Methods for treating and / or preventing graft-versus-host disease and / or diffuse alveolar hemorrhage and / or veno-occlusive disease associated with hematopoietic stem cell transplant

Abstract

To provide methods of inhibiting the effects of MASP-2-dependent complement activation in a human subject suffering from graft-versus-host disease and / or diffuse alveolar hemorrhage and / or veno-occlusive disease associated with a hematopoietic stem cell transplant.SOLUTION: The methods comprise the step of administering to a subject a composition comprising an amount of a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, effective to inhibit MASP-2-dependent complement activation. Preferably, the MASP-2 inhibitory antibody is a monoclonal antibody or a fragment thereof that specifically binds to human MASP-2.SELECTED DRAWING: Figure 61

Description

Technical Field

[0001] Description of the Sequence Listing The sequence listing related to this application is provided in text format instead of a hard copy and is incorporated herein by reference. The name of the text file containing the sequence listing is MP_1_0278_PCT_SequenceListingasFiled_20180807, this text file is 116 KB, was created on August 7, 2018, and has been submitted via EFS-Web together with the filing of this specification.

Background Art

[0002] Background The complement system provides an initial mechanism for initiating, amplifying, and organizing the immune response to microbial infections and other acute assaults in humans and other vertebrates (M.K. Liszewski and J.P. Atkinson, 1993, in Fundamental Immunology, Third Edition, edited by W.E, Paul, Raven Press, Ltd., New York). Complement activation provides a beneficial first line of defense against potential pathogens, but the activity of complement that promotes the defensive immune response can also pose a potential threat to the host (K.R, Kalli, et al., Springer Semin. Immunopathol. 15:417-431, 1994; B.P. Morgan, Eur. J. Clinical Investig. 24:219-228, 1994). For example, C3 and C5 proteolytic products mobilize and activate neutrophils. Activated neutrophils are essential for host defense but can release destructive enzymes without discrimination and cause organ damage. Furthermore, due to complement activation, soluble complement components can deposit on the surfaces of nearby host cells as well as microbial targets, and as a result, host cells may lyse.

[0003] The complement system is also involved in the development of a vast number of acute and chronic disease states, including myocardial infarction, stroke, ARDS, reperfusion injury, septic shock, capillary leak after burns, inflammation after cardiopulmonary bypass, graft rejection, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and Alzheimer's disease. In almost all of these states, complement is not the cause but is one of several factors involved in the development. Nevertheless, complement activation is considered to be a major pathological mechanism and represents an effective point for clinical management in many of these disease states. The increasing recognition of the importance of complement-mediated tissue damage in various disease states underscores the need for effective complement inhibitory drugs. To date, eculizumab (Solaris®), an anti-C5 antibody, 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 complement system activation is not inhibited even when C5 is blocked. Therefore, inhibitors of the initiation steps of complement activation are thought to be considerably superior to "downstream" complement inhibitors.

[0004] Currently, it is widely recognized that the complement system can be activated via three distinct pathways: the classical pathway, the lectin pathway, and the alternative pathway. The classical pathway is typically induced by complexes consisting of host antibodies bound to foreign particles (i.e., antigens) and thus requires prior exposure to the antigen to generate a specific antibody response. Since activation of the classical pathway depends on a previous acquired immune response by the host, the classical pathway is part of the adaptive immune system. In contrast, both the lectin pathway and the alternative pathway are independent of acquired immunity and are part of the innate immune system.

[0005] Activation of the complement system results in sequential activation of serine protease precursors. The first step in classical pathway activation is the binding of the specific recognition molecule C1q to IgG and IgM molecules bound to an antigen. C1q binds to the serine protease proenzymes C1r and C1s as a complex called C1. When 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 form a covalent bond with an adjacent hydroxyl or amino group and generate C3 convertase (C4b2a) through non-covalent interaction with the C2a fragment of activated C2. C3 convertase (C4b2a) activates C3 by protein cleavage into C3a and C3b subcomponents, leading to the generation of C5 convertase (C4b2a3b). C5 convertase (C4b2a3b) can cause cell lysis by disrupting the cell membrane by cleaving C5, resulting in the formation of a membrane attack complex (formed by the combination of C5b with C6, C7, C8, and C9, also called "MAC"). Activated forms of C3 and C4 (C3b and C4b) are deposited on the surface of foreign targets by covalent bonds and recognized by complement receptors on multiple phagocytic cells.

[0006] Independently, the first step in complement system activation via the lectin pathway is also the binding of specific recognition molecules and the subsequent activation of the associated serine protease zymogen. However, instead of 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 lectin (MBL), H-ficolin, M-ficolin, L-ficolin, and C-type lectin CL-11). See J. Lu et al., Biochim. Biophys. Acta 1572:387-400, (2002); Holmskov et al., Annu. Rev. Immunol. 21:547-578(2003); Teh et al., Immunology 101:225-232(2000)). See also 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 185(10):6096-6104(2010).

[0007] Ikeda et al. first demonstrated that, similar to C1q, MBL can activate the complement system in a C4-dependent manner when it binds to red blood cells coated with yeast mannan (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-hydroxy and 4-hydroxy groups are oriented on the equatorial binding plane of the pyranose ring. Thus, while the prominent ligands for MBL are D-mannose and N-acetyl-D-glucosamine, carbohydrates that do not meet these steric requirements have 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 single-digit millimolar range. MBL achieves tight and specific binding to glycan ligands by avidity, that is, by interacting simultaneously with multiple monosaccharide residues located in close proximity to each other (Lee et al., Archiv. Biochem. Biophys. 299:129-136, (1992)). MBL generally recognizes carbohydrate patterns that decorate microorganisms, such as bacteria, yeast, parasites, and certain viruses. In contrast, MBL usually does not recognize the penultimate and final sugars, D-galactose and sialic acid, which decorate the "mature" complex glycans present 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, MBL binds with high affinity to clusters of high-mannose "precursor" glycans in N-linked glycoproteins and glycolipids sequestered in the endoplasmic reticulum and Golgi of mammalian cells (Maynard et al., J. Biol. Chem. 257:3788-3794, (1982)). Thus, damaged cells are potential targets for lectin pathway activation via MBL binding.

[0008] Ficolin has a type of lectin domain different from MBL, called the fibrinogen-like domain. Ficolin binds to sugar residues in a Ca ++ -independent manner. In humans, three types of ficolin (L-ficolin, M-ficolin, and H-ficolin) have been identified. The two serum ficolins, L-ficolin and H-ficolin, commonly have specificity for N-acetyl-D-glucosamine. However, H-ficolin also binds to N-acetyl-D-galactosamine. The different sugar specificities of L-ficolin, H-ficolin, CL-11, and MBL mean that different lectins can complement each other and target different complex carbohydrates by overlap. This idea is supported by a recent report that among the known lectins in the lectin pathway, only L-ficolin 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)). Collectins (i.e., MBL) and ficolins do not have significant similarity in amino acid sequence. However, these two protein groups have similar domain architectures and, like C1q, assemble to construct an oligomeric structure that maximizes the potential for multi-site binding.

[0009] The serum concentration of MBL varies considerably in the healthy population, which is genetically controlled by polymorphisms / mutations in both the promoter region and the coding region of the MBL gene. As an acute-phase protein, MBL expression is further upregulated during inflammation. L-ficolin is present in serum at approximately the same concentration as MBL. Therefore, the L-ficolin branch of the lectin pathway can be comparable in strength to the MBL arm in some cases. MBL and ficolin can also function as opsonins. For this reason, phagocytes can target surfaces decorated by MBL and surfaces decorated by ficolin (see Jack et al., J Leukoc Biol., 77(3):328-36(2004), Matsushita and Fujita, Immunobiology, 205(4-5):490-7(2002), Aoyagi et al., J. Immunol, 174(1):418-25(2005)). This opsonization requires the interaction of these proteins with phagocyte receptors (Kuhlman et al., J. Exp. Med. 169:1733, (1989); Matsushita et al., J. Biol. Chem. 271:2448-54, (1996)). The identity of the phagocyte receptor has not been proven.

[0010] Human MBL gives rise to a specific and high-affinity interaction with a unique C1r / C1s-like serine protease called MBL-associated serine protease (MASP) via its collagen-like domain. To date, three MASPs have been described. First, a single enzyme, "MASP", was identified and characterized as the enzyme responsible for 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 revealed 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 proven that the MBL-MASP-2 complex alone is sufficient for complement activation (Vorup-Jensen et al., J. Immunol 165:2093-2100, (2000)). Furthermore, only MASP-2 cleaved C2 and C4 at a high rate (Ambrus et al., J. Immunol, 170:1374-1382, (2003)). Thus, MASP-2 is the protease responsible for activating C4 and C2 to generate C4b2a, the C3 convertase. This is a major difference from the C1 complex of the classical pathway, in which the cooperative action of two specific serine proteases (C1r and C1s) leads to complement system activation. Additionally, a third novel protease, MASP-3, has been isolated (Dahl, M.R. et al., Immunity 15:127-35, 2001). MASP-1 and MASP-3 are alternative splicing products of the same gene.

[0011] MASP has the same domain composition as that of 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 / urchin VEGF / bone morphogenetic protein (CUB) domain, an epidermal growth factor-like domain, a second CUB domain, a tandem array of complement control protein domains, and a serine protease domain. Similar to C1 protease, activation of MASP-2 occurs by cleavage of the Arg-Ile bond adjacent to the serine protease domain. This cleavage results in the enzyme being separated into disulfide-linked A and B chains. The latter consists of the serine protease domain.

[0012] MBL can also bind to an alternative splicing form of MASP-2 known as the 19 kDa MBL-associated protein (MAp19) or small MBL-associated protein (sMAP), which lacks the catalytic activity of MASP2 (Stover, J. Immunol. 162:3481-90, (1999); Takahashi et al., Int. Immunol. 11:859-863, (1999)). MAp19 contains an extra sequence consisting 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 gene and the MASP-2 gene are located on human chromosome 3 and chromosome 1, respectively (Schwaeble et al., Immunobiology 205:455-466, (2002)).

[0013] Evidence from several studies suggests that there are different MBL-MASP complexes and that most of the MASP in serum does not form a complex with MBL (Thiel. et al., J. Immunol. 165:878-887, (2000)). Both H-ficolin and L-ficolin bind to all MASPs as does MBL and activate the lectin complement pathway (Dahl et al., Immunity 15:127-35, (2001); Matsushita et al., J. Immunol. 168:3502-3506, (2002)). The lectin pathway and the classical pathway both form the common C3 convertase (C4b2a), and the two pathways converge at this stage.

[0014] The lectin pathway is widely thought to have a major role in host defense against infection in naive hosts. Strong evidence for the involvement of MBL in host defense has been obtained from the analysis of patients with low serum levels of functional MBL (Kilpatrick, Biochim. Biophys. Acta 1572:401-413, (2002)). Such patients are susceptible to recurrent bacterial and fungal infections. These symptoms typically appear during early childhood, at a time when maternally derived antibody titers are waning but before the full repertoire of the antibody response has developed, an apparent period of vulnerability. This syndrome is often due to mutations at several sites in the collagen portion of MBL, which interfere with the proper formation of MBL oligomers. However, since MBL can function as an opsonin independently of complement, it is not known to what extent the increased susceptibility to infection is due to impairment of complement activation.

[0015] In contrast to the classical and lectin pathways, the recognition functions performed by C1q and lectins in the other two stages are not found to be carried out by the initiating factors of the alternative pathway. Currently, it is widely recognized that the alternative pathway spontaneously undergoes low-level turnover activation. This turnover activation can be readily amplified on foreign surfaces or other abnormal surfaces (bacteria, yeast, virus-infected cells, or damaged tissues) that lack the appropriate molecular elements to suppress spontaneous complement activation. There are four plasma proteins that are directly involved in alternative pathway activation: C3, factor B and factor D, and properdin.

[0016] There is extensive evidence indicating that both the classical complement pathway and the alternative complement pathway are involved in the development of non-infectious human diseases, but the role of the lectin pathway has only recently begun to be evaluated. Recent studies have provided evidence that activation of the lectin pathway may be responsible for complement activation and associated inflammation in ischemia / reperfusion injury. Collard et al., (2000) reported that cultured endothelial cells subjected to oxidative stress bind to MBL and show C3 deposition when exposed to human serum (Collard et al., Am. J. Pathol 156:1549-1556, (2000)). Furthermore, treatment of human serum with blocking anti-MBL monoclonal antibodies inhibited MBL binding and complement activation. These findings were extended to a rat myocardial ischemia-reperfusion model. In this model, rats treated with a blocking antibody against rat MBL showed significantly less myocardial injury than control antibody-treated rats during coronary artery occlusion (Jordan et al., Circulation, 104:1413-1418, (2001)). The molecular mechanism of MBL binding to vascular endothelium after oxidative stress is unknown. Recent studies have suggested that activation of the lectin pathway after oxidative stress may be mediated by MBL binding to vascular endothelial cytokeratin, but not by complex carbohydrates (Collard e al., Am. J. Pathol. 159:1045-1054, (2001)). Other studies have shown that the classical and alternative pathways are involved in the development of ischemia / reperfusion injury, and the role of the lectin pathway in this disease remains controversial (Riedermann, N.C. et al., Am. J. Pathol. 162:363-367, 2003).

[0017] Recent studies have shown that MASP-1 (and perhaps also MASP-3) is required to convert factor D, a second pathway activation enzyme, from its zymogen form to its enzymatically active form (see Takahashi M. et al., J Exp Med 207(1):29-37(2010)). The physiological importance of this process is emphasized by the absence of functional alternative pathway activity in the plasma of MASP-1 / 3-deficient mice. The alternative pathway is required for the generation of C3b from native C3 by proteolysis. Since the alternative pathway C3 convertase (C3bBb) contains C3b, an essential subunit, questions regarding the origin of the first C3b via the alternative pathway have been intractable problems that have inspired extensive research.

[0018] 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 having a terminal carbonyl group that forms a thioester covalent bond with the sulfhydryl group of a cysteine three amino acids away. This bond is labile, and the electrophilic glutamyl-thioester can react with a nucleophilic moiety such as a hydroxyl group or an amino group and can thus form a covalent bond with another molecule. The thioester bond is quite stable when sequestered within the hydrophobic pocket of intact C3. However, when C3 is cleaved by proteolysis into C3a and C3b, the highly reactive thioester bond is exposed on C3b, and after nucleophilic attack by an adjacent moiety containing a hydroxyl group or an amino group, C3b covalently binds to the target. The C3 thioester is thought to have a central role not only in the well-documented role in the covalent binding of C3b to complement targets but also in the initiation of the alternative pathway. According to the widely accepted “tick-over theory,” the alternative pathway is initiated by the generation of the liquid-phase convertase iC3Bb, which is formed from C3 having a hydrolyzed thioester (iC3; C3(H2O)) and factor B (Lachmann, P.J., et al., Springer Semin. Immunopathol. 7:143-162, (1984)). C3b-like C3(H2O) is generated from native C3 by the slow spontaneous hydrolysis of an internal thioester in this protein (Pangburn, M.K., et al., J. Exp. Med. 154:856-867, 1981). The activity of the C3(H2O)Bb convertase deposits C3b molecules on the target surface, thereby initiating the alternative pathway.

[0019] Little is known about the initiators of alternative pathway activation. Activators are thought to include yeast cell wall (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, but due to the complexity and diversity of carbohydrate structures, it is difficult to demonstrate a recognized common molecular determinant. Alternative pathway activation is widely recognized to be controlled by a delicate balance between inhibitory regulatory components of this pathway, such as factor H, factor I, DAF, and CR1, and properdin, the only positive regulator of the alternative pathway (see Schwaeble W.J. and Reid K.B., Immunol Today 20(1):17-21(1999)).

[0020] In addition to the clearly disordered activation mechanism described above, since the generated C3b can participate in the formation of further alternative pathway C3 convertase (C3bBb) together with factor B, the alternative pathway can also provide a powerful amplification loop for lectin / classical pathway C3 convertase (C4b2a). The alternative pathway C3 convertase is stabilized by properdin binding. Properdin extends the half-life of the alternative pathway C3 convertase by 6 to 10 times. Addition of C3b to the alternative pathway C3 convertase results in the formation of the alternative pathway C5 convertase.

[0021] All three pathways (i.e., the classical pathway, the lectin pathway, and the alternative pathway) have been thought to converge at C5, which is cleaved to form products with multiple pro-inflammatory actions. The converged pathway has been called the terminal complement pathway. C5a is the most potent anaphylatoxin that induces changes in smooth muscle tone and vascular tension as well as vascular permeability. It is also a potent chemotactic and activating factor for neutrophils and monocytes. Cell activation via C5a can significantly amplify the inflammatory response by inducing the release of multiple additional inflammatory mediators, including cytokines, hydrolytic enzymes, arachidonic acid metabolites, and reactive oxygen species. When C5 is cleaved, C5b-9, also known as the membrane attack complex (MAC), is formed. There is now strong evidence that sublytic MAC deposition, which is not sufficient to cause lysis, can play an important role in inflammation in addition to its role as a lytic pore-forming complex.

[0022] In addition to its essential role in immune defense, the complement system contributes to tissue damage in many clinical conditions. Therefore, there is an urgent need to develop therapeutically effective complement inhibitors to prevent these side effects. SUMMARY OF THE INVENTION

[0023] Summary This summary is provided to introduce, in simplified form, a broad concept that will be further described in the following detailed description. This summary is not intended to identify key features of the claimed subject matter nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0024] In one aspect, the present invention provides a method of inhibiting microvascular endothelial cell damage and / or thrombosis in a subject suffering from thrombotic microangiopathy (TMA), the method comprising administering to the subject a composition comprising an MASP-2 inhibitory antibody in an amount effective to inhibit MASP-2-dependent complement activation. In some embodiments, the subject has or is at risk of developing TMA selected from the group consisting of atypical hemolytic uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP), and non-typical hemolytic uremic syndrome (HUS). In some embodiments, prior to administration of the composition, the subject is confirmed to exhibit one or more symptoms selected from the group consisting of (i) anemia, (ii) thrombocytopenia, (iii) renal insufficiency, and (iv) elevated creatinine, and the composition is administered in an amount effective to improve the one or more symptoms and over a sufficient period of time. In some embodiments, the MASP-2 inhibitor is an anti-MASP-2 antibody or a fragment thereof. In some embodiments, the MASP-2 inhibitor is an anti-MASP-2 monoclonal antibody or a fragment thereof that specifically binds to a portion of SEQ ID NO:6. In some embodiments, the MASP-2 inhibitor inhibits microvascular endothelial cell damage.

[0025] In another aspect, the present invention provides a method of inhibiting MASP-2-dependent complement activation in a subject suffering from or at risk of developing atypical hemolytic uremic syndrome (aHUS), the method comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation. In one aspect, prior to administration of the composition, the subject is confirmed to exhibit one or more symptoms selected from the group consisting of (i) anemia, (ii) thrombocytopenia, (iii) renal insufficiency, and (iv) high creatinine, and the composition is administered in an amount effective to improve the one or more symptoms and for a sufficient time. In one aspect, the subject suffers from or is at risk of developing non-factor H-dependent aHUS. In one aspect, the subject suffers from aHUS associated with factor I, factor B, or membrane cofactor CD46. In one aspect, the MASP-2 inhibitor is an anti-MASP-2 antibody or fragment thereof, e.g., an anti-MASP-2 monoclonal antibody or fragment thereof that specifically binds to a portion of SEQ ID NO:6. In one aspect, the MASP-2 inhibitor inhibits microvascular endothelial cell damage. In one aspect, the MASP-2 inhibitor inhibits thrombosis.

[0026] In another aspect, the present invention provides a method for reducing the likelihood that a subject at risk of developing atypical hemolytic uremic syndrome (aHUS) will develop clinical symptoms associated with aHUS. The method according to this aspect of the invention comprises: (a) ascertaining the presence in the subject of a genetic marker known to be associated with aHUS; (b) periodically monitoring the subject to ascertain the presence or absence of at least one symptom selected from the group consisting of anemia, thrombocytopenia, renal insufficiency, and elevated creatinine; and (c) administering to the subject a composition comprising an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation when at least one of anemia, thrombocytopenia, renal insufficiency, or elevated creatinine is ascertained to be present, wherein the composition is administered in an amount effective to ameliorate the one or more symptoms and for a sufficient period of time. In one embodiment, the MASP-2 inhibitor is an anti-MASP-2 antibody or a fragment thereof, for example, an anti-MASP-2 monoclonal antibody or a fragment thereof that specifically binds to a portion of SEQ ID NO:6. In one embodiment of the method, step (a) comprises performing a genetic screening test on a sample obtained from the subject and identifying the presence of at least one genetic marker associated with aHUS in a gene selected from the group consisting of complement factor H (CFH), factor I (CFI), factor B (CFB), membrane cofactor CD46, C3, complement factor H-related protein (CFHR1), anticoagulant protein thrombomodulin (THBD), complement factor H-related protein 3 (CFHR3), and complement factor H-related protein 4 (CFHR4). In one embodiment, the method further comprises monitoring the subject for the occurrence of an event known to be associated with the induction of aHUS clinical symptoms, and administering to the subject a composition comprising a MASP-2 inhibitor before, during, or after the occurrence of the triggering event. In one embodiment, the event associated with the induction of aHUS clinical symptoms is selected from the group consisting of drug exposure, infection, malignancy, injury, organ or tissue transplantation, and pregnancy. In one embodiment, the infection is a bacterial infection. In one embodiment, the composition is administered subcutaneously.In one aspect, the MASP-2 inhibitor inhibits microvascular endothelial cell damage. In one aspect, the MASP-2 inhibitor inhibits thrombus formation.

[0027] In another aspect, the present invention provides a method for inhibiting MASP-2-dependent complement activation in a subject suffering from or at risk of developing atypical hemolytic uremic syndrome (aHUS) secondary to infection, the method comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitor effective to inhibit MASP-2 complement activation. In one aspect, the subject is suffering from or at risk of developing non-enteric aHUS associated with Streptococcus pneumoniae (S. pneumoniae) infection. In one aspect, the MASP-2 inhibitor is an anti-MASP-2 antibody or a fragment thereof, such as an anti-MASP-2 monoclonal antibody or a fragment thereof that specifically binds to a portion of SEQ ID NO:6. In one aspect, the MASP-2 inhibitor inhibits microvascular endothelial cell damage. In one aspect, the MASP-2 inhibitor inhibits thrombus formation.

[0028] In another aspect, the present invention provides a method of treating a subject suffering from atypical hemolytic uremic syndrome (aHUS), the method comprising administering to the subject a composition comprising a MASP-2 inhibitor in an amount effective to inhibit MASP-2-dependent complement activation, wherein the administration of the MASP-2 inhibitor is via an intravenous catheter or other catheter delivery method. In one aspect, the method further comprises treating the patient by plasmapheresis. In one aspect, the composition comprising the MASP-2 inhibitor is administered in the absence of plasmapheresis. In one aspect, the composition comprising the MASP-2 inhibitor is administered via a catheter over a first period, and the method further comprises administering the composition comprising the MASP-2 inhibitor over a second period, wherein the composition is administered subcutaneously during the second period. In one aspect, the method further comprises periodically determining the level of at least one complement factor, wherein determining that the level of at least one complement factor is below a standard value or compared to a healthy subject indicates the need to continue treatment with the composition. In one aspect, the MASP-2 inhibitor is an anti-MASP-2 antibody or a fragment thereof, such as an anti-MASP-2 monoclonal antibody or a fragment thereof that specifically binds to a portion of SEQ ID NO:6. In one aspect, the MASP-2 inhibitor inhibits microvascular endothelial cell damage. In one aspect, the MASP-2 inhibitor inhibits thrombus formation.

[0029] In another aspect, the present invention provides a method of treating a subject suffering from thrombotic thrombocytopenic purpura (TTP) or a subject showing symptoms consistent with a diagnosis of TTP, the method comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation, wherein the administration of the MASP-2 inhibitor is administered to the subject via an intravenous catheter or other catheter delivery method. In one aspect, the subject exhibits at least one or more symptoms selected from the group consisting of central nervous system complications, thrombocytopenia, severe cardiac complications, severe pulmonary complications, gastrointestinal infarction, and gangrene. In one aspect, the subject is determined to have a positive presence of an ADAMTS13 inhibitor, and the method further comprises administering an immunosuppressive agent to the subject. In one aspect, the composition comprising the MASP-2 inhibitor is administered in the absence of plasmapheresis over a first period. In one aspect, the subject is determined to have a positive presence of an ADAMTS-13 inhibitor, and the method further comprises administering ADAMTS-13. In one aspect, the method further comprises treating the patient by plasmapheresis. In one aspect, the composition comprising the MASP-2 inhibitor is administered in the presence of plasmapheresis. In one aspect, the composition comprising the MASP-2 inhibitor is administered via a catheter over a first period, and the method further comprises administering the composition comprising the MASP-2 inhibitor over a second period, wherein the composition is administered subcutaneously during the second period. In one aspect, the method further comprises periodically determining the level of at least one complement factor, wherein determining that the level of at least one complement factor is below a standard value or compared to a healthy subject indicates the need to continue treatment with the composition. In one aspect, the MASP-2 inhibitor is an anti-MASP-2 antibody or fragment thereof, such as an anti-MASP-2 monoclonal antibody or fragment thereof that specifically binds to a portion of SEQ ID NO:6. In one aspect, the MASP-2 inhibitor inhibits microvascular endothelial cell damage. In one aspect, the MASP-2 inhibitor inhibits thrombus formation.

[0030] In another aspect, the present invention provides a method for treating a subject suffering from treatment-resistant thrombotic thrombocytopenic purpura (TTP), the method comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation. In one aspect, the composition is administered subcutaneously. In one aspect, the method further comprises periodically determining the level of at least one complement factor, wherein determining that the level of at least one complement factor is below a normal value or compared to a healthy subject indicates the need for continued treatment with the composition. In one aspect, the MASP-2 inhibitor is an anti-MASP-2 antibody or a fragment thereof, such as an anti-MASP-2 monoclonal antibody or a fragment thereof that specifically binds to a portion of SEQ ID NO:6. In one aspect, the MASP-2 inhibitor inhibits microvascular endothelial cell damage. In one aspect, the MASP-2 inhibitor inhibits thrombus formation.

[0031] In another aspect, the present invention provides a method of inhibiting MASP-2-dependent complement activation in a subject suffering from thrombotic microangiopathy (TMA) or at risk of developing TMA, the method comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation, wherein the TMA is at least one of (i) TMA secondary to cancer; (ii) TMA secondary to chemotherapy; or (iii) TMA secondary to transplantation. In some embodiments, the subject is suffering from or at risk of developing TMA secondary to cancer, and the MASP-2 inhibitor is systemically administered to the subject in an amount effective to reduce the risk of developing TMA or reduce the severity of TMA. In some embodiments, the subject is suffering from or at risk of developing TMA secondary to chemotherapy, and the MASP-2 inhibitor is systemically administered to the subject before, during, or after chemotherapy in an amount effective to reduce the risk of developing TMA or reduce the severity of TMA. In some embodiments, the subject is suffering from or at risk of developing TMA secondary to transplantation, and the MASP-2 inhibitor is systemically administered to the subject before, during, or after a transplantation procedure in an amount effective to reduce the risk of developing TMA or reduce the severity of TMA. In some embodiments, the transplantation procedure is an allogeneic hematopoietic stem cell transplantation. In some embodiments, the subject has previously received or is currently receiving treatment with a terminal complement inhibitor that inhibits cleavage of complement protein C5. In some embodiments, the method further comprises administering to the subject a terminal complement inhibitor that inhibits cleavage of complement protein C5, such as a humanized anti-C5 antibody or an antigen-binding fragment thereof, such as eculizumab.

[0032] In another aspect, the present invention provides a method for inhibiting MASP-2-dependent complement activation in a subject, comprising administering to the subject, who has Upshaw-Schulman syndrome (USS) or is at risk of developing USS, a composition comprising an MASP-2 inhibitor in an amount effective to inhibit MASP-2-dependent complement activation. In some embodiments, the method comprises treating a subject at risk of developing USS, and administering to the subject, for a period effective to alleviate or prevent one or more clinical symptoms associated with TTP, an amount of an MASP-2 inhibitor effective to alleviate or prevent the clinical symptoms. In some embodiments, the method further comprises periodically monitoring the subject and administering the MASP-2 inhibitor based on the presence of an event known to be associated with the induction of TTP clinical symptoms. In some embodiments, the method further comprises periodically monitoring the subject and administering the MASP-2 inhibitor based on a determination that anemia, thrombocytopenia, or increased creatinine is present. In some embodiments, the subject has previously received or is currently receiving treatment with a terminal complement inhibitor that inhibits cleavage of complement protein C5. In some embodiments, the method further comprises administering to the subject a terminal complement inhibitor that inhibits cleavage of complement protein C5, such as a humanized anti-C5 antibody or an antigen-binding fragment thereof, such as eculizumab.

