of covalent modification of the CRIg polypeptide within the scope of the invention includes alteration of the polypeptide's natural glycosylation pattern. “Modification of the glycosylation pattern in nature” for the purposes herein is the deletion of one or more carbohydrate moieties found in the CRIg of the naturally occurring sequence and / or Adding one or more glycosylation sites that are not present in the CRIg of the sequence present in and / or changing the ratio and / or composition of the sugar residues attached to the glycosylation site (s) Intended to mean letting. A predicted natural glycosylation site for mouse CRIg is found in the sequence NGTG at position 170.

  Glycosylation of polypeptides is usually 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. Tripeptide sequences (asparagine-X-serine and asparagine-X-threonine, where X is an amino acid other than proline) are recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. O-linked glycosylation refers to the linkage of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxy amino acid (most commonly serine or threonine), but 5-hydroxyproline or 5-hydroxy Lysine may also be included in O-linked glycosylation. The naturally occurring sequence CRIg has only a small amount of N-glycosylation. Addition of glycosylation sites to CRIg polypeptides can be accomplished by changing the amino acid sequence. This change can be caused, for example, by the addition of one or more serine or threonine residues to the CRIg of a naturally occurring sequence or substitution with one or more serine or threonine residues (for O-linked glycosylation sites). Alternatively, N-linked glycosylation can be created by the addition of a recognition sequence. The CRIg amino acid sequence is optionally altered by changes at the DNA level, in particular by mutating the DNA encoding the CRIg polypeptide at a preselected base so that a codon that can be translated into the desired amino acid occurs. be able to.

  Another means of increasing the number of carbohydrate moieties on the CRIg polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, for example, WO 87/05330 published September 11, 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem. 259-306 (1981).

  Removal of the carbohydrate moiety present in the CRIg polypeptide can be accomplished by chemical or enzymatic substitution of the codon encoding the amino acid residue targeted for glycosylation, or substitution by mutation. Chemical deglycosylation techniques are known in the art, see, eg, Hakimudin, et al., Arch. Biochem. Biophys. 259: 52 (1987), and Edge et al., Anal. Biochem. 118: 131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides is described by Thotkura et al., Meth. Enzymol. 138: 350 (1987) by the use of various endoglycosidases and exoglycosidases.

  Other types of covalent modification of CRIg include, for example, U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417. Of various non-proteinaceous polymers (eg, polyethylene glycol, polypropylene glycol, or polyoxyalkylene) in the manner described in US Pat. No. 4,791,192; or 4,179,337. Linking the CRIg polypeptide to one is included.

  The naturally occurring CRIg and variant CRIg of the present invention is also modified in a method to form a chimeric molecule comprising CRIg, comprising a fragment of CRIg fused to another heterologous polypeptide or amino acid sequence. In some cases. In one embodiment, such a chimeric molecule includes a fusion of CRIg with a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amino terminus or carboxyl terminus of the CRIg polypeptide. The presence of such epitope-tagged forms of CRIg polypeptide can be detected using an antibody against the tag polypeptide. The provision of the epitope tag also allows the CRIg polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; flu HA tag polypeptide and its antibody 12CA5 (Field et al., Mol. Cell. Biol., 8: 2159). -2165 (1988); c-myc tag and 8F9, 3C7, 6E10, G4, B7, and 9E10 antibodies thereto (Evan et al., Molecular and Cellular Biology, 5: 3610-3616 (1985)); and herpes simplex virus Glycoprotein D (gD) tag and its antibody (Paborsky et al., Protein Engineering, 3 (6): 547-553 (1990)) Other flag polypeptides include Flag- Peptides (Hopp et al., BioTechnology, 6: 1204-1210 (1988)); KT3 epitope peptides (Martin et al., Science, 255: 192-194 (1992)); and α-tubulin epitope peptides (Skinner et al., J. Biol). Chem., 266: 15163-15166 (1991)); and T7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87: 6393-6997 (1990)). .

  In another embodiment, a chimeric molecule can include a fusion of a CRIg polypeptide or fragment thereof with an immunoglobulin or a specific region of an immunoglobulin. For the bivalent form of the chimeric molecule, such a fusion can be to the Fc region of an IgG molecule. These fusion polypeptides are antibody-like molecules that combine the effector function of immunoglobulin constant domains with the binding specificity (adhesion) of heterologous proteins and are often referred to as immunoadhesins . Structurally, an immunoadhesive includes a fusion of an amino acid sequence (ie, heterologous) having a desired binding specificity other than the antigen recognition and binding site of an antibody with an immunoglobulin constant domain sequence. It is. The adhesion part of an immunoadhesive molecule is usually a contiguous amino acid sequence comprising at least a receptor or ligand binding site. The immunoglobulin constant domain sequence of the immunoadhesive can be any immunoglobulin (eg, IgG-1, IgG-2, IgG-3, or IgG-4 subtype, IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM).

  Chimeras constructed from receptor sequences linked to appropriate immunoglobulin constant domain sequences (immunoadhesives) are known in the art. Immunoadhesins reported in the literature include fusions of T cell receptors (Gascoigne et al., Proc. Natl. Acad. Sci. USA, 84: 2936-2940 (1987)); CD4 fusions (Capon et al., , Nature 337: 525-531 (1989); Traunecker et al., Nature, 339: 68-70 (1989); Zettmeissel et al., DNA Cell Biol. USA, 9: 347-353 (1990); Byrn et al., Nature, 344. : 667-670 (1990)); fusions of L-selectin (homing receptor) (Watson et al., J. Cell. Biol., 110: 2221-2229 (1990); Watson et al., Nature, 349: 164-167. (199 )); A fusion of CD44 (Aruffo et al., Cell, 61: 1303-1313 (1990)); a fusion of CD28 and B7 (Linsley et al., J. Exp. Med., 173: 721-730 (1991)); Fusion of CTLA-4 (Lisley et al., J. Exp. Med. 174: 561-569 (1991)); fusion of CD22 (Stamenkovic et al., Cell, 66: 1133-1144 (1991)); Fusions (Ashkenazi et al., Proc. Natl. Acad. Sci. USA, 88: 10535-10539 (1991); Lesslauer et al., Eur. J. Immunol., 27: 2883-2886 (1991); Peppel et al., J. Exp. Med., 174: 1483. 1489 (1991)); NP receptor fusions (Bennett et al., J. Biol. Chem. 266: 23060-23067 (1991)); and IgE receptor α fusions (Ridgway et al., J. Cell). Biol., 115: abstr.1448 (1991)).

  In the simplest and most direct immunoadhesive structure, the binding region (s) of the “adhesion” protein are combined with the hinge and Fc regions of an immunoglobulin heavy chain. Usually, when preparing the CRIg-immunoglobulin chimera of the present invention, the nucleic acid encoding the CRIg polypeptide or the extracellular domain of the CRIg polypeptide is compared to the nucleic acid encoding the N-terminus of the immunoglobulin constant domain sequence. Fused at the C-terminus. However, fusion at the N-terminus is also possible.

  Usually, in such fusions, the encoded chimeric polypeptide retains the hinge region of an immunoglobulin heavy chain and the CH2 and CH3 domains of the constant region. Fusions can also be made to the C-terminus of the Fc portion of the constant domain and immediately N-terminal to the heavy chain CH1 or to the corresponding region of the light chain.

  The exact site where the fusion takes place is not critical. Specific sites are well known and can be selected to optimize the biological activity, secretion or binding properties of the CRIg-immunoglobulin chimera.

  In some embodiments, the CRIg-immunoglobulin chimera is a monomer, or as a heterodimer or homomultimer, and specifically a dimer essentially as described in WO 91/08298. Or assembled as a tetramer.

In a preferred embodiment, the sequence of a naturally occurring human CRIg polypeptide (eg, huCRIg (long) (SEQ ID NO: 4) or huCRIg (short) (SEQ ID NO: 6), or the sequence of a CRIg extracellular domain (Including huCRIg (long) and huCRIg (short) ECDs) of the C-terminal portion of an antibody (specifically, an Fc domain) comprising the effector function of an immunoglobulin (eg, immunoglobulin G ₁ (IgG1)) Fused to the N-terminus, the entire heavy chain constant region can be fused to CRIg or the CRIg extracellular domain sequence, but more preferably a papain cleavage site (which chemically defines an IgG Fc) A residue at position 216, the first residue of the heavy chain constant region, 114 In other preferred embodiments, the amino acid sequence of CRIg is in the hinge region and CH2 and CH3, starting with the hinge region immediately upstream of the other immunoglobulin (similar sites). Or fused to the CH1, hinge, CH2, and CH3 domains of an IgG1, IgG2, or IgG3 heavy chain The exact site at which the fusion occurs is not critical and the optimal site can be determined by routine experimentation Specific CRIg-Ig immunoadhesive structures are shown in FIGS.

  In some embodiments, CRIg-immunoglobulin chimeras are assembled as multimers and specifically as homodimers or homotetramers. In general, these assembled immunoglobulins will have a known unit structure. The basic four chain structural unit is the form in which IgG, IgD, and IgE are present. The four units are repeated with a larger molecular weight immunoglobulin. IgM generally exists as a basic 4-unit pentamer held together by disulfide bonds, and IgA globulin (and possibly IgG globulin) can also exist in multimeric form in serum. . In the case of multimers, the four individual units may be the same or different.

  Alternatively, the CRIg or CRIg extracellular domain sequence can be inserted between the immunoglobulin heavy chain sequence and the light chain sequence, resulting in an immunoglobulin containing the chimeric heavy chain. In this embodiment, the CRIg sequence is fused to the 3 ′ end of the immunoglobulin heavy chain of each arm of the immunoglobulin, either between the hinge and CH2 domains or between the CH2 and CH3 domains. The A similar structure is described in Hoogenboom et al., Mol. Immunol. 28: 1027-1037 (1991).

  Although the presence of an immunoglobulin light chain is not required in the immunoadhesive of the present invention, the immunoglobulin light chain is present covalently linked to a CRIg immunoglobulin heavy chain fusion polypeptide or directly fused to the CRIg extracellular domain. There is a case. In the former case, usually the DNA encoding the immunoglobulin light chain is expressed together with the DNA encoding the CRIg-immunoglobulin heavy chain fusion protein. When secreted, the hybrid heavy and light chains are covalently linked to provide an immunoglobulin-like structure comprising two disulfide-linked immunoglobulin heavy chain-light chain pairs. A suitable method for the preparation of such a structure is disclosed, for example, in US Pat. No. 4,816,567 issued on Mar. 28, 1989.

  Nucleotide sequences encoding particular CRIg-Ig fusion proteins of the invention are shown in FIGS. 59, 60, and 67-70. As shown in FIGS. 67-70, for example, the fusion protein may include a linker (eg, a short peptide sequence (eg, DKTHT)) between the CRIg and the immunoglobulin sequence. In addition, in some constructs, the sequence between the CRIg transmembrane (TM) region and the immunoglobulin (Fc) region (referred to herein as the “stalk” sequence) is deleted. be able to. The amino acid positions where the linkers begin in the various CRIg constructs shown in FIGS. 67-70 are as follows: huCRIg-long-Fc + stalk; position 267; 173rd position; huCRIg-short-Fc-stalk: 140th position.

(2. Preparation of CRIg polypeptides and variant CRIg polypeptides of naturally occurring sequences)
DNA encoding a CRIg polypeptide of a naturally occurring sequence can be obtained from a cDNA library prepared from tissue that has CRIg mRNA and is believed to express it at detectable levels. Thus, human CRIg DNA may be conveniently obtained from a cDNA library prepared from human tissue, as described in Example 1. The gene encoding CRIg can also be obtained from a genomic library or by synthesis of oligonucleotides.

  The library can be screened with a probe designed to identify the gene of interest or the protein encoded thereby (eg, an antibody to CRIg, or an oligonucleotide of at least about 20-80 bases). Screening of cDNA or genomic libraries with selected probes is performed using standard procedures described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Press, 1989). be able to. Another means for isolating the gene encoding CRIg is to use the PCR method (Sambok et al., Supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)). .

Example 1 describes a technique for screening a cDNA library. The oligonucleotide sequence selected as the probe must be long enough and clear enough to minimize false positives. The oligonucleotide is preferably labeled such that it can be detected when hybridized to DNA in the library being screened. Labeling methods are well known in the art and include the use of radioisotope labeling such as ³² P-labeled ATP, biotinylation, or enzyme labeling. Hybridization conditions including moderate and high stringency are provided in Sambrook et al. (Supra).

  The sequences identified in such library screening methods are stored in public databases such as GenBank or other personal sequence databases and compared to other known sequences available. Can be aligned. Sequence identity (either at the amino acid level or at the nucleotide level) in a defined region of the molecule or over the full length sequence uses various algorithms to measure homology, BLAST, BLAST-2, ALIGN , DNAstar, and INHERIT can be determined by sequence alignment using computer software programs.

  Nucleic acids having protein-encoding sequences are selected by first screening a selected cDNA or genomic library using the deduced amino acid sequences disclosed herein, and as necessary. Obtained by detecting precursors and processing intermediates of mRNA that may not have been reverse transcribed into cDNA using conventional primer extension procedures such as those described in Sambrook et al. (Supra). Sometimes.

  A host cell is transfected or transformed with an expression vector or cloning vector described herein for the production of CRIg, a promoter encoding, selection of transformants, or a gene encoding the desired sequence. Cultured in conventional nutrient media modified to be suitable for amplification of Culture conditions (eg, medium, temperature, pH, etc.) can be selected by those skilled in the art without undue experimentation. In general, the principles, protocols, and practical techniques for maximizing cell culture productivity are described in Mammalian Cell Biotechnology: A Practical Approach, M .; Edited by Butler (IRL Press, 1991) and Sambrook et al. (Supra).

  Methods of transfection are known to those skilled in the art, for example CaPO4 and electroporation. Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. Calcium treatment using calcium chloride, as described in Sambrook et al. (Supra), or electroporation is usually used for prokaryotes, or other cells that contain a substantial cell wall barrier. Infection with Agrobacterium tumefaciens has been described in certain plants as described in Shaw et al., Gene, 23: 315 (1983) and WO 89/05858 published on 29 June 1989. Used for cell transformation. For mammalian cells that do not have such cell walls, the calcium phosphate precipitation method of Graham and von der Eb, Virology, 52: 456-457 (1978) can be used. General aspects of transformation of mammalian cell host systems are described in US Pat. No. 4,399,216. Transformation into yeast is generally performed by Van Solingen et al. Bact. , 130: 946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76: 3829 (1979). However, other methods for introducing DNA into cells (eg, nuclear microinjection, electroporation, fusion of bacterial protoplasts with complete cells, or polycations (eg, polybrene, polyornithine)) are also used can do. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymology, 185: 527-537 (1990) and Mansour 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 eukaryote cells. Suitable prokaryotes include, but are not limited to, eubacteria (eg, Gram negative or Gram positive bacteria, such as Enterobacteriaceae such as E. coli). Various E. coli strains (eg, E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325), and K5772 (ATCC 53,635). )) Is publicly available.

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

  Suitable host cells for the expression of glycosylated CRIg are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells. Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) cells and COS cells. As a more specific example, monkey kidney CV1 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. USA, 77: 4216 (1980)). Mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23: 243-251 (1980)); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary gland Tumor cells (MMT 060 62, ATCC CCL51) can be mentioned. The selection of an appropriate host cell is deemed to be within the ability of those skilled in the art.

  A nucleic acid encoding CRIg (eg, cDNA or genomic DNA) can be inserted into a replicable vector for cloning (amplification of DNA) or for expression. Various vectors are publicly available. The vector can be, for example, in the form of a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence can be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site (s) using techniques known in the art. Vector components typically include, but are not limited to, one or more signal sequences, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Standard ligation techniques known to those skilled in the art are used to construct a suitable vector containing one or more of these components.

  CRIg polypeptides can be produced not only directly by recombination, but also heterologous polypeptides that can be signal sequences or other polypeptides that have a specific cleavage site at the N-terminus of the mature protein or polypeptide. It can also be produced as a fusion polypeptide with a peptide. In general, the signal sequence can be a component of the vector, or it can be a part of the CRIg DNA that is inserted into the vector. The signal sequence can be, for example, a prokaryotic signal sequence selected from the group of alkaline phosphatase, penicillinase, lpp, or thermostable enterotoxin II leader. For selection in yeast, signal sequences include, for example, the yeast invertase leader, alpha factor leader (Saccharomyces and Kluyveromyces "-factor leader, 010,182), or the leader of acid phosphatase, the leader of C. albicans glucoamylase (EP 362,179 published April 4, 1990), or November 1990 The signal described in WO 90/13646 published on May 15. For expression in mammalian cells, the mammalian signal sequence can be used to direct protein secretion, For example, the same species or It can be a signal sequence derived from a secreted polypeptide of a linked species, as well as a viral secretion leader.

  Both expression and cloning vectors contain a nucleic acid sequence that enables 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 derived from plasmid pBR322 is suitable for gram-negative bacteria. 2: Plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, SV, or BPV) are useful for cloning vectors in mammalian cells.

  Expression and cloning vectors usually contain a selection gene, also called a selectable marker. Typical selection genes include (a) proteins that confer resistance to antibiotics or other toxins (eg, ampicillin, neomycin, methotrexate, or tetracycline), (b) proteins that compensate for auxotrophic deficiencies, or (c ) A gene that encodes a protein that supplies important nutrients that cannot be obtained from the complex medium, for example, a gene encoding the Bacilli D-alanine racemase.

  Examples of selectable markers suitable for mammalian cells are those that can identify a cell's ability to take up CRIg nucleic acids, for example, DHFR or thymidine kinase. Suitable host cells are those described in Urlaub et al., Proc. When wild type DHFR is used. Natl. Acad. Sci. USA, 77: 4216 (1980), prepared and expanded CHO cells deficient in DHFR activity. A selection gene suitable for use in yeast is the trp1 gene present in the yeast plasmid TRp7 (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 a mutant strain of yeast (ATCC No. 44076 or PEP4-1) lacking the ability to grow in tryptophan (Jones, Genetics, 85:12 (1977)). .

  Expression and cloning vectors usually contain a promoter operably linked to the CRIg nucleic acid sequence to direct the synthesis of mRNA. Promoters recognized by a variety of potential host cells are well known. Suitable promoters for use with prokaryotic hosts include □ -lactamase and lactose promoter systems (Chang et al., Nature, 275: 615 (1978); Goeddel et al., Nature, 281: 544 (1979)), alkaline phosphatase, tryptophan (Trp) promoter system (Goeddel, Nucleic Acids Res., 8: 4057 (1980); EP 36, 776), and hybrid promoters such as the tac promoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21). -25 (1983)). Promoters used in bacterial systems will also include a Shine-Dalgarno (SD) sequence operably linked to DNA encoding CRIg.

  Examples of promoter sequences suitable for use in yeast hosts include 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)), for example, enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phospho Fructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase, and glucokinase .

  Other yeast promoters are inducible promoters that have the additional advantage of transcription controlled by growth conditions, such as alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradation enzymes involved in nitrogen metabolism, metallothionein , Glyceraldehyde-3-phosphate dehydrogenase, and an enzyme responsible for the use of maltose and galactose. Suitable vectors and promoters used for expression in yeast are further described in EP 73,657.

  Transcription of CRIg from a vector in mammalian host cells is, for example, viral (eg, polyoma virus, chicken poxvirus (UK 2,211,504 published July 5, 1989)), adenovirus (eg, Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and simian virus 40 (SV40)) by promoters derived from heterologous mammalian promoters (eg actin Such as promoters or immunoglobulin promoters) and by heat shock promoters, provided that such promoters are compatible with the host cell system.

  Transcription of DNA encoding a CRIg polypeptide by higher eukaryotes can be increased by insertion of an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Usually, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer (bp 100-270) located behind the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer behind the origin of replication, and the adenovirus enhancer. The enhancer can be spliced into the vector at a position 5 'or 3' to the CRIg encoding sequence, but is preferably located 5 'from the promoter.

  Expression vectors used in eukaryotic host cells (nucleated cells derived from yeast, fungi, insects, plants, animals, humans, or other multicellular organisms) can also be used to terminate and stabilize mRNA transcription. Necessary sequences will also be included. Such sequences are generally available from the 5 'and optionally 3' untranslated region of eukaryotic or viral DNA or cDNA. These regions include nucleotide segments that are transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding CRIg.

  Still other methods, vectors, and host cells suitable for application to the synthesis of CRIg in recombinant vertebrate cell culture are described in Gething et al., Nature, 293: 620-625 (1981); Mantei et al., Nature. 281: 40-46 (1979); EP 117,060; and EP 117,058.

  Amplification and / or expression of the gene can be performed, for example, by conventional Southern blotting or Northern blotting for quantifying mRNA transcription (Thomas, Proc. Natl. Acad. Sci. USA, 77: 5201-5205 (1989)), Measurements can be made directly in the sample by dot blotting (DNA analysis) or in situ hybridization using appropriately labeled probes based on the sequences provided herein. Alternatively, antibodies that can specifically recognize specific duplexes (including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes) or DNA-protein duplexes are used. be able to. The antibody can then be labeled and the assay performed. Here, the presence of an antibody bound to the double chain can be detected when the double chain binds to the surface and, as a result, forms a double chain on the surface.

  Alternatively, gene expression may be measured by immunological methods (eg, immunohistochemical staining of cells or tissue sections, and cell culture or body fluid assays) to directly quantify gene product expression. it can. Antibodies useful for immunohistochemical staining and / or sample liquid assays can be either monoclonal or polyclonal, and can be prepared in any mammal. Typically, the antibody is fused to a CRIg polypeptide of a naturally occurring sequence, or to a synthetic peptide based on the DNA sequence provided herein, or fused to a CRIg DNA and specific. Can be prepared against exogenous sequences encoding specific antibody epitopes.

  CRIg forms can be recovered from culture medium or host cell lysate. If bound to the membrane, it can be detached from the membrane using a suitable surfactant solution (eg, Triton-X 100) or by enzymatic cleavage. Cells used for CRIg expression can be disrupted by various physical or chemical means (eg, freeze-thaw cycles, sonication, mechanical disruption, or cytolytic agents).

  It may be desired to purify CRIg from recombinant cell proteins or polypeptides. The following procedure is an example of a suitable purification procedure: fractionation on an ion exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or a cation exchange resin (eg DEAE); chromatographic fractionation; SDS -PAGE; ammonium sulfate precipitation; gel filtration (eg, using Sephadex G-75); protein A Sepharose column to remove contaminants such as IgG; and metal chelation column that binds to epitope-tagged forms of CRIg polypeptide by. Various protein purification methods can be used. Such methods are well 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 (s) selected will vary depending on, for example, the nature of the production process used and the particular CRIg produced.

(3. Agonists of CRIg polypeptides)
An agonist of a CRIg polypeptide can mimic the qualitative biological activity of a CRIg polypeptide of a naturally occurring sequence. Preferably, the biological activity is the ability to bind to C3b, the ability to affect complement or complement activation, specifically the ability to inhibit alternative complement pathways and / or C3 convertase. It is. Agonists include, for example, immunoadhesives, peptidomimetics, and small organic molecules that are non-peptides that mimic the qualitative biological activity of naturally occurring CRIg.

  CRIg-Ig immunoadhesives are discussed above.

  Another group of CRIg agonists are peptidomimetics of CRIg polypeptides with naturally occurring sequences. Peptidomimetics include, for example, peptides containing amino acids that do not occur in nature, provided that the compound retains the biological activity of CRIg described herein. Similarly, peptidomimetics and analogs include non-amino acid chemical structures that mimic the structure of key structural elements of the CRIg polypeptides of the invention and retain the biological activity of CRIg. obtain. The term “peptide” as used herein is a constrained (ie, initiates a β-turn or β-pleated sheet of less than about 50 amino acid residues, preferably less than about 40 amino acid residues, or, for example, Means an amino acid sequence that has several structural elements, such as the presence of an amino acid that is cyclized by the presence of a disulfide-linked Cys residue), or is unconstrained (eg, linear) This includes multimers such as the dimer or dimer between them. Of peptides having less than about 40 amino acid residues, peptides of between about 10 and about 30 amino acid residues, particularly about 20 amino acid residues are preferred. However, upon reading this disclosure, those of skill in the art will recognize that the peptide is not the length of a particular peptide, but its ability to bind to C3b and inhibit C3 convertase, particularly the alternative complement pathway. It will be readily understood that its ability to inhibit the C3 convertase.

  Peptides can usually be prepared using solid phase peptide synthesis (Merifield, J. Am. Chem. Soc. 85: 2149 (1964); Houghten, Proc. Natl. Acad. Sci. USA, 82. : 5132 (1985)). Solid phase synthesis is initiated by coupling a protected amino acid to an inert solid support at the carboxyl terminus of the putative peptide. The inert solid support can be any macromolecule that can act as an anchor for the C-terminus of the first amino acid. Typically, the macromolecular support is a crosslinked polymer resin (eg, a polyamide resin or polystyrene, as shown in FIGS. 1-1 and 1-2 on pages 2-4 of Stewart and Young (supra). Resin). 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 linkage of the peptide anchor is stable under the conditions used to deprotect the α-amino group of the blocked amino acid during peptide synthesis. Where base labile α-protecting groups are used, it may be desirable to use an acid labile linkage between the peptide and the solid support. For example, acid labile ether resins are effective for the synthesis of base labile Fmoc-amino acid peptides, as described on page 16 of Stewart and Young (supra). Alternatively, peptide anchor linkages and α-protecting groups that exhibit various instabilities to acid degradation can be used. For example, aminomethyl resins (eg, phenylacetamidomethyl (Pam) resins) function well in combination with Boc-amino acid peptide synthesis as described on pages 11-12 of Stewart and Young (supra). .

  After the first amino acid is attached to the inert solid support, the α-amino protecting group of the first amino acid is removed, for example, with trifluoroacetic acid (TFA) in methylene chloride and, for example, with triethylamine (TEA). Neutralized. After deprotection of the α-amino group of the first amino acid, the next α-amino and side chain protected amino acids are added during synthesis. The remaining α-amino acids, and optionally the side chain protected amino acids, are then sequentially joined together by condensation in the desired order, resulting in an intermediate compound attached to the solid support. Alternatively, several amino acids are joined together so that the desired peptide fragment is formed, and then the peptide fragment is added for growth of the solid phase peptide chain.

  The condensation reaction between two amino acids, or an amino acid and a peptide, or a peptide and a peptide can be carried out using an axide method, a mixed acid anhydride method, DCC (N, N′-dicyclohexylcarbodiimide), or DIC (N, N′— Diisopropylcarbodiimide) method, active ester method, p-nitrophenyl ester method, BOP (benzotriazol-1-yl-oxy-tris (dimethylamino) phosphonium hexafluorophosphate) method, N-hydroxysuccinimide ester method and the like, and The reaction can be carried out according to a usual condensation method such as Woodward reagent K method.

  In chemical synthesis of peptides, it is common to protect any reactive side chain group of an amino acid with a suitable protecting group. Ultimately, these protecting groups are removed after the desired polypeptide chain is continuously assembled. Protection of the α-amino group of an amino acid or peptide fragment is also common, but 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 and then , The α-amino group is selectively removed, allowing the addition of the next amino acid or peptide fragment to the solid phase polypeptide chain. Therefore, in the synthesis of polypeptides, it is common to produce intermediate compounds that contain each of the amino acid residues arranged in the desired sequence in the peptide chain. In this case, the individual residues still have side chain protecting groups. These protecting groups can be removed substantially simultaneously after removal from the solid phase to yield the desired polypeptide product.

The α- and ε-amino side chains are benzyloxycarbonyl (abbreviated as Z), isonicotinyloxycarbonyl (iNOC), o-chlorobenzyloxycarbonyl (Z (2Cl)), p-nitrobenzyloxycarbonyl ( Z _(NO 2)), p- methoxybenzyloxycarbonyl (Z (OMe))), t- butoxycarbonyl (Boc), t-amyloxycarbonyl (Aoc), isobornyloxycarbonyl, adamantyloxycarbonyl, 2- (4-biphenyl) -2-propyloxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl (Fmoc), methylsulfonylethoxycarbonyl (Msc), trifluoroacetyl, phthalyl, formyl, 2-nitrophenylsulfenyl (NPS) ), Diphenylphosphine Nochioiru (Ppt), and dimethylphosphinothioyl Ji oil (Mpt) can be protected with such groups.

