RU2837284C2 - HYBRID PROTEINS OF HUMAN PROTEIN FRAGMENTS FOR CREATING ORDERLY MULTIMERIZED COMPOSITIONS OF Fc REGIONS OF IMMUNOGLOBULINS WITH ENHANCED BINDING TO COMPLEMENT SYSTEM - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, specifically to obtaining peptide homodimers and multimers for binding to a component of the complement system C1q, and can be used in medicine for treating or preventing rheumatoid arthritis. Constructed is a peptide homodimer consisting of two identical monomers, wherein each monomer consists, in the direction from the amino-to-carboxyl end, of a multimerisation domain of the hinge region of IgG2, consisting of an amino acid sequence under SEQ ID NO: 4, and an IgG1 Fc domain consisting of the amino acid sequence under SEQ ID NO: 2 with point mutations including S267E, H268F and S324T based on the EC index according to Kabat.

EFFECT: invention provides obtaining a peptide homodimer which binds to complement C1q and inhibits CDC.

9 cl, 63 dwg, 12 tbl, 14 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 62/196,478, filed July 24, 2015, the contents of which are incorporated herein by reference in their entirety.

DESCRIPTION OF THE TEXT FILE PROVIDED IN ELECTRONIC FORM

[0002] The contents of the text file provided in electronic form are incorporated herein by reference in their entirety: copy of the sequence listing in machine-readable format (file name: GLIK_015_01WO_SeqList_ST25.txt, accession date: July 22, 2016, file size 193 kilobytes).

AREA OF TECHNOLOGY

[0003] The present invention generally relates to the fields of immunology, autoimmunity, inflammation and tumor immunology. More particularly, the present invention relates to biologically active biomimetic molecules comprising immunoglobulin Fc domains that exhibit altered binding to an Fc receptor and preserved or enhanced binding to elements of the complement system, to compositions containing said biomimetics, as well as to methods for producing and using said biomimetics. The present invention also relates to the treatment or prevention of pathological conditions such as complement-mediated diseases, autoimmune diseases, inflammatory diseases, blood diseases and various types of cancer.

LEVEL OF TECHNOLOGY

[0004] The complement system is a part of the immune system that is involved in the lysis of target cells and the phagocytosis of antigens. Currently, three major complement pathways are recognized: the classical pathway, the alternative pathway, and the lectin-binding pathway. The classical pathway of the complement system is activated by binding of the C1q protein to one or more intact IgM immunoglobulin molecules or at least two intact IgG1, IgG2, or IgG3 immunoglobulin molecules (Janeway's Immunobiology, ^8th Ed., Murphy ed., Garland Science, 2012, Chapter 10). Activation of the complement system results in complement-dependent cytotoxicity (CDC). Alterations in the Fc region of monoclonal antibodies have been shown to increase or decrease complement binding affinity (Moore et al., MAbs. 2(2): 181-9 (2010). However, this work was performed with a monoclonal antibody and therefore relied at least in part on the Fab's target specificity, and did not address binding avidity.

[0005] Excessive complement activation and/or deposition can be deleterious and has been associated with many diseases, including myasthenia gravis, hemolytic uremic syndrome (HUS), and paroxysmal nocturnal hemoglobinuria (PNH). Significantly elevated levels of the complement component C1q have been found in the brains of elderly subjects (Stephan et al., J. Neuroscience, 14 August 2013, 33(33): 13460-13474). The complement system is significantly involved in the pathophysiology of myasthenia gravis, which is caused by the production of antibodies to the acetylcholine receptor (Tűzűn and Christadoss, Autoimmun Rev. 2013 Jul; 12(9):904-11. doi: 10.1016/j.autrev.2013.03.003). A number of immunological, genetic, and protein biochemical studies indicate that the complement system plays an important role in the etiology of age-related macular degeneration (Weber et al., Dtsch Arztebl Int., 2014 Feb; 111(8): 133-138. doi:10.3238/arztebl.2014.0133). There is strong evidence that the classical and alternative complement pathways are abnormally activated in rheumatoid arthritis and in animal models of rheumatoid arthritis (Okroj et al., Ann Med. 2007;39(7):517-30).

[0006] The classical, alternative, and lectin pathways are activated sequentially, and all three pathways are involved in systemic disease. Activation of the complement system has been implicated in the pathophysiology of systemic autoimmune diseases. Activation via the classical pathway has long been implicated in the pathophysiology of immune complex-mediated diseases such as cryoglobulinemic vasculitis and systemic lupus erythematosus (Chen et al., Journal of Autoimmunity, 2009; doi:10.1016/j.jaut.2009.11.014). Activation of the complement system via the alternative and lectin pathways has been found in patients with Henoch-Schönlein purpura nephritis and IgA nephropathy (Hisano et al., Am J Kidney Dis 2005;45:295e302). The importance of the complement system in membranous nephropathy, one of the most common causes of adult nephrotic syndrome, has been well described. Membranous nephropathy is characterized by glomerular subepithelial immune deposits. Studies in Heymann nephritis, an experimental model of membranous nephropathy, have shown that these deposits locally activate the complement system with subsequent podocyte damage, resulting in cytoskeletal reorganization, loss of slit diaphragms, and proteinuria (Beck et al., The Role of Complement in Membranous Nephropathy. Semin Nephrol 2013 Nov;33(6):531–42).

[0007] Complement activation is an extremely complex process, and even many decades after the mechanism was thought to be fully established, new components of the cascade continue to be discovered. The first step in the classical complement pathway is the binding of C1q to antibody. (Nature. 1988 Apr 21;332(6166):738-40; The binding site for C1q on IgG. Duncan AR, Winter G.).

[0008] Binding of non-aggregated antibody to C3 or C3b appears to occur without complement activation in plasma, but is thought to be supported primarily by complexes of the C3b activation product ₂ with IgG (Mol Immunol. 2006 Jan;43(1-2):2-12. Complement amplification revisited. Lutz HU, Jelezarova E). However, during activation of the alternative pathway of the complement system by IgG antibody-containing immune aggregates, the C3b alpha chain can covalently link at one or two sites to the Fd portion of the IgG heavy chain (i.e., the portion of the heavy chain that is part of the Fab fragment) (Biochem J. May 1, 1981; 195(2): 471-480). KJ Gadd and KB Reid)). Traditionally, the complement components C4a, C3a, C5a, and the membrane attack complex have been generally referred to as complement cleavage products or anaphylotoxins. However, evidence from the literature clearly indicates that in some cases C4a (Xie et al., International Immunopharmacology 12 (2012) 158–168), C3a (Coulthard and Woodruff, J Immunol 2015; 194:3542–3548), and C5a (Nishise et al., Therapeutic Apheresis and Dialysis 13(6):509–514 and Gerard et al., J. Biol Chem. 280(48):39677–39680, December 2, 2005) are anti-inflammatory and unrelated to the propagation of the cascade.

[0009] The predominant therapeutic agent currently available commercially for the treatment of complement-mediated diseases is the anti-C5 antibody, eculizumab, which binds to C5 and inhibits its cleavage into C5a and C5b-9, partially inhibiting downstream reactions of the complement cascade and the associated inflammatory and thrombotic reactions. However, the antibody does not directly affect upstream reactions involving components of the complement system, such as C1q, C1R, C1S, C1-like complex, or C4, C4a, C4b, C2, C1, C4b2, C2b, C4b2a, C3, C3a, or C3b, which are components of the lectin pathway, classical pathway, and alternative pathway, the reactions of which precede reactions involving the complement component C5 and which may be preferred targets for disease treatment. In contrast, normal immunoglobulins and monoclonal antibodies bind to C1q and other targets that are involved in pre-C5 reactions. Accordingly, there is a need in the art for improved methods of treating complement-mediated diseases by binding to components of the complement cascade involved in initial activation reactions and subsequently modulating the complement cascade. There is now a further need for improved methods of treating complement-mediated diseases using a compound comprising an Fc region of an immunoglobulin that binds hexameric C1q with avidity, rather than affinity alone, and that does not bind with significant avidity to low-affinity Fc receptors.

[00010] The native Fc region of IgG1 binds naturally to more than a dozen ligands, one of which is complement factor C1q. Other ligands of the IgG1 Fc region include, but are not limited to, canonical Fc receptors, neonatal receptor FcRn, iron, protein A, FcRL1-6, TRIM21, and DC-SIGN. Soluble homodimeric IgG1 naturally binds to C1q with an affinity that is too low to be functional (CA Diebolder et. al. Complement Is Activated by IgG Hexamers Assembled at the Cell Surface. Science Mar 2014; 343(6176):1260–1263) and with a high dissociation rate (Gaboriaud et. al. The crystal structure of the globular head of complement protein C1q provides a basis for its versatile recognition properties. J. Biol. Chem. 2003 Nov 21; 278(47):46974–82). However, when IgG1 is bound to the target antigen via its Fab, a conformational change occurs (M. Oda et. al. Evidence of allosteric conformational changes in the antibody constant region upon antigen binding. Int. Immunol. (2003) 15 (3): 417–426), and the binding affinity for C1q increases.

[00011] Furthermore, because IgG1 accumulates at the site of antigen accumulation, multiple Fc regions of the immunoglobulin can be presented by C1q, resulting in more avid binding at the antigen binding site. Because C1q is a hexamer with six Fc binding sites (KB Reid. Chemistry and molecular genetics of C1q. Behring Inst Mitt. 1989 Jul;(84):8-19), binding of aggregated IgG1 is highly avid (Diebolder, 2014). Therefore, tightly bound IgG1 binds to C1q not only with homodimeric immunoglobulin affinity but also with avidity, resulting in complement-dependent cytotoxicity, in addition to being available for cross-linking of Fc receptors, resulting in antibody-dependent cytotoxicity (ADCC) and antibody-dependent phagocytosis of cells (ADCP). The resulting functional binding response of the Fc region clusters associated with hexameric C1q is a signal for activation of the classical complement pathway to form C1qC1rC1s and cleavage of C4 into C4a and C4b. Soluble IgG1 aggregates, which are found in very low concentrations in nature (Soltis and Hasz. Spontaneous aggregation of native immunoglobulins in hypoalbuminemic serum. J Clin Lab Immunol. 1982 Oct;9(1):13-7) and in pooled human intravenous immunoglobulin (IVIG) (A. Herrera et. al. Immunoglobulin composition of three commercially available intravenous immunoglobulin preparations. J. All Clin Immunol, 84(1):556-561), represent the multivalent Fc region of IgG1 that is unbound to its ligands, including the low-affinity Fc receptors and C1q. Soluble (i.e., non-cell-bound) IgG1 aggregates are therefore capable of avidly binding to hexameric C1q. Binding of soluble C1q to soluble IgG1 aggregates (i.e., not cell-bound) may inhibit subsequent activation of the complement cascade, including inhibition of CDC.

[00012] Pooled human IVIG, which is pooled from samples from tens of thousands of blood donors, contains a very small and variable proportion (0.1-5%) of IgG1 aggregates that mimic the natural action of soluble native IgG1 aggregates. IVIG binds to C1q and has been shown to be useful in clinical settings for complement-mediated diseases such as myasthenia gravis. However, IVIG is associated with all the associated risks that come with being a blood product, not being produced recombinantly, having poorly controlled amounts of IgG1 aggregates, and consisting primarily of inactive fractions for the treatment of complement-mediated diseases. Moreover, IVIG treatment also often results in an undesirable proinflammatory response due to binding to low-affinity Fc receptors (Andresen et. al. Product equivalence study comparing various human immunoglobulin-G formulations. J Clin Pharmacol 2000; 40:722-730 and Ghielmetti et. al. Gene expression profiling of the effects of intravenous immunoglobulin in human whole blood. Mol Immunol 43 (2006) 939-949). There is a need for new, uniform therapeutics produced by recombinant methods that mimic the natural process of the aggregated soluble Fc region of IgG1 acting as a complement acceptor through avid binding to C1q, while avoiding undesirable pro-inflammatory responses due to preferential binding to components of the complement system compared to other natural ligands, including low-affinity Fc receptors.

BRIEF DESCRIPTION OF THE INVENTION

[00013] The present invention relates to biologically active biomimetic hybrid protein molecules comprising one or more human immunoglobulin Fc domains and one or more multimerization domains, wherein the Fc domain of the hybrid protein comprises one or more point mutations. According to one aspect of the present invention, the biomimetic molecules preferentially bind to one or more components of the complement system, compared to a normal, non-aggregated human immunoglobulin Fc region. According to some embodiments of the present invention, preferential binding to one or more components of the complement system is achieved by an Fc domain of the biomimetic having a reduced ability to bind to canonical Fc receptors, compared to a normal, non-aggregated immunoglobulin Fc region or, in another embodiment, a normal aggregated immunoglobulin Fc region. According to some embodiments of the present invention, the biologically active biomimetic hybrid protein molecules comprise stradomer units. The present invention provides compositions containing biologically active hybrid protein molecules of biomimetics and methods for using them.

[00014] According to one aspect, the present invention provides a stradomer unit comprising at least one IgG1 Fc domain comprising one or more point mutations corresponding to at least one of positions 267, 268 and/or 324 of the IgG1 Fc domain. In some embodiments, the stradomer unit also comprises at least one multimerization domain. In some embodiments, the amino acid at position 267 is mutated from serine (Ser, S) to any other amino acid. In other embodiments, the amino acid at position 267 is mutated to glutamic acid (Glu; E). In some embodiments, the amino acid at position 268 is mutated from histidine (His; H) to any other amino acid. In other embodiments, the amino acid at position 268 is mutated to phenylalanine (Phe; F). In some embodiments, the amino acid at position 324 is mutated to serine and any other amino acid. In other embodiments, the amino acid at position 324 is mutated to threonine (Thr; T). In some embodiments, the Fc domain of the stratomeric unit comprises point mutations at positions 267, 268, and 324. In other embodiments, the Fc domain comprises the point mutations S267E, H268F, and S324T.

[00015] According to some embodiments of the present invention, the stradomer unit comprises an IgG1 Fc domain comprising an amino acid sequence comprising point mutations at positions 267, 268 and/or 324 and further comprising at least one point mutation at position 233 and/or 234 and/or 235 and/or 236 and/or 238 and/or 265 and/or 297 and/or 299 and/or 328. In one embodiment of the present invention, the amino acid at position 297 is mutated from asparagine (Asn; N) to any other amino acid. In another embodiment of the present invention, the amino acid at position 297 is mutated to alanine (Ala; A). According to one embodiment of the present invention, the amino acid at position 299 is mutated from threonine (T) to any other amino acid except serine or cysteine. According to another embodiment of the present invention, the amino acid at position 299 is mutated to alanine (Ala; A). According to one embodiment of the present invention, the amino acid at position 238 is mutated from proline (Pro; P) to any other amino acid. According to another embodiment of the present invention, the amino acid at position 238 is mutated to aspartic acid (Asp; D). According to one embodiment of the present invention, the amino acid at position 233 is mutated from glutamic acid (Glu; E) to any other amino acid. According to another embodiment of the present invention, the amino acid at position 233 is mutated to proline (Pro; P). According to another embodiment of the present invention, the amino acid at position 236 is mutated from glycine (Gly; G) to any other amino acid. According to another embodiment of the present invention, the amino acid at position 236 is mutated to arginine (Arg; R). According to one embodiment of the present invention, the amino acid at position 236 is deleted. According to one embodiment of the present invention, the amino acid at position 234 is mutated from leucine (Leu; L) to any other amino acid. According to another embodiment of the present invention, the amino acid at position 234 is mutated from valine (Val; V) or alanine (Ala; A). According to another embodiment of the present invention, the amino acid at position 235 is mutated from leucine (Leu; L) to any other amino acid. According to another embodiment of the present invention, the amino acid at position 235 is mutated to alanine (Ala; A). According to one embodiment of the present invention, the amino acid at position 265 is mutated from aspartic acid (Asp; D) to any other amino acid. According to another embodiment of the present invention, the amino acid at position 265 is mutated to alanine (Ala; A). According to another embodiment of the present invention, the amino acid at position 265 is mutated to tryptophan (Trp; W). According to one embodiment of the present invention, the amino acid at position 297 is mutated from asparagine (Asn; N) to any other amino acid. According to one embodiment of the present invention, the amino acid at position 297 is mutated to alanine (Ala; A). According to one embodiment of the present invention, the amino acid at position 297 is mutated to glutamine (Gln; Q). According to one embodiment of the present invention, the amino acid at position 299 is mutated from threonine (Thr; T) to any amino acid other than serine (Ser; S) or cysteine (Cys; C). According to one embodiment of the present invention, the amino acid at position 299 is mutated to alanine. According to one embodiment of the present invention, the amino acid at position 298 is mutated to any amino acid other than proline. According to one embodiment of the present invention, the amino acid at position 328 is mutated from leucine (Leu; L) to any amino acid other than serine (Ser; S) or cysteine (Cys; C). According to one embodiment of the present invention, the amino acid at position 328 is mutated to phenylalanine (Phe; F).

[00016] According to some embodiments of the present invention, the Fc domain of the stradomeric unit comprises point mutations at positions 267, 268, 297, and 324. According to other embodiments of the present invention, the Fc domain comprises point mutations S267E, H268F, N297A, and S324T.

[00017] According to some embodiments of the present invention, the Fc domain of the stradomeric unit comprises point mutations at positions 234, 235, 267, 268, and 324. According to other embodiments of the present invention, the Fc domain comprises point mutations L234V, L235A, S267E, H268F, and S324T.

[00018] According to some embodiments of the present invention, the Fc domain of the stradomeric unit comprises point mutations at positions 234, 235, 267, 268, 297, and 324. According to other embodiments of the present invention, the Fc domain comprises point mutations L234V, L235A, S267E, H268F, N297A, and S324T.

[00019] According to some embodiments of the present invention, the Fc domain of the stradomeric unit comprises point mutations at positions 233, 234, 235, 267, 268, and 324 and an amino acid deletion at position 236. According to other embodiments of the present invention, the Fc domain comprises point mutations E233P, L234A, L235A, S267E, H268F, and S324T and an amino acid deletion at position 236.

[00020] According to some embodiments of the present invention, the Fc domain of the stradomeric unit comprises point mutations at positions 233, 234, 235, 267, 268, 297, and 324. According to other embodiments of the present invention, the Fc domain comprises point mutations E233P, L234A, L235A, S267E, H268F, N297A, and S324T.

[00021] According to some embodiments of the present invention, the Fc domain of the stradomeric unit comprises a point mutation at positions 265, 267, 268, and 324. According to other embodiments of the present invention, the Fc domain comprises the point mutations D265A, S267E, H268F, and S324T.

[00022] According to some embodiments of the present invention, the Fc domain of the stradomeric unit comprises a point mutation at positions 238, 267, 268, and 324. According to other embodiments of the present invention, the Fc domain comprises the point mutations P238D, S267E, H268F, and S324T.

[00023] According to some embodiments of the present invention, the Fc domain of the stradomeric unit comprises a point mutation at positions 238, 267, 268, 297, and 324. According to other embodiments of the present invention, the Fc domain comprises the point mutations P238D, S267E, H268F, N297A, and S324T.

[00024] According to some embodiments of the present invention, the Fc domain of the stradomeric unit comprises a point mutation at positions 236, 267, 268, and 324. According to other embodiments of the present invention, the Fc domain comprises the point mutations G236R, S267E, H268F, and S324T.

[00025] According to some embodiments of the present invention, the Fc domain of the stradomeric unit comprises a point mutation at positions 233, 236, 267, 268, and 324. According to other embodiments of the present invention, the Fc domain comprises the point mutations E233P, G236R, S267E, H268F, and S324T.

[00026] According to some embodiments of the present invention, the Fc domain of the stradomeric unit comprises a point mutation at positions 233, 236, 267, 268, 324, and 328. According to other embodiments of the present invention, the Fc domain comprises the point mutations E233P, G236R, S267E, H268F, S324T, and L328F.

[00027] In some embodiments of the present invention, the Fc domain of the stradomer unit comprises a point mutation at positions 238, 265, 267, 268, and 324. In other embodiments of the present invention, the Fc domain comprises the point mutations P238D, D265G, S267E, H268F, and S324T. In other embodiments of the present invention, the Fc domain comprises the point mutations P238D, D265W, S267E, H268F, and S324T.

[00028] According to some embodiments of the present invention, the Fc domain of the stradomeric unit comprises a point mutation at positions 267, 268, 324, and 328. According to other embodiments of the present invention, the Fc domain comprises the point mutations S267E, H268F, S324T, and L328F.

[00029] According to some embodiments of the present invention, the Fc domain of the stradomer unit comprises a point mutation at positions 233, 234, 235, 267, 268, 297, 324, and 328 and an amino acid deletion at position 236. According to other embodiments of the present invention, the Fc domain comprises the point mutations E233P, L234V, L235A, S267E, H268F, N297A, S324T, and L328F and an amino acid deletion at position 236.

[00030] According to some embodiments of the present invention, the Fc domain of the stradomeric unit comprises a point mutation at positions 233, 268, and 324. According to other embodiments of the present invention, the Fc domain comprises the point mutations E233P, H268F, and S324T.

[00031] According to some embodiments of the present invention, the Fc domain of the stradomeric unit comprises a point mutation at positions 233, 268, 297, and 324. According to other embodiments of the present invention, the Fc domain comprises the point mutations E233P, H268F, S324T, and a point mutation at position 297 other than N297A or N297Q.

[00032] In some embodiments of the present invention, the Fc domain of the stradomer unit comprises a point mutation at positions 233, 268, 299, and 324. In other embodiments of the present invention, the Fc domain comprises the point mutations E233P, H268F, S324T, and a point mutation at position 299 other than T299S and T299C. In some embodiments of the present invention, the Fc domain of the stradomer unit comprises the point mutations E233P, H268F, S324T, and T299A.

[00033] According to some embodiments of the present invention, the Fc domain of the stradomer box comprises point mutations at positions 233, 268, 324, and 267. According to some embodiments of the present invention, the Fc domain of the stradomer box comprises point mutations E233P, H268F, S324T, and a point mutation at position 267 other than the point mutation S267E.

[00034] According to some embodiments of the present invention, the Fc domain of the stradomer box comprises point mutations at positions 233, 268, 324, 267, and 297. According to other embodiments of the present invention, the Fc domain of the stradomer box comprises point mutations E233P, H268F, S324T, a point mutation at position 267 other than the point mutation S267E, and a point mutation at position 297 other than the point mutations N297A and N297Q. In other embodiments of the present invention, the Fc domain of the stradomer box comprises mutations at positions 233, 268, 324, 267 and 297. In other embodiments of the present invention, the Fc domain of the stradomer box comprises the point mutations E233P, H268F, S324T, a point mutation at position 267 other than the point mutation S267E, a point mutation at position 297 other than the point mutations N297A and N297Q, and a point mutation at position 299 other than the point mutations T299S and T299C. In other embodiments of the present invention, the mutation at position 299 is T299A.

[00035] According to some embodiments of the present invention, the Fc domain of the stradomer box comprises point mutations at positions 233, 268, 324, 267, and 236. According to some embodiments of the present invention, the Fc domain of the stradomer box comprises point mutations E233P, H268F, S324T, a point mutation at position 267 other than S267E, and a point mutation at position 236 other than G236R.

[00036] According to some embodiments of the present invention, the Fc domain of the stradomer box comprises point mutations at positions 233, 268, 324, 267, 236, and 297. According to some embodiments of the present invention, the Fc domain of the stradomer box comprises point mutations E233P, H268F, S324T, a point mutation at position 267 other than S267E, a point mutation at position 236 other than G236R, and a point mutation at position 297 other than N297A and N297Q.

[00037] In some embodiments of the present invention, the Fc domain of the stradomer box comprises point mutations at positions 233, 268, 324, 267, 236, and 299, wherein the point mutation at position 299 is a point mutation other than T299S and T299C. In some embodiments of the present invention, the Fc domain of the stradomer box comprises point mutations E233P, H268F, S324T, a point mutation at position 267 other than S267E, a point mutation at position 236 other than G236R, and T299A.

[00038] In some embodiments, the Fc domain of the stradomer box comprises point mutations at at least one of positions 267, 268, and/or 324, and further comprises a point mutation at three or more of positions 233, 235, 236, 267, 268, 297, 299, and/or 324. In some embodiments, the Fc domain of the stradomer box comprises point mutations at at least one of positions 267, 268, and/or 324, and further comprises a point mutation at three or more of positions 233, 235 other than L235H, 236 other than 236R, 267 other than S267E, 268, 297 other than N297A, or, in another embodiment, 299 and/or 324.

[00039] According to one embodiment of the present invention, the stradomer comprises a multimerization domain, wherein said multimerization domain is selected from the group consisting of an IgG2 hinge region, an isoleucine zipper, and a GPP domain, and is capable of multimerizing said stradomer units. According to some embodiments of the present invention, the multimerization domain creates multimers of said stradomer units. According to other embodiments of the present invention, the multimers of said stradomer units are highly ordered multimers.

[00040] According to some embodiments of the present invention, a stradomer comprises, in amino- to carboxyl-terminal direction, a leader sequence; an Fc domain comprising an IgG1 hinge region, IgG1 CH2, and IgG1 CH3; and an IgG2 hinge region, wherein the stradomer comprises one or more point mutations according to the present invention. According to another embodiment of the present invention, the leader sequence is cleaved upon expression. Therefore, according to another embodiment, the present invention provides a stradomer comprising, in amino- to carboxyl-terminal direction, an Fc domain comprising an IgG1 hinge region, IgG1 CH2, and IgG1 CH3; and an IgG2 hinge region, wherein the stradomer comprises one or more point mutations according to the present invention. According to one embodiment of the present invention, the stradomer comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-16, 18-63 and 65-77, or a functional variant thereof.

[00041] According to some embodiments of the present invention, a stradomer comprises, in amino- to carboxyl-terminal direction, a leader sequence; an IgG2 hinge region; and an Fc domain comprising an IgG1 hinge region, IgG1 CH2, and IgG1 CH3, wherein the stradomer comprises one or more point mutations according to the present invention. According to another embodiment of the present invention, the leader sequence is cleaved upon expression. Therefore, according to another embodiment, the present invention provides a stradomer comprising, in amino- to carboxyl-terminal direction, an IgG2 hinge region; and an Fc domain comprising an IgG1 hinge region, IgG1 CH2, and IgG1 CH3, wherein said stradomer comprises one or more point mutations according to the present invention. According to one embodiment of the present invention, the stradomer comprises an amino acid sequence selected from SEQ ID NO: 17 and SEQ ID NO: 64, or a functional variant thereof.

[00042] According to some embodiments of the present invention, a stradomer comprises, in an amino- to carboxyl-terminal direction, a leader sequence; an IgG2 hinge region; and an Fc domain comprising IgG1 CH2 and IgG1 CH3, wherein said stradomer comprises one or more point mutations according to the present invention. According to another embodiment of the present invention, the leader sequence is cleaved upon expression. Therefore, according to another embodiment, the present invention provides a stradomer comprising, in an amino- to carboxyl-terminal direction, an IgG2 hinge region; and an Fc domain comprising IgG1 CH2 and IgG1 CH3, wherein said stradomer comprises one or more point mutations according to the present invention.

[00043] According to one embodiment of the present invention, the stradomer unit also comprises one or more amino acid linker sequences. According to another embodiment of the present invention, the linker is cleavable. According to another embodiment of the present invention, the linker is protease sensitive. According to one embodiment of the present invention, the linker is cleaved by a protease that is predominantly intracellular. According to another embodiment of the present invention, the linker is cleaved by a protease that is predominantly present in the Golgi apparatus and the endoplasmic reticulum. According to one embodiment of the present invention, the linker is cleaved by furin. According to another embodiment of the present invention, the linker is cleaved by a protease that is predominantly an organ-specific protease, such as, for example, the protease neuropsin, which is found in the brain. According to another embodiment of the present invention, the linker is cleaved by a protease, which is preferably a tumor-specific protease, such as, for example, metalloproteinase 9 and urokinase plasminogen activator.

[00044] In some embodiments of the present invention, the stradomer unit preferentially binds to complement compared to FcγRI, FcγRII including FcγRIIa and/or FcγRIIb, and/or FcγRIII. In some embodiments of the present invention, the stradomer unit exhibits reduced binding to a low affinity Fcγ receptor. In other embodiments of the present invention, the stradomer unit exhibits reduced binding to FcγRI, FcγRII including FcγRIIa and/or FcγRIIb, and/or FcγRIII, compared to a stradomer of similar structure that does not contain a point mutation at one or more positions 267, 268, and/or 324.

[00045] According to some embodiments of the present invention, the stradomer unit comprises a mutation at 297, 298, or 299 and retains binding to C1q, inhibits CDC, and retains binding to FcγRI or a low affinity Fcγ receptor, including FcγRIIa, FcγRIIb, and/or FcγRIII.

[00046] In one aspect, the present invention provides a clustered stradomer comprising two or more stradomer units disclosed herein. For example, in some embodiments, the present invention provides a clustered stradomer comprising two or more stradomer units comprising an IgG1 Fc domain comprising an amino acid sequence comprising point mutations at positions 267, 268, and/or 324. In other embodiments, the two or more stradomer units further comprise at least one point mutation at position 233 and/or 234 and/or 235 and/or 236 and/or 238 and/or 265 and/or 297 and/or 299 and/or 328. In some embodiments, the two or more stradomer units comprise an amino acid deletion at position 236.

[00047] According to some embodiments of the present invention, the stradomers provided by the present invention are used to treat or prevent diseases and disorders, including, but not limited to, complement-mediated diseases, autoimmune diseases, inflammatory diseases, allergies, B-cell-mediated diseases, antibody-mediated diseases, kidney disorders, and blood disorders.

[00048] In some embodiments, the antibody-mediated disease is selected from the group consisting of Goodpasture's disease; solid organ transplant rejection; neuromyelitis optica; neuromyotonia; limbic encephalitis; Morvan's fibrillary chorea syndrome; myasthenia gravis; Lambert-Eaton myasthenic syndrome; autonomic neuropathy; Alzheimer's disease; atherosclerosis; Parkinson's disease; stiff person syndrome or hyperekplexia; recurrent spontaneous abortion; Hughes syndrome; systemic lupus erythematosus; autoimmune cerebellar ataxia; connective tissue diseases including scleroderma, Sjogren's syndrome; polymyositis; rheumatoid arthritis; polyarteritis nodosa; Thibierge-Weissenbach syndrome; endocarditis; Hashimoto's thyroiditis; mixed connective tissue disease; channelopathies; pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS); clinical conditions associated with antibodies to N-methyl-D-aspartate receptors, in particular NR1, contactin-associated protein 2, AMPA receptors, GluR1/GluR2, glutamate decarboxylase, Gly-alpha-1a receptors, acetylcholine receptor, VGCC type P/Q, VGKC, MuSK, GABA _B receptors; aquaporin-4; and pemphigus. According to some embodiments of the present invention, the autoimmune disease is arthritis.

[00049] According to some embodiments of the present invention, the complement system-mediated disease is selected from the group consisting of myasthenia gravis, hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), neuromyelitis optica, antibody-mediated allograft rejection, nephropathy, including membranous nephropathy and nephritis, including membranoproliferative glomerulonephritis (MPGN), and lupus nephritis.

[00050] According to some embodiments of the present invention, the blood disorder is anemia, such as sickle cell diseases, including hemoglobin SS, hemoglobin SC, hemoglobin Sβ ⁰ thalassemia, hemoglobin Sβ ⁺ thalassemia, hemoglobin SD, and hemoglobin SE thalassemia. According to some embodiments of the present invention, the inflammatory disease is age-related macular degeneration, Alzheimer's disease, amyotrophic lateral sclerosis, or Parkinson's disease.

[00051] In some embodiments, the stradomets of the present invention are administered to a subject in need thereof. In other embodiments, the stradomets of the present invention are administered intravenously, subcutaneously, orally, intraperitoneally, sublingually, bucally, transdermally, via a subcutaneous implant, or intramuscularly.

[00052] According to some embodiments, the present invention provides stradomets that can be used in the treatment or prevention of autoimmune disease-related vision loss or hearing loss, such as noise-induced or age-related hearing loss. According to another embodiment, the present invention provides stradomets that can be used in reducing inflammation or autoimmune reactions associated with the implantation of a device, such as a cochlear implant or other hearing aids.

