JP2010505743A - Crystal structure of CRIg and C3b: CRIg complex - Google Patents

Abstract

  The present invention relates to the crystal structure of the macrophage-specific receptor CRIg (previously called STIgMA) and its complex with the C3b and C3c subunits of complement C3 (C3b: CRIg and C3c: CRIg complex). Concerning the decision. The invention further relates to the use of the crystal structure of CRIg or C3b: CRIg complex for screening and identifying molecules structurally and / or functionally related to CRIg, including CRIg agonists and antagonists.

Description

  The present invention relates to the crystal structure of the macrophage-specific receptor CRIg (previously called STIgMA) and its complex with the C3b and C3c subunits of complement C3 (C3b: CRIg and C3c: CRIg complex). Concerning the decision. The invention further relates to the use of the crystal structure of CRIg or C3b: CRIg complex for screening and identifying molecules structurally and / or functionally related to CRIg, including CRIg agonists and antagonists.

  The complement system is a complex enzyme cascade composed of a series of serum glycoproteins that normally exist in an inactive proenzyme form. C3 plays an important role in complement activation because it represents the convergence of three major pathways: the classical, lectin, and alternative pathways of complement. C3 is composed of more than 20 proteins (MJ Walport, N Engl J Med 344, 1058 (2001) and (MJ Walport, N Engl J Med 344, 1140 (2001)) and a proteolytic enzyme C3 convertase (J. Janssen et al., Nature 437, 505 (2005)) interacts through a number of distinct binding sites that are only exposed after its cleavage.The first proteolytic step is a small (9 kDa) C3a peptide, also called anaphylatoxin, and a large (177 kDa) C3b. Cleavage of C3 to C3a and C3b induces a conformational change that exposes an embedded thio-ester bond in C3b that can covalently attach the molecule to the particle surface. This process, which is said to target the particles to bind to macrophage specific receptors, then removes the particles from the system.

  Recently, CRIg was identified as a receptor for C3b / iC3b opsonized particles (K. Y. Helmy et al., Cell 124, 915 (2006)). CRIg binds to C3b, iC3b, and C3c, but cannot form a complex with the inactive precursor C3.

In addition to its function as opsonin, C3b is a subunit of complement C3 and C5 convertases. C3b binds to serine protease factor B (W. Vogt, G. Schmidt, B. Von Buttlar, L. Dieminger, Immunology 34, 29 (1978); Z. Fishelson, MK Pangburn, HJ Muller-Eberhard, J Biol Chem. 258, 741 1 (1983)). This complex forms a C3bBb complex which is an active C3 convertase of the alternative pathway (AP) after replenishment of factor D and activation by factor D. Addition of a second C3b molecule to C3 convertase has been shown to mediate inflammation in experimental animal models and play an important role in mediating human autoimmunity and inflammatory disease (MJ Walport, KA Davies, BJ Morley, M. Botto, Ann N YAcadSci 815, 267 (1997)) resulting in the formation of (C3b) ₂ Bb, the C5 convertase of the alternative pathway of complement. The C3b subunit of the alternative pathway convertase becomes a docking site for each substrate C3 or C5 and is required for the catalytic activity of the convertase (N. Rawal, MK Pangburn, J Immunol 164, 1379 (2000)). The alternative pathway of complement amplifies complement activation initiated through any of three pathways: alternative, classical and mannose-binding lectin pathways. As a result, loss of complement factor H, which is a regulator of alternative pathways, leads to amplification of inflammation (N. Rougier et al., J Am Soc Nephrol 9, 2318 (1998); MA Abrera-Abeleda et al. J Med Genet (Nov 18 , 2005)). Thus, cleavage of C3 and C5 by alternative pathway complement convertases results in opsonization, conversion enzyme production and inflammation.

  As a central component of the convertase, C3b is the target of many complement regulators. These include complement receptor 1 (CR1), membrane cofactor protein (MCP), decay accelerating factor (DAF), complement-related receptor y (Crry), factor H and factor I (D. Hourcade, VM Holers, JP Atkinson , Adv Immunol 45, 381 (1989)). Binding of factor I results in inactivation of C3b via proteolysis to form C3b and C3f (2 kDa), including a thio-ester domain (TED) and C3dg (40 kDa) that remains bound to the target; C3c (135 kDa) is finally generated. Recent reports on the crystal structures of C3 and C3c have significantly improved our understanding of the mechanism by which C3 is activated (B. J. Janssen et al., 2005, supra). C3c shows significant structural changes compared to C3, reflecting the results of multiple proteolytic cleavage steps. These structural studies suggest a conformation-dependent mechanism of C3 activation and regulation, but do not provide an answer to the conformation of the activated species C3b.

  The present disclosure provides the crystal structure of the macrophage complement receptor CRIg required for pathogen elimination. Also provided are C3b and C3c crystal structures in complex with CRIg. This first structure of the convertase subunit in complex with the natural cellular receptor and a regulator of complement activation improves our understanding of the mechanism of complement activation and regulation. The present invention also relates to CRIg C3b binding regions and domains and sequences within C3b involved in such binding that can be used to identify molecules functionally related to CRIg, including CRIg agonists and antagonists. Providing information.

  In one aspect, the invention relates to crystals formed by native sequence CRIg polypeptides or functional fragments or conservative amino acid substitution variants thereof.

In one embodiment, the crystal has a space group of lattice constant (cell constant) a = 30.3 Å, b = 50.8 Å, c = 62.0 Å, and P2 ₁ 2 ₁ 2 ₁ .

  In other embodiments, the native sequence CRIg polypeptide comprises a human CRIg polypeptide of SEQ ID NOs: 1, 2, and 3; a mouse CRIg polypeptide of SEQ ID NO: 4; a rat CRIg polypeptide of SEQ ID NO: 5; a bovine CRIg of SEQ ID NO: 6 A monkey CRIg polypeptide of SEQ ID NO: 7.

  In yet another embodiment, the functional fragment of CRIg is the extracellular domain (ECD) sequence of native sequence CRIg.

  In further embodiments, the crystal diffracts X-rays to determine atomic coordinates with a resolution of about 1 to 40 inches, or a resolution of at least about 5 inches, or at least 4 or at least 3 inches.

  In yet a further embodiment, the present invention relates to a CRIg crystal having the structural coordinates shown in Appendix 1.

  In another aspect, the invention relates to a composition comprising a crystal as described above.

  In yet another aspect, the invention relates to a crystalline form of a complex of a native sequence CRIg polypeptide or functional fragment or conservative amino acid substitution variant thereof and complement factor C3b.

In one embodiment, the crystalline form of the composite has approximately the lattice constants a = 97.6 Å, b = 255.7 Å, c = 180.3 Å, and the C222 ₁ space group.

  In other embodiments, the crystalline form of the complex comprises a human CRIg polypeptide of SEQ ID NO: 1, 2 and 3; a mouse CRIg polypeptide of SEQ ID NO: 4; a rat CRIg polypeptide of SEQ ID NO: 5; a bovine CRIg of SEQ ID NO: 6 A polypeptide; and one of the monkey CRIg polypeptides of SEQ ID NO: 7.

  In a further embodiment, the functional CRIg fragment is the extracellular domain (ECD) sequence of native sequence CRIg.

  In still further embodiments, the crystalline form of the complex diffracts X-rays to determine atomic coordinates with a resolution of about 1 to 40 inches, or a resolution of at least about 5 inches, or at least 4 or at least 3 inches.

  In yet another embodiment, the crystalline form of the CRIg: C3b complex has the structural coordinates shown in Appendix 2.

  In a further aspect, the invention relates to a crystalline form of a complex of a native sequence CRIg polypeptide or functional fragment or conservative amino acid substitution variant thereof and complement factor C3c.

  In certain embodiments, the crystalline form of the CRIg: C3b complex has approximately a space group of lattice constants a = 382.8 Å, b = 65.0 Å, c = 147.2 Å, β = 102.7, and C2. is doing.

  In other embodiments, the native sequence CRIg polypeptide present in the CRIg: C3C complex is a human CRIg polypeptide of SEQ ID NOs: 1, 2, and 3; a mouse CRIg polypeptide of SEQ ID NO: 4; a rat CRIg of SEQ ID NO: 5 Selected from the group consisting of: a polypeptide; a bovine CRIg polypeptide of SEQ ID NO: 6; and a monkey CRIg polypeptide of SEQ ID NO: 7.

  In yet another embodiment, the CRIg functional fragment present in the complex is the extracellular domain (ECD) sequence of native sequence CRIg.

  In further embodiments, the crystalline form of the CRIg: C3c complex diffracts X-rays to determine atomic coordinates with a resolution of about 1 to 40 inches, or at least about 5 inches, or at least 4 or at least 3 inches. .

  In a particular embodiment, the CRIg: C3c complex has the structural coordinates shown in Appendix 3.

  In a further aspect, the invention provides a molecule or molecular complex comprising at least a portion of a CRIg polypeptide of SEQ ID NO: 2 or a conservative substitution C3b binding site thereof, wherein the binding site is 8, 14- At least selected from the group consisting of 15, 41-42, 44, 45-47, 50, 52, 54-61, 62, 64, 85-87, 89, 95, 99, 105, 107-110, and 111 The present invention relates to a molecule or molecular complex that includes one amino acid residue and whose binding site is determined by a set of points having a distance of less than 4.7 mm from a point representing an amino acid atom represented by the structural coordinates described in Appendix 2.

  In another aspect, the invention relates to a three-dimensional conformation of a point where at least part of the point is derived from the structure coordinates of appendix 2 representing the position of the skeleton atom of at least the core amino acid that defines the CRIg binding site for C3b. . The three-dimensional structure of the points can be shown as, but not limited to, a holographic image, a stereo diagram, a model, or a computer display image where the CRIg binding site for C3b forms a crystal with space group symmetry C2221.

  In yet another aspect, the invention provides a machine readable data storage medium comprising data storage material encoded with machine readable data, wherein the machine programmed with instructions for using the data is C3b. A three-dimensional graphic diagram of at least one molecule or molecular complex comprising at least a part of the binding site is displayed, and the binding site determined by the set of points is represented by the structural coordinates described in Appendix 2. Relates to a medium having a distance of less than about 4.7 mm from the point representing the atoms.

  In a further aspect, the invention relates to a method of identifying a CRIg agonist comprising designing a compound that mimics the C3b binding site of CRIg.

  The invention further comprises (a) performing a fitting operation between the three-dimensional structure of the chemical and the CRIg polypeptide or C3b: CRIg complex using computational or experimental means; (b) obtained in step (a) It relates to chemicals identified by analyzing the data to characterize the binding between the chemical and the native CRIg or C3b: CRIg complex.

  In certain embodiments, the chemical entity interferes with the binding between CRIg and C3b in vivo or in vitro.

  The invention further relates to a molecule or molecular complex comprising at least part of the C3b binding region of a native sequence CRIg molecule or a conservative amino acid substitution variant thereof.

This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application in color drawings will be provided by the Patent Office upon request and payment of the necessary fee.
Domain structure and overall structure of C3 and C3b: CRIg and C3c: CRIg complexes. (A) C3 domain structure, β chain is shown in green, α chain is shown in blue green (ANA domain), orange (CUB), blue (TED), and purple. The red sphere indicates the position of the thio-ester. (B) Ribbon diagram of natural C3 (left side), C3b (middle) as a complex with CRIg, and C3c as a complex with CRIg. Using the color scheme of FIG. 1A, the surface of CRIg is shown in yellow. Note the significant movement of the CUB and TED domains when comparing C3 and C3b. After activation, the TED moves and rotates relative to the rest of C3b, and Cys988 moves beyond 80 cm. (C) Schematic of FIG. 1B; potential covalent binding of C3b to pathogens is shown in the C3B: CRIg complex.

CRIg binds to the center of the C3b and C3c β-strand keyrings. (A) C3c and CRIg complex. The surface of C3c is shown with the α chain residues whitened, and the MG1, MG2, MG3, MG4, MG6, and LNK domains of the β chain are white, orange, red, light green, blue, and dark green, respectively. CRIg is shown as a ribbon diagram and labeled all β chains and the N- and C-termini. (B) Surface view of CRIg rotated 180 degrees about the Y axis from FIG. 2A. All atoms that were less than 4.5 A distance to C3c / C3b were colored similarly to the residue they are in contact with in FIG. 2A. (C) Relative affinity of CRIg wild type and color encoded CRIg variants. Affinity was determined by competition of various CRIg proteins with CRIg-LFH for binding to coated C3b on the plate. CRIg-LFH binding was detected using an anti-FLAG antibody. (D) CRIg surface showing the CRIg mutation used in the binding experiment shown in FIG. 2C. Mutated residues were colored according to the binding curve they represent in FIG. 2C.

CRIg inhibits the alternative (AP) pathway of complement but not the classical (CP) pathway. (A) CRIg inhibits AP-mediated C5b-9 deposition, but not the CP or MBL pathway. The complement pathway was reconstituted on plates coated with LPS (AP) or IgM (CP) and incubated with C1q deficient (AP) or fB deficient (CP) serum. Complement activation was determined by chromogen for detection of bound HRPO-conjugated antibody to C5b-9. The IC50 value of CRIg in the AP assay is 1.49 ± 0.19 μM (n = 3). (B) CRIg inhibits liquid phase C3 convertase activity. C3, fB and fD were incubated in veronal buffer in the presence of increasing concentrations of huCRIg (L) -ECD. The reaction was stopped with EDTA and the cleavage products were visualized with Coomassie stained gel (left panel) or ELISA (right panel). The IC50 value for CRIg-ECD is 0.39 ± 0.14 μM (n = 3). (C) CRIg inhibits C5 convertase collected on the surface of zymosan particles. Zymosan particles are opsonized with C3b in the presence of fB and fD to produce C5 convertase. A fixed concentration of C5 was mixed with convertase in the presence of increasing concentrations of CRIg wt or CRIg variant C (CRIg (C)). Convertase activity was determined by measuring C5b6 produced in the liquid phase using a hemolysis assay. IC50 values: CRIg (Wt) 1.63 ± 0.62 μM (n = 3), CRIg (E) 6.52 ± 3.21 μM (n = 3).

Systemically administered CRIg-Fc inhibits local and systemic complement activation associated with antibody-induced arthritis. (A) Reduced inflammation (histological score) and decreased bone deformity (articular cortical bone volume JCBV in mice treated with muCRIg-Fc of an already identified disease 7 days after secondary immunization with collagen increase). On day 64, metatarsal joints were prepared for routine H & E histology or micro computed tomography (micro CT). (B) Clinical score in mice receiving 3 weekly doses of 12 mg / kg muCRIg-Fc starting 3 days (left panel) or 5 days (right panel) after passive immunization with anti-collagen type II antibody Decrease. (C) Treatment with CRIg-Fc inhibits the accumulation of C3 fragments but not IgG2a on the cartilage surface of the joint. In the combined image, the red color indicates the presence of IgG2a anti-collagen antibody and the green color indicates the presence of complement C3 product on the cartilage surface. (D) Binding of CRIg-Fc to C3 (green) in the joints of mice treated with the previously identified disease CRIg-Fc. Scale bar = 20 um. (E) Upper panel: Inhibition of C3a production in joints in CRIg-Fc treated mice initiated the day before passive immunization. Lower panel: significant reduction in C5a levels in the serum of mice treated with CRIg-Fc. Statistical analysis (Student t test): * p <0.01, ** p <0.001, ** p <0.0001.

CRIg inhibits C5 convertase by inhibiting C5 binding to the non-catalytic subunit of the convertase. (A) CRIg blocks the binding of C5 to C3b2, but does not block the binding to C3bC4b. C3b, C3b2 and C3bC4b were captured with a polyclonal anti-C3 antibody and incubated with a mixture of C5 and increasing concentrations of CRIg (wt) or mutant CRIg (C). C5 bound to the convertase subunit was detected with a polyclonal anti-C5 antibody and a HRPO-conjugated secondary antibody. The IC50 for CRIg inhibition of C5 binding was 0.88 ± 0.22 μM. (B) CRIg decreases the Vmax of purified C5 convertase. A fixed concentration of CRIg was mixed with zymosan-conjugated C5 convertase in the presence of increasing concentrations of C5. The Km and Vmax values calculated based on the Michaelis-Menten equation are 0.99 and 4.01 in the absence of CRIg (wt) and 1.70 and 2.1 in the presence of 0.8 uM RIg (wt). 16.

Difference in domain arrangement between C3 and C3b. Shown are ribbon diagrams of CUB, TED and MG8-domains. (A) Overlay based on CUB domains of C3 and C3b (both orange). This superposition indicates that the CUB domain in C3b migrates with respect to the C3 MG8 domain (shown in light purple) while the TED domain undergoes a large rotation upon C3 activation. (C) Superposition based on TED domain. C3b is dark and C3 is light blue. Note the very compact domain arrangement in C3 that is completely open in the C3b structure. Cys988 [Position is indicated by a red sphere.