  Protecting groups for carboxyl functional groups are exemplified by benzyl ester (OBzl), cyclohexyl ester (Chx), 4-nitrobenzyl ester (ONb), t-butyl ester (Obut), 4-pyridylmethyl ester (Opic), etc. The In many cases, specific amino acids having functional groups other than amino and carboxyl groups (eg, arginine, cysteine, and serine) are protected by appropriate protecting groups. For example, the guanidino group of arginine is nitro, p-toluenesulfonyl, benzyloxycarbonyl, adamantyloxycarbonyl, p-methoxybenzsulfonyl, 4-methoxy-2,6-dimethylbenzenesulfonyl (Nds), 1,3,5- It can be protected with trimethylphenylsulfonyl (Mts) or the like. The thiol group of cysteine can be protected with p-methoxybenzyl, trityl and the like.

  Many of the blocked amino acids described above are available from commercial sources such as Novabiochem (San Diego, Calif), Bachem CA (Torrance, Calif), or Peninsula Labs (Belmont, Calif). .

  Stewart and Young (supra) provide detailed information on procedures for preparing peptides. The protection of the α-amino group is described on pages 14 to 18 and the side chain block is described on pages 18 to 28. Tables of protecting groups for amine, hydroxyl, and sulfhydryl functions are described on pages 149-151.

  After the desired amino acid sequence is complete, the peptide can be cleaved from the solid support, recovered, and purified. The peptide is cleaved from the solid support by a reagent that can break the peptide-solid phase bond and optionally deprotect the blocked side chain functional group on the peptide. In one embodiment, the peptide is cleaved from the solid phase by acidolysis with liquid hydrogen fluoride (HF). This also removes any remaining side chain protecting groups. Preferably, the acidolysis reaction mixture includes thio-cresol and cresol scavenger to avoid alkylation of residues in the peptide (eg, alkylation of methionine, cysteine, and tyrosine residues). After cleavage with HF, the resin is washed with ether and the free peptide is extracted from the solid phase by successive washes with acetic acid solution. The mixed washings are frozen and the peptide is purified.

(4. CRIg polypeptide antagonists)
Naturally occurring CRIg polypeptide antagonists have found utility in the treatment of conditions (including tumor treatment) where excessive complement activation is effective.

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

  Methods for preparing polyclonal antibodies are known to those skilled in the art. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and optionally an adjuvant. Usually, the immunizing agent and / or adjuvant will be injected into the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent can include a CRIg polypeptide of the present invention or a fragment or fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein that is known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants that can be used include Freud's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). The immunization protocol can be selected by one skilled in the art without undue experimentation.

  An antibody that recognizes and binds to the polypeptide of the present invention or acts as an antagonist thereto may alternatively be a monoclonal antibody. Monoclonal antibodies can be prepared using hybridoma methods such as those described in Kohler and Milstein, Nature, 256: 495 (1975). In hybridoma methods, a mouse, hamster, or other suitable host animal usually produces an immunizing agent to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Immunized. Alternatively, lymphocytes can be immunized in vitro.

  The immunizing agent will typically include a CRIg polypeptide of the invention, an antigenic fragment thereof or a fusion protein. Generally, any of the peripheral blood lymphocytes ("PBL") are used where cells of human origin are desired, and the spleen cells or lymph node cells are non-human mammals Used when a source is desired. The lymphocytes are then fused with an immortal cell line using an appropriate fusion agent (eg, polyethylene glycol) to form hybridoma cells (Goding. Monoclonal Antibodies: Principles and Practice, Academic Press, (1986). 59-103). Immortal cell lines are usually transformed mammalian cells, specifically myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are used. The hybridoma cells can be cultured in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of unfused, immortalized cells. For example, if the original cell lacks the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium of the hybridoma usually contains hypoxanthine, aminopterin, and thymidine. (“HAT medium”). These substances prevent the growth of HGPRT deficient cells.

  Preferred immortal cell lines are those that fuse efficiently, support stable high level expression of antibodies by selected antibody producing cells, and are sensitive to media such as HAT media. A more preferred immortal cell line is the mouse myeloma line, which can be obtained from, for example, the Salk Institute Cell Distribution Center, San Diego, California, and the American Type Culture Collection, Rockville, Maryland. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Production Technologies and Applications. 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 the polypeptides of the invention or having similar activity as the polypeptides 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, eg, by radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). . Such techniques and assays are known in the art. The binding affinity of a monoclonal antibody is described, for example, in Munson and Pollard, Anal Biochem. 107: 220 (1980).

  After the desired hybridoma cells have been identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, hybridoma cells can be grown in vivo, such as mammalian ascites.

  Monoclonal antibodies secreted by subclones are isolated from culture medium or ascites fluid by conventional immunoglobulin purification procedures (eg, protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography). Or it can be purified.

  Monoclonal antibodies can also be made by recombinant DNA methods (eg, those described in US Pat. No. 4,816,567). DNA encoding the monoclonal antibodies of the invention can be obtained using conventional procedures (eg, by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of mouse antibodies). ) Can be easily isolated and sequenced. The hybridoma cells of the present invention are a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector, after which monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins, etc. In a recombinant host cell to obtain monoclonal antibody synthesis. DNA can also be substituted, for example, with human heavy and light chain constant domain coding sequences instead of homologous mouse sequences (US Pat. No. 4,816,567; Morrison et al., Supra). Alternatively, it can be modified by covalent attachment to the entire immunoglobulin coding sequence or part of the coding sequence of a non-immunoglobulin polypeptide. Such non-immunoglobulin polypeptides can be replaced with the constant domains of the antibodies of the present invention, and can be replaced with the variable domains of the antigen binding sites of the antibodies of the present invention to generate chimeric bivalent antibodies. can do.

  The antibody is preferably a monovalent antibody. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of an immunoglobulin light chain and a modified heavy chain. The heavy chain is generally truncated at any site in the Fc region to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted to prevent crosslinking.

  In vitro methods are also suitable for preparing monovalent antibodies. Digestion to generate fragments of the antibody (especially Fab fragments) can be performed using routine techniques known in the art.

The antibodies of the present invention may further include humanized antibodies or human antibodies. Humanized forms of non-human (eg, murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (eg, Fv, Fab, Fab ′, f (ab ′) ₂ ), or other antigen binding of an antibody. Subsequence), which includes minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibodies), wherein the residues derived from the recipient's complementarity determining regions (CDRs) have the desired specificity, affinity, and ability for mice, rats, Alternatively, it is replaced with a residue derived from the CDR of a non-human species (donor antibody) such as rabbit. In some examples, human immunoglobulin Fv framework residues are replaced by corresponding non-human residues. Humanized antibodies may also contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody includes substantially all of at least one, usually two variable domains, all or substantially all of the CDR regions corresponding to those of non-human immunoglobulin, and All or substantially all of the FR region is of human immunoglobulin consensus sequence. Humanized antibodies optimally also include at least a portion of an immunoglobulin constant region (Fc), which is usually that of a human immunoglobulin (Jones et al., Nature, 321: 522-525 (1986)). Riechmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992)).

  Methods for humanizing non-human antibodies are well known in the art. Generally, 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 and are usually derived from “import” variable domains. Humanization is in principle according to 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)), by replacing a rodent CDR or CDR sequence with the corresponding sequence of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (US Pat. No. 4,816,567). In this case, substantially less than the entire human variable domain is replaced with the corresponding sequence from a non-human species. In fact, humanized antibodies are usually human antibodies in which some CDR residues and possibly some FR residues are substituted with residues from similar sites in rodent antibodies. It is.

  Human antibodies also include various phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581 (1991)). It can be produced using techniques known in the art. The techniques of Cole et al. And Boerner et al. Can also be utilized for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Lis, p. 77 (1985); Boerner et al., J. Immunol. 147 (1): 86-95 (1991); U.S. 5,750,373). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals (eg, mice). In this case, the endogenous immunoglobulin genes are either partially inactivated or completely inactivated. When challenged, production of human antibodies is observed. This is very similar to that seen in humans in all embodiments, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425. No. 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); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

  Bispecific antibodies are monoclonal antibodies that have binding specificities for at least two different antigens, preferably human antibodies or humanized antibodies. In this case, one of the binding specificities may be for a polypeptide of the invention and the other is for any other antigen, preferably a cell surface protein or receptor or receptor subunit.

  Methods for making bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs. In this case, the two heavy chains have different specificities (Milstein and Cuello, Nature, 305: 537-539 (1983)). Due to the random combination of immunoglobulin heavy and light chains, these hybridomas (quadromas) can give a mixture of 10 different antibody molecules, only one of which is accurate bispecific It has a typical structure. Accurate molecular purification is usually performed by an affinity chromatography step. A similar procedure is described in WO 93/08829 published May 13, 1993, and Traunecker et al., EMBO J. et al. 10: 3655-3659 (1991).

  Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to sequences of immunoglobulin constant domains. The fusion is preferably with an immunoglobulin heavy chain constant domain comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have a first heavy chain constant region (CH1) containing the site necessary for light chain binding present in at least one of the fusions. The immunoglobulin heavy chain fusion, and optionally the DNA encoding the immunoglobulin light chain, is inserted into another expression vector and cotransfected into a suitable host organism. For further details on making bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

  Heteroconjugate antibodies are composed of two covalently linked antibodies. Such antibodies are used, for example, to target cells of the immune system to unwanted cells (US Pat. No. 4,676,980) and for the treatment of HIV infection (WO 91/00360; WO 92). / 200373; EP03089). It is envisioned that antibodies can be prepared in vitro using methods known in synthetic protein chemistry, including methods involving cross-linking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate, and those disclosed in US Pat. No. 4,676,980.

  With regard to effector function, it may be desirable to modify the antibodies of the invention, for example, to increase the effectiveness of the antibodies in the treatment of diseases involving immunity. For example, cysteine residue (s) can be introduced into the Fc region, thereby forming an interchain disulfide bond in this region. Homodimeric antibodies thus generated may have improved internalization capabilities and / or high complement-mediated cell killing and antibody-dependent cytotoxicity (ADCC). Caron et al. Exp. Med. 176: 1191-1195 (1992) and Shopes, B .; , J .; Immunol. 148: 2918-2922 (1992). Homodimeric antibodies with high anti-tumor activity can also be prepared using heterobifunctional crosslinkers as described in Wolff et al., Cancer Research 53: 2560-2565 (1993). . Alternatively, the antibody can be engineered so that it has a dual Fc region, thereby having high complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).

  The invention also includes cytotoxic agents (eg, chemotherapeutic agents, toxins (eg, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof) or radioisotopes (ie, radioactive It also relates to an immunoconjugate comprising an antibody conjugated to a conjugate)).

Chemotherapeutic agents useful for the production of such immunoconjugates are described above. Usable enzymatically active toxins and fragments thereof include diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A, abrin A chain, modesin A chain, α-sarcin, Aurelites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPIII, and PAP-S), bitter gourd (momordicica charantia) inhibitor, crusin, saponin, saponin Factors, gelonin, mitgelonin, restrictocin, phenomycin, enomycin, and trichothese Can be mentioned. A variety of radionuclides are useful for the production of radioconjugated antibodies. Examples include ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y, and ¹⁸⁶ Re.

  Conjugates of antibodies and cytotoxic agents include various bifunctional protein coupling reagents such as N-succinimidyl-3- (2-pyridyldithiol) propionic acid (SPDP), iminothiolane (IT), imidoesters. Bifunctional derivatives (eg dimethyl adipimidate HCL), active esters (eg disuccinimidyl suberate), aldehydes (eg glutaraldehyde), bis-azide compounds (eg bis (p-azidobenzoyl)) Hexanediamine), bis-diazonium derivatives (eg, bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (eg, tolylene 2,6-diisocyanate), and bis-active fluorine compounds (eg, 1,5-difluoro- 2,4-dinitrobenzene ) Are created using. For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanate benzoyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for radionuclide binding to antibodies. See WO94 / 11026.

  In another embodiment, the antibody can bind to a “receptor” (eg, streptavidin) for use in tissue pre-targeting. In this case, the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a chelator, followed by cytotoxic substances (eg, radionuclides). A “ligand” (eg, avidin) bound to is administered.

(5. Target disease)
(5.1 Diseases and symptoms related to complement)
The CRIg polypeptides and agonists thereof (particularly CRIg-Ig immunoadhesins) of the present invention have found utility in the prevention and / or treatment of diseases and conditions involving complement. Such diseases and conditions include, but are not limited to, inflammatory diseases and autoimmune diseases.

  Specific examples of diseases involving complement include rheumatoid arthritis (RA), acute respiratory distress syndrome (ARDS), distant tissue injury after ischemia reperfusion, complement activity during cardiopulmonary bypass surgery Dermatomyositis, pemphigus, lupus nephritis and resulting glomerulonephritis and vasculitis, cardiopulmonary bypass surgery, coronary artery endothelial dysfunction induced by heart failure, type II membranoproliferative glomerulonephritis, IgA nephropathy , Acute renal failure, cryoglobulinemia, antiphospholipid syndrome, degenerative diseases of the skin and other eye conditions involving complement, such as age-related macular degeneration (AMD), chronic choroidal neovascularization (CNV) ), Uveitis, diabetic retinopathy and other ischemia-related retinopathy, endophthalmitis, and other intraocular neovascular disorders such as diabetic macular edema, pathological myopia, von Hippel Lind Disease, ocular histoplasmosis, central retinal vein occlusion (CRVO), corneal neovascularization, retinal neovascularization, and allograft, hyperacute rejection, hemodialysis, chronic obstructive pulmonary disease (COPD), asthma, and aspiration Examples include, but are not limited to pneumonia.

(5.2 Eye diseases involving complement)
CRIg polypeptides and their agonists (especially CRIg-Ig immunoadhesins) include ocular symptoms involving complement (including the classical and alternative complement pathways in their development, in particular alternative complement pathways). All eye conditions and diseases involving complements of the body pathways are included, for example, all stages of ocular degenerative diseases such as age-related macular degeneration (AMD) (dry and wet (non-exudative forms) ), Chronic choroidal neovascularization (CNV), uveitis, diabetic retinopathy and other ischemia-related retinopathy, endophthalmitis, and other intraocular neovascular disorders, For example, particularly useful in the prevention and / or treatment of diabetic macular edema, pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, central retinal vein occlusion (CRVO), corneal neovascularization, and retinal neovascularization) It is. Preferred groups of ocular conditions involving complement include age-related macular degeneration (AMD) (including wet (wet) AMD and non-wet (dry or atrophic) AMD), chronic choroidal neovascularization (CNV), diabetic retinopathy (DR), and endophthalmitis).

  AMD is a degeneration associated with aging of the eye, which is a major cause of irreversible visual impairment in individuals over 60 years of age. There are two types of AMD, non-wetting (dry) AMD and wet (wetting) AMD. Dry, or non-wetting, includes the atrophic and hypertrophic changes (plaque) of the underlying central retinal pigment epithelium (RPE) and the accumulation of RPE (drose). Non-wetting AMD patients can progress to wet, or wet AMD, in which abnormal blood vessels called choroidal neovascularization (CNVM) occur under the retina, causing fluid and blood leakage. This eventually results in discoid scars in the retina and under the retina. Non-wetting AMD is usually a precursor to wet AMD and is more common. Non-wetting AMD symptoms vary; hard drusen, soft drusen, PRE geographic atrophy, and pigment aggregation can develop. Complement components accumulate in RPE early in AMD and are the major component of drusen.

  The invention particularly relates to the treatment of high risk AMD. This includes Category 3 and Category 4 AMD. Category 3 AMD has no advanced AMD in any eye and at least one eye has a visual acuity of 20/32 or greater, or at least one large drusen (eg, 125 μm), a wide range of (druse Have moderate drusen (as measured by area), or geographic atrophy (GA) (not including the center of the plaque), or are characterized by any combination thereof. Category 3 AMD (also considered “dry” AMD) has a high risk of conversion to choroidal neovascularization (CNV).

  Category 4 high-risk AMD (classified as “wet” AMD) has a visual acuity of 20/32 or more and progressed in the eye index (center of plaque, or choroidal neovascularization) It is characterized by not having GA including the above-mentioned features. Other eyes are characterized by visual acuity of less than 20/32 due to advanced AMD or AMD macular degeneration. Typically, high-risk AMD, if untreated, progresses rapidly to choroidal neovascularization (CNV) at a rate approximately 10-30 times faster than that of category 1 or 2 (not high-risk) AMD. To do.

  CRIg and its agonists (eg, CRIg-Ig immunoadhesins) prevent the progression of AMD (especially in Category 3 or Category 4 AMD) to CNV and / or unaffected other eyes or severely Particular utility has been found in preventing the onset / progress of AMD or CNV in other unaffected eyes. In this context, the term “prevention” is used in the broadest sense, including complete or partial block and delay of disease progression, and delay of worsening of the disease to a more severe form. . This aspect of the invention is particularly useful for patients at high risk of developing or progressing to high risk AMD (category 4) or CNV.

  It is known that complement factor H (CFH) polymorphisms are associated with an individual's risk of developing AMD and / or CNV. CFH mutations can activate complement, which in turn can lead to AMD / CNV. It has recently been reported that 50% of the risk of AMD is due to complement factor H (CFH) polymorphism (Klein et al., Science 308: 385-9 (2005)). The general halpotype of CFH (HF1 / CFH) has been shown to make individuals susceptible to age-related macular degeneration (Hageman et al., Proc. Natl. Acad. Sci. USA, 102 (2): 7227-7232 (2003)). AMD has been isolated as an autosomal dominant trait, with a maximum rod score of about 3.20, and chromosome 1q25-q31, between markers D1S466 and D1S413 (Klein et al., Arch Optalmol. 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 (2001); Iyengar et al., Am. J. Hum. Genet. 74 (1): 20-39 (2004)); A marker D1 on chromosome 2q3 / 2q32 with a maximum rod score of 32 / 2.03 Between S1319 and D2S1384 (Seddon et al., Supra); chromosome 3q13 with a maximum rod score of 2.19 between markers D12S1300 and D12S1763 (Majewski et al., Supra; Sick et al., Am. J. Hum. Genet. 72 (6): 1412-24 (2003)); with a maximum rod score of 3.59 / 3.17, between chromosome 6q14 and between markers D6S1056 and DS249 (Knizeva et al., Am. J. Ophthmol. 130 (2): 197-202 (2000)); with a maximum rod score of 2.06, on chromosome 9q33, on marker D9S934 (Mejwski et al., Supra); Rod score on chromosome 10q26, marker D10S1230 (M ajewski et al., supra, Iyengar et al., supra; Keneally et al., Mol. Vis. 10: 57-61 (2004); 3.16 with a maximum rod score on chromosome 17q25, on marker D17S928 (Weeks et al., supra And having a disease locus mapped to chromosome 22q12, to marker D22S1045 (Seddon et al., Supra), with a maximum rod score of 2.0. It is an important part of identifying patients who are particularly good candidates for prevention of disease progression to more severe forms such as progression to CNV).

  In addition, given the strong evidence for the association between complement activation and age-related macular degeneration (AMD), the present invention provides for the inhibition of complement (particularly by inhibiting the alternative complement pathway). ) Novel methods for the prevention and treatment of CNV and AMD are provided. Inhibitors of alternative complement pathways other than CRIg include fusion proteins (eg, immunoadhesins), agonist anti-CRIg antibodies, and peptide and non-peptide small molecules.

(5.3 Inflammatory symptoms and autoimmune diseases)
A more extensive list of inflammatory conditions as examples of diseases involving complement includes, for example, inflammatory bowel disease (IBD), systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathy, systemic Systemic sclerosis (scleroderma), idiopathic inflammatory myopathy (dermatomyositis, polymyositis), Sjogren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, seizures) Nocturnal hemoglobinuria), autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura, immune thrombocytopenia), thyroiditis (Graves' disease, Hashimoto's disease thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroid) Inflammation), diabetes mellitus, immune kidney disease (glomerulonephritis, tubulointerstitial nephritis), demyelinating diseases of the central and peripheral nervous system (eg, multiple sclerosis, idiopathic multiple neuropathy), Hepatobiliary disease (Eg, infectious hepatitis (A, B, C, D, hepatitis E, and other non-liver tropic viruses)), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and Sclerosing cholangitis, inflammatory and fibrotic lung disease (eg cystic fibrosis), gluten-sensitive bowel disease, Whipple disease, autoimmune or immune skin disease (vesicular skin disease, erythema multiforme, and Contact dermatitis), psoriasis, allergic diseases of the lung (eg, eosinophilic pneumonia, idiopathic pulmonary fibrosis, and hypersensitivity pneumonia), transplant-related diseases (transplant rejection and graft-versus-host disease) Included).

  In systemic lupus erythematosus, the central mediator of the disease is the production of autoreactive antibodies against self proteins / tissues, followed by the development of inflammation mediated by immunity. Antibodies mediate 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 generation of autoreactive antibodies. Therefore, disease development is dependent on T lymphocytes. Multiple organs and systems are clinically affected, including kidney, lung, skeletal muscle system, cutaneous mucosa, eye, central nervous system, cardiovascular system, gastrointestinal tract, bone marrow, and blood.

  Rheumatoid arthritis (RA) is a chronic systemic autoimmune inflammatory disease mainly involving multiple joint synovial membranes with consequent damage to articular cartilage. Onset is T lymphocyte dependent and involves the production of rheumatoid factor, an autoantibody against self IgG, and the resulting formation of immune complexes that reach high levels in synovial fluid and blood. These complexes in the joints can induce significant lymphocyte and monocyte infiltration into the synovium, followed by significant synovial fluid changes. The joint space / fluid is infiltrated by similar cells, which is accompanied by the addition of multiple neutrophils. Affected tissues are mainly joints, often in a symmetric pattern. However, diseases other than joints also occur in two major forms. One form is the development of ongoing joint disease and non-joint lesions that have typical lesions of pulmonary fibrosis, vasculitis, and skin ulcers. A second form of disease other than joints is the so-called Ferti syndrome, which causes a delay in the course of RA disease, often in a quiescent phase after joint disease, with neutropenia, thrombocytopenia, and splenomegaly. Existence is related. This can involve vasculitis in multiple organs where infarctions, skin ulcers, and gangrene are formed. Patients often also develop rheumatoid nodules in the subcutaneous tissue overlying the affected joint; late stages of the nodules have a necrotic center surrounded by mixed inflammatory cell infiltration. Other symptoms that can occur in RA include pericarditis, pleurisy, coronary arteritis, interstitial pneumonia with pulmonary fibrosis, dry keratoconjunctivitis, and rheumatoid nodules.

  Juvenile chronic arthritis is a chronic idiopathic inflammatory disease, which often occurs before the age of 16. This phenotype has some similarities to RA; some patients who are positive for rheumatoid factor are classified as juvenile rheumatoid arthritis. The disease is subdivided into three main categories: pautical type, polytypic type, and systemic type. This arthritis can be severe and is usually destructive, leading to joint stiffness and slow growth. Other symptoms can include chronic anterior uveitis and systemic amyloidosis.

  Spondyloarthropathies are a group of disorders that have a common relationship between several common clinical features and the expression of the HLA-B27 gene product. This disorder includes ankylosing spondylitis, Reiter's syndrome (reactive arthritis), arthritis associated with inflammatory bowel disease, spondylitis associated with psoriasis, juvenile spondyloarthropathy, and anaplastic spondyloarthropathy. Prominent features include sacroiliac with or without spondylitis; inflammatory asymmetric arthritis; association with HLA-B27 (a serologically defined allele of the HLA-B locus of class I MHC) The absence of autoantibodies associated with ocular inflammation and other rheumatic diseases. The most probable cell for the induction of this disease is CD8 + T lymphocytes, which target cells presented by class I MHC molecules. CD8 + T cells can respond to the class I MHC allele HLA-B27 as if it were a foreign peptide expressed by MHC class I molecules. It has been hypothesized that epitopes of HLA-B27 can mimic antigenic epitopes of bacteria or other microorganisms and thus induce a CD8 + T cell response.

  The etiology of systemic sclerosis (scleroderma) has not been clarified. Characteristic of this disease is skin hardening, possibly induced by an active inflammatory process. Scleroderma may be localized or systemic; vascular lesions are common, and microvascular endothelial cell damage is an early critical factor in the development of systemic sclerosis A vascular injury can be mediated by immunity. The immunological basis is implied by the presence of mononuclear cell infiltration into skin lesions and the presence of antinuclear antibodies in many patients. ICAM-1 is often upregulated on the cell surface of fibroblasts in skin lesions, which means that the interaction of T cells with these cells has a role in the pathogenesis of the disease Suggests that you may have. Other organs involved are: gastrointestinal tract: smooth muscle atrophy and fibrosis resulting in abnormal peristalsis / motility; kidney: small arcuate and interlobar arteries resulting in decreased blood flow in the renal cortex Intensive subendothelial vascular intimal cell proliferation affecting protein causes proteinuria, hypernitrogenemia, and hypertension; skeletal muscle; atrophy, intestinal fibrosis; inflammation; lung: interstitial pneumonia and interstitial Qualitative fibrosis; and heart: necrosis of the contraction zone, scarring / fibrosis.

  Idiopathic inflammatory myopathy, including dermatomyositis, polymyositis, etc., is a chronic muscular inflammatory disorder with unknown etiology causing muscle weakness. Muscle damage / inflammation is often systemic and progressive. Autoantibodies are involved in most forms. These myositis-specific autoantibodies are directed against and inhibit the function of components, proteins, and RNA involved in protein synthesis.

  Sjogren's syndrome is caused by immune inflammation followed by disruption of lacrimal and salivary gland function. The disease may be associated with an inflammatory connective tissue disease, or the disease may be accompanied by an inflammatory connective tissue disease. This disease is associated with the production of autoantibodies against Ro and La antigens. Both of these antigens are small RNA-protein complexes. The lesion results in psoriatic keratoconjunctivitis, xerostomia, which is accompanied by other signs or associations including biliary cirrhosis, peripheral and sensory neuropathy, and overt purpura.

  In systemic vasculitis, the main lesion is inflammation and subsequent vascular damage, which in some cases results in ischemia / necrosis / degeneration of tissue supplied by the affected blood vessels, and ultimately In particular, dysfunction of the target organ occurs. Vasculitis may also occur as a secondary lesion and also other immune-inflammatory diseases such as rheumatoid arthritis, systemic sclerosis (especially involving the formation of immune complexes) It may also occur as a sequelae of disease)). As a disease in the group of primary systemic sclerosis: systemic necrotizing vasculitis: nodular polyarteritis, allergic vasculitis and granulomatosis, polyangiitis; Wegener's granulomatosis; lymphoma-like granulation Mastosis; and giant cell arteritis. A wide range of vasculitis includes mucocutaneous lymph node syndrome (MLNS or Kawasaki disease), isolated CNS vasculitis, Behcet's disease, obstructive thromboangiitis (Berger's disease), and necrotizing phlebitis of the skin Can be mentioned. Most pathological mechanisms of the listed types of vasculitis are primarily the accumulation of immunoglobulin complexes in the vascular wall, followed by either ADCC, complement activation, or both This is thought to be due to the induction of an inflammatory response.

  Sarcoidosis is a condition whose etiology is unclear and is characterized by the presence of epithelioid granulomas in almost every tissue in the body; most commonly it includes the lungs. Chronic sequelae resulting from the persistence of activated macrophages and lymphocytes at the site of disease, followed by the release of local and systemic active products released by these types of cells Is mentioned.

  Autoimmune hemolytic anemia, including autoimmune hemolytic anemia, immune pancytopenia, and paroxysmal nocturnal hemoglobinuria, erythrocytes (and in some cases other blood cells, including platelets) Cells coated with such antibodies by the production of antibodies that react with antigens expressed on the surface of and of complement-mediated lysis and / or mechanisms mediated by ADCC / Fc receptors It is a reflection of the removal.

  Autoimmune thrombocytopenia, including thrombocytopenic purpura, and immune thrombocytopenia in other clinical situations, platelet destruction / removal, attachment of either antibody or complement to platelets, and subsequent complement Occurs as a result of lysis, ADCC, or removal by Fc-receptor mediated mechanisms.