[0033] In another aspect, the present invention provides a method for inhibiting MASP-2-dependent complement activation in a subject, comprising administering to the subject, who has Degos disease, a composition comprising an MASP-2 inhibitor in an amount effective to inhibit MASP-2-dependent complement activation. In some embodiments, the subject has previously received or is currently receiving treatment with a terminal complement inhibitor that inhibits cleavage of complement protein C5. In some embodiments, the method further comprises administering to the subject a terminal complement inhibitor that inhibits cleavage of complement protein C5, such as a humanized anti-C5 antibody or an antigen-binding fragment thereof, such as eculizumab.

[0034] In another aspect, the present invention provides a method of inhibiting MASP-2-dependent complement activation in a subject suffering from catastrophic antiphospholipid syndrome (CAPS), the method comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation. In some embodiments, the subject has previously received or is currently receiving treatment with a terminal complement inhibitor that inhibits cleavage of complement protein C5. In some embodiments, the method further comprises administering to the subject a terminal complement inhibitor that inhibits cleavage of complement protein C5, such as a humanized anti-C5 antibody or an antigen-binding fragment thereof, such as eculizumab.

[0035] In some embodiments of any of the disclosed methods of the present invention, the MASP-2 inhibitor is a MASP-2 inhibitory antibody or a fragment thereof. In some embodiments, the MASP-2 inhibitory antibody has reduced effector function. In some embodiments, the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. In some embodiments, the MASP-2 inhibitor is an anti-MASP-2 monoclonal antibody or a fragment thereof that specifically binds to a portion of SEQ ID NO:6. In some embodiments, the anti-MASP-2 antibody or a fragment thereof is selected from the group consisting of recombinant antibodies, antibodies having reduced effector function, chimeric antibodies, humanized antibodies, and human antibodies. In some embodiments, the MASP-2 inhibitory antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2. In some embodiments, the MASP-2 inhibitory antibody is a single-chain molecule. In some embodiments, the MASP-2 inhibitory antibody is selected from the group consisting of IgG1 molecules, IgG2 molecules, and IgG4 molecules. In some embodiments, the MASP-2 inhibitory antibody is an IgG4 molecule comprising an S228P mutation. In some embodiments, the MASP-2 inhibitory antibody has a K of 10 nM or less DBind to human MASP-2. In some embodiments, the MASP-2 inhibitory antibody binds to an epitope in the CCP1 domain of MASP-2. In some embodiments, the MASP-2 inhibitory antibody inhibits C3b deposition in 1% human serum in an in vitro assay method with an IC 50 of 10 nM or less. In some embodiments, the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum with an IC 50 of 30 nM or less.

[0036] In some embodiments of any of the disclosed methods of the present invention, the MASP-2 inhibitory monoclonal antibody or its antigen-binding fragment comprises: (a) (i) a heavy-chain CDR-H1 comprising the amino acid sequence of positions 31-35 of SEQ ID NO: 67; and (ii) a heavy-chain CDR-H2 comprising the amino acid sequence of positions 50-65 of SEQ ID NO: 67; and (iii) a heavy-chain CDR-H3 comprising the amino acid sequence of positions 95-102 of SEQ ID NO: 67, a heavy-chain variable region, and (b) (i) a light-chain CDR-L1 comprising the amino acid sequence of positions 24-34 of SEQ ID NO: 70; and (ii) a light-chain CDR-L2 comprising the amino acid sequence of positions 50-56 of SEQ ID NO: 70; and (iii) a light-chain CDR-L3 comprising the amino acid sequence of positions 89-97 of SEQ ID NO: 70, a light-chain variable region. In some embodiments, the MASP-2 inhibitory monoclonal antibody comprises the heavy-chain variable region shown in SEQ ID NO: 67 and the light-chain variable region shown in SEQ ID NO: 70. In some embodiments, the MASP-2 inhibitory antibody or its antigen-binding fragment specifically recognizes at least a part of the epitope recognized by a reference antibody comprising the heavy-chain variable region shown in SEQ ID NO: 67 and the light-chain variable region shown in SEQ ID NO: 70.

[0037] In another aspect of the invention, provided is a method for inhibiting thrombosis in a subject, comprising the step of administering to the subject an effective amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof that inhibits MASP-2-dependent complement activation. In some embodiments, the MASP-2 inhibitory antibody inhibits thrombosis in serum from a subject suffering from atypical hemolytic uremic syndrome (aHUS) by at least 40% compared to untreated serum. In some embodiments, the MASP-2 inhibitory antibody inhibits thrombosis in serum from a subject suffering from aHUS at a level that is at least 20% higher (e.g., at least 30% higher, at least 40% higher, or at least 50% higher) than the inhibitory effect of the antibody on C5b-9 deposition in serum from the same subject. In some embodiments, the subject is in the acute phase of aHUS. In some embodiments, the subject is in the remission phase of aHUS. In some embodiments, the MASP-2 inhibitory antibody is a monoclonal antibody or a fragment thereof that specifically binds to a portion of SEQ ID NO:6. In some embodiments, the MASP-2 inhibitory antibody or fragment thereof is selected from the group consisting of recombinant antibodies, antibodies with reduced effector function, chimeric antibodies, humanized antibodies, and human antibodies. In some embodiments, the MASP-2 inhibitory antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2. In some embodiments, the MASP-2 inhibitory antibody is a single-chain molecule. In some embodiments, the MASP-2 inhibitory antibody is selected from the group consisting of IgG1 molecules, IgG2 molecules, and IgG4 molecules. In some embodiments, the MASP-2 inhibitory antibody is an IgG4 molecule comprising the S228P mutation. In some embodiments, the MASP-2 inhibitory antibody has a K D d for binding to human MASP-2. In some embodiments, the MASP-2 inhibitory antibody binds to an epitope in the CCP1 domain of MASP-2. In some embodiments, the MASP-2 inhibitory antibody inhibits C3b deposition in 1% human serum in an in vitro assay at an IC 50 of 10 nM or less. In some embodiments, the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum at an IC of 30 nM or less50 Inhibits. In some embodiments, the MASP-2 inhibitory monoclonal antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising (i) a heavy chain CDR-H1 comprising the amino acid sequence of amino acids 31-35 of SEQ ID NO: 67; and (ii) a heavy chain CDR-H2 comprising the amino acid sequence of amino acids 50-65 of SEQ ID NO: 67; and (iii) a heavy chain CDR-H3 comprising the amino acid sequence of amino acids 95-102 of SEQ ID NO: 67, and (b) a light chain variable region comprising (i) a light chain CDR-L1 comprising the amino acid sequence of amino acids 24-34 of SEQ ID NO: 70; and (ii) a light chain CDR-L2 comprising the amino acid sequence of amino acids 50-56 of SEQ ID NO: 70; and (iii) a light chain CDR-L3 comprising the amino acid sequence of amino acids 89-97 of SEQ ID NO: 70. In some embodiments, the MASP-2 inhibitory monoclonal antibody comprises the heavy chain variable region shown in SEQ ID NO: 67 and the light chain variable region shown in SEQ ID NO: 70. In some embodiments, the MASP-2 inhibitory antibody or antigen-binding fragment thereof specifically recognizes at least a portion of the epitope recognized by a reference antibody comprising the heavy chain variable region shown in SEQ ID NO: 67 and the light chain variable region shown in SEQ ID NO: 70.

[0038] In another aspect, the present invention provides a method of treating a subject suffering from plasma therapy-resistant aHUS, the method comprising administering to the subject a composition comprising an MASP-2 inhibitory antibody in an amount effective to inhibit MASP-2-dependent complement activation. In one aspect, the MASP-2 inhibitor is an anti-MASP-2 monoclonal antibody or a fragment thereof that specifically binds to human MASP-2. In one aspect, the antibody or fragment thereof is selected from the group consisting of recombinant antibodies, antibodies having reduced effector function, chimeric antibodies, humanized antibodies, and human antibodies. In one aspect, the subject has previously been treated or is currently being treated with a terminal complement inhibitor that inhibits cleavage of complement protein C5; for example, the terminal complement inhibitor is a humanized anti-C5 antibody or an antigen-binding fragment thereof. In one aspect, the method further comprises treating the patient by plasmapheresis. In one aspect, the composition comprising the MASP-2 inhibitory antibody is administered in the absence of plasmapheresis.

[0039] In another aspect, the present invention provides a method of treating a subject suffering from TMA associated with hematopoietic stem cell transplantation, the method comprising administering to the subject a composition comprising an MASP-2 inhibitory antibody in an amount effective to inhibit MASP-2-dependent complement activation. In one aspect, the MASP-2 inhibitor is an anti-MASP-2 monoclonal antibody or a fragment thereof that specifically binds to human MASP-2. In one aspect, the antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody having reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In one aspect, the subject has previously received or is currently receiving treatment with a terminal complement inhibitor that inhibits cleavage of complement protein C5. In one aspect, the subject is suffering from TMA associated with hematopoietic stem cell transplantation that is resistant to treatment with platelet transfusion and / or resistant to treatment with plasma exchange. In one aspect, the MASP-2 inhibitory antibody is administered in an amount effective to improve at least one or more clinical parameters associated with TMA associated with hematopoietic stem cell transplantation, such as increasing the platelet count (e.g., increasing at least 2-fold, at least 3-fold, at least 4-fold the platelet count prior to treatment), increasing haptoglobin, and / or decreasing lactate dehydrogenase.

[0040] In another aspect, the present invention provides a method for treating a human subject suffering from persistent thrombotic microangiopathy associated with hematopoietic stem cell transplantation (HSCT-TMA), the method comprising administering to the subject a composition comprising an MASP-2 inhibitory antibody or an antigen-binding fragment thereof in an amount effective to inhibit MASP-2-dependent complement activation. In one aspect, the method further comprises identifying a human subject having persistent TMA associated with hematopoietic stem cell transplantation prior to the step of administering to the subject a composition comprising an MASP-2 inhibitory antibody or an antigen-binding fragment thereof in an amount effective to inhibit MASP-2-dependent complement activation. In one aspect, the MASP-2 inhibitory antibody is a monoclonal antibody or a fragment thereof that specifically binds to human MASP-2. In one aspect, the antibody or antigen-binding fragment thereof is selected from the group consisting of recombinant antibodies, antibodies having reduced effector function, chimeric antibodies, humanized antibodies, and human antibodies. In one aspect, the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. In one aspect, the MASP-2 inhibitory antibody reduces C3b deposition in 90% human serum to an IC 50It inhibits. In one aspect, the MASP-2 inhibitory antibody or its antigen-binding fragment comprises a heavy chain variable region containing CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO:67, and a light chain variable region containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:70. In one aspect, the MASP-2 inhibitory antibody is administered to a patient in the absence of plasmapheresis. In one aspect, the subject has previously received or is currently receiving treatment with a humanized anti-C5 antibody or its antigen-binding fragment. For example, the terminal complement inhibitor is a humanized anti-C5 antibody or its antigen-binding fragment. In one aspect, the MASP-2 inhibitory antibody is systemically delivered to the subject. In one aspect, the MASP-2 inhibitory antibody or its antigen-binding fragment is administered in an amount effective to improve at least one or more of the following clinical parameters associated with persistent TMA associated with hematopoietic stem cell transplantation: (i) increasing the platelet count (e.g., increasing at least 2-fold, at least 3-fold, at least 4-fold the platelet count before treatment); (ii) increasing haptoglobin; (iii) decreasing lactate dehydrogenase (LDH); and / or (iv) decreasing creatinine.

[0041] In another aspect, the present invention provides a method of treating a human subject suffering from or at risk of developing graft-versus-host disease (GVHD), the method comprising administering to the subject a composition comprising an MASP-2 inhibitory antibody or an antigen-binding fragment thereof in an amount effective to inhibit MASP-2-dependent complement activation. In one aspect, the subject has previously received, is currently receiving, or is scheduled to receive a hematopoietic stem cell transplant. In one aspect, the method further comprises identifying a human subject suffering from or at risk of developing graft-versus-host disease prior to the step of administering to the subject a composition comprising an MASP-2 inhibitory antibody or an antigen-binding fragment thereof in an amount effective to inhibit MASP-2-dependent complement activation. In one aspect, the MASP-2 inhibitory antibody is a monoclonal antibody or a fragment thereof that specifically binds to human MASP-2. In one aspect, the MASP-2 inhibitory antibody or fragment thereof is selected from the group consisting of recombinant antibodies, antibodies with reduced effector function, chimeric antibodies, humanized antibodies, and human antibodies. In one aspect, the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. In one aspect, the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum at an IC 50 of 30 nM or less. In one aspect, the MASP-2 inhibitory antibody is systemically delivered to the subject. In one aspect, the subject is suffering from acute GVHD. In one aspect, the subject is suffering from steroid-resistant GVHD. In one aspect, the MASP-2 inhibitory antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO:67, and a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:70.

[0042] In another aspect, the present invention provides a method of treating, preventing, or alleviating one or more neurological symptoms associated with graft-versus-host disease or TMA, the method comprising administering to a subject in need thereof a composition comprising an MASP-2 inhibitory antibody or an antigen-binding fragment thereof in an amount effective to inhibit MASP-2-dependent complement activation. In one aspect, the one or more neurological symptoms associated with graft-versus-host disease or TMA are selected from the group consisting of weakness, abnormal sensation, quadriplegia, sensorimotor impairment, dysautonomic polyneuropathy, and / or neurogenic bladder. In one aspect, the subject has undergone a hematopoietic stem cell transplantation and suffers from one or more neurological symptoms selected from the group consisting of abnormal sensation, quadriplegia, and neurogenic bladder. In one aspect, the subject has undergone a hematopoietic stem cell transplantation and suffers from graft-versus-host disease. In one aspect, the subject has undergone a hematopoietic stem cell transplantation and suffers from HSCT-TMA. In one aspect, the MASP-2 inhibitory antibody is a monoclonal antibody or a fragment thereof that specifically binds to human MASP-2. In one aspect, the antibody or fragment thereof is selected from the group consisting of recombinant antibodies, antibodies with reduced effector function, chimeric antibodies, humanized antibodies, and human antibodies. In one aspect, the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. In one aspect, the MASP-2 inhibitory antibody reduces C3b deposition in 90% human serum to 30 nM or less of the IC 50It inhibits. In one aspect, the MASP-2 inhibitory antibody is systemically delivered to the subject. In one aspect, the method further comprises identifying a human subject suffering from one or more neurological symptoms associated with hematopoietic stem cell transplantation prior to administering to the subject a composition comprising an MASP-2 inhibitory antibody or an antigen-binding fragment thereof in an amount effective to inhibit MASP-2-dependent complement activation. In one aspect, the MASP-2 inhibitory antibody or an antigen-binding fragment thereof comprises a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO:67, and a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:70.

[0043] In another aspect, the present invention provides a method of treating a human subject suffering from or at risk of developing diffuse alveolar hemorrhage (DAH) associated with hematopoietic stem cell transplantation, the method comprising administering to the subject a composition comprising an MASP-2 inhibitory antibody or an antigen-binding fragment thereof in an amount effective to inhibit MASP-2-dependent complement activation. In one aspect, the subject has previously received, is currently receiving, or is scheduled to receive a hematopoietic stem cell transplantation. In one aspect, the method further comprises identifying a human subject suffering from or at risk of developing diffuse alveolar hemorrhage (DAH) prior to administering to the subject a composition comprising an MASP-2 inhibitory antibody or an antigen-binding fragment thereof in an amount effective to inhibit MASP-2-dependent complement activation. In one aspect, the MASP-2 inhibitory antibody is a monoclonal antibody or a fragment thereof that specifically binds to human MASP-2. In one aspect, the MASP-2 inhibitory antibody or a fragment thereof is selected from the group consisting of recombinant antibodies, antibodies with reduced effector function, chimeric antibodies, humanized antibodies, and human antibodies. In one aspect, the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. In one aspect, the MASP-2 inhibitory antibody reduces C3b deposition in 90% human serum to an IC of 30 nM or less 50It inhibits. In one aspect, the MASP-2 inhibitory antibody is systemically delivered to the subject. In one aspect, the MASP-2 inhibitory antibody or its antigen-binding fragment comprises a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO:67, and a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:70. In another aspect, the present invention provides a composition for inhibiting side effects of MASP-2-dependent complement activation, comprising a therapeutically effective amount of a MASP-2 inhibitor such as a MASP-2 inhibitory antibody and a pharmaceutically acceptable carrier. Also provided is a method for manufacturing a medicament for use in inhibiting side effects of MASP-2-dependent complement activation in a living subject in need thereof, the medicament comprising a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier. Also provided is a method for manufacturing a medicament for use in inhibiting MASP-2-dependent complement activation for the treatment of each of the conditions, diseases, and disorders described hereinbelow.

[0044] In another aspect, the present invention provides a method of treating a human subject who has or is at risk of developing veno-occlusive disease (VOD) associated with hematopoietic stem cell transplantation, the method comprising administering to the subject a composition comprising an MASP-2 inhibitory antibody or antigen-binding fragment thereof in an amount effective to inhibit MASP-2-dependent complement activation. In one aspect, the subject has previously received, is currently receiving, or is scheduled to receive a hematopoietic stem cell transplantation. In one aspect, the method further comprises identifying a human subject who has or is at risk of developing veno-occlusive disease (VOD) prior to the step of administering to the subject a composition comprising an MASP-2 inhibitory antibody or antigen-binding fragment thereof in an amount effective to inhibit MASP-2-dependent complement activation. In one aspect, the MASP-2 inhibitory antibody is a monoclonal antibody or fragment thereof that specifically binds to human MASP-2. In one aspect, the MASP-2 inhibitory antibody or fragment thereof is selected from the group consisting of recombinant antibodies, antibodies with reduced effector function, chimeric antibodies, humanized antibodies, and human antibodies. In one aspect, the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. In one aspect, the MASP-2 inhibitory antibody has an IC 50It inhibits. In one aspect, the MASP-2 inhibitory antibody is systemically delivered to a subject. In one aspect, the MASP-2 inhibitory antibody or its antigen-binding fragment comprises a heavy-chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO:67, and a light-chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:70. In another aspect, the present invention provides a composition for inhibiting side effects of MASP-2-dependent complement activation, comprising a therapeutically effective amount of a MASP-2 inhibitor such as a MASP-2 inhibitory antibody and a pharmaceutically acceptable carrier. Also provided is a method for manufacturing a medicament for use in inhibiting side effects of MASP-2-dependent complement activation in a living subject in need thereof, the medicament comprising a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier. Also provided is a method for manufacturing a medicament for use in inhibiting MASP-2-dependent complement activation for the treatment of each of the conditions, diseases, and disorders described hereinbelow.

[0045] The methods, compositions, and medicaments of the present invention are useful for inhibiting side effects of in vivo MASP-2-dependent complement activation in mammalian subjects, including humans who may have or are at risk of developing thrombotic microangiopathy (TMA), and / or subjects who may have or are at risk of developing graft-versus-host disease (GVHD), and / or subjects who may have or are at risk of developing diffuse alveolar hemorrhage after stem cell transplantation, and / or subjects who may have or are at risk of developing veno-occlusive disease (VOD), as further described herein. [Inventive concept 1001] A method of treating a human subject who has or is at risk of developing graft-versus-host disease (GVHD), the method comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or its antigen-binding fragment effective to inhibit MASP-2-dependent complement activation. [Inventive concept 1002] The method of the present invention 1001, wherein the MASP-2 inhibitory antibody is a monoclonal antibody or a fragment thereof that specifically binds to human MASP-2. [The present invention 1003] The method of the present invention 1001, wherein the antibody or a fragment thereof is selected from the group consisting of a recombinant antibody, an antibody having reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. [The present invention 1004] The method of the present invention 1001, wherein the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. [The present invention 1005] The method of the present invention 1001, wherein the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum with an IC 50 of 30 nM or less. [The present invention 1006] The method of the present invention 1001, wherein the MASP-2 inhibitory antibody is systemically delivered to the subject. [The present invention 1007] The method of the present invention 1001, further comprising the step of identifying a human subject who has or is at risk of developing graft-versus-host disease prior to administering to the subject a composition comprising an effective amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof that inhibits MASP-2-dependent complement activation. [The present invention 1008] The method of the present invention 1001, wherein the subject has previously received, is currently receiving, or is scheduled to receive a hematopoietic stem cell transplantation. [The present invention 1009] The method of the present invention 1001, wherein the subject has acute GVHD. [The present invention 1010] The method of the present invention 1001, wherein the subject has chronic GVHD. [The present invention 1011] The method of the present invention 1001, wherein the subject has steroid-resistant GVHD. [The present invention 1012] The method of the present invention 1001, wherein the MASP-2 inhibitory antibody or an antigen-binding fragment thereof comprises a heavy-chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO:67, and a light-chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:70. [The present invention 1013] The method according to any one of the present inventions 1001 to 1012, comprising the step of administering to a subject a composition comprising the MASP-2 inhibitory antibody at least once a week at a dose of 1 mg / kg to 10 mg / kg. [The present invention 1014] A method for treating, preventing, or alleviating one or more neurological symptoms associated with graft-versus-host disease or HSCT-TMA, the method comprising administering to a subject a composition comprising an MASP-2 inhibitory antibody or an antigen-binding fragment thereof in an amount effective to inhibit MASP-2-dependent complement activation before, during, or after the subject undergoes hematopoietic stem cell transplantation. [The present invention 1015] The method of the present invention 1014, wherein one or more neurological symptoms associated with graft-versus-host disease or HSCT-TMA are selected from the group consisting of weakness, abnormal sensation, quadriplegia, sensorimotor disorder, autonomic neuropathic polyneuropathy, and / or neurogenic bladder. [The present invention 1016] The method of the present invention 1014, wherein the subject has undergone hematopoietic stem cell transplantation and the subject suffers from one or more neurological symptoms selected from the group consisting of weakness, abnormal sensation, quadriplegia, sensorimotor disorder, autonomic neuropathic polyneuropathy, and / or neurogenic bladder. [The present invention 1017] The method according to any one of the present inventions 1014 to 1016, wherein the subject has undergone hematopoietic stem cell transplantation and suffers from graft-versus-host disease. [The present invention 1018] The method according to any one of the present inventions 1014 to 1017, wherein the subject has undergone hematopoietic stem cell transplantation and suffers from HSCT-TMA. [The present invention 1019] The method of the present invention 1014, wherein the MASP-2 inhibitory antibody is a monoclonal antibody or a fragment thereof that specifically binds to human MASP-2. [The present invention 1020] The method of the present invention 1014, wherein the antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody having reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. [The present invention 1021] The method of the present invention 1014, wherein the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. [The present invention 1022] The method of the present invention 1014, wherein the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum with an IC 50 of 30 nM or less. [The present invention 1023] The method of the present invention 1014, wherein the MASP-2 inhibitory antibody is systemically delivered to the subject. [The present invention 1024] The method of the present invention 1014, further comprising identifying a human subject suffering from one or more neurological symptoms associated with hematopoietic stem cell transplantation prior to administering to the subject a composition comprising an MASP-2 inhibitory antibody or an antigen-binding fragment thereof in an amount effective to inhibit MASP-2-dependent complement activation. [The present invention 1025] The method of the present invention 1014, wherein the MASP-2 inhibitory antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO:67, and a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:70. [The present invention 1026] A method of treating a human subject suffering from or at risk of developing diffuse alveolar hemorrhage, the method comprising administering to the subject a composition comprising an MASP-2 inhibitory antibody or an antigen-binding fragment thereof in an amount effective to inhibit MASP-2-dependent complement activation. [The present invention 1027] The method of the present invention 1026, wherein the MASP-2 inhibitory antibody is a monoclonal antibody or a fragment thereof that specifically binds to human MASP-2. [The present invention 1028] The method of the present invention 1026, wherein the antibody or a fragment thereof is selected from the group consisting of a recombinant antibody, an antibody having reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. [The present invention 1029] The method of the present invention 1026, wherein the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. [The present invention 1030] The method of the present invention 1026, wherein the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum with an IC 50 of 30 nM or less. [The present invention 1031] The method of the present invention 1026, wherein the MASP-2 inhibitory antibody is systemically delivered to the subject. [The present invention 1032] The method of the present invention 1026, further comprising identifying a human subject who has or is at risk of developing diffuse alveolar hemorrhage prior to administering to the subject a composition comprising an effective amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof that inhibits MASP-2-dependent complement activation. [The present invention 1033] The method of the present invention 1026, wherein the subject has previously received, is currently receiving, or is scheduled to receive a hematopoietic stem cell transplantation. [The present invention 1034] The method of the present invention 1026, wherein the MASP-2 inhibitory antibody or an antigen-binding fragment thereof comprises a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO:67, and a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:70. [The present invention 1035] The method according to any one of the present inventions 1026 to 1034, wherein the subject has received a hematopoietic stem cell transplantation and has diffuse alveolar hemorrhage. [The present invention 1036] A method of treating a human subject suffering from or at risk of developing venous occlusion, the method comprising administering to the subject a composition comprising an MASP-2 inhibitory antibody or an antigen-binding fragment thereof in an amount effective to inhibit MASP-2-dependent complement activation. [Inventive concept 1037] The method of inventive concept 1036, wherein the MASP-2 inhibitory antibody is a monoclonal antibody or a fragment thereof that specifically binds to human MASP-2. [Inventive concept 1038] The method of inventive concept 1036, wherein the antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody having reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. [Inventive concept 1039] The method of inventive concept 1036, wherein the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. [Inventive concept 1040] The method of inventive concept 1036, wherein the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum with an IC 50 of 30 nM or less. [Inventive concept 1041] The method of inventive concept 1036, wherein the MASP-2 inhibitory antibody is systemically delivered to the subject. [Inventive concept 1042] The method of inventive concept 1036, further comprising identifying a human subject suffering from or at risk of developing venous occlusion prior to the step of administering to the subject a composition comprising an MASP-2 inhibitory antibody or an antigen-binding fragment thereof in an amount effective to inhibit MASP-2-dependent complement activation. [Inventive concept 1043] The method of inventive concept 1036, wherein the subject has previously received, is currently receiving, or is scheduled to receive a hematopoietic stem cell transplant. [Inventive concept 1044] The method of the present invention 1036, wherein the MASP-2 inhibitory antibody or an antigen-binding fragment thereof comprises a heavy chain variable region containing CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO: 67, and a light chain variable region containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO: 70. [The present invention 1045] Any of the methods of the present invention 1036 to 1044, wherein the subject has received a hematopoietic stem cell transplantation and suffers from veno-occlusive disease. [Brief Description of the Drawings]

[0046] Many of the foregoing aspects and attendant advantages of the present invention will become more readily appreciated and understood by reference to the following detailed description when taken in conjunction with the accompanying drawings.

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Mode for Carrying Out the Invention

[0047] Explanation of Sequence Listing 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-2 gDNA (exons 1-6) Antigen: (based on MASP-2 mature protein) SEQ ID NO:8 CUBI sequence (aa1-121) SEQ ID NO:9 CUBEGF sequence (aa1-166) SEQ ID NO:10 CUBEGFCUBII (aa1-293) SEQ ID NO:11 EGF region (aa122 - 166) SEQ ID NO:12 Serine protease domain (aa429 - 671) SEQ ID NO:13 Inactive serine protease domain (aa610 - 625 with mutation from Ser618 to Ala) TIFF2025108730000002.tif63138 Peptide inhibitor: SEQ ID NO:20 MBL full - length cDNA SEQ ID NO:21 MBL full - length protein SEQ ID NO:22 OGK - X - GP (consensus binding) TIFF2025108730000003.tif71134 Expression inhibitor: SEQ ID NO:30 cDNA of CUBI - EGF domain (nucleotides 22 - 680 of SEQ ID NO:4) SEQ ID NO:31 TIFF2025108730000004.tif5134 Nucleotides 12 - 45 (sense) of SEQ ID NO:4 containing the MASP - 2 translation start point SEQ ID NO:32 TIFF2025108730000005.tif5134 Nucleotides 361 - 396 (sense) of SEQ ID NO:4 encoding the region containing the MASP - 2 MBL binding site SEQ ID NO:33 TIFF2025108730000006.tif5134 Nucleotides 610 - 642 of SEQ ID NO:4 encoding the region containing the CUBII domain Cloning primers: TIFF2025108730000007.tif41134 SEQ ID NOs: 38 - 47 are cloning primers for humanized antibodies. SEQ ID NO:48 is a 9aa peptide bond. Expression vectors: SEQ ID NO:49 is a MASP - 2 mini - gene insert. SEQ ID NO:50 is mouse MASP-2 cDNA. SEQ ID NO:51 is mouse MASP-2 protein (w / leader). SEQ ID NO:52 is mature mouse MASP-2 protein. SEQ ID NO:53 is rat MASP-2 cDNA. SEQ ID NO:54 is rat MASP-2 protein (w / leader). SEQ ID NO:55 is mature rat MASP-2 protein. SEQ ID NOs:56 - 59 are oligonucleotides for site-directed mutagenesis of human MASP-2 used to generate human MASP-2A. SEQ ID NOs:60 - 63 are oligonucleotides for site-directed mutagenesis of mouse MASP-2 used to generate mouse MASP-2A. SEQ ID NOs:64 - 65 are oligonucleotides for site-directed mutagenesis of rat MASP-2 used to generate rat MASP-2A. SEQ ID NO:66 DNA encoding the heavy chain variable region (VH) of 17D20_dc35VH21N11VL (OMS646) (without signal peptide) SEQ ID NO:67 Heavy chain variable region (VH) polypeptide of 17D20_dc35VH21N11VL (OMS646) SEQ ID NO:68 Heavy chain variable region (VH) polypeptide of 17N16mc SEQ ID NO:69 DNA encoding the light chain variable region (VL) of 17D20_dc35VH21N11VL (OMS646) SEQ ID NO:70 Light chain variable region (VL) polypeptide of 17D20_dc35VH21N11VL (OMS646) SEQ ID NO:71 Light chain variable region (VL) polypeptide of 17N16_dc17N9

[0048] Detailed Description The present invention is based on the surprising discovery by the inventors that it is possible to inhibit the lectin-mediated MASP-2 pathway while leaving the classical pathway intact. The present invention also describes the use of MASP-2 as a therapeutic target for inhibiting cytotoxicity associated with lectin-mediated complement pathway activation while leaving the components of the classical (C1q-dependent) pathway of the immune system intact.