Interaction between TED and Arg80 in C3b. The figure shows a surface view of TED colored according to the electrostatic potential calculated by the program pymol (DeLano, MC and Cao, Y., Neuroimaging Clin. N. Am. 12 (1): 21-34 (2002)). Yes. Red and blue indicate negatively and positively charged patches, respectively. The side chain atom of R80 is represented by a sphere. Note that Arg 80 is in close proximity to the highly negatively charged grooves on the surface of the TED.

Sequence alignment of CRIg from different species. Residues in contact with C3b were colored according to FIG. 2A. Residue number means human CRIg after signal sequence. Secondary structural elements are shown in the sequence alignment above as arrows (strands) and boxes (helixes).

CRIg has the same affinity as C3b and C3c. Affinity was determined by competition of increasing concentrations of C3b or C3c with a fixed concentration of CRIg-LFH. CRIg-LFH binding was detected using an anti-FLAG antibody conjugated to HRPO, and the absorbance of the TNB reaction product was measured spectrophotometrically at a wavelength of 415 nm.

Local difference between C3 and C3b in the vicinity of the CRIg binding site. The superposition of C3 and C3b shown here is based on the Cα atom of the MG6 domain. A part of C3 is shown in white, and the α′NT domain is shown in blue. The C3b: CRIg complex is shown in green (C3b β chain), purple (C3b α chain), and the α′NT domain is shown in blue. CRIg is shown in yellow. Large conformational changes that occur upon C3 activation are indicated by red arrows. Note the large movement of the α'NT domain. The rotation of MG3 relative to MG6 and the movement of the helical segment (577: 590) is required for CRIg binding to C3b and C3c.

CRIg selectively inhibits alternative pathways in human and mouse serum. (A) CRIg blocks hemolysis of rabbit erythrocytes in C1q-deficient serum (alternate pathway, left panel), but does not block hemolysis of IgM opsonized sheep erythrocytes in fB-deficient serum (classical pathway, right panel). E-IgM and Er were incubated in 5% serum containing different concentrations of CRIg-Fc or CRIg-ECD fusion protein. Hemolysis was stopped by adding ice-cold EDTA and O. The amount of hemolysis was measured by determining D. IC50 values: CRIg-Fc 127 ± 49 nM (n = 6), CRIg-ECD 640 ± 123 nM. (B) CRIg inhibits the alternative pathway of complement disclosed by the classical pathway. The complement components of the classical pathway are collected on SRBC to generate EAC34 (AP, left panel) or EAC1234 (CP, right panel). Hemolysis was determined as described in (A). (C) Mouse CRIg-Fc inhibits alternative pathway hemolysis in mouse serum. Er was incubated with 30% mouse serum containing increasing concentrations of mouse CRIg-Fc. Hemolysis was determined as described in (A). (D) CRIg does not inhibit the mannose-binding lectin pathway. C5b-9 deposition induced by MBL pathway activation was measured spectrophotometrically at a wavelength of 415 nm.

CRIg inhibits collagen-induced arthritis. (A) Arthritis was induced by immunizing mice twice on days 0 and 23 with bovine collagen. Mice were treated with muCRIg-Fc three times weekly starting on day 24 (prophylactic) or day 45 (arrow) when disease was already confirmed. The clinical score reflects the thickness of the ankle. On day 70 after induction of arthritis, mice were euthanized, articular cortical bone volume (JCBV) was measured (B), and signs of inflammation were assessed on H & E stained sections (C). (D) Reduction of IL-6 (left panel) and IL-1b (right panel) in the joints of mice treated with CRIg-Fc. At the indicated times after induction of arthritis, mice were euthanized and the joints were dissected and processed for cytokine measurements. The increase in cytokine levels with LPS treatment (first time point) is not affected by muCRIg-Fc treatment. Statistics: * p <0.01, ** p <0.001, student t test.

(A) CRIg does not affect the cofactor activity of factor H, nor does it have an intrinsic cofactor activity. Left panel: A fixed concentration of C3b was mixed with increasing concentrations of Factor H in the presence of 4 μM CRIg (wt) or CRIg (C). Cofactor activity was monitored by visualizing reaction products on a Coomassie-stained SDS gel to distinguish C3b from iC3b. Right panel: Fixed concentrations of C3b and fI were mixed with fH or increasing concentrations of CRIg. (B) CRIg does not inhibit factor B binding to C3b. A fixed concentration of factor B was mixed with increasing concentrations of CRIg. The amount of bound factor B was determined using a polyclonal antibody against fB and a secondary HRPO conjugate ab. (C) CRIg does not disrupt C3 convertase. C3 convertase was formed on maxisoap plates by incubating C3b, factor B and factor D. The plates were then incubated with increasing concentrations of CRIg or factor H. The remaining bound factor B was detected with anti-factor B antibody and HRPO-conjugated secondary antibody. The results shown are representative of 3 independent experiments.

CRIg ECD can compete with CRIg-LFH for binding to C3b, but not C5. Mixtures of CRIg-LFH and increasing concentrations of CRIg-ECD or C5 were added to maxisolv plates coated with C3b. Remaining CRIg-LFH binding was detected using an anti-flag antibody.

Detailed Description of the Preferred Embodiments Definitions Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd edition, J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). See For the purposes of the present invention, the following terms are defined as follows:

  “CRIg”, “PRO362”, “JAM4” and “STIgMA” are used interchangeably and mean native sequence and mutant CRIg polypeptides.

  A “native sequence” CRIg is a polypeptide having the same amino acid sequence as a naturally occurring CRIg, regardless of its mode of preparation. Thus, the native sequence CRIg can be isolated from nature or can be produced by recombinant and / or synthetic means. The term “native sequence CRIg” specifically refers to naturally occurring truncated or secreted forms of CRIg (eg, extracellular domain sequences), naturally occurring mutant forms (eg, alternative splicing forms) and naturally occurring allelic variants of CRIg. Include. The native sequence CRIg polypeptide and fragments thereof are human CRIg polypeptides of SEQ ID NO: 1, 321 amino acids long, with or without the N-terminal signal sequence, with or without the first methionine position, sequence Specifically encompassed are those that contain or do not contain any or all of the transmembrane domain of amino acid positions from approximately 277 to 307 of number 1. The native sequence CRIg polypeptide and fragments thereof are the full-length 399 amino acid long human CRIg polypeptide (huCRIg or huCRIg-long) of SEQ ID NO: 2, with or without an N-terminal signal sequence, and the starting methionine at position 1. Including or not including, including or not including any or all of the transmembrane domain at approximately amino acid position 277 to 307 of SEQ ID NO: 2. In yet a further embodiment, the native sequence CRIg polypeptide and fragments thereof are a 305 amino acid short form of human CRIg polypeptide (huCRIg-short, SEQ ID NO: 3), with or without an N-terminal signal sequence, Those containing or not including the starting methionine at position 1 and those containing or not including any or all of the transmembrane domain at amino acid positions of approximately 183 to 213 of SEQ ID NO: 3. In a different embodiment, the native sequence CRIg polypeptide or fragment thereof is the 280 amino acid long full length mouse CRIg polypeptide (muCRIg) of SEQ ID NO: 4, with or without an N-terminal signal sequence. Those with or without an initiating methionine, those with or without any or all of the transmembrane domain at amino acid positions approximately 181 to 211 of SEQ ID NO: 4. The native sequence CRIg polypeptide or fragment thereof is a rat CRIg polypeptide of SEQ ID NO: 5, with or without an N-terminal signal sequence, with or without an initiation methionine, and any of the transmembrane domains or It can be included or not included. The terms further include the bovine CRIg polypeptide of SEQ ID NO: 6 and the monkey CRIg polypeptide of SEQ ID NO: 7, each with or without an N-terminal signal sequence, with or without an initiating methionine, and in each sequence In particular, those including or not including any or all of the transmembrane domain are included.

  "CRIg variant" means an active CRIg polypeptide as defined below having at least about 80% amino acid sequence identity with a native sequence CRIg polypeptide. In certain embodiments, the CRIg variant has at least about 80% amino acid sequence homology with the sequence of a native sequence mature full-length CRIg polypeptide. Such CRIg polypeptides include, for example, CRIg polypeptides in which one or more amino acid residues have been inserted, substituted, and / or deleted at the N- or C-terminus of the native sequence. Other variants are those in which one or more amino acids have been inserted, substituted, and / or deleted within the transmembrane region of the native polypeptide sequence.

  Typically, the CRIg variant is at least about 80% amino acid sequence identity, or at least about 85% amino acid sequence identity, or at least about 90% amino acid sequence identity with the mature amino acid sequence from any of SEQ ID NOs: 1-7. Or at least about 95% amino acid sequence identity, or at least about 98% amino acid sequence identity, or at least about 99% amino acid sequence identity. Preferably, the highest degree of sequence identity is within the extracellular domain (ECD) (amino acid 1 or about 21 to X of SEQ ID NO: 1 or 2, wherein X is any amino acid residue from 271 to 281; or sequence Amino acid 1 of number 3 or about 21 to X, wherein X is any amino acid residue from 178 to 186, or amino acid 1 or about 21 to X of SEQ ID NO: 4, wherein X is any amino acid of 176 to 184 Is a residue).

  CRIg (PRO362) “Extracellular domain” or “ECD” means the form of CRIg polypeptide essentially free of the transmembrane and cytoplasmic domains of each full-length molecule. CRIg ECD typically has less than 1% of such transmembrane and / or cytoplasmic domains, preferably less than 0.5% of such domains. As discussed above, in some cases, the CRIg ECD comprises amino acid residues 1 or about 21 to X of SEQ ID NO: 1, 2, 3, or 4 where X is about 1 of SEQ ID NO: 1 or 2. It includes any amino acid residue from 271 to 281, any amino acid from about 178 to 186 of SEQ ID NO: 3, and any amino acid from about 176 to 184 of SEQ ID NO: 4.

  “Percent (%) amino acid sequence identity” with respect to CRIg sequences refers to aligning sequences and introducing gaps as necessary to obtain maximum percent sequence identity, and any conservative substitutions are part of sequence identity. Defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues of the CRIg sequence. Alignments for the purpose of determining percent amino acid sequence identity are publicly available in various ways within the skill of the art, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software This can be achieved by using simple computer software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Unless otherwise stated, alignment is performed using the default parameters of the selected software. The acid sequence identity is then calculated for longer sequences. That is, even if a shorter sequence shows 100% sequence identity with a portion of a longer sequence, the overall sequence identity will be less than 100%.

  “Percent (%) nucleic acid sequence identity” to a CRIg coding sequence is a candidate that is identical to the nucleotides of the CRIg coding sequence after aligning the sequences and introducing gaps if necessary to obtain maximum percent sequence identity. Defined as the percentage of nucleotides in the sequence. Alignments for the purpose of determining percent nucleic acid sequence identity can be obtained from various methods within the purview of those skilled in the art, such as publicly available computers such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. This can be achieved by using software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The acid sequence identity is then calculated for longer sequences. That is, even if a shorter sequence shows 100% sequence identity with a portion of a longer sequence, the overall sequence identity will be less than 100%.

  An “isolated” nucleic acid molecule is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule normally associated with the natural source of the nucleic acid. An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules are therefore distinguished from nucleic acid molecules present in natural cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the encoded polypeptide where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.

  An “isolated” CRIg polypeptide-encoding nucleic acid molecule is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule normally associated with a natural source of the CRIg-encoding nucleic acid. An isolated CRIg polypeptide-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Thus, an isolated CRIg polypeptide-encoding nucleic acid molecule is distinguished from nucleic acid molecules present in natural cells. However, an isolated CRIg-encoding nucleic acid molecule includes, for example, CRIg-encoding nucleic acid molecules contained in cells that normally express CRIg where the nucleic acid molecule is in a chromosomal location different from that of natural cells.

  The term “complement-related disease” is used herein in the broadest sense and includes all diseases and pathological conditions whose etiology, such as complement deficiency, includes abnormalities in the activation of the complement system. The term includes diseases and pathological conditions that benefit from inhibition of C3 convertase. The term further includes diseases and pathological conditions that benefit from inhibition, including selective inhibition of alternative complement pathways. Complement-related diseases include, but are not limited to, inflammatory and autoimmune diseases such as rheumatoid arthritis (RA), acute respiratory distress syndrome (ARDS), remote tissue injury after ischemia and reperfusion, cardiopulmonary Complement activation during bypass surgery, dermatomyositis, pemphigus, lupus nephritis and resultant glomerulonephritis and vasculitis, cardiopulmonary bypass, cardioplegia-induced coronary endothelial dysfunction, type II membranoproliferative glomeruli Nephritis, IgA nephropathy, acute renal failure, cryoglobulinemia, antiphospholipid syndrome, macular degenerative diseases and other complement related ocular symptoms such as age-related macular degeneration (AMD), choroidal neovascularization (CNV) ), Uveitis, diabetes and other ischemia-related retinopathy, endophthalmitis, and other intraocular neovascular diseases such as diabetic macular edema, pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis , Film center vein occlusion (CRVO), corneal neovascularization, retinal neovascularization, as well as allotransplantation, hyperacute rejection, hemodialysis, chronic obstructive pulmonary distress syndrome (COPD), include asthma, and aspiration pneumonia.

  The term “complement-related ocular condition” is used in the broadest sense and includes all ocular conditions in which the etiology, including the classical and alternative pathways, particularly the alternative pathway of complement, is associated with complement. Complement-related ocular symptoms include, but are not limited to, macular degenerative diseases such as age-related macular degeneration (AMD), including dry and wet (non-exudative and exudative) forms, choroidal neovascularization ( CNV), uveitis, diabetes and other ischemia-related retinopathy, and other intraocular neovascular diseases such as diabetic macular edema, pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, retinal center Venous occlusion (CRVO), corneal neovascularization, and retinal neovascularization are included. Preferred groups of complement-related ocular symptoms include age-related macular degeneration (AMD), including non-wetting (wet) and exudative (dry or atrophic) AMD, choroidal neovascularization (CNV), diabetic Retinopathy (DR) and endophthalmitis are included.

  The terms “inflammatory disease” and “inflammatory disorder” are used interchangeably herein and a component of the mammalian immune system produces, mediates or contributes to an immune response that contributes to the prevalence of the mammal Means a disease or disorder. Also included are diseases in which a decrease in inflammatory response has a ameliorative effect on disease progression. Included within this term are immune-mediated inflammatory diseases, including autoimmune diseases.

  The term “T cell mediated” disease refers to a disease in which T cells directly or indirectly mediate or contribute to a mammalian morbidity. T cell mediated diseases are associated with cell mediated effects, lymphokine mediated effects, etc., and even B cell related effects if, for example, B cells are stimulated by lymphokines secreted by T cells.

  Examples of immune-related and inflammatory diseases, some of which are T cell mediated include, but are not limited to, inflammatory bowel disease (IBD), systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, Spondyloarthropathy, systemic sclerosis (scleroderma), 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-mediated thrombocytopenia), thyroiditis (Graves disease, Hashimoto thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroid) Inflammation), diabetes mellitus, immune-mediated renal disease (glomerulonephritis, tubulointerstitial nephritis), demyelinating diseases of the central and peripheral nervous systems, such as multiple sclerosis, multiple neuropathy, hepatobiliary disease, For example, infectious hepatitis (type A , B, C, D, E, and other nonhepatotropic viruses), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, Autoimmune or immune-mediated skin diseases including inflammatory and fibrotic lung diseases (eg cystic fibrosis), gluten irritable bowel disease, Whipple disease, bullous skin disease, erythema multiforme and contact dermatitis, psoriasis, Included are allergic diseases of the lung, such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and hypersensitivity pneumonia, transplantation related diseases including rejection and graft versus host disease. Alzheimer's disease and atherosclerosis are included.

  “Tumor” as used herein means any neoplastic cell growth and proliferation, whether malignant or benign, and any pre-cancerous cells and tissues.

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

  “Treatment” is an intervention performed by the present invention to prevent or alter the progression of disease pathology. Thus, “treatment” refers to both therapeutic treatment and prophylactic or protective measures. Those in need of treatment include those already suffering from the disease as well as those in which the disease is to be prevented. In the treatment of immune related diseases, the therapeutic agent directly alters the magnitude of the response of the components of the immune response or against treatment with other therapeutic agents such as antibiotics, antifungal agents, anti-inflammatory agents, chemotherapeutic agents, etc. It can increase the susceptibility of the disease.

  The “pathology” of diseases such as complement-related diseases includes all phenomena that endanger the good health of the patient. This includes, but is not limited to, abnormal or uncontrollable cell proliferation (neutrophils, eosinophils, monocytic, lymphoid cells), antibody production, autoantibody production, complement production, normal Interference with functioning neighboring cells, abnormal level release of cytokines or other secreted products, suppression or exacerbation of any inflammatory or immune response, inflammatory cells (neutrophils, eosinophils, monocytic , Lymphatic) infiltration into the tissue space.

