Compositions and methods of inhibiting MASP-1 and / or MASP-2 and or MASP-3 for the treatment of various diseases and disorders

Abstract

To provide methods and compositions for inhibiting MASP-3-dependent complement activation in a subject suffering from a disease or disorder or at risk of developing the disease or disorder by administering to the subject a composition comprising an amount of a MASP-3 inhibitory agent effective to inhibit MASP-3-dependent complement activation.SOLUTION: A MASP-2 inhibitory agent and a MASP-1 inhibitory agent are administered, a MASP-2 inhibitory agent and a MASP-3 inhibitory agent are administered, a MASP-3 inhibitory agent and a MASP-1 inhibitory agent are administered, or a MASP-1 inhibitory agent, a MASP-2 inhibitory agent, and a MASP-3 inhibitory agent are administered.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 61 / 661,167, filed June 18, 2012.

[0002] Sequence Listing Description The sequence listing associated with this application is provided in text form in lieu of hard copy and is incorporated herein by reference. The text file containing the sequence listing is named MP_1_0176_PCT_SequenceListingFiled_20130614.txt and has been submitted via EFS-Web with the application hereof. [Background technology]

[0003] background The complement system provides an early mechanism for initiating, amplifying, and orchestrating immune responses to microbial infections and other acute insults in humans and other vertebrates (M.K. Liszewski and J.P. Atkinson, 1993, in Fundamental Immunology, Third Edition, edited by W.E. Paul, Raven Press, Ltd., New York). Although complement activation provides a valuable first line of defense against potential pathogens, complement activity, which promotes a protective immune response, can also pose a potential threat to the host (K.R., Kalli, et al., Springer Semin. Immunopathol. 15:417-431, 1994; B.P. Morgan, Eur. J. Clinical Investig. 24:219-228, 1994). For example, C3 and C5 proteolytic products recruit and activate neutrophils. Activated neutrophils, while essential for host defense, can indiscriminately release destructive enzymes, causing organ damage. Furthermore, complement activation can result in the deposition of lytic complement components on the surfaces of nearby host cells as well as microbial targets, resulting in host cell lysis.

[0004] The complement system has also been implicated in the development of numerous acute and chronic disease states, including myocardial infarction, stroke, ARDS, reperfusion injury, septic shock, capillary leak after burn injury, post-cardiopulmonary bypass inflammation, transplant rejection, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and Alzheimer's disease. In nearly all of these conditions, complement is not the cause but is one of several factors involved in their development. Nevertheless, complement activation may be a major pathological mechanism and is an important point for clinical management of many of these disease states. The growing recognition of the importance of complement-mediated tissue damage in various disease states highlights the need for effective complement-inhibiting drugs. To date, eculizumab (Solaris®), an antibody against C5, is the only complement-targeting drug approved for human use. However, C5 is one of several effector molecules "downstream" of the complement system, and blocking C5 does not inhibit complement system activation. Thus, inhibitors of the initiation steps of complement activation appear to have a significant advantage over "downstream" complement inhibitors.

[0005] It is now widely accepted that the complement system can be activated through three different pathways: the classical pathway, the lectin pathway, and the alternative pathway. The classical pathway is usually triggered by a complex consisting of a host antibody bound to a foreign particle (i.e., an antigen) and therefore requires prior exposure to the antigen to generate a specific antibody response. Because activation of the classical pathway depends on a previous adaptive immune response by the host, the classical pathway is part of the adaptive immune system. In contrast, both the lectin pathway and the alternative pathway are independent of adaptive immunity and are part of the innate immune system.

[0006] Activation of the complement system involves the sequential activation of serine protease proenzymes. The first step in classical pathway activation is the binding of the specific recognition molecule C1q to antigen-bound IgG and IgM molecules. C1q then binds to the C1r and C1s serine protease proenzymes, forming a complex called C1. Upon binding of C1q to the immune complex, autoproteolytic cleavage of the Arg-Ile site of C1r occurs, followed by C1r-mediated cleavage and activation of C1s, thereby acquiring the ability to cleave C4 and C2. C4 is cleaved into two fragments, C4a and C4b, and C2 is similarly cleaved into C2a and C2b. The C4b fragment can form a covalent bond with the adjacent hydroxyl or amino group and generate the C3 convertase (C4b2a) through noncovalent interaction with the C2a fragment of activated C2. C3 convertase (C4b2a) activates C3 by proteolytic cleavage into the smaller components C3a and C3b, thereby generating C5 convertase (C4b2a3b). C5 convertase (C4b2a3b) cleaves C5, resulting in the formation of the membrane attack complex (C5b in combination with C6, C7, C8, and C9, also known as the "MAC"). The membrane attack complex disrupts cell membranes and can lead to cell lysis. The activated forms of C3 and C4 (C3b and C4b) are covalently deposited on foreign target surfaces and are recognized by complement receptors on multiple phagocytes.

[0007] Independently, the first step in complement activation via the lectin pathway is also the binding of specific recognition molecules and subsequent activation of associated serine protease proenzymes. However, rather than immune complex binding by C1q, the recognition molecules of the lectin pathway include a group of carbohydrate-binding proteins collectively called lectins (mannan-binding lectin (MBL), H-ficolin, M-ficolin, L-ficolin, and C-type lectin CL-11). See J. Lu et al., Biochim. Biophys. Acta 1572:387-400, (2002); Holmskov et al., Annu. Rev. Immunol. 21:547-578(2003); Teh et al., Immunology 101:225-232(2000)). J. Luet et al., Biochim Biophys Acta 1572:387-400(2002); Holmskov et al., Annu. Rev. Immunol. 21:547-578(2003); Teh et al., Immunology 101:225-232(2000); Hansen et al., J, Immunol See also 185(10):6096-6104 (2010).

[0008] Ikeda et al. were the first to demonstrate that, like C1q, MBL can activate the complement system in a C4-dependent manner when bound to yeast mannan-coated red blood cells (Ikeda et al., J. Biol. Chem. 262:7451-7454, (1987)). MBL, a member of the collectin protein family, is a calcium-dependent lectin that binds to carbohydrates in which the 3- and 4-hydroxy groups are oriented toward the equatorial binding face of the pyranose ring. Therefore, the most prominent ligands for MBL are D-mannose and N-acetyl-D-glucosamine, whereas carbohydrates that do not meet this steric requirement have undetectable affinity for MBL (Weis et al., Nature 360:127-134, (1992)). The interaction of MBL with monovalent sugars is extremely weak, with dissociation constants typically in the single-digit millimolar range. MBL achieves tight and specific binding to glycan ligands through avidity, i.e., by simultaneously interacting with multiple monosaccharide residues located close to each other (Lee et al., Archiv. Biochem. Biophys. 299:129-136, (1992)). MBL generally recognizes carbohydrate patterns that decorate microorganisms, such as bacteria, yeast, parasites, and certain viruses. In contrast, MBL does not recognize D-galactose and sialic acid, the penultimate and final sugars that typically decorate "mature" glycoconjugates present on mammalian plasma glycoproteins and cell surface glycoproteins. This binding specificity is thought to facilitate recognition of "foreign" surfaces and help protect against "autoactivation." However, MBL binds with high affinity to high-mannose "precursor" glycan clusters on N-linked glycoproteins and glycolipids sequestered in the endoplasmic reticulum and Golgi of mammalian cells (Maynard et al., J. Biol. Chem. 257:3788-3794, (1982)).Furthermore, it has been shown that MBL can bind polynucleotides, DNA, and RNA, which may be exposed to necrotic and apoptotic cells (Palaniyar et al., Ann. NY Acad. Sci., 1010:467-470 (2003); Nakamura et al., J. Leuk. Biol. 86:737-748 (2009)). Therefore, damaged cells are potential targets for lectin pathway activation via MBL binding.

[0009] Ficolins contain a type of lectin domain called a fibrinogen-like domain, which is different from that of MBL. ++ Three ficolins (L-ficolin, M-ficolin, and H-ficolin) have been identified in humans. Two serum ficolins, L-ficolin and H-ficolin, share specificity for N-acetyl-D-glucosamine. However, H-ficolin also binds N-acetyl-D-galactosamine. The different carbohydrate specificities of L-ficolin, H-ficolin, CL-11, and MBL imply that different lectins may complement each other and, through overlap, target different glycoconjugates. This idea is supported by a recent report that, of the known lectins in the lectin pathway, only L-ficolin specifically binds lipoteichoic acid, a cell wall glycoconjugate found in all Gram-positive bacteria (Lynch et al., J. Immunol. 172:1198-1202, (2004)). In addition to acetylated sugar moieties, ficolins can also bind acetylated amino acids and polypeptides (Thomsen et al., Mol. Immunol. 48(4):369-81 (2011)). Collectins (i.e., MBL) and ficolins do not share significant amino acid sequence similarity. However, these two protein groups have similar domain organization and, like C1q, assemble into oligomeric structures that maximize the potential for multisite binding.

[0010] Serum concentrations of MBL are highly variable in healthy populations and are genetically controlled by polymorphisms / mutations in both the promoter and coding regions of the MBL gene. As an acute-phase protein, MBL expression is further upregulated during inflammation. L-ficolin is present in serum at concentrations similar to those of MBL. Thus, the L-ficolin branch of the lectin pathway potentially rivals the strength of the MBL branch. MBL and ficolin can also function as opsonins, enabling phagocytes to target surfaces decorated with MBL and ficolin (see Jack et al., J. Leukoc. Biol., 77(3):328-36(2004); Matsushita and Fujita, Immunobiology, 205(4-5):490-7(2002); Aoyagi et al., J. Immunol., 174(1):418-25(2005)). This opsonization requires the interaction of these proteins with phagocyte receptors (Kuhlman et al., J. Exp. Med. 169:1733, (1989); Matsushita et al., J. Biol. Chem. 271:2448-54, (1996)). The identity of the phagocyte receptors remains to be determined.

[0011] Human MBL interacts specifically and with high affinity via its collagen-like domain with a unique C1r / C1s-like serine protease called MBL-associated serine protease (MASP). To date, three types of MASP have been described. First, a single enzyme, "MASP," was identified and characterized as the enzyme responsible for initiating the complement cascade (i.e., cleaving C2 and C4) (Matsushita et al., J Exp Med 176(6):1497-1502(1992); Ji et al., J. Immunol 150:571-578, (1993)). Later, it was determined that MASP activity is actually a mixture of two types of proteases: MASP-1 and MASP-2 (Thiel et al., Nature 386:506-510, (1997)). However, it has been demonstrated that the MBL-MASP-2 complex alone is sufficient for complement activation (Vorup-Jensen et al., J. Immunol 165:2093-2100, (2000)). Furthermore, only MASP-2 cleaved C2 and C4 at a high rate (Ambrus et al., J. Immunol, 170:1374-1382, (2003)). Thus, MASP-2 is the protease responsible for activating C4 and C2 to generate the C3 convertase C4b2a. This is in stark contrast to the C1 complex of the classical pathway, in which the coordinated action of two specific serine proteases (C1r and C1s) leads to complement activation. Furthermore, a third novel protease, MASP-3, has been isolated (Dahl, MR et al., Immunity 15:127-35, 2001). MASP-1 and MASP-3 are alternative splicing products of the same gene.

[0012] MASPs have a domain organization identical to that of the C1r and C1s enzyme components of the C1 complex (Sim et al., Biochem. Soc. Trans. 28:545, (2000)). These domains include an N-terminal C1r / C1s / sea urchin VEGF / bone morphogenetic protein (CUB) domain, an epidermal growth factor-like domain, a second CUB domain, a tandem array of complement regulatory protein domains, and a serine protease domain. Like the C1 protease, activation of MASP-2 occurs through cleavage of an Arg-Ile bond adjacent to the serine protease domain. This cleavage separates the enzyme into disulfide-linked A and B chains, the latter of which consists of the serine protease domain.

[0013] MBL can also bind to an alternatively spliced ​​form of MASP-2 known as the 19-kDa MBL-associated protein (MAp19) or small MBL-associated protein (sMAP), which lacks the catalytic activity of MASP2 (Stover, J. Immunol. 162:3481-90, (1999); Takahashi et al., Int. Immunol. 11:859-863, (1999)). MAp19 contains an extra sequence of four unique amino acids following the first two domains of MASP-2. The function of MAp19 is unknown (Degn et al., J. Immunol. Methods. 2011). The MASP-1 and MASP-2 genes are located on human chromosomes 3 and 1, respectively (Schwaeble et al., Immunobiology 205:455-466, (2002)).

[0014] Several lines of evidence suggest that there are distinct MBL-MASP complexes, and that the majority of MASPs in serum do not form complexes with MBL (Thiel et al., J. Immunol. 165:878-887, (2000)). Both H-ficolin and L-ficolin, like MBL, bind to all MASPs and activate the lectin complement pathway (Dahl et al., Immunity 15:127-35, (2001); Matsushita et al., J. Immunol. 168:3502-3506, (2002)). Both the lectin and classical pathways form a common C3 convertase (C4b2a), and the two pathways merge at this stage.

[0015] The lectin pathway is widely believed to play a major role in host defense against infection in naive hosts. Strong evidence for the involvement of MBL in host defense came from the analysis of patients with low serum levels of functional MBL (Kilpatrick, Biochim. Biophys. Acta 1572:401-413, (2002)). Such patients are susceptible to recurrent bacterial and fungal infections. These symptoms usually appear early in life, when maternal antibody titers are declining, during a period of apparent vulnerability before the full repertoire of antibody responses has developed. This syndrome is often due to mutations in several sites in the collagen moiety of MBL that prevent the proper formation of MBL oligomers. However, because MBL can function as an opsonin independently of complement, it is unclear to what extent the increased susceptibility to infection is due to impaired complement activation.

[0016] In contrast to the classical and lectin pathways, initiators of the alternative pathway have not previously been found to perform the recognition functions performed by C1q and lectins in the other two steps. It is now widely accepted that the alternative pathway spontaneously undergoes low-level turnover activation. This turnover activation can be easily amplified on foreign or other abnormal surfaces (bacteria, yeast, virus-infected cells, or damaged tissue) that lack the appropriate molecular elements that suppress spontaneous complement activation. There are four plasma proteins directly involved in alternative pathway activation: C3, factors B and D, and properdin.

[0017] Although extensive evidence links the classical and alternative complement pathways to the development of noninfectious human diseases, the role of the lectin pathway has only just begun to be evaluated. Recent studies have provided evidence that activation of the lectin pathway may be responsible for complement activation and associated inflammation in ischemia / reperfusion injury. Collard et al. (2000) reported that cultured endothelial cells subjected to oxidative stress bound MBL and exhibited C3 deposition upon exposure to human serum (Collard et al., Am. J. Pathol 156:1549-1556, (2000)). Furthermore, treatment of human serum with a blocking anti-MBL monoclonal antibody inhibited MBL binding and complement activation. These findings were extended to a rat myocardial ischemia-reperfusion model. In this model, rats treated with a blocking antibody against rat MBL showed significantly less myocardial damage during coronary artery occlusion than rats treated with a control antibody (Jordan et al., Circulation, 104:1413-1418, (2001)). The molecular mechanism of MBL binding to the vascular endothelium after oxidative stress is unknown. Recent studies suggest that activation of the lectin pathway after oxidative stress may be mediated by MBL binding to endothelial cytokeratins but not by glycoconjugates (Collard et al., Am. J. Pathol. 159:1045-1054, (2001)). Other studies have linked the classical and alternative pathways to the development of ischemia / reperfusion injury, and the role of the lectin pathway in this disease remains controversial (Riedermann, NC et al., Am. J. Pathol. 162:363-367, 2003).

[0018] Recent studies have shown that MASP-1 and MASP-3 convert the alternative pathway-activating enzyme, factor D, from its proenzyme form to its enzymatically active form (see Takahashi M. et al., J Exp Med 207(1):29-37(2010); Iwaki et al., J. Immunol. 187:3751-58 (2011)). The physiological importance of this process is highlighted by the absence of functional alternative pathway activity in the plasma of MASP-1 / 3-deficient mice. The proteolytic generation of C3b from native C3 requires the function of the alternative pathway. Because the alternative pathway C3 convertase (C3bBb) contains the essential subunit C3b, the origin of the initial C3b produced via the alternative pathway remains a puzzling question that has stimulated extensive research.

[0019] C3 (along with C4 and α-2 macroglobulin) belongs to a family of proteins containing a rare post-translational modification known as a thioester bond. The thioester group consists of a glutamine with a terminal carbonyl group that forms a covalent thioester bond with the sulfhydryl group of a cysteine ​​three amino acids away. This bond is unstable, and the electrophilic glutamyl-thioester can react with nucleophilic moieties, such as hydroxyl or amino groups, and thus form covalent bonds with other molecules. The thioester bond is fairly stable when sequestered within the hydrophobic pocket of intact C3. However, upon proteolytic cleavage of C3 into C3a and C3b, the highly reactive thioester bond is exposed on C3b, which then covalently binds to its target after nucleophilic attack by an adjacent moiety containing a hydroxyl or amino group. In addition to its well-documented role in covalently binding C3b to complement targets, the C3 thioester is also thought to play a central role in triggering the alternative pathway. According to the widely accepted "tick-over theory," the alternative pathway is initiated by the generation of the fluid-phase convertase iC3Bb, which is formed from C3 with a hydrolyzed thioester (iC3; C3(H2O)) and factor B (Lachmann, PJ, et al., Springer Semin. Immunopathol. 7:143-162, (1984)). The C3b-like C3(H2O) is generated from native C3 by the slow spontaneous hydrolysis of an internal thioester in the protein (Pangburn, MK, et al., J. Exp. Med. 154:856-867, 1981). The activity of the C3(H2O)Bb convertase deposits C3b molecules on target surfaces, thereby initiating the alternative pathway.

[0020] Prior to the discoveries described herein, little was known about the initiators of alternative pathway activation. Activators were thought to include yeast cell walls (zymosan), many pure polysaccharides, rabbit erythrocytes, certain immunoglobulins, viruses, fungi, bacteria, animal tumor cells, parasites, and damaged cells. The only common feature shared by these activators is the presence of carbohydrates, but the complexity and diversity of carbohydrate structures makes it difficult to establish a recognized common molecular determinant. Alternative pathway activation is controlled by a delicate balance between inhibitory regulatory components of the pathway, such as factor H, factor I, DAF, and CR1, and properdin, with the latter widely recognized as the only positive regulator of the alternative pathway (see Schwaeble WJ and Reid KB, Immunol Today 20(1):17-21 (1999)).

[0021] In addition to the apparently promiscuous activation mechanism described above, the alternative pathway can also provide a powerful amplification loop for the lectin / classical pathway C3 convertase (C4b2a), since the generated C3b can participate with factor B in the formation of additional alternative pathway C3 convertases (C3bBb). The alternative pathway C3 convertase is stabilized by properdin binding, which extends the half-life of the alternative pathway C3 convertase by 6-10-fold. Addition of C3b to the alternative pathway C3 convertase results in the formation of the alternative pathway C5 convertase.

[0022] All three pathways (i.e., the classical, lectin, and alternative pathways) have been thought to converge at C5. C5 is cleaved to form products with multiple proinflammatory effects. This convergent pathway has been called the terminal complement pathway. C5a is the most potent anaphylatoxin, inducing changes in smooth muscle tone and vascular tone and vascular permeability. C5a is also a potent chemotaxin and activator of neutrophils and monocytes. C5a-mediated cell activation can significantly amplify the inflammatory response by inducing the release of multiple additional inflammatory mediators, including cytokines, hydrolases, arachidonic acid metabolites, and reactive oxygen species. C5 cleavage leads to the formation of C5b-9, also known as the membrane attack complex (MAC). There is now strong evidence that sublytic MAC deposition may play an important role in inflammation in addition to its role as a lytic pore-forming complex.

[0023] In addition to its essential role in immune defense, the complement system contributes to tissue damage in many clinical conditions. Therefore, there is an urgent need to develop therapeutically effective complement inhibitors to prevent these side effects. Summary of the Invention

[0024] overview In one aspect, the present invention provides a method for inhibiting MASP-3-dependent complement activation in a subject suffering from paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy, asthma, dense deposit disease, pauci-immune necrotizing crescentic glomerulonephritis, traumatic encephalopathy, aspiration pneumonia, endophthalmitis, neuromyelitis optica, or Behçet's disease. The method comprises administering to the subject a composition comprising a MASP-3 inhibitor in an amount effective to inhibit MASP-3-dependent complement activation. In some embodiments, the method further comprises administering to the subject a composition comprising a MASP-2 inhibitor.

[0025] In another aspect, the present invention provides a method for inhibiting MASP-2-dependent complement activation in a subject suffering from or at risk of developing a disease or disorder selected from the group consisting of dense deposit disease, microimmune necrotizing crescentic glomerulonephritis, traumatic encephalopathy, aspiration pneumonia, endophthalmitis, neuromyelitis optica, or Behçet's disease. The method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation. In some embodiments, the MASP-2 inhibitor is a MASP-2 antibody or a fragment thereof. In some embodiments, the MASP-2 inhibitor is a MASP-2 monoclonal antibody or a fragment thereof, which specifically binds to a portion of SEQ ID NO:5. In some embodiments, the MASP-2 antibody is a chimeric, humanized, or human antibody.

[0026] In another aspect, the present invention provides a pharmaceutical composition comprising at least one inhibitor, including a MASP-2 inhibitor and a MASP-3 inhibitor, and a pharmaceutically acceptable carrier.

[0027] In another aspect, the present invention provides a pharmaceutical composition comprising a MASP-3 inhibitor that binds to a portion of MASP-1 (SEQ ID NO:10, full length) and also binds to a portion of MASP-3 (SEQ ID NO:8), and a pharmaceutical carrier.

[0028] In another aspect, the present invention provides a pharmaceutical composition comprising a MASP-3 inhibitor that binds to a portion of MASP-2 (SEQ ID NO:5, full length) and also binds to a portion of MASP-3 (SEQ ID NO:8), and a pharmaceutical carrier.

[0029] In another aspect, the present invention provides a pharmaceutical composition comprising a MASP-3 inhibitor that binds to a portion of MASP-1 (SEQ ID NO:10, full length) and also binds to a portion of MASP-2 (SEQ ID NO:5), and a pharmaceutical carrier.

[0030] In another aspect, the present invention provides a pharmaceutical composition comprising a MASP-3 inhibitor that binds to a portion of MASP-1 (SEQ ID NO:10, full length), a portion of MASP-2 (SEQ ID NO:5, full length), and a portion of MASP-3 (SEQ ID NO:8), and a pharmaceutical carrier.

[0031] In another aspect, the present invention provides a method for producing a medicament for use in inhibiting the effects of MASP-3-dependent complement activation in a living subject in need thereof, the method comprising the step of combining a therapeutically effective amount of a MASP-3 inhibitor with a pharmaceutical carrier. In some embodiments, the method of this aspect of the present invention comprises the step of producing a medicament for use in inhibiting the effects of MASP-3-dependent complement activation in a subject suffering from or at risk of developing a disease or disorder selected from the group consisting of paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy, asthma, dense deposit disease, microimmune necrotizing crescentic glomerulonephritis, traumatic encephalopathy, aspiration pneumonia, endophthalmitis, neuromyelitis optica, or Behcet's disease. In some embodiments, the method further comprises the step of combining a therapeutically effective amount of a MASP-2 inhibitor with or in a medicament comprising a MASP-3 inhibitor.

[0032] In another aspect, the present invention provides a method for producing a medicament for use in inhibiting the effects of MASP-2-dependent complement activation in a living subject in need thereof, the method comprising the step of mixing a therapeutically effective amount of a MASP-2 inhibitor with a pharmaceutical carrier. In some embodiments, the method of this aspect of the present invention comprises the step of producing a medicament for use in inhibiting the effects of MASP-2-dependent complement activation in a subject suffering from or at risk of developing a disease or disorder selected from the group consisting of dense deposit disease, microimmune necrotizing crescentic glomerulonephritis, traumatic encephalopathy, aspiration pneumonia, endophthalmitis, neuromyelitis optica, or Behcet's disease. In some embodiments, the method further comprises the step of mixing a therapeutically effective amount of a MASP-3 inhibitor with or in a medicament comprising a MASP-2 inhibitor.

[0033] As described herein, various embodiments of the MASP-3 inhibitor and / or various embodiments of the MASP-2 inhibitor may be used in the pharmaceutical compositions of the present invention.

[0034] As described herein, the pharmaceutical compositions of the present invention can be used in accordance with the methods of the present invention.

[0035] These and other aspects and embodiments of the invention described herein will become apparent upon reference to the following detailed description and drawings, in which: All U.S. patents, U.S. published patent applications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications cited herein are incorporated by reference in their entirety, as if each were individually incorporated. [Brief explanation of the drawings]