BRIEF DESCRIPTION OF THE FIGURES

[00053] Figure 1 shows the binding of GL-2045 stradomer to FcγRI, FcγRIIb, FcγRIIIa, FcγRIIa, or FcRn as measured by biolayer interferometry (ForteBio Octet).

[00054] Figure 2 shows a radial plot of maximum response units (RU _max ) for each Fc receptor, ELISA data for assessing C1q binding and CDC inhibition for G045c and the complement preferential binding stradomer G997. Each radial plot in these descriptions was generated by visual inspection of the binding curves derived from Octet biolayer interferometry readings.

[00055] Figure 3 shows the results of the binding of G997 stradomer to FcγRI, FcγRIIb, FcγRIIIa, FcγRIIa and FcRn as measured by biolayer interferometry.

[00056] Figure 4A shows a radial plot of RU _max values for each Fc receptor, ELISA data for C1q binding, and CDC inhibition data for G045c and the complement preferential binding stradomer G998. Figure 4B shows a radial plot of RU values at 300 s (RU300s) for each Fc receptor for G045c and the complement preferential binding stradomers G997 and G998.

[00057] Figure 5 shows the results of the binding of G998 stradomer to FcγRI, FcγRIIb, FcγRIIIa, FcγRIIa and FcRn as measured by biolayer interferometry.

[00058] Figure 6A shows a radial plot of RU _max values for each Fc receptor, ELISA data for assessing binding to C1q, and CDC inhibition data for G045c and the complement preferential binding stradomers G1022 and G1033. Figure 6B shows a radial plot of RU values at 300 s (RU300s) for each Fc receptor for G045c and the complement preferential binding stradomers G1022 and G1033.

[00059] Figure 7A shows a radial plot of RU _max values for each Fc receptor, ELISA data for assessing C1q binding, and CDC inhibition data for G045c and the universal stradomer G1032. Figure 7B shows a radial plot of RU values at 300 s (RU300s) for each Fc receptor for G045c and the universal stradomer G1032.

[00060] Figure 8A shows a radial plot of RU _max values for each Fc receptor, ELISA data for assessing C1q binding, and CDC inhibition data for G045c and the universal stradomer G1023. Figure 8B shows a radial plot of RU values at 300 s (RU300s) for each Fc receptor for G045c and the universal stradomer G1023.

[00061] Figure 9A shows a radial plot of RU _max values for each Fc receptor, ELISA data for assessing C1q binding, and CDC inhibition data for G045c and the complement preferential binding stradomer G1006. Figure 9B shows a radial plot of RU values at 300 s (RU300s) for each Fc receptor for G045c and the complement preferential binding stradomer G1006.

[00062] Figure 10A shows a radial plot of RU _max values for each Fc receptor, ELISA data for assessing C1q binding, and CDC inhibition data for G045c and the complement preferential binding stradomer G1027. Figure 10B shows a radial plot of RU values at 300 s (RU300s) for each Fc receptor for G045c and the complement preferential binding stradomer G1027.

[00063] Figure 11A shows a radial plot of RU _max values for each Fc receptor, ELISA data for assessing C1q binding, and CDC inhibition data for G045c and the complement preferential binding stradomer G1003. Figure 11B shows a radial plot of RU values at 300 s (RU300s) for each Fc receptor for G045c and the complement preferential binding stradomer G1003.

[00064] Figure 12A shows a radial plot of RU _max values for each Fc receptor, ELISA data for assessing C1q binding, and CDC inhibition data for G045c and the complement preferential binding stradomer G989. Figure 12B shows a radial plot of RU at 300 s (RU300s) values for each Fc receptor for G045c and the complement preferential binding stradomer G989.

[00065] Figure 13 shows the results of the binding of G989 stradomer to FcγRI, FcγRIIb, FcγRIIIa, FcγRIIa and FcRn as measured by biolayer interferometry.

[00066] Figure 14A shows a radial plot of RU _max values for each Fc receptor, ELISA data for C1q binding, and CDC inhibition data for G045c and the complement preferential binding stradomer G990. Figure 14B shows a radial plot of RU at 300 s (RU300s) values for each Fc receptor for G045c and the complement preferential binding stradomer G990.

[00067] Figure 15 shows the results of the binding of G990 stradomer to FcγRI, FcγRIIb, FcγRIIIa, FcγRIIa and FcRn as measured by biolayer interferometry.

[00068] Figure 16A shows a radial plot of RU _max values for each Fc receptor, ELISA data for assessing C1q binding, and CDC inhibition data for G045c and the complement preferential binding stradomer G994. Figure 16B shows a radial plot of RU at 300 s (RU300s) values for each Fc receptor for G045c and the complement preferential binding stradomer G994.

[00069] Figure 17 shows the results of the binding of G994 stradomer to FcγRI, FcγRIIb, FcγRIIIa, FcγRIIa and FcRn as measured by biolayer interferometry.

[00070] Figure 18A shows a radial plot of RU _max values for each Fc receptor, ELISA data for assessing C1q binding, and CDC inhibition data for G045c and the complement preferential binding stradomer G996. Figure 18B shows a radial plot of RU values at 300 s (RU300s) for each Fc receptor for G045c and the complement preferential binding stradomer G996.

[00071] Figure 19 (top row of panels) shows the results of the binding of G996 stradomer to FcγRI, FcγRIIb, FcγRIIIa, FcγRIIa, or FcRn as measured by biolayer interferometry. Figure 19 (bottom row of panels) also shows the results of the binding of G996 stradomer to mouse FcγRIIb, FcγRIII, and FcγRIV.

[00072] Figure 20A shows a radial plot of RU _max values for each Fc receptor, ELISA data for assessing C1q binding, and CDC inhibition data for G045c and the complement preferential binding stradomer G1042. Figure 20B shows a radial plot of RU values at 300 s (RU300s) for each Fc receptor for G045c and the complement preferential binding stradomer G1042.

[00073] Figure 21 shows the results of the binding of G1042 stradomer to FcγRI, FcγRIIb, FcγRIIIa, and FcγRIIa as measured by biolayer interferometry.

[00074] Figure 22A shows a radial plot of RU _max values for each Fc receptor, ELISA data for assessing C1q binding, and CDC inhibition data for G045c and the complement preferential binding stradomer G1043. Figure 22B shows a radial plot of RU values at 300 s (RU300s) for each Fc receptor for G045c and the complement preferential binding stradomer G1043.

[00075] Figure 23 shows the results of the assessment of the binding of the G1043 stradomer to FcγRI, FcγRIIb, FcγRIIIa, or FcγRIIa, as measured by biolayer interferometry.

[00076] Figure 24A shows a radial plot of RU _max values for each Fc receptor, ELISA data for assessing C1q binding, and CDC inhibition data for G045c and the complement preferential binding stradomer G1046. Figure 24B shows a radial plot of RU values at 300 s (RU300s) for each Fc receptor for G045c and the complement preferential binding stradomer G1046.

[00077] Figure 25 shows the results of the assessment of the binding of the G1046 stradomer to FcγRI, FcγRIIb, FcγRIIIa, or FcγRIIa, as measured by biolayer interferometry.

[00078] Figure 26A shows a radial plot of RU _max values for each Fc receptor, ELISA data for assessing C1q binding, and CDC inhibition data for G045c and the complement preferential binding stradomer G1050. Figure 26B shows a radial plot of RU values at 300 s (RU300s) for each Fc receptor for G045c and the complement preferential binding stradomer G1050.

[00079] Figure 27 shows the results of the binding of G1050 stradomer to FcγRI, FcγRIIb, FcγRIIIa, and FcγRIIa as measured by biolayer interferometry.

[00080] Figure 28A shows a radial plot of RU _max values for each Fc receptor, ELISA data for assessing C1q binding, and CDC inhibition data for G045c and the universal stradomer G1049. Figure 28B shows a radial plot of RU values at 300 s (RU300s) for each Fc receptor for G045c and the universal stradomer G1049.

[00081] Figure 29 shows the results of the binding of G1049 stradomer to FcγRI, FcγRIIb, FcγRIIIa, and FcγRIIa as measured by biolayer interferometry.

[00082] Figure 30A shows a radial plot of RU _max values for each Fc receptor, ELISA data for assessing C1q binding, and CDC inhibition data for G045c and the complement preferential binding stradomer G1025. Figure 30B shows a radial plot of RU values at 300 s (RU300s) for each Fc receptor for G045c and the complement preferential binding stradomer G1025.

[00083] Figure 31A shows the results of the binding of the negative control G001, the parent stradomer GL-2045, G993, G997, G998, G996, and G994 to C1q as measured by absorbance at increasing stradomer concentrations using ELISA. Figure 31B shows the log-transformed data and _EC50 values for each of the stradomers tested. _EC50 values are presented in μg/mL.

[00084] Figure 32 shows the results of the binding of the negative control G001, the original stradomer GL-2045, G994, G996, G997 and G998 to the complement component C3, as measured by absorbance measurements at increasing concentrations of the stradomer using ELISA. The upper panel shows the logarithmically transformed absorbance values and the lower panel shows the non-logarithmically transformed absorbance values.

[00085] Figure 33 shows the results of the binding assessment of the negative control G001, the original stradomer GL-2045, G997, G998 and G994 to the complement component C3b, as measured by absorbance at increasing concentrations of the stradomer using ELISA.

[00086] Figure 34 shows the results of the binding assessment of the negative control G001, the original stradomer GL-2045, G996, G997, G998 and G994 to the complement component C4, as measured by the absorbance at increasing concentrations of the stradomer using ELISA

[00087] Figure 35 shows the results of the binding assessment of the negative control G001, the original stradomer GL-2045, G996, G997, G998 and G994 to the complement component C5, as measured by absorbance at increasing concentrations of stradomer using ELISA.

[00088] Figures 36A and 36B show the sequences of the polymorphic forms of human IgG1, DEL (Figure 36A; SEQ ID NO: 3) and EEM (Figure 36B; SEQ ID NO: 2).

[00089] Figure 37 shows a significant reduction in proteinuria in animals administered the test stradomets in an animal model of nephritis.

[00090] Figures 38A and 38B show the histological examination results of a lesioned control animal treated with phosphate-buffered saline (PBS) after induction of glomerulonephritis with an anti-Thy 1 antibody (Figure 38A) and an animal that received 40 mg/kg G998 (Figure 36B). In Figure 38A, (G) refers to glomeruli with thickened basement membrane; (B) refers to basophilic tubules and interstitial infiltrates; and the arrow points to dilated tubules with proteinaceous fluid. Figure 36B shows unlesioned glomeruli and tubules from an animal treated with G998.

[00091] Figure 39 graphically shows the results of histological examination of kidney tissue from animals receiving PBS as a control treatment (Group 2) or 40 mg/kg G0998 (Group 4).

[00092] Figure 40 shows blood urea nitrogen (BUN) concentrations in control animals treated with PBS, healthy control animals that did not receive the anti-Thy1 antibody, and animals that received G994 (40 mg/kg), G998 (40 mg/kg or 80 mg/kg), or G1033 (40 mg/kg). P values presented in the graph are compared to diseased control animals treated with PBS.

[00093] Figure 41 shows the results of proteinuria assessment (mg/24 hours) in control animals that received only anti-Fx1a antibody, animals that received anti-Fx1a antibody and G998 on day 0, and animals that received anti-Fx1a antibody on day 0 and G998 on day 1.

[00094] Figure 42 shows the levels (mcg/mL) of G994, G998, or G1033 in rats following administration of a single dose of the compound as a function of time.

[00095] Figure 43 shows the results of a study of the activity of the complement system in the blood of rats after administration of a single dose of G994 (left panel), G998 (middle panel) or G1033 (right panel).

[00096] Figures 44A and 44B show the correlation between drug concentrations (μg/mL on the x-axis) and complement activity (% on the y-axis) for each of G994 (left panels), G998 (middle panels), and G1033 (right panels). Figure 44A shows all data points collected in the study for each complement-binding stradomer. In Figure 44, more attention is given to the lower portion of the curve (lower drug concentrations).

[00097] Figure 45 shows the results of drug concentrations (μg/mL) of G994, G998, or G1033 in cynomolgus monkeys following administration of a single dose of a compound with preferential binding to the complement system at time 0, as measured at 0, 1, 2, 4, 12, 24, 48, 72, 144, 216, and 312 hours post-dose.

[00098] Figure 46 shows the results of the assessment of complement activity (% cell death) depending on time after administration of a single dose of G994 (left panel), G998 (middle panel), or G1033 (right panel).

[00099] Figures 47A and 47B show the correlation between drug level and complement activity for each of the test compounds in the study. Figure 47A shows all data points collected during the experiment, and Figure 47B shows the first portion of the curve with lower drug concentrations. For G994, the intercept value on the abscissa is 144 and 168 μg/mL for 2% and 4% serum. The R ² values for the correlation are 0.866 and 0.835. For G998, the intercept value on the abscissa is 425 and 347 μg/mL, and the R ² values are 0.925 and 0.743 for 2% and 4% serum. For G1033, the intercept value on the abscissa axis is 346 and 379 μg/mL with R2 values ^of 0.584 and 0.714.

[000100] Figures 48A-G are gels showing that, similar to the parent compound GL-2045, from which the tested stradomer derivative compound (G994 or G998) was designed, the stradomer derivative compounds form multimers. Compounds GL-2045, G994, G1103, G1088, G1089, G1104, G1082, G1105, and G1106 are shown in Figure 48A. Compounds GL-2045, G994, G1102, G1100, G1101, G1125, G1108, G1109, and G1084 are shown in Figure 48B. Compounds GL-2045, G998, and G1107 are shown in Figure 48C. Compounds GL-2045, G994, G1110, G1111, G1112, G1114, G1115, G1116, G1117, G1118, and G1119 are shown in Fig. 48D. Compounds GL-2045, G994, G1120, G1121, G1122, G1123, G1124, G1128, G1129, G1130, and G1131 are shown in Fig. 48E. Compounds GL-2045, G998, G1071d2, G1068, G1094, G1092, G1096, G1093, and G1095 are shown in Fig. 48F. Compounds GL-2045, G998, G1069, G1070, G1132, G1075 and G1075 are shown in Fig. 48G.

[000101] Figure 49 shows the results of the binding assay of the negative control G001, which is IgG1 Fc, GL-2045 stradomer, or the complement-preferential stradomer G994, G998, or G1033, to complement component C3 (upper left panel), C3b (upper right panel), C5 (lower left panel), or C4 (lower right panel), as measured by absorbance at increasing stradomer concentrations using ELISA.

[000102] Figures 50A-50C show the results of the binding of G1103, G1088, G1089, G1104, G1082, G1105, and G1106 to FcγRI, FcγRIIa, FcγRIIb, or FcγRIII as measured by biolayer interferometry (ForteBio Octet).

[000103] Figures 51A-51C show the results of the binding of G1102, G1100, G1101, G1125, G1108, G1109, and G1084 to FcγRI, FcγRIIa, FcγRIIb, or FcγRIII as measured by biolayer interferometry (ForteBio Octet).

[000104] Figures 52A-52G show the results of the binding of stradomer G1100, G1111, G1112, G1113, G1114, G1115, G1116, G1117, G1118, G1119, G1120, G1121, G1122, G1123, G1124, G1128, G1129, G1130, and G1131 to FcγRI, FcγRIIa, FcγRIIb, or FcγRIII as measured by biolayer interferometry (ForteBio Octet).

[000105] Figures 53A-53C show the results of the binding of G1071d2, G1068, G1094, G1092, G1096, G1107, G1093, and G1095 to FcγRI, FcγRIIa, FcγRIIb, or FcγRIII as measured by biolayer interferometry (ForteBio Octet).

[000106] Figures 54A-54B show the results of the binding of G1069, G1070, G1132, G1074, and G1075 to FcγRI, FcγRIIa, FcγRIIb, or FcγRIII as measured by biolayer interferometry (ForteBio Octet).

[000107] Figure 55A shows data on the induction of IL-1Rα from isolated peripheral blood mononuclear cells (PBMCs) following 20 hours of treatment with vehicle control (0 μg/mL G019, G994, or G1033) or doses of G019, G994, and G1033 ranging from 0.02 μg/mL to 2000 μg/mL. G019, which binds to all canonical receptors, was used as a positive control.

[000108] Figure 55B shows data on the induction of TNF-α from isolated PBMCs following 4 hours of treatment with vehicle control (0 μg/mL G019, G994, or G1033) or doses of G019, G994, and G1033 ranging from 0.02 μg/mL to 2000 μg/mL. G019, which binds to all canonical receptors, was used as a positive control.

[000109] Figure 56 shows data on C1q binding by stradomers with preferential binding to the complement system, as measured by ELISA.

[000110] Figure 57 shows the mean ankle diameter of Lewis rats in the collagen-induced arthritis (CIA) model treated with G994, G998, G1033, control PBS, dexamethasone, or untreated controls.

[000111] Figures 58A-58B show C1q binding data for some of the compounds with preferential binding to the complement system shown in Figure 56, G1088, G1129, G1082, G1092, and G1102 (Figure 58A), as well as G1069, G1114, and G1075 (Figure 58B).

[000112] Figure 59 shows the results of the binding of the G999 and G996 stradomer to FcγRI, FcγRIIb and FcγRIIIa as measured by biolayer interferometry.

[000113] Figure 60 shows CDC inhibition data for G999 and G996. CD20 denotes the addition of anti-CD20 antibody. “6%” denotes the addition of 6% serum complement. Controls include cells with G996 alone (50 and 100 μg/mL), cells with serum (“+6%”), and cells with serum and antibody (“cells + CD20 +6%”).

[000114] Figure 61 shows CDC inhibition data for G994. CD20 denotes the addition of anti-CD20 antibody. “6%” denotes the addition of 6% serum complement. Controls include cells with G996 alone (50 and 100 μg/mL), cells with serum (“+6%”), and cells with serum and antibody (“cells + CD20 +6%”).

[000115] Figure 62 shows CDC inhibition data for G998. CD20 denotes the addition of anti-CD20 antibody. “6%” denotes the addition of 6% serum complement. Controls include cells with G996 alone (50 and 100 μg/mL), cells with serum (“+6%”), and cells with serum and antibody (“cells + CD20 +6%”).

[000116] Figure 63 shows CDC inhibition data for G1033. CD20 denotes the addition of anti-CD20 antibody. “6%” denotes the addition of 6% serum complement. Controls include cells with G996 alone (50 and 100 μg/mL), cells with serum (“+6%”), and cells with serum and antibody (“cells + CD20 +6%”).

DETAILED DESCRIPTION OF THE INVENTION

[000117] An approach to rational molecular design of the immune system modulating compounds described herein involves the creation, using recombinant and/or biochemical methods, of an immunologically active biomimetic(s) that exhibits retained, preferential, or enhanced binding to the complement system. In one embodiment, the compositions of the invention enhance binding to the complement system by increasing the degree of binding to the complement system, as compared to binding to Fc receptors. In one embodiment, the compositions of the invention exhibit reduced or no binding to one or more FcγRs. In another embodiment, the compositions of the invention exhibit reduced or no binding to FcRn. In one embodiment, the compositions of the invention bind to the complement system and exhibit reduced or no binding to certain FcγRs as well as FcRn. The compositions of the present invention can be used to treat, for example, complement-mediated diseases and complement-associated diseases. The compositions of the present invention are also capable of multimerization. According to one embodiment of the present invention, the compositions of the present invention exhibit preserved or enhanced multimerization compared to previously described biomimetics.

[000118] WO 2008/151088 discloses the use of linked immunoglobulin Fc domains to create ordered multimerized hIVIG immunoglobulin Fc biomimetics (biologically active ordered multimers known as stradomers) that contain short sequences, including restriction sites and affinity tags, between the individual components of the stradomer, which are intended for the treatment of pathological conditions, including autoimmune diseases and other inflammatory conditions. See WO 2008/151088 and U.S. Patent No. 8,680,237, the contents of each of which are incorporated herein by reference in their entirety. WO 2012/016073 discloses stradomers in which the individual components are directly linked rather than separated by restriction enzyme sites or affinity tags. See WO 2012/016073 and U.S. Patent Application Publication No. 2013/0156765, the entire contents of which are incorporated herein by reference. WO 2012/016073 also specifically discloses a multimerizing stradomer (GL-2045) comprising an IgG1 Fc domain and an IgG2 hinge region multimerization domain fused directly to its C-terminus, which exhibits improved multimerization and complement binding compared to an N-terminal tethered construct (G019, described in WO2008/151088). The stratum corneal units described herein contain one or more point mutations in the Fc region of GL-2045 that result in enhanced binding to the complement system and/or selective or increased complement binding compared to canonical Fc-gamma receptor binding, when compared to previously described molecules.

[000119] G045c and G019 have been previously described (see WO 2012/016073). G045c has the following structure: IgG1 hinge region - IgG1 CH2 CH3 IgG1 - IgG2 hinge region, and G045c is shown in SEQ ID NOs: 7 and 8.

[000120] In this application, the terms "G045c", "GL-2045" are used interchangeably. G045c has the following structure: IgG1 hinge region - CH2 IgG1 CH3 IgG1 - IgG2 hinge region. In this application, the term "G045c-based stradomer" and the like refers to a stradomer having the structure: IgG1 hinge region - CH2 IgG1 CH3 IgG1 - IgG2 hinge region (SEQ ID NO: 7 or 8). G019 has the following structure: IgG2 hinge region - IgG1 hinge region - CH2 IgG1 - CH3 IgG1. In this application, the term "G019-based stradomer" and the like refers to a stradomer having the structure: IgG1 hinge region - CH2 IgG1 CH3 IgG1 - IgG2 hinge region (SEQ ID NO: 7 or 8). refers to a stradomer having the structure: IgG2 hinge region - IgG1 hinge region - IgG1 CH2 - IgG1 CH3 (SEQ ID NO: 9). One skilled in the art will appreciate that the amino acid sequences of GL-2045 and G019 contain an N-terminal leader sequence (SEQ ID NO: 1) that is cleaved upon expression of the mature protein.

[000121] Mutations in the Fc regions of full-length antibody molecules have predictable effects on antibody performance and function. See, e.g., Shields et al., Journal of Biological Chemistry, 276; 6591 (2001). In particular, Moore et al. showed that the triple mutation S267E, H268F, and/or S324T in the Fc region of a monoclonal antibody significantly increased the binding of the monoclonal antibody to complement, compared to binding to the canonical Fcγ receptor or FcRn (Mabs 2:2; 18 (2010)). However, the present inventors have disclosed compounds in which the S267E, H268F and/or S324T mutations in the Fc region of the multimerizing stradomer, including the S267E/H268F/S324T triple mutation, are in fact and quite unexpectedly associated with a lack of binding to C1q and, therefore, do not increase the degree of binding to the complement system, compared to binding to canonical Fcγ receptors or FcRn (e.g., G999, G1024, G1030, G1031, G1040, G1044 and G1048).

[000122] It is generally recognized that specific mutations in monoclonal antibodies that increase binding to C1q (e.g., S267E, H268F, and/or S324T described in Moore et al.) thereby increase CDC (Moore et al. 2010). The present inventors describe herein numerous compounds that, by incorporating the aforementioned mutations, as applied to the multimerizing stradomer, are associated with not only a lack of increase in CDC, but also a lack of inherent CDC at all. In addition, given that these mutations are associated with an increase in CDC, as applied to a monoclonal antibody, the present inventors disclose herein that compounds of the present invention containing the aforementioned mutations actually inhibit CDC in a concentration-dependent manner (e.g., G994, G998, and many others, Figures 61-63).

[000123] In addition, with respect to the antibody, it was found that a point mutation introduced at position 297 of the Fc domain reduces binding to high-affinity and low-affinity canonical Fcγ receptors (Robert L. Shields, et al. High Resolution Mapping of the Binding Site on Human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and Design of IgG1 Variants with Improved Binding to the FcγR. J. Biol. Chem., Feb 2001; 276: 6591 - 6604). Similarly, for a monoclonal antibody, a point mutation introduced at position 299 of the Fc domain was shown to differentially affect FcγR binding (e.g., reduce binding to FcγRIIIa and retain binding to FcγRIIa) and additionally inhibit binding to C1q (Sazinsky et al. Aglycosylated immunoglobulin G ₁ variants productively engage activating Fc receptors. Proc Natl Acad Sci US A. 2008 Dec 23; 105(51): 20167–20172; PCT/US2008/085757). For specific multimerizing stradomers, point mutations at position 297 of the Fc domain resulted in decreased binding to low-affinity canonical Fcγ receptors. However, in some stradomers, the mutation at position 299 did result in the expected reduction in FcγR binding, but still maintained or enhanced Fc binding to all canonical FcγRs.

[000124] Additionally, a double mutation at positions 236 and 328 (Tai et al., Blood 119; 2074 (2012)) or a mutation at position 233 (Shields, et al. J. Biol. Chem., 276(9):6591 (2001)) has been shown to reduce antibody or immunoglobulin Fc binding to canonical FcγRs. A double mutation at positions 236 and 328 has also been shown to abolish antibody or immunoglobulin binding to FcγRI (Tai et al. 2012); However, the inventors found, unexpectedly, that in the case of a multimerizing stradomer and a complement binding enhancing mutation at position 267 and/or 268 and/or 324, binding to FcγRI is retained in an unpredictable manner in some multimerizing stradomers. In addition, a double mutation at positions 233 and 236 (E233P and G236R) would be expected to reduce binding to all canonical Fc receptors; however, the inventors found that several combinations of mutations, including mutations at these two positions, resulted in binding to one or more Fc receptors that was retained in an unpredictable manner or increased, compared to the corresponding unmutated stradomer. In addition, the mutation at position 328 (L328F) was expected to increase binding to FcγRIIb only (Chu et al., Molecular Immunology 45; 3926 (2008)); however, the present inventors unexpectedly found that a stradomer containing a mutation at position 328, when applied to a stradomer containing additional mutations at at least positions 267, 268, and 324, exhibits increased binding to one or more other canonical Fc gamma receptors in an unpredictable manner. In addition, the mutation at position 238 was expected to increase binding to FcγRIIb and decrease binding to FcγRI and FcγRIIa (Mimoto et al., Protein Engineering, Design, and Selection p. 1-10 (2013)), while the mutation at position 265 was expected to decrease binding to all canonical FcγRs. However, we unexpectedly found that, for the multimerizing stradomer and the complement binding enhancing mutation at position 267 and/or 268 and/or 324, the mutations at positions 238 and 265 resulted in strong binding to FcγRIIa in some multimerizing stradomers. Therefore, the present inventors have discovered the surprising fact that mutations described in the literature that modify antibody function, for example to reduce or eliminate canonical binding of the monoclonal antibody or to alter binding to C1q, do not have a similar effect in the case of the multimerizing stradomer.

[000125] Moreover, even in the case of a stradomer, the effect of mutations is also unpredictable. For example, as described herein, in the case where two stradomers are identical to each other except for one or more mutations at one or more specific positions, even if the mutation involves substitution of structurally similar amino acids, the two stradomers may have very different functional characteristics. Furthermore, WO 2012/016073 discloses that despite the fact that GL-2045 and G019 contain similar components and are essentially identical molecules except for the position of the hinge region of IgG2 relative to the Fc domain of IgG1, these molecules unexpectedly have very different complement binding activities. Both molecules are capable of multimerization and binding to Fc receptors. An unexpected finding was that GL-2045 exhibits strong binding to all Fc receptors and also binds to the complement system and inhibits complement-dependent cytotoxicity (CDC), whereas G019 does not bind to complement or inhibit CDC. Likewise, if similar mutations were introduced into GL-2045 and G019, the outcome could be quite different and, in fact, opposite. As an example, compounds 996 and 999 carry the triple mutation disclosed in Moore, et al., which is expected to increase binding to C1q (S267E/H268F/S324T), as well as an additional mutation G236R. However, in the case of GL-2045, this mutation retains preferential binding to C1q over canonical Fcγ receptors (G996), whereas G019 does not bind to C1q (G999). This discrepancy in the functional impact of a well-characterized mutation highlights the unpredictability of the effects of mutations introduced into the multimerizing stradomer.

[000126] Furthermore, while monoclonal antibodies have affinity for FcγRs and complement target components that can be increased or decreased by introducing mutations, stradomers present a polyvalent Fc region to Fcγ receptors and complement, and therefore rely more on avidity to bind to their targets. In contrast, monoclonal antibodies are not capable of avid binding via their Fc domains. These features highlight the fact that stradomers and monoclonal antibodies are fundamentally different not only in structure but also in function and applications.

[000127] Therefore, the effect of any mutation or set of mutations within any region of the molecule on the activity of the multimerizing stradomer cannot be predicted from the literature relating to monoclonal antibodies. Accordingly, the effect of amino acid mutations that are known in the art to have a specific effect on antibodies, such as mutations that will, for example, increase or decrease binding to a particular FcγR, when applied to an antibody having antigen specificity, cannot be predicted for stradomers. Similarly, the point mutations L234A and L235A have been described as decreasing Fc binding to C1q (See WO 2015/132364; Arduin et al, Molecular Immunology, 65(2):456-463 (2015); Boyle et al, Immunity, 42(3):580-590 (2015)). However, the present inventors have discovered the unexpected fact that introducing these mutations into the biomimetics of the present invention results in retention or enhancement of binding to C1q (e.g. G1033 and G1032).

[000128] The present inventors have identified immunologically active biomimetics in which the degree of binding to the complement system is increased, compared to binding to the Fc receptor, when compared to the parent biomimetic (e.g., GL-2045 or G019). In one embodiment, the biomimetics of the present invention exhibit preserved or enhanced binding to C1q. In one embodiment, the biomimetics of the present invention bind to components of the complement system, including, but not limited to, C1q, C1r, C1s, C4, C4a, C3, C3a, C4b2a3b, C3b, C5, C5a, C5b, C6, C7, C8, and/or C9, and can therefore act as a "complement acceptor." In the present application, the term "complement acceptor" refers to the phenomenon of binding to C1q or another component involved in the reactions of the complement cascade preceding activation and preventing subsequent activation of the complement system. In one embodiment of the present invention, the biomimetics of the present invention bind to components of the complement system that enter the reaction cascade before the membrane attack complex C5b-9. In one embodiment of the present invention, the biomimetics of the present invention bind to components of the complement system that enter the reaction cascade before C5a. In one embodiment of the present invention, the biomimetics of the present invention exhibit reduced binding to C5a and the membrane attack complex. In one embodiment of the present invention, the biomimetics of the present invention exhibit reduced binding to FcγRI and/or FcγRIIa and/or FcγRIIb and/or FcγRIII and maintain or enhance binding to the complement system. According to another embodiment of the present invention, the biomimetics of the present invention exhibit reduced binding to FcRn and preserved or enhanced binding to the complement system. According to another embodiment of the present invention, the biomimetics of the present invention exhibit preserved or enhanced binding to the complement system and reduced binding to canonical FcγRs, as well as reduced binding to FcRn. According to one embodiment of the present invention, the biomimetics of the present invention exhibit preserved or enhanced binding to the complement system and selective binding to one or more canonical Fcγ (e.g., bind to FcγRI but not to FcγRIIb, or bind to FcγRIIb but not to FcγRI) or enhanced binding to the complement system and selective binding to FcRn.

[000129] According to some embodiments of the present invention, the advantage of the biomimetics and compositions of the present invention is enhanced binding to components of the complement pathway, compared to innate immunoglobulin IgG1. The degree of enhanced binding to components of the complement pathway, relative to innate immunoglobulin IgG1, can in fact be quite significant, practically comparable to or superior to the binding of components of the complement pathway to aggregated IgG1, which may be present in the human body under certain circumstances. According to some embodiments of the present invention, the advantage of the biomimetics and compositions of the present invention is maintaining binding to components of the complement pathway, compared to innate immunoglobulin IgG1, with a decrease in binding to Fc receptors and other ligands.