  As used herein, the term “mammal” refers to any animal classified as a mammal, including humans, household and agricultural animals, zoos, sports, or pet animals, such as horses, pigs, cows, dogs, cats, and beaks. Means animals. In a preferred embodiment of the invention, the mammal is a human.

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

  The term “cytokine” is a generic term for proteins released from one cell population that act as intercellular mediators on other cells. Examples of such cytokines include lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, follicle stimulating hormone (FSH) and other glycoproteins Hormone, parathyroid stimulating hormone (TSH), luteinizing hormone (LH), liver growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-α and -β, Mueller inhibitor, mouse gonadotropin Related peptides, inhibin, activin, vascular endothelial growth factor, integrin, thrombopoietin (TPO), nerve growth factors such as NGF-β platelet growth factor, transforming growth factor (TGF) such as TGF-α and TGF-β, insulin-like Growth factors I and I Erythropoietin (EPO), osteoinductive factor, interferon such as interferon α, β, γ; colony stimulating factor (CSF) such as macrophage CSF (M-CSF), granulocyte macrophage CSF (GM-CSF), And granulocyte CSF (G-CSF); IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, Interleukins (IL) such as IL-11, IL-12, tumor necrosis factors such as TNF-α or TNF-β, and other polypeptide factors including LIF and kit ligand (KL) are included. As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of native sequence cytokines.

  A “therapeutically effective amount” refers to the active CRIg, CRIg agonist and CRIg required to achieve a measurable improvement in the target disease or condition, eg, a complement-related disease or condition, or cancer state, eg, pathology The amount of antagonist.

  The term “control sequence” refers to a DNA sequence necessary to express an operably linked coding sequence in a particular host organism. For example, suitable control sequences for prokaryotes 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 in a functional relationship with another nucleic acid sequence. For example, if a presequence or secretory leader DNA is expressed as a preprotein that participates in the secretion of the polypeptide, it is operably linked to the DNA of the polypeptide; Is operably linked to the coding sequence; or the ribosome binding site is operably linked to the coding sequence if it is positioned so as to facilitate translation. In general, “operably linked” means that the bound DNA sequences are in close proximity and, in the case of a secretory leader, in close proximity and in the reading phase. However, enhancers do not necessarily have to be close together. Binding is achieved by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to conventional techniques.

  The “stringency” of a hybridization reaction is readily determined by those skilled in the art and is generally an empirical calculation that depends on probe length, wash temperature, and salt concentration. In general, the longer the probe, the higher the temperature for proper annealing, and the shorter the probe, the lower the temperature. Hybridization generally depends on the ability of denatured DNA to reanneal when the complementary strand is present in an environment close to but below its 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, higher relative temperatures make the reaction conditions more stringent, while lower temperatures reduce stringency. 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 defined herein are (1) those using low ionic strength and high temperature for washing, eg, 0.015 M sodium chloride / 50 ° C. Using 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate; (2) using a denaturing agent such as formamide during hybridization, eg 50% (v / v) formamide at 42 ° C. Using 0.1% bovine serum albumin / 0.1% ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer pH 6.5 and 750 mM sodium chloride, 75 mM sodium citrate; or ( 3) 50% formamide at 42 ° C., 5 × SSC (0.75 M NaC , 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 × Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1 After washing in 0.2% SSC (sodium chloride / sodium citrate) at 42 ° C. in 50% formamide at 55 ° C. with 0% SDS and 10% dextran sulfate, 0% containing EDTA at 55 ° C. Can be identified by using a high stringency wash consisting of 1 × SSC.

  “Moderate stringent conditions” are specified as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and have lower stringency and higher stringency than those described above. Includes the use of hybridization conditions (eg, temperature, ionic strength and% SDS). Moderate stringent conditions are: 20% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, and 20 mg After overnight incubation at 37 ° C. in a solution containing / mL denatured sheared salmon sperm DNA, the filter was washed in 1 × SSC at 37-50 ° C. One skilled in the art will know how to adjust temperature, ionic strength, etc. to adapt to factors such as probe length as needed.

  The term “epitope tag” as used herein refers to a chimeric polypeptide comprising a polypeptide of the invention fused to a “tag polypeptide”. A tag polypeptide has a sufficient number of residues to provide an epitope from which the antibody can be produced, but its length is short enough not to inhibit the activity of the fused polypeptide. Also, the tag polypeptide is preferably fairly unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least 6 amino acid residues, usually about 8 to about 50 amino acid residues (preferably about 10 to about 20 residues).

  With respect to variants of the CRIg polypeptides of the invention, “active” or “activity” refers to the form of the polypeptide that retains the biological and / or immunological activity of the naturally occurring or naturally occurring polypeptide of the invention. means. A preferred biological activity is the ability to bind to C3b and / or affect complement or complement activation, in particular to inhibit alternative complement pathways and / or C3 convertase. Inhibition of 3 convertase can be measured, for example, by measuring inhibition of C3 turnover in normal serum during collagen- or antibody-induced arthritis, or inhibition of C3 deposition indirectly in arthritis.

  “Biological activity” for an antibody, polypeptide or other molecule (eg, organic or inorganic small molecule, peptide, etc.) that mimics the biological activity of CRIg and can be identified by the screening assays disclosed herein. Refers to the ability of such molecules to bind to C3b and / or affect complement or complement activation and specifically inhibit alternative complement pathways and / or C3 convertase.

  The term CRIg “agonist” is used in the broadest sense and includes any molecule that mimics the qualitative biological activity (described above) of a native sequence CRIg polypeptide.

  The term CRIg “antagonist” is used in the broadest sense and is any that partially or fully blocks, inhibits or neutralizes the qualitative biological activity of a natural polypeptide, such as a native sequence CRIg polypeptide. Including molecules.

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

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

  The term “antibody” is used in the broadest sense and includes, but is not limited to, a single anti-CRIg monoclonal antibody (including agonists, antagonists, and neutralizing antibodies), and anti-CRIg antibodies with multi-epitope specificity. Includes the composition. The term “monoclonal antibody” as used herein is identical except for a substantially homogeneous population of antibodies, ie, naturally occurring mutations in which the individual antibodies that make up may be present in minor amounts. It means an antibody obtained from a population.

  “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 increased levels by myeloma. The term “antibody” is used in the broadest sense and includes, but is not limited to, an intact monoclonal antibody, a polyclonal antibody, a multispecific antibody (eg, a bispecific antibody) formed from at least two intact antibodies, And specifically include antibody fragments so long as they exhibit the desired biological activity.

  “Natural antibodies” and “natural immunoglobulins” are heterotetrameric glycoproteins of approximately 150,000 daltons, usually composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy chain and light chain has regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the light chain constant domain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is the heavy chain Aligned with the variable domain. Certain amino acid residues are thought to form an interface between the light and heavy chain variable domains.

  The term “variable” means that a portion of the variable domain varies widely in sequence between antibodies and is used for the binding and specificity of each particular antibody to that particular antigen. However, variability is not uniformly distributed across the variable domains of antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions of both the light and heavy chain variable domains. The more highly conserved portions of variable domains are called the framework region (FR). The natural heavy and light chain variable domains are primarily four in a β-sheet arrangement linked by three CDRs that bind the β-sheet structure and in some cases form a loop that forms part of it. FR region is included. The CDRs of each chain are held close together by the FR region, and contribute to the formation of the antigen-binding site of the antibody together with the CDRs of the other chains (Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant domain is not directly involved in binding of the antibody to the antigen, but indicates the involvement of the antibody in various effector functions, such as antibody-dependent cytotoxic activity.

“Antibody fragments” comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments are Fab, Fab ′, F (ab ′) ₂ , and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8 (10): 1057-1062 [1995]); Single chain antibody molecules; and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments called “Fab” fragments, each with a single antigen-binding site and a residue “Fc” fragment. The name “Fc” reflects the ability to crystallize easily. Pepsin treatment yields an F (ab ′) ₂ fragment that has two antigen binding sites and is still capable of cross-linking antigen.

“Fv” is the minimum antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy chain and one light chain variable region in tight, non-covalent association. Three CDR of the variable domain in this structure forms the antigen-binding site on the surface of the V _H -V _L dimer interact. Collectively, the six CDRs provide antigen binding specificity for the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for the antigen) has a lower affinity than the entire binding site, but has the ability to recognize and bind to the antigen. is doing.

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 from the antibody hinge region. Fab′-SH is the notation here for Fab ′ in which the cysteine residue of the constant domain has a free thiol group. F (ab ′) ₂ antibody fragments originally were produced as pairs of Fab ′ fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

  The “light chain” of antibodies (immunoglobulins) from any vertebrate species is based on the amino acid sequence of their constant domains into one of two clearly distinct types called kappa (κ) and lambda (λ). Can be classified.

  Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be classified into different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which are further classified into subclasses (isotypes) such as 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 conformations of different classes of immunoglobulins are well known.

  The term “monoclonal antibody” as used herein is identical except for a substantially homogeneous population of antibodies, ie, naturally occurring mutations in which the individual antibodies that make up may be present in minor amounts. Refers to an antibody obtained from a population. Monoclonal antibodies are highly specific and correspond to a single antigenic site. Furthermore, unlike normal (polyclonal) antibodies that typically include different antibodies corresponding to different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. In addition to specificity, monoclonal antibodies are advantageous because they are synthesized by hybridoma cultures that are not contaminated by other immunoglobulins. The form “monoclonal” indicates the nature of the antibody as being obtained from a substantially homogeneous population of antibodies and does not imply that the antibody must be produced in any specific way. For example, monoclonal antibodies used in the present invention can be made by the hybridoma method first described by Kohler et al. In Nature 256: 495 [1975], or by recombinant DNA methods (see, eg, US Pat. No. 4,816,567). Reference) can be made. “Monoclonal antibodies” can be isolated from phage antibody libraries using the techniques described, for example, by Clackson et al., Nature 352: 624-628 (1991) and Marks et al., J. Mol. Biol. 222: 581-597 (1991). May be separated. See also US Pat. Nos. 5,750,373, 5,571,698, 5,403,484 and 5,223,409, which describe the preparation of antibodies by using phagemids and phage vectors.

  In this context, monoclonal antibodies are particularly those in which part of the heavy and / or light chain is derived from a particular species or is identical or homologous to the corresponding sequence of an antibody belonging to a particular antibody class or subclass. The remaining portions are "chimeric" antibodies (immunoglobulins) that are identical or homologous to the corresponding sequences of antibodies derived from other species or belonging to a particular antibody class or subclass, as well as those that have the desired biological activity. As far as indicated, fragments of those antibodies are included (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 refer to chimeric immunoglobulins, immunoglobulin chains or fragments thereof (eg, Fv, Fab, Fab ′, F (ab ′) ₂ or other antigen-binding subsequences of an antibody. ), Which contains a minimal sequence derived from non-human immunoglobulin. Mostly, humanized antibodies are CDRs of non-human species (donor antibodies) in which the complementarity determining region (CDR) residues of the recipient have the desired specificity, affinity and ability, such as mouse, rat or rabbit. Is a human immunoglobulin (recipient antibody) substituted by In some cases, Fv framework region (FR) residues of the human immunoglobulin can also be replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These changes are made to further refine and maximize antibody performance. In general, a humanized antibody has at least one, typically all or almost all CDR regions corresponding to those of non-human immunoglobulin and all or almost all FR regions of human immunoglobulin sequences. Contains substantially all of the two variable domains. A humanized antibody optimally comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see 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). Humanized antibodies include “primatized” antibodies in which the antigen-binding region of the antibody is derived from an antibody generated by immunizing a macaque monkey with an antigen of interest. Antibodies containing residues from Old World monkeys are also possible in the present invention. See, for example, U.S. Patent Nos. 5,658,570, 5,693,780, 5,681,722, 5,750,105, and 5,756,096.

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

The term “diabody” refers to a small antibody fragment having two antigen binding sites, the fragment of which is a heavy chain bound to a light chain variable domain (V _L ) within the same polypeptide chain (V _H -V _L ). Contains the variable domain (V _H ). By using a linker that is too short to pair between two domains of the same chain, the domains are forced to pair with the complementary domains of the other chain to create two antigen binding sites. Diabodies are described in more detail, for example, in EP 404097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

  By “isolated” polypeptide, such as an antibody, is meant one that has been identified and separated and / or recovered from a component of its natural environment. The natural environmental contaminants are substances that typically interfere with the diagnostic or therapeutic use of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the polypeptide comprising the antibody is (1) up to a proportion greater than 95% by weight of the compound as measured by the Lowry method, most preferably greater than 99% by weight, and (2) spinning. By using a cup sequenator, to an extent sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues, or (3) under reducing or non-reducing conditions using Coomassie blue or preferably silver staining To homogeneity by SDS-PAGE. Isolated compound, such as an antibody or other polypeptide, includes the compound in situ within recombinant cells since at least one component of the compound's natural environment will not be present. Ordinarily, however, isolated compound will be prepared by at least one purification step.

  The term “label” when used herein refers to a detectable compound or composition that is conjugated directly or indirectly to a compound, eg, an antibody or polypeptide, such that a “labeled” compound is produced. To do. The label may itself be detectable (eg, a radioactive label or a fluorescent label) or, in the case of an enzyme label, may catalyze chemical conversion of the detectable substrate compound or composition.

  “Solid phase” means a non-aqueous matrix to which a compound of the invention can be attached. Examples of solid phases encompassed herein include those formed, in part or in whole, from glass (eg, controlled pore glass), polysaccharides (eg, agarose), polyacrylamide, polystyrene, polyvinyl alcohol and silicone. . In certain embodiments, depending on the content, the solid phase can include the wells of an assay plate; in others, it can be a purification column (eg, an affinity chromatography column). The term also encompasses a discontinuous solid phase of discrete particles, such as those described in US Pat. No. 4,275,149.

  “Liposomes” are small forms of various types of lipids, phospholipids and / or surfactants that are useful for delivery of drugs (anti-ErbB2 antibodies and optionally chemotherapeutic agents disclosed herein) to mammals. It is a vesicle. The components of the liposome are usually arranged in a bilayer form similar to the lipid arrangement of biological membranes.

  As used herein, the term “immunoadhesin” refers to an antibody-like molecule that combines the binding specificity of a heterologous protein (“adhesin”) with the effector function of an immunoglobulin constant domain. Structurally, an immunoadhesin comprises a fusion of an immunoglobulin constant domain sequence with the desired binding specificity and other than the antigen recognition and binding site of an antibody (ie, a “heterologous”) and an immunoglobulin constant domain sequence. . The adhesin portion of an immunoadhesin molecule is typically a contiguous amino acid sequence that includes at least a receptor or ligand binding site. The immunoglobulin constant domain sequence of the immunoadhesin can be any of IgG-1, IgG-2, IgG-3 or IgG-4 subtype, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM. It can be obtained from the immunoglobulins.

  “Chronic” administration means that the drug is administered in a continuous mode that is different from the acute mode so as to maintain the desired effect for an extended period of time.

  “Intermittent” administration is a treatment of periodic nature, rather than continuous without interruption.

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

  A “patient” is a vertebrate, preferably a mammal, more preferably a human.

  The term “mammal” includes, but is not limited to, mammals including humans, livestock, livestock, and zoo animals, sports, or pet animals such as sheep, dogs, horses, cats, cows, and the like. Used to refer to any animal that is classified.

  An “effective amount” is an amount sufficient to obtain a beneficial or desired therapeutic (including prophylactic) result. An effective amount can be administered in one or more doses.

(Mode for carrying out the invention)
The practice of the present invention, unless otherwise stated, is a general technique of molecular biology, microbiology, cell biology, biochemistry and immunology that is within the skill of the artisan (including recombinant techniques). Is used. Such techniques are described, for example, in “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al., 1989); “Oligonucleotide Synthesis” (MJ Gait, 1984); “Animal Cell Culture” (RI Freshney, 1987); in Enzymology ”(Academic Press, Inc.);“ Handbook of Experimental Immunology ”, 4th edition (DM Weir & CC Blackwell, Blackwell Science Inc., 1987);“ Gene Transfer Vectors for Mammalian Cells ”(JM Miller & MP Calos 1987); “Current Protocols in Molecular Biology” (FM Ausubel et al., 1987); “PCR: The Polymerase Chain Reaction” (Mullis et al., 1994); and “Current Protocols in Immunology” (JE Coligan et al. , 1991).

Crystal Structure of CRIg and C3b: CRIh and C3c Complexes Polypeptides containing CRIg molecules have a three-dimensional structure determined by the primary amino acid sequence and the environment surrounding the polypeptide. This three-dimensional structure establishes the activity, stability, binding affinity, binding specificity, and other biochemical attributes of the polypeptide. Thus, knowledge of the three-dimensional structure of a protein provides great guidance in designing drugs that mimic, inhibit or improve its biological activity.

