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

Abstract

Provided are methods and compositions for inhibiting MASP-3-dependent complement activation in a subject by administering to the subject suffering from a disease or disorder or at risk of developing the disease or disorder a composition comprising an amount of a MASP-3 inhibitor effective to inhibit MASP-3-dependent complement activation. [Solution] The patient is administered a MASP-2 inhibitor and a MASP-1 inhibitor, or a MASP-2 inhibitor and a MASP-3 inhibitor, or a MASP-3 inhibitor and a MASP-1 inhibitor, or a MASP-1 inhibitor, a MASP-2 inhibitor, and a MASP-3 inhibitor.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Patent Application No. 61 / 661,167, filed on 18 June 2012.

[0002] Description regarding sequence listings The sequence listing relating to this application is provided in text format instead of as a hard copy and is incorporated herein by reference. The name of the text file containing the sequence listing is MP_1_0176_PCT_SequenceListingFiled_20130614.txt and was submitted via EFS-Web together with the application herein. [Background technology]

[0003] background The complement system provides the initial mechanisms for initiating, amplifying, and organizing immune responses to microbial infections and other acute injuries in humans and other vertebrates (MK Liszewski and JP Atkinson, 1993, in Fundamental Immunology, Third Edition, WE, Paul (ed.), Raven Press, Ltd., New York). While complement activation provides a beneficial first line of defense against potential pathogens, complement activity that promotes a protective immune response can also be a potential threat to the host (KR, Kalli, et al., Springer Semin. Immunopathol. 15:417-431, 1994; BP Morgan, Eur. J. Clinical Investig. 24:219-228, 1994). For example, C3 and C5 protein degradation products recruit and activate neutrophils. Activated neutrophils are essential for host defense, but they can indiscriminately release destructive enzymes, potentially causing organ damage. Furthermore, complement activation can lead to the deposition of soluble complement components on the surface of nearby host cells and microbial targets, potentially resulting in the lysis of host cells.

[0004] The complement system has also been linked to the development of a great many acute and chronic disease conditions, including myocardial infarction, stroke, ARDS, reperfusion injury, septic shock, capillary leakage after burns, inflammation after cardiopulmonary bypass, graft rejection, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and Alzheimer's disease. In almost all of these conditions, complement is not the cause, but one of several factors involved in its development. Nevertheless, complement activation may be the primary pathological mechanism and is an effective point for clinical management in many of these disease conditions. The growing recognition of the importance of complement-mediated tissue injury in various disease conditions highlights the need for effective complement inhibitory drugs. To date, eculizumab (Solaris®), an antibody against C5, is the only complement-targeted drug approved for use in humans. However, C5 is one of several effector molecules located "downstream" of the complement system, and blockade of C5 does not inhibit complement system activation. Therefore, inhibitors of the complement activation initiation process are considered to have a significant advantage over "downstream" complement inhibitors.

[0005] It is now widely accepted that the complement system can be activated via three distinct pathways: the classical pathway, the lectin pathway, and the secondary pathway. The classical pathway is typically triggered by a complex consisting of host antibodies bound to an exogenous particle (i.e., an antigen), and therefore requires prior exposure to the antigen to produce a specific antibody response. Since the activation of the classical pathway depends on a previous adaptive immune response by the host, the classical pathway is part of the acquired immune system. In contrast, both the lectin pathway and the secondary pathway are unrelated to adaptive immunity and are part of the innate immune system.

[0006] When the complement system is activated, serine protease enzyme precursors are successively activated. The first step in classical pathway activation is the binding of the specific recognition molecule C1q to antigen-bound IgG and IgM molecules. C1q binds to the C1r and C1s serine protease proenzymes as a complex called C1. Once C1q binds to the immune complex, the Arg-Ile site of C1r is cleaved by autoproteolysis, followed by cleavage and activation of C1s mediated by C1r, thereby acquiring the ability to cleave C4 and C2. C4 is cleaved into two fragments called C4a and C4b, and similarly, C2 is cleaved into C2a and C2b. The C4b fragment can covalently bond to an adjacent hydroxyl or amino group and, through non-covalent interactions with the C2a fragment of activated C2, can produce C3 convertase (C4b2a). C3 convertase (C4b2a) activates C3 by proteolytic cleavage into C3a and C3b subcomponents, thereby generating C5 convertase (C4b2a3b). C5 convertase (C4b2a3b) cleaves C5, forming a membrane invasion complex (also known as "MAC" when C5b is combined with C6, C7, C8, and C9). The membrane invasion complex can disrupt the cell membrane and lead to cell lysis. Activated forms of C3 and C4 (C3b and C4b) are covalently deposited on the surface of foreign targets and recognized by complement receptors on multiple phagocytic cells.

[0007] Independently, the first step in complement system activation via the lectin pathway is also the binding of specific recognition molecules, followed by the activation of associated serine protease proenzymes. However, rather than the binding of immune complexes by C1q, the recognition molecules of the lectin pathway include a group of carbohydrate-binding proteins collectively called lectins (mannan-binding lectins (MBL), H-phycoline, M-phycoline, L-phycoline, 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, similar to C1q, MBL can activate the complement system in a C4-dependent manner when it binds to yeast mannan-coated erythrocytes (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-hydroxyl and 4-hydroxyl groups are oriented to the equatorial plane of the pyranose ring. Therefore, while D-mannose and N-acetyl-D-glucosamine are common ligands for MBL, carbohydrates that do not meet this stereochemical requirement have an undetectable affinity for MBL (Weis et al., Nature 360:127-134, (1992)). The interaction between MBL and monosaccharides is extremely weak, and the dissociation constant is typically in the millimolar range of a single order of magnitude. MBLs achieve tight and specific binding to glycan ligands through avidity, that is, by simultaneously interacting with multiple monosaccharide residues located close to each other (Lee et al., Archiv. Biochem. Biophys. 299:129-136, (1992)). MBLs generally recognize carbohydrate patterns that adorn microorganisms, such as bacteria, yeasts, parasites, and certain viruses. In contrast, MBLs do not recognize D-galactose and sialic acid, the second-to-last and last sugars that adorn "mature" complex carbohydrates typically found on mammalian plasma glycoproteins and cell surface glycoproteins. This binding specificity is thought to facilitate the recognition of "foreign" surfaces and help protect against "self-activation." However, MBLs bind with high affinity to high-mannose "precursor" glycan clusters on N-linked glycoproteins and glycolipids sequestered within the endoplasmic reticulum and Golgi apparatus of mammalian cells (Maynard et al., J. Biol. Chem. 257:3788-3794, (1982)).Furthermore, it has been shown that MBLs can bind polynucleotides, DNA, and RNA that can 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 a potential target for lectin pathway activation via MBL binding.

[0009] Ficolin has a lectin domain of a different type from MBL, called a fibrinogen-like domain. Ficolin is Ca ++ They bind to sugar residues independently. In humans, three types of phycolin (L-phycolin, M-phycolin, and H-phycolin) have been identified. Two serum phycolins, L-phycolin and H-phycolin, share specificity for N-acetyl-D-glucosamine. However, H-phycolin also binds to N-acetyl-D-galactosamine. The differing sugar specificities of L-phycolin, H-phycolin, CL-11, and MBL suggest that different lectins can complement each other and, through overlap, target different complex carbohydrates. This idea is supported by a recent report that, of the known lectins in the lectin pathway, only L-phycolin specifically binds to lipoteichoic acid, a cell wall complex carbohydrate found in all Gram-positive bacteria (Lynch et al., J. Immunol. 172:1198-1202, (2004)). In addition to acetylated sugar moieties, phycolin can also bind acetylated amino acids and polypeptides (Thomsen et al., Mol. Immunol. 48(4):369-81 (2011)). Collectin (i.e., MBL) and phycolin do not have significant similarities in their amino acid sequences. However, these two protein groups have similar domain configurations and, like C1q, assemble to form oligomeric structures that maximize the potential for multi-site binding.

[0010] Serum concentrations of MBL are highly variable in healthy populations, and this is 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-phycoline is present in serum at concentrations nearly identical to those of MBL. Therefore, the L-phycoline branch of the lectin pathway is potentially comparable in strength to the MBL branch. Both MBL and phycoline can also function as opsonins. For this reason, phagocytic cells can target surfaces decorated with MBL and surfaces decorated with phycoline (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 interaction between these proteins and phagocytic receptors (Kuhlman et al., J. Exp. Med. 169:1733, (1989); Matsushita et al., J. Biol. Chem. 271:2448-54, (1996)). The identity of the phagocytic receptors has not been proven.

[0011] Human MBL forms specific and high-affinity interactions with a unique C1r / C1s-like serine protease called MBL-associated serine protease (MASP) via its collagen-like domain. To date, three types of MASP have been described. Firstly, a single enzyme, "MASP," was identified and characterized as the enzyme responsible for the initiation of the complement cascade (i.e., cleavage of C2 and C4) (Matsushita et al., J Exp Med 176(6):1497-1502(1992):Ji et al., J. Immunol 150:571-578, (1993)). Subsequently, it was determined that MASP activity is actually a mixture of two proteases: MASP-1 and MASP-2 (Thiel et al., Nature 386:506-510, (1997)). However, it has been shown 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)). Therefore, MASP-2 is a protease responsible for activating C4 and C2 to produce C4b2a, a C3 converter. This is a significant difference from the classical C1 complex, where the coordinated action of two specific serine proteases (C1r and C1s) leads to complement system activation. In addition, 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] MASP has the same domain structure as C1r and C1s, the enzymatic 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 / osteogenesis imperfecta (CUB) domain, an epidermal growth factor-like domain, a second CUB domain, a tandem arrangement of complement regulatory protein domains, and a serine protease domain. Similar to C1 proteases, activation of MASP-2 occurs by cleavage of the Arg-Ile bond adjacent to the serine protease domain. This cleavage separates the enzyme into disulfide-bonded A and B chains, the latter consisting of the serine protease domain.

[0013] MBL can also bind to an alternative splicing form of MASP-2, known as the 19kDa 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 chromosome 3 and chromosome 1, respectively (Schwaeble et al., Immunobiology 205:455-466, (2002)).

[0014] Several pieces of evidence suggest that there are different MBL-MASP complexes, and that the majority of MASP in serum does not form a complex with MBL (Thiel. et al., J. Immunol. 165:878-887, (2000)). Both H-ficorin and L-ficorin bind to all MASP, similar to MBL, 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 pathway and the classical pathway form a common C3 convertase (C4b2a), and the two pathways merge at this stage.

[0015] The lectin pathway is widely considered to play a major role in host defense against infection in naive hosts. Strong evidence of MBL involvement in host defense has been obtained from analyses 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 typically appear in adolescence, when maternal antibody titers are reduced, but during an apparent period of vulnerability before the full repertoire of antibody responses has developed. This syndrome is often attributed to mutations in several sites within the MBL collagen region that interfere with the proper formation of MBL oligomers. However, since 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, it had not been previously found that the initiators of the second pathway perform the recognition functions that C1q and lectins perform in the other two steps. It is now widely accepted that the second pathway spontaneously undergoes low levels of turnover activation. This turnover activation can be readily amplified on foreign or other abnormal surfaces (bacteria, yeast, virus-infected cells, or damaged tissue) lacking the appropriate molecular elements to suppress spontaneous complement activation. Four plasma proteins are directly involved in the activation of the second pathway: C3, factor B, factor D, and propergine.

[0017] While there is broad evidence linking the classical and secondary complement pathways to the development of non-infectious human diseases, the role of the lectin pathway has only recently begun to be evaluated. Recent studies have provided evidence that lectin pathway activation 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 bind to MBL and show 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 an antiblocking antibody against rat MBL showed significantly less myocardial damage during coronary artery occlusion compared to control antibody-treated rats (Jordan et al., Circulation, 104:1413-1418, (2001)). The molecular mechanism of MBL binding to vascular endothelium after oxidative stress is unknown. Recent studies suggest that activation of the lectin pathway after oxidative stress is mediated by MBL binding to vascular endothelial cytokeratin, but may not be mediated by complex carbohydrates (Collard e al., Am. J. Pathol. 159:1045-1054, (2001)). Other studies link the development of ischemia / reperfusion injury to the classical and secondary pathways, leaving the role of the lectin pathway in this disease open to debate (Riedermann, NC et al., Am. J. Pathol. 162:363-367, 2003).

[0018] Recent studies have shown that MASP-1 and MASP-3 convert factor D, a secondary pathway activator enzyme, from its precursor form to its 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 secondary pathway activity in the plasma of MASP-1 / 3-deficient mice. Functional secondary pathway activity is necessary for the production of C3b from native C3 via proteolysis. Since secondary pathway C3 convertase (C3bBb) contains the essential subunit C3b, the question of the origin of the initial C3b via secondary pathway is a perplexing one that has fueled a vast amount of research.

[0019] C3 belongs to a family of proteins that contain a rare post-translational modification known as a thioester bond (along with C4 and α-2 macroglobulin). The thioester group consists of glutamine with a terminal carbonyl group that forms a thioester covalent bond with a cysteine ​​sulfhydryl group three amino acids away. This bond is unstable, and the electrophilic glutamyl-thioester can react with a nucleophilic moiety such as a hydroxyl or amino group, and thus form a covalent bond with other molecules. The thioester bond is fairly stable when sequestered within the hydrophobic pocket of intact C3. However, when C3 is cleaved into C3a and C3b by proteolysis, the highly reactive thioester bond is exposed on C3b, and after nucleophilic attack by an adjacent moiety containing a hydroxyl or amino group, C3b covalently bonds with a target. In addition to its detailed recorded role in the covalent bonding of C3b to complement targets, the C3 thioester is also thought to play a central role in the induction of the secondary pathway. According to the widely accepted "tick-over theory," the second pathway is initiated by the generation of the liquid-phase combaturase iC3Bb, formed from C3 (iC3; C3(H2O)) with a hydrolyzable thioester and factor B (Lachmann, PJ, et al., Springer Semin. Immunopathol. 7:143-162, (1984)). C3b-like C3(H2O) is produced from native C3 by the slow, spontaneous hydrolysis of the internal thioester in this protein (Pangburn, MK, et al., J. Exp. Med. 154:856-867, 1981). The activity of C3(H2O)Bb combaturase causes the C3b molecule to be deposited on the target surface, thereby initiating the second pathway.

[0020] Prior to the discovery described herein, little was known about the initiators of the second 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 feature common to these activators is the presence of carbohydrates; however, due to the complexity and diversity of carbohydrate structures, it is difficult to prove a recognized common molecular determinant. Second pathway activation is regulated by a delicate balance between inhibitory regulatory components of this pathway, such as factor H, factor I, DAF, and CR1, as well as propergine, the latter being widely recognized as the sole positive regulator of the second pathway (see Schwaeble WJ and Reid KB, Immunol Today 20(1):17-21(1999)).

[0021] In addition to the aforementioned seemingly disordered activation mechanism, the generated C3b, along with factor B, can participate in the formation of a further secondary C3 convertase (C3bBb), thus the secondary pathway can also provide a strong amplification loop for the lectin / classical C3 convertase (C4b2a). The secondary C3 convertase is stabilized by propergine binding. Propergine extends the half-life of the secondary C3 convertase by 6 to 10 times. Adding C3b to the secondary C3 convertase leads to the formation of the secondary C5 convertase.

[0022] It has been thought that all three pathways (i.e., the classical pathway, the lectin pathway, and the secondary pathway) merge at C5. C5 is cleaved to form multiple pro-inflammatory products. The merged pathway has been called the terminal complement pathway. C5a is the most potent anaphylatoxin, inducing changes in smooth muscle tone and vascular tone, as well as vascular permeability. C5a is also a potent chemotaxin and activator of neutrophils and monocytes. Cellular activation via C5a can significantly amplify the inflammatory response by inducing the release of multiple further inflammatory mediators, including cytokines, hydrolases, arachidonic acid metabolites, and reactive oxygen species. When C5 is cleaved, C5b-9, also known as membrane invasion complexes (MACs), is formed. There is now strong evidence that sublytic MAC deposition, insufficient to cause lysis, may play a significant role in inflammation in addition to its role as a soluble 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 counteract these side effects. [Overview of the project]

[0024] overview In one aspect, the present invention provides a method for inhibiting MASP-3-dependent complement activation in subjects 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 brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, or Behçet's disease. The method comprises administering a composition containing a MASP-3 inhibitor in an amount effective to inhibit MASP-3-dependent complement activation to the subject. In some embodiments, the method further comprises administering a composition containing a MASP-2 inhibitor to the subject.

[0025] In another aspect, the present invention provides a method for inhibiting MASP-2-dependent complement activation in subjects 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 brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, or Behçet's disease. The method comprises administering to a subject a composition containing an amount effective in inhibiting MASP-2-dependent complement activation of a MASP-2 inhibitor. 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, the fragment of 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 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 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 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 pharmaceutical for use in inhibiting the effects of MASP-3-dependent complement activation in a living subject requiring such inhibition, the method comprising the step of mixing a therapeutically effective amount of a MASP-3 inhibitor with a pharmaceutical carrier. In some embodiments, the method in this aspect of the present invention comprises the step of producing 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 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 brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, or Behçet's disease. In some embodiments, the method further comprises the step of mixing a therapeutically effective amount of a MASP-2 inhibitor with or in combination with a pharmaceutical containing a MASP-3 inhibitor.

[0032] In another aspect, the present invention provides a method for producing a pharmaceutical for use in inhibiting the effects of MASP-2-dependent complement activation in a living subject requiring such inhibition, 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 in this aspect of the present invention comprises the step of 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, microimmune necrotizing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, or Behçet's disease. In some embodiments, the method further comprises the step of mixing a therapeutically effective amount of a MASP-3 inhibitor with or with a pharmaceutical containing a MASP-2 inhibitor.

[0033] As described herein, various embodiments of MASP-3 inhibitors and / or various embodiments of MASP-2 inhibitors can 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] [Invention 1001] 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 brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, and Behçet's disease, comprising the step of administering to the subject a composition containing an amount effective in inhibiting MASP-3-dependent complement activation of a MASP-3 inhibitor. [Invention 1002] The method of the present invention 1001, wherein the MASP-3 inhibitor is a MASP-3 monoclonal antibody or a fragment thereof, and the fragment specifically binds to a portion of SEQ ID NO:8. [Invention 1003] The method of the present invention 1001 further comprises the step of administering a composition containing a MASP-1 inhibitor to the subject. [Invention 1004] The method of the present invention 1003, wherein the MASP-1 inhibitor is a MASP-1 monoclonal antibody or a fragment thereof, and the fragment specifically binds to a portion of SEQ ID NO:10. [Invention 1005] The method of the present invention 1001 further comprises the step of administering a composition containing a MASP-2 inhibitor to the subject. [Invention 1006] The method of the present invention 1005, wherein the MASP-2 inhibitor is a MASP-2 monoclonal antibody or a fragment thereof, and the fragment specifically binds to a portion of SEQ ID NO:5. [Invention 1007] The method of the present invention 1001 further comprises the step of administering a composition containing a MASP-1 inhibitor and a MASP-2 inhibitor to the subject. [Invention 1008] The method of the present invention 1001, wherein the MASP-3 inhibitor inhibits second pathway-driven C3b deposition. [Invention 1009] The method of the present invention 1001, wherein the MASP-3 inhibitor inhibits factor D maturation. [Invention 1010] The method of the present invention 1003, wherein the MASP-1 inhibitor specifically binds to a portion of MASP-1 with at least 10 times greater affinity than when it binds to MASP-3 (SEQ ID NO:8). [Invention 1011] The method of the present invention 1003, wherein the MASP-1 inhibitor specifically binds to the serine protease domain of MASP-1 (aa449~694 of SEQ ID NO:10). [Invention 1012] The method of the present invention 1001, wherein the MASP-3 inhibitor also binds to a portion of MASP-1 (SEQ ID NO:10). [Invention 1013] The method of the present invention 1012, wherein the MASP-3 inhibitor is a dual MASP-1 / MASP-3 inhibitor that binds to the consensus region within the CUBI-CCP2 domain. [Invention 1014] The method of the present invention 1012, wherein the MASP-3 inhibitor is a dual MASP-1 / MASP-3 inhibitor that binds to the consensus region within the CCP2 domain. [Invention 1015] The method of the present invention 1012, wherein the MASP-3 inhibitor is a bispecific monoclonal antibody, and the bispecific monoclonal antibody 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). [Invention 1016] The method of the present invention 1001, wherein the MASP-3 inhibitor also binds to a portion of MASP-2 (SEQ ID NO:5). [Invention 1017] The method of the present invention 1016, wherein the MASP-3 inhibitor is a dual MASP-3 / MASP-2 inhibitor that binds to a conserved region within the serine protease domains of MASP-3 and MASP-2. [Invention 1018] The method of the present invention 1016, wherein the MASP-3 inhibitor is a bispecific monoclonal antibody, and the bispecific monoclonal antibody binds to the serine protease domain of MASP-3 (aa450~711 of SEQ ID NO:8) and also binds to at least one of the serine protease domain of MASP-2 (aa445~682 of SEQ ID NO:5) or the CCP-1-CCP2 domain of MASP-2 (aa300~431 of SEQ ID NO:5). [Invention 1019] The method of the present invention 1001, 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. [Invention 1020] The method of the present invention 1001, wherein the MASP-3 inhibitor specifically binds to a portion of MASP-3 with at least 10 times greater affinity than when it binds to MASP-1 (SEQ ID NO:10). [Invention 1021] The method of the present invention 1001, wherein the MASP-3 inhibitor specifically binds to the serine protease domain of MASP-3 (aa450~711 of SEQ ID NO:8). [Invention 1022] The method of the present invention 1001, 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. [Invention 1023] The method of the present invention 1001, wherein the MASP-3 inhibitor is a trispecific inhibitor of MASP-1, MASP-2, and MASP-3. [Invention 1024] The method of the present invention 1002, wherein the composition further comprises a MASP-2 antibody. [Invention 1025] The method of the present invention 1002, wherein the composition further comprises a MASP-1 antibody. [Invention 1026] The method of the present invention 1007, wherein the composition comprises MASP-1 antibody, MASP-2 antibody, and MASP-3 antibody. [Invention 1027] The method of the present invention 1007, comprising the simultaneous administration of a composition comprising at least one of MASP-2 antibody, MASP-1 antibody, or MASP-3 antibody. [Invention 1028] The method of the present invention 1012, wherein the composition further comprises a MASP-2 antibody. [Invention 1029] The method of the present invention 1016, wherein the composition further comprises a MASP-1 antibody. [Invention 1030] The method of the present invention 1001, wherein the antibody or fragment thereof is selected from the group consisting of recombinant antibodies, antibodies having reduced effector function, chimeric antibodies, and humanized antibodies or human antibodies. [Invention 1031] The method of the present invention 1001, wherein the composition is administered systemically. [Invention 1032] The method of the present invention 1031, wherein the composition is administered subcutaneously, intramuscularly, intravenously, intraarterially, or as an inhalant. [Invention 1033] The method of the present invention 1001, wherein the subject is suffering from or at risk of developing age-related macular degeneration. [Invention 1034] The method of the present invention 1001, wherein the subject is suffering from arthritis or is at risk of developing arthritis. [Invention 1035] The method of the present invention 1034, wherein the arthritis is selected from the group consisting of osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, and psoriatic arthritis. [Invention 1036] The method of the present invention 1001, wherein the subject is suffering from or at risk of developing disseminated intravascular coagulation. [Invention 1037] The method of the present invention 1036, wherein the disseminated intravascular coagulation occurs secondary to sepsis, trauma, infection (bacteria, viruses, fungi, parasites), malignant tumor, transplant rejection, transfusion reaction, complications of childbirth, vascular aneurysm, liver failure, heatstroke, burns, radiation exposure, shock, or severe poisoning reaction. [Invention 1038] The method of the present invention 1001, wherein the subject is suffering from or at risk of developing thrombotic microangiopathy. [Invention 1039] The method of the present invention 1038, 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). [Invention 1040] The method of the present invention 1001, wherein the subject is suffering from asthma or is at risk of developing asthma. [Invention 1041] The method of the present invention 1001, wherein the subject is suffering from dense deposit disease or is at risk of developing dense deposit disease. [Invention 1042] The method of the present invention 1001, wherein the subject is suffering from or at risk of developing microimmune necrotizing crescent-forming glomerulonephritis. [Invention 1043] The method of the present invention 1001, wherein the subject is suffering from or at risk of developing traumatic brain injury. [Invention 1044] The method of the present invention 1001, wherein the subject is suffering from aspiration pneumonia or is at risk of developing aspiration pneumonia. [Invention 1045] The method of the present invention 1001, wherein the subject is suffering from endophthalmitis or is at risk of developing endophthalmitis. [Invention 1046] The method of the present invention 1001, wherein the subject is suffering from neuromyelitis optica or is at risk of developing neuromyelitis optica. [Invention 1047] The method of the present invention 1001, wherein the subject is suffering from Behcet's disease or is at risk of developing Behcet's disease. [Invention 1048] 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 brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, and Behçet's disease, the method comprising the step of administering to the subject a composition containing an amount of a MASP-2 inhibitor effective in inhibiting MASP-2-dependent complement activation. [Invention 1049] The method of the present invention 1048, wherein the MASP-2 inhibitor is a MASP-2 antibody or a fragment thereof. [Invention 1050] The method of the present invention 1048, wherein the MASP-2 inhibitor is a MASP-2 monoclonal antibody or a fragment thereof, and the fragment specifically binds to a portion of SEQ ID NO:5. [Invention 1051] The method of the present invention 1046, wherein the MASP-2 antibody is a chimeric antibody, a humanized antibody, or a human antibody. [Invention 1052] A method for manufacturing a pharmaceutical product for use in subjects 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 brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, and Behçet's disease. [Invention 1053] The method of the present invention 1052 further comprises the step of mixing a therapeutically effective amount of a MASP-2 inhibitor with the pharmaceutical product containing a MASP-3 inhibitor. [Invention 1054] A method for producing a pharmaceutical product for use in subjects 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 brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, and Behçet's disease, comprising the step of mixing a therapeutically effective amount of a MASP-2 inhibitor with a pharmaceutical carrier. [Invention 1055] The method of the present invention 1054, further comprising the step of mixing a therapeutically effective amount of a MASP-3 inhibitor with the pharmaceutical product containing a MASP-2 inhibitor. These and other aspects and embodiments of the inventions described herein will be made apparent by reference to the following detailed description and drawings. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications cited herein are incorporated herein by reference as together, each individually. [Brief explanation of the drawing]