[0036] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein: [Figure 1] New understanding of lectins and the alternative pathway is presented. [Figure 2] Schematic diagram from Schwaeble et al., Immunobiol 205:455-466 (2002) modified by Yongqing et al., BBA 1824:253 (2012) showing MASP-2 and MAp19 protein domains and the exons that encode them. [Figure 3] Schematic diagram modified from Schwaeble et al., Immunobiol 205:455-466 (2002) by Yongqing et al., BBA 1824:253 (2012) showing MASP-1, MASP-3, and MAp44 protein domains and the exons that encode them. [Figure 4] An alignment of the amino acid sequences of MASP-1, MASP-2, and MASP-3 proteins is shown, indicating the consensus regions between them. [Figure 5] An alignment of the amino acid sequences of MASP-1, MASP-2, and MASP-3 alpha chains is shown. [Figure 6] An alignment of the amino acid sequences of MASP-1, MASP-2, and MASP-3 beta chains is shown. [Figure 7A] Pairwise alignment of the amino acid sequences of the MASP-1 and MASP-2 protease domains (beta chains) is shown. [Figure 7B] Pairwise alignment of the amino acid sequences of the MASP-1 and MASP-3 protease domains (beta chains) is shown. [Figure 7C] Pairwise alignment of the amino acid sequences of the MASP-2 and MASP-3 protease domains (beta chains) is shown. [Figure 8]1 is a Kaplan-Meyer plot graphically illustrating the survival of MASP-2 KO and WT mice following administration of an infectious dose of 2.6 x 107 cfu of N. meningitidis serogroup A Z2491, as described in Example 1, demonstrating that MASP-2-deficient mice are protected from meningococcal-induced death. [Figure 9] 1 is a Kaplan-Meier plot graphically illustrating the survival of MASP-2 KO and WT mice following challenge with an infectious dose of 6 x 10 cfu of Neisseria meningitidis serogroup B strain MC58, as described in Example 1, demonstrating that MASP-2-deficient mice are protected from meningococcal-induced death. [Figure 10] As described in Example 1, a graph shows the log cfu / mL of meningococcal serogroup B strain MC58 in 1 mL of blood collected from MASP-2 KO mice and WT mice at various time points after intraperitoneal infection with 6 x 106 cfu of meningococcal serogroup B strain MC58 (n = 3 at various time points for both mouse groups), demonstrating that although MASP-2 KO mice were infected with the same amount of meningococcal serogroup B strain MC58 as WT mice, MASP-2 KO mice had enhanced clearance of bacteremia compared to WT mice. [Figure 11] As described in Example 1, the graph shows the average disease scores of MASP-2 KO mice and WT mice 3, 6, 12, and 24 hours after infection with 6 x 106 cfu of meningococcal serogroup B strain MC58, demonstrating that MASP-2-deficient mice showed significantly reduced disease scores at 6, 12, and 24 hours post-infection compared to WT mice. [Figure 12] This is a Kaplan-Meier plot graphically showing the survival rate of mice administered either an inhibitory MASP-2 antibody (1 mg / kg) or a control isotype antibody 3 hours after administration of an infectious dose of 4 x 106 cfu of Neisseria meningitidis serogroup B strain MC58, as described in Example 2, demonstrating that MASP-2 antibodies are effective in treating and improving survival rates in subjects infected with Neisseria meningitidis. [Figure 13]FIG. 5 graphically depicts the log cfu / mL viable counts of Neisseria meningitidis serogroup B strain MC58 recovered at various time points in human serum samples shown in Table 5, recorded at various time points after incubation with Neisseria meningitidis serogroup B strain MC58, as described in Example 3. [Figure 14] As described in Example 3, the log cfu / mL viable counts of meningococcal serogroup B-MC58 recovered at various time points in the human serum samples shown in Table 7 are shown graphically, demonstrating that complement-dependent killing of meningococci in 20% (v / v) human serum is MASP-3 and MBL dependent. [Figure 15] As described in Example 3, the log cfu / mL of viable meningococcal serogroup B-MC58 bacteria recovered at various time points in the mouse serum samples shown in Table 9 is shown in graph form, demonstrating that MASP-2- / - knockout mouse (referred to as "MASP-2- / -") serum has a higher level of bactericidal activity against meningococci than WT mouse serum, while in contrast, MASP-1 / 3- / - mouse serum does not have any bactericidal activity. [Figure 16] Graph showing the kinetics of C3 activation in WT, C4− / −, MASP-1 / 3− / −, factor B− / −, and MASP-2− / − mouse serum under lectin pathway-specific conditions (1% plasma), as described in Example 4. [Figure 17] The levels of alternative pathway-driven (AP-driven) C3b deposition on zymosan-coated microtiter plates under "conventional" alternative pathway-specific (AP-specific) conditions (i.e., BBS / EGTA / Mg++ without Ca++) are shown graphically as a function of serum concentration in serum samples taken from MASP-3-deficient, C4-deficient, and MBL-deficient human subjects, as described in Example 4. [Figure 18] The level of AP-driven C3b deposition on zymosan-coated microtiter plates under "conventional" AP-specific conditions (i.e., BBS / EGTA / Mg++ without Ca++) as described in Example 4 is shown graphically as a function of time in 10% human serum samples taken from MASP-3-deficient, C4-deficient, and MBL-deficient human subjects. [Figure 19A] The level of C3b deposition on mannan-coated microtiter plates is shown graphically as a function of serum concentration in serum samples taken from WT, MASP-2-deficient, and MASP-1 / 3-deficient mice under "traditional" AP-specific conditions (i.e., BBS / EGTA / Mg++ without Ca++) or under physiological conditions that allow the lectin pathway and alternative pathway (AP) to function (BBS / Mg++ / Ca++), as described in Example 4. [Figure 19B] The level of C3b deposition on zymosan-coated microtiter plates is shown graphically as a function of serum concentration in serum samples taken from WT, MASP-2-deficient, and MASP-1 / 3-deficient mice under conventional AP-specific conditions (i.e., BBS / EGTA / Mg++ without Ca++) or under physiological conditions that allow the lectin and alternative pathways to function (BBS / Mg++ / Ca++), as described in Example 4. [Figure 19C] The level of C3b deposition on S. pneumoniae D39-coated microtiter plates is shown graphically as a function of serum concentration in serum samples taken from WT, MASP-2-deficient, and MASP-1 / 3-deficient mice under conventional AP-specific conditions (i.e., BBS / EGTA / Mg++ without Ca++) or under physiological conditions that allow the lectin and alternative pathways to function (BBS / Mg++ / Ca++), as described in Example 4. [Figure 20A] 10A-10C graphically show the results of a C3b deposition assay in highly diluted serum performed on mannan-coated microtiter plates using serum concentrations ranging from 0% to 1.25% under conventional AP-specific conditions (i.e., BBS / EGTA / Mg++ without Ca++) or physiological conditions that allow the lectin and alternative pathways to function (BBS / Mg++ / Ca++), as described in Example 4. [Figure 20B]Graph showing the results of a C3b deposition assay performed on zymosan-coated microtiter plates using serum concentrations ranging from 0% to 1.25% under conventional AP-specific conditions (i.e., BBS / EGTA / Mg++ without Ca++) or physiological conditions that allow the lectin and alternative pathways to function (BBS / Mg++ / Ca++), as described in Example 4. [Figure 20C] Graph showing the results of a C3b deposition assay performed on S. pneumoniae D39-coated microtiter plates using serum concentrations ranging from 0% to 1.25% under conventional AP-specific conditions (i.e., BBS / EGTA / Mg++ without Ca++) or physiological conditions that allow the lectin and alternative pathways to function (BBS / Mg++ / Ca++), as described in Example 4. [Figure 21] As described in Example 5, the graph shows the level of hemolysis of mannan-coated mouse red blood cells by human serum (measured by photometry of hemoglobin release from lysed mouse red blood cells (Crry / C3- / -) into the supernatant) over a range of serum dilutions in serum from MASP-3- / -, heat-inactivated normal human serum (HI NHS), MBL- / -, NHS+MASP-2 monoclonal antibody and NHS control under physiological conditions (i.e., in the presence of Ca++). [Figure 22] As described in Example 5, the graph shows the level of hemolysis of mannan-coated mouse red blood cells by human serum (measured by photometry of hemoglobin release from lysed mouse red blood cells (Crry / C3- / -) into the supernatant) over a range of serum concentrations in serum from MASP-3- / -, heat-inactivated (HI) NHS, MBL- / -, NHS+MASP-2 monoclonal antibody, and NHS control under physiological conditions (i.e., in the presence of Ca++). [Figure 23]As described in Example 5, the graph shows the level of hemolysis of uncoated mouse red blood cells by human serum (measured by photometry of hemoglobin release from lysed WT mouse red blood cells into the supernatant) over a range of serum concentrations in serum from 3MC (MASP-3- / -), heat-inactivated (HI) NHS, MBL- / -, NHS+MASP-2 monoclonal antibody, and NHS control under physiological conditions (i.e., in the presence of Ca++). [Figure 24] As described in Example 5, the graph shows hemolysis of uncoated mouse red blood cells by human serum (measured by photometry of hemoglobin release from lysed mouse red blood cells (CD55 / 59- / -) into the supernatant) over a range of serum concentrations in serum from heat-inactivated (HI) NHS, MBL- / -, NHS+MASP-2 monoclonal antibody, and NHS control under physiological conditions (i.e., in the presence of Ca++). [Figure 25] As described in Example 6, the graph shows the hemolysis (measured by photometry of hemoglobin release from lysed rabbit red blood cells into the supernatant) of mannan-coated rabbit red blood cells by MASP-1 / 3- / - mouse serum and WT control mouse serum over a range of serum concentrations under physiological conditions (i.e., in the presence of Ca++). [Figure 26] The level of C3b deposition (OD 405 nm) on zymosan-coated microtiter plates in a C3 deposition assay performed under AP-specific conditions as described in Example 7 is shown graphically as a function of serum concentration in serum samples from Factor D- / -, MASP-2- / -, and WT mice serum. [Figure 27] The level of C3b deposition (OD 405 nm) on zymosan-coated microtiter plates in a C3 deposition assay performed under physiological conditions (in the presence of Ca++) as described in Example 7 is shown graphically as a function of serum concentration in serum samples from Factor D- / -, MASP-2- / -, and WT mice. [Figure 28]The graph shows the level of C3b deposition (OD 405 nm) on zymosan-coated microtiter plates as a function of serum incubation time (min) in mouse serum collected from Factor D- / -, Factor B- / -, and with and without MASP-2 monoclonal antibody in a C3b deposition assay performed under physiological conditions (in the presence of Ca++) as described in Example 7. [Figure 29] Figure 29A graphically shows lectin pathway-specific C4b deposition on zymosan-coated microtiter plates measured ex vivo in undiluted serum samples collected from mice (n=3 mice / group) at various time points after subcutaneous administration of either 0.3 mg / kg or 1.0 mg / kg of murine MASP-2 MoAb, as described in Example 13. Figure 29B graphically shows the time course of lectin pathway recovery over 3 weeks after a single intraperitoneal administration of 0.6 mg / kg of murine MASP-2 MoAb in mice, as described in Example 13. [Figure 30] Figure 30A is a FACS histogram of MASP-3 antigen / antibody binding for clone M3J5, as described in Example 15. Figure 30B is a FACS histogram of MASP-3 antigen / antibody binding for clone M3M1, as described in Example 15. [Figure 31] Graph showing the saturation binding curve of clone M3J5 (clone 5) for the MASP-3 antigen, as described in Example 15. [Figure 32] Figure 32A is an amino acid sequence alignment of the VH regions of M3J5, M3M1, D14, and 1E10 to the chicken DT40 VH sequence, where dots represent amino acid identity with the DT40 sequence and dashes indicate spacing introduced to maximize alignment, as described in Example 15. Figure 32B is an amino acid sequence alignment of the VL regions of M3J5, M3M1, D14, and 1E10 to the chicken DT40 VL sequence, where dots represent amino acid identity with the DT40 sequence and dashes indicate spacing introduced to maximize alignment, as described in Example 15. [Figure 33] A bar graph showing the inhibitory activity of mAb1E10 in the Wieslab Complement System Screen, MBL Pathway, compared to the positive serum and isotype control antibody provided with the assay kit, as described in Example 15, demonstrating that mAb1E10 partially inhibits LEA-2-dependent activation (by inhibiting MASP-1-dependent activity of MASP-2), whereas the isotype control antibody does not. [Figure 34] As described in Example 16, the levels of C3b deposition for 1% normal human serum plus isotype control, SGMI-1Fc, or SGMI-2Fc over a concentration range of 0.15 to 1000 nM are graphically shown, demonstrating that both SGMI-1Fc and SGMI-2Fc inhibited C3b deposition from normal serum in mannan-coated ELISA wells with IC50 values ​​of approximately 27 nM and 300 nM, respectively. [Figure 35A] As described in Example 17, flow cytometry analysis of C3b deposition on heat-killed Staphylococcus aureus is presented, demonstrating the absence of C3b deposition in normal human serum in the presence of lectins and EDTA, which is known to inactivate the alternative pathway (panel 1), alternative pathway-driven C3b deposition in normal human serum treated with Mg++ / EGTA (panel 2), and the absence of alternative pathway-driven C3b deposition in factor B-, factor D-, and properdin (factor P)-depleted serum, as shown in panels 3, 4, and 5, respectively. [Figure 35B]As described in Example 17, the results of flow cytometry analysis of C3b deposition in heat-killed S. aureus are presented. Similar to EDTA-treated normal serum (panel 1), AP-driven C3b deposition was not observed in 3MC serum in the presence of Mg++ / EGTA (panel 3). Meanwhile, panels 4 and 5 show that both active full-length rMASP-3 (panel 4) and active rMASP-3(CCP1-CCP2-SP) (panel 5) restored AP-driven C3b deposition in 3MC serum to the level observed in normal human serum treated with Mg++ / EGTA (panel 2), whereas neither inactive rMASP-3(S679A) (panel 6) nor wild-type rMASP-1 (panel 7) was able to restore AP-driven C3b deposition in 3MC serum. [Figure 36] Figure 1 shows the results of Western blot analysis to determine factor B cleavage in response to Staphylococcus aureus in 3MC serum in the presence or absence of rMASP-3, demonstrating that normal human serum in the presence of EDTA (negative control, lane 1) shows very little factor B cleavage compared to normal human serum in the presence of Mg++ / EGTA, as shown in lane 2 (positive control), and that 3MC serum also shows very little factor B cleavage in the presence of Mg++ / EGTA, as shown in lane 3. However, as described in Example 17, factor B cleavage is restored by the addition of full-length recombinant MASP-3 protein to 3MC serum and pre-incubation, as shown in lane 4. [Figure 37] Coomassie staining of a protein gel in which factor B cleavage was analyzed, as described in Example 17, is shown, demonstrating that factor B cleavage is optimal in the presence of C3, MASP-3, and pro-factor D (lane 1), and that either MASP-3 or pro-factor D alone can mediate factor B cleavage as long as C3 is present, as shown in lanes 4 and 5. [Figure 38]As described in Example 17, the mean fluorescence intensity (MFI) of C3b staining of Staphylococcus aureus obtained from mAbD14 (binding to MASP-3), mAb1A5 (negative control antibody), and isotype control antibody is shown graphically as a function of mAb concentration in 3MC serum in the presence of rMASP-3, demonstrating that mAbD14 inhibits MASP-3-dependent C3b deposition in a concentration-dependent manner. [Figure 39] Western blot analysis of pro-factor D substrate cleavage shows that full-length wild-type recombinant MASP-3 (lane 2) and MASP-1 (lane 5) both completely or partially cleave pro-factor D to generate mature factor D, compared to pro-factor D alone (lane 1) or inactive full-length recombinant MASP-3 (S679A; lane 3) or MASP-1 (S646A; lane 4), as described in Example 18. [Figure 40] Western blot showing the inhibitory activity of MASP-3 binding to mAb D14 (lane 2) and M3M1 (lane 3) in MASP-3-dependent pro-factor D cleavage, compared to a control reaction containing only MASP-3 and pro-factor D (no mAb, lane 1) and a control reaction containing a mAb obtained from the DTLacO library that binds to MASP-1 but not MASP-3 (lane 4), as described in Example 18. [Figure 41] As described in Example 19, the level of AP-driven C3b deposition on zymosan-coated microtiter plates is shown graphically as a function of serum concentration in serum samples taken from MASP-3-deficient subjects (3MC), C4-deficient subjects, and MBL-deficient subjects, demonstrating that MASP-3-deficient sera from patient 2 and patient 3 have residual AP activity at high serum concentrations (25%, 12.5%, 6.25% serum concentrations), but have significantly higher AP50s (i.e., 8.2% and 12.3% of serum required to achieve 50% of maximum C3 deposition). [Figure 42A]The level of AP-driven C3b deposition on zymosan-coated microtiter plates under "conventional" AP-specific conditions (i.e., BBS / EGTA / Mg++ without Ca++) as described in Example 19 is shown graphically as a function of time in 10% human serum samples taken from MASP-3-deficient, C4-deficient, and MBL-deficient human subjects. [Figure 42B] Western blots showing human profactor D (25,040 Da) and / or mature factor D (24,405 Da) detected by a human factor D-specific antibody using plasma collected from 3MC patient #2 (MASP-3(- / -), MASP-1(+ / +)), 3MC patient #3 (MASP-3(- / -), MASP-1(- / -)), and serum from a normal donor (W), as described in Example 19. [Figure 42C] 10 graphically depicts the results of Weislab classical, lectin, and alternative pathway assays using plasma collected from 3MC patient #2, 3MC patient #3, and normal human serum, as described in Example 19. [Figure 43] A graph shows the hemolysis rate (measured photometrically as hemoglobin release from lysed rabbit red blood cells into the supernatant) of mannan-coated rabbit red blood cells over a range of serum concentrations in serum from two normal human subjects (NHS) and two 3MC patients (patient 2 and patient 3), measured in the absence of Ca++, as described in Example 19, demonstrating that MASP-3 deficiency reduces the rate of complement-mediated lysis of mannan-coated red blood cells compared to normal human serum. [Figure 44] As described in Example 19, the level of AP-driven C3b deposition on zymosan-coated microtiter plates is shown graphically as a function of the concentration of recombinant full-length MASP-3 protein added to serum samples collected from human 3MC patient 2 (MASP-3- / -), demonstrating that active recombinant MASP-3 protein reconstitutes AP-driven C3b deposition on zymosan-coated plates in a concentration-dependent manner, compared to negative control inactive recombinant MASP-3 (MASP-3A; S679A). [Figure 45] The graph shows the hemolysis rate (measured by photometry of hemoglobin release from lysed rabbit red blood cells into the supernatant) of mannan-coated rabbit red blood cells measured in the absence of Ca++ over a range of serum concentrations in (1) normal human serum (NHS); (2) 3MC patient serum; (3) 3MC patient serum + active full-length recombinant MASP-3 (20 μg / ml); and (4) heat-inactivated human serum (HIS), as described in Example 19, demonstrating that the lysis rate of rabbit red blood cells in 3MC serum containing rMASP-3 is significantly increased (p=0.0006) compared to the lysis rate in 3MC serum without recombinant MASP-3. [Figure 46] As described in Example 19, the graph shows the rabbit erythrocyte lysis rate in 7% human serum from 3MC patient 2 and 3MC patient 3 containing active recombinant MASP-3 at concentrations of 0 to 110 μg / ml (in BBS / Mg++ / EGTA), demonstrating that the rabbit erythrocyte lysis rate increases in a concentration-dependent manner with the amount of recombinant MASP-3. [Figure 47] The level of LEA-2-driven C3b deposition on mannan-coated ELISA plates is shown graphically as a function of the concentration of human serum diluted in BBS buffer for sera from a normal human subject (NHS), two 3MC patients (patient 2 and patient 3), the parents of patient 3, and an MBL-deficient subject. [Figure 48] Figure 48A presents results showing baseline VEGF protein levels in the RPE-choroid complex isolated from wild-type (WT) (+ / +) and MASP-2(- / -) mice, as described in Example 20. Figure 48B presents results showing VEGF protein levels in the RPE-choroid complex in WT (+ / +) and MASP-2(- / -) mice 3 days after laser-induced damage in a macular degeneration model, as described in Example 20. [Figure 49] As described in Example 20, results are presented showing the mean choroidal neovascularization (CNV) volume at 7 days after laser-induced injury in WT (+ / +) and MASP-2 (- / -) mice. [Figure 50]The graph shows the mean choroidal neovascularization (CNV) area at 7 days after laser-induced injury in WT(+ / +) mice pretreated with a single intraperitoneal injection of 0.3 mg / kg or 1.0 mg / kg of mouse MASP-2 monoclonal antibody, as described in Example 21. [Figure 51] Figure 51A presents results showing reduced infarct size in WT(+ / +) mice and MASP-2(- / -) mice after injury in a coronary artery occlusion and reperfusion model, as described in Example 22. Figure 51B presents results showing the distribution of individual mice tested in the coronary artery occlusion and reperfusion model, as described in Example 22. [Figure 52A] The graph shows the mean ischemic area at risk (AAR) and infarct volume (INF) as a percentage of total myocardial volume in WT(+ / +) and MASP-2(- / -) mice after left anterior descending coronary artery occlusion and reperfusion, as described in Example 23. [Figure 52B] This graph shows the infarct volume (INF) plotted against the average ischemic area (AAR) as a percentage of the left ventricular myocardial volume in WT (+ / +) and MASP-2 (- / -) mice after coronary artery occlusion and reperfusion, as described in Example 23. [Figure 52C] This graph shows the infarct volume (INF) in buffer-perfused hearts of WT (+ / +) and MASP-2 (- / -) mice prepared according to the Langendorff isolated perfused mouse heart model, in which global ischemia and reperfusion were performed in the absence of serum, as described in Example 23. [Figure 52D] This graph shows the relationship between infarct volume (INF) and risk zone (RZ) in buffer-perfused hearts of WT (+ / +) and MASP-2 (- / -) mice prepared according to the Langendorff isolated perfused mouse heart model, as described in Example 23. [Figure 53]Figure 53A graphically shows the results of a C3b deposition assay on immune complex-coated plates, demonstrating that MASP-2(- / -) mice retain a functional classical pathway, as described in Example 24. In the figure, the symbol "*" indicates serum from WT (MASP-2(+ / +)), the symbol "●" indicates serum from WT (C1q-depleted), the symbol "□" indicates serum from MASP-2(- / -), and the symbol "△" indicates serum from MASP-2(- / -) (C1q-depleted). Figure 53B graphically shows the results of a C3b deposition assay on zymosan-coated plates, demonstrating that MASP-2(- / -) mice retain a functional alternative pathway, as described in Example 24. In the figure, the symbol "*" indicates serum from WT (MASP-2(+ / +)), and the symbol "□" indicates serum from MASP-2(- / -). [Figure 54] Figure 54A graphically depicts myocardial ischemia / reperfusion injury (MIRI)-induced tissue loss after ligation of the left anterior descending coronary artery (LAD) and reperfusion in C4(- / -) mice (n=6) and matched WT littermate controls (n=7), showing the ischemic area (AAR) and infarct size (INF), as described in Example 24. Figure 54B graphically depicts infarct size (INF) as a function of ischemic area (AAR) in C4(- / -) and WT mice treated as described in Figure 42A, demonstrating that C4(- / -) mice are as susceptible to MIRI as WT controls (dashed line). [Figure 55A] 10 graphically depicts the results of a C3b deposition assay using serum from WT mice, serum from C4(- / -) mice, and serum from C4(- / -) mice preincubated with mannan, as described in Example 24. [Figure 55B] The graph shows the results of a C3b deposition assay using serum from WT mice, serum from C4(- / -) mice, and serum from MASP-2(- / -) mice mixed with various concentrations of murine MASP-2 mAb (mAbM11), as described in Example 24. [Figure 55C]10 graphically depicts the results of a C3b deposition assay using human serum from a WT (C4-sufficient) subject, human serum from a C4-deficient subject, and serum from a C4-deficient subject preincubated with mannan, as described in Example 24. [Figure 55D] The graph shows the results of a C3b deposition assay using human serum from a WT (C4-sufficient) subject and human serum from a C4-deficient subject mixed with human MASP-2 mAb (mAbH3), as described in Example 24. [Figure 56] Figure 56A graphically depicts a comparative analysis of C3 convertase activity in plasma from various complement-deficient mouse strains tested under either lectin- or classical-activation pathway-specific assay conditions, as described in Example 24. Figure 56B graphically depicts the time-resolved kinetics of C3 convertase activity in plasma from various complement-deficient mouse strains tested under lectin-activation pathway-specific conditions, as described in Example 24. [Figure 57A] The graph shows the extent of tissue damage in WT and MASP-2(- / -) mice after inducing transient ischemia / reperfusion injury (GIRI) in the gastrointestinal tract, as described in Example 25, demonstrating that MASP-2(- / -) mice have a significant degree of protection compared to WT controls. [Figure 57B] A graph showing the results of a C4b deposition assay performed using serum collected from mice (n=3) over time after a single intraperitoneal bolus injection of recombinant mouse MASP-2 antibody (mAbM11), as described in Example 25, demonstrating in vivo loss of lectin pathway functional activity. [Figure 57C]As described in Example 25, the effect of MASP-2 mAb treatment on the severity of GIRI pathology is graphically shown, demonstrating that mice administered murine MASP-2 mAb (mAbM11) 24 hours before being subjected to transient ischemia / reperfusion injury in the gastrointestinal tract (GIRI) showed significantly reduced tissue damage compared to mice administered saline (*p<0.05 when compared to mice treated with either the MASP-2 inhibitor antibody mAbM11 or an irrelevant isotype control antibody). [Figure 57D] This shows histological presentation of GIRI-mediated pathology in the small intestine in mice pretreated with a single intraperitoneal injection of saline, isotype control antibody, or recombinant mouse MASP-2 antibody (mAbM11) 12 hours before GIRI induction, as described in Example 25. [Figure 58] As described in Example 26, a graph shows cerebral infarction volume in WT (MASP-2(+ / +)) and MASP-2(- / -) mice after 30 minutes of ischemia and 24 hours of reperfusion. [Figure 59A] Figure 52A shows a series of photographs of stained brain sections from a WT (MASP-2+ / +) mouse after 30 minutes of ischemia and 24 hours of reperfusion. As described in Example 26, panels 1-8 of Figure 52A show different brain section areas corresponding to bregma 1-8, respectively, relative to the exit of the auditory nerve (bregma 0). [Figure 59B] A series of photographs of stained brain sections from a MASP-2(- / -) mouse after 30 minutes of ischemia and 24 hours of reperfusion are shown. As described in Example 26, panels 1-8 of Figure 52B show different brain section areas corresponding to bregma 1-8, respectively, relative to the exit of the auditory nerve (bregma 0). [Figure 60] As described in Example 27, results are presented showing the mean clinical arthritis scores of WT (+ / +) and MASP-2 (- / -) mice over time following Col2 mAb-induced rheumatoid arthritis. [Figure 61] 10 graphically depicts the results of a C3 deposition assay on serum samples taken from WT mice in the presence of dust mites or zymosan, as described in Example 28. [Figure 62] Figures 62A and 62B present dose-response curves of inhibition of C4b deposition (Figure 62A) and inhibition of thrombin activation in normal rat serum after administration of MASP-2 Fab2 antibody (H1), as described in Example 29. [Figure 63] Figures 63A and 63B show measured platelet aggregation (shown as aggregation area) in MASP-2(- / -) mice (Figure 63B) compared to platelet aggregation in untreated wild-type mice and wild-type mice in which the complement pathway has been inhibited by the depletion substances cobra venom factor (CVF) and terminal pathway inhibitor (C5aR antagonist) (Figure 63A) in a focal Schwartzman reaction model of disseminated intravascular coagulation, as described in Example 30. [Figure 64] 1 shows the results of Western blot analysis demonstrating the activation of human C3 by the thrombin substrates FXIa and FXa, as indicated by the presence of the a' chain, as described in Example 31. [Figure 65] 10A-10C are graphs showing the results of a C3b deposition assay on serum samples collected from WT, MASP-2(- / -), F11(- / -), F11(- / -) / C4(- / -), and C4(- / -) mice, as described in Example 31, demonstrating that a functional lectin pathway is present even in the complete absence of C4 or F11, whereas mice with combined F11-(- / -) / C4(- / -) deficiency lack a functional lectin pathway. [Figure 66] As described in Example 32, a graph shows the time to onset of microvascular occlusion after LPS injection in MASP-2- / - and WT mice, and the percentage of mice that exhibited thrombus formation measured over 60 minutes, demonstrating that thrombus formation was detected after 15 minutes in WT mice, with up to 80% of WT mice exhibiting thrombus formation at 60 minutes; in contrast, none of the MASP-2- / - mice exhibited thrombus formation during the 60-minute period (log-rank: p=0.0005). [Figure 67]As described in Example 33, the graph shows the survival rate over time (in hours) of saline-treated control mice (n=5) and MASP-2 antibody-treated mice (n=5) in the STX / LPS-induced HUS model, demonstrating that all control mice died within 42 hours, whereas 100% of MASP-2 antibody-treated mice survived throughout the entire experimental period. [Figure 68] The graph shows the percentage of mice that developed microvascular occlusion in the FITC / dextran UV model as a function of time after injury induction after treatment with isotype control or human MASP-2 antibody mAbH6 (10 mg / kg) administered 16 hours and 1 hour before FITC / dextran injection, as described in Example 34. [Figure 69] A graph shows the occlusion time in minutes for mice treated with human MASP-2 antibody (mAbH6) and an isotype control antibody. Data are reported as scattered dots with mean values ​​(horizontal bars) and standard error bars (vertical bars). The statistical test used for analysis was an unpaired t-test, as described in Example 34. The symbol "*" indicates p=0.0129. [Figure 70] A graph showing the time to occlusion in minutes for wild-type mice, MASP-2 KO mice, and wild-type mice pretreated with 10 mg / kg of human MASP-2 antibody (mAbH6) administered intraperitoneally 16 hours and again 1 hour before thrombus induction in the FITC-dextran / light-induced endothelial cell injury model of thrombosis using low light intensity (800-1500) as described in Example 34. DETAILED DESCRIPTION OF THE INVENTION

[0037] Sequence Listing Description SEQ ID NO:1 Human MAp19 cDNA SEQ ID NO:2 Human MAp19 protein (with leader) SEQ ID NO:3 Human MAp19 protein (mature) SEQ ID NO:4 Human MASP-2 cDNA SEQ ID NO:5 Human MASP-2 protein (with leader) SEQ ID NO:6 Human MASP-2 protein (mature) SEQ ID NO:7 Human MASP-3 cDNA SEQ ID NO:8 Human MASP-3 protein (with leader) SEQ ID NO:9 Human MASP-1 cDNA SEQ ID NO:10 Human MASP-1 protein (with leader) SEQ ID NO:11 Human MAp44 protein (with leader) SEQ ID NO:12 Rat MASP-2 cDNA SEQ ID NO:13 Rat MASP-2 protein (with leader) SEQ ID NO: 14 17D20_dc35VH21N11VL (OMS646) DNA encoding the heavy chain variable region (VH) (without signal peptide) SEQ ID NO:15 17D20_dc35VH21N11VL (OMS646) heavy chain variable region (VH) polypeptide SEQ ID NO:16 17N16mc heavy chain variable region (VH) polypeptide SEQ ID NO: 17 17D20_dc21N11VL (OMS644) light chain variable region (VL) polypeptide SEQ ID NO: 18 17N16_dc17N9 (OMS641) DNA encoding the light chain variable region (VL) (without signal peptide) SEQ ID NO:19 17N16_dc17N9 (OMS641) light chain variable region (VL) polypeptide SEQ ID NO:20 scFv daughter clone 17N16m_d17N9 full-length polypeptide SEQ ID NO:21 scFv daughter clone 17D20m_d3521N11 full-length polypeptide SEQ ID NO:22 scFv daughter clone 17N16m_d17N9 DNA encoding full-length polypeptide (no signal peptide) SEQ ID NO:23 scFv daughter clone 17D20m_d3521N11 DNA encoding the full-length polypeptide (without signal peptide) SEQ ID NO:24 Parent DTLacO heavy chain variable region (VH) polypeptide SEQ ID NO:25 MASP-3 specific clone M3J5 heavy chain variable region (VH) polypeptide SEQ ID NO:26 MASP-3 specific clone M3M1 heavy chain variable region (VH) polypeptide SEQ ID NO:27 Parent DTLacO light chain variable region (VL) polypeptide SEQ ID NO:28 MASP-3 specific clone M3J5 light chain variable region (VL) polypeptide SEQ ID NO:29 MASP-3 specific clone M3M1 light chain variable region (VL) polypeptide SEQ ID NO:30 MASP-3 clone D14 heavy chain variable region (VH) polypeptide SEQ ID NO:31 MASP-3 clone D14 light chain variable region (VL) polypeptide SEQ ID NO:32 MASP-1 clone 1E10 heavy chain variable region (VH) polypeptide SEQ ID NO:33 MASP-1 clone 1E10 light chain variable region (VL) polypeptide SEQ ID NO:34 SGMI-1 peptide SEQ ID NO:35 SGMI-2 peptide SEQ ID NO:36 Human IgG1-Fc polypeptide SEQ ID NO:37 Peptide linker #1 (12 aa) SEQ ID NO:38 Peptide linker #2 (10 aa) SEQ ID NO:39 Nucleic acid encoding a polypeptide fusion comprising a human IL-2-signal sequence, SGMI-1, linker #1 and human IgG1-Fc SEQ ID NO:40 Mature polypeptide fusion comprising SGMI-1, linker #1 and human IgG1-Fc (SGMI-1Fc) SEQ ID NO:41 Nucleic acid encoding a polypeptide fusion comprising human IL-2-signal sequence, SGMI-2, linker #1 and human IgG1-Fc SEQ ID NO:42 Mature polypeptide fusion comprising SGMI-2, linker #1 and human IgG1-Fc (SGMI-2Fc)

[0038] Detailed Description I. Definition Unless otherwise defined herein, all terms used herein have the same meaning as would be understood by one of ordinary skill in the art of the present invention. The following definitions are provided to clarify the terms used in the specification and claims to describe the present invention.

[0039] As used herein, lectin pathway effector arm 1 ("LEA-1") refers to the lectin-dependent activation of factors B and D by MASP-3.

[0040] As used herein, lectin pathway effector arm 2 ("LEA-2") refers to MASP-2-dependent complement activation.

[0041] As used herein, the term "MASP-3-dependent complement activation" refers to two parts: (i) Ca ++ (ii) lectin MASP-3-dependent activation of factor B and factor D, which is involved in LEA-1-mediated complement activation, occurring in the presence of Ca and generally resulting in the conversion of C3bB to C3bBb and pro-factor D to factor D; and ++LEA-1-mediated complement activation and lectin-independent conversion of factors B and D have been shown to result in opsonization and / or lysis. While not wishing to be bound by any particular theory, it is believed that only when multiple C3b molecules associate and bind in close proximity does the C3bBb C3 convertase change its substrate specificity and cleave C5 as an alternative pathway C5 convertase termed C3bBb(C3b)n.

[0042] As used herein, the term "MASP-2-dependent complement activation," also referred to herein as LEA-2-mediated complement activation, refers to Ca ++ The lectin pathway involves MASP-2 lectin-dependent activation, which occurs in the presence of lectin-dependent ...

[0043] As used herein, the term "traditional understanding of the alternative pathway," also referred to as the "conventional alternative pathway," refers to the alternative pathway prior to the discoveries described herein, which was traditionally thought to result from the spontaneous proteolytic generation of C3b from complement factor C3, i.e., complement activation induced by, for example, zymosan from fungal and yeast cell walls, lipopolysaccharide (LPS) from Gram-negative outer membranes and rabbit erythrocytes, as well as many pure polysaccharides, viruses, bacteria, animal tumor cells, parasites, and damaged cells. As used herein, activation of the "traditional alternative pathway," also referred to as the "alternative pathway," is associated with the activation of Mg ++ in EGTA / EGTA buffer (i.e., Ca ++ (in the absence of

[0044] The term "lectin pathway," as used herein, refers to complement activation that occurs through the specific binding of serum and non-serum carbohydrate-binding proteins, including mannan-binding lectin (MBL), CL-11, and ficolins (H-ficolin, M-ficolin, or L-ficolin). As described herein, the inventors have discovered that the lectin pathway is driven by two effector arms: lectin pathway effector arm 1 (LEA-1), which is now known to be MASP-3 dependent, and lectin pathway effector arm 2 (LEA-2), which is MASP-2 dependent. Activation of the lectin pathway, as used herein, is defined as the activation of the Ca2+ receptor by Ca2+ receptors. ++ The assay is performed using a buffer containing

[0045] As used herein, the term "classical pathway" refers to complement activation that is triggered by antibodies bound to foreign particles and requires the binding of the recognition molecule C1q.

[0046] As used herein, the term "HTRA-1" refers to the serine peptidase high temperature requirement serine protease A1.

[0047] As used herein, the term "MASP-3 inhibitor" refers to any agent that directly or indirectly inhibits MASP-3-dependent complement activation, including agents that bind to or directly interact with MASP-3, including MASP-3 antibodies and their MASP-3-binding fragments, natural and synthetic peptides, competitive substrates, small molecules, expression inhibitors, and isolated natural inhibitors, and also encompasses peptides that compete with MASP-3 for binding to other recognition molecules in the lectin pathway (e.g., MBL, CL-11, H-ficolin, M-ficolin, or L-ficolin). In one embodiment, the MASP-3 inhibitor is specific for MASP-3 and does not bind to MASP-1 or MASP-2. Inhibitors that directly inhibit MASP-3 can be referred to as direct MASP-3 inhibitors (e.g., MASP-3 antibodies), while inhibitors that indirectly inhibit MASP-3 can be referred to as indirect MASP-3 inhibitors (e.g., MASP-1 antibodies that inhibit MASP-3 activation). An example of a direct MASP-3 inhibitor is a MASP-3-specific inhibitor, e.g., a MASP-3 inhibitor that specifically binds to a portion of MASP-3 (SEQ ID NO:8) with a binding affinity that is at least 10-fold greater than that for other components in the complement system. In one embodiment, the MASP-3 inhibitor indirectly inhibits MASP-3 activity, e.g., inhibitors of MASP-3 activation, including inhibitors of MASP-1-mediated MASP-3 activation (e.g., MASP-1 antibodies or MASP-1-binding fragments thereof, natural and synthetic peptides, small molecules, expression inhibitors, and isolated natural inhibitors, as well as peptides that compete with MASP-1 for binding to MASP-3). In another embodiment, the MASP-3 inhibitor inhibits MASP-3-mediated maturation of factor D. In another embodiment, the MASP-3 inhibitor inhibits MASP-3-mediated activation of factor B. MASP-3 inhibitors useful in the methods of the present invention may reduce MASP-3-dependent complement activation by more than 10%, for example, more than 20%, more than 50%, or more than 90%.In one embodiment, the MASP-3 inhibitor reduces MASP-3-dependent complement activation by more than 90% (i.e., results in only 10% or less MASP-3 complement activation). MASP-3 inhibition is expected to completely or partially block lectin-independent conversion of LEA-1-associated lysis and opsonization and factor B- and factor D-associated lysis and opsonization.

[0048] As used herein, the term "MASP-1 inhibitor" refers to any agent that binds to or directly interacts with MASP-1 and inhibits at least one of (i) MASP-3-dependent complement activation and / or (ii) MASP-2-dependent complement activation and / or (iii) lectin-independent or lectin-dependent MASP-1-mediated maturation of factor D. Lectin-dependent MASP-1-mediated maturation of factor D includes direct activation of factor D, including MASP-1 antibodies and their MASP-1-binding fragments, natural and synthetic peptides, small molecules, expression inhibitors, and isolated natural inhibitors, and also encompasses peptides that compete with MASP-1 for binding to another recognition molecule in the lectin pathway (e.g., MBL, CL-11, H-ficolin, M-ficolin, or L-ficolin). In one embodiment, a MASP-1 inhibitor useful in the methods of the present invention reduces MASP-3-dependent complement activation by more than 10%, e.g., more than 20%, more than 50%, or more than 90%. In one embodiment, the MASP-1 inhibitor reduces MASP-3-dependent complement activation by more than 90% (i.e., results in only 10% or less MASP-3 complement activation). In another embodiment, the MASP-1 inhibitor useful in the methods of the present invention reduces MASP-2-dependent complement activation by more than 10%, for example, more than 20%, more than 50%, or more than 90%. In one embodiment, the MASP-1 inhibitor reduces MASP-2-dependent complement activation by more than 90% (i.e., results in only 10% or less MASP-2 complement activation).

[0049] In another embodiment, MASP-1 inhibitors useful in the methods of the present invention reduce MASP-3-dependent complement activation (LEA-1), lectin-independent factor B and D conversion, and MASP-2-dependent complement activation (LEA-2) by more than 10%, for example, more than 20%, more than 50%, or more than 90%. In one embodiment, MASP-1 inhibitors reduce MASP-3-dependent complement activation (LEA-1), lectin-independent factor B and D conversion, and MASP-2-dependent complement activation (LEA-2) by more than 90% (i.e., result in only 10% or less MASP-3 complement activation and only 10% or less MASP-2 complement activation).

[0050] An example of a direct MASP-1 inhibitor is a MASP-1-specific inhibitor, e.g., a MASP-1 inhibitor that specifically binds to a portion of MASP-1 (SEQ ID NO:10) with a binding affinity at least 10-fold greater than that for other components of the complement system. In many cases, inhibition of MASP-1 would be expected to be effective in inhibiting MASP-3 and / or MASP-2, provided that MASP-1 can activate MASP-3 and MASP-1 can activate MASP-2. However, in some cases, inhibition of MASP-1 or MASP-3 or MASP-2 may be preferable over inhibition of other MASP targets. For example, in the context of Staphylococcus aureus (S. aureus) infection, MASP-3 has been shown to be activated and responsible for S. aureus opsonization in the absence of MASP-1 (see Iwaki D. et al., J Immunol 187(7):3751-8 (2011)). Thus, for example, in the treatment of paroxysmal nocturnal hemoglobinuria (PNH), it may be advantageous to directly inhibit MASP-1 rather than MASP-3 during LEA-1 inhibitory treatment of PNH, thereby reducing potential susceptibility to S. aureus.

[0051] As used herein, the term "MASP-2 inhibitor" refers to any agent that binds to or directly interacts with MASP-2 and inhibits at least one of (i) MASP-2-dependent complement activation and / or (ii) MASP-1-dependent complement activation, including MASP-2 antibodies and their MASP-2-binding fragments, natural and synthetic peptides, small molecules, expression inhibitors, and isolated natural inhibitors, as well as peptides that compete with MASP-2 for binding to other recognition molecules in the lectin pathway (e.g., MBL, CL-11, H-ficolin, M-ficolin, or L-ficolin). MASP-2 inhibitors useful in the methods of the present invention can reduce MASP-2-dependent complement activation by more than 10%, e.g., more than 20%, more than 50%, or more than 90%. In one embodiment, the MASP-2 inhibitor reduces MASP-2-dependent complement activation by more than 90% (i.e., causes only 10% or less MASP-2 complement activation). An example of a direct MASP-2 inhibitor is a MASP-2-specific inhibitor, e.g., a MASP-2 inhibitor that specifically binds to a portion of MASP-2 (SEQ ID NO:5) with a binding affinity that is at least 10 times greater than for other components in the complement system.