  Thyroiditis, including Graves' disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, and atrophic thyroiditis, is associated with the production of antibodies that react with proteins that are present in the thyroid and often are specific to the thyroid. Concomitant autoimmune response to thyroid antigen. Includes naturally occurring models: (rats (BUF and BB rats) and chickens (obese chicken strains)); inducible models: (immunization of animals with either thyroglobulin, thyroid microsomal antigen (thyroid peroxidase)) There are several experimental models.

  Type I diabetes mellitus or insulin-dependent diabetes is the autoimmune destruction of islet β cells. This destruction is mediated by autoantibodies and autoreactive T cells. Antibodies to insulin or the insulin receptor may also produce a non-insulin responsive phenotype.

  Immune kidney disease (including glomerulonephritis and tubulointerstitial nephritis) is the result of damage to kidney tissue mediated by antibodies or T lymphocytes and is the result of autoreactive antibodies or T cells against kidney antigens. Either directly as a result of production or indirectly as a result of accumulation of antibodies and / or immune complexes in the kidney which reacts to other non-renal antigens. Thus, other immune diseases that result in the formation of immune complexes can also induce immune kidney disease as an indirect sequelae. Both direct and indirect immune mechanisms produce an inflammatory response that leads to / induces the development of lesions in the kidney tissue, with organ function failure, and in some cases with progression of kidney failure. Both humoral and cellular immune mechanisms may be involved in the development of lesions.

  Central and peripheral demyelinating diseases (including multiple sclerosis, idiopathic multiple neuropathy, or Guillain-Barre syndrome) and chronic inflammatory demyelinating polyneuropathy have reasons for autoimmunity And result in demyelination of the nerve as a direct result of oligodendrocyte or myelin damage. There is evidence in MS that suggests that disease induction and progression is T lymphocyte dependent. Multiple sclerosis is a demyelinating disease that is T lymphocyte dependent and has either a relapsing-remitting course or a chronic progressive course. The etiology is unknown. However, viral infection, genetic predisposition, environment, and autoimmunity are all involved. Lesions include infiltration of microglia and infiltrating macrophages mediated primarily by T lymphocytes. CD4 + T lymphocytes are the most dominant cell type in the lesion. The mechanism of oligodendrocyte death and subsequent demyelination is not known, but is probably driven by T lymphocytes.

  Inflammation and fibrotic lung disease (including eosinophilic pneumonia, idiopathic pulmonary fibrosis, and hypersensitivity pneumonia) may involve a deregulated immune inflammatory response. Inhibiting the response is therapeutically effective.

  Autoimmune or immune skin diseases (including bullous skin disease, erythema multiforme, and contact dermatitis) are mediated by autoantibodies. Its onset is T lymphocyte dependent.

  Psoriasis is an inflammatory disease mediated by T lymphocytes. Lesions include infiltration of T lymphocytes, macrophages, and antigen-treated cells, and sometimes neutrophils. Allergic diseases (including asthma; allergic rhinitis; atopic dermatitis; food hypersensitivity; and urticaria) are T lymphocyte dependent. These diseases are primarily mediated by inflammation induced by T lymphocytes, inflammation mediated by IgE, or a combination of both.

  Diseases related to transplantation (including graft rejection and graft-versus-host disease (GVHD)) are T lymphocyte dependent; inhibition of T lymphocyte function is ameliorating.

(6. Treatment method)
For the prevention, treatment, or reduction in severity of a complement-related (immune) disease, an appropriate dose of a compound of the invention is a disease to be treated as defined above. Type of disease, severity and course of the disease, whether the drug is administered for prophylactic or therapeutic purposes, prior treatment, patient history, response to the compound, and It will vary depending on the judgment of the doctor. The compound is suitably administered to the patient in a single treatment or series of treatments. Preferably, it is desirable to determine dose response curves and pharmaceutical compositions of the invention in useful animal models first in vitro and then prior to human testing.

  For example, depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (eg, 0.1-20 mg / kg) of the polypeptide may be administered, for example, by one or more separate doses or sustained. It is the first candidate dose for administration to a patient, whether by infusion. A typical daily dose can range from about 1 μg / kg to 100 mg / kg or more, depending on the above factors. For repeated administrations over several days or longer depending on the condition, the treatment is maintained until a desired suppression of disease symptoms occurs. However, other dosage resumes may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

  The compounds of the invention for the prevention or treatment of eye diseases or conditions are usually administered by ocular injection, intraocular injection, and / or intravitreal injection. Other methods of administration are also used, including but not limited to topical, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal, and intralesional administration. Parenteral injection includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

  Formulations for ocular, intraocular and / or intravitreal administration can be prepared by methods known in the art using ingredients known in the art. The main requirement for efficient treatment is proper penetration through the eye. Unlike diseases in front of the eye where drugs can be administered locally, retinal diseases require a more site-specific approach. Eye drops and ointments rarely penetrate the back of the eye, and the blood-eye barrier prevents penetration of systemically administered drugs into ocular tissue. Thus, usually the method chosen for delivery of drugs to treat retinal diseases (eg AMD and CNV) is direct intravitreal injection. Intravitreal injection is usually repeated at intervals depending on the patient's condition and the characteristics and half-life of the drug being administered. For penetration into the eye (eg into the vitreous), small molecules are usually preferred. In the case of CRIg, all forms, including short and long forms of huCRIg, their Ig (Fc) fusions, long and short forms of full-length huCRIg, and their Ig (Fc) fusions All are suitable for intraocular (intravitreal) administration.

  The treatment efficiency of an ocular condition (eg, AMD or CNV) involving complement can be measured by various endpoints commonly used to assess intraocular disease. For example, blindness can be assessed. Blindness, for example, measures the average change in best corrected visual acuity (BCVA) from baseline to the desired point in time (eg, BCVA is an early treatment diabetic study (ETDRS) vision chart and a 4 meter test) Measuring the proportion of subjects who lost less than 15 characters in the desired visual acuity relative to the baseline (based on distance assessment), the desired visual acuity relative to the baseline Measuring the percentage of subjects who have acquired at least 15 characters, and measuring the percentage of subjects having visual-acoustic equivalent of 20/2000 or worse at the desired time. , NEI Visual Functionin Measuring questionnaire Questionnaire, for example, as assessed by fluorescein angiography, it can be evaluated, such as by evaluating the CNV size and CNV leakage amount at a desired time point, but are not limited to. Eye evaluation includes, but is not limited to, for example, performing eye examinations, measuring intraocular pressure, evaluating visual acuity, measuring slit pressure pressure, evaluating intraocular inflammation, and the like. Can be done in a way that is not.

  CRIg antagonists (eg, antibodies to CRIg) can be used in immunoadjuvant therapy for the treatment of tumors (cancer). It is now well established that T cells recognize human tumor-specific antigens. One group of tumor antigens encoded by the MAGE, BAGE, and GAGE families of genes is silent in all adult normal tissues, but tumors (eg, melanoma, lung tumors, head and neck tumors, and bladder) In cancer, it is expressed in a significant amount. DeSmet, C.I. (1996) Proc. Natl. Acad. Sci. USA, 93: 7149. It has been shown both in vitro and in vivo that co-stimulation of T cells induces tumor suppression and anti-tumor responses. Melero, I.M. Et al., Nature Medicine (1997) 3: 682; Kwon, E. et al. D. Et al., Proc. Natl. Acad. Sci. USA (1997) 94: 8099; Lynch, D .; H. Et al., Nature Medicine (1997) 3: 625; Finn, O .; J. et al. and Lotze, M .; T.A. , J .; Immunol. (1998) 21: 114. The CRIg antagonists of the invention are administered as adjuvants, alone or together with growth regulators, cytotoxic agents, or chemotherapeutic agents to stimulate T cell proliferation / activation and anti-tumor responses to tumor antigens. be able to. Growth regulators, cytotoxic agents, or chemotherapeutic agents can be administered in conventional amounts using known dosage regimes. Immunostimulatory activity with the CRIg antagonists of the present invention can reduce the amount of growth regulators, cytotoxic agents, or chemotherapeutic agents, thereby reducing toxicity to the patient.

  Although some macrophages are involved in tumor eradication, many solid tumors are known to contain macrophages that support tumor growth (Bingle et al., J. Pathol 196: 254-265 (2002). Mantovani et al., Trends Immunol.23: 549-555 (2002)). These macrophages can 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 aid in eradication of the tumor by complement-mediated lysis. This approach is particularly useful in tumors containing CRIg positive macrophages.

  In the treatment methods of the invention, the compositions herein can be combined with one or more additional treatment modalities for the prevention or treatment of the target disease or condition. Thus, for example, if the objective is prevention or treatment of ocular conditions involving complement, administration of CRIg (including all forms and their EC regions and / or Ig fusions) In combination with administration of the anti-VEGF-A antibody ranibizumab (Lucenitis ™, Genentech, Inc.) or administration of CRIg can supplement the administration of the anti-VEGF-A antibody ranibizumab. The anti-VEGF-A antibody ranibizumab is in clinical development for the treatment of AMD. In a recent phase III clinical trial, 25% of patients treated with 0.3 mg Lucentis (59) in addition to meeting the primary efficacy goal of the study on maintaining visual acuity in patients with wet AMD , 238), and 34% (81/240) of patients treated with 0.5 mg of Lucentis ™, as measured by the Early Treatment of Diabetic Retinopathy (ETDRS) vision chart, Compared with approximately 5% (11/238), the visual acuity improved with the acquisition of 15 characters or more. Approximately 40% of patients treated with Lucentis ™ reached a visual acuity score of 20/40 or better at 12 months compared to 11 percent (26/238) of the control group. At 12 months, patients treated with Lucentis ™ gained an average of 7 characters in visual acuity compared to the start of the experiment, while the control group lost an average of 10.5 characters.

  If the goal is to treat inflammatory or autoimmune diseases involving complement, administration of CRIg (including all forms) can be combined with other therapies for such indications . Thus, for example, if the target is rheumatoid arthritis (RA), other arthritic drugs (eg salicialate (eg aspirin), conventional non-steroidal anti-inflammatory molecules (NSAIDs) (eg Asaid, Arthrotec) , Cataflam, ibuprofen, Naproxen, etc.), COX-2 inhibitors (eg Celebrex, Vioxx), in this context, “combinations” are the same or different administrations in any order and in any dosage form. Mean simultaneous or sequential administration by route.

(7. Screening assays and animal models)
CRIg and CRIg agonists (including Ig fusions of CRIg and CRIg ECD) can be evaluated in various cell-based assays and animal models of diseases or conditions in which complement is implicated.

  Thus, for example, the efficiency of prevention and / or treatment of arthritis can be assessed in a model of collagen-induced arthritis, as shown in Example 7 below (Terato et al., Brit. J. Rheum. 35: 828- 838 (1966)). Potential prophylactic / therapeutic agents for arthritis are also described in Terato et al. Immunol. 148: 2103-8 (1992); Terato et al., Autoimmunity 22: 137-47 (1995), and antibodies induced by intravenous injection of a mixture of four monoclonal antibodies, as described in Example 8 below. Can be screened in a model of arthritis mediated by. Candidates for prevention and / or treatment of arthritis can also be studied in transgenic animal models (eg, TNF-α transgenic mice (Taconic)). These animals express human tumor necrosis factor (TNF-α), a cytokine involved in the pathogenesis of human rheumatoid arthritis. Expression of TNF-α in these mice results in severe chronic arthritis in the fore and hind legs, providing a simple mouse model of inflammatory arthritis.

During the last few years, animal models of psoriasis have also been provided. Thus, Asebia (ab), flaky skin (fsn), and chronic proliferative dermatitis (cpd) are spontaneous mouse mutations with psoriatic-like skin changes. Transgenics in which cytokines (eg, interferon-γ, interleukin-1α, keratinocyte growth factor, transforming growth factor-α, interferon-6, vascular endothelial growth factor, or bone morphogenetic protein-6) are overexpressed in the skin Mice can also be used for in vivo studies of psoriasis and for the identification of therapeutic agents for the treatment of psoriasis. Psoriasis-like lesions were reconstructed using CD4 ⁺ / CD45RB ^hi T lymphocytes in β ₂ -integrin hypomorphic mice backcrossed to the PL / J strain, in β ₁ -integrin transgenic mice. / Scid mice, as well as in HLA-B27 / hβ ₂ m transgenic rats. Xenograft models using human skin transplanted into immunodeficient mice are also known. Accordingly, the compounds of the present invention are disclosed in Schoon, M .; P. Nat. Med. (1997) 3: 183, which can be tested in the scid / scid mouse model. In this case, the mouse exhibits a histopathological skin lesion similar to psoriasis. Another suitable model is Nickoloff, B .; J. et al. Et al., Am. J. et al. Path. (1995) 146: 580. A human skin / scid mouse chimera prepared. For further details see, for example, Schoon, M. et al. P. , J .; See Invest Dermatology 112: 405-410 (1999).

Recombinant (transgenic) animal models can be manipulated by introducing the coding portion of the gene of interest into the genome of the animal of interest using standard techniques for the production of transgenic animals. . Animals that can be targeted for transgenic manipulation include mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates such as baboons, chimpanzees, and other monkeys, These are not limited. Techniques known in the art for introducing transgenes into such animals include pronucleic microinjection (Hope and Wanger, US Pat. No. 4,873,191); retrovirus-mediated gene transfer into the reproductive system ( For example, Van der Putten et al., Proc. Natl. Acad. Sci. USA, 82, 6148-615 (1985)); Gene targeting in embryonic stem cells (Thompson et al., Cell 56.313-321 (1989)) Electroporation of the embryo ((Lo, Mol. Cell. Biol. 3: 1803-1814 (1983)); sperm-mediated gene transfer (Lavitrano et al., Cell 57, 717-73 (1989)). For example, See the country Patent No. 4,736,866.

  For the purposes of the present invention, transgenic animals include animals having a transgene only in a portion of their cells (“mosaic animals”). The transgene can be incorporated either as a single transgene or as a concatamer (eg, head-to-head or head-to-tail tandem). Selective introduction of transgenes into specific cell types is described, for example, in Lasko et al., Proc. Natl. Acad. Sci. USA, 89, 623-636 (1992).

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

  Animals can be further tested for signs of immune disease pathology, for example, by histological experiments to determine the infiltration of immune cells into specific tissues. Blocking experiments can also be performed, in which case the transgenic animals have a degree of effect on complement and complement activation (including traditional mild and alternative complement pathways), or T cell proliferation. To determine, treat with CRIg or a candidate agonist. In these experiments, a blocking antibody that binds to a polypeptide of the invention is administered to an animal and the desired biological effect is monitored.

  Alternatively, a “knockout” animal that is deficient in CRIg or has another gene encoding CRIg can be transferred to an endogenous gene encoding the CRIg polypeptide and the same polypeptide introduced into the embryonic cells of the animal. It can be constructed as a result of homologous recombination between different genomic DNA encoding. For example, cDNA encoding CRIg can be used to clone genomic DNA encoding CRIg according to established techniques. The portion of genomic DNA encoding CRIg can be deleted or replaced with another gene (eg, a gene encoding a selectable marker that can be used to monitor integration). Usually, several kilobases of unaltered DNA (both 5 'and 3' ends) are included in the vector (see, for example, Thomas and Capecchi, Cell, 51: 503 for descriptions of homologous recombination vectors). (See 1987). The vector is introduced into an embryonic stem cell line (eg, by electroporation), and cells are selected in which the introduced DNA is homologously recombined with endogenous DNA (eg, Li et al., Cell, 69: 915 (1992)). The selected cells are then injected into undifferentiated embryonic cells of an animal (eg, mouse or rat) to form a chimeric population (see, eg, Bradley, Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. et al. J. Robertson, ed. (IRL, Oxford, 1987), pages 113-152). The chimeric embryo can then be transferred to a suitable pseudopregnant female foster animal, resulting in an embryo for creating a “knockout” animal. Progeny having DNA homologously recombined in their germline cells can be identified by standard techniques, including cells homologously recombined in all cells of the animal Can be used to breed animals. Knockout animals can be characterized, for example, for their ability to defend against certain pathological conditions and for the development of pathological conditions due to their absence of CRIg polypeptides.

  Thus, the biological activity of CRIg or its potential agonist can be further tested in mouse CRIg knockout mice as described in Example 7 below.

  Antigen-induced airway hypersensitivity, pulmonary eosinophilia, and inflammation are induced by animal sensitization with ovalbumin and subsequent animal challenge with the same protein administered by aerosol Asthma models have been described. Some animal models (guinea pigs, rats, non-human primates) exhibit symptoms similar to atopic asthma in humans when challenged with aerosol antigens. The mouse model has many of the characteristics of human asthma. Suitable procedures for testing CRIg and CRIg agonists for activity and efficacy in treating asthma are described by Wollynie, W. et al. W. Et al., Am. J. et al. Respir. Cell Mol. Biol. (1998) 18: 777 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, thereby producing a delayed type hypersensitivity reaction that is measured and quantified. Contact hypersensitivity initially has a sensitization phase followed by an induction phase. The induction phase occurs when epidermal cells encounter an antigen that they have previously contacted. Swelling and inflammation occur, which makes it an excellent model for human allergic contact dermatitis. Suitable procedures are described in Current Protocols in Immunology, J. MoI. E. Cologan, A .; M.M. Kruisbeek, D.M. H. Margulies, E .; M.M. Shevach and W.M. Strober, Hen, John Wiley & Sons, Inc. 1994, unit 4.2. Grabbe, S .; and Schwartz, T .; Immun. See also Today 19 (1): 37-44 (1998).

  Graft-versus-host disease occurs when immunocompetent cells are transplanted into patients who are immunosuppressed or tolerated. Donor cells recognize and respond to host antigens. Responses can vary from life-threatening severe inflammation to mild symptoms of diarrhea and weight loss. Some models of graft-versus-host disease provide a means of assessing T cell reactivity to MHC antigens and non-major transplant antigens. Suitable means are described in detail in Current Protocols in Immunology, supra, unit 4.3.

  Animal models of skin allograft rejection are a means of testing the ability of T cells to mediate tissue destruction in vivo, which is an indicator and measure of their role in antiviral and antitumor immunity is there. In the most common and accepted model, mouse tail-skin transplantation is used. Repeated experiments show that skin allograft rejection is mediated by T cells, helper T cells, and killer effector T cells, but not by antibodies. Auchincross, H.M. Jr. and Sachs, D.A. H. Fundamental Immunology, 2nd edition, W.M. E. Edited by Paul. , Raven Press, NY, 1989, 889-992. A suitable procedure is described in detail in Current Protocols in Immunology, supra, unit 4.4. Other transplant rejection models that can be used to test CRIg and CRIg agonists are described in Tanabbe, M .; Et al., Transplantation (1994) 58:23 and Tinubu, S. et al. A. Et al. Immunol. (1994) 4330-4338.

  An animal model of delayed type hypersensitivity provides a sufficient assay for cell-mediated immune function. A delayed type hypersensitivity reaction is an in vivo immune response mediated by T cells that is characterized by inflammation that does not reach a peak until a certain amount of time has elapsed after challenge with an antigen. These reactions also occur in tissue-specific autoimmune diseases such as multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE, model of MS). A suitable procedure is described in detail in Current Protocols in Immunology, supra, unit 4.5.

  EAE is an autoimmune disease mediated by T cells characterized by inflammation of T cells and mononuclear cells, followed by demyelination of the central nervous system axons. EAE is generally considered a relevant animal model for human MS. Bolton, C.I. , Multiple Sclerosis (1995) 1: 143. Both acute and relapsing remission models have been developed. CRIg and its agonists and antagonists have been tested for T cell stimulatory or inhibitory activity against immune-mediated demyelinating disease using the protocol described in Current Protocols in Immunology, above, 15.1 and 15.2. can do. Oligodendrocytes or Schwann cells are described in Duncan, I. et al. D. Et al., Molec. Med. See also a model for myelin disease transplanted into the central nervous system as described in Today (1997) 554-561.

  An animal model of age-related macular degeneration (AMD) consists of mice that have a null mutation in the Ccl-2 or Ccr-2 gene. These mice develop AMD cardiac features, including lipofuscin accumulation in the retinal pigment epithelium (RPE) and the druses beneath it, photoreceptor atrophy, and choroidal neovascularization (CNV) It is. These features develop after 6 months of age. CRIg and CRIg agonists can be tested for drusen formation, photoreceptor atrophy, and choroidal neovascularization.

  CNV can be tested in various models of laser-induced choroidal neovascularization. Thus, for example, CNV can be induced in rats and cynomolgus monkeys by intense laser photocoagulation resulting in choroidal neovascularization. The progression and treatment of this symptom may include, for example, fluorescein angiography, histopathological and immunohistochemical evaluation, and pharmacokinetics of serum collected from pre- and post-treatment animals at various time intervals, It can be assessed by hemolysis, antibody screening, and complement activation assays. The effectiveness of prophylactic administration is monitored for vascular leakage by fluorescein angiography, inhibition of complement accumulation at the site of laser incineration, eye testing, eye photography, taking vitreous and retinal tissue, etc. Can be monitored by similar methods. Further details are provided in the examples below.

  A model of myocardial ischemia reperfusion can be performed in mice or rats. Animals are tracheotomized and oxygenated with a small animal ventilator. A polyethylene catheter is inserted into the internal carotid artery and the external jugular vein for measurement of mean arterial blood pressure. Myocardial ischemia reperfusion is initiated by ligation of the left anterior descending artery (LAD) with a 6-O suture. Ischemia was caused by suturing a reversible ligation around the LAD to completely occlude the blood vessel. The ligature was removed after 30 minutes and the heart was perfused for 4 hours. CRIg and CRIg agonists can be tested for their efficacy by measuring cardiac infarct size, cardiac creatine kinase activity, myeloperoxidase activity, and immunohistochemistry using anti-C3 antibodies.

  Models of diabetic retinopathy include treatment of mice or rats with streptozotocin. CRIg and CRIg agonists can be tested for their effects on venous dilation, intraretinal microvascular abnormalities, and neovascularization of the retina and vitreous cavity.

  A model of membranoproliferative glomerulonephritis can be established as follows: female mice are injected i.p. with 0.5 mg control rabbit IgG in CFA. p. Immunize with (-7 days). After 7 days (day 0), 1 mg of rabbit anti-mouse glomerular basement membrane (GBM) antibody was injected i. v. Inject. The increase in anti-rabbit IgG antibody in the serum is measured by ELISA. A 24-hour urine sample is taken from the mouse in a metabolic cage and the renal function of the mouse is assessed by measuring urine protein in addition to blood urine nitrogen.

(7. Pharmaceutical composition)
The active molecules of the present invention (including polypeptides and their agonists), as well as other molecules identified by the screening assays disclosed above, may be used in pharmaceutical compositions for the treatment of inflammatory diseases. It can be administered in the form.

  The therapeutic form of an active molecule (preferably a CRIg polypeptide or CRI agonist of the present invention) is the optimal pharmaceutically acceptable carrier, excipient, or stabilizer for the active molecule of the desired purity. And are prepared for storage by mixing in the form of a lyophilized formulation or an aqueous solution (Reminoton's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are not toxic to the recipient at the dosages and concentrations used, such as phosphates, citrates, and other organic acids Buffers; antioxidants including ascorbic acid and methionine; preservatives (eg octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benztonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens (eg Catechol; resorcinol; cyclohexanol: 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; protein (eg, serum albumin, gelatin, or immunoglobulin) ; Hydrophilicity Limers (eg, polyvinylpyrrolidone); amino acids (eg, glycine, glutamine, asparagine, histidine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrin); chelating agents (Eg EDTA); sugars (eg sucrose, mannitol, trehalose or sorbitol); salt forming counterions (eg sodium); metal complexes (eg Zn-protein complexes): and / or non-ionic Surfactants such as TWEEN ™, PLURONICS ™, or polyethylene glycol (PEG).

  The 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 polypeptides, antibodies, or antibody fragments to cells. Where antibody fragments are used, the smallest fragment that specifically binds to the binding domain of the target protein is preferred. For example, peptide molecules that retain the ability to bind to a target protein sequence can be designed based on the variable region sequence of the antibody. Such peptides can be synthesized chemically and / or produced by recombinant DNA technology (eg, Marasco et al., Proc. Natl. Acad. Sci. USA, 90, 7889-7893 ( 1993)).

  The formulations herein may also contain one or more active compounds that are essential for the particular indication being treated, and preferably have complementary activities that do not adversely affect each other. It is what you are doing. Alternatively or additionally, the composition may include a cytotoxic agent, cytokine or growth inhibitory factor. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

  The active molecules are, for example, microcapsules prepared by coacervation technology or by interfacial polymerization (eg colloidal drug delivery systems (eg liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or It can also be entrapped in hydroxymethylcellulose or gelatin microcapsules and poly- (methyl methacrylate) microcapsules, respectively, in microemulsions. Such techniques are described in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. et al. Ed., (1980).

  Formulations used for in vivo administration must be sterile. This can be easily done by filtration through a sterile filtration membrane.

  Sustained release preparations may be prepared. Suitable examples of sustained release preparations include solid hydrophobic polymer semipermeable matrices containing antibodies. This matrix is in the form of a shaped material (eg, a membrane or microcapsule). Examples of sustained release matrices include polyesters, hydrogels (eg, poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactic acid (US Pat. No. 3,773,919), L-glutamic acid and □ Copolymers with ethyl L-glutamic acid, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers (eg LUPRON DEPOT ™ (injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly -D-(-)-3-hydroxybutyric acid, although polymers (eg, ethylene-vinyl acetate and lactic acid-glycolic acid) can release molecules for over 100 days, certain hydrogels have shorter time Protein can be released only between If the cellized antibodies remain in the body for extended periods of time, they may denature or aggregate as a result of exposure to moisture at 37 ° C., which results in loss of biological activity and immunogenicity. A rational method for stabilization depending on the mechanism involved can be considered, for example, the aggregation mechanism is the formation of intermolecular SS bonds by thio-disulfide exchange. Once found, stabilization is achieved by modifying sulfhydryl residues, lyophilization from acid solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions. It can be carried out.

  For intraocular administration, typical injectable formulations are used and are usually administered at intervals of about 6 weeks. The eye is anesthetized before each injection.

  However, it is also possible to use transplantation of sustained release formulations of CRIg or agonist (eg CRIg-Ig or CRIg ECD-Ig fusion) for intravitreal release.

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

  All patents and academic references cited herein are hereby incorporated by reference in their entirety.

  Commercially available reagents referred to in the examples were used according to manufacturer's instructions unless otherwise indicated. The source of these cells is identified in the examples below and throughout the specification by the ATCC registration number (the ATCC registration number is American Type Culture Collection, 10801, Universality Boulevard, Manassan, VA 20110-2209). .

Example 1
(Isolation of cDNA clone (PRO362) encoding human CRIg)
Sequence tags that are expressed using the extracellular domain (ECD) sequence of about 950 known secreted proteins (including secretion signals, if present) from the Swiss-Prot public protein database. EST) Prosecuted the database. The EST database includes a public EST database (eg, GenBank) and a private EST DNA database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, Calif.). The search was performed using the computer program BLAST or BLAST-2 (eg, Altshul et al., Methods in Enzymology 266: 460-480 (1996)) as a comparison of the ECD protein to a six-frame translation of the EST sequence. . These comparisons that produced a BLAST score of 70 (or 90 in some cases) that did not encode a known protein were collected and used with the program “phrap” (Phil Green, University of Washington, Seattle, Wash.). Assembled into consensus DNA.

  Consensus DNA sequences were assembled using phrap compared to other EST sequences. This consensus sequence was designated herein as DNA42257 (SEQ ID NO: 9) (see FIG. 32). Based on the DNA42257 (SEQ ID NO: 9) consensus sequence shown in Figure 32, oligonucleotides were identified 1) to identify a cDNA library containing the sequence of interest by PCR, and 2) a clone of the full-length coding sequence of CRIg Was synthesized for use as a probe for isolation. Forward and reverse PCR primers are generally in the range of 20 to 30 nucleotides and are often designed to produce PCR products that are approximately 100-1000 bp in length. The probe sequence is usually 40-55 bp in length. In some cases, additional oligonucleotides are synthesized if the consensus sequence is longer than about 1-1.5 kbp. In order to screen several libraries for full length clones, DNA from the libraries was screened by PCR amplification using pairs of PCR primers according to Ausubel et al., Current Protocols in Molecular Biology. Thereafter, using a positive library, a clone encoding a gene of interest was isolated using a probe oligonucleotide and a pair of primers.