[0036] Many of the aforementioned aspects and associated advantages of the present invention will be more readily understood by referring to the following detailed description in conjunction with the accompanying drawings. [Figure 1] This study provides a new understanding of lectins and the secondary pathway. [Figure 2]This is a schematic diagram showing the MASP-2 and MAp19 protein domains and their encoding exons, modified by Yongqing et al., BBA 1824:253 (2012) from a figure taken from Schwaeble et al., Immunobiol 205:455-466 (2002). [Figure 3] This is a schematic diagram showing the MASP-1, MASP-3, and MAp44 protein domains and their encoding exons, modified by Yongqing et al., BBA 1824:253 (2012) from a figure in Schwaeble et al., Immunobiol 205:455-466 (2002). [Figure 4] This shows the amino acid sequence alignment of the MASP-1, MASP-2, and MASP-3 proteins, and indicates the consensus region between them. [Figure 5] The amino acid sequence alignment of the MASP-1, MASP-2, and MASP-3 alpha chains is shown. [Figure 6] The amino acid sequence alignment of the MASP-1, MASP-2, and MASP-3 beta chains is shown. [Figure 7A] The pairwise alignment of the amino acid sequences of the MASP-1 and MASP-2 protease domains (beta chain) is shown. [Figure 7B] The pairwise alignment of the amino acid sequences of the MASP-1 and MASP-3 protease domains (beta chain) is shown. [Figure 7C] The pairwise alignment of the amino acid sequences of the MASP-2 and MASP-3 protease domains (beta chain) is shown. [Figure 8] As described in Example 1, this is a Kaplan-Meyer plot showing the survival rates of MASP-2 KO mice and WT mice after administration of N. meningitidis serogroup A Z2491 at an infectious dose of 2.6 × 10⁷ cfu, demonstrating that MASP-2 deficient mice are protected from meningococcal-induced death. [Figure 9] As described in Example 1, this is a Kaplan-Meier plot showing the survival rates of MASP-2 knockout (KO) and wild-type (WT) mice after administration of meningococcal serogroup B strain MC58 at an infectious dose of 6 × 10⁶ cfu, demonstrating that MASP-2 deficient mice are protected from meningococcal-induced death. [Figure 10] As described in Example 1, after intraperitoneal infection with 6 × 10⁶ cfu of Neisseria meningitidis serogroup B strain MC58, the log cfu / mL of Neisseria meningitidis serogroup B strain MC58 in 1 mL of blood recovered from MASP-2 KO mice and WT mice at various time points was graphed (n=3 at various time points for both mouse groups). This demonstrates that MASP-2 KO mice, when infected with the same amount of Neisseria meningitidis serogroup B strain MC58 as WT mice, exhibit enhanced bacteremia clearance compared to WT mice. [Figure 11] As described in Example 1, the mean disease scores of MASP-2 KO mice and WT mice 3, 6, 12, and 24 hours after infection with 6 × 10⁶ cfu of Neisseria meningitidis serogroup B strain MC58 are shown graphically, demonstrating that MASP-2-deficient mice showed significantly lower disease scores at 6, 12, and 24 hours post-infection compared to WT mice. [Figure 12] As described in Example 2, this is a Kaplan-Meier plot showing the survival rate of mice administered either inhibitory MASP-2 antibody (1 mg / kg) or control isotype antibody 3 hours after being administered an infectious dose of 4 × 10⁶ cfu of Neisseria meningitidis serogroup B strain MC58. This demonstrates that the MASP-2 antibody is effective in treating and improving the survival rate of subjects infected with Neisseria meningitidis. [Figure 13] As described in Example 3, the log cfu / mL of viable cells of meningococcal 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 meningococcal serogroup B strain MC58, is shown graphically. [Figure 14]As described in Example 3, the log cfu / mL of viable meningococcal serogroup B-MC58 cells recovered at various time points in human serum samples shown in Table 7 is graphed, demonstrating that complement-dependent death of meningococcal bacteria in human 20% (v / v) serum is MASP-3 and MBL-dependent. [Figure 15] As described in Example 3, the log cfu / mL of viable meningococcal serogroup B-MC58 cells recovered at various time points in mouse serum samples shown in Table 9 is graphed, demonstrating that MASP-2- / - knockout mouse serum (referred to as "MASP-2- / -") has higher levels of bactericidal activity against meningococcus than WT mouse serum, while MASP-1 / 3- / - mouse serum, in contrast, has no bactericidal activity whatsoever. [Figure 16] As described in Example 4, the dynamics of C3 activation in WT, C4- / -, MASP-1 / 3- / -, factor B- / -, and MASP-2- / - mouse serum under lectin pathway-specific conditions (1% plasma) are shown graphically. [Figure 17] As described in Example 4, the level of second-pathway-driven (AP-driven) C3b deposition on zymosan-coated microtiter plates under "conventional" second-pathway-specific (AP-specific) conditions (i.e., BBS / EGTA / Mg++ without Ca++) is graphically shown as a function of serum concentration in serum samples taken from MASP-3-deficient, C4-deficient, and MBL-deficient human subjects. [Figure 18] As described in Example 4, the level of AP-driven C3b deposition on zymosan-coated microtiter plates under "conventional" AP-specific conditions (i.e., BBS / EGTA / Mg++ without Ca++) is graphically shown as a function of time in 10% human serum samples taken from MASP-3-deficient, C4-deficient, and MBL-deficient human subjects. [Figure 19A]As described in Example 4, under "conventional" AP-specific conditions (i.e., BBS / EGTA / Mg++ without Ca++) or physiological conditions that allow the lectin pathway and secondary pathway (AP) to function (BBS / Mg++ / Ca++), the level of C3b deposition on mannan-coated microtiter plates is graphically shown as a function of serum concentration in serum samples taken from WT, MASP-2 deficient, and MASP-1 / 3 deficient mice. [Figure 19B] As described in Example 4, the level of C3b deposition on zymosan-coated microtiter plates is graphically shown as a function of serum concentration in serum samples taken from WT, MASP-2 deficient, and MASP-1 / 3 deficient mice, either under conventional AP-specific conditions (i.e., BBS / EGTA / Mg++ without Ca++) or physiological conditions that allow the lectin pathway and secondary pathway to function (BBS / Mg++ / Ca++). [Figure 19C] As described in Example 4, the level of C3b deposition on microtiter plates coated with Streptococcus pneumoniae (S. pneumoniae) D39 is graphically shown as a function of serum concentration in serum samples taken from WT, MASP-2 deficient, and MASP-1 / 3 deficient mice, either under conventional AP-specific conditions (i.e., BBS / EGTA / Mg++ without Ca++) or physiological conditions that allow the lectin pathway and secondary pathway to function (BBS / Mg++ / Ca++). [Figure 20A] As described in Example 4, the results of a C3b deposition assay in highly diluted serum performed on mannan-coated microtiter plates using conventional AP-specific conditions (i.e., BBS / EGTA / Mg++ without Ca++) or physiological conditions that allow the lectin pathway and secondary pathway to function (BBS / Mg++ / Ca++), with serum concentrations ranging from 0% to 1.25%, are shown graphically. [Figure 20B]As described in Example 4, the results of the C3b deposition assay performed on zymosan-coated microtiter plates using conventional AP-specific conditions (i.e., BBS / EGTA / Mg++ without Ca++) or physiological conditions that allow the lectin pathway and secondary pathway to function (BBS / Mg++ / Ca++), with serum concentrations ranging from 0% to 1.25%, are shown graphically. [Figure 20C] As described in Example 4, the results of the C3b deposition assay performed on microtiter plates coated with Streptococcus pneumoniae D39 are graphically shown, 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 pathway and secondary pathway to function (BBS / Mg++ / Ca++). [Figure 21] As described in Example 5, the level of hemolysis of mannan-coated mouse erythrocytes by human serum (measured by photometric analysis of hemoglobin release of lysed mouse erythrocytes (Crry / C3- / -) into the supernatant) across a range of serum dilutions in MASP-3- / -, thermoinactivated normal human serum (HI NHS), MBL- / -, NHS+MASP-2 monoclonal antibody, and NHS control, under physiological conditions (i.e., in the presence of Ca++), is shown graphically. [Figure 22] As described in Example 5, the level of hemolysis of mannan-coated mouse erythrocytes by human serum (measured by photometric method by hemoglobin release of lysed mouse erythrocytes (Crry / C3- / -) into the supernatant) over a range of serum concentrations in serum from MASP-3- / -, thermally inactivated (HI) NHS, MBL- / -, NHS+MASP-2 monoclonal antibody, and NHS control under physiological conditions (i.e., in the presence of Ca++) is shown graphically. [Figure 23]As described in Example 5, the level of hemolysis of uncoated mouse erythrocytes with human serum (measured by photometric analysis of hemoglobin release from WT mouse erythrocytes dissolved in 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++) is shown graphically. [Figure 24] As described in Example 5, the graph shows the hemolysis of uncoated mouse erythrocytes with human serum (hemoglobin release of lysed mouse erythrocytes (CD55 / 59- / -) into the supernatant measured by photometric method) over a certain range of serum concentrations in serum from thermoinactivated (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 hemolysis of mannan-coated rabbit erythrocytes with MASP-1 / 3- / - mouse serum and WT control mouse serum over a certain range of serum concentrations under physiological conditions (i.e., in the presence of Ca++) is shown graphically (hemoglobin release of lysed rabbit erythrocytes into the supernatant is measured by photometry). [Figure 26] As described in Example 7, the level of C3b deposition (OD 405 nm) on a zymosan-coated microtiter plate in a C3 deposition assay performed under AP-specific conditions is graphically shown as a function of serum concentration in serum samples from factor D- / -, MASP-2- / -, and WT mouse serum. [Figure 27] As described in Example 7, the level of C3b deposition (OD 405 nm) on a zymosan-coated microtiter plate in a C3 deposition assay performed under physiological conditions (in the presence of Ca++) is graphically shown as a function of serum concentration in serum samples from factor D- / -, MASP-2- / -, and WT mouse serum. [Figure 28]As described in Example 7, the level of C3b deposition (OD 405 nm) on a zymosan-coated microtiter plate in a C3b deposition assay performed under physiological conditions (in the presence of Ca++) is graphically shown as a function of serum incubation time (minutes) in mouse serum collected from mice with and without factor D- / -, factor B- / -, and with and without MASP-2 monoclonal antibody. [Figure 29] Figure 29A graphs lectin pathway-specific C4b deposition on zymosan-coated microtiter plates, measured ex vivo in undiluted serum samples taken 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 mouse MASP-2 MoAb, as described in Example 13. Figure 29B graphs the time course of lectin pathway recovery over 3 weeks after a single intraperitoneal administration of 0.6 mg / kg of mouse 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] As described in Example 15, the saturated binding curve of clone M3J5 (clone 5) for the MASP-3 antigen is shown graphically. [Figure 32] Figure 32A shows the amino acid sequence alignment of the VH region of M3J5, M3M1, D14, and 1E10 to the chicken DT40 VH sequence, as described in Example 15, where dots represent amino acid identity with the DT40 sequence and dashes indicate spaces introduced to maximize the alignment. Figure 32B shows the amino acid sequence alignment of the VL region of M3J5, M3M1, D14, and 1E10 to the chicken DT40 VL sequence, as described in Example 15, where dots represent amino acid identity with the DT40 sequence and dashes indicate spaces introduced to maximize the alignment. [Figure 33] As described in Example 15, this is a bar graph showing the inhibitory activity of mAb1E10 in the Wieslab Complement System Screen, MBL Pathway compared to positive serum and isotype control antibodies provided with the assay kit, demonstrating that mAb1E10 partially inhibits LEA-2-dependent activation (by inhibiting MASP-1-dependent activity of MASP-2), while the isotype control antibody does not inhibit it. [Figure 34] As described in Example 16, the levels of C3b deposition for 1% normal human serum + isotype control, SGMI-1Fc, or SGMI-2Fc are graphically shown over the concentration range of 0.15 to 1000 nM, 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, we present the results of flow cytometry analysis of C3b deposition on heat-sterilized Staphylococcus aureus, demonstrating that no C3b deposition was observed in normal human serum in the presence of EDTA, which is known to inactivate lectins and the secondary pathway (Panel 1), secondary pathway-driven C3b deposition was observed in normal human serum treated with Mg++ / EGTA (Panel 2), and secondary pathway-driven C3b deposition was not observed in factor B-depleted serum, factor D-depleted serum, and propagin (factor P)-depleted serum, respectively, as shown in Panels 3, 4, and 5. [Figure 35B]As described in Example 17, we present the results of flow cytometry analysis of C3b deposition in heat-sterilized Staphylococcus aureus. Similar to EDTA-treated normal serum (Panel 1), no AP-driven C3b deposition was observed in 3MC serum in the presence of Mg++ / EGTA (Panel 3). On the other hand, Panels 4 and 5 show that both active full-length rMASP-3 (Panel 4) and active rMASP-3 (CCP1-CCP2-SP) (Panel 5) restore AP-driven C3b deposition in 3MC serum to levels observed in normal human serum treated with Mg++ / EGTA (Panel 2), but demonstrate that neither inactive rMASP-3 (S679A) (Panel 6) nor wild-type rMASP-1 (Panel 7) can restore AP-driven C3b deposition in 3MC serum. [Figure 36] 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 are shown, 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 shown in lane 2 (positive control), and further demonstrating that 3MC serum shows very little factor B cleavage in the presence of Mg++ / EGTA, as shown in lane 3. However, as described in Example 17, as shown in lane 4, factor B cleavage is restored by adding full-length recombinant MASP-3 protein to 3MC serum and pre-incubating. [Figure 37] As described in Example 17, Coomassie staining of the protein gel in which factor B cleavage is analyzed is shown, demonstrating that factor B cleavage is optimal in the presence of C3, MASP-3, and pro-factor D (lane 1), and that, as shown in lanes 4 and 5, as long as C3 is present, either MASP-3 or pro-factor D can mediate factor B cleavage on its own. [Figure 38]As described in Example 17, the mean fluorescence intensity (MFI) of Staphylococcus aureus C3b staining obtained from mAbD14 (bound to MASP-3), mAb1A5 (negative control antibody), and isotype control antibody is graphed 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] As described in Example 18, Western blot analysis of pro-D factor substrate cleavage is shown, demonstrating that full-length wild-type recombinant MASP-3 (lane 2) and MASP-1 (lane 5) both completely or partially cleave pro-D factor to produce mature D factor, compared to pro-D factor alone (lane 1) or inactive full-length recombinant MASP-3 (S679A; lane 3) or MASP-1 (S646A; lane 4). [Figure 40] As described in Example 18, this Western blot shows the inhibitory activity of MASP-3 that binds to mAb D14 (lane 2) and M3M1 (lane 3) in MASP-3-dependent pro-D factor cleavage, compared to a control reaction containing only MASP-3 and pro-D factor (no mAb, lane 1) and a control reaction containing an mAb obtained from a DTLacO library that binds to MASP-1 but not to MASP-3 (lane 4). [Figure 41] As described in Example 19, the level of AP-driven C3b deposition on zymosan-coated microtiter plates is graphed 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 serum from patients 2 and 3 has residual AP activity at high serum concentrations (25%, 12.5%, and 6.25% serum concentrations), but with significantly higher AP50 (i.e., 8.2% and 12.3% of the serum required to achieve 50% of maximum C3 deposition). [Figure 42A]As described in Example 19, the level of AP-driven C3b deposition on zymosan-coated microtiter plates under "conventional" AP-specific conditions (i.e., BBS / EGTA / Mg++ without Ca++) is graphically shown as a function of time in 10% human serum samples taken from MASP-3-deficient, C4-deficient, and MBL-deficient human subjects. [Figure 42B] As described in Example 19, Western blots are shown in which human pro-factor D (25,040 Da) and / or mature factor D (24,405 Da) were detected by human factor D-specific antibodies using plasma collected from 3MC patient #2 (MASP-3 (- / -), MASP-1 (+ / +)) and 3MC patient #3 (MASP-3 (- / -), MASP-1 (- / -)) and serum from a normal donor (W). [Figure 42C] As described in Example 19, the results of the Weislab classical, lectin, and second pathway assays using plasma and normal human serum collected from 3MC patient #2 and 3MC patient #3 are shown graphically. [Figure 43] As described in Example 19, the hemolysis rate of mannan-coated rabbit erythrocytes (measured by photometric method of hemoglobin release of lysed rabbit erythrocytes into the supernatant) was graphed 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++, demonstrating that MASP-3 deficiency reduces the rate of complement-mediated lysis of mannan-coated erythrocytes compared to normal human serum. [Figure 44] As described in Example 19, the level of AP-driven C3b deposition on a zymosan-coated microtiter plate is graphed as a function of the concentration of recombinant full-length MASP-3 protein added to a serum sample taken from human 3MC patient 2 (MASP-3- / -), demonstrating that active recombinant MASP-3 protein reconstitutes AP-driven C3b deposition on the zymosan-coated plate in a concentration-dependent manner, compared to the negative control inactive recombinant MASP-3 (MASP-3A; S679A). [Figure 45] As described in Example 19, the hemolysis rate of mannan-coated rabbit erythrocytes (hemoglobin release of lysed rabbit erythrocytes into the supernatant measured by photometric method) over a certain 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), measured in the absence of Ca++, is shown graphically, demonstrating that the lysis rate of rabbit erythrocytes in 3MC serum containing rMASP-3 is significantly increased compared to the lysis rate in 3MC serum without recombinant MASP-3 (p=0.0006). [Figure 46] As described in Example 19, the erythrolysis rates of rabbits in 7% human serum from 3MC patients 2 and 3, containing activated recombinant MASP-3 at concentrations of 0 to 110 μg / ml (in BBS / Mg++ / EGTA), are shown graphically, demonstrating that the erythrolysis rates of rabbits increase in a concentration-dependent manner with the amount of recombinant MASP-3. [Figure 47] The levels of LEA-2-driven C3b deposition on mannan-coated ELISA plates are graphically shown as a function of the concentration of human serum diluted in BBS buffer for serum 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 the results showing baseline VEGF protein levels in the RPE-choroidal complex isolated from wild-type (WT)(+ / +) and MASP-2(- / -) mice, as described in Example 20. Figure 48B presents the results showing VEGF protein levels in the RPE-choroidal complex in WT(+ / +) and MASP-2(- / -) mice 3 days after laser-induced injury in a macular degeneration model, as described in Example 20. [Figure 49] As described in Example 20, we present results showing the mean choroidal neovascularization (CNV) volume 7 days after laser-induced injury in WT(+ / +) and MASP-2(- / -) mice. [Figure 50]As described in Example 21, 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 is shown in the graph. [Figure 51] Figure 51A presents results showing the reduction in infarct size in WT(+ / +) mice and MASP-2(- / -) mice after injury in the 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] As described in Example 23, the mean ischemic area (area-at-risk) (AAR) and infarct volume (INF) in WT(+ / +) and MASP-2(- / -) mice after left anterior descending coronary artery occlusion and reperfusion are shown graphically as a percentage of total myocardial volume. [Figure 52B] As described in Example 23, the graph shows the infarct volume (INF) plotted against the mean ischemic area (AAR) as a percentage of left ventricular myocardial volume in WT(+ / +) and MASP-2(- / -) mice after coronary artery occlusion and reperfusion. [Figure 52C] The graph shows the infarct volume (INF) in buffer-perfused hearts of WT(+ / +) and MASP-2(- / -) mice, prepared according to the Langendorf isolated perfused heart mouse model, in which total ischemia and reperfusion were performed in the absence of serum, as described in Example 23. [Figure 52D] As described in Example 23, the relationship between infarct volume (INF) and risk zone (RZ) in buffer-perfused hearts of WT(+ / +) and MASP-2(- / -) mice prepared according to the Langendorf isolated perfusion mouse cardiac model is shown graphically. [Figure 53]Figure 53A graphically shows the results of a C3b deposition assay on an immunocomplex-coated plate, demonstrating that MASP-2(- / -) mice retain the functional classical pathway, as described in Example 24. In the figure, the symbol "*" represents serum from WT (MASP-2(+ / +)), the symbol "●" represents serum from WT (C1q depleted), the symbol "□" represents serum from MASP-2(- / -), and the symbol "△" represents serum from MASP-2(- / -)(C1q depleted). Figure 53B graphically shows the results of a C3b deposition assay on a zymosan-coated plate, demonstrating that MASP-2(- / -) mice retain the functional secondary pathway, as described in Example 24. In the figure, the symbol "*" represents serum from WT (MASP-2(+ / +)), and the symbol "□" represents serum from MASP-2(- / -). [Figure 54] Figure 54A graphically shows myocardial ischemia / reperfusion injury (MIRI)-induced tissue loss after ligation and reperfusion of the left anterior descending coronary artery (LAD) in C4(- / -) mice (n=6) and corresponding WT littermates (n=7), showing ischemic area (AAR) and infarct size (INF), as described in Example 24. Figure 54B graphically shows infarct size (INF) as a function of ischemic area (AAR) in C4(- / -) and WT mice treated as described in Figure 42A, as described in Example 24, demonstrating that C4(- / -) mice are as sensitive to MIRI as WT controls (dashed line). [Figure 55A] The results of the C3b deposition assay using serum from WT mice, serum from C4(- / -) mice, and serum from C4(- / -) mice pre-incubated with mannan are shown in the graph, as described in Example 24. [Figure 55B] As described in Example 24, the results of the C3b deposition assay using serum from WT mice, serum from C4(- / -) mice, and serum from MASP-2(- / -) mice mixed with various concentrations of mouse MASP-2 mAb (mAbM11) are shown in the graph. [Figure 55C]As described in Example 24, the results of a C3b deposition assay using human serum from WT (C4-sufficient) subjects, human serum from C4-deficient subjects, and serum from C4-deficient subjects pre-incubated with mannan are shown in graph form. [Figure 55D] As described in Example 24, the results of a C3b deposition assay using human serum from WT (sufficient C4) subjects and human serum from C4-deficient subjects mixed with human MASP-2 mAb (mAbH3) are shown in the graph. [Figure 56] Figure 56A is a graph showing a comparative analysis of plasma C3 convertase activity from various complement-deficient mouse lines tested under either lectin activation pathway-specific assay conditions or classical activation pathway-specific assay conditions, as described in Example 24. Figure 56B is a graph showing the time-resolved dynamics of plasma C3 convertase activity from various complement-deficient mouse lines tested under lectin activation pathway-specific conditions, as described in Example 24. [Figure 57A] As described in Example 25, the degree of tissue damage in WT and MASP-2(- / -) mice after inducing transient ischemia / reperfusion injury (GIRI) in the gastrointestinal tract is graphically shown, demonstrating that MASP-2(- / -) mice have a significantly greater degree of protection compared to WT controls. [Figure 57B] As described in Example 25, 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) are shown in a graph to demonstrate the in vivo disappearance of lectin pathway functional activity. [Figure 57C]As described in Example 25, the effect of MASP-2 mAb treatment on the severity of GIRI is shown graphically, demonstrating that mice administered with mouse MASP-2 mAb (mAbM11) 24 hours prior to exposure to transient ischemia / reperfusion injury (GIRI) in the gastrointestinal tract showed significantly reduced tissue damage compared to mice administered with saline (*p<0.05 when compared to mice treated with either the MASP-2 inhibitory antibody mAbM11 or an unrelated isotype control antibody). [Figure 57D] As described in Example 25, this shows the tissue presentation of GIRI-mediated pathology in the small intestine of mice pretreated with a single intraperitoneal injection of saline, isotype control antibody, or recombinant mouse MASP-2 antibody (mAbM11) 12 hours prior to GIRI induction. [Figure 58] As described in Example 26, the cerebral infarct volume in WT (MASP-2(+ / +)) and MASP-2(- / -) mice after 30 minutes of ischemia and 24 hours of reperfusion is shown graphically. [Figure 59A] A series of photographs of stained brain sections from WT (MASP-2+ / +) mice after 30 minutes of ischemia and 24 hours of reperfusion are shown. As described in Example 26, panels 1–8 of Figure 52A show different brain section regions corresponding to bregmas 1–8, respectively, with respect to the auditory nerve exit (bregma 0). [Figure 59B] A series of photographs of stained brain sections from MASP-2(- / -) mice 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 regions corresponding to bregmas 1–8, respectively, with respect to the auditory nerve exit (bregma 0). [Figure 60] As described in Example 27, we present results showing the mean clinical arthritis scores over time in WT(+ / +) and MASP-2(- / -) mice after Col2 mAb-induced rheumatoid arthritis. [Figure 61] As described in Example 28, the results of the C3 deposition assay on serum samples collected from WT mice in the presence of dust mites or zymosan are shown in the graph. [Figure 62] Figures 62A and 62B show dose-response curves for inhibition of C4b deposition (Figure 62A) and inhibition of thrombin activation after administration of MASP-2 Fab2 antibody (H1) in normal rat serum, as described in Example 29. [Figure 63] Figures 63A and 63B show the measured platelet aggregation (expressed 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 was inhibited by the depleted substance cobra venom factor (CVF) and terminal pathway inhibitor (C5aR antagonist) (Figure 63A) in a localized Schwartzman reaction model of disseminated intravascular coagulation, as described in Example 30. [Figure 64] As described in Example 31, the results of a Western blot analysis showing activation of human C3 by thrombin substrates FXIa and FXa, indicated by the presence of the a' chain, are shown. [Figure 65] As described in Example 31, the results of the C3b deposition assay for serum samples taken from WT, MASP-2(- / -), F11(- / -), F11(- / -) / C4(- / -), and C4(- / -) mice are shown graphically, demonstrating that the functional lectin pathway is present even in the complete absence of C4 or F11, but mice with the F11-(- / -) / C4(- / -) compound deficiency lack the functional lectin pathway. [Figure 66] As described in Example 32, the time to the onset of microvascular occlusion after LPS injection in MASP-2- / - and WT mice is shown graphically, and the percentage of mice that showed thrombus formation measured over 60 minutes is shown. In WT mice, thrombus formation was detected after 15 minutes, and up to 80% of WT mice showed thrombus formation within 60 minutes. In contrast, none of the MASP-2- / - mice showed thrombus formation during the 60-minute period (log-rank: p=0.0005). [Figure 67]As described in Example 33, the time-course (hourly) survival rates of saline-treated control mice (n=5) and MASP-2 antibody-treated mice (n=5) in an STX / LPS-induced HUS model are shown graphically. All control mice died within 42 hours, while in contrast, 100% of the MASP-2 antibody-treated mice survived throughout the entire experimental period. [Figure 68] As described in Example 34, the percentage of mice that developed microvascular occlusion in the FITC / dextran UV model after treatment with isotype control or human MASP-2 antibody mAbH6 (10 mg / kg) administered 16 hours and 1 hour prior to FITC / dextran injection is shown graphically as a function of time after injury induction. [Figure 69] The graph shows the occlusion time in minutes for mice treated with human MASP-2 antibody (mAbH6) and isotype control antibodies. The data are reported as variance dots, along with the mean (horizontal bars) and standard error bars (vertical bars). As described in Example 34, the statistical test used for the analysis was an unpaired t-test. The symbol "*" indicates p=0.0129. [Figure 70] As described in Example 34, the time to occlusion in minutes is shown graphically for wild-type mice, MASP-2 knockout mice, and wild-type mice pre-treated with intraperitoneal administration of human MASP-2 antibody (mAbH6) 10 mg / kg 16 hours and again 1 hour before thrombosis in a FITC-dextran / photo-induced endothelial cell injury model of thrombosis using low light intensity (800-1500). [Modes for carrying out the invention]