[0052] As used herein, the term "antibody" encompasses antibodies and antibody fragments thereof derived from any antibody-producing mammal (e.g., mouse, rat, rabbit, and primate, including human) or derived from hybridoma, phage selection, recombinant expression, or transgenic animal (or other method of producing antibodies or antibody fragments) that specifically bind to a target polypeptide, e.g., a MASP-1, MASP-2, or MASP-3 polypeptide or portion thereof. The term "antibody" is not intended to be limited in terms of the source of the antibody or the manner in which the antibody is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animal, peptide synthesis, etc.). Exemplary antibodies include polyclonal antibodies, monoclonal antibodies, and recombinant antibodies; panspecific antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies); humanized antibodies; murine antibodies; chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies; and anti-idiotypic antibodies, and may be any intact antibody or fragment thereof. As used herein, the term "antibody" encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (e.g., dAb, Fab, Fab', F(ab')2, Fv), single chain (ScFv), synthetic variants thereof, naturally occurring variants, fusion proteins comprising antibody portions and antigen-binding fragments of the required specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of an immunoglobulin molecule that contains an antigen-binding site or fragment (epitope recognition site) of the required specificity.

[0053] "Monoclonal antibody" refers to a homogeneous antibody population. Monoclonal antibodies are composed of amino acids (natural and non-natural) that are involved in selective binding of an epitope. Monoclonal antibodies are highly specific for their target antigen. The term "monoclonal antibody" encompasses not only intact and full-length monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single-chain (ScFv), variants thereof, fusion proteins containing the antigen-binding portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of an immunoglobulin molecule containing an antigen-binding fragment (epitope recognition site) with the required specificity and ability to bind to the epitope. The term is not intended to be limited in terms of the source of the antibody or the manner in which the antibody is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins and fragments, etc., as described above in the definition of "antibody."

[0054] As used herein, the term "antibody fragment" refers to a portion derived from or related to a full-length antibody, e.g., an MASP-1, MASP-2, or MASP-3 antibody, generally comprising the antigen-binding or variable region thereof. Illustrative examples of antibody fragments include Fab, Fab', F(ab)2, F(ab')2, and Fv fragments, scFv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0055] As used herein, a "single-chain Fv" or "scFv" antibody fragment refers to a fragment of an antibody V H Domain or V L These domains are present in a single polypeptide chain. Generally, an Fv polypeptide comprises a V H Domains and V L It further comprises a polypeptide linker between the domains which enables the scFv to form the desired structure for antigen binding.

[0056] As used herein, a "chimeric antibody" is a recombinant protein that contains the variable domains and complementarity determining regions derived from an antibody of a non-human species (e.g., rodent), while the remainder of the antibody molecule is derived from a human antibody.

[0057] As used herein, a "humanized antibody" is a chimeric antibody that contains minimal sequence matching specific complementarity-determining regions derived from a non-human immunoglobulin grafted onto a human antibody framework. Humanized antibodies are typically recombinant proteins in which only the antibody complementarity-determining regions are of non-human origin (including phage-display or yeast-produced antibodies).

[0058] As used herein, the term "mannan-binding lectin" ("MBL") is synonymous with mannan-binding protein ("MBP").

[0059] As used herein, "membrane attack complex" ("MAC") refers to a complex of five terminal complement components (C5b in combination with C6, C7, C8, and C9) (also called C5b-9) that inserts into and destroys membranes.

[0060] As used herein, a "subject" includes all mammals, including, but not limited to, humans, non-human primates, dogs, cats, horses, sheep, goats, cattle, rabbits, pigs, and rodents.

[0061] The abbreviations for amino acid residues used herein are as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0062] In the broadest sense, natural amino acids can be divided into groups based on the chemical properties of their side chains. "Hydrophobic" amino acids refer to Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys, or Pro. "Hydrophilic" amino acids refer to Gly, Asn, Gln, Ser, Thr, Asp, Glu, Lys, Arg, or His. This group of amino acids can be further divided into subgroups as follows: "Uncharged hydrophilic" amino acids refer to Ser, Thr, Asn, or Gln. "Acidic" amino acids refer to Glu or Asp. "Basic" amino acids refer to Lys, Arg, or His.

[0063] The term "conservative amino acid substitutions," as used herein, is exemplified by substitutions between amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine; (2) phenylalanine, tyrosine, and tryptophan; (3) serine and threonine; (4) aspartic acid and glutamic acid; (5) glutamine and asparagine; and (6) lysine, arginine, and histidine.

[0064] As used herein, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term covers oligonucleobases composed of naturally occurring nucleotides, sugars, and covalent internucleoside (backbone) linkages, as well as oligonucleotides containing non-naturally occurring modifications.

[0065] As used herein, "epitope" refers to a site on a protein (e.g., human MASP-3 protein) to which an antibody binds. "Overlapping epitopes" include at least one (e.g., two, three, four, five, or six) common amino acid residues, including linear and non-linear epitopes.

[0066] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably and refer to any peptide-linked amino acid chain, regardless of length or post-translational modification. The MASP proteins (MASP-1, MASP-2, or MASP-3) described herein may comprise a wild-type protein or may be a variant having 50 or fewer conservative amino acid substitutions (e.g., 1 or fewer, 2 or fewer, 3 or fewer, 4 or fewer, 5 or fewer, 6 or fewer, 7 or fewer, 8 or fewer, 9 or fewer, 10 or fewer, 12 or fewer, 15 or fewer, 20 or fewer, 25 or fewer, 30 or fewer, 35 or fewer, 40 or fewer, or 50 or fewer). Conservative substitutions typically include substitutions 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.

[0067] The human MASP-1 protein (designated SEQ ID NO:10), human MASP-2 protein (designated SEQ ID NO:5), and human MASP-3 protein (designated SEQ ID NO:8) described herein also include "peptide fragments" of proteins shorter than the full-length and / or immature (pre-pro) MASP protein, including peptide fragments of MASP proteins including terminal and internal deletion mutants of the protein. Deletion mutants can be missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid segments (of two or more amino acids) or single non-contiguous amino acids. In some embodiments, the human MASP-1 protein may have an amino acid sequence that is 70 (e.g., 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100)% or more identical to the human MASP-1 protein having the amino acid sequence set forth in SEQ ID NO:10.

[0068] In some embodiments, the human MASP-3 protein may have an amino acid sequence that is 70 (e.g., 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100)% or more identical to the human MASP-3 protein having the amino acid sequence set forth in SEQ ID NO:8.

[0069] In some embodiments, the human MASP-2 protein may have an amino acid sequence that is 70 (e.g., 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100)% or more identical to the human MASP-2 protein having the amino acid sequence set forth in SEQ ID NO:5.

[0070] In some embodiments, the peptide fragments are at least 6 (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, or 600 or more amino acid residues (e.g., at least 6 consecutive amino acid residues of any one of SEQ ID NOs:5, 8, or 10). In some embodiments, the antigenic peptide fragment of a human MASP protein is less than 500 amino acids in length (e.g., less than 450, less than 400, less than 350, less than 325, less than 300, less than 275, less than 250, less than 225, less than 200, less than 190, less than 180, less than 170, less than 160, less than 150, less than 140, less than 130, less than 120, less than 110, less than 100, less than 95, less than 90, less than 85, less than 80, less than 75, less than 70, less than 65, less than 60, less than 55, less than 50, less than 49, less than 48, less than 47, less than 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 amino acid residues (e.g., less than 500 consecutive amino acid residues of any one of SEQ ID NOs: 5, 8, or 10).

[0071] In some embodiments, for generating antibodies that bind to MASP-1, MASP-2 and / or MASP-3, the peptide fragments are antigenic and retain at least 10% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% or more) of the ability of the full-length protein to elicit an antigen response in a mammal (see "Methods for producing antibodies" below).

[0072] Amino acid sequence identity percentage (%) is defined as the percentage of amino acids in candidate sequence that are identical to the amino acids in reference sequence, after aligning sequences to achieve maximum sequence identity percentage, and introducing gaps if necessary.The alignment for determining sequence identity percentage can be achieved in various ways within the skill of the art, for example, by using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.Appropriate parameters for measuring alignment can be determined by known methods, including any algorithm required to achieve maximum alignment over the full length of the sequence to be compared.

[0073] In a representative embodiment, human MASP-1 protein (SEQ ID NO:10) is encoded by the cDNA sequence designated SEQ ID NO:9, human MASP-2 protein (SEQ ID NO:5) is encoded by the cDNA sequence designated SEQ ID NO:4, and human MASP-3 protein (SEQ ID NO:8) is encoded by the cDNA sequence designated SEQ ID NO:7. Those skilled in the art will recognize that the cDNA sequences disclosed in SEQ ID NO:9, SEQ ID NO:4, and SEQ ID NO:7 represent single alleles of human MASP-1, MASP-2, and MASP-3, respectively, and that allelic variation and alternative splicing are expected to occur. Allelic variations of the nucleotide sequences set forth in SEQ ID NO:9, SEQ ID NO:4, and SEQ ID NO:7, including silent mutations and mutations resulting in amino acid sequence changes, are within the scope of the present invention. Allelic variants of the MASP-1, MASP-2 or MASP-3 sequences can be cloned by probing cDNA or genomic libraries from different individuals according to standard techniques, or can be identified by homology comparison searches (e.g., BLAST searches) of databases containing such information.

[0074] II. Lectin pathway: A new understanding i. Overview: The lectin pathway has been redefined As described herein, the inventors have made the surprising discovery that the lectin pathway of complement has two effector arms for activating complement, both driven by the lectin pathway activation complex formed by carbohydrate-recognition components (MBL, CL-11, and ficolin): (i) an effector arm formed by the lectin pathway-associated serine proteases MASP-1 and MASP-3, termed "lectin pathway effector arm 1" or "LEA-1"; and (ii) a MASP-2-driven activation effector arm, termed "lectin pathway effector arm 2" or "LEA-2" herein. Both LEA-1 and LEA-2 can perform lysis and / or opsonization.

[0075] In addition, both Ca ++ It has been found that lectin-independent factor B conversion by MASP-3 and lectin-independent factor D conversion by HTRA-1, MASP-1, and MASP-3, which can occur in the absence of LEA-1, generally result in the conversion of C3bB to C3bBb and pro-factor D to factor D. Thus, inhibiting MASP-3 can inhibit both LEA-1 and lectin-independent factor B and / or factor D activation, which can result in the inhibition of lysis and / or opsonization.

[0076] Figure 1 illustrates this new understanding of the pathway of complement activation. As shown in Figure 1, LEA-1 is driven by lectin-bound MASP-3, which can activate the factor D zymogen to its active form and / or cleave C3b- or C3b(HO)-bound factor B, converting the C3bB zymogen complex to its enzymatically active form, C3bBb. Activated factor D generated by MASP-3 can also convert the C3bB or C3b(HO) zymogen complex to its enzymatically active form. MASP-1 can rapidly autoactivate, whereas MASP-3 cannot. In many cases, MASP-1 is an activator of MASP-3.

[0077] While in many instances lectins (i.e., MBL, CL-11, or ficolins) can target activity to the cell surface, Figure 1 also outlines the lectin-independent functions of MASP-3, MASP-1, and HTRA-1 in factor B activation and / or factor D maturation. Similar to the lectin-associated form of MASP-3 in LEA-1, the lectin-independent form of MASP-3 can mediate the conversion of C3bB or C3b(H20) to C3bBb (see also Figures 36 and 37) and the conversion of pro-factor D to factor D (see Figure 39). MASP-1 (see also Figure 39) and the non-MASP-related protein HTRA-1 can also activate factor D in a manner that does not require a lectin component (Stanton et al., Evidence That the HTRA1 Interactome Influences Susceptibility to Age-Related Macular Degeneration, presented at the Association for Research in Vision and Ophthalmology 2011 conference on May 4, 2011).

[0078] Thus, MASP-1 (via LEA-1 and lectin-independent forms), MASP-3 (via LEA-1 and lectin-independent forms), and HTRA-1 (lectin-independent only) can be activated either directly or indirectly at one or more points along the MASP-3-factor D-factor B axis. In doing so, they generate C3bBb, the alternative pathway C3 convertase, stimulating the production and deposition of C3b on the microbial surface. C3b deposition plays a key role in opsonization, marking the surface of microorganisms for destruction by host phagocytes such as macrophages. As an example herein (Figure 35), MASP-3 is important in the opsonization of Staphylococcus aureus. C3b deposition occurs rapidly in a MASP-3-dependent manner on S. aureus exposed to human serum (Figure 35).

[0079] However, the contribution of LEA-1 and the lectin-independent functions of MASP-3, MASP-1, or HTRA-1 are not limited to opsonization. As shown in Figure 1, these three components can also result in cell lysis and C3b production by indirect or direct factor B activation. These components also mediate the production of C3bBb (C3b) through the alternative pathway C5 convertase, i.e., C3bBb (C3b). n As further described herein, the requirement for MASP-3 and MBL, but not MASP-2 (and therefore not LEA-2 in this example), in the lysis of meningococci (see Figures 13, 14, and 15) demonstrates the role of LEA-1 in lysis. In summary, the opsonization results obtained from the S. aureus study and the lysis results observed in the meningococcal study support the role of LEA-1 in both processes (as shown in Figure 1). Furthermore, these studies demonstrate that both opsonization and lysis can result from the conversion of C3bB or C3b(H20) and / or the conversion of pro-factor D to factor D. Thus, both processes can be the result of lectin-independent roles of MASP-3, MASP-1, or HTRA-1. Thus, the model developed by the inventors in Figure 1 supports the use of inhibitors primarily of MASP-3 and of MASP-1 and / or HTRA-1 to block opsonization and / or lysis and treat diseases caused by dysregulation of these processes.

[0080] 1. Lectin pathway effector arm (LEA-1) The first effector arm of the lectin pathway, LEA-1, is formed by the lectin pathway-associated serine proteases MASP-1 and MASP-3. As described herein, we have previously shown that the alternative pathway is not substantially activated on a surface structure in the absence of MASP-3 but the presence of MASP-1. These results demonstrate a previously undiscovered role for MASP-3 in initiating the alternative pathway, confirmed using MASP-3-deficient 3MC serum collected from a patient with a rare 3MC autosomal recessive disorder harboring a mutation that disables the serine protease domain of MASP-3 (Rooryck C, et al., Nat Genet. 43(3):197-203 (2011)). Based on these novel findings, complement activation involving the traditionally defined alternative pathway is predicted to be MASP-3 dependent. Indeed, MASP-3 and its activation of LEA-1 may represent a previously elusive initiator of the alternative pathway.

[0081] As further described in Examples 1-4 herein, the inventors observed higher activity of lectin-dependent alternative pathway activation in MASP-2-deficient serum, resulting in higher bactericidal activity (i.e., lytic activity) against meningococci. While not wishing to be bound by any particular theory, it is likely that in the absence of MASP-2, carbohydrate-recognition complexes containing MASP-1 tightly associate with carbohydrate-recognition complexes containing MASP-3, activating MASP-3. It is known that MASP-3 activation is dependent on MASP-1 activity, as MASP-3 is not a self-activating enzyme and, in many cases, requires the activity of MASP-1 to convert its proenzyme form to its enzymatically active form. While MASP-1 (like MASP-2) is an auto-activating enzyme, MASP-3 is not self-activating and, in many cases, requires the enzymatic activity of MASP-1 to convert to its enzymatically active form. See Zundel S, et al., J. Immunol., 172(7):4342-50 (2004). In the absence of MASP-2, all lectin pathway recognition complexes are loaded with either MASP-1 or MASP-3. Thus, the absence of MASP-2 promotes MASP-1-mediated conversion of MASP-3 to its enzymatically active form. Once activated, activated MASP-3 initiates alternative pathway activation, now referred to as "LEA-1" activation, via MASP-3-mediated conversion of C3bB to C3bBb and / or pro-factor D to factor D. C3bBb, also referred to as alternative pathway C3 convertase, cleaves additional C3 molecules, resulting in the deposition of opsonic C3b molecules. If several C3b fragments bind closely to the C3bBb convertase complex, the alternative pathway C5 convertase C3bBb(C3b)n forms, which promotes MAC formation. In addition, surface-deposited C3b molecules form new sites for factor B binding, which in turn are cleaved by factor D and / or MASP-3 to create additional sites at which the alternative pathway C3 and C5 convertase complexes can form.This latter process is necessary for effective lysis and does not require lectins once initial C3b deposition has occurred. A recent publication (Iwaki D. et al., J Immunol 187(7):3751-8 (2011)) and data generated by the present inventors (Figure 37) demonstrate that activated MASP-3 converts the alternative pathway C3 convertase proenzyme complex C3bB into its enzymatically active form. We have previously found that MASP-3-mediated cleavage of factor B represents a subcomponent of the newly described LEA-1, which promotes the lectin-dependent formation of the alternative pathway C3 convertase C3bBb.

[0082] 2. Lectin pathway effector arm (LEA-2) The second effector arm of the lectin pathway, LEA-2, is formed by the lectin pathway-associated serine protease MASP-2. MASP-2 is activated when the recognition component binds to its respective pattern and can also be activated by MASP-1, subsequently cleaving complement component C4 into C4a and C4b. After the cleavage product C4b binds to plasma C2, C4b-bound C2 becomes the substrate for a second MASP-2-mediated cleavage step that converts C4b-bound C2 into the enzymatically active complex C4bC2a and small C2b cleavage fragments. C4b2a is the C3 convertase of the lectin pathway that converts the abundant plasma component C3 into C3a and C3b. C3b binds to any nearby surface via a thioester bond. If several C3b fragments bind closely to the C3 convertase complex C4b2a, this convertase changes its specificity to convert C5 to C5b and C5a, forming the C5 convertase complex C4b2a(C3b)n. Although this C5 convertase can initiate MAC formation, this process alone is thought to be insufficient to promote lysis. Rather, initial C3b opsonins produced by LEA-2 nucleate the formation of new alternative pathway C3 convertase and C5 convertase sites, which ultimately result in abundant MAC formation and lysis. The latter event is mediated by factor D activation of factor B associated with LEA-2-formed C3b and is therefore dependent on LEA-1 due to the essential role of MASP-1 in factor D maturation. Furthermore, C4-deficient mice are not protected from ischemia-reperfusion injury, whereas MASP-2-deficient mice are (Schwaeble et al., PNAS, 2011), suggesting that the MASP-2-dependent C4 bypass activation pathway for activating C3 in the absence of C4 plays an important role in the pathophysiology of ischemia-reperfusion injury. LEA-2 is also involved in the coagulation pathway, including the cleavage of prothrombin to thrombin (common pathway) and the cleavage of factor XII (Hagemann factor) to its enzymatically active form, XIIa. Factor XIIa, in turn, cleaves factor XI to XIa (intrinsic pathway).Activation of the intrinsic pathway of the coagulation cascade results in fibrin formation, which is crucial for thrombus formation.

[0083] Figure 1 illustrates our new understanding of the lectin and alternative pathways based on the results provided herein. Figure 1 details the role of LEA-2 in both opsonization and lysis. Physiologically, MASP-2 is the initiator of "downstream" C3b deposition (and consequent opsonization) in multiple lectin-dependent contexts (Figures 20A, 20B, and 20C), but it also plays a role in the lysis of serum-susceptible bacteria. As shown in Figure 1, the proposed molecular mechanism responsible for the increased bactericidal activity of MASP-2-deficient or MASP-2-depleted serum / plasma for serum-susceptible pathogens such as N. meningitidis is that, for bacterial lysis, the lectin pathway recognition complex associated with MASP-1 and MASP-3 must bind to the bacterial surface in close proximity, thereby allowing MASP-1 to cleave MASP-3. In contrast to MASP-1 and MASP-2, MASP-3 is not an autoactivating enzyme but often requires activation / cleavage by MASP-1 to be converted to its enzymatically active form.

[0084] As further shown in Figure 1, activated MASP-3 can then cleave C3b-bound factor B on the pathogen surface to initiate the alternative activation cascade by forming the enzymatically active alternative pathway C3 and C5 convertases C3bBb and C3bBb(C3b)n, respectively. The lectin pathway activation complex with MASP-2 plays a role in MASP-3 activation, and in the absence of MASP-2 or after MASP-2 depletion, all lectin pathway activation complexes are loaded with either MASP-1 or MASP-3. Thus, in the absence of MASP-2, lectin pathway activation complexes with MASP-1 and MASP-3 are positioned in close proximity to each other on the microbial surface, activating more MASP-3 and thereby significantly increasing the likelihood of MASP-3-mediated cleavage of C3b-bound factor B to form the alternative pathway C3 and C5 convertases C3bBb and C3bBb(C3b)n on the microbial surface. This results in activation of the terminal activation cascade C5b-C9, which forms the membrane attack complex, consisting of surface-bound C5b associated with C6, C5bC6 associated with C7, C5bC6C7 associated with C8, and C5bC6C7C8, resulting in polymerization of C9, which inserts into bacterial surface structures and forms pores in the bacterial wall, which results in osmotic killing of complement-targeted bacteria.

[0085] Central to this novel concept is that the data provided herein clearly demonstrate that the lectin pathway activation complex drives two distinct activation pathways, as shown in Figure 1: (i) LEA-1: A MASP-3-dependent activation pathway that initiates and drives complement activation by generating the alternative pathway convertase C3bBb through the initial cleavage and activation of factor B on the activator surface, which then catalyzes C3b deposition and the formation of the alternative pathway convertase C3bBb. The MASP-3-driven activation pathway plays an essential role in the opsonization and lysis of microorganisms, driving the alternative pathway on the bacterial surface to generate an optimal activation rate for generating the membrane attack complex. (ii) LEA-2: A MASP-2-dependent activation pathway that results in the formation of the C3 convertase C4b2a, and subsequently the C5 convertase C4b2a (C3b)n upon accumulation of the C3 cleavage product C3b. In the absence of complement C4, MASP-2 can form a second C3 convertase complex containing C2 and coagulation factor XI.

[0086] In addition to its role in lysis, the MASP-2-driven activation route plays a key role in bacterial opsonization, leading to microorganisms being coated with covalently bound C3b and its cleavage products (i.e., iC3b and C3dg), which are targeted for uptake and killing by C3 receptor-bearing phagocytes, such as granulocytes, macrophages, monocytes, microglia, and the reticuloendothelial system. This is the most effective route for clearance of bacteria and microorganisms that are resistant to complement lysis, including the majority of Gram-positive bacteria.

[0087] In addition to LEA-1 and LEA-2, there is the possibility of lectin-independent activation of factor D by MASP-3, MASP-1, and / or HTRA-1, and there is the possibility of lectin-independent activation of factor B by MASP-3.

[0088] Without wishing to be bound by any particular theory, it is believed that (i) LEA-1, (ii) LEA-2, and (iii) lectin-independent factor B and / or factor D activation each result in opsonization and / or MAC formation and consequent lysis.

[0089] ii. Background of MASP-1, MASP-2, and MASP-3 Currently, three mannan-binding lectin-associated serine proteases (MASP-1, MASP-2, and MASP-3) are known to associate with mannan-binding lectin (MBL) in human serum. Mannan-binding lectin has also been referred to as "mannose-binding protein" or "mannose-binding lectin" in recent literature. The MBL-MASP complex plays an important role in innate immunity thanks to the binding of MBL to carbohydrate structures present on a variety of microorganisms. Interaction of MBL with specific arrays of carbohydrate structures results in activation of MASP zymogens, which in turn activate complement by cleaving complement components C4 and C2 to form the C3 convertase C4b2b (Kawasaki et al., J. Biochem 106:483-489 (1989); Matsushita & Fujita, J. Exp Med. 176:1497-1502 (1992); Ji et al., J. Immunol. 150:571-578 (1993)).

[0090] Until recently, the MBL-MASP zymogen complex was thought to contain only one type of protease (MASP-1), but it is now clear that there are two other distinct proteases associated with MBL (i.e., MASP-2 and MASP-3) (Thiel et al., Nature 386:506-510 (1997); Dahl et al., Immunity 15:127-135 (2001)) and an additional 19-kDa serum protein termed "MAp19" or "sMAP" (Stover et al., J. Immunol. 162:3481-3490 (1999); Stover et al., J. Immunol. 163:6848-6859 (1999); Takahashi et al., Int. Immunol 11:859-63 (1999)).

[0091] MAp19 is an alternatively spliced ​​gene product of the structural gene of MASP-2 and lacks the four C-terminal domains of MASP-2, including the serine endopeptidase domain. Abundantly expressed truncated mRNA transcripts encoding MAp19 are generated by alternative splicing / polyadenylation events of the MASP-2 gene. By a similar mechanism, the MASP-1 / 3 gene produces three major gene products: two serine proteases, MASP-1 and MASP-3, and a 44-kDa truncated gene product called "MAp44" (Degn et al., J. Immunol 183(11):7371-8 (2009); Skjoedt et al., J. Biol Chem 285:8234-43 (2010)).

[0092] MASP-1 was originally described as the P-100 protease component of serum Ra-reactive factor, but it is now recognized as a complex composed of MASP in addition to MBL (Matsushita et al., Collections and Innate Immunity, (1996); Ji et al., J Immunol 150:571-578 (1993). The ability of the MBL-associated endopeptidase within the MBL-MASP complex to act on complement components C4 and C2 in a manner apparently identical to the C1s enzyme within the C1q-(C1r)2-(C1s)2 complex of the classical complement pathway suggests the existence of an MBL-MASP complex functionally similar to the C1q-(C1r)2-(C1s)2 complex. The C1q-(C1r)2-(C1s)2 complex is activated by the interaction of C1q with the Fc region of antibodies IgG or IgM present in immune complexes. This results in autoactivation of the C1r zymogen, which in turn activates the C1s zymogen, which then acts on complement components C4 and C2.

[0093] The stoichiometry of MBL-MASP complexes differs from that seen in C1q-(C1r)2-(C1s)2 complexes in that different MBL oligomers appear to be associated with different proportions of MASP-1 / MAp19 or MASP-2 / MASP-3 (Dahl et al., Immunity 15:127-135 (2001)). The majority of MASPs and MAp19 found in serum are not complexed with MBL (Thiel et al., J Immunol 165:878-887 (2000)) and may be associated, in part, with ficolins, a recently described group of lectins with fibrinogen-like domains that can bind to N-acetylglucosamine residues on microbial surfaces (Le et al., FEBS Lett 425:367 (1998); Sugimoto et al., J. Biol Chem 273:20721). (1998)). Among these, human L-ficolin, H-ficolin, and M-ficolin are associated with MASPs and MAp19, and can activate the lectin pathway upon binding to specific carbohydrate structures recognized by ficolins (Matsushita et al., J Immunol 164:2281-2284 (2000); Matsushita et al., J Immunol 168:3502-3506 (2002)). In addition to ficolins and MBL, a collectin, an MBL-like lectin called CL-11, has been identified as a lectin pathway recognition molecule (Hansen et al. J Immunol 185:6096-6104 (2010); Schwaeble et al. PNAS 108:7523-7528 (2011)). There is overwhelming evidence highlighting the physiological importance of these alternative carbohydrate recognition molecules; therefore, it is important to understand that MBL is not the only recognition component of the lectin activation pathway, and MBL deficiency should not be mistaken for a lectin pathway deficiency. Perhaps the existence of an array of alternative carbohydrate recognition complexes structurally related to MBL could broaden the spectrum of microbial structures that initiate a direct response of the innate immune system via complement activation.

[0094] All lectin pathway recognition molecules feature a specific MASP-binding motif within their collagen homology stalk region (Wallis et al. J. Biol Chem 279:14065-14073 (2004)). The MASP-binding sites in MBL, CL-11, and ficolins feature a distinct motif within this domain: Hyp-Gly-Lys-Xaa-Gly-Pro, where Hyp is hydroxyproline and Xaa is generally an aliphatic residue. Point mutations in this sequence disrupt MASP binding.

[0095] 1. Structure, sequence, chromosomal localization, and splice variants of each Figure 2 is a schematic diagram showing the domain structure of the MASP-2 polypeptide (SEQ ID NO:5) and MAp19 polypeptide (SEQ ID NO:2) and the exons encoding them. Figure 3 is a schematic diagram showing the domain structure of the MASP-1 polypeptide (SEQ ID NO:10), MASP-3 polypeptide (SEQ ID NO:8), and MAp44 polypeptide (SEQ ID NO:11) and the exons encoding them. As shown in Figures 2 and 3, the serine proteases MASP-1, MASP-2, and MASP-3 consist of six distinct domains arranged as found in C1r and C1s: (I) an N-terminal C1r / C1s / sea urchin VEGF / bone morphogenetic protein (or CUBI) domain; (II) an epidermal growth factor (EGF)-like domain; (III) a second CUB domain (CUBII); (IV and V) two complement control protein (CCP1 and CCP2) domains; and (VI) a serine protease (SP) domain.

[0096] Human and mouse MASP-1 (Sato et al., Int Immunol 6:665-669 (1994); Takada et al., Biochem Biophys Res Commun 196:1003-1009 (1993); Takayama et al., J. Immunol. 152:2308-2316 (1994)), human, mouse, and rat MASP-2 (Thiel et al., Nature 386:506-510 (1997); Endo et al., J. Immunol 161:4924-30 (1998); Stover et al., J. Immunol. 162:3481-3490 (1999); Stover et al., J. Immunol. 163:6848-6859 (1999)), and human MASP-3 (Dahl et al., Immunity 15:127-135 (2001)), cDNA-derived amino acid sequences indicate that these proteases are serine peptidases with a characteristic triad of His, Asp, and Ser residues within their putative catalytic domains (Genbank accession numbers as accessed in Genbank on February 15, 2012: human MASP-1: BAA04477.1; mouse MASP-1: BAA03944; rat MASP-1: AJ457084; human MASP-3: AAK84071; mouse MASP-3: AB049755, each incorporated herein by reference).

[0097] As further shown in Figures 2 and 3, when the zymogen is converted to its active form, the heavy chain (alpha or A chain) and the light chain (beta or B chain) are split to generate a disulfide-linked A chain and a smaller B chain corresponding to the serine protease domain. Cleavage of the Arg-Ile bond located between the second CCP domain (domain V) and the serine protease domain (domain VI) activates the single-chain zymogen MASP-1 (as do zymogens C1r and C1s). Zymogens MASP-2 and MASP-3 are thought to be activated in a manner similar to MASP-1. Each MASP protein forms a homodimer and binds Ca. ++ It associates with MBL and ficolins individually in a dependent manner.

[0098] 2. MASP-1 / 3 Human MASP-1 polypeptide (SEQ ID NO:10) and MASP-3 polypeptide (SEQ ID NO:8) arise from a single structural gene (Dahl et al., Immunity 15:127-135 (2001)), and the gene has been mapped to the 3q27-28 region of the long arm of chromosome 3 (Takada et al., Genomics 25:757-759 (1995)). MASP-3 and MASP-1 mRNA transcripts are generated from the primary transcript by alternative splicing / polyadenylation processes. The MASP-3 translation product consists of an alpha chain that is common to both MASP-1 and MASP-3, and a beta chain (serine protease domain) that is unique to MASP-3. As shown in Figure 3, the human MASP-1 gene encompasses 18 exons. The human MASP-1 cDNA (SEQ ID NO:10) The human MASP-3 cDNA (denoted as SEQ ID NO:9) is encoded by exons 2, 3, 4, 5, 6, 7, 8, 10, 11, 13, 14, 15, 16, 17, and 18. As further shown in Figure 3, the human MASP-3 gene encompasses 12 exons. The human MASP-3 cDNA (denoted as SEQ ID NO:7) is encoded by exons 2, 3, 4, 5, 6, 7, 8, 10, 11, and 12. Alternative splicing produces a protein called MBL-associated protein 44 ("MAp44") (denoted as SEQ ID NO:11), which is derived from exons 2, 3, 4, 5, 6, 7, 8, and 9.

[0099] The human MASP-1 polypeptide (SEQ ID NO:10, from Genbank BAA04477.1) has 699 amino acid residues, including a 19-residue leader peptide. Excluding the leader peptide, the calculated molecular weight of MASP-1 is 76,976 Da. As shown in Figure 3, the MASP-1 amino acid sequence contains four N-linked glycosylation sites. The domains of the human MASP-1 protein (see SEQ ID NO:10) are shown in Figure 3 and include an N-terminal C1r / C1s / sea urchin VEFG / bone morphogenetic protein (CUBI) domain (aa 25-137 of SEQ ID NO:10), an epidermal growth factor-like domain (aa 139-181 of SEQ ID NO:10), a second CUB domain (CUBII) (aa 185-296 of SEQ ID NO:10) and a tandem of complement control protein domains (CCP1 aa 301-363 and CCP2 aa 367-432 of SEQ ID NO:10), and a serine protease domain (aa 449-694 of SEQ ID NO:10).

[0100] The human MASP-3 polypeptide (SEQ ID NO:8 from Genbank AAK84071) has 728 amino acid residues, including a 19-residue leader peptide. Excluding the leader peptide, the calculated molecular weight of MASP-3 is 81,873 Da. As shown in Figure 3, there are seven N-linked glycosylation sites in MASP-3. The domains of the human MASP-3 protein (see SEQ ID NO:8) are shown in Figure 3 and include an N-terminal C1r / C1s / sea urchin VEGF / bone morphogenetic protein (CUBI) domain (aa 25-137 of SEQ ID NO:8), an epidermal growth factor-like domain (aa 139-181 of SEQ ID NO:8), a second CUB domain (CUBII) (aa 185-296 of SEQ ID NO:8), and a tandem of complement control protein domains (CCP1 aa 301-363 and CCP2 aa 367-432 of SEQ ID NO:8) and a serine protease domain (aa 450-711 of SEQ ID NO:8).

[0101] The MASP-3 translation product consists of an alpha chain (heavy chain) (alpha chain: aa 1-448 of SEQ ID NO:8) containing the CUB-1-EGF-CUB-2-CCP-1-CCP-2 domains, which are common to both MASP-1 and MASP-3, and a light chain (beta chain: aa 449-728 of SEQ ID NO:8) containing a serine protease domain, which is unique to MASP-3 and MASP-1.

[0102] 3. MASP-2 The human MASP-2 gene is located on chromosome 1p36.3-2 (Stover et al., Cytogenet and Cell Genet. 84:148-149 (1999)) and encompasses 12 exons, as shown in Figure 2. MASP-2 (SEQ ID NO:5) and MAp19 (SEQ ID NO:2) are encoded by a single structural gene transcript generated by alternative splicing / polyadenylation (Stover et al., Genes and Immunity 2:119-127 (2001)). Human MASP-2 cDNA (SEQ ID NO:4) is encoded by exons 2, 3, 4, 6, 7, 8, 9, 10, 11, and 12. A 20 kDa protein (SEQ ID NO:2) called MBL-associated protein 19 (also called "MAp19" or "sMAP"), encoded by (SEQ ID NO:1), arises from exons 2, 3, 4, and 5. MAp19 is a non-enzymatic protein containing the N-terminal CUB1-EGF region of MASP-2 with four additional residues (EQSL) derived from exon 5, as shown in Figure 2.