  The three-dimensional structure of a polypeptide can be determined in a number of ways. Many of the most accurate methods use x-ray crystallography (Van Holde, Physical Biochemistry (Prentice Hall: NJ, 1971), pp. 221-239). This technique relies on the ability of the crystal lattice to diffract x-rays or other forms of radiation. Diffraction experiments suitable for determining the three-dimensional structure of macromolecules typically require high quality crystals. Unfortunately, such crystals were not available for IGF-1 as well as many other proteins of interest. Crystals are described for example against M-CSF (EP668914B1), CD40 ligand (WO97 / 00895), and BC2 Fab fragment (WO99 / 01476).

  Various methods for preparing crystalline proteins and polypeptides are known in the art (McPherson et al., “Preparation and Analysis of Protein Crystals,” McPherson (Robert E. Krieger Publishing Company, Malabar, FL, 1989 Weber, Advances in Protein Chemistry, 41: 1-36 (1991); US Pat. Nos. 4,672,108 and 4,833,233). Multiple approaches exist for crystallizing a polypeptide, but a single set that offers reasonable success potential, especially if the crystal must be suitable for x-ray diffraction studies This condition does not exist. Significant effort is required to obtain crystals of sufficient size and resolution that provide accurate information about the structure. For example, once a sufficiently pure protein is obtained, it must be crystallized to a size and transparency useful for x-ray diffraction and subsequent structure elucidation. Furthermore, even if the amino acid sequence of the target protein is known, the crystal structure of the protein cannot be accurately predicted from this sequence information. Nor does sequence information provide an understanding of the structural, conformational and chemical interactions between a protein and a binding pair with which it interacts, such as CRIg or C3b. Thus, while crystal structures can provide a wealth of valuable information in the field of drug design and discovery, crystals of certain biologically relevant compounds such as CRIg are not readily available to those skilled in the art. CRIg's high quality diffractive crystals further understand its biological role, and its three dimensions become important for designing CRIg variants, agonists and pale tagists, including but not limited to agonists and antagonist antibodies Helps determine the structure.

  In addition to providing structural information, crystalline polypeptides provide other advantages. For example, the crystallization method itself further purifies the polypeptide and meets one of the classical conditions of homogeneity. Indeed, crystallization often results in unmatched purification quality that removes impurities that are not removed by other purification methods such as HPLC, dialysis, general column chromatography, and the like. In addition, crystalline polypeptides are often stable at ambient temperatures and are not subject to protease contamination or other degradation associated with solution storage. Crystalline polypeptides can also be useful as pharmaceutical formulations. Finally, crystallization techniques generally are generally free of problems such as denaturation associated with other stabilization methods (eg, lyophilization).

  The present invention provides a crystal structure of a complex of CRIg alone and C3b and C3c, respectively. The structure of the C3b: CRIg complex completely reveals the conformational change that occurs upon C3 activation and defines the binding site for the complement receptor CRIg. We will also examine the results of this interaction on the proteolytic activity of alternative pathway convertases.

That is, the present invention provides a CRIg crystal having approximately the following dimensions: a = 30.3 Å, b = 50.8 Å, c = 62.0 Å. The crystal has a symmetry of P2 ₁ 2 ₁ 2 ₁ or a space group. The structural coordinates of the CRIg crystal are provided in Appendix 1.

The present invention further provides a crystal structure of a C3b: CRIg complex having approximately the following dimensions: a = 97.6 Å, b = 255.7 Å, c = 180.3 Å, and a C222 ₁ space group. The structural coordinates of the CRIg crystal are provided in Appendix 2, and the ribbon structure is shown in FIG. 1B.

  The present invention also provides crystals of the C3c: CRIg complex having approximately the following dimensions: a = 382.8 Å, b = 65.0 Å, c = 147.2 β, β = 102.7, and C2. Provide structure. The structural coordinates of the C3c: CRIg crystal are provided in Appendix 3, and the ribbon structure is shown in FIG. 1C.

  C3b, consisting of residues 1-645, is folded into five macroglobulin-like domains (MG1-MG5) and the N-terminal half of the sixth MG domain, the linker region (LNK) (FIGS. 1A and 1B). The immunoglobulin topology MG domain is arranged in a “key ring” -like format and circles around a central groove that is about 10 mm wide and 30 mm long. The α chain of C3b consists of residues 729: 1641 and lacks the C3a or ANA domain compared to C3. The N-terminal residue (729: 745) of the C3bα chain, called α′NT domain, adopts an extended conformation and is linked to the residue that forms the second half of the sixth MG domain. Following this, the α chain contains two additional MG domains (MG7 and MG8) with a CUB domain and a thio-ester domain (TED) inserted in between. Finally, the C-terminal 170 residues of the α chain form a C345C domain with the so-called “anchor region”.

  As described in Example 1, the C3b conformation undergoes dramatic changes upon activation. The TED rotates away from the CUB and NG8 domains, leaving residues that link the TED and CUB domains in a completely different conformation. As part of this conformational change, the Cys residue at position 988 of C3b is moved from its first protected nest in the C3 structure to an exposed position.

  The crystallization study described in Example 1 revealed that the binding site for CRIg is on the opposite side of C3b's CUB and TED domains, and that CRIg is located approximately 40 mm from the TED domain. (See FIGS. 1A and 1B).

Bonding interface between CRIg and C3c or C3b is large, discontinuous, fills the solvent-exposed area of about 2670A ² in total (Fig. 2A). CRIg contacts C3b and the residues from the A ′, G, G, C, C ′, C ″ sheets are the major interaction formed by the residues and loops near the strands C ′ and C ″. The hairpin loop connecting C ′ and C ″ projects into a cleft formed in the center of the C3b key ring-shaped β chain. On the C3b side of the interface, domains MG3, MG4, MG5, MG6, LNK and MG6 all contribute to CRIg binding, with MG3 and MG6 being responsible for about 30% and 40% of the buried interface, respectively ( (See FIG. 2B). The MG3 domain is thought to play an important role in gating CRIg binding to C3b (and C3c). Also, the relatively small but still significant 15 ° rotation of MG3 compared to the rest of the C3 molecule is responsible for the relative reorientation of MG3 with respect to the rings of other MG domains that make up the CRIg binding site. Thus, in conjunction with the movement of the helical portion of the LNK region, this rotation couples CRIg to C3b and C3c. Without these moderate changes, C3 would not be latched into CRIg.

  These structure-function findings were confirmed by testing several amino acid substitution variants of CRIg. It was confirmed that mutations in the NG6 and MG3 binding domains effectively abrogated CRIg binding to C3b. In contrast, mutation of the opposite CRIg surface residue had no effect on the activity of wild type CRIg.

Design, preparation and screening of CRIg agonists and antagonists The present invention has utility in the treatment of various inflammatory conditions including, for example, complement-related diseases or disorders, such as immune complexes and autoimmune diseases, and complement-mediated inflammatory tissue damage. Includes screening assays that identify certain CRIg agonists. The pathology of complement-related diseases varies, including long-term or short-term complement activation, the entire cascade, only one of the cascades (eg classical or alternative pathways), activation of only a few components of the cascade, etc. May include. In certain diseases, the complement biological activity of the complement fragment causes tissue damage and disease. Thus, complement inhibitors have a high therapeutic potential. Alternative pathway selective inhibitors would be particularly useful because removal of pathogens and other organisms from the blood by the classical pathway remains untouched.

  An agonist of a CRIg polypeptide mimics the qualitative biological activity of a native sequence CRIg polypeptide. Preferably, the biological activity is the ability to bind to C3b and / or affect complement or complement activation, in particular to inhibit alternative complement pathways and / or C3 convertase. Agonists include, for example, immunoadhesins, peptidomimetics, agonist antibodies including non-peptide small organic molecules that mimic the qualitative biological activity of natural CRIg, as well as antibody fragments.

  Immunoadhesins are antibody-like molecules that combine the binding specificity of heterologous proteins (“adhesins”) and the effector functions of immunoglobulin constant domains. Structurally, an immunoadhesin comprises a fusion of an immunoglobulin constant domain sequence with an amino acid sequence having a desired binding specificity other than the antigen recognition and binding site of an antibody (ie, "heterologous"). It becomes. The adhesin portion of an immunoadhesin molecule is typically a contiguous amino acid sequence comprising at least a receptor or ligand binding site. The immunoglobulin constant domain sequence of the immunoadhesin can be any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE. , IgD or IgM.

  Peptidomimetics include, for example, peptides containing non-naturally occurring amino acids if the compound retains the CRIg biological activity described herein. Similarly, peptidomimetics and analogs can include non-amino acid chemical structures that mimic the structure of key structural elements of the CRIg polypeptides of the invention and retain CRIg biological activity. The term “peptide” is constrained to be less than about 50 amino acid residues, and preferably less than 40 amino acid residues (ie, for example, the presence of an amino acid that leads to a β-turn or β-pleated sheet, or, for example, a disulfide bond Cys A non-constrained (eg, linear) amino acid sequence that includes a multimer such as a dimer or intermediate thereof, having a structural element that cyclizes by the presence of residues) used. For peptides of less than about 40 amino acid residues, peptides between about 10 and about 30 amino acid residues are preferred, especially peptides of about 20 amino acid residues. However, after reading this disclosure, one of skill in the art will recognize that a peptide is not the length of a particular peptide, but binds to its C3b and inhibits the C3 convertase, particularly the alternative complement pathway C3 convertase. You will admit that it is an ability.

  Screening and identification of CRIg agonists is greatly facilitated by disclosure of the crystal structure of CRIg and C3b: CRIg complex and identification of the binding interface between CRIg and C3b. This information enables the design of compounds that mimic CRIg C3b binding sites.

  The CRIg agonist can also be obtained by (a) performing a fitting operation between the three-dimensional structure of a chemical substance and a CRIg polypeptide or C3b: CRIg complex using calculation or experimental means; (b) obtained in step (a) The obtained data can be analyzed to identify the binding characteristics between the chemical and native CRIg or C3b: CRIg complex. Based on this information, agonist candidates can be synthesized and their agonist properties can be demonstrated in biological assays of CRIg activity.

  In certain embodiments, the agonist will be a chemical comprising at least a portion of the C3b binding region of a native sequence CRIg molecule or a conservative amino acid substitution variant thereof.

  Peptidomimetics can be conveniently prepared using solid phase peptide synthesis (Merrifield, J. Am. Chem. Soc., 85: 2149 (1964); Houghten, Proc. Natl. Acad. Sci. USA 82: 5132 ( 1985)). The solid phase synthesis method is initiated at the carboxy terminus of the putative peptide by attaching a protected amino acid to an inert solid support. The inert solid carrier can be any macromolecule that can serve as the C-terminal anchor of the starting amino acid. Typically, the polymeric carrier is a crosslinked polymeric resin (eg, polyamide or polystyrene resin). In one embodiment, the C-terminal amino acid is linked to a polystyrene resin to form a benzyl ester. The polymeric support is selected such that the peptide anchor bond is stable under the conditions used to deprotect the α-amino group of the amino acid that inhibits peptide synthesis. If a base labile alpha protecting group is used, it is desirable to use an acid labile bond between the peptide and the solid support. For example, acid labile ether resins are effective for base labile Fmoc amino acid peptide synthesis. Alternatively, peptide anchor linkages and α-protecting groups that differentially enter acid degradation can be used. For example, aminomethyl resins such as phenylacetamidomethyl (Pam) resin work well in connection with Boc-amino acid peptide synthesis. After the starting amino acid is linked to an inert solid support, the alpha amino acid protecting group of the starting amino acid is removed, for example with trifluoroacetic acid (TFA) in methylene chloride and neutralized with, for example, triethylamine (TEA). Following deprotection of the α-amino group of the starting amino acid, the next α-amino and side chain protected amino acids in the synthesis are added. Next, the remaining α-amino acid and, if necessary, side-chain protected amino acids are successively bonded in a predetermined order by condensation to obtain an intermediate compound bonded to the solid support. Alternatively, before adding the peptide fragment to the extending solid phase peptide chain, several amino acids may be linked together to form the desired peptide fragment.

  The condensation reaction between two amino acids, or an amino acid and a peptide, or a peptide and a peptide can be carried out by a conventional condensation method such as an azide 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 Woodward Reagent K method.

  Common to chemical synthesis of peptides is the protection of 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 has been assembled sequentially. More common is that the α-amino group on the amino acid or peptide fragment is protected, 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, followed by the α-amino group. Selectively remove and add the next amino acid or peptide fragment to the solid phase peptide chain. Thus, in peptide synthesis, it is common to produce an intermediate compound that includes each of the amino acid residues located in the desired sequence of the peptide chain, each individual residue carrying a side chain protecting group. These protecting groups can be removed at substantially the same time to produce the desired polypeptide product after removal from the solid phase.

The α- and ε-amino acid 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-propyl-oxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl (Fmoc), methylsulfoneethoxycarbonyl (Msc), trifluoroacetyl, phthalyl, formyl, 2-nitrophenylsulfenyl (NPS), Diphenylphosphinothioyl oil (Ppt) and dimethylophosphine It can be protected in such Nochioiru (Mpt) groups.

  Examples of the protecting group for the carboxy functional group include benzyl ester (OBzl), cyclohexyl ester (Chx), 4-nitrobenzyl ester (ONb), t-butyl ester (Obut), 4-pyridylmethyl ester (OPic) and the like. The It is often desirable to protect certain amino acids such as arginine, cysteine, and serine having functional groups other than amino and carboxyl groups with a suitable protecting group. For example, the guanidino group of arginine is nitro, p-toluenesulfonyl, benzyloxycarbonyl, adamantyloxycarbonyl, p-methoxybenzenesulfonyl, 4-methoxy-2,6-dimethylbenzenesulfonyl (Nds), 1,3,5- 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 inhibitory amino acids described above can be obtained from commercial sources such as Novabiochem (San Diego, Calif.), Bachem CA (Terrence, Calif.) Or Peninsula Labs (Belmont, Calif.).

  After the desired amino acid sequence is complete, the peptide can be cleaved from the solid support, recovered and purified. The peptide is removed from the solid support by a reagent that can break the peptide solid phase bond and, in some cases, deprotect the inhibitory side-chain functionality of the peptide. In one embodiment, the peptide is removed from the solid phase by acidolysis with liquid hydrofluoric acid (HF) which also removes any remaining side chain protecting groups. Preferably, in order to avoid alkylation of residues in the polypeptide (eg, alkylation of methionine, cysteine, and tyrosine residues), the acidolysis reaction mixture includes a thio-cresol and a cresol scavenger. After HF cleavage, the resin is washed with ether and the free peptide is extracted from the solid phase with a continuous wash of acetic acid solution. The combined washings are lyophilized and the peptide is purified.

  Native sequence CRIg polypeptide antagonists find utility in treating conditions that benefit from excessive complement activation and enhanced complement-mediated host defense mechanisms. CRIg antagonists such as antibodies to CRIg can be used for immunoadjuvant treatment in tumor (cancer) treatment. It is now well established that T cells recognize human tumor-specific antigens. A group of tumor antigens encoded by the MAGE, BAGE and GAGE families of genes are silent in all adult normal tissues, but in significant amounts in tumors such as melanoma, lung tumors, head and neck tumors, and bladder cancer Expressed. DeSmet, C. et al. (1996) Proc. Natl. Acad. Sci. 93: 7149. Co-stimulation of T cells has been shown to induce tumor regression and anti-tumor responses both in vitro and in vivo. Melero. I. et al., Nature Medicine (1997) 3: 682; Kwon. ED et al., Proc. Natl. Acad. Sci. USA (1997) 94: 8099; Lynch, DH et al., Nature Medicine (1997) 3: 625; Finn, OJ and Lotze. MT, J. Immunol. (1998) 21: 114. CRIg antagonists designed and identified according to the present invention can be administered alone or in conjunction with growth regulators, cytotoxic agents or chemotherapeutic agents to stimulate T cell proliferation / activation and anti-tumor responses to tumor antigens . Growth regulators, cytotoxic agents or chemotherapeutic agents can be administered in conventional amounts using known methods of administration. Immunostimulatory activity with the CRIg antagonists of the present invention can reduce the amount of growth regulators, cytotoxic agents or chemotherapeutic agents, thus potentially 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 could activate complement on tumor cells and help eradicate the tumor by complement-mediated lysis. This approach is expected to be particularly useful for tumors containing CRIg positive macrophages.

  A preferred group of antagonists includes antibodies that specifically bind to native CRIg and inhibit its biological activity.

  Examples of antibodies (both agonists and antagonists) include polyclonal, monoclonal, humanized, bispecific and heteroconjugate antibodies, and antibody fragments.