[0049] I. Definitions Unless otherwise defined herein, all terms used herein have the same meaning as would be understood by one of ordinary skill in the art of the present invention. To clarify the terms used in the specification and claims for the purpose of describing the present invention, the following definitions are provided.

[0050] As used herein, the term "MASP-2-dependent complement activation" refers to the formation of the lectin pathway C3 convertase C4b2a and the subsequent formation of the C5 convertase C4b2a(C3b)n upon accumulation of the C3 cleavage product C3b, which occurs under physiological conditions (i.e., in the presence of Ca ++ ), and which has been found to mainly cause opsonization.

[0051] As used herein, the term "alternative pathway" refers to complement activation induced by, for example, zymosan derived from fungal cell walls and yeast cell walls, lipopolysaccharide (LPS) derived from the outer membrane of Gram-negative bacteria, and rabbit erythrocytes, as well as many pure polysaccharides, rabbit erythrocytes, viruses, bacteria, animal tumor cells, parasites, and damaged cells, and which has conventionally been thought to result from the spontaneous proteolytic generation of C3b from complement factor C3.

[0052] As used herein, the term "lectin pathway" refers to complement activation that occurs via the 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).

[0053] As used herein, the term "classical pathway" refers to complement activation induced by an antibody bound to a foreign particle and requiring the binding of the recognition molecule C1q.

[0054] As used herein, the term "MASP-2 inhibitor" refers to any agent that binds to MASP-2 or directly interacts with MASP-2 to effectively inhibit MASP-2-dependent complement activation, including anti-MASP-2 antibodies and their MASP-2-binding fragments, natural and synthetic peptides, small molecules, soluble MASP-2 receptors, expression inhibitors, and isolated natural inhibitors, and also includes peptides that compete with MASP-2 in the binding to another recognition molecule (e.g., MBL, H-ficolin, M-ficolin, or L-ficolin) in the lectin pathway, but does not include antibodies that bind to such other recognition molecules. A MASP-2 inhibitor useful in the methods of the present invention can reduce MASP-2-dependent complement activation by more than 20%, e.g., more than 50%, e.g., more than 90%. In one embodiment, the MASP-2 inhibitor reduces MASP-2-dependent complement activation by more than 90% (i.e., results in no more than 10% or less of MASP-2 complement activation).

[0055] As used herein, the term "antibody" includes any antibody-producing mammal (e.g., primates including mouse, rat, rabbit, and human) that specifically binds to a target polypeptide, e.g., a MASP-2 polypeptide or a part thereof, or an antibody and antibody fragment derived from a hybridoma, phage selection, recombinant expression, or transgenic animal (or other methods for producing an antibody or antibody fragment). The term "antibody" 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 animal, peptide synthesis, etc.). Exemplary antibodies include polyclonal antibodies, monoclonal antibodies, and recombinant antibodies; pan-specific, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies); humanized antibodies; mouse antibodies; chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies; and anti-idiotypic antibodies, and may be any intact antibody or fragment thereof. As used herein, the term "antibody" includes not only intact polyclonal or monoclonal antibodies, but also fragments thereof (e.g., dAb, Fab, Fab', F(ab')2, Fv), single-chain (ScFv), synthetic variants, natural variants, antibody moieties, and fusion proteins containing an antigen-binding fragment of the required specificity, humanized antibodies, chimeric antibodies, and any other modified constructs of immunoglobulin molecules containing an antigen-binding site or fragment (epitope recognition site) of the required specificity.

[0056] "Monoclonal antibody" refers to a homogeneous population of antibodies, and 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 fragments thereof (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) that have the required specificity and ability to bind to the epitope. This term is not intended to be limited in terms of the antibody source or the way in which the antibody is produced (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".

[0057] As used herein, the term "antibody fragment" refers to a portion derived from or related to a full-length antibody, e.g., an anti-MASP-2 antibody, and generally includes its antigen-binding region or variable region. 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.

[0058] As used herein, the "single-chain Fv" or "scFv" antibody fragment contains the V H domain or V L domain of an antibody, and these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further contains a polypeptide linker between the V H domain and the V L domain, so that the scFv can form a structure desirable for antigen binding.

[0059] As used herein, a "chimeric antibody" is a recombinant protein that contains variable domains and complementarity-determining regions derived from non-human species (e.g., rodents) antibodies, but the remainder of the antibody molecule is derived from human antibodies.

[0060] As used herein, a "humanized antibody" is a chimeric antibody that contains a minimal sequence that matches a specific complementarity-determining region derived from a non-human immunoglobulin, transplanted into a human antibody framework. A humanized antibody is typically a recombinant protein in which only the antibody complementarity-determining regions are derived from non-human sources.

[0061] As used herein, the term "mannan-binding lectin" ("MBL") is synonymous with mannan-binding protein ("MBP").

[0062] As used herein, "membrane attack complex" ("MAC") refers to a complex of five terminal complement components (a combination of C5b and C6, C7, C8, and C9) (also called C5b-9) that enters the membrane and disrupts the membrane.

[0063] As used herein, "subject" includes, but is not limited to, all mammals, including humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs, and rodents.

[0064] 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).

[0065] In the broadest sense, natural amino acids can be grouped based on the chemical properties of the side chains of each amino acid. "Hydrophobic" amino acids mean Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys or Pro. "Hydrophilic" amino acids mean Gly, Asn, Gln, Ser, Thr, Asp, Glu, Lys, Arg, or His. This amino acid group can be further divided into sub-groups as follows. "Uncharged hydrophilic" amino acids mean Ser, Thr, Asn, or Gln. "Acidic" amino acids mean Glu or Asp. "Basic" amino acids mean Lys, Arg, or His.

[0066] As used herein, the term "conservative amino acid substitution" is exemplified by substitutions between amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartic acid and glutamic acid, (5) glutamine and asparagine, and (6) lysine, arginine, and histidine.

[0067] As used herein, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term covers oligonucleobases consisting of natural nucleotides, sugars, and internucleoside (backbone) covalent bonds, as well as oligonucleotides with non-natural modifications.

[0068] As used herein, "epitope" refers to a site on a protein (e.g., human MASP-2 protein) to which an antibody binds. "Overlapping epitopes" include at least 1 (e.g., 2, 3, 4, 5, or 6) common amino acid residues, including linear and non-linear epitopes.

[0069] As used herein, the terms "polypeptide", "peptide", and "protein" are used interchangeably and mean any peptide-bonded amino acid chain, regardless of length or post-translational modification. The MASP-2 protein described herein may contain a wild-type protein, may be a wild-type protein, or may be a variant 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 within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine, and threonine; lysine, histidine, and arginine; and phenylalanine and tyrosine.

[0070] In some embodiments, the human MASP-2 protein may have an amino acid sequence that is 70 (e.g., 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100)% identical, or more than 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)% identical, to the human MASP-2 protein having the amino acid sequence set forth in SEQ ID NO:5.

[0071] In some embodiments, the peptide fragment may be at least 6 (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, or 600, or more) amino acid residues (e.g., at least 6 contiguous amino acid residues of SEQ ID NO:5). In some embodiments, the antigenic peptide fragment of the human MASP-2 protein is 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, less than 46, less than 45, less than 44, less than 43, less than 42, less than 41, less than 40, less than 39, less than 38, less than 37, less than 36, less than 35, less than 34, less than 33, less than 32, less than 31, less than 30, less than 29, less than 28, less than 27, less than 26, less than 25, less than 24, less than 23, less than 22, less than 21, less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, or less than 6) amino acid residues (e.g., any one of SEQ ID NO:5 having less than 500 contiguous amino acid residues).

[0072] Percent (%) amino acid sequence identity is defined as the percent of amino acids in a candidate sequence that are identical to the amino acids in a reference sequence, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps if necessary. For the purpose of determining percent sequence identity, alignment can be achieved in various ways within the skill in 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 the maximum alignment over the full length of the sequences being compared, can be determined by known methods.

[0073] II. Summary of the Invention Lectins (MBL, M-ficolin, H-ficolin, L-ficolin, and CL-11) are specific recognition molecules that induce the innate complement system, which includes the lectin-initiated pathway and associated terminal pathway amplification loops that amplify the activation of terminal complement effector molecules initiated by the lectin. C1q is a specific recognition molecule that induces the adaptive complement system, which includes the classical-initiated pathway and associated terminal pathway amplification loops that amplify the activation of terminal complement effector molecules initiated by C1q. The inventors refer to these two major complement activation systems as the lectin-dependent complement system and the C1q-dependent complement system, respectively.

[0074] In addition to its essential role in immune defense, the complement system contributes to tissue damage in many clinical conditions. Therefore, there is an urgent need to develop therapeutically effective complement inhibitors to prevent these side effects. Recognizing that it is possible to inhibit the lectin-mediated MASP-2 pathway while leaving the classical pathway intact, it has become evident that it is highly desirable to specifically inhibit only the complement activation system that causes a particular pathological condition without completely blocking the immune defense ability of the complement. For example, in disease states where complement activation is mainly mediated by the lectin-dependent complement system, it is considered advantageous to specifically inhibit only this system. For this purpose, the C1q-dependent complement activation system is thought to remain intact in order to handle immune complex processing and assist host defense against infections.

[0075] In the development of therapeutic substances that specifically inhibit the lectin-dependent complement system, the preferred protein component of interest is MASP-2. Among all known protein components of the lectin-dependent complement system (MBL, H-ficolin, M-ficolin, L-ficolin, MASP-2, C2-C9, factor B, factor D, and properdin), MASP-2 is the only one that is unique to the lectin-dependent complement system and is required for the system to function. Lectins (MBL, H-ficolin, M-ficolin, L-ficolin, and CL-11) are also components unique to the lectin-dependent complement system. However, it is thought that the activation of the system is not necessarily inhibited even if any one of these lectin components is missing due to lectin redundancy. To ensure inhibition of the lectin-dependent complement activation system, it is considered necessary to inhibit all five types of lectins. Furthermore, since MBL and ficolin are known to have opsonin activity independent of complement, it is thought that inhibiting the lectin function will result in the loss of this beneficial host defense mechanism against infectious diseases. In contrast, when MASP-2 is the inhibition target, this complement-independent lectin opsonin activity is thought to remain intact. An additional benefit of MASP-2 as a therapeutic target for inhibiting the lectin-dependent complement activation system is that the MASP-2 plasma concentration (about 500 ng / ml) is among the lowest plasma concentrations of complement proteins. Therefore, a low concentration of a high-affinity MASP-2 inhibitor corresponding to that may be sufficient to obtain complete inhibition (Moller-Kristensen, M., et al., J. Immunol Methods 282:159-167, 2003).

[0076] III. Role of MASP-2 in thrombotic microangiopathy and treatment methods using MASP-2 inhibitors Overview Thrombotic microangiopathy (TMA) is a condition characterized by blood clots in small blood vessels (Benz. K.; et al., Curr Opin Nephrol Hypertens 19(3):242-7(2010)). Stress or injury to the underlying vascular endothelium is thought to be the primary driver. Clinical and laboratory findings in TMA include thrombocytopenia, anemia, purpura, and renal failure. Classic TMA is hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). Characteristic underlying pathologic features of TMA are platelet activation and microthrombus formation in arterioles and venules. Complement activation, at least partially initiated by injury or stress to the microvascular endothelium, is also involved in other TMAs, including catastrophic antiphospholipid syndrome (CAPS), systemic sclerosis, and TMA secondary to cancer, cancer chemotherapy, and transplantation.

[0077] Studies of patients with genetic deficiencies in specific complement components have provided direct evidence of the pathogenic role of complement in the nephritic host. In many reports, the relationship between kidney injury and deficiency of complement regulatory factor H has been described (Ault, B.H., Nephrol. 14:1045-1053, 2000; Levy, M., et al., Kidney Int. 30:949-56, 1986; Pickering, M.C., et al., Nat. Genet, 31:424-8, 2002). Due to the consumption of factor B and C3 associated with activation by factor H deficiency, the plasma levels of these components are reduced. The circulating levels of C5b-9 in the sera of these patients are also increased. This indicates complement activation. Membranoproliferative glomerulonephritis (MPGN) and atypical hemolytic uremic syndrome (HUS) are associated with factor H deficiency or factor H mutations. Factor H-deficient pigs (Jansen, J.H., et al., Kidney Int. 53:331-49, 1998) and factor H knockout mice (Pickering, M.C., 2002) exhibit MPGN-like symptoms. This supports the importance of factor H in complement regulation. Deficiencies of other complement components are associated with kidney diseases secondary to the development of systemic lupus erythematosus (SLE) (Walport, M.J., Davies, et al., Ann. N.Y. Acad. Sci. 815:267-81, 1997). Deficiencies of C1q, C4, and C2 are strong predisposing factors for the development of SLE through mechanisms related to the incomplete clearance of immune complexes and apoptotic materials. In many of these SLE patients, lupus nephritis characterized by the deposition of immune complexes throughout the glomeruli occurs.

[0078] aHUS Atypical hemolytic uremic syndrome (aHUS) is part of a group of conditions called "thrombotic microangiopathies." In atypical HUS (aHUS), the disease is associated with defective complement regulation and can be sporadic or familial. Familial aHUS cases are associated with mutations in genes encoding complement activation or complement regulatory proteins, including complement factor H, factor I, factor B, membrane cofactor CD46, and complement factor H-related protein 1 (CFHR1) and complement factor H-related protein 3 (CFHR3) (Zipfel, P.F., et al., PloS Genetics 3(3):e41(2007)). The integrated characteristics of this broad range of gene mutations associated with aHUS are predisposing factors for enhanced complement activation at the cell surface or tissue surface. Accordingly, one aspect of the present invention includes treating a patient suffering from aHUS associated with factor H deficiency by administering an effective amount of a MASP-2 inhibitor. Another aspect of the present invention includes treating a patient suffering from HUS associated with a deficiency of factor I, factor B, membrane cofactor CD46, CFHR1, or CFHR3 by administering an effective amount of a MASP-2 inhibitor.

[0079] Recently, there has been considerable progress towards understanding the molecular pathophysiology underlying the enhanced complement activation in aHUS caused by diverse mutant complement factor sets. This mechanism is best understood for factor H mutations. Factor H is an abundant serum protein containing 20 short consensus repeat (SCR) domains that acts as a negative regulator of complement activation both in the fluid phase and on host cell surfaces. It targets activated C3 together with factor I and other cofactors, promotes its inactivation, and prevents further complement activation. To effectively control complement activation on host cell surfaces, factor H needs to interact with host cells, which is mediated by SCR domains 16 - 20. All factor H mutations associated with aHUS described to date cluster in the C-terminal region containing SCR domains 16 - 20. These mutant factor H proteins function fully in C3 activation control in the fluid phase but are unable to interact with host cell surfaces and, as a result, cannot control C3 activation on the cell surface (J Exp Med 204(6):1249 - 56 (2007)). Thus, certain factor H mutations are associated with aHUS because the mutant factor H proteins do not interact with host cell surfaces and thus cannot effectively reduce complement activation on host cell surfaces, including microvascular endothelium. As a result, when initial C3 activation occurs, subsequent complement activation on the microvascular endothelium surface proceeds unabated in patients with factor H mutations. This uncontrolled complement activation ultimately leads to progressive damage to the vascular endothelium, subsequent platelet aggregation and microvascular thrombosis, and hemolysis caused by shear stress of RBC passage through partially occluded microvessels. Thus, aHUS disease manifestations as well as clinical and laboratory findings are directly related to defects in negative complement regulation on microvascular endothelium surfaces.

[0080] Similar to the H factor mutations, loss-of-function mutations in factor I and membrane cofactor protein (CD46), which are negative complement regulators, are also associated with aHUS. Since aHUS has been found to be associated with gain-of-function mutations in factor B and C3 protein, the opposite has been observed for these proteins (Pediatr Nephrol 25(12):2431-42(2010)). Thus, a large body of consolidated data indicates that complement activation is involved in the development of aHUS. This idea is most convincingly supported by the therapeutic efficacy of eculizumab, a monoclonal antibody that blocks the terminal complement protein C5, in the treatment of aHUS.

[0081] The central role of complement as an effector mechanism in aHUS is widely recognized, but the triggers for initiating complement activation and the molecular pathways involved remain unresolved. Not all individuals with the above mutations develop aHUS. In fact, family studies suggest that the penetrance of aHUS is only about 50% (Ann Hum Genet 74(1):17-26(2010)). From the natural course of this disease, aHUS is suggested to develop in most cases after initiating events such as infectious episodes or injury. Infectious agents are well known to activate the complement system. In the absence of existing acquired immunity, complement activation by infectious agents can be initiated mainly via the lectin pathway. Thus, lectin pathway activation induced by infection may serve as an initiating trigger for subsequent pathological amplification of complement activation in individuals predisposed to aHUS, and ultimately may lead to disease progression. Accordingly, another aspect of the present invention includes treating a patient suffering from aHUS secondary to an infection by administering an effective amount of a MASP-2 inhibitor.

[0082] Other forms of injury to host tissue, particularly injury to vascular endothelium, activate complement via the lectin pathway. Human vascular endothelial cells under oxidative stress respond, for example, by binding to lectins and expressing surface moieties that activate the complement lectin pathway (Am J. Pathol 156(6):1549-56(2000)). Vascular injury after ischemia / reperfusion also activates complement via the lectin pathway in vivo (Scand J Immunol 61(5):426-34(2005)). Lectin pathway activation in this context causes pathological consequences to the host, and inhibition of the lectin pathway by MASP-2 blockade prevents further host tissue injury and adverse events (Sehwaeble PNAS 2011).

[0083] Thus, it is known that other processes that acutely cause aHUS also activate the complement lectin pathway. Thus, the lectin pathway may be an initial complement activation mechanism that is inappropriately amplified in a disordered manner in individuals with a genetic predisposition to aHUS and thus initiates aHUS pathogenesis. By inference, agents that block complement activation via the lectin pathway, including anti-MASP-2 antibodies, are expected to prevent disease progression or reduce exacerbation in aHUS-susceptible individuals.

[0084] As further support for this idea, recent studies have identified Streptococcus pneumoniae (S. pneumoniae) as an important etiological agent in pediatric cases of aHUS (Nephrology (Carlton), 17:48-52 (2012); Pediatr Infect Dis J. 30(9):736-9 (2011)). This specific etiology appears to be associated with an unfavorable prognosis, high mortality, and a prolonged pathological state. In particular, these cases were accompanied by non-intestinal infections leading to the manifestation of microangiopathy, uremia, and hemolysis, without evidence of concurrent complement gene mutations known to be predisposing factors for aHUS. S. pneumoniae is particularly effective in complement activation, and it is important to note that it mainly activates complement via the lectin pathway. Therefore, in the case of non-intestinal HUS associated with pneumococcal infection, the manifestation of microangiopathy, uremia, and hemolysis is expected to be mainly driven by lectin pathway activation, and agents that block the lectin pathway, including anti-MASP-2 antibodies, are expected to prevent the progression of aHUS and reduce disease severity in these patients. Accordingly, another aspect of the present invention includes treating a patient suffering from non-intestinal aHUS associated with S. pneumoniae infection by administering an effective amount of a MASP-2 inhibitor.

[0085] According to the foregoing, in some embodiments, in the context of a subject having a risk of developing kidney failure associated with aHUS, administering to the subject an effective amount of a MASP-2 inhibitor over a period effective to remit or prevent the kidney failure, thereby providing a method for reducing the likelihood of developing aHUS or developing kidney failure associated with aHUS. In some embodiments, the method further comprises determining whether the subject has a risk of developing aHUS prior to the manifestation of symptoms associated with aHUS. In other embodiments, the method comprises determining whether the subject has a risk of developing aHUS based on the manifestation of at least one or more symptoms indicative of aHUS (e.g., the presence of anemia, thrombocytopenia, and / or renal insufficiency in the subject), and / or the presence of thrombotic microangiopathy in a biopsy sample obtained from the subject. Determining whether the subject has a risk of developing aHUS includes determining whether the subject has a genetic predisposition to develop aHUS, which can be done by evaluating genetic information (e.g., from a database containing the subject's genotype) or by performing at least one genetic screening test on the subject to determine the presence or absence of genetic markers associated with aHUS (i.e., by genomic sequencing or gene-specific analysis (e.g., PCR analysis) to determine the presence or absence of genetic mutations associated with aHUS in genes encoding complement factor H (CFH), factor I (CFI), factor B (CFB), membrane cofactor CD46, C3, complement factor H-related protein 1 (CFHR1), or THBD (encoding the anticoagulant protein thrombomodulin), or complement factor H-related protein 3 (CFHR3), or complement factor H-related protein 4 (CFHR4)), and / or by determining whether the subject has a family history of aHUS. Methods for genetic screening for the presence or absence of genetic mutations associated with aHUS are well established.For example, see Noris M et al., "Atypical Hemolytic-Uremic Syndrome," November 16, 2007 [updated May 10, 2011]. In: Pagon RA, Bird TD, Dolan CR, et al., eds. GeneReviews (trademark), Seattle (WA): University of Washington, Seattle.

[0086] For example, overall, the disease penetrance among individuals with complement factor H (CFH) mutations is 48%, the penetrance of CD46 mutations is 53%, the penetrance of CFI mutations is 50%, the penetrance of C3 mutations is 56%, and the penetrance of THBD mutations is 64% (Caprioli J. et al., Blood, 108:1267-79 (2006); Noris et al., Clin J Am Soc Nephrol 5:1844-59 (2010)). As described above, Caprioli et al., (2006), a significant number of individuals with complement factor H (CFH) mutations never develop aHUS, and it is assumed that suboptimal CFH activity in these individuals is sufficient to protect the host from the effects of complement activation under physiological conditions. However, when exposed to agents that activate complement and produce more C3b than normal, suboptimal CFH activity is not sufficient to prevent C3b from depositing on vascular endothelial cells.

[0087] Accordingly, in one aspect, there is provided a method for inhibiting MASP-2-dependent complement activation in a subject suffering from non-H factor-dependent atypical hemolytic uremic syndrome or a subject at risk of developing non-H factor-dependent atypical hemolytic uremic syndrome, the method comprising administering to the subject a composition comprising an effective amount of a MASP-2 inhibitor for inhibiting MASP-2-dependent complement activation. In another aspect, there is provided a method for inhibiting MASP-2-dependent complement activation in a subject at risk of developing H factor-dependent atypical hemolytic uremic syndrome, the method comprising periodically monitoring the subject to determine the presence or absence of anemia, thrombocytopenia, or increased creatinine, and treating the subject with a MASP-2 inhibitor based on a determination that anemia, thrombocytopenia, or increased creatinine is present. In another aspect, there is provided a method for reducing the likelihood that a subject at risk of developing H factor-independent aHUS will develop clinical symptoms associated with aHUS, the method comprising administering a MASP-2 inhibitor before, during, or after an event known to be associated with the induction of aHUS clinical symptoms, such as drug exposure (e.g., chemotherapy), infection (e.g., bacterial infection), malignancy, injury, organ transplantation or tissue transplantation, or pregnancy.

[0088] In one aspect, there is provided a method for reducing the likelihood that a subject at risk of developing aHUS will develop clinical symptoms associated with aHUS, the method comprising periodically monitoring the subject to determine the presence or absence of anemia, thrombocytopenia, or increased creatinine, and treating the subject with a MASP-2 inhibitor based on a determination that anemia, thrombocytopenia, or increased creatinine is present.

[0089] In another aspect, there is provided a method for reducing the likelihood that a subject at risk of developing aHUS will develop clinical symptoms associated with aHUS, the method comprising administering a MASP-2 inhibitor before, during, or after an event known to be associated with the induction of aHUS clinical symptoms, such as drug exposure (e.g., chemotherapy), infection (e.g., bacterial infection), malignancy, injury, organ transplantation or tissue transplantation, or pregnancy.

[0090] In some embodiments, the MASP-2 inhibitor is administered for at least 1 day, 2 days, 3 days, 4 days, or a longer period before, during, or after an event associated with the induction of aHUS clinical symptoms, and may be repeated as determined by a physician until the condition has recovered or is managed. In the context of pre-aHUS, the MASP-2 inhibitor may be administered systemically to a subject by, for example, intra-arterial administration, intravenous administration, intramuscular administration, inhalation administration, nasal administration, subcutaneous administration, or other parenteral administration.

[0091] In some embodiments, in the context of an initial diagnosis of aHUS, or in a subject presenting with one or more symptoms consistent with a diagnosis of aHUS (e.g., the presence of anemia, thrombocytopenia, and / or renal insufficiency), the subject is treated with an effective amount of a MASP-2 inhibitor (e.g., an anti-MASP-2 antibody) in the absence of plasmapheresis or in combination with plasmapheresis as a first-line therapy. As a first-line therapy, the MASP-2 inhibitor may be administered systemically to a subject by, for example, intra-arterial administration, intravenous administration, intramuscular administration, inhalation administration, nasal administration, subcutaneous administration, or other parenteral administration. In some embodiments, in the absence of plasmapheresis to avoid potential plasmapheresis complications including bleeding, infections, and exposure to disorders and / or allergies specific to plasma donors, or in a subject who otherwise dislikes plasmapheresis, or in a situation where plasmapheresis is not available, the MASP-2 inhibitor is administered to the subject as a first-line therapy.

[0092] In some embodiments, the method comprises administering a MASP-2 inhibitor to a subject suffering from aHUS (e.g., intravenously) via a catheter for a first period (e.g., at least 1 day to 1 or 2 weeks), and then administering the MASP-2 inhibitor subcutaneously to the subject for a second period (e.g., at least 2 weeks or a longer chronic phase). In some embodiments, the administration in the first period and / or the second period is performed in the absence of plasmapheresis. In some embodiments, the method further comprises determining, before and optionally during treatment, the level of at least one complement factor (e.g., C3, C5) in the subject, and determination of the level of at least one complement factor that is decreased compared to a standard value or healthy control subjects indicates the need for continued treatment with a MASP-2 inhibitor.

[0093] In some embodiments, the method comprises administering a MASP-2 inhibitor, e.g., an anti-MASP-2 antibody, to a subject suffering from aHUS or at risk of developing aHUS, either intravenously, intramuscularly, or preferably subcutaneously. The treatment is chronic and may be performed daily to monthly, preferably every 2 weeks. The anti-MASP-2 antibody may be administered alone or in combination with a C5 inhibitor such as eculizumab.

[0094] HUS Similar to atypical HUS, typical HUS exhibits all the clinical and laboratory findings of TMA. However, typical HUS is, in many cases, a pediatric disease and usually has no family element or direct association with complement gene mutations. The cause of typical HUS is closely related to the infection of certain enteric pathogens. Patients typically present with acute renal failure, hemoglobinuria, and thrombocytopenia and typically follow an episode of bloody diarrhea. This condition is caused by enteric infections with Shigella dysenteriae, Salmonella, or Shiga toxin-producing enterohemorrhagic Escherichia coli strains, such as E. coli O157:H7. These pathogens are obtained from contaminated food or water supplies. HUS is a medical emergency, and the mortality rate is 5-10%. A significant portion of survivors develop chronic kidney disease (Corrigan and Boineau, Pediatr Rev 22(11):365-9(2011)) and may require kidney transplantation.

