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

Abstract

In one aspect, the invention provides a method 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 hematopoietic stem cell transplantation. The method comprises the step of administering to a subject in need thereof an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2-dependent complement activation.

Description

[0001] Notes on sequence listing

[0002] The sequence listing attached to this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into this specification. The name of the text file containing the sequence listing is MP_1_0278_PCT_SequenceListingasFiled_20180807; the text file is 116KB; it was created on August 7, 2018, and was submitted with the application for this specification via EFS-Web.

[0003] background

[0004] The complement system provides an early mechanism of action that initiates, amplifies, and coordinates immune responses to microbial infections and other acute injuries in humans and other vertebrates (MK Liszewski and JP Atkinson, 1993, Fundamental Immunology, 3rd edition, edited by WE Paul, Raven Press, Ltd., New York). Although complement activation provides an important first line of defense against potential pathogens, complement activity that promotes protective immune responses can also represent a potential threat to the host (KR Kalli et al., Springer Semin. Immunopathol. 15: 417-431, 1994; BP Organ, Eur. J. Clinical Investig. 24: 219-228, 1994). For example, C3 and C5 proteolytic products recruit and activate neutrophils. Although essential for host defense, activated neutrophils indiscriminately release destructive enzymes and can cause organ damage. Additionally, complement activation can cause deposition of lytic complement components on adjacent host cells as well as on microbial targets, leading to host cell lysis.

[0005] The complement system has also been implicated in the pathogenesis of many acute and chronic disease states, including: myocardial infarction, stroke, ARDS, reperfusion injury, septic shock, capillary leak following thermal burns, inflammation following cardiopulmonary bypass, graft rejection, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and Alzheimer's disease. In almost all of these conditions, complement is not the cause, but rather one of several factors involved in the pathogenesis. However, complement activation may be the primary pathological mechanism and represents a point of clinical control in many of these disease states. The increasing recognition of the importance of complement-mediated tissue damage in a variety of disease states underscores the need for effective complement inhibitory drugs. To date, eculizumab has been used to treat complement-mediated tissue damage in a variety of disease states. An antibody against C5 is the only complement-targeted drug that has been approved for human use. However, C5 is one of several effector molecules located "downstream" in the complement system, and blockade of C5 does not inhibit activation of the complement system. Therefore, inhibitors of the initial step of complement activation would have significant advantages over "downstream" complement inhibitors.

[0006] At present, it is widely accepted that the complement system can be activated by three different pathways: the classical pathway, the lectin pathway, and the alternative pathway. The classical pathway is usually triggered by a complex containing host antibodies bound to foreign particles (i.e., antigens), and therefore requires prior exposure to antigens to produce a specific antibody response. Because the activation of the classical pathway depends on a prior adaptive immune response by the host, the classical pathway is part of the acquired immune system. In contrast, both the lectin and alternative pathways are independent of adaptive immunity and are part of the innate immune system.

[0007] The activation of the complement system leads to the sequential activation of serine protease zymogens. The first step of the classical pathway activation is the combination of a specific recognition molecule C1q with antigen-bound IgG and IgM molecules. C1q associates with Clr and Cls serine protease zymogens as a complex called Cl. After C1q binds to the immune complex, the autoproteolytic cleavage of the Arg-Ile site of Clr is followed by the cleavage and activation of Cls mediated by Clr-, which thus requires 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 produce a C3 convertase (C4b2a) by non-covalent interaction with the C2a fragment of activated C2. C3 convertase (C4b2a) activates C3 by proteolytic cleavage into C3a and C3b subcomponents, leading to the production of C5 convertase (C4b2a3b), which cleaves C5 leading to the formation of a membrane attack complex (C5b in combination with C6, C7, C8, and C-9, also known as "MAC"), which can damage cell membranes, leading to cell lysis. The activated forms of C3 and C4 (C3b and C4b) are covalently deposited on the surface of foreign targets, which are recognized by complement receptors on multiple phagocytes.

[0008] Independently, the first step of complement system activation through the lectin pathway is also the binding of specific recognition molecules, which is followed by the activation of the associated serine protease zymogen. However, instead of binding of immune complexes through C1q, the recognition molecules in the lectin pathway include a group of sugar-binding proteins (mannan-binding lectin (MBL), H-ficolin, M-ficolin, L-ficolin and C-type lectin CL-11), collectively referred to as lectins. 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).

[0009] Ikeda et al. first demonstrated that, like C1q, MBL is able to activate the complement system when bound to yeast mannan-coated erythrocytes in a C4-dependent manner (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 sugars with 3- and 4-hydroxyl groups located in the equatorial plane of the pyranose ring. Thus, the major ligands for MBL are D-mannose and N-acetyl-D-glucosamine, while sugars that do not meet this steric requirement have undetectable affinity for MBL (Weis et al., Nature 360:127-134, (1992)). The interaction between MBL and monovalent sugars is very weak, with dissociation constants typically in the single-digit millimolar range. MBL achieves tight, specific binding to glycan ligands by avidity, i.e., by simultaneously interacting with multiple monosaccharide residues in close proximity to one another (Lee et al., Archiv. Biochem. Biophys. 299: 129-136, (1992)). MBL recognizes sugar patterns that commonly decorate microorganisms such as bacteria, yeast, parasites, and certain viruses. In contrast, MBL does not recognize D-galactose and sialic acid, penile and terminal sugars that commonly decorate "mature" complex glycoconjugates present on mammalian plasma and cell surface glycoproteins. This binding specificity is thought to facilitate recognition of "foreign" surfaces and help protect against "self-activation". However, MBL binds with high affinity to high-mannose "precursor" glycan clusters on N-linked glycoproteins and glycolipids sequestered in the endoplasmic reticulum and Golgi apparatus of mammalian cells (Maynard et al., J. Biol. Chem. 257: 3788-3794, (1982)). Therefore, damaged cells are potential targets for lectin pathway activation via MBL binding.

[0010] Ficolin has a different type of lectin domain than MBL, called a fibrinogen-like domain. Ficolin binds sugar residues in a Ca++-independent manner. In humans, three classes of ficolins have been identified (L-ficolin, M-ficolin, and H-ficolin). Two classes of serum ficolins, L-ficolin and H-ficolin, share a specificity for N-acetyl-D-glucosamine; however, H-ficolin also binds N-acetyl-D-galactosamine. The differences in sugar specificity of L-ficolin, H-ficolin, CL-11, and MBL mean that different lectins can be complementary and target different targets through interleaved glycoconjugates. This concept is supported by the recent report that, of the known lectins of the lectin pathway, only L-ficolin binds specifically to lipoteichoic acid, a cell wall glycoconjugate present on all Gram-positive bacteria (Lynch et al., J. Immunol. 172: 1198-1202, (2004)). Collectins (i.e., MBL) and ficolin have insignificant similarities in amino acid sequence. However, both classes of proteins have similar domain organizations and, like C1q, assemble into oligomeric structures that maximize the potential for multisite binding.

[0011] Serum concentrations of MBL are highly variable in healthy populations, which is genetically controlled by polymorphisms / mutations in the promoter and coding regions of the MBL gene. As an acute phase protein, expression of MBL is also upregulated during inflammation. L-ficolin is present in serum at concentrations similar to MBL. Therefore, the L-ficolin branch of the lectin pathway is potentially comparable in strength to the MBL arm. MBL and ficolins may also function as opsonins, which allow phagocytes to target MBL- and ficolin-decorated surfaces (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 which has not yet been determined.

[0012] Human MBL forms specific and high-affinity interactions through its collagen-like domain with unique C1r / Cls-like serine proteases, termed MBL-associated serine proteases (MASPs). To date, three MASPs have been described. First, a single enzyme, "MASP," was identified and characterized as the enzyme responsible for the initiation of the complement cascade (i.e., cleavage of C2 and C4) (Matsushita et al., J Exp Med 176(6):1497-1502 (1992); Ji et al., J. Immunol. 150:571-578, (1993)). It was subsequently determined that MASP activity was in fact a mixture of two proteases: MASP-1 and MASP-2 (Thiel et al., Nature 386:506-510, (1997)). However, it has been demonstrated that the MBL-MASP-2 complex alone is sufficient for complement activation (Vorup-Jensen et al., J. Immunol. 165:2093-2100, (2000)). In addition, MASP-2 alone cleaves 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 produce the C3 convertase C4b2a. This is significantly different from the C1 complex of the classical pathway, in which the coordinated action of two specific serine proteases (C1r and C1s) leads to activation of the complement system. In addition, a third novel protease, MASP-3, has been isolated (Dahl, MR et al., Immunity 15:127-35, 2001). MASP-1 and MASP-3 are alternative splicing products of the same gene.

[0013] MASPs share the same domain organization with the enzyme components Clr and Cls of the Cl complex (Sim et al., Biochem. Soc. Trans. 28:545, (2000)). These domains include an N-terminal Clr / Cls / sea urchin VEGF / bone morphogenetic protein (CUB) domain, an epidermal growth factor-like domain, a second CUB domain, tandem complement control protein domains, and a serine protease domain. As in the Cl protease, activation of MASP-2 occurs by cleavage of an Arg-Ile bond adjacent to the serine protease domain, which separates the enzyme into disulfide-linked A and B chains, the latter consisting of the serine protease domain.

[0014] MBL can also associate with an alternatively spliced ​​form of MASP-2, called MBL-associated protein of 19 kDa (MAp19) or small MBL-associated protein (sMAP), which lacks catalytically active MASP2 (Stover, J. Immunol. 162:3481-90, (1999); Takahashi et al., Int. Immunol. 11:859-863, (1999)). MAp19 includes the first two domains of MASP-2, followed by an additional sequence of four unique amino acids. The function of Map19 is unclear (Degn et al., J Immunol. Methods, 2011). The MASP-1 and MASP-2 genes are located on human chromosomes 3 and 1, respectively (Schwaeble et al., Immunobiology 205:455-466, (2002)).

[0015] Several lines of evidence indicate that there are different MBL-MASP complexes and that the majority of MASPs in serum are not complexed with MBL (Thiel et al., J. Immunol. 165:878-887, (2000)). Both H- and L-ficolin bind all MASPs and activate the lectin complement pathway, as does MBL (Dahl et al., Immunity 15:127-35, (2001); Matsushita et al., J. Immunol. 168:3502-3506, (2002)). Both the lectin and classical pathways form a common C3 convertase (C4b2a), and the two pathways converge at this step.

[0016] It is widely believed that the lectin pathway plays an important role in host defense against infection in the first infected host. Strong evidence for the involvement of MBL in host defense comes from the analysis of patients with reduced serum levels of functional MBL (Kilpatrick, Biochim. Biophys. Acta 1572: 401-413, (2002)). Such patients show susceptibility to recurrent bacterial and fungal infections. These symptoms are usually evident early in life, during the apparent window of vulnerability, but before the full repertoire of antibody responses develops, as maternal antibody titers diminish. The syndrome is often caused by mutations at several sites in the collagenous portion of MBL that interfere with the correct formation of MBL oligomers. However, because MBL can act as an opsonin independent of complement, it is unclear to what extent the increased susceptibility to infection is due to impaired complement activation.

[0017] In contrast to the classical and lectin pathways, no initiators of the alternative pathway have been found to fulfill the recognition function performed by CIq and lectins in the other two pathways. It is now widely accepted that the alternative pathway spontaneously undergoes a low level of turnover activation that can be easily amplified on foreign or otherwise abnormal surfaces (bacteria, yeast, virus-infected cells, or damaged tissues) that lack the appropriate molecular elements to control spontaneous complement activation. There are four plasma proteins that are directly involved in the activation of the alternative pathway: C3, factors B and D, and properdin.

[0018] Although extensive evidence indicates that both the classical and alternative complement pathways are involved in the pathogenesis of non-infectious human diseases, the role of the lectin pathway is only beginning to be evaluated. Recent studies provide 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 MBL and show C3 deposition when exposed to human serum (Collard et al., Am. J. Pathol. 156: 1549-1556, (2000)). In addition, treatment of human serum with a blocking anti-MBL monoclonal antibody inhibited MBL binding and complement activation. These findings extend to a rat model of myocardial ischemia-reperfusion, in which rats treated with a blocking antibody to rat MBL showed significantly lower myocardial injury after coronary artery occlusion compared to rats treated with a control antibody (Jordan et al., Circulation 104: 1413-1418, (2001)). The molecular mechanism of MBL binding to vascular endothelium after oxidative stress is unclear; recent studies have shown that activation of the lectin pathway after oxidative stress can be mediated by MBL binding to vascular endothelial cytokeratin and not binding to glycoconjugates (Collard et al., Am. J. Pathol. 159: 1045-1054, (2001)). Other studies have implicated the classical and alternative pathways in the pathogenesis of ischemia / reperfusion injury, and the role of the lectin pathway in this disease remains controversial (Riedermann, NC et al., Am. J. Pathol. 162: 363-367, 2003).