  An antibody that recognizes and binds to the polypeptide of the invention or acts as an antagonist thereto may alternatively be a monoclonal antibody. Monoclonal antibodies can be prepared using the hybridoma method as described in Kohler and Milstein, Nature, 256: 495 (1975). In hybridoma methods, mice, hamsters or other suitable host animals are typically immunized with an immunizing agent to generate antibodies that specifically bind to the immunizing agent or to induce lymphocytes that can be generated. . Alternatively, lymphocytes can be immunized in vitro.

  The immunizing agent typically comprises a CRIg polypeptide of the invention, an antigenic fragment thereof or a fusion protein. In general, peripheral blood lymphocytes ("PBL") are used when human-derived cells are desired, or spleen cells or lymph node cells are used when non-human mammalian sources are desired. Subsequently, lymphocytes are fused with immortalized cell lines using an appropriate fusion agent such as polyethylene glycol to form hybridoma cells [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59- 103]. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells from rodents, cows and humans. Usually, rat or mouse myeloma cell lines are used. The hybridoma cells are preferably cultured in a suitable medium containing one or more substances that inhibit the survival or proliferation of unfused, immortalized cells. For example, if the parent cell lacks the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma medium typically contains hypoxatin, aminopterin and thymidine (“HAT medium”), This substance prevents the growth of HGPRT deficient cells.

  Preferred immortalized cell lines are those that fuse efficiently, support stable high levels of antibody expression by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. A more preferred immortalized cell line is the mouse myeloma line, which is available, for example, from the Salk Institute Cell Distribution Center in San Diego, California or the American Type Culture Collection in Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines for generating human monoclonal antibodies have also been disclosed [Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications , Marcel Dekker, Inc., New York, (1987) pp. 51-63].

  The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies that have activity similar to or against the polypeptides of the invention. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is measured by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunoassay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can be measured, for example, by the Scatchard analysis method by Munson and Pollard, Anal. Biochem., 107: 220 (1980).

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

  The monoclonal antibody secreted by the subclone can be obtained from the culture medium or ascites fluid by a general immunoglobulin purification method such as protein A-Sepharose method, hydroxylapatite chromatography method, gel electrophoresis method, dialysis method or affinity chromatography. It can be isolated or purified.

  Monoclonal antibodies can also be made by recombinant DNA methods such as those described in US Pat. No. 4,816,567. The DNA encoding the monoclonal antibody of the present invention can be easily obtained using a conventional method (for example, using an oligonucleotide probe capable of specifically binding to the gene encoding the heavy and light chains of the mouse antibody). Can be isolated and sequenced. The hybridoma cells of the present invention are a preferred source of such DNA. Once isolated, the DNA can be placed in an expression vector that can be placed in a host cell, such as a monkey COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell that otherwise does not produce immunoglobulin protein. Transfected and can synthesize monoclonal antibodies in recombinant host cells. DNA can also be replaced, for example, by replacing the coding sequences of human heavy and light chain constant domains in place of homologous mouse sequences [US Pat. No. 4,816,567; Morrison et al., Supra], or non-immune to immunoglobulin coding sequences. Modification can be made by covalently binding part or all of the coding sequence of a globulin polypeptide. Such non-immunoglobulin polypeptides can be substituted for the constant domains of the antibodies of the invention, or can be substituted for the variable domains of one antigen binding site of the antibodies of the invention, producing chimeric bivalent antibodies.

  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 immunoglobulin light chains and modified heavy chains. The heavy chain is generally cleaved at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted or deleted with other amino acid residues to prevent crosslinking.

  In vitro methods are also suitable for preparing monovalent antibodies. Generation of fragments, particularly Fab fragments, by antibody digestion can be accomplished using conventional techniques known in the art.

The antibody of the present invention may further comprise a humanized antibody or a human antibody. A humanized form of a non-human (eg mouse) antibody is a chimeric immunoglobulin, immunoglobulin chain or fragment thereof (eg Fv, Fab, Fab ′, F (ab ′) ₂ or other antigen binding subsequence of an antibody). And contains the minimum sequence derived from non-human immunoglobulin. A humanized antibody is a CDR residue of a non-human species (donor antibody) in which the complementarity determining region (CDR) of the recipient has the desired specificity, affinity and ability, such as mouse, rat or rabbit. Human immunoglobulin (recipient antibody) substituted by In some examples, human immunoglobulin Fv framework residues are replaced by corresponding non-human residues. Humanized antibodies may also contain residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody has at least one, typically all or almost all CDR regions corresponding to those of a non-human immunoglobulin and all or almost all FR regions of a human immunoglobulin consensus sequence, typically Contains substantially all of the two variable domains. Humanized antibodies optimally comprise at least part of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op Struct. Biol., 2: 593-596 (1992)].

  Methods for humanizing non-human antibodies are well known in the art. Generally, one or more amino acid residues derived from non-human are introduced into a humanized antibody. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization is basically performed by 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 using a rodent CDR or CDR sequence instead of the corresponding sequence of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (US Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

  Human antibodies also include phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581 (1991)]. It can also be created using various methods known in The methods of Cole et al. And Boerner et al. Can also be used for the preparation of human monoclonal antibodies [Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol. ., 147 (1): 86-95 (1991)]. Similarly, human antibodies can be produced by introducing human immunoglobulin loci into transgenic animals, eg, mice in which endogenous immunoglobulin genes have been partially or completely inactivated. Upon administration, human antibody production is observed that is very similar to that found in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in US Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; and the following scientific literature: 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, preferably human or humanized antibodies, that have binding specificities for at least two different antigens. In the case of the present invention, one of the binding specificities is for a polypeptide of the present invention and the other may be 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 where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305: 537-539 [1983]). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is usually achieved by an affinity chromatography step. Similar procedures are disclosed in WO 93/08829 published May 13, 1993 and Traunecker et al., EMBO J., 10: 3655-3656 (1991).

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

  Heteroconjugate antibodies consist of two covalently bound antibodies. Such antibodies are used, for example, to target immune system cells to unwanted cells (US Pat. No. 4,676,980) and for the treatment of HIV infection (WO 91/00360; WO 92/003733). European Patent No. 03089) has been proposed. This antibody could be prepared in vitro using known methods in synthetic protein chemistry, including those related to crosslinkers. For example, immunotoxins can be made 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, for example, in US Pat. No. 4,676,980.

  It may be desirable to modify the antibodies of the present invention for effector function, for example to improve the efficacy of the antibody in the treatment of immune related diseases. For example, a cysteine residue may be introduced into the Fc region, thereby forming an interchain disulfide bond in this region. Allogeneic dimeric antibodies so produced may have improved internalization capabilities and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp. Med. 176: 1191-1195 (1992) and Shopes, B. J. Immunol. 148: 2918-2922 (1992). In addition, homodimeric antibodies with improved antitumor activity can be prepared using heterobifunctional cross-linking as described in Wolff et al., Cancer Research 53: 2560-2565 (1993). Alternatively, the antibody can be engineered to have two Fc regions, thereby improving complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3: 219-230 (1989).

  The present invention also includes chemotherapeutic agents, cytotoxic agents such as toxins (eg, enzyme-active toxins derived from bacteria, fungi, plants or animals, or fragments thereof), or radioisotopes (ie, radioconjugates). It also relates to immune complexes that include conjugated antibodies.

Chemotherapeutic agents useful for generating such immune complexes are as described above. Enzyme-active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A Chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, Curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and trichothecene ( tricothecene). A variety of radioactive nucleotides are available for the production of radioconjugated antibodies. Examples include ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y, and ¹⁸⁶ Re.

  The conjugates of antibodies and cytotoxic agents are various bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), imidoester bifunctional Derivatives (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- Using diazonium derivatives (such as bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as triene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene) Can be produced. For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for conjugation of radionucleotide to the antibody. See WO94 / 11026.

  In other embodiments, the antibody may be conjugated to a “receptor” (such as streptavidin) for use in tissue pre-targeting, and the antibody-receptor complex is administered to the patient, followed by a clarifying agent. Used to remove unbound complexes from the circulation, followed by administration of a “ligand” (eg, avidin) conjugated to a cytotoxic agent (eg, a radionucleotide, etc.).

  CRIg and its agonists and antagonists can be tested in various in vitro and in vivo assays to determine whether they mimic or inhibit the biological activity of the native CRIg molecule.

  As a result of its ability to inhibit complement activation, particularly the alternative complement pathway, CRIg polypeptides find utility in the prevention and / or treatment of complement-related diseases and pathological conditions. Such diseases and conditions include, but are not limited to, complement-related inflammatory and autoimmune diseases.

  Specific examples of complement-related diseases include, but are not limited to, rheumatoid arthritis (RA), acute respiratory distress syndrome (ARDS), remote tissue injury after ischemia and reperfusion, supplementation during cardiopulmonary bypass surgery Body activation, dermatomyositis, pemphigus, lupus nephritis and the resulting glomerulonephritis and vasculitis, cardiopulmonary bypass, cardioplegia-induced coronary endothelial dysfunction, type II membranoproliferative glomerulonephritis, IgA nephropathy , Acute renal failure, cryoglobulinemia, antiphospholipid syndrome, macular degenerative diseases and other complement-related ocular symptoms, age-related macular degeneration (AMD), including non-exudative and exudative AMD, diabetic Retinopathy (DR), endophthalmitis, uveitis, allograft, foreign body transplant, hyperacute rejection, hemodialysis, chronic obstructive pulmonary distress syndrome (COPD), asthma, hereditary angioedema, paroxysmal nocturnal hemoglobinuria , Alzheimer's disease, Rohm arteriosclerosis, aspiration pneumonia, urticaria such as chronic idiopathic urticaria, hemolytic uremic syndrome, endometriosis, cardiogenic shock, ischemia reperfusion injury, multiple sclerosis (MS ) Is included.

  AMD is age-related degeneration of the macula that is a major cause of irreversible visual impairment in individuals over 60 years of age. There are two types of AMD, non-exudable (dry) and exudable (wet) AMD. Dry or non-wetting forms include atrophy and abnormal hypertrophy changes in the central retina (macular) as well as the retinal pigment epithelium (RPE) underlying the deposits on the RPE (Druze). Patients with non-wetting ARMD have discoid scars in which abnormal blood vessels called choroidal neovascular membranes (CNVM) develop under the retina, leakage fluid and blood, and eventually lead to blindness within and under the retina Can progress to a wet or exudative form of ARMD that results in Non-wetting ARMD, which is usually a precursor of wet ARMD, is more common. The presentation of non-wetting ARMD varies; there may be hard drusen, soft drusen, RPE geographic atrophy, and pigment aggregation. The complement component is deposited on the RPE early in AMD and is a major component of drusen. Complement factor H (CFH) polymorphisms have recently been reported to account for 50% of the risk attributable to AMD (Klein et al., Science 308: 385-9 (2005)).

  CRIg is particularly useful in the treatment of high-risk AMD, including category 3 and category 4 AMD. Category 3 AMD has no progressive AMD in both eyes, at least one eye has 20/32 or better vision, at least one large drusen (eg 125 μm), extensive (measured by drusen area) intermediate drusen, or macular Characterized by having a map-free atrophy (GA) or any combination thereof. Category 4 high-risk AMD is characterized by visual acuity of 20/32 or greater, and there is no advanced AMD in the index eye (GA containing features of macular center or choroidal neovascularization). The other eye is characterized by progressive AMD, or vision less than 20/32 attributed to AMD maculopathy. Typically, if untreated, high-risk AMD rapidly progresses to choroidal neovascularization (CNV) at a rate approximately 10-30 times higher than that of category 1 or 2 (not high-risk) AMD. . Therefore, CRIg finds utility in the prevention and treatment of CNV and AMD.

  A broader list of inflammatory conditions as examples of complement-related diseases includes, for example, inflammatory bowel disease (IBD), systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthritis, systemic sclerosis ( Scleroderma), idiopathic inflammation myopathy (dermatomyositis, polymyositis), Sjogren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immuneplastic anemia, paroxysmal nocturnal hemoglobinuria), autoimmunity Thrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis (Graves disease, Hashimoto thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediated renal disease ( Glomerulonephritis, tubulointerstitial nephritis), demyelinating diseases of the central and peripheral nervous systems, such as multiple sclerosis, idiopathic discontinuous polyneuropathy, or Guillain-Barre syndrome, and chronic inflammation Voluntary polyneuropathy, hepatobiliary disease, such as infectious hepatitis (A, B, C, D, hepatitis E and other nonhepatotropic viruses), autoimmune chronic active hepatitis, Primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, inflammatory bowel disease (ulcerative colitis; Crohn's disease), gluten-sensitive bowel disease, and Whipple disease, bullous skin disease, polymorphism Autoimmune or immune-mediated skin diseases including erythema and contact dermatitis, psoriasis, allergic diseases such as asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity and urticaria, pulmonary immune diseases such as eosinophils Includes transplant-related diseases including pneumonia, idiopathic pulmonary fibrosis and hypersensitivity pneumonia, rejection and graft-versus-host disease.

  In systemic lupus erythematosus, the central mediator of disease is the production of autoreactive antibodies against self-proteins / tissues, followed by the development of immune-mediated inflammation. Antibodies mediate tissue injury 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. Thus, disease development is dependent on T lymphocytes. Multiple organs and systems are clinically affected, including kidney, lung, musculoskeletal, mucocutaneous, eye, central nervous system, cardiovascular system, gastrointestinal tract, bone marrow and blood.

  Rheumatoid arthritis (RA) is a chronic systemic autoimmune inflammatory disease that primarily involves the synovium of multiple joints, resulting in damage to articular cartilage. The pathogen is T lymphocyte dependent and is associated with the production of rheumatoid factors, autoantibodies to self IgG, resulting in immune complexes reaching high levels in synovial fluid and blood. These complexes in the joints induce significant infiltration of lymphocytes and monocytes into the synovium followed by significant synovial changes; infiltration of similar cells with the addition of multiple neutrophils If done, it is also true for joint space / fluid. The affected tissues are often symmetric patterns, mainly joints. However, two main forms of extra-articular disease also occur. One form is the development of extra-articular disorders with ongoing progressive joint disease and typical lesions of pulmonary fibrosis, vasculitis, and skin ulcers. The second form of extra-articular disease is the so-called Felty syndrome, which occurs at the end of the RA disease process, sometimes after the joint disease has subsided, and is responsible for the presence of neutropenia, thrombocytopenia and splenic hypertrophy. This is accompanied by vasculitis in multiple organs with the formation of infarcts, skin ulcers and gangrene. Often, patients develop rheumatoid nodules in the subcutaneous tissue on the diseased joint; the nodules have a necrotic center surrounded by mixed inflammatory cell infiltration at the end. Other signs that may occur in RA include: epicarditis, pleurisy, coronary arteritis, interstitial pneumonia with pulmonary fibrosis, dry keratoconjunctivitis, and rheumatoid nodules.

  Juvenile chronic arthritis is a chronic idiopathic inflammatory disease that often begins before the age of 16 years. Its phenotype has some similarities to RA; some patients positive for rheumatoid factor are classified as juvenile rheumatoid arthritis. The disease is subdivided into three main categories: pauarticular, polyarticular and systemic. Arthritis is severe and typically devastating, leading to arthrosis and slow growth. Other signs include chronic anterior uveitis and systemic amyloidosis.

  Spondyloarthropathies are a group of diseases that have a common association between several common clinical features and expression of the HLA-B27 gene product. The diseases include: 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 arthritis with or without spondylitis; asymmetric arthritis with inflammation; association with HLA-B27 (a serologically defined allele at the HLA-B locus of class I MHC); This includes the absence of autoantibodies associated with eye inflammation and other rheumatic diseases. The most implicated cell as a key to disease induction is CD8 + T lymphocytes, which target cells presented by class I MHC molecules. CD8 + T cells 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 postulated that the epitope of HLA-B27 mimics the antigenic epitope of bacterial or other microorganisms and thus elicits a CD8 + cell response.

  Systemic sclerosis (scleroderma) is not well known for its etiology. A prominent feature of the disease is skin induration; this appears to be triggered by an active inflammatory process. Scleroderma is local or systemic: vascular lesions are common, and endothelial cell injury in the microvasculature is an early critical event in the development of systemic sclerosis; vascular injury can be immune-mediated . Immunological criteria are guided by the presence of mononuclear cell infiltration in skin lesions and the presence of anti-nuclear antibodies in many patients. In many cases, ICAM-1 is up-regulated on the cell surface of skin foci fibroblasts, suggesting that T cell interactions with these cells play a role in disease pathogenesis. Other organs involved: Gastrointestinal tract: resulting abnormal peristalsis / motility Smooth muscle atrophy and fibrosis: Kidney: affects arch and interlobular arteries, resulting in renal cortical blood flow Concentric subendothelial proliferation that results in decreased proteinuria, high nitrogen hematuria and hypertension: skeletal muscle: atrophy, interstitial fibrosis: inflammation: lung: interstitial pneumonia and interstitial fibrosis: and heart : Contraction band necrosis, scar / fibrosis.