PCR primers (forward and reverse) were synthesized:
Forward PCR primer 1 (42257.f1)

（配列番号１０）
正方向ＰＣＲプライマー２（４２２５７．ｆ２）

(SEQ ID NO: 10)
Forward PCR primer 2 (42257.f2)

（配列番号１１）
逆方向ＰＣＲプライマー１（４２２５７．ｒ１）

(SEQ ID NO: 11)
Reverse PCR primer 1 (42257.r1)

（配列番号１２）
逆方向ＰＣＲプライマー２（４２２５７．ｒ１）

(SEQ ID NO: 12)
Reverse PCR primer 2 (42257.r1)

（配列番号１３）。

(SEQ ID NO: 13).

In addition, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA42257 sequence. This had the following nucleotide sequence:
Hybridization probe (42257.p1)

（配列番号１４）
全長クローンの供給源についていくつかのライブラリーをスクリーニングするために、ライブラリーに由来するＤＮＡを、上記のＰＣＲプライマーの対を用いたＰＣＲ増幅によってスクリーニングした。その後、ポジティブライブラリーを使用して、プローブオリゴヌクレオチドと一対のＰＣＲプライマーとを使用してＣＲＩｇ遺伝子をコードするクローンを単離した。

(SEQ ID NO: 14)
To screen several libraries for a source of full-length clones, DNA from the libraries was screened by PCR amplification using the PCR primer pairs described above. A positive library was then used to isolate a clone encoding the CRIg gene using a probe oligonucleotide and a pair of PCR primers.

  RNA for construction of the cDNA library was isolated from human fetal brain tissue (LIB153). The cDNA library used to isolate the cDNA clones was constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA. The cDNA is primed with oligo dT containing a NotI site blunt-ended to a SalI to mikinase adapter, cut with NotI, sized by gel electrophoresis and properly cloned in a defined orientation. PRKB or pRKD: pRK5B is a precursor of pRK5D and does not contain an SfiI site; Holmes et al., Science 253: 1278-1280 (1991), cloned at unique XhoI and NotI sites.

  The DNA sequence of the isolated RIg polypeptide was obtained by the DNA sequence of the clone isolated as described (designated herein as UNQ317 (DNA45416-1251) (SEQ ID NO: 1)).

  The entire nucleotide sequence of UNQ317 (DNA45416-1251) is shown in FIG. 1 (SEQ ID NO: 1). Clone UNQ367 (DNA45416-1251) (SEQ ID NO: 1) contains one open reading frame with an apparent translation start site at nucleotide positions 1082-1084 (FIG. 1, SEQ ID NO: 1). The predicted polypeptide precursor is 321 amino acids long (Figure 1, SEQ ID NO: 2). The CRIg protein shown in FIG. 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 (SEQ ID NO: 2) as shown in FIG. 1 revealed that the glycosaminoglycan binding site from about amino acid position 149 to about 152 amino acid and about 306 amino acid to about 306 amino acid. The presence of a transmembrane domain up to the amino acid at the position was revealed. Clone UNQ317 (DNA45416-1251) has been deposited under ATCC Deposit No. 209620.

  Similar to members of the JAM family, CRIg (PRO362) (more recently referred to as CRIg) is a type 1 transmembrane molecule and a member of the immunoglobulin superfamily. The extracellular domain of the long form of human CRIg (huCRIg (L)) encodes both V and C2 terminal Ig domains (Smith and Xue, J. Mol. Biol., 274: 530-545). (1997)), the short form (huCRIg (S)) encodes only one V-type Ig and is similar to mouse CRIg (muCRIg) (FIG. 42A). The C-terminal cytoplasmic domain of human and mouse CRIg contains the consensus AP-2 internalization motif (YARL and DSQALI, Bonafacino & Traub Ann Rev Biochem 72, p395 (2003)). huCRIg and muCRIg have 67% overall sequence homology and 83% homology in the IgV domain. Among members of the JAM family, huCRIg is most closely related to JAM-A. Sequence similarity confirmed that the stretch of residues forming the Ig domain fold was conserved (FIG. 42A). Both human and mouse CRIg were present at the Xq12 position on the X chromosome, and they had a synteny position on the chromosome adjacent to hephaestin and moesin.

(Example 2)
(Protein production and purification)
The extracellular domains of huCRIg and muCRIg were cloned into a modified pRK5 expression vector encoding a human or mouse IgG1 Fc region downstream of the CRIg sequence. The Fc portion of mouse IgG1 contains a double mutation (D265A, N297A), thereby preventing Fc receptor binding (Gong et al., J. Immunol. 174: 817-826 (2005)). Used to control regulation: human IREM-1 and mouse CLM-1 Fc fusion protein or mouse anti-gp120 IgG antibody were used as controls.LFH-tagged CRIg was used in yeast leucine zipper, Flag, and C-terminus ( 6) Created by fusing CRIg ECD to histidine, protein was overexpressed in CHO cells by transient transfection, cells based on F-12 / Dulbecco modified Eagle medium , Ultra-Low IgG serum (Invitrolog n) and in a fully automated bioreactor using medium supplemented with Primatone HS (Sigma) Cultures were maintained for 7-12 days until harvested Fc fusion proteins were analyzed by protein A affinity chromatography. Subsequently, it was purified by Sephacryl S-300 gel filtration, LFH fusion protein was purified on a nickel column, and human CRIg-ECD protein was affinity purified on a Millipore Glyceryl-CPG (173700404) column adsorbed with monoclonal antibody 3C9. The protein is eluted at pH 3.0, and huCRIg-HIS and muCRIg-HIS are cloned into a baculovirus expression vector containing a C-terminal (6) histidine and CRIg ECD is cloned. Plasmid DNA was transfected into Sf9 cells, the supernatant was used to infect H5 cells, and the protein was purified on a nickel column.All purified protein entities were analyzed by N-terminal sequence analysis. The lipopolysaccharide concentration was <5 Eu / mg for human or mouse CRIg preparations.

(Example 3)
(Preparation of antibody)
Polyclonal antibodies were generated by immunization of New Zealand rabbits with 200 μg huCRIg (L) -His in complete Freud's adjuvant, followed by a boost 6 weeks after the first immunization. Monoclonal antibodies against muCRIg and huCRIg were generated by immunization of Wistar rats and Balb / c mice with 50 μg of his-tagged CRIg fusion protein by injection into the leg pad. Clones were selected by ELISA, FACS, Western blot, and immunohistochemistry based on reactivity with human and mouse CRIg-ECD. The resulting antibody was used in subsequent studies unless otherwise stated.

Example 4
(Infiltration of inflammatory cells into guinea pig skin)
The following examples show that huCRIg (PRO362) is pro-inflammatory in that it stimulates the infiltration of inflammatory cells (ie, neutrophils, eosinophils, monocytes, or lymphocytes) into the skin of guinea pigs. Indicates that there is. The assay described herein monitors the ability of this protein to induce inflammatory cell infiltration into guinea pig skin. Compounds that stimulate inflammatory infiltrates are therapeutically useful when enhancing the inflammatory response is effective. Compounds that inhibit lymphocyte proliferation are therapeutically useful when suppression of the inflammatory response is effective. The therapeutic agent may be in the form of, for example, a mouse-human chimera, a humanized or human antibody against CRIg, a small molecule, a peptide, etc. (which mimics the biological activity of CRIg), a CRIg fusion protein, a CRIg extracellular region, etc. it can.

  Hairless guinea pigs (Charles River Labs) weighing 350 grams or more were anesthetized intramuscularly with ketamine (75-80 mg / kg body weight) and xylazine (5 mg / kg body weight). Protein samples of huCRIg protein and control protein were injected intradermally into the back of each animal in a volume of 100 μl per injection site. One animal was injected at approximately 16-24 sites. 1 mL of Evans blue dye (1% physiological buffered saline) was injected into the endocardium. The animals were anesthetized 6 hours later and individual skin injection sites were biopsied and fixed in formalin. The skin was prepared for histopathological evaluation. Each site was evaluated for inflammatory cell infiltration into the skin. Sites with viable inflammatory cells were scored as positive. Samples that induced inflammatory cell infiltration were scored as pro-inflammatory substances. The CRIg tested is positive in this assay, indicating anti-inflammatory activity.

(Example 5)
(Expression of CRIg (PRO362) mRNA and polypeptide)
(A. In situ hybridization and immunohistochemistry)
CRIg mRNA expression was assessed in various tissue types by in situ hybridization, immunohistochemistry, and RT-PCR.

For in situ hybridization, tissues were fixed (4% formalin), embedded in paraffin, sectioned (3-5 μm thick), deparaffinized, and proteinase K slow protein (20 μg / ml) (37 For 15 minutes) and processed for in situ hybridization. Probes for the polypeptides of the invention were generated by PCR. Primers included a T7 or T3 RNA polymerase initiation site to allow in vitro transcription of sense or antisense probes from the amplified product. Sense and antisense probes labeled with ³³ P-UTP were hybridized overnight (55 ° C.), washed (0.1 × SSC, 2 hours at 55 ° C.), and NBT2 nuclear track emulsion (Eastman Kodak, Rochester, NY) ), Baked (4-6 weeks at 4 ° C.), developed, and counterstained with hematoxylin and eosin. A representative pair of bright field and dark field images is typically shown.

  Immunohistochemical staining was performed on 5 mm thick frozen sections using a DAKO Autostainer. Endogenous peroxidase activity was blocked with Kirkegaard and Perry Blocking Solution (1:10, 4 min at 20 ° C.). 10% NGS (DAKO) in TBS / 0.05% Tween-20 was used for dilution and blocking. MAb 4F722.2 anti-CRIg (anti-PRO362) 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 with Vector Labs Standard ABC Elite Kit (Vector Labs, Burlingame, Calif.). Slides were developed using Pierce metal-enhanced diaminobenzidine (Pierce Chemicals, Rockford, IL). Dual immunohistochemistry for CRIg (PRO362) and CD68 expression was performed on frozen sections to reveal localization of CRIg expression on macrophages. (DAKO) mAb 4F7.22.2 anti-CRIg and anti-CD68 mAb KP-1 were utilized and detected by phycoerythrin and FITC markers, respectively.

  Expression was tested in a wide variety of different tissue and cell types from humans and other mammals.

(A. Normal tissue)
Normal adult human tissues tested include tonsils, lymph nodes, spleen, kidney, urine, bladder, lung, heart, aorta, coronary artery, liver, gallbladder, prostate, stomach, small intestine, colon, pancreas, thyroid, The skin, adrenal gland, placenta, uterus, ovary, testis, retina, and brain (cerebellum, brain stem, cerebral cortex) were included. Normal human fetal tissues, including E12-16 week old brain, spleen, intestine, and thyroid were also examined. In addition, expression in mouse liver was also examined.

(B. Inflammatory tissue)
Inflamed tissues examined by in situ hybridization include chronic asthma, chronic bronchitis, lung with chronic bronchitis / chronic obstructive pulmonary disease, kidney with chronic lymphocytic interstitial nephritis, and chronic inflammation and chronic Tissues with chronic inflammatory diseases such as liver due to hepatitis caused by hepatitis C infection, autoimmune hepatitis or alcoholic cirrhosis were included.

(C. Primary neoplasm)
Primary human neoplasms tested by in situ hybridization for expression of PRO362 included breast cancer, lung squamous cell cancer, lung adenocarcinoma, prostate cancer, and colon adenocarcinoma.

(2. Results)
CRIg (PRO362) is a cryosection of mouse liver (FIG. 6), a cryosection of human liver (FIG. 7), and a number of tissue macrophage-like cells (colon macrophages (FIG. 8A), Kupffer cells (FIG. 8B), Includes adrenal macrophages (FIG. 8C), Hofbauer cells (FIG. 8D), synovial cells (FIG. 9), alveolar macrophages, resident macrophages in the lamina propria, and stromal macrophages in many tissues ) Was revealed. CRIg was also significantly expressed in brain microglial cells (FIG. 10). CRIg expression may include neoplasms or inflammatory diseases (including rheumatoid arthritis (Figure 9), inflammatory bowel disease, chronic hepatitis (Figure 12), pneumonia, chronic asthma (Figure 11), glioma, and bronchitis) When activated by the presence of, it increased significantly in these tissues.

  In order to further test the expression of CRIg, immunohistochemical staining was performed on various tissue types. Double immunohistochemical staining for CRIg and CD68 was performed on tissue macrophages (including adrenal macrophages, liver Kupffer cells, brain microglia, placental Hofbauer cells), and CRIg and CD68 were the same tissue It was determined whether it was expressed in.

  CRIg is co-expressed with CD68 in adrenal macrophages (FIG. 13), liver Kupffer cells (FIG. 14), brain microglia (FIG. 15), and placental Hofbauer cells (FIG. 16). It was revealed.

(Example 6)
(Involvement of CRIg (PRO362) in chronic inflammation)
A novel macrophage-related receptor having homology to the A33 antigen and JAM1 was cloned as described in Example 1 and below, which was associated with the macrophage association of one transmembrane Ig superfamily member. Identified as a polypeptide (CRIg or PRO362).

  CRIg is expressed as two types of spliced variants. One variant is a 399 amino acid polypeptide containing an N-terminal IgV-like domain and a C-terminal IgC2-like domain, called huCRIg or huCRIg-long (SEQ ID NO: 4). The spliced form (which is 355 amino acids long and lacks the C-terminal domain) is called huCRIg-short (SEQ ID NO: 6). Both receptors have a transmembrane domain and a cytoplasmic domain containing tyrosine residues that are structurally phosphorylated in macrophages in vitro.

  This study revealed that CRIg is selectively expressed in a subset of tissue-resident macrophages and is associated with chronic inflammation.

(Materials and methods)
(cell)
Blood was obtained from healthy adult volunteers by venipuncture after informed consent and separated using Ficoll-Paque PLUS (Amersham Pharmacia Biotech) according to the manufacturer's instructions. PBMC were obtained from the interface, washed with chilled PBS, dissolved in 0.2% NaCl for 30 minutes and neutralized with 1.6% NaCl. Cells were counted and kept on ice until use. An unused MACS kit (Miltenyi Biotech, Auburn, Calif.) Was used according to the manufacturer's instructions to isolate peripheral blood subsets. Differentiation to macrophage phenotype was performed for up to 2 weeks in HG-DMEM medium containing 10% (v / v) autologous human serum, 20% fetal bovine serum, and 10 mM L-glutamine, penicillin, and streptomycin. CD14 ⁺ monocytes were induced by culturing. On day 5, the medium was changed. For flow cytometric analysis, cells were dissociated from the culture dish using ice-cold cell dissociation solution (Sigma). Lysates for Western blot analysis were prepared by adding 0.5 ml lysis buffer directly to the wells. Lysates were mixed with sample buffer containing SDS and β-mercaptoethanol, run on a Tris-Glycine gel and transferred to a nitrocellulose membrane. Cell viability was assessed by trypan blue dye exclusion.

(Flow cytometry)
Cells for use in flow cytometric analysis were blocked with PBS containing 2% fetal calf serum and 5 μg / ml human IgG (Calbiochem, San Diego, Calif.) At 4 ° C. for 30 minutes. The cells were then incubated with 3C9 (anti-CRIg (anti-PRO362) monoclonal antibody). After washing with PBS, cells were stained with phycoerythrin (PE) -conjugated antibodies against CD11b, CD14, CD163, CD15, CD68 obtained from Pharmingen.

(Cell-cell adhesion experiment)
The pRK expression vector containing full length CRIg was stably expressed in human Jurkat T cell line using neomycin selection and autoclone sorting as described elsewhere. Cells were preloaded with the fluorescent dye BCECF (Molecular Probes, Oregon) and 96-well Maxisorb plates (CORNIG) coated with a monolayer of human umbilical vein endothelial cells (HUVEC) treated or not with 10 ng / ml TNFα. (Trademark)). Cells are generally loaded into wells with incubation buffer (HBSS containing 10 mM CaCl, 10 mM magnesium, and 1.5 mM NaCl), and then the plate is placed upside down on a piece of blotting paper. By gentle washing. After three washes, fluorescence was counted with a fluorescence spectrometer. The fluorescence reading represents the number of cells that remain attached to the HUVEC cells.

(Northern blot analysis)
Multiple tissue northern blots (CLONTECH) were probed with ³² P-labeled full length CRIg cDNA randomly primed using the Ambion kit according to manufacturer's recommendations. Blots were exposed to a phosphoraging screen at 22 ° C. for 4 hours. Blots are stripped and pre-probed with a commercially available probe (Clontech) for human or mouse β-actin to assess RNA loading and quantity in individual lanes and Storm® phosphorImager (Molecular Dynamics). , Sunnyvale, CA).

(Real-time RtPCR analysis)
For quantitative PCR analysis (TAQMAN ™), total mRNA (100 ng) from human tissues or primary cells is recommended (PerkinElmer Life Sciences) with primers based on the coding sequence of CRIg.

(Production of Fc and His fusion proteins)
Human CRIg was cloned into the baculovirus expression vector pHIF (Pharmingen). The HIS-tagged CRIg fusion protein is composed of the extracellular domain of CRIg fused to 8 histidines. HIS-tagged fusion protein was purified from the supernatant of baculovirus infected insect cells grown in suspension using nickel affinity resin.

(Production of monoclonal and polyclonal antibodies)
For this experiment, BALBc females were treated with Ghilardi et al. Biol. Chem. 277: 16831-16836 (2002). Immunization and boosting were performed with 10 μg CRIg-His8 by injection into the leg pad as previously described. Single clones were screened against CRIg-His by ELISA. Selected clones were tested against JAM family members and human IgG Fc. Clones were titrated to a density of 1 cell and prescreened. Clone 3C9 (IgG1) was found to be selectively reactive to CRIg. Multiple clones were used to generate ascites and purified with Protein G (Amersham Pharmacia Biotech). Protein concentration was determined using Pierce BCA reagent (Pierce, Rockford, IL).

  Polyclonal antibodies were made by injecting 150 μg CRIg-His into New Zealand Rabbit. Serum titers were determined by ELISA. Serum was collected at the peak of circulating IgG levels and purified on a protein A column.

(In situ hybridization)
PCR primer (upstream primer:

（配列番号１８）、および下流プライマー：

(SEQ ID NO: 18), and downstream primers:

（配列番号１９））を、ｈｕＪＡＭ４の７００ｂｐの断片を増幅するように設計した。プライマーには、Ｔ７またはＴ３ ＲＮＡポリメラーゼ開始部位を含め、これによって、それぞれ、増幅産物からセンスプローブまたはアンチセンスプローブがインビトロで転写されるようにした。正常なヒト組織には、扁桃腺、リンパ節、脾臓、腎臓、肺、および心臓を含めた。慢性的な炎症性疾患を有している組織には、慢性喘息、慢性気管支炎による肺、慢性的なＣ型肝炎感染が原因で慢性的な炎症および肝炎を有している肝臓を含めた。組織を、４％のホルマリン中に固定し、パラフィンで包埋し、切片とし（３〜５μｍの厚み）、脱パラフィン化し、２０μｇ／ｍｌのプロテイナーゼＫで徐タンパクし（３７℃で１５分間）、別に記載したようにインサイチュハイブリダイゼーションのために処理した。

(SEQ ID NO: 19) was designed to amplify a 700 bp fragment of huJAM4. The primers included a T7 or T3 RNA polymerase start site, which allowed the sense or antisense probe to be transcribed in vitro from the amplification product, respectively. Normal human tissues included tonsils, lymph nodes, spleen, kidneys, lungs, and heart. Tissues with chronic inflammatory disease included chronic asthma, lung with chronic bronchitis, and liver with chronic inflammation and hepatitis due to chronic hepatitis C infection. Tissues were fixed in 4% formalin, embedded in paraffin, sectioned (3-5 μm thickness), deparaffinized, slowly proteinated with 20 μg / ml proteinase K (15 minutes at 37 ° C.), Processed for in situ hybridization as described elsewhere.

(Immunohistochemistry)
Human liver was obtained from Ardais Corporation, Lexington, MA. Immunohistochemical staining was performed on frozen liver sections 5-6 μm thick using a DAKO autosteiner. Endogenous peroxidase activity was blocked with Kirkegaard and Perry blocking solution (1:10, 4 min at 20 ° C.). 10% normal goat serum (NGS) in TBS / 0.05% Tween-20 was used for dilution and blocking. Mab 3C9 was used at 1 μg / ml. Slides were developed using metal-enhanced diaminobenzidine (Pierce Chemicals).

  For immunofluorescence staining of sections, sections were blocked with PBS / 10% NGS and incubated with mAb 3C9 for 1 hour at 20 ° C. Rabbit anti-mouse FITC labeled secondary antibody conjugated to FITS was used as a detection reagent. Subsequently, sections were stained with a PE-conjugated monoclonal antibody against human CD68 for the double staining procedure.

(result)
As described in Example 1, huCRIg was cloned from a human fetal cDNA library using degenerate primers that recognize the conserved Ig domain of human JAM1. Sequencing of several clones revealed an open reading frame of 321 amino acids (Figure 1, SEQ ID NO: 2). Blast search confirmed similarity to Z39Ig (type 1 transmembrane protein) (Langnaese et al., Biochim Biophys Acta 1492: 522-525 (2000)). It was later revealed that this 321 amino acid protein lacks several C-terminal amino acid residues. The full-length huSIgMA protein has been determined to have 399 amino acid residues as shown in FIG. 2 (SEQ ID NO: 4). The extracellular region of CRIg consisted of two Ig-like domains, including an N-terminal V-set domain and a C-terminal C2-set domain. CRIg-short (305 amino acids, FIG. 3, SEQ ID NO: 6) (which lacks the membrane-proximal IgG domain), a splicing variant of CRIg, using 3 ′ and 5 ′ primers Cloned.

(Cloning and sequence of mouse CRIg compared to human CRIg)
The mouse expressed sequence tag (EST) database was searched using the full-length open reading frame of huCRIg and the tblastn algorithm. DNA sequencing of the three clones yielded the same complete 280 amino acid open reading frame. The full-length transcript was cloned from a mouse spleen library using primers for the 3 prime region. The mouse clone resembled the spliced form of huCRIg in that it lacked the C-terminal Ig-like domain. The extracellular IgV domain was well conserved between the human and mouse receptors and had 93% identity. The mouse cytoplasmic domain was less conserved, 20 amino acids shorter than its human counterpart, and 40% identity. The nucleic acid encoding mouse CRIg (muCRIg) and the deduced amino acid sequence are shown in FIG. 4 as SEQ ID NOS: 7 and 8, respectively.

(CRIg is expressed in a subset of resident macrophages in various tissues and its expression is increased in inflammation)
Northern blot analysis of huCRIg shows two transcripts, 1.5 kb and 1.8 kb (FIG. 17), with the highest expression in the adrenal gland, lung, heart, and placenta, and other organs ( For example, it had low expression in the spinal cord, thyroid, mammary gland, and lymph nodes). In all tissues, the 1.8 kb transcript is the most abundantly expressed transcript, which probably encodes the long form of CRIg. One transcript of approximately 1.4 kb was detected in mouse liver and heart.

(TAOMAN ™ real-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 seen in the myeloid cell line HL-60 and the monocyte cell line THP-1 treated with PMA. Expression was absent from B and T cell lines (FIG. 18A).

(Expression of CRIg in differentiated monocytes)
In order to establish details when CRIg is expressed in differentiating monocytes / macrophages, we have developed non-adherent monocytes induced to differentiate in the presence of human autologous serum and The level of CRIg mRNA in adherent monocytes was determined. CRIg mRNA levels increased stepwise with time and reached a maximum level 7 days after plating (FIG. 18B). At this differentiation stage, mRNA levels were 100 times higher than mRNA levels in undifferentiated monocytes.

  Western blotting of monocyte / macrophage lysates showed increased expression of CRIg protein in parallel with increased expression of CRIg mRNA (FIG. 18C), indicating that CRIg was able to form monocytes into macrophages. It is expressed during differentiation. A 48 kDa band and a 40 kDa band are evident on the blot, which probably indicate the long and short forms of human CRIg.

(CRIg molecule characterization)
CRIg migrates similarly under reducing and non-reducing conditions, indicating that it is expressed as a monomer (FIG. 19A). Even when CRIg was deglycosylated using PNGase F, only a slight change in the migration pattern was observed, indicating that N-glycosylation is not important. CRIg was phosphorylated when cells overexpressing CRIg were treated with pervanadate (FIG. 19B). Phosphorylated CRIg migrated as a slightly larger Mw protein (55 kDa). In human HEK293 cells, the tyrosine phosphorylated CRIg cytoplasmic domain does not recruit Syk kinase (results not shown).

(Flow cytometric analysis of CRIg expression in peripheral blood mononuclear cells)
To determine the expression pattern of CRIg in circulating leukocytes, flow cytometric analysis was performed on lymphocytes isolated from blood from healthy donors using monoclonal anti-human CRIg antibody 3C9. Antibodies were generated by immunizing Balb / C mice with octa-His tagged human CRIg extracellular domain. The antibody is a non-blocking antibody that can be used to detect naturally occurring proteins in cryosections fixed with acetone directly linked to ALEXA ™ A488. Counterstaining was performed with PE-conjugated antibodies against several immune cell surface antigens. CRIg was not present on the surface of all leukocytes (including B cells, T cells, NK cells, monocytes, and granulocytes) (FIG. 20). However, CRIg was expressed in monocytes cultured for 7 days in macrophage differentiation medium.

(Regulation of CRIg expression in monocytes)
To study the regulation of CRIg expression, macrophages were cultured for 7 days in the presence of various pro- and anti-inflammatory cytokines and the expression level of CRIg was determined by real-time PCR or flow analysis. CRIg mRNA expression increased after 2 days of treatment with macrophages with IL-10 and TGF-β and was down-regulated by IL-4, IL13, and LPS (FIG. 21A). Treatment with dexamethasone increased expression 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 monocytes treated with various cytokines and dexamethasone for 5 days. CRIg was detected using monoclonal antibody clone 3C9 conjugated to ALEXA ™ A488. Cells were co-stained with anti-CD14 antibody. High surface expression of CRIg was seen after 5 days of monocyte treatment with IL-10 and LPS (FIG. 21B). A dramatic increase in the expression of CRIg on the surface was seen after treatment with dexamethasone.

(Intracellular distribution of CRIg)
To study the intracellular distribution of CRIg, monocyte-derived macrophages (MDM) were maintained in culture for 15 days, after which they were fixed and then monoclonal antibody (clone 3C9) or polyclonal antibody 4F7, then Stained with FITC-conjugated secondary antibody and PE-labeled anti-CD63 antibody. Confocal microscopy showed high expression of CRIg in the specific cytoplasm, which overlapped with the expression of lysosomal membrane protein CD63 (FIG. 22). CRIg was also expressed at the tip and end of macrophages whose staining pattern did not overlap with the CD63 staining pattern.

(Expression of CRIg in normal and diseased tissues)
The expression of CRIg in tissue resident macrophages and its expression in tissues of chronic inflammatory disease were studied. Using in situ hybridization, changes in the expression of CRIg mRNA were determined on a panel of human tissues fixed with paraformaldehyde. High expression levels were seen in alveolar macrophages obtained by autopsy of lungs of patients with pneumonia or chronic asthma (FIGS. 23, A, B, C, and D). High mRNA expression was found in Kupffer cells in the liver of patients with chronic hepatitis (FIGS. 23, E and F).

  In previous studies (Walker, Biochimica et al., Biophysica Acta 1574: 387-390 (2002)) and library electrical screening, high expression of CRIg mRNA was found in the synovium of patients with rheumatoid arthritis. Therefore, the expression pattern of CRIg in the synovium obtained from patients with rheumatoid arthritis, osteoarthritis, and degenerative bone disease was studied. High expression of CRIg mRNA was seen in synovial cells obtained from osteoarthritis patients (Figure 24, B). Surface synovial cells had the highest CRIg expression (FIG. 24, D). In addition, polyclonal antibody 6F1 was used to study the expression of CRIg in frozen sections of human synovium obtained from patients with rheumatoid arthritis. CRIg was expressed in a subset of synovial cells (20-40%) and tissue macrophages in the synovium (FIGS. 25, A, B, C). These cells were most likely type A macrophage-like synoviocytes. Staining was absent in the control synovium (FIG. 25D).