[0019] Recent studies have shown that MASP-1 (and possibly MASP-3) are required to convert the alternative pathway activating enzyme Factor D 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 illustrated by the lack of alternative pathway functional activity in the plasma of MASP-1 / 3-deficient mice. Proteolytic generation of C3b from native C3 is essential for the alternative pathway to function. Because the alternative pathway C3 convertase (C3bBb) contains C3b as a basic subunit, questions about the source of the first C3b to pass through the alternative pathway have raised puzzling questions and prompted a great deal of research.

[0020] C3 belongs to a family of proteins (as well as C4 and alpha-2 macroglobulin) that contain a rare post-translational modification called a thioester bond. The thioester group contains glutamine, whose terminal carbonyl forms a covalent thioester bond with the sulfhydryl group of cysteine ​​three amino acids away. This bond is unstable and the electrophilic glutamyl-thioester can react with nucleophilic moieties such as hydroxyl or amino groups, thus forming covalent bonds with other molecules. When isolated in the hydrophobic pocket of intact C3, the thioester bond is quite stable. However, the proteolytic cleavage of C3 into C3a and C3b results in the highly reactive thioester bond being exposed on C3b, and after nucleophilic attack by adjacent moieties including hydroxyl or amino groups, C3b becomes covalently attached to the target. In addition to its well-documented role in the covalent attachment of C3b to complement targets, it is also believed that the C3 thioester plays a key role in triggering the alternative pathway. According to the widely accepted "tick-over theory", the alternative pathway begins by generating a fluid-phase invertase iC3Bb from C3(iC3; C3(H 2 O)) and factor B formation (Lachmann, PJ et al., Springer Semin. Immunopathol. 7:143-162, (1984)). C3b-like C3 (H 2 O) is generated from natural C3 by slow spontaneous hydrolysis of the protein's internal thioester (Pangburn, MK et al., J. Exp. Med. 154: 856-867, 1981). 2 O)Bb convertase activity, C3b molecules are deposited on the target surface, thereby initiating the alternative pathway.

[0021] Little is known about the initiators of activation of the alternative pathway. It is believed that activators include yeast cell walls (zymosan), many pure polysaccharides, rabbit erythrocytes, certain immunoglobulins, viruses, fungi, bacteria, animal tumor cells, parasites and damaged cells. The only feature common to these activators is the presence of sugars, but the complexity and diversity of sugar structures make it difficult to establish a common identified molecular determinant. It is widely recognized that the activation of the alternative pathway is controlled by a fine balance between the inhibitory regulatory components of the pathway (e.g., factor H, factor I, DAF and CR1 and properdin as the only positive regulator of the alternative pathway) (see Schwaeble WJ and Reid K.B., Immunol Today 20 (1): 17-21 (1999)).

[0022] In addition to the apparently unregulated activation mechanisms described above, the alternative pathway can also provide a powerful amplification loop for the lectin / classical pathway C3 convertase (C4b2a), since any C3b produced can participate with factor B in the formation of an additional alternative pathway C3 convertase (C3bBb). The alternative pathway C3 convertase is stabilized by binding properdin. Properdin prolongs the half-life of the alternative pathway C3 convertase 6-10 fold. Addition of C3b to the alternative pathway C3 convertase results in the formation of the alternative pathway C5 convertase.

[0023] All three pathways (i.e., classical, lectin, and alternative) are thought to converge at C5, which is cleaved to form products with a variety of proinflammatory effects. The convergent pathway is known as the terminal complement pathway. C5a is the most potent anaphylatoxin, inducing changes in smooth muscle and vascular tone and vascular permeability. It is also a potent chemoattractant and activator of neutrophils and monocytes. C5a-mediated cell activation can significantly amplify the inflammatory response by inducing the release of a variety of additional inflammatory mediators, including cytokines, hydrolases, arachidonic acid metabolites, and reactive oxygen species. C5 cleavage results in the formation of C5b-9, also known as the membrane attack complex (MAC). There is now strong evidence that, in addition to its role as a lytic pore-forming complex, sublytic MAC deposition can also play an important role in inflammation.

[0024] In addition to its fundamental role in immune defense, the complement system also contributes to tissue damage in many clinical conditions. Therefore, there is an urgent need to develop therapeutically effective complement inhibitors to prevent these adverse effects.

[0025] Brief Description

[0026] This Summary is provided to introduce selected concepts in a simplified form that are further described below in the 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.

[0027] In one aspect, the invention provides a method of inhibiting microvascular endothelial cell damage and / or thrombus formation in a subject suffering from thrombotic microangiopathy (TMA), comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2-dependent complement activation. In some embodiments, the subject suffers from or is at risk for developing a TMA selected from hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP), and atypical hemolytic uremic syndrome (aHUS). In some embodiments, prior to administering the composition, the subject is determined 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 effective amount and for a sufficient time to ameliorate the one or more symptoms. In some embodiments, the MASP-2 inhibitory agent is an anti-MASP-2 antibody or fragment thereof. In some embodiments, the MASP-2 inhibitory agent is an anti-MASP-2 monoclonal antibody or fragment thereof that specifically binds to a portion of SEQ ID NO: 6. In some embodiments, the MASP-2 inhibitory agent inhibits microvascular endothelial cell damage.

[0028] In another aspect, the invention provides a method of inhibiting MASP-2-dependent complement activation in a subject having or at risk for developing atypical hemolytic uremic syndrome (aHUS), comprising administering to the subject a composition comprising a MASP-2 inhibitory agent in an amount effective to inhibit MASP-2-dependent complement activation. In one embodiment, prior to administering the composition, the subject is determined 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 and for a sufficient time to ameliorate the one or more symptoms. In one embodiment, the subject has or is at risk for developing non-factor H dependent aHUS. In one embodiment, the subject has aHUS associated with factor I, factor B, or the membrane cofactor CD46. In one embodiment, the MASP-2 inhibitory agent is an anti-MASP-2 antibody or fragment thereof that specifically binds to a portion of SEQ ID NO:6, such as an anti-MASP-2 monoclonal antibody or fragment thereof. In one embodiment, the MASP-2 inhibitory agent inhibits microvascular endothelial cell damage. In one embodiment, the MASP-2 inhibitory agent inhibits thrombosis.

[0029] In another aspect, the invention provides methods of reducing the likelihood that a subject at risk for developing atypical hemolytic uremic syndrome (aHUS) will suffer from clinical symptoms associated with aHUS. The methods according to this aspect of the invention comprise: (a) determining the presence of a genetic marker in a subject known to be associated with aHUS; (b) periodically monitoring the subject to determine the presence or absence of at least one symptom selected from anemia, thrombocytopenia, renal insufficiency, and elevated creatinine; and (c) upon determining the presence of at least one of anemia, thrombocytopenia, renal insufficiency, or elevated creatinine, administering to the subject a composition comprising a MASP-2 inhibitory agent in an amount effective to inhibit MASP-2-dependent complement activation, wherein the composition is administered in an amount effective and for a time sufficient to ameliorate the one or more symptoms. In one embodiment, the MASP-2 inhibitory agent is an anti-MASP-2 antibody or fragment thereof that specifically binds to a portion of SEQ ID NO:6, such as an anti-MASP-2 monoclonal antibody or fragment thereof. In one embodiment of the method, step (a) comprises performing a genetic screening assay 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 triggering clinical symptoms of aHUS, and administering to the subject a composition comprising a MASP-2 inhibitory agent before, during, or after the occurrence of the triggering event. In one embodiment, the event associated with triggering clinical symptoms of aHUS 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 embodiment, the MASP-2 inhibitory agent inhibits microvascular endothelial cell damage. In one embodiment, the MASP-2 inhibitory agent inhibits thrombosis.

[0030] In another aspect, the invention provides a method of inhibiting MASP-2-dependent complement activation in a subject having or at risk for developing atypical hemolytic uremic syndrome (aHUS) secondary to an infection, comprising administering to the subject a composition comprising a MASP-2 inhibitory agent in an amount effective to inhibit MASP-2 complement activation. In one embodiment, the subject has or is at risk for non-intestinal aHUS associated with an infection with Streptococcus pneumoniae (S. pneumoniae). In one embodiment, the MASP-2 inhibitory agent is an anti-MASP-2 antibody or fragment thereof that specifically binds to a portion of SEQ ID NO:6, such as an anti-MASP-2 monoclonal antibody or fragment thereof. In one embodiment, the MASP-2 inhibitory agent inhibits microvascular endothelial cell damage. In one embodiment, the MASP-2 inhibitory agent inhibits thrombosis.

[0031] In another aspect, the invention provides a method of treating a subject suffering from atypical hemolytic uremic syndrome (aHUS) comprising administering to the subject a composition comprising a MASP-2 inhibitory agent in an amount effective to inhibit MASP-2-dependent complement activation, wherein the MASP-2 inhibitory agent is administered via an intravenous catheter or other catheter delivery method. In one embodiment, the method further comprises treating the patient with plasmapheresis. In one embodiment, the composition comprising the MASP-2 inhibitory agent is administered in the absence of plasmapheresis. In one embodiment, the composition comprising the MASP-2 inhibitory agent is administered via a catheter for a first period of time, further comprising administering the composition comprising the MASP-2 inhibitory agent for a second period of time, wherein the composition is administered subcutaneously during the second period of time. In one embodiment, the method further comprises periodically determining the level of at least one complement factor, wherein determining a decreased level of at least one complement factor compared to a standard value or a healthy subject indicates a need for continued treatment with the composition. In one embodiment, the MASP-2 inhibitory agent is an anti-MASP-2 antibody or fragment thereof that specifically binds to a portion of SEQ ID NO:6, such as an anti-MASP-2 monoclonal antibody or fragment thereof. In one embodiment, the MASP-2 inhibitory agent inhibits microvascular endothelial cell damage. In one embodiment, the MASP-2 inhibitory agent inhibits thrombosis.

[0032] In another aspect, the present invention provides a method of treating a subject having thrombotic thrombocytopenic purpura (TTP) or displaying symptoms consistent with a diagnosis of TTP, comprising administering to the subject a composition comprising a MASP-2 inhibitory agent in an amount effective to inhibit MASP-2-dependent complement activation, wherein the MASP-2 inhibitory agent is administered to the subject via an intravenous catheter or other catheter delivery method. In one embodiment, the subject displays at least one or more symptoms selected from central nervous system involvement, thrombocytopenia, severe cardiac involvement, severe lung involvement, gastrointestinal infarction, and gangrene. In one embodiment, the subject tests positive for the presence of an inhibitor of ADAMTS13, and the method further comprises administering an immunosuppressant to the subject. In one embodiment, the composition comprising the MASP-2 inhibitory agent is administered for a first period of time in the absence of plasmapheresis. In one embodiment, the subject tests positive for the presence of an inhibitor of ADAMTS-13, and the method further comprises administering ADAMTS-13. In one embodiment, the method further comprises treating the patient with plasmapheresis. In one embodiment, the composition comprising the MASP-2 inhibitory agent is administered in the presence of plasmapheresis. In one embodiment, the composition comprising the MASP-2 inhibitory agent is administered via a catheter for a first period of time, further comprising administering the composition comprising the MASP-2 inhibitory agent for a second period of time, wherein the composition is administered subcutaneously during the second period of time. In one embodiment, the method further comprises periodically determining the level of at least one complement factor, wherein a decrease in the level of at least one complement factor as compared to a standard value or a healthy subject indicates a need for continued treatment with the composition. In one embodiment, the MASP-2 inhibitory agent is an anti-MASP-2 antibody or fragment thereof that specifically binds to a portion of SEQ ID NO:6, such as an anti-MASP-2 monoclonal antibody or fragment thereof. In one embodiment, the MASP-2 inhibitory agent inhibits microvascular endothelial cell damage. In one embodiment, the MASP-2 inhibitory agent inhibits thrombosis.

[0033] In another aspect, the invention provides a method of treating a subject with refractory thrombotic thrombocytopenic purpura (TTP) comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2-dependent complement activation. In one embodiment, the composition is administered subcutaneously. In one embodiment, the method further comprises periodically determining the level of at least one complement factor, wherein a decrease in the level of at least one complement factor as compared to a standard value or a healthy subject indicates a need for continued treatment with the composition. In one embodiment, the MASP-2 inhibitory agent is an anti-MASP-2 antibody or fragment thereof that specifically binds to a portion of SEQ ID NO:6, such as an anti-MASP-2 monoclonal antibody or fragment thereof. In one embodiment, the MASP-2 inhibitory agent inhibits microvascular endothelial cell damage. In one embodiment, the MASP-2 inhibitory agent inhibits thrombus formation.