[0095] Microvascular coagulation in typical HUS mainly occurs in the renal microvasculature, but does not occur only in the renal microvasculature. The underlying pathophysiology is mediated by Shiga toxin (STX). STX is excreted into the intestinal lumen by enteric pathogenic microorganisms, crosses the intestinal barrier, enters the bloodstream, and binds to vascular endothelial cells via the globotriaosyl ceramide receptor CD77 (Boyd and Lingwood Nephron 51:207 (1989)). CD77 is preferentially expressed on glomerular endothelium and mediates the toxic effects of STX. When STX binds to the endothelium, it induces a series of events that damage the vascular endothelium, activate leukocytes, and cause vWF-dependent thrombosis (Forsyth et al., Lancet 2:411-414 (1989); Zoja et al., Kidney Int. 62:846-856 (2002); Zanchi et al., J. Immunol 183:1460-1469 (2008); Morigi et al., Blood 98:1828-1835 (2001); Guessou et al., Infect. Immun., 73:8306-8316 (2005)). These microthrombi obstruct or occlude the arterioles and capillaries of the kidney and other organs. As RBCs squeeze through the narrowed vessels, the shear stress applied to RBCs increases due to the occlusion of blood flow in arterioles and capillaries by microthrombi. As a result, RBCs may be destroyed by shear stress, forming RBC fragments called schistocytes. The presence of schistocytes is a characteristic finding in HUS. This mechanism is known as microangiopathic hemolysis. Furthermore, blood flow occlusion causes ischemia, initiating a complement-mediated inflammatory response that causes further damage to the affected organs.

[0096] The lectin pathway of complement contributes to the development of HUS through two main mechanisms: (1) direct activation of the coagulation cascade via MASP-2, induced by endothelial damage, and (2) subsequent complement activation via lectin, induced by ischemia resulting from initial occlusion of microvascular blood flow.

[0097] STX damages microvascular endothelial cells, and the damaged endothelial cells are known to activate the complement system. As detailed above, complement activation after endothelial cell injury is mainly driven by the lectin pathway. Human vascular endothelial cells under oxidative stress react by binding to lectins and expressing surface moieties that activate the complement lectin pathway (Collard et al., Am J Pathol. 156(5):1549-56(2000)). Vascular injury after ischemia-reperfusion also activates complement in vivo via the lectin pathway (Scand J Immunol 61(5):426-34(2005)). Activation of the lectin pathway in this context results in pathological consequences for the host, and inhibition of the lectin pathway by MASP-2 blockade prevents further host tissue damage and adverse events (Schwaeble et al., PNAS(2011)). In addition to complement activation, lectin-dependent activation of MASP-2 has been shown to cleave prothrombin to form thrombin and promote coagulation. Thus, activation of the complement lectin pathway by damaged endothelial cells can directly activate the coagulation system. Therefore, the complement lectin pathway via prothombin activation through MASP-2 is likely to be the major molecular pathway linking the initial endothelial injury by STX to the coagulation and microvascular thrombosis that occur in HUS. Thus, lectin pathway inhibitors, including but not limited to antibodies that block MASP-2 function, are expected to prevent or reduce microvascular coagulation, thrombosis, and hemolysis in patients suffering from HUS. Indeed, mice in a typical HUS model are significantly protected by administration of anti-MASP-2 antibodies. As described in Example 36 and as shown in FIG. 45, all control mice exposed to STX and LPS developed severe HUS and became moribund or died within 48 hours. On the other hand, as also shown in FIG. 45, all mice treated with anti-MASP-2 antibody and then exposed to STX and LPS survived (Fisher's exact test p<0.01; N=5). Thus, mice in this HUS model are significantly protected by anti-MASP-2 therapy.Administration of MASP-2 inhibitors, such as MASP-2 antibodies, is expected to be effective in the treatment of HUS patients and to provide protection from microvascular coagulation, thrombosis, and hemolysis caused by infections with enteropathogenic E. coli or other STX-producing pathogens.

[0098] Although HUS caused by STX is shown herein, anti-MASP-2 therapy is expected to be beneficial also for HUS-like syndromes due to endothelial damage caused by other toxic substances. This includes agents such as mitomycin, ticlopidine, cycplatin, quinine, cyclosporine, bleomycin, and other chemotherapeutic and immunosuppressive drugs. Accordingly, anti-MASP-2 antibody therapy or other modalities that inhibit MASP-2 activity are expected to effectively prevent or limit coagulation, thrombosis, and RBC destruction and to prevent renal failure in HUS and other TMA-related diseases (i.e., aHUS and TTP).

[0099] Patients suffering from HUS often present with diarrhea and vomiting, and usually the patient has a low platelet count (thrombocytopenia) and few RBCs (anemia). The pre-HUS diarrheal phase typically lasts about 4 days, during which time subjects at risk of developing HUS typically exhibit, in addition to severe diarrhea, one or more of the following symptoms: a hematocrit level of less than 30% with smear evidence of intravascular RBC destruction, thrombocytopenia (platelet count < 150×10 3 / mm 3) and / or the presence of renal dysfunction (serum creatinine concentration above the upper limit of the reference range for age). The presence of oliguria (> 0.5 mL / kg / h urine output over > 1 day) can be used as a measure of progression to the development of HUS (see C. Hickey et al., Arch Pediatr Adolesc Med 165(10):884 - 889(2011)). Typically, tests are performed for the presence of an infection with Escherichia coli bacteria (E. coli O157:H7) or Shigella or Salmonella species. In subjects who test positive for an infection with enterogenic E. coli (e.g., E. coli O157:H7), the use of antibiotics is contraindicated. This is because the use of antibiotics may increase the risk of developing HUS due to increased STX production (see Wong C. et al., N Engl J. Med 342:1930 - 136(2000)). In the case of subjects who test positive for Shigella or Salmonella, antibiotics are typically administered to eliminate the infection. Other well - established first - line therapies for HUS include volume expansion, dialysis, and plasma exchange.

[0100] According to the foregoing, in some embodiments, a subject suffering from one or more symptoms associated with the pre - HUS phase and a subject having a risk of developing HUS (i.e., the subject exhibits one or more of the following: diarrhea, a hematocrit level of less than 30% with schistocyte evidence of intravascular hemolysis, thrombocytopenia (150×10 3 / mm 3In the situation of a low platelet count), and / or the presence of renal dysfunction (serum creatinine concentration exceeding the upper limit of the age-based reference range)), administering an effective amount of a MASP-2 inhibitor over a period effective to alleviate or prevent the renal dysfunction, to reduce the risk of developing HUS or the likelihood of renal failure in the subject. In some embodiments, the MASP-2 inhibitor is administered over a period of at least 1 day, 2 days, 3 days, 4 days, or more, and may be repeated as determined by a physician until the condition has recovered or is managed. In the pre-HUS situation, the MASP-2 inhibitor may be administered systemically to the subject, for example, by intra-arterial administration, intravenous administration, intramuscular administration, inhalation administration, nasal administration, oral administration, subcutaneous administration, or other parenteral administration.

[0101] The treatment of Escherichia coli O157:H7 infection with bactericidal antibiotics, particularly β-lactams, has been associated with an increased risk of developing HUS (Smith et al., Pediatr Infect Dis J 31(1):37-41(2012)). In some embodiments, in the situation of a subject suffering from symptoms associated with the pre-HUS stage, who has been found to be infected with an Escherichia coli (e.g., Escherichia coli O157:H7) for which the use of antibiotics is contraindicated, administering an effective amount of a MASP-2 inhibitor to suppress or prevent the presence of oliguria in the subject over a first period effective to suppress or prevent the presence of oliguria (e.g., at least 1 day, 2 days, 3 days, or 4 days), to reduce the risk of developing HUS or the likelihood of renal failure in the subject, and the administration of the MASP-2 inhibitor during the first period is carried out in the absence of antibiotics. In some embodiments, the method further comprises administering the MASP-2 inhibitor in combination with an antibiotic to the subject over a second period (e.g., at least 1-2 weeks).

[0102] In another aspect, in the context of a subject who has symptoms associated with the pre-HUS period and is known to be infected with Shigella or Salmonella, a method for reducing the risk of developing HUS or a method for reducing the likelihood of renal failure in the subject, comprising administering to the subject an effective amount of a MASP-2 inhibitor over a period effective to suppress or prevent the presence of oliguria, wherein the administration of the MASP-2 inhibitor is in the presence or absence of a suitable antibiotic.

[0103] In some aspects, in the context of an initial diagnosis of HUS, or in a subject presenting with one or more symptoms consistent with a diagnosis of HUS (e.g., renal failure, or microangiopathic hemolytic anemia in the absence of hypofibrinogen, or the presence of thrombocytopenia), the subject is treated with an effective amount of a MASP-2 inhibitor (e.g., an anti-MASP-2 antibody) in the absence of plasmapheresis or in combination with plasmapheresis as a first-line therapy. As a first-line therapy, the MASP-2 inhibitor may be administered systemically to the subject, for example, by intraarterial, intravenous, intramuscular, inhaled, nasal, subcutaneous, or other parenteral administration. In some aspects, in order to avoid plasmapheresis complications such as bleeding, infection, and exposure to disorders, and / or allergies specific to plasma donors, in the absence of plasmapheresis, or in a subject who otherwise dislikes plasmapheresis, or in situations where plasmapheresis is not available, the MASP-2 inhibitor is administered to the subject as a first-line therapy.

[0104] In some embodiments, the method includes administering a MASP-2 inhibitor to a subject suffering from HUS (e.g., intravenously) via a catheter for a first period (e.g., an acute phase lasting at least 1 day to 1 week or 2 weeks), and then subcutaneously administering the MASP-2 inhibitor to the subject for a second period (e.g., a chronic phase of at least 2 weeks or more). In some embodiments, the administration in the first period and / or the second period is performed in the absence of plasmapheresis. In some embodiments, the method further includes determining, prior to treatment and optionally during treatment, the level of at least one complement factor (e.g., C3, C5) in the subject, and determination of a decreased level of the at least one complement factor compared to a standard value or a healthy control subject indicates a need for treatment, and determination of a normal level indicates improvement.

[0105] In some embodiments, the method includes subcutaneously or intravenously administering a MASP-2 inhibitor, e.g., an anti-MASP-2 antibody, to a subject suffering from HUS or at risk of developing HUS. The treatment is preferably daily, but may be less frequent, such as weekly or monthly. The treatment lasts at least 1 week and as long as 3 months. The anti-MASP-2 antibody may be administered alone or in combination with a C5 inhibitor such as eculizumab.

[0106] TTP: Thrombotic thrombocytopenic purpura (TTP) is a life-threatening blood coagulation disorder caused by autoimmune or genetic dysfunction that activates the coagulation cascade or complement system (George, JN, N Engl J Med; 354:1927-35(2006)). It causes numerous very small blood clots, i.e., thrombosis, in small blood vessels throughout the body. Red blood cells are subjected to shear stress that damages the red blood cell membrane, which causes intravascular hemolysis. The resulting reduced blood flow and endothelial damage cause organ damage, including to the brain, heart, and kidneys. Clinically, TTP is characterized by thrombocytopenia, microangiopathic hemolytic anemia, neurological changes, renal failure, and fever. In the pre-plasma exchange era, the mortality rate during acute episodes was 90%. Even with plasma exchange, survival at 6 months is approximately 80%.

[0107] TTP can result from genetic or acquired inhibition of the enzyme ADAMTS-13, a metalloprotease 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 regulates vWF activity. In its absence, vWF forms large multimers that are likely to bind to platelets, making the patient prone to platelet aggregation and thrombosis in the microvasculature.

[0108] Upshaw-Schulman syndrome (USS, also known as congenital TTP) is a congenital ADAMTS13 activity deficiency due to ADAMTS13 gene mutations (Schulman et al., Blood, 16(1):943-57, 1960; Upshaw et al., New Engl. J. Med, 298 (24):1350-2, 1978). A very large number of ADAMTS13 mutations have been identified in individuals with congenital TTP (Kinoshita et al., International Journal of Hematology, 74:101-108 (2001); Levy et al., Nature, 413 (6855):488-494 (2001); Kokame et al., PNAS 99(18):11902-11907 (2002); Savasan et al., Blood, 101:4449-4451 (2003); Matsumoto et al., Blood, 103:1305-1310 (2004) and Fujimura et al., Brit. J. Haemat 144:742-754 (2008)). Subjects with USS typically have 5-10% of normal ADAMTS13 activity (Kokame et al., PNAS 99(18):11902-11907, 2002). There are some similarities between acquired TTP and USS, but there are some important differences in clinical features in USS. USS usually appears in infancy or childhood and is characterized by severe hyperbilirubinemia, negative Coombs test immediately after birth, response to fresh plasma infusion, and frequent relapses (Savasan et al., Blood, 101:4449-4451, 2003). In some cases, patients with this hereditary ADAMTS13 deficiency have a mild phenotype at birth and develop symptoms related to TTP only in clinical situations with high von Willebrand factor levels, such as during infection or pregnancy. For example, Fujimura et al. reported nine Japanese women diagnosed with disorders during their first pregnancy from six families in which USS was genetically confirmed.In each of 15 pregnancies, thrombocytopenia occurred during the second to third trimesters, and in many cases, TTP subsequently developed. All of these women were found to have severely deficient ADAMTS13 activity (Fujimura et al., Brit. J. Haemat 144:742-754, 2008).

[0109] According to the foregoing, in some embodiments, in the context of a subject having Upshaw - Schulman syndrome (USS) (i.e., the subject is known to lack ADAMTS13 activity and / or the subject is known to have one or more ADAMTS13 gene mutations), administering an effective amount of a MASP - 2 inhibitor (e.g., a MASP - 2 antibody) over a period effective to relieve or prevent one or more clinical symptoms associated with TTP, to provide a method for reducing the likelihood of developing clinical symptoms associated with congenital TTP (e.g., thrombocytopenia, anemia, fever, and / or renal insufficiency). In some embodiments, the method further comprises determining whether the subject has a risk of developing symptoms associated with congenital TTP before the subject exhibits any symptoms associated with TTP or based on the manifestation of at least one or more symptoms indicative of TTP (e.g., the presence of anemia, thrombocytopenia, and / or renal insufficiency). The step of determining whether the subject has a risk of developing symptoms associated with congenital TTP (i.e., whether the subject has USS) comprises determining whether the subject has a mutation in the gene encoding ADAMTS13 and / or determining whether the subject lacks ADAMTS13 activity and / or determining whether the subject has a family history of USS. Methods for genetic screening for the presence or absence of gene mutations associated with USS are well - established. See, for example, Kinoshita et al., International Journal of Hematology, 74:101 - 108 (2001); Levy et al., Nature, 413(6855):488 - 494 (2001); Kokame et al., PNAS 99(18):11902 - 11907 (2002); Savasan et al., Blood, 101:4449 - 4451 (2003); Matsumoto et al., Blood, 103:1305 - 1310 (2004) and Fujimura et al., Brit. J. Haemat 144:742 - 754 (2008).

[0110] In one aspect, there is provided a method for reducing the likelihood that a subject diagnosed with USS will develop clinical symptoms associated with TTP, the method comprising the steps of periodically monitoring the subject to determine the presence or absence of anemia, thrombocytopenia, or an increase in creatinine, and treating with a MASP-2 inhibitor (e.g., a MASP-2 antibody) based on a determination that anemia, thrombocytopenia, or an increase in creatinine is present, or based on the presence of an event known to be associated with the induction of TTP clinical symptoms, such as drug exposure (e.g., chemotherapy), infection (e.g., bacterial infection), malignancy, injury, transplantation, or pregnancy.

[0111] In another aspect, there is provided a method for treating a subject having USS and a subject suffering from clinical symptoms associated with TTP, the method comprising administering an effective amount of a MASP-2 inhibitor (e.g., a MASP-2 antibody) over a period of time effective to remit or prevent one or more clinical symptoms associated with TTP.

[0112] TTP can be caused by autoantibodies against ADAMTS-13. Additionally, TTP can occur during breast cancer, gastrointestinal cancer, or prostate cancer (George. JN., Oncology(Wiiliston Park). 25:908-14(2011)), pregnancy (second trimester or after childbirth), George JN., Curr Opin Hematol 10:339-344(2003)), or be associated with diseases such as autoimmune diseases like 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 components, cancer chemotherapeutic agents (bleomycin, cisplatin, cytosine arabinoside, daunomycin, gemcitabine, mitomycin C, and tamoxifen), cyclosporine A, oral contraceptives, penicillin, rifampin, and antiplatelet drugs including ticlopidine and clopidogrel (Azarm, T. et al., J Res Med Sci., 16:353-357(2011)). Other factors or conditions associated with TTP are toxins such as bee venom, sepsis, splenic sequestration, transplantation, vasculitis, vascular surgery, and infections such as Streptococcus pneumoniae and cytomegalovirus (Moake JL., N Engl J Med., 347:589-600(2002)). As a result of endothelial cell damage associated with Streptococcus pneumoniae infection, TTP due to transient functional ADAMTS-13 deficiency can occur (Pediair Nephrol., 26:631-5(2011)).

[0113] Plasma exchange is the standard treatment for TTP (Rock GA, et al., N Engl J Med 325:393-397(1991)). By plasma exchange, ADAMTS-13 activity is replaced in patients with genetic defects and ADAMTS-13 autoantibodies are removed in patients with acquired autoimmune TTP (Tsai, H-M, Hematol Oncol Clin North Am., 21(4):609-v(2007)). Additional agents, such as immunosuppressive drugs, are routinely added to the therapy (George, JN, N Engl J Med, 354:1927-35(2006)). However, plasma exchange is successful in only about 20% of patients, recurrence occurs in more than one-third of patients, plasmapheresis is expensive, and technically very laborious. Furthermore, many patients cannot tolerate plasma exchange. As a result, there is still a great need for additional and better treatments for TTP.

[0114] Since TTP is a disorder of the blood coagulation cascade, treatment with complement antagonists may help to stabilize the disease and assist in treatment. Although pathological activation of the alternative complement pathway has been linked to aHUS, the role of complement activation in TTP is less clear. Functional deficiency of ADAMTS13 is important for susceptibility to TTP but is not sufficient to trigger acute episodes. Environmental factors and / or other genetic mutations may contribute to the development of TTP symptoms. For example, genes encoding proteins involved in the coagulation cascade, vWF, platelet function, components of the endothelial vascular surface, or regulation of the complement system may be involved in the development of acute thrombotic microangiopathy (Galbusera, M. et al., Haematologica, 94; 166-170(2009)). In particular, complement activation has been shown to play an important role. Serum from thrombotic microangiopathy associated with ADAMTS-13 deficiency has been shown to cause C3 and MAC deposition and subsequent neutrophil activation, which can be inhibited by complement inactivation (Ruiz-Torres MP, et al., Thromb Haemost, 93:443-52(2005)). Furthermore, it has recently been shown that levels of C4d, C3bBbP, and C3a increase during acute episodes of TTP, consistent with activation of the classical / lectin and alternative pathways (M. Reti et al., J Thromb Haemost. Feb 28.(2012) doi:10.1111 / j.1538-7836.2012.04674.x.[Electronic publication ahead of print]). This increase in the amount of complement activation during acute episodes initiates terminal pathway activation and may contribute to further exacerbation of TTP.

[0115] The roles of ADAMTS-13 and vWF in TTP clearly play a role in platelet activation and aggregation in microangiopathy and their subsequent role in shear stress and deposition. Activated platelets interact with and induce the classical and alternative pathways of complement. Complement activation mediated by platelets increases the inflammatory mediators C3a and C5a (Peerschke E et al., Mol Immunol, 47:2170-5(2010)). Thus, platelets can serve as targets for classical complement activation in hereditary or autoimmune TTP.

[0116] As described above, the complement lectin pathway by prothrombin activation via MASP-2 is the main molecular pathway linking endothelial damage, coagulation, and microvascular thrombosis that occurs in HUS. Similarly, activation of the complement lectin pathway can directly drive the coagulation system in TTP. Lectin pathway activation can be initiated in response to the initial endothelial damage caused by ADAMTS-13 deficiency in TTP. Thus, lectin pathway inhibitors, including but not limited to antibodies that block MASP-2 function, are expected to reduce microvascular coagulation, thrombosis, and microangiopathy associated with hemolysis in patients with TTP.

[0117] Patients suffering from TTP typically present to the emergency room with one or more of the following: purpura, renal failure, thrombocytopenia, anemia, and / or thrombosis including stroke. The current standard of care for TTP involves intravascular catheter delivery (e.g., intravenous catheter or other forms of catheter) of exchange plasma pheresis, typically three times a week but up to daily, over a period of two weeks or more. If the subject is determined to be positive in a test for the presence of an inhibitor of ADAMTS13 (i.e., an endogenous antibody to ADAMTS13), plasma pheresis can be performed in combination with immunosuppressive therapy (e.g., corticosteroids, rituxan, or cyclosporine). Subjects with refractory TTP (about 20% of TTP patients) do not respond to plasma pheresis therapy for at least two weeks.

[0118] According to the foregoing, in one aspect, in the context of an initial diagnosis of TTP, or in a subject presenting with one or more symptoms consistent with a diagnosis of TTP (e.g., central nervous system complications, severe thrombocytopenia (less than 5000 / μL or a platelet count of 5000 / μL if not taking aspirin, less than 20,000 / μL or a platelet count of 20,000 / μL if taking aspirin), severe cardiac complications, severe pulmonary complications, gastrointestinal infarction, or gangrene), a method is provided for treating the subject with an effective amount of a MASP-2 inhibitor (e.g., an anti-MASP-2 antibody) as a first-line therapy in the absence of plasma exchange, or in combination with plasma exchange. As a first-line therapy, the MASP-2 inhibitor may be administered systemically to the subject, for example, by intra-arterial, intravenous, intramuscular, inhaled, nasal, subcutaneous, or other parenteral administration. In some aspects, in order to avoid potential plasma exchange complications such as bleeding, infections, and exposure to disorders and / or allergies specific to plasma donors, in the absence of plasma exchange, or in a subject who otherwise dislikes plasma exchange, or in a situation where plasma exchange is not available, the MASP-2 inhibitor is administered to the subject as a first-line therapy. In some aspects, the MASP-2 inhibitor is administered to a subject suffering from TTP in combination with (including co-administration) an immunosuppressant (e.g., a corticosteroid, rituxan, or cyclosporine), and / or in combination with a high concentration of ADAMTS-13.

[0119] In some embodiments, the method comprises administering, via a catheter (e.g., intravenously), a MASP-2 inhibitor to a subject suffering from TTP for a first period (e.g., an acute phase lasting at least 1 day to 1 or 2 weeks), and then subcutaneously administering the MASP-2 inhibitor to the subject for a second period (e.g., a chronic phase of at least 2 weeks or more). In some embodiments, the administration in the first period and / or the second period is performed in the absence of plasmapheresis. In some embodiments, the method is used to maintain the subject such that the subject does not suffer from one or more symptoms associated with TTP.

[0120] In another embodiment, a method for treating a subject suffering from refractory TTP (i.e., a subject who does not respond to plasma exchange therapy for at least 2 weeks) is provided by administering an amount of a MASP-2 inhibitor effective to alleviate one or more symptoms of TTP. In one embodiment, the MASP-2 inhibitor (e.g., an anti-MASP-2 antibody) is administered to a subject having refractory TTP via subcutaneous or other parenteral administration over a chronic period of at least 2 weeks or more. The administration may be repeated as determined by a physician until the condition has recovered or is managed.

[0121] In some embodiments, the method further comprises determining, prior to treatment and optionally during treatment, the level of at least one complement factor (e.g., C3, C5) in the subject, and determination of a decreased level of the at least one complement factor as compared to a standard value or healthy control subjects indicates the need for continued treatment with a MASP-2 inhibitor.

[0122] In one aspect, the method includes subcutaneously or intravenously administering a MASP-2 inhibitor, such as an anti-MASP-2 antibody, to a subject suffering from TTP or a subject at risk of developing TTP. The treatment is preferably daily, but may be less frequent, such as bi-weekly. The treatment is continued until the subject's platelet count exceeds 150,000 / ml for at least two consecutive days. The anti-MASP-2 antibody may be administered alone or in combination with a C5 inhibitor such as eculizumab.

[0123] In one aspect, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: the antibody binds human MASP-2 with a K D of 10 nM or less, the antibody binds an epitope in the CCP1 domain of MASP-2, the antibody inhibits C3b deposition in 1% human serum in an in vitro assay method with an IC 50 of 10 nM or less, the antibody inhibits C3b deposition in 90% human serum with an IC 50 of 30 nM or less, the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2, the antibody is a single-chain molecule, the antibody is an IgG2 molecule, the antibody is an IgG1 molecule, the antibody is an IgG4 molecule containing the S228P mutation, and / or the antibody does not substantially inhibit the classical pathway. In one aspect, the antibody binds to MASP-2, selectively inhibits the lectin pathway, and does not substantially inhibit the alternative pathway. In one aspect, the antibody binds to MASP-2, selectively inhibits the lectin pathway, and does not substantially inhibit either the classical pathway or the alternative pathway (i.e., inhibits the lectin pathway while leaving the classical pathway and the complement alternative pathway intact).

[0124] In one aspect, the MASP-2 inhibitory antibody inhibits thrombus formation in serum from a subject suffering from TTP by at least 30%, for example, at least 40%, for example, at least 50%, for example, at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95% up to a maximum of 99%, as compared to untreated serum. In some aspects, the MASP-2 inhibitory antibody inhibits thrombus formation in serum from a subject suffering from TTP at a level that is at least 20 percentage points or more (e.g., at least 30%, at least 40%, at least 50%) higher than the inhibitory effect on C5b-9 deposition in serum.

[0125] In one aspect, the MASP-2 inhibitory antibody inhibits thrombus formation in serum from a TTP patient by at least 30%, for example, at least 40%, for example, at least 50%, for example, at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95% up to a maximum of 99%, as compared to untreated serum.

[0126] In one aspect, the MASP-2 inhibitory antibody is administered to a subject via an intravenous catheter or other catheter delivery method.

[0127] In one aspect, the present invention is a method for inhibiting thrombosis in a subject suffering from TTP, comprising the step of administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof, wherein the antibody or its antigen-binding fragment comprises (I) (a) (i) a heavy chain CDR-H1 comprising the amino acid sequence of positions 31-35 of SEQ ID NO: 67; and (ii) a heavy chain CDR-H2 comprising the amino acid sequence of positions 50-65 of SEQ ID NO: 67; and (iii) a heavy chain CDR-H3 comprising the amino acid sequence of positions 95-102 of SEQ ID NO: 67, a heavy chain variable region, and, (b) (i) a light chain CDR-L1 comprising the amino acid sequence of positions 24-34 of SEQ ID NO: 70; and (ii) a light chain CDR-L2 comprising the amino acid sequence of positions 50-56 of SEQ ID NO: 70; and (iii) a light chain CDR-L3 comprising the amino acid sequence of positions 89-97 of SEQ ID NO: 70, a light chain variable region, or (II) variants thereof comprising a heavy chain variable region having at least 90% identity with SEQ ID NO: 67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with SEQ ID NO: 67) and a light chain variable region having at least 90% identity with SEQ ID NO: 70 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity).

[0128] In some aspects, the method comprises the step of administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 67. In some aspects, the method comprises the step of administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0129] In one aspect, the method includes administering to a subject a composition comprising a MASP-2 inhibitory antibody or an antigen-binding fragment thereof that specifically recognizes at least a portion of a human MASP-2 epitope recognized by a reference antibody OMS646 comprising a heavy chain variable region shown in SEQ ID NO:67 and a light chain variable region shown in SEQ ID NO:70.

[0130] Dego's disease Degos disease, also known as malignant atrophic papulosis, is a very rare TMA that affects the endothelium of small blood vessels in the skin, gastrointestinal tract, and CNS. This vasculopathy causes occlusion of venules and arterioles, resulting in skin lesions, intestinal ischemia, and CNS disorders including stroke, epilepsy, and cognitive impairment. Connective tissue necrosis in the skin is due to thrombotic occlusion of small arteries. However, the cause of Degos disease is unknown. Vasculitis, coagulation disorders, or primary endothelial cell dysfunction have been associated. The 50% survival rate of Degos disease is only 2 to 3 years. There is no effective treatment for Degos disease, but antiplatelet drugs, anticoagulants, and immunosuppressants are used to alleviate symptoms.