  Idiopathic inflammatory myopathy, including dermatomyositis, polymyositis and others, is a chronic muscular inflammatory disease of unknown etiology that leads to muscle weakness. Muscle injury / inflammation is often contrasting and progressive. Autoantibodies are associated with many forms. These myositis-specific autoantibodies are produced against components involved in protein synthesis, proteins and RNA, and inhibit their functions.

  Sjogren's syndrome is due to immune-mediated inflammation and subsequent functional disruption of the lacrimal and salivary glands. The disease may be associated with or associated with inflammatory connective tissue disease. The disease is associated with autoantibody production against Ro and La antigens, both small RNA-protein complexes. The lesions are psoriatic keratoconjunctivitis, xerostomia, with biliary cirrhosis, peripheral or sensory neuropathy, and other signs or associations including overt purpura.

  Systemic vascular inflammation is an inflammation of the primary lesion, which subsequently damages the blood vessels, resulting in ischemia / necrosis / degeneration in the tissue supplied by the affected vessels, and in some cases the final end It is a disease that causes organ dysfunction. Vasculitides may also occur as a secondary or secondary lesion of other immune inflammation-mediated diseases such as rheumatoid arthritis, systemic sclerosis, especially diseases related to immune complex formation . For diseases of the primary systemic vascular inflammation group: systemic necrotizing vasculitis: polyarteritis nodosa, allergic vasculitis and granulomatosis, polyvasculitis: Wegener's granulomatosis; lymphoma-like granulation Tumors: and giant cell arteritis. Other vasculitis: mucocutaneous lymph node syndrome (MLNS or Kawasaki disease), isolated CNS vasculitis, Behet's disease, obstructive thrombotic vasculitis (Berger's disease) and skin necrotizing phlebitis ( venulitis). The pathogenesis mechanism of most types of listed vasculitis is thought to be due mainly to the attachment of immunoglobulin complexes to the vessel wall followed by the induction of an inflammatory response via ADCC, complement activity or both. ing.

  Sarcoidosis is a condition with little known etiology characterized by the presence of epithelioid granulomas in almost all tissues in the body; pulmonary involvement is the most common. Etiology is associated with residual active macrophages and lymphocytes at the disease site, followed by chronic sequelae as a result of the release of local or systemic active products released from these cell types.

  Autoimmune hemolytic anemia, including autoimmune hemolytic anemia, immunoregenerative anemia, and paroxysmal nocturnal hemoglobinuria, are antigens expressed on the surface of red blood cells (in some cases other blood cells that also contain platelets). This is a result of the production of an antibody that reacts with and is reflected in the removal of the antibody-coated cells via complement-mediated lysis and / or ADCC / Fc-receptor-mediated mechanisms.

  In autoimmune thrombocytopenia, including thrombocytopenic purpura and immune-mediated thrombocytopenia in other clinical settings, platelet destruction / removal involves antibody or complement conjugation to platelets followed by complement lysis, ADCC or Fc As a result of being removed by a -receptor-mediated mechanism.

  Thyroiditis, including Graves' disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, and atrophic thyroiditis, is a thyroid that involves the production of antibodies that are present in the thyroid and often react with thyroid-specific proteins. This is due to the result of an autoimmune response to the antigen. There are experimental models that include immunization of animals with either natural models: rats (BUF and BB rats) and chicken (obese chicken species); inducible models: thyroglobulin, thyroid microsomal antigen (thyroid peroxidase).

  Type I diabetes mellitus or insulin-dependent diabetes is the autoimmune destruction of pancreatic islets of Langerhans; this destruction is mediated by autoantibodies and autoreactive T cells. Also, antibodies against insulin or insulin receptors can create an insulin-non-reactive phenotype.

  Immune-mediated renal disease, including glomerulonephritis and tubulointerstitial nephritis, can occur directly as a result of the production of autoreactive antibodies or T cells to the kidney antigen or to other non-renal antigens It is due to antibody or T cell mediated injury in the kidney tissue indirectly as a result of the deposition of certain antibodies and / or immune complexes in the kidney. Thus, immune-mediated renal disease can also be induced as an indirect sequelae by other immune-mediated diseases resulting in the production of immune complexes. Both direct and indirect immune mechanisms result in an inflammatory response that causes / induces lesion development in the renal tissue, impairing organ function and in some cases progressing to renal dysfunction. Both humoral and cellular immune mechanisms can be involved in the pathogenesis of the disorder.

  Demyelinating diseases of the central and peripheral nervous system, including multiple sclerosis; idiopathic and isolated polyneuropathy or Guillain-Barre syndrome; and chronic inflammatory degenerative polyneuropathy have causes in autoimmunity and It is believed that neuronal demyelination occurs as a result of damage directly caused by dendrocytes or myelin. In MS, there is evidence to suggest that disease induction and progression is dependent on T lymphocytes. Multiple sclerosis is a demyelinating disease that is T lymphocyte dependent and has either a recurrent relaxation pathway or a chronic progression pathway. The etiology is not well known, but viral infection, genetic predisposition, environment and autoimmunity all contribute. The lesion contains the predominant T cell-mediated microglia wetting and infiltrating microphages; CD4 + T lymphocytes are the predominant cell type in the lesion. The mechanism of oligodendrocyte cell death and subsequent demyelination is not well known, but appears to be driven by T lymphocytes.

  Uncontrolled immune inflammatory responses are associated with inflammation and fibrotic lung disease, including eosinophilic pneumonia; idiopathic pulmonary fibrosis and hypersensitivity pneumonia. Inhibiting the response would be therapeutically beneficial.

  Autoimmune or immune-mediated skin diseases, including bullous skin disease, erythema multiforme and contact dermatitis, are mediated by autoantibodies, the pathogenesis of which is T lymphocyte dependent.

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

  Transplant-related diseases, including rejection and graft-versus-host disease (GVHD), are T lymphocyte dependent; they are ameliorated by inhibiting T lymphocyte function.

  In view of the therapeutic applications listed above CRIg, potential agonists of CRIg can be evaluated in various cell-based assays and animal models of complement-related, inflammation and / or autoimmune diseases or disorders.

  Thus, for example, efficacy in the prevention and / or treatment of arthritis can be assessed in a collagen-induced arthritis model (Terato et al. Brit. J. Rheum. 35: 828-838 (1966)). Potential arthritis prevention / treatment drugs include four monoclonal antibodies as described in Terato et al., J. Immunol. 148: 2103-8 (1992); Terato et al., Autoimmunity 22: 137-47 (1995). Can be screened in an antibody-mediated arthritis model induced by intravenous injection of See also Example 4 herein. Candidates for the prevention and / or treatment of arthritis can also be studied in transgenic animal models such as, for example, TNF-α transgenic mice (Taconic). These animals express human tumor necrosis factor (TNF-α), a cytokine that has been linked to the pathogenesis of human rheumatoid arthritis. Expression of TNF-α in these mice results in severe chronic arthritis of the forelimbs and hind limbs, providing a simple mouse model of inflammatory arthritis.

Recently, animal models of psoriasis have also been developed. Thus, Asebia (ab), scaly skin (fsn) and chronic proliferative dermatitis (cpd) are natural mouse mutations with psoriasis-like skin changes. For example, transgenic mice having skin overexpression of cytokines such as interferon-γ, interleukin-1α, keratinocyte growth factor, transforming growth factor-α, interferon-6, vascular endothelial growth factor, or bone morphogenetic protein 6. It can also be used to study psoriasis in vivo and identify therapeutic agents for the treatment of infection. Psoriatic-like lesions are also β ₂ -integrin hypomorphic mice and β ₁ -integrin transgenic mice backcrossed to the PL / J strain, scid / scid mice reconstituted with CD4 ⁺ / CD45RB ^hi T lymphocytes, and HLA-B27. / Hβ ₂ m transgenic rat. Xenograft models using human skin transplanted into immunodeficient mice are also known. Thus, the compounds of the present invention are tested in the scid / scid mouse model described by Schon, MP et al., Nat. Med. (1997) 3: 183, where mice exhibit histopathological skin lesions similar to psoriasis. be able to. Another suitable model is the human skin / scid mouse chimera prepared as described in Nickoloff, BJ et al., Am. J. Path. (1995) 146: 580. For further details see, for example, Schon, MP, J 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 subject animal using standard techniques for producing transgenic animals. Animals that can be targeted for transgenic manipulation include, but are not limited to, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates such as baboons, chimpanzees and other monkeys. Techniques known in the art for introducing transgenes into such animals include pronuclear microinjection (Hoppe and Wanger, US Pat. No. 4,873,191); retrovirus-mediated gene transfer to the embryonic lineage (eg, 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]); embryo electroporation (Lo, Mol. Cel. Biol. 3, 1803-1814 [1983]); including sperm-mediated gene transfer (Lavitrano et al., Cell 57, 717-73 [1989]). For review, see, for example, US Pat. No. 4,736,866.

  For the purposes of the present invention, transgenic animals include those that have the transgene only in a portion of their cells ("mosaic animals"). The transgene can be incorporated as a single transgene or as a concatamer, eg, head-to-head or head-to-tail tandem. Selective introduction of a transgene into a specific cell type is also possible, for example, according to the technique of Lasko et al., Proc. Natl. Acad. Sci. USA 89, 6232-636 (1992).

  Transgene expression in transgenic animals can be monitored by standard techniques. For example, Southern blot analysis or PCR amplification is 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 immunohistochemistry.

  The animal can be further examined for signs of immune disease pathology, for example by histological examination to determine infiltration of immune cells into specific cells. Blocking experiments can also be performed in which transgenic animals are treated with CRIg or candidate agonists to determine the degree of effect on complement activation or T cell proliferation, including complement and classical and alternative pathways. In these experiments, blocking antibodies that bind to the polypeptides of the invention are administered to animals and the biological effects of interest are monitored.

  Alternatively, a “knockout” animal may undergo CRIg recombination as a result of homologous recombination between an endogenous gene encoding a CRIg polypeptide and a modified genomic DNA encoding the polypeptide introduced into the animal's embryonic cells. Can be constructed as having a defective or modified gene. For example, cDNA encoding CRIg can be used for cloning genomic DNA encoding CRIg by established techniques. A portion of the genomic DNA encoding CRIg can be replaced or removed by other genes, such as genes encoding selectable markers that can be used to monitor integration. Typically, the vector contains several kilobases of intact flanking DNA (both 5 'and 3' ends) [eg Thomas and Capecchi, Cell, 51: 503 for a description of homologous recombination vectors. (1987)]. A vector is introduced into an embryonic stem cell line (for example, by electroporation), and a cell in which the introduced DNA is homologously recombined with endogenous DNA is selected [eg, Li et al., Cell, 69: 915 ( 1992)]. The selected cells are then injected into the blastocyst of the animal (eg mouse or rat) to form an aggregate chimera [eg Bradley, Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, edited by EJ Robertson (IRL, Oxford 1987), pp. 113-152]. The chimeric embryo is then transplanted into an appropriate pseudopregnant female nanny and given birth to create a “knockout” animal. Offspring having DNA homologously recombined into the embryonic cells can be identified by standard techniques and used to breed animals that contain DNA in which all cells of the animal have been homologously recombined. Knockout animals can be characterized, for example, by their ability to defend against certain pathological conditions and the development of those pathological conditions due to the absence of CRIg polypeptides.

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

  An asthma model is described in which antigen-induced hyperrespiratory hyperresponsiveness, eosinophilia and inflammation are sensitized to animals with ovalbumin and the animals are exposed to the same protein delivered by aerosol. Some animal models (guinea pigs, rats, non-human primates) show signs similar to human atopic asthma upon exposure to aerosol antigen. The mouse model has many of the characteristics of human asthma. Suitable procedures for testing CRIg and CRIg agonists for activity and efficacy in the treatment of asthma are cited therein as Wolyniec, WW et al., Am. J. Respir. Cell Mol. Biol. (1998) 18: 777. It is described in the literature.

  Contact hypersensitivity is a simple in vivo assay of cell-mediated immune function. In this procedure, epidermal cells are exposed to an exogenous hapten that produces a delayed type hypersensitivity reaction, and the response is measured and quantified. Contact hypersensitivity is the first sensitization stage followed by the manifestation stage. The manifestation phase occurs when epidermal cells encounter an antigen that they have previously contacted. Swelling and inflammation occur, creating an excellent model of human allergic contact dermatitis. Suitable procedures are described in detail in Current Protocols in Immunology, J.E. Coligan, A.M.Kruisbeek, D.H.Marglies, E.M.Shevach, and W. Strober, John Wiley & Sons, Inc, unit 4.2. See also Grabbe, S. and Schwarz, T. Immun. Today 19 (1): 37-44 (1998).

  Graft-versus-host disease occurs when immunocompetent cells are transplanted into immunosuppressed or resistant patients. Donor cells recognize and react to host antigens. Responses range from severe life-threatening inflammation to mild cases such as diarrhea and weight loss. The graft versus host disease model provides a means of assessing T cell reactivity against MHC antigens and small amounts of transplant antigen. A suitable procedure is described in detail in Current Protocols in Immunology, above, unit 4.3.

  Animal models for skin allograft rejection are a means of testing the ability of T cells, an indicator and measure of their role in antiviral and tumor immunity, to mediate tissue destruction in vivo. The most common and accepted model uses mouse tail-skin grafts. Repeated experiments have shown that skin allograft rejection is mediated by T cells, helper T cells and killer-effector T cells, but not by antibodies. Auchincloss, H. Jr. and Sachs, D.H., Fundamental Immunology, 2nd ed., W.E. Paul ed., 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 by Tanabe, M. et al., Transplantation (1994) 58:23 and Tinubu, SA et al., J. Immunol. (1994) 4330-4338. There is an allocardial graft model.

  Animal models of delayed type hypersensitivity also provide assays for cell-mediated immune function. A delayed-type hypersensitivity reaction is a T cell-mediated in vivo immune response characterized by inflammation that does not peak until the elapsed time after challenge. These reactions also occur in tissue-specific autoimmune diseases such as multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE, a model for MS). A suitable procedure is described in detail in Current Protocols in Immunology, above, unit 4.5.

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

  An animal model of age-related macular degeneration (AMD) consists of mice with non-expressing mutations in the Ccl-2 or Ccr-2 gene. These mice develop major features of AMD, including the accumulation of lipofuscin and underlying drusen in the retinal pigment epithelium (RPE), photoreceptor atrophy and choroidal neovascularization (CNV). These features develop beyond the age of 6 months. CRIg and CRIg agonists can be tested for drusen formation, photoreceptors and choroidal neovascularization.

  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 placed in the internal carotid artery and external jugular vein for measurement of mean arterial pressure. Myocardial ischemia reperfusion is initiated by ligating the left anterior descending coronary artery (LAD) with a 6-O suture. Ischemia is created by tightening a reversible ligature around the LAD to completely occlude the blood vessel. The ligature is removed after 30 minutes and the heart is 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 venule dilation, intraretinal microvascular abnormalities, and retinal and vitreous cavity angiogenesis.

  A model of membranoproliferative glomerulonephritis can be established as follows: female mice are immunized intraperitoneally with 0.5 mg control rabbit IgG in CFA (day -7). Seven days later (day 0), 1 mg of rabbit anti-mouse glomerular basement membrane (GBM) antibody is injected intravenously from the tail vein. Elevated serum anti-rabbit IgG antibody is measured by ELISA. 24-hour urine samples are collected from mice in metabolic cages, and the kidney function of the mice is assessed by measuring urinary protein in addition to blood urea nitrogen.

Pharmaceutical Compositions CRIg agonists and antagonists identified in accordance with the present invention can be administered in the form of pharmaceutical compositions for the treatment of inflammatory diseases.

A therapeutic formulation is prepared and stored by mixing the active molecule of the desired degree of purity with an optimal pharmaceutically acceptable carrier, excipient or stabilizer in the form of a lyophilized formulation or aqueous solution. (Remington's Pharmaceutical Science 16th edition, Osol, A. Ed. [1980]). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations used, and buffers such as phosphate, citrate, and other organic acids; ascorbic acid and Antioxidants including methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclo Hexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; protein such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymer such as polyvinylpyrrolidone; glycine, glutamine, Asparagine, histidine Amino acids such as arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming pairs such as sodium Ions; including metal complexes (eg, Zn-protein complexes) and / or nonionic surfactants such as TWEEN ^™ , Pluronics ^™ , and polyethylene glycol (PEG).

  Lipofection or liposomes can be used to deliver a polypeptide, antibody, or antibody fragment into a cell. 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 (Marasco et al., Proc. Natl. Acad. Sci. USA 90: 7889-7893 [1993]).