[0034] In another aspect, the invention provides a method of inhibiting MASP-2-dependent complement activation in a subject having or at risk for developing thrombotic microangiopathy (TMA), wherein the TMA is at least one of: (i) TMA secondary to cancer; (ii) TMA secondary to chemotherapy; or (iii) TMA secondary to transplantation, comprising administering to the subject a composition comprising a MASP-2 inhibitory agent in an amount effective to inhibit MASP-2-dependent complement activation. In some embodiments, the subject has or is at risk for developing TMA secondary to cancer, and the MASP-2 inhibitory agent is systemically administered to the subject in an amount effective to reduce the risk of developing TMA or to reduce the severity of TMA. In some embodiments, the subject has or is at risk for developing TMA secondary to chemotherapy, and the MASP-2 inhibitory agent is systemically administered to the subject before, during, or after chemotherapy in an amount effective to reduce the risk of developing TMA or to reduce the severity of TMA. In some embodiments, the subject has or is at risk for developing TMA secondary to transplantation, and the MASP-2 inhibitory agent is systemically administered to the subject before, during, or after the transplant procedure in an amount effective to reduce the risk of developing TMA or to reduce the severity of TMA. In some embodiments the transplant procedure is an allogeneic hematopoietic stem cell transplant. In some embodiments, the subject has previously undergone or is currently undergoing 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 antigen-binding fragment thereof, such as eculizumab.

[0035] In another aspect, the invention provides methods of inhibiting MASP-2-dependent complement activation in a subject having or at risk for developing Upshaw-Schulman syndrome (USS), comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2-dependent complement activation. In some embodiments, the method comprises treating a subject at risk for developing USS, wherein the method comprises administering an amount of a MASP-2 inhibitory agent for a period of time effective to improve or prevent one or more clinical symptoms associated with TTP. In some embodiments, the method further comprises periodically monitoring the subject and administering the MASP-2 inhibitory agent in the presence of an event known to be associated with triggering clinical symptoms of TTP. In some embodiments, the method further comprises periodically monitoring the subject and administering the MASP-2 inhibitory agent in the presence of anemia, thrombocytopenia, or elevated creatine. In some embodiments, the subject has previously undergone or is currently undergoing 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 antigen-binding fragment thereof, such as eculizumab.

[0036] In another aspect, the invention provides methods of inhibiting MASP-2-dependent complement activation in a subject having Degos' disease, comprising administering to the subject a composition comprising a MASP-2 inhibitory agent in an amount effective to inhibit MASP-2-dependent complement activation. In some embodiments, the subject has previously undergone or is currently undergoing 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 antigen-binding fragment thereof, such as eculizumab.

[0037] In another aspect, the invention provides methods of inhibiting MASP-2-dependent complement activation in a subject suffering from catastrophic antiphospholipid syndrome (CAPS), comprising administering to the subject a composition comprising a MASP-2 inhibitory agent in an amount effective to inhibit MASP-2-dependent complement activation. In some embodiments, the subject has previously undergone or is currently undergoing 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 antigen-binding fragment thereof, such as eculizumab.

[0038] In some embodiments of any of the methods disclosed herein, the MASP-2 inhibitory agent is a MASP-2 inhibitory antibody or 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 inhibitory agent is an anti-MASP-2 monoclonal antibody or fragment thereof that specifically binds to a portion of SEQ ID NO:6. In some embodiments, the anti-MASP-2 antibody or fragment thereof is selected from a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In some embodiments, the MASP-2 inhibitory antibody is 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 an IgG1 molecule, an IgG2, and an IgG4 molecule. In some embodiments, the MASP-2 inhibitory antibody is an IgG4 molecule comprising an S228P mutation. In some embodiments, the MASP-2 inhibitory antibody binds to human MASP-2 with a KD of 10 nM or less. 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 with an IC50 of 10 nM or less in 1% human serum in an in vitro assay. In some embodiments, the MASP-2 inhibitory antibody inhibits C3b deposition with an IC50 of 30 nM or less in 90% human serum.

[0039] In some embodiments of any of the methods disclosed herein, 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 31-35 of SEQ ID NO:67; and ii) a heavy chain CDR-H2 comprising the amino acid sequence of 50-65 of SEQ ID NO:67; and iii) a heavy chain CDR-H3 comprising the amino acid sequence of 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 24-34 of SEQ ID NO:70; and ii) a light chain CDR-L2 comprising the amino acid sequence of 50-56 of SEQ ID NO:70; and iii) a light chain CDR-L3 comprising the amino acid sequence of 89-97 of SEQ ID NO:70. In some embodiments, the MASP-2 inhibitory monoclonal antibody comprises a heavy chain variable region set forth in SEQ ID NO: 67 and a light chain variable region set forth 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 an epitope recognized by a reference antibody comprising a heavy chain variable region set forth in SEQ ID NO: 67 and a light chain variable region set forth in SEQ ID NO: 70.

[0040] In another aspect of the invention, methods of inhibiting thrombosis in a subject with atypical hemolytic uremic syndrome (aHUS) are provided, comprising administering to the subject an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof effective to inhibit MASP-2-dependent complement activation. In some embodiments, the MASP-2 inhibitory antibody inhibits thrombosis in serum from a subject with aHUS by at least 40% compared to untreated serum. In some embodiments, the MASP-2 inhibitory antibody inhibits thrombosis in serum from a subject with 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 its inhibitory effect 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 remission of aHUS. In some embodiments, the MASP-2 inhibitory antibody is a monoclonal antibody or 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 a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In some embodiments, the MASP-2 inhibitory antibody is selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab') 2In some embodiments, the MASP-2 inhibitory antibody is a single chain molecule. In some embodiments, the MASP-2 inhibitory antibody is selected from an IgG1 molecule, an IgG2, and an IgG4 molecule. In some embodiments, the MASP-2 inhibitory antibody is an IgG4 molecule comprising an S228P mutation. In some embodiments, the MASP-2 inhibitory antibody binds to human MASP-2 with a KD of 10 nM or less. 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 with an IC50 of 10 nM or less in 1% human serum in an in vitro assay. In some embodiments, the MASP-2 inhibitory antibody inhibits C3b deposition with an IC50 of 30 nM or less in 90% human serum. 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 31-35 of SEQ ID NO: 67; and ii) a heavy chain CDR-H2 comprising the amino acid sequence of 50-65 of SEQ ID NO: 67; and iii) a heavy chain CDR-H3 comprising the amino acid sequence of 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 24-34 of SEQ ID NO: 70; and ii) a light chain CDR-L2 comprising the amino acid sequence of 50-56 of SEQ ID NO: 70; and iii) a light chain CDR-L3 comprising the amino acid sequence of 89-97 of SEQ ID NO: 70. In some embodiments, the MASP-2 inhibitory monoclonal antibody comprises a heavy chain variable region set forth in SEQ ID NO: 67 and a light chain variable region set forth 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 an epitope recognized by a reference antibody comprising a heavy chain variable region set forth in SEQ ID NO:67 and a light chain variable region set forth in SEQ ID NO:70.

[0041] In another aspect, the invention provides a method of treating a subject having plasma therapy resistant aHUS comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2-dependent complement activation. In one embodiment, the MASP-2 inhibitory antibody is an anti-MASP-2 monoclonal antibody or fragment thereof that specifically binds to human MASP-2. In one embodiment, the antibody or fragment thereof is selected from a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In one embodiment, the subject has previously undergone, or is currently undergoing, treatment with a terminal complement inhibitor that inhibits cleavage of complement protein C5, e.g., wherein the terminal complement inhibitor is a humanized anti-C5 antibody or antigen-binding fragment thereof. In one embodiment, the method further comprises treating the patient with plasmapheresis. In one embodiment, the composition comprising the MASP-2 inhibitory antibody is administered in the absence of plasmapheresis.

[0042] In another aspect, the invention provides a method of treating a subject suffering from TMA associated with hematopoietic stem cell transplantation, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2-dependent complement activation. In one embodiment, the MASP-2 inhibitory antibody is an anti-MASP-2 monoclonal antibody or fragment thereof that specifically binds to human MASP-2. In one embodiment, the antibody or fragment thereof is selected from a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In one embodiment, the subject has previously undergone, or is currently undergoing, treatment with a terminal complement inhibitor that inhibits cleavage of complement protein C5. In one embodiment, the subject is suffering from TMA associated with hematopoietic stem cell transplantation that is resistant to treatment with platelet transfusions, and / or resistant to treatment with plasmapheresis. In one embodiment, a 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 an increase in platelet count (e.g., at least 2-fold, at least 3-fold, at least 4-fold the platelet count prior to treatment), an increase in haptoglobin, and / or a decrease in lactate dehydrogenase.

[0043] In another aspect, the invention provides a method of treating a human subject having persistent TMA associated with hematopoietic stem cell transplantation (HSCT-TMA) comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or antigen binding fragment thereof effective to inhibit MASP-2-dependent complement activation. In one embodiment, 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 amount of a MASP-2 inhibitory antibody or antigen binding fragment thereof effective to inhibit MASP-2-dependent complement activation. In one embodiment, the MASP-2 inhibitory antibody is a monoclonal antibody or fragment thereof that specifically binds to human MASP-2. In one embodiment, the antibody or antigen binding fragment thereof is selected from a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In one embodiment, the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. In one embodiment, the MASP-2 inhibitory antibody has an IC of 30 nM or less in 90% human serum. 50 Inhibition of C3b deposition. In one embodiment, 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 set forth in SEQ ID NO:67; and a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequence set forth in SEQ ID NO:70. In one embodiment, the MASP-2 inhibitory antibody is administered to the patient in the absence of plasmapheresis. In one embodiment, the subject has previously undergone, or is currently undergoing, treatment with a humanized anti-C5 antibody or antigen-binding fragment thereof, e.g., wherein the terminal complement inhibitor is a humanized anti-C5 antibody or antigen-binding fragment thereof. In one embodiment, the MASP-2 inhibitory antibody is delivered to the subject systemically. In one embodiment, a MASP-2 inhibitory antibody or antigen-binding fragment thereof 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) an increase in platelet count (e.g., at least 2-fold, at least 3-fold, at least 4-fold the platelet count prior to treatment); (ii) an increase in haptoglobin; (iii) a decrease in lactate dehydrogenase (LDH); and / or (iv) a decrease in creatinine.

[0044] 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) comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof effective to inhibit MASP-2-dependent complement activation. In one embodiment, the subject has previously undergone, is currently undergoing, or will undergo a hematopoietic stem cell transplant. In one embodiment, 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 amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof effective to inhibit MASP-2-dependent complement activation. In one embodiment, the MASP-2 inhibitory antibody is a monoclonal antibody or fragment thereof that specifically binds to human MASP-2. In one embodiment, the MASP-2 inhibitory antibody or fragment thereof is selected from a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In one embodiment, the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. In one embodiment, the MASP-2 inhibitory antibody has an IC of 30 nM or less in 90% human serum. 50 Inhibits C3b deposition. In one embodiment, the MASP-2 inhibitory antibody is delivered systemically to the subject. In one embodiment, the subject suffers from acute GVHD. In one embodiment, the subject suffers from steroid-resistant GVHD. In one embodiment, 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 set forth in SEQ ID NO:67, and a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequence set forth in SEQ ID NO:70.

[0045] In another aspect, the present invention provides a method for treating, preventing or ameliorating one or more neurological symptoms associated with graft-versus-host disease or TMA, comprising administering to a subject in need thereof a composition comprising an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof effective to inhibit MASP-2-dependent complement activation. In one embodiment, the one or more neurological symptoms associated with graft-versus-host disease or TMA are selected from weakness, paresthesia, quadriplegia, sensorimotor deficits, autonomic insufficiency polyneuropathy, and / or neurogenic bladder. In one embodiment, the subject has received a hematopoietic stem cell transplant and the subject is suffering from one or more neurological symptoms selected from paresthesia, quadriplegia, and neurogenic bladder. In one embodiment, the subject has received a hematopoietic stem cell transplant and is suffering from graft-versus-host disease. In one embodiment, the subject has received a hematopoietic stem cell transplant and is suffering from HSCT-TMA. In one embodiment, the MASP-2 inhibitory antibody is a monoclonal antibody or fragment thereof that specifically binds to human MASP-2. In one embodiment, the antibody or fragment thereof is selected from a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In one embodiment, the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. In one embodiment, the MASP-2 inhibitory antibody has an IC of 30 nM or less in 90% human serum. 50 Inhibition of C3b deposition. In one embodiment, the MASP-2 inhibitory antibody is delivered to the subject systemically. In one embodiment, the method further comprises identifying a human subject suffering from one or more neurological symptoms associated with hematopoietic stem cell transplantation prior to the step of administering to the subject a composition comprising an amount of the MASP-2 inhibitory antibody or antigen-binding fragment thereof effective to inhibit MASP-2-dependent complement activation. In one embodiment, 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 set forth in SEQ ID NO:67, and a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequence set forth in SEQ ID NO:70.