[0131] The mechanism of Degos disease is unknown, but the complement pathway has been implicated. Margo et al. confirmed prominent C5b-9 deposition in the cutaneous, gastrointestinal, and cerebral blood vessels of four end-stage Degos disease patients (Margo et al., Am J Clin Pathol 135(4):599-610, 2011). Experimental treatment with eculizumab was initially effective in treating skin and intestinal lesions but did not halt the progression of the systemic disease (see Garrett-Bakelman F. et al., 「C5b-9 is a potential effector in the pathophysiology of Degos disease; a case report of treatment with eculizumab」 (Abstract), Jerusalem: International Society of Hematology; 2010, Poster #156; and Polito J. et al, 「Early detection of systemic Degos disease (DD) or malignant atrophic papulosis (MAP) may increase survival」 (Abstract), San Antonio, TX: American College of Gastroenterology; 2010, Poster #1205).

[0132] Many patients with Degos disease have a blood coagulation defect. Thrombotic occlusion of small arteries in the skin is characteristic of this disease. Since the complement pathway is associated with this disease, lectin pathway inhibitors, including but not limited to antibodies that block MASP-2 function, are expected to be beneficial in the treatment of patients with Degos disease, as described herein for other TMAs.

[0133] Accordingly, in another aspect, the present invention provides a method for treating Degos disease by administering to a subject suffering from Degos disease or a condition resulting from Degos disease a composition comprising a therapeutically effective amount of a MASP-2 inhibitor such as a MASP-2 antibody in a pharmaceutical carrier. The MASP-2 inhibitor is systemically administered to a subject suffering from Degos disease or a condition resulting from Degos disease, for example, by intra-arterial administration, intravenous administration, intramuscular administration, inhalation administration, subcutaneous administration, or other parenteral administration, or in the case of non-peptidic agents, by oral administration. The anti-MASP-2 antibody may be administered alone or in combination with a C5 inhibitor, such as eculizamab.

[0134] In one aspect, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: the antibody binds human MASP-2 with a K D of 10 nM or less, the antibody binds an epitope in the CCP1 domain of MASP-2, the antibody inhibits C3b deposition in 1% human serum in an in vitro assay method with an IC 50 of 10 nM or less, the antibody inhibits C3b deposition in 90% human serum with an IC 50 of 30 nM or less, the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2, the antibody is a single-chain molecule, the antibody is an IgG2 molecule, the antibody is an IgG1 molecule, the antibody is an IgG4 molecule containing the S228P mutation, and / or the antibody does not substantially inhibit the classical pathway. In one aspect, the antibody binds to MASP-2, selectively inhibits the lectin pathway, and does not substantially inhibit the alternative pathway. In one aspect, the antibody binds to MASP-2, selectively inhibits the lectin pathway, and does not substantially inhibit either the classical pathway or the alternative pathway (i.e., inhibits the lectin pathway while leaving the classical pathway and the alternative complement pathway intact).

[0135] In one aspect, the MASP-2 inhibitory antibody inhibits thrombus formation in serum from a subject suffering from Gougerot disease by at least 30%, for example, at least 40%, for example, at least 50%, for example, at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95% up to a maximum of 99%, as compared to untreated serum. In some aspects, the MASP-2 inhibitory antibody inhibits thrombus formation in serum from a subject suffering from Gougerot disease at a level that is at least 20 percent (e.g., at least 30%, at least 40%, at least 50%) higher than the inhibitory effect on C5b-9 deposition in the serum.

[0136] In one aspect, the MASP-2 inhibitory antibody inhibits thrombus formation in serum from a patient with Gougerot disease by at least 30%, for example, at least 40%, for example, at least 50%, for example, at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95% up to a maximum of 99%, as compared to untreated serum.

[0137] In one aspect, the MASP-2 inhibitory antibody is administered to a subject via an intravenous catheter or other catheter delivery method.

[0138] In one aspect, the present invention provides a method of inhibiting thrombosis in a subject suffering from Degos disease, the method comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises: (I) (a) (i) a heavy-chain CDR-H1 comprising the amino acid sequence of amino acids 31 to 35 of SEQ ID NO: 67; and (ii) a heavy-chain CDR-H2 comprising the amino acid sequence of amino acids 50 to 65 of SEQ ID NO: 67; and (iii) a heavy-chain CDR-H3 comprising the amino acid sequence of amino acids 95 to 102 of SEQ ID NO: 67, a heavy-chain variable region, and (b) (i) a light-chain CDR-L1 comprising the amino acid sequence of amino acids 24 to 34 of SEQ ID NO: 70; and (ii) a light-chain CDR-L2 comprising the amino acid sequence of amino acids 50 to 56 of SEQ ID NO: 70; and (iii) a light-chain CDR-L3 comprising the amino acid sequence of amino acids 89 to 97 of SEQ ID NO: 70, a light-chain variable region, or (II) variants thereof comprising a heavy-chain variable region having at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity) to SEQ ID NO: 67 and a light-chain variable region having at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity) to SEQ ID NO: 70.

[0139] In some aspects, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof comprising a heavy-chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 67. In some aspects, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof comprising a light-chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0140] In one aspect, the method comprises administering to a subject a composition comprising a MASP-2 inhibitory antibody or an antigen-binding fragment thereof that specifically recognizes at least a portion of a human MASP-2 epitope recognized by a reference antibody OMS646 comprising the heavy chain variable region shown in SEQ ID NO:67 and the light chain variable region shown in SEQ ID NO:70.

[0141] Severe antiphospholipid antibody syndrome (CAPS) Severe antiphospholipid antibody syndrome (CAPS) is an extreme variant of antiphospholipid antibody (APLA) syndrome. CAPS is characterized by venous and arterial thrombosis due to pathogenic antibodies. CAPS is a TMA accompanied by multi-organ thrombosis, ischemia, and organ failure. Similar to other TMAs, it is characterized by occlusion of small blood vessels in various organs. The mortality rate of CAPS is as high as about 50% and is often associated with infections or trauma. Patients have antiphospholipid antibodies, generally IgG.

[0142] Clinically, CAPS involves at least three organs or tissues showing histological evidence of small vessel occlusion. Peripheral thrombosis may involve veins and arteries in the CNS, cardiovascular, renal, or pulmonary systems. Patients are treated with antibiotics, anticoagulants, corticosteroids, plasma exchange, and intravenous immunoglobulins. Nevertheless, they may die due to multi-organ failure.

[0143] The complement pathway is associated with CAPS. For example, studies in animal models have shown that complement inhibition can be an effective means of preventing thrombosis associated with CAPS (Shapira L. et al., Arthritis Rheum 64(8):2719-23, 2012). Further, as further reported by Shapira et al., administration of a dose of eculizumab that blocks the complement pathway to subjects suffering from CAPS halted acute progressive thrombotic events and reversed thrombocytopenia (see also Lim W., Curr Opin Hematol 18(5):361-5, 2011). Thus, as described herein for other TMAs, lectin pathway inhibitors, including but not limited to antibodies that block MASP-2 function, are expected to be beneficial in the treatment of patients suffering from CAPS.

[0144] Accordingly, in another aspect, the present invention provides a method for treating CAPS by administering to a subject suffering from CAPS or a condition resulting from CAPS a composition comprising a therapeutically effective amount of a MASP-2 inhibitor, such as a MASP-2 antibody, in a pharmaceutical carrier. The MASP-2 inhibitor is systemically administered to a subject suffering from CAPS or a condition resulting from CAPS, for example, by intra-arterial administration, intravenous administration, intramuscular administration, inhalation administration, subcutaneous administration, or other parenteral administration, or, in the case of non-peptidic agents, by oral administration. The anti-MASP-2 antibody may be administered alone or in combination with a C5 inhibitor, such as eculizumab.

[0145] In one aspect, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: the antibody binds human MASP-2 with a K D of 10 nM or less, the antibody binds an epitope in the CCP1 domain of MASP-2, the antibody inhibits C3b deposition in 1% human serum in an in vitro assay with an IC 50 of 10 nM or less, the antibody inhibits C3b deposition in 90% human serum with an IC of 30 nM or less50 inhibiting, the antibody being an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2, the antibody being a single-chain molecule, the antibody being an IgG2 molecule, the antibody being an IgG1 molecule, the antibody being an IgG4 molecule containing the S228P mutation, and / or the antibody not substantially inhibiting the classical pathway. In one aspect, the antibody binds to MASP-2, selectively inhibits the lectin pathway, and does not substantially inhibit the alternative pathway. In one aspect, the antibody binds to MASP-2, selectively inhibits the lectin pathway, and does not substantially inhibit either the classical pathway or the alternative pathway (i.e., inhibits the lectin pathway while leaving the classical pathway and the alternative complement pathway intact).

[0146] In one aspect, the MASP-2 inhibitory antibody inhibits thrombosis in the serum from a subject suffering from CAPS by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% up to a maximum of 99% compared to untreated serum. In some aspects, the MASP-2 inhibitory antibody inhibits thrombosis in the serum from a subject suffering from CAPS at a level that is at least 20 percentage points or more (e.g., at least 30%, at least 40%, at least 50%) higher than the inhibitory effect on C5b-9 deposition in the serum.

[0147] In one aspect, the MASP-2 inhibitory antibody inhibits thrombosis in the serum from a CAPS patient by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% up to a maximum of 99% compared to untreated serum.

[0148] In one aspect, the MASP-2 inhibitory antibody is administered to a subject via an intravenous catheter or other catheter delivery method.

[0149] In one aspect, the present invention provides a method for inhibiting thrombosis in a subject suffering from CAPS, the method comprising administering to the subject a composition comprising an amount of an anti-MASP-2 inhibitory antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises: (I) (a) (i) a heavy chain CDR-H1 comprising the amino acid sequence of amino acids 31-35 of SEQ ID NO: 67; and (ii) a heavy chain CDR-H2 comprising the amino acid sequence of amino acids 50-65 of SEQ ID NO: 67; and (iii) a heavy chain CDR-H3 comprising the amino acid sequence of amino acids 95-102 of SEQ ID NO: 67, a heavy chain variable region, and (b) (i) a light chain CDR-L1 comprising the amino acid sequence of amino acids 24-34 of SEQ ID NO: 70; and (ii) a light chain CDR-L2 comprising the amino acid sequence of amino acids 50-56 of SEQ ID NO: 70; and (iii) a light chain CDR-L3 comprising the amino acid sequence of amino acids 89-97 of SEQ ID NO: 70, a light chain variable region, or (II) variants thereof comprising a heavy chain variable region having at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity) to SEQ ID NO: 67 and a light chain variable region having at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity) to SEQ ID NO: 70.

[0150] In some aspects, the method comprises administering to the subject a composition comprising an amount of an anti-MASP-2 inhibitory antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 67. In some aspects, the method comprises administering to the subject a composition comprising an amount of an anti-MASP-2 inhibitory antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0151] In one aspect, the method includes administering to a subject a composition comprising a MASP-2 inhibitory antibody or an antigen-binding fragment thereof that specifically recognizes at least a portion of a human MASP-2 epitope recognized by a reference antibody OMS646 comprising a heavy chain variable region shown in SEQ ID NO:67 and a light chain variable region shown in SEQ ID NO:70.

[0152] TMA secondary to cancer All types of systemic malignancies can cause clinical and pathological manifestations of TMA (see, for example, Batts and Lazarus, Bone Marrow Transplantation 40:709-719, 2007). Cancer-associated TMA is often observed in the lungs and appears to be associated with tumor emboli (Francis KK et al., Commun Oncol 2:339-43, 2005). Tumor emboli reduce blood flow and can thus mimic a state of low perfusion in the affected arterioles and venules. The resulting tissue stress and damage are expected to locally activate the lectin pathway of complement. Next, the activated lectin pathway can activate the coagulation cascade via MASP-2-dependent cleavage of prothrombin to thrombin, mimicking a prothrombotic state characteristic of TMA. Inhibiting MASP-2 in this context is expected to reduce local thrombin activation and thereby mitigate the prothrombotic state.

[0153] Thus, as described herein for other TMAs, lectin pathway inhibitors, including but not limited to, antibodies that block MASP-2 function, are expected to be beneficial in the treatment of patients suffering from TMA secondary to cancer.

[0154] Accordingly, in another aspect, the present invention provides a method for treating or preventing TMA secondary to cancer by administering to a subject suffering from TMA secondary to cancer or at risk of developing such TMA a composition comprising a therapeutically effective amount of a MASP-2 inhibitor such as a MASP-2 antibody in a pharmaceutical carrier. The MASP-2 inhibitor is systemically administered to a subject suffering from TMA secondary to cancer or at risk of developing such TMA, for example, by intra-arterial administration, intravenous administration, intramuscular administration, inhalation administration, subcutaneous administration, or other parenteral administration, or, in the case of non-peptidic agents, by oral administration. The anti-MASP-2 antibody may be administered alone or in combination with a C5 inhibitor such as eculizumab.

[0155] In one aspect, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: the antibody binds human MASP-2 with a K D of 10 nM or less, the antibody binds an epitope in the CCP1 domain of MASP-2, the antibody inhibits C3b deposition in 1% human serum in an in vitro assay with an IC 50 of 10 nM or less, the antibody inhibits C3b deposition in 90% human serum with an IC 50 of 30 nM or less, the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2, the antibody is a single-chain molecule, the antibody is an IgG2 molecule, the antibody is an IgG1 molecule, the antibody is an IgG4 molecule containing the S228P mutation, and / or the antibody does not substantially inhibit the classical pathway. In one aspect, the antibody binds to MASP-2, selectively inhibits the lectin pathway, and does not substantially inhibit the alternative pathway. In one aspect, the antibody binds to MASP-2, selectively inhibits the lectin pathway, and does not substantially inhibit either the classical pathway or the alternative pathway (i.e., inhibits the lectin pathway while leaving the classical pathway and the alternative complement pathway intact).

[0156] In one aspect, the MASP-2 inhibitory antibody inhibits thrombosis in serum from a subject suffering from TMA secondary to cancer by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, up to a maximum of 99% compared to untreated serum.

[0157] In one aspect, the MASP-2 inhibitory antibody inhibits thrombosis in serum from a subject suffering from TMA secondary to cancer by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, up to a maximum of 99% compared to untreated serum.

[0158] In one aspect, the MASP-2 inhibitory antibody is administered to a subject via an intravenous catheter or other catheter delivery method.

[0159] In one aspect, the present invention is a method of inhibiting thrombosis in a subject suffering from TMA secondary to cancer, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises: (I) (a) (i) a heavy chain CDR-H1 comprising the amino acid sequence of amino acids 31-35 of SEQ ID NO: 67; and (ii) a heavy chain CDR-H2 comprising the amino acid sequence of amino acids 50-65 of SEQ ID NO: 67; and (iii) a heavy chain CDR-H3 comprising the amino acid sequence of amino acids 95-102 of SEQ ID NO: 67, a heavy chain variable region, and (b) (i) a light chain CDR-L1 comprising the amino acid sequence of amino acids 24-34 of SEQ ID NO: 70; and (ii) a light chain CDR-L2 comprising the amino acid sequence of amino acids 50-56 of SEQ ID NO: 70; and (iii) a light chain CDR-L3 comprising the amino acid sequence of amino acids 89-97 of SEQ ID NO: 70, a light chain variable region, or (II) variants thereof comprising a heavy chain variable region having at least 90% identity to SEQ ID NO: 67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO: 67) and a light chain variable region having at least 90% identity to SEQ ID NO: 70 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity).

[0160] In some aspects, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 67. In some aspects, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0161] In one aspect, the method includes administering to a subject a composition comprising a MASP-2 inhibitory antibody or an antigen-binding fragment thereof that specifically recognizes at least a portion of a human MASP-2 epitope recognized by a reference antibody OMS646 comprising the heavy chain variable region shown in SEQ ID NO:67 and the light chain variable region shown in SEQ ID NO:70.

[0162] TMA secondary to cancer chemotherapy Chemotherapy-associated thrombotic microangiopathy (TMA) is a condition associated with thrombocytopenia, microangiopathic hemolytic anemia, and renal insufficiency that develops in 2-10% of patients with a history of malignant neoplasms treated with chemotherapeutic agents such as gemcitabin, mitomycin, oxaliplatin, etc. Chemotherapy-associated TMA is associated with high mortality and poor clinical outcomes (see, e.g., Blake-Haskins et al., Clin Cancer Res 17(18):5858-5866, 2011).

[0163] The cause of TMA after chemotherapy is thought to include non-specific and toxic injury to the microvascular endothelium. Direct damage to endothelial cells has been shown in an animal model of mitomycin-induced TMA (Dlott J. et al., Ther Apher Dial 8:102-11, 2004). Endothelial cell injury via various mechanisms has been shown to activate the lectin pathway of complement. For example, Stahl et al. have shown that endothelial cells exposed to oxidative stress activate the lectin pathway of complement both in vitro and in vivo (Collard et al., Am J Pathol. 156(5):1549-56, 2000; La Bonte et al., J Immunol. 15;188(2):885-91, 2012). In vivo, this process leads to thrombosis, and it has been shown that thrombosis is prevented by inhibiting the lectin pathway (La Bonte et al. J Immunol. 15;188(2):885-91, 2012). Furthermore, as demonstrated in Examples 37-39 herein, in a TMA mouse model in which local injury to the microvasculature and subsequent local photoexcitation of FITC-Dex are used to induce the development of a TMA response, the inventors have shown that TMA can be prevented by MASP-2 inhibition. Thus, the lectin pathway of complement may be activated by microvascular endothelial injury caused by chemotherapeutic agents, and then activation of the lectin pathway of complement leads to a state that promotes local thrombosis, which in turn promotes the TMA response. Since the activation of the lectin pathway and the generation of a state that promotes thrombosis are MASP-2-dependent, MASP-2 inhibitors, including but not limited to antibodies that block MASP-2 function, are expected to reduce the TMA response and lower the risk of TMA after cancer chemotherapy.

[0164] Accordingly, in another aspect, the present invention provides a method for treating or preventing TMA secondary to chemotherapy by administering to a subject suffering from TMA secondary to chemotherapy or at risk of developing said TMA a composition comprising a therapeutically effective amount of a MASP-2 inhibitor such as a MASP-2 antibody in a pharmaceutical carrier. The MASP-2 inhibitor is systemically administered to a subject who has undergone chemotherapy, is undergoing chemotherapy, or is scheduled to undergo chemotherapy, for example, by intra-arterial administration, intravenous administration, intramuscular administration, inhalation administration, subcutaneous administration, or other parenteral administration, or, in the case of non-peptidic agents, by oral administration. The anti-MASP-2 antibody may be administered alone or in combination with a C5 inhibitor, such as eculizumab.

[0165] In one aspect, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: the antibody binds human MASP-2 with a K D of 10 nM or less, the antibody binds an epitope in the CCP1 domain of MASP-2, the antibody inhibits C3b deposition in 1% human serum in an in vitro assay method with an IC 50 of 10 nM or less, the antibody inhibits C3b deposition in 90% human serum with an IC 50 of 30 nM or less, the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2, the antibody is a single-chain molecule, the antibody is an IgG2 molecule, the antibody is an IgG1 molecule, the antibody is an IgG4 molecule comprising the S228P mutation, and / or the antibody does not substantially inhibit the classical pathway. In one aspect, the antibody binds to MASP-2, selectively inhibits the lectin pathway, and does not substantially inhibit the alternative pathway. In one aspect, the antibody binds to MASP-2, selectively inhibits the lectin pathway, and does not substantially inhibit either the classical pathway or the alternative pathway (i.e., inhibits the lectin pathway while leaving the classical pathway and the alternative complement pathway intact).

[0166] In one aspect, the MASP-2 inhibitory antibody inhibits thrombus formation in serum from a subject suffering from TMA secondary to cancer chemotherapy by at least 30%, for example, at least 40%, for example, at least 50%, for example, at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95% up to a maximum of 99% as compared to untreated serum.

[0167] In one aspect, the MASP-2 inhibitory antibody inhibits thrombus formation in serum from a subject suffering from TMA secondary to cancer chemotherapy by at least 30%, for example, at least 40%, for example, at least 50%, for example, at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95% up to a maximum of 99% as compared to untreated serum.

[0168] In one aspect, the MASP-2 inhibitory antibody is administered to a subject via an intravenous catheter or other catheter delivery method.

[0169] In one aspect, the present invention is a method of inhibiting thrombosis in a subject suffering from TMA secondary to cancer chemotherapy, the method comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises: (I) (a) (i) a heavy chain CDR-H1 comprising the amino acid sequence of amino acids 31-35 of SEQ ID NO: 67; and (ii) a heavy chain CDR-H2 comprising the amino acid sequence of amino acids 50-65 of SEQ ID NO: 67; and (iii) a heavy chain CDR-H3 comprising the amino acid sequence of amino acids 95-102 of SEQ ID NO: 67, a heavy chain variable region, and, (b) (i) a light chain CDR-L1 comprising the amino acid sequence of amino acids 24-34 of SEQ ID NO: 70; and (ii) a light chain CDR-L2 comprising the amino acid sequence of amino acids 50-56 of SEQ ID NO: 70; and (iii) a light chain CDR-L3 comprising the amino acid sequence of amino acids 89-97 of SEQ ID NO: 70, a light chain variable region, or, (II) variants thereof comprising a heavy chain variable region having at least 90% identity to SEQ ID NO: 67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO: 67) and a light chain variable region having at least 90% identity to SEQ ID NO: 70 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity).

[0170] In some aspects, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 67. In some aspects, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0171] In one aspect, the method includes administering to a subject a composition comprising a MASP-2 inhibitory antibody or an antigen-binding fragment thereof that specifically recognizes at least a portion of a human MASP-2 epitope recognized by a reference antibody OMS646 comprising a heavy chain variable region shown in SEQ ID NO:67 and a light chain variable region shown in SEQ ID NO:70.

[0172] TMA secondary to transplantation Transplantation-associated thrombotic microangiopathy (TA-TMA) is a devastating syndrome that can occur in transplant patients, such as allogeneic hematopoietic stem cell transplant recipients (see, e.g., Batts and Lazarus, Bone Marrow Transplantation 40:709-719, 2007). The cause of this condition is not fully understood, but it is likely that a collection of responses resulting in endothelial cell injury is involved (Laskin B.L. et al., Blood 118(6):1452-62, 2011). As discussed above, endothelial cell injury is a typical stimulus for activation of the lectin pathway and generation of a pro-thrombotic environment.

[0173] Recent data further support the role of complement activation via the lectin pathway in the development of TA-TMA. Laskin et al. demonstrated that renal arteriolar C4d deposition (75%) in subjects with histological TA-TMA was much more frequently seen compared to controls (8%) (Laskin B.L., et al., Transplantation, 27; 96(2):217-23, 2013). Thus, C4d may be a pathological marker of TA-TMA, meaning local complement fixation via the lectin or classical pathway.

[0174] Since activation of the lectin pathway and generation of a pro-thrombotic state are MASP-2 dependent, MASP-2 inhibitors, including but not limited to antibodies that block MASP-2 function, are expected to reduce the TMA response and lower the risk of transplantation-associated thrombotic microangiopathy (TA-TMA).

[0175] Accordingly, in another aspect, the present invention provides a method for treating or preventing TMA secondary to transplantation by administering to a subject suffering from TMA secondary to transplantation or at risk of developing said TMA a therapeutically effective amount of a MASP-2 inhibitor, such as a MASP-2 antibody, in a pharmaceutical carrier. The MASP-2 inhibitor is systemically administered to a subject who has undergone, is undergoing, or is scheduled to undergo a transplantation procedure, for example, by intra-arterial, intravenous, intramuscular, inhaled, subcutaneous, or other parenteral administration, or, in the case of non-peptidic agents, by oral administration. The anti-MASP-2 antibody may be administered alone or in combination with a C5 inhibitor, such as eculizumab. In some aspects, the present invention provides a method for treating or preventing TMA secondary to allogeneic stem cell transplantation, comprising administering to the subject, before, during, or after receiving an allogeneic stem cell transplantation, a composition comprising an amount of a MASP-2 inhibitor, such as a MASP-2 inhibitory antibody.

[0176] In one aspect, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: the antibody binds human MASP-2 with a K D of 10 nM or less, the antibody binds an epitope in the CCP1 domain of MASP-2, the antibody inhibits C3b deposition in 1% human serum in an in vitro assay with an IC 50 of 10 nM or less, the antibody inhibits C3b deposition in 90% human serum with an IC 50inhibiting; the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2; the antibody is a single-chain molecule; the antibody is an IgG2 molecule; the antibody is an IgG1 molecule; the antibody is an IgG4 molecule containing an S228P mutation; and / or the antibody does not substantially inhibit the classical pathway. In one aspect, the antibody binds to MASP-2, selectively inhibits the lectin pathway, and does not substantially inhibit the alternative pathway. In one aspect, the antibody binds to MASP-2, selectively inhibits the lectin pathway, and does not substantially inhibit either the classical pathway or the alternative pathway (i.e., inhibits the lectin pathway while leaving the classical pathway and the alternative complement pathway intact).

[0177] In one aspect, the MASP-2 inhibitory antibody inhibits thrombosis in the serum from a subject suffering from TMA secondary to transplantation by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% up to a maximum of 99% compared to untreated serum.

[0178] In one aspect, the MASP-2 inhibitory antibody inhibits thrombosis in the serum from a subject suffering from TMA secondary to transplantation by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% up to a maximum of 99% compared to untreated serum.

[0179] In one aspect, the MASP-2 inhibitory antibody is administered to a subject via an intravenous catheter or other catheter delivery method.

[0180] In one aspect, the present invention provides a method for inhibiting thrombosis in a subject suffering from TMA secondary to transplantation, the method comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises: (I) (a) (i) a heavy chain CDR-H1 comprising the amino acid sequence of amino acids 31-35 of SEQ ID NO: 67; and (ii) a heavy chain CDR-H2 comprising the amino acid sequence of amino acids 50-65 of SEQ ID NO: 67; and (iii) a heavy chain CDR-H3 comprising the amino acid sequence of amino acids 95-102 of SEQ ID NO: 67, a heavy chain variable region, and (b) (i) a light chain CDR-L1 comprising the amino acid sequence of amino acids 24-34 of SEQ ID NO: 70; and (ii) a light chain CDR-L2 comprising the amino acid sequence of amino acids 50-56 of SEQ ID NO: 70; and (iii) a light chain CDR-L3 comprising the amino acid sequence of amino acids 89-97 of SEQ ID NO: 70, a light chain variable region, or (II) variants thereof comprising a heavy chain variable region having at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity) to SEQ ID NO: 67 and a light chain variable region having at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity) to SEQ ID NO: 70.

[0181] In some aspects, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 67. In some aspects, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0182] In one aspect, the method includes administering to a subject a composition comprising a MASP-2 inhibitory antibody or an antigen-binding fragment thereof that specifically recognizes at least a portion of a human MASP-2 epitope recognized by a reference antibody OMS646 comprising a heavy chain variable region shown in SEQ ID NO:67 and a light chain variable region shown in SEQ ID NO:70.

[0183] IV. Role of MASP-2 in other diseases and conditions, and methods of treatment using MASP-2 inhibitors Renal conditions Activation of the complement system is associated with the development of a variety of kidney diseases, including mesangial proliferative glomerulonephritis (IgA nephropathy, Berger's disease) (Endo, M., et al., Clin. Nephrology 55:185-191, 2001), membranous glomerulonephritis (Kerjashki, D., Arch B Cell Pathol. 58:253-71, 1990; Brenchley, P.E., et al., Kidney Int., 41:933-7, 1992; Salant, D.J., et al., Kidney Int. 55:976-84, 1989), membranoproliferative glomerulonephritis (mesangiocapillary glomerulonephritis) (Bartlow, B.G.. et al., Kidney Int. 15:294-300, 1979; Meri, S. et al., J. Exp. Med. 175:939-50, 1992), postinfectious acute glomerulonephritis (poststreptococcal glomerulonephritis), cryoglobulinemic glomerulonephritis (Ohsawa, I., et al., Clin Immunol, 101:59-66, 2001), lupus nephritis (Gatenby, P.A., Autoimmunity 11:61-6, 1991), and Henoch-Schönlein purpura nephritis (Endo, M., et al., Am. J. Kidney Dis. 35:401-407, 2000). Although the involvement of complement in kidney diseases has been recognized for decades, its exact role in the onset, development, and recovery phases of kidney diseases remains a major topic of debate. The contribution of complement under normal conditions is beneficial to the host, but inappropriate activation and deposition of complement can contribute to tissue damage.