[0037] Explanation of the sequence list 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 a full-length polypeptide (without signal peptide) SEQ ID NO:23 scFv daughter clone 17D20m_d3521N11 DNA encoding a 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 (12aa) SEQ ID NO:38 Peptide Linker #2 (10aa) SEQ ID NO:39 Nucleic acid encoding a polypeptide fusion containing human IL-2 signaling sequence, SGMI-1, linker #1, and human IgG1-Fc. SEQ ID NO:40 Mature polypeptide fusion containing SGMI-1, linker #1, and human IgG1-Fc (SGMI-1Fc) SEQ ID NO:41 Nucleic acid encoding a polypeptide fusion containing human IL-2 signal sequence, SGMI-2, linker #1, and human IgG1-Fc. SEQ ID NO:42 Mature polypeptide fusion (SGMI-2Fc) containing SGMI-2, linker #1, and human IgG1-Fc

[0038] Detailed explanation I. Definition Unless otherwise defined herein, all terms used herein have the same meaning as those understood by those skilled in the art. To clarify the terms used in the specification and claims to describe the present invention, the following definitions are provided.

[0039] As used herein, lectin pathway effector arm 1 ("LEA-1") refers to lectin-dependent activation of factor B and factor 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” is two-part: (i) Ca ++ (ii) Ca ++This can occur in the absence of LEA-1 and generally involves lectin-independent conversion of factors B and D, resulting in the conversion of C3bB to C3bBb and pro-factor D to factor D. 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 we do not wish to be constrained by any particular theory, it is thought that only when multiple C3b molecules are involved and bind in close proximity does the C3bBb C3 convertase change its substrate specificity and cleave C5 as a second-pathway C5 convertase called C3bBb(C3b)n.

[0042] The term "MASP-2-dependent complement activation," also referred to herein as LEA-2-mediated complement activation, is used herein to mean Ca ++ This involves MASP-2 lectin-dependent activation, which occurs in the presence of C3, leading to the formation of lectin pathway C3 convertase C4b2a, and subsequently, upon accumulation of the C3 cleavage product C3b, to the formation of C5 convertase C4b2a(C3b)n, which is known to cause opsonization and / or lysis.

[0043] The term “conventional understanding of the second pathway,” as used herein, also known as the “conventional second pathway,” refers to the pre-discovery second pathway described herein, which was traditionally thought to arise from the spontaneous proteolytic production of C3b from complement factor C3. This pathway is characterized by complement activation induced by, for example, zymosan from fungal and yeast cell walls, lipopolysaccharide (LPS) from Gram-negative outer membranes, rabbit erythrocytes and many pure polysaccharides, viruses, bacteria, animal tumor cells, parasites, and damaged cells. The activation of the “conventional second pathway,” also known as the “second pathway,” as used herein, is Mg ++ / EGTA buffer (i.e., Ca ++ (Measured in the absence of)

[0044] As used herein, the term "lectin pathway" refers to complement activation that occurs through specific binding of serum and non-serum carbohydrate-binding proteins including mannose-binding lectin (MBL), CL-11, and ficolin (H-ficolin, M-ficolin, or L-ficolin). As described herein, the inventors have found that the lectin pathway is driven by two effector arms, namely, 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 evaluated using a buffer containing Ca ++ containing buffer.

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

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

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

[0049] In another embodiment, a MASP-1 inhibitor useful in the method of the present invention reduces 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, a MASP-1 inhibitor reduces 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., resulting in only 10% or less of MASP-3 complement activation and only 10% or less of MASP-2 complement activation).

[0050] An example of a direct MASP-1 inhibitor is a MASP-1 specific inhibitor, such as a MASP-1 inhibitor that specifically binds to a portion of MASP-1 (SEQ ID NO:10) with a binding affinity at least 10 times greater than that for other components in the complement system. In many cases, given that MASP-1 can activate MASP-3 and MASP-2, inhibition of MASP-1 is expected to be effective in inhibiting MASP-3 and / or MASP-2. However, in some cases, inhibition of MASP-1, MASP-3, or MASP-2 may be preferable to 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 to be responsible for Staphylococcus aureus opsonization in the absence of MASP-1 (see Iwaki D. et al., J Immunol 187(7):3751-8 (2011)). Therefore, 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 therapy for PNH, thereby reducing potential susceptibility to Staphylococcus aureus.

[0051] As used herein, the term “MASP-2 inhibitor” refers to any active 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, and includes MASP-2 antibodies and their MASP-2 binding fragments, native and synthetic peptides, small molecules, expression inhibitors, and isolated native inhibitors, and also includes peptides that compete with MASP-2 for binding to other recognition molecules in the lectin pathway (e.g., MBL, CL-11, H-phycoline, M-phycoline, or L-phycoline). MASP-2 inhibitors useful in the methods of the present invention can reduce MASP-2-dependent complement activation by more than 10%, for example, more than 20%, more than 50%, or more than 90%. In one embodiment, a MASP-2 inhibitor reduces MASP-2-dependent complement activation by more than 90% (i.e., resulting in only 10% or less of MASP-2 complement activation). An example of a direct MASP-2 inhibitor is a MASP-2 specificity inhibitor, such as a MASP-2 inhibitor that specifically binds to a portion of MASP-2 (SEQ ID NO:5) with a binding affinity at least 10 times greater than that for other components in the complement system.

[0052] As used herein, the term “antibody” includes antibodies and antibody fragments derived from any antibody-producing mammal (e.g., primates including mice, rats, rabbits, and humans) or from hybridomas, phage selection, recombinant expression, or transgenic animals (or other methods for producing antibodies or antibody fragments) that specifically bind to the target polypeptide, e.g., MASP-1, MASP-2, or MASP-3 polypeptide or a portion thereof. The term “antibody” is not intended to be limited in terms of the antibody source or the way the antibody is produced (e.g., by hybridomas, phage selection, recombinant expression, transgenic animals, peptide synthesis, etc.). Exemplary antibodies include polyclonal antibodies, monoclonal antibodies, and recombinant antibodies; pan-specific antibodies, multispecific antibodies (e.g., bispecific antibodies, tripspecific antibodies); humanized antibodies: mouse antibodies; chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies; and anti-idiotype antibodies, which may also be any intact antibody or fragment thereof. As used herein, the term “antibody” encompasses not only intact polyclonal or monoclonal antibodies, but also their fragments (e.g., dAb, Fab, Fab', F(ab')2, Fv), single chains (ScFv), their synthetic variants, native variants, fusion proteins containing the antibody moiety and antigen-binding fragments of the required specificity, humanized antibodies, chimeric antibodies, and any other modified configurations of immunoglobulin molecules containing antigen-binding sites or fragments (epitope recognition sites) of the required specificity.

[0053] "Monoclonal antibody" refers to a homogeneous antibody population. Here, monoclonal antibodies consist of amino acids (natural and non-natural) involved in the selective binding of epitopes. Monoclonal antibodies are highly specific for the target antigen. The term "monoclonal antibody" includes not only intact monoclonal antibodies and full-length monoclonal antibodies, but also their fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain (ScFv), variants thereof, fusion proteins containing antigen-binding portions, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified constructs of immunoglobulin molecules containing antigen-binding fragments (epitope recognition sites) capable of binding to the required specificity and epitope. This term is not intended to be limited in terms of the antibody source or the way the antibody is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). This term includes the entire immunoglobulin and fragments as described above in the definition of "antibody".

[0054] As used herein, the term "antibody fragment" refers to a portion generally containing the antigen-binding region or variable region, derived from or related to a full-length antibody, e.g., an antibody to MASP-1, MASP-2, or MASP-3. Exemplary 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, the "single-chain Fv" or "scFv" antibody fragment contains the V H domain or V L domain of an antibody. These domains exist in a single polypeptide chain. Generally, the Fv polypeptide further contains a polypeptide linker between the V H domain and the V L domain, such that the scFv can form a structure desirable for antigen binding.

[0056] The "chimeric antibody" as used herein is a recombinant protein that contains a variable domain and complementarity-determining region derived from a non-human species (e.g., rodent) antibody, but the remainder of the antibody molecule is derived from a human antibody.

[0057] As used herein, “humanized antibody” is a chimeric antibody that contains a minimal sequence corresponding to a specific complementarity-determining region derived from a non-human immunoglobulin, transplanted into a human antibody framework. Humanized antibodies are typically recombinant proteins in which only the antibody complementarity-determining region is non-human (including phage display or antibodies produced from yeast).

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

[0059] As used herein, “membrane invasion complex” (“MAC”) refers to a complex of five terminal complement components (combinations of C5b with C6, C7, C8, and C9) (also known as C5b-9) that penetrate and disrupt membranes.

[0060] As used herein, “subjects” 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 its broadest sense, natural amino acids can be grouped based on the chemical properties of their side chains. "Hydrophobic" amino acids are Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys, or Pro. "Hydrophilic" amino acids are 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 are Ser, Thr, Asn, or Gln. "Acidic" amino acids are Glu or Asp. "Basic" amino acids are Lys, Arg, or His.

[0063] As used herein, the term “conservative amino acid substitution” is exemplified by substitutions between amino acids in 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 oligomers or polymers of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or their mimics. This term also covers oligonucleotides that are naturally occurring, consisting of nucleotides, sugars, and oligonucleobases with nucleoside-to-nucleoside (backbone) covalent bonds, as well as oligonucleotides with unnatural modifications.

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

[0066] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used synonymously and refer to any peptide-bonded amino acid chain, regardless of length or post-translational modifications. The MASP proteins described herein (MASP-1, MASP-2, or MASP-3) may contain wild-type proteins, or may be wild-type proteins, or may be variants having 50 or fewer (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 amino acid substitutions. Conservative substitutions typically include substitutions in the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine, and threonine; lysine, histidine, and arginine; as well as phenylalanine and tyrosine.

[0067] The human MASP-1 protein (denoted as SEQ ID NO: 10), human MASP-2 protein (denoted as SEQ ID NO: 5), and human MASP-3 protein (denoted as SEQ ID NO: 8) described herein also include peptide fragments of MASP proteins that are shorter than the full-length and / or pre-pro MASP proteins, including peptide fragments of MASP proteins containing terminal and internal deletion mutants. Deletion mutants may delete 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 a non-adjacent single amino acid. 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 described in SEQ ID NO:10.

[0068] In some embodiments, the human MASP-3 protein can 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 can 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 have a length of at least 6 (for example, 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, It can be 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 (for example, at least six consecutive amino acid residues of any one of SEQ ID NO: 5, 8 or 10). In some aspects, the antigenic peptide fragments of human MASP protein have a length of less than 500 (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, These are amino acid residues with fewer 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 (for example, fewer than 500 consecutive amino acid residues with SEQ ID NO: 5, 8, or 10).

[0071] In some embodiments, with respect to producing antibodies that bind to MASP-1, MASP-2, and / or MASP-3, the peptide fragment is antigenic and retains 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 induce an antigen response in mammals (see "Methods for Producing Antibodies" below).

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

[0073] In a typical embodiment, the human MASP-1 protein (SEQ ID NO:10) is encoded by the cDNA sequence denoted as SEQ ID NO:9, the human MASP-2 protein (SEQ ID NO:5) is encoded by the cDNA sequence denoted as SEQ ID NO:4, and the human MASP-3 protein (SEQ ID NO:8) is encoded by the cDNA sequence denoted as 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 allele mutations and alternative splicing are expected to occur. Allele mutations of the nucleotide sequences shown in SEQ ID NO:9, SEQ ID NO:4, and SEQ ID NO:7 are within the scope of the present invention, including silent mutations and mutations that result in amino acid sequence changes. Allele variants of the MASP-1, MASP-2, or MASP-3 sequences can be cloned by searching cDNA or genomic libraries from different individuals according to standard methods, or they can be identified by homology comparison searches (e.g., BLAST search) of databases containing such information.

[0074] II. The Lectin Pathway: A New Understanding i. Summary; the lectin pathway has been redefined. As described herein, the inventors have made the remarkable discovery that the complement lectin pathway has two effector arms for activating complement, both driven by lectin pathway activation complexes formed of carbohydrate recognition components (MBL, CL-11, and phycolin): (i) an effector arm formed by lectin pathway-related serine proteases MASP-1 and MASP-3, referred to herein as "lectin pathway effector arm 1" or "LEA-1"; and (ii) a MASP-2-driven activation effector arm referred herein as "lectin pathway effector arm 2" or "LEA-2". Both LEA-1 and LEA-2 can perform lysis and / or opsonization.

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

[0076] Figure 1 illustrates this new understanding of the complement activation pathway. As shown in Figure 1, LEA-1 is driven by lectin-binding MASP-3, which activates the enzyme precursor of factor D into its active form and / or cleaves C3b- or C3b(H2O)-binding factor B, converting the C3bB enzyme precursor complex to its enzymatically active form, C3bBb. The activated factor D produced by MASP-3 can also convert the C3bB or C3b(H2O) enzyme precursor complex to its enzymatically active form. MASP-1 can rapidly self-activate, while MASP-3 cannot. In many cases, MASP-1 is the activator of MASP-3.

[0077] In many cases, lectins (i.e., MBL, CL-11, or phycolin) can direct their activity toward the cell surface, but 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(H2O) 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 lectin components (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] Therefore, 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 directly or indirectly at one or more sites along the MASP-3-factor-D-factor-B axis. In doing so, they generate C3bBb, i.e., the C3 convertase of the second pathway, stimulating the production and deposition of C3b on the microbial surface. C3b deposition plays a crucial role in opsonization, labeling the microbial surface in preparation for destruction by host phagocytic cells 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 in Staphylococcus aureus exposed to human serum (Figure 35).

[0079] However, the contribution of LEA-1 and the lectin-independent function of MASP-3, MASP-1, or HTRA-1 are not limited to opsonization. As shown in Figure 1, these three components can also cause cell lysis and C3b production via indirect or direct factor B activation. These components are linked to the second pathway C5 converter, i.e., C3bBb(C3b) n It forms a complex that generates [the substance]. As further described herein, the need for MASP-3 and MBL (not MASP-2, and therefore not LEA-2 in this example) in the lysis of meningococcus demonstrates the role of LEA-1 in lysis. In summary, the opsonization results obtained from Staphylococcus aureus studies and the lysis results observed in meningococcus studies 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(H2O) and / or the conversion of pro-D factor to D factor. Thus, both processes can be the result of a lectin-independent role of MASP-3, MASP-1, or HTRA-1. Therefore, the model shown in Figure 1, developed by the inventors, supports the use of inhibitors primarily of MASP-3, as well as inhibitors of MASP-1 and / or HTRA-1, to block opsonization and / or lysis and to treat diseases resulting from dysregulation of these processes.

[0080] 1. Lectin pathway effector arm (LEA-1) The first effector arm of the lectin pathway, namely LEA-1, is formed by the lectin pathway-related serine proteases MASP-1 and MASP-3. As described herein, we have so far shown that in the absence of MASP-3 and in the presence of MASP-1, the second pathway is substantially unactivated on the planar structure. These results demonstrate that MASP-3 plays a previously undisclosed role in initiating the second pathway, which has been confirmed using MASP-3-deficient 3MC serum collected from patients with a rare 3MC autosomal recessive disorder who have mutations that cause dysfunction of 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 with the conventionally defined second pathway is expected to be MASP-3 dependent. In fact, MASP-3, and its LEA-1 activation, may be previously unclear initiators of the second pathway.