[000130] According to some embodiments of the present invention, the advantage of the biomimetics and compositions of the present invention is preferential binding to the complement system, compared to binding to canonical Fc receptors, as observed for innate immunoglobulin IgG1, intravenous immunoglobulin (IVIG), human plasma enriched with immunoglobulin aggregates, or the parent biomimetic composition that does not contain point mutations. Such preferential binding may be characterized by, but is not limited to, enhanced association, reduced dissociation, avidity features, or a similar degree of binding at a lower concentration. In some embodiments of the present invention, the advantage of the biomimetics and compositions of the present invention is enhanced binding to the complement system, compared to binding to canonical Fc receptors observed for innate immunoglobulin IgG1, intravenous immunoglobulin (IVIG), human plasma enriched with immunoglobulin aggregates, or the parent composition of the biomimetic that does not contain point mutations. In some embodiments of the present invention, the biomimetics that bind to the complement system and compositions of the present invention inhibit complement-dependent cytotoxicity (CDC). In some embodiments of the present invention, the biomimetics that bind to the complement system and compositions of the present invention inhibit CDC with reduced binding or functionally absent binding to FcγRI and/or FcγRIIa and/or FcγRIIb and/or FcγRIII. In some embodiments of the present invention, the complement binding biomimetics and compositions of the present invention bind to the complement component(s) C1q and/or C4 and/or C4a and/or C3 and/or C3a and/or C5 and/or C5a. In some embodiments of the present invention, the complement binding biomimetics and compositions of the present invention bind to C3b. In some embodiments of the present invention, the complement binding biomimetics and compositions of the present invention bind to a complement molecule, such as C1q, C3 or C3b, preventing or reducing complement activation and preventing or reducing subsequent complement-mediated functional reactions such as cell lysis, inflammation or thrombosis. According to some embodiments of the present invention, the biomimetics and compositions of the present invention are associated with elevated levels of C4a, C3a and/or C5a, and said elevated levels are associated with anti-inflammatory or antithrombotic clinical profiles.

[000131] In some embodiments of the present invention, an additional advantage of the complement-binding biomimetics and compositions of the present invention is enhanced multimerization relative to intact immunoglobulins and/or the previously described complement-binding compositions. In another embodiment, an additional advantage of the complement-binding biomimetics and compositions of the present invention is reduced or absent binding to all or some canonical FcγRs. In some embodiments of the present invention, an additional advantage of the complement-binding biomimetics and compositions of the present invention is similar or enhanced binding to the complement system compared to intact immunoglobulins, enhanced multimerization, and reduced binding to one or more canonical FcγRs compared to intact immunoglobulins or a mutated parent compound. In other embodiments of the present invention, an additional advantage of the complement-binding biomimetics and compositions of the present invention is enhanced binding to the complement system, enhanced multimerization, and reduced binding to FcγRIIIa. In some embodiments of the present invention, the complement-binding biomimetics and compositions of the present invention exhibit preserved or enhanced binding to the complement system and retain binding to FcγRI, FcγRIIa, FcγRIIb, or FcγRIII. In one embodiment of the present invention, the complement-binding biomimetics and compositions of the present invention exhibit preserved or enhanced binding to the complement system and retain binding to the neonatal Fc receptor (FcRn), but not to any other canonical low affinity FcγR to any significant extent (e.g., the G1033 stradomer described herein). In one embodiment of the present invention, the complement-binding biomimetics and compositions of the present invention exhibit retained or enhanced binding to the complement system and retain binding to FcRn and FcγRI, but not to any activating low affinity canonical FcγR to any significant extent (e.g., the G994 and G998 stradomers described herein). In one embodiment of the present invention, the complement-binding biomimetics and compositions of the present invention exhibit retained or enhanced binding to the complement system and retain binding to FcγRI and/or FcγRIIb, but not to any other canonical FcγR (e.g., the G994 stradomer described herein). In one embodiment of the present invention, the complement-binding biomimetics and compositions of the present invention exhibit preserved or enhanced binding to the complement system and retain binding to FcRn and FcγRI, but have reduced binding to the low affinity activating receptors FcγRIIa and/or FcγRIIIa (e.g., stradomers G997, G1003, and G1022 described herein). In another embodiment of the present invention, the complement-binding biomimetics and compositions of the present invention exhibit preserved or enhanced binding to the complement system and retain binding to FcγRIIb, but have reduced binding to other canonical FcγRs comprising FcγRI, FcγRIIa, or FcγRIIIa (e.g., stradomers G996 and G1003 described herein).

[000132] According to another embodiment of the present invention, the complement-binding biomimetics and compositions of the present invention exhibit retained or enhanced binding to the complement system and retain binding to FcγRIIb but not to any other canonical low affinity FcγR (e.g., G994 and G998). Therefore, in one aspect, the biomimetics and compositions of the present invention have an advantage of retained or enhanced binding to the complement system with relatively reduced or absent binding to FcγR, or an advantage of binding to selected activating or inhibitory FcγRs, in each case relative to native non-aggregated IgG1 immunoglobulin and/or relative to the parent biomimetic. According to one embodiment of the present invention, the biomimetics and compositions of the present invention exhibit preserved or enhanced binding to the complement system, relative to the innate immunoglobulin IgG1 or the parent biomimetic.

[000133] According to one embodiment of the present invention, the biomimetics and compositions of the present invention may have modified effector functions, such as complement-dependent cytotoxicity, compared to innate immunoglobulin IgG1 or the parent biomimetic or composition. According to one embodiment of the present invention, the biomimetics and compositions of the present invention exhibit preserved or enhanced binding to molecules of the complement system, compared to innate immunoglobulin IgG1, IVIG, or the parent biomimetic or composition. According to another embodiment of the present invention, the biomimetics and compositions exhibit preserved or enhanced binding to C1q, compared to innate immunoglobulin IgG1, IVIG, or the parent biomimetic or composition. In some embodiments, the present invention provides biomimetics that are capable of binding to C1q, C4, C4a, C3, C3a, C3b, C5, or C5a to reduce or prevent subsequent functional reactions mediated by the complement system, such as complement-dependent cytotoxicity or cell lysis. In other embodiments, the present invention provides biomimetics that exhibit preserved or enhanced binding to C1q, as well as equivalent or enhanced CDC with altered binding to canonical Fc receptors, compared to innate immunoglobulin IgG1, IVIG, or the parent biomimetic.

[000134] According to one embodiment of the present invention, the biomimetics and compositions of the present invention exhibit preserved or enhanced binding to the complement system and similar binding to FcRn with reduced binding to one or more canonical Fc receptors, compared to innate immunoglobulin IgG1 or the parent biomimetic. According to one embodiment of the present invention, the biomimetics and compositions of the present invention exhibit preserved or enhanced binding to the complement system and a longer half-life, compared to innate immunoglobulin IgG1 or the parent biomimetic. According to one embodiment of the present invention, and without wishing to be bound by any particular theory, the inventors believe that the biomimetics of the present invention have a longer functional half-life than innate immunoglobulin IgG1 or the parent biomimetic, which is made possible by internalization by FcRn, providing a delayed release and a delayed or prolonged biological effect of the biomimetic. According to another embodiment of the present invention, the biomimetics and compositions of the present invention exhibit preserved or enhanced binding to the complement system and a shorter half-life than innate immunoglobulin IgG1 or the parent biomimetic. According to one embodiment of the present invention, the biomimetics and compositions of the present invention exhibit preserved or enhanced clearance of complement, compared to innate immunoglobulin IgG1 or the parent biomimetic. Therefore, in some embodiments, the present invention provides biomimetics with a reduced half-life compared to innate immunoglobulin IgG1, IVIG, or the parent biomimetic, which are additionally capable of binding to C1q or other components of the complement system to reduce or prevent membrane attack complex formation and subsequent complement-mediated functional reactions such as cell lysis.

[000135] The C1q component contains six heads linked by collagen-like rods to a central core (Reid KBM & Porter, RR Biochem. J. 155, 19-23 (1976)), and the isolated heads bind relatively weakly to the Fc portion of the antibody with an affinity of 100 μM (Hughes-Jones, NC & Gardner, B. Molec Immun. 16, 697-701 (1979)). Binding of the antibody to multiple epitopes on the antigen surface results in aggregation of the antibody. Such aggregation facilitates binding of several of the heads of the C1q molecule, resulting in an increased affinity of approximately 10 nM (Burton, DR Molec. Immun. 22, 161-206 (1985)). In contrast, the complement-preferential multimerizing stradomers and universal multimerizing stradomers of the present invention present a polyvalent Fc component of C1q, thereby providing avid binding of complement components to the stradomer unit comprising the multimerized stradomer, wherein the Fc preferentially binds to complement components compared to one or more canonical Fc receptors. Therefore, the complement-preferential multimerizing stradomers and universal stradomers of the present invention can exhibit preserved or enhanced binding affinity and/or avidity for C1q and act as a complement acceptor, even though said stradomers do not contain a Fab (and therefore do not contain the F _D portion of the Fc region) and cannot bind to multiple epitopes on the antigen surface, unlike aggregated antibodies. In one embodiment of the present invention, the multimerizing complement-binding preferential stradomers of the present invention present 4, 5, 6, 7, 8 or more functional Fcs to hexameric C1q, resulting in avidity binding with a low off-rate and functional inhibition of CDC. Similarly, the multimerizing complement-binding preferential stradomers and universal stradomers of the present invention may exhibit preserved or increased binding affinity and/or avidity for C4, C4a, C3, C3a, C3b, C5 or C5a, serving as a complement acceptor. Therefore, the biomimetics of the present invention, similar to the Fc region of IVIG aggregates or aggregated antibodies, can bind to complement components C1q, C4, C4a, C3, C3a, C3b, C5 or C5a, while the Fc region of intact isolated immunoglobulin has low binding affinity and lacks avidity for these complement components.

[000136] Specific single, double, or triple mutations at positions 267, 268, and/or 324 of the Fc domain of monoclonal antibodies, and in particular the S267E/H268F/S324T triple mutation, have been reported to increase the extent of binding of the monoclonal antibody to the complement system, compared to binding to the canonical Fcγ receptor or FcRn (Moore et al., MAbs 2; 181 (2010)), and it has previously been suggested that the increased binding to the complement system depends on prior binding of the antibody's Fab to the target antigen, followed by binding to C1q. However, the inventors of the present invention have unexpectedly found that a mutation at position S267E, H268F and/or S324T, including a triple mutation S267E/H268F/S324T, in relation to the multimerizing stradomer, unpredictably decreased binding to the complement system, increased binding to the complement system or selectively retained binding to the complement system, indicating that the described effects of these mutations in the monoclonal antibody cannot be predicted for their effect in relation to the multimerizing stradomer. Furthermore, in cases where unexpected binding to the complement system was demonstrated for the multimerizing stradomers of the present invention, this occurred despite the absence of Fab in the multimerizing stradomer. In one embodiment of the present invention, a complement-preferential multimerizing stradomer of the present invention binds to C1q independently of targeting of the Fab to an antigen. In one embodiment of the present invention, in contrast to a monoclonal antibody, a complement-preferential multimerizing stradomer of the present invention binds to C1q without the stradomer binding to a cell. Therefore, in one aspect, the present invention provides multimerizing stradomers comprising point mutations at positions S267E, H268F and/or S324T. Therefore, the present invention provides multimerizing stradomers comprising point mutations at positions S267E, H268F and/or S324T that effectively bind to C1q and inhibit CDC (e.g., 1102). However, in addition, the present invention also provides multimerizing stradomers containing point mutations at positions S267E, H268F and/or S324T that are incapable of effective binding to C1q or inhibition of CDC (e.g., G1106, G999, G1024, G1030, G1031, G1040, G1044 and G1048). Therefore, point mutations S267E, H268F and/or S324T that increase binding to C1q in a monoclonal antibody have an unpredictable effect on the multimerizing stradomer, in particular with respect to additional mutations. According to some embodiments, the present invention also provides multimerizing stradomers comprising point mutations at positions S267E, H268F, and S324T, as well as point mutations at one or more of positions 233, 234, 235, 236, 238, 265, 297, 299, 328, and/or an amino acid deletion at position 236.

[000137] According to one aspect, the present invention provides biomimetics that exhibit retained or enhanced binding to the complement system, wherein said biomimetics comprise an Fc domain comprising a point mutation at position 267 and/or 268 and/or 324. According to another embodiment of the present invention, the Fc region comprises one or more of the following point mutations: S267E and/or H268F and/or S324T. According to another embodiment of the present invention, the Fc region comprises the following point mutations: S267E, H268F, and S324T.

[000138] According to one embodiment of the present invention, a complement fixing biomimetic comprises an Fc domain, wherein said Fc domain comprises a point mutation at position 267 and/or 268 and/or 324, and wherein said Fc domain further comprises a point mutation at position 297, 298 and/or 299. Fc domains comprising a mutation at position 297, 298 and/or 299 are referred to herein as "aglycosylated mutated variants" or "non-glycosylation mutated variants" because the mutations at these positions alter the normal glycosylation pattern of IgG Fc. With respect to the monoclonal antibody in which said mutations were first characterized, the aglycosylated mutated variants reduce or eliminate the binding of normal immunoglobulin Fc to canonical FcγRs due to the altered glycosylation pattern. However, when applied to the multimerizing stradomer, the effects of these mutations are not predictable. In some multimerizing stradomers, FcγR binding is reduced compared to the parent biomimetic (e.g., G998). However, in some other multimerizing stradomers, FcγR binding is retained or enhanced in the aglycosylated mutated variants compared to the unmutated parent (e.g., G1068).

[000139] According to one embodiment of the present invention, a complement-binding biomimetic comprises an Fc domain, wherein said Fc domain comprises a point mutation at position 267 and/or 268 and/or 324, and wherein said Fc domain further comprises a point mutation at position 233 and/or 234 and/or 235 and/or 236 and/or 238 and/or 265 and/or 297 and/or 299.

[000140] In this application, the term “a” or “an,” when used in combination with the term “comprising” in the claims and/or description, may mean “one,” but this is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[000141] As used herein, the terms "biomimetic," "biomimetic molecule," "biomimetic compound," and related terms refer to a compound made by man that mimics the function of another compound, such as human intravenous immunoglobulin ("hIVIG"), a monoclonal antibody, or an Fc fragment of an antibody. "Biologically active" biomimetics are compounds that exhibit various biological activities that are analogous to or similar to those of natural analogs. The term "natural" refers to a molecule or portion thereof that is normally found in the body. The term "natural" also means substantially natural. "Immunologically active" biomimetics are biomimetics that exhibit immunological activity that is analogous to or similar to that of natural immunologically active molecules, such as antibodies, cytokines, interleukins, and other immunological molecules known in the art. In preferred embodiments, the biomimetics of the present invention are stradomers as defined herein. As used herein, the term "parent biomimetic" refers to unmutated biomimetics used as the basis for the compounds described herein (e.g., GL-2045 and G019).

[000142] As used herein, the term "complement" refers to any of the small proteins of the complement cascade, which are sometimes referred to in the literature as the complement system or the complement cascade. As used herein, the terms "complement fixation" or "complement binding" refer to the binding of any of the components of the complement system. The components of the complement cascade are known in the art and are described, for example, in Janeway's Immunobiology, ^8th Ed., Murphy ed., Garland Science, 2012. Three major complement pathways are currently known: the classical pathway, the alternative pathway, and the lectin binding pathway. The classical complement pathway is activated by binding of the C1q protein to one or more intact IgM immunoglobulin molecules or at least two intact IgG1, IgG2, or IgG3 immunoglobulin molecules. Activation of the complement system results in complement-dependent cytolysis. Excessive activation of the complement system can be detrimental and has been associated with several diseases, including myasthenia gravis, hemolytic uremic syndrome (HUS), and paroxysmal nocturnal hemoglobinuria (PNH). As used herein, the term "preferentially binding complement" refers to binding one or more components of the complement system to a greater extent than binding to other receptors (e.g., FcγR or FcRn). In some embodiments, the stradomers of the invention are preferentially binding complement stradomers. In some embodiments, the stradomers of the invention are universal stradomers. As used herein, the term "universal stradomers" refers to stradomers that bind to one or more components of the complement system and also bind to any of the canonical Fc receptors and/or the neonatal FcRn receptor.

[000143] As used herein, the terms "complement-mediated disease" and "complement-associated disease" refer to diseases and conditions in which the complement system plays a role. For example, complement-mediated diseases include diseases associated with defects in the activation of the complement system. According to some embodiments of the present invention, complement-mediated diseases can be treated, prevented, or reduced by inhibiting the complement cascade. Complement-related diseases are known in the art and include, but are not limited to, myasthenia gravis, hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), Shiga toxin-induced hemolytic uremic syndrome of E. coli (STEC-HUS), systemic thrombotic microangiopathy (TMA), paroxysmal nocturnal hemoglobinuria (PNH), neuromyelitis optica, relapsing neuromyelitis optica (NMO), antibody-mediated allograft rejection, Barraquer-Simons syndrome, asthma, lupus, autoimmune heart disease, multiple sclerosis, inflammatory bowel disease, ischemic reperfusion injury, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, spinal cord injury, macular degeneration associated with factor H (Y402H), age-related macular degeneration (AMD), hereditary angioedema and membranoproliferative glomerulonephritis (MPGN), membranous nephropathy, rheumatoid arthritis (RA), acute respiratory distress syndrome (ARDS), complement activation during cardiopulmonary bypass, dermatomyositis, pemphigus, lupus nephritis, glomerulonephritis and vasculitis, IgA-mediated nephropathy, acute renal failure, cryoglobulinemia, antiphospholipid antibody syndrome, uveitis, diabetic retinopathy, hemodialysis, chronic occlusive pulmonary distress syndrome (COPD), and aspiration pneumonia. Complement-related diseases may also include various other autoimmune, inflammatory, immunological, neurological, rheumatic, or infectious agent-associated diseases.

[000144] By "directly linked" is meant two sequences joined together without intervening or extraneous sequences, for example, amino acid sequences obtained by introducing restriction enzyme recognition sites into DNA or by cloning fragments. One skilled in the art will understand that the term "directly linked" includes the addition or deletion of amino acids as long as the ability to multimerize is not substantially altered.

[000145] The term "homologous" refers to full-length sequence identity of a particular nucleic acid or amino acid sequence. For example, "80% homology" means that a particular sequence is approximately 80% identical to a claimed sequence and may contain insertions, deletions, substitutions, and open reading frame shifts. One skilled in the art will appreciate that sequence alignment may be made to take into account insertions and deletions to determine full-length sequence identity.

[000146] As used herein, the numbering of residues in the IgG heavy chain is according to the EU numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), expressly incorporated herein by reference. "EU index according to Kabat" refers to the numbering of residues of a human IgG1 antibody according to the EU system. See Figures 36A and 36B.

[000147] There are two polymorphic forms of human IgG1, designated the DEL and EEM polymorphs. The DEL polymorph contains a D at position 356 and an L at position 358; the EEM polymorph contains an E at position 356 and an M at position 358 (numbering according to Kabat, see Figures 36A and 36B). Stradomers of the present invention may contain either the DEL or EEM polymorphic forms of IgG1. Therefore, even if a proposal for a particular mutated variant is explicitly given in relation to the DEL polymorph, one skilled in the art will recognize that the same mutations can be introduced into the EEM polymorph to obtain similar results. For example, compounds G994 and G998 were designed to include both the DEL and EEM polymorphs and then assessed for functional differences. No functional differences were observed. In particular, C1q binding and CDC inhibition were essentially the same for each polymorphic form of the respective compounds.

[000148] According to one embodiment of the present invention, the immunologically active biomimetics of the present invention are stradomers. The immunologically active compounds of the present invention are multimers of homodimers. According to one embodiment of the present invention, each homodimer has the ability to bind to complement. Therefore, in multimerized form, the immunologically active biomimetics comprise at least two homodimers, each of which has the ability to bind to the complement system.

[000149] The following subsections provide a definition of the building blocks of the biomimetics of the present invention, both structural and functional, and then provide a definition of the biomimetics themselves. However, it should first be noted that, as noted above, each of the biomimetics of the present invention comprises at least one Fc domain and one multimerization domain. Each of the Fc domain and the multimerization domain is at least a dimeric polypeptide (or a dimeric region of a larger polypeptide) that comprises two peptide chains or two arms (monomers) that associate to form a functional Fc receptor or complement binding site and a multimerization domain capable of multimerizing the resulting homodimer. Accordingly, the functional form of the individual fragments and domains discussed herein typically exists in a dimeric (or multimeric) form. The monomers of the individual fragments and domains discussed herein are single chains or arms that must associate with a second chain or arm to form a functional dimeric structure.

Fragment Fc

[000150] As used herein, the term "Fc fragment" is used to describe a region of a protein or a folded protein structure that is typically found at the carboxyl terminus of immunoglobulins. The Fc fragment can be isolated from the Fab fragment of a monoclonal antibody by enzymatic cleavage, such as with papain, but this process is incomplete and has disadvantages (see Mihaesco C and Seligmann M. Papain Digestion Fragments Of Human IgM Globulins. Journal of Experimental Medicine, Vol 127, 431-453 (1968)). When combined with the Fab fragment (containing the antigen-binding domain), the Fc fragment constitutes a holoantibody, i.e., a complete antibody as used herein. The Fc fragment consists of the carboxyl terminal regions of the heavy chains of the antibody. Each of the chains in an Fc fragment is approximately 220-265 amino acids in length, and the chains are often linked via a disulfide bond. An Fc fragment often contains one or more independent structural folds or functional subdomains. In particular, an Fc fragment comprises an Fc domain, defined herein as the minimal structure that binds to an Fcγ receptor. An isolated Fc fragment is comprised of two monomeric Fc fragments (e.g., two carboxy-terminal portions of antibody heavy chains; further defined herein) that are dimerized. Upon binding of two monomeric Fc fragments, the resulting Fc fragment is capable of binding to the complement system and/or an Fc receptor.

Incomplete Fc fragment

[000151] A "partial Fc fragment" is a domain that comprises a partial fragment of an Fc region of an antibody while retaining sufficient structure to exhibit activity similar to that of an Fc fragment, including activity with respect to binding to an Fc receptor and/or the complement system. In this regard, a partial Fc fragment may lack all or a portion of the hinge region, all or a portion of the CH2 domain, all or a portion of the CH3 domain, and/or all or a portion of the CH4 domain, depending on the antibody isotype from which the Fc domain is derived. Another example of a partial Fc fragment includes a molecule comprising the CH2 and CH3 domains of IgG1. In this example, the partial Fc fragment lacks the hinge domain present in IgG1. Partial Fc fragments are composed of two partial Fc fragment monomers. As further defined herein, upon binding of two of said partial Fc fragment monomers, the resulting partial Fc fragment is capable of binding to an Fc receptor and/or the complement system.

Fc domain

[000152] As used herein, the term "Fc domain" describes the minimal region (with respect to a larger polypeptide) or the smallest folded structure of a protein (with respect to an isolated protein) that can bind or be bound by an Fc receptor (FcR). In an Fc fragment and a partial Fc fragment, the Fc domain is the minimal binding site that allows the molecule to bind to an Fc receptor. While an Fc domain may be limited to a discrete homodimeric polypeptide that binds an Fc receptor, it is understood that an Fc domain may be a portion or an entire Fc fragment, as well as a portion or an entire partial Fc fragment. When using the term "Fc domains" herein, one skilled in the art will recognize that the term refers to more than one Fc domain. An Fc domain is composed of two Fc domain monomers. As further defined herein, the Fc domain formed by the association of two of said Fc domain monomers is capable of binding to an Fc receptor and/or binding to the complement system. Accordingly, the Fc domain is a dimeric structure that can bind to complement and/or an Fc receptor. The stradomers described herein comprise an Fc domain containing one or more mutations that alter the ability of the stradomer to bind to complement and/or an Fc receptor.

Incomplete Fc domain

[000153] As used herein, the term "partial Fc domain" describes a portion of an Fc domain. Partial Fc domains comprise individual heavy chain constant region domains (e.g., CH1, CH2, CH3, and CH4 domains) and hinge regions of various immunoglobulin classes and subclasses. Accordingly, the human partial Fc domains of the present invention comprise an IgG1 CH1 domain, an IgG1 CH2 domain, an IgG1 CH3 domain, and IgG1 and IgG2 hinge regions. The corresponding partial Fc domains in other species will depend on the immunoglobulins present in those species and their name. Preferably, the partial Fc domains of the present invention comprise the IgG1 CH1, CH2, and hinge regions and the IgG2 hinge region. A partial Fc domain of the present invention may further comprise a combination of more than one of these domains and hinge regions. However, individual partial Fc domains of the present invention and combinations thereof do not have the ability to bind to an FcR. In this regard, partial Fc domains and combinations thereof comprise a partial Fc domain sequence. Partial Fc domains can be linked together to form a peptide that is capable of binding to the complement system and/or an Fc receptor, thereby forming an Fc domain. In the present invention, partial Fc domains are used together with Fc domains as building blocks to create the biomimetics of the present invention, as defined herein. Each partial Fc domain is composed of two partial Fc domain monomers. The association of these two partial Fc domain monomers results in the formation of a partial Fc domain.

[000154] As noted above, each of the Fc fragments, partial Fc fragments, and partial Fc domains is a dimeric protein or domain. Thus, each of these molecules consists of two monomers that join to form a dimeric protein or domain. Although the characteristics and activities of the homodimeric forms have been described above, monomeric peptides are discussed below.

Fc fragment monomer

[000155] As used herein, an "Fc fragment monomer" is a single protein chain that, when associated with another Fc fragment monomer, forms an Fc fragment. An Fc fragment monomer, therefore, is the carboxy-terminal portion of one of the antibody heavy chains that comprise the Fc fragment of a holoantibody (e.g., a contiguous portion of a heavy chain that comprises a hinge region, a CH2 domain, and a CH3 domain of an IgG). In one embodiment, an Fc fragment monomer comprises at least one hinge region chain (a hinge monomer), one CH2 domain chain (a CH2 domain monomer), and one CH3 domain chain (a CH3 domain monomer), which are joined directly to form a peptide. In one embodiment, the CH2, CH3, and hinge domains are derived from different isotypes. In a specific embodiment, the Fc fragment monomer comprises the hinge domain of IgG2 and the CH2 and CH3 domains of IgG1.

Fc domain monomers

[000156] As used herein, the term "Fc domain monomer" describes a single-chain protein that, when joined with another Fc domain monomer, forms an Fc domain that can bind to the complement system. The joining of two Fc domain monomers results in the formation of a single Fc domain.

[000157] In one embodiment of the present invention, the Fc domain monomers of the present invention do not contain extraneous sequences, unlike the Fc domain monomers previously described in WO 2008/151088. In contrast, the Fc domain monomers of the present invention are linked directly with a leader sequence (e.g., SEQ ID NO: 1) at one end (e.g., the N-terminus of the Fc monomer) and a multimerization domain (e.g., SEQ ID NO: 4, 5, or 6) at the other end (e.g., the C-terminus of the Fc monomer). One skilled in the art will appreciate that, although the constructs are designed with a leader sequence, the sequence is subsequently cleaved. Accordingly, in preferred embodiments of the present invention, the mature protein does not contain a leader sequence.

[000158] According to one embodiment of the present invention, an Fc domain monomer comprises, in amino- to carboxyl-terminal direction, an Fc domain comprising an IgG1 hinge region, IgG1 CH2, and IgG1 CH3, and an IgG2 hinge region (G045c base), wherein said monomer comprises one or more point mutations in the Fc domain. According to another embodiment of the present invention, an Fc domain monomer comprises, in amino- to carboxyl-terminal direction, an Fc domain comprising an IgG2 hinge region, an IgG1 hinge region, IgG1 CH2, and IgG1 CH3 (G019 base), wherein said Fc monomer comprises one or more point mutations in the Fc domain. According to another embodiment of the present invention, an Fc domain monomer comprises, in the amino to carboxyl terminus direction, an Fc domain comprising an IgG2 hinge region, an IgG1 CH2, and an IgG1 CH3 (base G051), wherein said Fc monomer comprises one or more point mutations in the Fc domain.

Stradomometers

[000159] In particular embodiments of the present invention, the biomimetics of the present invention include stradomers. The stradomers of the present invention are biomimetic compounds capable of binding to complement that preferably exhibit significantly improved binding to the complement system. In a preferred embodiment, the stradomers of the present invention exhibit an increased degree of binding to the complement system compared to binding to one or more canonical Fc receptors. A stradomer can have four different physical conformations: sequential, cluster, core, or Fc fragment. As will be appreciated, the Fc fragments, partial Fc fragments, Fc domains, and partial Fc domains described above can be used to construct different conformations of stradomers. In addition, there are individual Fc domain monomers and partial Fc domain monomers, also discussed above, which are produced first by forming homodimeric stradomer units and then multimerize by incorporation of a multimerization domain (e.g., an IgG2 hinge region) to produce multimeric structures that are clustered stradomers according to the present invention. Specific stradomers are described in detail in WO 2008/151088 and WO 2012/016073, the contents of which are incorporated herein by reference in their entirety. The degree of binding to the Fcγ receptor and/or FcRn can be further increased by additional mutations at one or more positions 233 and/or 234 and/or 235 and/or 236 and/or 238 and/or 265 and/or 297 and/or 299.

Stradomer block monomer

[000160] As used herein, the term "stradomer unit monomer" refers to a single, continuous peptide molecule that, when linked to at least a second stradomer unit monomer, forms a homodimeric stradomer unit comprising at least one Fc domain. While in preferred embodiments, stradomer units are comprised of two linked stradomer monomers, a stradomer may also comprise three or more stradomer monomers.

[000161] A monomer of a stradomer unit may comprise an amino acid sequence that will form one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more Fc domains when linked to another monomer of a stradomer unit to form a "stradomer unit." The monomer of a stradomer unit may further comprise an amino acid sequence that will form one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more partial Fc domains when linked to another monomer of a stradomer unit to form a stradomer unit.

[000162] The regions of the stradomer block monomers that will form the Fc domains and partial Fc domains, with respect to the stradomer block, may be arranged in a carboxy-terminal to amino-terminal direction of successive portions of the stradomer block monomer molecule. The arrangement of the individual Fc domain monomers and the partial Fc domain monomers comprising the stradomer block monomer is not critical. However, the arrangement should allow for the formation of two functional Fc domains when two stradomer block monomers are joined. In one embodiment, a stradomer of the present invention comprises a direct linkage between the N-terminus of an IgG1 Fc monomer and the C-terminus of the leader peptide (SEQ ID NO: 1), and the C-terminus of an IgG1 Fc region and the N-terminus of the IgG2 hinge region multimerization domain (SEQ ID NO: 4).

[000163] As an illustrative example, one skilled in the art will appreciate that the stradomer molecules of the present invention containing the said point mutations can be constructed by obtaining a polynucleotide molecule that encodes an Fc domain monomer with the desired point mutations and a multimerization region. The said polynucleotide molecule can be inserted into an expression vector that can be used to transform a population of bacteria or transfect a population of mammalian cells. Stradomer block monomers can then be obtained by culturing the transformed bacteria or transfecting the mammalian cells under appropriate culture conditions. For example, a clonal cell line extending a pool of stably transfected cells can be obtained by selecting cells using geneticin/G418. In another embodiment, the cells can be transfected with DNA encoding a stradomer of the present invention (e.g., DNA encoding a stradomer according to any of the sequences set forth in SEQ ID NOs: 10-77) under the control of a CMV promoter. The expressed stradomer monomers can then form functional stradomer blocks and stradomers by self-aggregation of stradomer monomers or blocks or by joining stradomer monomers using linkages between stradomer monomers. The expressed stradomers can then be purified from the cell culture medium by affinity chromatography using, for example, protein A or protein G columns. One skilled in the art will appreciate that the leader peptide included in the nucleic acid construct is used only to facilitate the production of stradomer block monomer peptides and is cleaved upon expression of the mature protein. Therefore, the biologically active biomimetics according to the present invention do not contain a leader peptide.

Cluster Stradomer

[000164] According to one embodiment of the present invention, the stradomer used in accordance with the present invention is a cluster stradomer. A "cluster stradomer" is a biomimetic that has a radial shape with a central "head" portion and two or more "legs" in which each leg contains one or more Fc domains that are capable of binding to at least one Fc gamma receptor and/or the complement system. A cluster stradomer is also known as a "multimerizing stradomer" due to the presence of a multimerization domain, which results in multimerization of the stradomer. Therefore, consecutive stradomers that contain multiple Fc domains on a single stradomer monomer molecule can still be classified as a cluster stradomer or a multimerizing stradomer as long as the molecule also contains at least one multimerization domain. Each cluster stradomer consists of more than one homodimeric protein, each of which is called a "cluster stradomer unit". Each cluster stradomer unit consists of at least one region that multimerizes and a "stalk" region that contains at least one functional Fc domain. The multimerizing region creates the "head" of the cluster stradomer after multimerization with another cluster stradomer unit. The stalk region can be capable of binding to a number of complement molecules that corresponds to the number of Fc domains in each stalk. For example, the stalk region can bind to a number of C1q molecules that corresponds to the number of Fc domains in each stalk region. Therefore, a cluster stradomer is a biomimetic compound capable of binding to two or more C1q molecules, thereby preventing subsequent complement-mediated lysis reactions. Particularly active biomimetics according to the present invention can bind all six heads of the C1q molecule.