[0103] The MASP-2 polypeptide (SEQ ID NO:5) has 686 amino acid residues, including a 15-residue leader peptide that is cleaved after secretion to yield the mature form of human MASP-2 (SEQ ID NO:6). As shown in Figure 2, the MASP-2 amino acid sequence does not contain any N-linked glycosylation sites. The MASP-2 polypeptide exhibits a molecular structure similar to that of MASP-1, MASP-3, and C1r and C1s, i.e., proteases of the C1 complement system. The domains of the human MASP-2 protein (numbered with reference to SEQ ID NO:5) are shown in Figure 2 and include an N-terminal C1r / C1s / sea urchin VEGF / bone morphogenetic protein (CUBI) domain (aa 24-136 of SEQ ID NO:5), an epidermal growth factor-like domain (aa 138-180 of SEQ ID NO:5), a second CUB domain (CUBII) (aa 184-295 of SEQ ID NO:5), and a tandem of complement control protein domains (CCP1 aa 300-359 and CCP2 aa 364-431 of SEQ ID NO:5) and a serine protease domain (aa 445-682 of SEQ ID NO:5).

[0104] As shown in Figure 2, the MASP-2 polypeptide has an alpha chain (heavy chain) containing the CUB-1-EGF-CUB-2-CCP-1-CCP-2 domains (alpha chain: aa 1-443 of SEQ ID NO:5) and a beta chain (light chain) containing a serine protease domain (beta chain: aa 444-686). The CUB-1, EGF, and CUB-2 domains are required for dimerization, and the CUB-1, EGF, CUB-2, and CCP-1 domains contain the binding site for MBP. As described in Wallis et al., J. Biol Chem 279:14065-14073 (2004), each MASP-2 dimer binds to two MBL subunits.

[0105] 4. Comparison of the amino acid sequences of MASP-1, MASP-2, and MASP-3 Figure 4 is an amino acid alignment of the protein sequences of MASP-1 (SEQ ID NO:10), MASP-2 (SEQ ID NO:6), and MASP-3 (SEQ ID NO:8), showing the conserved catalytic triad residues (H, D, S) in the CUBI, EGF, CUBII, CCP1, CCP2, and serine protease (SP) domains. The symbol "." indicates identical amino acid sequences.

[0106] Figure 5 shows an amino acid alignment of the alpha chain sequences, including CUBI-EGF-CUBII-CCP1-CCP2, of MASP-1 (alpha chain: aa 1-447 of SEQ ID NO:10), MASP-2 (alpha chain: aa 1-443 of SEQ ID NO:5), and MASP-3 (alpha chain: aa 1-448 of SEQ ID NO:8). As indicated by the dotted boxes in Figure 5, there are numerous identities in the CUBI, EGF, and CUBII domains. The CCP1 and CCP2 domains are indicated by dark boxes. The total percent identity between the alpha chains of human MASP1 / 3 and human MASP-2 is presented in Table 1 below.

[0107] Figure 6 shows an amino acid alignment of the beta chain sequences (including the serine protease domain) of MASP-1 (beta chain: aa 448-699 of SEQ ID NO:10), MASP-2 (beta chain: aa 444-686 of SEQ ID NO:5), and MASP-3 (beta chain: aa 449-728 of SEQ ID NO:8). Figure 7A shows the pairwise amino acid alignment between the beta chain sequences of MASP-1 (beta chain: aa 448-699 of SEQ ID NO:10) and MASP-2 (beta chain: aa 444-686 of SEQ ID NO:5). Figure 7B shows the pairwise amino acid alignment between the beta chain sequence of MASP-1 (beta chain: aa 448-699 of SEQ ID NO:10) and the beta chain sequence of MASP-3 (beta chain: aa 449-728 of SEQ ID NO:8). Figure 7C shows the pairwise amino acid alignment between the beta chain sequence of MASP-2 (beta chain: aa 444-686 of SEQ ID NO:5) and the beta chain sequence of MASP-3 (beta chain: aa 449-728 of SEQ ID NO:8). Regions of identity in Figures 5-7 are shown as dotted boxes surrounding identical amino acids (shown as "." symbols).

[0108] The percent identity between the alpha and beta chains of human MASP-1, MASP-2, and MASP-3 proteins is presented in Table 1 below.

[0109] Table 1. Percent identity between human MASP proteins TIFF2026021601000002.tif38157

[0110] With regard to the alpha chain (heavy chain), the MASP-1 alpha chain and the MASP-3 alpha chain are identical (except for the 15 amino acid sequence at the 3' end), as shown in Table 1 above. The overall percent identity between the MASP-2 alpha chain and the MASP-3 alpha chain is 45.4%, with numerous identical regions in the CUBI-EGF-CUBII domain, as shown in Figure 5.

[0111] Regarding the beta chains (light chains), the overall percent identity among the three beta chains is low, ranging from 27% to 28%. However, although the overall identity among the three B chains is low, there are numerous areas of identity, as shown in Figure 6. As further shown in Figures 7A-C, the identical portions of the sequences are more widely distributed between MASP-2 and MASP-3 than between MASP-1 and MASP-2 or between MASP-1 and MASP-3.

[0112] All cysteine ​​residues present in MASP-2, MASP-3, C1r, and C1s align with the equivalent residues in MASP-1. However, MASP-1 has two cysteine ​​residues (positions 465 and 481 in the L chain) that are not found in MASP-2, MASP-3, C1r, and C1s. These two cysteine ​​residues in MASP-1 are in the predicted positions used to form "histidine loop" disulfide bridges, as found in trypsin and chymotrypsin. This suggests that MASP-2, MASP-3, C1r, and C1s may have evolved from MASP-1 by gene duplication and diversification (Nonaka & Miyazawa, Genome Biology 3 Reviews 1001.1-1001.5 (2001)).

[0113] 5. Biological function / activity of each, including associated human genetic data The role of the MBL / ficolin-MASP complex in innate immunity is mediated by calcium-dependent binding of the C-type lectin domain (present in MBL molecules) or the fibrinogen-like domain (present in ficolin molecules) to carbohydrate structures found in yeast, bacteria, viruses, and fungi. This recognition step leads to activation of the proenzyme MASP-2, which then cleaves C4 and C2 to form the C3 convertase C4b2b, mimicking the action of activated C1 in the C1q-(C1r)2-(C1s)2 complex. This allows deposition of C4b and C3b on target pathogens, thereby facilitating their killing and clearance by phagocytosis.

[0114] Recent evidence in the literature suggests that the lectin pathway activation complex requires only the activity of MASP-2 to cleave C4 and C2: (i) reconstitution of the minimal lectin pathway activation complex using recombinant MBL and recombinantly expressed MASP-2 appears sufficient to efficiently cleave both C4 and C2 in vitro (Vorup-Jensen et al., J. Immunol. 165:2093-2100 (2000); Rossi et al., J. Biol Chem 276:40880-40887 (2001); Ambrus et al., J. Immunol 170:1374-1382 (2003); Gal et al., J. Biol Chem 280:33435-33444 (2005)); and (ii) serum from mice with a genetically targeted deficiency of MASP-2 lacks any lectin pathway functional activity (Schwaeble et al., J. Immunol. 165:2093-2100 (2000); Rossi et al., J. Biol Chem 276:40880-40887 (2001); Ambrus et al., J. Immunol. 170:1374-1382 (2003); Gal et al., J. Biol Chem 280:33435-33444 (2005)). al., PNAS 108:7523-7528 (2011)). Recently, a genetically determined defect in MASP-2 has been described (Stengaard-Pedersen et al., New Eng. J. Med. 349:554-560, (2003)). A single nucleotide mutation causes an Asp-Gly exchange in the CUB1 domain, rendering MASP-2 unable to bind to MBL.

[0115] In addition, functional characterization of serum from mice lacking both MASP-1 and MASP-3 revealed that serum from wild-type mice and MASP-1 / MASP-3 knockout (MASP-1 / 3 - / -) mice serum under physiological conditions shows that lectin pathway activity is slower, but not absent (Takahashi et al., J. Immunol. 180:6132-6138 (2008); Schwaeble et al., PNAS (2011)). These studies suggest that, in contrast to the classical pathway effector endopeptidase C1s, activation of MASP-2 does not involve or require the activity of any of the other MBL-related serine endopeptidases (i.e., MASP-1 or MASP-3), and that the proteolytic activity of MASP-2 is sufficient to translate binding of lectin pathway carbohydrate recognition molecules (i.e., MBL, ficolin, or CL-11) into complement activation. However, more recent studies have demonstrated that although MASP-2 is capable of autoactivation, the catalytic rate of MASP-1 activation by the MASP-2 zymogen exceeds the rate of MASP-2 cleavage of its own zymogen form by approximately 85,000-fold (Heja et al., PNAS 106:10498-503 (2011); Megyeri et al., J. Biol. Chem. 288(13):8922-34 (2013)). Therefore, it is likely that the primary activator of MASP-2 in physiological situations is MASP-1. Based on the size of the C4 fragments produced and the functional C3 convertase activity generated, activated MASP-2 likely cleaves C4 and C2 in a manner identical to that performed by activated Cls, i.e., at a single arginyl bond in the alpha chain of C4 (Arg76 A1a77) and a single arginyl bond in the zymogen chain of C2 (Arg223 Lys224). It has also been reported that mouse MASP (in the form of a mouse MBL-MASP complex called Ra-reactive factor), but not C1s, can cleave the alpha chain of complement component C3 to generate the biologically active fragments C3a and C3b (Ogata et al., J. Immunol. 154:2351-2357 (1995)).If this occurs in the human system, it is thought to require cleavage of a single arginyl bond (Arg77-Ser78) within the alpha chain of C3. Activated MASP-2, like activated C1s, is unable to cleave complement component C5. The proteolytic activity of MASP-1 and MASP-2 is inhibited by C1 inhibitor (Matsushita et al., J Immunol 165:2637-2642 (2000)), whereas C1 inhibitor does not react with MASP-3 (Dahl et al., Immunity 15:127-135 (2001); Zundel et al., J Immunol 172:4342-4350 (2004)).

[0116] The biological functions of MASP-1 and MASP-3 have emerged slowly. The substrate specificity and physiological role of MASP-1 have been the subject of debate since its discovery. In recent years, numerous potential substrates have been identified. MASP-1 can slowly cleave native C3, and it was suggested that this direct C3 cleavage could initiate the complement cascade, possibly with the contribution of the alternative pathway (Matsushita et al., J Immunol 165:2637-2642 (2000)). Later, recombinant MASP-1 was shown to cleave the inactive (thioester hydrolyzed) form of C3, which is nonproductive in initiating the complement cascade (Ambrus et al., J Immunol 170:1374-1382 (2003)). The lack of lectin pathway activity in serum dilutions from MASP-2-deficient mice unequivocally demonstrated the absence of a MASP-1-driven C3 bypass mechanism (Schwaeble et al., PNAS 108:7523-7528 (2011)). Complement components cleaved with considerable efficiency by MASP-1 are C2 (Rossi et al., J Biol Chem 276:40880-40887 (2001); Ambrus et al., J Immunol 170:1374-1382 (2003)) and the proenzyme form of factor D (Takahashi et al., J Exp Med 207:29-37 (2010)). Therefore, regarding the ability of MASP-1 to cleave C2, it is conceivable that MASP-1 may enhance the ability of MASP-2 to form the C3 convertase (C4b2a) by cleaving C2. This suggestion is supported by the observation that lectin pathway activity is reduced in MASP-1-depleted human serum and in serum from MASP-1 / 3-deficient mice (Takahashi et al., J Immunol 180:6132-6138 (2008)), which also suggests that MASP-1 has a role in activating MASP-2.Furthermore, all C4b deposited by the MBL-MASP complex can form the C4b2a convertase, whereas only one in four C4b deposited by the classical pathway C1 complex can do so (Rawal et al., J Biol Chem 283 (12):7853-63 (2008)).

[0117] MASP-1 also cleaves MASP-2 and MASP-3 (Megyeri M., et al., J. Biol. Chem. 2013 Mar. 29;288(13):8922-34). Recent experiments suggest that MASP-2 can autoactivate, but MASP-1 is the primary activator of the proenzyme MASP-2. MASP-2 activation is delayed in the serum of MASP-1 knockout mice (Takahashi et al., J. Immunol. 180:6132-6138 (2008)), and similar results were obtained when MASP-1 activity was blocked by a specific inhibitor in normal human serum (Kocsis et al., J. Immunol. 185(7):4169-78 (2010)). Furthermore, Degn et al. (J. Immunol. 189(8):3957-69 (2012)) found that MASP-1 is crucial for MASP-2 activation and subsequent C4 cleavage in human serum. The catalytic rate of conversion of proenzyme MASP-2 to active MASP-2 is more than 85,000 times greater than the rate at which MASP-2 can self-activate (Megyeri et al., J. Biol. Chem. 288:8922-8934 (2013); Heja et al., J. Biol. Chem. 287(24):20290-300 (2012); Heja et al., PNAS 109:10498-503 (2012)).

[0118] Recent discoveries have also implicated MASP-1 in the alternative pathway. MASP-1 can convert the zymogen factor D to its enzymatically active form (Figure 39; Takahashi et al., J Exp Med 207:29-37 (2010)). Furthermore, MASP-1 activates the zymogen form of MASP-3 (Megyeri et al., J. Biol. Chem. 288:8922-8934 (2013); Degn et al. J. Immunol. 189(8):3957-69 (2012)), itself can activate the zymogen factor D (Figure 39), and can cleave factor B, another essential component of the alternative pathway, to its active form (Iwaki et al., J. Immunol. 187:3751-58 (2011)). However, conversion of pro-D and pro-B factors is likely independent of the activation state of LEA-2 and can occur through uncomplexed MASP-1.

[0119] Several lines of evidence indicate that MASP-1 is a thrombin-like enzyme and is important in activating the coagulation pathway. MASP-1 can cleave several thrombin substrates, including fibrinogen (Hajela K. et al., Immunobiology 205(4-5):467-75 (2002)), factor XIII (Krarup et al., Biochim Biophys Acta 1784(9):1294-1300 (2008)), and protease-activated receptor 4 (PAR4) (Megyeri et al., J Immunol 183(5):3409-16 (2009)). Furthermore, antithrombin in the presence of heparin is a more efficient inhibitor of MASP-1 than C1 inhibitor (Dobo et al., J Immunol 183:1207-1214 (2009)). The connection between complement and coagulation pathways is also emphasized by the observation that MASP-2 can activate prothrombin (Krarup A. et al., PLoS One 2(7):e623 (2007)). Limited coagulation represents an ancient type of innate immunity when the spread of invading pathogens is prevented by a fibrin clot. The released fibrinopeptide B has proinflammatory activity. MASP-1-mediated cleavage of PAR4 activates endothelial cell-initiated inflammatory responses (Megyeri et al., J Immunol 183(5):3409-16 (2009)).

[0120] MASP-3 does not have proteolytic activity against C4, C2, or C3 substrates. Conversely, MASP-3 has been reported to act as an inhibitor of the lectin pathway (Dahl et al., Immunity 15:127-135 (2001)). This conclusion can be derived from the fact that MASP-3, in contrast to MASP-1 and MASP-2, is not an autoactivating enzyme (Zundel S. et al., J Immunol 172:4342-4350 (2004); Megyeri et al., J. Biol. Chem. 288:8922-8934 (2013)).

[0121] Recently, transgenic mouse studies using mouse strains deficient in both MASP-1 and MASP-3 have provided evidence of possible physiological functions of MASP-1 and MASP-3. Although MASP-1 / 3 knockout mice have a functional lectin pathway (Schwaeble et al., PNAS 108:7523-7528 (2011)), they appear to lack alternative pathway activity (Takahashi et al., JEM 207(1):29-37 (2010)). The lack of alternative pathway activity is thought to be due to defective processing of complement factor D, which is required for alternative pathway activity. In MASP-1 / 3 knockout mice, all factor D circulates as a proteolytically inactive precursor form, whereas in normal mouse serum, virtually all factor D is in its active form. Biochemical analysis suggests that MASP-1 can convert complement factor D from its zymogen form to its enzymatically active form (Figure 39; Takahashi et al., JEM 207(1):29-37 (2010)). MASP-3 also cleaves the pro-factor D zymogen in vitro to produce active factor D (Figure 39; Takahashi et al., JEM 207(1):29-37 (2010)). Factor D exists as an active enzyme while circulating in normal individuals, and MASP-1, MASP-3, and HTRA-1 may be responsible for this activation. Furthermore, mice with both MBL and ficolin deficiency still produce normal levels of factor D and have a fully functional alternative pathway. Thus, these physiological functions of MASP-1 and MASP-3 do not necessarily involve lectins and are therefore independent of the lectin pathway. Recombinant mouse and human MASP-3 also cleave factor B in vitro and appear to support C3 deposition on Staphylococcus aureus (Figure 36; Iwaki D. et al., J Immunol 187(7):3751-8 (2011)).

[0122] Recent studies of patients with 3MC syndrome (formerly known as Carnevale, Mingarelli, Malpuech, and Michels syndrome; OMIM #257920) have revealed an unexpected physiological role for MASP-3. These patients exhibit severe developmental abnormalities, including cleft palate, cleft lip, cranial anomalies, and mental retardation. Genetic analysis identified 3MC patients who were homozygous for a dysfunctional MASP-3 gene (Rooryck et al., Nat. Genet. 43(3):197-203 (2011)). Another group of 3MC patients was found to be homozygous for a mutation in the MASP-1 gene that resulted in the absence of functional MASP-1 and MASP-3 proteins. Yet another group of 3MC patients lacked a functional CL-11 gene (Rooryck et al., Nat. Genet. 43(3):197-203 (2011)). Therefore, it is thought that the CL-11-MASP-3 axis plays a role during embryonic development. The molecular mechanism of this developmental pathway is unclear. However, because individuals with a deficiency in the common complement component C3 do not develop this syndrome, it is unlikely to be mediated by a conventional complement-driven process. Therefore, prior to our discovery described herein, the functional role of MASP-3 in lectin-dependent complement activation had not been previously established.

[0123] The structures of the catalytic fragments of MASP-1 and MASP-2 have been determined by X-ray crystallography. Structural comparison of the MASP-1 protease domain with that of other complement proteases revealed the basis for its relaxed substrate specificity (Dobo et al., J. Immunol. 183:1207-1214 (2009)). While the accessibility of the substrate-binding groove of MASP-2 is restricted by surface loops (Harmat et al., J. Mol. Biol. 342:1533-1546 (2004)), MASP-1 possesses an open substrate-binding pocket that resembles that of trypsin more than that of other complement proteases. A thrombin-like feature of the MASP-1 structure is an unusually large 60-amino acid loop (loop B) that may interact with substrates. Another intriguing feature of the MASP-1 structure is the internal salt bridge between S1 Asp189 and Arg224. Similar salt bridges, which can regulate the protease activity of factor D, are also found in the substrate-binding pocket. C1s and MASP-2 have nearly identical substrate specificity. Surprisingly, some of the eight surface loops of MASP-2 that determine substrate specificity have completely different conformations compared to those of C1s. This means that the two functionally related enzymes interact differently with the same substrate. The structure of zymogen MASP-2 reveals an inactive protease domain with a split oxyanion hole and substrate-binding pocket (Gal et al., J Biol Chem 280:33435-33444 (2005)). Surprisingly, zymogen MASP-2 exhibits considerable activity toward the large protein substrate C4. The structure of zymogen MASP-2 is highly flexible, likely allowing for transitions between inactive and active forms. This flexibility, reflected in the structure, may play a role in the autoactivation process.

[0124] Northern blot analysis indicates that the liver is the major source of MASP-1 and MASP-2 mRNA. Using a 5'-specific cDNA probe for MASP-1, a large MASP-1 transcript was found at 4.8 kb and a smaller one at approximately 3.4 kb, both of which were present in human and mouse liver (Stover et al., Genes Immunity 4:374-84 (2003)). MASP-2 mRNA (2.6 kb) and MAp19 mRNA (1.0 kb) are abundantly expressed in liver tissue. MASP-3 is expressed in the liver and many other tissues, including neural tissue (Lynch NJ et al., J Immunol 174:4998-5006 (2005)).

[0125] Patients with a history of infection and chronic inflammatory disease have been found to have mutant forms of MASP-2 that are unable to form active MBL-MASP complexes (Stengaard-Pedersen et al., N Engl J Med 349:554-560 (2003)). Some researchers have determined that MBL deficiency leads to a tendency to frequent infections in childhood (Super et al., Lancet 2:1236-1239 (1989); Garred et al., Lancet 346:941-943 (1995)) and a reduced resistance to HIV infection (Nielsen et al., Clin Exp Immunol 100:219-222 (1995); Garred et al., Mol Immunol 33 (suppl 1):8 (1996)). However, other studies have not demonstrated a significant correlation between low MBL levels and increased infections (Egli et al., PLoS One. 8(1):e51983 (2013); Ruskamp et al., J Infect Dis. 198(11):1707-13 (2008); Israels et al., Arch Dis Child Fetal Neonatal Ed. 95(6):F452-61 (2010)). Although the literature is conflicting, a deficiency, or non-availability, of MASPs can adversely affect an individual's ability to mount a rapid, non-antibody-dependent defense against certain pathogens.

[0126] iii. Ca ++ Traditional assay conditions lack supporting data for new understanding of Ca ++ Results obtained using a more physiological set of conditions including Provided herein are several independent lines of strong experimental evidence pointing to the conclusion that the lectin pathway activation route of complement activates complement via two independent effector mechanisms: (i) LEA-2: a MASP-2-driven pathway that mediates complement-driven opsonization, chemotaxis (Schwaeble et al., PNAS 108:7523-7528 (2011)), and cell lysis; and (ii) LEA-1: a novel MASP-3-dependent activation route that initiates complement activation by cleaving and activating factor B on the activator surface to generate the alternative pathway convertase C3bBb, which then catalyzes C3b deposition and the formation of the alternative pathway convertase C3bBb, which can result in cell lysis and microbial opsonization. In addition, as described herein, separate lectin-independent activation of factor B and / or factor D by MASP-1, MASP-3 or HTRA-1, or any combination of these three, can also result in complement activation via the alternative pathway.

[0127] Lectin pathway-dependent MASP-3-driven activation of the alternative pathway is thought to contribute to the well-established factor D-mediated C3b-bound factor B cleavage to achieve optimal activation rates for complement-dependent lysis via the terminal activation cascade to lyse bacterial cells via the formation of the C5b-9 membrane attack complex (MAC) on the cell surface (Figures 14-15). This rate-limiting event appears to require optimal coordination, as it is incomplete in the absence of MASP-3 functional activity and factor D functional activity. As described in Examples 1-4 herein, the inventors discovered this MASP-3-dependent lectin pathway function when studying the phenotypes of MASP-2 deficiency and MASP-2 inhibition in experimental mouse models of meningococcal infection. Genetically targeted MASP-2-deficient mice and wild-type mice treated with antibody-based MASP-2 inhibitors were highly resistant to experimental meningococcal infection (see Figures 8-12). When the infection dose was adjusted to achieve approximately 60% mortality among wild-type littermates, all MASP-2-deficient or MASP-2-depleted mice cleared the infection and survived (see Figures 8 and 12). This extremely high level of resistance was reflected in the significantly increased serum bactericidal activity in the sera of MASP-2-deficient or MASP-2-depleted mice. Further experiments demonstrated that this bactericidal activity was dependent on alternative pathway-driven bacteriolysis. Sera from mice lacking factor B, factor D, or C3 did not exhibit bactericidal activity against meningococci, indicating that the alternative pathway is essential for driving the terminal activation cascade. A surprising result was that sera from mice lacking MBL-A and MBL-C (both of which are lectin pathway recognition molecules that recognize meningococci) and lectin pathway-associated serine proteases MASP-1 and MASP-3 lost all lytic activity against meningococci (Figure 15). A recent paper (Takahashi M. et al., JEM 207:29-37 (2010)) and the research presented therein (Figure 39) demonstrate that MASP-1 can convert the proenzyme form of factor D to its enzymatically active form and may partially explain the loss of lytic activity due to the absence of enzymatically active factor D in these sera.This does not explain the lack of bactericidal activity in MBL-deficient mice, because these mice have normal, enzymatically active factor D (Banda et al., Mol Immunol 49(1-2):281-9 (2011)). Surprisingly, when human sera from patients with the rare 3MC autosomal recessive disorder, which harbors a mutation that renders the serine protease domain of MASP-3 dysfunctional (Rooryck C, et al., Nat. Genet. 43(3):197-203), were tested, bactericidal activity against meningococci was not detected (note: these sera contain MASP-1 and factor D, but not MASP-3).

[0128] The hypothesis that human serum requires lectin pathway-mediated MASP-3-dependent activity to express bactericidal activity is further supported by the observation that MBL-deficient human serum is also unable to lyse meningococci (Figures 13-14). MBL is the only human lectin pathway recognition molecule that binds to this pathogen. Because MASP-3 does not autoactivate, we hypothesize that the higher bacteriolytic activity in MASP-2-deficient serum may also be explained by favorable activation of MASP-3 through MASP-1. This is because, in the absence of MASP-2, all lectin pathway activation complexes that graft onto the bacterial surface are loaded with either MASP-1 or MASP-3. Because activated MASP-3 cleaves both factor D (Figure 39) and factor B in vitro to generate their respective enzymatically active forms (Figure 37 and Iwaki D., et al., J. Immunol. 187(7):3751-3758 (2011)), the most likely function of MASP-3 is to promote the formation of the alternative pathway C3 convertase (i.e., C3bBb).

[0129] Although the data for a lectin-dependent role are compelling, several experiments suggest that MASP-3 and MASP-1 are not necessarily required to function in complexes with lectin molecules. Experiments such as those shown in Figure 35B demonstrate the ability of MASP-3 to activate the alternative pathway (as demonstrated by C3b deposition on Staphylococcus aureus) in the absence of lectin complexes (i.e., in the presence of EGTA). Figure 35A demonstrates that deposition under these conditions depends on factors B, D, and P, all of which are key components of the alternative pathway. In addition, factor D activation by MASP-3 and MASP-1 (Figure 39) and factor B activation by MASP-3 (Figure 37) can occur in vitro in the absence of lectins. Finally, hemolysis studies of mouse erythrocytes in the presence of human serum demonstrate a clear role for MBL and MASP-3 in cell lysis. However, MBL deficiency does not fully reproduce the severity of MASP-3 deficiency, in contrast to what would be expected if all functional MASP-3 were complexed with MBL. Therefore, we do not wish to be constrained by the notion that all of the roles of MASP-3 (and MASP-1) demonstrated herein can be attributed solely to lectin-associated functions.

[0130] The identification of the two effector arms of the lectin pathway and the possible lectin-independent functions of MASP-1, MASP-3, and HTRA-1 represent novel opportunities for therapeutic intervention to effectively treat certain human diseases caused by excessive complement activation in the presence of microbial pathogens or altered host cell or metabolic deposits. As described herein, we have previously found that the alternative pathway is not activated on surface structures in the absence of MASP-3 and the presence of MASP-1 (see Figures 17-18, 35B, 41-42, 45-46). Because the alternative pathway is important for driving the rate-limiting events that result in bacteriolysis and cell lysis (Mathieson PW, et al., J Exp Med 177(6):1827-3 (1993)), our results demonstrate that activated MASP-3 plays a key role in the lytic activity of complement. As shown in Figures 14-15, 21-23, 43-44, and 46-47, the terminal complement activation cascade is defective in the serum of 3MC patients deficient in MASP-3, but not MASP-1. The data shown in Figures 14 and 15 demonstrate the loss of lytic activity in the absence of MASP-3 and / or MASP-1 / MASP-3 functional activity. Similarly, the loss of hemolytic activity in MASP-3-deficient human serum (Figures 21-23, 43-44, and 46-47), combined with the ability to reconstitute hemolysis by adding recombinant MASP-3 (Figures 46-47), strongly supports the conclusion that activation of the alternative pathway on target surfaces (essential for driving complement-mediated lysis) depends on the presence of activated MASP-3. Therefore, based on the new understanding of the lectin pathway detailed above, alternative pathway activation on target surfaces depends on lectin-independent factor B and / or factor D activation, also mediated by LEA-1 and / or MASP-3, and therefore, agents that block MASP-3-dependent complement activation will prevent alternative pathway activation on target surfaces.

[0131] The disclosure of the essential role of MASP-3-dependent initiation of alternative pathway activation, as described in essentially all current medical textbooks and recent review articles on complement, implies that the alternative pathway is not a stand-alone complement activation pathway. The widely held current scientific view is that the alternative pathway is activated by the amplification of spontaneous "tickover" C3 activation on the surfaces of specific particulate targets (microorganisms, zymosan, and rabbit erythrocytes). However, the absence of any alternative pathway activation in the sera of MASP-1 and MASP-3 double-deficient mice and human 3MC patient sera on zymosan-coated plates and two different bacteria (Neisseria meningitidis and Staphylococcus aureus), as well as the reduced hemolysis of erythrocytes in MASP-3-deficient sera from humans and mice, indicates that initiation of alternative pathway activation on these surfaces requires functional MASP-3. The required role of MASP-3 may be lectin-dependent or lectin-independent, resulting in the formation of alternative pathway C3 convertase and C5 convertase complexes, i.e., C3bBb and C3bBb(C3b)n, respectively. Thus, we disclose herein the existence of a previously elusive alternative pathway initiation route that relies on (i) the newly discovered lectin pathway activation arm, LEA-1, and / or (ii) the lectin-independent role of the proteins MASP-3, MASP-1, and HTRA-1.

[0132] III. Role of MASP-2 and MASP-3 in Paroxysmal Nocturnal Hemoglobinuria and Treatment Methods Using MASP-2 and MASP-3 Inhibitors i. Overview of PNH Paroxysmal nocturnal hemoglobinuria (PNH), sometimes called Marchiafava-Micheli syndrome, is an acquired, potentially life-threatening blood disorder. PNH can occur spontaneously, referred to as "primary PNH," or in the setting of other bone marrow disorders, such as aplastic anemia, referred to as "secondary PNH." The majority of cases are primary PNH. PNH is characterized by complement-induced red blood cell destruction (hemolysis), a low red blood cell count (anemia), thrombosis, and bone marrow failure. Laboratory findings of PNH show changes consistent with intravascular hemolytic anemia: low hemoglobin, high lactate dehydrogenase levels, a high reticulocyte count (immature blood cells released by the bone marrow to replace destroyed cells), and high bilirubin (a breakdown product of hemoglobin), in the absence of autoreactive RBC-binding antibodies as a possible cause.

[0133] A hallmark of PNH is chronic complement-mediated hemolysis caused by unregulated activation of terminal complement components, including the membrane attack complex, on the surface of circulating RBCs. PNH RBCs undergo uncontrolled complement activation and hemolysis due to the absence of complement regulators CD55 and CD59 on their surface (Lindorfer, MA, et al., Blood 115(11):2283-91(2010); Risitano, et al., Mini-Reviews in Medicinal Chemistry, 11:528-535(2011)). CD55 and CD59 are abundantly expressed on normal RBCs and regulate complement activation. CD55 acts as a negative regulator of the alternative pathway, inhibiting the assembly of the alternative pathway C3 convertase (C3bBb) complex and promoting the decay of preformed convertases, thus blocking the formation of the membrane attack complex (MAC). CD59 inhibits the complement membrane attack complex by directly binding to the C5b678 complex and preventing C9 from binding and polymerizing.

[0134] Although hemolysis and anemia are the primary clinical features of PNH, this disease is a complex hematological disorder that also includes thrombosis and bone marrow failure as part of its clinical manifestations (Risitano et al., Mini Reviews in Med Chem 11:528-535(2011)). At the molecular level, PNH is caused by the abnormal clonal expansion of hematopoietic stem cells lacking a functional PIG A gene. PIG A is an X-linked gene encoding a glycosyl-phosphatidylinositol transferase required for stable surface expression of GPI-anchored class A glycoproteins, including CD55 and CD59. For reasons currently under investigation, hematopoietic stem cells with a dysfunctional PIG A gene resulting from spontaneous somatic mutations are capable of clonally expanding to the point where their progeny constitute a significant portion of the peripheral hematopoietic cell pool. Both erythroid and lymphoid progeny of mutant stem cell clones lack CD55 and CD59, yet only RBCs undergo significant lysis after entering the circulation.

[0135] Current treatments for PNH include blood transfusions for anemia, anticoagulation for thrombosis, and the use of the monoclonal antibody eculizumab (Soliris®), which protects blood cells from immune destruction by inhibiting the complement system (Hillmen P. et al., N. Engl. J. Med. 350(6):552-559(2004)). Eculizumab (Soliris®) is a humanized monoclonal antibody that targets complement component C5 and blocks C5 cleavage by C5 convertase, thereby preventing the production of C5a and the assembly of MAC. Treatment of PNH patients with eculizumab reduced intravascular hemolysis as measured by lactate dehydrogenase (LDH) and led to hemoglobin stabilization and transfusion independence in approximately half of patients (Risitano et al., Mini-Reviews in Medicinal Chemistry, 11(6)(2011)). Although LDH levels became normal or near-normal in almost all patients treated with eculizumab (due to control of intravascular hemolysis), only about one-third of patients achieved hemoglobin levels of approximately 11 gr / dL, and the remaining patients receiving eculizumab continued to exhibit moderate to severe (i.e., transfusion-dependent) anemia at roughly the same rate (Risitano AM et al., Blood 113:4094-100(2009)). As described in Risitano et al., Mini-Reviews in Medicinal Chemistry 11:528-535(2011), it was demonstrated that PNH patients receiving eculizumab (but not untreated patients) contained large amounts of C3 fragments bound to PNH red blood cells. This finding led to the recognition that in PNH patients treated with Soliris, PNH RBCs, which were no longer hemolyzed due to C5 blockade, were now able to accumulate large amounts of membrane-bound C3 fragments. Membrane-bound C3 fragments act as opsonins, resulting in their capture within reticuloendothelial cells via specific C3 receptors, with subsequent extravascular hemolysis.Thus, while eculizumab therapy prevents intravascular hemolysis and the resulting sequelae, it merely changes the nature of these RBCs from intravascular to extravascular hemolysis, resulting in significant untreated anemia in many patients (Risitano AM et al., Blood 113:4094-100(2009)). Therefore, patients who develop C3 fragment-mediated extravascular hemolysis continue to require red blood cell transfusions, and these patients require a treatment strategy in addition to the use of eculizumab. Such C3 fragment-targeting approaches have proven useful in experimental systems (Lindorfer et al., Blood 115:2283-91, 2010).