[0046] In another aspect, the invention provides a method of treating a human subject having or at risk of developing diffuse alveolar hemorrhage (DAH) associated with hematopoietic stem cell transplantation, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof effective to inhibit MASP-2-dependent complement activation. In one embodiment, the subject has previously undergone, is currently undergoing, or will undergo a hematopoietic stem cell transplantation. In one embodiment, the method further comprises identifying a human subject having or at risk of developing diffuse alveolar hemorrhage (DAH) prior to the step of administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof effective to inhibit MASP-2-dependent complement activation. In one embodiment, the MASP-2 inhibitory antibody is a monoclonal antibody or fragment thereof that specifically binds to human MASP-2. In one embodiment, the MASP-2 inhibitory 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. In one embodiment, the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. In one embodiment, the MASP-2 inhibitory antibody has an IC of 30 nM or less in 90% human serum. 50 Inhibition of C3b deposition. In one embodiment, the MASP-2 inhibitory antibody is delivered systemically to the subject. In one embodiment, 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 set forth in SEQ ID NO:67, and a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequence set forth in SEQ ID NO:70. In another aspect, the present invention provides a composition for inhibiting the adverse effects of MASP-2-dependent complement activation, comprising a therapeutically effective amount of a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, and a pharmaceutically acceptable carrier. Also provided is a method of preparing a medicament for inhibiting the adverse effects of MASP-2-dependent complement activation in a living subject in need thereof, comprising a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutically acceptable carrier. Also provided is a method of preparing a medicament for inhibiting MASP-2-dependent complement activation for treating each of the conditions, diseases and disorders described below.

[0047] In another aspect, the invention provides a method of treating a human subject suffering from or at risk of developing veno-occlusive disease (VOD) associated with hematopoietic stem cell transplantation, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof effective to inhibit MASP-2-dependent complement activation. In one embodiment, the subject has previously undergone, is currently undergoing, or will undergo a hematopoietic stem cell transplantation. In one embodiment, the method further comprises identifying a human subject suffering from or at risk of developing veno-occlusive disease (VOD) prior to the step of administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof effective to inhibit MASP-2-dependent complement activation. In one embodiment, the MASP-2 inhibitory antibody is a monoclonal antibody or fragment thereof that specifically binds to human MASP-2. In one embodiment, the MASP-2 inhibitory 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. In one embodiment, the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. In one embodiment, the MASP-2 inhibitory antibody has an IC of 30 nM or less in 90% human serum. 50 Inhibition of C3b deposition. In one embodiment, the MASP-2 inhibitory antibody is delivered systemically to the subject. In one embodiment, 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 set forth in SEQ ID NO:67, and a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequence set forth in SEQ ID NO:70. In another aspect, the present invention provides a composition for inhibiting the adverse effects of MASP-2-dependent complement activation, comprising a therapeutically effective amount of a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, and a pharmaceutically acceptable carrier. Also provided is a method of preparing a medicament for inhibiting the adverse effects of MASP-2-dependent complement activation in a living subject in need thereof, comprising a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutically acceptable carrier. Also provided is a method of preparing a medicament for inhibiting MASP-2-dependent complement activation for treating each of the conditions, diseases and disorders described below.

[0048] The methods, compositions and medicaments of the invention are useful for inhibiting the adverse effects of MASP-2-dependent complement activation in vivo in mammalian subjects (including humans) suffering from or at risk for developing thrombotic microangiopathy (TMA), and / or subjects suffering from or at risk for developing GVHD, and / or subjects suffering from or at risk for developing diffuse alveolar hemorrhage following stem cell transplantation, and / or subjects suffering from or at risk for developing veno-occlusive disease (VOD), as further described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The foregoing aspects and many of the attendant advantages of the present invention will become more readily understood as will become better understood with reference to the following detailed description when taken in conjunction with the accompanying drawings, in which:

[0051] Figure 1 is a diagram illustrating the genomic structure of human MASP-2;

[0052] Figure 2A is a schematic diagram illustrating the domain structure of human MASP-2 protein;

[0053] Figure 2B is a schematic diagram illustrating the domain structure of human MAp19 protein;

[0054] Figure 3 is a diagram illustrating the knockout strategy for murine MASP-2;

[0055] Figure 4 is a diagram illustrating a human MASP-2 minigene construct;

[0056] Figure 5A Presents results demonstrating that MASP-2-deficiency results in loss of lectin-pathway-mediated C4 activation as measured by lack of C4b deposition on mannan as described in Example 2;

[0057] Figure 5B Presents results demonstrating that MASP-2-deficiency results in loss of lectin-pathway-mediated C4 activation as measured by lack of C4b deposition on zymosan as described in Example 2;

[0058] Figure 5C Shows results demonstrating relative C4 activation levels of serum samples obtained from MASP-2+ / -, MASP-2- / - and wild-type strains as measured by C4b deposition on mannan and zymosan as described in Example 2;

[0059] Figure 6Presents results demonstrating that addition of murine recombinant MASP-2 to MASP-2- / - serum samples restores lectin-pathway-mediated C4 activation in a protein concentration-dependent manner as measured by C4b deposition on mannan as described in Example 2;

[0060] Figure 7 represents the results demonstrating that the classical pathway is operative in the MASP-2- / - strain, as described in Example 8;

[0061] Fig. 8A represents the results demonstrating that anti-MASP-2 Fab2 antibody #11 inhibits C3 convertase formation, as described in Example 10;

[0062] Figure 8B represents the results demonstrating that anti-MASP-2 Fab2 antibody #11 binds to native rat MASP-2, as described in Example 10;

[0063] Figure 8C represents the results demonstrating that anti-MASP-2 Fab2 antibody #41 inhibits C4 cleavage, as described in Example 10;

[0064] Fig. 9 represents the results demonstrating that all tested anti-MASP-2 Fab2 antibodies that were also found to inhibit C3 convertase formation inhibited C4 cleavage, as described in Example 10;

[0065] Fig.10 is a diagram illustrating recombinant polypeptides derived from rat MASP-2 for epitope mapping of anti-MASP-2 blocking Fab2 antibodies, as described in Example 11;

[0066] Fig.11 represents the results demonstrating that anti-MASP-2 Fab2 #40 and #60 bind to rat MASP-2 polypeptide, as described in Example 11;

[0067] Fig.12 Shows results demonstrating blood urea nitrogen clearance in wild type (+ / +) and MASP-2 (- / -) mice at 24 and 48 hours after reperfusion in a renal ischemia / reperfusion injury model, as described in Example 12;

[0068] Fig.13A represents results showing baseline VEGF protein levels in RPE-choroidal complexes isolated from wild type (+ / +) and MASP-2 (- / -) mice, as described in Example 13;

[0069] Fig. 13BShows results showing VEGF protein levels in the RPE-choroidal complex in wild type (+ / +) and MASP-2 (- / -) mice at day 3 after laser-induced injury in a macular degeneration model, as described in Example 13;

[0070] Fig.14 Shows the results showing the mean choroidal neovascularization (CNV) volume at day 7 after laser-induced injury in wild-type (+ / +) and MASP-2 (- / -) mice, as described in Example 13;

[0071] Fig.15A and 15B Shows dose response curves for inhibition of C4b deposition (FIG.15A) and inhibition of thrombin activation (FIG.15B) following administration of MASP-2 Fab2 antibodies in normal rat serum, as described in Example 14;

[0072] Fig.16A and 16B In the local Schwartzman reaction model of disseminated intravascular coagulation, the complement pathway was inhibited by the phlebotomist cobra venom factor (CVF) and the terminal pathway inhibitor (C5aR antagonist) compared with untreated wild-type mice and wild-type mice in which the complement pathway was inhibited by the phlebotomist cobra venom factor (CVF) and the terminal pathway inhibitor (C5aR antagonist) ( Fig.16A ) compared to platelet aggregation in MASP-2(- / -) mice (FIG.16B) (expressed as aggregate area), as described in Example 15;

[0073] Fig.17 Graph illustrating blood urea nitrogen (BUN) levels measured in WT (+ / +) (B6) or MASP-2 (- / -) transplant recipient mice of WT (+ / +) donor kidneys, as described in Example 16;

[0074] Fig.18 Graph illustrating the percent survival of WT (+ / +) and MASP-2 (- / -) mice as a function of days following microbial infection in the cecal ligation and puncture (CLP) model, as described in Example 17;

[0075] Fig.19 Graph illustrating bacterial numbers measured in WT (+ / +) and MASP-2 (- / -) following microbial infection in the cecal ligation and puncture (CLP) model, as described in Example 17;

[0076] Fig. 20is a Kaplan-Mayer curve illustrating the percent survival of WT (+ / +), MASP-2 (- / -), and C3 (- / -) mice 6 days after challenge with intranasal administration of P. aeruginosa, as described in Example 18;

[0077] Fig.21 Graph illustrating the levels of C4b deposition measured as % of control in samples obtained at various time points following subcutaneous administration of 0.3 mg / kg or 1.0 mg / kg of mouse anti-MASP-2 monoclonal antibody in WT mice, as described in Example 19;

[0078] Fig. 22 Graph illustrating the levels of C4b deposition measured as % of control in samples obtained at various time points following ip administration of 0.6 mg / kg of mouse anti-MASP-2 monoclonal antibody in WT mice, as described in Example 19;

[0079] Fig.23 The graph illustrates the mean choroidal neovascularization (CNV) volume at day 7 after laser-induced injury in WT (+ / +) mice pretreated with a single ip injection of 0.3 mg / kg or 1.0 mg / kg mouse anti-MASP-2 monoclonal antibody, as described in Example 20;

[0080] Fig.24A Graph illustrating the percent survival of MASP-2 (- / -) and WT (+ / +) mice following infection with 5 x 108 / 100 μl cfu N. meningitidis, as described in Example 21;

[0081] Fig. 24B Graph illustrating the log cfu / ml of N. meningitidis recovered at various time points in blood samples taken from MASP-2 KO (- / -) and WT (+ / +) mice infected with 5 x 108 cfu / 100 μl N. meningitidis, as described in Example 21;

[0082] Fig.25A Graph illustrating the percent survival of MASP-2 KO (- / -) and WT (+ / +) mice following infection with 2x108 cfu / 100 μl N. meningitidis, as described in Example 21;

[0083] Fig.25B Graph illustrating the log cfu / ml of N. meningitidis recovered at different time points in blood samples taken from WT (+ / +) mice infected with 2 x 108 cfu / 100 μl N. meningitidis, as described in Example 21;

[0084] Fig.25CGraph illustrating the log cfu / ml of N. meningitidis recovered at different time points in blood samples taken from MASP-2(- / -) mice infected with 2 x 108 cfu / 100 μl N. meningitidis, as described in Example 21;

[0085] Fig.26A Graph illustrating the results of a C3b deposition assay demonstrating that MASP-2(- / -) mice retain a functional classical pathway, as described in Example 22;

[0086] Fig.26B Graph illustrating the results of a C3b deposition assay on zymosan-coated plates demonstrating that MASP-2(- / -) mice retain a functional alternative pathway, as described in Example 22;

[0087] Fig.27A Graphs illustrate myocardial ischemia / reperfusion injury (MIRI)-induced tissue loss following ligation and reperfusion of the left anterior descending artery (LAD) in C4(- / -) mice (n=6) and matched WT littermate controls (n=7), indicating the area at risk (AAR) and infarct size (INF), as described in Example 22;

[0088] Fig.27B The diagram shows how to Fig.27A Infarct size (INF) as a function of area at risk (AAR) in C4(- / -) and WT mice treated as described in Example 22, demonstrating that C4(- / -) mice are as susceptible to MIRI as WT controls (dashed line);

[0089] Fig.28A The graph illustrates the results of a C3b deposition assay using sera from WT mice, C4(- / -) mice, and sera from C4(- / -) mice pre-incubated with mannan, as described in Example 22;

[0090] Fig.28B Graph illustrating the results of a C3b deposition assay on sera from WT, C4(- / -) and MASP-2(- / -) mice mixed with various concentrations of anti-murine MASP-2 mAb (mAbM11), as described in Example 22;

[0091] Fig.28C Graph illustrating the results of a C3b deposition assay on human sera from WT (C4 sufficient) and C4 deficient sera and sera from C4 deficient subjects pre-incubated with mannan, as described in Example 22;

[0092] Fig.28DGraph illustrating the results of a C3b deposition assay on human sera from WT (C4 sufficient) and C4 deficient subjects mixed with anti-human MASP-2 mAb (mAbH3), as described in Example 22;

[0093] Fig.29A Graph illustrating comparative analysis of C3 convertase activity in plasma from various complement-deficient mouse strains tested under lectin activation pathway specific assay conditions or under classical activation pathway specific assay conditions, as described in Example 22;

[0094] Fig.29B Graph illustrating the time-resolved kinetics of C3 convertase activity in plasma from various complement-deficient mouse strains tested under conditions specific for the lectin activation pathway, as described in Example 22;

[0095] Fig.30 illustrates the results of a Western blot analysis showing activation of human C3 as evidenced by the presence of the thrombin substrates FXIa and the a' chain of FXa, as described in Example 23;

[0096] Fig.31 Shown are the results of a C3 deposition assay on serum samples obtained from WT, MASP-2(- / -), F11(- / -), F11(- / -) / C4(- / -), and C4(- / -), as described in Example 23;