  CRIg protein expression was found in macrophages in a number of different tissues. Cryosections prepared from CHO cells stably expressing CRIg showed membrane localization of CRIg (FIG. 26A). CRIg protein is expressed in alveolar macrophages (FIG. 26, B), histocytes in the lamina propria (FIG. 26, C), placental Hofbauer cells (FIG. 26, D), and adrenal macrophages (FIG. 26, E). And in Kupfer cells of the liver (FIG. 26, F).

  Atheromatous plaques contained a large number of macrophages or macrophage foam cells and were firmly attached to the lumen wall of the aorta. Considering the role of CRIg in macrophage-endothelial adhesion, the expression of CRIg in atheromatous plaques was studied. Another section of plaque was stained with anti-CD63 (FIG. 27, A and B) or anti-CRIg (FIG. 27, C and D). Overlapping staining patterns of anti-CD63 and CRIg are seen in foam cells parallel to the vessel wall, indicating a role for CRIg in atherosclerosis.

  To determine whether CRIg is selectively expressed in macrophages, double immunofluorescent staining was performed on cardiac stromal macrophages (FIG. 28). As shown in the overlay (FIG. 28, third panel), most of the stromal macrophages that were positive for CRIg were also positive for CD68. Not all CD68 positive macrophages are also positive for CRIg, indicating that CRIg positive macrophages are specific for tissue resident macrophage subtypes.

  In order to quantitatively determine the expression level of mRNA in inflammatory bowel disease (IBD) syndrome, mRNA was obtained from colon tissue obtained from patients with ulcerative colitis, Crohn's disease, or patients who did not develop IBD. Extracted from. Real-time PCR was performed using primers specific for CRIg to determine relative expression levels. The expression level was 16 times higher in patients with ulcerative colitis and 5 times higher in patients with Crohn's disease compared to control tissues (FIG. 29, A). Similarly, relative RNA equivalents were determined in lung tissue and were found to be highest in tissues from patients with chronic obstructive pulmonary disease (COPD) (14 times normal tissue) In asthmatic patients, there was no significant difference from normal tissues (FIG. 29, B).

  Ig superfamily of molecules are well known to mediate cell surface recognition and cell-cell adhesion. Since CRIg expression was high in stromal macrophages parallel to blood vessels, the involvement of CRIg in macrophage-endothelial cell adhesion was studied. A Jurkat cell line stably transfected with full length CRIg-long (FIG. 30A) was loaded with the fluorescent dye BCECF and added to the wells of a 96-well maxisorb plate to culture a monolayer of HUVEC cells. Adhesion was measured by the amount of fluorescence retained after 3 gentle washes. Jurkat cells expressing CRIg were more adherent to both control and TNFα-stimulated endothelial cells compared to Jurkat cells stably transfected with the control plasmid (FIG. 30B). .

(Discussion)
In this study, we first described the tissue distribution, regulation of expression and molecular characterization of CRIg / Z39Ig, a member of a novel Ig superfamily, and confirmed its selective expression in tissue-resident macrophages.

  CRIg expression was seen in resident macrophages with a fully differentiated phenotype. Its expression was increased in tissues with chronic inflammation such as rheumatoid arthritis and inflammatory bowel disease. Increased expression of CRIg in these diseases (often characterized as Th2-type disease) may be consistent with the regulation of its expression by Th2 cytokines in vitro. Whether this high expression is due to increased presence of CRIg positive macrophages or due to increased expression in inflammatory macrophages has not yet been determined.

  CRIg can mediate one of the effector functions of human macrophages, including bacterial recognition, phagocytosis, antigen presentation, and cytokine release. These results indicate the role of CRIg in macrophage adhesion to blood vessel endothelial cell walls and possibly motility.

  CRIg expression was increased in non-microbial inflammatory diseases such as ulcerative colitis and chronic obstructive pulmonary disease (COPD), but LPS or other such as lipoteichoic acid or bacterial lipoproteins Were down-regulated with isolated macrophages. Long-term treatment with LPS for 2 days resulted in increased expression of CRIg. This may be due to the autocrine effect of IL-10 secreted by LPS stimulated macrophages. Significant CRIg up-regulation at both mRNA and protein levels was observed when monocytes or macrophages were treated with dexamethasone. A small number of monocyte / macrophage surface receptors have been shown to increase expression when treated with dexamethasone. One example is CD163, but its induction by dexamethasone is less dramatic. Up-regulation of CRIg by anti-inflammatory cytokines IL10 and TGFβ is of considerable interest, indicating that CRIg may mediate the anti-inflammatory role of glucocorticoids.

  As described herein, CRIg was expressed in a subset of CD68 positive macrophages capable of presenting activated macrophages. Blocking and activating antibodies against CRIg and CRIg-Fc fusion proteins were used to study their role in macrophage effector function, adhesion and migration, and their role in chronic inflammatory disease and described in Example 7 did.

  Only a few surface markers were specifically expressed on differentiated macrophages such as CD68 and CD163. CD68 was clearly expressed in all human macrophage populations, but the antigen could also be detected in other bone marrow cells and in certain non-myeloid cells. Thus, CRIg presents a first cell surface antigen that is selectively expressed on a subset of stromal mature macrophages.

(Example 7)
(CRIg fusion protein in collagen-induced arthritis (CIA) in DBA-1J mice)
The purpose of this experiment was to compare CRIg fusion protein against control mouse IgG1 in the onset and progression of disease of CIA (collagen induced arthritis, experimental animal model system for rheumatoid arthritis).

  As discussed in Example 4, CRIg is highly and specifically expressed in a subset of macrophages and is increased in tissues with chronic inflammation. Murine CRIg is highly expressed in macrophages and in synovial cells of inflamed joints of collagen-induced arthritic mice. In vitro experiments have shown that CRIg is involved in macrophage adhesion to endothelial cells. CRIg-Fc fusion proteins either by affecting the properties of tissue macrophages or by affecting the immune response of other cells (eg, T cells, B cells, epithelial cells, endothelial cells) Affects the course of autoimmune disease (in this case, collagen-induced arthritis in mice). This can result in reduced joint inflammation, swelling, and prolonged bone loss.

  A muCRIg-Fc fusion protein was made by fusing the mouse IgG1 hinge, CH2, and CH3 domains to the extracellular domain of mouse CRIg (aa1-200). A fusion containing a double mutation to prevent Fc receptor binding was used as a control for Fc receptor modulation. The nucleotide sequence of the muCRIg-Fc fusion protein is shown in SEQ ID NO: 17. (Similar huCRIg-Ig and huCRIg-short-Ig coding sequences are shown in sequences 15 and 16, respectively). Protein was produced in CHO cells by transient transfection of plasmid DNA. The fusion protein was purified by placing the cell supernatant on a protein A column followed by ion exchange chromatography to remove aggregates. Serum half-life was estimated by injection of a single dose of 4 mg / kg CRIg-Fc into C57B6 mice, followed by collection of serum from the mice at specified time intervals. Serum levels of mouse CRIg-Fc were determined by sandwich ELISA using anti-CRIg mAbs that recognize various epitopes on the extracellular domain of CRIg.

Animal model species: mouse strain (s): DBA-1J
Supplier (s): JACKSON
Age range: 7-8 weeks old Collagen-induced arthritis (CIA) is inflammatory polyarthritis and has clinical and pathological features similar to human rheumatoid arthritis (RA) Mice were selected as species for studying CIA. This animal model is used in many laboratories, and the histopathology of CIA is that of synovial fluid that progresses to pannus formation, cartilage degeneration / disintegration, and loss of bone in the surrounding area, followed by joint deformation. Similar to that seen in RA with proliferation. The mouse is also the phylogenetic lowest mammal. In addition, there are no in vitro models that can be used to mimic the complex multiple etiology of RA.

(Experiment design)
Treatment group:
1) 6 mg / kg mIgG1 isoform in 200 μl saline 3 times subcutaneously (SC) for 7 weeks (n = 8).

    2) 4 mg / kg muCRIg in 100 μl saline, 3 times per week for 7 weeks (n = 8).

  Mice were immunized intradermally with bovine CII (100 μg, Sigma, St Louis) emulsified in CFS (Difco). Mice were challenged again with CII in IFA (Difco) after 21 days. Starting on day 24, one group of mice (n = 7) received 100 μg muCRIg (PRO362) Fc 3 times a week for 6 weeks. The second group (n = 8) received 100 μg mouse IgG1 as a control. Mice were tested daily for signs of joint inflammation and scored as follows: 0, normal; 1, erythema and mild swelling confined to the heel joint; 2, heel to metatarsal and metacarpal bones Erythema and mild swelling extending to the joint; 3, Erythema and moderate swelling extending from the heel to the metatarsal and metacarpal joint; 4, Erythema and severe swelling extending from the heel to the finger. The highest arthritic score for one paw was 4, and the highest score for one mouse was 16 (FIG. 31).

  All mice were immunized on day 0 with 100 μg bovine type II collagen in 100 μl complete Freund's adjuvant (CFA). Type II collagen in CFA was injected intradermally at the right base of the tail. On day 21, a second immunization with 100 μg bovine type II collagen in 100 μl incomplete Freud's adjuvant was performed i. d. Administered. The animals were checked by the researcher daily (MF). Nestlets were used as an enrichment device to provide animals with extra padding. If necessary, moistened food was provided at the bottom of the cage. Debilitated animals were slaughtered after consultation with veterinary staff. Terminal faxitron X-Ray and microCT were taken at the end of the experiment to assess joint lesion / loss. In addition, animals were weighed before and at the end of treatment.

  On day 35 and at the end of the experiment, groups 1-8 mice were bled for serum pK and anti-type II collagen antibody titers were determined (100 μl of orbital blood).

  On day 70, all mice were finally intracardially under 3% isoflurane for the last hemogram, for various leukocyte counts, and for the evaluation of serum pK (G3). Blood was collected from.

  Mice were euthanized 70 days after induction of arthritis. Radiographs, 5CT, and histopathology were collected for all four limbs.

(result)
Systemic injection of muCRIg-Fc, a CRIg fusion protein, into mice with collagen-induced arthritis (animal model of rheumatoid arthritis) administered CRIg fusion protein to a control group (circles) of mice that received IgG1. The tested group of mice (squares) showed a significant (see FIG. 31; p-value = 0.004) reduction in CIA progression. Collagen-induced arthritis was induced by injection of bovine type II collagen emulsified in complete Freud's adjuvant. A booster was administered 21 days after the first immunization. Animals were treated 3 times a week with either mouse CRIg-FC fusion protein or anti-gp120 IgG1. The dose was 4 mg / kg in 100 μl PBS and was subcutaneous. Treatment started on day 21 and continued until day 70. Mice were observed daily for hind limb swelling as a sign of arthritis. The severity of arthritis was graded on a scale of 1-16 as follows: 0 = no signs of erythema and swelling, 1 = erythema and mild swelling limited to tarsal bones or heels, 2 = Erythema and mild swelling extending from the heel to tarsal bone, 3 = erythema and moderate swelling extending from the heel to the metatarsal joint, 4 = erythema and severe swelling involving the heel, foot, and fingers.

(Repeated experiment)
Modifications to the above protocol were repeated to confirm the results of previous experiments in the collagen-induced arthritis (CIA) model. The improved protocol included a study of the potential effects of radiation as a result of in vivo microCT imaging on disease and onset progression.

  70 DBA1 / J7 mice (7-8 weeks old, Jackson Laboratories) were divided into 5 treatment groups, 2 groups (G1 and G3) had 15 mice per group and 2 groups (G4 And G5) contained 10 mice per group and one group (G2) contained 20 mice.

Treatment group G1: 4 mg / kg MuIgG1 isoform in 100 μl saline was s. c. 3 times a week for 7 weeks (n = 15).

    G2: 4 mg / kg MuCRIg-IgG1 in 100 μl physiological saline was s. c. 3 times a week for 7 weeks (n = 20).

    G3: 4 mg / kg MuTNFRII-IgG1 isoform in 100 μl saline was s. c. 3 times a week for 7 weeks (n = 15).

    G4: 4 mg / kg MuIgG1 isoform in s. c. Anesthesia with in vivo microCT three times a week for 7 weeks (n = 10).

    G5: 4 mg / kg MuTNFRII-IgG1 in 100 μl saline was s. c. Anesthesia with in vivo microCT three times a week for 7 weeks (n = 10).

  TNF is a cytokine secreted by mononuclear phagocytes, Ag-stimulated T cells, NK cells, and mast cells. This is related to normal inflammatory and immune responses. TNF-α plays an important role in the pathogenesis of rheumatoid arthritis (RA). High levels of TNF were seen in synovial fluid of RA patients. In this protocol, mTNFRII-Fc was used as a positive control to block the interaction between TNF and its cell surface receptor.

  All mice from G1 to G5 were immunized on day) with 100 μg bovine type II collagen in 100 μl complete Freund's adjuvant. Type II collagen in CFA was injected intradermally at the right base of the tail. On day 21, a second immunization with 100 μg bovine type II collagen in 100 μl incomplete Freud's adjuvant was administered intradermally on the left side of the tail.

  Animals were checked daily. G4-5 groups of mice were anesthetized with isoflurane and in vivo microCT was performed weekly. Terminal faxitron X-Ray and microCT were taken at the end of the experiment to assess joint lesion / loss. In addition, animals were weighed before and at the end of treatment.

  On day 35 and at the end of the experiment, G1-5 mice were bled for serum pK and anti-type II collagen antibody titers were determined (100 μl of orbital blood). On day 70, all mice were finally intracardially under 3% isoflurane for the last hemogram, for various leukocyte counts, and for the evaluation of serum pK (G3). Blood was collected from.

  Mice were euthanized 70 days after induction of arthritis. Radiographs, microCT, and histopathology were collected for all four limbs.

  FIG. 33 shows that joint swelling was significantly reduced in mice treated with CRIg-Fc.

  Immunohistochemistry (H & E staining) performed on formalin-fixed and paraffin-embedded tissues obtained from animals treated with muCRIg-Fc on day 70 demonstrated inhibition of joint inflammation as a result of treatment. Show. FIG. 34 shows H & E stained sections of metatarsal joints of DBA1 / J mice 70 days after immunization with type II collagen. A. A large amount of inflammatory cell infiltration is seen in the area around the tendon sheath and the area around the joint cavity; Details of A; Low degree of inflammation infiltration in the joints of mice treated with CRIg-Fc. Few inflammatory cells were found in the area around the tendon sheath and the area around the joint cavity; Details of B.

  FIG. 35 shows that cortical bone volume was maintained in the joints of mice treated with muCRIg-Fc. Groups of mice treated with control IgG-Fc and CRIg-Fc were sacrificed 70 days after collagen injection and joints were scanned by μCT. The loss of bone and decreased bone density in the typical mouse joints of the CRIg-Fc group and the control IgG group are shown in the left figure compared to animals treated with muIgG1. The preservation of cortical bone volume was significantly greater in animals treated with muCRIg-Fc. The image is a 3D surface rendering created from μCT data using Analyze image analysis software.

  FIG. 36 shows that treatment with CRIg-Fc did not change the number or morphology of tissue resident macrophages. Livers and lungs from mice treated with either anti-gp120 IgG1 (left panel) or CRIg-Fc (right panel) were removed, fixed in formalin and embedded in paraffin wax. Seven micron sections were stained using an antibody against F4 / 80. Careful examination of the sections shows an equal number of F4 / 80 positive macrophages in both treatment groups. In addition, no difference in macrophage morphology was observed.

  FIG. 37 shows that treatment with muCRIg-Fc did not affect serum anti-collagen antibody titers. Serum titers of anti-collagen antibodies were determined 70 days after immunization. There was no difference in serum titers of antibody IgG1, IgG2, and IgM subclasses in animals treated with CRIg-Fc versus animals treated with gp120. This means that CRIg-Fc does not affect the antibody response in mice immunized with type II collagen. FIG. 38 shows that muCRIg-Fc reduces the number of inflammatory macrophages circulating. Peripheral blood was obtained from animals treated with CRIg-Fc and anti-gp-120 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 the number of inflammatory monocytes and a decrease in the number of non-inflammatory monocytes compared to the group treated with anti-gp120.

  In conclusion, the results of the experiment described in this example indicate that the muCRIg-Fc fusion protein inhibits collagen-induced arthritis. Specifically, these results indicate that CRIg-Fc inhibits joint swelling, inhibits inflammation, preserves cortical bone volume in joints, and reduces the number of circulating inflammatory macrophages. Show.

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

(Example 8)
(CRIg fusion protein in mouse antibody-derived CIA)
Antibody-induced arthritis differs from collagen-induced arthritis in that an antibody that recognizes type II collagen is injected instead of injection of an antigen (bovine type II collagen). In this way, adaptive B and T cell responses are avoided to directly induce effector function on macrophages and neutrophils by activation mediated by Fc receptors and complement.

  Antibody-derived CIA is an i.V. of a mixture of four monoclonal antibodies generated by the Arthrogen-CIA® mouse B-hybridoma cell line. v. It can be induced by injection (Terato et al., J. Immunol. 148: 2103-8 (1992)). Three of the monoclonal antibodies recognize the autoantigenic epitope clustered in LyC2 of CB11, a fragment of 84 amino acid residues (fragment that causes minimal type II collagen arthritis), and the fourth monoclonal antibody. The antibody reacts with LyC1. All four antibodies recognize conserved epitopes shared by various type II collagen species and cross-react with homologous and heterologous type II collagens (Terato et al., Supra); Terato et al., Autoimmunity 22: 137-47 (1995)). A mixture of monoclonal antibodies that induce Arthrogen-CIA® arthritis is commercially available (Chemicon International. Inc., Temecula, Calif., Catalog number 90035).

(Protocol)
Ten BALB-c mice (CR / Hollister), 4-5 weeks old, were divided into two groups, each group containing 5 mice.

  Animals were treated daily with 100 μg muCRIg-Fc or 100 μg control-Fc (anti-gp120 IgG1) starting the day before (1 day) injection of the antibody mixture and continued until day 14. On day 14, animals were checked at least twice a day and observations continued to be recorded in writing. The extent of the disease was scored by visual inspection.

Visual scoring system:
0 = no signs of erythema and swelling 1 = erythema and mild swelling limited to tarsal bone 2 = erythema and mild swelling extending from heel to tarsal bone 3 = erythema and spreading from heel to metatarsal joint Moderate swelling 4 = erythema and severe swelling involving the heels, feet, and fingers.

  Nestlets were used as an enrichment device to provide animals with extra padding.

  All animals were sacrificed on day 14 and joints were collected for immunohistochemical staining or hematoxylin-eosin staining. Blood was sampled for hematological analysis.

(result)
FIG. 39 shows macrophage infiltration in joints after antibody-induced arthritis (AIA) made by F4 / 80 staining in frozen joints that have not been decalcified. Female Balb / C mice were treated with 2 mg of anti-collagen antibody (arthrogen) i. v. 3 days later, 25 μg LPS was injected i.p. p. Injected. Fourteen days after antibody injection, mice were euthanized and their paws were collected and embedded in polyvinyl alcohol. Frozen joints were cut into 7 μm thick sections and stained with an antibody against mouse CRIg and an antibody against F4 / 80, a macrophage specific marker.

  FIG. 40 shows that muCRIg prevents joint swelling after antibody-induced arthritis in Balb / c mice. Arthritis recognizes a conserved epitope on type II collagen by the method of Terato and co-workers (Terato et al., (1992), supra; Terato et al., (1995), supra) 4 Derived using a mixture of different monoclonal antibodies (Chemicon). Female Balb / C mice (6 weeks old) received 2 mg of anti-CII antibody i. v. 3 days later, 25 μg LPS was injected i.p. p. Injected by injection. Animals were treated daily with either mouse CRIg-Fc fusion protein or control-Fc fusion protein. The dose was 4 mg / kg in 100 μl PBS and was subcutaneous. Treatment started on the day before the injection of anti-collagen antibody and continued until mice were euthanized on day 14. Mice were observed daily after LPS injection for swelling of the hind limbs as a sign of arthritis. The severity of arthritis was graded on a scale of 1-16 as follows: 0 = no signs of erythema and swelling, 1 = erythema and mild swelling limited to tarsal bones or heels, 2 = Erythema and mild swelling extending from the heel to tarsal bone, 3 = erythema and moderate swelling extending from the heel to the metatarsal joint, 4 = erythema and severe swelling including the heel, foot, and fingers.

  Therapeutic treatment was performed in the same way as prophylactic treatment except that treatment started on day 4 instead of day -1. Treatment with muCRIg-Fc reduced the level of inflammatory cytokines in the paws of AIA mice. Measurement of the concentrations of cytokines, C3a and C5a in the hind limbs of arthritis has been reported by Kagari et al. Immuol. 169: 1459-66 (2002). Briefly, at the indicated time after induction of antibody-induced arthritis, the paw was collected and frozen in liquid nitrogen. Subsequently, the feet were crushed on a metal plate cooled with liquid nitrogen and dispersed in ice-cold PBS containing 0.1% PMSF (Sigma). Samples were removed by homogenizing on ice using a Vitatron (NL) homogenizer, spinning the insoluble portion at 14000 g for 10 minutes and collecting the supernatant. Cytokines in the supernatant were measured using a cytokine ELISA by BD Pharmingen.

  Treatment with muCRIg-Fc inhibited complement C3 accumulation, but did not inhibit IgG2a accumulation on AIA cartilage. Female Balb / C mice were given 2 mg of anti-collagen antibody (arthrogen) i. v. 3 days later, 25 μg LPS was injected i.p. p. Injected. Fourteen days after antibody injection, mice were euthanized, paws were collected, embedded in polyvinyl alcohol, and frozen in dry water on ispentane. Frozen joints were cut into 7 μm thick sections and stained with FITC-conjugated polyclonal antibody against mouse C3 (Calbiochem) and polyclonal A594-conjugated antibody against mouse IgG2a (Jackson Immunoresearch). Photographs of the sections were taken with a Leitz fluorescence microscope.

  The results of immunohistochemistry performed using H & E staining are shown in FIG. Mice treated with control (muIgG1) had moderate to severe arthritis (left panel). Mice treated with muCRIg had negligible arthritis to no arthritis (right panel). The results show that muCRIg inhibits joint inflammation in antibody-induced arthritis.

  In conclusion, animals treated with mouse CRIg-Fc had significantly reduced clinical scores when compared to animals treated with anti-gp120 IgG1. CRIg showed both prophylactic and therapeutic efficacy in this animal model. The reduced severity of arthritis was also reflected by a decrease in joint inflammatory cells, particularly neutrophils. There is an increase in the number of neutrophils in the circulation, probably reflecting a decrease in neutrophil movement into the joint. muCRIg-Fc inhibited local IL-1β and IL-6 production in parallel with the clinical symptoms of RA. Treatment with muCRIg did not affect immune complex accumulation but inhibited complement C3 accumulation on cartilage. It was revealed that effector function is dependent on Fc receptor binding. huCRIg-short-Fc was also found to have significant prophylactic activity.

Example 9
(Mouse CRIg-Fc binds to C3 opsonized sheep erythrocytes (E-IgM))
SRBC (MP Biomedicals, ICN / Cappel) was coated with rat IgM (E-IgM) (Forssman Ag, Pharmingen). E-IgM was opsonized with normal mouse serum and serum from C3 knockout mice. Opsonized E-IgM was incubated with various concentrations of mouse CRIg-Fc. Binding of the fusion protein to E-IgM was monitored by flow cytometry using a FITC-labeled antibody against the Fc portion of the fusion protein.

  As shown in FIG. 42, mouse CRIg bound dose-dependently to E-IgM opsonized with normal mouse serum, but not to E-IgM opsonized with C3-deficient serum. It was. This indicates selective binding of CRIg to mouse C3 or a fragment of C3.

(Example 10)
(The binding of human CRIg to E-IgM is C3-dependent)
SRBC (MP Biomedicals, ICN / Cappel) was coated with rat IgM (E-IgM) (Forssman Ag, Pharmingen). E-IgM was opsonized with C3-deficient or C5-deficient human serum. Opsonized E-IgM was incubated with various concentrations of human CRIg-Fc. Binding of the fusion protein to E-IgM was monitored by flow cytometry using a FITC-labeled antibody against the Fc portion of the fusion protein.

  As shown in FIG. 43, human CRIg bound to E-IgM opsonized with C5-deficient serum in a dose-dependent manner, but did not bind to E-IgM opsonized with C3-deficient serum. This indicates the selective binding of CRIg to human C3 or a fragment of C3. Similar results were obtained using human CRIg ECD.

(Example 11)
(Binding of serum opsonized particles to CHO cells expressing CRIg)
50 μl fresh C57B6 female serum + 20 μg / ml mCRIg-mFc (PUR5270-B) or mPIGR-mFc (4699) were mixed together. A488 particles, zymosan, S. aureus, or E. coli (by Molecular Probes), PBS / 0.2% gelatin / 0.18% glucose / 1 mM MgCl2 (PBSgg ++) In addition at 37 ° C. over 60 minutes. Opsonized particles were washed twice with PBS and against CHO cells expressing mouse CRIg (clone 5C10) or CHO cells expressing human JAM2, or in the presence of CRIg-Fc or control-Fc protein Was added over a period of 30 minutes at 37 ° C. The cells were washed twice with PBS and analyzed for particle binding to the cell surface in a FACS Caliber.

  As shown in FIG. 44, particles opsonized with C3 sufficient serum bound to CHO cells expressing CRIg but not to CHO cells expressing JAM2. Binding was withdrawn in the presence of the CRIg-Fc fusion protein, but not in the presence of the control-Fc fusion protein. This indicates that the CRIg binding site for C3b exists in the extracellular domain.

(Example 12)
(MuCRIg Fc binds to C3b)
Real-time monitored surface plasmon resonance assays were performed using a Biocore®-2000 instrument and data were analyzed using BiaEvaluation 3.0 software (Biacore AB, Uppsala, Sweden). Carboxylated dextran chips (sensor chip CM5, research grade by Biacore AB) were used for all assays. A CM5 chip flow cell was used for a standard amine coupling procedure or a direct activation of C3b by using a standard activation-deactivation procedure without adding any protein during the process. Either prepared for enzymatic coupling was performed. The activation step is performed using a new solution containing N-hydroxysuccinimide and N-ethyl-N ′-(dimethylaminopropyl) -carbodiimide (Biacore AB, 7-15 min injection at a flow rate of 5 μl / min). And then quenched with ethanolamine-HCl (1.0 M, pH 8.5) (Biacore AB, 7-15 min infusion). Hepes buffered saline (Biagrade, Biacore AB) or VBS was used as the flow buffer throughout. After these initial steps, VBS or VBS was used as a continuous flow buffer at 5 μl / min; just degassed buffer was used.

  Protein Amine Coupling on Biacore® Chips—C3b, iC3b, C3c, and C3d are coupled onto a CM5 chip using standard amine coupling procedures recommended by the manufacturer. It was. The protein to be coupled was dialyzed against 10 mM acetate buffer (pH 5.0-5.7) to obtain a net negative charge for amine coupling. Briefly, the surface of the chip was activated with N-ethyl-N ′-(dimethylaminopropyl) -carbodiimide (7-15 min injection, 5 μl / min) and either purified C3b (50 μg / Ml, 20 μl), C3c (70 μg / ml, 30 μl), or C3d (130 μg / ml, 20 μl) at a level of coupling suitable for binding experiments (ie 1,000-5,000 resonance units). ) (RU) until injection. The flow cells were then inactivated as described above. Prior to the experiment, the flow cells were washed thoroughly with 3M NaCl (pH 4.6) in VBS and 10 mM acetate buffer.

  Binding assay using Biacore®—We tested CRIg-Fc binding to amine-coupled C3b, C3c, and C3d. For Biacore® injection, the reagent is dialyzed against VBS, diluted with VBS, and filtered (0.20 μm, Minisart®, Sartorius Corp., Edgewood, NY) or centrifuged ( 14,000 × g for 10 minutes). The protein concentration of the dialyzed reagent was measured using BCA Protein Assay (Pierce). Fusion protein is flowed through control flow cells (flow cells activated and inactivated without using any coupled protein, “blank channel”) and at 22 ° C. at a flow rate of 5 μl / min. Were injected separately through the flow cells with the coupled protein. All binding assays were performed in at least duplicates using separately prepared sensor chips.

  As shown in FIG. 45, mouse CRIg-Fc showed specific binding of C3b to the sensor chip and used a calculated 250 nM Kd.