[0184] Glomerulonephritis, which is an inflammation of the glomeruli, often begins with the deposition of immune complexes in the glomerular or tubular structures, and then there is much evidence that this deposition induces complement activation, inflammation, and tissue damage. Kahn and Sinniah demonstrated increased C5b-9 deposition in the tubular basement membrane in biopsy specimens taken from patients with various forms of glomerulonephritis (Kahn, T.N., et al., Histopath. 26:351-6, 1995). In a study of patients with IgA nephropathy (Alexopoulos, A., et al., Nephrol. Dial. Transplant 10:1166-1172, 1995), C5b-9 deposition in the tubular epithelial / basement membrane structure was correlated with plasma creatinine levels. Another study of membranous nephropathy demonstrated a relationship between clinical outcome and urinary sC5b-9 levels (Kon, S. P., et al., Kidney Int. 48:1953-58, 1995). High sC5b-9 levels were positively correlated with a poor prognosis. Lehto et al. measured high levels of CD59, a complement regulatory factor that inhibits the membrane attack complex in the plasma membrane, as well as C5b-9, in urine derived from patients with membranous glomerulonephritis (Lehto, T., et al., Kidney Int. 47; 1403-11, 1995). Histopathological analysis of biopsy specimens taken from these same patients demonstrated deposition of C3 and C9 proteins in the glomeruli, whereas CD59 expression in these tissues was decreased compared to normal kidney tissue. From these various studies, it was suggested that ongoing complement-mediated glomerulonephritis results in urinary excretion of complement proteins that correlates with the degree of tissue damage and disease prognosis.

[0185] Inhibition of complement activation in various animal models of glomerulonephritis has also demonstrated the importance of complement activation in the pathogenesis of this disease. In a membranoproliferative glomerulonephritis (MPGN) model, injection of anti-Th1 antiserum into C6-deficient rats (which cannot form C5b-9) resulted in a 90% reduction in glomerular cell proliferation, an 80% reduction in platelet and macrophage infiltration, a decrease in type IV collagen synthesis (a marker of mesangial matrix expansion), and a 50% reduction in proteinuria compared to C6+ normal rats (Brandt. J., et al., Kidney Int. 49:335-343, 1996). These results imply C5b-9 as the major tissue-damaging mediator by complement in this rat anti-thymocyte serum model. In another glomerulonephritis model, stepwise administration of rabbit anti-rat glomerular basement membrane resulted in a dose-dependent influx of polymorphonuclear leukocytes (PMNs), which was attenuated by pretreatment with cobra venom factor (which consumes complement) (Scandrett, A.L., et al., Am. J. Physiol 268:F256-F265, 1995). Rats treated with cobra venom factor also showed reduced histopathological changes, reduced long-term proteinuria, and lower creatinine levels compared to control rats. Using three types of GN models (anti-thymocyte serum, ConA anti-ConA, and passive Heymann nephritis) in rats, Couser et al. demonstrated the potential therapeutic efficacy of an approach to inhibit complement by using recombinant sCR1 protein (Couser, W.G., et al., J. Am. Soc. Nephrol. 5:1888-94, 1995). Rats treated with sCR1 showed a significant reduction in PMN, platelet, and macrophage influx, as well as a reduction in mesangiolysis and proteinuria compared to control rats. Further evidence of the importance of complement activation in glomerulonephritis has been obtained by using anti-C5 MoAb in the NZB / W F1 mouse model. Anti-C5 MoAb inhibits C5 cleavage and thus blocks the generation of C5a and C5b-9. Continuous therapy with anti-C5 MoAb for 6 months significantly ameliorated the course of glomerulonephritis.The humanized anti-C5 MoAb monoclonal antibody (5G1.1), which blocks the cleavage of human complement component C5 into inflammatory promoting components, is currently being developed by Alexion Pharmaceuticals, Inc., New Haven, Connecticut as a potential treatment for glomerulonephritis.

[0186] Studies of patients with genetic deficiencies of specific complement components have provided direct evidence of the pathogenic role of complement in kidney injury. Many reports have demonstrated an association between kidney disease and deficiency of the complement regulatory factor H (Ault, B.H.. Nephrol. 14:1045-1053, 2000; Levy, M., et al., Kidney Int. 30:949-56, 1986; Pickering, M.C., et al., Nat. Genet, 31:424-8, 2002). Deficiency of factor H results in decreased plasma levels of factor B and C3 and consumption of C5b-9. Both atypical membranoproliferative glomerulonephritis (MPGN) and idiopathic hemolytic uremic syndrome (HUS) have been associated with factor H deficiency. Factor H-deficient pigs (Jansen, J.H., et al., Kidney Int. 55:331-49, 1998) and factor H knockout mice (Pickering, M.C., 2002) exhibit MPGN-like symptoms. This supports the importance of factor H in complement regulation. Deficiencies of other complement components have been associated with kidney diseases that follow the development of systemic lupus erythematosus (SLE) (Walport, M.J., Davies, et al., Ann. N. Y, Acad. Sci. 815:267-81, 1997). Deficiencies of C1q, C4, and C2 are strong predisposing factors for the development of SLE through mechanisms related to the incomplete clearance of immune complexes and apoptotic materials. In many of these SLE patients, lupus nephritis, characterized by immune complex deposition throughout the glomeruli, develops.

[0187] The identification of autoantibodies against complement components in patients has provided further evidence linking complement activation to kidney disease. Some of these autoantibodies have been directly associated with kidney disease (Trouw, L.A., et al., Mol. Immunol. 38:199-206, 2001). Since these numerous autoantibodies show a fairly high correlation with kidney disease, the term nephritis factor (NeF) has been introduced to denote this activity. In clinical studies, approximately 50% of patients positive for nephritis factor developed MPGN (Spitzer, R.E. et al., Clin. Immunol. Immunopathol. 64:177-83, 1992). C3NeF is an autoantibody against the alternative pathway C3 convertase (C3bBb), which stabilizes this convertase and thereby promotes alternative pathway activation (Daha, M.R., et al., J. Immunol. 116:1-7, 1976). Similarly, autoantibodies specific for the classical pathway C3 convertase (C4b2a) are called C4NeF, which stabilizes this convertase and thereby promotes classical pathway activation (Daha, M.R. et al., J. Immunol. 125:2051-2054, 1980; Halbwachs, L., et al., J, Clin. Invest. 65:1249-56, 1980). Anti-C1q autoantibodies have been reported to be associated with nephritis in SLE patients (Hovath, L. et al., Clin. Exp. Rheumatol, 19:667-72, 2001 ; Siegert, C, et al., J. Rheumatol 18:230-34, 1991; Siegert, C, et al., Clin. Exp. Rheumatol. 10:19-23, 1992). An increase in the titer of these anti-C1q autoantibodies has been reported to predict the progression of nephritis (Coremans, I.E. et al., Am. J. Kidney Dis. 26:595-601, 1995).The accumulation of these anti-C1q autoantibodies was revealed by immunoprecipitation eluted from the postmortem kidneys of SLE patients (Mannick, M, et al., Arthritis Rheumatol. 40:1504-11, 1997). All these facts suggest the pathogenic role of these autoantibodies. However, not all patients with anti-C1q autoantibodies develop kidney disease, and some healthy individuals also have low-titer anti-C1q autoantibodies (Siegert, C.E., et al., Clin. Immunol. Immunopathol. 67:204-9, 1993).

[0188] In addition to the alternative and classical pathways of complement activation, the lectin pathway may also have an important pathogenic role in kidney disease. High levels of MBL, MBL-associated serine protease, and complement activation products have been detected by immunohistochemical techniques in kidney biopsy materials obtained from patients diagnosed with several different kidney diseases, including Henoch-Schönlein purpura nephritis (Endo, M. et al., Am. J. Kidney Dis. 35:401-407, 2000), cryoglobulinemic glomerulonephritis (Ohsawa, I., et al., Clin. Immunol. 101:59-66, 2001), and IgA neuropathy (Endo, M., et al., Clin. Nephrology 55:185-191, 2001). Therefore, despite the fact that the relationship between complement and kidney disease has been known for decades, the data on exactly how complement affects these kidney diseases are far from complete.

[0189] Blood disorder Sepsis is caused by an overwhelming reaction of the patient to invading microorganisms. The main function of the complement system is to organize the inflammatory response against invading bacteria and other pathogens. Consistent with this physiological role, in a very large number of studies, complement activation has been shown to have a major role in the development of sepsis (Bone, R.C., Annals. Internal, Med. 115:457-469, 1991). The definition of the clinical symptoms of sepsis has been constantly evolving. Sepsis is usually defined as a systemic host response to an infectious disease. However, in many patients with sepsis symptoms, clinical evidence of an infectious disease (e.g., positive bacterial blood cultures) has not been found. This contradiction was first considered at the Consensus Conference in 1992, at which time the term "systemic inflammatory response syndrome" (SIRS) was established, and the definable presence of a bacterial infectious disease was no longer required (Bone, R.C., et al., Crit. Care Med. 20:724-726, 1992). Currently, there is a general consensus that sepsis and SIRS are associated with dysregulation of the inflammatory response. To briefly review this, the inventors consider the clinical definition of sepsis to include severe sepsis, septic shock, and SIRS as well.

[0190] Prior to the late 1980s, the most predominant source of infection in sepsis patients was Gram-negative bacteria. Lipopolysaccharide (LPS), the main component of the Gram-negative bacterial cell wall, was known to stimulate the release of inflammatory mediators from various cell types and induce acute infectious symptoms when injected into animals (Haeney, M.R., et al., Antimicrobial Chemotherapy 41(Suppl. A):41-6, 1998). Interestingly, the spectrum of causative microorganisms has seemingly changed from mainly Gram-negative bacteria in the late 1970s and 1980s to mainly Gram-positive bacteria currently, for reasons that are not yet clear (Martin, G.S., et al., N. Eng. J. Med. 348:1546-54, 2003).

[0191] Numerous studies have demonstrated the importance of complement activation in mediating inflammation and contributing to the characteristics of shock, particularly septic shock and hemorrhagic shock. Usually, both Gram-negative and Gram-positive organisms can cause septic shock. LPS is a potent activator of complement mainly via the alternative pathway, but classical pathway activation mediated by antibodies also occurs (Fearon, D.T., et al., N. Engl. J. Med. 292:937-400, 1975). The major components of the Gram-positive cell wall are peptidoglycan and lipoteichoic acid, both of which are potent activators of the alternative complement pathway, but the classical complement pathway can also be activated in the presence of specific antibodies (Joiner, K.A., et al., Ann. Rev. Immunol. 2:461-2, 1984).

[0192] When researchers noticed that anaphylatoxins C3a and C5a, which are components of the complement system, mediate various inflammatory responses that can also occur in sepsis, the complement system was first associated with the development of sepsis. These anaphylatoxins induce vasodilation and increased microvascular permeability, events that play a central role in septic shock (Schumacher, W.A., et al., Agents Actions 34:345-349, 1991). In addition, anaphylatoxins induce bronchospasm, histamine release from mast cells, and platelet aggregation. Furthermore, they exert a number of actions on granulocytes, such as chemotaxis, aggregation, adhesion, release of lysosomal enzymes, generation of toxic superoxide anions, and leukotriene formation (Shin, H.S., et al., Science 162:361-363, 1968; Vogt, W., Complement 3:177-86, 1986). These biological effects are thought to play a role in the development of sepsis complications such as shock or acute respiratory distress syndrome (ARDS) (Hammerschmidt, D.E., et al., Lancet 1:947-949, 1980; Slotman, G.T., et al., Surgery 99:744-50, 1986). In addition, high levels of anaphylatoxin C3a have been associated with a lethal outcome in sepsis (Hack, C.E., et al., Am. J. Med. 86:20-26, 1989). In some shock animal models, certain complement-deficient strains (e.g., C5-deficient strains) are more resistant to the effects of LPS injection (Hseuh, W, et al., Immunol. 70:309-14, 1990).

[0193] Blocking C5a generation by antibodies during the onset of sepsis in rodents has been shown to significantly improve survival (Czermak, B.J., et al., Nat. Med. 5:788-792, 1999). Similar findings were made when the C5a receptor (C5aR) was blocked using antibodies or small molecule inhibitors (Huber-Lang, M.S., et al., FASEB J. 16:1567-74, 2002; Riedemann, N.C., et al., J. Clin. Invest. 110:101-8, 2002). Initial experimental studies in monkeys have suggested that antibody blockade of C5a attenuated Escherichia coli (E. coli)-induced septic shock and adult respiratory distress syndrome (Hangen, D.H. et al., J. Surg. Res. 46:195-9, 1989; Stevens, J.H., et al., J. Clin. Invest. 77:1812-16, 1986). In humans with sepsis, C5a was increased and associated with a significantly lower survival rate with multiple organ failure compared to patients and survivors with less severe sepsis (Nakae, H,, et al., Res. Commun. Chem. Pathol. Pharmacol. 84:189-95, 1994; Nakae, et al., Surg. Today 26:225-29, 1996; Bengtson, A., et al., Arch. Surg. 123:645-649, 1988). The mechanism by which C5a exerts a harmful effect during sepsis has not yet been investigated in detail, but recent data suggest that when C5a is generated during sepsis, the innate immune functions of blood neutrophils (Huber-Lang, M.S., et al., J. Immunol. 169:3223-31, 2002), the ability to express respiratory burst, and the ability to produce cytokines (Riedemann, N.C., et al., Immunity 19:193-202, 2003) are significantly impaired.Furthermore, C5a generation during sepsis appears to have a procoagulant effect (Laudes, I.J., et al., Am. J. Pathol. 160:1867-75, 2002). The complement regulatory protein CI INH has also shown efficacy in animal models of sepsis and ARDS (Dickneite, G., BehringIns. Mitt. 93:299-305, 1993).

[0194] The lectin pathway may also have a role in the development of sepsis. MBL has been shown to bind to a clinically important range of microorganisms, including Gram-negative and Gram-positive bacteria, and activate the lectin pathway (Neth, O., et al., Infect. Immun, 68:688, 2000). Lipoteichoic acid (LTA) is increasingly regarded as the Gram-positive counterpart of LPS. It is a potent immunostimulant that induces cytokine release from monocytes and whole blood (Morath, S., et al., J. Exp. Med. 195:1635, 2002; Morath, S., et al., Infect. Immun. 70:938, 2002). Recently, L-ficolin has been demonstrated to specifically bind to LTA isolated from a very large number of Gram-positive bacterial species, including Staphylococcus aureus, and activate the lectin pathway (Lynch, N.J., et al., J. Immunol. 172:1198-02, 2004). MBL has also been shown to bind to LTA derived from species of the genus Enterococcus in which the polyglycerol phosphate chain is substituted with glycosyl groups, but not to LTA derived from nine other species, including Staphylococcus aureus (Polotsky, V.Y., et al., Infect. Immun. 64:380, 1996).

[0195] Accordingly, one aspect of the present invention provides a method for treating sepsis or a condition resulting from sepsis by administering to a subject suffering from sepsis or a condition resulting from sepsis, including but not limited to severe sepsis, septic shock, acute respiratory distress syndrome resulting from sepsis, and systemic inflammatory response syndrome, a composition comprising a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier. A related method for treating such conditions is provided by administering to a subject suffering from other blood disorders including hemorrhagic shock, hemolytic anemia, autoimmune thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), or other bone marrow / blood destructive conditions, a composition comprising a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier. The MASP-2 inhibitor is systemically administered to the subject, for example, by intraarterial administration, intravenous administration, intramuscular administration, inhalation (particularly in the case of ARDS), subcutaneous administration, or other parenteral administration, or in the case of non-peptide agents, by oral administration. The MASP-2 inhibitor composition may be combined with one or more additional therapeutic agents to combat the sequelae of sepsis and / or shock. In the case of advanced sepsis or shock or the resulting critical condition, the MASP-2 inhibitor composition may be administered, as appropriate, in a rapid-acting dosage form, for example, by bolus intravenous delivery or intraarterial delivery of a solution containing the MASP-2 inhibitor composition. Repeated dosing may be carried out as determined by a physician until the condition has resolved.

[0196] Coagulation disorder Evidence has been obtained regarding the role of the complement system in disseminated intravascular coagulation ("DIC"), for example, DIC following severe physical trauma.

[0197] Previous studies have shown that C4- / - mice are not protected from renal reperfusion injury (Zhou, W., et al., "Predominant role for C5b-9 in renal ischemia / reperfusion injury", J Clin Invest 105:1363-1371 (2000)). To investigate whether C4- / - mice may be able to activate complement via either the classical or lectin pathway, C3 turnover in C4- / - plasma was measured in assays specific for the classical pathway activation pathway or the lectin pathway activation pathway. C3 cleavage was not observed upon induction of activation via the classical pathway, but highly efficient lectin pathway-dependent C3 activation was observed in C4-deficient serum (Figure 30). According to many previously published papers on alternative pathway activation, it can be seen that C3b deposition on mannan and zymosan is greatly impaired in MASP-2- / - mice even under experimental conditions acceptable for all three pathways. When the same serum was used in wells coated with immune globulin complexes instead of mannan or zymosan, C3b deposition and factor B cleavage were seen in MASP-2+ / + mouse serum and MASP-2- / - serum, but not in C1q-depleted serum. This indicates that alternative pathway activation is promoted in MASP-2- / - serum when initial C3b is provided via classical activation. Figure 30C shows the surprising finding that C3 can be efficiently activated in a lectin pathway-dependent manner in C4-deficient plasma.

[0198] This "C4 bypass" is lost by inhibiting lectin pathway activation by preincubating plasma with soluble mannan or mannose.

[0199] Abnormal non-immune activation of the complement system can be potentially harmful to humans and may play an important role in the activation of hematological pathways, particularly in severe trauma situations where both inflammatory and hematological pathways are activated. In a normal healthy state, C3 conversion is <5% of total plasma C3 protein. In severe infections including sepsis and immune complex diseases, C3 conversion spontaneously returns to approximately 30%, and complement levels are often lower than normal due to increased utilization and changes in pool distribution. Rapid C3 pathway activation above 30% generally shows obvious clinical evidence of vasodilation and fluid loss to tissues. In C3 conversion above 30%, the initiating mechanism is mainly non-immune, and the resulting clinical symptoms are harmful to the patient. Complement C5 levels in healthy states and controlled diseases appear to be considerably more stable than C3. Marked decreases and / or conversion of C5 levels are associated with the patient's response to the likely onset of abnormal multiple trauma (e.g., traffic accidents) and shock lung syndrome. Thus, any evidence regarding complement C3 activation above 30% of the vascular pool, or complement C3 activation involving any C5, or both, can be considered likely to be a harbinger of harmful pathological changes in the patient.

[0200] Both C3 and C5 act on mast cells and basophils to release anaphylatoxins (C3a and C5a) that cause the release of vasoactive chemicals. These provide a chemotactic gradient that directs polymorphonuclear cells (PMNs) to the center of the immunological disturbance (beneficial response), but differ in that C5a has a specific clamping (aggregation) action on these phagocytes, preventing the phagocytes from randomly leaving the reaction site. In the normal control of infection, C3 activates C5. However, in multiple trauma, C5 is widely activated and C5a anaphylatoxin appears to occur systemically. Due to this uncontrolled activity, polymorphs cluster within the vascular system, and these masses are then washed into the pulmonary capillaries, occluding them and causing local damaging effects as a result of superoxide release. Although not bound by theory, this mechanism is probably important in the development of acute respiratory distress syndrome (ARDS). However, recently, this idea has been challenged. C3a anaphylatoxins can be shown to be potent platelet aggregators in vitro, but in vivo, their involvement is less clear, and the release of platelet substances and plasmin in wound healing may only be secondarily involved with complement C3. A long-term increase in C3 activation may be required to produce DIC.

[0201] In addition to the cellular and vascular effects of the activated complement components outlined above, which can explain the relationship between trauma and DIC, new and emerging scientific discoveries have identified direct molecular relationships and functional crosstalk between the complement and coagulation systems. The supporting data are derived from studies in C3-deficient mice. Since C3 is a common component of each complement pathway, C3-deficient mice are expected to lack all complement functions. However, surprisingly, C3-deficient mice are able to fully activate the terminal complement components (Huber-Lang, M., et al., 「Generation of C5a in the absence of C3: a new complement activation pathway」, Nat. Med 12:682-687(2006)). In-depth studies have revealed that the C3-independent activation of the terminal complement components is mediated by thrombin, the rate-limiting enzyme of the coagulation cascade (Huber et al., 2006). The molecular components mediating thrombin activation after initial complement activation remain enigmatic.

[0202] The present inventors have elucidated what is thought to be the molecular basis of the crosstalk between the complement cascade and the coagulation cascade, and have identified MASP-2 as a central control point connecting the two systems. From biochemical studies on the substrate specificity of MASP-2, in addition to the well-known C2 and C4 complement proteins, prothrombin was identified as a potential substrate. MASP-2 specifically cleaves a functional-related site of prothrombin to generate thrombin, which is the rate-limiting enzyme of the coagulation cascade (Krarup, A., et al., 「Simultaneous Activation of Complement and Coagulation by MBL-Associated Serine Protease 2」, PLoS. ONE. 2;e623(2007)). Thrombin generated by MASP-2 can promote fibrin deposition in a defined reconstituted in vitro system. From this, the functional relevance of MASP-2 cleavage is demonstrated (Krarup et al., 2007). As discussed in the following examples of this specification, the present inventors further confirmed the physiological significance of this discovery by recording thrombin activation in normal rodent serum after lectin pathway activation, and demonstrated that this process is blocked by a neutralizing MASP-2 monoclonal antibody.

[0203] MASP-2 may be a central branching point in the lectin pathway that can promote the activation of both the complement and coagulation systems. Since lectin pathway activation is a physiological response to many types of traumatic injury, the inventors believe that the concurrent systemic inflammation (mediated by complement components) and disseminated coagulation (mediated via the coagulation pathway) can be explained by MASP-2's ability to activate both pathways. These findings clearly suggest the role of MASP-2 in the development of DIC and the therapeutic benefit of MASP-2 inhibition in the treatment or prevention of DIC. MASP-2 may provide a molecular link between the complement and coagulation systems, and lectin pathway activation, as occurs in trauma situations, can directly initiate coagulation system activation via the MASP-2-thrombin axis, thus providing a mechanistic link between trauma and DIC. According to one aspect of the present invention, it is believed that inhibition of MASP-2 inhibits lectin pathway activation and reduces the production of anaphylatoxins C3a and C5a. A long-term increase in C3 activation is thought to be required for the development of DIC.

[0204] Microcirculatory coagulation (blood clots in capillaries and small vessels) occurs in such septic shock situations. As demonstrated by the protective phenotype of the septic MASP-2(- / -) mouse model as described in Example 17 and FIGS. 18 and 19, the role of the lectin pathway in septic shock has been elucidated. Furthermore, as demonstrated in Example 15 and FIGS. 16A and 16B, MASP-2(- / -) mice are protected in the localized Schwartzman reaction model of disseminated intravascular coagulation (DIC), a model of localized coagulation in the microvasculature.

[0205] V. MASP-2 Inhibitors In one aspect, the present invention provides a method of inhibiting MASP-2-dependent complement activation in a subject suffering from thrombotic microangiopathy or at risk of developing thrombotic microangiopathy. The MASP-2 inhibitor is administered in an amount effective to inhibit MASP-2-dependent complement activation in a living subject. In the practice of this aspect of the invention, representative MASP-2 inhibitors include molecules that inhibit the biological activity of MASP-2 (e.g., small molecule inhibitors, anti-MASP-2 antibodies, or blocking peptides that interact with MASP-2 or interfere with protein-protein interactions), as well as molecules that reduce MASP-2 expression, thereby preventing MASP-2 from activating the lectin complement pathway (e.g., MASP-2 antisense nucleic acid molecules, MASP-2-specific RNAi molecules, and MASP-2 ribozymes). The MASP-2 inhibitor may be used alone as primary therapy or in combination with other therapeutic agents as adjuvant therapy to improve the therapeutic benefit of other medical treatments.

[0206] Inhibition of MASP-2-dependent complement activation is characterized by at least one of the following changes in complement system components resulting from administration of a MASP-2 inhibitor according to the method of the invention: inhibition of the generation or production of MASP-2-dependent complement activation system products C4b, C3a, C5a, and / or C5b-9 (MAC) (e.g., as measured as described in Example 2), a decrease in complement activation as evaluated in a hemolytic assay using non-sensitized rabbit or guinea pig erythrocytes (e.g., as measured as described in Example 33), a decrease in C4 cleavage and C4b deposition (e.g., as measured as described in Example 2), or a decrease in C3 cleavage and C3b deposition (e.g., as measured as described in Example 2).

[0207] According to the present invention, a MASP-2 inhibitor effective in inhibiting the MASP-2-dependent complement activation system is used. MASP-2 inhibitors useful in the practice of this aspect of the present invention include, for example, anti-MASP-2 antibodies and fragments thereof, MASP-2 inhibitory peptides, small molecules, MASP-2 soluble receptors, and expression inhibitors. The MASP-2 inhibitor may inhibit the MASP-2-dependent complement activation system by blocking the biological function of MASP-2. For example, the inhibitor may effectively block MASP-2 protein-protein interactions, may interfere with MASP-2 dimerization or aggregation, may block Ca 2+ binding, may interfere with the MASP-2 serine protease active site, or may reduce MASP-2 protein expression.

[0208] In some embodiments, the MASP-2 inhibitor selectively inhibits MASP-2 complement activation without impairing the function of the C1q-dependent complement activation system.

[0209] In one embodiment, the MASP-2 inhibitor useful in the method of the present invention is a specific MASP-2 inhibitor that binds specifically to the polypeptide containing SEQ ID NO:6 with an affinity at least 10-fold higher than other antigens of the complement system. In another embodiment, the MASP-2 inhibitor binds specifically to the polypeptide containing SEQ ID NO:6 with an affinity at least 100-fold higher than other antigens of the complement system. The binding affinity of the MASP-2 inhibitor can be determined using an appropriate binding assay.

[0210] The MASP-2 polypeptide exhibits a molecular structure similar to MASP-1, MASP-3, which are proteases of the C1 complement system, and C1r and C1s. The cDNA molecule shown in SEQ ID NO:4 encodes a representative example of MASP-2 (consisting of the amino acid sequence shown in SEQ ID NO:5), and provides a human MASP-2 polypeptide having a leader sequence (aa1-15) that is cleaved after secretion to yield mature human MASP-2 (SEQ ID NO:6). As shown in Figure 2, the human MASP2 gene contains 12 exons. The human MASP-2 cDNA is encoded by exons B, C, D, F, G, H, I, J, K, and L. As shown in Figure 2, alternative splicing gives rise to a 20 kDa protein called MBL-associated protein 19 (also called "MAp19", "sMAP") (SEQ ID NO:2) encoded by (SEQ ID NO:1) arising from exons B, C, D, and E. The cDNA molecule shown in SEQ ID NO:50 encodes a mouse MASP-2 (consisting of the amino acid sequence shown in SEQ ID NO:51), and provides a mouse MASP-2 polypeptide having a leader sequence that is cleaved after secretion to yield mature mouse MASP-2 (SEQ ID NO:52). The cDNA molecule shown in SEQ ID NO:53 encodes a rat MASP-2 (consisting of the amino acid sequence shown in SEQ ID NO:54), and provides a rat MASP-2 polypeptide having a leader sequence that is cleaved after secretion to yield mature rat MASP-2 (SEQ ID NO:55).

[0211] One of ordinary skill in the art would recognize that the sequences disclosed in SEQ ID NO:4, SEQ ID NO:50, and SEQ ID NO:53 are each a single allele of human MASP-2, mouse MASP-2, and rat MASP-2, respectively, and would expect that allelic variation and alternative splicing would occur. Allelic variants of the nucleotide sequences shown in SEQ ID NO:4, SEQ ID NO:50, and SEQ ID NO:53, including allelic variants containing silent mutations and allelic variants in which the amino acid sequence is changed by mutations, are within the scope of the present invention. Allelic variants of the MASP-2 sequence can be cloned by probing cDNA libraries or genomic libraries derived from different individuals according to standard procedures.

[0212] The domains of the human MASP-2 protein (SEQ ID NO:6) are shown in FIGS. 1 and 2A and include an N-terminal C1r / C1s / urchin Vegf / bone morphogenetic protein (CUBI) domain (aa 1-121 of SEQ ID NO:6), an epidermal growth factor-like domain (aa 122-166), another CUBI domain (aa 167-293), and a tandem array of complement control protein domains and a serine protease domain. MAp19 shown in FIG. 1 is obtained from alternative splicing of the MASP2 gene. MAp19 is a non-enzymatic protein containing the N-terminal CUBI-EGF region of MASP-2 and four additional residues (EQSL) derived from exon E, as shown in FIG. 1.

[0213] Several proteins have been shown to bind to or interact with MASP-2 via protein-protein interactions. For example, MASP-2 binds to the lectin proteins MBL, H-ficolin, and L-ficolin and, together with these, Ca 2+It is known to form a dependency complex. Each MASP-2 / lectin complex has been shown to activate complement via MASP-2-dependent cleavage of proteins C4 and C2 (Ikeda, K., et al., J. Biol Chem. 262:7451-7454, 1987; Matsushita, M., et al., J. Exp. Med 176:197-2284, 2000; Matsushita, M., et al., J. Immunol.168:3502-3506, 2002). Studies have shown that the CUB1-EGF domain of MASP-2 is essential for the binding of MASP-2 to MBL (Thielens, N.M., et al., J. Immunol. 166:5068, 2001). The CUB1EGFCUBII domain has also been shown to mediate the MASP-2 dimerization required for the formation of the active MBL complex (Wallis, R., et al., J. Biol. Chem. 275:30962-30969, 2000). Therefore, a MASP-2 inhibitor that binds to the MASP-2 target region known to be important for MASP-2-dependent complement activation, or a MASP-2 inhibitor that interferes with the MASP-2 target region, can be identified.