[0097] Fig.32A is a Kaplain-Meier survival curve showing percent survival over time following exposure to 7.0 Gy of radiation in control mice and in mice treated with an anti-murine MASP-2 antibody (mAbM11) or an anti-human MASP-2 antibody (mAbH6), as described in Example 29;

[0098] Fig.32B is a Kaplain-Meier survival curve showing percent survival over time following exposure to 6.5 Gy of radiation in control mice and in mice treated with an anti-murine MASP-2 antibody (mAbM11) or an anti-human MASP-2 antibody (mAbH6), as described in Example 29;

[0099] Fig.33 is a Kaplan-Meyer curve illustrating the percent survival of MASP-2 KO and WT mice following administration of an infectious dose of 2.6 x 107 cfu of N. meningitidis serotype A Z2491, demonstrating that MASP-2-deficient mice are protected from N. meningitidis-induced mortality, as described in Example 30;

[0100] Fig.34 is a Kaplan-Meyer curve illustrating the percent survival of MASP-2 KO and WT mice following administration of an infectious dose of 6 x 106 cfu of N. meningitidis serotype B strain MC58, demonstrating that MASP-2-deficient mice are protected from N. meningitidis serotype B strain MC58-induced mortality, as described in Example 30;

[0101] Fig.35 The graph illustrates the log cfu / ml of N. meningitidis serotype B strain MC58 recovered in blood samples taken from MASP-2 KO and WT mice at different time points after ip infection with 6 x 106 cfu of N. meningitidis serotype B strain MC58 (n=3 for both groups of mice at different time points, results are expressed as mean ± SEM), demonstrating that although MASP-2 KO mice were infected with the same dose of N. meningitidis serotype B strain MC58 as WT mice, MASP-2 KO mice had improved clearance of bacteremia compared to WT, as described in Example 30;

[0102] Fig.36 Graph illustrating mean disease scores of MASP-2 and WT mice at 3, 6, 12 and 24 hours after infection with 6 x 106 cfu / 100 μl of Neisseria meningitidis serotype B strain MC58, demonstrating that MASP-2 deficient mice display high resistance to infection with much lower disease scores at 6 hours, as described in Example 30;

[0103] Fig.37 is a Kaplan-Meyer curve illustrating the percent survival of mice following administration of an infective dose of 4 x 106 / 100 μl cfu of Neisseria meningitidis serotype B strain MC58 followed by administration of an inhibitory anti-MASP-2 antibody (1 mg / kg) or a control isotype antibody 3 hours post infection, demonstrating that anti-MASP-2 antibodies are effective in treating and improving the survival of subjects infected with Neisseria meningitidis, as described in Example 31;

[0104] Fig.38Graph illustrating the log cfu / ml of viable counts of N. meningitidis serotype B-MC58 recovered at different time points following ip infection with 6.5 x 106 cfu / 100 μl N. meningitidis serotype B strain MC58 at 20% human serum concentration at 0, 30, 60 and 90 minutes post incubation in the presence of: (A) normal human serum (NHS) plus human anti-MASP-2 antibody; (B) normal human serum (NHS) plus isotype control antibody; (C) MBL- / - human serum; (D) normal human serum (NHS) and (E) heat-inactivated normal human serum (NHS), showing that complement-dependent killing of N. meningitidis in human serum is significantly enhanced by the addition of human anti-MASP-2 antibody, as described in Example 32;

[0105] Fig.39 Graph illustrating the log cfu / ml of viable counts of Neisseria meningitidis serotype B-MC58 recovered in mouse serum samples at different time points, demonstrating that MASP-2- / - mouse serum has a higher level of bactericidal activity against Neisseria meningitidis than WT mouse serum, as described in Example 32;

[0106] Fig.40 The graph illustrates hemolysis of mannan-coated murine erythrocytes (as measured by the release of hemoglobin from lysed mouse erythrocytes (Crry / C3- / -) into the supernatant, measured photometrically) by human sera over a range of serum concentrations. Sera tested included heat-inactivated (HI) NHS, MBL- / -, NHS+anti-MASP-2 antibody, and NHS control, as described in Example 33;

[0107] Fig.41 The graph illustrates hemolysis of uncoated murine erythrocytes (as measured by release of hemoglobin from lysed WT mouse erythrocytes into the supernatant, measured photometrically) by human serum over a range of serum concentrations. The sera tested included heat-inactivated (HI) NHS, MBL- / -, NHS+anti-MASP-2 antibody, and NHS control, demonstrating that inhibition of MASP-2 inhibits complement-mediated lysis of non-sensitized WT mouse erythrocytes, as described in Example 33;

[0108] Fig.42 The graph illustrates hemolysis of uncoated murine erythrocytes (as measured by release of hemoglobin from lysed mouse erythrocytes (CD55 / 59- / -) into the supernatant, measured photometrically) by human sera over a range of serum concentrations. Sera tested included heat-inactivated (HI) NHS, MBL- / -, NHS+anti-MASP-2 antibody, and NHS control, as described in Example 33;

[0109] Fig.43 Graph illustrating the percent survival over time (days) following exposure to 8.0 Gy of radiation in control mice and in mice treated with an anti-human MASP-2 antibody (mAbH6), as described in Example 34;

[0110] Fig.44 Graph illustrating the time to onset of microvascular occlusion following LPS injection of MASP-2- / - and WT mice, showing the percentage of mice with thrombosis measured over 60 minutes, demonstrating that thrombosis was detected in WT mice after 15 minutes, with up to 80% of WT mice demonstrating thrombosis at 60 minutes; in contrast, no MASP-2- / - mice showed any thrombosis during the 60 minutes (log rank: p=0.0005), as described in Example 35;

[0111] Fig.45 Graph illustrating the percent survival over time (hours) of saline-treated control mice (n=5) and anti-MASP-2 antibody-treated mice (n=5) in the STX / LPS-induced HUS model, demonstrating that all control mice died by 42 hours, while in contrast, 100% of the anti-MASP-2 antibody-treated mice survived over the entire time course of the experiment, as described in Example 36;

[0112] Fig.46 Graph illustrating the percentage of mice with microvascular obstruction in the FITC / dextran UV model following treatment by administration of isotype control or human MASP-2 antibody mAbH6 (10 mg / kg) 16 hours and 1 hour prior to injection of FITC / dextran as a function of time after injury induction, as described in Example 37;

[0113] Fig.47 The graph illustrates the blocking time (minutes) of mice treated with human MASP-2 antibody (mAbH6) and isotype control antibody, wherein the data are reported as scatter plots with mean (horizontal bars) and standard error bars (vertical bars). The statistical test used for the analysis was an unpaired t-test; wherein the symbol "*" indicates p=0.0129, as described in Example 37; and

[0114] Fig.48 Graph illustrating the time until occlusion (minutes) in a FITC-dextran / light-induced endothelial cell injury thrombosis model using low light intensity (800-1500) for wild-type mice, MASP-2 KO mice, and wild-type mice pretreated with 10 mg / kg human MASP-2 antibody (mAbH6) ip 16 hours and again 1 hour prior to induction of thrombosis, as described in Example 37;

[0115] Fig.49 is a Kaplan-Meier curve showing the percentage of mice with thrombi as a function of time in FITC-dextran-induced thrombotic microangiopathy in mice treated with increasing doses of human MASP-2 inhibitory antibody (mAbH6) or isotype control antibody, as described in Example 39;

[0116] Fig.50 Graph illustrating the median time (minutes) to initiation of thrombus formation as a function of mAbH6 dose (*p<0.01 compared to control), as described in Example 39;

[0117] Fig.51 is a Kaplan-Meier curve showing the percentage of mice with microvascular obstruction as a function of time in mice treated with increasing doses of human MASP-2 inhibitory antibody (mAbH6) or isotype control antibody in FITC-dextran-induced thrombotic microangiopathy, as described in Example 39;

[0118] Fig.52 Graph illustrating median time to microvascular occlusion as a function of mAbH6 dose (*p<0.05 compared to control), as described in Example 39;

[0119] Fig.53A The graph illustrates the level of MAC deposition in the presence or absence of human MASP-2 monoclonal antibody (OMS646) under lectin pathway-specific assay conditions, demonstrating that OMS646 inhibits lectin-mediated MAC deposition, IC 50 The value is about 1 nM, as described in Example 40;

[0120] Fig.53B Graph illustrating the level of MAC deposition under classical pathway-specific assay conditions in the presence or absence of a human MASP-2 monoclonal antibody (OMS646), demonstrating that OMS646 does not inhibit classical pathway-mediated MAC deposition, as described in Example 40;

[0121] Fig.53C Graph illustrating the level of MAC deposition under alternative pathway-specific assay conditions in the presence or absence of a human MASP-2 monoclonal antibody (OMS646), demonstrating that OMS646 does not inhibit alternative pathway-mediated MAC deposition, as described in Example 40;

[0122] Fig.54Graph illustrating the pharmacokinetic (PK) profile of a human MASP-2 monoclonal antibody (OMS646) in mice showing OMS646 concentrations (mean, n=3 animals / group) as a function of time after administration at the indicated doses, as described in Example 40;

[0123] Fig.55A Graph illustrating the pharmacodynamic (PD) response of a human MASP-2 monoclonal antibody (OMS646) as measured by the reduction in systemic lectin pathway activity in mice following intravenous administration, as described in Example 40;

[0124] Fig.55B Graph illustrating the pharmacodynamic (PD) response of a human MASP-2 monoclonal antibody (OMS646) as measured by the reduction in systemic lectin pathway activity in mice following subcutaneous administration, as described in Example 40;

[0125] Fig.56 Graph illustrating the inhibitory effect of MASP-2 antibody (OMS646) on aHUS serum-induced C5b-9 deposition on ADP-activated HMEC-1 cells compared to sCR1, as described in Example 41;

[0126] Fig.57 Graph illustrating the inhibitory effect of MASP-2 antibody (OMS646) on aHUS serum-induced thrombosis on ADP-activated HMEC-1 cells compared to sCR1, as described in Example 42;

[0127] Fig.58 Graph illustrating the mean change in platelet count from baseline over time (weeks) in subjects with persistent hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA) following treatment with a MASP-2 inhibitory antibody (OMS646), as described in Example 46;

[0128] Fig.59 Graph illustrating the mean change in LDH from baseline over time (weeks) in subjects with persistent (HSCT-TMA) following treatment with a MASP-2 inhibitory antibody (OMS646), as described in Example 46;

[0129] Fig.60 Graph illustrating the mean change from baseline in haptoglobin over time (weeks) in subjects with persistent (HSCT-TMA) following treatment with a MASP-2 inhibitory antibody (OMS646), as described in Example 46;

[0130] Fig.61Describes the clinical course of patients developing HSCT-TMA and graft-versus-host disease (GVHD) following hematopoietic stem cell transplantation (HSCT), and demonstrates improvement in HSCT-TMA, GVHD, and improvement in neurological symptoms following treatment with a MASP-2 inhibitory antibody (OMS646), as described in Example 47;

[0131] Fig.62A The graphic illustrates creatinine levels over time in compassionate use patient #1, wherein the vertical line represents the initiation of treatment with a MASP-2 inhibitory antibody (OMS646), as described in Example 48;

[0132] Fig.62B Graph illustrating haptoglobin levels in compassionate use patient #1 over time, wherein the vertical line represents the initiation of treatment with a MASP-2 inhibitory antibody (OMS646), as described in Example 48;

[0133] Fig.62C The graphic illustrates the levels of hemoglobin over time in compassionate use patient #1, wherein the vertical line represents the initiation of treatment with a MASP-2 inhibitory antibody (OMS646), as described in Example 48;

[0134] Fig.62D graphically illustrates the levels of LDH in compassionate use patient #1 over time, wherein the vertical line represents the initiation of treatment with a MASP-2 inhibitory antibody (OMS646), as described in Example 48; and

[0135] Fig.62E The graphic illustrates the levels of platelets in compassionate use patient #1 over time, with the vertical line representing the initiation of treatment with a MASP-2 inhibitory antibody (OMS646), as described in Example 48.