[000165] The multimerization region may be a peptide sequence that causes further multimerization of dimeric proteins or, alternatively, the multimerization region may be glycosylated, which enhances multimerization of dimeric proteins. Examples of peptide multimerization regions include the IgG2 hinge region, the IgE CH2 domain, the isoleucine zipper, the collagen glycine-proline-proline ("GPP") repeat, and zinc fingers. Changes in glycosylation, whether resulting from amino acid substitution or culture conditions, may also affect the multimerization of the biomimetics of the present invention. The influence of glycosylation on peptide multimerization is well described in the art (e.g., Role of Carbohydrate in Multimeric Structure of Factor VIII/V on Willebrand Factor Protein. Harvey R. Gralnick, Sybil B. Williams and Margaret E. Rick. Proceedings of the National Academy of Sciences of the United States of America, Vol. 80, No. 9, [Part 1 : Biological Sciences] (May 1, 1983), pp. 2771-277 '4; Multimerization and collagen binding of vitronectin is modulated by its glycosylation. Kimie Asanuma, Fumio Arisaka and Haruko Ogawa. International Congress Series Volume 1223, December 2001, Pages 97-101).

[000166] The multimerizing region may be a peptide sequence that induces dimerization or multimerization of peptides and contains the IgG2 hinge region, the IgE CH2 domain, the isoleucine zipper, the collagen GPP, and the zinc finger. It is well known that the human IgG2 hinge region can form covalent dimers (Yoo, E. M. et al. J. Immunol. 170, 3134-3138 (2003); Salfeld Nature Biotech. 25, 1369-1372 (2007)). IgG2 dimer formation is potentially mediated by the structure of the IgG2 hinge region via C-C bonds (Yoo et al 2003), suggesting that the hinge structure alone can mediate dimer formation. However, the number of IgG2 dimers detected in human serum is limited. The amount of IgG2 existing as a dimer of homodimers is estimated to be less than 10% of total IgG2 (Yoo et al. 2003). Moreover, there are no quantitative data on the multimerization of IgG2 beyond the dimer of homodimers (Yoo et al. 2003). In other words, native IgG2 has not been shown to form higher order multimers in human serum. The IgG2 hinge region-containing stradomers (i.e., G045c and G019, described in WO 2012/016073) are present in highly ordered multimers and, in contrast to native IgG2 in human serum, in which IgG2 hinge region interactions are variable and dynamic, G045c has been shown to form highly stable multimers, as confirmed by non-reducing SDS-PPAG electrophoresis, analytical ultracentrifugation, and 3-month stability studies at 100% humidity and 37°C. Furthermore, it is also surprising that the amount of multimers in the IgG2 hinge region-containing stradomer preparations is significantly higher than the approximately 10% of IgG2 dimers in human serum, whereas no IgG2 multimers have been detected in human serum. For example, the percentage of stradomers that preferentially bind to the complement system that are multimers, including dimers, trimers, tetramers, and higher order multimers of homodimers, exceeds 20% and may exceed 30%, 40%, 50%, 60%, 70%, 80%, or even 90%.

[000167] The amino acid sequence of a human IgG2 hinge monomer is ERKCCVECPPCP (SEQ ID NO: 4). Mutation of any one of the 4 cysteines in SEQ ID NO: 4 can be associated with significantly reduced stradomer multimerization. The IgG2 hinge monomer comprises two C-X-X-C regions. Therefore, the stradomer monomers of the present invention can comprise the entire 12 amino acid IgG2 hinge monomer sequence or any or both of the four amino acid core regions, along with Fc domain monomers. While the X-X of the core structures can be any amino acid, in a preferred embodiment the X-X sequence is V-E or P-P. One skilled in the art will appreciate that the IgG2 hinge region monomer may consist of any portion of the hinge region sequence, in addition to the four amino acid core structures, including the entire IgG2 hinge region sequence and some or all of the CH2 and CH3 domain monomer sequences of IgG2. Without wishing to be bound by any theory, the inventors believe that the multimerization domain of the IgG2 hinge region can form multimers by interacting with any portion of the stradomer monomer. That is, the IgG2 hinge region of one stradomer monomer can associate with the IgG2 hinge region of another stradomer monomer, thereby forming a dimer from a homodimer or higher order multimers while maintaining enhanced functional binding to Fc receptors compared to the native IgG1 Fc region. In another embodiment, the IgG2 hinge region domain of one stradomer monomer may bind to the IgG1 hinge region of another stradomer monomer, thereby forming a dimer from the homodimer or higher order multimers while maintaining increased functional binding to Fc receptors compared to the native IgG1 Fc region. It is also possible that the IgG2 hinge region of one stradomer monomer binds to another portion of the IgG1 Fc domain, i.e., the CH2 or CH3 domain of another stradomer monomer, thereby forming a dimer from the homodimers or higher order multimers while maintaining increased functional binding to Fc receptors compared to the native IgG1 Fc region.

[000168] Leucine and isoleucine zippers can also be used as a multimerizing region. Leucine and isoleucine zippers (coiled coiled coil domains) are known to facilitate the formation of protein dimers, trimers, and tetramers (Harbury et al. Science 262:1401-1407 (1993); O'Shea et al. Science 243:538 (1989)). Clustered stradomers can be generated by exploiting the natural tendency of the isoleucine zipper to form a trimer.

[000169] Although one skilled in the art will appreciate that various types of leucine and isoleucine zippers can be used, a preferred embodiment utilizes an isoleucine zipper from the transcriptional regulator GCN4, modified as described in the literature (Morris et al., Mol. Immunol. 44:3112-3121 (2007); Harbury et al. Science 262:1401-1407 (1993)): GGGS IKQIEDKIEEILSKIYHIENEIARIKKL IGERGHGGG (SEQ ID NO: 5). This isoleucine zipper sequence is only one of several possible sequences that can be used to multimerize Fc domain monomers. Although the entire sequence as set forth in SEQ ID NO: 5 may be used, the underlined portion of the sequence is the core sequence of the isoleucine zipper that may be used in the cluster stradomer of the present invention. Therefore, the monomers of the stradomers of the present invention may comprise the entire amino acid sequence of the isoleucine zipper or the 28 amino acid core sequence, along with one or more Fc domain monomers. One skilled in the art will also appreciate that the isoleucine zipper may consist of any portion of the zipper in addition to the 28 amino acid core structure and may therefore comprise more than 28 amino acids but not the entire sequence.

[000170] GPP is an amino acid sequence found in human collagen that causes collagen protein:protein binding. While one skilled in the art will appreciate that various types of GPP repeats can be used as the multimerization domain, a preferred embodiment utilizes the glycine-proline-proline repeat described in the literature (Fan et al FASEB Journal 3796 vol. 22 2008) (SEQ ID NO: 6). This glycine-proline-proline repeat sequence is only one of several possible sequences that can be used to multimerize Fc domain monomers. While the entire sequence set forth in SEQ ID NO: 6 can be used, repeats of varying lengths can also be used to multimerize Fc domain monomers. Similarly, repeats containing different amino acids in the GPP repeats can also be substituted.

[000171] It should be understood that the stradomers and other biomimetic molecules described herein can be derived from any of a variety of species, including humans. Indeed, the Fc domains, or partial Fc domains, of any of the biomimetic molecules of the present invention can be derived from immunoglobulin from more than one (e.g., two, three, four, five, or more) species. However, they will typically be derived from a single species. It should also be understood that any of the methods described herein (e.g., methods of treatment) can be administered to any species. Typically, all components of a biomimetic administered to a species of interest will be derived from that species. However, biomimetics in which all components are derived from different species or from more than one species (including and excluding the species in which the method is administered) can also be used.

[000172] The particular CH1, CH2, CH3, and CH4 domains and hinge regions that comprise the Fc domains and partial Fc domains of the stradomers and other biomimetics of the present invention may be independently selected based on the immunoglobulin subclass as well as the organism from which they are derived. Accordingly, the stradomers and other biomimetics disclosed herein may comprise Fc domains and partial Fc domains that are independently derived from different types of immunoglobulins, such as human IgG1, IgG2, IgG3, IgG4, IgA, IgA1, IgD, IgE, and IgM, mouse IgG2a, or dog IgA or IgB. Similarly, each Fc domain and partial Fc domain can be obtained from a variety of species, preferably mammalian species, including non-human primates (e.g., monkeys, baboons, and chimpanzees), humans, mice, rats, cattle, horses, cats, dogs, pigs, rabbits, goats, sheep, deer, ferrets, rodents, guinea pigs, hamsters, bats, birds (e.g., chickens, turkeys, and ducks), fish, and reptiles, to produce species-specific or chimeric stradomer molecules.

[000173] Individual Fc domains and partial Fc domains may also be humanized. One skilled in the art will appreciate that different Fc regions and partial Fc domains will provide different types of functionality. For example, FcRn binds specifically to IgG immunoglobulins, but binds poorly to other classes of immunoglobulins. One skilled in the art will also appreciate that various adverse effects may be associated with the use of certain Ig domains, such as anaphylaxis associated with IgA infusions. The biomimetics disclosed herein will generally be designed to avoid these effects, although such effects may be desirable under certain circumstances.

[000174] Also included within the scope of the present invention are stradomers comprising Fc domains and partial Fc domains comprising amino acids that differ from the naturally occurring amino acid sequences of an Fc domain or partial Fc domain. Preferred Fc domains for inclusion in the biomimetic compounds of the present invention have measurable specific binding affinity for the complement system. Primary amino acid sequences and structures of numerous Fc domains and Fc domain monomers obtained by X-ray crystallography are available in the art. See, for example, Woof JM, Burton DR. Human antibody-Fc receptor interactions illuminated by crystal structures. Nat Rev Immunol. 2004 Feb;4(2):89-99. Exemplary Fc domains capable of binding to Fcγ receptors include the Fc domains from human IgG1 (SEQ ID NO: 2 or 3). These native sequences have been subjected to extensive structural and functional studies, including functional sequence mapping using site-directed mutagenesis. Based on the results of these preliminary structural and functional studies and available crystallographic data, one skilled in the art can design functional variants of the Fc domain sequence while maintaining the ability to bind to the complement system. For example, cysteine residues can be added to enhance disulfide bond formation between monomers or removed to alter the interaction between stradomer homodimers. In addition, one skilled in the art can design functional variants of the Fc domain sequence while maintaining increased ability to bind to the complement system, or can design functional variants of the Fc domain sequence with even greater ability to bind to the complement system.

[000175] Amino acid substitutions may be found throughout the Fc domain sequence or may be defined for specific partial Fc domains that comprise the Fc domain. Functional Fc domain variants used in stradomers and other biomimetics of the present invention will have at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a native Fc domain. Similarly, functional variants of partial Fc domains used in stradomers and other biomimetics according to the present invention will have at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a native partial Fc domain.

[000176] One skilled in the art will appreciate that the present invention also includes the use of functional variants of Fc domain monomers in the construction of Fc fragment monomers, partial Fc fragment monomers, stradomer monomers, and other monomers of the present invention. Functional variants of Fc domain monomers will have at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a native Fc domain monomer.

[000177] Similarly, the present invention also includes within its scope the use of functional variants of partial Fc domain monomers in the construction of Fc fragment monomers, partial Fc fragment monomers, Fc domain monomers, stradomer monomers, and other monomers of the present invention. The functional variants of partial Fc domain monomers will have at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a native partial Fc domain monomer sequence.

[000178] Amino acid substitutions may decrease, increase, or leave unchanged the binding affinity of the stradomer for FcRn or canonical Fcγ receptors. Preferably, such amino acid changes will be conservative amino acid substitutions, however, suitable changes include deletions, additions, and other substitutions. Conservative amino acid substitutions typically include changes within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine, and threonine; lysine, histidine, and arginine; and phenylalanine and tyrosine. Additionally, an amino acid substitution may enhance the strength of multimerization, such as by adding cysteine residues.

[000179] As used herein, the term "functional variant" refers to a sequence that is homologous to a reference sequence that is capable of mediating the same biological effects as the reference sequence (in the case of a polypeptide), or that encodes a polypeptide that is capable of mediating the same biological effects as the polypeptide encoded by the reference sequence (in the case of a polynucleotide). For example, a functional variant of any of the biomimetics described herein will have a certain degree of homology or identity, and will be capable of immunomodulating monocytes or APCs. Functional sequence variants include polynucleotides and polypeptides. Sequence identity is typically assessed using BLAST 2.0 (Basic Local Alignment Search Tool) using default parameters: Filter - enabled, BLOSUM62 scoring matrix, word size -3, E value - 10, gap penalty - 11.1, and alignments - 50. According to some embodiments of the present invention, a functional variant comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to an amino acid sequence provided herein.

[000180] In addition to the amino acid sequence composition of native Fc domains, the carbohydrate content of the Fc region is known to play an important role in Fc domain structure. See, e.g., Robert L. Shields, et al. Lack of Fucose on Human IgG1 N-Linked Oligosaccharide Improves Binding to Human FcγRIII and Antibody-dependent Cellular Toxicity. J. Biol. Chem., Jul 2002; 277: 26733 - 26740 (doi:10.1074/jbc.M202069200); Ann Wright and Sherie L. Morrison. Effect of C2- Associated Carbohydrate Structure on Ig Effector Function: Studies with Chimeric Mouse-Human IgG1 Antibodies in Glycosylation Mutants of Chinese Hamster Ovary Cells. J. Immunol, Apr 1998; 160: 3393-3402. The carbohydrate content can be controlled using, for example, specific protein expression systems, including specific cell lines or enzymatic modification in vitro. Therefore, stradomers comprising Fc domains with the native carbohydrate content of the holoantibody from which the domains were derived, as well as biomimetic compounds with altered carbohydrate content, are included within the scope of the present invention. In another embodiment of the present invention, the multimeric components of a stradomer are characterized by a different glycosylation pattern compared to the homodimeric component of a similar stradomer. In one embodiment, a complement-binding stradomer comprises mutations that do not alter the glycosylation pattern, but reduce or eliminate binding to canonical Fc receptors and/or binding to the FcRn receptor.

Preferred embodiments of stradomers that preferentially bind to the complement system

[000181] The complement-binding preferential stradomers described herein provide an enhanced degree of binding to the complement system compared to binding to Fc receptors. Accordingly, the complement-binding preferential stradomers described herein are referred to in some embodiments as "complement-binding preferential stradomers" or "complement-binding stradomers." In some embodiments, the stradomers of the present invention are "universal stradomers" that bind to one or more components of the complement cascade and also bind to any of the FcγRs.

[000182] A G045c-based stradomer containing point mutations at positions 267, 268, and 324 (SEQ ID NO: 13) is referred to herein as G997. In some embodiments of the present invention, G997 unexpectedly exhibits strong binding to C1q and inhibition of CDC, while maintaining binding to FcγRI, the inhibitory receptor FcγRII, and FcRn, and significantly reduced binding to the activating receptors FcγRIIa and FcγRIIIa.

[000183] A G045c-based stradomer containing point mutations at positions 267, 268, 297, and 324 (SEQ ID NO: 14 or 15) is designated herein as G998. The multimerizing stradomer containing the FcγN297A mutation (but not containing the mutations at position 267, 268, or 324) binds to C1q with low affinity and avidity, exhibits moderate inhibition of CDC, and has no detectable binding to FcγRIIa, FcγRIIb, or FcγRIIIa. In contrast, the present inventors have found the surprising fact that the introduction of additional Fc mutations S267E, H268F and/or S324T, applied to the GL-2045-based stradomer containing the N297A mutation (stradomer designated G998), further significantly enhances binding to C1q and inhibition of CDC. Even more surprisingly, the resulting compound binds strongly to FcγRIIb. Therefore, in some embodiments, G998 unexpectedly exhibits even stronger C1q binding and CDC inhibition than the parent G045c stradomer, and also fully retains FcRn binding and substantially retains FcγRI binding compared to the parent G045c, and also partially retains binding to the inhibitory receptor FcγRIIb with significantly reduced binding to the activating receptors FcγRIIa and FcγRIIIa. To highlight the unpredictability of these mutations, when a series of similar mutations are introduced into the stradomer with an N-terminal multimerization domain (G019), compared to the stradomer with a C-terminal multimerization domain (GL-2045), this preferential binding to C1q is completely abolished (see compound G999). In some embodiments, the present invention provides a stradomer comprising point mutations at positions 267, 268, 297, and 324, and further comprising mutations at positions 253, 310, and 435. In other embodiments, the present invention provides stradomers comprising mutations at positions 233, 234, 235, 253, 267, 268, 297, 299, 310, 324, and 435, and an amino acid deletion at position G236. Thus, in some embodiments, the present invention provides a stradomer comprising the following mutations: I253A, S267E, H268F, N297A, H310A, S324T, and H435A; or a stradomer comprising the following mutations: E233P, L234V, L235A, I253A, S267E, H268F, N297A, T299A, H310A, S324T, and H435A, as well as a G236 deletion. According to some embodiments of the present invention, the resulting stradomers retain binding to C1q and inhibition of CDC similar to the stradomer according to the sequences presented in SEQ ID NO: 14 or 15, and exhibit reduced binding to FcγRIIb and/or FcγRI, compared to the stradomer according to the sequences presented in SEQ ID NO: 14 or 15.

[000184] A G045c-based stradomer comprising point mutations at positions 234, 235, 267, 268, 297, and 324 (SEQ ID NO: 21 or 22) is referred to herein as G1033. In some embodiments of the present invention, G1033 (e.g., with respect to the N297A and L234A/L235A mutations) unexpectedly retains some degree of binding to FcγRI and FcγRIIa, in addition to strong binding to C1q and inhibition of CDC.

[000185] A G045c-based stradomer containing point mutations at positions 233, 236, 267, 268, and 324 (SEQ ID NO: 10 or 11) is designated herein as G994. In some embodiments of the present invention, G994 retains strong binding to FcγRI and FcRn and minimal binding to FcγRIIb, while not exhibiting significant binding to the activating receptors FcγRIIa and FcγRIIIa. These results were particularly unexpected based on the data in Shields et al. (Shields, et al. J. Biol. Chem., 276(9):6591 (2001)), which reported that mutations at positions 233 or 236 resulted in abolition of binding to FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcRn. These results also highlight the unpredictability of this point mutation in the stradomer. A multimerizing stradomer containing the FcE233P and G236R mutations (but not the mutation at position 267, 268, or 324) binds C1q with low affinity and avidity, but does not significantly inhibit CDC and does not exhibit significant binding to FcγRIIa, FcγRIIb, or FcγRIIIa. In contrast, the present inventors have unexpectedly discovered that introducing additional Fc mutations S267E, H268F and/or S324T into a stradomer containing the E233P and G236R mutations (stradomer designated G994) further enhances C1q binding and CDC inhibition while maintaining some degree of FcγRIIb binding.

[000186] A G045c-based stradomer comprising point mutations at positions 233, 234, 235, 267, 268, 297, and 324 and a deletion of G236 (SEQ ID NO: 19) is designated herein as G1022. In some embodiments of the invention, G1022 unexpectedly binds to C1q, inhibits CDC, and retains strong binding to FcγRIIa and weak binding to FcγRIIb, but unexpectedly does not retain binding to high affinity FcγRI.

[000187] A G045c-based stradomer containing point mutations at positions 234, 235, 267, 268, and 324 (SEQ ID NO: 63) is referred to herein as G1032. In some embodiments of the present invention, G1032 unexpectedly binds to C1q and inhibits CDC while maintaining strong binding to all canonical Fc receptors and exhibits moderately strong binding to FcRn.

[000188] A G045c-based stradomer containing point mutations at positions 233, 234, 235, 267, 268, and 324 and a deletion of G236 (SEQ ID NO: 62) is designated herein as G1023. In some embodiments of the present invention, G1023 unexpectedly binds to C1q and inhibits CDC while maintaining strong binding to all canonical Fc receptors.

[000189] A G045c-based stradomer containing point mutations at positions 265, 267, 268, and 324 (SEQ ID NO: 18) is referred to herein as G1006. In some embodiments of the present invention, G1006 unexpectedly binds to C1q and inhibits CDC while maintaining strong binding to FcRn and FcγRI and moderate binding to FcγRIIa, despite the presence of a mutation at position 265 that would be expected to reduce binding to all canonical Fc receptors.

[000190] A G045c-based stradomer comprising point mutations at positions 238, 267, 268, 297, and 324 (SEQ ID NO: 20) is referred to herein as G1027. In some embodiments, G1027 unexpectedly binds to C1q and inhibits CDC, but retains the ability to bind to a canonical Fc receptor despite the mutations at positions 238 and 297, and retains strong binding to FcγRI and FcRn and weak binding to FcγRIIa and FcγRIIb.

[000191] A G045c-based stradomer containing point mutations at positions 236, 267, 268, and 324 (SEQ ID NO: 12) is referred to herein as G996. In some embodiments of the present invention, G996 unexpectedly binds to FcγRI, FcγRIIa, and FcγRIIb, in addition to binding to C1q and inhibiting CDC. Also unexpectedly, G996 did not bind to canonical receptors in mice, making it an ideal compound for assessing true CDC inhibition in rodents. In contrast to G996, a multimerizing stradomer containing the Fc G236R mutation (but not the mutation at position 267, 268, or 324), designated G990, binds to C1q with low affinity and avidity but does not significantly inhibit CDC. G990 also does not exhibit significant binding to FcγRIIa, FcγRIIb, or FcγRIIIa. The inventors have unexpectedly found that introducing additional Fc mutations S267E, H268F, and/or S324T into G990 further enhances C1q binding and CDC inhibition. Even more surprisingly, G996 binds to FcγRIIb with high avidity.

[000192] A G045c-based stradomer containing point mutations at positions 233, 236, 267, 268, 324, and 328 (SEQ ID NO: 23) is referred to herein as G1042. In some embodiments of the present invention, G1042 unexpectedly binds tightly to FcγRI, FcγRIIa, and FcγRIIb. Therefore, the addition of a mutation at position 328 (comparing G1042 with G994 containing point mutations at positions 233, 236, 267, 268 and 324) unexpectedly resulted in strong binding to both receptors, FcγRIIa and FcγRIIb, despite the fact that the mutation at position 328 was expected to increase binding only to FcγRIIb.

[000193] A G045c-based stradomer comprising the point mutations P238D, D265G, S267E, H268F, and S324T (SEQ ID NO: 24) is referred to herein as G1043. In some embodiments of the present invention, G1043 unexpectedly binds strongly to FcγRIIa, FcγRIIb, and FcγRI, despite the fact that mutations at positions 238 and 265 would be expected to reduce binding to FcγRIIa and FcγRI.

[000194] A G045c-based stradomer containing the point mutations P238D, D265W, S267E, H268F, and S324T (SEQ ID NO: 25) is referred to herein as G1046. In some embodiments of the present invention, G1046 unexpectedly binds strongly to FcγRI and also exhibits increased binding to FcγRIIa. This result was unexpected because the mutation at position 238 was expected to increase binding to FcγRIIb but not binding to FcγRIIa.

[000195] A G045c-based stradomer containing point mutations at positions 233, 234, 235, 267, 268, 297, 324, and 328, and an amino acid deletion at position 236 (SEQ ID NO: 26) is designated herein as G1050. In some embodiments of the present invention, G1050 binds strongly to FcγRIIa and FcγRIIb. Surprisingly, binding to low-affinity FcγRII was retained, while binding to high-affinity FcγRI was absent.

[000196] A G045c-based stradomer containing the point mutations P238D, S267E, H268F, and S324T (SEQ ID NO: 27) is referred to herein as G1025. In some embodiments of the present invention, G1025 unexpectedly binds strongly to FcγRI, FcγRIIa, and FcγRIIb. This result was unexpected because the mutation at position 238 was expected to reduce binding to FcγRI, FcγRIIa, and FcγRIIIa, but only reduced binding to FcγRIIIa. Even more surprisingly, when a similar set of mutations was introduced into the stradomer with an N-linked multimerization domain (G1024), in contrast to the C-linked stradomer GL-2045, the said preferential binding to the complement system was completely eliminated. The obtained results once again emphasize the unpredictable nature of these mutations as applied to the multimerizing stradomer.

[000197] In some embodiments, the present invention provides stradomers comprising a G019 backbone (IgG2 hinge - IgG1 hinge - IgG1 CH2 CH3, in an amino to carboxyl direction) and comprising one or more point mutations in the Fc domain. The resulting stradomer, comprising point mutations at positions 267, 268, and 324 (SEQ ID NO: 17), is designated herein as G1003. In some embodiments, G1003 unexpectedly bound to C1q and robustly inhibited CDC, while the parent stradomer exhibited minimal binding to C1q and did not inhibit CDC, despite the fact that the parent G019 compound formed higher order multimers capable of avid interaction with the receptors.

[000198] A G019-based stradomer containing point mutations at positions 267, 268, 324, and 328 (SEQ ID NO: 64) is referred to herein as G1049. In some embodiments of the present invention, G1049 unexpectedly exhibited strong binding to all canonical FcγRs, along with complement inhibition and the ability to bind to C1q.

[000199] The present inventors have discovered, surprisingly, that when certain Fc mutations, which have been described to increase the affinity of a monoclonal antibody for the complement system, are introduced into the Fc region of a multimerizing stradomer that does not significantly bind to FcγRIIb, the resulting compound restores binding to FcγRIIb. For example, G994, G996, and G998 each bind to FcγRIIb, in some cases with avidity, despite the fact that the corresponding stradomers containing the same mutation, with the exception of the S267E, H268F, and S324T mutations, do not exhibit avid binding to FcγRIIb.

[000200] Additional stradomers were generated based on each of the complement-binding preferential stradomers, G994 and G998. In the first set of stradomers generated from G994, the G994 mutations E233P, H268F, and S324T were retained, the residue at position 267 was the wild-type residue (serine), and various mutations were also introduced at position 236: arginine (as in G994) or an amino acid similar to arginine. The resulting stradomers were named G1103, G1088, G1089, G1104, G1082, G1105, and G1106. The extent to which these stradomers bind to FcγR and complement component C1q was highly variable and unpredictable, suggesting that the mutation at position 236 of the G994 stradomer has unpredictable effects. In a second set of stradomers derived from G994, the G994 mutations E233P, H268F, and S324T were retained, the residue at position 236 was the wild-type residue (glycine), and various mutations were also introduced at position 267: glutamic acid (as in G994) or an amino acid similar to glutamic acid. The resulting stradomers were named G1102, G1100, G1101, G1125, G1108, G1109, and G1084. Similarly, the extent to which stradomers bind to FcγR and complement component C1q varied substantially and was unpredictable, indicating that, similar to position 236, the mutation at position 267 of the G994 stradomer has unpredictable effects. In a further set of G994-based stradomers, combinations of mutations at positions 236 and 267 were tested and named G1110, G1111, G1112, G1114, G1115, G1116, G1117, G1118, G1119, G1120, G1121, G1122, G1123, G1124, G1128, G1129, G1130, and G1131. The FcγR and complement binding profiles of these stradomers were unpredictable.

[000201] Derivatives of G998 were also prepared and tested. In the first set of G998 derivatives, the G998 H268F and S324T mutations were retained, the residue at position 267 was the wild-type residue (serine) or was replaced with an amino acid similar to serine, and a mutation was added at position 299 (T299A). The resulting compounds were named G1071d2, G1068, G1094, G1092, G1096, G1107, G1093, and G1095. The T299A mutation was designed to eliminate the deglycosylation site. The extent to which this mutation reduced FcγR binding was unpredictable given the stradomer structure and other mutations. In addition, derivatives of G998 were prepared containing the H268F, S324T and N297A mutations (for deglycosylation) of G998, but different mutations at position 267. The resulting compounds were named G1069, G1070, G1132, G1074 and G1075 and also exhibited unpredictable FcγR and complement binding profiles. Surprisingly, only one of the compounds in this group (G1069) retained complement binding while eliminating FcγR binding. This compound was therefore a compound with preferential complement binding.

[000202] According to some embodiments of the present invention, G994, G998, G1033, G1022, G1032, G1023, G1006, G1027, G996 or another multimerizing stradomer, preferably binding to the complement system, is administered to a subject that has an acute condition, such as, for example, acute hemolysis (e.g., hemolytic disease of the newborn), autoimmune hemolytic anemia, drug-induced hemolytic anemia, acquired hemolytic anemia, transfusion reactions/alloimmune hemolytic anemia, infectious hemolytic anemia (e.g., anemias that are associated with tick-borne diseases, including bacterial (Lyme disease, typhus, Rocky Mountain spotted fever, tularemia, ehrlichiosis), viral (meningoencephalitis and hemorrhagic fever), and protozoal (piroplasmosis) tick-borne infections; or those associated with mosquito-borne infectious diseases such as malaria, including hemoglobinuric fever, dengue fever, chikungunya, Zika virus fever or Ebola virus), toxin-associated hemolytic anemias (e.g., snake venom toxins, toxic chemicals or tick toxins), malignant hypertension, burns or Reye's syndrome. According to some embodiments of the present invention, the multimerizing stradomer, preferably binding to the complement system, G998, G994, G1033, G1022, G1032, G1023, G1006, G1027, G996 is administered to a subject in need thereof by intravenous administration.

[000203] According to some embodiments of the present invention, a multimerizing stradomer, preferably binding to the complement system, G998, G994, G1033, G1022, G1032, G1023, G1006, G1027, G996 is administered to a subject who has a chronic disease or condition. Chronic diseases and conditions include, for example, kidney disease, macular degeneration, chronic hemolysis (e.g., paroxysmal nocturnal hemoglobinuria), mechanical hemolysis (e.g., due to an artificial heart valve, cardiopulmonary bypass device, or other device used in blood vessels, hemodialysis with or without cellophane membranes, preeclampsia or eclampsia, or thrombotic thrombocytopenic purpura), hereditary spherocytosis, ovalocytosis, pyruvate kinase deficiency, G6PD deficiency, sickle cell anemia, thalassemia, hereditary hemolytic anemia, hemolytic uremic syndrome, immune disorder complex involving the complement system (e.g., hepatitis B, hepatitis C, mixed cryoglobulinemia, lepromatous leprosy, and bacteremic shock), primary biliary cirrhosis, celiac disease, multiple myeloma and urticaria-induced vasculitis. According to some embodiments of the present invention, G994 is administered to a subject in need thereof by subcutaneous administration.

[000204] According to some embodiments of the present invention, a multimerizing stradomer, preferably binding to the complement system, G998, G994, G1033, G1022, G1032, G1023, G1006, G1027 and G996 is administered to a subject who has a kidney disease. Kidney diseases include, but are not limited to, membranoproliferative glomerulonephritis, mesangial proliferative glomerulonephritis, idiopathic proliferative glomerulonephritis, focal sclerosing glomerulonephritis, focal segmental glomerulosclerosis, thin basement membrane disease, nephritis, lupus nephritis, fibrillary glomerulonephritis, immunotactoid glomerulonephritis, Bright's disease, Berger's disease/IgA-mediated nephropathy, nephrosclerosis, nephrosis, nephrotic syndrome, membranous nephropathy, membranous nephritis, post-infectious glomerulonephritis, tubular necrosis, drug-induced nephrotoxicity, and Goodpasture's syndrome.

[000205] Thus, the inventors have found that individual stradomers can be prepared that have activity that preferentially binds to the complement system, exhibiting strong binding to C1q and reduced binding to FcγR, compared to the parent stradomer containing the native Fc domain sequence, but the activity that each individual stradomer has with respect to binding to C1q and FcγR cannot be predicted based on the effect of the said mutation in the monoclonal antibody.

[000206] The amino acid sequences of representative stradomers included within the scope of the present invention are shown in Table 1. Mutated amino acids are indicated in the table (text highlighted in gray).