[0214] Anti-MASP-2 antibody In some embodiments of this aspect of the invention, the MASP-2 inhibitor comprises an anti-MASP-2 antibody that inhibits the MASP-2-dependent complement activation system. Anti-MASP-2 antibodies useful in this aspect of the invention include polyclonal antibodies, monoclonal antibodies, or recombinant antibodies derived from any antibody-producing mammal, and may be multispecific, chimeric, humanized, anti-idiotypic, and antibody fragments. Antibody fragments include Fab, Fab', F(ab)2, F(ab')2, Fv fragments, scFv fragments, and single-chain antibodies, as further described herein.

[0215] Several anti-MASP-2 antibodies have been described in the literature, some of which are listed in Table 1 below. The ability of these previously described anti-MASP-2 antibodies to inhibit the MASP-2-dependent complement activation system can be screened using the assay methods described herein. For example, as further detailed in Examples 10 and 11 herein, an anti-rat MASP-2 Fab2 antibody that blocks MASP-2-dependent complement activation has been identified. Once an anti-MASP-2 antibody that functions as a MASP-2 inhibitor is identified, it can be used to generate anti-idiotype antibodies and to identify other MASP-2 binding molecules, as further described below.

[0216] (Table 1) MASP-2 specific antibodies from the literature TIFF2025108730000008.tif166155

[0217] Anti-MASP-2 antibody with reduced effector function In some aspects of this aspect of the invention, the anti-MASP-2 antibody has reduced effector function to alleviate inflammation that may result from activation of the classical complement pathway. The ability of IgG molecules to induce the classical complement pathway has been shown to be within the Fc portion of this molecule (Duncan, A.R.. et al., Nature 332:738-740 1988). IgG molecules in which the Fc portion of this molecule has been removed by enzymatic cleavage do not have this effector function (see Harlow, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). Thus, antibodies with reduced effector function as a result of lacking the Fc portion of this molecule can be generated by having a genetically engineered Fc sequence that minimizes effector function or by making it a human IgG2 or IgG4 isotype.

[0218] Antibodies with reduced effector function can be made by standard molecular biology manipulations of the Fc portion of the IgG heavy chain as described in Example 9 herein and also as described in Jolliffe et al., Int'l Rev. Immunol. 10:241-250, 1993 and Rodrigues et al., J. Immunol. 151:6954-6961, 1998. Antibodies with reduced effector function also include human IgG2 and IgG4 isotypes with reduced ability to activate complement and / or interact with Fc receptors (Ravetch, J. V., et al., Annu. Rev. Immunol. 9:457-492, 1991 ; Isaacs, J.D., et al., J. Immunol. 148:3062-3071, 1992; van de Winkel, J.G., et al., Immunol Today 14:215-221, 1993). Humanized or fully human antibodies specific for human MASP-2 consisting of the IgG2 or IgG4 isotype can be made by one of several methods known to those of skill in the art as described in Vaughan, T.J., et al., Nature Biotechnical 16:535-539, 1998.

[0219] Production of anti-MASP-2 antibodies Anti-MASP-2 antibodies can be prepared using an MASP-2 polypeptide (e.g., full-length MASP-2) or a peptide containing an antigenic MASP-2 epitope (e.g., a part of the MASP-2 polypeptide). The immunogenic peptide may be as small as 5 amino acid residues. For example, an MASP-2 polypeptide comprising the entire amino acid sequence of SEQ ID NO:6 may be used to induce anti-MASP-2 antibodies useful in the methods of the present invention. Certain MASP-2 domains known to be involved in protein-protein interactions, such as the CUBI and CUBIEGF domains, as well as the region containing the serine-protease active site, may be expressed as recombinant polypeptides and used as antigens as described in Example 3. Furthermore, peptides comprising at least 6 amino acid portions of the MASP-2 polypeptide (SEQ ID NO:6) are also useful for inducing MASP-2 antibodies. Further examples of MASP-2-derived antigens useful for inducing MASP-2 antibodies are shown in Table 2 below. The MASP-2 peptides and polypeptides used to produce the antibodies may be native polypeptides or recombinant or synthetic peptides and catalytically inactive recombinant polypeptides, such as those isolated as MASP-2A, as further described in Examples 5-7. In some embodiments of this aspect of the invention, anti-MASP-2 antibodies are obtained using transgenic mouse lines as described in Examples 8 and 9 and further described below.

[0220] Antigens useful in the preparation of anti-MASP-2 antibodies also include fusion polypeptides, such as fusions of MASP-2 or a part thereof with an immunoglobulin polypeptide or maltose-binding protein. The polypeptide immunogen may be a full-length molecule or a part thereof. When the polypeptide portion is hapten-like, such a portion may be conveniently conjugated or linked to a macromolecular carrier (e.g., keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or tetanus toxoid) for immunization.

[0221] (Table 2) MASP-2-derived antigens TIFF2025108730000009.tif186153

[0222] Polyclonal antibody Polyclonal antibodies against MASP-2 can be prepared by immunizing an animal with an MASP-2 polypeptide or an immunogenic portion thereof using methods well known to those skilled in the art. See, for example, Green et al., “Production of Polyclonal Antisera”, Immunochemical Protocols (Manson, ed.), page 105. Further described in Example 6. The immunogenicity of the MASP-2 polypeptide can be enhanced using adjuvants including mineral gels such as aluminum hydroxide or Freund's adjuvant (complete or incomplete), surfactants such as lysophosphatidylcholine, pluronic polyols, polyanions, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Polyclonal antibodies are typically produced in animals such as horses, cows, dogs, chickens, rats, mice, rabbits, guinea pigs, goats, or sheep. Alternatively, anti-MASP-2 antibodies useful in the present invention may also be derived from primates close to humans. General techniques for producing antibodies useful for diagnosis and treatment in baboons can be found, for example, in Goldenberg et al., International Patent Publication WO91 / 11465, and Losman, M.J., et al., Int. J. Cancer 46:310, 1990. Serum containing immunologically active antibodies is then generated from the blood of such immunized animals using standard procedures well known in the art.

[0223] Monoclonal antibody In some embodiments, the MASP-2 inhibitor is an anti-MASP-2 monoclonal antibody. Anti-MASP-2 monoclonal antibodies are highly specific because they are generated against a single MASP-2 epitope. As used herein, the modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous population of antibodies and should not be construed to require production of the antibody by a particular method. Monoclonal antibodies can be obtained using any technique that provides for production of antibody molecules by continuous cell lines, such as, for example, the hybridoma method described in Kohler, G., et al., Nature 256:495, 1975. Alternatively, monoclonal antibodies may be produced by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567 to Cabilly). Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described in Clackson, T., et al., Nature 352:624-628, 1991, and Marks, J.D., et al., J. Mol Biol. 222:581-597, 1991. Such antibodies may be antibodies of any immunoglobulin class and any subclass thereof, including IgG, IgM, IgE, IgA, and IgD.

[0224] For example, monoclonal antibodies can be obtained by injecting a composition comprising a MASP-2 polypeptide or a portion thereof into a suitable mammal (e.g., a BALB / c mouse). After a predetermined period of time, spleen cells are removed from the mouse and suspended in cell culture medium. The spleen cells are then fused with an immortal cell line to form hybridomas. The resulting hybridomas are grown in cell culture and screened for the ability to produce monoclonal antibodies against MASP-2. An example further describing the production of anti-MASP-2 monoclonal antibodies is shown in Example 7 (see also Current Protocols in Immunology, Vol.1., John Wiley & Sons, pages 2.5.1-2.6.7, 1991).

[0225] Human monoclonal antibodies can be obtained using transgenic mice engineered to produce specific human antibodies in response to antigen exposure. In this technique, elements of the human immunoglobulin heavy and light chain loci are introduced into a mouse strain derived from an embryonic stem cell line containing targeted disruption of the endogenous immunoglobulin heavy and light chain loci. Such transgenic mice can synthesize human antibodies specific for human antigens, such as the MASP-2 antigen described herein, and, as further described in Example 7, can be used to generate human MASP-2 antibody-secreting hybridomas by fusing B cells derived from such animals with an appropriate myeloma cell line using conventional Köhler-Milstein techniques. Transgenic mice having the human immunoglobulin genome are commercially available (e.g., from Abgenix, Inc., Fremont, CA and Medarex, Inc., Annandale, NJ). Methods for obtaining human antibodies from transgenic mice are described, for example, by Green, L.L., et al., Nature Genet. 7:13, 1994; Lonberg, N., et al., Nature 368:856, 1994; and Taylor, L.D., et al., Int. Immun. 6:579, 3994.

[0226] Monoclonal antibodies can be isolated and purified from the hybridoma cultures by a variety of well-established techniques. Such isolation methods include affinity chromatography using protein A Sepharose, size exclusion chromatography, and ion exchange chromatography (see, e.g., pages 2.7.1-2.7.12 and 2.9.1-2.9.3 of Coligan; Baines et al., "Purification of Immunoglobulin G (IgG)", Methods in Molecular Biology, The Humana Press, Inc., Vol. 10, pages 79-104, 1992).

[0227] Once a polyclonal antibody, monoclonal antibody, or phage-derived antibody has been produced, it is first tested for specific MASP-2 binding. To detect antibodies that specifically bind to MASP-2, various assay methods known to those skilled in the art can be used. Exemplary assay methods include Western blot or immunoprecipitation analysis by standard methods (e.g., as described in Ausubel et al.), immunoelectrophoresis, enzyme-linked immunosorbent assay, dot blot, inhibition assay or competition assay, and sandwich assay (as described in Harlow and Land, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988). Once an antibody that specifically binds to MASP-2 has been identified, the anti-MASP-2 antibody is tested for its ability to function as a MASP-2 inhibitor in one of several assay methods, such as the lectin-specific C4 cleavage assay (described in Example 2), the C3b deposition assay (described in Example 2), or the C4b deposition assay (described in Example 2).

[0228] The affinity of the anti-MASP-2 monoclonal antibody can be readily determined by those skilled in the art (see, for example, Scatchard, A., NY Acad. Sci. 51:660-672, 1949). In one embodiment, the anti-MASP-2 monoclonal antibody useful in the methods of the present invention binds to MASP-2 with a binding affinity of <100 nM, preferably <10 nM, and most preferably <2 nM. In some embodiments, the MASP-2 inhibitory monoclonal antibody useful in the methods of the present invention comprises: (I) (a) (i) a heavy chain CDR-H1 comprising the amino acid sequence of amino acids 31-35 of SEQ ID NO:67; and (ii) a heavy chain CDR-H2 comprising the amino acid sequence of amino acids 50-65 of SEQ ID NO:67; and (iii) a heavy chain CDR-H3 comprising the amino acid sequence of amino acids 95-102 of SEQ ID NO:67, a heavy chain variable region, and (b) (i) a light chain CDR-L1 comprising the amino acid sequence of amino acids 24-34 of SEQ ID NO:70; and (ii) a light chain CDR-L2 comprising the amino acid sequence of amino acids 50-56 of SEQ ID NO:70; and (iii) a light chain CDR-L3 comprising the amino acid sequence of amino acids 89-97 of SEQ ID NO:70, a light chain variable region, or (II) a heavy chain variable region having at least 90% identity to SEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:67) and a light chain variable region having at least 90% identity to SEQ ID NO:70 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity), and variants thereof, which are MASP-2 inhibitory monoclonal antibodies or antigen-binding fragments thereof.

[0229] Chimeric / humanized antibody Monoclonal antibodies useful in the method of the present invention include chimeric antibodies in which a portion of the heavy and / or light chains is identical or homologous to the corresponding sequences of antibodies from a particular species or antibodies belonging to a particular antibody class or subclass, but the remaining portions of the chains are identical or homologous to the corresponding sequences of antibodies from another species or antibodies belonging to another antibody class or subclass, as well as fragments of such antibodies (U.S. Patent No. 4,816,567 to Cabilly; and Morrison, S.L., et al., Proc. Nat'l Acad. Sci. USA 81:6851-6855, 1984).

[0230] One form of chimeric antibody useful in the present invention is a humanized monoclonal anti-MASP-2 antibody. A humanized form of a non-human (e.g., mouse) antibody is a chimeric antibody containing minimal sequences derived from the non-human immunoglobulin. A humanized monoclonal antibody is produced by introducing non-human (e.g., mouse) complementarity determining regions (CDRs) derived from the variable heavy and variable light chains of a mouse immunoglobulin into human variable domains. Then, typically, human antibody residues are substituted in the framework regions of the non-human counterparts. Further, the humanized antibody may contain residues not found in either the recipient antibody or the donor antibody. These modifications are made to further enhance the performance of the antibody. Generally, a humanized antibody contains substantially all of at least one, and typically two, variable domains. All or substantially all of the hypervariable loops correspond to the hypervariable loops of the non-human immunoglobulin, and all or substantially all of the Fv framework regions correspond to the Fv framework regions of human immunoglobulin sequences. The humanized antibody may also optionally contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the immunoglobulin constant region (Fc) of a human immunoglobulin. For further details, see Jones, P.T, et al., Nature 321:522-525, 1986; Reichmann, L., et al., Nature 332:323-329, 1988; and Presta, Curr. Op. Struct. Biol. 2:593-596, 1992.

[0231] Humanized antibodies useful in the present invention include human monoclonal antibodies containing at least the MASP-2 binding CDR3 region. Further, the Fc portion may be exchanged to produce IgA or IgM antibodies as well as human IgG antibodies. Such humanized antibodies specifically recognize human MASP-2 and are considered particularly useful clinically because they do not elicit an immune response against the antibody itself in humans. As a result, such humanized antibodies are more suitable for in vivo administration in humans, particularly when repeated or long-term administration is required.

[0232] An example of the production of a humanized anti-MASP-2 antibody from a mouse anti-MASP-2 monoclonal antibody is shown in Example 6 of this specification. Techniques for producing humanized monoclonal antibodies are also described, for example, in Jones, P.T, et al., Nature 321:522, 1986; Carter, P., et al., Proc. Nat'l Acad. Sci. USA 89:4285, 1992; Sandhu, J.S., Crit. Rev. Biotech. 12:437, 1992; Singer, I.I., et al., J. Immun. 150:2844, 1993; Sudhir (ed.), Antibody Engineering Protocols, Humana Press, Inc., 1995; Kelley, 「Engineering Therapeutic Antibodies」, Protein Engineering:Principles and Practice, Cleland et al.(eds.), John Wiley & Sons, Inc., pages 399 - 434, 1996; and U.S. Patent No. 5,693,762 to Queen, 1997. Further, there are commercial entities such as Protein Design Labs (Mountain View, CA) that synthesize humanized antibodies from specific mouse antibody regions.

[0233] Recombinant antibody Anti-MASP-2 antibodies can also be produced using recombinant methods. For example, human antibodies can be produced using a human immunoglobulin expression library (available, for example, from Stratagene, Corp., La Jolla, CA) to produce human antibody fragments (V H , V L , Fv, Fd, Fab, or F(ab')2). Then, techniques similar to those for producing chimeric antibodies are used to construct whole human antibodies using these fragments.

[0234] Anti-idiotype antibody Once anti-MASP-2 antibodies with desirable inhibitory activity are identified, anti-idiotypic antibodies that resemble a portion of MASP-2 can be generated using these antibodies and techniques well known in the art. See, for example, Greenspan, N.S., et al., FASEB J. 7:437, 1993. For example, an antibody that binds to MASP-2 and completely inhibits the MASP-2 protein interactions required for complement activation can be used to generate an anti-idiotype that resembles the MBL binding site on the MASP-2 protein and thus binds to and neutralizes a binding ligand of MASP-2, such as MBL.

[0235] Immunoglobulin fragment MASP-2 inhibitors useful in the methods of the present invention include not only intact immunoglobulin molecules but also well-known fragments formed from antibody fragments, including Fab, Fab', F(ab)2, F(ab')2, and Fv fragments, scFv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies.

[0236] It is well known in the art that only the paratope, a small portion of the antibody molecule, is involved in the binding of an antibody to its epitope (see, for example, Clark, W.R., The Experimental Foundations of Modern Immunology, Wiley & Sons, Inc., NY, 1986). The pFc' and Fc regions of an antibody are effectors of the classical complement pathway but are not involved in antigen binding. Antibodies in which the pFc' region has been enzymatically cleaved or that are made without a pFc' region are called F(ab')2 fragments and retain both antigen-binding sites of the intact antibody. Isolated F(ab')2 fragments are called bivalent monoclonal fragments because of their two antigen-binding sites. Similarly, antibodies in which the Fc region has been enzymatically cleaved or that are made without an Fc region are called Fab fragments and retain one of the antigen-binding sites of the intact antibody molecule.

[0237] Antibody fragments can be obtained by proteolytic cleavage of the whole antibody by conventional methods, e.g., pepsin digestion or papain digestion. For example, antibody fragments can be prepared by enzymatically cleaving the antibody with pepsin to obtain a 5S fragment called F(ab')2. This fragment can be further cleaved using a thiol reducing agent that yields 3.5S Fab' monovalent fragments. Optionally, the cleavage reaction can be carried out using a blocking group for sulfhydryl groups that cleaves disulfide bonds. Alternatively, enzymatic cleavage with pepsin directly generates two monovalent Fab fragments and one Fc fragment. These methods are described, for example, in U.S. Patent No. 4,331,647 to Goldenberg; Nisonoff, A., et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, R.R., Biochem, J. 73:119, 1959; Edelman, et al., Methods in Enzymology 1:422, Academic Press, 1967; and pages 2.8.1 - 2.8.10 and 2.10. - 2.10.4 of Coligan.

[0238] In some embodiments, it is preferred to use antibody fragments lacking an Fc region to avoid activation of the classical complement pathway that begins when Fc binds to the Fcγ receptor. There are several ways to generate MoAbs that avoid Fcγ receptor interaction. For example, the Fc region of a monoclonal antibody can be chemically removed using partial proteolytic digestion (e.g., ficin digestion) with a proteolytic enzyme, thereby generating, for example, an antigen - binding antibody fragment, e.g., a Fab fragment or an F(ab)2 fragment (Mariani, M., et al., Mol. Immunol. 28:69 - 71, 1991). Or, a human γ4 IgG isotype that does not bind to the Fcγ receptor can be used during the construction of humanized antibodies as described herein. Antibodies without an Fc domain, single - chain antibodies, and antigen - binding domains can also be engineered using the recombinant methods described herein.

[0239] Single-chain antibody fragment Alternatively, a peptide single-chain binding molecule specific for MASP-2 can be prepared in which the heavy-chain Fv region and the light-chain Fv region are connected. The Fv fragments may be connected by a peptide linker so as to form a single-chain antigen-binding protein (scFv). These single-chain antigen-binding proteins are prepared by constructing a structural gene containing DNA sequences encoding the V H and V L domains. The structural gene is inserted into an expression vector and then introduced into a host cell such as Escherichia coli. The recombinant host cell synthesizes a single polypeptide chain having a linker peptide that bridges the two V domains. Methods for producing scFv are described, for example, in Whitlow, et al., "Methods: A Companion to Methods in Enzymology" 2:97, 1991; Bird, et al., Science 242:423, 1988; U.S. Patent No. 4,946,778 to Ladner; Pack, P., et al., Bio / Technology 11:1271, 1993.

[0240] As an illustrative example, MASP-2 specific scFv can be obtained by exposing lymphocytes to MASP-2 polypeptide in vitro and selecting an antibody display library in a phage vector or a similar vector (e.g., by using immobilized or labeled MASP-2 protein or peptide). Genes encoding polypeptides having potential MASP-2 polypeptide binding domains can be obtained by screening a random peptide library displayed on phage or bacteria, e.g., E. coli. Using these random peptide display libraries, peptides that interact with MASP-2 can be screened. Techniques for generating and screening such random peptide display libraries are well known in the art (U.S. Patent No. 5,223,409 to Lardner; U.S. Patent No. 4,946,778 to Ladner; U.S. Patent No. 5,403,484 to Ladner; U.S. Patent No. 5,571,698 to Ladner; and Kay et al., Phage Display of Peptides and Proteins Academic Press, Inc., 1996). Random peptide display libraries and kits for screening such libraries are commercially available from, for example, CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly, Mass.), and Pharmacia LKB Biotechnology Inc. (Piscataway, N.J.).

[0241] Another form of the anti-MASP-2 antibody fragment useful in this aspect of the invention is a peptide encoding a single complementarity-determining region (CDR) that binds to an epitope on the MASP-2 antigen and inhibits MASP-2-dependent complement activation. The CDR peptide (“minimal recognition unit”) can be obtained by constructing a gene encoding the CDR of the antibody of interest. Such genes are prepared, for example, by synthesizing the variable region from the RNA of antibody-producing cells using the polymerase chain reaction (see, for example, Larrick et al., Methods; A Companion to Methods in Enzymology 2:106, 1991; Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies”, Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al., (eds.), page 166, Cambridge University Press, 1995; and Ward et al., “Genetic Manipulation and Expression of Antibodies”, Monoclonal Antibodies: Principles and Applications, Birch et al., (eds,), page 137, Wiley-Liss, Inc., 1995).

[0242] To inhibit MASP-2-dependent complement activation, the MASP-2 antibodies described herein are administered to a subject in need thereof. In some embodiments, the MASP-2 inhibitor is a high-affinity human or humanized monoclonal anti-MASP-2 antibody with reduced effector function.

[0243] Peptide inhibitor In some aspects of this aspect of the present invention, the MASP-2 inhibitor comprises an isolated MASP-2 peptide inhibitor comprising an isolated natural peptide inhibitor and a synthetic peptide inhibitor that inhibits the MASP-2-dependent complement activation system. As used herein, the term "isolated MASP-2 peptide inhibitor" binds to MASP-2, competes with MASP-2 for binding to another recognition molecule of the lectin pathway (e.g., MBL, H-ficolin, M-ficolin, or L-ficolin), and / or interacts directly with MASP-2 to inhibit MASP-2-dependent complement activation. By inhibiting, a peptide that inhibits MASP-2-dependent complement activation is meant, the peptide being substantially pure and essentially free of other substances that may be found together in nature to a degree that is practical and suitable for the intended use.

[0244] Peptide inhibitors have been successfully used in vivo to interfere with protein-protein interactions and catalytic sites. For example, recently, a peptide inhibitor against an adhesion molecule structurally related to LFA-1 was approved for clinical use in coagulation disorders (Ohman, E.M., et al., European Heart J. 16:50-55, 1995). Short linear peptides (<30 amino acids) that block or interfere with integrin-dependent adhesion have been described (Murayama, O., et al., J. Biochem. 120:445-51, 1996). Long peptides ranging in length from 25 to 200 amino acid residues have also been successfully used to block integrin-dependent adhesion (Zhang, L., et al., J. Biol. Chem. 271(47):29953-57, 1996). Generally, long peptide inhibitors have higher affinity and / or a slower off-rate than short peptides and can thus be potent inhibitors. Cyclic peptide inhibitors have also been shown to be effective integrin inhibitors in vivo for the treatment of human inflammatory diseases (Jackson, D.Y., et al., J. Med. Chem. 40:3359-68, 1997). One method of producing cyclic peptides involves peptide synthesis in which the terminal amino acids of the peptide are cysteine, thereby allowing the peptide to exist in a cyclic form through a disulfide bond between the terminal amino acids. This cyclic form has been shown to improve in vivo affinity and half-life for the treatment of hematopoietic neoplasms (e.g., U.S. Patent No. 6,649,592 to Larson).

[0245] Synthetic MASP-2 peptide inhibitor The MASP-2 inhibitory peptides useful in the methods of this aspect of the invention are exemplified by amino acid sequences that mimic target regions important for MASP-2 function. Inhibitory peptides useful in the practice of the methods of the invention range in size from about 5 amino acids to about 300 amino acids. Table 3 provides a list of exemplary inhibitory peptides that may be useful in the practice of this aspect of the invention. For example, in one of several assay methods, including the lectin-specific C4 cleavage assay (described in Example 2) and the C3b deposition assay (described in Example 2), the ability of a candidate MASP-2 inhibitory peptide to function as a MASP-2 inhibitor can be tested.

[0246] In some embodiments, the MASP-2 inhibitory peptide is derived from the MASP-2 polypeptide and is selected from the full-length mature MASP-2 protein (SEQ ID NO:6), or specific domains of the MASP-2 protein, such as the CUBI domain (SEQ ID NO:8), the CUBIEGF domain (SEQ ID NO:9), the EGF domain (SEQ ID NO:11), and the serine protease domain (SEQ ID NO:12). As noted above, the CUBEGFCUBII region has been shown to be required for dimerization and binding to MBL (Thielens et al., supra). In particular, the peptide sequence TFRSDYN (SEQ ID NO:16) in the CUBI domain of MASP-2 has been shown to be involved in binding to MBL in studies identifying humans with a homozygous mutation from Asp105 to Gly105, resulting in the loss of MASP-2 from the MBL complex (Stengaard-Pedersen, K., et al., New England J. Med. 349:554-560, 2003).

[0247] In some embodiments, the MASP-2 inhibitory peptide binds to MASP-2 and is derived from a lectin protein involved in the lectin complement pathway. Several different lectins involved in this pathway have been identified, including mannose-binding lectin (MBL), L-ficolin, M-ficolin, and H-ficolin (Ikeda, K., et al., J. Biol. Chem. 262:7451-7454, 1987; Matsushita, M., et al., J. Exp. Med. 176:1497-2284, 2000; Matsushita, M., et al., J. Immunol. 168:3502-3506, 2002). These lectins exist in serum as oligomers of homotrimeric subunits that each have an N-terminal collagen-like fiber with a carbohydrate recognition domain. These different lectins have been shown to bind to MASP-2, and the lectin / MASP-2 complex activates complement by cleaving the proteins C4 and C2. H-ficolin has a 24 amino acid amino-terminal region, a collagen-like domain with 11 Gly-Xaa-Yaa repeats, a 12 amino acid neck domain, and a 207 amino acid fibrinogen-like domain (Matsushita, M., et al., J. Immunol. 168:3502-3506, 2002). H-ficolin binds to GlcNAc and agglutinates human erythrocytes coated with LPS derived from Salmonella typhimurium, S. minnesota, and Escherichia coli. H-ficolin has been shown to bind to MASP-2 and MAp19 and activate the lectin pathway. That is, L-ficolin / P35 has also been shown to bind to GlcNAc and to MASP-2 and MAp19 in human serum. This complex has been shown to activate the lectin pathway (Matsushita, M., et al., J. Immunol. 164:2281, 2000).Accordingly, the MASP-2 inhibitory peptides useful in the present invention may include a region of at least 5 amino acids selected from MBL protein (SEQ ID NO:21), H-ficolin protein (GenBank accession number NM_173452), M-ficolin protein (GenBank accession number O00602), and L-ficolin protein (GenBank accession number NM_015838).

[0248] More specifically, scientists have identified that the MASP-2 binding site on MBL is located between the hinge and the neck in the C-terminal portion of the collagen-like domain of MBP, within 12 Gly-X-Y triplets within TIFF2025108730000010.tif12128 (Wallis, R. et al., J. Biol Chem. 279:14065, 2004). This MASP-2 binding site region is also highly conserved in human H-ficolin and human L-ficolin. A consensus binding site has been described that is present in all three lectin proteins and contains the amino acid sequence "OGK-X-GP" (SEQ ID NO:22), where the letter "O" represents hydroxyproline and the letter "X" is a hydrophobic residue (Wallis et al., 2004, supra). Accordingly, in some embodiments, the MASP-2 inhibitory peptides useful in this aspect of the present invention are at least 6 amino acids in length and include SEQ ID NO:22. The amino acid sequence TIFF2025108730000011.tif4128-containing peptides derived from MBL have been shown to bind to MASP-2 in vitro (Wallis, et al., 2004, supra). To enhance binding to MASP-2, peptides can be synthesized with two GPO triplets adjacent to each end that enhance the formation of triple helices as seen in the native MBL protein TIFF2025108730000012.tif5132 (further described in Wallis, R., et al., J. Biol. Chem. 279:14065, 2004).

[0249] The MASP-2 inhibitory peptide may also be derived from human H-ficolin and contain a sequence derived from the consensus MASP-2 binding region of H-ficolin. TIFF2025108730000013.tif4145. Peptides derived from human L-ficolin containing a sequence derived from the consensus MASP-2 binding region of L-ficolin are also included. TIFF2025108730000014.tif12153 are also included.