[0136] Sequence Listing Description

[0137] SEQ ID NO: 1 Human MAp19 cDNA

[0138] SEQ ID NO:2 Human MAp19 protein (with leader sequence)

[0139] SEQ ID NO:3 Human MAp19 protein (mature)

[0140] SEQ ID NO:4 human MASP-2 cDNA

[0141] SEQ ID NO: 5 Human MASP-2 protein (with leader sequence)

[0142] SEQ ID NO:6 Human MASP-2 protein (mature)

[0143] SEQ ID NO:7 Human MASP-2 gDNA (exons 1-6)

[0144] Antigen: (refer to MASP-2 mature protein)

[0145] SEQ ID NO:8 CUBI sequence (aa 1-121)

[0146] SEQ ID NO:9 CUBEGF sequence (aa 1-166)

[0147] SEQ ID NO:10CUBEGFCUII(aa 1-293)

[0148] SEQ ID NO:11 EGF region (aa 122-166)

[0149] SEQ ID NO:12 Serine protease domain (aa429–671)

[0150] SEQ ID NO: 13 Inactive serine protease domain (aa 610-625 with Ser618 to Ala mutation)

[0151] SEQ ID NO:14 TPLGPKWPEPVFGRL (CUB1 peptide)

[0152] SEQ ID NO:15

[0153] TAPPGYRLRLYFTHFDLELSHLCEYDFVKLSSGAKVLATLCGQ (CUBI peptide)

[0154] SEQ ID NO:16 TFRSDYSN (MBL binding region core)

[0155] SEQ ID NO:17 FYSLGSSLDITFRSDYSNEKPFTGF (MBL binding region)

[0156] SEQ ID NO:18 IDECQVAPG (EGF peptide)

[0157] SEQ ID NO: 19 ANMLCAGLESGGKDSCRGDSGGALV (serine protease binding core)

[0158] Peptide inhibitors:

[0159] SEQ ID NO:20 MBL full-length cDNA

[0160] SEQ ID NO:21 MBL full-length protein

[0161] SEQ ID NO:22 OGK-X-GP (common binding)

[0162] SEQ ID NO:23 OGKLG

[0163] SEQ ID NO:24 GLR GLQ GPO GKL GPO G

[0164] SEQ ID NO:25 GPO GPO GLR GLQ GPO GKL GPO GPO GPO

[0165] SEQ ID NO:26

[0166] GKDGRDGTKGEKGEPGQGLRGLQGPOGKLGPOG

[0167] SEQ ID NO:27

[0168] GAOGSOGEKGAOGPQGPOGPOGKMGPKGEOGDO (human h-ficolin)

[0169] SEQ ID NO:28

[0170] GCOGLOGAOGDKGEAGTNGKRGERGPOGPOGKAGPOGPNGAOGEO (human ficarin p35)

[0171] SEQ ID NO:29 LQRALEILPNRVTIKANRPFLVFI (C4 cleavage site) expression inhibitor:

[0172] SEQ ID NO:30 cDNA of CUBI-EGF domain (nucleotides 22-680 of SEQ ID NO:4)

[0173] SEQ ID NO:31

[0174] 5'CGGGCACACCATGAGGCTGCTGACCCTCCTGGGC 3' Nucleotides 12-45 of SEQ ID NO:4, including the MASP-2 translation start site (sense)

[0175] SEQ ID NO:32

[0176] 5'GACATTACCTTCCGCTCCGACTCCAACGAGAAG3' Nucleotides 361-396 of SEQ ID NO:4, encoding a region including the MASP-2 MBL binding site (sense)

[0177] SEQ ID NO:33

[0178] 5'AGCAGCCCTGAATACCCACGGCCGTATCCCAAA3' Nucleotides 610-642 of SEQ ID NO:4, encoding a region including the CUBII domain. Primer for cloning:

[0179] SEQ ID NO:34 CGGGATCCATGAGGCTGCTGACCCTC (5' PCR for CUB)

[0180] SEQ ID NO:35 GGAATTCCTAGGCTGCATA (for CUB3' PCR)

[0181] SEQ ID NO:36 GGAATTCCTACAGGGCGCT (3' PCR for CUBIEGF)

[0182] SEQ ID NO:37 GGAATTCCTAGTAGTGGAT (3' PCR for CUBIEGFCUBII)

[0183] SEQ ID NOS:38-47 are cloning primers for humanized antibodies

[0184] SEQ ID NO:48 is a 9aa peptide bond

[0185] Expression vector:

[0186] SEQ ID NO:49 is the MASP-2 minigene insert

[0187] SEQ ID NO:50 is mouse MASP-2 cDNA

[0188] SEQ ID NO:51 is mouse MASP-2 protein (including leader sequence)

[0189] SEQ ID NO:52 is the mature murine MASP-2 protein

[0190] SEQ ID NO:53 is rat MASP-2 cDNA

[0191] SEQ ID NO:54 is rat MASP-2 protein (including leader sequence)

[0192] SEQ ID NO:55 is the mature rat MASP-2 protein

[0193] SEQ ID NOs: 56-59 are oligonucleotides for site-directed mutagenesis of human MASP-2, which are used to generate human MASP-2A

[0194] SEQ ID NOs: 60-63 are oligonucleotides for site-directed mutagenesis of murine MASP-2, which were used to generate murine MASP-2A

[0195] SEQ ID NOs:64-65 are oligonucleotides for site-directed mutagenesis of rat MASP-2, which were used to generate rat MASP-2A

[0196] SEQ ID NO:66 DNA encoding the heavy chain variable region (VH) of 17D20_dc35VH21N11VL (OMS646) (without signal peptide)

[0197] SEQ ID NO:67 17D20_dc35VH21N11VL(OMS646) heavy chain variable region (VH) polypeptide

[0198] SEQ ID NO:68 17N16mc heavy chain variable region (VH) polypeptide

[0199] SEQ ID NO:69: DNA encoding the light chain variable region (VL) of 17D20_dc35VH21N11VL (OMS646)

[0200] SEQ ID NO:70:17D20_dc35VH21N11VL(OMS646) light chain variable region (VL) polypeptide

[0201] SEQ ID NO:71:17N16_dc17N9 light chain variable region (VL) polypeptide

[0202] Detailed Description

[0203] The present invention is based on the unexpected discovery of 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 cellular damage associated with lectin-mediated complement pathway activation while leaving components of the classical (C1q-dependent) pathway of the immune system intact.

[0204] I. Definitions

[0205] Unless explicitly defined herein, all terms used herein have the same meaning as understood by one of ordinary skill in the art of the present invention. To provide clarity regarding the terms as they are used in the specification and claims to describe the present invention, the following definitions are provided.

[0206] As used herein, the term "MASP-2-dependent complement activation" includes MASP-2-dependent activation of the lectin pathway, which occurs under physiological conditions (i.e., in the presence of Ca ++ ), leading to the formation of the lectin pathway C3 convertase C4b2a and upon accumulation of the C3 cleavage product C3b, the subsequent formation of the C5 convertase C4b2a(C3b)n, which has been determined to be primarily responsible for opsonization.

[0207] As used herein, the term "alternative pathway" refers to complement activation that is triggered, for example, by zymosan from the cell walls of fungi and yeast, lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria, and rabbit erythrocytes, as well as a number of pure polysaccharides, rabbit erythrocytes, viruses, bacteria, animal tumor cells, parasites, and damaged cells, and which is traditionally believed to arise from the spontaneous proteolysis of complement factor C3 to produce C3b.

[0208] As used herein, the term "lectin pathway" refers to complement activation that occurs through specific binding of serum and non-serum carbohydrate binding proteins, including mannan-binding lectin (MBL), CL-11, and ficolins (H-ficolin, M-ficolin, or L-ficolin).

[0209] As used herein, the term "classical pathway" refers to complement activation, which is triggered by antibodies bound to foreign particles and requires binding of the recognition molecule CIq.

[0210] As used herein, the term "MASP-2 inhibitory agent" refers to any agent that binds to or directly interacts with MASP-2 and is effective in inhibiting MASP-2-dependent complement activation, including anti-MASP-2 antibodies and MASP-2 binding fragments thereof, 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 for binding to another recognition molecule in the lectin pathway (e.g., MBL, H-ficolin, M-ficolin, or L-ficolin), but does not include antibodies that bind to such other recognition molecules. MASP-2 inhibitory agents useful in the methods of the invention can reduce MASP-2-dependent complement activation by more than 20%, such as more than 50%, such as more than 90%. In one embodiment, the MASP-2 inhibitory agent reduces MASP-2-dependent complement activation by more than 90% (i.e., results in only 10% or less MASP-2 complement activation).

[0211] As used herein, the term "antibody" includes antibodies and antibody fragments thereof derived from any mammal that produces antibodies (e.g., mouse, rat, rabbit, and primate, including human) or hybridoma, phage screening, recombinant expression, or transgenic animals (or other methods of producing antibodies or antibody fragments) that specifically bind to a target polypeptide, such as MASP-2, a polypeptide, or a portion thereof. The term "antibody" is not intended to be limited by the source of the antibody or the manner in which it is prepared (e.g., by hybridoma, phage screening, recombinant expression, transgenic animals, peptide synthesis, etc.). Exemplary antibodies include polyclonal, monoclonal, and recombinant antibodies; pan-specific, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies); humanized antibodies; murine antibodies; chimeric mouse-human, mouse-primate, primate-human monoclonal antibodies; and anti-idiotypic antibodies, and may be any intact antibody or fragment thereof. As used herein, the term "antibody" 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 thereof, naturally occurring variants, fusion proteins comprising an antibody portion of an antigen-binding fragment having the desired specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of an immunoglobulin molecule comprising an antigen-binding site or fragment (epitope recognition site) having the desired specificity.

[0212] "Monoclonal antibody" refers to a homogeneous antibody population, wherein the monoclonal antibody comprises amino acids (naturally occurring and non-naturally occurring) that participate in the selective binding of an epitope. Monoclonal antibodies have a high degree of specificity 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), its variants, fusion proteins including antigen-binding portions, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of immunoglobulin molecules including antigen-binding fragments (epitope recognition sites) with the desired specificity and ability to bind epitopes. It is not intended to limit the source of the antibody or the way it is prepared (e.g., by hybridoma, phage screening, recombinant expression, transgenic animals, etc.). The term includes intact immunoglobulins as well as the fragments and the like under the definition of "antibody" as described above.

[0213] As used herein, the term "antibody fragment" refers to a portion derived from or associated with a full-length antibody, such as an anti-MASP-2 antibody, generally including the antigen binding or variable regions thereof. Illustrative examples of antibody fragments include Fab, Fab', F(ab)2, F(ab')2 and Fv fragments, scFv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0214] As used herein, "single-chain Fv" or "scFv" antibody fragments include the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide also includes a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0215] As used herein, a "chimeric antibody" is a recombinant protein comprising the variable domains and complementarity determining regions of an antibody derived from a non-human species (eg, a rodent), while the remainder of the antibody molecule is derived from a human antibody.

[0216] As used herein, a "humanized antibody" is a chimeric antibody that includes minimal sequences corresponding to specific complementary determining regions derived from non-human immunoglobulins grafted into a human antibody framework. Humanized antibodies are typically recombinant proteins in which only the antibody complementary determining regions are of non-human origin.

[0217] As used herein, the term "mannan-binding lectin" ("MBL") is equivalent to mannan-binding protein ("MBP").

[0218] As used herein, "membrane attack complex" ("MAC") refers to a complex (also known as C5b-9) of the terminal five complement components (C5b in combination with C6, C7, C8, and C-9) that inserts into and disrupts membranes.

[0219] As used herein, "subject" includes all mammals, including, without limitation, humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs, and rodents.

[0220] As used herein, the amino acid residues are abbreviated 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).

[0221] In the broadest sense, naturally occurring amino acids can be divided into multiple groups according to the chemical properties of the side chains of the individual amino acids. "Hydrophobic" amino acids refer to Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys or Pro. "Hydrophilic" amino acids refer to Gly, Asn, Gln, Ser, Thr, Asp, Glu, Lys, Arg or His. This grouping of amino acids can be further subdivided as follows. "Uncharged hydrophilic" amino acids refer to Ser, Thr, Asn or Gln. "Acidic" amino acids refer to Glu or Asp. "Basic" amino acids refer to Lys, Arg or His.

[0222] As used herein, the term "conservative amino acid substitution" is described by substitutions among amino acids within 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.

[0223] As used herein, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or its mimetics. The term also encompasses those oligonucleoside bases comprising naturally occurring nucleotides, sugars, and covalent internucleoside (backbone) bonds as well as oligonucleotides with non-naturally occurring modifications.

[0224] 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 one (e.g., 2, 3, 4, 5, or 6) common amino acid residues and include linear and non-linear epitopes.

[0225] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably to refer to any peptide-linked amino acid chain, regardless of length or post-translational modification. The MASP-2 protein described herein may comprise or may be a wild-type protein, or may be a variant having no more than 50 (e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40 or 50) conservative amino acid substitutions. Conservative substitutions generally 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.

[0226] In some embodiments, the human MASP-2 protein can have an amino acid sequence that is equal to or greater 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.

[0227] In some embodiments, the peptide fragment can 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 in length (e.g., at least 6 consecutive amino acid residues of SEQ ID NO: 5). In some embodiments, the antigenic peptide fragments of the human MASP-2 protein are less than 500 (e.g., less than 450, 400, 350, 325, 300, 275, 250, 225, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 7, 8, 9, 10, 15, 14, 13, 12, 1 ...5

[0228] Percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to those in a reference sequence, after alignment of the sequences and introduction of gaps (if necessary) to achieve maximum percent sequence identity. For purposes of determining percent sequence identity, alignment can be achieved by the various methods of the art, for example using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Suitable parameters for measuring alignment, including any algorithm required for achieving maximum alignment over the full length of the sequence to be compared, can be determined by known methods.