(Example 13)
(Mouse and human CRIg-Fc bind to complement C3b)
Maxisorb plates were coated with 3 μg / ml C1, C3a, b, c, d, C4, C6 in PBS. Plates were blocked in PBS + 4% BSA for 2 hours and incubated with various concentrations of mouse or human CRIg-Fc fusion protein for 1 hour at room temperature in PBS + 4% BSA + 0.1% Tween. Plates were washed and incubated with goat anti-mouse Fc antibody or goat anti-human Fc antibody conjugated to peroxidase. After washing, the plate was incubated with TNB substrate and the OD was read with a plate reader.

  The results shown in FIG. 46 show increased concentration-dependent binding of mouse and human CRIg to C3b, C3c, and C3bi, and the absence of binding to C1, C2, C4, C3a, and C3d.

(Example 14)
(Mouse and human CRIg-Fc inhibits C3 accumulation in zymosan)
Inhibition of the alternative complement pathway was studied using a method that utilizes flow cytometric analysis of C3 accumulation in zymosan A particles (Sigma) (Quig et al., J. Immunol. 160: 4553-4560 (1998). ). Briefly, 50 mg of zymosan particles in 10 ml of 0.15 M NaCl were first activated by boiling for 60 minutes and then washed twice with PBS. In each alternative complement pathway assay condition, 2 × 10 ⁷ particles were added to a reaction tube containing a final concentration of 10 mM EGTA and 5 mM MgCl ₂ . The sample described in the text was then added containing either 10 mM EDTA (negative control) or increasing amounts of mouse CRIg-Fc. 10 μl of BALB / c serum was added as a complement source and all samples were made up to 100 μl with PBS. Samples were incubated at 37 ° C. for 20 minutes and the reaction was stopped by adding 10 mM EDTA. The particles were centrifuged and the supernatant was removed and frozen for later analysis. The particles were then washed twice with cold PBS, 1% BSA and then incubated for 1 hour on ice with FITC-conjugated goat anti-mouse C3 (Cappel, Durham, NC). Samples were then washed twice with cold PBS, 1% BSA, resuspended in PBS, and then analyzed by flow cytometry using an EPICS cytometer (Coulter, Hialeah, FL). Percentage inhibition was expressed as: [1- [average channel fluorescence of sample−background (conditions of 10 mM EDTA) / average channel fluorescence of positive control (no Cry-Ig) −background]] × 100. Calculated using.

  The reaction supernatant was also analyzed by Western blotting to determine the extent of C3 cleavage. For this analysis, 5 μl of 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, Hybond high sensitivity chemiluminescence (ECL) paper (Amersham, Arlington Heights, IL), 0.19 M Tris, 0.025 M glycine, 20 Transferred in% methanol buffer overnight. After this, the membrane was blocked with PBS containing 10% milk, 0.1% Tween for 1 hour. Subsequently, pre-titrated anti-C3 mAb RmC11H9 (Quig et al., Supra) was 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 to mouse IgG) was added over 1 hour, after which the blot was washed and sensitive chemiluminescence (ECL) Developed using the system (Amersham).

  Inhibition of complement activation by CRIg-Fc on zymosin particles was analyzed by flow cytometry to detect surface bound C3 (Figure 47A) or aliquots of zymosin reaction supernatants were analyzed by Western blotting When detected, anti-C3 mAb was used to detect (FIG. 47B). The position of the complete C3 and C3 'chains in B is indicated by the right arrow. The 10 mM EDTA lane represents a negative control, and the top of lanes 2-7 shows increasing doses of CRIg-Fc.

(Example 15)
(CRIg inhibits hemolysis of the SRBC alternative complement pathway)
For the alternative complement route, rabbit erythrocytes (RRBC) were washed with veronal buffer (Bio Whittacker) containing 0.1% gelatin and resuspended in GVB to 1 × 10e9 cells / ml. . 10 μl of cell suspension was added to 10 μl of C1q depleted serum containing the inhibitor. The mixture was incubated at 37 ° C. for 35 minutes in a greenhouse with shaking. 200 μl GVB containing 10 mM EDTA was added, the cells were centrifuged at 2500 rpm for 5 minutes, and 100 μl aliquots were read at a wavelength of 412 nm.

  For the classical complement pathway, sheep red blood cells (E-IgM) opsonized with IgM were incubated with fB-deficient serum. The method is similar to the measurement for the alternative complement pathway.

  The results shown in FIG. 48 indicate that mouse CRIg inhibits hemolysis induced by the alternative complement pathway but does not affect the hemolysis of the classical complement pathway. Similar results were obtained with human CRIg.

(Example 16)
(CRIg selectively inhibits alternative complement pathway)
(Hemolysis assay using whole serum)
The alternative complement pathway was evaluated using rabbit erythrocytes (Er) as described in Kostavasili et al. (J. Immunol. 158: 1763-71 (1997)). Briefly, Er (Colorado Serum, Denver, CO) was washed 3 times with GVB and resuspended to 1 × 10 9 / ml. 10 μl Er was added to 10 μl GVB / EGTA (0.1 M EGTA / 0.1 M MgCl 2), inhibitor, 10 μl C1q-depleted human serum and the volume adjusted to 100 μl with GVB, then Incubated for 30 minutes at 37 ° C. The reaction was stopped by adding 250 μl GVB / 10 mM EDTA and centrifuged at 500 × g for 5 minutes. Hemolysis was determined by the absorbance at 412 nm of 200 μg of supernatant. The percent lysis was normalized by considering 100% lysis equal to the lysis that occurs in the absence of inhibitor.

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

(Measurement of cleavage mediated by C3 convertase of C3)
The effect of CRIg on the cleavage of C3 in the liquid phase by C3 convertase (C3b.Bb) (Kostavasili et al., Supra) was determined by comparing 0.4 μM purified C3 with huCRIg − in GVB (20 μl volume). Tested by incubating with long, huCRIg-short, muCRIg, or factor H for 15 minutes at 37 ° C. Subsequently, 0.4 μM Factor B and 0.04 μM Factor D were added in a total volume of 30 μl in the presence of 50 mM MgEGTA to activate the pathway. After 30 minutes at 37 ° C., the reaction mixture was quenched with 30 μl Laemmli's sample buffer (BioRad) containing 2-ME, boiled for 3 minutes, and run on an 8% SDS-PAGE gel (Invitrogen). Electrophoresis was performed. Proteins were visualized by staining the gel with SimplyBlue staining (Invitrogen, Carlsbad, CA). The gel was scanned for densitometric analysis and the percentage of C3 cleaved was calculated. Controls were incubated with GVBE (GVB containing 10 mM EDTA) to inhibit cleavage.

  A microtiter plate assay for the alternative complement pathway DAA was performed as described (Krych-Goldberg et al., J. Biol. Chem. 274: 31160-8 (1999)). Microtiter plates were coated overnight with 5 μg / ml C3b (Advanced Research Technologies) in phosphate buffered saline. 1. Plates are blocked for 2 hours at 37 ° C. in phosphate buffered saline containing 1% bovine serum albumin and 0.1% Tween 20 and containing 71 mM NaCl and 0.05% Tween 20. Incubated with 10 ng factor B, 1 ng factor D, and 0.8 mM NiCl 2 in 5 mM veronal buffer (pH 7.4) for 15 minutes at 37 ° C. A 1: 15,000 dilution of anti-goat antibody conjugated to 0.01-1 μg CRIg-Fc, 0.129 μg goat anti-human factor B antibody and 100 μg horseradish peroxidase using the same buffer. A continuous 1 hour incubation was performed with (Jackson Immunoresearch Laboratories, West Grove, PA). Color was developed with O-phenylenediamine. In this assay, DAF and Factor H, as expected, acted as mediators of decoy accelerated activity and C3a release was detected using the Amersham Pharmacia Biotech des-Arg RIA kit.

(C5 convertase assay)
C3b was accumulated in zymosan by resuspending 1 × 10 ¹⁰ zymosan particles in 0.2 ml 10 mg / ml C3 and adding 5 μg trypsin followed by incubation at 22 ° C. for 10 minutes. It was. C3b accumulation with trypsin was repeated and the cells were washed 6 times with 5 ml GVB. Zymosan particles were resuspended in 100 μl GVB and mixed with 50 μl GVB and 50 μl 10 mM NiCl 2 containing Factor B (35 μg) and Factor D (0.5 μg). After 5 minutes incubation at 22 ° C., 5 μl of 0.2M EDTA was added. Bound C3b was amplified by adding 50 μl C3 (500 μg) and incubating the cells for 30 minutes at 22 ° C. The zymosan particles with C3b were washed and the amplification procedure was repeated until the desired number of C3b / zymosan was obtained.

  Since the formation of C5 convertase took less than a minute, the enzyme was formed in the same reaction mixture that was assayed. Enzyme velocities were determined in microcentrifuge tubes treated with 0.5 ml of silicon under saturated concentrations of Factor B and Factor D and C6 as described above. The assay mixture contained various concentrations of C5 (preincubated for 20 minutes at 37 ° C. to exclude background C5b, 6-like activity caused by freeze / thaw), factor B (1.2 μg, 516 nM). , Factor D (0.1 μg, 167 nM), C6 (2.5 μg, 833 nM), and 0.5 mM NiCl 2. The reaction was initiated by the addition of ZymC3b, ESC3b, or ERC3b. Depending on the density of C3b per cell, the concentration of cells has 9-35 ng of bound C3b in GVB in a final volume of 25 μg, resulting in an enzyme concentration of 2-8 nM and It adjusted so that it might become. After 15 minutes incubation at 37 ° C., further cleavage of C5 was prevented by transferring the assay tube to an ice bath and adding ice-cold GVBE. Appropriately diluted assay mixtures were rapidly titrated for the formation of C5b, 6 by hemolysis assay using EC. C5b and 6 were quantified using a calibration curve prepared using purified C5b and 6. It was established by the control that the low temperature and this dilution was sufficient to reduce C5 cleavage during the next step to undetectable levels. Rabbit red blood cell (ER) or sheep red blood cell (ES) lysis was shown to lead to <2% for C5b, 6 titers, using EC lysis as the endpoint.

  C5b, 6 was measured by hemolytic activity using the sensitivity of EC to hemolysis by human C5b-9. For an aliquot (25 μl) of the diluted sample from the C5 convertase assay, 1.2 × 10 7 EC and 5 μl of pooled normal human serum (NHS) as a source of complement proteins C7-C9 The mixture was added in a final volume of 225 μl GVBE. The reaction mixture was incubated at 37 ° C. for 10 minutes, after which unlysed cells were removed by centrifugation at 10,000 × g for 1 minute. The amount of hemoglobin released was quantified by spectroscopic altitude at 414 nm. 100% lysis was measured as EC dissolved in 2% Nonidet P-40. A control containing C5 and C6 but no C5 convertase was subtracted as background. Controls containing C5 convertase but no purified C5 or C6 did not form significant amounts of C5b, 6 from NHS used as the source of C7-9 during EC lysis. Was showing.

(result)
The results are shown in FIGS. 49 (A) to (E).

  FIG. 49 (A) shows that CRIg inhibits hemolysis of rabbit erythrocytes (alternative complement pathway) in C1q-deficient serum, but hemolysis of IgM opsonized sheep erythrocytes (classical complement pathway) in fB-deficient serum. It showed no inhibition. This indicates that CRIg selectively inhibits the alternative complement pathway.

  As shown in FIG. 49 (B), CRIg inhibits C3 convertase activity in the liquid phase. The gel shows inhibition of cleavage of the 115 kDa α chain of C3 with increasing concentrations of human CRIg-ECD (10-100 nM).

  FIGS. 49 (C) and (D) show that CRIg not only does not function as a cofactor for C3 factor I-mediated cleavage but also as a promoter of C3 convertase decay.

  The data shown in FIG. 49 (E) shows that CRIg inhibits the alternative complement pathway C5 convertase formed on zymosan particles.

(Example 17)
(CRIg is expressed in a subset of tissue macrophages)
Monoclonal antibodies specific for human CRIg and mouse CRIg were generated and utilized to define CRIg expression as described in Example 3. CRIg was not present in peripheral blood C14 + monocytes, but it was readily detected in monocyte-derived macrophages by flow cytometry (FIG. 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, muCRIg was not present in peripheral blood and spleen leukocytes (including CD11b ⁺ bone marrow cells) but was detected in liver Kupffer cells (KC, FIG. 50B). Expression of huCRIg (L) and (S) proteins was confirmed as 55K and 48K Mr proteins, as monocytes differentiated into macrophages (FIG. 50C). Similarly, mouse CRIg was detected as a 48K Mr glycoprotein in peripheral macrophages (PM). muCRIg has a putative N-linked glycosylation site that is glycosylated and causes a migration shift of approximately 5 kDa in the gel.

  Since CRIg mRNA is highly detected in the liver, the expression of CRIg in the liver was further analyzed by immunohistochemistry. CRIg was expressed in CD68 + KC in human and mouse liver, but was also detected in adrenal, placenta, synovium, intestine, and peritoneal macrophages (data not shown). CRIg is present in human spleen macrophages, Langerhans cells, microglia cells, and bone marrow derived macrophages, as well as various human and mouse macrophage cell lines (THP-1, RAW275, PU1.1, J774; results not shown). Did not exist. Taken together, these results indicate that CRIg is highly expressed in populations of resident macrophages in various tissues.

(Example 18)
(CRIg binds to C3b and iC3b)
(Materials and methods)
(Complement protein)
Human and mouse C3 can be separated from another protein A column to remove contaminating IgG according to the method of Hammer et al. (J. Biol. Chem. 256 (8): 3955-4006 (1981)). Isolated. To obtain hC3b, hC3 was incubated with CVF, hfB, ug, hfD at a 10: 10: 1 molar ratio at 37 ° C. for 1 hour in the presence of 10 mM MgCl2. The hC3b fragment was subsequently subjected to strong anion exchange mono Q 5/50 (Amersham Biosciences, Piscataway, NJ) and Superdex S-200 10/300 GL gel filtration column (Amersham Biosciences, Piscataway gel, Sea Blue bear, NJ). Isolated with a purity of> 95%. To make the C3b dimer, C3b prepared as described above was combined with bismaleimide hexane (Pierce) in methanol at a molar ratio of 2.2: 1 in PBS (pH 7.0). The reaction was performed at 4 ° C. for 3 days. Crosslinks were created through free sulfhydryl groups by breaking thioether bonds. When this procedure was used, the yield exceeded 50%. The dimer was purified by Superdex S-200 10/300 GL gel filtration column (Amersham Biosciences, Piscataway, NJ). The dimer was 95% pure according to Coomassie blue stained gel. Hydrolyzed C3 was generated by adding 2 M methylamine (pH 7.0) to C3 in PBS with 10 mM EDTA to a final concentration of 50 mM in the reaction volume. The reaction was carried out at 37 ° C. for 4 hours, after which this was performed on a Superdex S-200 10/300 GL gel filtration column (Amersham Biosciences, Piscataway, NJ), iC3b and C3c (Advanced Research Technologies 10 / Sex), Purification on a 300 GL gel filtration column separated the monomer from the dimer. C3d, Factor B, Factor D, and Factor P, complement components C1-9, sheep erythrocytes and cobra venom factor sensitized with antibodies were obtained from Advanced Research Technologies (San Diego, Calif.).

(result)
Expression of CRIg on a high population of phagocytes prompted us to study in detail whether CRIg is involved in opsonized particle binding. Complement and Fc receptors have been shown to mediate phagocytosis (Aderem and Underhill, Annu. Rev. Immunol. 17: 593-623 (1999), Underhill and Ozinsky, Annu Rev. Immunol. 20). : 825-852 (2002)). To determine whether CRIg binds to complement C3, sheep erythrocytes coated with either rabbit IgG (E-IgG) or mouse IgM (E-IgM) are treated in the presence of C3 or C5-deficient human serum. Were analyzed for their ability to rosette with the Jurkat T cell line expressing CRIgL. Jurkat cells expressing CRIg (L) (but not control Jurkat cells) formed rosettes with E-IgM in the presence of C3 (C3 +), but in the absence of C3 (C3-) (FIG. 52A). Since ES opsonized with IgG did not form rosettes with Jurkat CRIg cells, CRIg did not appear to be involved in Fc receptor mediated binding (results not shown).

  To test whether CRIg can bind directly to complement components on the cell surface, a soluble form of human CRIg was created in which the ECD of CRIg was fused to the Fc portion of human IgG1. The huCRIg-long-Fc fusion protein (but not the control Fc fusion protein) bound to opsonized E-IgM in the presence of C3, but not in the absence of C3 (FIG. 52B). Binding was restored when C3-deficient serum was reconstituted with purified human C3. V-type Ig domain was sufficient for binding. This is because both huCRIg (S) -Fc and muCRIg-Fc could bind to E-Igm (results not shown).

  As a result of complement activation that induces a cascade of enzymatic reactions, C3 is responsible for its multiple decay products C3b, iC3b, C3c, C3dg, and C3d, each of which can serve as a binding partner for CRIg. ). When using plate-bound ELISA, huCRIg (L) and huCRIg (S) -Fc (but not the control Fc) showed saturable binding to C3b and iC3b (FIG. 52C) , C3, C3a, C3c, or C3d showed no binding (data not shown). Similar binding was observed for huCRIgL-ECD (which lacks the Fc portion) and muCRIg-Fc, and binding to iC3b was greater than binding to C3b (results not shown). Conversely, soluble C3b also bound to plate-coated huCRIg (L) -Fc and competed with huCRIg (L) -ECD (results not shown). Thus, CRIg can bind to C3b and iC3b in solution or when C3b and iC3b are bound to a substrate. Since C3b exists as a multimeric form when accumulated on the cell surface, the binding of CRIg to an artificially assembled C3b dimer (C3b2) was further evaluated. C3b2 bound to huCRIg (L) with 131 nM Kd (FIG. 52D) and to huCRIg (S) with 44 nM Kd as measured by surface plasmon resonance (FIG. 52D).

  To supplement these biochemical studies, we evaluated the binding specificity of cell surface CRIg for products derived from C3. A488-labeled dimeric form of C3b2 bound to the surface of CRIg + but not to CRIg−, THP-1 cells (FIG. 52E). The binding was specific. This is because it was competed by the addition of soluble unlabeled C3b2, C3b monomer, and huCRIG (L) -ECD, but not by naturally occurring C3. In addition to binding to soluble complement fragments, muCIg expressed on the surface of CHO cell lines is also present in various opsonized particles in C3 sufficient serum (but not in C3 deficient serum). Combined (FIG. 51B). Taken together, these experiments indicate that CRIg is expressed on the cell surface and that soluble CRIg (CRIg-FC) is a receptor for iC3b and C3b.

(Example 19)
(Expression of CRIg in Kupffer cells is essential for binding of C3 fragments bound to particles)
(Materials and methods)
(1. Generation of CRIg knockout (ko) mice)
All animals were kept under sterile pathogen-free conditions and animal experiments were approved by the Genentech research institution's institutional animal care and use committee. CRI gko embryonic stem cells were generated by electroporation of a linear targeting vector in which exon 1 was replaced with a nemycin resistance gene (FIG. 53A) into C2B6 embryonic stem (ES) cells. Neomycin resistant clones were selected and homologous recombination was confirmed by Southern blotting. Seven of the 100 clones screened were positive for homologous recombination. Two targeted clones were injected into C57VL / 6 blastocysts, transferred to pseudopregnant foster parents, and the resulting male chimeric mice were mated with C57BL / 6 females to obtain +/- mice. . Germline transmission was confirmed for the cloned 2ES by Southern blot analysis of tail DNA from F1 progeny (FIG. 42B). Cross-breeding of +/− mice was performed to generate − / − CRIg mice. The phenotype of the two clones was the same. For routine genotyping by the PCR method, a common sense primer 5'-CCACTGGTCCCCAGAGAAGT-3 '(SEQ ID NO: 22) and a wild type specific antisense primer (5'-CACTATTTAGGGGCCCAGGA-3') (sequence) No. 23) and a knockout specific antisense primer (5′-GGGAGGATTGGGAAGACAAT-3 ′) (SEQ ID NO: 24) were used to obtain a 306 bp fragment for the wild type allele and a 406 bp fragment for the mutant allele. Amplified. The generation of C3 ko mice has been previously described (Naughton et al., Immunol. 156: 3051-3056 (1996)). To create CRIg / C3 double knockout mice, C3 ko mice (F2) with mixed s129 / B6 background were mated with CRIg ko mice. F1 females that are heterozygous for either allele were then mated with C3g heterozygous males that were hemizygous for the CRIg allele. The offspring from this mating were used in the experiment. C57B6 mice used for analysis of CRIg expression by flow cytometry were purchased from Jackson Laboratories (Bar Harbor).

(2. Western blotting and deglycosylation)
Human and mouse macrophages were lysed in PBS containing 1% SDS, 0.1% Triton X-100, and protease inhibitor mixture (Boehringer). After centrifugation at 10,000 g, the soluble fraction was run on an SDS gel and transferred to a nitrocellulose membrane. CRIg protein was visualized using anti-CRIg antibody and HRPO-conjugated secondary antibody, followed by detection of chemiluminescence of antibody bound by ECL (Amersham). For determination of glycosylation status of CRIg, cells expressing CRIg-gD are immunoprecipitated with anti-gD antibody and treated with PNGase, O-glycosidase, and neuraminidase according to the manufacturer's instructions (Biolabs, NE). And Western blot analysis was performed using a biotinylated anti-gD antibody.

(result)
In order to study the biological function of CRIg, mice carrying null mutations in the CRIg gene were generated by homologous recombination as described above and shown in FIG. 42A. Deletion was confirmed by Southern blotting (FIG. 53B), Western blotting of peripheral leached cell lysates (FIG. 54A), and flow cytometry (FIG. 54B). Mice were born with the expected Mendelian ratio and showed no overall phenotype or histopathological abnormalities. The absolute number of immune cells in the various lymph compartments was similar in blood, spleen, and lymph nodes from wt and ko animals (FIG. 53C). In addition, there was no difference in F4 / 80 + KC and cardiac macrophage numbers when analyzed by flow cytometry and immunohistochemistry, respectively (results not shown). The expression levels of CR3 α and β chains and other components including proteins including complement receptor-related gene y (Crry) at KC were unchanged (FIG. 54C). Similarly, low or undetectable expression of the β chain of CR1, CR2, or CD11c, CR4 was comparable between wt KC and ko KC (FIG. 53D).

  Next, the binding ability of CRIg wt KC and CRIg ko KC to C3 degradation products was tested. C3 fragments (C3b, C3b2, and iC3b) readily accumulated on the surface of CRIg wt KC (FIG. 54B). In contrast, C3b, C3b2, iC3b, or iC3b2 binding was not detected with CRIg ko KC. It was detected that there was little or no binding of C3 and C3c to either wt KC or ko KC (FIG. 53E).

  To extend the analysis from binding of soluble C3 fragments to binding of C3 fragments bound to the cell surface, the role of CRIg at KC to bind to red blood cells coated with C3 opsonized IgM was examined. CRIg ko KC showed an approximately 60% reduction in E-IgM rosette formation compared to CRIg wt KC (FIG. 54D). CR3 had a small contribution to the overall binding activity as a further decrease in rosette formation (<20%) was observed when CR3 blocking antibody was added. Thus, CRIg expression is essential for binding of C3 opsonized particles and C3 degradation products to Kupffer cells.

(Example 20)
(CRIg is internalized and expressed in recycled endosomes)
Their subsequent endocytosis can be induced by the binding of C3 opsonized particles to their receptors (Fearon et al., J. Exp. Med. 153: 1615-1628 (1981); Sengelov, Crit Rev. Immunol.15: 107-131 (1995)), a polyclonal antibody that suppresses Alexa488 fluorescent dye (Austin et al., Mol. Biol. Cell 15: 5268-5282 (2004)), CRIg and C3b were internalized in KC. Analyzed whether to rise. A488-conjugated anti-CRIg mAb was preincubated with KC at 4 ° C. Addition of anti-A488 antibody at 4 ° C. suppressed the fluorescence of the anti-CRIg antibody bound to the surface, as shown in FIG. 47A, panel 1. When A488-conjugated anti-CRIg mAb was incubated with KC at 37 ° C. for 30 minutes and then with anti-A488 antibody, fluorescence was not suppressed (FIG. 55A, panel 4), which increased from 4 ° C. to 37 ° C. As cells migrate, the anti-CRIg antibody internalizes, thus indicating that it was unable to access the suppressed anti-A488 antibody. Similar results were seen for C3b (FIG. 55A, panels 3 and 6). Anti-CRIg antibody internalization was independent of the presence of C3. This is because antibody uptake occurred with KC isolated from C3 ko 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 KC from CRIg wt mice (but not ko mice) (FIG. 55B). Over time, K488 coated with A488-conjugated anti-CRIg antibody was incubated in the presence of extracellular anti-A488 antibody and the decrease in fluorescence over time was observed. This suggests that the anti-CRIg antibody is recycled back to the cell surface (FIG. 55C). The time course of reuse was also independent of C3. This is because the kinetics of inhibition were similar to those in the presence of C3 and in the absence of C3 (data not shown). In contrast, antibodies against the lysosomal protein Lamp1 remained in the cells and did not decrease with time. These results indicate that CRIg plays a role as a receptor for C3b present on the structurally reused pool of membranes.

To further determine the intracellular compartment in which CRIg is recycled, human monocyte-derived macrophages (MDMs) are deconjugated using transferrin as a marker for the reused endosome and Lam1 as the marker for the endosome. Visualized using a volume microscope. MDM cultured for 7 days exhibited C3b saturation binding that could compete with the extracellular domain of huCRIg (L) (FIG. 55A) expressed CRIg on 60% of the cells (data not shown) . Macrophages coated with anti-CRIg antibody at 4 ° C. showed local CRIg expression in the extension of filpodia rich in F-actin (arrowhead, FIG. 56A, panels 1-3). In addition, CRIg antibody was localized with C3b on the cell surface (results not shown). Cell migration from 4 ° C. to 37 ° C. followed by a 10 minute incubation at 37 ° C. (FIG. 56B) leads to the transferrin ⁺ endosomal compartment of the CRIg antibody and C3b present around the cell (FIG. 56B, Panels 1-4, arrows) and rapid internalization of Lamp1 ⁺ compartment edges (arrows, FIG. 57D, panels 1-4) occurred. CRIg remained localized in the endosomal compartment and was not degraded in lysosomes during follow-up times up to 24 hours (results not shown). Incubation of macrophages with anti-CRIg antibody had no effect on CRIg distribution. This is because the internalized CRIg antibody completely overlaps with all pools of CRIg detected after fixation with polyclonal antibody (FIG. 57C, panels 1-3) and what is the presence of C3 in the medium? This is because it is irrelevant (FIG. 57C, panel 4). Taken together, these results indicate that CRIg is present in the early endosomes that are being recycled and that CRIg internalization occurs in the absence of ligand or cross-linked antibody.

Since the majority of C3b and iC3b accumulated on the serum-exposed particles (Brown, Curr. Opin. Immunol. 3: 76-82 (1991)), we next introduced C3 opsonized particles. The localization of CRIg positive endosomes in macrophages during phagocytosis was studied. When encountering iC3b opsonized sheep erythrocytes (E-IgM), CRIg rapidly (10 minutes) redistributes from transferrin positive vesicles to form phagosomes that can be seen as a ring around swallowed erythrocytes. (FIG. 56C, panels 1 and 4, arrows). After 2 hours of macrophage incubation with C3 opsonized particles, the phagosomes grew well as indicated by their migration into the lysosomal compartment (FIG. 56C, panels 5-8). CRIg was highly expressed in the phagosomal membrane surrounding the C3 opsonized particles (FIG. 56C, panels 5 and 8, arrows) and was no longer in the transferrin ⁺ endosomal compartment in most macrophages. CRIg remained present in a subset of phagosomes in the lysosomal compartment, but its expression did not overlap with LAMP-1 (FIG. 56C, panels 7 and 8, arrowhead). The absence of CRIg in the LAMP-1 ⁺ membrane did not appear to be a result of lysosomal degradation of CRIg. This is because the protease inhibitor was continuously present during the incubation. In some macrophages that have taken up E-IgM but lacked CRIg ⁺ phagosomes, CRIg ⁺ is localized with the transferrin ⁺ compartment (thick arrow, FIG. 56C5, panels 5 and 8). , Thick arrows), suggesting that CRIg ⁺ returns to the reclaimed compartment after transfer of E-IgM to the lysosomal compartment.