[0081] As further described in Examples 1-4 of this specification, the inventors observed higher activity of lectin-dependent secondary pathway activation in MASP-2-deficient serum, resulting in higher bactericidal activity (i.e., lytic activity) against Neisseria meningitidis. While we do not wish to be constrained by any particular theory, it is likely that in the absence of MASP-2, carbohydrate recognition complexes containing MASP-1 tightly bind to carbohydrate recognition complexes containing MASP-3, thereby activating MASP-3. In many cases, MASP-3 is not a self-activating enzyme and very often requires the activity of MASP-1 to be converted from its enzyme precursor form to its enzymatically active form; therefore, it is known that the activation of MASP-3 depends on MASP-1 activity. MASP-1 is a self-activating enzyme (like MASP-2), but MASP-3 is not self-activating and often requires the enzymatic activity of MASP-1 to be converted 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 ligated with either MASP-1 or MASP-3. Therefore, the absence of MASP-2 promotes MASP-1-mediated sex conversion from MASP-3 to its enzymatically active form. Once MASP-3 is activated, activated MASP-3 initiates activation of the second pathway, now called "LEA-1" activation, via MASP-3-mediated sex conversion from C3bB to C3bBb and / or pro-factor D to factor D. C3bBb, also called the second pathway C3 convertase, cleaves further C3 molecules, resulting in the deposition of opsonin C3b molecules. If several C3b fragments bind in close proximity to the C3bBb convertase complex, the result is the formation of the secondary C5 convertase C3bBb(C3b)n, which promotes MAC formation. In addition, C3b molecules deposited on the surface form new sites for factor B binding, which are then cleaved by factor D and / or MASP-3 to form further sites where the secondary C3-C5 convertase complex can be formed.This latter process is necessary for effective lysis and does not require lectins once initial C3b deposition has occurred. Recent publications (Iwaki D. et al., J Immunol 187(7):3751-8 (2011)) and data generated by the inventors (Figure 37) demonstrate that activated MASP-3 converts the secondary pathway C3 convertase enzyme precursor complex C3bB into its enzymatically active form. To date, the inventors have found that MASP-3-mediated cleavage of factor B represents a newly described subcomponent of LEA-1 that promotes the lectin-dependent formation of the secondary pathway C3 convertase C3bBb.

[0082] 2. Lectin pathway effector arm (LEA-2) The second effector arm of the lectin pathway, namely LEA-2, is formed by the lectin pathway-associated serine protease MASP-2. MASP-2 is activated when recognition components bind to their respective patterns and can also be activated by MASP-1, and subsequently cleaves complement component C4 into C4a and C4b. After the cleavage product C4b binds to plasma C2, C4b-bound C2 becomes a substrate for a second MASP-2-mediated cleavage step, which converts C4b-bound C2 into the enzymatically active complex C4bC2a and small C2b cleavage fragments. C4b2a is the lectin pathway's C3 conversion C3 convertase, which converts the abundant plasma component C3 into C3a and C3b. C3b binds to any adjacent surface via thioester bonds. If several C3b fragments bind in close proximity to the C3 convertase complex C4b2a, this convertase alters its specificity to convert C5 to C5b and C5a, forming the C5 convertase complex C4b2a(C3b)n. While this C5 convertase can initiate MAC formation, this process alone is considered insufficient to promote lysis. Rather, the initial C3b opsonin produced by LEA-2 forms a nucleus for the formation of new secondary C3 convertase and C5 convertase sites, which ultimately lead to abundant MAC formation and lysis. The latter event is mediated by LEA-2-mediated activation of factor D by factor B associated with C3b formation, and is therefore LEA-1 dependent thanks to the essential role of MASP-1 in factor D maturation. Furthermore, since C4-deficient mice are not protected from ischemia-reperfusion injury, while MASP-2-deficient mice are (Schwaeble et al., PNAS, 2011), there is a MASP-2-dependent C4 bypass activation pathway for activating C3 in the absence of C4, which plays an important role in the pathophysiology of ischemia-reperfusion injury. LEA-2 is also linked to coagulation pathways that include cleavage from prothrombin to thrombin (common pathway) and cleavage of factor XII (Hagemann factor) to its enzymatically active form XIIa. Factor XIIa, on the other hand, cleaves factor XI to XIa (endogenous pathway).Activation of the intrinsic pathways of the coagulation cascade leads to fibrin formation, which is crucial for thrombus formation.

[0083] Figure 1 illustrates a new understanding of the lectin pathway and secondary pathway based on the results provided herein. Figure 1 details the role of LEA-2 in both opsonization and lysis. MASP-2 is physiologically an initiator of “downstream” C3b deposition (and consequently opsonization) in several lectin-dependent situations (Figures 20A, 20B, 20C), but also plays a role in the lysis of serosensitive 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 against serosensitive pathogens such as Neisseria meningitidis is that, in the case of bacterial lysis, the lectin pathway recognition complex associated with MASP-1 and MASP-3 must bind to the bacterial surface in close proximity to each other, thereby enabling MASP-1 to cleave MASP-3. In contrast to MASP-1 and MASP-2, MASP-3 is not a self-activating enzyme, but in many cases 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-binding factor B on the pathogen surface, initiating a second activation cascade by forming enzymatically active second pathway C3 and C5 convertases C3bBb and C3bBb(C3b)n, respectively. Lectin pathway activation complexes containing MASP-2 play a role in the activation of MASP-3, and in the absence of MASP-2, or after MASP-2 depletion, all lectin pathway activation complexes are affixed with either MASP-1 or MASP-3. Therefore, in the absence of MASP-2, lectin pathway activation complexes containing MASP-1 and MASP-3 are positioned in close proximity to each other on the microbial surface, leading to the activation of more MASP-3, which in turn significantly increases the likelihood of faster MASP-3-mediated cleavage of C3b-binding factor B and the formation of second pathway C3 and C5 convertases C3bBb and C3bBb(C3b)n on the microbial surface. This consists of surface-bound C5b associated with C6, C5bC6 associated with C7, and C5bC6C7 and C5bC6C7C8 associated with C8, which leads to the activation of the terminal activation cascade C5b-C9, forming a membrane invasion complex that causes polymerization of C9. This C9 then penetrates the bacterial surface structure and forms pores in the bacterial wall, which leads to the osmotic death of complement-targeted bacteria.

[0085] The core of 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 secondary pathway convertase C3bBb through initial cleavage and activation of factor B on the activator surface, which then catalyzes C3b deposition and the formation of secondary pathway convertase C3bBb. The MASP-3-driven activation pathway plays an essential role in microbial opsonization and lysis, driving the secondary pathway on the bacterial surface to produce an optimal activation rate for the formation of membrane invasion complexes. (ii) LEA-2: A MASP-2-dependent activation pathway that induces the formation of the lectin pathway C3 convertase C4b2a, and when the C3 cleavage product C3b accumulates, it subsequently produces the C5 convertase C4b2a(C3b)n. 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 crucial role in bacterial opsonization, which leads to microorganisms being coated with covalent C3b and its cleavage products (i.e., iC3b and C3dg), and is targeted for uptake and death by phagocytic cells possessing C3 receptors, such as granulocytes, macrophages, monocytes, microglial cells, and the reticuloendothelial system. This is the most effective route for the clearance of bacteria and microorganisms resistant to complement lysis. These include the majority of Gram-positive bacteria.

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

[0088] While we do not wish to be constrained by any particular theory, it is thought that (i) LEA-1, (ii) LEA-2, and (iii) lectin-independent factor B and / or factor D activation each lead to opsonization and / or MAC formation, and consequently 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 be associated with mannan-binding lectins (MBLs) in human serum. Mannan-binding lectins are also referred to as "mannose-binding proteins" or "mannose-binding lectins" in recent literature. The MBL-MASP complex plays a crucial role in innate immunity thanks to the binding of MBLs to carbohydrate structures present on a variety of microorganisms. The interaction between MBL and specific arrays of carbohydrate structures leads to the activation of the MASP enzyme precursor, which in turn activates 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 enzyme precursor complex was thought to contain only one type of protease (MASP-1), but it is now clear that there are two other separate 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 a further 19kDa serum protein called "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 the alternative spliced ​​gene product of the structural gene MASP-2, lacking the four C-terminal domains of MASP-2, including the serine endopeptidase domain. The abundantly expressed cleaved mRNA transcript encoding MAp19 is produced by the alternative splicing / polyadenylation event of the MASP-2 gene. Through a similar mechanism, the MASP-1 / 3 genes produce three major gene products: the two serine proteases MASP-1 and MASP-3, as well as a 44kDa cleaved 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 initially 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., Collectins and Innate Immunity, (1996); Ji et al., J Immunol 150:571-578). (1993). The ability of MBL-associated endopeptidases in the MBL-MASP complex to act on complement components C4 and C2 in a manner clearly identical to that of the C1s enzyme in the classical complement pathway C1q-(C1r)2-(C1s)2 complex 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 between C1q and the Fc region of antibody IgG or IgM present in the immune complex. This leads to the self-activation of the C1r enzyme precursor, which in turn activates the C1s enzyme precursor, which then acts on complement components C4 and C2.

[0093] The stoichiometry of the MBL-MASP complex differs from that of the C1q-(C1r)2-(C1s)2 complex in that various MBL oligomers appear to be associated with varying proportions of MASP-1 / MAp19 or MASP-2 / MASP-3 (Dahl et al., Immunity 15:127-135 (2001)). The majority of MASP and MAp19 found in serum are not complexed with MBL (Thiel et al., J Immunol 165:878-887 (2000)) and may be partially associated with phycolin, a recently described group of lectins that have fibrinogen-like domains capable of binding to N-acetylglucosamine residues on microbial surfaces (Le et al., FEBS Lett 425:367 (1998); Sugimoto et al., J. Biol Chem 273:20721). (1998)). Of these, human L-phycoline, H-phycoline, and M-phycoline are associated with MASP and MAp19, and can activate the lectin pathway when they bind to specific carbohydrate structures recognized by phycoline (Matsushita et al., J Immunol 164:2281-2284 (2000); Matsushita et al., J Immunol 168:3502-3506 (2002)). In addition to phycoline and MBL, a 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, and therefore it is important to understand that MBL is not the sole recognizing component of the lectin activation pathway, and that MBL deficiency should not be mistaken for a lectin pathway deficiency. Perhaps the presence 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 by complement activation.

[0094] All lectin pathway-recognizing molecules are characterized by specific MASP-binding motifs within their collagen homologous stem domains (Wallis et al. J. Biol Chem 279:14065-14073 (2004)). The MASP-binding sites in MBL, CL-11, and phycolin are characterized by 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. Confirmation of the structure, sequence, chromosomal location, and splice mutations of each. Figure 2 is a schematic diagram showing the domain structures of the MASP-2 polypeptide (SEQ ID NO: 5) and MAp19 polypeptide (SEQ ID NO: 2) and their encoding exons. Figure 3 is a schematic diagram showing the domain structures of the MASP-1 polypeptide (SEQ ID NO: 10), MASP-3 polypeptide (SEQ ID NO: 8), and MAp44 polypeptide (SEQ ID NO: 11) and their encoding exons. As shown in Figures 2 and 3, the serine proteases MASP-1, MASP-2, and MASP-3 consist of six distinct domains arranged as seen in C1r and C1s; namely, (I) the N-terminal C1r / C1s / sea urchin VEGF / osteogenesis imperfecta (or CUBI) domain; (II) the epidermal growth factor (EGF)-like domain; (III) the second CUB domain (CUBII); (IV and V) the two complement regulatory protein (CCP1 and CCP2) domains; and (VI) the 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., The amino acid sequences derived from the cDNA of Immunity 15:127-135 (2001)) indicate that these proteases are serine peptidases possessing a characteristic triple structure of His, Asp, and Ser residues within their putative catalytic domains (Genbank accession numbers as of February 15, 2012: Human MASP-1: BAA04477.1; Mouse MASP-1: BAA03944; Rat MASP-1: AJ457084; Human MASP-3: AAK84071; Mouse MASP-3: AB049755, respectively, are incorporated herein by reference).

[0097] As further shown in Figures 2 and 3, when the enzyme precursor is converted to its active form, the heavy chain (alpha or A chain) and light chain (beta or B chain) are separated, producing a disulfide-bonded A chain and a smaller B chain corresponding to the serine protease domain. The 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 enzyme precursor MASP-1 (as with enzyme precursors C1r and C1s). Enzyme precursors MASP-2 and MASP-3 are thought to be activated in a similar manner to MASP-1. Each MASP protein forms a homodimer, and Ca ++ It is dependent on MBL and Ficolin, and is related to them individually.

[0098] 2. MASP-1 / 3 Human MASP-1 polypeptide (SEQ ID NO:10) and MASP-3 polypeptide (SEQ ID NO:8) originate from a single structural gene (Dahl et al., Immunity 15:127-135 (2001)), which is 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 common to both MASP-1 and MASP-3, and a beta chain (serine protease domain) unique to MASP-3. As shown in Figure 3, the human MASP-1 gene contains 18 exons. Human MASP-1 cDNA (SEQ ID NO:10) The exon (denoted as 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 contains 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 results in a protein called MBL-related protein 44 ("MAp44") (denoted as SEQ ID NO:11), which arises 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 the N-terminal C1r / C1s / sea urchin VEFG / osteogenesis imperfecta (CUBI) domain (aa25-137 in SEQ ID NO: 10), epidermal growth factor-like domains (aa139-181 in SEQ ID NO: 10), a second CUB domain (CUBII) (aa185-296 in SEQ ID NO: 10), a tandem complement regulatory protein domain (CCP1 aa301-363 and CCP2 aa367-432 in SEQ ID NO: 10), and serine protease domains (aa449-694 in 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, MASP-3 contains seven N-linked glycosylation sites. The domains of the human MASP-3 protein (see SEQ ID NO: 8) are shown in Figure 3 and include the N-terminal C1r / C1s / sea urchin VEGF / osteogenesis imperfecta (CUBI) domain (aa25-137 in SEQ ID NO: 8), epidermal growth factor-like domains (aa139-181 in SEQ ID NO: 8), a second CUB domain (CUBII) (aa185-296 in SEQ ID NO: 8), a tandem complement regulatory protein domain (CCP1 aa301-363 and CCP2 aa367-432 in SEQ ID NO: 8), and serine protease domains (aa450-711 in SEQ ID NO: 8).

[0101] The MASP-3 translation product consists of an alpha chain (heavy chain) containing the CUB-1-EGF-CUB-2-CCP-1-CCP-2 domain (alpha chain: aa1~448 of SEQ ID NO: 8), which is common to both MASP-1 and MASP-3, and a light chain (beta chain: aa449~728 of SEQ ID NO: 8) containing the 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 contains 12 exons, as shown in Figure 2. MASP-2 (SEQ ID NO: 5) and MAp19 (SEQ ID NO: 2) are encoded by the transcript of a single structural gene produced by alternative splicing / polyadenylation (Stover et al., Genes and Immunity 2:119-127 (2001)). The human MASP-2 cDNA (SEQ ID NO: 4) is encoded by exons 2, 3, 4, 6, 7, 8, 9, 10, 11, and 12. A 20kDa protein called MBL-related protein 19 (also known as "MAp19" or "sMAP"), encoded by (SEQ ID NO:1), arises from exons 2, 3, 4, and 5 (SEQ ID NO:2). MAp19 is a non-enzymatic protein containing the N-terminal CUB1-EGF region of MASP-2, which has 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, which is cleaved after secretion to produce 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 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 the N-terminal C1r / C1s / sea urchin VEGF / bone morphogenetic protein (CUBI) domain (aa24-136 in SEQ ID NO: 5), epidermal growth factor-like domains (aa138-180 in SEQ ID NO: 5), a second CUB domain (CUBII) (aa184-295 in SEQ ID NO: 5), a tandem complement regulatory protein domain (CCP1 aa300-359 and CCP2 aa364-431 in SEQ ID NO: 5), and serine protease domains (aa445-682 in 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 domain (alpha chain: aa1~443 of SEQ ID NO: 5) and a beta chain (light chain) containing the serine protease domain (beta chain: aa444~686). The CUB-1, EGF, and CUB-2 domains are required for dimerization, and the CUB-1, EGF, CUB-2, and CCP-1 domains contain binding sites for MBP. Each MASP-2 dimer binds to two MBL subunits, as described in Wallis et al., J. Biol Chem 279:14065-14073 (2004).

[0105] 4. Comparison of the amino acid sequences of MASP-1, MASP-2, and MASP-3 Figure 4 shows the 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), indicating the conserved catalytic triplicate residues (H, D, S) in the CUBI, EGF, CUBII, CCP1, CCP2 domains, and serine protease (SP) domain. The symbol "." indicates identical amino acid sequences.

[0106] Figure 5 shows the amino acid alignment of the alpha chain sequences containing CUBI-EGF-CUBII-CCP1-CCP2 in MASP-1 (alpha chain: aa1-447 of SEQ ID NO: 10), MASP-2 (alpha chain: aa1-443 of SEQ ID NO: 5), and MASP-3 (alpha chain: aa1-448 of SEQ ID NO: 8). As shown by the dotted boxes in Figure 5, there are numerous identical regions within the CUBI, EGF, and CUBII domains. The CCP1 and CCP2 domains are indicated by the dark boxes. The total identity percentage between the alpha chains of human MASP1 / 3 and human MASP-2 is shown in Table 1 below.

[0107] Figure 6 shows the amino acid alignment of the beta-chain sequences (including the serine protease domain) of MASP-1 (beta-chain: aa448-699 of SEQ ID NO: 10), MASP-2 (beta-chain: aa444-686 of SEQ ID NO: 5), and MASP-3 (beta-chain: aa449-728 of SEQ ID NO: 8). Figure 7A shows the pairwise amino acid sequence between the beta-chain sequence of MASP-1 (beta-chain: aa448-699 of SEQ ID NO: 10) and the beta-chain sequence of MASP-2 (beta-chain: aa444-686 of SEQ ID NO: 5). Figure 7B shows the pairwise amino acid sequence between the beta-chain sequence of MASP-1 (beta-chain: aa448~699 in SEQ ID NO: 10) and the beta-chain sequence of MASP-3 (beta-chain: aa449~728 in SEQ ID NO: 8). Figure 7C shows the pairwise amino acid sequence between the beta-chain sequence of MASP-2 (beta-chain: aa444~686 in SEQ ID NO: 5) and the beta-chain sequence of MASP-3 (beta-chain: aa449~728 in SEQ ID NO: 8). Identical regions in Figures 5-7 are shown as dotted boxes surrounding identical amino acids (indicated by the symbol ".").

[0108] The percentage of alpha-chain and beta-chain identity of human MASP-1, MASP-2, and MASP-3 proteins is shown in Table 1 below.

[0109] (Table 1) Identity percentage between human MASP proteins TIFF0007839502000001.tif38157

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

[0111] Regarding the beta chains (light chains), the overall identity percentage 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 identical regions, as shown in Figure 6. As further shown in Figures 7A to 7C, identical regions 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 their equivalent residues in MASP-1. However, MASP-1 possesses two cysteine ​​residues not found in MASP-2, MASP-3, C1r, and C1s (positions 465 and 481 in the light chain). These two cysteine ​​residues in MASP-1 are in predicted positions used to form a "histidine loop" disulfide bridge, similar to those found in trypsin and chymotrypsin. This suggests that MASP-2, MASP-3, C1r, and C1s may have evolved from MASP-1 through gene replication and diversification (Nonaka & Miyazawa, Genome Biology 3 Reviews 1001.1-1001.5 (2001)).

[0113] 5. Relevant human gene data, including their respective biological functions / activities. The role of the MBL / ficolin-MASP complex in innate immunity is mediated by calcium-dependent binding of the type C lectin domain (present in the MBL molecule) or by the binding of the fibrinogen-like domain (present in the ficolin molecule) to carbohydrate structures found in yeast, bacteria, viruses, and fungi. This recognition step leads to the activation of the enzyme precursor MASP-2, which then mimics the action of activated C1 in the C1q-(C1r)2-(C1s)2 complex by cleaving C4 and C2 to form the C3 convertase C4b2b. This enables the deposition of C4b and C3b on target pathogens, thereby promoting phagocytic death and clearance.

[0114] Evidence in recent 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 is considered sufficient to effectively 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 MASP-2 gene targeting deficiency lacks any lectin pathway functional activity (Schwaeble et al. al., PNAS 108:7523-7528 (2011). More recently, a genetically determined MASP-2 deficiency 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, making it impossible for MASP-2 to bind to MBL.

[0115] In addition, functional characterization of serum from mice lacking both MASP-1 and MASP-3 was performed using the serum of wild-type mice and MASP-1 / MASP-3 knockout mice (MASP-1 / 3 - / -When compared with mouse serum under physiological conditions, 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 classical pathway effector endopeptidases C1s, MASP-2 activation does not involve or require the activity of any other MBL-related serine endopeptidases (i.e., MASP-1 or MASP-3), and that the proteolytic activity of MASP-2 is sufficient to translate the binding of carbohydrate recognition molecules in the lectin pathway (i.e., MBL, phycolin, or CL-11) to complement activation. However, more recent studies have demonstrated that while MASP-2 has the ability to self-activate, the catalytic rate of MASP-1 activation of the MASP-2 enzyme precursor is approximately 85,000 times faster than the rate of MASP-2 cleavage in its own enzyme precursor form (Heja et al., PNAS 106:10498-503 (2011); Megyeri et al., J. Biol. Chem. 288(13):8922-34 (2013)). Therefore, it is highly probable that MASP-1 is the primary activator of MASP-2 in physiological situations. Judging from the size of the C4 fragments produced and the functional C3 convertase activity generated, activated MASP-2 is likely to cleave C4 and C2 in the same manner as activated Cls, namely at a single arginyl bond in the alpha chain of C4 (Arg76 A1a77) and a single arginyl bond in the enzyme precursor chain of C2 (Arg223 Lys224). Furthermore, it has been reported that mouse MASP (in the form of the mouse MBL-MASP complex called Ra-reactive factor), unlike C1s, can cleave the alpha chain of complement component C3 to produce 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 the cleavage of a single arginyl bond (Arg77 Ser78) within the alpha chain of C3. Activated MASP-2, like activated C1s, cannot cleave complement component C5. The proteolytic activity of MASP-1 and MASP-2 is inhibited by C1 inhibitors (Matsushita et al., J Immunol 165:2637-2642 (2000)), while C1 inhibitors do 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 a subject of debate since its discovery. In recent years, numerous potential substrates have been identified. MASP-1 can slowly cleave native C3, and it has been suggested that this direct C3 cleavage may initiate the complement cascade, possibly with the contribution of a secondary pathway (Matsushita et al., J Immunol 165:2637-2642 (2000)). Later, it was shown that recombinant MASP-1 cleaves an inactive (thioester hydrolysis) form of C3, which is not productive in initiating the complement cascade (Ambrus et al., J Immunol 170:1374-1382 (2003)). The absence 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 enzyme precursor form of factor D (Takahashi et al., J Exp Med 207:29-37 (2010)). Therefore, regarding MASP-1's ability to cleave C2, it is thought that MASP-1 can enhance MASP-2's ability to form C3 convertase (C4b2a) by cleaving C2. This suggestion is supported by observations of reduced lectin pathway activity in MASP-1 depleted human serum and MASP-1 / 3 deficient mouse serum (Takahashi et al., J Immunol 180:6132-6138 (2008)), which also suggests that MASP-1 plays a role in MASP-2 activation.Furthermore, while all C4b deposited by the MBL-MASP complex can form C4b2a convertase, only one out of four C4b deposited by the classical C1 complex can form it (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 while MASP-2 can self-activate, MASP-1 is the primary activator of the enzyme precursor MASP-2. MASP-2 activation was 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 specific inhibitors 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 extremely important for MASP-2 activation and subsequent C4 cleavage in human serum. The catalytic rate of conversion of the enzyme precursor 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] Furthermore, recent discoveries have linked MASP-1 to the second pathway. MASP-1 can convert the enzyme precursor factor D into its enzymatically active form (Figure 39; Takahashi et al., J Exp Med 207:29-37 (2010)). In addition, MASP-1 activates the enzyme precursor form of MASP-3 (Megyeri et al., J. Biol. Chem. 288:8922-8934 (2013); Degn et al. J. Immunol. 189(8):3957-69 (2012)), and can itself activate the enzyme precursor factor D (Figure 39), and can also cleave factor B, another essential component of the second pathway, into its active form (Iwaki et al., J. Immunol. 187:3751-58 (2011)). However, the conversion of pro-factor D and pro-factor B is likely independent of the activation state of LEA-2 and may occur via non-complex-bound MASP-1.