[0229] II. Summary of the Invention

[0230] Lectins (MBL, M-ficolin, H-ficolin, L-ficolin, and CL-11) are specific recognition molecules that trigger the innate complement system, and the system includes a lectin initiation pathway and an associated terminal pathway amplification loop that amplifies lectin-triggered activation of terminal complement effector molecules. C1q is a specific recognition molecule that triggers the acquired complement system, and the system includes a classical initiation pathway and an associated terminal pathway amplification loop that amplifies C1q-triggered activation of terminal complement effector molecules. We call these two major complement activation systems the lectin-dependent complement system and the C1q-dependent complement system, respectively.

[0231] In addition to its fundamental role in immune defense, the complement system also contributes to tissue damage in many clinical situations. Therefore, there is an urgent need to develop therapeutically effective complement inhibitors to prevent these adverse effects. With the realization that it is possible to inhibit the lectin-mediated MASP-2 pathway while leaving the classical pathway intact, it is particularly desirable to recognize that it is possible to specifically inhibit only the complement activation system that causes a specific pathology without completely shutting down the immune defense capabilities of complement. For example, in disease states where complement activation is primarily mediated by the lectin-dependent complement system, it would be advantageous to specifically inhibit only this system. This would keep the C1q-dependent complement activation system intact to handle immune complex processing and aid in host defense against infection.

[0232] A preferred targeted protein component in the development of therapeutic agents that specifically inhibit the lectin-dependent complement system is MASP-2. Of all the 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), only MASP-2 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 unique components of the lectin-dependent complement system. However, the loss of any one of the lectin components will not necessarily inhibit activation of the system due to lectin redundancy. It would be necessary to inhibit all five lectins to ensure inhibition of the lectin-dependent complement activation system. Furthermore, because MBL and ficolins are also known to have opsonic activity that is independent of complement, inhibition of lectin function would result in loss of this beneficial host defense mechanism against infection. In contrast, if MASP-2 were the inhibitory target, the complement-independent lectin opsonic activity would remain intact. An additional benefit of MASP-2 as a therapeutic target for inhibition of the lectin-dependent complement activation system is that the plasma concentration of MASP-2 is the lowest of any complement protein (approximately 500 ng / ml); therefore, relatively low concentrations of high-affinity inhibitors of MASP-2 may be sufficient to obtain complete inhibition (Moller-Kristensen, M. et al., J. Immunol Methods 282: 159-167, 2003).

[0233] III. Role of MASP-2 in thrombotic microangiopathy and therapeutic approaches using MASP-2 inhibitors

[0234] Overview

[0235] Thrombotic microangiopathy (TMA) is a pathology characterized by blood clots in small blood vessels (Benz, K. et al., Curr Opin Nephrol Hypertens 19(3):242–7 (2010)). Stress or damage to the underlying vascular endothelium is believed to be the primary driver. Clinical and laboratory findings of TMA include thrombocytopenia, anemia, purpura, and renal failure. Classic TMAs are hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). The characteristic underlying pathological features of TMA are platelet activation and the formation of small thrombi in small arteries and venules. Other TMAs are also implicated in complement activation, at least in part, triggered by damage or stress to the microvascular endothelium, including catastrophic antiphospholipid syndrome (CAPS), systemic Degos' disease, and TMA secondary to cancer, cancer chemotherapy, and transplantation.

[0236] Direct evidence for the pathological role of complement in nephritic hosts is provided by studies of patients with genetic defects in specific complement components. Many reports have demonstrated that renal damage is associated with defects in complement regulatory factor H (Ault, BH, Nephrol. 14: 1045-1053, 2000; Levy, M. et al., Kidney Int. 30: 949-56, 1986; Pickering, MC et al., Nat. Genet. 31: 424-8, 2002). Factor H deficiency leads to low plasma levels of factor B and C3, which is due to activation-related consumption of these components. The circulating level of C5b-9 is also increased in the serum of these patients, indicating complement activation. Membrane proliferative glomerulonephritis (MPGN) and idiopathic hemolytic uremic syndrome (HUS) are associated with factor H deficiency or factor H mutation. Factor H-deficient pigs (Jansen, JH et al., Kidney Int. 53:331-49, 1998) and factor-H knockout mice (Pickering, MC, 2002) show MPGN-like symptoms, confirming the importance of factor H in complement regulation. Defects in other complement components are associated with the development of systemic lupus erythematosus (SLE) and subsequent renal disease (Walport, MJ, Davies et al., Ann. NY Acad. Sci. 815:267-81, 1997). Defects in C1q, C4, and C2 strongly contribute to the development of SLE through mechanisms involving incomplete clearance of immune complexes and apoptotic material. Lupus nephritis occurs in many of these SLE patients, characterized by the deposition of immune complexes throughout the glomeruli.

[0237] AHr

[0238] Atypical hemolytic uremic syndrome (aHUS) is part of a group of conditions known as "thrombotic microangiopathy". In atypical forms of HUS (aHUS), the disease is associated with incomplete complement regulation, and can be sporadic or familial. Familial cases of aHUS 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, PF et al., PloS Genetics 3(3):e41 (2007)). The unifying feature of this diverse set of gene mutations associated with aHUS is an increased propensity for complement activation on the surface of cells or tissues. Thus, one aspect of the invention includes treating a patient suffering from aHUS associated with factor H deficiency by administering an effective amount of a MASP-2 inhibitory agent. Another aspect of the invention involves treating a patient suffering from HUS associated with a deficiency in Factor I, Factor B, membrane cofactor CD46, CFHR1 or CFHR3 by administering an effective amount of a MASP-2 inhibitory agent.

[0239] There has been significant progress in the understanding of the molecular pathophysiology that underlies the enhanced complement activation in aHUS caused by different groups of mutant complement factors. This mechanism is best understood for factor H mutations. Factor H is an abundant serum protein that includes 20 short consensus repeat (SCR) domains that act as a negative regulator of complement activation in solution and on the surface of host cells. It targets the activated form of C3 and, together with factor I and other cofactors, promotes its inactivation, preventing further complement activation. To effectively control complement activation on the host cell surface, factor H needs to interact with the host cell, which is mediated by the SCR domains 16-20. All factor H mutations associated with aHUS described to date are clustered in the C-terminal region that includes the (SCR) domains 16-20. These mutated factor H proteins are fully functional in controlling C3 activation in solution, but are unable to interact with the host cell surface and therefore cannot control C3 activation on the cell surface (Exp Med 204(6):1249-56(2007)). Thus, certain mutations in factor H are associated with aHUS because the mutated factor H protein is unable to interact with the host cell surface and, therefore, is unable to effectively downregulate complement activation on the host cell surface, including the microvascular endothelium. As a result, once initial C3 activation occurs, subsequent complement activation on the microvascular endothelial surface proceeds and is not attenuated in patients with factor H mutations. This uncontrolled complement activation ultimately leads to progressive damage to the vascular endothelium, subsequent platelet aggregation and microvascular coagulation, and hemolysis caused by complete stress of RBCs through partially occluded microvessels. Thus, aHUS disease manifestations and clinical and laboratory findings are directly related to defects in negative complement regulation on the microvascular endothelial surface.

[0240] Similar to factor H mutations, loss-of-function mutations in the negative complement regulator factor I and the membrane cofactor protein (CD46) have also been associated with aHUS. Conversely, there have been observations for factor B, with the C3 protein being found to be associated with gain-of-function mutations in these proteins in aHUS (Pediatr Nephrol 25(12):2431-42(2010)). Thus, much of the converging data implicate complement activation in the pathogenesis of aHUS. This concept 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.

[0241] Although the central role of complement as an effector mechanism in aHUS is widely recognized, the triggers for initiating complement activation and the molecular pathways involved have not been determined. Not all individuals with the above mutations develop aHUS. In fact, familial studies have shown that the penetrance of aHUS is only about 50% (Ann Hum Genet 74(1):17-26 (2010)). The natural history of the disease suggests that aHUS most often develops following an initiating event, such as an infectious episode or injury. Infectious agents that activate the complement system are well known. In the absence of pre-existing adaptive immunity, complement activation by infectious agents may be initiated primarily through the lectin pathway. Therefore, infection-triggered lectin pathway activation may represent an initiating trigger for the subsequent pathological amplification of complement activation in aHUS-susceptible individuals, which may ultimately lead to disease progression. Therefore, another aspect of the invention includes treating a patient suffering from aHUS secondary to infection by administering an effective amount of a MASP-2 inhibitory agent.

[0242] Other forms of host tissue damage will activate complement via the lectin pathway, particularly damage to the vascular endothelium. Human vascular endothelial cells, which are susceptible to oxidative stress, respond, for example, by expressing surface moieties of the lectin pathway that bind lectins and activate complement (Am J. Pathol 156(6):1549-56(2000)). Vascular damage following ischemia / reperfusion also activates complement via the lectin pathway in vivo (Scand J Immunol 61(5):426-34(2005)). Activation of the lectin pathway in this setting has pathological consequences for the host, and inhibiting the lectin pathway by blocking MASP-2 prevents further host tissue damage and adverse outcomes (Schwaeble PNAS 2011).

[0243] Thus, other processes that contribute to aHUS are also known to activate the lectin pathway of complement. Thus, it is likely that the lectin pathway may represent an initiating complement activation mechanism that is inappropriately amplified in a deregulated manner in individuals genetically susceptible to aHUS, thereby initiating aHUS pathogenesis. By inference, agents that block complement activation through the lectin pathway, including anti-MASP-2 antibodies, are expected to prevent disease progression or reduce exacerbations in individuals susceptible to aHUS.

[0244] In further support of this concept, recent studies have identified S. pneumoniae as an important pathogen in pediatric aHUS cases (Nephrology (Carlton), 17:48-52 (2012); Pediatr Infect Dis J. 30(9):736-9 (2011)). This particular etiology has been shown to have a poor prognosis, with significant mortality and long-term morbidity. In particular, these cases involve non-intestinal infections that result in manifestations of microangiopathy, uremia, and hemolysis, without evidence of concurrent complement gene mutations known to be causative of aHUS. It is important to note that S. pneumoniae is particularly efficient in activating complement, and it does so primarily through the lectin pathway. Therefore, in cases of non-intestinal HUS associated with pneumococcal infection, manifestations of microangiopathy, uremia, and hemolysis are expected to be driven primarily through activation of the lectin pathway, and agents that block the lectin pathway, including anti-MASP-2 antibodies, are expected to prevent the progression of aHUS or reduce disease severity in these patients. Thus, another aspect of the invention involves treating a patient suffering from non-intestinal aHUS associated with S. pneumoniae infection by administering an effective amount of a MASP-2 inhibitory agent.

[0245] In accordance with the foregoing, in some embodiments, in the context of a subject at risk for developing renal failure associated with aHUS, methods are provided for reducing the likelihood of developing aHUS or developing renal failure associated with aHUS, comprising administering an amount of a MASP-2 inhibitory agent for a period of time effective to improve or prevent renal failure in the subject. In some embodiments, the methods further comprise the step of determining whether the subject is at risk for developing aHUS prior to the onset of any symptoms associated with aHUS. In other embodiments, the methods comprise determining whether the subject is at risk for developing aHUS upon the onset of at least one or more symptoms indicative of aHUS (e.g., the presence of anemia, thrombocytopenia, and / or renal insufficiency) and / or the presence of thrombotic microangiopathy in a biopsy obtained from the subject. Determining whether a subject is at risk for developing aHUS includes determining whether the subject has a genetic predisposition to developing aHUS, which can be performed by genomic sequencing or gene-specific analysis (e.g., PCR analysis), evaluating genetic information (e.g., from a database containing the subject's genotype), or performing at least one genetic screening test on the subject to determine the presence or absence of genetic markers associated with aHUS (i.e., determining the presence or absence of genetic mutations associated with aHUS in genes encoding complement factor H (CFH), factor I (CFI), factor B (CFB), membrane cofactors 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 determining whether the subject has a family history of aHUS. Methods for genetic screening for the presence or absence of gene mutations associated with aHUS are well established; see, for example, Noris M et al., “Atypical Hemolytic-Uremic Syndrome,” Nov 16, 2007 [updated Mar 10, 2011], in: Pagon RA, Bird TD, Dolan CR et al., eds., Gene Reviews TM ,Seattle (WA): University of Washington, Seattle.

[0246] For example, overall, the penetrance of the disease in those individuals with mutations in complement factor H (CFH) is 48%, and the penetrance for CD46 mutations is 53%, for mutations in CFI is 50%, for mutations in C3 is 56%, and for mutations in THBD 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 in Caprioli et al., (2006), supra, a large number of individuals with mutations in complement factor H (CFH) never develop aHUS, and it is assumed that in these individuals, suboptimal CFH activity is sufficient to protect the host from the effects of complement activation under physiological conditions, however, when exposure to complement-activating agents produces higher than normal amounts of C3b, suboptimal CFH activity is insufficient to prevent C3b deposition on vascular endothelial cells.