  Collectively, these results indicate that CRIg is recruited from endosomes to the site of particle engulfment and is involved in the initial stages of phagosome formation, but escapes from phagosomes when phagosome-lysosome fusion occurs. And return to the endosomal compartment.

(Example 21)
(Mice lacking CRIg are susceptible to infection with Listeria monocytogenes)
(Materials and methods)
(1. Evaluation of Listeria growth by microbial infection of mice and determination of CFU number)
Infectious Listeria monocytogenes (LM) (ATCC 43251 strain) was used for all experiments. Bacterial virulence was maintained by serial passage in BALB / c mice. New isolates were obtained from infected spleens and grown on brain heart infusion (liquid) or brain heart infusion plates (Difco Laboratories, Detroit, MI). Bacteria were washed repeatedly, resuspended in sterile phosphate buffered saline (PBS), and then stored at −80 ° C. in small aliquots in PBS containing 40% glycerol. Mice were inoculated intravenously into the tail vein with various doses of Listeria monocytogenes. For observation of bacterial growth in various organs, we intravenously transferred non-lethal doses of 1 × 10 ⁴ colony forming unit (CFU) listeria to either CRIg ko or CRIg wt mice. Internal injection. The number of bacteria surviving in inoculum, in liver and spleen homogenates, and in infected cells, was determined by plating 10-fold dilutions on brain-heart infusion agar (Difco Laboratories) plates. did. The number of CFU was counted after 24 hours incubation at 37 ° C.

2. Determination of Listeria-A488 uptake in Kupffer cells
Surviving Listeria monocytogenes was labeled with the A-488 labeling kit according to the manufacturer's instructions (Molecular Probes, Oregon). The number of Listeria surviving after the labeling procedure was assessed by colony count. CRIg wt or CRIg ko mice were injected intravenously with 10 million CFU LM. After 1 hour, the liver was perfused and Kupffer cells were isolated according to the method described above. Cells are stained with a PE-labeled antibody against F4 / 80 and positive cells are sorted using anti-PE beads (Miletnyi) followed by a MoFlo flow cytometer (DakoCytomation, Ft. Collins, CO). Isolated. F4 / 80 positive cells were collected on cover slides and the number of internalized labeled bacteria was estimated using a confocal light microscope. The number of bacteria per cell was counted in 400 cells from 4 fields per slide. The phagocytic index was calculated by multiplying the percentage of Kupffer cells containing at least one bacterium by the average number of bacteria per cell. The results show the mean and standard deviation of the phagocytic index obtained from 4 animals.

(result)
To study the role of CRIg in phagocytosis of complement opsonized particles in vivo based on the binding of CRIg to C3b / iC3b opsonized particles, CRIg wt mice and CRIg KO mice were treated with Gram-positive facultative bacteria. A variety of doses of Listeria monocytogenes (LM) (which, when exposed to serum, activate an alternative complement pathway that preferentially accumulates C3b and iC3b on bacterial surfaces (Croize et al., Infec Immunol.61: 5134-5139 (1993))). CRIg KO mice were clearly susceptible to LM as shown by high mortality (FIG. 58A). Conversely, prior treatment with CRIg-Ig fusion protein increased the sensitivity of CRIg wt mice, but not CRIg ko mice (FIG. 62).

  Consistent with the role of CRIg in complement C3 opsonized particle binding and phagocytosis, CRIg ko mice have reduced clearance of LM from the blood resulting in high LM loading in the spleen and lung (FIG. 58B). ). The LM load in the liver and heart of infected mice is also reduced, probably reflecting the presence of macrophages expressing CRIg in these tissues (FIG. 58B). The inflammatory response was elevated in CRIg ko mice, which was reflected by increased serum concentrations of IFN-γ, TNF-α, and IL-6 (FIG. 58C). Consistent with the need for CRIg in clearance of O3 opsonized particles, CRIg ko KC showed a significant decrease in LM binding and phagocytosis compared to CRIg wt KC (FIG. 58D). Finally, the high Listeria loading detected in the blood of CRIg ko mice was C3 dependent, as infection of C3 ko mice reversed the difference in bacterial titers in CRIg ko mice versus CRIg wt mice (FIG. 58E). Interestingly, bacterial circulating levels are significantly lower in C3 ko mice compared to C3 well mice, probably reflecting an increased involvement of C3-dependent mechanisms responsible for clearance of Listeria in C3 ko mice. is doing. However, rapid clearance in the absence of C3 did not result in sufficient pathogen clearance over a long period of time, as C3-deficient mice die within 2 days after infection with Gram-positive bacteria (Cunnion et al., J Lab.Clin.Med.143: 358-365 (2004)). These results strongly indicate that CRIg expressed in liver Kupffer cells plays an important role in the rapid clearance of complement C3 opsonized pathogens from the circulation.

(Example 22)
Inhibition of complement-mediated immunohemolysis with huCRIg molecules
It is well established that rabbit erythrocytes specifically activate the alternative complement pathway, resulting in cell lysis by the C5b-9 complex (Pollill, et al., J. Immunol. 121 (1), 363). -370 (1978)). Specifically, rabbit erythrocytes initiate a cascade of alternative complement pathways and cause lysis of these cells by the formation of the resulting MAC. If the test compound can inhibit the alternative complement pathway, the addition of reagents to rabbit erythrocytes batched in serum (in this case, in cynomolgus serum or in human C1q-depleted serum) should prevent cell lysis. It is. This can be assayed by monitoring the change in light absorbance at a wavelength of 412 nm caused by the release of hemoglobin from lysed red blood cells. In the cyno serum experiment, blood was collected from the cynomolgus monkey's general vein. No anticoagulant was used. The sample was allowed to solidify at room temperature. Samples were centrifuged, serum collected and stored in a freezer set to maintain -60 to -80 ° C. Rabbit erythrocytes (RRBC) were washed 3 times with GVB (1 × Verontal Buffer (Biowhittaker), 0.1% gelatin) and resuspended in GVB to 1 × 10 ⁹ / ml. . GVB, huCRIg (short, long, or long ECD) was added followed by 10 μl GVB + / EGTA (GVB, 0.1 M EGTA, 0.1 M MgCl 2). 10 μl of cyno or C1q depleted serum (Quidel) was added, followed by 10 μl of RRBC and mixed by flicking the mixture. After 45 minutes incubation at 37 ° C. in a greenhouse with shaking, 250 μl GVB / 10 mM EDTA was added and the mixture was centrifuged at 2500 rpm for 5 minutes. Read at 412 nm using 250 μl aliquots. The results shown in FIGS. 63A and B (cyno serum), and FIGS. 64-66 (human serum) show that the tested CRIg compounds inhibited complement hST-L: human CRIG-long hST-S: Human CRIg-short
hST-L ECD: Human CRIg-long ECD
hPIGR: human multimeric immunoglobulin receptor fH: complement factor H
(Example 23)
(Test of mouse CRIg-Fc fusion protein in a mouse model of choroidal neovascularization)
Choroidal neovascularization (CNV) can be induced experimentally by causing laser burns in the retina. In this experiment, 40 C57BL-6 mice (Charles River Laboratory) were divided into two treatment arms.

  Group 1 (control): On day 1, day 1, day 3, and day 5, 12 mg / kg gp120 mIgG1 was administered i. p. injection.

  Group 2: 12 mg / ml mouse CIRg (mCRIg) was administered i.d. on days -1, 1, 3, and 5. p. injection.

  In each arm, animals were anesthetized by subcutaneous (sc) injection of a mixture of ketamine (25 mg / g) and xylazine (1.28 mg / g). The pupil was dilated using 1 drop of 1% tropicamide. The animal was then fixed in a plastic mold. Using a diode laser (100 μm pore size), OcuLight GL Diode Laser (532 nm), Zeiss 30W slit lamp, and micromanipulator, 3 lasers in the eye around the optic nerve I made a spot to hit. The right eye was irradiated with a laser with a slit size of 120 mW, 0.1 second, and 100 μm. Bubbles generated in the laser spot indicate rupture of the Bruch's membrane.

  Laser spots were evaluated using a confocal microscope 7 days after laser treatment. At this point, the animals were anesthetized with isoflurane and perfused from the heart with 0.5 ml PBS containing 50 mg / ml fluorescein labeled dextran (Sigma). The eyes were removed and fixed in 10% phosphate buffered formalin, the retina was discarded, and the remaining eye cup was fixed flat on the slide. Histopathological examination included immunohistochemical staining of choroidal flat-fixed ones for complement fragment and elastin and the CNV complex by monitoring the eye's FITC-dextran stained vessels by confocal microscopy. Including size analysis.

  The results are shown in FIGS. 71A and B. Here, burn holes in the right eye were scored on a scale of 0-3 and 0-5, respectively.

(Example 24)
(Testing of CRIg ECD and CRIg-Fc fusion proteins in cynomolgus monkeys with laser-induced retinal damage)
Twenty-four cynomolgus monkeys (either male or female, 12 males and 12 females) were used in this experiment. The animals were 2-7 years old and weighed 2-5 kg.

投与は、橈側皮静脈からの静脈内注射である。動物に、レーザー処理の前に少なくとも１回投与し、残りの実験の間には、１週間に３回投与した。用量は最近に記録された体重に基づき、１０〜１５ｍｇ／ｋｇの範囲である。

Administration is intravenous injection from the cephalic vein. Animals were dosed at least once prior to laser treatment and three times per week for the remainder of the experiment. The dose is in the range of 10-15 mg / kg based on the recently recorded body weight.

  On the fourth day, each eye spot of all animals was subjected to a 532 nm diode green laser burn (using a slit lamp delivery system and a Kaufmann-Wallow (Ocular Instruments Inc, Bellevue, Wash) plan fundus contact lens). Laser processing was performed by CORL on a diode green laser (OcuLight GL, IRIDEX Corp Inc. Mountain View, Calif.). Laser and support equipment were supplied by CORL. The animals were anesthetized with ketamine and xylazine. Nine regions were placed symmetrically at each eye spot. Laser parameters included a 75 micron pore size and a 0.1 second duration. The power used was evaluated by their ability to produce blisters and small bleedings. If bleeding was not observed by the first laser treatment, the second laser spot was placed near the first spot (except for adjusting the wattage) according to the same laser procedure. For the region not adjacent to the small indentation, the initial power setting was 500 mW, and when placing the second spot, the power was set to 650 mW. For the area adjacent to the small indentation, the power settings were 400 mW (first) and 550 mW (second). During surgical judgment of the retina, the power setting was adjusted based on observations at the time of laser treatment.

(Clinical ophthalmic study)
A clinical ophthalmic study was performed on each animal once prior to the start of treatment and on days 8, 15, 22, and 29. The animals were anesthetized with ketamine and the eyes were opened with mydriatic drugs. The appendages and anterior portion of both eyes were examined using a slit lamp bioscope. The fundus of both eyes was examined using an indirect ophthalmoscope. At the discretion of the ophthalmologist, the eyes could be examined using other suitable devices and photographs could be taken.

(Eye photo)
Eye photographs (OP) were taken on day 1 of laser treatment (after laser treatment), for administration phase 1 on days 10, 17, 24 and 31 and administration phase 2 Was taken on day 6 (day of autopsy). When fluorescein angiography was performed simultaneously during dosing phase 1, OP was performed first.

  When performed concurrently with fluorescein angiography, animals were anesthetized with ketamine, maintained with isoflurane anesthesia, and anesthetized with ketamine and xylazine when performed alone (ie, the following laser treatment). I opened my eyes with mydriatic. Color photographs were taken for each eye, including eye abnormalities associated with the retina, posterior stereophotographs, and non-stereophotographs of the two central peripheral visions (temporal and nose).

(Fluorescein angiography)
Fluorescein angiography was performed once for all animals before the start of treatment, and for dosing phase 1, on days 10, 17, 24, and 31 (6 and 13 days after laser irradiation). Eyes, 20th day, and 27th day).

  Animals were fasted prior to fluorescein angiography. The animals were anesthetized with ketamine and maintained with isoflurane and the eyes were opened with mydriatics. The animals were intubated because of the possibility of vomiting after fluorescein infusion. Animals received an intravenous injection of fluorescein. Pictures were taken at the beginning and end of the fluorescein injection. After injection of fluorescein, a series of stereoscopic photographs of the posterior pole of the right eye were taken rapidly, and then a pair of stereoscopic photographs of the posterior pole of the left eye was taken within 1 minute. Thereafter, each eye was photographed at about 1-2 minutes and 5 minutes. Between about 2 and 5 minutes, non-stereoscopic photographs were taken of the two central peripheral fields (temporal and nose) of each eye. If leakage of fluorescein was observed at 5 minutes, a pair of stereoscopic photographs was taken in about 10 minutes.

  Evaluation of fluorescein angiography was performed according to the following scoring system for evidence of excessive permeability (fluorescein leakage) or any other abnormality.

（材料の寄託）
以下の材料を、アメリカンタイプカルチャーコレクション（Ａｍｅｒｉｃａｎ Ｔｙｐｅ Ｃｕｌｔｕｒｅ Ｃｏｌｌｅｃｔｉｏｎ），１０８０１，Ｕｎｉｖｅｒｓｉｔｙ Ｂｏｕｌｅｖａｒｄ，Ｍａｎａｓｓａｎ，ＶＡ２０１１０−２２０９，ＵＳＡ（ＡＴＣＣ）に寄託した。

(Deposition of materials)
The following materials were deposited with the American Type Culture Collection, 10801, Universality Boulevard, Manassan, VA 20110-2209, USA (ATCC).

この寄託は、特許手続きのための微生物寄託の国際認識に関するブダペスト条約とその規制（ブダペスト条約）の規定のもとで行った。これによって、寄託日から３０年間の間、寄託した生存している培養物の維持が確実となる。寄託物は、ブダペスト条約の条項にしたがってＡＴＣＣによって入手することができ、そしてＧｅｎｅｎｔｅｃｈ，Ｉｎｃ．およびＡＴＣＣの合意が得られる。これにより、該当する米国特許が発行されると、または任意の米国出願もしくは外国出願が出願公開されると、どれでも、まずそのような状態となりさえすれば、一般の人々が寄託された培養物の子孫を永久的に制限なく入手できることが確実となり、そして３５ ＵＳＣ’１２２とそれに続く委員会の規則（３７ ＣＦＲ（米国特許法施行規則）§１．１４を含む、特に、８８６ ＯＧ ６３８を参照のこと）にしたがってそれらに与えられる、米国特許庁長官によって決定されるものの子孫を確実に入手できるようになる。

This deposit was made under the provisions of the Budapest Treaty and its regulations (Budapest Treaty) on the international recognition of microbial deposits for patent procedures. This ensures maintenance of the deposited living culture for 30 years from the date of deposit. Deposits can be obtained by the ATCC in accordance with the terms of the Budapest Treaty and can be obtained from Genentech, Inc. And the agreement of ATCC is obtained. This ensures that when a relevant US patent is issued or any US or foreign application is published, the culture in which the general public has been deposited, as long as it is in such a state. Of the United States, including 35 USC'122 and subsequent Commission Rules (including 37 CFR § 1.14, in particular, see 886 OG 638) To ensure that the descendants of those determined by the US Patent Commissioner are given to them.

  The designated agent of this application will be notified if the deposited material culture has been killed, lost or destroyed even if cultured under appropriate conditions. As soon as I agree to replace the material with the same different one. The availability of deposited materials is not to be construed as a permit to practice the invention in violation of the rights granted under the authority of any relevant Office in accordance with its patent rights.

  The above description herein is considered sufficient to enable one skilled in the art to practice the invention. The scope of the invention is not limited by the deposited construct. This is because the deposited embodiment is intended as an explanation of a particular aspect of the invention, and any construct that is functionally equivalent is within the scope of the invention. The deposit of materials herein is insufficient to allow the description contained herein to practice any embodiment of the invention, including its best mode, and it also demonstrates It is not intended to be interpreted as limiting the scope of the claims to the specific description.

  Indeed, in addition to those shown and described herein, various modifications of the present invention will become apparent to those skilled in the art from the foregoing description and will be encompassed by the appended claims.