[0119] Several lines of evidence suggest that MASP-1 is a thrombin-like enzyme and is important in the activation of the coagulation pathway. MASP-1 can cleave several substrates of thrombin, 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-activating 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 inhibitors (Dobo et al., J Immunol 183:1207-1214 (2009)). The connection between complement and the coagulation pathway is also highlighted by the observation that MASP-2 can activate prothrombin (Krarup A. et al., PLoS One 2(7):e623 (2007)). Limited coagulation constitutes an ancient type of innate immunity when the transmission of invading pathogens is prevented by a fibrin clot. The released fibrinopeptide B has pro-inflammatory activity. MASP-1-mediated cleavage of PAR4 activates an endothelial cell-initiated inflammatory response (Megyeri et al., J Immunol 183(5):3409-16 (2009)).

[0120] MASP-3 does not possess 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 is not a self-activating enzyme, in contrast to MASP-1 and MASP-2 (Zundel S. et al., J Immunol 172:4342-4350 (2004); Megyeri et al., J. Biol. Chem. 288:8922-8934 (2013)).

[0121] Recent transgenic mouse studies using mouse strains with both MASP-1 and MASP-3 deficiencies have provided evidence of possible physiological functions of MASP-1 and MASP-3. MASP-1 / 3 knockout mice possess a functional lectin pathway (Schwaeble et al., PNAS 108:7523-7528 (2011)), but they appear to lack secondary pathway activity (Takahashi et al., JEM 207(1):29-37 (2010)). This lack of secondary pathway activity is thought to be due to a processing defect of complement factor D, which is required for secondary pathway activity. In MASP-1 / 3 knockout mice, all factor D circulates as a proteolytically inactive precursor, whereas in the serum of normal mice, virtually all factor D is in an active form. Biochemical analysis suggests that MASP-1 can convert complement factor D from its enzyme precursor 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 enzyme precursor in vitro, producing active factor D (Figure 39; Takahashi et al., JEM 207(1):29-37 (2010)). Factor D exists as an active enzyme circulating in normal individuals, and MASP-1, MASP-3, and HTRA-1 can be responsible for this activation. Furthermore, mice with both MBL deficiency and phycolin deficiency still produce normal levels of factor D and possess a fully functional secondary pathway. Therefore, these physiological functions of MASP-1 and MASP-3 are not necessarily associated with lectins and are thus independent of the lectin pathway. Recombinant mouse and human MASP-3 also cleave factor B in vitro and are thought to support C3 deposition in 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 of MASP-3. These patients exhibit severe developmental abnormalities, including cleft palate, cleft lip, cranial malformations, and intellectual disability. Genetic analysis identified 3MC patients who were homozygous for the 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 mutations in the MASP-1 gene that result in the absence of functional MASP-1 and MASP-3 proteins. Yet another group of 3MC patients were deficient in the functional CL-11 gene (Rooryck et al., Nat. Genet. 43(3):197-203 (2011)). Therefore, the CL-11 MASP-3 axis is thought to play a role during embryonic development. The molecular mechanism of this developmental pathway is unclear. However, individuals with a deficiency in the common complement component C3 do not develop this syndrome, making it unlikely to be mediated by conventional complement-driven processes. Thus, prior to our findings described herein, the functional role of MASP-3 in lectin-dependent complement activation was not previously established.

[0123] The structures of the catalytic fragments of MASP-1 and MASP-2 have been determined by X-ray crystallography. Structural comparisons of the MASP-1 protease domain with those 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 in MASP-2 is limited by a surface loop (Harmat et al., J Mol Biol 342:1533-1546 (2004)), MASP-1 has an open substrate binding pocket that is more similar to that of trypsin than to that of other complement proteases. The thrombin-like properties of the MASP-1 structure are due to the unusually large 60-amino acid loop (loop B) that can interact with the substrate. Another interesting feature of the MASP-1 structure is the internal salt bridge between S1 Asp189 and Arg224. Similar salt bridges capable of regulating its protease activity can be found within the substrate-binding pocket of factor D. C1s and MASP-2 exhibit nearly identical substrate specificity. Surprisingly, some of the eight surface loops of MASP-2 that determine substrate specificity have entirely different conformations compared to those of C1s. This means that the two functionally related enzymes interact with the same substrate in different ways. The structure of the enzyme precursor MASP-2 shows an inactive protease domain with a fragmented oxyanion hole and substrate-binding pocket (Gal et al., J Biol Chem 280:33435-33444 (2005)). Surprisingly, the enzyme precursor MASP-2 exhibits considerable activity against the large protein substrate C4. The structure of the enzyme precursor 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 self-activation process.

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

[0125] Patients with a history of infectious diseases and chronic inflammatory diseases were found to have a mutant form of MASP-2 that is unable to form the active MBL-MASP complex (Stengaard-Pedersen et al., N Engl J Med 349:554-560 (2003)). Some researchers have determined that MBL deficiency is linked to a tendency towards frequent infections in early childhood (Super et al., Lancet 2:1236-1239 (1989); Garred et al., Lancet 346:941-943 (1995)) and 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). The literature is divided, but MASP deficiency, or non-utilization, may negatively affect an individual's ability to develop rapid, non-antibody-dependent defenses against certain pathogens.

[0126] iii. Ca ++ Supporting data and Ca for a new understanding highlight the lack of conventional assay conditions. ++ Results obtained using a more physiological set of conditions including Several independent lines of strong experimental evidence are provided herein to point to the conclusion that the complement lectin pathway activation route 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 generating secondary pathway convertase C3bBb through cleavage and activation of factor B on the activator surface, which then catalyzes C3b deposition and the formation of secondary pathway convertase C3bBb, resulting 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 thereof, can also lead to complement activation via a secondary pathway.

[0127] The lectin pathway-dependent MASP-3-driven activation of the second pathway is thought to contribute to the well-established factor-D mediated C3b-binding factor-B cleavage, achieving the optimal activation rate of complement-dependent lysis via a terminal activation cascade for lysing bacterial cells by forming the C5b-9 membrane invasion complex (MAC) on the cell surface (Figures 14-15). This rate-limiting event is incomplete in the absence of MASP-3 functional activity and factor-D functional activity, suggesting that optimal coordination is required. As described in Examples 1-4 of this specification, we investigated the phenotypes of MASP-2 deficiency and MASP-2 inhibition in experimental mouse models of meningococcal infection and discovered this MASP-3-dependent lectin pathway function. Genetically targeted MASP-2-deficient mice and wild-type mice treated with antibody-based MASP-2 inhibitors showed high resistance to experimental meningococcal infection (see Figures 8-12). When the infection dose was adjusted to achieve approximately 60% mortality in 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 a significant increase in serum bactericidal activity in the serum of MASP-2-deficient or MASP-2-depleted mice. Further experiments showed that this bactericidal activity depends on secondary pathway-driven lysis. Serum from mice deficient in factor B, factor D, or C3 showed no bactericidal activity against Neisseria meningitidis, indicating that the secondary pathway is essential for driving the terminal activation cascade. A surprising result was that serum from mice deficient in MBL-A and MBL-C (both lectin pathway-recognizing molecules that recognize Neisseria meningitidis), as well as from mice deficient in lectin pathway-related serine proteases MASP-1 and MASP-3, lost all lytic activity against Neisseria meningitidis (Figure 15). Recent papers (Takahashi M. et al., JEM 207:29-37 (2010)) and the studies presented therein (Figure 39) demonstrate that MASP-1 can convert factor D in its enzyme precursor form into its enzymatically active form, and that this can partially explain the loss of lytic activity due to the absence of enzymatically active factor D in serum.This does not explain the lack of bactericidal activity in MBL-deficient mice, because these mice possess normal, enzymatically active factor D (Banda et al., Mol Immunol 49(1-2):281-9 (2011)). Surprisingly, when human serum from patients with a rare 3MC autosomal recessive disorder carrying a mutation that impairs the serine protease domain of MASP-3 was tested (Rooryck C, et al., Nat. Genet. 43(3):197-203), no bactericidal activity against Neisseria meningitidis was detected (Note: these serums possess 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 bacterial activity is further supported by the observation that MBL-deficient human serum cannot lyse Neisseria meningitidis (Figures 13-14). MBL is the only human lectin pathway-recognizing molecule that binds to this pathogen. Since MASP-3 does not self-activate, we hypothesize that the higher lytic activity in MASP-2-deficient serum can also be explained by the favorable activation of MASP-3 via MASP-1. This is because, in the absence of MASP-2, all lectin pathway activation complexes that bind to the bacterial surface are affixed with either MASP-1 or MASP-3. Since activated MASP-3 cleaves both factor D (Figure 39) and factor B in vitro to produce 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 secondary C3 converter (i.e., C3bBb).

[0129] While the data on lectin-dependent roles are compelling, multiple experiments suggest that MASP-3 and MASP-1 are not necessarily compelled to function in complex with lectin molecules. Experiments, such as those shown in Figure 35B, demonstrate MASP-3's ability to activate the secondary pathway in the absence of lectin complexes (i.e., in the presence of EGTA) (as demonstrated by C3b deposition against Staphylococcus aureus). Figure 35A demonstrates that deposition under these conditions is dependent on factors B, D, and P, which are key components of the secondary 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 of 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 combined with MBL. Therefore, we do not wish that all the roles of MASP-3 (and MASP-1) demonstrated herein be limited by the notion that they can be attributed solely to lectin-related 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 interventions to effectively treat certain human diseases caused by excessive complement activation in the presence of microbial pathogens or altered host cells or metabolites. As described herein, we have so far found that the second pathway is not activated on surface structures in the absence of MASP-3 and in the presence of MASP-1 (see Figures 17-18, 35B, 41-42, 45-46). Since the second pathway is important for driving the rate-limiting events that result in lysis and cytolysis (Mathieson PW, et al., J Exp Med 177(6):1827-3 (1993)), our results demonstrate that activated MASP-3 plays a crucial role in complement lysis activity. As shown in Figures 14-15, 21-23, 43-44, and 46-47, the complement lysis terminal activation cascade is incomplete in the serum of 3MC patients who lack 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 secondary pathway on the target surface (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, activation of the secondary pathway on the target surface depends on lectin-independent factor B and / or factor D activation mediated by LEA-1 and / or MASP-3, and thus, agents that inhibit MASP-3-dependent complement activation prevent activation of the secondary pathway on the target surface.

[0131] The disclosure of the essential role of MASP-3-dependent initiation of secondary pathway activation implies that secondary pathway is not an independent complement activation pathway, as is essentially stated in all current medical textbooks and recent commentaries on complement. The widely supported current scientific view is that secondary pathway is activated by the amplification of spontaneous "tick-over" C3 activation on the surface of specific particulate targets (microorganisms, zymosan, and rabbit erythrocytes). However, the absence of any secondary pathway activation in the serum of MASP-1 and MASP-3 double-deficient mice and human 3MC patient serum on zymosan-coated plates and two different bacteria (Meningococcus and Staphylococcus aureus), as well as the reduced hemolysis of erythrocytes in MASP-3-deficient serum from humans and mice, indicates that the initiation of secondary pathway activation on these surfaces requires functional MASP-3. The role required of MASP-3 may be lectin-dependent or lectin-independent, resulting in the formation of secondary pathway C3 convertase and C5 convertase complexes, namely C3bBb and C3bBb(C3b)n, respectively. Accordingly, the inventors hereby disclose the existence of a previously unclear initiation route for a second pathway. This initiation route relies on (i) a newly discovered lectin pathway activation arm, LEA-1, and / or (ii) the lectin-independent roles of the proteins MASP-3, MASP-1, and HTRA-1.

[0132] III. The roles of MASP-2 and MASP-3 in paroxysmal nocturnal hemoglobinuria and treatment methods using MASP-2 and MASP-3 inhibitors i. PNH Overview Paroxysmal nocturnal hemoglobinuria (PNH), sometimes also known as Marchia-Fava-Micheli syndrome, is an acquired, potentially life-threatening blood disorder. PNH can occur spontaneously and is called “primary PNH,” or, in the context of other bone marrow disorders such as aplastic anemia, it is called “secondary PNH.” The majority of cases are primary PNH. PNH is characterized by complement-induced erythrolysis (hemolysis), low red blood cell count (anemia), thrombosis, and bone marrow dysfunction. Laboratory findings of PNH, in the absence of autoreactive RBC-binding antibodies as a possible cause, show changes consistent with intravascular hemolytic anemia: low hemoglobin, high lactate dehydrogenase, high reticulocyte count (immature blood cells released by the bone marrow to replace destroyed cells), and high bilirubin (hemoglobin breakdown products).

[0133] A prominent feature of PNH is chronic complement-mediated hemolysis caused by the disordered activation of terminal complement components, including the membrane invasion complex, on the surface of circulating red blood cell (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 in normal RBCs and regulate complement activation. CD55 acts as a negative regulator of the secondary pathway, inhibiting the assembly of the secondary pathway C3 convertase (C3bBb) complex, promoting the disintegration of pre-formed convertases, and thus blocking the formation of the membrane invasion complex (MAC). CD59 inhibits the complement membrane invasion complex by directly binding to the C5b678 complex and preventing C9 from binding and polymerizing.

[0134] While hemolysis and anemia are the primary clinical features of PNH, the disease is a complex hematological disorder that further includes thrombosis and bone marrow dysfunction as part of its clinical findings (Risitano et al., Mini Reviews in Med Chem 11:528-535 (2011)). At the molecular level, PNH is caused by the abnormal clonal proliferation of hematopoietic stem cells lacking the functional PIG A gene. PIG A is an X-linked gene that encodes a glycosyl-phosphatidylinositol transferase required for the 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 cell mutations can clonalize to the point where their offspring constitute a significant portion of the peripheral hematopoietic cell pool. Both the erythrocyte and lymphocyte offspring of the mutant stem cell clones lack CD55 and CD59, but only RBCs undergo apparent lysis after entering 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, blocking C5 cleavage by C5 convertase, thereby inhibiting C5a production and MAC assembly. Treatment of PNH patients with eculizumab reduced intravascular hemolysis as measured by lactate dehydrogenase (LDH) and led to hemoglobin stabilization and transfusion independence in about half of the patients (Risitano et al., Mini-Reviews in Medicinal Chemistry, 11(6)(2011)). In almost all patients treated with eculizumab, LDH levels became normal or near normal (for the management of intravascular hemolysis), but only about one-third of patients reached hemoglobin levels of approximately 11 gr / dL, and the remaining patients who took 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 who took eculizumab contained a large amount of C3 fragments bound to PNH red blood cells (whereas untreated patients did not). This finding has led to the understanding that in PNH patients treated with Soliris, PNH RBCs that are no longer hemolyzed due to C5 blockade can now accumulate a large amount of membrane-bound C3 fragments. The membrane-bound C3 fragment acts as an opsonin, and as a result, is captured within reticuloendothelial cells via specific C3 receptors, subsequently leading to extravascular hemolysis.Therefore, while eculizumab therapy prevents intravascular hemolysis and the resulting complications, it merely changes the properties of these RBCs from intravascular to extravascular hemolysis, resulting in a significant amount of untreated anemia remaining in many patients (Risitano AM et al., Blood 113:4094-100 (2009)). Consequently, patients who develop extravascular hemolysis mediated by C3 fragments continue to require red blood cell transfusions, and these patients require treatment strategies beyond the use of eculizumab. Such approaches targeting C3 fragments have demonstrated usefulness in experimental systems (Lindorfer et al., Blood 115:2283-91, 2010).

[0136] ii. Complement Initiation Mechanism in PNH The causal relationship between the incomplete 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 complement-mediated condition. While this paradigm is widely accepted, the events that initiate complement activation and the complement activation pathways involved remain unresolved. CD55 and CD59 negatively modulate the terminal amplification step in the complement cascade common to all complement initiation pathways; therefore, regardless of whether complement activation is initiated by the lectin pathway, the classical pathway, or the spontaneous turnover of the secondary pathway, deficiency in these molecules impairs the formation and integration of membrane invasion complexes. Consequently, in PNH patients, any complement activation event leading to C3b deposition on the RBC surface can trigger subsequent amplification and pathological hemolysis (intravascular and / or extravascular hemolysis), potentially causing sudden hemolytic episodes. The precise mechanisms of molecular events that induce hemolytic episodes in PNH patients are still not fully understood. Since the complement initiation event in PNH patients experiencing hemolytic episodes is completely unclear, the prevailing view is that complement activation in PNH may spontaneously occur through low-level secondary pathway "tick-over" activation, followed by amplified activation due to inadequate control of terminal complement activation caused by CD55 and CD59 deficiencies.

[0137] However, it is important to note that in the natural course of PNH, PNH typically 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 is independent of prior host immunity to the irritating pathogen and is therefore likely not involved in the classical pathway. More precisely, this complement activation response is thought to be initiated by lectin binding to exogenous carbohydrate patterns or "altered self" carbohydrate patterns expressed on the surface of microbial agents or damaged host tissue. Thus, the events that suddenly trigger hemolytic episodes in PNH are closely related to lectin-mediated complement activation. For this reason, the lectin activation pathway is very likely to provide the initiation trigger that ultimately leads to hemolysis in PNH patients.

[0138] To analyze the activation cascade in detail at the molecular level, the inventors, using a sufficiently clear pathogen that activates complement via lectins as an experimental model, fully demonstrate that complement activation can be initiated by either LEA-2 or LEA-1, depending on the inducing microorganism, leading to opsonization and / or lysis. The principle of this same dual response (i.e., opsonization and / or lysis) to lectin-initiated events is likely to apply to other types of infectious pathogens or other lectin-driven complement activation events that can cause lectin-mediated complement activation or PNH after tissue damage to the host. Based on this duality in the lectin pathway, the inventors hypothesize that LEA-2 and / or LEA-1-initiated complement activation in PNH patients promotes C3b-mediated opsonization and / or lysis of RBCs, as well as subsequent extravascular and intravascular hemolysis. Therefore, in the context of PNH, inhibition of both LEA-1 and LEA-2 can be expected to address both extravascular and intravascular hemolysis and offer a significant advantage over the C5 inhibitor eculizumab.

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

[0140] In investigating the role of LEA-2 in the innate host response to microorganisms, the inventors tested further pathogens. When studying Neisseria meningitidis as a model organism, dramatically different results were observed. Neisseria meningitidis also activates complement via lectins, and complement activation is necessary to contain meningococcal infection within a naive host. 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 removal of MASP-2 did not reduce survival rates after meningococcal infection. On the contrary, in these studies, blocking LEA-2 by MASP-2 removal significantly improved survival rates (Figures 8 and 9) and disease scores (Figure 11). Blocking LEA-2 by administration of MASP-2 antibody yielded the same results (Figure 12), ruling out secondary or compensatory effects in the knockout mouse line as a possible cause. These favorable results in LEA-2-deficient animals were associated with more rapid removal of Neisseria meningitidis from the blood (Figure 10). Furthermore, incubation of Neisseria meningitidis with normal human serum, as described herein, killed the Neisseria meningitidis (Figure 13). The addition of a functional monoclonal antibody specific to human MASP-2 that blocks LEA-2 could enhance this killing response, but the administration of isotype-controlled monoclonal antibodies could not. Nevertheless, since MBL-deficient human serum or thermo-inactivated human serum failed to kill Neisseria meningitidis, this process is dependent on lectins and, at least partially, the functional complement system (Figure 13). Overall, these novel findings suggest that Neisseria meningitidis infection in the presence of the functional complement system is suppressed by a lectin-dependent but LEA-2-independent complement activation pathway.

[0141] Using serum samples from a 3MC patient, we tested the hypothesis that LEA-1 may be a complement pathway responsible for lectin-dependent meningococcal meningitis. 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 otherwise had sufficient complement (exon 12 is specific to 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 meningococci, but thermally inactivated serum lacking MBL (one of the lectin pathway recognition molecules) and MASP-3-deficient serum failed to kill meningococci (Figure 14). Therefore, LEA-1 is thought to mediate meningococcal meningitis. This finding was confirmed using serum samples from a knockout mouse strain. Complement containing normal mouse serum readily killed Neisseria meningitidis, but MBL-deficient or MASP-1 / 3-deficient mouse serum was as ineffective as thermally inactivated serum lacking functional complement (Figure 15). Conversely, MASP-2-deficient serum showed efficient killing of Neisseria meningitidis.

[0142] These findings provide evidence of previously unknown lectin pathway duality 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 non-overlapping and mediate separate 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, leading to opsonization, while others (e.g., Neisseria meningitidis) preferentially initiate complement activation via LEA-1 and promote the cytolytic process. However, since both pathways can mediate opsonization and / or lysis in other contexts, the data do not necessarily limit LEA-2 to opsonization and LEA-1 to the cytolytic process.

[0143] In the context of lectin-dependent complement activation by Neisseria meningitidis, LEA-2 blockade enhanced LEA-1-dependent lytic disruption of the organism in vivo, suggesting that the LEA-2 and LEA-1 arms compete with each other (Figure 15). As mentioned above, this finding can be explained by the increased possibility that, in the absence of MASP-2, the lectin MASP-1 complex is located in close proximity to the lectin MASP-3 complex, thereby enhancing LEA-1 activation and consequently promoting more effective meningococcal lysis. Since meningococcal lysis is a major protective mechanism in naive hosts, in vivo blockade of LEA-2 increases meningococcal clearance and enhances death.

[0144] The above examples show that LEA-2 and LEA-1 have opposite effects on outcomes after meningococcal infection, but there may be other situations in which LEA-2 and LEA-1 can work synergistically to produce specific outcomes. As detailed below, in other situations of lectin-mediated pathological complement activity, such as those present in PNH, LEA-2 and LEA-1-driven complement activation can work synergistically to contribute to the overall pathogenesis of PNH. In addition, as described herein, MASP-3 also contributes to the lectin-independent conversion of factor B and factor D, which is Ca ++ This can occur in the absence of [the substance], generally resulting in conversion from C3bB to C3bBb and from pro-D factor to D factor, which may further contribute to the pathogenesis of PNH.

[0145] iii. Biological and expected 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 finding supports the usefulness 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 their MASP-1-mediated activation) for treating patients with one or more aspects of PNH, as well as the use of LEA-2 and / or LEA-1 and / or MASP-3-dependent lectin-independent complement activation inhibitors (including MASP-2 inhibitors, MASP-3 inhibitors, and MASP-2 / MASP-3 or MASP-1 / MASP-2 bispecific or bispecific inhibitors, as well as panspecific MASP-1 / MASP-2 / MASP-3 inhibitors) to mitigate the effects of C3 fragment-mediated extravascular hemolysis in PNH patients receiving treatment with C5 inhibitors such as eculizumab.