[000207] Complement binding proteins, such as the monoclonal antibody eculizumab (an anti-C5 antibody), have been used in the art to block the complement pathway as a method for treating complement-mediated diseases. The biomimetics of the present invention provide increased binding to components of the complement system, such as C1q, through certain mutations of the Fc domains. For example, the biomimetics of the present invention comprise a point mutation at position 267 and/or 268 and/or 324 of the Fc domain of IgG1. The complement-binding biomimetics of the present invention also exhibit altered binding to FcRn, FcγRI, FcγRII, and/or FcγRIII, compared to the Fc domains of wild-type IgG1, generally in a manner that is not predictable based on literature data describing mutations contained in the biomimetics of the present invention. Accordingly, in one embodiment of the present invention, the biomimetics of the present invention are complement-binding preferential stradomers that are capable of multimerization and exhibit an increased degree of complement binding, compared to binding to one or more canonical FcγR and/or FcRn, when compared to normal non-aggregated immunoglobulin. In another embodiment, the biomimetics of the present invention are complement-binding preferential stradomers that are capable of multimerization and exhibit an increased degree of complement binding compared to binding to one or more canonical FcγR and/or FcRn when compared to normal aggregated immunoglobulin. In another embodiment, the biomimetics of the present invention are complement-binding preferential stradomers that are capable of multimerization and exhibit an increased degree of complement binding compared to binding to one or more canonical FcγR and/or FcRn when compared to a monoclonal antibody comprising similar mutations in its Fc domain. In another embodiment, the biomimetics of the present invention have an altered half-life compared to native IgG, IVIG, or the parent stradomer.

[000208] As used herein, the terms "FcγR" and "Fcγ receptor" include all members of the Fcγ RI, RII, and RIII families. Fcγ receptor includes low-affinity and high-affinity Fcγ receptors, including, but not limited to, human FcγRI (CD64), FcγRII (CD32) and its isotypes and allotypes FcγRIIa LR, FcγRIIa HR, FcγRIIb, and FcγRIIc; FcγRIII (CD 16) and its isotypes FcγRIIIa and FcγRIIIb. One of skill in the art will appreciate that the description herein of FcγR and FcγR homologs, such as those described in Davis, et al. (2004) “Differential B cell expression of mouse Fc receptor homologs,” Int. Immunol., 16(9):1343–1353, will be applicable to future FcγRs and their associated isotypes and allotypes that have not yet been discovered.

[000209] Specific binding is typically defined as the amount of labeled ligand that can then be replaced by excess unlabeled ligand in a binding assay. However, this does not exclude other methods for assessing specific binding that are well known in the art (e.g., Mendel CM, Mendel DB, 'Non-specific' binding. The problem, and a solution. Biochem J. 1985 May 15;228(l):269-72). Specific binding can be measured by various methods well known in the art, such as surface plasmon resonance (SPR) technology (commercially available from BIACORE®) or biolayer interferometry (commercially available from ForteBio®) to characterize the association and dissociation constants of immunologically active biomimetics (Asian K, Lakowicz JR, Geddes C. Plasmon light scattering in biology and medicine: new sensing approaches, visions and perspectives. Current Opinion in Chemical Biology 2005, 9:538-544).

[000210] "Immunological activity of aggregated native IgG" refers to the properties of multimerized IgG that influence the functioning of the immune system when IgG aggregates are exposed to the immune system. Specific properties of native multimerized IgG include altered specific binding to FcγR, cross-linking of FcγR on immune cell surfaces, or effector functional capacity of multimerized IgG such as antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis (ADCP), or complement fixation (see, e.g., Nimmerjahn F, Ravetch JV. The anti-inflammatory activity of IgG: the intravenous IgG paradox. J Exp Med. 2007; 204:11–15; Augener W, Friedman B, Brittinger G. Are aggregates of IgG the effective part of high-dose immunoglobulin therapy in adult idiopathic thrombocytopenic purpura (ITP) Blut. 1985;50:249–252; Arase N, Arase H, Park SY, Ohno H, Ra C, Saito T. Association with FcRgamma is essential for activation signal through NKR-Pl (CD161) in natural killer (NK) cells and NKl.1+ T cells. J Exp Med. 1997;186:1957-1963; Teeling JL, Jansen-Hendriks T, Kuijpers TW, et al. Therapeutic efficacy of intravenous immunoglobulin preparations depends on the immunoglobulin G dimers: studies in experimental immune thrombocytopenia. Blood. 2001;98: 1095-1099; Anderson CF, Mosser DM. Cutting edge: biasing immune responses by directing antigen to macrophage Fc gamma receptors. J Immunol. 2002; 168:3697-3701; Jefferis R, Lund J. Interaction sites on human IgG-Fc for FcγR: current models. Immunology Letters. 2002;82:57; Banki Z, Kacani L, Mullauer B, et al. Cross-Linking of CD32 Induces Maturation of Human Monocyte-Derived Dendritic Cells Via NF- {kappa} B Signaling Pathway. J Immunol. 2003;170:3963-3970; Siragam V, Brine D, Crow AR, Song S, Freedman J, Lazarus AH. Can antibodies with specificity for soluble antigens mimic the therapeutic effects of intravenous IgG in the treatment of autoimmune disease? J Clin Invest. 2005;l 15:155- 160). These properties are usually assessed by comparison with the properties of homodimeric IgG.

[000211] Although higher order multimers have been found to be effective in altering the immune response as described herein, homodimers are also effective immunomodulators. Without wishing to be bound by any particular theory, the present inventors believe that homodimers modulate the avidity of highly ordered multimers over time and can form more ordered multimers in vivo. Without wishing to be bound by any particular theory, the present inventors also believe that the multimers of the present invention slowly dissociate from the bound target and are internalized into immune cells expressing such targets, possibly altering the activation status or maturation rate of said immune cell over an extended period of time or possibly permanently. The results of the multimerization experiments described herein indicate that an otherwise pure population of homodimers is capable of multimerization in the presence of small amounts of blood or fetal bovine serum. Accordingly, while highly ordered multimers are more effective than the homodimeric fraction in modulating the immune response, the homodimeric fraction of the naturally associated stradomers of the present invention may also be an effective immunomodulator, in part by multimerizing the homodimer in the presence of small amounts of blood or serum. In this regard, the term "highly ordered multimers" is intended to include multimers other than homodimers that are formed in solution prior to injection into a subject, as well as multimers other than homodimers that are formed in vivo.

[000212] The terms "immunomodulatory activity,""modulation of the immune response,""modulation of the immune system," and "immune modulation" mean altering immune systems by altering the activities, capabilities, and relative abundance of one or more immune cells, including maturation of a cell of a particular type while maintaining that type or becoming another cell type. For example, immune modulation can be suppression or activation of an immune response. For example, in one aspect, immune modulation can mean inducing insensitivity or tolerance in a T cell or B cell. As used herein, the term "tolerance" refers to a state of a T cell or B cell, or of the immune response in general, in which the T cell or B cell or other immune cell does not respond to its cognate antigen or to an antigen, epitope, or other signal to which it normally responds. As another example, immune modulation of memory B cells can result in selective apoptosis of certain memory B cells with a concomitant decrease in the production of certain antibodies. As another example, immunomodulatory activities can result in a decrease in the levels of proinflammatory cytokines or cytokines that are typically elevated in autoimmune diseases, such as IL-6 and IL-8. As another example, immunomodulatory activities can result in the activation of NKT cells with subsequent secretion and cleavage of FGF-beta. Blocking immune cell receptors to prevent receptor activation is also included within the term "immune modulation" and may be separately referred to as "inhibitory immune modulation." In another aspect, immune modulation can be an enhancement or activation of an immune response. For example, immune modulation can mean the activation of T cells or B cells. As another example, immune modulation of immature monocytes can lead to an increase in populations of more mature monocytes, dendritic cells, macrophages, or osteoclasts, all of which are derived from immature monocytes. As another example, immune modulation of NK cells can lead to enhanced antibody-dependent cell-mediated cytotoxicity (ADCC). As another example, immunomodulatory activity can lead to an increase in the population of cells with phenotypes that may not otherwise be expressed at high levels, such as CD8 ⁺ /CD11c ⁺ cells. For example, immune cell receptors can be coupled to immunologically active biomimetics and activate intracellular signaling to induce a variety of immune cell changes, which is separately referred to as “activating immune modulation.”

[000213] Modulation of dendritic cells can promote or inhibit antigen presentation to T cells, such as by inducing expression of CD86 and/or CD1a on the surface of dendritic cells. CD1a is an MHC class I-related glycoprotein that is expressed on the surface of antigen-presenting cells, particularly dendritic cells. CD1a is involved in the presentation of lipid antigens to T cells. CD86 is also expressed on the surface of antigen-presenting cells and provides a costimulatory signal for T cells. CD86 is a ligand for CD28 and CTLA-4 on the surface of T cells to transmit activating and inhibitory signals, respectively. Therefore, the level of expression of CD86 and its cognate receptors determines whether resistance or a specific immune response will be induced. In a preferred embodiment, the stradomers of the present invention are capable of modulating the immune response, in part by inducing expression of CD86 and CD1a on the surface of antigen-presenting cells, in particular dendritic cells. According to one embodiment of the present invention, the modulated dendritic cell interacts with immune cells specific for the antigen of the antigen-specific stradomer.

[000214] Modulation of monocyte maturation refers to the differentiation of a monocyte into a mature APC, macrophage, or osteoclast. Differentiation can be modulated to increase the rate or direction of maturation and/or increase the number of monocytes that differentiate. Alternatively, differentiation can be decreased in terms of the rate of differentiation and/or the number of cells that undergo differentiation.

[000215] As used herein, the term "isolated" polypeptide or peptide refers to a polypeptide or peptide that either does not have a naturally occurring analog or has been separated or purified from components of the natural environment, such as in tissues such as pancreas, liver, spleen, ovary, testis, muscle tissue, joint tissue, neural tissue, gastrointestinal tissue, or breast tissue or tumor tissue (e.g., breast cancer tissue) or body fluids such as blood, serum, or urine. Generally, a polypeptide or peptide is considered "isolated" if it is at least 70% by dry weight, free of proteins and other natural organic molecules with which it is naturally associated. Preferably, the preparation of the polypeptide (or peptide) according to the present invention constitutes at least 80%, more preferably at least 90% and most preferably at least 99% by dry weight of the polypeptide (peptide), respectively, according to the present invention. Since the polypeptide or peptide, which is chemically synthesized, is by its nature separated from the components of the natural environment, the synthetic polypeptide or peptide is "isolated".

[000216] An isolated polypeptide (or peptide) of the present invention may be obtained, for example, by extraction from a natural source (e.g., from body tissues or fluids); by expression of a recombinant nucleic acid encoding the polypeptide or peptide; or by chemical synthesis. A polypeptide or peptide that is produced in a cellular system different from the source from which it naturally originates is "isolated" because it will necessarily be free of components of the natural environment. In a preferred embodiment, an isolated polypeptide of the present invention comprises only sequences corresponding to the IgG1 Fc region monomer and the IgG2 hinge multimerization domain (SEQ ID NO: 4), the isoleucine multimerization domain (SEQ ID NO: 5), or the GPP (SEQ ID NO: 6), and does not comprise additional sequences that may facilitate cloning or purification of the protein (i.e., introduced restriction enzyme sites or purification tags). The degree of recovery or purity may be measured by any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC.

Pharmaceutical compositions

[000217] Administration of the stradomer compositions described herein will be by any conventional route, orally, parenterally, or topically. Typical routes of administration include, but are not limited to, oral, nasal, buccal, rectal, vaginal, ophthalmic, subcutaneous, intramuscular, intraperitoneal, intravenous, intraarterial, intratumoral, spinal, intrathecal, intraarticular, intraarterial, subarachnoid, sublingual, oral mucosal, bronchial, lymphatic, intrauterine, subcutaneous, intratumoral, via integration on an implantable device such as a suture or an implantable device such as an implantable polymer, intradural, intracortical, or dermal. Suitable compositions are typically administered as pharmaceutically acceptable compositions as described herein. In a preferred embodiment, the isolated stradomer is administered intravenously or subcutaneously.

[000218] As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, and the like. The use of suitable media and agents for pharmaceutically active substances is well known in the art. Except where any conventional media or agent is incompatible with the vectors or cells of the present invention, the use of said agent in therapeutic compositions is within the scope of the present invention. Additional ingredients may also be included in the compositions.

[000219] The stradomer compositions of the present invention can be prepared in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of the protein) and those formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with free carboxyl groups can also be prepared from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine, and the like.

[000220] Sterile injectable solutions are prepared by incorporating the stradomer in the required amount in a suitable solvent with various other ingredients listed above, if necessary, followed by filter sterilization. According to some embodiments of the present invention, sterile injectable solutions are prepared for intramuscular, subcutaneous or intravenous administration. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from the above. In the case of sterile powders for the preparation of sterile injectable solutions, vacuum drying and freeze-drying techniques are preferred methods of preparation, which allow obtaining a powder of the active ingredient with any additional desired ingredient from a solution already sterilized by filtration.

[000221] In addition, according to one embodiment, the present invention provides a composition of stradomer suitable for oral administration, which is provided in a pharmaceutically acceptable carrier, with or without the addition of an inert diluent. The carrier should be assimilable or edible and includes liquid, semi-solid, i.e. pastes, or solid carriers. Except in cases where any conventional media, agent, diluent or carrier is deleterious to the recipient or to the therapeutic effectiveness of the stradomer preparation contained therein, their use in the composition for oral administration for practicing the methods of the present invention is allowed. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof. In this application, the term "oral administration" includes oral, buccal, enteral or intragastric administration.

[000222] According to one embodiment of the present invention, the stradomer composition is combined with the carrier in any convenient and appropriate manner, i.e., by dissolution, suspension, emulsification, mixing, encapsulation, microencapsulation, absorption, and the like. Suitable procedures are standard for those skilled in the art.

[000223] According to a specific embodiment, the stradomers composition in powder form is combined or thoroughly mixed with a semi-solid or solid carrier. Mixing can be accomplished by any convenient method, such as crushing. Stabilizers can also be added during mixing to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers for use in a composition intended for oral administration include buffers, gastric acid secretion antagonists, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, proteolytic enzyme inhibitors, and the like. More preferably, the stabilizer for a composition intended for oral administration can also include gastric acid secretion antagonists.

[000224] Additionally, the stradomer composition intended for oral administration, which is combined with a semi-solid or solid carrier, can be further prepared as hard or soft gelatin capsules, tablets or pills. More preferably, the gelatin capsules, tablets or pills are coated with an enteric coating. Enteric coatings prevent denaturation of the composition in the stomach or upper intestine, where the pH is acidic. See, for example, U.S. Patent No. 5,629,001. Upon entering the small intestine, the coating dissolves at basic pH values, which provides release of the composition for interaction with intestinal cells, such as M-cells of Peyer's patches.

[000225] According to another embodiment of the present invention, the stradomer powder composition is combined or intimately mixed with materials that create a nanoparticle that encapsulates the immunologically active biomimetic or to which the immunologically active biomimetic is attached. Each nanoparticle will have a size of less than or equal to 100 microns. The nanoparticle may have mucoadhesive properties that provide for absorption of the immunologically active biomimetic in the gastrointestinal tract, which would otherwise not have bioavailability for oral administration.

[000226] According to another embodiment of the present invention, the powder composition is combined with a liquid carrier, such as water or saline, with or without the addition of a stabilizing agent.

[000227] A specific formulation of the stradomer that can be used is a solution of an immunologically active biomimetic protein in a hypotonic phosphate-based buffer that does not contain potassium, wherein the buffer composition comprises: 6 mM sodium dihydrogen phosphate monohydrate, 9 mM sodium monohydrogen phosphate heptahydrate, 50 mM sodium chloride, pH=7.0±0.1. The concentration of the immunologically active biomimetic protein in the hypotonic buffer can be from 10 μg/mL to 100 mg/mL. The formulation can be administered by any route of administration, such as, but not limited to, intravenous.

[000228] In addition, the topical stradomer composition that is combined with a semi-solid carrier can also be formulated as a cream or gel ointment. A preferred carrier for forming a gel ointment is a gel polymer. Preferred polymers that are used to prepare the gel composition of the present invention include, but are not limited to, carbopol, carboxymethylcellulose, and pluronic acid polymers. In particular, the Fc multimer powder composition is combined with an aqueous gel containing a polymerizing agent such as carbopol 980 with an active agent content of 0.5 to 5% w/v for application to the skin for treating a disease of the skin or subcutaneous tissues. In the present application, the term "topical administration" includes application to the surface of the dermis, epidermis, subcutaneous tissue, or mucous membrane.

[000229] In addition, the stradomer composition can be manufactured in the form of a polymer for subcutaneous or subdermal implantation. A preferred formulation for the implantable drug-loading polymer is an agent that is generally regarded as safe and may comprise, for example, cross-linked dextran (Samantha Hart, Master of Science Thesis, “Elution of Antibiotics from a Novel Cross-Linked Dextran Gel: Quantification” Virginia Polytechnic Institute and State University, June 8, 2009), dextran-tyramine (Jin, et al. (2010) Tissue Eng. Part A. 16(8):2429-40), dextran-polyethylene glycol (Jukes, et al. (2010) Tissue Eng. Part A., 16(2):565-73), or dextran-glutaraldehyde (Brondsted, et al. (1998) J. Controlled Release, 53:7-13). One skilled in the art will recognize that many similar polymers and hydrogels can be formed by incorporating a pore sizer anchored within the polymer or hydrogel and controlling the pore size to achieve a desired diameter.

[000230] Once prepared, the solutions are administered in a manner compatible with the dosage form and in an amount that is therapeutically effective to result in improvement or elimination of symptoms. The formulations can be readily administered in a variety of dosage forms, such as edible solutions, drug release capsules, and the like. Some variation in dosage may be made depending on the patient's condition. The person responsible for administering the drug can, in any case, determine the appropriate dose for an individual subject. In addition, preparations for human administration meet the standards of sterility, general safety, and purity required by the Center for Biologics Evaluation and Study of the U.S. Food and Drug Administration (FDA).

[000231] It is understood that the route of administration depends on the location and nature of the disease being treated and may include, for example, intradermal, transdermal, subdermal, parenteral, nasal, intravenous, intramuscular, intranasal, subcutaneous, transdermal, intratracheal, intraperitoneal, intratumoral, perfusion, lavage, direct injection, and oral routes of administration.

[000232] According to one embodiment of the present invention, the stradomer is administered intravenously, subcutaneously, orally, intraperitoneally, sublingually, buccally, transdermally, rectally, via a subcutaneous implant, or intramuscularly. In particular embodiments, the stradomer is administered intravenously, subcutaneously, or intramuscularly. According to one embodiment of the present invention, the stradomer is administered at a dose of from about 0.01 mg/kg to about 1000 mg/kg. According to another embodiment of the present invention, the stradomer is administered at a dose of from about 0.1 mg/kg to about 100 mg/kg. According to another embodiment, the stradomer is administered at a dose of from about 0.5 mg/kg to about 50 mg/kg. According to another embodiment, the stradomer is administered at a dose of from about 1 mg/kg to about 25 mg/kg. According to another embodiment, the stradomer is administered at a dose of about 5 mg/kg to about 15 mg/kg. The stradomer may be administered at least once daily, weekly, biweekly, or monthly. A two-phase dosing regimen may be used, wherein the first dosing phase comprises a dose of about 0.1% to about 300% of the second dosing phase.

[000233] According to another embodiment of the present invention, the stradomer is administered before, during, or after the administration of one or more additional pharmaceutical and/or therapeutic agents. According to another embodiment of the present invention, the additional pharmaceutically active agent comprises a steroid; a biological anti-autoimmune drug such as a monoclonal antibody, a fusion protein, or an anti-cytokine; a non-biological anti-autoimmune drug; an immunosuppressant; an antibiotic; and an antiviral agent; a cytokine; or an agent that may otherwise act as an immunomodulator. According to another embodiment, the steroid is prednisone, prednisolone, cortisone, dexamethasone, mometasone, testosterone, estrogen, oxandrolone, fluticasone, budesonide, beclomethasone, albuterol, or levalbuterol. According to another embodiment of the present invention, the monoclonal antibody is eculizumab, infliximab, adalimumab, rituximab, tocilizumab, golimumab, ofatumumab, LY2127399, belimumab, veltuzumab, mepolizumab, necitumumab, nivolumab, dinutuximab, secukinumab, evolocumab, blinatumomab, pembrolizumab, ramucirumab, vedolizumab, siltuximab, obinutuzumab, adotrastuzumab, raxibacumab, pertuzumab, brentuximab, ipilumumab, denosumab, canakinumab, ustekinumab, catumaxomab, ranibizumab, panitumumab, natalizumab, bevacizumab, cetuximab, efalizumab, omalizumab, toitumomab-I131, alemtuzumab, gemtuzumab, trastuzumab, palivizumab, basilixumab, daclizumab, abciximab, murononomab or certolizumab. According to another embodiment of the present invention, the fusion protein is etanercept or abatacept. According to another embodiment of the present invention, the anti-cytokine biologic is anakinra. According to another embodiment of the present invention, the anti-rheumatic non-biologic agent is cyclophosphamide, methotrexate, azathioprine, hydroxychloroquine, leflunomide, minocycline, organogold compounds, fostamatinib, tofacitinib, etoricoxib or sulfasalazine. According to another embodiment of the present invention, the immunosuppressant is cyclosporin A, tacrolimus, sirolimus, mycophenolate mofetil, everolimus, OKT3, antithymocyte globulin, basiliximab, daclizumumab or alemtuzumab. According to another embodiment of the present invention, the stradomer is administered before, during or after the administration of the chemotherapeutic agent. According to another embodiment of the present invention, the stradomer and the additional therapeutic agent exhibit a therapeutic synergistic effect when administered together. According to one embodiment of the present invention, the stradomer is administered before the administration of the additional therapeutic agent. According to another embodiment of the present invention, the stradomer is administered simultaneously with the administration of the additional therapeutic agent. According to another embodiment of the present invention, the stradomer is administered after the administration of the additional therapeutic agent.

[000234] According to one embodiment of the present invention, the stradomer is administered in a form covalently attached to an implantable device. According to one embodiment of the present invention, the stradomer is attached to a suture. According to another embodiment of the present invention, the stradomer is attached to a graft or stent. According to another embodiment of the present invention, the stradomer is attached to a heart valve, an artificial orthopedic joint, or an implanted electrical wire. According to another embodiment of the present invention, the stradomer is attached to and embedded in an implantable matrix. In a preferred embodiment, the stradomer is attached to and embedded in an implantable hydrogel. According to one embodiment of the present invention, the hydrogel is comprised of dextran, polyvinyl alcohol, sodium polyacrylate, or acrylate polymers. According to another embodiment of the present invention, the stradomer is administered attached to a hydrogel with pore sizes large enough to allow immune cells to penetrate to interact with the immobilized stradomer and then return to the bloodstream. According to another embodiment of the present invention, the pore size of the hydrogel is from 5 to 50 microns. In a preferred embodiment, the pore size of the hydrogel is 25-30 microns.

[000235] According to another embodiment of the present invention, the stradomer is administered to treat humans, non-human primates (e.g., monkeys, baboons, and chimpanzees), mice, rats, cows, horses, cats, dogs, pigs, rabbits, goats, deer, sheep, ferrets, rodents, guinea pigs, hamsters, bats, birds (e.g., chickens, turkeys, and ducks), fish, and reptiles using species-specific or chimeric stradomer molecules. According to another embodiment of the present invention, the human is an adult or a child. According to another embodiment of the present invention, the stradomer is administered to prevent a complement-mediated disease. According to another embodiment of the present invention, the stradomer is administered to prevent vaccine-associated autoimmune conditions in companion animals and livestock.

[000236] As used herein, the term "parenteral administration" includes any form of administration in which the compound is absorbed by the subject without involving intestinal absorption. Examples of parenteral routes of administration that are useful in the present invention include, but are not limited to, intramuscular, intravenous, intraperitoneal, intratumoral, intraocular, nasal, or intraarticular administration.

[000237] Furthermore, the stradomer of the present invention may optionally be administered before, during or after the administration of another pharmaceutical agent. For example, it has been surprisingly found that concomitant administration of the stradomer of the present invention and prednisolone provides synergistically superior results compared to those observed with the use of a stradomer composition or prednisolone alone (WO 2012/016073).

[000238] The following are specific examples of various categories of pharmaceuticals and preferred routes of administration, as indicated, for specific illustrative diseases:

[000239] Buccal or sublingual dissolving tablet: angina pectoris, polyarteritis nodosa.

[000240] Intravenous, intramuscular, or subcutaneous route: myasthenia gravis, hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), membranous nephropathy, neuromyelitis optica, antibody-mediated allograft rejection, membranoproliferative glomerulonephritis (MPGN), lupus nephritis, idiopathic thrombocytopenic purpura, inclusion body myositis, paraproteinemic demyelinating polyneuropathy associated with IgM secretion, necrotizing fasciitis, pemphigus, gangrene, dermatomyositis, granuloma, lymphoma, sepsis, aplastic anemia, multiple organ failure, multiple myeloma, and monoclonal gammopathy of unknown significance, chronic inflammatory demyelinating polyradiculoneuropathy, inflammatory myopathies, thrombotic thrombocytopenic purpura, myositis, anemia, neoplasia, hemolytic anemia, encephalitis, myelitis, myelopathy especially associated with human T-cell lymphotropic virus 1, leukemia, multiple sclerosis and optic neuritis, asthma, epidermal necrolysis, Lambert-Eaton myasthenic syndrome, myasthenia gravis, neuropathy, uveitis, Guillain-Barré syndrome, graft-versus-host disease, stiff person syndrome, paraneoplastic cerebellar degeneration with anti-Yo antibodies, paraneoplastic encephalomyelitis and sensory neuropathy with anti-Hu antibodies, systemic vasculitis, systemic lupus erythematosus, autoimmune diabetic neuropathy, acute idiopathic dysautonomia neuropathy, Vogt-Koyanagi-Harada syndrome, multifocal motor neuropathy, anti-/GM1-associated peripheral motor neuron syndrome, demyelination, membranoproliferative glomerulonephritis, cardiomyopathy, Kawasaki disease, rheumatoid arthritis and Evans syndrome IM - ITP, CIDP, MS, dermatomyositis, myasthenia gravis, muscular dystrophy. In this application, the term "intravenous administration" includes all techniques for delivering a compound or composition of the present invention into the systemic circulation by intravenous injection or infusion.

[000241] Skin gel, lotion, cream or patch: vitiligo, shingles, acne, cheilitis.

[000242] Rectal suppository, gel or infusion: ulcerative colitis, hemorrhoidal inflammation.

[000243] Oral form as a pill, lozenge, encapsulated or enteric-coated form: Crohn's disease, celiac disease, irritable bowel syndrome, inflammatory liver disease, Barrett's esophagus.

[000244] Intracortical form: epilepsy, Alzheimer's disease, multiple sclerosis, Parkinson's disease, Huntington's disease.

[000245] Intra-abdominal infusion or implant: endometriosis.

[000246] Intravaginal gel or suppository: bacterial, trichomonal or fungal vaginitis.

[000247] Medical devices: coronary artery stent coating, joint prostheses.

Methods of therapeutic use of stradomers that preferentially bind to the complement system

[000248] According to one embodiment, the present invention provides a method of treating or preventing a disease or condition, such as a complement-mediated disease or condition, comprising administering to a subject in need thereof a stradomer comprising an IgG1 Fc domain and a multimerization domain, wherein said stradomer exhibits an increased degree of binding to the complement system, as compared to binding to an Fc receptor. According to another embodiment of the present invention, the stradomer exhibits a decrease in or absence of binding to FcγR and/or a decrease in or absence of binding to FcRn and exhibits enhanced or preferential binding to the complement system. According to another embodiment of the present invention, the stradomer exhibits enhanced or preferential binding to C1q.

[000249] Based on the rational design and the results of in vitro and in vivo testing, the stradomers of the present invention will serve as important biopharmaceuticals for the treatment of inflammatory diseases and disorders, in particular complement-mediated diseases and disorders; as well as for altering immune function in various other situations, such as biological immune therapy of allergies, cancer, autoimmune diseases, infectious diseases and inflammatory diseases. Medical conditions suitable for treatment using the immunologically active, preferably complement-binding biomimetics disclosed herein include any disease caused by or associated with complement activation or complement-mediated effector functions, including increased or inappropriate complement activity. Suitable medical conditions include those conditions that are currently treated or have been previously treated using complement-binding drugs such as eculizumab. Eculizumab binds to complement protein C5 (a complement protein that reacts after C1 and C1q in the classical complement pathway), inhibiting its cleavage and subsequent complement-mediated cell lysis. The biomimetics of the present invention provide a safe and effective alternative to other complement-fixing drugs known in the art. For example, in some embodiments, the biomimetics of the present invention bind to C1q, the first subunit in the C1 complex of the classical complement pathway. Medical conditions suitable for treatment with immunologically active biomimetics that preferentially bind to the complement system include, but are not limited to, myasthenia gravis, hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), neuromyelitis optica, antibody-mediated allograft rejection, macular degeneration, sickle cell anemia, and membranoproliferative glomerulonephritis (MPGN). Additional medical conditions suitable for treatment with the immunologically active, complement-binding biomimetics described herein include those conditions that are currently commonly treated with non-specific immunosuppressive therapy, including hIVIG, or conditions in which hIVIG has been found to provide clinical benefit, such as autoimmune cytopenias, chronic inflammatory demyelinating polyneuropathy, Guillain-Barré syndrome, myasthenia gravis, factor VIII-directed autoimmune disease, dermatomyositis, vasculitis, and uveitis (see, F. G. van der Meche, P. I. Schmitz, N. Engl. J. Med. 326, G1123 (1992); P. Gajdos et al, Lancet i, 406 (1984); Y. Sultan, M. D. Kazatchkine, P. Maisonneuve, U. E. Nydegger, Lancet ii, 765 (1984); M. C. Dalakas et al., N. Engl. J. Med. 329, 1993 (1993); D. R. Jayne, M. J. Davies, C. J. Fox, C. M. Black, C. M. Lockwood, Lancet 337, 1137 (1991); P. LeHoang, N. Cassoux, F. George, N. Kullmann, M. D. Kazatchkine, Ocul. Immunol. Inflamm. 8, 49 (2000)), and those cancers or inflammatory pathological conditions for which a monoclonal antibody can be used or is already used in clinical practice. Also suitable are conditions that can be effectively treated with the compounds of the present invention, which include inflammatory diseases with an imbalance of cytokine networks, an autoimmune disorder mediated by pathogenic autoantibodies or autoaggressive T cells, or the acute or chronic phase of a chronic relapsing autoimmune, anti-inflammatory or infectious disease or process.

[000250] In addition, other medical conditions that have an inflammatory component involving the complement system would benefit clinically from treatment with stradomers, such as amyotrophic lateral sclerosis, Huntington's disease, Alzheimer's disease, Parkinson's disease, myocardial infarction, stroke, hepatitis B, hepatitis C, human immunodeficiency virus-associated inflammation, adrenoleukodystrophy, and epileptic disorders, particularly those thought to be associated with postviral encephalitis, including Rasmussen syndrome, West syndrome, and Lennox-Gastaut syndrome.

[000251] A general approach to therapy using the isolated stradomers described herein is to administer to a subject having a disease or condition a therapeutically effective amount of an isolated immunologically active biomimetic to effect treatment. According to some embodiments of the present invention, the diseases or conditions can be broadly classified as inflammatory diseases with an imbalance of cytokine networks, an autoimmune disorder mediated by pathogenic autoantibodies or autoaggressive T cells, or an acute or chronic phase of a chronic disease or relapsing process.

[000252] As used herein, the terms "treat" and "treatment" refer to administering to a subject a therapeutically effective amount of a stradomer of the present invention such that the subject has an improvement in a disease or condition, or a symptom of a disease or condition. An improvement is any improvement or reversal of the disease or condition, or a symptom of a disease or condition. An improvement is an observable or measurable improvement, or may be an improvement in the general condition of the subject. Accordingly, one skilled in the art will appreciate that a treatment may improve a disease state, but may not provide a complete cure for said disease. In particular, an improvement in subjects may include one or more of the following: a reduction in inflammation; a reduction in levels of laboratory markers of inflammation, such as C-reactive protein; reduction in autoimmunity as evidenced by one or more of the following: improvement in levels of autoimmune markers such as autoantibodies or platelet, white blood cell, or red blood cell counts; reduction in rash or purpura; reduction in weakness, numbness, or tingling; improvement in glucose levels in patients with hyperglycemia; reduction in pain, inflammation, swelling, or joint destruction; reduction in cramping and in the frequency and extent of diarrhea; reduction in angina, reduction in tissue inflammation, or reduction in the frequency of attacks; reduction in tumor burden, increase in time to tumor progression, decrease in cancer pain, increase in survival, or improve quality of life; or slowing the progression of or improving osteoporosis.