1A-1B show the nucleotide and amino acid sequences (SEQ ID NOs: 1 and 2, respectively) of a 321 amino acid human CRIg polypeptide. 1A-1B show the nucleotide and amino acid sequences (SEQ ID NOs: 1 and 2, respectively) of a 321 amino acid human CRIg polypeptide. 2A-2B show the nucleotide and amino acid sequences (SEQ ID NOs: 3 and 4, respectively) of the full-length form of 399 amino acids of human CRIg (huCRIg or huCRIg-long) occurring in nature. 2A-2B show the nucleotide and amino acid sequences (SEQ ID NOs: 3 and 4, respectively) of the full-length form of 399 amino acids of human CRIg (huCRIg or huCRIg-long) occurring in nature. FIGS. 3A-3B show the nucleotide and amino acid sequences of 305 amino acid short form (huCRIg-short) of human CRIg that occurs in nature (SEQ ID NOs: 5 and 6, respectively). FIGS. 3A-3B show the nucleotide and amino acid sequences of 305 amino acid short form (huCRIg-short) of human CRIg that occurs in nature (SEQ ID NOs: 5 and 6, respectively). FIGS. 4A-4C show the nucleotide and amino acid sequences (SEQ ID NOs: 7 and 8, respectively) of the naturally occurring mouse CRIg (muCRIg) of 280 amino acids. FIGS. 4A-4C show the nucleotide and amino acid sequences (SEQ ID NOs: 7 and 8, respectively) of the naturally occurring mouse CRIg (muCRIg) of 280 amino acids. FIGS. 4A-4C show the nucleotide and amino acid sequences (SEQ ID NOs: 7 and 8, respectively) of the naturally occurring mouse CRIg (muCRIg) of 280 amino acids. FIG. 5 shows the amino acid sequences of full length huCRIg (SEQ ID NO: 4) and huCRIg-short (SEQ ID NO: 6) aligned with muCRIg (SEQ ID NO: 8). The hydrophobic signal sequences IgV, IgC, and the transmembrane region are shown. muCRIg has one putative N-linked glycosylation site at position 170 (NGTG). The Ig domain boundaries presumed to be derived from the exon-intron boundaries of the human CRIg gene are shown. FIG. 6 shows in situ hybridization of CRIg in mouse liver frozen sections. FIG. 7 shows in situ hybridization of CRIg on human liver frozen sections. FIG. 8 shows in situ hybridization of CRIg in activated colon and adrenal macrophages, Kupfer cells, and placental Hofbauer cells. FIG. 9 shows in situ hybridization of CRIg mRNA in RA synovial cells. FIG. 10 shows in situ hybridization of CRIg mRNA in brain microglial cells. FIG. 11 shows in situ hybridization of CRIg mRNA in cells derived from human asthmatic tissue. FIG. 12 shows in situ hybridization of CRIg mRNA in cells derived from human chronic hepatitis tissue. FIG. 13 shows an immunohistochemical analysis of CRIg in adrenal macrophages. FIG. 14 shows an immunohistochemical analysis of CRIg in liver Kupffer cells. FIG. 15 shows an immunohistochemical analysis of CRIg in brain microglial cells. FIG. 16 shows immunohistochemical analysis of CRIg in placental Hofbauer cells. Northern blot analysis showing expression of huCRIg in various tissues. Two transcripts, 1.5 kb and 1.8 kb, were present in human tissues expressing CRIg. (A) TAQMAN® PCR analysis showing increased expression of huCRIg in bone marrow mononuclear cell lines HL60 and THP-1 and differentiated macrophages. Low level expression was seen in Jurkat T cells, MOLT3, MOLT4, and RAMOS B cell lines. (B) Increased expression of huCRIg mRNA during monocyte differentiation in vitro. Monocytes isolated from human peripheral blood were differentiated by adhering to plastic for 7 days. Total RNA was extracted at various times during differentiation. (C) Increased expression of huCRIg protein during monocyte to macrophage differentiation. Monocytes were treated as indicated in (B) and all of the cell lysate was run on a gel, transferred to a nitrocellulose membrane, which was incubated with a polyclonal antibody against huCRIg (4F7). Polyclonal antibodies recognize the 48 kDa and 38 kDa bands, probably indicating the long and short forms of huCRIg. Characterization of huCRIg protein molecules in cell lines. (A) huCRIg-gd was transiently expressed in 293E cells, immunoprecipitated with anti-gd, and the blots were incubated with polyclonal antibodies against the extracellular domain of anti-gd or CRIg. (B) huCRIg expressed in 293 cells is a monomeric N-glycosylated protein. CRIg is a tyrosine phosphorylated by treating HEK293 cells with sodium pervanadate, but does not recruit Syk kinase. Phosphorylated CRIg migrated to a slightly higher molecular weight compared to unphosphorylated CRIg. FIG. 20 shows selective expression of huCRIg on human monocyte-derived macrophages. Peripheral blood mononuclear cells were stained with antibodies specific for B cells, T cells, NK cells, monocytes, and an ALEXA ™ A488-conjugated monoclonal antibody against CRIg (3C9). Expression was absent in all peripheral blood leukocytes, as well as in monocyte-derived dendritic cells, but was expressed in macrophages differentiated in vitro. CRIg mRNA and protein expression was increased by IL-10 and dexamethasone. (A) Real-time PCR showed increased expression of CRIg mRNA after treatment with IL-10, and TGF-β was greatly induced by dexamethasone but was down-regulated by treatment with LPS, IFNγ, and TNFα. It was rate. (B) Peripheral blood mononuclear cells separated with Ficoll were treated with various cytokines and dexamethasone for 5 days and double-stained with anti-CD14 and anti-CRIg. Flow analysis shows a dramatic increase in the expression of CRIg on the surface of monocytes after treatment with dexamethasone and treatment with IL-10 and LPS. CRIg mRNA and protein expression was increased by IL-10 and dexamethasone. (A) Real-time PCR showed increased expression of CRIg mRNA after treatment with IL-10, and TGF-β was greatly induced by dexamethasone but was down-regulated by treatment with LPS, IFNγ, and TNFα. Rated. (B) Peripheral blood mononuclear cells separated with Ficoll were treated with various cytokines and dexamethasone for 5 days and double-stained with anti-CD14 and anti-CRIg. Flow analysis shows a dramatic increase in the expression of CRIg on the surface of monocytes after treatment with dexamethasone and treatment with IL-10 and LPS. Intracellular 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 examined in a confocal microscope. CRIg was found in the cytoplasm and localized with the lysosomal membrane protein CD63. CRIg was also expressed at the beginning and end of macrophages in a pattern similar to that of F-actin. Scale bar = 10 μm. Localization of CRIg mRNA in chronic inflammatory diseases. In situ hybridization indicated the presence of CRIg mRNA in alveolar macrophages obtained from tissues of pneumonia patients (A, B) or chronic asthma patients (C, D). CRIg mRNA was also expressed in liver Kupffer cells (E, F) in tissues obtained by liver biopsy from patients with chronic hepatitis. CRIg mRNA expression was increased in inflamed synovium. CRIg mRNA was low or absent in the synovium of the joints obtained by knee replacement in patients with non-inflamed joints (A, C), but patients with osteoarthritis (B, D) pannus was highly expressed in cells (probably synovial cells or synovial macrophages). Detection of CRIg protein using polyclonal antibody 6F1 in cells lining the synovium of patients with degenerative joint disease (A, B, C). No immunohistochemical detection of CRIg was seen in the control synovium (D). CRIg protein was expressed in a subtype of tissue resident macrophages, and its expression was increased in chronic inflammatory diseases. (A) CRIg was expressed on the membrane of CHO cells that stably express CRIg. High expression of CRIg protein was seen in alveolar macrophages (B) in tissues obtained from patients with chronic asthma. (C) CRIg expression in human small intestinal histocytes. Sections were obtained from tissue removed by surgery, which could contain tumors. (D) Expression of CRIg protein in placental Hofbauer cells prior to the expected date of human birth. High expression of CRIg protein in macrophages was present in the adrenal glands (E) and in human Kupffer cells (F). Staining was performed on sections fixed with 5 μm thick acetone using DAB as the chromogen. Images were taken at 20 × and 40 × magnification. Immunohistochemical staining of CD68 and CRIg on vascular plaques obtained from patients with atherosclerosis. Serial sections were fixed and stained with monoclonal antibody (A, B) against human CD68 and polyclonal antibody 6F1 (C, D) raised against human CRIg. CRIg was evident in macrophage populations and phoam cell populations present in atheromatous plaques and overlapped with CD68 positive macrophages as judged by staining on serial sections. Magnification: 10x (A, C) and 20x (B, D). Co-staining of CRIg and CD68 on cardiac stromal macrophages. 5 μm sections were obtained from a human heart (cadaveric autopsy) and stained with a monoclonal antibody against CRIg (3C9) and a secondary anti-mouse FITC-labeled antibody. CD68 was detected by staining with a PE-labeled monoclonal antibody against CD68. Magnification: 20x. CRIg mRNA levels were significantly higher in colon tissue from patients with ulcerative colitis, Crohn's disease, chronic obstructive pulmonary disease (COPD), and asthma. Real-time PCR was performed on total RNA extracted from various tissues. CRIg mRNA was significantly increased in tissues from patients with ulcerative colitis, Crohn's disease, and COPD. Statistical analysis was performed using the Mann-Whitney U test. Cells expressing human CRIg showed increased adhesion to human endothelial cells. (A) CRIg was stably expressed in the human Jurkat T cell line. (B) 96-well plate coated with a monolayer of human umbilical vein endothelial cells (HUVEC) pre-loaded with fluorescent dye BCECF (Molecular Probes, Oregon) and treated or not with 10 ng / ml TNFα Added to. After three washes, fluorescence was counted with a fluorescence spectrometer, which revealed the number of cells that remained attached to HUVEC cells. The graph is typical of 4 independent experiments. Inhibition of the progression of a mouse model of collagen-induced arthritis (CIA) by muCRIg IgG-Fc fusion protein. (CIA) One group of mice (n = 7) was administered 100 μg of muCRIg IgG-Fc fusion protein (square), while a control group of CIA mice (n = 8) was administered with 100 μg of mouse IgG1 ( Circle) was administered 3 times a week for 6 weeks. Mice were observed daily for signs of inflammation, scored on a scale of 0-16 (described in detail in Example 25), and results plotted on a graph (mean ± SD, student T-test control IgG1 vs. test muCRIg protein For p value = 0.0004). FIG. 32 is the nucleotide sequence (SEQ ID NO: 9) of DNA42257 (consensus sequence). FIG. 33 shows reduction of joint swelling in mice treated with CRIg-Fc. FIG. 34 shows that muCRIg inhibits joint inflammation. FIG. 35 shows the preservation of cortical bone mass in the joints of mice treated with muCRIg-Fc. FIG. 36 shows that treatment with CRIg-Fc does not change the number or morphology of tissue resident macrophages. FIG. 37 shows that treatment with muCRIg has no effect on serum anti-collagen antibody titers. FIG. 38 shows that muCRIg does not alter T-dependent B cell responses in vivo. FIG. 39 shows macrophage infiltration in joints after antibody-induced arthritis (AIA) caused by F4 / 80 staining in frozen decalcified joints. FIG. 40 shows that CRIg-Fc prevents joint swelling after antibody-induced arthritis in Balb / c mice. FIG. 41 shows that muCIRg inhibits joint inflammation in antibody-induced arthritis. FIG. 42 shows that mouse CRIg-Fc fusion protein binds to C3 opsonized sheep erythrocytes (E-IgM). FIG. 43 shows that the binding of human CRIg-Fc to E-IgM is C3-dependent. FIG. 44 shows the binding of serum opsonized particles to CHO cells expressing CRIg. FIG. 45 shows that mouse CRIg-Fc binds complements C3b and iC3b but not C2, C4, C3c, and C3d. FIG. 46 shows that mouse and human CRIg-Fc binds complements C3b, C3bi, and C3c but not C1, C2, C4, C3a, and C3d. FIG. 47A shows that mouse and human CRIg-Fc inhibit zymosan C3 accumulation. FIG. 47B shows that mouse CRIg-Fc inhibits C3 activation in serum. FIG. 48 shows that mouse CRIg inhibits the hemolytic response induced by the alternative complement pathway but does not affect the hemolysis response of the classical complement pathway. FIG. 48 shows that mouse CRIg inhibits the hemolytic response induced by the alternative complement pathway but does not affect the hemolysis response of the classical complement pathway. FIG. 49 shows that CRIg ECD inhibits C3 and C5 alternative complement pathway convertases. (A) CRIg inhibits the hemolytic reaction of rabbit erythrocytes (alternative complement pathway) in C1q-deficient serum, but does not inhibit the hemolytic reaction of IgM opsonized sheep erythrocytes (classical complement pathway) in fB-deficient serum. (B) CRIg inhibits liquid phase C3 convertase activity. (C) CRIg does not function as a cofactor for C3 cleavage mediated by factor I. (D) CRIg does not function as an acceleration factor for C3 convertase decoy. (E) CRIg inhibits the alternative complement pathway C5 convertase formed on zymosin particles. CRIg is selectively expressed on a subpopulation of tissue resident macrophages. (A) CRIg is a member of a transmembrane immunoglobulin superfamily consisting of one (human CRIg short (muCRIg (S)) and mouse CRIg (muCRIg), or two (huCRIgL) immunoglobulin domains. The scale at the top of the left panel shows the size of the amino acids.The right panel shows that huCRIg and muCRIg are closely related to the joint adhesion molecule-A (JAM-A) and A33 antigens. The upper scale of the right panel shows the percentage of amino acid similarity (B) CRIg is expressed in macrophages but not in monocytes. Human CD14 + monocytes and CD cultured for 7 days in 10% autologous serum and 20% fetal calf serum 4+ monocytes were analyzed for huCRIg staining by flow cytometry using anti-human CRIg MAb (3C9) Mouse CD11b + and F4 / 80 + liver Kupffer cells were muCRIg stained using anti-muCRIg MAb (14G6). (C) Western blot analysis of human and mouse macrophages, lysates from human CD14 + monocytes or mouse peripheral macrophages cultured for the indicated times were boiled in reducing SDS buffer and 4-4 Loaded on 10% Tris-Glycine gel and incubated with 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) was used as an isotype control, the arrows on the left panel indicate the positions of the 57 kDa and 50 kDa bands, which are probably huCRIg (L) and huCRIg (S). (D) Co-localization of CRIg and CD68 on liver Kupffer cells, immunostaining for sections obtained from human and mouse liver, monoclonal anti-CRIg (3C9 human and 14G6 mice), and monoclonal This was done using an anti-CD68 antibody. Flow cytometric analysis of CRIg expression on peripheral blood leukocytes and analysis of binding of C3 fragment C3 opsonized particles to CHO cells expressing CRIg. (A) Flow cytometric analysis of human and mouse expression of CRIg on human and mouse peripheral leukocytes. (B) Binding of soluble C3 fragment or complement opsonized pathogen to CHO cells expressing mouse CRIg. It does not bind to CHO cells that express JAM-2. Cells in suspension were incubated with A488-labeled complement opsonized particles for 30 minutes at room temperature under continuous rotation. Cells were washed three times and particle binding was monitored by flow cytometric analysis. The results are typical of 3 separate experiments. Soluble CRIg and cell surface expressed CRIg bind to C3 fragments deposited in solution or on the cell surface. (A) Jurkat cells (Jurkat-CRIg) transfected with CRIg (L) form rosettes with C3 and IgM opsonized sheep erythrocytes (E-IgM), but Jurkat transfected with an empty vector Cells (Jurkat-control) do not form this. The column chart (left panel) shows the expression of CRIg on Jurkat cells stably transfected with human CRIg (L). E-IgM opsonized with C3-deficient (C3-) serum or C3-enriched (C3 +) serum was mixed for 1 hour with Jurkat transfected with CRIg or control vector. The experiment is representative of three separate experiments. (B) CIg (L) -Fc binding 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 supplemented with increasing concentrations of purified human C3. Subsequently, E-IgM was incubated with huCRIg (L) -Fc protein, which was subsequently detected with an anti-human Fc polyclonal antibody detected by flow cytometry. The experiment is representative of three separate experiments. (C) ELISA showing binding of CRIg (L) -Fc and CRIg (S) -Fc to C3b and iC3b. Increasing concentrations of huCRIg (L) -Fc and huCRIg (S) -Fc fusion protein were added to purified C3b and iC3b coated maxisorb plates. Binding was detected using an HRPO-conjugated anti-huFc antibody. The results shown are representative of 4 separate experiments using different batches of fusion protein and purified complement components. (D) Kinetic binding data showing soluble C3b dimer binding to huCRIg (L) -Fc. The affinity of C3b for CRIg fusion protein was determined using surface plasmon resonance. CRIg protein was captured on a CM5 sensor chip by amine coupling of antibodies directed against the Fc fusion tag. The dimer C3b was then injected over a period of time sufficient to reach saturation. Kd was calculated from a binding curve showing the response at equilibrium, plotted against concentration. The C3b dimer bound with a calculated affinity of 44 nM to huCRIg (S) and with an affinity of 131 nM to huCRIg (L). (E) CRIg expressed on the cell surface binds to A488-labeled C3b dimer (C3b) 2) but does not bind to C3 present in nature. The left panel shows the expression level of huCRIg (L) on transfected THP-1 cells by flow cytometric analysis. (C3b) 2 shows saturable binding to THP-1 cells transfected with CRIg. Binding of (C3b) 2 to THP-1 CRIg competed with (C3b) 2, C3b, and the extracellular domain of CRIg (CRIg-ECD), but not with C3. The results are representative of 3 separate experiments. Creation and characterization of CRIg ko mice. (A) Creation of targeting vector used for homologous recombination in ES cells. (B) Confirmation by Southern blot of homologous recombination of SRIg alleles in heterozygous female offspring derived from chimeric mice mated with wt mice. (C) Comparison of leukocyte counts in peripheral blood of wt and ko male and female mice. (D) FACS analysis showing no expression of CR1, CR2, and CD11c in KC. (E) FACS analysis of C3-A488 and C3c-A488 binding to wt KC and ko KC. CRIg expression on Kupffer cells is essential for C3b and iC3b binding. (A) CRIg protein is not present on macrophages obtained from CRIg KO mice. Peripheral macrophages obtained from CRIg wt, CRIg het, or CRIg ko mice were incubated with anti-muCRIg mAb (14G6: left panel). Kupffer cells (KC) obtained from CRIg wt and CRIg ko mice were incubated with antibody 14G6 and analyzed by flow cytometry. (B) The expression levels of CD11b and CD18, complement receptor 3 α and β chains, and Cry were similar for Kupffer cells obtained from CRIg we and CRIg ko mice. Kupffer cells isolated from CRIg wt or CRIg ko mice were incubated with antibodies against CD11b, CD18, and Crry and analyzed by flow cytometry. (C) Kupffer cells isolated from CRIg wt or ko mice were incubated with activated mouse serum (activated by incubation at 37 ° C. for 30 minutes), C3b, (C3b) 2, and iC3b. Binding of the purified complement component to the cell surface was detected with polyclonal antibodies that recognize various C3-derived fragments. Results are typical of 4 experiments. (D) KC isolated from CRIg ko mice showed low reset by sheep red blood cells (E-IgM) coated with IgM opsonized in C3 well mouse serum. KCs isolated from livers of CRIg wt and CRIg ko mice were incubated with complement C3 opsonized E-IgM in the presence of control IgG or anti-CR3 blocking antibody (M1 / 70) for 30 minutes. Cells were fixed and the number of KCs that formed rosettes with E-IgM were counted and expressed as a percentage of the total number of KCs. ^* = P <0.05. The results are typical of two separate experiments. Recycling of CRIg on Kupffer cells (A) Kupfer cells (KC) from C3 wt (panels 1, 3, 4, and 6) or C3 ko mice (panels 2, 5) were transformed into A488-labeled anti-CRIg antibody (14G6). ) And (C3b) 2 were incubated at 4 ° C. for 1 hour (panels 1-3) or at 37 ° C. for 10 minutes (panels 4-6). The cells are then transferred to 4 ° C. and incubated with anti-A488 antibody (red column) or with no antibody (black column) and cytoplasm derived from anti-CRIg or C3b expressed on the cell surface. Distinguished. (B) Internalization and colocalization of CRIg and C3b at CRIg wt. This does not occur with CRIg ko, KC. KCs isolated from livers of CRIg wt and CRIg ko mice were cultured on chamber slides for 2 days, incubated with A455-conjugated anti-CRIg antibody and A488-conjugated C3b for 30 minutes at 37 ° C., mounted, and photographed. (C) CRIg (not Lamp1) antibody is recycled on the cell surface. Kupffer cells were loaded with A488-conjugated anti-muCRIg antibody or anti-muLamp1 antibody for 10 minutes at 37 ° C., washed, and then incubated at 37 ° C. in the presence of anti-A488 stop antibody for the time indicated. The results shown are typical of 3 independent experiments. CRIg is expressed on recycled endosomes that are recruited to particle uptake sites. (A) CRIg expressed on the cell surface is localized in the F-actin positive membrane raffle. Monocyte-derived macrophages were cultured for 7 days with A488-conjugated anti-CRIg A488 mAb 3C9 (green channel in A1 and A3), and Alexa 546-phalloidin (red channel in A2 and A3). Incubated at 0 ° C. The arrow tip shows a membrane raffle where both CRIg and actin staining are stronger than the rest of the cell surface (yellow in the superimposed image in A3). The scale bar is 20 μm. (B) CRIg and C3b are localized together with transferrin in the recycled endosome. Macrophages were incubated for 1 hour on ice with CRIg-A488 (green channel in B1, B4) or C30A488 (red channel in B2, B4), then A647-transferrin (B3, B4 in Followed for 10 minutes at 37 ° C. in the presence of blue channel). Scale bar = 20 μm. (C) CRIg is recruited to the phagocytic cup and the phagosome membrane. Macrophages were incubated for 10 minutes at 37 ° C. (C1-4) or 2 with IgM-coated erythrocytes opsonized with C3-enriched serum in the presence of A647-labeled transferrin (C2,6, and blue channels C4,8) or 2 Incubated for time (C5-8). The cells were then fixed, permeabilized, and red in the anti-CRIg polyclonal antibody (C1,2, and C4, 5 green channels) and the A555 binding antibody against LAMP-1 (C3,7, and C4,8) Channel). Transport of CRIg in human monocyte-derived macrophages (A) FACS plot showing saturable binding of C3b-A488 to CRIg on day 7 MDM. (B) MDM was pulsed with anti-CRIg antibody and C3b-A488 for 10 minutes at 37 ° C. in the presence of a 10-fold molar excess of huCRIg (L) -ECD. Anti-CRIg antibody binding and uptake was specific for CRIg. This could be avoided by co-incubation of the antibody with a 10-fold molar excess of CRIg-ECD (panel 1), while transferrin uptake could remain intact (panel 2). is there. (C) MDM was 20 hours at 37 ° C. in the presence of a lysosomal protease inhibitor, then the cells were washed and fixed with 1% PFA, and the incorporated antibody was detected with Cy3-labeled anti-mouse IgG (C panel 1 and the red channel in panel 3). Cells were stained simultaneously with 10 μg / ml rabbit anti-CRIg 6F1, followed by FITC anti-rabbit to detect the distribution of all CRIg (green panel in C panel 2 and C panel 3). The incorporated antibody almost completely overlaps with the endogenous CRIg signal (yellow in the superimposed image in C panel 3), indicating that antibody uptake has an effect on CRIg transport. It shows that it does not reach. The scale bar was 20 μm, and 5 μm was shown in the lower right of each channel in the 4 × enlarged inset of the portion enclosed by the square. In C panel 4, human macrophages were incubated with C3-depleted serum for 13 hours, then fixed and labeled with rabbit anti-CRIg F1 and FITC anti-rabbit. The distribution of CRIg was essentially the same as the distribution in C3-enriched serum, both almost completely overlapping transferrin, a reused endosomal marker (data not shown). The scale bar is 20 μm. (D) MDM was incubated with 1 μg / ml anti-CRIg-A488 (panel 1), transferrin-A647 (panel 2) for 10 minutes at 37 ° C., fixed in 4% PFA, and permeated with saponin buffer. And incubated with mouse anti-human Lamp-1-A555 (panel 3). Arrows indicate co-localization of CRIg and transferrin to the compartment being reused. (E) MDM was added at 37 ° C. with 1 μg / ml anti-CRIg-A488 (green channel in panel 1, panel 4), transferrin-A647 (blue channel in panel 2, panel 4). Incubated with compartmental C3 opsonized sheep erythrocytes (SRBC, red channel in panel 3, panel 4) stained with PKH at a 1:10 macrophage: SRBC ratio. Mice deficient in CIRg are susceptible to infection with Listeria Monocytogenes (LM). (A) Survival curves of female CRIg wt and CRIg ko mice (n = 5-7 per group) infected with the doses of LM indicated by injection into the tail branch. Statistical analysis (Wilcoxon): p <0.005 for wt vs. ko 2 × 10e4 colony forming units (CFU), p <0.0001 for 5 × 10e4 and 2 × 10e5 CFU. (B) Analysis of the number of bacteria in the heart, liver, blood, and spleen 10 minutes after LM infection (2 × 10e7 CFU, n = 5 per group). Statistical analysis ( ^paired t-test): ^** p <0.01, ^* p <0.05. (C) Increased concentrations of cytokines and chemokines in serum of CRIg ko mice one day after LM infection. Statistical analysis (unpaired t-test): ^*** p <0.001. (D) Reduced LM-A488 uptake in KCs of CRIg ko mice. Mice were infected with 2 × 10e7 LM-A488. After 1 hour, the liver was perfused, incubated with antibodies to F4 / 80 and analyzed by flow cytometry. F4 / 80 positive KCs were then sorted by FACS and collected on poly-lysine coated slides for observation by fluorescence microscopy. The number of internalized LM-A488 was counted with a confocal microscope and the phagocytic index was calculated. The results are typical of at least two experiments. (E) CRIg mice have reduced clearance of LM from circulation. CRIg and C3 double or single ko mice were i.v. with 2 × 10e7 CFU LM. v. Injected. CFU in the blood was counted 10 minutes after infection. In the presence of C3, CRIg ko mice had significantly lower clearance of LM from circulation (p <0.001). In the absence of C3, there was no significant decrease in LM clearance in CRI wt or CRIg ko mice. FIG. 59 shows the nucleotide sequence of a human CRIg (short) -IgG fusion (SEQ ID NO: 20). FIG. 60 shows the nucleotide sequence of the human CRIg (long) -IgG fusion (SEQ ID NO: 21). FIG. 61 shows the CRIg (STIgMA) -Fc binding moiety in the two constructs. Both of these are inserted into the CRK1-XbaI site in the pRK5 vector. FIG. 62 shows that muCRIg-Fc fusion protein (not the control Fc fusion protein) inhibits LM clearance from circulation in wt (not in CRIg ko cells). CRIg wt mice and CRIg ko mice were injected with 2 × 10 ⁷ CFU of LM i.v. with 2 injections of 12 mg / kg muCRIg-Fc or control-Fc fusion protein. v. Treated 24 and 16 hours prior to injection. CFU in the blood was counted 10 minutes after injection. CRIg wt mice treated with muCRIg-Fc had significantly lower clearance of LM from the circulation when compared to wt mice treated with control-Fc (p <0.001, unmatched student t Test). In CRIg ko mice, treatment with muCRIg-Fc had no effect on LM clearance. Inhibition of complement-mediated immune hemolysis with huCRIg molecules. (A) Inhibition of hemolysis of Cyno serum RRBC using hCRIg-short and hCRIg-long fusion proteins. (B) Inhibition of cyno serum RRBC hemolysis using hCRIg-long ECD. Inhibition of the hemolysis of human serum using hCRIg-long in two experiments. Inhibition of hemolysis of human serum using hCRIg-short-Fc and hCRIg-long-Fc fusion proteins. Inhibition of hemolysis of human serum using hCRIg-long-ECD and hCRIg-short-ECD, respectively. FIG. 67 shows the nucleic acid sequence encoding huCRIg-long-Fc (“empty” construct) (SEQ ID NO: 25). FIG. 68 shows the nucleic acid sequence encoding huCRIg-long-Fc with a “stem” inserted between the transmembrane domain of CRIg and the Fc portion (SEQ ID NO: 26). FIG. 69 shows the nucleic acid sequence encoding huCRIg-short-Fc (a “no-pattern” construct) (SEQ ID NO: 27). FIG. 70 shows a nucleic acid sequence encoding huCRIg-short-Fc in which a “handle” is inserted between the transmembrane domain of CRIg and the Fc portion (SEQ ID NO: 28). 71A and B show the results of the murine CNV experiment described in Example 23. FIG.

Claims (82)

A method for the prevention or treatment of an ocular condition involving complement, comprising the step of administering to a subject in need thereof a prophylactically effective amount or a therapeutically effective amount of a complement inhibitor. 2. The method of claim 1, wherein the complement inhibitor is a selective inhibitor of an alternative complement pathway. 3. The method of claim 2, wherein the complement inhibitor is a CRIg polypeptide or an agonist thereof. 4. The method of claim 3, wherein the CRIg polypeptide is selected from the group consisting of the CRIg polypeptide of SEQ ID NOs: 2, 4, 6, 8, and the extracellular domain (ECD) of the polypeptide. 5. The method of claim 4, wherein the CRIg polypeptide is an ECD of a CRIg polypeptide of SEQ ID NO: 2, 4, 6, or 8. 6. The method of claim 5, wherein the CRIg polypeptide is an ECD of a CRIg polypeptide of SEQ ID NO: 4 or 6. 4. The method of claim 3, wherein the CRIg polypeptide is fused to an immunoglobulin sequence. 8. The method of claim 7, wherein the immunoglobulin sequence is an immunoglobulin constant region sequence. 9. The method of claim 8, wherein the immunoglobulin constant region sequence is that of an immunoglobulin heavy chain. 10. The immunoglobulin heavy chain constant region sequence is fused to the extracellular region of the CRIg polypeptide of SEQ ID NO: 2, 4, 6, or 8 so that a CRIg-Ig fusion protein is produced. the method of. 11. The method of claim 10, wherein the immunoglobulin constant region sequence is that of IgG. The method according to claim 11, wherein the IgG is IgG-1 or IgG-3. 13. The method of claim 12, wherein the IgG-1 heavy chain constant region sequence includes at least a hinge, CH2, and CH3 region. 13. The method of claim 12, wherein the IgG-1 heavy chain constant region sequence includes a hinge, CH1, CH2, and CH3 region. 11. The method of claim 10, wherein the CRIg-Ig fusion protein includes a linker between the CRIg sequence and the Ig sequence. 11. The method of claim 10, wherein the CRIg-Ig fusion protein is encoded by a nucleic acid selected from the group consisting of SEQ ID NOs: 20, 21, 25, 26, 27, and 28. Retinopathy in which the complement symptoms are related to age-related macular degeneration (AMD), chronic choroidal neovascularization (CNV), uveitis, diabetic retinopathy and other ischemia Selected from the group consisting of: endophthalmitis, diabetic macular edema, pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, central retinal vein occlusion (CRVO), corneal neovascularization, and retinal neovascularization, The method of claim 1. 18. The method of claim 17, wherein the eye disease to which the complement is related is age-related macular degeneration (AMD) or chronic choroidal neovascularization (CNV). 19. The method of claim 18, wherein the method includes prevention of CNV. 19. The method of claim 18, wherein the method includes prevention of AMD progression. 21. The method of claim 20, wherein the method includes prevention of progression of AMD to CNV. A method for the prevention of the onset or progression of age-related macular degeneration (AMD), wherein the subject is at risk of developing AMD in at least one eye or diagnosed with AMD in at least one eye. Administering an effective amount of a CRIg polypeptide or agonist thereof. 23. The method of claim 22, wherein the CRIg polypeptide is the extracellular domain of the polypeptide of SEQ ID NO: 2, 4, 6, or 8. 24. The method of claim 23, wherein the CRIg polypeptide is the extracellular domain of the polypeptide of SEQ ID NO: 4 or 6. 23. The method of claim 22, wherein the agonist is a fusion polypeptide comprising a CRIg polypeptide sequence fused to an immunoglobulin sequence. 26. The method of claim 25, wherein the fusion polypeptide comprises the extracellular domain of the polypeptide of SEQ ID NO: 4 or 6, fused to an immunoglobulin heavy chain constant region sequence. 27. The method of claim 26, wherein the fusion polypeptide is selected from the group consisting of fusion polypeptides encoded by the nucleotide sequences of SEQ ID NOs: 20, 21, 25, 26, 27, and 28. 28. The method of any one of claims 22 to 27, wherein the subject is a human. 30. The method of claim 28, wherein the human subject has been diagnosed with AMD in at least one eye. 30. The method of claim 29, wherein the AMD is Category 3 or Category 4 dry AMD. 32. The method of claim 30, wherein the subject has been identified as being at risk of developing CNV. 32. The method of claim 31, wherein the subject is genetically at risk of developing CNV. 32. The method of claim 30, wherein the human subject has been diagnosed as having AMD in both eyes. 34. The method of claim 33, wherein the human subject has category 3 or category 4 AMD in both eyes. 30. The method of claim 28, wherein the administration slows progression of AMD. 29. The method of claim 28, wherein the administration slows progression of AMD to CNV. 30. The method of claim 28, wherein the administration prevents AMD from progressing to CNV. 30. The method of claim 29, wherein the human subject has been diagnosed with AMD in only one eye. 40. The method of claim 38, wherein the administration delays the onset of AMD in the other eye. 40. The method of claim 38, wherein said administration prevents the development of AMD in the other eye. 30. The method of claim 28, wherein the administration is performed by intravitreal injection. 30. The method of claim 28, further comprising administration of an additional agent for prevention or treatment of AMD or CNV. 43. The method of claim 42, wherein the additional agent is an anti-VEGF-A antibody. A method for the treatment of dry age-related macular degeneration (AMD) comprising administering to a subject in need thereof a prophylactically effective amount or a therapeutically effective amount of a CRIg polypeptide or an agonist thereof. 45. The method of claim 44, wherein the CRIg polypeptide is selected from the group consisting of a CRIg polypeptide of SEQ ID NOs: 2, 4, 6, 8, and an extracellular domain (ECD) of the polypeptide. 46. The method of claim 45, wherein the CRIg polypeptide is an extracellular region of a CRIg polypeptide of SEQ ID NO: 2, 4, 6, or 8. 47. The method of claim 46, wherein the CRIg polypeptide is the extracellular region of the CRIg polypeptide of SEQ ID NO: 4 or 6. 46. The method of claim 45, wherein the CRIg polypeptide is fused to an immunoglobulin sequence. 49. The method of claim 48, wherein the immunoglobulin sequence is an immunoglobulin constant region sequence. 50. The method of claim 49, wherein the immunoglobulin constant domain sequence is that of an immunoglobulin heavy chain. 51. The method of claim 50, wherein the immunoglobulin heavy chain constant region sequence is fused to the extracellular domain (ECD) of the CRIg polypeptide of SEQ ID NO: 2, 4, 6, or 8. 52. The method of claim 51, wherein the immunoglobulin constant region sequence is that of IgG. 53. The method of claim 52, wherein the IgG is IgG-1 or IgG-3. 54. The method of claim 53, wherein the IgG-1 heavy chain constant region sequence includes at least a hinge, CH2, and CH3 region. 54. The method of claim 53, wherein the IgG-1 heavy chain constant region sequence includes a hinge, CH1, CH2, and CH3 regions. A method for the prevention or treatment of a disease or condition involving complement, wherein a subject in need of such treatment is treated with a prophylactically or therapeutically effective amount of a CRIg polypeptide or an agonist thereof. A method comprising the step of: 57. The method of claim 56, wherein the CRIg polypeptide is selected from the group consisting of CRIg polypeptides of SEQ ID NOs: 2, 4, 6, 8 and the extracellular region of such polypeptides. 58. The method of claim 57, wherein the CRIg polypeptide is fused to an immunoglobulin sequence. 59. The method of claim 58, wherein the immunoglobulin sequence is an immunoglobulin constant region sequence. 60. The method of claim 59, wherein the immunoglobulin constant region sequence is that of an immunoglobulin heavy chain. 61. The method of claim 60, wherein the immunoglobulin heavy chain constant region sequence is fused to the extracellular domain (ECD) of the CRIg polypeptide of SEQ ID NO: 2, 4, 6, or 8. 62. The method of claim 61, wherein the immunoglobulin heavy chain constant region sequence is that of an IgG. 64. The method of claim 62, wherein the IgG is selected from IgG-1 and IgG-3. 64. The method of claim 62, wherein said IgG-1 heavy chain constant region sequence comprises at least a hinge, CH2, and CH3 region. 64. The method of claim 62, wherein the IgG-1 heavy chain constant region sequence includes a hinge, CH1, CH2, and CH3 regions. 57. The method of claim 56, wherein the complement-related disease is an inflammatory disease or an autoimmune disease. Diseases with which the complement is concerned include rheumatoid arthritis (RA), adult respiratory distress syndrome (ARDS), distant tissue injury after ischemia reperfusion, complement activation during cardiopulmonary bypass surgery, dermatomyositis , Pemphigus, lupus nephritis and resulting glomerulonephritis and vasculitis, cardiopulmonary bypass surgery, coronary artery endothelial dysfunction induced by heart failure, 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 distress syndrome (COPD), asthma, aspiration Selected from the group consisting of pneumonia, urticaria, chronic idiopathic urticaria, hemolytic uremic syndrome, endometriosis, cardiogenic shock, ischemia reperfusion injury, and multiple sclerosis (MS), Item 66 The method described. Diseases related to the complement include inflammatory bowel disease (IBD), systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathy, systemic sclerosis (scleroderma), idiopathic inflammatory muscle Disease (dermatomyositis, polymyositis), Sjogren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria) autoimmune thrombocytopenia (idiopathic) Thrombocytopenic purpura, immune thrombocytopenia), thyroiditis (Graves' disease, Hashimoto's disease thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, immune kidney disease (glomerulonephritis, Tubulointerstitial nephritis), demyelinating diseases of the central and peripheral nervous system (eg, multiple sclerosis, idiopathic polyneuropathy), hepatobiliary disease (eg, infectious hepatitis (A, B, C, D, hepatitis E, And other non-liver tropic viruses)), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, inflammatory and fibrotic lung disease (eg, cystic fibrosis) ), Gluten-sensitive bowel disease, Whipple disease, autoimmune or immune skin disease (including vesicular skin disease, polymorphic erythema, and contact dermatitis), psoriasis, lung allergic disease (eg, favorable Eosinophilic pneumonia, idiopathic pulmonary fibrosis, and hypersensitivity pneumonia), transplant-related diseases (including transplant rejection and graft-versus-host disease), Alzheimer's disease, paroxysmal nocturnal hemoglobinuria, hereditary angioedema, 68. The method of claim 66, wherein the method is selected from the group consisting of: atherosclerosis. 68. The method of claim 66, wherein the complement-related disease is rheumatoid arthritis (RA), psoriasis, or asthma. 58. The method of claim 57, wherein the subject is a mammal. 72. The method of claim 70, wherein the mammal is a human. A method for inhibiting the production of a C3b complement fragment in a mammal comprising administering to said mammal an effective amount of a CRIg polypeptide or an agonist thereof. 74. The method of claim 72, wherein the CRIg polypeptide is selected from the group consisting of CRIg polypeptides of SEQ ID NOs: 2, 4, 6, 8, and the extracellular region of such polypeptides. 74. The method of claim 73, wherein the CRIg polypeptide is fused to an immunoglobulin sequence. 75. The method of claim 74, wherein the immunoglobulin sequence is an immunoglobulin constant region sequence. 76. The method of claim 75, wherein the immunoglobulin constant region sequence is that of an immunoglobulin heavy chain. 77. The method of claim 76, wherein the immunoglobulin heavy chain constant region sequence is fused to the extracellular domain (ECD) of the CRIg polypeptide of SEQ ID NO: 2, 4, 6, or 8. 77. The method of claim 76, wherein the immunoglobulin heavy chain constant region sequence is that of an IgG. 79. The method of claim 78, wherein the IgG is selected from IgG-1 and IgG-3. 80. The method of claim 79, wherein said IgG-1 heavy chain constant region sequence comprises at least a hinge, CH2, and CH3 region. 80. The method of claim 79, wherein said IgG-1 heavy chain constant region sequence comprises a hinge, CH1, CH2, and CH3 regions. A method for selective inhibition of an alternative complement pathway in a mammal comprising the step of administering to said mammal an effective amount of a CRIg polypeptide or an agonist thereof.

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