[0146] iv. MASP-2 inhibitors that block opsonization of PNH RBCs and extravascular hemolysis via the reticuloendothelial system. As detailed above, PNH patients develop anemia due to two separate mechanisms of RBC clearance from circulation: intravascular hemolysis due to activation of the membrane invasion complex (MAC), and extravascular hemolysis after opsonization by C3b and subsequent clearance after complement receptor binding and uptake by the reticuloendothelial system. Intravascular hemolysis is generally prevented when patients are treated with eculizumab. Eculizumab does not block extravascular hemolysis because it blocks terminal lysis effector mechanisms that occur downstream of both the complement initiation activation event and subsequent opsonization (Risitano AM et. al., Blood 113:4094-100 (2009)). Instead, RBCs that are thought to have undergone hemolysis in untreated PNH patients can now accumulate activated C3b protein on their surface, which enhances uptake by the reticuloendothelial system and thus enhances extravascular hemolysis. Therefore, eculizumab treatment effectively changes the nature of RBCs from intravascular hemolysis to potential extravascular hemolysis. As a result, some PNH patients treated with eculizumab remain anemic. Thus, agents that block complement activation upstream and inhibit the 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 evaluated on three prototype lectin-activated surfaces (mannan, Figure 19A; zymosan, Figure 19B; and Streptococcus pneumoniae, Figure 19C), LEA-2 was found to be the dominant route of lectin-dependent opsonization under physiological conditions (i.e., when all complement pathways are activatable). ++In the presence of ( ), it is considered to be the dominant route of lectin-dependent opsonization. Under these experimental conditions, MASP-2 deficient serum (lacking LEA-2) showed substantially lower opsonization of the test surface than WT serum. MASP-1 / 3 deficient serum (lacking LEA-1) was also impaired, but this effect was far less pronounced compared to serum lacking LEA-2. The relative magnitudes of the contributions of LEA-2 and LEA-1 to lectin-driven opsonization are further shown in Figures 20A-20C. Although it has been reported that the second complement pathway supports opsonization of the lectin-activated surface in the absence of the lectin pathway or classical pathway (Selander et al., J Clin Invest 116(5):1425-1434 (2006)), the isolated second pathway (Ca ++Measurements under free assay conditions suggest that the effect is substantially less than that of the LEA-2 and LEA-1 initiation processes described herein. Extrapolation suggests that PNH RBC opsonization may be preferentially initiated by LEA-2, and only to a lesser extent by LEA-1 than as a result of lectin-independent secondary pathway activation (possibly amplified by the secondary pathway amplification loop). Therefore, LEA-2 inhibitors can be expected to be most effective in suppressing opsonization and preventing extravascular hemolysis in PNH. However, the recognition of the fact that lectins other than MBLs, e.g., phycoline, bind to non-carbohydrate structures, e.g., acetylated proteins, and that MASP-3 preferentially associates with H-phycoline (Skjoedt et al., Immunobiol. 215:921-931, 2010), leaves open the possibility of a significant role for LEA-1 in PNH-related RBC opsonization. Therefore, LEA-1 inhibitors are expected to have a further anti-opsonizing effect, and a 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 (lacking the ability to activate the second pathway in the fluid phase but possessing functional classical and functional LEA-1 and LEA-2 pathways) does not show opsonization deficiency compared to WT serum. Factor B-deficient serum lacking LEA-1 shows reduced opsonization, while 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 MASP-2 blockade or factor D blockade alone. Therefore, LEA-2 and LEA-1 are expected to act additively or synergistically to promote opsonization, and cross-reactive or bispecific LEA-1 / LEA-2 inhibitors are expected to be most effective in preventing opsonization and extravascular hemolysis in PNH.

[0148] v. The role of MASP-3 inhibitors in PNH Using an in vitro model of PNH, the inventors demonstrated that complement activation and resulting hemolysis in PNH are indeed initiated by LEA-2 and / or LEA-1 activation, and that this is not an independent function of the second 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 that is not present in PNH patients. When mannan-sensitized Crry-deficient RBCs were exposed to complement-sufficient human serum, the RBCs were substantially hemolytic at a 3% serum concentration (Figures 21 and 22), whereas complement-deficient serum (HI: heat-inactivated) was not hemolytic. Surprisingly, complement-sufficient serum with LEA-2 blocked by the addition of MASP-2 antibody showed reduced hemolytic activity, and 6% serum was required for effective hemolysis. Similar observations were obtained when CD55 / CD59-deficient RBCs were tested (Figure 24). Complement-sufficient human serum supplemented with MASP-2 monoclonal antibody (i.e., serum with suppressed LEA-2) was approximately twice as effective in supporting hemolysis compared to untreated serum. Furthermore, higher concentrations of LEA-2-blocked serum (i.e., treated with anti-MASP-2 monoclonal antibody) were required to effectively promote hemolysis of untreated WT RBCs compared to untreated serum (Figure 23).

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

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

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

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

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

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

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

[0156] Other activators that block the terminal amplification loop of the complement system that leads to C5 activation and MAC deposition (including, but not limited to, activators that block propagin, 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 activators are not expected to interfere with LEA-2-mediated opsonization in PNH patients. This leaves some PNH patients treated with such activators with substantial residual anemia due to untreated LEA-2-mediated extravascular hemolysis. Therefore, treatment with such activators that prevent intravascular hemolysis is expected to be more effective in preventing anemia that develops in PNH patients when combined with LEA-2 blockers that minimize extravascular hemolysis than either activator alone. In fact, the combination of such activators and LEA-2 blockers is expected to prevent all associated mechanisms of RBC destruction in PNH, and consequently prevent all symptoms of anemia in PNH.

[0157] viii. Use of LEA-1 and LEA-2 multispecific, bispecific, or panspecific antibodies for the treatment of PNH As detailed above, the use of a combination of pharmacological substances that individually block LEA-1 and LEA-2, and thus combine to block all complement activation events mediating intravascular and extravascular hemolysis, is expected to provide the best clinical outcomes for PNH patients. This outcome can be achieved, for example, by the simultaneous administration of an antibody having LEA-1 blocking activity and an antibody having 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 having LEA-1 and LEA-2 combined blocking activity effectively inhibits intravascular and extravascular hemolysis and prevents anemia in PNH. Such an entity may or may consist of a bispecific antibody in which one antigen-binding site specifically recognizes MASP-1, blocking LEA-1 and reducing LEA-2, and a second antigen-binding site specifically recognizes MASP-2 and further blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody, where 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, where one antigen-binding site specifically recognizes both MASP-1 and MASP-3, thereby blocking LEA-1 and reducing LEA-2, and furthermore, a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Furthermore, based on the similarity of the overall protein sequence and architecture, it is conceivable that conventional antibodies with two identical binding sites can be developed that functionally bind specifically to MASP-1, MASP-2, and MASP-3, thereby achieving functional blockade of LEA-1 and LEA-2. Such antibodies possessing pan-MASP inhibitory activity are expected to inhibit both intravascular and extravascular hemolysis, thereby effectively treating anemia in patients with PNH.

[0158] IV. The roles of MASP-2 and MASP-3 in age-related macular degeneration and therapies using MASP-2 and MASP-3 inhibitors Age-related macular degeneration (AMD) is a leading cause of visual impairment and blindness in older adults, accounting for up to 50% of blindness cases in developed countries. The prevalence of AMD in adults is approximately 3%, increasing with age, with nearly two-thirds of the population over 80 years old exhibiting some symptoms. In the United States, over 1.75 million individuals have progressive 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), which causes degeneration of photoreceptors in the central retina, i.e., the macula, and loss of central vision. Early and mid-stage AMD is characterized by the gradual deposition of drusen, a yellowish substance containing lipids, proteins, lipoproteins, and necrotic cell fragments, in the subretinal space adjacent to the RPE, as well as pigment irregularities in the retina. Advanced AMD comprises two clinical subtypes: non-neoangiogenic geographically atrophic ("dry") AMD and neoangiogenic exudative ("wet") AMD. Dry AMD accounts for 80–90% of advanced AMD cases, but 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 states. Currently, there are no treatments approved by the United States Food and Drug Administration (FDA) for dry AMD. FDA-approved treatment options for wet AMD include intravitreal injections of anti-angiogenic agents (ranibizumab, pegaptanib sodium, aflibercept), laser therapy, photodynamic laser therapy, and implantable telescopes.

[0159] The etiology and pathophysiology of AMD are complex and not fully understood. Several pieces of evidence support the role of complement system dysregulation in the pathogenesis of AMD. Gene 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 polymorphisms in the complement factor H (CFH) gene, with homozygous Y402H mutants having approximately a 6-fold increased risk of developing AMD compared to non-risk genotypes, and heterozygotes having approximately a 2.5-fold increased risk (Khandhadia, S., et al., Immunobiol. 217:127-146, 2012). Mutations in other complement pathway-coding genes, including complement factors B (CFB), C2, C3, 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 the eyes of donors from AMD patients have shown increased and localized complement cascade proteins in drusen (Issa, PC, et al., Graefes. Arch. Clin. Exp. Ophthalmol. 249:163-174, 2011). Furthermore, AMD patients have increased systemic complement activation as measured in peripheral blood (Issa et al., 2011).

[0160] In the pathogenesis of AMD, the complement secondary pathway is considered more relevant than the classical pathway. The essential recognition component C1q 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 suggested the involvement of the CFH and CFB genes. These proteins are involved in the secondary pathway amplification loop, with CFH being a fluid phase inhibitor and CFB being the activating protease component of the secondary pathway. The Y402H variant of CFH affects ligand binding interactions, including binding to C-reactive proteins, heparin, M proteins, and glycosaminoglycans. Binding to this altered ligand may reduce binding to the cell surface, which in turn may lead to a decrease in factor I-mediated denaturation of the C3b activating fragment and impaired regulation of the secondary C3 convertase, potentially resulting in hyperactivation of the secondary pathway (Khandhadia et al., 2012). Alterations in the CFB gene are associated with a protective effect against AMD expression. The functional mutant fB32Q has four times lower binding affinity to C3b than the risk mutant fB32R, resulting in reduced C3 convertase formation (Montes, T. et al., Proc. Natl. Acad. Sci. USA 106:4366-4371, 2009).

[0161] Complement Initiation Mechanism in AMD The aforementioned human genetic linkage studies suggest a crucial role for the complement system in AMD pathogenicity. Furthermore, complement activators are abundant in drusen, a characteristic pathological lesion in both wet and dry forms of 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 cell fragments and oxidized waste products derived from the retina that accumulate beneath the retinal pleoplasm (RPE) with aging of the eye. In addition, oxidative stress is thought to play a significant role (Cai et al; Front Biosci., 17:1976-95, 2012), and has been shown to induce 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 damage (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 inference, lectin-dependent complement activation may play a central role in AMD pathogenicity.

[0163] The role of the complement system has been evaluated in mouse models of AMD. In photoinjury mouse models, which are experimental models of oxidative stress-mediated photoreceptor degeneration, knockout mice with the classical pathway eliminated (C1qα on a C57BL / 6 background) - / - ) had the same sensitivity to photodamage compared to wild-type littermates, but lacked the second pathway of complement factor D elimination (CFD). - / -) provided protection from photodamage (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 Brook's membrane, complement factor B-deficient knockout mice (CFB) - / - In the same model, intravenous administration of a recombinant form of complement H (CR2-fH) targeted at 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). Furthermore, 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). Since fB is activated by LEA-1, and MASP-1 and MASP-3 contribute to the maturation of factor D, these findings suggest that LEA-1 inhibitors may have therapeutic benefits in AMD patients.

[0164] Initial experimental studies in rodent models of AMD using MBL-deficient mice did not support a crucial 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 other lectins may also induce complement activation in AMD. In fact, our previous research showed that MASP-2, a rate-limiting serine protease critically required for lectin pathway function, plays a crucial role in AMD. As described in U.S. Patent No. 7,919,094 (assigned to Omeros Corporation), 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 confirmed preclinical model of wet AMD (Ryan et al., Tr Am Opth Soc LXXVII:707-745, 1979). Therefore, inhibitors of LEA-2 are expected to effectively prevent CNV and improve outcomes in AMD patients.

[0165] Therefore, considering the above, LEA-1 inhibitors and LEA-2 inhibitors are expected to have independent therapeutic benefits in AMD. In addition, when LEA-1 inhibitors and LEA-2 inhibitors are used in combination, they may achieve further therapeutic benefits or provide effective treatment to a broader patient subset compared to either being used alone. Combined LEA-1 and LEA-2 inhibition can be achieved by the simultaneous administration of LEA-1 and LEA-2 blockers. Optimally, LEA-1 and LEA-2 inhibitory functions may be encompassed 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 in which each binding site can bind to and block MASP-1 / 3 or MASP-2.

[0166] Thus, in accordance with the foregoing, an aspect of the present invention provides a method for inhibiting LEA-1-dependent complement activation to treat age-related macular degeneration (wet and dry forms) by administering a composition containing 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 to a subject suffering from such a condition. The MASP-1, MASP-3, or MASP-1 / 3 inhibitor composition may be administered topically to the eye by irrigation, intravitreal administration, or application of the composition in the form of a gel, ointment, or drop. Alternatively, the MASP-1, MASP-3, or MASP-1 / 3 inhibitor may be administered systemically to the subject, for example by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or potentially by oral administration in the case of a non-peptidogenic substance. Administration may be repeated, as determined by a physician, until the condition is resolved or suppressed.

[0167] In one embodiment, the method of this aspect of the present invention further comprises the step of inhibiting LEA-2-dependent complement activation in a subject suffering from age-related macular degeneration, and includes the step of 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 substances that individually block LEA-1 and LEA-2 is expected to provide improved clinical outcomes in AMD patients compared to LEA-1 inhibition alone. This outcome can be achieved, for example, by the co-administration of an antibody having LEA-1 blocking activity and an antibody having LEA-2 blocking activity. In some embodiments, LEA-1 blocking activity and LEA-2 blocking activity are combined into 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 a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

[0168] MASP-2 inhibitory compositions may be administered topically to the eye by means of irrigation, intravitreal injection, or topical application of compositions in the form of gels, ointments, or drops. Alternatively, MASP-2 inhibitors may be administered systemically to the subject, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or potentially orally in the case of non-peptidogenic substances. Administration may be repeated at the discretion of the physician until the condition is resolved or suppressed.

[0169] The application of the MASP-3 inhibitory composition and any MASP-2 inhibitory composition of the present invention may, in the case of AMD treatment, be carried out by a single dose or a limited number of consecutive doses of the composition (e.g., a single composition or simultaneous administration of separate compositions containing MASP-2 and MASP-3 inhibitors or bispecific or bi-inhibitory inhibitors). Alternatively, in the case of AMD treatment, the composition may be administered over a long period at regular intervals such as once daily, twice weekly, once weekly, once every two weeks, once a month, or once every two months.

[0170] V. The roles of MASP-2 and MASP-3 in ischemia-reperfusion injury and therapies using MASP-2 and MASP-3 inhibitors Tissue ischemia is the basis of a wide range of clinical complications. 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 known as ischemia-reperfusion (I / R) injury (Eltzschig, HK and Tobias, E., Nat. Med. 17:1391-1401, 2011). I / R injuries can affect a single organ, such as the heart (acute coronary syndrome), kidneys (acute kidney injury), intestines (intestinal I / R), and brain (stroke). I / R injuries can also affect multiple organs, such as after major trauma and resuscitation (multiple organ failure), circulatory arrest (hypoxic brain injury, acute kidney injury), peripheral vascular disease, and sickle cell disease (acute thoracic 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 impairment. Currently, there are no specific therapies that target I / R impairment, and there is a need for effective treatments that maximize tissue salvage in the ischemic zone and improve functional outcomes in these common situations.

[0171] The pathophysiology of I / R dysfunction is complex and characterized by a strong inflammatory response after reperfusion. Complement system activation has been suggested to be a key component of I / R dysfunction, 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 secondary pathways in I / R dysfunction is 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 secondary pathways in I / R dysfunction.

[0172] The role of the secondary pathway in gastrointestinal irrigation / retrograde (IR) injury has been studied using factor D-deficient (- / -) mice and heterozygous (+ / -) mice (Stahl, GL, et al. Am. J. Pathol. 162:449-455, 2003). After transient gastrointestinal ischemia, intestinal and lung damage was reduced in factor D-deficient mice compared to heterozygous mice, but this was not preventive, and the addition of human factor D to - / - mice restored IR damage. When the same model was evaluated in C1q-deficient mice and MBL-A / C-deficient mice, the results showed that gastrointestinal irrigation / retrograde injury is independent of C1q and classical pathway activation, but intestinal injury requires MBL and lectin pathway activation (Hart, ML, et al. J. Immunol. 174:6373-6380, 2005). Conversely, C1q recognition molecules in the classical pathway were responsible for lung injury after intestinal induction / remission (Hart, ML, et al. J. Immunol. 174:6373-6380, 2005). One hypothesis is that complement activation during induction / remission occurs through spontaneous IgM binding to autoantigens present on the surface of ischemic (but not normal) tissue, such as non-muscle myosin heavy chain type II. In a mouse gastrointestinal induction / remission model, immune complexes from intestinal tissue have been evaluated for the presence of initiators in the classical (C1q) pathway, the lectin (MBL) pathway, or the second (factor B) pathway (Lee, H., et al., Mol. Immunol. 47:972-981, 2010). The results showed that C1q and MBL were detected in these immune complexes, but factor B was not, indicating involvement of the classical and lectin pathways, but not of the second pathway. In the same model, factor B-deficient mice were not protected from localized tissue damage, providing further support for the lack of involvement of the second pathway. The role of the lectin pathway in gastrointestinal I / R injury was directly evaluated in MASP-2-deficient mice, and the results showed reduced gastrointestinal injury in these mice compared to 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 of this specification. In summary, these results support the involvement of the lectin pathway in gastrointestinal I / R disorders, but there is conflicting data regarding the involvement of a second pathway.

[0173] In a mouse myocardial I / R injury model, the pathogenic role of the lectin pathway was demonstrated when MBL-deficient mice were protected from myocardial injury, while C1q-deficient 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 in this specification. Treatment of rats in a myocardial I / R model with a monoclonal antibody against rat MBL resulted in a reduction in post-ischemia-reperfusion injury (Jordan, JE, et al., Circulation 104:1413-18, 2001). In a study of myocardial infarction patients treated with angioplasty, MBL deficiency was associated with a reduced 90-day mortality rate compared to patients with adequate MBL management (M Trendelenburg et al., Eur Heart J. 31:1181, 2010). Furthermore, myocardial infarction patients who developed cardiac dysfunction after angioplasty had approximately three times higher MBL levels than patients who recovered (Haahr-Pedersen S., et al., J Inv Cardiology, 21:13, 2009). In addition, MBL antibodies reduced complement deposition on endothelial cells in vitro after oxidative stress, demonstrating the role of the lectin pathway in myocardial I / R injury (Collard, CD, et al., Am. J. Pathol. 156:1549-56, 2000). In a mouse ectopic syngeneic heart transplantation model with I / R impairment, the role of the secondary pathway has been studied 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 impairment to a level comparable to treatment with CR2-Crry, which inhibits the whole complement pathway, demonstrating the critical importance of the secondary pathway in this model.

[0174] In a mouse model of renal I / R impairment, B factor-deficient mice were protected from renal dysfunction and tubular damage compared to wild-type mice, suggesting the involvement of a second pathway (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 impairment 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 to 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 a C5 antibody did not show an effect in preventing renal injury, suggesting that the role of MBL in this model does not involve activation of terminal complement components. Rather, human proximal tubular cells incubated with MBL in vitro internalized MBL, and subsequently underwent cell death, suggesting that MBL has a direct toxic effect on tubular cells. Castellano G. et al. (Am J Pathol, 176(4):1648-59, 2010) tested C1 inhibitors that irreversibly inactivate 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 inhibitors reduced complement deposition in peritubular capillaries and glomeruli, thereby reducing tubular damage.

[0175] Factor B-deficient mice showed reduced systemic complement activation as measured by serum C5a levels and decreased post-traumatic neuronal cell death compared to wild-type mice, suggesting that the second pathway is involved in experimental traumatic brain injury (Leinhase, I., et al., BMC Neurosci. 7:55-67, 2006). In human stroke, immunohistochemical staining of ischemic lesions detected complement components C1q, C3c, and C4d, 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 yielded mixed results; some studies demonstrated protection, while others showed no benefit (Arumugam, TV, et al., Neuroscience 158:1074-1089, 2009). Experiments and clinical trials have provided strong evidence of lectin pathway involvement. In experimental stroke models, deficiency in either MBL or MASP-2 resulted 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 showed a better prognosis than patients with adequate MBL support (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, demonstrating the involvement of a secondary 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 pathogenicity and adverse outcomes. Based on the experimental and clinical findings described above, LEA-2 inhibitors are expected to be protective in most I / R situations. Lectin-dependent activation of LEA-1 can lead to complement activation via the secondary pathway in at least some situations. In addition, LEA-2 initiated complement activation can be further amplified by the secondary pathway amplification loop, potentially exacerbating I / R-related tissue damage. Therefore, LEA-1 inhibitors are expected to offer additional or supplemental therapeutic benefits in patients suffering from ischemia-related conditions.

[0178] Considering the above, LEA-1 inhibitors and LEA-2 inhibitors are expected to have independent therapeutic benefits in treating ischemia-reperfusion-related conditions, preventing ischemia-reperfusion-related conditions, or mitigating the severity of ischemia-reperfusion-related conditions. In addition, LEA-1 inhibitors and LEA-2 inhibitors, when used in combination, may achieve further therapeutic benefits compared to either one alone. Therefore, the optimally effective treatment for I / R-related conditions contains a pharmaceutically active ingredient that blocks both LEA-1 and LEA-2, either alone or in combination. Combined LEA-1 and LEA-2 inhibition can be achieved by the simultaneous administration of a LEA-1 blocker and a LEA-2 blocker. Preferably, LEA-1 and LEA-2 inhibitory functions can be encompassed 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 in which each binding site can bind to and block MASP-1 / 3 or MASP-2.

[0179] Thus, in accordance with the foregoing, an aspect of the present invention provides a method for inhibiting LEA-1-dependent complement activation to treat ischemia-reperfusion injury, prevent ischemia-reperfusion injury, or reduce the severity of ischemia-reperfusion injury, by administering a composition containing a therapeutically effective amount of a 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 experiencing ischemia-reperfusion. The MASP-1, MASP-3, or MASP-1 / 3 inhibitor composition may be administered to the subject by intra-arterial, intra-venous, intracranial, intramuscular, subcutaneous, or other parenteral administration, and potentially orally in the case of non-peptidogenic inhibitors, most preferably by intra-arterial or intravenous administration. Administration of the LEA-1 inhibitor composition of the present invention is preferably initiated immediately after or as soon as possible thereafter of the ischemia-reperfusion event. When reperfusion occurs in a controlled environment (e.g., after aortic aneurysm repair, organ transplantation, or reattachment of amputated or damaged limbs or fingers), LEA-1 inhibitors may be administered before, during, and / or after reperfusion. Administration may be repeated periodically as determined by the physician to obtain the 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 / finger replantation, stroke, myocardial infarction, and hemodynamic resuscitation after shock and / or surgical procedure.