  The formulations herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively or in addition, the composition may comprise a cytotoxic agent, cytokine or growth inhibitor. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

  The active molecules can also be used in colloidal drug delivery systems (e.g., in microcapsules prepared by coacervation techniques or by interfacial polymerization, e.g., hydroxymethylcellulose or gelatin-microcapsules and poly (methyl methacrylate) microcapsules, respectively. , Liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or microemulsions. Such techniques are disclosed in Remington's Pharmaceutical Science 16th edition, Osol, A. Ed. (1980).

  Formulations used for in vivo administration must be sterile. This is easily accomplished by filtration through sterile filtration membranes.

Sustained release formulations can also be prepared. Suitable examples of sustained release formulations include a semi-permeable matrix of solid hydrophobic polymer containing antibodies, which matrix is in the form of a molded article, such as a film or a microcapsule. Examples of sustained release matrices include polyesters, hydrogels (eg, poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactide (US Pat. No. 3,773,919), L-glutamic acid and γ-ethyl-L- Degradable lactic acid-glycolic acid copolymers such as glutamate copolymers, non-degradable ethylene-vinyl acetate, LUPRON DEPOT ^™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), poly- (D) -3- Contains hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules over 100 days, certain hydrogels release proteins in a shorter time. When encapsulated antibodies remain in the body for a long time, they denature or aggregate upon exposure to moisture at 37 ° C., resulting in decreased biological activity and possible changes in immunogenicity. . Reasonable methods can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is found to be intermolecular S—S bond formation through thio-disulfide exchange, stabilization may include modification of sulfhydryl residues, lyophilization from acidic solutions, control of water content, appropriate Addition of various additives and the development of specific polymer matrix compositions.

  The following examples are provided for illustration only and are in no way intended to limit the scope of the invention.

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

Example 1
Materials and methods for determining the crystal structure of CRIg and C3b: CRIg complexes a. Production and purification of mature human CRIg protein A DNA fragment encoding residues 1 to 119 of mature human CRIg of SEQ ID NO: 2 (SEQ ID NO: 8) was cloned into the NdeI / BamHI site of the pET28b expression vector (Novagen) and cleaved by thrombin A fusion was made with an N-terminal His tag followed by a site. After purification using Ni-affinity chromatography, the fusion protein was digested with thrombin and further purified using size exclusion chromatography. The final protein stock had a protein concentration of 20 mg / ml in 10 mM Hepes, 50 mM NaCl, pH 7.2. Selenomethionine labeled protein was obtained using standard protocols.

  Purified CRIg was mixed with C3c or C3b in a 5-fold molar excess and incubated on ice for 30 minutes. The sample was purified using size exclusion chromatography and concentrated to 10-20 mg / ml in 25 mM Hepes, pH 7.2, 50 mM NaCl.

b. Crystallization All crystals were grown at 19 ° C. using the hanging drop vapor diffusion method.

  CRIg was crystallized by equal mixing of the protein solution in an equal volume of a reservoir containing 30% PEG 4000, 0.1 M sodium acetate, and 0.2 M ammonium acetate. Crystals formed after 3 days. C3c: CRIg complex crystals were obtained by mixing a protein solution (20 mg / ml) in a 1: 1 ratio with a reservoir solution containing 12% PEG 20000, 0.1 M MES, pH 6.5. Crystals of the C3b: CRIg complex were obtained by mixing an equal volume of reservoir solution containing 12% PEG 20000, 0.1 M MES, pH 6.5 and protein solution (10 mg / ml). Crystals formed after 3 days.

c. Data collection, structure elucidation and densification For data collection, crystals were briefly immersed in a solution-containing reservoir supplemented with 20% glycerol and then flash frozen in liquid nitrogen. All data are from ALS beamline 5.0.2. And processed using HKL2000 (Otwinowski, Z. and Minor, W., Methods Enzymol. 276: 307-326 (1997)). The uncomplexed CRIg crystals diffract with a resolution of 1.2 Å and belong to the space group P2 ₁ 2 ₁ 2 ₁ with lattice parameters of a = 30.2, b = 50.7 and c = 61.9A. The structure is elucidated using several abnormal dispersions and the program auto-SHARP (G. Bricogne, C. Vonrhein, C. Flensburg, M. Schiltz, W. Paciorek, Acta Crystallogr D Biol Crystallogr 59, 2023 (2003)) did. Densification using Refmac (GN Murshudov, AA Vagin, EJ Dodson, Acta Crystallogr D Biol Crystallogr 53, 240 (1997)) and program O (TA Jones, JY Zou, SW Cowan, Kjeldgaard, Acta Crystallogr A 47 (Pt 2) , 110 (1991)), a model of 14.9% Rcryst and 17.7% Rfree was obtained. The crystal of C3c diffracts to 3.1Å and belongs to space group C2 with lattice parameters of a = 382Å, b = 65.0Å, c = 147.2Å and β = 102.7 °, and two asymmetric units Is included. The structure was solved using AMoRe (Navazza J., Acta Crystallogr. A 50: 157-163 (1994)) and unbound CRIg and C3C models (pdb code 2A74). The final Rcryst and Rfree after densification applying non-crystallographic symmetry constraints were 23.7% and 29.7%, respectively. C3b: crystals of CRIg complex is diffracted to 4.1Å resolution, a = 97.6Å, b = 255.7Å , belongs to the space group C2222 ₁ lattice parameters c = 180.3Å. After molecular replacement using the C3c: CRIg complex, the CUB domain (pdb code 2A73) and TED domain (1C3D) were manually docked into the electron density map. After rigid body densification, the region connecting the TED and CUB domains and the unambiguous density of the two glycosylation sites had interpretable densities and could fit into the electron density. The final model contains a complex of CRIg and C3b without the C345C domain; the latter domain has a very weak density and is included in the model with an occupancy set to 0.0 for FIG. It was. The final Rcyst and Rfree were 25.2% and 33.3%, respectively.

Results a. Determination of structure It was recently determined that CRIg forms a complex with C3b and iC3b but cannot bind to the parent molecule C3 (KY Helmy et al., Cell 124, 915 (2006)). In order to better understand the structural basis for the selectivity of CRIg, the crystal structure of CRIg in its unbound state was determined at high resolution and also for the complex with C3b and C3c (4.1Å each). And 3.2 cm). The structure of unbound CRIg was solved using three-wavelength anomalous dispersion (MAD) fading (Hendrickson, WA et al., Proteins 4: 77-88 (1988)). Subsequently, the structure of the complex was elucidated by molecular replacement using the coordinates of various domains and fragments of the CRIg and C3 (2A73), C3c (2A74), and C3d (1C3D) structures as a search model. The C3c: CRIg structure contained two molecules in the asymmetric unit, and after densification using non-crystallographic symmetry constraints, the final R and Rfree were 23.6% and 29.5%, respectively. Extensive positional densification is not possible due to the limited resolution of the C3c: CRIg complex. However, after various linker modeling and individual domain rigid body densification, the final model R and Rfree are 24.8% and 33.1%, respectively, which is good quality considering the limited resolution of the structure It is shown that.

b. The overall structure of C3b The structure of the individual domains of C3b is similar to the recently reported C3 structure (Janssen et al., Supra; D. Hourcade, VM Holers, JP Atkinson, Adv Immunol 45, 381 (1989)). The arrangement of these domains is quite different compared to C3. Briefly, the C3b β-strand consisting of residues 1-645 is located in five macroglobulin-like domains (MG1-MG5), the N-terminal half of the sixth MG domain, and the linker region (LNK) (FIGS. 1A, B). Folded. The immunoglobulin topology MG domain is arranged in a “key ring” -like format and circles around a central groove that is about 10 mm wide and 30 mm long. The α chain of C3b consists of residues 729: 1641 and lacks the C3a or ANA domain compared to C3. The N-terminal residue (729: 745) of the C3bα chain, called the α′NT domain, adopts an extended conformation and is linked to the residue that forms the second half of the sixth MG domain. Following this, the α chain contains two additional MG domains (MG7 and MG8) with a CUB domain and a thio-ester domain (TED) inserted in between. Finally, the C-terminal 170 residues of the α chain form a C345C domain with the so-called “anchor region”.

c. Conformational changes upon C3 activation Comparison of the structures of C3, C3b, and C3c shows that a large conformational change, ie, cleavage of C3 into C3b and C3a occurs immediately after activation (FIGS. 1A, B ). Release of the ANA domain intervening between the MG3 and MG8 domains in C3 (F. Fredslund, J, supra) causes MG3 and MG8 to rotate. These conformational changes further rotate the MG7 domain, and when C3 is compared to C3c, an interesting movement of the α′NT domain described by Jannsen et al. As in C3c, α'NT is more solvent-exposed in C3b than in C3 and has many potential binding sites for complement activation receptors and regulators, CR1, CFH and factor B. (JD Lambris et al., J Immunol 156, 4821 (1996); A. Taniguchi-Sidle, DE Isenman, J Immunol 153, 5285 (1994); AE Oran, DE Isenman, J Biol Chem 274, 5120 (1999)). However, the greatest movement induced by C3 activation relates to CUB and TED modules. CUB closely packed against TED and MG8 in C3 moves about 25 cm relative to MG8 (FIGS. 1B and 6) and forms only a loose interaction with MG2 in the C3b structure. In C3, TED is tightly buried in the MG8 domain and CUB that protects the thio-ester bond formed between residues Cys988 and Gln991 from solvents. Upon activation, the TED rotates away from the CUB and MG8 domains, leaving the residues that connect the TED and CUB domains in a completely different conformation; in the course of these changes, Cys988 Move over 80 cm from its initial protected nest to its significantly exposed position in the C3 structure.

  C3 exists in two common allotypes that affect the incidence of inflammatory diseases (J. E. Finn et al., Nephrol Dial Transplant 9, 1564 (1994)). These variants differ by a single amino acid. The more common allotype C3F has a large positively charged arginine at position 80, while C3S has a small uncharged glycine. The loop around residue R80 does not form an interaction in the C3 or C3c structure (Janssen et al., Supra), and no other protein that interacts with R80 has been identified so far. In C3b, this loop is responsible for many contacts between MG1 and the TED domain, and R80 is located in close proximity to the negatively charged groove on the surface of the TED (FIG. 7). Further experiments are needed to confirm whether the interaction between R80 and TED domains is responsible for the observed differences between C3S and C3F allotypes.

  The α-helical toroid TED module in C3d and C3 shows many significant differences (Janssen et al. Supra). The most important point is that -required for covalent attachment of the TED domain to the particle surface-residues His1104 and Glu1106 are separated from each other and from Cys988. However, in C3d, these residues are in close proximity, which is an important requirement for covalent reactions. While the resolution of the C3b: CRIg complex presented here does not allow for the precise location of individual amino acid side chains, the TED conformation in C3b is very similar to the C3d structure That is clear from our structure. Due to the significant conformational shift between C3 and C3b, TED residues important for the binding reaction become solvent-exposed and are located at the distal end of the C3b molecule like a spear tip.

d. CRIg binds to the β chain of C3b and C3c As predicted by sequence analysis (K. Langnaese et al., Biochim Biophys Acta 1492, 522 (2000)), the N-terminal domain of CRIg is topologically It belongs to the IgV family of immunoglobulin-like domains, but shares only about 20% sequence identity with other Ig domains currently deposited in the Protein Data Bank. Like all members of this folding family, the CRIg domain is formed by two β sheets. One of these sheets is composed of strands A ′, G, F, C, C ′ and C ″, and the other is composed of strands B, E and D (FIGS. 2A and 8). The domain is further stabilized by a canonical Ig-like disulfide bond formed between two cysteine residues that internally link strands B and F.

  CRIg engages C3b and C3c in the same manner, and the arrangement of all contact domains present in both CRIg complexes is very similar. In line with this, the affinity of CRIg for C3b and C3c can be compared (FIG. 9). The superposition of C3b: CRIg and C3c: CRIg structures is less than 0.6 Å for the 642 common Ca position of the β chain and less than 0.5 に 対 し て for the 479 Ca atom of the C3c and C3b α chains. Become RMSD.

  Unlike all other complement receptors identified so far (JD Lambris et al., J Immunol 156, 4821 (1996); A. Taniguchi-Sidle, DE Isenman, J Immunol 153, 5285 (1994); AE Oran DE Isenman, J Biol Chem 274, 5120 (1999)), CRIg mainly binds to the β chain of C3b. A comparison of the C3c and CRIg structures in their unbound state and each other's complex revealed that no significant conformational adaptation was required in either of the two molecules to enhance binding. The 963 common Ca position in both unbound (Janssen et al.) And bound C3c structures to CRIg overlaps with a 0.8 Å RMSD, with the largest movement occurring in the mobile region, and only one small loop Migration occurs in the MG6 domain (results not shown). The binding site for CRIg is on the opposite side of C3b from the CUB and TED domains, and CRIg is located approximately 40 cm away from the TED domain.

Bonding interface between CRIg and C3c or C3b is large, discontinuous, fills the solvent-exposed area of about 2670A ² in total (Fig. 2A). CRIg contacts C3b and the residues from the A ′, G, G, C, C ′, C ″ sheets are the major interaction formed by the residues and loops near the strands C ′ and C ″. The hairpin loop connecting C ′ and C ″ projects into a cleft formed in the center of the C3b key ring-shaped β chain. On the C3b side of the interface, domains MG3, MG4, MG5, MG6, LNK and MG6 all contribute to CRIg binding, with MG3 and MG6 being responsible for about 30% and 40% of the buried interface, respectively ( FIG. 2B). Comparison of the C3 and C3b structures suggests an important role for the MG3 domain in gating CRIg binding to C3b and C3c. Apart from the large-scale domain migration that occurs upon C3 activation, there are several smaller domain shifts that can have functional significance. The key between these is a relatively slight 15 ° MG3 rotation, yet relatively significant compared to the rest of the C3 molecule. This rotation is responsible for the relative reorientation of MG3 with respect to the rings of other MG domains that make up the CRIg binding site, and in conjunction with the movement of the helical portion of the LNK region (FIG. 9), CRIg becomes C3b and C3c. Without these moderate changes, C3 will not be latched into CRIg.

The important binding parameters are summarized in Table 1 below.

Example 2
CRIg variants To functionally verify the importance of the binding interface revealed by the structural complex, the following CRIg variants were designed and manufactured and tested for their ability to bind to C3b: In Table 2, the number indicates the start position of the signal sequence cleavage site.

Table 2
Mutant Amino Acid Substitution A E85A M86A
B H57A Q59A
C V107S D109R I111E
D L34S K36D L38H
E P91A T93A D95A
Control H21A T23A D25A

  Mutants were made using the QuikChange site-directed mutagenesis kit (Stratagene) and primers were designed according to the manufacturer's instructions. The results of binding experiments with double or triple mutants are in close agreement with the crystallographically defined protein-protein contacts (FIG. 2B). Mutations in the MG6 and MG3 binding domains (E85A M86A and H57A Q59A) most effectively abrogated CRIg binding to C3b (IC50 values were> 10000 and 100-fold higher compared to wtCRIg), and these in CRIg binding Shows the importance of domain interactions. In contrast, mutations in the CRIg surface residues located on the opposite side of the binding interface showed binding activity similar to that of the wild type molecule, confirming that these mutations did not change the overall folding of the molecule. In addition, circular dichroism spectroscopy was further used to confirm that all mutant proteins were correctly folded (results not shown).

Example 3
CRIg binding to C3b inhibits the alternative pathway of complement Since C5 convertase in both the AP and classical / MBL pathways contains a C3b subunit (Pangburn and Muller-Eberhard, Biochem J., 235: 723 ( 1986)), we asked whether CRIg could regulate any pathway of complement activation through its binding to C3b. CRIg blocked complement activation via the alternative pathway, but not the classical or MBL pathway (FIG. 3a). This inhibitory activity was lost in CRIg mutants lacking C3b binding activity, confirming that CRIg-C3b binding was the direct cause of disrupting convertase activity. Selective inhibition of AP by CRIg was further confirmed with cell-based assays that distinguish between alternative and classical / MBL pathways. IC50 was reduced by a factor of 2 when CRIg was fused to the Fc portion of IgG (FIG. S6A). Since CRIg is a selective inhibitor of the alternative pathway, CRIg can inhibit alternative pathway hemolysis initiated by the classical pathway (FIG. S6B). It was confirmed that the alternative pathway inhibition was retained in mouse CRIg (FIG. S3C), and the complement inhibitory activity of CRIg was systematically conserved.

  To further determine the molecular mechanism by which CRIg inhibits convertase, the interaction of CRIg with the C3b subunit of C3 and C5 convertases was studied using purified components. CRIg was able to inhibit liquid phase C3 convertase as indicated by decreased C3a production (FIG. 3B). CRIg also reduced the enzymatic activity of C5 convertase collected on the surface of zymosan particles (FIG. 3C). The inhibitory activity of various CRIg variants correlates with its affinity for the C3b subunit, indicating that CRIg binding to C3b directly modulates the activity of the C5 convertase. This indicates that CRIg binding to the C3bβ chain prevents cleavage of both C3 and C5 by alternative pathway convertases.