[0136] ii. Complement initiation mechanisms in PNH The causal relationship between the defective surface expression of the negative complement regulators CD55 and CD59 in PNH, combined with the efficacy of eculizumab in preventing intravascular hemolysis, clearly defines PNH as a condition mediated by the complement system. Although this paradigm is widely accepted, the events that initiate complement activation and the specific complement activation pathways involved remain unresolved. Because CD55 and CD59 negatively regulate the terminal amplification step in the complement cascade common to all complement initiation pathways, deficiencies in these molecules impair membrane attack complex formation and membrane integration, regardless of whether complement activation is initiated by the lectin pathway, the classical pathway, or the alternative pathway's spontaneous turnover. Therefore, in PNH patients, any complement activation event that leads to C3b deposition on the RBC surface can trigger subsequent amplification and pathological hemolysis (intravascular and / or extravascular hemolysis), resulting in a sudden hemolytic attack. A clear mechanistic understanding of the molecular events that trigger hemolytic episodes in PNH patients remains unclear. Because the complement-initiating events in PNH patients experiencing hemolytic episodes are completely unknown, the prevailing view is that complement activation in PNH may occur spontaneously through low-level alternative pathway "tickover" activation, which is then augmented by inappropriate regulation of terminal complement activation due to the absence of CD55 and CD59.

[0137] However, it is important to note that in the natural history of PNH, PNH usually develops or worsens after certain events, such as infection or injury, which have been shown to trigger complement activation (Risitano, Biologics 2:205-222 (2008)). This complement activation response does not depend on prior host immunity to the inciting pathogen and therefore likely does not involve the classical pathway. Rather, this complement activation response is thought to be initiated by lectin binding to foreign or "altered self" carbohydrate patterns expressed on the surface of microbial agents or damaged host tissue. Thus, the events that precipitate the hemolytic episode in PNH are closely related to lectin-initiated complement activation. Thus, the lectin activation pathway very likely provides the initiating trigger that ultimately leads to hemolysis in PNH patients.

[0138] To dissect the activation cascade at the molecular level, we used well-defined pathogens that activate complement via lectins as experimental models to demonstrate that, depending on the inducing microorganism, complement activation can be initiated by either LEA-2 or LEA-1, resulting in opsonization and / or lysis. This same principle of dual responses (i.e., opsonization and / or lysis) to lectin-initiated events likely applies to other types of infectious pathogens or to lectin-induced complement activation following tissue injury to the host, or to other lectin-driven complement activation events that can lead to PNH. Based on this duality in the lectin pathway, we hypothesize that LEA-2- and / or LEA-1-initiated complement activation in PNH patients promotes opsonization and / or lysis of RBCs by C3b and subsequent extravascular and intravascular hemolysis. Thus, in the setting of PNH, inhibition of both LEA-1 and LEA-2 would be expected to address both extravascular and intravascular hemolysis and offer significant advantages over the C5 inhibitor eculizumab.

[0139] Exposure to Streptococcus pneumoniae was found to preferentially trigger lectin-dependent LEA-2 activation, which resulted in opsonization of the microorganism by C3b. Because Streptococcus pneumoniae is resistant to MAC-mediated lysis, clearance from the circulation occurs through opsonization by C3b. This opsonization and subsequent removal from the circulation is LEA-2 dependent, as demonstrated by impaired bacterial suppression in MASP-2-deficient mice and mice treated with a MASP-2 monoclonal antibody (PLOS Pathog., 8:e1002793. (2012)).

[0140] In investigating the role of LEA-2 in the innate host response to microorganisms, we tested additional pathogens. When meningococcus was studied as a model organism, dramatically different results were observed. Neisseria meningitidis also activates complement via lectins, and complement activation is required to contain meningococcal infection in naive hosts. However, LEA-2 does not play a host-protective functional role in this response. As shown in Figures 8 and 9, blocking LEA-2 by genetic ablation of MASP-2 does not reduce survival after meningococcal infection. Conversely, in these studies, blocking LEA-2 by MASP-2 ablation significantly improved survival (Figures 8 and 9) and disease scores (Figure 11). LEA-2 blockade by administration of MASP-2 antibodies produced the same results (Figure 12), ruling out secondary or compensatory effects in knockout mouse strains as a possible cause. These favorable results in LEA-2-depleted animals correlated with more rapid clearance of meningococci from the blood (Figure 10). Also, as described herein, incubation of meningococci with normal human serum killed the meningococci (Figure 13). Addition of a functional monoclonal antibody specific for human MASP-2, which blocks LEA-2, could enhance this killing response, whereas administration of an isotype-control monoclonal antibody did not. Nevertheless, this process depends on lectins and, at least in part, a functional complement system, as MBL-deficient or heat-inactivated human serum failed to kill meningococci (Figure 13). Together, these novel findings suggest that meningococcal infection in the presence of a functional complement system is suppressed by a lectin-dependent but LEA-2-independent complement activation pathway.

[0141] We tested the hypothesis that LEA-1 may be a complement pathway responsible for lectin-dependent killing of Neisseria meningitidis using serum samples from patient 3MC. This patient was homozygous for a nonsense mutation in exon 12 of the MASP-1 / 3 gene. As a result, this patient lacked functional MASP-3 protein but was otherwise complement-competent (exon 12 is specific for the MASP-3 transcript, and the mutation does not affect MASP-1 function or expression levels) (see Nat Genet. 43(3):197-203 (2011)). Normal human serum efficiently killed Neisseria meningitidis, whereas heat-inactivated serum lacking MBL (one of the recognition molecules in the lectin pathway) and MASP-3-deficient serum failed to kill Neisseria meningitidis (Figure 14). Thus, LEA-1 appears to mediate killing of Neisseria meningitidis. This finding was confirmed using serum samples from knockout mouse strains. Complement containing normal mouse serum readily killed meningococci, whereas serum from MBL-deficient or MASP-1 / 3-deficient mice was as ineffective as heat-inactivated serum lacking functional complement (Fig. 15). Conversely, MASP-2-deficient serum showed efficient killing of meningococci.

[0142] These findings provide previously unknown evidence of the dual nature of the lectin pathway by revealing the existence of separate LEA-2 and LEA-1 pathways for lectin-dependent complement activation. In the examples described above, LEA-2 and LEA-1 are nonredundant and mediate distinct functional outcomes. The data suggest that certain types of lectin pathway activators (including, but not limited to, Streptococcus pneumoniae) preferentially initiate complement activation via LEA-2, resulting in opsonization, whereas others (e.g., Neisseria meningitidis) preferentially initiate complement activation via LEA-1 and promote the cytolytic process. However, the data do not necessarily restrict LEA-2 to opsonization and LEA-1 to the cytolytic process, as both pathways can mediate opsonization and / or lysis in other situations.

[0143] In the context of lectin-dependent complement activation by meningococci, the LEA-2 and LEA-1 arms appear to compete with each other, as blockade of LEA-2 enhanced LEA-1-dependent lytic destruction of the organism in vitro (Figure 15). As mentioned above, this finding can be explained by the increased likelihood that in the absence of MASP-2, the lectin-MASP-1 complex resides in close proximity to the lectin-MASP-3 complex, which enhances LEA-1 activation and thus promotes more effective meningococcal lysis. Because meningococcal lysis is a major protective mechanism in the naive host, blockade of LEA-2 in vivo increases meningococcal clearance and results in enhanced killing.

[0144] While the examples above demonstrate opposing effects of LEA-2 and LEA-1 on outcomes following meningococcal infection, there may be other situations in which LEA-2 and LEA-1 may act synergistically together to produce specific outcomes. As detailed below, in other situations of lectin-mediated pathological complement activation, such as those present in PNH, LEA-2- and LEA-1-driven complement activation may cooperate synergistically to contribute to the overall pathology of PNH. In addition, as described herein, MASP-3 also contributes to the lectin-independent conversion of factors B and D, which is related to Ca ++ This can occur in the absence of C3bB and generally results in the conversion of C3bB to C3bBb and pro-factor D to factor D, which may further contribute to the pathogenesis of PNH.

[0145] iii. Biology and predicted functional activity in PNH This section describes the inhibitory effects of LEA-2 and LEA-1 blockade on hemolysis in an in vitro model of PNH. This discovery supports the utility of LEA-2 blockers (including, but not limited to, antibodies that bind to and block the function of MASP-2) and LEA-1 blockers (including, but not limited to, antibodies that bind to MASP-3, MASP-3, or both and block the function of its MASP-1-mediated activation) to treat patients suffering from one or more aspects of PNH, as well as the use of inhibitors of LEA-2 and / or LEA-1 and / or MASP-3-dependent lectin-independent complement activation (including MASP-2 inhibitors, MASP-3 inhibitors, and MASP-2 / MASP-3 or MASP-1 / MASP-2 dual or bispecific inhibitors, as well as pan-specific MASP-1 / MASP-2 / MASP-3 inhibitors) to alleviate the effects of C3 fragment-mediated extravascular hemolysis in PNH patients receiving treatment with C5 inhibitors such as eculizumab.

[0146] iv. MASP-2 inhibitors to block reticuloendothelial system-mediated opsonization of PNH RBCs and extravascular hemolysis As detailed above, patients with PNH become anemic due to two separate mechanisms of RBC clearance from the circulation: intravascular hemolysis due to activation of the membrane attack complex (MAC), and extravascular hemolysis following opsonization with C3b and subsequent clearance following complement receptor binding and uptake by the reticuloendothelial system. Intravascular hemolysis is largely prevented when patients are treated with eculizumab. Because eculizumab blocks the terminal lytic effector mechanisms that occur downstream of both complement-initiated activation events and subsequent opsonization, eculizumab does not block extravascular hemolysis (Risitano AM et al., Blood 113:4094-100(2009)). Instead, RBCs that would otherwise be hemolyzed in untreated PNH patients can now accumulate activated C3b protein on their surface, which enhances uptake by the reticuloendothelial system and enhances their extravascular hemolysis. Thus, eculizumab treatment essentially changes the nature of RBCs from intravascular hemolysis to potentially extravascular hemolysis. As a result, some PNH patients treated with eculizumab remain anemic. Therefore, agents that block upstream complement activation and inhibit opsonization of PNH RBCs may be particularly suitable for blocking the extravascular hemolysis sometimes seen with eculizumab.

[0147] The microbial data presented herein suggest that LEA-2 is often the dominant route of lectin-dependent opsonization. Furthermore, when lectin-dependent opsonization (measured as C3b deposition) was assessed on three prototypic lectin-activating surfaces (mannan, Figure 19A; zymosan, Figure 19B, and S. pneumoniae, Figure 19C), LEA-2 was shown to be a key factor in the opsonization of lectins under physiological conditions (i.e., Ca2+, where all complement pathways are operational). ++In the presence of lectin, the MASP-2-deficient serum (lacking LEA-2) appears to be the dominant route of lectin-dependent opsonization. Under these experimental conditions, MASP-2-deficient serum (lacking LEA-2) is substantially less effective than WT serum at opsonizing the test surface. MASP-1 / 3-deficient serum (lacking LEA-1) is also impaired, but this effect is much less pronounced compared to serum lacking LEA-2. The relative magnitude of the contribution of LEA-2 and LEA-1 to lectin-driven opsonization is further illustrated in Figures 20A-20C. Although it has been reported that the alternative pathway of complement supports opsonization of lectin-activated surfaces in the absence of the lectin or classical pathway (Selander et al., J Clin Invest 116(5):1425-1434 (2006)), the isolated alternative pathway (Ca ++The LEA-2 pathway (measured under lectin-free assay conditions) is thought to be substantially less effective than the LEA-2- and LEA-1-initiated processes described herein. By extrapolation, these data suggest that opsonization of PNH RBCs may be preferentially initiated by LEA-2 and to a lesser extent by LEA-1 than the result of lectin-independent alternative pathway activation (possibly amplified by an alternative pathway amplification loop). Thus, LEA-2 inhibitors may be expected to be most effective in suppressing opsonization and preventing extravascular hemolysis in PNH. However, recognition that lectins other than MBL, such as ficolins, bind to non-carbohydrate structures, e.g., acetylated proteins, and that MASP-3 preferentially associates with H-ficolin (Skjoedt et al., Immunobiol. 215:921-931, 2010), leaves open the possibility of a significant role for LEA-1 in PNH-associated RBC opsonization. Therefore, LEA-1 inhibitors are expected to have an additional anti-opsonic effect, and the combination of LEA-1 and LEA-2 inhibitors is expected to be optimal and mediate the strongest therapeutic benefit in suppressing opsonization and extravascular hemolysis in PNH patients. This concept is further supported by the opsonization data shown in Figure 28. Serum from factor D-deficient mice (which lack the ability to activate the alternative pathway in fluid phase but have functional classical and LEA-1 and LEA-2 pathways) does not exhibit a defect in opsonization compared to WT serum. Factor B-deficient serum lacking LEA-1 exhibits reduced opsonization, whereas factor D-deficient serum treated with MASP-2 monoclonal antibody to block LEA-2-mediated complement activation produces stronger opsonization suppression (Figure 28). Importantly, the addition of MASP-2 monoclonal antibody to factor B-deficient serum suppressed opsonization more effectively than either MASP-2 blockade or factor D blockade alone. Thus, LEA-2 and LEA-1 may act additively or synergistically to promote opsonization, and cross-reactive or bispecific LEA-1 / LEA-2 inhibitors are predicted to be most effective in blocking opsonization and extravascular hemolysis in PNH.

[0148] v. Role of MASP-3 inhibitors in PNH Using an in vitro model of PNH, we demonstrated that complement activation and resulting hemolysis in PNH are indeed initiated by LEA-2 and / or LEA-1 activation and are not an independent function of the alternative pathway. These studies used mannan-sensitized RBCs from various mouse strains, including RBCs from Crry-deficient mice (a key negative regulator of the terminal complement pathway in mice) and RBCs from CD55 / CD59-deficient mice, which lack the same complement regulator absent in PNH patients. When mannan-sensitized Crry-deficient RBCs were exposed to complement-sufficient human serum, the RBCs were substantially hemolyzed at a serum concentration of 3% (Figures 21 and 22), whereas complement-deficient serum (HI: heat-inactivated) was not hemolytic. Surprisingly, complement-sufficient serum in which LEA-2 was blocked by the addition of an MASP-2 antibody had reduced hemolytic activity, and 6% serum was required for effective hemolysis. Similar observations were made when CD55 / CD59-deficient RBCs were tested (Figure 24). Complement-sufficient human serum supplemented with MASP-2 monoclonal antibody (i.e., LEA-2-inhibited serum) was approximately two-fold less effective than untreated serum in supporting hemolysis. Furthermore, a higher concentration of LEA-2-blocking serum (i.e., treated with anti-MASP-2 monoclonal antibody) was required to promote effective hemolysis of intact WT RBCs compared with untreated serum (Figure 23).

[0149] Even more surprisingly, serum from a 3MC patient homozygous for dysfunctional MASP-3 protein (and thus lacking LEA-1) was completely unable to hemolyze mannan-sensitized Crry-deficient RBCs (Figures 22 and 23). Similar results were observed when non-sensitized normal RBCs were used. As shown in Figure 23, LEA-1-deficient serum isolated from a 3MC patient had no effect on mediating hemolysis. In summary, these data indicate that while LEA-2 significantly contributes to the intravascular hemolytic response, LEA-1 is the dominant complement-initiated pathway leading to hemolysis. Thus, while LEA-2 blockers are expected to significantly reduce intravascular hemolysis of RBCs in PNH patients, LEA-1 blockers are expected to have a more profound effect, largely eliminating complement-driven hemolysis.

[0150] It should be noted that the serum of the LEA-1-deficient 3MC patient used in this study had a reduced but functional alternative pathway when tested under conventional alternative pathway assay conditions (Figure 17). This finding suggests that LEA-1 contributes to hemolysis to a greater extent than traditionally defined alternative pathway activity in this PNH experimental setting. By corollary, this implies that LEA-1 blockers will be at least as effective as agents that block other aspects of the alternative pathway in preventing or treating intravascular hemolysis in PNH patients.

[0151] vi. Role of MASP-2 inhibitors in PNH The data presented herein suggest the following pathogenic mechanisms for anemia in PNH: intravascular hemolysis due to unregulated activation of terminal complement components and lysis of RBCs via MAC formation, primarily, but not exclusively, initiated by LEA-1; and extravascular hemolysis resulting from opsonization of RBCs with C3b, thought to be primarily initiated by LEA-2. While a recognized role for LEA-2 in initiating complement activation and promoting MAC formation and hemolysis is clear, this process is thought to be substantially less effective than LEA-1-initiated complement activation, which results in hemolysis. Thus, while LEA-2 blockers are expected to significantly reduce intravascular hemolysis in PNH patients, this therapeutic activity is expected to be only partial. By comparison, a more substantial reduction in intravascular hemolysis in PNH patients is expected with LEA-1 blockers.

[0152] Extravascular hemolysis, a less dramatic but equally important mechanism of RBC destruction that results in anemia in PNH, is primarily the result of opsonization by C3b, which is thought to be mediated primarily by LEA-2. Therefore, LEA-2 blockers can be expected to preferentially prevent RBC opsonization and subsequent extravascular hemolysis in PNH. Because there are currently no treatments for PNH patients experiencing this pathogenic process, this unique therapeutic activity of LEA-2 blockers is expected to provide significant therapeutic benefit to all PNH patients.

[0153] vii. LEA-2 inhibitors as adjunctive therapy to LEA-1 inhibitors or terminal complement blockers The data presented herein demonstrate two pathogenic mechanisms of RBC clearance and anemia in PNH that can be targeted separately or in combination by distinct classes of therapeutic agents: intravascular hemolysis, which is primarily, but not exclusively, initiated by LEA-1 and is therefore expected to be effectively prevented by LEA-1 blockers; and extravascular hemolysis via C3b opsonization, which is primarily driven by LEA-2 and is therefore effectively prevented by LEA-2 blockers.

[0154] It is well documented that both intravascular and extravascular mechanisms of hemolysis result in anemia in PNH patients (Risitano et al., Blood 113:4094-4100 (2009)). Therefore, LEA-1 blockers, which prevent intravascular hemolysis, combined with LEA-2 blockers, which primarily prevent extravascular hemolysis, are expected to be more effective than either agent alone in preventing the anemia that develops in PNH patients. In fact, the combination of LEA-1 and LEA-2 blockers is expected to prevent all relevant mechanisms of complement initiation in PNH and thus all symptoms of anemia in PNH.

[0155] It is also known that C5 blockers (e.g., eculizumab) effectively prevent intravascular hemolysis but do not interfere with opsonization. This leaves some anti-C5-treated PNH patients with substantial residual anemia due to untreated extravascular hemolysis mediated by LEA-2. Therefore, a C5 blocker (e.g., eculizumab) that prevents intravascular hemolysis, combined with an LEA-2 blocker that reduces extravascular hemolysis, is expected to be more effective than either agent alone in preventing anemia from developing in PNH patients.

[0156] Other agents that block the terminal amplification loop of the complement system, which results in C5 activation and MAC deposition (including, but not limited to, agents that block properdin, factor B, or factor D or enhance the inhibitory activity of factor I, factor H, or other complement inhibitors), are also expected to inhibit intravascular hemolysis. However, these agents are not expected to interfere with LEA-2-mediated opsonization in PNH patients. This leaves some PNH patients treated with such agents with substantial residual anemia due to untreated extravascular hemolysis mediated by LEA-2. Therefore, treatment with such agents that prevent intravascular hemolysis, combined with LEA-2 blockers that minimize extravascular hemolysis, is expected to be more effective than either agent alone in preventing anemia from developing in PNH patients. In fact, the combination of such agents with LEA-2 blockers is expected to prevent all relevant mechanisms of RBC destruction in PNH and, therefore, all symptoms of anemia in PNH.

[0157] viii. Use of LEA-1 and LEA-2 Multispecific, Bispecific, or Panspecific Antibodies to Treat PNH As detailed above, the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 and thus, in combination, block all complement activation events that mediate intravascular and extravascular hemolysis is expected to provide the best clinical outcomes for PNH patients. This outcome can be achieved, for example, by coadministering an antibody with LEA-1 blocking activity and an antibody with LEA-2 blocking activity. In some embodiments, LEA-1 blocking activity and LEA-2 blocking activity are combined into a single molecular entity, and such an entity with combined LEA-1 and LEA-2 blocking activity effectively prevents intravascular and extravascular hemolysis and prevents anemia in PNH. Such an entity may comprise or consist of a bispecific antibody in which one antigen-binding site specifically recognizes MASP-1, blocks LEA-1, and reduces LEA-2, and a second antigen-binding site specifically recognizes MASP-2 and also blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody, with one antigen-binding site specifically recognizing MASP-3 and thus blocking LEA-1, and a second antigen-binding site specifically recognizing MASP-2 and blocking LEA-2. Optimally, such an entity may consist of a bispecific monoclonal antibody, with one antigen-binding site specifically recognizing both MASP-1 and MASP-3 and thus blocking LEA-1 and reducing LEA-2, and a second antigen-binding site specifically recognizing MASP-2 and further blocking LEA-2. Based on the similarity of protein sequence and overall architecture, it is conceivable that conventional antibodies with two identical binding sites could be developed that functionally bind specifically to MASP-1, MASP-2, and MASP-3, thereby achieving functional blocking of LEA-1 and LEA-2. Such antibodies with pan-MASP inhibitory activity are expected to block both intravascular and extravascular hemolysis and thus effectively treat anemia in PNH patients.

[0158] IV. The Role of MASP-2 and MASP-3 in Age-Related Macular Degeneration and Therapeutics Using MASP-2 and MASP-3 Inhibitors Age-related macular degeneration (AMD) is a leading cause of vision damage and blindness in the elderly, accounting for up to 50% of blindness cases in developed countries. The prevalence of AMD in adults is approximately 3% and increases with age, with nearly two-thirds of people over 80 years of age experiencing some form of the condition. More than 1.75 million individuals in the United States have advanced AMD, and the prevalence is expected to increase with the aging population, reaching nearly 3 million by 2020 (Friedman, DS, et al., Arch. Ophthalmol. 122:564-572, 2004). AMD is an abnormality of the retinal pigment epithelium (RPE) that overlies the central retina, i.e., the macula, resulting in degeneration of photoreceptors and loss of central vision. Early and intermediate forms of AMD are characterized by the gradual deposition of drusen, a yellowish substance containing lipids, proteins, lipoproteins, and necrotic cell debris, in the subretinal space adjacent to the RPE, as well as pigment irregularities in the retina. Advanced AMD consists of two clinical subtypes: non-neovascular geographic atrophic ("dry") AMD and neovascular exudative ("wet") AMD. While dry AMD accounts for 80-90% of advanced AMD cases, the majority of sudden and severe vision loss occurs in patients with wet AMD. It is unclear whether the two types of AMD represent different phenotypes arising from similar pathologies or two distinct conditions. Currently, no therapies have been approved by the United States Food and Drug Administration (FDA) to treat dry AMD. FDA-approved treatment options for wet AMD include intravitreal injections of antiangiogenic drugs (ranibizumab, pegaptanib sodium, aflibercept), laser therapy, photodynamic laser therapy, and implantable telescopes.

[0159] The etiology and pathophysiology of AMD are complex and incompletely understood. Several lines of evidence support the role of dysregulation of the complement system in the pathogenesis of AMD. Genetic association studies have identified multiple loci associated with AMD, including genes encoding a range of complement proteins, factors, and regulators. The strongest association is with a polymorphism in the complement factor H (CFH) gene; compared with non-risk genotypes, homozygotes for the Y402H variant have an approximately six-fold increased risk of developing AMD, and heterozygotes have an approximately 2.5-fold increased risk (Khandhadia, S., et al., Immunobiol. 217:127-146, 2012). Mutations in other complement pathway-encoding genes, including complement factor B (CFB), C2, C3, factor I, and CFH-related proteins 1 and 3, have also been associated with increased or decreased AMD risk (Khandhadia et al.). Immunohistochemical and proteomic studies in donor eyes from AMD patients have shown that complement cascade proteins are increased and localized in drusen (Issa, PC, et al., Graefes. Arch. Clin. Exp. Ophthalmol. 249:163-174, 2011). Furthermore, AMD patients have increased systemic complement activation measured in peripheral blood (Issa et al., supra, 2011).

[0160] The alternative complement pathway appears to be more relevant than the classical pathway in the pathogenesis of AMD. C1q, the essential recognition component for classical pathway activation, was not detected in drusen by immunohistochemical analysis (Mullins et al., FASEB J. 14:835-846, 2000; Johnson et al., Exp. Eye Res. 70:441-449, 2000). Genetic association studies have implicated the CFH and CFB genes. These proteins are involved in the alternative pathway amplification loop, with CFH being a fluid-phase inhibitor and CFB being the activating protease component of the alternative pathway. The Y402H variant of CFH affects ligand binding interactions, including binding to C-reactive protein, heparin, M protein, and glycosaminoglycans. This altered ligand binding may reduce cell surface binding, which in turn may lead to reduced factor I-mediated degradation of C3b activation fragments and impaired regulation of the alternative C3 convertase, resulting in overactivation of the alternative pathway (Khandhadia et al., 2012). Alterations in the CFB gene have been associated with a protective effect against AMD development. The functional variant fB32Q has a four-fold lower binding affinity to C3b than the risk variant fB32R, resulting in reduced C3 convertase formation (Montes, T. et al., Proc. Natl. Acad. Sci. USA 106:4366-4371, 2009).

[0161] Complement initiation mechanisms in AMD The human genetic linkage studies described above suggest an important role for the complement system in the pathogenesis of AMD. Furthermore, complement activation products are abundant in drusen, the characteristic pathological lesions in both wet and dry AMD (Issa, PC, et al., Graefes. Arch. Clin. Exp. Ophthalmol. 249:163-174, 2011). However, the nature of the events that initiate complement activation and the complement activation pathways involved remain incompletely understood.

[0162] It is important to note that drusen deposits are composed of necrotic cellular debris and oxidized waste products from the retina that accumulate under the RPE as the eye ages. Additionally, oxidative stress is thought to play an important role (Cai et al.; Front Biosci., 17:1976-95, 2012) and has been shown to cause complement activation in the RPE (J Biol. Chem., 284(25):16939-47, 2009). It is widely understood that both oxidative stress and cell or tissue damage activate complement system lectins. For example, Collard et al. demonstrated that endothelial cells exposed to oxidative stress induce abundant complement deposition mediated by lectins (Collard CD et al., Mol Immunol., 36(13-14):941-8, 1999; Collard CD et al., Am J Pathol., 156(5):1549-56, 2000) and that blocking lectin binding and lectin-dependent complement activation improves outcomes in experimental models of oxidative stress injury (Collard CD et al., Am J Pathol., 156(5):1549-56, 2000). Therefore, it is likely that oxidative waste products present in drusen also activate complement via lectins. By implication, lectin-dependent complement activation may play a pivotal role in the pathogenesis of AMD.

[0163] The role of the complement system has been evaluated in mouse models of AMD. In a mouse model of light injury, an experimental model of oxidative stress-mediated photoreceptor degeneration, classical pathway-ablated knockout mice (C1qα on a C57BL / 6 background) were used. - / - ) had the same sensitivity to light damage compared to wild-type littermates, but lacked the alternative pathway of complement factor D (CFD) - / -) provided protection from light damage (Rohrer, B. et al., Invest. Ophthalmol. Vis. Sci. 48:5282-5289, 2007). In a mouse model of choroidal neovascularization (CNV) induced by laser photocoagulation of Bruch's membrane, complement factor B-null knockout mice (CFB) - / - ) were protected against CNV compared with wild-type mice (Rohrer, B. et al., Invest. Ophthalmol. Vis. Sci. 50:3056-3064, 2009). In the same model, intravenous administration of a recombinant form of complement factor H (CR2-fH) targeted to the site of complement activation reduced the extent of CNV. This protective effect was observed whether CR2-fH was administered at the time of laser injury or therapeutically (after laser injury). In addition, a human therapeutic version of CR2-fH (TT30) was effective in a mouse CNV model (Rohrer, B. et al. J. Ocul. Pharmacol. Ther., 28:402-409, 2012). Because fB is activated by LEA-1, and because MASP-1 and MASP-3 contribute to factor D maturation, these findings suggest that LEA-1 inhibitors may have therapeutic benefit in AMD patients.

[0164] Initial experimental studies in rodent models of AMD using MBL-deficient mice did not support a critical role for the lectin pathway in pathogenic complement activation (Rohrer et al., Mol. Immunol. 48:e1-8, 2011). However, MBL is only one of several lectins, and lectins other than MBL may induce complement activation in AMD. In fact, our previous studies showed that MASP-2, a rate-limiting serine protease critically required for lectin pathway function, plays an important role in AMD. As described in U.S. Patent No. 7,919,094 (assigned to Omeros Corporation), which is incorporated herein by reference, and in Examples 20 and 21 herein, MASP-2-deficient mice and mice treated with MASP-2 antibodies were protected in a mouse model of laser-induced CNV, a validated preclinical model of wet AMD (Ryan et al., Tr Am Opth Soc LXXVII:707-745, 1979). Thus, inhibitors of LEA-2 are expected to effectively prevent CNV and improve outcomes in AMD patients.

[0165] Therefore, in view of the above, it is expected that LEA-1 inhibitors and LEA-2 inhibitors have independent therapeutic benefits in AMD.In addition, when LEA-1 inhibitors and LEA-2 inhibitors are used in combination, they can achieve additional therapeutic benefits compared to either inhibitor alone, or provide effective treatment for a wider range of patient subsets.Combined LEA-1 and LEA-2 inhibition can be achieved by simultaneous administration of LEA-1 blockers and LEA-2 blockers.Optimally, LEA-1 inhibitory function and LEA-2 inhibitory function can be included in a single molecular entity, for example, a bispecific antibody composed of MASP-1 / 3 and MASP-2 specific binding sites, or a bispecific antibody, each binding site of which can bind to and block MASP-1 / 3 or MASP-2.

[0166] Thus, in accordance with the foregoing, aspects of the present invention provide methods for inhibiting LEA-1-dependent complement activation to treat age-related macular degeneration (wet and dry forms) by administering to a subject suffering from such a condition a composition comprising a therapeutically effective amount of a MASP-1 inhibitor, a MASP-3 inhibitor, or a combination of MASP-1 / 3 inhibitors in a pharmaceutical carrier. The MASP-1, MASP-3, or MASP-1 / 3 inhibitor composition may be administered locally to the eye by irrigation, intravitreal administration, or application of the composition in the form of a gel, ointment, or drops. Alternatively, the MASP-1, MASP-3, or MASP-1 / 3 inhibitor may be administered to the subject systemically, e.g., by intra-arterial, intravenous, intramuscular, inhalation, intranasal, subcutaneous, or other parenteral administration, or potentially by oral administration in the case of non-peptidergic agents. Administration may be repeated as determined by a physician until the condition resolves or is suppressed.

[0167] In one embodiment, the method of this aspect of the present invention further comprises inhibiting LEA-2-dependent complement activation in a subject suffering from age-related macular degeneration, comprising administering a therapeutically effective amount of a MASP-2 inhibitor and a MASP-1, MASP-3, or MASP-1 / 3 inhibitor to a subject in need thereof. As described above, the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 is expected to provide improved clinical outcomes in AMD patients compared to inhibiting LEA-1 alone. This outcome can be achieved, for example, by co-administering an antibody with LEA-1 blocking activity and an antibody with LEA-2 blocking activity. In some embodiments, LEA-1 blocking activity and LEA-2 blocking activity are combined in a single molecular entity, and such an entity has combined LEA-1 and LEA-2 blocking activity. Such an entity may comprise or consist of a bispecific antibody in which one antigen-binding site specifically recognizes MASP-1 and blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody in which one antigen-binding site specifically recognizes MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Optimally, such an entity may consist of a bispecific monoclonal antibody in which one antigen-binding site specifically recognizes both MASP-1 and MASP-3 and thus blocks LEA-1, while the second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

[0168] The MASP-2 inhibitory composition can be administered locally to the eye, such as by irrigation, intravitreal injection, or topical application of the composition in the form of a gel, ointment, or drops. Alternatively, the MASP-2 inhibitor can be administered to the subject systemically, for example, by intra-arterial, intravenous, intramuscular, inhalation, intranasal, subcutaneous, or other parenteral administration, or potentially by oral administration in the case of non-peptidergic agents. Administration can be repeated as determined by a physician until the condition resolves or is suppressed.

[0169] The application of the MASP-3 inhibitory composition and any MASP-2 inhibitory composition of the present invention can be carried out by single administration or limited continuous administration of the composition (e.g., simultaneous administration of a single or separate composition comprising MASP-2 and MASP-3 inhibitors or bispecific or dual inhibitors) in the case of AMD treatment. Alternatively, the composition can be administered over a long period of time, such as once daily, twice weekly, once weekly, once every two weeks, once monthly, or once every two months, for AMD treatment.

[0170] V. Role of MASP-2 and MASP-3 in Ischemia-Reperfusion Injury and Therapeutics Using MASP-2 and MASP-3 Inhibitors Tissue ischemia is the basis of a wide range of clinical disorders. While timely restoration of blood flow is essential for preserving ischemic tissue, it has long been recognized that reperfusion, which can occur spontaneously or through therapeutic intervention, can lead to further tissue damage, a phenomenon called ischemia-reperfusion (I / R) injury (Eltzschig, HK and Tobias, E., Nat. Med. 17:1391-1401, 2011). I / R injury can affect a single organ, such as the heart (acute coronary syndrome), kidney (acute kidney injury), intestine (intestinal I / R), or brain (stroke). I / R injury can also affect multiple organs, such as after major trauma and resuscitation (multiple organ failure), circulatory arrest (hypoxic encephalopathy, acute kidney injury), peripheral vascular disease, and sickle cell disease (acute chest syndrome, acute kidney injury). Major surgeries, including cardiac surgery (acute heart failure after cardiopulmonary bypass), thoracic surgery (acute lung injury), peripheral vascular surgery (compartment syndrome), vascular surgery (acute kidney injury), and solid organ transplantation (acute graft failure), can also be associated with I / R injury. Currently, there are no specific treatments that target I / R injury, and there is a need for effective therapies to maximize tissue salvage in the ischemic zone and improve functional outcomes in these common conditions.