[0181] In some embodiments, the method is used to treat or prevent ischemia-reperfusion injury in subjects who are scheduled to receive, are receiving, or have received an organ transplant. In some embodiments, the method is used to treat or prevent ischemia-reperfusion injury in subjects who are scheduled to receive, are receiving, or have 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 the step of inhibiting LEA-2-dependent complement activation in a subject experiencing ischemia-reperfusion injury, and further comprises the step of administering a therapeutically effective amount of a MASP-2 inhibitor and a MASP-1, MASP-3, or MASP-1 / 3 inhibitor to the subject. As described above, the use of a combination of pharmacological substances that individually block LEA-1 and LEA-2 is expected to provide improved clinical outcomes in treating ischemia-reperfusion injury, preventing ischemia-reperfusion injury, or reducing the severity of ischemia-reperfusion injury, compared to LEA-1 inhibition alone. This outcome can be achieved, for example, by the co-administration of an antibody having LEA-1 blocking activity and an antibody having LEA-2 blocking activity. In some embodiments, LEA-1 blocking activity and LEA-2 blocking activity are combined into 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 a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

[0183] MASP-2 inhibitory compositions can be administered to subjects requiring them by intra-arterial, intravenous, intracranial, intramuscular, subcutaneous, or other parenteral administration, and potentially orally in the case of non-peptide-gated inhibitors, most preferably by intra-arterial or intravenous administration. Administration of the MASP-2 inhibitory compositions of the present invention is preferably initiated immediately after or as soon as possible thereafter of an ischemia-reperfusion event. If reperfusion occurs in a controlled environment (e.g., after aortic aneurysm repair, organ transplantation, or reattachment of amputated or damaged limbs or fingers), the MASP-2 inhibitor may be administered before and / or during and / or after reperfusion. Administration may be repeated periodically as determined by a physician to obtain the optimal therapeutic effect.

[0184] The application of the MASP-3 inhibitory composition and any MASP-2 inhibitory composition of the present invention may be carried out by a single dose or a limited number of consecutive doses of the composition (e.g., a single composition or simultaneous administration of separate compositions containing MASP-2 and MASP-3 inhibitors or bispecific or bi-inhibitory inhibitors) for the treatment or prevention of ischemia-reperfusion injury. Alternatively, the composition may be administered over a long period at regular intervals such as once daily, twice weekly, once weekly, once every two weeks, once a month, or once every two months for the treatment of subjects experiencing ischemia-reperfusion injury.

[0185] VI. The roles of MASP-2 and MASP-3 in inflammatory and non-inflammatory arthritis, and therapies using MASP-2 and MASP-3 inhibitors. Rheumatoid arthritis (RA) is a chronic inflammatory disease of the synovial joints that can also manifest as systemic symptoms. RA affects approximately 1% of the world's population, and is 2-3 times more common in women. 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. Treatment goals for RA 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 treatment for RA includes disease-modifying antirheumatic drugs (DMARDs), analgesics, and anti-inflammatory agents (glucocorticoids and nonsteroidal anti-inflammatory drugs). DMARDs are the most important treatments 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 biological 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] While adaptive immunity is clearly central to RA pathogenicity, as evidenced by its genetic association with T cell activating genes and the presence of autoantibodies, the involvement of innate immune mechanisms has also been suggested (McInnes, IB and Schett, G. New Engl. J. Med. 365:2205-2219, 2011). In human RA, synovial fluid levels of secondary pathway cleavage fragment Bb were several times higher than those from samples from patients with crystal-induced arthritis or osteoarthritis, suggesting the involvement of preferential activation of the secondary pathway in RA patients (Brodeur, JP, et al., Arthritis Rheum. 34:1531-1537, 1991). In an experimental passive transmission model of arthritis using anti-type II collagen antibodies, factor B-deficient mice showed reduced inflammation and joint damage compared to wild-type mice, while C4-deficient mice exhibited disease activity similar to wild-type mice, indicating the need for a secondary 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 active or only the lectin pathway active were unable to develop arthritis (Banda, NK et al., Clin. Exp. Immunol. 159:100-108, 2010). Data from this study suggested that either the classical or lectin pathway can activate low levels of C3 in vitro. However, in the absence of the secondary pathway amplification loop, the level of C3 joint deposition was insufficient to produce clinical disease.A key step in the activation of the secondary pathway is the conversion of factor D zymogen (profactor D) to maturation 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 to wild-type mice (Banda, NK, et al., J. Immunol. 185:5598-5606, 2010). In MASP-1 / 3-deficient mice, pro-factor D was detected in serum during CAIA evolution, but mature factor D was not. Addition of human factor D reconstituted C3 activation and C5a production in vitro using serum from these mice. In contrast, in a mouse model of the effector phase of arthritis, C3-deficient mice developed very mild arthritis compared to WT mice, while factor B-deficient mice still developed arthritis, demonstrating independent contributions from both the classical / lectin pathway and the secondary pathway (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 similarly to wild-type mice, while mice lacking factor B did not develop arthritis or showed only mild arthritis, demonstrating the necessity of a secondary 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 because the role of MBL-C was not studied, the potential role of the lectin pathway could not be ruled out (Ji et al., 2002).

[0187] Two independent research groups have proposed that lectin-dependent complement activation promotes inflammation in RA patients through the interaction of MBL and 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 arthritis is associated with a marked increase in galactose-deficient IgG glycoforms (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 arthritis and returns to normal when the patient enters remission. In vivo, IgG0 is deposited in synovial tissue, and MBL is present at increased 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 allele variants of MBL in RA patients suggest that MBL may play a pro-inflammatory role in this disease (Garred et al., J. Rheumatol. 27:26-34, 2000). Therefore, lectin-dependent complement activation mediated by LEA-1 and / or LEA-2 may play a significant 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). Similar to adult RA, elevated serum and synovial fluid levels of secondary pathway complement activator Bb compared to 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 a crucial role in psoriatic arthritis. Patients with this condition have elevated circulating complement activators, and their red blood cells are thought to have lower levels of the complement modulator CD59 (Triolo., Clin Exp Rheumatol., 21(2):225-8, 2003). Complement levels are associated with disease activity and are a high predictor of treatment outcomes (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). While the precise role of complement in psoriatic arthritis has not been determined, the presence of circulating C4d and Bb complement activators in these patients suggests a significant role in the pathogen. Based on the observed products, LEA-1 and possibly LEA-2 are thought to be responsible for 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 physical disability. The joints most commonly affected by OA are the hands, neck, hips, knees, and hips. The disease is progressive, and current treatments focus on alleviating symptomatic pain and do not alter the natural course of the disease. The etiology of OA is unknown, but the involvement of complement has been suggested. Proteomic and transcriptome analyses of synovial fluid from OA patients showed abnormal expression of several complement components, including the classical (C1s and C4A) and secondary (factor B) pathways, as well as C3, C5, C7, and C9, compared to samples from healthy individuals (Wang, Q., et al., Nat. Med. 17:1674-1679, 2011). Furthermore, in a mouse model of OA induced by medial meniscectomy, C5-deficient mice showed 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 second pathway, reduced the incidence of OA (Wang et al., 2011).

[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 diseases, these viruses can cause epidemics involving millions of infected individuals. Arthritis is thought to be initiated by viral replication in the joints and induction of a host inflammatory response, with the complement system 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 to wild-type mice, suggesting the involvement of complement (Morrison et al., 2007). Specific complement pathways involved have been studied, including mice with the inactivated lectin pathway (MBL-A). - / - and MBL-C - / - ) reduced arthritis compared to wild-type mice. In contrast, inactivating the classical pathway (C1q) reduced arthritis. - / - ) or the second pathway (factor B) - / - Mice possessing ) developed severe arthritis, demonstrating that the lectin pathway initiated by MBL plays an essential role in this model (Gunn, BM, et al., PLoS Pathog. 8:e1002586, 2012). Since arthritis involves joint damage, initial joint damage caused by various pathogenesis may induce a secondary wave of complement activation mediated by LEA-2. Supporting this concept, our research, as described in Example 27 herein, demonstrated that MASP-2 KO mice had reduced joint damage compared to WT mice in a collagen-induced model of RA.

[0192] Considering the evidence presented above, LEA-1 inhibitors and LEA-2 inhibitors are expected to be therapeutically useful in the treatment of arthritis, either alone or in combination. Therefore, the optimally effective treatment for arthritis may include a pharmaceutically active ingredient capable of blocking both LEA-1 and LEA-2, either alone or in combination. Combined inhibition of LEA-1 and LEA-2 can be achieved by the simultaneous administration of a LEA-1 blocker and a LEA-2 blocker. Preferably, LEA-1 inhibitory function and LEA-2 inhibitory function can be encompassed 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 in which each binding site can bind to and block MASP-1 / 3 or MASP-2. Thus, in accordance with the foregoing, an aspect of the present invention provides a method for inhibiting LEA-1-dependent complement activation to treat inflammatory or non-inflammatory arthritis, including osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, and psoriatic arthritis, to prevent inflammatory or non-inflammatory arthritis, or to reduce the severity of inflammatory or non-inflammatory arthritis, by administering a composition containing a therapeutically effective amount of a 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 may be administered systemically to the subject, for example, by intra-arterial, intra-venous, intramuscular, subcutaneous, or other parenteral or oral administration. Alternatively, administration may be by local delivery, for example, by intra-articular injection. LEA-1 inhibitors may be administered regularly over a long period of time for the treatment or suppression of chronic conditions, or as a single or repeated dose during the period before, during, and / or after acute trauma or injury, including surgical procedures performed on the joints.

[0193] In one embodiment, the method of this aspect of the present invention further comprises the step of inhibiting LEA-2-dependent complement activation in subjects suffering from inflammatory or non-inflammatory arthritis (including osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, and psoriatic arthritis) or subjects at risk of developing inflammatory or non-inflammatory arthritis by administering a therapeutically effective amount of a MASP-2 inhibitor and a MASP-1, MASP-3, or MASP-1 / 3 inhibitor to the subject. As described above, the use of a combination of pharmacological substances that individually block LEA-1 and LEA-2 is expected to provide improved therapeutic outcomes in treating or preventing arthritis compared to LEA-1 inhibition alone. This outcome can be achieved, for example, by the co-administration of an antibody having LEA-1 blocking activity and an antibody having LEA-2 blocking activity. In some embodiments, LEA-1 blocking activity and LEA-2 blocking activity are combined into 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 a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

[0194] MASP-2 inhibitory compositions may be administered systemically to subjects in need, for example, by intra-arterial, intravenous, intramuscular, subcutaneous, or other parenteral administration, or potentially by oral administration in the case of non-peptide-gated inhibitors. Alternatively, administration may be by local delivery, such as by intra-articular injection. MASP-2 inhibitors may be administered regularly over a long period for the treatment or suppression of chronic conditions, or by single or repeated doses before, during, and / or after acute trauma or injury, including surgical procedures performed on the joints.

[0195] The MASP-3 inhibitory composition and any MASP-2 inhibitory composition of the present invention may be applied by a single dose or a limited number of consecutive doses of the composition (e.g., a single composition or simultaneous administration of separate compositions containing MASP-2 and MASP-3 inhibitors or bispecific or bi-inhibitory inhibitors) to treat inflammatory or non-inflammatory arthritis, to prevent inflammatory or non-inflammatory arthritis, or to reduce the severity of inflammatory or non-inflammatory arthritis. Alternatively, in the case of treating subjects suffering from inflammatory or non-inflammatory arthritis, the composition may be administered over a long period at regular intervals such as once daily, twice weekly, once weekly, once every two weeks, once a month, or once every two months.

[0196] VII. The roles of MASP-2 and MASP-3 in disseminated intravascular coagulation (DIC) and therapies using MASP-2 and MASP-3 inhibitors Disseminated intravascular coagulation (DIC) is a syndrome of pathological hyperstimulation of the coagulation system that can clinically manifest as bleeding and / or thrombosis. DIC does not occur as a primary condition, but rather 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, pre-eclampsia), and miscellaneous conditions such as cardiogenic shock, drowning, fat embolism, and aortic aneurysm. Thrombocytopenia is a common abnormality in patients in the intensive care unit, occurring in 35%–44% of cases, but DIC accounts for approximately 25% of these cases. In other words, DIC develops in about 10% of critically ill patients (Levi, M. and Opal, SM Crit. Care 10:222-231, 2006). The pathophysiology of DIC is that the underlying disease process initiates a physiological coagulation response. However, when thrombosis-promoting substances overwhelm the normal counter-equilibrium mechanism, improper 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 in the appropriate underlying disease or process, along with abnormal laboratory values ​​(prothrombin time, partial thromboplastin time, fibrin degradation products, D-dimer, or platelet count). The primary treatment of DIC is to address the underlying disease that triggers it. In some cases, blood product support in the form of red blood cells, platelets, fresh frozen plasma, and cryoprecipitate may be necessary to treat or prevent clinical complications.

[0197] The role of the complement pathway in DIC has been studied in several research projects. Complement activation has been evaluated by comparing the clinical course of pediatric patients with meningococcal infection in relation to 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 complex, C4bc, and C3bBbP than MBL-sufficient patients, and exhibited lower degrees of normal complement, terminal complement, and secondary pathway activation. Furthermore, the degree of systemic complement activation correlated with disease severity and parameters in DIC and MBL-deficient patients showed a milder clinical course than in MBL-sufficient patients. Therefore, while 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 of this specification, experimental studies highlight the important contributions of MBL and MASP-1 / 3 to the innate immune response against *Neisseria meningitidis*, the causative agent of meningococcal infection. MBL-deficient serum from mice or humans, MASP-3-deficient human serum, or serum from MASP-1 / 3 knockout mice are less effective than wild-type serum in activating complement and lysing *Neisseria meningitidis* in vitro. Similarly, naive MASP-1 / 3 knockout mice are more susceptible to Neisseria infection than their wild-type counterparts. Therefore, 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 thrombosis compared to wild-type or C2 / B factor-null mice, and the deficiency was reconstituted with recombinant human MBL (La Bonte, LR, 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 to serum from wild-type or C2 / B factor-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 a significant role in thrombosis. Therefore, LEA-1 may play a crucial role in pathological thrombosis, including DIC.

[0200] Experimental studies have demonstrated an equally important role for LEA-2 in pathological thrombosis. As described in Example 30 herein, in a mouse model of focal DIC, we demonstrated that MASP-2 knockout mice were far 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 system and the coagulation system. As described in Examples 29 and 31 herein, MASP-2 has factor Xa-like activity and can activate prothrombin by cleavage to form thrombin, which then scavenges fibrinogen and promotes fibrin clot formation (see also Krarup et al., PLoS One, 18:2(7):e623, 2007).

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

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

[0203] The data described above suggest that LEA-1 and LEA-2 provide an independent link between lectin-dependent complement activation and coagulation. Therefore, considering the above, LEA-1 inhibitors and LEA-2 inhibitors are expected to have independent therapeutic benefits in treating subjects suffering from disseminated intravascular coagulation. In some embodiments, subjects suffer from disseminated intravascular coagulation secondary to sepsis, trauma, infection (bacterial, viral, fungal, parasitic), malignancy, transplant rejection, transfusion reaction, complications of childbirth, vascular aneurysm, hepatic failure, heatstroke, 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 meningococcal infection.

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

[0205] In addition, LEA-1 inhibitors and LEA-2 inhibitors, when used in combination, may achieve greater therapeutic benefits or provide effective treatment to a broader patient subset compared to either being used alone. Combined LEA-1 and LEA-2 inhibition can be achieved by the simultaneous administration of LEA-1 and LEA-2 blockers. Optimally, LEA-1 and LEA-2 inhibitory functions may be encapsulated 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 in which each binding site binds to and blocks MASP-1 / 3 or MASP-2.

[0206] Thus, in accordance with the foregoing, an aspect of the present invention provides a method for inhibiting LEA-1-dependent complement activation to treat, prevent, or reduce the severity of disseminated intravascular coagulation (DIC) in a subject in need thereof, the method comprising administering a composition containing a therapeutically effective amount of a LEA-1 inhibitor in a pharmaceutical carrier, including a combination of a MASP-1 inhibitor, a MASP-3 inhibitor, or a MASP-1 / 3 inhibitor, to a subject experiencing or at risk of developing DCI. The MASP-1, MASP-3, or MASP-1 / 3 inhibitor composition may be administered systemically to the subject, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or potentially by oral administration in the case of a non-peptidogenic substance. Administration may be repeated, at the discretion of a physician, until the condition is resolved or suppressed. For the treatment or prevention of DIC following trauma or other acute events, LEA-1 inhibitory compositions may be administered to patients considered to be at risk of DIC immediately after or prophylactically following a traumatic injury, 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, LEA-1 inhibitory compositions may be administered in a rapid-acting dosage form, for example, by intravenous or intra-arterial delivery of a bolus of a solution containing the LEA-1 inhibitory composition.

[0207] In one embodiment, a method of this aspect of the present invention further comprises the step of inhibiting LEA-2-dependent complement activation to treat, prevent, or reduce the severity of disseminated intravascular coagulation in a subject requiring such treatment, and further comprises the step of administering a therapeutically effective amount of a MASP-2 inhibitor and a MASP-1, MASP-3, or MASP-1 / 3 inhibitor to the subject. As described above, the use of a combination of pharmacological substances that individually block LEA-1 and LEA-2 is expected to provide improved therapeutic outcomes in treating or preventing disseminated intravascular coagulation compared to LEA-1 inhibition alone. This outcome can be achieved, for example, by the co-administration of an antibody having LEA-1 blocking activity and an antibody having LEA-2 blocking activity. In some embodiments, LEA-1 blocking activity and LEA-2 blocking activity are combined into 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 a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

[0208] MASP-2 inhibitory compositions may be administered systemically to subjects in need, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or potentially by oral administration in the case of non-peptidogenic substances. Administration may be repeated, at the discretion of the physician, until the condition is resolved or suppressed. In cases of DIC following trauma or other acute events, MASP-2 inhibitory compositions may be administered to patients considered to be at risk of DIC immediately after the traumatic injury, or prophylactically, before, during, immediately after, or within 1 to 7 days or longer, for example, within 24 to 72 hours, before, during, or immediately after a trauma-induced injury or surgery. In some embodiments, MASP-2 inhibitory compositions may be administered preferably in a rapid-acting dosage form, for example, by intravenous or intra-arterial delivery of a bolus of a solution containing the MASP-2 inhibitory substance composition.

[0209] The application of the MASP-3 inhibitory composition and any MASP-2 inhibitory composition of the present invention may be carried out by a single dose or a limited number of consecutive doses of the composition (e.g., a single composition or simultaneous administration of separate compositions containing MASP-2 and MASP-3 inhibitors or bispecific or bi-inhibitory inhibitors) to treat, prevent, or reduce the severity of disseminated intravascular coagulation in subjects requiring it. Alternatively, the composition may be administered over a long period at regular intervals such as once daily, twice weekly, once weekly, once every two weeks, once a month, or once every two months for the treatment of subjects experiencing or at risk of developing disseminated intravascular coagulation.

[0210] VIII. The roles 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 therapies using MASP-2 and MASP-3 inhibitors. Thrombotic microangiopathy (TMA) is a group of disorders clinically characterized by thrombocytopenia, microangiogenic hemolytic anemia, and variable organ ischemia. Characteristic pathological features of TMA include platelet activation and the formation of microthrombi in the arterioles and venules. Classical TMAs 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 without diarrhea (D-) or atypical HUS (aHUS).

[0211] HUS D+HUS, usually accompanied by 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. While human infection with Escherichia coli O157 is relatively common, the proportion of bloody diarrhea progressing to D+HUS ranges from 3% to 7% in sporadic cases and 20% to 30% in some 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 about two-thirds of children require dialysis during the acute phase of the disease. Treatment for D+HUS is supportive, as there is no specific treatment that has been shown to be effective. The prognosis for D+HUS is good, and the vast majority of patients regain renal function.

[0212] The pathogens of D+HUS include bacterial-produced Shiga toxins that bind to the membranes of microvascular endothelial cells, monocytes, and platelets. The renal microvascular system is most frequently affected. After binding, the toxins are internalized, leading to the release of pro-inflammatory mediators and eventual cell death. Endothelial cell damage is thought to induce renal microvascular thrombosis by promoting the 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 to normal controls and normalized 28 days after discharge (Thurman, JM et al., Clin. J. Am. Soc. Nephrol. 4:1920-1924, 2009). When activation proceeds in the presence of ethylene glycoltetraacetic acid, which blocks the classical pathway, Shiga toxin 2 (Stx2) was found to activate human complement in the fluid phase primarily via the second pathway in vitro (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 (Orth, D. et al, 2009). These results suggest that Shiga toxin may cause renal damage through multiple potential mechanisms, including direct toxic effects, and indirectly through complement activation or inhibition of complement modulochemicals. As demonstrated in Examples 21-23 of this specification, and as evidenced by the effectiveness of MASP-2 blockade in preventing complement-mediated reperfusion injury in various vascular beds, the toxic effect on vascular endothelial cells is expected to activate complement via LEA-2. 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 showed less thrombocytopenia and were protected from renal dysfunction compared to wild-type mice, suggesting the involvement of LEA-1-dependent activation of a secondary pathway in microvascular thrombosis (Morigi, M. et al., J. Immunol. 187:172-180, 2011). As described in Example 33 herein, administration of MASP-2 antibody was equally effective in the same model, increasing survival rates after STX challenge and suggesting the involvement of the LEA-2-dependent complement pathway in microvascular thrombosis.

[0214] Based on the above, LEA-1 inhibitors and LEA-2 inhibitors are expected to have independent therapeutic benefits in the treatment or prevention of HUS. In addition, when LEA-1 inhibitors and LEA-2 inhibitors are used in combination, they may achieve further therapeutic benefits or provide effective treatment to a broader patient subset compared to either being used alone. Combined LEA-1 and LEA-2 inhibition can be achieved by the simultaneous administration of LEA-1 blockers and LEA-2 blockers. Optimally, LEA-1 inhibitory and LEA-2 inhibitory functions may be encompassed 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 in which each binding site can bind to and block MASP-1 / 3 or MASP-2.

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

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

[0217] Factor H is a complement regulatory protein that protects host tissues from secondary complement attack. FH regulates the secondary complement amplification loop in three ways. FH is a cofactor of FI, which cleaves C3b; inhibits the formation of the secondary C3 convertase C3bBb; and blocks C3b deposition by binding to polyanions on the cell surface and tissue matrix (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 incomplete binding of FH to heparin, C3b, and endothelium, but without altering plasma C3 regulation between the N-terminal domains (Pickering, MC et al., J. Exp. Med. 204:1249-1256, 2007). FH-deficient mice exhibit unregulated plasma C3 activation and spontaneously develop membranoproliferative glomerulonephritis type II, but do not spontaneously develop aHUS. However, FH-deficient mice transgenically expressing mouse FH protein functionally equivalent to that of aHUS-associated human FH mutants spontaneously develop HUS but do not spontaneously develop membranoproliferative glomerulonephritis type II, providing in vivo evidence that incomplete regulation of secondary pathway activation in the renal endothelium is a key factor in the pathogenesis of FH-associated aHUS (Pickering et al., 2007). 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 genes encoding five FH-associated proteins (Loirat and Fremeaux-Bacchi, 2011).

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

[0219] While the central role of the deregulated secondary pathway amplification loop in aHUS is widely accepted, the triggers and molecular pathways involved in initiating complement activation remain unclear. Not all individuals with the above mutations develop aHUS. In fact, family studies have suggested that the penetrance of aHUS is only about 50% (Sullivan M. et al., Ann Hum Genet 74:17-26 2010). The natural course 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 pre-existing adaptive immunity, infectious agent-induced complement activation can be initiated primarily via LEA-1 or LEA-2. Therefore, infection-induced lectin-dependent complement activation can trigger the initiation of subsequent pathological amplification of complement activation in individuals predisposed to aHUS, which can ultimately lead to disease progression. Therefore, another aspect of the present invention includes treating patients suffering from aHUS secondary to an infection by administering an effective amount of 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 respond, for example, by expressing a surface portion that binds to lectins and activates the complement LEA-2 pathway (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 context has pathological consequences for the host, and inhibition of LEA-2 by blocking MASP-2, as shown in Examples 22 and 23, prevents further host tissue damage and adverse outcomes (see also Schwaeble PNAS, 2011).