[000253] As used herein, the term "therapeutically effective amount" refers to an amount that results in improvement or restoration of the symptoms of a disease or condition.

[000254] As used herein, the term "prevention" may mean the complete prevention of disease symptoms, a delay in the onset of disease symptoms, or a reduction in the severity of subsequent disease symptoms.

[000255] As used herein, the term "subject" refers to any mammalian subject to which the stradomers of the present invention are administered according to the methods described herein. In a particular embodiment, the methods of the present invention are used to treat a human subject. The methods of the present invention can also be used to treat non-human primates (e.g., monkeys, baboons, and chimpanzees), mice, rats, cows, horses, cats, dogs, pigs, rabbits, goats, deer, sheep, ferrets, rodents, guinea pigs, hamsters, bats, birds (e.g., chickens, turkeys, and ducks), fish, and reptiles to produce species-specific or chimeric stradomer molecules.

[000256] In one embodiment of the present invention, the stradomers of the present invention provide superior safety and efficacy compared to other complement-fixing molecules. In another embodiment of the present invention, the stradomers of the present invention exhibit superior safety and efficacy compared to the anti-C5 antibody, eculizumab.

[000257] Inhibition of the complement system has been shown to reduce antibody-mediated diseases (see, e.g., Stegall et al. Terminal Complement Inhibition Decreases Antibody-Mediated Rejection in Sensitized Renal Transplant Recipients. American Journal of Transplantation 2011 Nov; 11(1):2405-2413-Epub 2011 Sept 22; DOI: 10.1111/j.1600-6143.2011.03757.x). The stradomers of the present invention can also be used to treat an antibody-mediated disease or condition. Autoantibodies mediate many known autoimmune diseases and likely play a role in numerous other autoimmune diseases. Established antibody-mediated diseases in which the stradomers of the present invention may be used include, but are not limited to, anti-glomerular basement membrane antibody-mediated nephritis including Goodpasture's syndrome; anti-donor antigen antibodies (donor antigen-specific alloantibodies) in solid organ transplantation; anti-aquaporin-4 antibody in neuromyelitis optica; anti-VGKC antibodies in neuromyotonia, limbic encephalitis, and Morvan's syndrome; anti-nicotinic acetylcholine receptor antibodies and anti-MuSK antibodies in myasthenia gravis; anti-VGCC antibodies in Lambert-Eaton myasthenic syndrome; anti-AMPAR and GABA(B)R antibodies in limbic encephalitis, often associated with tumors; anti-GlyR antibodies in stiff person syndrome or hyperekplexia; Antibodies to fosoflipids, cardiolipin, and β2-glycoprotein I in recurrent spontaneous abortion, Hughes syndrome, and systemic lupus erythematosus; Antibodies to glutamate decarboxylase in stiff-person syndrome, autoimmune cerebellar ataxia, or limbic encephalitis; Antibodies to NMDA receptors in a recently described syndrome affecting limbic and subcortical functions with marked movement disorder, often in young adults and children, which is usually associated with ovarian teratoma but may not be paraneoplastic; Antibodies to double-stranded DNA, single-stranded DNA, RNA, SM, and C1q in systemic lupus erythematosus; Antibodies to core and nucleolar antigens in connective tissue diseases including scleroderma, Sjogren's syndrome, and polymyositis, including antibodies to Ro, La, Scl70, Jo-1; Anti-rheumatoid factor antibodies in rheumatoid arthritis; anti-hepatitis surface antigen antibodies in polyarteritis nodosa; anti-centromere antibodies in CREST syndrome; anti-streptococcal antibodies in endocarditis or at risk for endocarditis, anti-thyroglobulin antibodies, anti-thyroid peroxidase antibodies, and anti-TSH receptor antibodies in Hashimoto's thyroiditis; anti-U1 RNP antibodies in mixed connective tissue disease and systemic lupus erythematosus; and anti-desmogelin and anti-keratinocyte antibodies in pemphigus.

[000258] The stradomets of the present invention can be used to treat conditions including, but not limited to, congestive heart failure (CHF), vasculitis, rosacea, acne, eczema, myocarditis and other myocardial diseases, systemic lupus erythematosus, diabetes mellitus, spondylopathy, synovial fibroblasts and bone marrow stroma; bone loss; Paget's disease, osteoclastoma; multiple myeloma; breast cancer; dysfunctional osteogenesis; malnutrition, periodontal disease, Gaucher disease, Langerhans cell histiocytosis, spinal cord injury, acute septic arthritis, osteomalacia, Cushing's syndrome, monostotic fibrous osteodysplasia, polystotic fibrous dysplasia, periodontal remodeling and bone fractures; sarcoidosis; osteolytic bone cancer, lung cancer, renal cancer, and colorectal cancer; bone metastases, control of bone pain and humoral malignant hypercalcemia, ankylosing spondylitis and other spondyloarthropathies; transplant rejection, viral infections, hematologic neoplasia and neoplastic-like conditions such as Hodgkin's lymphoma; non-Hodgkin's lymphoma (Burkitt's lymphoma, small lymphocytic lymphoma/chronic lymphocytic leukemia, mycosis fungoides, mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, hair cell leukemia, and lymphoplasmacytic leukemia), lymphocyte precursor cell tumors including B-cell acute lymphoblastic leukemia/lymphoma and T-cell acute lymphoblastic leukemia/lymphoma, thymoma, mature T- and NK cell tumors including peripheral T-cell leukemias, adult T-cell leukemia/T-cell lymphomas, and large granular lymphocyte leukemia, Langerhans cell histiocytosis, myeloid neoplasias such as acute myeloid leukemias including AML with maturation, AML without differentiation, acute promyelocytic leukemia, acute myelomonocytic leukemia and acute monocytic leukemia, myelodysplastic syndromes and chronic myeloproliferative disorders including chronic myelogenous leukemia, tumors of the central nervous system such as brain tumors (glioma, neuroblastoma, astrocytoma, medulloblastoma, ependymoma and retinoblastoma), solid tumors (nasopharyngeal cancer, basal cell carcinoma, pancreatic cancer, bile duct cancer, Kaposi's sarcoma, testicular cancer, uterine cancer, vaginal cancer or cervical cancer, ovarian cancer, primary liver cancer or endometrial cancer, tumors of the vascular system (angiosarcoma and hemangiopericytoma)) or other cancers.

[000259] As used herein, the term "cancer" refers to or describes a physiological condition in mammals that is typically characterized by uncontrolled cell proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, leiomyosarcoma, chordoma, lymphangiosarcoma, lymphangioendotheliosarcoma, rhabdomyosarcoma, fibrosarcoma, myxosarcoma, chondrosarcoma), neuroendocrine tumors, mesothelioma, synovioma, schwannoma, meningioma, adenocarcinoma, melanoma, and leukemia and lymphoid malignancies. More specific examples of eligible cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and squamous cell lung cancer, small cell lung carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial carcinoma or uterine carcinoma, salivary gland carcinoma, renal cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, rectal carcinoma, penile carcinoma, testicular cancer, esophageal cancer, biliary tract tumors, Ewing tumor, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, myelodysplastic disease, myelodysplastic disease, severe myeloma chains, neuroendocrine tumors, schwannoma and other carcinomas, and head and neck cancer.

[000260] The stradomes of the present invention can be used to treat autoimmune diseases. As used herein, the term "autoimmune disease" refers to a diverse group of more than 80 diseases and conditions. The underlying problem with all of these diseases and conditions is that the body's immune system attacks the body itself. Autoimmune diseases affect all major systems of the body, including connective tissue, nerves, muscles, the endocrine system, skin, blood, the respiratory system, and the gastrointestinal tract. Autoimmune diseases include, for example, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and type 1 diabetes.

[000261] The disease or condition treatable using the compositions and methods of the present invention may be a hematoimmunological process, including, but not limited to, sickle cell anemia, idiopathic thrombocytopenic purpura, alloimmune/autoimmune thrombocytopenia, acquired idiopathic thrombocytopenic purpura, autoimmune neutropenia, autoimmune hemolytic anemia, parvovirus B19-associated red cell aplasia, acquired autoimmune reaction to factor VIII, acquired von Willebrand disease, multiple myeloma and monoclonal gammopathy of unknown significance, sepsis, aplastic anemia, pure red cell aplasia, Diamond-Blackfan syndrome, hemolytic disease of the newborn, immune-mediated neutropenia, refractoriness to platelet transfusion, neonatal post-transfusion purpura, hemolytic uremic syndrome, systemic vasculitis, thrombotic thrombocytopenic purpura or Evans syndrome.

[000262] The disease or condition may also be a neuroimmunological process including, but not limited to, Guillain-Barré syndrome, chronic inflammatory demyelinating polyradiculoneuropathy, paraproteinemic demyelinating polyneuropathy associated with IgM secretion, Lambert-Eaton myasthenic syndrome, myasthenia gravis, multifocal motor neuropathy, anti/GM1-associated peripheral motor neuron syndrome, demyelination, multiple sclerosis and optic neuritis, stiff person syndrome, anti-Yo paraneoplastic cerebellar degeneration, paraneoplastic encephalomyelitis, anti-Hu sensory neuropathy, epilepsy, encephalitis, myelitis, myelopathy, particularly associated with human T-cell lymphotropic virus 1, autoimmune diabetic neuropathy, Alzheimer's disease, Parkinson's disease, Huntington's disease, or acute idiopathic dysautonomia neuropathy.

[000263] The disease or condition may also be an inflammation or an autoimmune disease associated with hearing loss or vision loss. For example, the disease or condition may be an autoimmune hearing loss, such as noise-induced hearing loss or age-related hearing loss, or may be associated with the implantation of devices such as hearing aids (e.g., cochlear implants). In some embodiments of the present invention, the compositions of the present invention may be administered to the subject prior to, concurrently with, or following implantation of the device.

[000264] The disease or condition may also be a rheumatic disease, including, but not limited to, Kawasaki disease, rheumatoid arthritis, Felty's syndrome, ANCA-positive vasculitis, spontaneous polymyositis, dermatomyositis, antiphospholipid syndromes, recurrent spontaneous abortions, systemic lupus erythematosus, juvenile idiopathic arthritis, Raynaud's syndrome, CREST syndrome, or uveitis.

[000265] The disease or condition may also be a dermatoimmunologic disease including, but not limited to, toxic epidermal necrolysis, gangrene, granuloma, autoimmune cutaneous pemphigus including pemphigus vulgaris, bullous pemphigoid, exfoliative pemphigus, vitiligo, streptococcal toxic shock syndrome, scleroderma, systemic sclerosis including diffuse and limited cutaneous systemic sclerosis, or atopic dermatitis (particularly steroid-dependent).

[000266] The disease or condition may also be an immunologic disease of the musculoskeletal system, including, but not limited to, inclusion body myositis, necrotizing fasciitis, inflammatory myopathies, myositis, decorin antibody (BJ antigen) myopathy, paraneoplastic necrotizing myopathy, X-linked vacuolating myopathy, penicillamine-induced polymyositis, atherosclerosis, coronary artery disease, or cardiomyopathy.

[000267] The disease or condition may also be an immunological disease of the gastrointestinal tract, including, but not limited to, pernicious anemia, autoimmune chronic active hepatitis, primary biliary cirrhosis, celiac disease, dermatitis herpetiformis, cryptogenic cirrhosis of the liver, reactive arthritis, Crohn's disease, Whipple's disease, ulcerative colitis, or sclerosing cholangitis.

[000268] The disease or condition may also be a graft-versus-host disease, antibody-mediated graft rejection, graft rejection after bone marrow transplantation, inflammation following an infectious disease, lymphoma, leukemia, neoplasia, asthma, type 1 diabetes mellitus with beta-cell antibodies, Sjogren's syndrome, mixed connective tissue disease, Addison's disease, Vogt-Koyanagi-Harada syndrome, membranoproliferative glomerulonephritis, Goodpasture's syndrome, Graves' disease, Hashimoto's thyroiditis, Wegener's granulomatosis, micropolyarteritis, Churg-Strauss syndrome, polyarthritis nodosa, or multiple organ failure.

[000269] As used herein, the term "allergy" includes all IgE-mediated immune responses as well as those responses that mimic IgE-mediated responses. Allergic responses are induced by allergens, including proteins, peptides, carbohydrates, and combinations thereof, that elicit an IgE-mediated immune response or a response similar to an IgE-mediated response. Typical allergic responses include nut allergies, pollen allergies, and insect bite allergies. Typical allergens include urushiol in poison ivy and oak; house dust mite antigen; birch pollen components Bet v 1 and Bet v 2; 15 kD antigen in celery; apple antigen Mal d 1; Pru p3 in peaches; timothy grass pollen allergen Phl p 1; Lol p 3, Lol p I or Lol p V in ryegrass; Cyn d 1 in pigweed; dust mite allergens der p1, der p2 or der f1; alpha-gliadin and γ-gliadin epitopes in gluten; bee venom phospholipase A2; Ara h 1, Ara h 2 and Ara h 3 epitopes in peanuts.

[000270] The disease or condition may be a glomerular disease and/or nephritis. Mesangial proliferation is a common feature of many human glomerular diseases, including IgA-mediated nephropathy, recovery from infectious glomerulonephritis, and a number of secondary glomerular diseases such as lupus nephritis, Henoch-Schönlein purpura, rheumatoid arthritis, cirrhosis, Alport syndrome, and diabetic nephropathy. The disease is characterized by varying degrees of mesangial hypercellularity and expansion of the mesangial matrix. In cases of progressive disease, these cellular changes may lead to glomerular capillary constriction, sclerosis, and capsular adhesions as a result of damage caused by a variety of immunologic, toxic, metabolic, mechanical, and inflammatory mediators. Although several experimental models have been developed, the most widely used model for studying mesangial proliferation has been the anti-thymocyte (anti-Thy-1) model (Yamamoto and Wilson, “Quantitative and qualitative studies of antibody-induced mesangial cell damage in the rat.” Kidney International 32; 514-24 (1987); Jefferson JA et al, “Experimental mesangial proliferative glomerulonephritis (the anti Thy-1.1 model).” J. Nephrol 12; 297-307 (1999)). Another model for studying nephropathy involves immunization of animals with proximal tubular epithelial fraction (Fx1A) (Probetex, San Antonio, TX, USA). Immunization of rats with Fx1A induces an immune complex "membranous" nephritis characterized by subepithelial immune deposits and proteinuria that bear obvious similarities to the human disease (Heymann W et al., Proc Soc. Exp. Biol. Med. 100:660-64 (1959); Edgington TS et al., J Exp. Med. 127; 555 (1968)). Fx1A contains the large glycoprotein gp330 (megalin), a nephritogenic antigen produced by glomerular epithelial cells. Administration of anti-Fx1A antibody results in nephritis characterized by two phases: 1) a heterologous phase, which is an acute nephritis induced by exogenously administered antibody, and 2) a chronic autologous phase, which is characterized by the host response to exogenous (heterologous) sheep immunoglobulin injected into the glomerular structures. The anti-Fx1A antibody-induced membranous nephropathy model results in subepithelial deposits and proteinuria. The Heymann model of passive nephritis and the involvement of complement in this model have been reviewed elsewhere (Jefferson et al. “Experimental Models of Membranous Nephropathy,” Drug Discov. Today Dis. Models 7(1-2): 27-33 (2010)).

[000271] The present invention further includes methods and compositions effective for treating diseases caused by infectious agents. Infectious agents include, but are not limited to, bacterial, mycological, parasitic, and viral agents. Examples of suitable infectious agents include: Staphylococcus, Methicillin-Resistant Staphylococcus Aureus, Escherichia Coli, Streptococcaceae, Neisseriaaceae, Cocci, Enterobacteriaceae, Enterococcus, Vancomycin-Resistant Enterococcus, Cryptococcus, Histoplasmosis, Aspergillus, Pseudomonadaceae, Vibrionaceae, Campylobacter, Pasteurellaceae, Bordetella, Francisella, Brucella, Legionellaceae, Bacteroidaceae, Gram-Negative Bacilli, Clostridium, Corynebacterium, Propionibacterium, Gram-Negative Bacilli, Anthrax, Actinomyces, Nocardia, Mycobacterium, Treponema, Borrelia, Leptospira, Mycoplasma, Ureaplasma, Rickettsia, Chlamydiae, Candida, Systemic mycoses, opportunistic mycoses, Protozoa, Nematodes, Trematodes, Cestodes, Adenoviruses, Herpesviruses (including, for example, herpes simplex virus, Epstein-Barr virus, herpes zoster virus), poxviruses, papovaviruses, hepatitis viruses (including, for example, hepatitis B virus and hepatitis C virus), papillomaviruses, orthomyxoviruses (including, for example, influenza A, influenza B and influenza C), paramyxoviruses, coronaviruses, picornaviruses, reoviruses, togaviruses, flaviviruses, bunyaviruses, rhabdoviruses, rotavirus, respiratory syncytial virus, human immunodeficiency virus and retroviruses. Typical infectious diseases include, but are not limited to, candidiasis, candidemia, aspergillosis, streptococcal pneumonia, streptococcal skin and oropharyngeal diseases, sepsis caused by gram-negative bacteria, tuberculosis, mononucleosis, influenza, respiratory disease caused by respiratory syncytial virus, malaria, schistosomiasis and trypanosomiasis. The present invention includes methods and compositions effective for the treatment of infectious diseases, including, but not limited to, diseases caused by bacterial, mycological, parasitic and viral agents. Typical infectious diseases include, but are not limited to, candidiasis, candidemia, aspergillosis, streptococcal pneumonia, streptococcal skin and oropharyngeal diseases, gram-negative sepsis, tuberculosis, mononucleosis, influenza, respiratory syncytial virus disease, malaria, schistosomiasis, and trypanosomiasis.

[000272] According to another embodiment of the present invention, the stradomers of the present invention can be used in a priming system wherein blood is obtained from a patient and briefly contacted with the stradomer(s) for a period of time from about one half hour to about three hours before being reintroduced into the patient. In this form of cell therapy, the patient's own effector cells are exposed to the stradomer, which is attached to a matrix, in an ex vivo setting to modulate the effector cells by exposing the effector cells to the stradomer. The blood, including the modulated effector cells, is then infused back into the patient. Such a priming system can have numerous clinical and therapeutic applications.

[000273] The stradomers disclosed herein can also be readily used to alter the immune system response in a variety of situations to target specific changes in immune response profiles. Altering or modulating the immune response in a subject refers to increasing, decreasing, or altering the ratio or components of the immune response. For example, cytokine production or secretion levels can be increased or decreased as desired by targeting the complement system, along with an appropriate combination of FcγR with a stradomer that is designed to bind to the complement system and interact with said receptors. Antibody production can also be increased or decreased; the ratio of two or more cytokines or immune cell receptors can be altered; or the production of additional types of cytokines or antibodies can be induced.

[000274] In a preferred embodiment, the immune response of a subject with an autoimmune or inflammatory disease is altered by administering a therapeutically effective amount of a stradomer as described herein to said subject, wherein the therapeutically effective amount of the stradomer alters the immune response in said subject. In the best embodiment, said intervention allows for a cure of the disease or condition in the subject. The altered immune response may be an increased or decreased response and may include altered levels of cytokines, including levels of any of IL-6, IL-10, IL-8, IL-23, IL-7, IL-4, IL-12, IL-13, IL-17, TNF-alpha, and IFN-alpha. In a preferred embodiment of the present invention, the level of IL-6 or IL-8 is reduced in response to therapy. In a particularly preferred embodiment of the present invention, the levels of IL-6 and IL-8 are reduced in response to therapy. However, the present invention is not limited to any particular mechanism of action of the described biomimetics. The altered immune response may be an altered level of autoantibodies in a subject. The altered immune response may be an altered level of autoaggressive T cells in a subject.

[000275] For example, reducing the amount of TNF-alpha produced in autoimmune diseases may have a therapeutic effect. In practice, such a therapeutic effect can be applied to therapy with an antibody to TNF-alpha (e.g., REMICADE®), the clinical effectiveness of which has been demonstrated for the treatment of plaque psoriasis, rheumatoid arthritis, psoriatic arthritis, Crohn's disease, ulcerative colitis, and ankylosing spondylitis. These autoimmune diseases have different etiologies, but have common key immunological components of pathological processes associated with inflammation and immune cell activity. A stradomer designed to reduce the production of TNF-alpha will also be effective in the treatment of this and other autoimmune diseases. An altered immune response profile may also be a direct or indirect modulation to reduce the production of antibodies, such as autoantibodies directed against the subject's own tissues, or altered levels of autoaggressive T cells in the subject. For example, multiple sclerosis is an autoimmune disease involving autoreactive T cells that can be treated with interferon-beta therapy. See, for example, Zafranskaya M, et al., Interferon-beta therapy reduces CD4+ and CD8+ T-cell reactivity in multiple sclerosis, Immunology 2007 May;121(l):29-39-Epub 2006 Dec 18. A stradomer designed to reduce levels of autoreactive T cells would also be effective in the treatment of multiple sclerosis and other autoimmune diseases involving autoreactive T cells.

[000276] The stradomometers of the present invention can be used to modulate the expression of costimulatory molecules from immune cells, including dendritic cells, macrophages, osteoclasts, monocytes, or NK cells, or to inhibit the differentiation and maturation of said immune system cells or the secretion of cytokines, including interleukin-12 (IL-12), or to increase the secretion of cytokines, including interleukin-10 (IL-10) or interleukin-6 (IL-6) or IL-1-RA. One skilled in the art can also test the efficacy of immunologically active biomimetics by exposing an immune cell to an immunologically active biomimetic and measuring the modulation of immune cell function, wherein said immune cell is a dendritic cell, macrophage, osteoclast, or monocyte. According to one embodiment of the present invention, an immune cell is exposed to an immunologically active biomimetic in vitro, including an additional step of determining the amount of cell surface receptors or cytokine production, wherein a change in the amount of cell surface receptors or cytokine production indicates modulation of immune cell function. According to another embodiment of the present invention, an immune cell is exposed to an immunologically active biomimetic in vivo in an animal model of an autoimmune disease, including an additional step of assessing the degree of improvement in the autoimmune disease.

[000277] Stradomes according to the present invention can also be used as a component of a device. For example, in some embodiments, stradomes according to the present invention can be applied to a device, such as a medical implant. For example, stradomes can be applied to a coronary stent or as part of a nanoparticle therapy to enhance penetration and increase the duration of drug release, such as for intraophthalmic use for uveitis or macular degeneration. Stradomes according to the present invention can also be used as a component of a diagnostic. According to some embodiments of the present invention, one skilled in the art can personalize therapy by determining which patient will benefit most from the use of a stradomet. For example, one skilled in the art can expose a patient's immune cells to an immunologically active biomimetic and measure the modulation of immune cell activation or maturation using flow cytometry or a cytokine profile to identify patients with an appropriate therapeutic response.

[000278] All literature references cited herein are incorporated into this application by reference in their entirety.

EXAMPLES

EXAMPLE 1 - Stradomers preferentially binding to the complement system

[000279] Various approaches have been used to generate stradomers that preferentially bind to the complement system. Stradomers have been generated in which at least one point mutation that enhances binding to the complement system has been introduced into the Fc region. In particular, the following mutations were made at positions 267, 268, and 324 of the Fc domain of the GL-2045 stradomer described in WO 2012/016073: S267E, H268F, and S324T. In some stradomers, additional mutations were made to further enhance the degree of binding to the complement system, compared to binding to the canonical FcγR, by altering binding to FcγR and/or binding to FcRn. The amino acid sequences of representative stradomers are provided above in Table 1 and Table 2.

[000280] For each generated stradomer, the level of binding to canonical FcγR, FcRn at pH=7.4, binding to the C1q component, and CDC inhibition were determined, compared to the parent stradomer, G045c (IgG1 hinge region - CH2 IgG1 CH3 IgG1 - IgG2 hinge region). In addition, the binding of some compounds to other components of the complement cascade was assessed.

[000281] Binding of complement-preferential stradomers or the parent G045c stradomer to FcγRI, FcγRIIb, FcγRIIIa, FcγRIIa, or FcRn was assessed. RU values for dissociation were measured by biolayer interferometry using a ForteBio Octet instrument. Receptor proteins containing a polyhistidine tag were bound to a sensor tip in 1× kinetic assay buffer (ForteBio), and the rate of the forward receptor/protein reaction was measured by transferring the sensor tip into 1× kinetic assay buffer containing the selected purified stradomer. The rate of the reverse reaction was measured by transferring the sensor tip into 1× kinetic assay buffer, and the RU value was calculated from the measured maximal binding using ForteBio software. The biolayer interferometry method detects the binding of a ligand immobilized on the surface of a biosensor tip to an analyte in solution. The binding causes an increase in absorbance at the biosensor tip, resulting in a wavelength shift (detected as a response unit "RU"). The maximum binding level (RU _max ) is the maximum possible amount of sample bound when equilibrium is reached that saturates the amount of ligand on the sensor surface. RU 300 represents the binding of the residual sample after 300 seconds of dissociation and can be used to describe the rate of dissociation of the analyte from the ligand.

[000282] To characterize the compounds, this document presents the results of maximal binding by biolayer interferometry (RU _max ) compared to 5 Fc receptors, the results of binding to C1q by ELISA, and CDC inhibition. To illustrate the differences in dissociation rates, the RU values at 300 s for binding of the compounds compared to binding to 5 Fc receptors are also presented. For RU _max and RU 300, in each case, the absolute value was visually determined from the association and dissociation plot generated by ForteBio, and the response was then normalized to a 0-10 scale, where 0 represents no response and 10 represents the maximum response observed for a given receptor or ligand for all compounds tested at a given time, RU _max , or RU 300, respectively.

[000283] To assess binding to C1q, a 96-well plate was coated with C1q (Sigma, catalog #C1740, 1 μg/mL) overnight in PBS. After coating, the plates were washed 3 times with standard wash buffer (1× PBS + 0.05% Tween 20) and blocked with blocking buffer (1% BSA + 1× PBS + 0.05% Tween 20) for 2 h at room temperature. After blocking, the plates were incubated with compound diluted in blocking buffer at 100 μL/well and washed 3 times with standard wash buffer. C1q-linked binding was detected by incubation with biotinylated mouse anti-human IgG1 (catalog #555869, BD Biosciences) diluted 1:5000 and streptavidin-HRP (catalog #7100-05, Southern Biotech) (100 μl/well) for 1 h at room temperature, followed by three washes with wash buffer, then staining was developed for 15 min using a standard TMB-based method according to the manufacturer's protocol. Absorbance was recorded at 450 nm.

[000284] The results of the binding assay of G045c to an exemplary Fc receptor are shown in Figure 1.

[000285] G997 is a stradomer containing three mutations (S267E/H268F/S324T) introduced into the G045c backbone. As shown in Figure 2, the resulting stradomer exhibits increased binding to C1q compared to the parent G045c, as well as slightly decreased binding to FcγRIIa and FcγRIIIa compared to G045c, and has no effect on CDC inhibition. The results of the Fc receptor binding assessment of G997 are presented in Figure 3.

[000286] G998 was next constructed. G998 contains an additional mutation at amino acid position 297, compared to G997. Specifically, G998 contains four mutations (S267E/H268F/S324T/N297A) inserted into the G045c backbone. The triple mutation in G998 restored strong C1q binding and CDC inhibition; binding to the activating receptors FcγRIIa and FcγRIIIa was abolished, and binding to FcγRIIb was reduced, while binding to FcRn was not reduced, compared to the parental G045c stradomer (Figure 4A). Also unexpectedly, G998 binding to FcγRI was completely retained despite the N297A mutation. The results of a comparison of the RU300 values for binding of G997, G998, and G045c to canonical FcγR and FcRn are shown in Figure 4B. Given the aglycosylation that occurs with the introduction of the N297A mutation, the preservation of binding of G997 to the inhibitory receptor FcγRIIb was unexpected. Although binding to FcγRIIb was significantly reduced based on the RU _max values for G998 compared to G997, G998 dissociates very slowly from FcγRIIb based on RU300 values (Figure 4B). The results of G998 Fc receptor binding assessment are shown in Figure 5.

[000287] Specific compounds G1033 and G1022 were also created. Specifically, G1033 contains 6 mutations (S267E/H268F/S324T/N297A/L234A/L235A) introduced into the G045c backbone; and G1022 contains 7 mutations (S267E/H268F/S324T/N297A/E233P/L234V/L235A and G236 deletion) introduced into the G045c backbone. The RU _max results for G1033 and G1022 binding to canonical FcγR and FcRn receptors, binding to C1q, and CDC inhibition are shown in Figure 6A. G1033 binds to C1q and FcRn and inhibits CDC without significant binding to FcγRIIb or FcγRIIIa. Surprisingly, despite its deglycosylation due to the N297A mutation, it retains little binding to FcγRI and FcγRIIa. G1022 binds to C1q and inhibits CDC without significant binding to FcγRI or FcγRIIIa; it retains little binding to FcγRIIb and moderate binding to FcγRIIa and FcRn. The abolition of G1022 binding to FcγRI was unexpected due to the intrinsic avidity, even at low affinity, of stradomers for the high-affinity receptor FcγRI. Therefore, the abolition of FcγRI binding by the mutation combination in G1022 was unexpected based on the retained FcγRI binding in other stradomers that contain these mutations. For example, the mutation combination in G998 (S267E/H268F/S324T/N297A) does not result in loss of FcγRI binding. It is particularly unexpected that FcγRI binding can be abolished in a stradomer that retains low-affinity Fc receptor binding and CDC inhibition. A comparison of RU300 values for binding of G045c, G1022, and G1033 to canonical FcγR and FcRn receptors is shown in Figure 6B.

[000288] G1032 contains 5 mutations (S267E/H268F/S324T/L234A/L235A) introduced into the G045c backbone. The RU _max values for FcγR binding, RU _{max values} for FcRn binding, C1q binding, and CDC inhibition are shown in Figure 7A, and the RU300 values for FcR binding are shown in Figure 7B. Surprisingly, despite G1032 binding to C1q and CDC inhibition, strong binding was retained for all canonical Fc receptors despite the L234A and L235A mutations; moderately strong binding was retained for FcRn.

[000289] G1023 contains 6 mutations (S267E/H268F/S324T/E233P/L234V/L235A) and a deletion of G at position 236. The RU _max values for FcγR binding, RU _{max values} for FcRn binding, C1q binding, and CDC inhibition are shown in Figure 8A, and the RU300 values for FcR binding are shown in Figure 8B. G1023 bound C1q and inhibited CDC, while maintaining strong binding to all canonical Fc receptors, and maintaining moderately strong binding to FcRn. An unexpected finding was that despite G1023 binding to C1q and CDC inhibition due to the E223, L234V, and L235A mutations and G236 deletion (and in the absence of deglycosylation due to the N297A mutation), strong binding to all canonical Fc receptors was maintained.

[000290] G1006 contains 4 mutations (S267E/H268F/S324T/D265A). The RU _max values for FcγR binding, RU _{max values} for FcRn binding, C1q binding, and CDC inhibition are shown in Figure 9A, and the RU300 values for FcR binding are shown in Figure 9B. This stradomer bound C1q and inhibited CDC, while retaining binding to FcγRI, FcγRIIa, and to a lesser extent, FcγRIIb. These results were unexpected because, although the D265A mutation has been characterized as reducing binding to all canonical Fc receptors, it retained strong binding to FcγRI and retained moderate binding to FcγRIIa. Binding to FcγRIIIa was abolished and moderately tight binding to FcRn was retained (Figure 9A).

[000291] G1027 contains 5 mutations (P238D/S267E/H268F/S324T/N297A). The RU _max values of G1027 for FcγR binding, RU _{max values} for FcRn binding, C1q binding, and CDC inhibition are shown in Figure 10A, and the RU300 values for FcR binding are shown in Figure 10B. This stradomer bound C1q and inhibited CDC, maintained strong binding to FcγRI, exhibited reduced binding to FcγRIIa and FcγRIIb, and moderate binding to FcRn, while no binding to FcγRIIIa. An unexpected finding was that binding of this stradomer to canonical Fc receptors was not abolished despite the P238D and N297A mutations.