[0250] The MASP-2 inhibitory peptide may also be derived from a C4 cleavage site, such as TIFF2025108730000015.tif4128, that is linked to the C-terminal portion of antithrombin III (Glover, G.I., et al., Mol. Immunol. 25:1261(1988)). TIFF2025108730000015.tif4128 (Glover, G.I., et al., Mol. Immunol. 25:1261(1988)).

[0251] (Table 3) Exemplary MASP-2 inhibitory peptides TIFF2025108730000016.tif29155 TIFF2025108730000017.tif218155 TIFF2025108730000018.tif58155 Note: The letter "O" represents hydroxyproline. The letter "X" is a hydrophobic residue.

[0252] Peptides derived from the C4 cleavage site and other peptides that inhibit the MASP-2 serine protease site can be chemically modified to be irreversible protease inhibitors. For example, suitable modifications include halomethyl ketones (Br, Cl, I, F) at the C-terminus, Asp, or Glu, or attached to a functional side chain; haloacetyl (or other α-haloacetyl) groups on an amino group or other functional side chain; epoxide-containing or imine-containing groups at the amino terminus or carboxy terminus or functional side chain; or imidate esters at the amino terminus or carboxy terminus or functional side chain, but are not necessarily limited thereto. Performing such modifications is thought to provide the advantage of permanently inhibiting the enzyme by covalent binding of the peptide. This may result in a lower effective amount and / or may require a reduced dosing frequency of the peptide inhibitor.

[0253] In addition to the inhibitory peptides described above, MASP-2 inhibitory peptides useful in the methods of the present invention include peptides containing the MASP-2-binding CDR3 region of anti-MASP-2 MoAbs obtained as described herein. The sequences of the CDR regions for use in peptide synthesis can be determined by methods known in the art. The heavy chain variable region is generally a peptide about 100-150 amino acids in length. The light chain variable region is generally a peptide about 80-130 amino acids in length. The CDR sequences within the heavy chain variable region and within the light chain variable region include sequences of only about 3-25 amino acids that can be readily sequenced by one of ordinary skill in the art.

[0254] One of ordinary skill in the art would recognize that substantially homologous variations of the MASP-2 inhibitory peptide would also exhibit MASP-2 inhibitory activity. Exemplary variations include, but are not necessarily limited to, peptides having insertions, deletions, exchanges, and / or additional amino acids in the carboxy-terminal portion or amino-terminal portion of the peptide, as well as mixtures thereof. Accordingly, homologous peptides having MASP-2 inhibitory activity are considered useful in the methods of the present invention. The described peptides may also include other modifications by way of repetitive motifs and conservative substitutions. Conservative variants are described elsewhere herein and include exchanging one amino acid for an amino acid with similar charge, size, or hydrophobicity, among other characteristics.

[0255] To more closely mimic segments in intact proteins, the MASP-2 inhibitory peptides may be modified to increase solubility and / or to maximize positive or negative charge. The derivatives may or may not have the exact primary amino acid structure of the peptides disclosed herein, as long as they functionally retain the desired MASP-2 inhibitory properties. Modifications include amino acid substitutions using one or another of the 20 generally known amino acids, amino acid substitutions using derivatized amino acids or substituted amino acids or D-amino acids having auxiliary desirable features, such as resistance to enzymatic degradation, or substitutions using another molecule or compound that mimics the native conformation and function of one amino acid, a plurality of amino acids, or a peptide, such as a carbohydrate; amino acid deletions; amino acid insertions using one or another of the 20 generally known amino acids, amino acid insertions using derivatized amino acids or substituted amino acids or D-amino acids having auxiliary desirable features, such as resistance to enzymatic degradation, or substitutions using another molecule or compound that mimics the native conformation and function of one amino acid, a plurality of amino acids, or a peptide, such as a carbohydrate; or substitutions using another molecule or compound that mimics the native conformation, charge distribution, and function of the parent peptide, such as a carbohydrate or a nucleic acid monomer. The peptides may also be modified by acetylation or amidation.

[0256] The synthesis of derivative inhibitory peptides may rely on known techniques such as peptide biosynthesis and carbohydrate biosynthesis. As a starting point, one of ordinary skill in the art may rely on appropriate computer programs to determine the conformation of the peptide of interest. Once the conformation of the peptides disclosed herein is known, one of ordinary skill in the art can, in a rational design manner, determine what types of substitutions can be made at one or more sites to create derivatives that retain the basic conformation and charge distribution of the parent peptide but may have features not present in the parent peptide or features enhanced beyond those seen in the parent peptide. Once candidate derivative molecules are identified, the derivatives can be tested using the assay methods described herein to determine whether they function as MASP-2 inhibitors.

[0257] Screening of MASP-2 Inhibitory Peptides Molecular modeling and rational molecular design can also be used to generate and screen peptides that mimic the molecular structure of important binding regions of MASP-2 and inhibit the complement activity of MASP-2. As previously described, the molecular structures used in the modeling include target regions known to be important for MASP-2 function, including the CDR regions of anti-MASP-2 monoclonal antibodies, as well as regions required for dimerization, regions involved in binding to MBL, and serine protease active sites. Methods for identifying peptides that bind to a particular target are well known in the art. For example, molecular imprinting can be used for the de novo construction of peptides, such as for the construction of macromolecular structures that bind to a particular molecule. See, for example, Shea, K.J., "Molecular Imprinting of Synthetic Network Polymers: The De Novo synthesis of Macromolecular Binding and Catalytic Sties", TRIP 2(5) 1994.

[0258] As an illustrative example, one method of preparing mimetics of MASP-2 binding peptides is as follows. A known MASP-2 binding peptide, or a functional monomer (template) of the binding region of an anti-MASP-2 antibody that exhibits MASP-2 inhibition, is polymerized. The template is then removed, and subsequently, another class of monomers is polymerized into the voids left by the template to obtain a new molecule that is similar to the template and exhibits one or more desirable properties. In addition to preparing peptides in this manner, other MASP-2 binding molecules that are MASP-2 inhibitors, such as polysaccharides, nucleosides, drugs, nucleoproteins, lipoproteins, carbohydrates, glycoproteins, steroids, lipids, and other biologically active materials, can also be prepared. This method is useful for the design of a wide variety of biological mimetics that are more stable than their natural counterparts. This is because these biological mimetics are typically prepared by free radical polymerization of functional monomers, resulting in compounds having a non-biodegradable backbone.

[0259] Peptide synthesis MASP-2 inhibitory peptides can be prepared using techniques well known in the art, such as the solid-phase synthesis technique first described by Merrifield in J. Amer. Chem. Soc. 85:2149-2154, 1963. Automated synthesis can be achieved, for example, using an Applied Biosystems 431 A Peptide Synthesizer (Foster City, Calif.) according to the instructions provided by the manufacturer. Other techniques can be found, for example, in Bodanszky, M., et al., Peptide Synthesis, 2nd ed., John Wiley & Sons, 1976 as well as other references known to those skilled in the art.

[0260] Peptides can also be prepared using standard genetic engineering techniques known to those skilled in the art. For example, a peptide can be enzymatically produced by inserting a nucleic acid encoding the peptide into an expression vector, expressing the DNA, and translating the DNA into a peptide in the presence of the required amino acids. The peptide can then be purified using chromatography or electrophoresis methods, or the sequence encoding the peptide can be inserted into an expression vector in frame with a nucleic acid sequence encoding a carrier protein to fuse with the peptide and be purified by a carrier protein that can be cleaved later. The fusion protein peptide may be isolated using chromatography, electrophoresis, or immunological techniques (e.g., binding of the resin to the carrier protein via an antibody). The peptide can be cleaved using chemical methodology or enzymatically, for example, by a hydrolytic enzyme.

[0261] The MASP-2 inhibitory peptides useful in the method of the present invention can also be produced in recombinant host cells according to conventional techniques. To express a sequence encoding the MASP-2 inhibitory peptide, a nucleic acid molecule encoding the peptide must be operably linked to regulatory sequences that control transcriptional expression within an expression vector and then introduced into a host cell. In addition to transcriptional regulatory sequences such as promoters and enhancers, the expression vector may include translational regulatory sequences and a marker gene suitable for selection of cells having the expression vector.

[0262] The nucleic acid molecule encoding the MASP-2 inhibitory peptide can be synthesized by a protocol such as the phosphoramidite method using a "gene machine". When chemically synthesized double-stranded DNA is required for applications such as the synthesis of a gene or gene fragment, each complementary strand is prepared separately. The preparation of short genes (60-80 base pairs) is technically simple and can be achieved by synthesizing the complementary strands and then annealing them. To prepare larger genes, synthetic genes (double-stranded) are assembled from single-stranded fragments 20-100 nucleotides in length in a modular format. For reviews on polynucleotide synthesis, see, for example, Glick and Pasternak, "Molecular Biotechnology, Principles and Applications of Recombinant DNA", ASM Press, 1994; Itakura, K., et al., Annu. Rev, Biochem. 53:323, 1984; and Climie, S., et al., Proc. Nal'l Acad. Sci. USA 87:633, 1990.

[0263] Small molecule inhibitor In some embodiments, the MASP-2 inhibitor is a small molecule inhibitor, including natural and synthetic substances having a low molecular weight, such as peptides, peptidomimetics, and non-peptide inhibitors (including oligonucleotides and organic compounds). The MASP-2 small molecule inhibitor can be prepared based on the molecular structure of the variable region of the anti-MASP-2 antibody.

[0264] Low molecular weight inhibitors may also be designed and generated based on the MASP-2 crystal structure using computational drug design (Kuntz I.D., et al., Science 257:1078, 1992). The crystal structure of rat MASP-2 has been described (Feinberg, H., et al., EMBO J. 22:2348-2359, 2003). Using the method described by Kuntz et al., the MASP-2 crystal structure coordinates are used as input to a computer program, such as DOCK, that outputs a list of low molecular weight structures predicted to bind to MASP-2. The use of such computer programs is well known to those skilled in the art. For example, the crystal structure of HIV-1 protease has been used to identify unique non-peptide ligands that are HIV-1 protease inhibitors by evaluating the fit of compounds found in the Cambridge Crystallographic database to the enzyme binding site using the program DOCK (Kuntz, I.D., et al., J. Mol. Biol. 161:269-288, 1982; DesJarlais, R.L., et al., PNAS 87:6644-6648, 1990).

[0265] The list of low molecular weight structures identified as potential MASP-2 inhibitors by computer computational methods is screened using a MASP-2 binding assay method, such as the MASP-2 binding assay method described in Example 10. The low molecular weight compounds found to bind to MASP-2 are then assayed in a functional assay method, such as the functional assay method described in Example 2, to determine whether they inhibit MASP-2-dependent complement activation.

[0266] MASP-2 soluble receptor Other suitable MASP-2 inhibitors are thought to include MASP-2 soluble receptors that can be made using techniques known to those skilled in the art.

[0267] Expression inhibitors of MASP-2 In another aspect of this aspect of the present invention, the MASP-2 inhibitor is an MASP-2 expression inhibitor capable of inhibiting MASP-2-dependent complement activation. In the practice of this aspect of the present invention, typical MASP-2 expression inhibitors include MASP-2 antisense nucleic acid molecules (e.g., antisense mRNA, antisense DNA, or antisense oligonucleotides), MASP-2 ribozymes, and MASP-2 RNAi molecules.

[0268] Antisense RNA molecules and DNA molecules hybridize to MASP-2 mRNA and act to directly block the translation of MASP-2 mRNA by preventing the translation of the MASP-2 protein. Antisense nucleic acid molecules may be constructed in a number of different ways, provided that they are capable of interfering with the expression of MASP-2. For example, an antisense nucleic acid molecule can be constructed by reversing the coding region (or a portion thereof) of MASP-2 cDNA (SEQ ID NO:4) relative to the normal transcription direction so that the complementary strand of the coding region (or a portion thereof) of MASP-2 cDNA (SEQ ID NO:4) can be transcribed.

[0269] Antisense nucleic acid molecules are typically substantially identical to at least a portion of one or more target genes. However, the nucleic acids need not be completely identical to inhibit expression. In general, high homology can be used to compensate for the use of short antisense nucleic acid molecules. The minimum percent identity is typically greater than about 65%, but higher percent identities may more effectively suppress the expression of the endogenous sequence. A fairly high percent identity of greater than about 80% is typically preferred, but identities of about 95% to complete identity are typically most preferred.

[0270] Antisense nucleic acid molecules need not have the same intron or exon pattern as the target gene. Non-coding segments of the target gene may be equally effective as coding segments in terms of achieving antisense suppression of target gene expression. DNA sequences of at least about 8 or so nucleotides can be used as antisense nucleic acid molecules, but longer sequences are preferred. In the present invention, a representative example of a useful MASP-2 inhibitor is an antisense MASP-2 nucleic acid molecule that is at least 90 percent identical to the complementary strand of the MASP-2 cDNA consisting of the nucleic acid sequence shown in SEQ ID NO:4. The nucleic acid sequence shown in SEQ ID NO:4 encodes the MASP-2 protein consisting of the amino acid sequence shown in SEQ ID NO:5.

[0271] Targeting of antisense oligonucleotides that bind to MASP-2 mRNA is another mechanism that can be used to reduce the level of MASP-2 protein synthesis. For example, the synthesis of polygalacturonase and the muscarinic type 2 acetylcholine receptor are inhibited by antisense oligonucleotides against their respective mRNA sequences (U.S. Patent No. 5,739,119 to Cheng, and U.S. Patent No. 5,759,829 to Shewmaker). Furthermore, examples of antisense inhibition have been demonstrated using nuclear protein cyclin, the multidrug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABA A receptor, and human EGF (see, for example, U.S. Patent No. 5,801,154 to Baracchini; U.S. Patent No. 5,789,573 to Baker; U.S. Patent No. 5,718,709 to Considine; and U.S. Patent No. 5,610,288 to Reubenstein).

[0272] A system is described by which one of ordinary skill in the art can determine which oligonucleotides are useful in the present invention. This system involves probing appropriate sites in the target mRNA using RnaseH cleavage as an indicator of accessibility of sequences within the transcript. Scherr, M., et al., Nucleic Acids Res. 25:5079-5085, 1998; Lloyd, et al., Nucleic Acids Res. 29:3665-3673, 2001. To create sites vulnerable to RNAseH, a mixture of antisense oligonucleotides complementary to a specific region of the MASP-2 transcript is added to and hybridized with cell extracts expressing MASP-2, such as hepatocytes. This method can be combined with computer-aided sequence selection that can predict the optimal sequence selection of the antisense composition based on the relative ability to form dimers, hairpins, or other secondary structures that reduce or prevent specific binding to the target mRNA in the host cell. These secondary structure analyses and considerations of target site selection can be performed using OLIGO primer analysis software (Rychlik, I., 1997) and BLASTN 2.0.5 algorithm software (Altschul, S.F., et al., Nucl. Acids Res. 25:3389-3402, 1997). Antisense compounds to the target sequence preferably contain from about 8 to about 50 nucleotides. Antisense oligonucleotides containing from about 9 to about 35 nucleotides, such as from about 9 to about 35 nucleotides, are particularly preferred. The inventors contemplate that all oligonucleotide compositions within the range of 9 to 35 nucleotides (i.e., about 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, or 35 bases in length) are highly preferred for the practice of the methods of the present invention based on antisense oligonucleotides.Highly preferred target regions of MASP-2 mRNA are target regions that are at or near the AUG translation initiation codon and these sequences that are substantially complementary to the 5' region of the mRNA, for example, the -10 to +10 region of the MASP-2 gene nucleotide sequence (SEQ ID NO:4). Exemplary MASP-2 expression inhibitors are shown in Table 4.

[0273] (Table 4) Exemplary expression inhibitors of MASP-2 TIFF2025108730000019.tif72155

[0274] As described above, the term "oligonucleotide" as used herein refers to oligomers or polymers of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term also covers oligonucleobases consisting of natural nucleotides, sugars, and covalent internucleoside (backbone) linkages, as well as oligonucleotides having non-natural modifications. These modifications can introduce certain desirable properties not provided by natural oligonucleotides, such as low toxicity, high stability against nuclease degradation, and high amounts of cellular uptake. In an exemplary embodiment, the antisense compounds of the present invention differ from natural DNA only in a phosphodiester backbone modification that extends the lifetime of the antisense oligonucleotide in which the phosphate substituents are replaced by phosphorothioates. Similarly, one or both ends of the oligonucleotide may be substituted with one or more acridine derivatives that intercalate between adjacent base pairs within the nucleic acid strand.

[0275] An alternative to antisense is the use of "RNA interference" (RNAi). Double-stranded RNA (dsRNA) can induce gene silencing in mammals in vivo. The natural functions of RNAi and cosuppression appear to be protection of the genome from mobile genetic elements such as retrotransposons and invasion by viruses that produce abnormal RNA or dsRNA in host cells when activated (see, for example, Jensen, J., et al., Nat. Genet. 27:209-12, 1999). Double-stranded RNA molecules can be prepared by synthesizing two RNA strands that can form a double-stranded RNA molecule, each having a length of about 19-25 (e.g., 19-23 nucleotides). For example, dsRNA molecules useful in the methods of the present invention may include RNA corresponding to the sequences listed in Table 4 and their complementary strands. Preferably, at least one RNA strand has a 3' overhang of 1-5 nucleotides. The synthesized RNA strands are combined under conditions that form a double-stranded molecule. This RNA sequence may comprise at least 8 nucleotide portions of SEQ ID NO:4 with a total length of 25 nucleotides or less. The design of siRNA sequences for a particular target is within the scope of ordinary skill in the art. Commercial services are available for designing siRNA sequences to ensure at least 70% knockdown of expression (Qiagen, Valencia, Calif).

[0276] dsRNA is administered as a pharmaceutical composition and can be carried out by known methods in which the nucleic acid is introduced into the desired target cells. Commonly used gene transfer methods include calcium phosphate, DEAE-dextran, electroporation, microinjection, and viral methods. Such methods are disclosed in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 1993.

[0277] To reduce the amount and / or biological activity of MASP-2, ribozymes, such as ribozymes targeting MASP-2 mRNA, can also be used. A ribozyme is a catalytic RNA molecule that can cleave nucleic acid molecules having a sequence that is fully or partially homologous to the ribozyme sequence. An RNA ribozyme encoding ribozyme transgene can be designed that specifically pairs with a target RNA, cleaves the phosphodiester backbone at a specific position, thereby functionally inactivating the target RNA. When performing this cleavage, the ribozyme itself does not change and can thus be reused to cleave other molecules. Incorporating a ribozyme sequence into an antisense RNA confers RNA cleavage activity to the antisense RNA, thereby increasing the activity of the antisense construct.

[0278] Ribozymes useful in the practice of the present invention typically include a hybridizing region of at least about 9 nucleotides that is complementary in nucleotide sequence to at least a portion of the target MASP-2 mRNA, and a catalytic region adapted to cleave the target MASP-2 mRNA (see generally EPA No. 0321201; WO88 / 04300; Haseloff, J., et al., Nature 534:585-591, 1988; Fedor, M.J., et al., Proc. Natl. Acad, Sci. USA 87:1668-1672, 1990; Cech, T.R., et al., Ann. Rev, Biochem. 55:599-629, 1986).

[0279] Ribozymes may be targeted directly to cells in the form of RNA oligonucleotides incorporating the ribozyme sequence, or may be introduced into cells as an expression vector encoding the desired ribozyme RNA. Ribozymes can be used and applied in much the same way as described for antisense polynucleotides.

[0280] In the method of the present invention, antisense RNAs and DNAs, ribozymes, and RNAi molecules useful therein can be prepared by any method known in the art for synthesizing DNA molecules and RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art, such as solid-phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be produced by in vitro transcription and in vivo transcription of a DNA sequence encoding an antisense RNA molecule. Such DNA sequences may be incorporated into a variety of vectors incorporating appropriate RNA polymerase promoters, such as T7 or SP6 polymerase promoters. Alternatively, an antisense cDNA construct that synthesizes antisense RNA constitutively or inducibly depending on the promoter used can be stably introduced into a cell line.

[0281] As means for increasing stability and extending the half-life, various well-known modifications of the DNA molecule can be introduced. Useful modifications include, but are not limited to, the addition of adjacent sequences of ribonucleotides or deoxyribonucleotides to the 5' end and / or 3' end of the molecule, or the use of phosphorothioate or 2'O-methyl instead of phosphodiester bonds in the oligodeoxyribonucleotide backbone.

[0282] VI. Pharmaceutical Compositions and Delivery Methods Dosage In another aspect, the present invention provides a composition for inhibiting side effects of MASP-2-dependent complement activation in a subject suffering from a disease or condition disclosed herein, comprising administering to the subject a composition comprising a therapeutically effective amount of a MASP-2 inhibitor and a pharmaceutically acceptable carrier. To treat or alleviate a condition associated with MASP-2-dependent complement activation, a MASP-2 inhibitor can be administered to a subject in need thereof at a therapeutically effective dose. A therapeutically effective dose refers to an amount of the MASP-2 inhibitor sufficient to alleviate symptoms associated with the disease or condition.

[0283] The toxicity and therapeutic efficacy of MASP-2 inhibitors can be determined by standard pharmaceutical procedures using experimental animal models, such as the mouse MASP-2 - / - mouse model expressing the human MASP-2 transgene described in Example 1. Using such animal models, the NOAEL (no-observed-adverse-effect level) and MED (minimum effective dose) can be determined by standard methods. The dose ratio between the NOAEL effect and the MED effect is the cure ratio, expressed as the ratio NOAEL / MED. MASP-2 inhibitors showing a large cure ratio or index are most preferred. Data obtained from cell culture assay methods and animal studies can be used in the formulation of a range of dosages for use in humans. The dosage of the MASP-2 inhibitor is preferably within the range of circulating concentrations that include the MED with little or no toxicity. The dosage can vary within this range depending on the dosage form used and the route of administration utilized.

[0284] For any compound formulation, the therapeutically effective dose can be evaluated using an animal model. For example, the dose reaching the concentration range in circulating plasma including the MED can be formulated in an animal model. The quantitative level of the MASP-2 inhibitor in plasma can also be measured, for example, by high performance liquid chromatography.

[0285] In addition to toxicity studies, the effective dosage may also be evaluated based on the amount of MASP-2 protein present in a living subject and the binding affinity of the MASP-2 inhibitor. MASP-2 levels in normal human subjects are present in serum at low levels within the...

Claims

**Claim 1** A method of treating a human subject suffering from or at risk of developing graft-versus-host disease (GVHD), the method comprising administering to the subject a composition comprising an MASP-2 inhibitory antibody or antigen-binding fragment thereof in an amount effective to inhibit MASP-2-dependent complement activation. **Claim 2** The method of claim 1, wherein the MASP-2 inhibitory antibody is a monoclonal antibody or fragment thereof that specifically binds to human MASP-2. **Claim 3** The method of claim 1, wherein the antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody having reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. **Claim 4** The method of claim 1, wherein the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. **Claim 5** The method according to claim 1, wherein the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum at an IC of 30 nM or less. 50 for inhibiting. **Claim 6** The method of claim 1, wherein the MASP-2 inhibitory antibody is systemically delivered to the subject. **Claim 7** The method of claim 1, further comprising identifying a human subject suffering from or at risk of developing graft-versus-host disease prior to the step of administering to the subject a composition comprising an MASP-2 inhibitory antibody or antigen-binding fragment thereof in an amount effective to inhibit MASP-2-dependent complement activation. **Claim 8** The method of claim 1, wherein the subject has previously received, is currently receiving, or is scheduled to receive a hematopoietic stem cell transplant. **Claim 9** The method of claim 1, wherein the subject has acute GVHD. **Claim 10** The method of claim 1, wherein the subject has chronic GVHD. **Claim 11** The method of claim 1, wherein the subject has steroid-resistant GVHD. **Claim 12** The method of claim 1, wherein the MASP-2 inhibitory antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO:67, and a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:

70. **Claim 13** The method according to any one of claims 1 to 12, comprising administering to the subject the composition comprising the MASP-2 inhibitory antibody at a dose of 1 mg / kg to 10 mg / kg at least once a week. **Claim 14** A method of treating, preventing, or alleviating one or more neurological symptoms associated with graft-versus-host disease or HSCT-TMA, the method comprising administering to a subject, before, during, or after receiving a hematopoietic stem cell transplantation, a composition comprising an MASP-2 inhibitory antibody or an antigen-binding fragment thereof in an amount effective to inhibit MASP-2-dependent complement activation.

15. The method according to claim 14, wherein one or more neurological symptoms associated with graft-versus-host disease or HSCT-TMA are selected from the group consisting of weakness, abnormal sensation, quadriplegia, sensorimotor impairment, autonomic neuropathy polyneuropathy, and / or neurogenic bladder.

16. The method according to claim 14, wherein the subject has received a hematopoietic stem cell transplantation and the subject is suffering from one or more neurological symptoms selected from the group consisting of weakness, abnormal sensation, quadriplegia, sensorimotor impairment, autonomic neuropathy polyneuropathy, and / or neurogenic bladder.

17. The method according to any one of claims 14 to 16, wherein the subject has received a hematopoietic stem cell transplantation and the subject is suffering from graft-versus-host disease.

18. The method according to any one of claims 14 to 17, wherein the subject has received a hematopoietic stem cell transplantation and the subject is suffering from HSCT-TMA.

19. The method according to claim 14, wherein the MASP-2 inhibitory antibody is a monoclonal antibody or a fragment thereof that specifically binds to human MASP-2.

20. The method according to claim 14, wherein the antibody or its fragment is selected from the group consisting of a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody.

21. The method according to claim 14, wherein the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway.

22. The method according to claim 14, wherein the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum at an IC of 30 nM or less. 50 as described in claim 14.

23. The method according to claim 14, wherein the MASP-2 inhibitory antibody is systemically delivered to the subject.

24. The method according to claim 14, further comprising the step of identifying a human subject suffering from one or more neurological symptoms associated with hematopoietic stem cell transplantation before the step of administering to the subject a composition comprising an MASP-2 inhibitory antibody or an antigen-binding fragment thereof in an amount effective to inhibit MASP-2-dependent complement activation.

25. The method according to claim 14, wherein the MASP-2 inhibitory antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO:67, and a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:

70.

26. A method of treating a human subject suffering from or at risk of developing diffuse alveolar hemorrhage, the method comprising administering to the subject a composition comprising an MASP-2 inhibitory antibody or antigen-binding fragment thereof in an amount effective to inhibit MASP-2-dependent complement activation.

27. The method according to claim 26, wherein the MASP-2 inhibitory antibody is a monoclonal antibody or fragment thereof that specifically binds to human MASP-2.

28. The method according to claim 26, wherein the antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody.

29. The method according to claim 26, wherein the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway.

30. The method according to claim 26, wherein the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum at an IC of 30 nM or less. 50 as described in claim 26.

31. The method according to claim 26, wherein the MASP-2 inhibitory antibody is systemically delivered to the subject.

32. The method according to claim 26, further comprising identifying a human subject suffering from or at risk of developing diffuse alveolar hemorrhage prior to the step of administering to the subject a composition comprising an MASP-2 inhibitory antibody or antigen-binding fragment thereof in an amount effective to inhibit MASP-2-dependent complement activation.

33. The method according to claim 26, wherein the subject has previously received, is currently receiving, or is scheduled to receive a hematopoietic stem cell transplantation. ​ ​ ​ ​ ​ A method of treating a human subject suffering from or at risk of developing venous occlusion, the method comprising administering to the subject a composition comprising an MASP-2 inhibitory antibody or an antigen-binding fragment thereof in an amount effective to inhibit MASP-2-dependent complement activation.

37. The method according to claim 36, wherein the MASP-2 inhibitory antibody is a monoclonal antibody or a fragment thereof that specifically binds to human MASP-2.

38. The method according to claim 36, wherein the antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody having reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody.

39. The method according to claim 36, wherein the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway.

40. The method according to claim 36, wherein the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum at an IC of 30 nM or less. 50 as described in claim 36.

41. The method according to claim 36, wherein the MASP-2 inhibitory antibody is systemically delivered to the subject.

42. The method according to claim 36, further comprising identifying a human subject suffering from or at risk of developing venous occlusion prior to the step of administering to the subject a composition comprising an MASP-2 inhibitory antibody or an antigen-binding fragment thereof in an amount effective to inhibit MASP-2-dependent complement activation.

43. The method according to claim 36, wherein the subject has previously received, is currently receiving, or is scheduled to receive a hematopoietic stem cell transplant.

44. The method according to claim 36, wherein the MASP-2 inhibitory antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO:67, and a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:

70.

45. The method according to any one of claims 36 to 44, wherein the subject has received a hematopoietic stem cell transplant and suffers from venous occlusion.

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