[0221] Therefore, other processes that trigger aHUS are also known to activate LEA-1 or LEA-2. Thus, the LEA-1 and / or LEA-2 pathways may be early complement activation mechanisms that are dysregulated and inappropriately amplified in individuals genetically predisposed to aHUS, thereby likely initiating aHUS pathogenicity. By reason, agents that block complement activation via LEA-1 and / or LEA-2 are expected to prevent disease progression or mitigate exacerbation in aHUS-susceptible individuals.

[0222] Further supporting this concept, recent studies have identified Streptococcus pneumoniae as a significant causative agent in pediatric cases of aHUS (Lee, CS 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 were accompanied by non-intestinal infections leading to the manifestation of microangiopathy, uremia, and hemolysis, without evidence of co-occurring mutations in complement genes known to predispose individuals to aHUS. It is important to note that Streptococcus pneumoniae is particularly effective in activating complement, primarily via LEA-2. Therefore, in cases of non-intestinal HUS associated with Streptococcus pneumoniae infection, the manifestation of microangiopathy, uremia, and hemolysis is expected to be primarily driven by LEA-2 activation, and LEA-2-blocking agents, 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 involves 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 blood coagulation disorder resulting from autoimmune or hereditary dysfunction that activates the coagulation cascade or complement system (George, JN, N Engl J Med; 354:1927-35, 2006). It is characterized by the formation of numerous microclots, or thrombi, in the small blood vessels throughout the body. Red blood cells are subjected to shear stress, which damages the membranes and leads to intravascular hemolysis. The resulting reduced blood flow and endothelial damage cause damage to organs, including the brain, heart, and kidneys. Clinically, TTP is characterized by thrombocytopenia, microangiogenic hemolytic anemia, neurological changes, renal failure, and fever. Before plasmapheresis, the mortality rate during acute episodes was 90%. Even with plasmapheresis, the 6-month survival rate is approximately 80%.

[0224] TTP can result from genetic or acquired inhibition of the enzyme ADAMTS-13, a metalloproteinase responsible for cleaving large multimers of von Willebrand factor (vWF) into smaller units. ADAMTS-13 inhibition or deficiency ultimately leads to increased coagulation (Tsai, H. J Am Soc Nephrol 14:1072-1081, 2003). ADAMTS-13 modulates vWF activity. In the absence of ADAMTS-13, vWF forms larger multimers that are more readily bound to platelets, creating a patient predisposition to platelet aggregation in the microvascular system and thrombosis.

[0225] Numerous 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 develop during breast, gastrointestinal, or prostate cancer (George JN., Oncology (Williston Park). 25:908-14, 2011), during 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 drug therapies, including heparin, quinine, immune-mediated components, cancer chemotherapy agents (bleomycin, cisplatin, cytosine arabinoside, daunomycin gemcitabine, mitomycin C, and tamoxifen), cyclosporine A, oral contraceptives, penicillin, rifampin, and antiplatelet agents 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, spleen isolation, 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 leading to TTP can occur as a result of endothelial cell damage associated with Streptococcus pneumoniae infection (Pediatr Nephrol, 26:631-5, 2011).

[0226] Plasma exchange is the standard treatment for TTP (Rock GA, et al., N Engl J Med 325:393-397, 1991). Plasma exchange exchanges 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). Further agents such as immunosuppressants are routinely added to treatment (George, JN, N Engl J Med, 354:1927-35, 2006). However, plasma exchange is unsuccessful in about 20% of patients, relapse occurs in more than one-third of patients, plasmapheresis is expensive and technically demanding. Furthermore, many patients cannot tolerate plasma exchange. As a result, there remains an urgent need for further and better treatments for TTP.

[0227] Since TTP is a disorder of the blood coagulation cascade, treatment with complement system antagonists may help stabilize and cure the disease. Pathological activation of the second complement pathway is associated with aHUS, but the role of complement activation in TTP is not as clear. Functional deficiency of ADAMTS13 is important for susceptibility to TTP, but it is not sufficient to cause an acute episode. Environmental factors and / or other genetic mutations may contribute to the manifestation of TTP symptoms. For example, genes encoding proteins involved in regulating the coagulation cascade, vWF, platelet function, components of the endothelial vessel surface, 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 a crucial role. Serum from thrombotic microangiopathy associated with ADAMTS-13 deficiency has been shown to induce C3 and MAC deposition and subsequent neutrophil activation, which could be suppressed by complement inactivation (Ruiz-Torres MP, et al., Thromb Haemost, 93:443-52, 2005). In addition, recent studies have shown that during acute episodes of TTP, there are increases in C4d, C3bBbP, and C3a levels, consistent with the activation of classical, lectin, and secondary pathways (M. Reti et al., J Thromb Haemost. 10(5):791-798, 2012). This increase in complement activation during acute episodes may initiate terminal pathway activation and contribute to further exacerbation of TTP.

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

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

[0230] Patients with TTP typically present with one or more thrombosis, including purpura, renal failure, hypothrombocytopenia, anemia, and / or stroke, during emergency care. The current standard of care for TTP involves intracatheter delivery (e.g., intravenous or other forms of catheterization) of exchange plasmapheresis, generally three to daily, for a period of two weeks or longer. If a patient tests positive for the presence of ADAMTS13 inhibitors (i.e., endogenous antibodies against ADAMTS13), plasmapheresis may be performed in combination with immunosuppressive therapy (e.g., corticosteroids, rituximab, or cyclosporine). Patients with refractory TTP (approximately 20% of TTP patients) do not respond to plasmapheresis treatment for at least two weeks.

[0231] Accordingly, in one embodiment, a method is provided for treating a subject with an effective amount of an LEA-2 inhibitor (e.g., MASP-2 antibody) or an LEA-1 inhibitor (e.g., MASP-1 or MASP-3 antibody) as a first therapy, either in the absence of plasmapheresis or in combination with plasmapheresis, in a subject exhibiting one or more symptoms consistent with the diagnosis of TTP (e.g., central nervous system complications, severe thrombocytopenia (platelet counts less than 5,000 or 5,000 / μL if aspirin is off, or less than 20,000 or 20,000 / μL if aspirin is on), in the absence of plasmapheresis or in combination with plasmapheresis. As a first therapy, the LEA-1 inhibitor and / or LEA-2 inhibitor may be administered systemically to the subject, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous or other parenteral administration. In some embodiments, LEA-1 inhibitors and / or LEA-2 inhibitors may be administered to subjects 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 averse to plasmapheresis, or in situations where plasmapheresis is unavailable. In some embodiments, LEA-1 inhibitors and / or LEA-2 inhibitors may be administered to subjects suffering from TTP in combination with immunosuppressants (including co-administration) (e.g., corticosteroids, rituxan, or cyclosporine) and / or in combination with concentrated ADAMTS-13.

[0232] In some embodiments, the method includes administering LEA-1 and / or LEA-2 inhibitors to a subject suffering from TTP via a catheter (e.g., intravenously) during a first period (e.g., an acute phase lasting at least 1 day to 1 or 2 weeks), and then administering LEA-1 and / or LEA-2 inhibitors subcutaneously to the subject during a second period (e.g., a chronic phase lasting at least 2 weeks or longer). In some embodiments, the administrations during the first and / or second periods are carried out in the absence of plasmapheresis. In some embodiments, the method is used to maintain the subject in a manner that prevents them from suffering from one or more symptoms associated with TTP.

[0233] In another embodiment, a method is provided for treating subjects suffering from refractory TTP (i.e., subjects who have not responded to plasmapheresis treatment for at least two weeks) by administering an amount of LEA-1 and / or LEA-2 inhibitors effective in reducing one or more symptoms of TTP. In one embodiment, the LEA-1 and / or LEA-2 inhibitors are administered to subjects with refractory TTP by subcutaneous or other parenteral administration for a long-term period of at least two weeks or longer. Administration may be repeated at the discretion of a physician until the condition is resolved or suppressed.

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

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

[0236] In summary, LEA-1 inhibitors and LEA-2 inhibitors are expected to have independent therapeutic benefits in the treatment of TMA, including HUS, aHUS, and TTP. In addition, LEA-1 and LEA-2 inhibitors, when used in combination, are expected to achieve further therapeutic benefits compared to either inhibitor alone, or provide effective treatment for a broader subset of patients suffering from various forms of TMA. Combined LEA-1 and LEA-2 inhibition can be achieved by the simultaneous administration of LEA-1 and LEA-2 blockers. Optimally, LEA-1 and LEA-2 inhibitory functions can be encapsulated 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 in which each binding site can bind to and block MASP-1 / 3 or MASP-2.

[0237] Thus, in accordance with the foregoing, an aspect of the present invention provides a method 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 the step of administering a composition containing a therapeutically effective amount of a LEA-1 inhibitor, including a combination of a MASP-1 inhibitor, a MASP-3 inhibitor, or a combination of a MASP-1 / 3 inhibitor, in a pharmaceutical carrier to a subject suffering from or at risk of developing thrombotic microangiopathy. The MASP-1, MASP-3, or MASP-1 / 3 inhibitor composition can be administered to the subject systemically, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or potentially by oral administration in the case of a non-peptidogenic substance. Administration may be repeated, at the doctor's discretion, until the condition is resolved or suppressed.

[0238] In one embodiment, a method of this aspect of the present invention further comprises the step of inhibiting LEA-2-dependent complement activation for the treatment of, prevention of, or reduction of the severity of, thrombotic microangiopathy such as hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), or thrombotic thrombocytopenic purpura (TTP), and further comprises the step of administering a therapeutically effective amount 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 substances 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 LEA-1 inhibition alone. This outcome can be achieved, for example, by the co-administration of an antibody having LEA-1 blocking activity and an antibody having LEA-2 blocking activity. In some embodiments, LEA-1 blocking activity and LEA-2 blocking activity are combined into a single molecular entity, such entity having combined LEA-1 and LEA-2 blocking activity. Such an entity may include, 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. 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, thereby blocking LEA-1, while a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

[0239] MASP-2 inhibitors may be administered systemically to the subject, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or potentially orally in the case of non-peptide-gated substances. Administration may be repeated, at the discretion of the physician, until the condition is resolved or suppressed.

[0240] IX. The application of the MASP-3 inhibitory composition and any MASP-2 inhibitory composition of the present invention may be carried out by a single dose of the composition (e.g., a single composition containing MASP-2 and MASP-3 inhibitors or bispecific inhibitors or bispecific inhibitors, or simultaneous administration of separate compositions) or a limited number of consecutive doses to treat thrombotic microangiopathy, prevent thrombotic microangiopathy, or reduce the severity of thrombotic microangiopathy in subjects suffering from or at risk of developing thrombotic microangiopathy. Alternatively, the composition may be administered over a long period at regular intervals such as once daily, twice weekly, once weekly, once every two weeks, once a month, or once every two months for the treatment of subjects requiring it. The roles of MASP-2 and MASP-3 in asthma and therapies using MASP-2 and MASP-3 inhibitors Asthma is a common chronic inflammatory airway disease. In the United States, approximately 25 million people have asthma, including 7 million children under the age of 18. More than half experience at least one asthma attack per year, resulting in over 1.7 million emergency room 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 non-allergic asthma, aspirin-exacerbated respiratory disease, post-infection asthma, occupational asthma, airborne irritant-induced asthma, and exercise-induced asthma. Key features of allergic asthma include airway hyperresponsiveness (AHR) to a variety of specific and unspecific 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 goal of asthma treatment is to control the disease, minimize exacerbations and daily symptoms, and enable patients to be physically active. Current treatment guidelines encourage a stepwise approach until asthma control is achieved. The first treatment step, if necessary, is a rapid-acting inhaled β2 agonist followed by long-term control medications such as inhaled corticosteroids, long-acting inhaled β2 agonists, leukotriene modifiers, theophylline, oral glucocorticosteroids, and anti-IgE monoclonal antibodies.

[0241] Asthma is pathogenically multifactorial, but is generally recognized as resulting from an inadequate immune response to common environmental antigens in genetically susceptible individuals. Asthma is associated with complement activation, and anaphylatoxins (ATs) C3a and C5a have pro-inflammatory and immunomodulatory properties related to the development and modulation of allergic reactions (Zhang, X. and Kohl, J. Expert. Rev. Clin. Immunol., 6:269-277, 2010). However, the relative involvement of the classical, secondary, and lectin pathways of complement in asthma is not fully understood. The secondary pathway can be activated on the surface of allergens, and the lectin pathway can be activated by recognition of the polysaccharide structure of allergens; both processes lead to AT production. Complement can be activated by different pathways depending on the causative allergen involved. For example, highly allergic pollen from the Parietaria family is very 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 for asthma may activate complement via a secondary pathway. For example, in vitro exposure of human serum to cigarette smoke or diesel exhaust particles resulted in complement activation, and this effect was unaffected by the presence of EDTA, suggesting that the activation occurred via a secondary pathway rather than a classical one (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 mouse ovalbumin-sensitized and challenge models. Wild-type mice developed AHR and airway inflammation in response to airborne allergen challenge. A Crry-Ig fusion protein that inhibits all pathways of complement activation was effective in preventing AHR and pneumonia when administered systemically or topically by inhalation in a mouse ovalbumin model of allergic pneumonia (Taube et al., Am J Respir Crit Care Med., 168(11):1333-41, 2003).

[0243] Factor B-deficient mice showed less AHR and airway inflammation compared to wild-type mice, while C4-deficient mice showed 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 secondary pathway involvement, rather than classical pathway involvement, in the mouse air allergen challenge model. Further evidence of the importance of the secondary pathway was provided in a study 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 secondary pathway and works 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 recombinant competitive antagonists increased the degree of AHR and airway inflammation (Takeda et al., 2012). Therapeutic delivery of CR2-fH, a chimeric protein that links the iC3b / C3d binding domain of CR2 to the complement regulatory domain of FH, which targets the complement regulatory activity of fH to existing complement activation sites, prevented the development of AHR and eosinophil infiltration into the airways after allergen challenge (Takeda et al., 2012). The protective effect was demonstrated using ovalbumin and ragweed allergen, a relevant 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 lacking mannan-binding lectin A (MBL-A), a carbohydrate-binding protein that functions as a 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 sensitized MBL-A(- / -) mice was significantly attenuated at both time points after conidial challenge compared to the sensitized MBL-A(+ / +) group. Four days postconidial, pulmonary TH2 cytokine levels (IL-4, IL-5, and IL-13) were significantly lower in A. fumigatus-sensitized MBL-A(- / -) compared to the wild-type group. These results indicate that MBL-A and the lectin pathway play important roles in the development and maintenance of AHR during chronic fungal asthma.

[0245] The findings described above suggest the involvement of lectin-dependent complement activation in the pathogenesis of asthma. Experimental data suggest that factor B activation plays a central role. Given the fundamental role of LEA-1 in lectin-dependent factor B activation and subsequent secondary pathway activation, LEA-1 blockers are expected to be beneficial in treating certain forms of asthma mediated by the secondary 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] Considering the data described above, the inventors believe that LEA-1 and LEA-2 mediate pathological complement activation in asthma. Depending on the causative allergen, LEA-1 or LEA-2 may be preferentially involved. Therefore, LEA-1 blockers combined with LEA-2 blockers may be useful in treating multiple forms of asthma, regardless of the underlying etiology. LEA-1 blockers and LEA-2 blockers may have complementary, additive, or synergistic effects in preventing, treating, or improving lung inflammation and asthmatic symptoms.

[0247] Combined LEA-1 and LEA-2 inhibition can be achieved by the simultaneous administration of LEA-1 and LEA-2 blockers. Optimally, LEA-1 and LEA-2 inhibitory functions can be encompassed within a single molecular entity, for example, 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, an aspect of the present invention provides a method for inhibiting LEA-1-dependent complement activation to treat asthma, prevent asthma, or reduce the severity of asthma, comprising the step of administering a composition containing a therapeutically effective amount of a 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 asthma or at risk of developing asthma. The MASP-1, MASP-3, or MASP-1 / 3 inhibitor composition may be administered systemically to the subject, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or potentially by oral administration in the case of a non-peptidogenic substance. Administration may be repeated, as determined by a physician, until the condition is resolved or suppressed.

[0249] In one embodiment, a method of this aspect of the present invention further comprises the step of inhibiting LEA-2-dependent complement activation for the treatment of asthma, the prevention of asthma, or the reduction of asthma severity, and further comprises the step of administering a therapeutically effective amount of a MASP-2 inhibitor and a MASP-1, MASP-3, or MASP-1 / 3 inhibitor to a subject suffering from asthma or a subject at risk of developing asthma. As described above, the use of a combination of pharmacological substances that individually block LEA-1 and LEA-2 is expected to provide improved therapeutic outcomes in treating, preventing, or reducing asthma severity compared to LEA-1 inhibition alone. This outcome can be achieved, for example, by the co-administration of an antibody having LEA-1 blocking activity and an antibody having LEA-2 blocking activity. In some embodiments, LEA-1 blocking activity and LEA-2 blocking activity are combined into 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 a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

[0250] MASP-2 inhibitors may be administered systemically to the subject, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or potentially orally in the case of non-peptide-gated substances. Administration may be repeated, at the discretion of the physician, until the condition is resolved or suppressed.

[0251] The application of the MASP-3 inhibitory composition and any MASP-2 inhibitory composition of the present invention may be carried out by a single dose or a limited number of consecutive doses of the composition (e.g., a single composition or simultaneous administration of separate compositions containing MASP-2 and MASP-3 inhibitors or bispecific or bi-inhibitory inhibitors) to treat asthma, prevent asthma, or reduce the severity of asthma in subjects suffering from asthma or at risk of developing asthma. Alternatively, the composition may be administered over a long period of time at regular intervals such as once daily, twice weekly, once weekly, once every two weeks, once a month, or once every two months for the treatment of subjects in need.

[0252] X. The roles of MASP-2 and MASP-3 in dense deposit disease and therapies using 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 walls due to subendothelial extension of the mesangium. MPGN is classified as primary (also called idiopathic) or secondary and is associated with underlying conditions such as infections, systemic immune complex disorders, neoplasms, and chronic liver disease. Idiopathic MPGN includes three morphological types. Type I, or classical MPGN, is characterized by subendothelial deposits of immune complexes and activation of the classical complement pathway. Type II, or dense deposit disease (DDD), is characterized by further intramembrane high-density deposits. Type III is characterized by further subepithelial deposits. Idiopathic MPGN is rare, accounting for only about 4-7% of primary renal causes in 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 glomerulonephritis, asymptomatic proteinuria and hematuria, or recurrent gross hematuria. Renal failure occurs in the majority of patients, and the disease progresses slowly, with approximately 40% of patients developing end-stage renal disease within 10 years of diagnosis (Alchi and Jayne, 2010). Current treatment options include corticosteroids, immunosuppressants, antiplatelet regimens, and plasmapheresis.

[0253] DDD is diagnosed by immunofluorescence staining of renal biopsy material, characterized by the absence of immunoglobulins and the presence of C3, and electron microscopy reveals characteristic high-density osminate affinity deposits along the glomerular basement membrane. DDD is caused by dysregulation of the complement secondary 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 glomerulonephritis factor, an autoantibody against the secondary pathway C3 convertase (C3bBb), which increases its half-life and, consequently, the activation of the pathway (Smith, RJH et al., Mol. Immunol. 48:1604-1610, 2011). Other secondary pathway abnormalities include factor H autoantibodies that block the function of factor H, increased functional C3 mutations, and genetic defects of factor H (Smith et al., 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 outcomes.

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

[0255] Human genetic data suggest that improper regulation or excessive activation of the secondary pathway amplification loop plays a crucial role, but complement initiation events have not been identified. Immunohistochemical studies of renal biopsy material provide evidence of MBL deposition in affected tissue, suggesting 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 secondary pathway has been further confirmed in experimental models. Factor H-deficient mice develop progressive proteinuria and renal pathological lesions characteristic of the human condition (Pickering et al., Nat Genet., 31(4):424, 2002). Pickering et al. further demonstrated that the loss of factor B, which mediates LEA-1-dependent activation of the secondary pathway, completely protects factor H-deficient mice from DDD (Pickering et al., Nat Genet., 31(4):424, 2002).

[0256] Therefore, it is expected that activators that block LEA-1 will effectively block lectin-dependent activation of the second pathway, thereby providing an effective treatment for DDD. Given that the second pathway amplification loop is dysregulated in DDD patients, it can be further expected that activators that block the amplification loop will be effective. Since LEA-1 targeted activators that block MASP-1 or MASP-1 and MASP-3 inhibit factor D maturation, such activators are predicted to effectively block the second pathway amplification loop.

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

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

[0259] When used in combination, LEA-1 and LEA-2 blockers are expected to exhibit higher efficacy than either one alone, or to be useful in treating diseases at various stages. Therefore, LEA-1 and LEA-2 blockers may have complementary, additional, or synergistic effects in preventing, treating, or improving DDD-related renal failure.

[0260] Combined LEA-1 and LEA-2 inhibition can be achieved by the simultaneous administration of LEA-1 and LEA-2 blockers. Optimally, the LEA-1 and LEA-2 blockers with inhibitory function may be contained within a single molecular entity, for example, 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, an aspect of the present invention provides a method for inhibiting LEA-1-dependent complement activation to treat dense deposit disease, prevent dense deposit disease, or reduce the severity of dense deposit disease, comprising the step of administering a composition containing a therapeutically effective amount of a 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 dense deposit disease or at risk of developing dense deposit disease. The MASP-1, MASP-3, or MASP-1 / 3 inhibitor composition may be administered systemically to the subject, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or potentially by oral administration in the case of non-peptidogenic substances. Administration may be repeated, at the discretion of a physician, until the condition is resolved or suppressed.

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

[0263] In some embodiments, the method includes a step of inhibiting both LEA-1-dependent complement activation and LEA-2-dependent complement activation. As described above, the use of a combination of pharmacological substances 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 LEA-1 inhibition alone. This outcome can be achieved, for example, by the co-administration of an antibody having LEA-1 blocking activity and an antibody having LEA-2 blocking activity. In some embodiments, LEA-1 blocking activity and LEA-2 blocking activity are combined into a single molecula...

Claims

1. A pharmaceutical composition for treating a subject having symptoms of traumatic brain injury resulting from penetrating brain injury or occlusion brain injury, or a subject at risk of developing symptoms of said traumatic brain injury, wherein the pharmaceutical composition comprises a MASP-2 antibody or a MASP-2 conjugated fragment thereof that is effective in inhibiting MASP-2-dependent complement activation.

2. The pharmaceutical composition according to claim 1, wherein the MASP-2 antibody or its MASP-2 binding fragment is a MASP-2 monoclonal antibody or a fragment thereof, and the fragment specifically binds to a portion of SEQ ID NO:

5.

3. The pharmaceutical composition according to claim 1, wherein the MASP-2 antibody is a chimeric antibody, a humanized antibody, or a human antibody.

4. A pharmaceutical agent for use in inhibiting the effects of MASP-2-dependent complement activation in subjects having symptoms of traumatic brain injury resulting from penetrating brain injury or occlusive brain injury, or subjects at risk of developing symptoms of said traumatic brain injury, wherein the pharmaceutical agent comprises a therapeutically effective amount of a MASP-2 antibody or a MASP-2 conjugated fragment thereof, and a pharmaceutical carrier.

5. The pharmaceutical product according to claim 4, further comprising a therapeutically effective amount of MASP-3 antibody or a MASP-3 conjugated fragment thereof.

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