[000292] G1003 was constructed based on G019 (IgG2 hinge - IgG1 hinge - IgG1 CH2 IgG1 CH3) with the introduction of 3 mutations: S267E/H268F/S324T. The results for this stradomer are shown in Figures 11A and 11B. This stradomer bound C1q and inhibited CDC. Additionally, the stradomer exhibited strong binding to FcγRI and FcγRIIb; reduced binding to FcγRIIa; and moderately strong binding to FcRn. The parental G019 stradomer exhibited minimal binding to C1q and negligible inhibition of CDC. Therefore, the fact that introducing a triple mutation into this parent stradomer resulted in a compound with a similar overall structure and multimerization profile as the parent G019, but capable of binding to C1q and potently inhibiting CDC, was particularly surprising. Even more surprising is the fact that the same mutations (S267E/H268F/S324T) introduced into the GL-2045 backbone (to yield G998) showed significantly different binding potencies. G997 and G1003 contain similar domains with the same mutations and have similar binding profiles, yet the parent compounds (G045c and G019, respectively) differ significantly in C1q binding and CDC inhibition. The results of this comparison further highlight the unpredictability of this set of mutations when applied to a multimerizing stradomer.

[000293] G989 is based on G045c, contains 2 mutations (E233P/G236R) and was designed to reduce binding to canonical receptors. However, unexpectedly, this stradomer not only exhibited reduced binding to canonical receptors, but was also unable to bind to C1q and inhibit CDC (Figures 12A, 12B and 13).

[000294] Therefore, G990 was created, which is also based on G045c, but contains only one mutation (G236R). Surprisingly, the G236R mutation alone reduced binding to canonical receptors and maintained minimal binding to C1q (Figures 14A, 14B, and 15).

[000295] Based on these unexpected results, an attempt was made to further enhance C1q binding by engineering G994. G994 was engineered from G045c and contains 5 mutations: E233P/G236R/S267E/H268F/S324T. However, an unexpected finding was that while G994 restored the lost complement binding and CDC inhibition ability of its derived stradomer G989, the addition of the triple mutation S267E/H268F/S324T restored FcγRI and FcγRIIb binding (Figures 16A, 16B, and 17). Therefore, the addition of the triple mutation is reported to enhance complement binding of stradomer while simultaneously enhancing canonical Fc receptor binding.

[000296] Accordingly, G994 was then used as a platform to retain C1q binding and CDC inhibition and to achieve specificity for complement binding compared to canonical receptors. Specifically, G996 was generated, which is based on G045c and contains 4 mutations (G236R/S267E/H268F/S324T). Surprisingly, G996 acquired additional binding to FcγRIIa and FcγRIIb, but not binding to the activating receptor FcγRIIIa. Therefore, G996 binds C1q, inhibits CDC, and binds to all Fc receptors except FcγRIIIa (Figures 18A, 18B, and 19, upper panels). However, also unexpectedly, G996 did not exhibit binding to canonical receptors in mouse, making it the most suitable compound to assess true CDC inhibition in rodents (Figure 19, bottom three panels).

[000297] G1042 is based on G045 and contains 6 mutations (E233P, G236R, S267E, H268F, S324T and L328F). Therefore, G1042 differs from G994 by the presence of the L328F mutation, which is expected to increase binding to FcγRIIb. Surprisingly, G1042 not only strongly bound to FcγRIIb, but also to FcγRI and FcγRIIa (Figures 20A, 20B and 21).

[000298] G1043 and G1046 were generated from G045c and contain 5 mutations: P238D/D265G/S267E/H268F/S324T and P238D/D265W/S267E/H268F/S324T, respectively. For both of these stradomers, the addition of the P238D mutation to the other mutations unexpectedly resulted in binding to FcγRIIa (Figures 22-25). Both stradomers were expected to have increased binding to FcγRIIb and decreased binding to FcγRIIa due to the mutation at position 238; However, an unexpected finding was that G1043 exhibited binding to FcγRIIa and FcγRIIb (Figures 22A, 22B, and 23), and G1046 exhibited binding to FcγRIIa but not FcγRIIb (Figures 24A, 24B, and 25).

[000299] G1050 is based on G045c and contains 8 mutations and a deletion at position 236 (E233P/L234V/L235A/S267E/H268F/N297A/S324T/S328F and a deletion at position 236). These mutations were expected to reduce or eliminate binding to FcγRIIa and FcγRIII (due to the mutation at E233P/L234V/L235A and the deletion at position 236) but retain binding to FcγRIIb (due to the mutation at position 328); However, an unexpected finding was that G1050 exhibited binding to FcγRIIa and FcγRIIb but did not bind to FcγRI or FcγRIII (Figures 26A, 26B, and 27).

[000300] G1049 is based on the G019 backbone and contains 4 mutations (S267E/H268F/S324T/L328F). Therefore, compared to G1003, G1049 contains an additional mutation at L328F, which was expected to increase binding to FcγRIIb only. However, unexpectedly, G1049 exhibited strong binding to all canonical FcγR receptors (Figures 28A, 28B, and 29) and, also unexpectedly, exhibited strong binding to C1q and CDC inhibition, in contrast to G019. Therefore, it is particularly unexpected that strong binding to C1q and CDC inhibition can be achieved using point mutations in a stradomer containing the G019 backbone.

[000301] G1025 is based on G045c and contains 4 mutations (P238D/S267E/H268F/S324T). The mutation at position 238 was expected to increase binding to FγRIIb and decrease binding to FcγRI, FcγRIIa, and FcγRIIIa; however, unexpectedly, only binding to FcγRIIIa was decreased and the stradomer retained strong binding to FcγRIIb, FcγRI, and FcγRIIa (Figures 30A and 30B).

[000302] The summarized results of the study are presented in Table 3.

Table 3. Summary of the activity of stradomers that preferentially bind to the complement system

Binding to FcγRI Binding to FcγRIIa Binding to FcγRIIb Binding to FcγRIIIa Binding to FcRn Binding to C1q CDC inhibition G994 *** - * - *** *** * G996 *** *** *** - *** *** * G997 *** ** *** *** *** *** * G998 *** - * - *** *** * G1003 *** * *** ** *** *** * G1006 *** ** * - *** *** * G1023 *** *** *** *** *** *** * G1022 - ** * - *** *** * G1025 *** *** *** - *** *** * G1027 *** * * - *** *** * G1032 *** *** *** *** *** *** * G1033 * * - - *** *** * G1042 *** *** *** - *** *** * G1043 *** * ** - *** *** * G1046 *** * * - *** *** (*) G1049 *** *** *** ** *** *** * G1050 - *** *** - *** *** *

(*) Indicates marginal activity

(-) Indicates no binding.

EXAMPLE 2 - Stradomers that preferentially bind to the complement system exhibit preserved or enhanced binding to C1q

[000303] The results of the comparison of binding to C1q among stradomers G994, G996, G997, and G998, compared to parent GL-2045 or the negative Fc control, G001, are presented in Figure 31. Figure 31A presents the absorbance values with increasing concentration of these stradomers, and Figure 31B presents the log transformed data and _EC50 values for each of the stradomers tested. _EC50 values are presented in μg/mL. Taken together, the data presented indicate that stradomers G997, G998, G996, and G994 exhibit superior binding to C1q compared to G001 (the IgG1 Fc control region), and superior binding to C1q compared to the stradomer GL-2045 tested.

[000304] Taken together, the results of the study demonstrate that the stradomers of the present invention are capable of binding to C1q and exhibit minimal or no binding to FcγRI, FcγRII and/or FcγRIII.

EXAMPLE 3 - Binding of complement-preferential stradomers to component C3

[000305] The study was conducted to assess the binding of stradomers that preferentially bind to the complement system to the C3 component.

[000306] 96-well plates were coated with complement component C3 (Quidel, catalog #A401; 1 μg/ml in PBS) overnight at 4°C and then washed 3 times with 300 μl of 1×PBS + 0.1% Tween 20. Plates were blocked in 1×PBS + 2% BSA + 0.05% Tween 20 for 2 h at room temperature. The test compound (GL-2045, G001, G997, G998, G994, or G996) was incubated with bound C3 in a concentration range from 100 μg/mL and 1:1 to 0.78125 μg/mL in blocking buffer for 2 h at room temperature, followed by three washes (300 μL 1×PBS, 0.1% Tween-20). The compound interacting with C3 was detected using biotinylated mouse anti-human IgG1 (catalog #555869, BD) + streptavidin-HRP (catalog #7100-05, Southern Biotech) diluted 1:5000 (ea.) in PBS-BSA-T, which were mixed at a ratio of 1:10 and 1:50 at 100 μl/well, incubated at room temperature for 1 h, followed by four washes (300 μl 1× PBS, 0.1% Tween-20). Staining was developed using TMB substrate added at 100 µl per well for 20 min, the reaction was stopped by _adding 50 µl 1 M _H2SO4 , and the absorbance was recorded at 450/650 nm.

[000307] The results of the study are shown in Figure 32. The negative control G001 and the G996 stradomer did not bind to C3. G997 showed an intermediate level of binding to C3 compared to the negative control and the original stradomer GL-2045. G994, GL-2045, and G998 showed the highest level of binding to C3.

EXAMPLE 4 - Binding of stradomers that preferentially bind to the complement system to components C3b, C4 and C5

[000308] Studies were conducted to assess the binding of stradomers that preferentially bind to the complement system to components C3b, C4, and C5.

[000309] To assess C3b binding, 96-well plates were coated with complement component C3b (catalog #GWB-8BA994, GenWay Biotech; 1 μg/mL in PBS). 100 μL of complement component C3b was added to each well and incubated overnight at 4 °C, followed by three washes (300 μL 1× PBS, 0.1% Tween 20). Plates were blocked in blocking buffer (1× PBS + 2% BSA + 0.05% Tween 20) for 2 h at room temperature, followed by three washes (300 μL 1× PBS, 0.1% Tween 20). The test compound (G001, GL-2045, G997, G998, or G994) was reacted with C3b for 4 h at room temperature at concentrations ranging from 100 μg/mL and at a 1:1 dilution to 0.78125 μg/mL in blocking buffer, followed by three washes (300 μL 1× PBS, 0.1% Tween 20). Bound compound was detected using mouse anti-human IgG1 (catalog #555869, BD) + streptavidin-HRP (catalog #7100-05, Southern Biotech) diluted 1:5000 (ea.) in 100 μL blocking buffer for 1 h at room temperature. Staining was developed using TMB substrate for 20 min, the reaction was stopped by _adding 50 µl 1 M _H2SO4 , and absorbance was recorded at 450/650 nm.

[000310] To assess C4 binding, 96-well plates were coated with complement component C4 (catalog #A402, Quidel; 1 μg/ml in PBS). 100 μl of complement component C4 was added to each well and incubated overnight at 4 °C, followed by three washes (300 μl 1× PBS, 0.1% Tween 20). Plates were blocked in blocking buffer (1× PBS + 2% BSA + 0.05% Tween 20) for 2 h at room temperature, followed by three washes (300 μl 1× PBS, 0.1% Tween 20). The test compound (G001, GL-2045, G996, G997, G998, or G994) was reacted with C4 for 4 h at room temperature over a concentration range of 100 μg/mL and diluted 1:1 to 0.78125 μg/mL in blocking buffer, followed by three washes (300 μL 1× PBS, 0.1% Tween 20). Bound compound was detected using mouse anti-human IgG1 (catalog #555869, BD) + streptavidin-HRP (catalog #7100-05, Southern Biotech) diluted 1:5000 (ea.) in 100 μL blocking buffer for 1 h at room temperature. Staining was developed using TMB substrate for 20 min, the reaction was stopped by _adding 50 µl 1 M _H2SO4 , and absorbance was recorded at 450/650 nm.

[000311] To assess C5 binding, 96-well plates were coated with complement component C5 (catalog #A403, Quidel; 1 μg/ml in PBS). 100 μl of complement component C5 was added to each well and incubated overnight at 4 °C, followed by three washes (300 μl 1× PBS, 0.1% Tween 20). Plates were blocked in blocking buffer (1× PBS + 2% BSA + 0.05% Tween 20) for 2 h at room temperature, followed by three washes (300 μl 1× PBS, 0.1% Tween 20). The test compound (G001, GL-2045, G996, G997, G998, or G994) was reacted with C5 for 4 h at room temperature over a concentration range of 100 μg/mL and diluted 1:1 to 0.78125 μg/mL in blocking buffer, followed by three washes (300 μL 1× PBS, 0.1% Tween 20). Bound compound was detected using mouse anti-human IgG1 (catalog #555869, BD) + streptavidin-HRP (catalog #7100-05, Southern Biotech) diluted 1:5000 (ea.) in 100 μL blocking buffer for 1 h at room temperature. Staining was developed using TMB substrate for 20 min, the reaction was stopped by _adding 50 µl 1 M _H2SO4 , and absorbance was recorded at 450/650 nm.

[000312] The results of the studies are shown in Figure 33 (complement component C3b), Figure 34 (complement component C4), and Figure 35 (complement component C5). The negative control G001 did not bind to C3b, C4, or C5. The parent stradomer GL-2045 bound to C5 but did not exhibit binding to C3b or C4. In contrast, each of the complement-preferential stradomers G994, G997, and G998 bound to C4 as well as C5.

EXAMPLE 5 - Treatment of Nephritis Using Complement-Preferential Stradomers in a Thy 1 Antibody-Induced Nephritis Model

[000313] Stradomers that preferentially bind to the complement system were evaluated in a model of anti-Thy-1-induced nephritis (anti-Thy 1-induced mesangial proliferative glomerulonephritis). In this model, an anti-thymocyte antibody (ATS) reacts with the Thy-1 surface antigen present on rat mesangial cells (Yamamoto 1987 and Jefferson 1999). Administration of ATS induces complement-dependent mesangiolysis followed by rapid development of mesangial proliferative glomerulonephritis that peaks within 5 days after injection and then resolves over time.

[000314] Disease was induced on day 0 by injection of mouse anti-rat CD90 (Thy1.1) antibody (Cedar Lane) into Wistar rats (n=8) to induce glomerulonephritis. On days 0, 2, 4, and 6, animals were treated with 40 mg/kg G998, 80 mg/kg G998, 80 mg/kg G994, or 80 mg/kg G1033. Unaffected controls did not receive anti-Thy 1 antibody or other treatment. The positive control, tacrolimus, was administered at 1 mg/kg intramuscularly daily beginning on day -9 before antiserum injection. Day 0 dosing was administered 4 hours before antiserum injection. Urine samples were collected before dosing and on days 3, 5, 7, and 9 after antiserum injection. Kidneys were collected from rats at the end of the study and fixed in 10% formalin for histological analysis. Serum was collected for serum BUN analysis.

[000315] The results of the study are shown in Figures 37, 38A, 38B, 39, and 40; and Table 4. Figure 37, in the table below the graph, provides the P values relative to the PBS-treated control mice. Each of the three test compounds, G994, G998, and G1033, significantly reduced proteinuria. Figures 38A and 38B show the histological analysis results of the lesions in the PBS-treated control mice (Figure 38A) and in the mice treated with 40 mg/kg G998 (Figure 38B). Table 4 and Figure 39 show the quantitative histological analysis results for each animal in the PBS-treated control group (represented as Group 2 in Table 3) and in the group treated with 40 mg/kg G998 (represented as Group 4 in Table 3). The results shown in Table 4 are presented in graphical form in Figure 39.

Table 4. Results of quantitative histological study in the model of nephritis induced by antibodies to Thy-1

CKD-GLI-1 Kidneys Group Animals Glomerulonephritis Degeneration of tubules Dilation of tubules CD-68
glomeruli CD-68 Interstitium of tubules 2 2-9 4 3 3 3 3 2-10 4 2 2 3 2 2-11 2 0 0 3 1 2-12 3 2 2 3 3 2-13 4 3 2 4 4 2-14 4 1 1 4 2 2-15 4 4 3 4 4 2-16 4 3 2 4 3 Average
Std. dev. of mean 3.63
0.74 2.25
1.28 1.88
0.99 3.50
0.53 2.75
1.04 4 4-25 1 0 0 3 1 4-26 1 1 0 4 1 4-27 0 1 0 3 1 4-28 0 1 0 4 1 4-29 0 0 0 3 1 4-30 0 0 0 4 1 4-31 0 0 0 3 1 4-32 3 1 0 3 1 Average
Std. dev. of mean 0.63
1.06 0.50
0.53 0,00
0,00 3.38
0.52 1.00
0,00 t-test 2.20447·10 ^-5 0.00570721 0.00106271 0.641986775 0.002007834

0 = sample tested, no results, 1 = minimal, 2 = slight, 3 = moderate, 4 = severe, P = present, - = not present.

[000316] On day 9, serum was collected from each animal and analyzed for blood urea nitrogen (BUN). BUN levels were significantly reduced in each of the animals treated with the complement-preferential stradomers compared to the diseased control animals treated with PBS, indicating an increased glomerular filtration rate and, therefore, improvement in disease progression (Figure 40).

EXAMPLE 6 - Treatment of nephritis using stradomers that preferentially bind to the complement system in the Heymann model of passive nephritis

[000317] Disease was induced in rats on day 0 by injection of Fx1a antiserum (catalog number PTX-002S, Probetex). G998 was administered on day 0 at 60 mg/kg two hours before antiserum injection (day 0 dosing) or on day 1 one day after antiserum injection (day 1 dosing), with dosing continued twice weekly for 3 weeks. Control mice received Fx1a antiserum vehicle only. Urine was collected before dosing and at 1, 2, and 3 weeks after antiserum injection, and proteinuria was assessed.

[000318] The results of the study are shown in Figure 41. By day 21, both G998 groups showed a significant reduction in proteinuria compared to control animals (p = 0.0220 for day 0 in the G998 group and p = 0.0145 for day 1 in the G998 group).

EXAMPLE 7 - Drug levels and functional half-life of stradomer compounds preferentially binding to the complement system in rats

[000319] A study was conducted to evaluate the half-life of the complement-preferential stradomers G994, G998, and G1033 in rats following a single dose. Functional half-life was measured by assessing complement activity in blood samples collected at various time points. Blood samples were also measured to assess levels of target drug, complement factor C1q, and to assess the C1q-bound fraction of drug.

[000320] Sprague-Dawley rats (4 animals per group) received a single 60 mg/kg dose of G994, G998, or G1033 intravenously. Blood samples (1.2-1.4 mL) were collected from each animal by retro-orbital blood draw into pre-chilled heparinized tubes prior to dosing and at 1, 4, and 8 hours after test compound administration. Blood samples were also collected on days 1, 2, 4, 7, and 10.

[000321] Drug levels were measured in plasma samples using ELISA. In a quantitative ELISA assay, the IgG1 Fc region of G994, G998, G1033 was reacted with anti-human IgG Fc antibody (catalog # MA1-83240, Thermo) that was adsorbed on the plate surface. After removing unbound sample proteins by washing, biotinylated anti-human IgG1 antibody (catalog # 555869, BD) conjugated to streptavidin-HRP (catalog # 7100-05, Southern Biotech) was added. The HRP-conjugated antibody forms a complex with pre-bound IgG1. The complex was quantified by adding a chromogenic substrate (TMB, catalog # 555214, BD). The amounts of G994, G998, and G1033 in serum samples were interpolated from a standard curve obtained for the same compound mixed in monkey serum and corrected for sample dilution.

[000322] Complement activity was measured in rat plasma samples using an in vitro quantitative complement lysis assay. Briefly, CD20-expressing Will2 cells were reincubated at 37°C with anti-CD20 monoclonal antibody for 20 min in culture media, after which the cells were pelleted and resuspended in fresh media. The cells were re-distributed into 96-well plates, rat plasma from various time points was then added to the cell suspension, and the plates were incubated at 37°C for 3 h. CytoTox Quantitative Assay Reagent (Promega) was added to each well, and the plates were incubated in the dark for 15 min at room temperature. Luminescence was recorded on a Promega GloMax luminometer, and the percent cell death was calculated. Complement activity was measured at different plasma concentrations in a quantitative assay, and values for 2% and 4% plasma were used for analysis.

[000323] The percentage of complement activity in each sample was calculated relative to the complement activity in the pre-dose samples after subtracting the values for the no-antibody control sample.

[000324] The results of the study are shown in Figures 42-45. Figure 42 shows the half-life data for G994, G998, and G1033. G994 had a significantly longer half-life than the other two compounds tested in the same experiment. G994 abolished complement activity in the blood of rats for an extended period of time. Complement activity after dosing with G994 remained low for up to 96 hours (4 days) and was approximately 50% of normal levels by day 10. Compounds G998 and G1033 had slightly shorter half-lives in rats. Two days after dosing with G998, complement activity was approximately 50% of pre-dose activity. For G1033, the functional half-life ranged from 2 to 4 days.

[000325] Figure 43 shows data on complement activity in rat blood following administration of a single dose of G994, G998, or G1033. Complement activity is expressed as percent cell death as measured at 2% or 4% plasma concentration in a quantitative assay. Figures 44A and 44B show the correlation between drug levels and complement activity for each of the stradomers that preferentially bind to the complement system, G994, G998, and G1033. Figure 44A shows all data points collected in the study, and Figure 44B shows data points for low drug concentrations (0-250 μg/mL for G994 and G998, and 0-150 μg/mL for G1033). The drug levels required to eliminate complement activity in rat blood were estimated from the measured abscissa intercepts. For G994, the abscissa intercepts were 164 and 170 μg/mL for 2% and 4% serum. The R ² values for the correlation were 0.696 and 0.558. For G998, the abscissa intercepts were 158 and 193 μg/mL with R ² values of 0.519 and 0.560 for 2% and 4% serum. For G1033, the abscissa intercepts were 88 and 109 μg/mL with R ² values of 0.612 and 0.648.

[000326] Based on the calculated values of the segments on the abscissa axis, it was determined that approximately 100-200 μg/ml of compound G994 and G998 are required to eliminate the activity of the complement system. For compound G1033, the approximate amount required to eliminate the activity of the complement system is 100 μg/ml.

EXAMPLE 8 - Pharmacokinetic study in cynomolgus monkeys

[000327] The half-life of G994, G998 or G1033 in cynomolgus monkeys was studied following a single dose of stradomer compounds that preferentially bind to the complement system.

[000328] Cynomolgus monkeys (3 animals per group) were administered a single 28 mg/kg dose of G994, G998, or G1033 subcutaneously. Blood samples (approximately 3 mL) were collected in serum separator tubes from each animal prior to dosing and at 1, 2, 4, 12, 24, 48, 72, 144, 216, and 312 hours post-dose. Drug levels were measured in serum samples by ELISA. In this quantitative ELISA study, IgG1 Fc in G994, G998, G1033 reacted with anti-human IgG Fc antibody (catalog # MA1-83240, Thermo) that was adsorbed to the surface of the plates. After removing unbound sample proteins by washing, biotinylated anti-human IgG1 (catalog #555869, BD) conjugated to streptavidin-HRP (HRP, catalog #7100-05, Southern Biotech) was added. The HRP-conjugated antibody forms a complex with previously bound IgG1. The complex was quantified by adding a chromogenic substrate (TMB, catalog #555214, BD). The amounts of G994, G998, and G1033 in serum samples were interpolated from standard curves generated for the same compound mixed in monkey serum and corrected for sample dilution.

[000329] Complement activity was measured in cynomolgus macaque serum samples using an in vitro quantitative complement-dependent cell death assay. Briefly, CD-20-expressing Will2 cells were incubated at 37°C with anti-CD-20 monoclonal antibody for 20 min in culture media, after which the cells were pelleted and resuspended in fresh media. The cells were distributed into 96-well plates, serum samples collected at various time points were added to the cell suspension, and the plates were incubated at 37°C for 3 h. CytoTox Quantitative Assay Reagent (Promega) was added to each well, and the plates were incubated in the dark for 15 min at room temperature. Luminescence was recorded on a Promega GloMax luminometer, and the percent cell death was calculated. Complement activity was measured at different plasma concentrations in a quantitative assay, and values for 2% and 4% plasma were used for analysis.

[000330] The percentage of complement activity in the sample was calculated compared to the complement activity in the pre-dose samples after subtracting the value for the control sample containing no antibody.

[000331] The results of the study are shown in Figures 45-47. Figure 45 shows the drug level for each of the tested complement preferential stradomers following administration of a single dose of the compound. G994 achieved significantly lower peak concentrations but had a significantly longer half-life than G998 and G1033. Figure 46 shows the results of complement activity (% cell death) as a function of time following administration of a single dose of G994 (left panel), G998 (middle panel), or G1033 (right panel). Figures 47A and 47B show the correlation between drug level and complement activity for each of the tested compounds in the study. Figure 47A shows all data points collected in the experiment, and Figure 47B shows the first portion of the curve with lower drug concentrations.

[000332] Studies will also be conducted to determine the levels of C4a, C3a, and C5a, which are indicative of complement activation following a single dose of G994, G998, and G1033. The concentrations of each of C4a, C3a, and C5a will be determined from serum samples collected as described above using a commercially available ELISA kit. The results of these studies will show a decrease in complement activation as a function of time following a single dose of G994, G998, or G1033, as determined by a decrease in the concentration of C4a, C3a, and/or C5a.

[000333] Analysis of residual complement activity following a single dose of a compound with preferential binding to the complement system indicates that G994 eliminates complement activity in the blood of cynomolgus monkeys over an extended period of time. Complement activity after dosing with G994 remained at a low level for up to 312 hours (13 days). Compounds G998 and G1033 had slightly shorter half-lives in cynomolgus monkeys. For G998, approximately 50% of the pre-dose complement activity level remained at day 6. For G1033, the functional half-life appeared to be between 9 and 13 days. For all compounds, complement activity persisted in the blood for approximately 4 hours. Without wishing to be bound by any theory, the inventors believe that this result may be due to the relatively slow absorption of the compounds following subcutaneous dosing.

[000334] A drug-complement correlation analysis (Figure 47B) was used to estimate the drug levels required to abolish complement activity in the blood of cynomolgus monkeys. Based on the calculated intercept values, approximately 150 μg/mL of G994 was found to be required to abolish complement activity. For G998 and G1033, the approximate value was 300-400 μg/mL.

[000335] C1q levels were measured in serum samples from cynomolgus monkeys treated with G994, G998, or G1033 as described above. Quantification was performed using a C1q-specific ELISA. Serum samples were also assayed using co-immunoprecipitation to determine the fraction of C1q that was bound to the compounds. To determine the fraction of C1q bound to the drug, a C1q ELISA was performed using an anti-C1q capture antibody followed by coupling a detection antibody to the human IgG1 portion of the drugs with preferential binding to the complement system. This allows for the capture and quantification of the drug-C1q complex. The supernatant from the ELISA mentioned above was then run in a C1q ELISA using an anti-C1q antibody as both the capture and detection antibody to quantify unbound C1q in the samples.

EXAMPLE 9 - Binding of the complement-preferential stradomers G994, G996 and G1033 to components C3, C3b, C4 and C5

[000336] Additional studies were performed to assess the binding of complement-preferential stradomers to complement components C3, C4, C3b, and C5. Ninety-six-well plates were coated with complement component (catalog #A401, Quidel, 1 μg/mL for C3; catalog #GWB-8BA994, Genway Biotech, 1 μg/mL for C3b; catalog #A402, Quidel, 1 μg/mL for C4; and A403, Quidel, 1 μg/mL for C5) in 100 μl PBS per well overnight at 4°C, followed by three washes (300 μl 1× PBS, 0.1% Tween 20). The plates were blocked in blocking buffer (1× PBS + 2% BSA + 0.05% Tween-20) for 2 h at room temperature, followed by three washes (300 μl 1× PBS, 0.1% Tween-20). The compound was reacted with the complement component for 2 h at room temperature in the concentration range from 100 μg/ml and in a 1:1 dilution in blocking buffer, followed by three washes (300 μl 1× PBS, 0.1% Tween-20). Bound compound was detected using biotinylated mouse anti-human IgG1 (catalog #555869, BD) + streptavidin-HRP (catalog #7100-05, Southern Biotech) diluted 1:5000 in 100 µl blocking buffer for 1 h at room temperature. Color development was developed using TMB reagent for 20 min at room temperature, the reaction was stopped by adding 50 µl ₁ M _H2SO4 , and absorbance was recorded at 450/650 nm.

[000337] The results of the study are shown in Figure 49. All of the complement preferential binding stradomers, G994, G998, and G1033, bound to each of the complement factors C3, C3b, C4, and C5. In addition, G1033 exhibited significantly greater binding to these complement factors in a direct binding assay compared to GL-2045 or the other complement preferential binding stradomers, G994 and G998.

EXAMPLE 10. Induction of cytokine production by complement-preferential stradomers G994, G996 and G1033

[000338] Additional studies were performed to evaluate the induction of cytokines by G994 and G1033 from isolated peripheral blood mononuclear cells (PBMCs). Human blood was collected in 100 mL heparin-coated blood collection tubes. Blood samples were aliquoted in 10 mL aliquots into 50 mL conical tubes and diluted with 10 mL PBS, followed by gentle mixing. Twenty mL of the diluted blood sample was added to 15 mL Ficoll-Paque PLUS in 50 mL conical tubes and centrifuged at 400 g for 30-40 min at 18-20 °C. After centrifugation, the supernatant was removed with a Pasteur pipette, leaving an intact lymphocyte layer at the interface. The lymphocyte layer was transferred to a clean 50 mL conical tube and 30 mL PBS was added. The diluted lymphocyte layer was centrifuged at 60-100g for 10 min at 18-20°C. After centrifugation, the supernatant was removed and the cells were washed in 30 ml PBS and centrifuged again. The cell pellet was resuspended in 1-2 ml RPMI medium supplemented with 10% fetal bovine serum (FBS). Cells were counted, aliquoted into tubes at 5 × 10 ⁶ cells/tube and incubated with the test material (G019, G994 or G1033) for 4 or 24 h. Cytokine levels after 4 and 20 h were determined using commercial ELISA kits.

[000339] The results of this study are shown in Figures 55A and 55B. Neither G1033 nor G994 induced the production of the proinflammatory cytokine, TNF-α (Figure 55B). Likewise, neither of these two compounds induced the production of the anti-inflammatory cytokine IL-1Rα (Figure 55A). This result is likely due to the inability of G1033 and G994 to bind to canonical FcγRs, since G019 (which binds to canonical receptors but not complement) induced both cytokines.

EXAMPLE 11 - Use of complement-preferential stradomers to treat established arthritis

[000340] A collagen-induced arthritis (CIA) assay was conducted to determine the efficacy of G994, G998, and G1033 in inhibiting the edema that develops in established collagen type II-induced arthritis in female Lewis rats. Rats were injected intradermally/subcutaneously (i.d. or s.c.) with porcine collagen type II to induce arthritis. Rats were then injected intravenously (i.v.) on days 11, 13, and 14 with phosphate-buffered saline (PBS control), G994 (40 mg/rat), G998 (40 mg/rat), or G1033 (40 mg/rat). As a positive control, the reference compound dexamethasone (Dex, 0.075 mg/kg) was administered orally every day (QD) on days 11-16. The study was conducted with a mask of the treatment regimen, which was removed after completion of the study. Efficacy assessment was based on ankle caliper measurements (Figure 57).

[000341] The results of these studies indicate that the preferentially binding complement stradomers G994, G998, and G1033 reduce inflammation in a Lewis rat model of CIA.

EXAMPLE 12 - Stradometers derived from G994 or G998

[000342] Additional stradomers were generated using the G994 sequence as a base sequence to evaluate the function of mutations at specific residues and to identify and validate additional stradomers that preferentially bind to the complement system. G994 (SEQ ID NO: 10 or 11) is a G045c-based stradomer containing point mutations at positions 233, 236, 267, 268, and 324. Specifically, G994 contains the following mutations: E223P, G236R, S267E, H268F, and S324T.