[0171] The pathophysiology of I / R injury is complex and characterized by a strong inflammatory response after reperfusion. Activation of the complement system has been implicated as a key component of I / R injury, and inhibition of complement activity has been effective in various animal models (Diepenhorst, GMP, et al., Ann. Surg. 249:889-899, 2009). The relative importance of the classical, lectin, and alternative pathways in I / R injury remains largely unresolved and may vary depending on the affected organ. Recently, the availability of knockout mice lacking specific complement proteins and pathway-specific inhibitors has generated data suggesting the involvement of the lectin and alternative pathways in I / R injury.

[0172] The role of the alternative pathway in gastrointestinal I / R injury has been studied using factor D-deficient (- / -) and heterozygous (+ / -) mice (Stahl, GL, et al. Am. J. Pathol. 162:449-455, 2003). After transient gastrointestinal ischemia, intestinal and pulmonary injury was reduced, but not prevented, in factor D-deficient mice compared with heterozygous mice, and addition of human factor D to - / - mice reversed IR injury. When the same model was evaluated in C1q-deficient and MBL-A / C-deficient mice, the results showed that gastrointestinal I / R injury was independent of C1q and classical pathway activation, whereas intestinal injury required MBL and lectin pathway activation (Hart, ML, et al. J. Immunol. 174:6373-6380, 2005). Conversely, the classical pathway's C1q recognition molecule was responsible for lung injury after intestinal I / R (Hart, M.L., et al. J. Immunol. 174:6373-6380, 2005). One hypothesis is that complement activation during I / R injury occurs through natural IgM binding to autoantigens, such as nonmuscle myosin heavy chain type II, present on the surface of ischemic (but not normal) tissue. In a mouse gastrointestinal I / R injury model, immune complexes from intestinal tissue have been evaluated for the presence of initiating factors in the classical (C1q), lectin (MBL), or alternative (factor B) pathways (Lee, H., et al., Mol. Immunol. 47:972-981, 2010). Results showed that C1q and MBL, but not factor B, were detected in these immune complexes, indicating the involvement of the classical and lectin pathways, but not the alternative pathway. In the same model, factor B-deficient mice were not protected from local tissue injury, providing further support for the lack of involvement of the alternative pathway.The role of the lectin pathway in gastrointestinal I / R injury was directly assessed in MASP-2-deficient mice, and the results showed that gastrointestinal injury was reduced in these mice compared with wild-type controls.Treatment with MASP-2 monoclonal antibody was similarly protective (Schwaeble, WJ, et al., Proc. Natl. Acad. Sci. 108:7523-7528, 2011). See also Example 23 herein. In summary, these results provide support for the involvement of the lectin pathway in gastrointestinal I / R injury, although there are conflicting data regarding the involvement of the alternative pathway.

[0173] The pathogenic role of the lectin pathway was demonstrated in a mouse myocardial I / R injury model when MBL-deficient mice were protected from myocardial damage, whereas C1q- and C2 / fB-deficient mice were not (Walsh, MC et al., J. Immunol. 175:541-546, 2005). Protection from myocardial I / R injury was also observed in MASP-2-deficient mice (Schwaeble, WJ, et al., Proc. Natl. Acad. Sci. 108:7523-7528, 2011). See also Examples 22 and 23 herein. Treatment of rats in a myocardial I / R model with a monoclonal antibody against rat MBL resulted in reduced post-ischemic reperfusion injury (Jordan, JE, et al., Circulation 104:1413-18, 2001). In a study of patients with myocardial infarction treated with angioplasty, MBL deficiency was associated with reduced 90-day mortality compared with MBL-sufficient counterparts (Trendelenburg M et al., Eur Heart J. 31:1181, 2010). Furthermore, patients with myocardial infarction who developed cardiac dysfunction after angioplasty had approximately three times higher MBL levels than patients whose cardiac function recovered (Haahr-Pedersen S., et al., J Inv Cardiology, 21:13, 2009). Furthermore, MBL antibodies reduced complement deposition on endothelial cells in vitro after oxidative stress, indicating a role for the lectin pathway in myocardial I / R injury (Collard, CD, et al., Am. J. Pathol. 156:1549-56, 2000). The role of the alternative pathway has been investigated in a mouse heterotopic isogeneic heart transplant model of I / R injury using the pathway-specific fusion protein CR2-fH (Atkinson, C., et al., J. Immunol. 185:7007-7013, 2010). Systemic administration of CR2-fH immediately after transplantation reduced myocardial I / R injury to a degree comparable to treatment with CR2-Crry, which inhibits the entire complement pathway, demonstrating the critical importance of the alternative pathway in this model.

[0174] In a mouse model of renal I / R injury, the involvement of the alternative pathway was suggested when factor B-deficient mice were protected from decreased renal function and tubular damage compared with wild-type mice (Thurman, JM, et al., J. Immunol. 170:1517-1523, 2003). Treatment with an inhibitory monoclonal antibody against factor B prevented complement activation and reduced renal I / R injury in mice (Thurman, JM, et al., J. Am. Soc. Nephrol. 17:707-715, 2006). In a bilateral renal I / R injury model, MBL-A / C-deficient mice were protected from renal injury compared with wild-type mice, and recombinant human MBL reversed the protective effect in MBL-A / C-deficient mice, suggesting the involvement of MBL in this model (Moller-Kristensen, M., et al., Scand. J. Immunol. 61:426-434, 2005). In a rat bilateral renal I / R injury model, inhibition of MBL with a monoclonal antibody against MBL-A preserved renal function after I / R (van der Pol, P., et al., Am. J. Transplant. 12:877-887, 2010). Interestingly, treatment with C5 antibody did not prevent renal injury, suggesting that the role of MBL in this model does not involve activation of terminal complement components. Rather, MBL is thought to have a direct toxic effect on renal tubular cells, because human proximal tubular cells incubated with MBL in vitro internalized MBL, followed by cell death. Castellano G. et al. (Am J Pathol, 176(4):1648-59, 2010) tested C1 inhibitor, which irreversibly inactivates C1r and C1s proteases in the classical pathway and MASP-1 and MASP-2 proteases in the MBL complex of the lectin pathway, in a porcine model of renal I / R injury and found that C1 inhibitor reduced complement deposition in peritubular capillaries and glomeruli and reduced tubular injury.

[0175] The alternative pathway is thought to be involved in experimental traumatic brain injury because factor B-deficient mice showed reduced systemic complement activation, as measured by serum C5a levels, and reduced post-traumatic neuronal death compared with wild-type mice (Leinhase, I., et al., BMC Neurosci. 7:55-67, 2006). In human stroke, complement components C1q, C3c, and C4d were detected by immunohistochemical staining in the ischemic lesion, suggesting activation via the classical pathway (Pedersen, ED, et al., Scand. J. Immunol. 69:555-562, 2009). Targeting the classical pathway in animal models of cerebral ischemia has produced mixed results, with some studies demonstrating protection and others showing no benefit (Arumugam, TV, et al., Neuroscience 158:1074-1089, 2009). Experiments and clinical trials have provided strong evidence for the involvement of the lectin pathway. In experimental stroke models, deficiency of either MBL or MASP-2 results in reduced infarct size compared to wild-type mice (Cervera A, et al.; PLoS One 3;5(2):e8433, 2010; Osthoff M. et al., PLoS One, 6(6):e21338, 2011 and Example 26 herein). Furthermore, stroke patients with low MBL levels exhibit a better prognosis than their MBL-sufficient counterparts (Osthoff M. et al., PLoS One, 6(6):e21338, 2011).

[0176] In a baboon model of cardiopulmonary bypass, treatment with factor D monoclonal antibody inhibited systemic inflammation as measured by plasma levels of C3a, sC5b-9, and IL-6 and reduced myocardial tissue damage, indicating the involvement of the alternative pathway in this model (Undar, A., et al., Ann. Thorac. Surg. 74:355-362, 2002).

[0177] Therefore, depending on the organ affected by I / R, all three complement pathways can contribute to pathogenesis and adverse outcomes. Based on the above-mentioned experimental and clinical findings, LEA-2 inhibitors are expected to be protective in most I / R situations. Lectin-dependent activation of LEA-1 may result in complement activation via the alternative pathway in at least some situations. In addition, LEA-2-initiated complement activation may be further amplified by the alternative pathway amplification loop, thus exacerbating I / R-related tissue damage. Therefore, LEA-1 inhibitors are expected to provide additional or complementary therapeutic benefit in patients suffering from ischemia-related conditions.

[0178] In view of the above, LEA-1 and LEA-2 inhibitors are expected to have independent therapeutic benefits in treating, preventing, or reducing the severity of ischemia-reperfusion-related conditions. In addition, LEA-1 and LEA-2 inhibitors, when used in combination, may achieve additional therapeutic benefits compared to either inhibitor alone. Therefore, optimally effective treatments for I / R-related conditions include pharmaceutical active ingredients that block both LEA-1 and LEA-2, either alone or in combination. Combined LEA-1 and LEA-2 inhibition can be achieved by coadministration of an LEA-1 blocker and an LEA-2 blocker. Preferentially, LEA-1 and LEA-2 inhibitory functions can be encompassed in a single molecular entity, such as a bispecific antibody composed of MASP-1 / 3- and MASP-2-specific binding sites, or a bispecific antibody in which each binding site can bind to and block MASP-1 / 3 or MASP-2.

[0179] Thus, in accordance with the foregoing, aspects of the present invention provide methods for inhibiting LEA-1-dependent complement activation to treat, prevent, or reduce the severity of ischemia-reperfusion injury by administering to a subject experiencing ischemia-reperfusion a composition comprising a therapeutically effective amount of an LEA-1 inhibitor, including a MASP-1 inhibitor, a MASP-3 inhibitor, or a combination of MASP-1 / 3 inhibitors, in a pharmaceutical carrier. The MASP-1, MASP-3, or MASP-1 / 3 inhibitor composition may be administered to the subject intraarterially, intravenously, intracranially, intramuscularly, subcutaneously, or by other parenteral administration, or potentially orally in the case of non-peptidergic inhibitors, most preferably intraarterially or intravenously. Administration of the LEA-1 inhibitor composition of the present invention is preferably initiated immediately after or as soon as possible after the ischemia-reperfusion event. When reperfusion occurs in a controlled environment (e.g., after aortic aneurysm repair, organ transplantation, or reattachment of a severed or injured limb or digit), the LEA-1 inhibitor may be administered before, during, and / or after reperfusion. Administration may be repeated periodically as determined by a physician to obtain optimal therapeutic effect.

[0180] In some embodiments, the method is used to treat or prevent ischemia-reperfusion injury associated with at least one of aortic aneurysm repair, cardiopulmonary bypass, vascular reanastomosis associated with organ transplantation and / or limb / digit replantation, stroke, myocardial infarction, and shock and / or hemodynamic resuscitation following surgical procedures.

[0181] In some embodiments, the method is used to treat or prevent ischemia-reperfusion injury in a subject who is about to receive, is receiving, or has received an organ transplant. In some embodiments, the method is used to treat or prevent ischemia-reperfusion injury in a subject who is about to receive, is receiving, or has received an organ transplant, provided that the organ transplant is not a kidney transplant.

[0182] In one embodiment, the method of this aspect of the present invention further comprises inhibiting LEA-2-dependent complement activation in a subject experiencing ischemia-reperfusion injury, comprising administering to the subject a therapeutically effective amount of a MASP-2 inhibitor and a MASP-1, MASP-3, or MASP-1 / 3 inhibitor. As described above, the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 is expected to provide improved clinical outcomes in treating, preventing, or reducing the severity of ischemia-reperfusion injury compared to inhibition of LEA-1 alone. This outcome can be achieved, for example, by coadministering an antibody with LEA-1 blocking activity and an antibody with LEA-2 blocking activity. In some embodiments, LEA-1 blocking activity and LEA-2 blocking activity are combined in a single molecular entity, which has combined LEA-1 and LEA-2 blocking activity. Such an entity may comprise or consist of a bispecific antibody in which one antigen-binding site specifically recognizes MASP-1 and blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody in which one antigen-binding site specifically recognizes MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Optimally, such an entity may consist of a bispecific monoclonal antibody in which one antigen-binding site specifically recognizes both MASP-1 and MASP-3 and thus blocks LEA-1, while the second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

[0183] The MASP-2 inhibitory composition may be administered to a subject in need thereof by intraarterial, intravenous, intracranial, intramuscular, subcutaneous, or other parenteral administration, and potentially orally in the case of non-peptidergic inhibitors, most preferably by intraarterial or intravenous administration. Administration of the MASP-2 inhibitory composition of the present invention is preferably initiated immediately after or as soon as possible after an ischemic reperfusion event. When reperfusion occurs in a controlled environment (e.g., after aortic aneurysm repair, organ transplantation, or reattachment of a severed or injured limb or digit), the MASP-2 inhibitor may be administered before, during, and / or after reperfusion. Administration may be repeated periodically, as determined by a physician, to achieve optimal therapeutic effect.

[0184] The application of the MASP-3 inhibitory composition and any MASP-2 inhibitory composition of the present invention can be carried out by single administration or limited number of consecutive administrations of the composition (e.g., co-administration of a single or separate composition comprising a MASP-2 and MASP-3 inhibitor or a bispecific or dual inhibitor) in the treatment or prevention of ischemia-reperfusion injury. Alternatively, the composition can be administered over a long period of time, such as once daily, twice weekly, weekly, biweekly, monthly, or bimonthly, in the treatment of a subject experiencing ischemia-reperfusion injury.

[0185] VI. The Role of MASP-2 and MASP-3 in Inflammatory and Non-inflammatory Arthritis and Therapeutics Using MASP-2 and MASP-3 Inhibitors Rheumatoid arthritis (RA) is a chronic inflammatory disease of synovial joints that can have systemic manifestations. RA affects approximately 1% of the global population, with women two to three times more likely to be affected. Joint inflammation manifests as swelling, pain, and stiffness. As the disease progresses, joint erosion and destruction can occur, resulting in impaired range of motion and deformity. The goals of RA treatment include preventing and controlling joint damage, preventing joint function loss and disease progression, reducing symptoms and improving quality of life, and achieving drug-free remission. Pharmacological treatments for RA include disease-modifying antirheumatic drugs (DMARDs), analgesics, and anti-inflammatory agents (glucocorticoids and nonsteroidal anti-inflammatory drugs). DMARDs are the most important treatment because they can induce long-term remission and slow or halt the progression of irreversible joint destruction. Examples of traditional DMARDs include small molecules such as methotrexate, sulfasalazine, hydroxychloroquine, gold salts, leflunomide, D-penicillamine, cyclosporine, and azathioprine. If traditional DMARDs are insufficient to control the disease, several biologic agents that target inflammatory cells or mediators, such as tumor necrosis factor inhibitors (etanercept, infliximab, adalimumab, certolizumab pegol, and golimumab), cytokine antagonists (anakinra and tocilizumab), rituximab, and abatacept, are available treatment options.

[0186] Although adaptive immunity is clearly central to RA pathogenesis, as evidenced by genetic associations with T cell activation genes and the presence of autoantibodies, the involvement of innate immune mechanisms has been suggested (McInnes, IB and Schett, G. New Engl. J. Med. 365:2205-2219, 2011). In human RA, synovial fluid levels of the alternative pathway cleavage fragment Bb were several-fold higher than those in samples from patients with crystal-induced arthritis or osteoarthritis, suggesting preferential activation of the alternative pathway in RA patients (Brodeur, JP, et al., Arthritis Rheum. 34:1531-1537, 1991). In an experimental anti-type II collagen antibody passive transfer model of arthritis, factor B-deficient mice showed reduced inflammation and joint damage compared with wild-type mice, whereas C4-deficient mice had disease activity similar to that of wild-type mice, demonstrating the requirement for the alternative pathway, rather than the classical pathway, in this model (Banda, NK et al., J. Immunol. 177:1904-1912, 2006). In the same experimental model of collagen antibody-induced arthritis (CAIA), mice with only the classical pathway or only the lectin pathway active failed to develop arthritis (Banda, NK et al., Clin. Exp. Immunol. 159:100-108, 2010). Data from this study suggested that either the classical pathway or the lectin pathway could activate low levels of C3 in vitro. However, in the absence of the alternative pathway amplification loop, the level of C3 deposition in the joints was insufficient to produce clinical disease.A key step in alternative pathway activation is the conversion of factor D zymogen (pro-factor D) to mature factor D, mediated by MASP-1 and / or MASP-3 (Takahashi, M., et al., J. Exp. Med. 207:29-37, 2010) and / or HTRA1 (Stanton et al., Evidence That the HTRA1 Interactome Influences Susceptibility to Age-Related Macular Degeneration, presented at the Association for Research in Vision and Ophthalmology 2011 conference on May 4, 2011). The role of MASP-1 / 3 was evaluated in mouse CAIA, and the results showed that MASP-1 / 3-deficient mice were protected from arthritis compared with wild-type mice (Banda, N.K., et al., J. Immunol. 185:5598-5606, 2010). In MASP-1 / 3-deficient mice, pro-factor D, but not mature factor D, was detected in serum during the development of CAIA, and addition of human factor D reconstituted C3 activation and C5a generation in vitro using serum from these mice. In contrast, in a mouse model of the effector phase of arthritis, C3-deficient mice developed much milder arthritis than WT mice, whereas factor B-deficient mice still developed arthritis, indicating independent contributions of both the classical / lectin and alternative pathways (Hietala, MA et al., Eur. J. Immunol. 34:1208-1216, 2004). In a K / BxN T cell receptor transgenic mouse model of inflammatory arthritis, mice lacking C4 or C1q developed arthritis similar to wild-type mice, whereas mice lacking factor B did not develop arthritis or showed mild arthritis, demonstrating the requirement for the alternative pathway rather than the classical pathway in this model (Ji H. et al., Immunity 16:157-168, 2002).In the K / BxN model, mice lacking MBL-A were not protected from serum-induced arthritis, but the role of MBL-C was not studied and a potential role for the lectin pathway could not be excluded (Ji et al., 2002 supra).

[0187] Two research groups have independently proposed that lectin-dependent complement activation promotes inflammation in RA patients through the interaction of MBL with specific IgG glycoforms (Malhotra et al., Nat. Med. 1:237-243, 1995; Cuchacovich et al., J. Rheumatol. 23:44-51, 1996). It has been noted that rheumatoid conditions are associated with a significant increase in IgG glycoforms lacking galactose (called IgG0 glycoforms) in the Fc region of the molecule (Rudd et al., Trends Biotechnology 22:524-30, 2004). The proportion of IgG0 glycoforms increases with disease progression in rheumatoid conditions and returns to normal when patients enter remission. In vivo, IgG0 is deposited in synovial tissue, and MBL is present at elevated levels in the synovial fluid of individuals with RA. Aggregated agalactosyl IgG (IgG0) associated with RA can bind to MBL and thus initiate lectin-dependent complement activation via LEA-1 and / or LEA-2. Furthermore, results from clinical studies examining MBL allelic variants in RA patients suggest that MBL may have an inflammation-promoting role in this disease (Garred et al., J. Rheumatol. 27:26-34, 2000). Thus, lectin-dependent complement activation via LEA-1 and / or LEA-2 may play an important role in the pathogenesis of RA.

[0188] Complement activation also plays an important role in juvenile rheumatoid arthritis (Mollnes, TE, et al., Arthritis Rheum. 29:1359-64, 1986). As in adult RA, elevated serum and synovial fluid levels of the alternative pathway complement activation product Bb compared with C4d (a marker of classical or LEA-2 activation) in juvenile rheumatoid arthritis indicate that complement activation is primarily mediated by LEA-1 (El-Ghobarey, AF et al., J. Rheumatology 7:453-460, 1980; Agarwal, A., et al., Rheumatology 39:189-192, 2000).

[0189] Similarly, complement activation plays an important role in psoriatic arthritis. Patients with this condition have increased complement activation products in the circulation, and their red blood cells are thought to have lower levels of the complement regulator CD59 (Triolo., Clin Exp Rheumatol., 21(2):225-8, 2003). Complement levels are associated with disease activity and have a high predictive value for determining treatment outcome (Chimenti et al., Clin Exp Rheumatol., 30(1):23-30, 2012). In fact, recent studies suggest that the effectiveness of anti-TNF therapy for this condition is due to complement modulation (Ballanti et al., Autoimmun Rev., 10(10):617-23, 2011). Although the exact role of complement in psoriatic arthritis remains to be determined, the presence of C4d and Bb complement activation products in the circulation of these patients suggests an important role in pathogenesis. Based on the products observed, it is believed that LEA-1 and possibly also LEA-2 are responsible for the pathological complement activation in these patients.

[0190] Osteoarthritis (OA) is the most common form of arthritis, affecting over 25 million people in the United States. OA is characterized by the destruction and eventual loss of articular cartilage, accompanied by new bone formation and synovial proliferation, leading to pain, stiffness, loss of joint function, and disability. The joints most commonly affected by OA are the hands, neck, lower back, knees, and hips. The disease is progressive, and current treatments are aimed at relieving symptomatic pain but do not alter the natural history of the disease. The pathogenesis of OA is unknown, but a role for complement has been implicated. Proteome and transcriptome analyses of synovial fluid from OA patients revealed abnormal expression of several components of the classical (C1s and C4A) and alternative (factor B) pathways, as well as complement components C3, C5, C7, and C9, compared with samples from healthy individuals (Wang, Q., et al., Nat. Med. 17:1674-1679, 2011). Moreover, in a mouse model of OA induced by medial meniscectomy, C5-deficient mice exhibited less cartilage loss, osteophyte formation, and synovitis than C5-positive mice, and treatment of wild-type mice with the fusion protein CR2-fH, which inhibits the alternative pathway, reduced the development of OA (Wang et al., 2011, supra).

[0191] Ross River virus (RRV) and chikungunya virus (CHIKV) belong to a group of mosquito-borne viruses that can cause acute and persistent arthritis and myositis in humans. In addition to causing endemic disease, these viruses can cause epidemics with millions of infected individuals. Arthritis is thought to be initiated by viral replication in the joint and the induction of a host inflammatory response, and the complement system has been activated as a key component in this process. Synovial fluid from humans with RRV-induced polyarthritis contains higher levels of C3a than synovial fluid from humans with OA (Morrison, TE, et al., J. Virol. 81:5132-5143, 2007). In a mouse model of RRV infection, C3-deficient mice developed less severe arthritis compared with wild-type mice, suggesting the role of complement (Morrison et al., 2007). The specific complement pathway involved has been investigated, and mice with an inactivated lectin pathway (MBL-A) were shown to be more susceptible to C3a than mice with OA. - / - and MBL-C - / - ) reduced arthritis compared to wild-type mice. In contrast, inactivated classical pathway (C1q - / - ) or alternative pathway (factor B - / - Mice bearing MASP-2 KO mice developed severe arthritis, indicating that the lectin pathway initiated by MBL plays an essential role in this model (Gunn, BM, et al., PLoS Pathog. 8:e1002586, 2012). Because arthritis involves joint damage, initial joint damage caused by various etiologies may trigger a secondary wave of LEA-2-mediated complement activation. In support of this concept, our study demonstrated that MASP-2 KO mice had reduced joint damage compared with WT mice in a collagen-induced model of RA, as described in Example 27 herein.

[0192] In view of the body of evidence described above, LEA-1 and LEA-2 inhibitors, alone or in combination, are expected to be therapeutically useful in treating arthritis. Therefore, an optimally effective treatment for arthritis may include a pharmaceutical active ingredient capable of blocking both LEA-1 and LEA-2, alone or in combination. Combined LEA-1 and LEA-2 inhibition may be achieved by coadministration of an LEA-1 blocker and an LEA-2 blocker. Preferentially, LEA-1 and LEA-2 inhibitory functions may be encompassed in a single molecular entity, such as a bispecific antibody composed of MASP-1 / 3 and MASP-2-specific binding sites, or a bispecific antibody in which each binding site can bind to and block MASP-1 / 3 or MASP-2. Thus, in accordance with the foregoing, aspects of the present invention provide methods for inhibiting LEA-1-dependent complement activation to treat, prevent, or reduce the severity of inflammatory or non-inflammatory arthritis, including osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, and psoriatic arthritis, by administering a therapeutically effective amount of an LEA-1 inhibitor, including a MASP-1 inhibitor, a MASP-3 inhibitor, or a combination of MASP-1 / 3 inhibitors, in a pharmaceutical carrier to a subject suffering from or at risk of developing inflammatory or non-inflammatory arthritis. The MASP-1, MASP-3, or MASP-1 / 3 inhibitor composition can be administered to a subject systemically, for example, intra-arterially, intravenously, intramuscularly, subcutaneously, or by other parenteral or oral route. Alternatively, administration can be by local delivery, for example, by intra-articular injection. LEA-1 inhibitors may be administered periodically over an extended period of time to treat or suppress a chronic condition, or may be administered in single or repeated doses before, during, and / or after acute trauma or injury, including surgical procedures performed on a joint.

[0193] In one embodiment, the method of this aspect of the present invention further comprises inhibiting LEA-2-dependent complement activation in a subject suffering from or at risk of developing inflammatory or non-inflammatory arthritis (including osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, and psoriatic arthritis) by administering to the subject a therapeutically effective amount of a MASP-2 inhibitor and a MASP-1, MASP-3, or MASP-1 / 3 inhibitor. As described above, the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 is expected to provide improved therapeutic outcomes in treating or preventing arthritis compared to inhibition of LEA-1 alone. This outcome can be achieved, for example, by coadministering an antibody with LEA-1 blocking activity and an antibody with LEA-2 blocking activity. In some embodiments, LEA-1 blocking activity and LEA-2 blocking activity are combined in a single molecular entity, which has combined LEA-1 and LEA-2 blocking activity. Such an entity may comprise or consist of a bispecific antibody in which one antigen-binding site specifically recognizes MASP-1 and blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody in which one antigen-binding site specifically recognizes MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Optimally, such an entity may consist of a bispecific monoclonal antibody in which one antigen-binding site specifically recognizes both MASP-1 and MASP-3 and thus blocks LEA-1, while the second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

[0194] The MASP-2 inhibitory composition may be administered to a subject in need thereof systemically, for example, by intra-arterial, intravenous, intramuscular, subcutaneous, or other parenteral administration, or potentially orally in the case of non-peptidergic inhibitors. Alternatively, administration may be by local delivery, such as by intra-articular injection. The MASP-2 inhibitor may be administered periodically over an extended period of time to treat or suppress a chronic condition, or may be administered in single or repeated doses before, during, and / or after acute trauma or injury, including surgical procedures performed on a joint.

[0195] The application of the MASP-3 inhibitory composition and any MASP-2 inhibitory composition of the present invention can be carried out by single administration or limited continuous administration of the composition (e.g., co-administration of a single or separate composition comprising an MASP-2 and MASP-3 inhibitor or a bispecific or dual inhibitor) to treat, prevent, or reduce the severity of inflammatory or non-inflammatory arthritis. Alternatively, in the case of treating a subject suffering from inflammatory or non-inflammatory arthritis, the composition can be administered over a long period of time, at regular intervals such as once daily, twice weekly, weekly, biweekly, monthly, or bimonthly.

[0196] VII. Role of MASP-2 and MASP-3 in Disseminated Intravascular Coagulation (DIC) and Therapy Using MASP-2 and MASP-3 Inhibitors Disseminated intravascular coagulation (DIC) is a syndrome of pathological overstimulation of the coagulation system that can manifest clinically as hemorrhage and / or thrombosis. DIC does not occur as a primary condition; rather, it occurs in association with a variety of disease processes, including tissue injury (trauma, burns, heatstroke, transfusion reactions, acute transplant rejection), neoplasms, infections, obstetric conditions (placenta previa, amniotic fluid embolism, preeclampsia), and miscellaneous conditions such as cardiogenic shock, drowning, fat embolism, and aortic aneurysm. Although thrombocytopenia is a frequent abnormality in intensive care unit patients, with an incidence of 35%–44%, DIC is the etiology of approximately 25% of these cases. DIC develops in approximately 10% of critically ill patients (Levi, M. and Opal, SM Crit. Care 10:222–231, 2006). The pathophysiology of DIC is that an underlying disease process initiates a physiological coagulation response. However, when prothrombotic substances overwhelm normal counterbalancing mechanisms, inappropriate deposition of fibrin and platelets occurs in the microcirculation, leading to organ ischemia, hypofibrinogenemia, and thrombocytopenia. The diagnosis of DIC is based on clinical findings of the appropriate underlying disease or process, along with abnormalities in laboratory values ​​(prothrombin time, partial thromboplastin time, fibrin split products, D-dimer, or platelet count). Primary treatment of DIC is to address the underlying precipitating disease. Blood product support in the form of red blood cells, platelets, fresh frozen plasma, and cryoprecipitate may be required to treat or prevent clinical complications.

[0197] The role of the complement pathway in DIC has been investigated in several studies. Complement activation has been evaluated in pediatric patients with meningococcal infection by comparing the clinical course with MBL genotype (Sprong, T. et al., Clin. Infect. Dis. 49:1380-1386, 2009). At admission, MBL-deficient patients showed lower circulating levels of C3bc, terminal complement complexes, C4bc, and C3bBbP than MBL-sufficient patients, indicating lower levels of conventional complement, terminal complement, and alternative pathway activation. Furthermore, the degree of systemic complement activation correlated with disease severity and parameters in DIC and MBL-deficient patients, and they showed a milder clinical course than MBL-sufficient patients. Therefore, although MBL deficiency is a risk factor for susceptibility to infection, MBL deficiency during septic shock may be associated with lower disease severity.

[0198] As demonstrated in Examples 1-4 herein, experimental studies highlight the important contribution of MBL and MASP-1 / 3 to the innate immune response to Neisseria meningitidis, the etiological agent of meningococcal infection. MBL-deficient sera from mice or humans, MASP-3-deficient human sera, or sera from MASP-1 / 3 knockout mice are less effective at activating complement and lysing Neisseria meningitidis in vitro than wild-type sera. Similarly, naive MASP-1 / 3 knockout mice are more susceptible to Neisseria infection than their wild-type counterparts. Thus, in the absence of adaptive immunity, the LEA-1 pathway contributes to innate host resistance to Neisseria infection. Conversely, LEA-1 enhances pathological complement activation, which induces adverse host responses, including DIC.

[0199] In a mouse model of arterial thrombosis, MBL-null and MASP-1 / -3 knockout mice showed reduced FeCl3-induced thrombus formation compared with wild-type or C2 / factor B-null mice, and the defect was reconstituted by recombinant human MBL (La Bonte, L.R., et al., J. Immunol. 188:885-891, 2012). In vitro, serum from MBL-null or MASP-1 / -3 knockout mice showed reduced thrombin substrate cleavage compared with serum from wild-type or C2 / factor B-null mice. Addition of recombinant human MASP-1 restored thrombin substrate cleavage in serum from MASP-1 / -3 knockout mice (La Bonte et al., 2012). These results suggest that the MBL / MASP complex, particularly MASP-1, may play an important role in thrombus formation. Therefore, LEA-1 may play an important role in pathological thrombosis, including DIC.

[0200] Experimental studies have demonstrated the equally important role of LEA-2 in pathological thrombosis. As described in Example 30 herein, in a mouse model of focal DIC, the inventors demonstrated that MASP-2 knockout mice were much less susceptible to LPS-induced microvascular coagulation than wild-type mice. In vitro studies further demonstrate that LEA-2 provides a molecular link between the complement and coagulation systems. As described in Examples 29 and 31 herein, MASP-2 has factor Xa-like activity and activates prothrombin by cleavage to form thrombin, which can then scavenge fibrinogen and promote fibrin clot formation (see also Krarup et al., PLoS One, 18:2(7):e623, 2007).

[0201] Separate studies have shown that lectin-MASP complexes can promote clot formation, fibrin deposition, and fibrinopeptide release in a MASP-2-dependent process (Gulla et al., Immunology, 129(4):482-95, 2010). Thus, LEA-2 simultaneously promotes lectin-dependent activation of the complement and coagulation systems.

[0202] Furthermore, in vitro studies have shown that MASP-1 has thrombin-like activity (Presanis JS, et al., Mol Immunol, 40(13):921-9, 2004) and cleaves fibrinogen and factor XIII (Gulla KC et al., Immunology, 129(4):482-95, 2010), suggesting that LEA-1 may activate the coagulation pathway independently of or in concert with LEA-2.

[0203] The above-mentioned data suggest that LEA-1 and LEA-2 provide an independent link between lectin-dependent complement activation and coagulation. Therefore, in view of the above, LEA-1 inhibitors and LEA-2 inhibitors are expected to have independent therapeutic benefits when treating subjects suffering from disseminated intravascular coagulation. In some embodiments, the subject suffers from disseminated intravascular coagulation secondary to sepsis, trauma, infection (bacterial, viral, fungal, parasitic), malignant tumor, transplant rejection, transfusion reaction, birth complications, vascular aneurysm, liver failure, heat stroke, burns, radiation exposure, shock, or severe toxic reactions (e.g., snake bite, insect bite, transfusion reaction). In some embodiments, the trauma is neurological trauma. In some embodiments, the infection is a bacterial infection, such as a meningococcal infection.

[0204] In addition, LEA-1 and LEA-2 inhibitors, when used in combination, may achieve additional therapeutic benefits compared to either inhibitor alone. Because both LEA-1 and LEA-2 are known to be activated by conditions that cause DIC (e.g., infection or trauma), LEA-1 and LEA-2 blockers, either separately or in combination, are expected to have therapeutic utility in treating DIC. LEA-1 and LEA-2 blockers may prevent different crosstalk mechanisms between complement and coagulation. Thus, LEA-1 and LEA-2 blockers may have complementary, additive, or synergistic effects in preventing DIC and other thrombotic disorders.