Example 4
CRIg is a material and method that inhibits complement activation in a mouse model of arthritis a. Induction of arthritis in mice Collagen-induced arthritis was performed as described elsewhere (KH Barck et al., Arthritis Rheum 50, 3377 (2004)). Mice were injected subcutaneously with 4 mg / kg CRIg-Ig fusion protein starting on day 21 or 12 mg / kg fusion protein starting on day 36 after primary immunization. In the CRIg-Ig fusion protein, the mouse CRIg extracellular domain is fused to the mouse Fc portion of the IgG1 molecule. Antibody-induced arthritis was induced by the method of Terao and colleagues (K. Terato et al., J Exp Med 162, 637 (1985)) using an arthritis-inducing mixture. Briefly, mice were injected intravenously with 2 mg of anti-CIIAb (2 mg / 500 μl / body) from the tail vein and 3 days later, 25 μg LPS (25 μg / 100 μl / body) was injected intraperitoneally. Bone volume measurements and histology were performed as described elsewhere (Barck et al., Supra).

b. Measurement slaughtered hind footpad of mice cytokine concentrations in hind arthritis was cut with hair hairline, frozen in liquid N _2. The footpads were homogenized in ice-cold RIPA solubilization buffer supplemented with one complete protease inhibitor tablet (Roche) per 25 ml using a Polytron homogenizer (KINEMATICA). The volume of PBS used for homogenization was adjusted to 75 mg tissue per milliliter of buffer. The homogenate was centrifuged at 1870 × g for 15 minutes and the supernatant was centrifuged at 13230 × g for 5 minutes. The supernatant was subjected to ELISA analysis. ELISA kits for mouse IL-1 ^beta and IL-6 (BD DuoSet) and mC3a (Bachem), was used according to the protocol of each ELISA kit. C5a was measured by ELISA using a set of specific antibodies against the neo-epitope of C5a-desarg according to the manufacturer's protocol (BD). The total protein concentration in the supernatant was measured using a BCA kit (Pierce). Cytokine and chemokine concentrations were expressed in picograms per milligram of protein.

c. ELISA analysis C3a and C5a ELISAs were established using capture and detection antibodies from BD or using a mouse ELISA kit. Weilisa Whole Complement Screen: The protocol followed the instructions provided by the kit. To evaluate the potential CRIg inhibition of CP, factor B depleted serum was used. C2-depleted serum was used to determine CRIg inhibition of AP.

d. Measurement slaughtered hind footpad of mice cytokine concentrations in hind arthritis was cut with hair hairline, frozen in liquid N _2. The footpads were homogenized in ice-cold RIPA solubilization buffer supplemented with one complete protease inhibitor tablet (Roche) per 25 ml using a Polytron homogenizer (KINEMATICA). The volume of PBS used for homogenization was adjusted to 75 mg tissue per milliliter of buffer. The homogenate was centrifuged at 1870 × g for 15 minutes and the supernatant was centrifuged at 13230 × g for 5 minutes. The supernatant was subjected to ELISA analysis. ELISA kits for mouse IL-1 ^beta and IL-6 (BD DuoSet) and mC3a (Bachem), was used according to the protocol of each ELISA kit. C5a was measured by ELISA using a set of specific antibodies against the neo-epitope of C5a-desarg according to the manufacturer's protocol (BD). The total protein concentration in the supernatant was measured using a BCA kit (Pierce). Cytokine and chemokine concentrations were expressed in picograms per milligram of protein.

Results Soluble regulators of complement activation have proven to be powerful therapeutic tools that inhibit complement activation leading to inflammation (HF Weisman et al., Science 249, 146 (1990); BP Morgan, CL Harris, Mol Immunol 40, 159 (2003)). The therapeutic potential of CRIg was tested in two mouse models of rheumatoid arthritis where collagen autoantibodies and complement activation contribute to the cause of the disease (A. Aggarwal et al., Rheumatology (Oxford) 39, 189 (2000 S. Solomon et al., Arthritis Res Ther 7, 129 (2005); K. Terato et al., J Immunol 148, 2103 (Apr 1, 1992)). To extend its pharmacological half-life in vivo, the extracellular domain of muCRIg was fused to the Fc portion of mouse IgG1. CRIg significantly reduced joint swelling, histological score and bone loss when given before or after clinical signs of arthritis (FIGS. 4A and B, S12). Along with its inhibition of convertase activity, CRIg significantly reduced complement activation in the joints as shown by a decrease in C3 deposition on the cartilage surface (FIG. 4C). In mice with an already identified disease, CRIg is co-localized with C3 behind the joint (FIG. 4D), indicating that it can bind to and inhibit the convertase locally. ing. Also, local levels of C3a and systemic levels of C5a were significantly lower in CRIg-Fc treatment than in control-Fc treated mice (FIG. 4E). These results indicate that CRIg inhibits arthritis in the effector phase of the disease by inhibiting local and systemic complement activation. The protective activity of CRIg was independent of Fc receptor function because mutations in the Fc part of CRIg-Fc that abolish Fc receptor binding had activity equivalent to CRIg fused to wtFc protein (results are Not shown). Based on these results, the single IgV-like domain of CRIg can be a promising protein therapeutic that inhibits alternative pathways in complement-mediated diseases.

Example 5
CRIg is a material and method that inhibits AP convertase by inhibiting substrate-enzyme binding a. Disintegration Acceleration Assay An alternative pathway DAA microtiter plate assay was performed as previously described (M. Krych-Goldberg et al., J Biol Chem 274, 31160 (1999)). Microtiter plates were coated overnight with 4 μg / ml C3b in phosphate buffered saline. Plates were blocked with phosphate buffered saline containing 1% bovine serum albumin for 2 hours at 37 ° C. and 40 ng in veronal buffer containing 71 mM NaCl, 0.05% Tween 20 and 4% BSA. Of factor B, 10 ng factor D, and 0.8 mM NiCl ₂ for 2 hours at room temperature (buffer concentration and composition checked by protocol). The wells were then incubated for 15 minutes at room temperature with Factor H or CRIg-His ECD in PBS containing 0.05% Tween 20 (PBST) to dissociate Bb from C3b on the plate. Bb was then incubated for 1 hour with 1: 5000 dilution of goat anti-human factor B polyclonal antibody (Kent) in PBST and 1: 3000 dilution of horseradish peroxidase-conjugated donkey anti-goat antibody (Caltag) in PBST. Detected (dilution and secondary antibody checked by protocol). The color was developed with O-phenylenediamine. In this assay, factor H behaved as a mediator of decay-accelerating activity as expected.

b. C3b-C4b and C3b-C3b dimer preparation and binding / competition assay The C3bC4b heterodimer was modified with the following modifications (S. Meri, MK Pangburn, Eur. J. Immunol. 20: 2555 (1990)) As described elsewhere (S. Meri, MK Pangburn, Eur J Immunol 20, 2555 (1990)). To determine the binding of CRIg to C3b-C4b heterodimer and C3b homodimer, Maxisorp 96 well microtiter plates were incubated at 4 ° C. with 5 ug / ml goat anti-C3 polyclonal antibody (ICN) in PBS. Overnight and washed 3 times with PBST. The plates were then blocked with 250 μl PBS / 1% BSA for 2 hours at room temperature and a titration amount of hCRIg-L LFH was added for 1 hour in PBST containing 1% BSA. Plates were again washed 3 times with PBST and CRIg binding was detected with mouse anti-FLAG M2 antibody (SIGMA) conjugated to 1: 20000 dilution of HRP in PBST / 1% BSA. The plates were then washed 3 times with PBST, developed with 100 ul TMB substrate solution (KPL) and stopped with 50 ul 2N H2SO4. Absorbance was read at 450 nm. It was also determined that C5 binds to both C3b-C4b heterodimers and C3b homodimers in this form. To determine whether CRIg can block C5 binding to C3b-C4b or C3b-C3b, complement dimers were captured and blocked as described above. Dilute in a buffer containing 20 mM TRIS pH 7.5, 20 mM MgCl 2, 20 mM CaCl 2, 150 mM NaCl, 0.05% Tween 20 and 1% BSA to give 400 nM C5 at a titrated concentration of hCRIg-S HisECD or This fusion variant that does not bind C3b was premixed and added to the captured dimer for 1 hour at room temperature. The wells were then washed 3 times with PBST and C5 was detected by stepwise addition of 1: 5000 PBST dilution of mouse anti-human C5 (Quidel) and 1: 3000 goat F′ab anti-mouse antibody (Caltag). . The plate was developed as above and the two wells A415 were averaged and considered as a single point.

Results All known regulators of complement activation act as cofactors for factor I-mediated cleavage of C3 or are competitors of Bb for binding to C3b, thus eliminating convertase activity (D Hourcade et al., Adv Immunol 45, 381 (1989); T. Seya, JP Atkinson, Biochem J 264, 581 (1989); X. Sun et al., Proc Natl Acad Sci USA 96, 628 (1999)). In contrast, CRIg exhibits neither disintegration activity nor cofactor activity. CRIg does not interfere with the binding of factor H, factor B or properdin to C3b (FIGS. S13A-C, results not shown). To further explore the mechanism by which CRIg inhibits AP complement activation, we determined whether CRIg interferes with C3b function rather than with the catalytic Bb subunit of C5 convertase. The non-catalytic C3b subunit of the alternative pathway convertase is responsible for the binding of substrates C3 and C5. The Bb factor subunit of the convertase can then act as a protease, cleaving the bound substrate (W. Vogt et al., Immunology 34, 29 (1978)). As a result, the activity of the convertase is directly proportional to the affinity of the substrate for C3b (N. Rawal, MK Pangburn, J Immunol 164, 1379 (2000)). CRIg prevents binding of C5 to the C3b ₂ subunit of AP convertase, but consistent with its selectivity for AP, C5 convertase (FIG. 5A) has no effect on the C3bC4b subunit of CP convertase. I don't have it. The importance of CRIg binding to the activity of C5 convertase was further determined with purified C5 convertase (N. Rawal, MK Pangburn, J Biol Chem 273, 16828 (1998)). In the presence of CRIg, the Vmax of the enzyme was reduced by ˜50% (FIG. 5B), indicating that CRIg inhibits the maximum binding capacity of C5 to C3b independently of the substrate concentration. This is an indication of steric or allosteric inhibition of C5 binding to C3b by CRIg and not a direct competition of CRIg with C5 for binding to C3b. This is further supported by results from binding assays where C5 cannot compete with CRIg for binding to C3b (FIG. 14). Thus, CRIg bound to the center of the C3b key ring-like β chain structure can block the binding of C5 to the convertase, thus inhibiting the activity of the enzyme. This indicates the importance of the C3b β chain for the interaction of C5-convertase with its substrate (N. Rawal, MK Pangburn, J Biol Chem 273, 16828 (1998)).

Claims (37)

  A crystal formed by a native sequence CRIg polypeptide or functional fragment or conservative amino acid substitution variant thereof. The crystal according to claim 1, which has a lattice constant of a = 30.3Å, b = 50.8Å, c = 62.0Å and a space group of P2 ₁ 2 ₁ 2 ₁ .   The native sequence CRIg polypeptide is a human CRIg polypeptide of SEQ ID NO: 1, 2 and 3; a mouse CRIg polypeptide of SEQ ID NO: 4; a rat CRIg polypeptide of SEQ ID NO: 5; a bovine CRIg polypeptide of SEQ ID NO: 6; The crystal of claim 1 selected from the group consisting of No. 7 monkey CRIg polypeptide.   The crystal according to claim 1, wherein the functional fragment is an extracellular domain (ECD) sequence of a native sequence CRIg.   The crystal of claim 1, wherein the crystal diffracts X-rays to determine atomic coordinates with a resolution of about 1 to 40 Å.   The crystal of claim 1 wherein the crystal diffracts x-rays to determine atomic coordinates with a resolution of at least about 5 Å.   The crystal according to claim 6, wherein the resolution is at least 4 少 な く と も.   The crystal according to claim 7, wherein the resolution is at least 3 少 な く と も.   CRIg crystal having the structural coordinates shown in appendix 1.   A composition comprising the crystal according to any one of claims 1 to 9.   Crystal form of complex of native sequence CRIg polypeptide or functional fragment or conservative amino acid substitution variant thereof and complement factor C3b. The crystal form according to claim 11, having a lattice constant of a = 97.6 ＝, b = 255.7 Å, c = 180.3 Å and a C222 ₁ space group.   The native sequence CRIg polypeptide is a human CRIg polypeptide of SEQ ID NO: 1, 2 and 3; a mouse CRIg polypeptide of SEQ ID NO: 4; a rat CRIg polypeptide of SEQ ID NO: 5; a bovine CRIg polypeptide of SEQ ID NO: 6; 12. The crystalline form of claim 11 selected from the group consisting of the monkey CRIg polypeptide of number 7.   12. The crystalline form of claim 11, wherein the functional fragment is the extracellular domain (ECD) sequence of native sequence CRIg.   The crystalline form of claim 11 diffracts X-rays to determine atomic coordinates with a resolution of about 1 to 40 Å.   The crystalline form of claim 11 diffracts X-rays to determine atomic coordinates with a resolution of at least about 5 Å.   The crystal according to claim 16, wherein the resolution is at least 4 少 な く と も.   The crystal according to claim 17, wherein the resolution is at least 3 少 な く と も.   The crystal form according to claim 11, having the structure coordinates shown in Appendix 2.   Crystal form of a complex of a native sequence CRIg polypeptide or functional fragment or conservative amino acid substitution variant thereof and complement factor C3c.   21. The crystalline form of claim 20, having approximately a space group of lattice constants a = 382.8 Å, b = 65.0 Å, c = 147.2 Å, β = 102.7, and C2.   The native sequence CRIg polypeptide is a human CRIg polypeptide of SEQ ID NO: 1, 2 and 3; a mouse CRIg polypeptide of SEQ ID NO: 4; a rat CRIg polypeptide of SEQ ID NO: 5; a bovine CRIg polypeptide of SEQ ID NO: 6; 21. The crystalline form of claim 20, selected from the group consisting of No. 7 monkey CRIg polypeptide.   21. The crystalline form of claim 20, wherein the functional fragment is the extracellular domain (ECD) sequence of native sequence CRIg.   21. The crystalline form of claim 20, wherein the crystal form is diffracted to determine atomic coordinates with a resolution of about 1 to 40 inches.   21. The crystalline form of claim 20, wherein the crystal form is diffracted to determine atomic coordinates with a resolution of at least about 5 mm.   The crystal according to claim 25, wherein the resolution is at least 4 Å.   The crystal according to claim 26, wherein the resolution is at least 3 少 な く と も.   21. Crystalline form according to claim 20, having the structural coordinates shown in appendix 3.   A molecule or molecular complex comprising at least a part of the CRIg polypeptide of SEQ ID NO: 2 or a conservative substitution C3b binding site thereof, wherein the binding site is 8, 14-15, 41-42, 44, 45 -47, 50, 52, 54-61, 62, 64, 85-87, 89, 95, 99, 105, 107-110, and at least one amino acid residue selected from the group consisting of 111 and 111, and binding A molecule or molecular complex determined by a set of points having a distance of less than 4.7 mm from a point representing an amino acid atom represented by the structural coordinates described in Appendix 2.   30. The molecule or molecular complex of claim 29, wherein the binding site comprises substantially all of the amino acid residues.   A three-dimensional structure of a point in which at least part of the point is derived from the structure coordinates of appendix 2 representing the position of the skeleton atom of at least the core amino acid that defines the CRIg binding site for C3b   32. The three-dimensional structure of a point according to claim 31, shown as a holographic image, stereo diagram, model, or computer display image, wherein the CRIg binding site for C3b forms a crystal with space group symmetry C2221.   A machine readable data storage medium comprising a data storage material encoded with machine readable data, wherein the machine programmed with instructions for using the data comprises at least a portion of a C3b binding site. A three-dimensional graphic diagram of at least one molecule or molecular complex is displayed, and the binding site determined by the set of points is about 4.7 cm from the point representing the amino acid atom represented by the structural coordinates described in Appendix 2. A medium having a distance of less than.   A method of identifying a CRIg agonist comprising designing a compound that mimics the C3b binding site of CRIg.   (A) performing a fitting operation between the chemical substance and the three-dimensional structure of the CRIg polypeptide or C3b: CRIg complex using calculation or experimental means; (b) analyzing the data obtained in step (a) A chemical identified by characterizing the binding between the chemical and the native CRIg or C3b: CRIg complex.   36. The chemical entity of claim 35 that interferes with binding between CRIg and C3b in vivo or in vitro.   A molecule or molecular complex comprising at least part of the C3b binding region of a native sequence CRIg molecule or a conservative amino acid substitution variant thereof.

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