[000343] To investigate the function of the mutations at position 236, stradomers were generated in which the wild-type amino acid (serine) was present at position 267, position 236 was mutated to arginine (as in G994) or an arginine-like residue, and the remaining G994 mutations (E233P, H268F, and S324T) were also present. The generated compounds are presented in Table 5 below:

Table 5. Stradomers preferentially binding to the complement system containing the G994 backbone and additional mutations at position 236

Stradometer SEQ ID NO Mutated amino acids 1103 65 E233P, G236E, 267S, H268F, S324T 1088 28 E233P, G236Q, 267S, H268F, S324T 1089 29 E233P, G236R, 267S, H268F, S324T 1104 66 E233P, G236D, 267S, H268F, S324T 1082 30 E233P, G236H, 267S, H268F, S324T 1105 31 E233P, G236N, 267S, H268F, S324T 1106 32 E233P, G236K, 267S, H268F, S324T

[000344] An unexpected finding was that mutation at position 236 to arginine or an amino acid similar to arginine altered canonical receptor binding and complement binding compared to G994. Mutation at position 236 to glutamic acid or aspartic acid (G1103 and 1104, respectively) resulted in high levels of canonical FcγR binding, retention of high levels of C1q binding, and inhibition of CDC. Therefore, G1103 and G1104 can be considered universal stradomers with increased canonical and C1q binding compared to the parent stradomer (G045c). In contrast, mutation at position 236 to glutamine, histidine, or asparagine (G1088, 1082, and 1105, respectively) resulted in decreased canonical binding while maintaining high levels of C1q binding and CDC inhibition. Compounds 1088, 1082, and 1105 can therefore be considered complement-preferential stradomers with similar therapeutic utility as G994, G998, and G1033. Mutation at position 236 to lysine (G1106) abolished C1q and canonical binding except for FcγRI and resulted in a failure to inhibit CDC.

[000345] To investigate the function of the mutations at position 267, stradomers were generated in which the wild-type amino acid (glycine) was present at position 236, position 236 was mutated to glutamic acid (as in G994) or a residue similar to glutamic acid, and the remaining G994 mutations (E233P, H268F, and S324T) were also present. The generated compounds are presented in Table 6 below.

Table 6. Stradomers preferentially binding to the complement system containing the G994 backbone and additional mutations at position 267

Stradometer SEQ ID NO Mutated amino acids 1102 67 E233P 236G, S267Q, H268F, S324T 1100 33 E233P 236G, S267R, H268F, S324T 1101 68 E233P 236G, S267D, H268F, S324T 1125 69 E233P 236G, S267H, H268F, S324T 1108 34 E233P 236G, S267N, H268F, S324T 1109 70 E233P 236G, S267E, H268F, S324T 1084 35 E233P 236G, S267K, H268F, S324T

[000346] Mutation at position 267 to asparagine (G1108) resulted in decreased canonical binding while maintaining high C1q binding and CDC inhibition. Therefore, G1108 can be considered a complement-preferential stradomer with similar therapeutic utility as G994, G998, and G1033. However, mutation at position 267 to glutamine, aspartic acid, histidine, or glutamic acid (G1102, 1101, 1125, and 1109, respectively) resulted in increased canonical binding while maintaining high C1q binding and CDC inhibition. Therefore, G1102 1101, 1125 and 1109 can be considered as universal stradomers with increased binding to C1q compared to the parent stradomer. Alternatively, mutation at position 267 to arginine or lysine (1100 and 1084, respectively) resulted in decreased canonical binding, decreased binding to C1q and failure to inhibit CDC.

[000347] To examine the function of the combination of mutations at positions 236 and 267, stradomers were generated in which position 236 was mutated to arginine (as in G994) or an amino acid structurally similar to arginine, and position 267 was mutated to glutamic acid (as in G994) or an amino acid structurally similar to glutamic acid, with the remaining G994 mutations (E233P, H268F, and S324T) also present. The generated compounds are presented in Table 7 below.

Table 7. Stradomers preferentially binding to the complement system containing the G994 backbone and additional mutations at positions 236 and 267

Stradometer SEQ ID NO Mutated amino acids G1110 36 E233P, G236D, S267R, H268F, S324T G1111 71 E233P, G236D, S267Q, H268F, S324T G1112 37 E233P, G236R, S267R, H268F, S324T G1113 38 E233P, G236R, S267D, H268F, S324T G1114 72 E233P, G236Q, S267D, H268F, S324T G1115 39 E233P, G236E, S267R, H268F, S324T G1116 40 E233P, G236H, S267K, H268F, S324T G1117 73 E233P, G236D, S267D, H268F, S324T G1118 41 E233P, G236Q, S267Q, H268F, S324T G1119 42 E233P, G236R, S267K, H268F, S324T G1120 43 E233P, G236R, S267Q, H268F, S324T G1121 44 E233P, G236D, S267K, H268F, S324T G1122 45 E233P, G236H, S267Q, H268F S324T G1123 46 E233P, G236Q, S267R, H268F, S324T G1124 47 E233P, G236K, S267K, H268F, S324T G1128 48 E233P, G236K, S267N, H268F, S324T G1129 49 E233P, G236N, S267E, H268F, S324T G1130 50 E233P, G236N, S267K, H268F, S324T G1131 51 E233P, G236R, S267N, H268F, S324T

[000348] An unexpected finding was that simultaneous mutations at both positions 236 and 267 had different effects compared to mutations at either position independently. The combination G263N/S267E, when applied to E233P/H268F/S324T (G1129), resulted in decreased binding to FcγRIIIa and preserved or improved binding to FcγRI, FcγRIIa, and FcγRIIb. Therefore, G1129 can be considered a stradomer that preferentially binds to the complement system. The combination of G236D/S267Q, G236Q/S267D and G236D/S267D point mutations (G1111, G1114 and G1117, respectively) resulted in increased canonical binding compared to the parent stradomer or G994, and also resulted in maintenance of high binding to C1q and inhibition of CDC. Therefore, G1111, G1114 and G1117 can be considered as universal stradomers with the potential to enhance therapeutic efficacy compared to the parent stradomer G045c. In another embodiment, all combinations of G236D/S267R, G236R/S267R, G236E/S267R, G236H/S267K, G236R/S267K, G236R/S267Q, G236D/S267K, G236H/S267Q, G236Q/S267R, G236N/S267K, or G236R/S267N (G1110, G1112, G1115, G1116, G1119, G1120, G1121, G1122, G1123, G1130, and G1131, respectively) resulted in decreased canonical binding and decreased binding to C1q, as well as decreased CDC inhibition. In some cases, such as the G236K/S267K and G236K/S267N combination (G1124 and G1128, respectively), point mutations increased binding to canonical receptors and decreased binding to C1q.

[000349] Additional stradomers were also generated using the G998 sequence as a base sequence and a mutation at position 299 (T299A) to assess the function of mutations at specific residues as applied to this stradomer. G998 (SEQ ID NO: 14 or 15) is a stradomer containing a G045c backbone and point mutations at positions 267, 268, 297, and 324. Specifically, G998 contains the following mutations: S267E, H268F, N297A, and S324T. The consensus glycosylation site is NXT, in which N is residue 297. The residue at position 297 covalently links sugar residues. The T299A mutation of the additional stradomers described herein was designed to produce an aglycosylated protein; The mutation of the amino acid at position 299 is intended to eliminate the consensus glycosylation site such that the protein will not be glycosylated but will contain an intact residue 297. Accordingly, the compounds shown in Table 8 below (in which the amino acid at position 267 was replaced by glutamic acid (as in G998) or an amino acid sequence similar to glutamic acid, or the residue was not mutated and was a wild-type serine residue; the amino acids at positions 268 and 324 were mutated as in G998; the amino acid at position 297 was not mutated, and a mutation from threonine to alanine was added at position 299) were designed and tested.

Table 8. Stradomers containing the G998 backbone, a mutation at position 299, and additional mutations at position 267

Stradometer SEQ ID NO Mutated amino acids 1071d2 52 267S, H268F, S324T, T299A 1068 74 S267E, H268F, S324T, T299A 1094 75 S267Q, H268F, S324T, T299A 1092 76 S267D, H268F, S324T, T299A 1096 53 S267R, H268F, S324T, T299A 1107 77 S267H, H268F, S324T, T299A 1093 54 S267K, H268F, S324T, T299A 1095 55 S267N, H268F, S324T, T299A

[000350] Unexpectedly, inclusion of the T299A mutation, which should result in abolition of canonical binding, affected only FcγR binding, which was characteristic of the S267R (G1096) or S267K (G1093) mutation. In both G1096 and G1093, binding to C1q was also reduced, and neither compound had the ability to inhibit CDC, likely due to a lack of binding to C1q. Other compounds containing the T299A mutations (1071d2, G1068, G1094, G1092, and G1107) exhibited enhanced binding to FcγR and C1q. Therefore, G1068, G1094, G1092 and G1107 can be considered as universal stradomers with the potential to enhance therapeutic efficacy compared to the parent stradomer G045c. An unexpected finding was that strong C1q binding does not necessarily correlate with CDC inhibition, since 1712d2 could not inhibit CDC. G1095 contains the T299A and S267N mutations, the combination of which resulted in slightly reduced binding to canonical receptors while maintaining C1q binding and CDC inhibition. Therefore, G1095 can be considered as a complement-preferential stradomer with similar therapeutic utility as G994, G998 and G1033.

[000351] Additional G998-based stradomers were generated and tested to further evaluate the function of the 267 mutations as applied to these stradomers. These mutated variants contain mutations at positions 268, 324, and 297, as in G998, and contain a wild-type serine residue or amino acid substitution at position 267, as shown in Table 9 below. These stradomers do not contain a mutation at position 299.

Table 9. Stradomers containing the G998 backbone, a mutation at position 299, and additional mutations at position 267

Stradometer SEQ ID NO Mutated amino acids 1069 56 S267S, H268F, S324T, N297A 1070 57 S267K, H268F, S324T, N297A 1132 58 S267R, H268F, S324T, N297A 1074 59 S267D, H268F, S324T, N297A 1075 60 S267Q, H268F, S324T, N297A

[000352] As expected, the stradomers in Table 9 exhibited reduced binding to the canonical receptors due to the N297A mutation resulting in aglycosylation. Surprisingly, the wild-type serine residue at position 267 (G1069), S267D, or S267Q point mutations (G1074 and G1075, respectively) also resulted in increased binding to C1q and decreased canonical binding. Therefore, G1069, G1074, and G1075 can be considered as complement-preferential stradomers with similar therapeutic utility as G994, G998, and G1033. Mutations S267K (G1070) and S267R (1132) resulted in not only decreased binding to FcγR, but also decreased binding to C1q and failure to inhibit CDC.

[000353] Compounds derived from G994 and G998 were separated on a gel to assess multimerization. 3 μg of each sample was separated at 150 V for approximately 1.2 h. 20 mM iodoacetamide was added and the samples were then incubated for 10 min before loading onto the gel. The results are shown in Figures 48A-G and indicate that, similar to G994 and G998, compounds G1103, G1088, G1089, G1104, G1082, G1105, G1106, G1102, G1100, G1101, G1125, G1108, G1109, G1084, G1107, G1110, G1111, G1112, G1114, G1115, G1116, G1117, G1118, G1119, G1120, G1121, G1122, G1123, G1124, G1128, G1129, G1130, G1131, G1071d2, G1068, G1094, G1092, G1096, G1093, G1095, G1069, G1070, G1132, G1075 and G1075 form multimers.

[000354] For each stradomer in Tables 5-9, the level of canonical FcγR binding, complement component C1q binding, and CDC inhibition were determined according to the methods described in Example 1. The results are summarized in Table 10.

Table 10. Summary of the activity of stradomers derived from G994 or G998

Binding to FcγRI Binding to FcγRIIa Binding to FcγRIIb Binding to FcγRIIIa Binding to C1q CDC inhibition (EC ₅₀ , μg/ml) G1103 *** *** *** *** High 5 G1088 *** ** * ** High 5 G1089 ** - * - Low 100 G1104 *** *** *** *** High 5 G1082 *** ** - *** High 5 G1105 *** ** ** ** High 5 G1106 *** - - - Low 100 G1102 *** *** *** *** High 7.5 G1100 *** - - *** Low NO G1101 *** *** *** *** High 5 G1125 *** ** ** ** High 5 G1108 *** * * *** High 5 G1109 *** *** *** *** High ND G1084 *** - - * Low NO G1110 *** - - - Low 25 G1111 *** *** *** *** High 5 G1112 * - - - Low NO G1113 ND ND ND ND ND ND G1114 *** *** *** *** High 5 G1115 *** - - *** Low 50 G1116 ** - - - Low NO G1117 *** *** *** *** High 5 G1118 *** ** ** ** Low 10 G1119 * - - - Low NO G1120 ** - - - Low 100 G1121 ** - - - Low 100 G1122 *** *** ** ** Low 5 G1123 ** - - * Low NO G1124 *** *** *** *** Low 10 G1128 *** *** *** *** Low 10 G1129 *** *** *** * High 5 G1130 *** *** *** - Low 10 G1131 *** - - *** Low 15 G1071d2 *** *** *** *** High NO G1068 *** *** *** * High 10 G1094 *** *** *** *** High 12.5 G1092 *** *** *** *** High 10 G1096 ** - - ** Low NO G1107 *** *** *** ** High 12.5 G1093 *** - - - Low NO G1095 *** ** ** ** High 15 G1069 *** - - * High 10 G1070 *** - - - Low NO G1132 *** - - - Low NO G1074 *** ** ** * High 10 G1075 *** - - - High 15

NI= No inhibition

ND = No data

EXAMPLE 13 - Stradomers with reduced binding to C1q and canonical Fc γ R receptors

[000355] Additional compounds containing the S267E/H268F/S324T triple mutation were generated to generate additional stradomers that preferentially bind to the complement system.

[000356] G1001 (SEQ ID NO: 78) is based on the G019 backbone and contains four mutations (S267E/H268F/S324T/N297A). Surprisingly, relative to the parent compound G019, abolition of glycosylation by introducing the N297A mutation into G1003 to yield G1001 was unexpectedly associated with loss of C1q binding in addition to the expected loss of canonical binding, even in the presence of the S267E/H268F/S324T triple mutation (Moore et al.). This unexpected result was not observed for the parent compound G045c, where abolition of glycosylation by introducing the N297A mutation into G997 to yield G998 was associated with the expected loss of canonical binding, while maintaining full C1q binding and CDC inhibition. In this regard, despite G998 and G1001 containing similar domains with the same mutations and differing only in the position of the IgG2 hinge region, they exhibit essentially similar canonical Fc receptor binding but significantly different C1q binding and CDC inhibition. These comparisons further highlight the unpredictability of this set of mutations when applied to the multimerizing stradomer.

[000357] G999 (SEQ ID NO: 79) is a G019-based stradomer containing four point mutations (G236R, S267E, H268F, and S324T) that exhibits reduced binding to C1q and an inability to inhibit CDC. This result is unexpected since, as described above, G999 differs from G996 only in the location of the IgG2 hinge region relative to the Fc region. However, the two compounds exhibit opposite potencies with respect to C1q binding and CDC inhibition (G999 neither binds nor inhibits, while G996 exhibits high binding to C1q and inhibits CDC). This comparison further highlights the unpredictability of this set of mutations in a multimerizing stradomer.

[000358] G1024 (SEQ ID NO: 80) is a G019-based stradomer containing 4 point mutations (P238D, S267E, H268F, S324T).

[000359] G1030 (SEQ ID NO: 81) is a GL-2045-based stradomer containing 7 point mutations and a deletion at 236 (E233P, L234V, L235A, G236Del, N297A, S267E, H268F, S324T).

[000360] G1031 (SEQ ID NO: 82) is a GL-2045-based stradomer containing 6 point mutations (E233P, G236R, D265A, S267E, H268F, S324T, N297A).

[000361] G1040 (SEQ ID NO: 83) is a GL-2045-based stradomer containing 8 point mutations and a deletion at position 236 (E233P, L234V, L235A, G236Del, S267E, H268F, S324T, L328F, N297A).

[000362] G1044 (SEQ ID NO: 84) is a GL-2045-based stradomer containing 5 point mutations (P238D, D265C, S267E, H268F, S324T).

[000363] G1045 (SEQ ID NO: 85) is a GL-2045-based stradomer containing 5 point mutations (P238D, D265P, S267E, H268F, S324T).

[000364] G1048 (SEQ ID NO: 86) is a G019-based stradomer containing 5 point mutations (G236R, L328F, S267E, H268F, S324T).

[000365] G1047 (SEQ ID NO: 87) is a GL-2045-based stradomer containing 5 point mutations (P238D, L328F, S267E, H268F, S324T).

Table 11. Stradomers with reduced binding to C1q and canonical Fc γ R receptors

Stradometer SEQ ID NO Mutated amino acids G1001 78 S267E, H268F, S324T, N297A G999 79 G236R, S267E, H268F, S324T G1024 80 P238D, S267E, H268F, S324T G1030 81 E233P, L234V, L235A, G236Del, N297A, S267E, H268F, S324T G1031 82 E233P, G236R, S267E, H268F, S324T, N297A G1040 83 E233P, L234V, L235A, G236Del, S267E, H268F, S324T, L328F, N297A G1044 84 P238D, D265C, S267E, H268F, S324T G1045 85 P238D, D265P, S267E, H268F, S324T G1048 86 G236R, L328F, S267E, H268F, S324T G1047 87 P238D, L328F, S267E, H268F, S324T

[000366] An unexpected finding was that, despite the presence of the S267E/H268F/S324T triple mutation, the stradomers listed in Table 11 did not bind C1q or inhibit CDC, thus further highlighting the unpredictability of this set of point mutations when applied to a multimerizing stradomer. The results of an experiment determining binding to FcγR and FcRn, binding to C1q, and inhibition of CDC are presented in Table 12.

Table 12. Summary of the activity of stradomers with reduced binding to C1q and canonical Fc γ R receptors

Binding to FcγRI Binding to FcγRIIa Binding to FcγRIIb Binding to FcγRIIIa Binding to FcRn (pH=7.4) Binding to C1q* CDC inhibition G1001 *** ** - - *** ND (*) G999 *** - ** - *** ND - G1024 *** *** *** - *** - - G1030 - - - - *** - - G1031 - - - - ** - - G1040 *** *** *** - *** ND - G1044 *** (*) - - *** ND - G1045 *** - - - *** - - G1048 *** *** *** *** *** ND - G1047 *** *** *** * *** ND -

EXAMPLE 14 - Use of complement-preferential stradomers for the treatment of sickle cell disease

[000367] The complement preferential binding stradomers G994, G998, G1033, G1022, G1032, G1023, G1006, G1027, and G996 were evaluated in a mouse model of sickle cell anemia described in detail in Turhan et al (2004) Blood 103:2397 and Chang et al (2008) Blood 111:915. Briefly, 12- to 16-week-old male SCD mice (Townes SS mice homozygous for Hbatm1(HBA)Tow and homozygous for Hbbtm2(HBG1,HBB*)Tow (genotype M21)) were randomly assigned to groups and injected intravenously with saline, IVIG (400 mg/kg), hIgG1 (400 mg/kg), albumin (400 mg/kg), or stradomer (G045c, G994, G998 G1003, and G1033, 60 mg/kg) 20 min before surgical preparation. Neutrophil adhesion and rolling as well as platelet and neutrophil aggregates were examined in cremasteric venules. Plasma was collected after terminal blood collection for marker assays including sE-selectin, sP-selectin, sVCAM-1, and sICAM-1.

[000368] The results of the study demonstrate that G994, G998, G1033, G1022, G1032, G1023, G1006, G1027, and G996 significantly reduce SSRBC-WBC interactions/WBC/min, significantly increase rolling flux per WBC/min or fractional percentage, and significantly improve cumulative survival for the treated group compared to the control. Therefore, stradomers that preferentially bind to the complement system are effective in curing sickle cell anemia.

--->

LIST OF SEQUENCES

<110> Gliknick Inc.

Block, David S.

Olsen, Henrik

<120> HYBRID PROTEINS OF HUMAN PROTEIN FRAGMENTS FOR

CREATION OF ORDERED MULTIMEDIA COMPOSITIONS

FC AREAS OF IMMUNOGLOBULINS WITH ENHANCED

BY BINDING TO THE COMPLEMENT SYSTEM

<130> GLIK-015/01WO 310975-2150

<150> US 62/196,478

<151> 2015-07-24

<160> 87

<170> PatentIn version 3.5

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<213> Homo sapiens

<400> 11

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Arg Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 12

<211> 264

<212> Protein

<213> Homo sapiens

<400> 12

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Leu Leu Arg Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 13

<211> 264

<212> Protein

<213> Homo sapiens

<400> 13

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 14

<211> 264

<212> Protein

<213> Homo sapiens

<400> 14

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 15

<211> 264

<212> Protein

<213> Homo sapiens

<400> 15

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 16

<211> 264

<212> Protein

<213> Homo sapiens

<400> 16

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Arg Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 17

<211> 264

<212> Protein

<213> Homo sapiens

<400> 17

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro

20 25 30

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

35 40 45

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

50 55 60

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

65 70 75 80

Val Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

85 90 95

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

100 105 110

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

115 120 125

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Thr Asn Lys Ala

130 135 140

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

145 150 155 160

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

165 170 175

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

180 185 190

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

195 200 205

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

210 215 220

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

225 230 235 240

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

245 250 255

Ser Leu Ser Leu Ser Pro Gly Lys

260

<210> 18

<211> 264

<212> Protein

<213> Homo sapiens

<400> 18

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Ala Val Glu Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 19

<211> 264

<212> Protein

<213> Homo sapiens

<400> 19

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Val Ala Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 20

<211> 264

<212> Protein

<213> Homo sapiens

<400> 20

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Asp Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 21

<211> 264

<212> Protein

<213> Homo sapiens

<400> 21

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 22

<211> 264

<212> Protein

<213> Homo sapiens

<400> 22

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 23

<211> 264

<212> Protein

<213> Homo sapiens

<400> 23

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Arg Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 24

<211> 264

<212> Protein

<213> Homo sapiens

<400> 24

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Asp Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Gly Val Glu Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 25

<211> 264

<212> Protein

<213> Homo sapiens

<400> 25

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Asp Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Trp Val Glu Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 26

<211> 263

<212> Protein

<213> Homo sapiens

<400> 26

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro

35 40 45

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

50 55 60

Cys Val Val Val Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe Asn

65 70 75 80

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

85 90 95

Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

100 105 110

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Thr

115 120 125

Asn Lys Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

130 135 140

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

145 150 155 160

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

165 170 175

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

180 185 190

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

195 200 205

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

210 215 220

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

225 230 235 240

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys Cys

245 250 255

Val Glu Cys Pro Pro Cys Pro

260

<210> 27

<211> 264

<212> Protein

<213> Homo sapiens

<400> 27

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Asp Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 28

<211> 264

<212> Protein

<213> Homo sapiens

<400> 28

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Gln Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Ser Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 29

<211> 264

<212> Protein

<213> Homo sapiens

<400> 29

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Arg Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Ser Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 30

<211> 264

<212> Protein

<213> Homo sapiens

<400> 30

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu His Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Ser Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 31

<211> 264

<212> Protein

<213> Homo sapiens

<400> 31

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Asn Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Ser Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 32

<211> 264

<212> Protein

<213> Homo sapiens

<400> 32

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Lys Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Ser Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 33

<211> 264

<212> Protein

<213> Homo sapiens

<400> 33

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Arg Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 34

<211> 264

<212> Protein

<213> Homo sapiens

<400> 34

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Asn Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 35

<211> 264

<212> Protein

<213> Homo sapiens

<400> 35

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Lys Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 36

<211> 264

<212> Protein

<213> Homo sapiens

<400> 36

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Asp Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Arg Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 37

<211> 264

<212> Protein

<213> Homo sapiens

<400> 37

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Arg Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Arg Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 38

<211> 264

<212> Protein

<213> Homo sapiens

<400> 38

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Arg Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Asp Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 39

<211> 264

<212> Protein

<213> Homo sapiens

<400> 39

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Glu Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Arg Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 40

<211> 264

<212> Protein

<213> Homo sapiens

<400> 40

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu His Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Lys Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 41

<211> 264

<212> Protein

<213> Homo sapiens

<400> 41

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Gln Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Gln Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 42

<211> 264

<212> Protein

<213> Homo sapiens

<400> 42

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Arg Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Lys Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 43

<211> 264

<212> Protein

<213> Homo sapiens

<400> 43

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Arg Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Gln Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 44

<211> 264

<212> Protein

<213> Homo sapiens

<400> 44

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Asp Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Lys Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 45

<211> 264

<212> Protein

<213> Homo sapiens

<400> 45

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu His Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Gln Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 46

<211> 264

<212> Protein

<213> Homo sapiens

<400> 46

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Gln Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Arg Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 47

<211> 264

<212> Protein

<213> Homo sapiens

<400> 47

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Lys Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Lys Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 48

<211> 264

<212> Protein

<213> Homo sapiens

<400> 48

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Lys Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Asn Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 49

<211> 264

<212> Protein

<213> Homo sapiens

<400> 49

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Asn Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 50

<211> 264

<212> Protein

<213> Homo sapiens

<400> 50

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Asn Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Lys Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 51

<211> 264

<212> Protein

<213> Homo sapiens

<400> 51

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Arg Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Asn Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 52

<211> 264

<212> Protein

<213> Homo sapiens

<400> 52

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Ser Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Ala Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 53

<211> 264

<212> Protein

<213> Homo sapiens

<400> 53

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Arg Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Ala Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 54

<211> 264

<212> Protein

<213> Homo sapiens

<400> 54

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Lys Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Ala Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 55

<211> 264

<212> Protein

<213> Homo sapiens

<400> 55

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Asn Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Ala Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 56

<211> 264

<212> Protein

<213> Homo sapiens

<400> 56

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Ser Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 57

<211> 264

<212> Protein

<213> Homo sapiens

<400> 57

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Lys Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 58

<211> 264

<212> Protein

<213> Homo sapiens

<400> 58

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Arg Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 59

<211> 264

<212> Protein

<213> Homo sapiens

<400> 59

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Asp Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 60

<211> 264

<212> Protein

<213> Homo sapiens

<400> 60

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Gln Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 61

<211> 264

<212> Protein

<213> Homo sapiens

<400> 61

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Leu Leu Arg Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 62

<211> 264

<212> Protein

<213> Homo sapiens

<400> 62

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Val Ala Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 63

<211> 264

<212> Protein

<213> Homo sapiens

<400> 63

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 64

<211> 264

<212> Protein

<213> Homo sapiens

<400> 64

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro

20 25 30

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

35 40 45

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

50 55 60

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

65 70 75 80

Val Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

85 90 95

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

100 105 110

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

115 120 125

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Thr Asn Lys Ala

130 135 140

Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

145 150 155 160

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

165 170 175

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

180 185 190

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

195 200 205

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

210 215 220

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

225 230 235 240

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

245 250 255

Ser Leu Ser Leu Ser Pro Gly Lys

260

<210> 65

<211> 264

<212> Protein

<213> Homo sapiens

<400> 65

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Glu Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Ser Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 66

<211> 264

<212> Protein

<213> Homo sapiens

<400> 66

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Asp Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Ser Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 67

<211> 264

<212> Protein

<213> Homo sapiens

<400> 67

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Gln Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 68

<211> 264

<212> Protein

<213> Homo sapiens

<400> 68

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Asp Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 69

<211> 264

<212> Protein

<213> Homo sapiens

<400> 69

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val His Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 70

<211> 264

<212> Protein

<213> Homo sapiens

<400> 70

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 71

<211> 264

<212> Protein

<213> Homo sapiens

<400> 71

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Asp Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Gln Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 72

<211> 264

<212> Protein

<213> Homo sapiens

<400> 72

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Gln Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Asp Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 73

<211> 264

<212> Protein

<213> Homo sapiens

<400> 73

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Asp Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Asp Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 74

<211> 264

<212> Protein

<213> Homo sapiens

<400> 74

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Ala Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 75

<211> 264

<212> Protein

<213> Homo sapiens

<400> 75

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Gln Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Ala Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 76

<211> 264

<212> Protein

<213> Homo sapiens

<400> 76

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Asp Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Ala Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 77

<211> 264

<212> Protein

<213> Homo sapiens

<400> 77

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val His Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Ala Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 78

<211> 264

<212> Protein

<213> Homo sapiens

<400> 78

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro

20 25 30

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

35 40 45

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

50 55 60

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

65 70 75 80

Val Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

85 90 95

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

100 105 110

Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

115 120 125

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Thr Asn Lys Ala

130 135 140

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

145 150 155 160

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

165 170 175

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

180 185 190

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

195 200 205

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

210 215 220

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

225 230 235 240

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

245 250 255

Ser Leu Ser Leu Ser Pro Gly Lys

260

<210> 79

<211> 264

<212> Protein

<213> Homo sapiens

<400> 79

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro

20 25 30

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

35 40 45

Pro Glu Leu Leu Arg Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

50 55 60

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

65 70 75 80

Val Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

85 90 95

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

100 105 110

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

115 120 125

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Thr Asn Lys Ala

130 135 140

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

145 150 155 160

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

165 170 175

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

180 185 190

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

195 200 205

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

210 215 220

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

225 230 235 240

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

245 250 255

Ser Leu Ser Leu Ser Pro Gly Lys

260

<210> 80

<211> 263

<212> Protein

<213> Homo sapiens

<400> 80

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Val Pro Gly

1 5 10 15

Ser Thr Gly Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Glu

20 25 30

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

35 40 45

Glu Leu Leu Gly Gly Asp Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

50 55 60

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

65 70 75 80

Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

85 90 95

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

100 105 110

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

115 120 125

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Thr Asn Lys Ala Leu

130 135 140

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

145 150 155 160

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys

165 170 175

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

180 185 190

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

195 200 205

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

210 215 220

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

225 230 235 240

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

245 250 255

Leu Ser Leu Ser Pro Gly Lys

260

<210> 81

<211> 264

<212> Protein

<213> Homo sapiens

<400> 81

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Val Ala Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 82

<211> 264

<212> Protein

<213> Homo sapiens

<400> 82

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Leu Leu Arg Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Ala Val Glu Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 83

<211> 264

<212> Protein

<213> Homo sapiens

<400> 83

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Pro Val Ala Gly Gly Pro Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 84

<211> 264

<212> Protein

<213> Homo sapiens

<400> 84

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Asp Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Cys Val Glu Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 85

<211> 264

<212> Protein

<213> Homo sapiens

<400> 85

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Asp Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Pro Val Glu Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<210> 86

<211> 263

<212> Protein

<213> Homo sapiens

<400> 86

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Val Pro Gly

1 5 10 15

Ser Thr Gly Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Glu

20 25 30

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

35 40 45

Glu Leu Leu Arg Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

50 55 60

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

65 70 75 80

Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

85 90 95

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

100 105 110

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

115 120 125

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Thr Asn Lys Ala Phe

130 135 140

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

145 150 155 160

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys

165 170 175

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

180 185 190

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

195 200 205

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

210 215 220

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

225 230 235 240

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

245 250 255

Leu Ser Leu Ser Pro Gly Lys

260

<210> 87

<211> 264

<212> Protein

<213> Homo sapiens

<400> 87

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

20 25 30

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Asp Ser Val Phe Leu Phe

35 40 45

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

50 55 60

Thr Cys Val Val Val Asp Val Glu Phe Glu Asp Pro Glu Val Lys Phe

65 70 75 80

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

85 90 95

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

100 105 110

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

115 120 125

Thr Asn Lys Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

130 135 140

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

145 150 155 160

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

165 170 175

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

180 185 190

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

195 200 205

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

210 215 220

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

225 230 235 240

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys

245 250 255

Cys Val Glu Cys Pro Pro Cys Pro

260

<---

Claims (10)

1. A peptide homodimer for binding to the complement system component C1q, consisting of two identical monomers, wherein each monomer consists, in the direction from the amino- to the carboxyl-terminus, of a multimerization domain of the hinge region of IgG2 consisting of the amino acid sequence of SEQ ID NO: 4, and an Fc domain of IgG1 consisting of the amino acid sequence of SEQ ID NO: 2 with point mutations including S267E, H268F and S324T based on the EU index according to Kabat. 2. The peptide homodimer of claim 1, wherein the IgG2 hinge region multimerization domain creates multimers of peptide homodimers. 3. The peptide homodimer of claim 2, wherein the multimers of peptide homodimers are highly ordered multimers. 4. The peptide homodimer of claim 1, wherein the peptide homodimer inhibits complement-dependent cytotoxicity and maintains binding to FcγRI, FcγRIIa, FcγRIIb and/or FcγRIII. 5. The peptide homodimer of claim 1, wherein the peptide homodimer exhibits increased binding to complement C1q compared to a peptide homodimer of similar structure that does not contain point mutations at positions S267, H268 and S324. 6. The peptide homodimer of claim 1, wherein each monomer of the peptide homodimer comprises amino acids 21-264 of the sequence SEQ ID NO: 17. 7. A multimer for binding to the complement system component C1q, consisting of two or more peptide homodimers according to any one of claims 1-6. 8. Use of a peptide homodimer according to any one of claims 1-6 or a multimer according to claim 7 for the treatment or prevention of rheumatoid arthritis. 9. The use according to claim 8, wherein the peptide homodimer or multimer is administered intravenously, subcutaneously, orally, intraperitoneally, sublingually, buccally, transdermally, via a subcutaneous implant, or intramuscularly.