[0205] In addition, LEA-1 and LEA-2 inhibitors, when used in combination, may achieve additional therapeutic benefit or provide effective treatment for a broader range of patient subsets than either inhibitor alone. Combined LEA-1 and LEA-2 inhibition may be achieved by co-administration of an LEA-1 blocker and an LEA-2 blocker. Optimally, LEA-1 and LEA-2 inhibitory functions may be encompassed in a single molecular entity, such as a bispecific antibody composed of MASP-1 / 3- and MASP-2-specific binding sites, or a bispecific antibody in which each binding site binds to and blocks MASP-1 / 3 or MASP-2.

[0206] Thus, in accordance with the foregoing, aspects of the present invention provide methods for inhibiting LEA-1-dependent complement activation to treat, prevent, or reduce the severity of disseminated intravascular coagulation in a subject in need thereof, comprising administering to a subject experiencing or at risk of developing disseminated intravascular coagulation a therapeutically effective amount of a composition comprising an LEA-1 inhibitor, including a MASP-1 inhibitor, a MASP-3 inhibitor, or a combination of MASP-1 / 3 inhibitors, in a pharmaceutical carrier. The MASP-1, MASP-3, or MASP-1 / 3 inhibitor composition may be administered to the subject systemically, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or potentially orally in the case of non-peptidergic agents. Administration may be repeated as determined by a physician until the condition resolves or is suppressed. For the treatment or prevention of DIC secondary to trauma or other acute event, the LEA-1 inhibitor composition may be administered immediately after the traumatic injury or prophylactically before, during, immediately after, or within 1 to 7 days or more, e.g., within 24 to 72 hours, of a trauma-induced injury or situation such as surgery, in a patient considered at risk for DIC. In some embodiments, the LEA-1 inhibitor composition may be suitably administered in a rapid-release dosage form, e.g., by intravenous or intra-arterial delivery of a bolus of a solution containing the LEA-1 inhibitor composition.

[0207] In one embodiment, the method of this aspect of the invention further comprises inhibiting LEA-2-dependent complement activation to treat, prevent, or reduce the severity of disseminated intravascular coagulation in a subject in need thereof, comprising administering to the subject therapeutically effective amounts of a MASP-2 inhibitor and a MASP-1, MASP-3, or MASP-1 / 3 inhibitor. As described above, the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 is expected to provide improved therapeutic outcomes in treating or preventing disseminated intravascular coagulation compared to inhibition of LEA-1 alone. This outcome can be achieved, for example, by coadministering an antibody with LEA-1 blocking activity and an antibody with LEA-2 blocking activity. In some embodiments, LEA-1 blocking activity and LEA-2 blocking activity are combined in a single molecular entity, such entity having combined LEA-1 and LEA-2 blocking activity. Such an entity may comprise or consist of a bispecific antibody in which one antigen-binding site specifically recognizes MASP-1 and blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody in which one antigen-binding site specifically recognizes MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Optimally, such an entity may consist of a bispecific monoclonal antibody in which one antigen-binding site specifically recognizes both MASP-1 and MASP-3 and thus blocks LEA-1, while the second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

[0208] The MASP-2 inhibitory composition may be administered to a subject in need thereof systemically, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or potentially orally in the case of non-peptidergic substances. Administration may be repeated, as determined by a physician, until the condition resolves or is suppressed. In the case of DIC secondary to trauma or other acute events, the MASP-2 inhibitory composition may be administered immediately after the traumatic injury or prophylactically in patients considered at risk for DIC, either before, during, or immediately after a trauma-induced injury or surgery, or within 1 to 7 days or longer, for example, within 24 to 72 hours. In some embodiments, the MASP-2 inhibitory composition may be administered in a rapid-release dosage form, for example, by intravenous or intra-arterial delivery of a bolus of a solution containing the MASP-2 inhibitory composition.

[0209] The application of the MASP-3 inhibitory composition and any MASP-2 inhibitory composition of the present invention can be carried out by a single administration or a limited number of consecutive administrations of the composition (e.g., co-administration of a single composition or separate compositions comprising MASP-2 and MASP-3 inhibitors or bispecific or dual inhibitors) to treat, prevent, or reduce the severity of disseminated intravascular coagulation in a subject in need thereof. Alternatively, in the case of treatment of a subject experiencing disseminated intravascular coagulation or at risk of developing disseminated intravascular coagulation, the composition may be administered over an extended period of time, at regular intervals such as once daily, twice weekly, weekly, biweekly, monthly, or bimonthly.

[0210] VIII. Role of MASP-2 and MASP-3 in Thrombotic Microangiopathy (TMA), Including Hemolytic Uremic Syndrome (HUS), Atypical Hemolytic Uremic Syndrome (aHUS), and Thrombotic Thrombocytopenic Purpura (TTP), and Therapeutics Using MASP-2 and MASP-3 Inhibitors Thrombotic microangiopathy (TMA) refers to a group of disorders characterized clinically by thrombocytopenia, microangiopathic hemolytic anemia, and variable organ ischemia. The characteristic pathological features of TMA are platelet activation and the formation of microthrombi in small arterioles and venules. Classic TMA are hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). HUS is distinguished from TTP by the presence of acute renal failure. HUS occurs in two forms: diarrhea-associated (D+) or typical HUS and diarrhea-free (D-) or atypical HUS (aHUS).

[0211] HUS D+HUS, usually associated with a prodromal diarrheal illness caused by Escherichia coli O157 or another Shiga toxin-producing bacterial strain, accounts for over 90% of HUS cases in children and is the most common cause of acute renal failure in children. Although human infection with Escherichia coli O157 is relatively common, the rate of bloody diarrhea progressing to D+HUS ranges from 3% to 7% in sporadic cases to 20% to 30% in several outbreaks (Zheng, XL and Sadler, JE, Annu. Rev. Pathol. 3:249-277, 2008). HUS usually develops 4 to 6 days after the onset of diarrhea, and approximately two-thirds of children require dialysis during the acute phase of the disease. Treatment for D+HUS is supportive, as no specific treatment has been shown to be effective. The prognosis for D+HUS is favorable, with the majority of patients recovering renal function.

[0212] The pathogenesis of D+HUS involves bacterially produced Shiga toxins that bind to membranes on microvascular endothelial cells, monocytes, and platelets. The renal microvasculature is most frequently affected. After binding, the toxins are internalized, leading to the release of proinflammatory mediators and eventual cell death. Endothelial cell injury is thought to induce renal microvascular thrombosis by promoting activation of the coagulation cascade. There is evidence of complement system activation in D+HUS. In children with D+HUS, plasma levels of Bb and SC5b-9 were elevated at admission compared with normal controls and normalized 28 days after discharge (Thurman, JM et al., Clin. J. Am. Soc. Nephrol. 4:1920-1924, 2009). Shiga toxin 2 (Stx2) was found to activate human complement in vitro in the fluid phase primarily via the alternative pathway when activation proceeded in the presence of ethylene glycol tetraacetic acid, which blocks the classical pathway (Orth, D. et al., J. Immunol. 182:6394-6400, 2009). Furthermore, Stx2 bound factor H, but not factor I, and delayed the cofactor activity of factor H on the cell surface (similar to Orth, D. et al., 2009). These results suggest that Shiga toxin can cause renal injury through multiple potential mechanisms, including direct toxic effects, and indirectly through complement activation or inhibition of complement regulators. As demonstrated in Examples 21-23 herein, its toxic effects on vascular endothelial cells are expected to activate complement via LEA-2, as evidenced by the effectiveness of MASP-2 blockade in preventing complement-mediated reperfusion injury in various vascular beds. See also Schwaeble, WJ, et al., Proc. Natl. Acad. Sci. 108:7523-7528, 2011.

[0213] In a mouse model of HUS induced by co-injection of Shiga toxin and lipopolysaccharide, factor B-deficient mice exhibited less thrombocytopenia and were protected from renal dysfunction compared with wild-type mice, suggesting the involvement of LEA-1-dependent activation of the alternative pathway in microvascular thrombosis (Morigi, M. et al., J. Immunol. 187:172-180, 2011). As described in Example 33 herein, in the same model, administration of an MASP-2 antibody was also effective, increasing survival after STX challenge, suggesting the involvement of the LEA-2-dependent complement pathway in microvascular thrombosis.

[0214] Based on the foregoing, LEA-1 and LEA-2 inhibitors are expected to have independent therapeutic benefits in the treatment or prevention of HUS. Additionally, when used in combination, LEA-1 and LEA-2 inhibitors may achieve additional therapeutic benefit or provide effective treatment for a broader range of patient subsets than either inhibitor alone. Combined LEA-1 and LEA-2 inhibition can be achieved by coadministration of an LEA-1 blocker and an LEA-2 blocker. Optimally, LEA-1 and LEA-2 inhibitory functions can be encompassed in a single molecular entity, such as a bispecific antibody composed of MASP-1 / 3- and MASP-2-specific binding sites, or a bispecific antibody in which each binding site can bind to and block MASP-1 / 3 or MASP-2.

[0215] aHUS Atypical HUS is an orphan disease with an estimated incidence of 2 per million in the United States (Loirat, C. and Fremeaux-Bacchi, V. Orphanet J. Rare Dis. 6:60-90, 2011). Atypical HUS can occur at any age, but the majority of patients present during childhood. Atypical HUS is heterogeneous. Some cases are familial, some are recurrent, and some are triggered by infection, typically upper respiratory or gastroenteritis. The onset of aHUS is usually sudden, and most patients require dialysis upon hospitalization. Additional renal manifestations occur in approximately 20% of patients and may include central nervous system involvement, myocardial infarction, ischemic distal gangrene, or multiple organ failure. Treatment for aHUS includes supportive care in the event of organ failure, plasma infusion or plasma exchange, and eculizumab, a humanized monoclonal antibody targeting C5 recently approved for use in the United States and the European Union. The prognosis in aHUS is not as good as in D+HUS, with approximately 25% dying in the acute phase and most survivors developing end-stage renal disease.

[0216] Atypical HUS is characterized as a disease of complement dysregulation in that approximately 50% of patients have mutations in genes encoding complement regulatory proteins (Zheng and Sadler, 2008, supra). Most mutations are found in factor H (FH). Other mutations include membrane cofactor protein (MCP), factor I (FI), factor B, and C3. Functional studies have shown that mutations in FH, MCP, and FI result in loss of function and therefore increased complement activation, while mutations in factor B are gain-of-function. The effects of these mutations primarily affect the alternative pathway. These genetic abnormalities are risk factors rather than the sole cause of the disease, as approximately 50% of families with mutations do not develop the disease by age 45 years (Loirat and Fremeaux-Bacchi, 2011, supra).

[0217] Factor H is a complement regulatory protein that protects host tissues from alternative pathway complement attack. FH regulates the alternative pathway amplification loop in three ways: it is a cofactor for factor H, which cleaves C3b; it inhibits the formation of the alternative pathway C3 convertase C3bBb; and it binds to polyanions on cell surfaces and tissue matrices, blocking C3b deposition (Atkinson, JP and Goodship, THJ, J. Exp. Med. 6:1245-1248, 2007). The majority of FH mutations in aHUS patients occur in the C-terminal short consensus repeat domain of the protein, resulting in defective binding of FH to heparin, C3b, and endothelium, but do not alter plasma C3 regulation within the N-terminal domain (Pickering, MC et al., J. Exp. Med. 204:1249-1256, 2007). FH-deficient mice exhibit uncontrolled plasma C3 activation and spontaneously develop membranoproliferative glomerulonephritis type II, but not aHUS. However, FH-deficient mice transgenically expressing a mouse FH protein functionally equivalent to an aHUS-associated human FH mutant spontaneously develop HUS, but not membranoproliferative glomerulonephritis type II, providing in vivo evidence that defective regulation of alternative pathway activation in the renal endothelium is a key event in the pathogenesis of FH-associated aHUS (Pickering et al., 2007, supra). Another form of FH-associated aHUS occurs in patients with anti-FH autoantibodies, resulting in loss of FH functional activity. The majority of these patients have deletions in the genes encoding five FH-related proteins (Loirat and Fremeaux-Bacchi, 2011, supra).

[0218] Like FH, MCP inhibits complement activation by regulating C3b deposition on target cells. MCP mutations result in proteins with reduced C3b binding and cofactor activity, thereby enabling dysregulated alternative pathway activation. FI is a serine protease that cleaves C3b and C4b in the presence of cofactors such as FH and MCP, thereby preventing the formation of C3 and C5 convertases and inhibiting both the alternative and classical complement pathways. Most FI-associated aHUS mutations result in reduced FI activity for the degradation of C3b and C4b (Zheng and Stadler, 2008, supra). FB is a zymogen that contains the catalytic site for the alternative pathway convertase C3bBb. Functional analysis has shown that aHUS-associated FB mutations lead to increased alternative pathway activation (Loirat and Fremeaux-Bacchi, 2011, supra). Heterozygous mutations in C3 are associated with aHUS. Most C3 mutations cause a defect in C3 binding to MCP, leading to an increased ability of FB to bind C3b and increased C3 convertase formation (Loirat and Fremeaux-Bacchi, 2011, supra). Therefore, aHUS is a disease closely associated with mutations in complement genes that result in insufficient regulation of the alternative pathway amplification loop. Because the alternative pathway amplification loop depends on factor B proteolytic activity and because LEA-1 is required for factor B activation (either by MASP-3-dependent cleavage or factor D-mediated cleavage in which MASP-1 contributes to factor D maturation), LEA-1 blockers are expected to prevent uncontrolled complement activation in susceptible individuals. Consequently, LEA-1 blockers are expected to effectively treat aHUS.

[0219] Although the central role of a deregulated alternative pathway amplification loop in aHUS is widely accepted, the triggers initiating complement activation and the molecular pathways involved remain unclear. Not all individuals with the above mutations develop aHUS. In fact, family studies have suggested that the penetrance of aHUS is only approximately 50% (Sullivan M. et al., Ann Hum Genet 74:17-26 2010). The natural history of the disease suggests that aHUS most frequently develops after an initiating event, such as an infectious episode or injury. It is well known that infectious agents activate the complement system. In the absence of preexisting adaptive immunity, complement activation by infectious agents can be initiated primarily via LEA-1 or LEA-2. Therefore, infection-induced lectin-dependent complement activation may trigger the subsequent pathological amplification of complement activation in individuals predisposed to aHUS, which may ultimately lead to disease progression. Thus, another aspect of the present invention involves treating a patient suffering from aHUS secondary to an infection by administering an effective amount of an LEA-1 or LEA-2 inhibitor.

[0220] Other forms of host tissue damage, particularly vascular endothelial damage, activate complement via LEA-2. Human vascular endothelial cells subjected to oxidative stress, for example, respond by expressing surface moieties that bind to lectins and activate the LEA-2 pathway of complement (Collard et al., Am J. Pathol 156(5):1549-56, 2000). Vascular injury after ischemia / reperfusion also activates complement via LEA-2 in vivo (Moller-Kristensen et al., Scand J Immunol 61(5):426-34, 2005). Lectin pathway activation in this situation has pathological consequences for the host, and as shown in Examples 22 and 23, inhibiting LEA-2 by blocking MASP-2 prevents further host tissue damage and adverse outcomes (see also Schwaeble, PNAS, 2011, supra).

[0221] Therefore, other processes that cause aHUS are also known to activate LEA-1 or LEA-2. Therefore, it is likely that the LEA-1 and / or LEA-2 pathways may be the initial complement activation mechanism that is deregulated and inappropriately amplified in individuals genetically predisposed to aHUS, thereby initiating aHUS pathogenesis. By implication, agents that block complement activation via LEA-1 and / or LEA-2 are expected to prevent or mitigate disease progression in aHUS-susceptible individuals.

[0222] Further supporting this concept, recent studies have identified Streptococcus pneumoniae as a significant etiologic agent in pediatric cases of aHUS (Lee, C.S. et al., Nephrology, 17(1):48-52 (2012); Banerjee R. et al., Pediatr Infect Dis J., 30(9):736-9 (2011)). This particular etiology appears to have an unfavorable prognosis, including significant mortality and long-term morbidity. Notably, these cases involved non-intestinal infections leading to the manifestations of microangiopathy, uremia, and hemolysis, without evidence of concomitant mutations in complement genes known to predispose to aHUS. It is important to note that Streptococcus pneumoniae is particularly effective at activating complement, primarily via LEA-2. Therefore, in cases of non-intestinal HUS associated with Streptococcus pneumoniae infection, the manifestations of microangiopathy, uremia, and hemolysis are expected to be primarily driven by activation of LEA-2, and agents that block LEA-2, including MASP-2 antibodies, are expected to prevent the progression of aHUS or reduce disease severity in these patients. Accordingly, another aspect of the present invention includes treating patients suffering from non-intestinal aHUS associated with Streptococcus pneumoniae infection by administering an effective amount of a MASP-2 inhibitor.

[0223] TTP Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder of the blood coagulation system caused by autoimmune or hereditary dysfunction that activates the coagulation cascade or complement system (George, JN, N Engl J Med; 354:1927-35, 2006). This leads to the formation of numerous microscopic blood clots, or thrombi, in small blood vessels throughout the body, which is the hallmark of TTP. Red blood cells are subjected to shear stress, which damages their membranes and leads to intravascular hemolysis. The resulting reduced blood flow and endothelial damage cause organ damage, including the brain, heart, and kidneys. TTP is clinically characterized by thrombocytopenia, microangiopathic hemolytic anemia, neurological changes, renal failure, and fever. In the era before plasma exchange, the fatality rate during acute episodes was 90%. Even with plasma exchange, the 6-month survival rate is approximately 80%.

[0224] TTP can result from genetic or acquired inhibition of the enzyme ADAMTS-13, a metalloprotease responsible for cleaving large von Willebrand factor (vWF) multimers into smaller units. ADAMTS-13 inhibition or deficiency ultimately results in increased coagulation (Tsai, H. J Am Soc Nephrol 14:1072-1081, 2003). ADAMTS-13 regulates the activity of vWF. In the absence of ADAMTS-13, vWF forms large multimers that are more likely to bind to platelets, predisposing patients to platelet aggregation and thrombosis in the microvasculature.

[0225] Many mutations in ADAMTS13 have been identified in individuals with TTP. This disease can also be caused by autoantibodies against ADAMTS-13. In addition, TTP can occur during breast, gastrointestinal, or prostate cancer (George JN., Oncology (Williston Park). 25:908-14, 2011), pregnancy (second trimester or postpartum) (George JN., Curr Opin Hematol 10:339-344, 2003), or is associated with autoimmune diseases such as HIV or systemic lupus erythematosus (Hamasaki K, et al., Clin Rheumatol. 22:355-8, 2003). TTP can also be caused by certain medications, including heparin, quinine, immune-mediated components, cancer chemotherapy drugs (bleomycin, cisplatin, cytosine arabinoside, daunomycin gemcitabine, mitomycin C, and tamoxifen), cyclosporin A, oral contraceptives, penicillin, rifampin, and antiplatelet drugs, including ticlopidine and clopidogrel (Azarm, T. et al., J Res Med. Sci., 16:353-357, 2011). Other factors or conditions associated with TTP include toxins, such as bee venom, sepsis, splenic sequestration, transplantation, vasculitis, vascular surgery, and infections, such as Streptococcus pneumoniae and cytomegalovirus (Moake JL., N Engl J. Med., 347:589-600, 2002). Transient functional ADAMTS-13 deficiency can result in TTP as a result of endothelial cell injury associated with Streptococcus pneumoniae infection (Pediatr Nephrol, 26:631-5, 2011).

[0226] Plasmapheresis is the standard treatment for TTP (Rock GA, et al., N Engl J Med 325:393-397, 1991). Plasmapheresis replaces ADAMTS-13 activity in patients with genetic defects and removes ADAMTS-13 autoantibodies in patients with acquired autoimmune TTP (Tsai, HM, Hematol Oncol Clin North Am., 21(4):609-v, 2007). Additional agents, such as immunosuppressants, are routinely added to treatment (George, JN, N Engl J Med, 354:1927-35, 2006). However, plasmapheresis is unsuccessful in approximately 20% of patients, relapse occurs in more than one-third of patients, and plasmapheresis is expensive and technically demanding. Furthermore, many patients cannot tolerate plasmapheresis. Consequently, there remains an urgent need for further and better treatments for TTP.

[0227] Because TTP is a disorder of the blood coagulation cascade, treatment with complement system antagonists may help stabilize and reverse the disease. While pathological activation of the alternative complement pathway has been associated with aHUS, the role of complement activation in TTP is less clear. Although functional deficiency of ADAMTS13 is important for susceptibility to TTP, it is not sufficient to cause an acute episode. Environmental factors and / or other genetic mutations may contribute to the manifestation of TTP. For example, genes encoding proteins involved in regulating the coagulation cascade, vWF, platelet function, endothelial vascular surface components, or the complement system may be involved in the development of acute thrombotic microangiopathy (Galbusera, M. et al., Haematologica, 94:166-170, 2009). In particular, complement activation has been shown to play an important role. Serum from thrombotic microangiopathy associated with ADAMTS-13 deficiency has been shown to result in C3 and MAC deposition and subsequent neutrophil activation, which could be prevented by complement inactivation (Ruiz-Torres MP, et al., Thromb Haemost, 93:443-52, 2005). Additionally, it has recently been shown that during acute episodes of TTP, there are increased levels of C4d, C3bBbP, and C3a, consistent with activation of the classical, lectin, and alternative pathways (M. Reti et al., J Thromb Haemost. 10(5):791-798, 2012). This increased complement activation during acute episodes may initiate terminal pathway activation and contribute to further exacerbation of TTP.

[0228] The role of ADAMTS-13 and vWF in TTP is apparently responsible for platelet activation and aggregation and their subsequent role in shear stress and deposition in microangiopathy. Activated platelets interact with and induce both the classical and alternative pathways of complement. Platelet-mediated complement activation increases the inflammatory mediators C3a and C5a (Peerschke E. et al., Mol Immunol, 47:2170-5 (2010)). Therefore, platelets may serve as targets for classical complement activation in hereditary or autoimmune TTP.

[0229] As described above, lectin-dependent activation of complement, which occurs due to the thrombin-like activity of MASP-1 and LEA-2-mediated prothrombin activation, is the dominant molecular pathway linking endothelial injury to coagulation and microvascular thrombosis in HUS. Similarly, activation of LEA-1 and LEA-2 may directly drive the coagulation system in TTP. LEA-1 and LEA-2 pathway activation may be initiated in response to the initial endothelial injury caused by ADAMTS-13 deficiency in TTP. Therefore, LEA-1 and LEA-2 inhibitors, including but not limited to antibodies that block MASP-2 function, MASP-1 function, MASP-3 function, or MASP-1 and MASP-3 function, are expected to alleviate the microvascular disease associated with microvascular coagulation, thrombosis, and hemolysis in patients with TTP.

[0230] Patients with TTP typically present with one or more of the following symptoms during emergency room care: purpura, renal failure, low platelets, anemia, and / or thrombosis, including stroke. The current standard of care for TTP involves intracatheter delivery (e.g., intravenous or other forms of catheter) of exchange plasmapheresis, typically three times weekly to daily, for two weeks or longer. If a subject tests positive for the presence of an inhibitor of ADAMTS13 (i.e., endogenous antibodies against ADAMTS13), plasmapheresis may be performed in combination with immunosuppressive therapy (e.g., corticosteroids, Rituxan, or cyclosporine). Subjects with refractory TTP (approximately 20% of TTP patients) do not respond to plasmapheresis treatment for at least two weeks.

[0231] In accordance with the foregoing, in one embodiment, in the setting of an initial diagnosis of TTP or in a subject exhibiting one or more symptoms consistent with a diagnosis of TTP (e.g., central nervous system complications, severe thrombocytopenia (platelet count less than 5000 / μL or 5000 / μL if aspirin is off, or less than 20,000 / μL or 20,000 / μL if aspirin is on), severe cardiac complications, severe pulmonary complications, gastrointestinal infarction, or gangrene), a method is provided for treating a subject with an effective amount of a LEA-2 inhibitor (e.g., a MASP-2 antibody) or a LEA-1 inhibitor (e.g., a MASP-1 or MASP-3 antibody) as a first therapy in the absence of, or in combination with, plasmapheresis. As a first therapy, the LEA-1 inhibitor and / or LEA-2 inhibitor may be administered to the subject systemically, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration. In some embodiments, a LEA-1 inhibitor and / or a LEA-2 inhibitor may be administered to a subject as a first-line therapy in the absence of plasmapheresis to avoid potential complications of plasmapheresis, such as bleeding, infection, and exposure to plasma donor-specific disorders and / or allergies, or in subjects who are otherwise unwilling to undergo plasmapheresis, or in situations where plasmapheresis is unavailable. In some embodiments, a LEA-1 inhibitor and / or a LEA-2 inhibitor is administered to a subject suffering from TTP in combination (including coadministration) with an immunosuppressant (e.g., a corticosteroid, Rituxan, or cyclosporine) and / or in combination with concentrated ADAMTS-13.

[0232] In some embodiments, the method comprises administering a LEA-1 and / or LEA-2 inhibitor via catheter (e.g., intravenously) to a subject suffering from TTP for a first period (e.g., an acute phase lasting at least 1 day to 1 or 2 weeks), followed by administering a LEA-1 and / or LEA-2 inhibitor subcutaneously to the subject for a second period (e.g., a chronic phase lasting at least 2 weeks or longer). In some embodiments, the administration during the first and / or second period is performed in the absence of plasmapheresis. In some embodiments, the method is used to prevent or maintain a subject from suffering from one or more symptoms associated with TTP.

[0233] In another embodiment, a method is provided for treating a subject suffering from refractory TTP (i.e., a subject who has not responded to at least two weeks of plasmapheresis treatment) by administering an LEA-1 and / or LEA-2 inhibitor in an amount effective to alleviate one or more symptoms of TTP. In one embodiment, the LEA-1 and / or LEA-2 inhibitor is administered chronically to a subject with refractory TTP by subcutaneous or other parenteral administration for at least two weeks or longer. Administration may be repeated as determined by a physician until the condition resolves or is controlled.

[0234] In some embodiments, the method further comprises measuring the level of at least one complement factor (e.g., C3, C5) in the subject before treatment and optionally during treatment, wherein measuring a decrease in the level of the at least one complement factor compared to baseline or a healthy control subject indicates the need for continued treatment with the LEA-1 and / or LEA-2 inhibitor.

[0235] In some embodiments, the method comprises administering a LEA-1 and / or LEA-2 inhibitor subcutaneously or intravenously to a subject suffering from or at risk of developing TTP. Treatment is preferably daily, but can also be monthly. Treatment is continued until the subject's platelet count exceeds 150,000 / ml for at least two consecutive days.

[0236] In summary, LEA-1 and LEA-2 inhibitors are expected to have independent therapeutic benefits in the treatment of TMA, including HUS, aHUS, and TTP. Additionally, when used in combination, LEA-1 and LEA-2 inhibitors are expected to achieve additional therapeutic benefit or provide effective treatment for a broader range of patient subsets suffering from various forms of TMA than either inhibitor alone. Combined LEA-1 and LEA-2 inhibition can be achieved by coadministration of an LEA-1 blocker and an LEA-2 blocker. Optimally, LEA-1 and LEA-2 inhibitory functions can be incorporated into a single molecular entity, such as a bispecific antibody composed of MASP-1 / 3 and MASP-2-specific binding sites, or a bispecific antibody in which each binding site can bind to and block MASP-1 / 3 or MASP-2.

[0237] Thus, in accordance with the foregoing, aspects of the present invention provide methods for inhibiting LEA-1-dependent complement activation to treat, prevent, or reduce the severity of thrombotic microangiopathy, such as hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), or thrombotic thrombocytopenic purpura (TTP), comprising administering to a subject suffering from or at risk of developing thrombotic microangiopathy a composition comprising a therapeutically effective amount of an LEA-1 inhibitor, including a MASP-1 inhibitor, a MASP-3 inhibitor, or a combination of MASP-1 / 3 inhibitors, in a pharmaceutical carrier. The MASP-1, MASP-3, or MASP-1 / 3 inhibitor composition may be administered to the subject systemically, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or potentially orally in the case of non-peptidergic agents. Administration may be repeated as determined by a physician until the condition resolves or is suppressed.

[0238] In one embodiment, the method of this aspect of the invention further comprises inhibiting LEA-2-dependent complement activation to treat, prevent, or reduce the severity of thrombotic microangiopathy, such as hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), or thrombotic thrombocytopenic purpura (TTP), and comprises administering therapeutically effective amounts of a MASP-2 inhibitor and a MASP-1, MASP-3, or MASP-1 / 3 inhibitor to a subject suffering from or at risk of developing thrombotic microangiopathy. As described above, the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 is expected to provide improved therapeutic outcomes in treating, preventing, or reducing the severity of thrombotic microangiopathy compared to inhibition of LEA-1 alone. This outcome can be achieved, for example, by coadministering an antibody with LEA-1 blocking activity and an antibody with LEA-2 blocking activity. In some embodiments, LEA-1 blocking activity and LEA-2 blocking activity are combined into a single molecular entity, such that the entity has combined LEA-1 and LEA-2 blocking activity. Such an entity may comprise or consist of a bispecific antibody in which one antigen-binding site specifically recognizes MASP-1 and blocks LEA-1, and the second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody in which one antigen-binding site specifically recognizes MASP-3 and thus blocks LEA-1, and the second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Such an entity may optimally consist of a bispecific monoclonal antibody in which one antigen-binding site specifically recognizes both MASP-1 and MASP-3 and thus blocks LEA-1, while the second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

[0239] The MASP-2 inhibitor may be administered to the subject systemically, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous or other parenteral administration, or, in the case of non-peptidergic agents, potentially by oral administration. Administration may be repeated as determined by a physician until the condition resolves or is suppressed.

[0240] IX. The application of the MASP-3 inhibitory composition and any MASP-2 inhibitory composition of the present invention can be carried out by a single administration of the composition (e.g., a single composition containing MASP-2 and MASP-3 inhibitors or bispecific or dual inhibitors, or simultaneous administration of separate compositions) or a limited number of consecutive administrations to treat, prevent, or reduce the severity of thrombotic microangiopathy in subjects suffering from or at risk of developing thrombotic microangiopathy. Alternatively, the composition can be administered at regular intervals, such as once daily, twice weekly, weekly, biweekly, monthly, or bimonthly, over an extended period of time to treat a subject in need thereof. The Role of MASP-2 and MASP-3 in Asthma and Therapeutics Using MASP-2 and MASP-3 Inhibitors Asthma is a common chronic inflammatory airway disease. In the United States, approximately 25 million people, including 7 million children under the age of 18, have asthma. Over half experience at least one asthma attack per year, resulting in over 1.7 million emergency department visits and 450,000 hospitalizations annually (World Wide Web: gov / health / prof / lung / asthma / naci / asthma-info / index.htm, accessed May 4, 2012). The disease is heterogeneous and has multiple clinical phenotypes. The most common phenotype is allergic asthma. Other phenotypes include nonallergic asthma, aspirin-exacerbated respiratory disease, postinfectious asthma, occupational asthma, airborne irritant-induced asthma, and exercise-induced asthma. Cardinal features of allergic asthma include airway hyperresponsiveness (AHR) to a variety of specific and nonspecific stimuli, excessive airway mucus production, pulmonary eosinophilia, and elevated serum IgE levels. Symptoms of asthma include cough, wheezing, chest tightness, and shortness of breath. The goals of asthma treatment are to control the disease, minimize exacerbations and daily symptoms, and allow patients to be physically active. Current treatment guidelines encourage a stepwise approach to treatment until asthma control is achieved. The initial treatment step is a rapid-acting inhaled beta-2 agonist, if needed, followed by long-term controller medications, such as inhaled corticosteroids, long-acting inhaled beta-2 agonists, leukotriene modifiers, theophylline, oral glucocorticosteroids, and anti-IgE monoclonal antibodies.

[0241] Asthma is pathologically multifactorial but is generally recognized to result from an inappropriate immune response to common environmental antigens in genetically susceptible individuals. Asthma is associated with complement activation, and the anaphylatoxins (ATs) C3a and C5a have proinflammatory and immunomodulatory properties related to the development and modulation of allergic responses (Zhang, X. and Kohl, J. Expert. Rev. Clin. Immunol., 6:269-277, 2010). However, the relative contributions of the classical, alternative, and lectin complement pathways in asthma are not fully understood. The alternative pathway can be activated on the surface of allergens, and the lectin pathway can be activated by recognition of the allergen's polysaccharide structure; both processes lead to the production of ATs. Complement can be activated by different pathways depending on the causative allergen involved. For example, highly allergenic pollen from the Parietaria family is highly effective in promoting MBL-dependent activation of C4, suggesting the involvement of LEA-2. Conversely, dust mite allergens do not require MBL for complement activation (Varga et al. Mol Immunol., 39(14):839-46, 2003).

[0242] Environmental triggers of asthma can activate complement via the alternative pathway. For example, in vitro exposure of human serum to cigarette smoke or diesel exhaust particles resulted in complement activation, and the effect was unaffected by the presence of EDTA, suggesting that activation was via the alternative pathway rather than the classical pathway (Robbins, RA et al., Am. J. Physiol. 260:L254-L259, 1991; Kanemitsu, H., et al., Biol. Pharm. Bull. 21:129-132, 1998). The role of the complement pathway in allergic airway inflammation was evaluated in a mouse ovalbumin sensitization and challenge model. Wild-type mice developed AHR and airway inflammation in response to aeroallergen challenge. A Crry-Ig fusion protein that inhibits all pathways of complement activation was effective in preventing AHR and pulmonary inflammation when administered systemically or locally by inhalation in a murine ovalbumin model of allergic pulmonary inflammation (Taube et al., Am J Respir Crit Care Med., 168(11):1333-41, 2003).

[0243] Factor B-deficient mice exhibited reduced AHR and airway inflammation compared with wild-type mice, whereas C4-deficient mice exhibited similar effects to wild-type mice (Taube, C., et al., Proc. Natl. Acad. Sci. USA 103:8084-8089, 2006). These results support the role of the alternative pathway, rather than the classical pathway, in the mouse aeroallergen challenge model. Further evidence of the importance of the alternative pathway was provided in studies of factor H (FH) using the same mouse model (Takeda, K., et al., J. Immunol. 188:661-667, 2012). FH is a negative regulator of the alternative pathway and acts to prevent autologous damage to self-tissues. Endogenous FH was found to be present in the airways during allergen challenge, and inhibition of FH with a recombinant competitive antagonist increased the severity of AHR and airway inflammation (Takeda et al., 2012, supra). Therapeutic delivery of CR2-fH, a chimeric protein linking the iC3b / C3d-binding region of CR2 to the complement regulatory region of FH, which targets the complement regulatory activity of fH to sites of existing complement activation, prevented the development of AHR and eosinophil infiltration into the airways after allergen challenge (Takeda et al., 2012, supra). The protective effect was demonstrated using ovalbumin and ragweed allergen, a related allergen in humans.

[0244] The role of lectin-dependent complement activation in asthma was evaluated in a mouse model of fungal asthma (Hogaboam et al., J. Leukocyte Biol. 75:805-814, 2004). These studies used mice genetically deficient in mannan-binding lectin A (MBL-A), a carbohydrate-binding protein that functions as the recognition component for activation of the lectin complement pathway. MBL-A(+ / +) and MBL-A(- / -) Aspergillus fumigatus-sensitized mice were tested on days 4 and 28 after it challenge with A. fumigatus conidia. AHR in the sensitized MBL-A(- / -) mice was significantly attenuated at both time points after conidial challenge compared with the sensitized MBL-A(+ / +) group. On day 4 after inoculation, pulmonary TH2 cytokine levels (IL-4, IL-5, and IL-13) were significantly lower in A. fumigatus-sensitized MBL-A(- / -) mice compared with the wild-type group. These results indicate that MBL-A and the lectin pathway play an important role in the development and maintenance of AHR during chronic fungal asthma.

[0245] The above findings suggest the involvement of lectin-dependent complement activation in the pathogenesis of asthma. Experimental data suggest that factor B activation plays a pivotal role. Given the fundamental role of LEA-1 in lectin-dependent factor B activation and subsequent alternative pathway activation, LEA-1 blockers are expected to be beneficial in treating certain forms of asthma mediated by the alternative pathway. Therefore, such treatment may be particularly useful in dust mite-induced asthma or asthma caused by environmental triggers such as cigarette smoke or diesel exhaust. On the other hand, pollen-induced asthmatic responses are likely to result in LEA-2-dependent complement activation. Therefore, LEA-2 blockers are expected to be particularly useful in treating asthmatic conditions in this subset of patients.

[0246] In light of the data described above, the inventors believe that LEA-1 and LEA-2 mediate pathological complement activation in asthma. Depending on the triggering allergen, LEA-1 or LEA-2 may be preferentially involved. Thus, LEA-1 blockers in combination with LEA-2 blockers may have utility in treating multiple forms of asthma, regardless of the underlying etiology. LEA-1 and LEA-2 blockers may have complementary, additive, or synergistic effects in preventing, treating, or ameliorating pulmonary inflammation and asthma symptoms.

[0247] Combined LEA-1 and LEA-2 inhibition can be achieved by co-administration of an LEA-1 blocker and an LEA-2 blocker. Optimally, the LEA-1 inhibitory function and the LEA-2 inhibitory function can be encompassed in a single molecular entity, such as a bispecific antibody composed of MASP-1 / 3- and MASP-2-specific binding sites, or a bispecific antibody in which each binding site can bind to and block MASP-1 / 3 or MASP-2.

[0248] Thus, in accordance with the foregoing, aspects of the present invention provide methods for inhibiting LEA-1-dependent complement activation to treat, prevent, or reduce the severity of asthma, comprising administering to a subject suffering from or at risk of developing asthma a composition comprising a therapeutically effective amount of an LEA-1 inhibitor, including a MASP-1 inhibitor, a MASP-3 inhibitor, or a combination of MASP-1 / 3 inhibitors, in a pharmaceutical carrier. The MASP-1, MASP-3, or MASP-1 / 3 inhibitor composition may be administered to the subject systemically, for example, by intra-arterial, intravenous, intramuscular, inhalation, intranasal, subcutaneous, or other parenteral administration, or, in the case of non-peptidergic agents, potentially by oral administration. Administration may be repeated, as determined by a physician, until the condition resolves or is suppressed.

[0249] In one embodiment, the method of this aspect of the present invention further comprises inhibiting LEA-2-dependent complement activation to treat, prevent, or reduce the severity of asthma, and comprises administering therapeutically effective amounts of a MASP-2 inhibitor and a MASP-1, MASP-3, or MASP-1 / 3 inhibitor to a subject suffering from or at risk of developing asthma. As described above, the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 is expected to provide improved therapeutic outcomes in treating, preventing, or reducing the severity of asthma compared to inhibition of LEA-1 alone. This outcome can be achieved, for example, by coadministering an antibody with LEA-1 blocking activity and an antibody with LEA-2 blocking activity. In some embodiments, LEA-1 blocking activity and LEA-2 blocking activity are combined in a single molecular entity, which has combined LEA-1 and LEA-2 blocking activity. Such an entity may comprise or consist of a bispecific antibody in which one antigen-binding site specifically recognizes MASP-1 and blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody in which one antigen-binding site specifically recognizes MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Optimally, such an entity may consist of a bispecific monoclonal antibody in which one antigen-binding site specifically recognizes both MASP-1 and MASP-3 and thus blocks LEA-1, while the second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

[0250] The MASP-2 inhibitor may be administered to the subject systemically, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous or other parenteral administration, or, in the case of non-peptidergic agents, potentially by oral administration. Administration may be repeated as determined by a physician until the condition resolves or is suppressed.

[0251] The application of the MASP-3 inhibitory composition and any MASP-2 inhibitory composition of the present invention can be carried out by single administration or limited continuous administration of the composition (e.g., co-administration of a single composition or separate compositions comprising MASP-2 and MASP-3 inhibitors or bispecific or dual inhibitors) to treat, prevent, or reduce the severity of asthma in subjects suffering from or at risk of developing asthma. Alternatively, the composition may be administered over an extended period of time at regular intervals, such as once daily, twice weekly, weekly, biweekly, monthly, or bimonthly, for the treatment of a subject in need thereof.

[0252] X. The Role of MASP-2 and MASP-3 in Dense Deposit Disease and Therapeutic Use of MASP-2 and MASP-3 Inhibitors Membranoproliferative glomerulonephritis (MPGN) is a renal disorder characterized morphologically by mesangial cell proliferation and thickening of the glomerular capillary wall due to subendothelial extension of the mesangium. MPGN is classified as primary (also called idiopathic) or secondary to underlying conditions such as infection, systemic immune complex disease, neoplasms, or chronic liver disease. Idiopathic MPGN includes three morphological types: Type I, or classic MPGN, is characterized by subendothelial deposition of immune complexes and activation of the classical complement pathway; Type II, or dense deposit disease (DDD), is characterized by additional intramembranous dense deposits; and Type III, characterized by additional subepithelial deposits. Idiopathic MPGN is rare, accounting for only approximately 4–7% of primary renal causes of nephrotic syndrome (Alchi, B. and Jayne, D. Pediatr. Nephrol. 25:1409–1418, 2010). MPGN primarily affects children and young adults and can manifest as nephrotic syndrome, acute nephritic syndrome, asymptomatic proteinuria and hematuria, or recurrent gross hematuria. Renal failure occurs in the majority of patients, and the disease follows a slowly progressive course, with approximately 40% of patients developing end-stage renal disease within 10 years of diagnosis (Alchi and Jayne, supra, 2010). Current treatment options include corticosteroids, immunosuppressants, antiplatelet regimens, and plasma exchange.

[0253] DDD is diagnosed by immunofluorescence staining of renal biopsy specimens, revealing the absence of immunoglobulins and the presence of C3, and electron microscopy reveals characteristic high-density osmiophilic deposits along the glomerular basement membrane. DDD results from dysregulation of the alternative complement pathway, which can arise from many different mechanisms (Sethi et al., Clin J Am Soc Nephrol. 6(5):1009-17, 2011). The most common complement system abnormality in DDD is the presence of C3 nephritic factor, an autoantibody against the alternative pathway C3 convertase (C3bBb), which increases its half-life and thus pathway activation (Smith, RJH et al., Mol. Immunol. 48:1604-1610, 2011). Other alternative pathway abnormalities include factor H autoantibodies that block factor H function, increased functional C3 mutations, and genetic deficiencies of factor H (Smith et al., supra, 2011). Recent case reports have shown that eculizumab (anti-C5 monoclonal antibody) treatment was associated with improved renal function in two patients with DDD (Daina, E. et al., New Engl. J. Med. 366:1161-1163, 2012; Vivarelli, M. et al., New Engl. J. Med. 366:1163-1165, 2012), suggesting a causal role of complement activation in renal outcome.

[0254] Considering the above genetic, functional, immunohistochemical, and anecdotal clinical data, the central role of complement in the pathogenesis of DDD is well established. Therefore, treatments that block the disease-causing mechanism of complement activation or the subsequent complement activation products are expected to be therapeutically useful in treating this condition.

[0255] Although human genetic data suggest that inappropriate regulation or excessive activation of the alternative pathway amplification loop plays a key role, the complement initiation event has not been identified. Immunohistochemical studies of renal biopsies have shown evidence of MBL deposition in affected tissue, suggesting the involvement of the lectin pathway in the initiation of pathological complement activation in DDD (Lhotta et al., Nephrol Dial Transplant., 14(4):881-6, 1999). The importance of the alternative pathway has been further confirmed in experimental models. Factor H-deficient mice develop progressive proteinuria and renal pathology characteristic of the human condition (Pickering et al., Nat Genet., 31(4):424, 2002). Pickering et al. further demonstrated that loss of factor B, which mediates LEA-1-dependent activation of the alternative pathway, completely protects factor H-deficient mice from DDD (Pickering et al., Nat Genet., 31(4):424, 2002).

[0256] Therefore, agents that block LEA-1 are expected to effectively block lectin-dependent activation of the alternative pathway, thereby providing an effective treatment for DDD. Considering that the alternative pathway amplification loop is dysregulated in DDD patients, agents that block the amplification loop can further be expected to be effective. Because LEA-1-targeting agents that block MASP-1 or MASP-1 and MASP-3 inhibit the maturation of factor D, such agents are expected to effectively block the alternative pathway amplification loop.

[0257] As mentioned above, significant MBL deposition was found in affected kidney specimens, highlighting the possible involvement of lectin-driven activation events in DDD pathogenesis. Once initial tissue damage to the glomerular capillaries is established, further MBL binding to the damaged glomerular endothelium and underlying mesangial structures is likely to occur. It is well known that such tissue damage can lead to LEA-2 activation, which in turn can result in further complement activation. Therefore, LEA-2 blockers may also be useful in preventing further complement activation in damaged glomerular structures, thereby preventing further disease progression toward end-stage renal failure.

[0258] The above data suggest that LEA-1 and LEA-2 promote distinct pathological complement activation processes in DDD. Therefore, LEA-1 and LEA-2 blockers, either alone or in combination, are expected to be useful in treating DDD.

[0259] When used in combination, LEA-1 and LEA-2 blockers are expected to be more effective than either alone or to be useful in treating various stages of the disease. Thus, LEA-1 and LEA-2 blockers may have complementary, additive, or synergistic effects in preventing, treating, or reversing DDD-associated renal dysfunction.

[0260] Combined LEA-1 and LEA-2 inhibition can be achieved by simultaneous administration of an LEA-1 blocker and an LEA-2 blocker. Optimally, the LEA-1 and LEA-2 blockers with inhibitory function can be contained in a single molecular entity, such as a bispecific antibody composed of MASP-1 / 3 and MASP-2 specific binding sites, or a bispecific antibody in which each binding site can bind to and block MASP-1 / 3 or MASP-2.

[0261] Thus, in accordance with the foregoing, aspects of the present invention provide methods for inhibiting LEA-1-dependent complement activation to treat, prevent, or reduce the severity of dense deposit disease, comprising administering to a subject suffering from or at risk of developing dense deposit disease a composition comprising a therapeutically effective amount of an LEA-1 inhibitor, including a MASP-1 inhibitor, a MASP-3 inhibitor, or a combination of MASP-1 / 3 inhibitors, in a pharmaceutical carrier. The MASP-1, MASP-3, or MASP-1 / 3 inhibitor composition may be administered to the subject systemically, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or potentially orally in the case of non-peptidergic agents. Administration may be repeated as determined by a physician until the condition resolves or is suppressed.

[0262] In another aspect, a method is provided for inhibiting LEA-2-dependent complement activation to treat, prevent, or reduce the severity of dense deposit disease, comprising administering a therapeutically effective amount of a MASP-2 inhibitor to a subject suffering from or at risk of developing dense deposit disease.In another aspect, a method is provided for inhibiting both LEA-1-dependent complement activation and LEA-2-dependent complement activation to treat, prevent, or reduce the severity of dense deposit disease, comprising administering therapeutically effective amounts of a MASP-2 inhibitor and a MASP-1, MASP-3, or MASP-1 / 3 inhibitor to a subject suffering from or at risk of developing dense deposit disease.

[0263] In some embodiments, the method includes inhibiting both LEA-1-dependent complement activation and LEA-2-dependent complement activation. As described above, the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 is expected to provide improved therapeutic outcomes in treating, preventing, or reducing the severity of dense deposit disease compared to inhibiting LEA-1 alone. This outcome can be achieved, for example, by coadministering an antibody with LEA-1 blocking activity and an antibody with LEA-2 blocking activity. In some embodiments, LEA-1 blocking activity and LEA-2 blocking activity are combined in a single molecular entity, such an entity having combined LEA-1 and LEA-2 blocking activity. Such an entity may comprise or consist of a bispecific antibody in which one antigen-binding site specifically recognizes MASP-1 and blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody in which one antigen-binding site specifically recognizes MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Optimally, such an entity may consist of a bispecific monoclonal antibody in which one antigen-binding site specifically recognizes both MASP-1 and MASP-3 and thus blocks LEA-1, while a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

[0264] The LEA-1 and / or LEA-2 inhibitors can be administered to a subject systemically, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or potentially orally in the case of non-peptidergic agents. Administration can be repeated as determined by a physician until the condition resolves or is suppressed.

[0265] The application of the MASP-3 inhibitory composition and / or MASP-2 inhibitory composition of the present invention can be carried out by a single administration or a limited number of consecutive administrations of the composition (e.g., co-administration of a single composition or separate compositions containing a MASP-2 and / or MASP-3 inhibitor or bispecific or dual inhibitor) to treat, prevent, or reduce the severity of dense deposit disease in a subject in need thereof. Alternatively, the composition may be administered over an extended period of time at regular intervals, such as once daily, twice weekly, weekly, biweekly, monthly, or bimonthly, for treatment of a subject in need thereof.

[0266] XI. Role of MASP-2 and MASP-3 in Microimmune Necrotizing Crescentic Glomerulonephritis and Therapy Using MASP-2 and MASP-3 Inhibitors Microimmune necrotizing crescentic glomerulonephritis (NCGN) is a form of rapidly progressive glomerulonephritis in which the glomerular capillary walls show signs of inflammation but have minimal detectable immune complex deposits or antibodies against the glomerular basement membrane. The condition is accompanied by a rapid decline in renal function. Most NCGN patients are known to have antineutrophil cytoplasmic autoantibodies (ANCA) and therefore belong to a group of diseases called ANCA-associated vasculitis. Vasculitis is a vascular disorder characterized by inflammation and fibrinoid necrosis of the vessel wall. Systemic vasculitis is classified based on vessel size: large, medium, or small. Several forms of small-vessel vasculitis are associated with the presence of ANCA, namely Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, and renal-confined vasculitis (NCGN). They can also be a manifestation of underlying conditions such as systemic lupus erythematosus. Target antigens of ANCA include proteinase-3 (PR3) and myeloperoxidase (MPO). Oligoimmune NCGN is a rare disease, with a reported incidence of approximately 4 per million in Wessex, UK (Hedger, N. et al., Nephrol. Dial. Transplant. 15:1593-1599, 2000). In a Wessex study of 128 patients with oligoimmune NCGN, 73% were ANCA-positive, 59% required initial dialysis, and 36% required long-term dialysis. Treatment for oligoimmune NCGN includes corticosteroids and immunosuppressants such as cyclophosphamide and azathioprine. Additional treatment options for ANCA-associated vasculitis include rituximab and plasma exchange (Chen, M. and Kallenberg, CGM Nat. Rev. Rheumatol. 6:653-664, 2010).

[0267] NCGN is characterized by minimal complement deposition, and the alternative complement pathway has been implicated in its pathogenesis. Evaluation of renal biopsies from seven patients with MPO-ANCA-associated minimally immunized NCGN detected the presence of membrane attack complex, C3d, factor B, and factor P (not detected in biopsies from normal controls or patients with minimal change disease), whereas C4d and mannose-binding lectin were not detected, suggesting selective activation of the alternative pathway (Xing, GQ et al. J. Clin. Immunol. 29:282-291, 2009). Experimental NCGN can be induced by transferring anti-MPO IgG into wild-type mice or anti-MPO splenocytes into immune-deficient mice (Xiao, H. et al. J. Clin. Invest. 110:955-963, 2002). In this mouse model of NCGN, the role of specific complement activation pathways has been studied using knockout mice. After injection of anti-MPO IgG, C4 - / - Mice developed kidney disease comparable to wild-type mice, whereas C5 - / - and factor B - / - Mice did not develop kidney disease, indicating that the alternative pathway, but not the classical or lectin pathway, was involved in this model (Xiao, H. et al. Am. J. Pathol. 170:52-64, 2007). Furthermore, incubation of MPO-ANCA or PR3-ANCA IgG from patients with TNF-α-primed human neutrophils resulted in the release of factors that lead to complement activation in normal human serum, as detected by the production of C3a. This effect was not observed with IgG from healthy subjects, suggesting a potential pathogenic role for ANCA in neutrophil and complement activation (Xiao et al., supra, 2007).

[0268] Based on the role outlined above for the alternative pathway in this condition, blocking alternative pathway activation is expected to have utility in the treatment of ANCA-positive NCGN. Given the requirement for fB activation for pathogenesis, inhibitors of LEA-1 are expected to be particularly useful in treating this condition and preventing further decline in renal function in these patients.

[0269] Yet another subset of patients develops progressive renal vasculitis manifesting as crescent formation accompanied by a rapid decline in renal function in the absence of ANCA. This form of the condition is called ANCA-negative NCGN and accounts for approximately one-third of all patients with oligoimmune NCGN (Chen et al., JASN 18(2):599-605, 2007). These patients tend to be relatively young, and renal outcomes tend to be particularly severe (Chen et al., Nat Rev Nephrol., 5(6):313-8, 2009). A distinctive pathological feature of these patients is the deposition of MBL and C4d in renal lesions (Xing et al., J Clin Immunol. 30(1):144-56, 2010). The intensity of MBL and C4d staining in renal biopsies negatively correlated with renal function (Xing et al., supra, 2010). These findings suggest an important role for lectin-dependent complement activation in pathogenesis. The fact that C4d, but not factor B, is commonly found in diseased tissue specimens indicates the involvement of LEA-2.

[0270] Based on the role of lectin-dependent complement activation in ANCA-negative NCGN described above, it is predicted that blocking activation of the LEA-2 pathway will have utility in the treatment of ANCA-negative NCGN.

[0271] The data described above suggest that LEA-1 and LEA-2 mediate pathological complement activation in ANCA-positive and ANCA-negative NCGN, respectively. Therefore, LEA-1 blockers in combination with LEA-2 blockers are expected to be useful in treating all forms of oligoimmune NCGN, regardless of the underlying etiology. Thus, LEA-1 and LEA-2 blockers may have complementary, additive, or synergistic effects in preventing, treating, or reversing NCGN-associated renal dysfunction.

[0272] When LEA-1 and LEA-2 inhibitors are used in combination, they may achieve additional therapeutic benefit or provide effective treatment for a broader range of patient subsets than either inhibitor alone. Combined LEA-1 and LEA-2 inhibition can be achieved by coadministration of an LEA-1 blocker and an LEA-2 blocker. Optimally, LEA-1 and LEA-2 inhibitory functions can be encompassed in a single molecular entity, such as a bispecific antibody composed of MASP-1 / 3- and MASP-2-specific binding sites, or a bispecific antibody in which each binding site can bind to and block MASP-1 / 3 or MASP-2.

[0273] Thus, in accordance with the above, an aspect of the present invention provides a method of inhibiting LEA-1-dependent complement activation to treat, prevent, or reduce the severity of oligoimmune necrotizing crescentic glomerulonephritis, comprising administering a composition comprising a therapeutically effective amount of an LEA-1 inhibitor, including a MASP-1 inhibitor, a MASP-3 inhibitor, or a combination of MASP-1 / 3 inhibitors, in a pharmaceutical carrier to a subject suffering from or at risk of developing oligoimmune necrotizing crescentic glomerulonephritis. The MASP-1, MASP-3, or MASP-1 / 3 inhibitory composition may be administered to a subject systemically, for example, by intra-arterial, intravenous, intramuscular, inhalation, intranasal, subcutaneous, or other parenteral administration, or, in the case of non-peptidergic agents, potentially by oral administration. Administration may be repeated as determined by a physician until the condition resolves or is suppressed.

[0274] In another aspect, a method is provided for inhibiting LEA-2-dependent complement activation to treat, prevent, or reduce the severity of oligoimmune necrotizing crescentic glomerulonephritis, comprising administering a therapeutically effective amount of a MASP-2 inhibitor to a subject suffering from or at risk of developing oligoimmune necrotizing crescentic glomerulonephritis. In another aspect, a method is provided which comprises inhibiting both LEA-1-dependent complement activation and LEA-2-dependent complement activation to treat, prevent, or reduce the severity of oligoimmune necrotizing crescentic glomerulonephritis, and which comprises administering therapeutically effective amounts of a MASP-2 inhibitor and a MASP-1, MASP-3, or MASP-1 / 3 inhibitor to a subject in need thereof.

[0275] In some embodiments, the method includes inhibiting both LEA-1-dependent complement activation and LEA-2-dependent complement activation. As described above, the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 is expected to provide improved therapeutic outcomes in treating, preventing, or reducing the severity of oligoimmune necrotizing crescentic glomerulonephritis compared to inhibition of LEA-1 alone. This outcome can be achieved, for example, by coadministering an antibody with LEA-1 blocking activity and an antibody with LEA-2 blocking activity. In some embodiments, LEA-1 blocking activity and LEA-2 blocking activity are combined in a single molecular entity, which has combined LEA-1 and LEA-2 blocking activity. Such an entity may comprise or consist of a bispecific antibody in which one antigen-binding site specifically recognizes MASP-1 and blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody in which one antigen-binding site specifically recognizes MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Optimally, such an entity may consist of a bispecific monoclonal antibody in which one antigen-binding site specifically recognizes both MASP-1 and MASP-3 and thus blocks LEA-1, while the second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

[0276] The MASP-2 inhibitor may be administered to the subject systemically, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous or other parenteral administration, or, in the case of non-peptidergic agents, potentially by oral administration. Administration may be repeated as determined by a physician until the condition resolves or is suppressed.

[0277] The application of the MASP-3 inhibitory composition and / or MASP-2 inhibitory composition of the present invention can be carried out by a single administration or a limited number of consecutive administrations of the composition (e.g., co-administration of a single composition or separate compositions containing a MASP-2 and / or MASP-3 inhibitor or a bispecific or dual inhibitor) to treat, prevent, or reduce the severity of oligoimmune necrotizing crescentic glomerulonephritis. Alternatively, the composition may be administered over an extended period of time at regular intervals, such as once daily, twice weekly, weekly, biweekly, monthly, or bimonthly, for the treatment of a subject in need thereof.

[0278] XII. Role of MASP-2 and MASP-3 in Traumatic Brain Injury and Therapeutics Using MASP-2 and MASP-3 Inhibitors Traumatic brain injury (TBI) is a major global health problem resulting in at least 10 million deaths or hospitalizations each year (Langlois, JA et al., J. Head Trauma Rehabil. 21:375-378, 2006). In 2003, there were an estimated 1.6 million cases of TBI in the United States, including 1.2 million emergency department visits, 290,000 hospitalizations, and 51,000 deaths (Rutland-Brown, W. et al., J. Head Trauma Rehabil. 21:544-548, 2006). The majority of TBIs in the United States result from falls and motor vehicle accidents. TBI can result in long-term or lifelong physical, cognitive, behavioral, and emotional sequelae. More than 5 million Americans live with long-term or lifelong TBI-related disabilities (Langlois et al., supra, 2006).

[0279] TBI can involve penetration of the brain parenchyma ("penetrating" injury) or injury that does not penetrate the brain ("closed" injury). The injury profile and associated neurobehavioral sequelae can differ significantly between penetrating and closed TBI. While each injury is unique, certain brain regions are particularly vulnerable to trauma-induced damage, including the frontal and anterior basal white matter, the basal ganglia and diencephalon, the rostral brainstem, and the temporal lobe, including the hippocampus (McAllister, TW Dialogues Clin. Neurosci. 13:287-300, 2011). TBI can lead to alterations in several neurotransmitter systems, including the acute release of glutamate and other excitatory amino acids and chronic changes in the catecholaminergic and cholinergic systems, which can be accompanied by neurobehavioral disorders (McAllister, supra, 2011). Survivors of severe TBI often suffer from cognitive impairment, personality changes, and increased psychiatric disorders, particularly depression, anxiety, and post-traumatic stress disorder. Despite intense research, no clinically effective treatment for TBI has been found that can reduce mortality and morbidity and improve functional outcome.

[0280] Complement Factors and TBI Numerous studies have identifi...

Claims

1. A method for inhibiting MASP-3-dependent complement activation in a subject suffering from or at risk of developing a disease or disorder selected from the group consisting of age-related macular degeneration, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy, asthma, dense deposit disease, microimmune necrotizing crescentic glomerulonephritis, traumatic encephalopathy, aspiration pneumonia, endophthalmitis, neuromyelitis optica, and Behcet's disease, comprising administering to the subject a composition comprising an amount of a MASP-3 inhibitor effective to inhibit MASP-3-dependent complement activation.

2. The method of claim 1, wherein the MASP-3 inhibitor is a MASP-3 monoclonal antibody or a fragment thereof, which specifically binds to a portion of SEQ ID NO:

8.

3. The method of claim 1, further comprising administering to the subject a composition comprising a MASP-1 inhibitor.

4. The method of claim 3, wherein the MASP-1 inhibitor is a MASP-1 monoclonal antibody or a fragment thereof, which specifically binds to a portion of SEQ ID NO:

10.

5. The method of claim 1, further comprising administering to the subject a composition comprising a MASP-2 inhibitor.

6. The method of claim 5, wherein the MASP-2 inhibitor is a MASP-2 monoclonal antibody or a fragment thereof, which specifically binds to a portion of SEQ ID NO:

5.

7. The method of claim 1, further comprising administering to the subject a composition comprising a MASP-1 inhibitor and a MASP-2 inhibitor.

8. The method of claim 1, wherein the MASP-3 inhibitor inhibits alternative pathway-driven C3b deposition.

9. The method of claim 1, wherein the MASP-3 inhibitor inhibits factor D maturation.

10. The method of claim 3, wherein the MASP-1 inhibitor specifically binds to a portion of MASP-1 with an affinity that is at least 10 times greater than it binds to MASP-3 (SEQ ID NO:8).

11. The method of claim 3, wherein the MASP-1 inhibitor specifically binds to the serine protease domain of MASP-1 (aa449-694 of SEQ ID NO:10).

12. The method of claim 1, wherein the MASP-3 inhibitor also binds to a portion of MASP-1 (SEQ ID NO:10).

13. The method of claim 12, wherein the MASP-3 inhibitor is a dual MASP-1 / MASP-3 inhibitor that binds to a consensus region within the CUBI-CCP2 domain.

14. The method of claim 12, wherein the MASP-3 inhibitor is a dual MASP-1 / MASP-3 inhibitor that binds to a consensus region within the CCP2 domain.

15. The method described in claim 12, wherein the MASP-3 inhibitor is a bispecific monoclonal antibody that binds to the serine protease domain of MASP-1 (aa449-694 of SEQ ID NO:10) and also binds to the serine protease domain of MASP-3 (aa450-711 of SEQ ID NO:8).

16. The method of claim 1, wherein the MASP-3 inhibitor also binds to a portion of MASP-2 (SEQ ID NO:5).

17. The method of claim 16, wherein the MASP-3 inhibitor is a dual MASP-3 / MASP-2 inhibitor that binds to a conserved region within the serine protease domain of MASP-3 and MASP-2.

18. The method of claim 16, wherein the MASP-3 inhibitor is a bispecific monoclonal antibody that binds to the serine protease domain of MASP-3 (aa 450 to 711 of SEQ ID NO:8) and also binds to at least one of the serine protease domain of MASP-2 (aa 445 to 682 of SEQ ID NO:5) or the CCP-1-CCP2 domain of MASP-2 (aa 300 to 431 of SEQ ID NO:5).

19. The method of claim 1, wherein the MASP-3 inhibitor binds to a portion of MASP-3 (SEQ ID NO:8) and does not inhibit MASP-1 or MASP-2.

20. The method of claim 1, wherein the MASP-3 inhibitor specifically binds to a portion of MASP-3 with an affinity that is at least 10 times greater than it binds to MASP-1 (SEQ ID NO:10).

21. The method of claim 1, wherein the MASP-3 inhibitor specifically binds to the serine protease domain of MASP-3 (aa450-711 of SEQ ID NO:8).

22. The method of claim 1, wherein the MASP-3 inhibitor is a pan-inhibitor for MASP-1, MASP-2, and MASP-3 and binds to a conserved region in the CUB1-EGF-CUB2 domain.

23. The method of claim 1, wherein the MASP-3 inhibitor is a trispecific inhibitor of MASP-1, MASP-2, and MASP-3.

24. The method of claim 2, wherein the composition further comprises a MASP-2 antibody.

25. The method of claim 2, wherein the composition further comprises a MASP-1 antibody.

26. The method of claim 7, wherein the composition comprises an MASP-1 antibody, an MASP-2 antibody, and an MASP-3 antibody.

27. The method of claim 7, comprising simultaneous administration of a composition comprising at least one of a MASP-2 antibody, a MASP-1 antibody, or a MASP-3 antibody.

28. The method of claim 12, wherein the composition further comprises a MASP-2 antibody.

29. The method of claim 16, wherein the composition further comprises a MASP-1 antibody.

30. The method of claim 1, wherein the antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, and a humanized or human antibody.

31. The method of claim 1, wherein the composition is administered systemically.

32. 32. The method of claim 31, wherein the composition is administered subcutaneously, intramuscularly, intravenously, intraarterially, or as an inhalant.

33. The method of claim 1, wherein the subject has or is at risk of developing age-related macular degeneration.

34. 10. The method of claim 1, wherein the subject has or is at risk of developing arthritis.

35. 35. The method of claim 34, wherein the arthritis is selected from the group consisting of osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, and psoriatic arthritis.

36. 10. The method of claim 1, wherein the subject has or is at risk of developing disseminated intravascular coagulation.

37. 37. The method of claim 36, wherein the disseminated intravascular coagulation is secondary to sepsis, trauma, infection (bacterial, viral, fungal, parasitic), malignancy, transplant rejection, transfusion reaction, obstetric complications, vascular aneurysm, liver failure, heat stroke, burns, radiation exposure, shock, or a severe toxic reaction.

38. The method of claim 1, wherein the subject has or is at risk of developing thrombotic microangiopathy.

39. 39. The method of claim 38, wherein the thrombotic microangiopathy is selected from the group consisting of hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), and thrombotic thrombocytopenic purpura (TTP).

40. 10. The method of claim 1, wherein the subject has asthma or is at risk of developing asthma.

41. The method of claim 1, wherein the subject has dense deposit disease or is at risk of developing dense deposit disease.

42. 10. The method of claim 1, wherein the subject has or is at risk of developing oligoimmune necrotizing crescentic glomerulonephritis.

43. 10. The method of claim 1, wherein the subject has suffered from or is at risk of developing a traumatic brain injury.

44. 10. The method of claim 1, wherein the subject has or is at risk of developing aspiration pneumonia.

45. The method of claim 1, wherein the subject has or is at risk of developing endophthalmitis.

46. 10. The method of claim 1, wherein the subject has or is at risk of developing neuromyelitis optica.

47. 10. The method of claim 1, wherein the subject has Behcet's disease or is at risk of developing Behcet's disease.

48. A method for inhibiting MASP-2-dependent complement activation in a subject suffering from or at risk of developing a disease or disorder selected from the group consisting of dense deposit disease, microimmune necrotizing crescentic glomerulonephritis, traumatic encephalopathy, aspiration pneumonia, endophthalmitis, neuromyelitis optica, and Behcet's disease, the method comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation.

49. The method of claim 48, wherein the MASP-2 inhibitor is a MASP-2 antibody or a fragment thereof.

50. The method of claim 48, wherein the MASP-2 inhibitor is a MASP-2 monoclonal antibody or a fragment thereof, which specifically binds to a portion of SEQ ID NO:

5.

51. The method of claim 46, wherein the MASP-2 antibody is a chimeric antibody, a humanized antibody, or a human antibody.

52. A method for manufacturing a pharmaceutical for use in inhibiting the effects of MASP-3-dependent complement activation in a subject suffering from or at risk of developing a disease or disorder selected from the group consisting of age-related macular degeneration, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy, asthma, dense deposit disease, microimmune necrotizing crescentic glomerulonephritis, traumatic encephalopathy, aspiration pneumonia, endophthalmitis, neuromyelitis optica, and Behcet's disease.

53. The method of claim 52, further comprising the step of combining a therapeutically effective amount of a MASP-2 inhibitor with the pharmaceutical comprising a MASP-3 inhibitor.

54. A method for producing a pharmaceutical for use in inhibiting the effects of MASP-2-dependent complement activation in a subject suffering from or at risk of developing a disease or disorder selected from the group consisting of dense deposit disease, oligoimmune necrotizing crescentic glomerulonephritis, traumatic encephalopathy, aspiration pneumonia, endophthalmitis, neuromyelitis optica, and Behcet's disease, the method comprising the step of mixing a therapeutically effective amount of a MASP-2 inhibitor with a pharmaceutical carrier.

55. The method of claim 54, further comprising the step of combining a therapeutically effective amount of a MASP-3 inhibitor with the pharmaceutical comprising a MASP-2 inhibitor.

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