[0247] Thus, in one embodiment, a method is provided for inhibiting MASP-2-dependent complement activation in a subject having or at risk for developing non-factor H-dependent atypical hemolytic uremic syndrome, comprising administering to the subject a composition comprising a MASP-2 inhibitory agent in an amount effective to inhibit MASP-2-dependent complement activation. In another embodiment, a method is provided for inhibiting MASP-2-dependent complement activation in a subject at risk for developing factor H-dependent atypical hemolytic uremic syndrome, comprising periodically monitoring the subject to determine the presence or absence of anemia, thrombocytopenia, or elevated creatinine, and treating with a MASP-2 inhibitory agent when the presence of anemia, thrombocytopenia, or elevated creatinine is determined. In another embodiment, a method is provided for reducing the likelihood that a subject at risk for developing factor H-dependent aHUS will suffer clinical symptoms associated with aHUS, comprising administering a MASP-2 inhibitory agent before, during, or after an event known to be associated with triggering clinical symptoms of aHUS (e.g., drug exposure (e.g., chemotherapy), infection (e.g., bacterial infection), malignancy, injury, organ or tissue transplant, or pregnancy).

[0248] In one embodiment, methods are provided for reducing the likelihood that a subject at risk for developing aHUS will experience clinical symptoms associated with aHUS, comprising periodically monitoring the subject to determine the presence or absence of anemia, thrombocytopenia, or elevated creatinine, and treating with a MASP-2 inhibitory agent when the presence of anemia, thrombocytopenia, or elevated creatinine is determined.

[0249] In another embodiment, methods are provided for reducing the likelihood that a subject at risk for developing aHUS will experience clinical symptoms associated with aHUS, comprising administering a MASP-2 inhibitory agent before, during, or after an event known to be associated with triggering clinical symptoms of aHUS (e.g., drug exposure (e.g., chemotherapy), infection (e.g., bacterial infection), malignancy, injury, organ or tissue transplant, or pregnancy).

[0250] In some embodiments, the MASP-2 inhibitory agent is administered for a period of at least 1 day, 2 days, 3 days, 4 days or longer, and may be repeated as determined by the physician until the condition is resolved or controlled, before, during, or after an event associated with triggering clinical symptoms of aHUS. In the pre-aHUS setting, the MASP-2 inhibitory agent may be administered to the subject systemically, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous or other parenteral administration.

[0251] In some embodiments, in the setting of an initial diagnosis of aHUS or in a subject displaying 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 inhibitory agent (e.g., an anti-MASP-2 antibody) as a first line therapy in the absence of plasmapheresis or in combination with plasmapheresis. As a first line therapy, the MASP-2 inhibitory agent may be administered to the subject systemically, e.g., by intra-arterial, intravenous, intramuscular, inhaled, nasal, subcutaneous or other parenteral administration. In some embodiments, the MASP-2 inhibitory agent is administered to a subject as a first line therapy in the absence of plasmapheresis to avoid potential plasmapheresis complications including bleeding, infection, and exposure to conditions inherent to plasma donors and / or allergic reactions, or in a subject who is otherwise unwilling to undergo plasmapheresis, or in a setting where plasmapheresis is not available.

[0252] In some embodiments, the methods comprise administering a MASP-2 inhibitory agent to a subject with aHUS via a catheter (e.g., intravenously) for a first period of time (e.g., at least 1 day to 1 or 2 weeks), followed by administering the MASP-2 inhibitory agent subcutaneously to the subject for a second period of time (e.g., at least 2 weeks or longer in the chronic phase). In some embodiments, the administering during the first and / or second period of time occurs in the absence of plasmapheresis. In some embodiments, the methods further comprise determining the level of at least 1 complement factor (e.g., C3, C5) in the subject prior to treatment and optionally during treatment, wherein determining a decrease in the level of at least 1 complement factor compared to a standard value or healthy control subjects indicates a need for continued treatment with the MASP-2 inhibitory agent.

[0253] In some embodiments, the methods comprise administering a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, to a subject having or at risk for developing aHUS intravenously, intramuscularly, or preferably subcutaneously. Treatment may be long-term, and administered daily to monthly, but preferably every two weeks. The anti-MASP-2 antibody may be administered alone or in combination with a C5 inhibitor, such as eculizumab.

[0254] HUS

[0255] Like atypical HUS, the classic form of HUS shows all clinical and laboratory findings of TMA. However, classic HUS is often a pediatric disease, usually without familial elements or direct association with complement gene mutations. The etiology of classic HUS is closely related to infection with certain enteric pathogens. Patients usually present with acute renal failure, hemoglobinuria and thrombocytopenia, usually followed by bloody diarrhea. The condition is caused by intestinal infection with Shigella dissenteriae, Salmonella, or Shiga toxin-producing enterohemorrhagic Escherichia coli strains such as Escherichia coli O157:H7. The pathogen is obtained from contaminated food or water supply. HUS is a medical emergency with a mortality rate of 5-10%. A considerable portion of survivors develop chronic kidney disease (Corrigan and Boineau, Pediatr Rev 22 (11): 365–9 (2011)) and may require a kidney transplant.

[0256] In typical HUS, microvascular coagulation mainly but not exclusively occurs in the renal microvascular system. Basic pathophysiology is mediated by Shiga toxin (STX). Secreted into the intestinal cavity by enteropathic microorganisms, STX passes through the intestinal barrier, enters the bloodstream and is bound to vascular endothelial cells (Boyd and Lingwood Nephron 51:207 (1989)) by blobotriaosylceramide receptor CD77, and the receptor is preferably expressed on glomerular endothelium and mediates the toxic effects of STX. Once bound to the endothelium, STX induces a series of events that damage the vascular endothelium, activates leukocytes and causes 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. 181: 1460-1469 (2008); Morigi et al., Blood 98: 1828-1835 (2001); Guessou et al., Infect. Immun., 73: 8306-8316 (2005)). These small thrombi block or occlude the arterioles and capillaries of the kidneys and other organs. The blockage of blood flow in the arterioles and capillaries by small thrombi increases the shear force applied to the RBCs because they squeeze through the narrow blood vessels. This can lead to the destruction of RBCs by shear forces and the formation of RBC fragments called schistocytes. The presence of schistocytes is a characteristic finding of HUS. This mechanism is known as microangiopathic hemolysis. In addition, the obstruction of blood flow leads to ischemia, triggering a complement-mediated inflammatory response, which causes additional damage to the affected organs.

[0257] The lectin pathway of complement contributes to the pathogenesis of HUS through two principle mechanisms: 1) direct MASP-2-mediated activation of the coagulation cascade resulting from endothelial injury; and 2) subsequent lectin-mediated complement activation resulting from ischemia caused by an initial obstruction of microvascular blood flow.

[0258] STX damages microvascular endothelial cells, and damaged endothelial cells are known to activate the complement system. As described in detail above, complement activation after endothelial cell injury is mainly driven by the lectin pathway. Human vascular endothelial cells susceptible to oxidative stress respond by expressing surface parts of the lectin pathway that bind lectins and activate complement (Collard et al., Am J Pathol. 156 (5): 1549-56 (2000)). Vascular injury after ischemia-reperfusion also activates complement through the lectin pathway in vivo (Scand J Immunol 61 (5): 426-34 (2005)). In this context, lectin pathway activation has pathological consequences for the host, and inhibiting the lectin pathway by blocking MASP-2 prevents further host tissue damage and adverse outcomes (Schwaeble et al., PNAS (2011)). In addition to complement activation, lectin-dependent activation of MASP-2 has been shown to cause prothrombin cleavage to form thrombin and promote coagulation. Therefore, the lectin pathway of complement activation by damaged endothelial cells can directly activate the coagulation system. Therefore, based on MASP-2-mediated prothrombin activation, the lectin pathway of complement may be the main molecular pathway connecting the initial endothelial damage caused by STX to the coagulation and microvascular thrombosis that occurs in HUS. It is therefore expected that lectin pathway inhibitors, including but not limited to antibodies that block MASP-2 function, will prevent or reduce microvascular coagulation, thrombosis and hemolysis in patients with HUS. In fact, administration of anti-MASP-2 antibodies significantly protected mice in a model of typical HUS. As described in Example 36 and Fig.45 As shown, all control mice exposed to STX and LPS developed severe HUS and became moribund or died within 48 hours. Fig.45 As further shown in Figure 1, all mice treated with anti-MASP-2 antibodies and then exposed to STX and LPS survived (Fisher's exact test p<0.01; N=5). Thus, anti-MASP-2 therapy significantly protected mice in this HUS model. It is expected that administration of MASP-2 inhibitory agents, such as MASP-2 antibodies, will effectively treat HUS patients and provide protection against microvascular coagulation, thrombosis, and hemolysis caused by infection with enteropathogenic E. coli or other STX-producing pathogens.

[0259] Although shown here for STX-induced HUS, it is expected that anti-MASP-2 therapy will also be beneficial for HUS-like syndromes due to endothelial damage caused by other toxic agents. This includes agents such as mitomycin, ticlopidine, cycplatin, quinine, cyclosporine, bleomycin, and other chemotherapeutic and immunosuppressive drugs. Therefore, it is expected that anti-MASP-2 antibody therapy or other modalities that inhibit MASP-2 activity will be effective in preventing or limiting coagulation, thrombosis, and RBC destruction, and preventing renal failure in HUS and other TMA-related diseases (i.e., aHUS and TTP).

[0260] Patients with HUS often present with diarrhea and vomiting, and they often have a decreased platelet count (thrombocytopenia) and a decrease in RBCs (anemia). The pre-HUS diarrheal phase usually lasts about 4 days, during which subjects at risk for developing HUS often display one or more of the following symptoms in addition to severe diarrhea: hematocrit levels less than 30% (smears show destruction of intravascular red blood cells), thrombocytopenia (platelet count <150 x 10 3 / mm 3 ) and / or the presence of impaired renal function (serum creatinine concentration greater than the upper limit of the age reference range). The presence of oligoanuria (urine excretion ≤ 0.5 mL / kg / h for > 1 day) can be used as a measure of progression toward the development of HUS (see C. Hickey et al., Arch Pediatr Adolesc Med 165(10):884-889(2011)). Testing is usually performed for the presence of Escherichia coli bacteria (E. coli O157:H7) or Shigella or Salmonella species infection. In subjects who test positive for enterogenic E. coli (e.g., E. coli 0157:H7) infection, antibiotic use is contraindicated because antibiotic use can increase the risk of developing HUS through increased STX production (see Wong C. et al., N Engl J. Med 342: 1930-1936 (2000). For subjects who test positive for Shigella or Salmonella, antibiotics are usually given to clear the infection. Other well-established first-line therapies for HUS include volume expansion, dialysis, and plasmapheresis.

[0261] In accordance with the foregoing, in some embodiments, in subjects who have one or more symptoms associated with the pre-HUS stage and are at risk for developing HUS (i.e., the subject exhibits one or more of the following symptoms: diarrhea, hematocrit level less than 30% (smear showing destruction of intravascular red blood cells), thrombocytopenia (platelet count less than 150 x 10 3 / mm 3In the setting of pro-HUS, a MASP-2 inhibitory agent is provided for reducing the risk of developing HUS or reducing the likelihood of renal failure in said subject, comprising administering an amount of a MASP-2 inhibitory agent for a period of time effective to improve or prevent the impairment of renal function. In some embodiments, the MASP-2 inhibitory agent is administered for a period of time of at least 1, 2, 3, 4 or more days, and may be repeated as determined by the physician until the condition has resolved or is controlled. In the setting of pro-HUS, the MASP-2 inhibitory agent may be administered to the subject systemically, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, oral, subcutaneous or other parenteral administration.

[0262] Treatment of E. coli 0157:H7 infection with bactericidal antibiotics, particularly beta-lactams, is 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 context of a subject with symptoms associated with a pre-HUS phase, wherein the subject is known to have an enterogenic E. coli infection (e.g., E. coli 0157:H7) for which antibiotic use is contraindicated, methods are provided for reducing the risk of developing HUS or reducing the likelihood of renal failure in the subject, comprising administering an amount of a MASP-2 inhibitory agent for a first period of time that is effective to inhibit or prevent the presence of oligoanuria in the subject (e.g., for at least 1, 2, 3, or 4 days), wherein administration of the MASP-2 inhibitory agent during the first period of time occurs in the absence of an antibiotic. In some embodiments, the method further comprises administering the MASP-2 inhibitory agent in combination with an antibiotic to the subject for a second period of time (e.g., at least 1-2 weeks).

[0263] In other embodiments, in the context of a subject suffering from symptoms associated with the pre-HUS stage, wherein the subject is known to have a Shigella or Salmonella infection, methods are provided for reducing the risk of developing HUS or reducing the likelihood of renal failure in said subject, comprising administering an amount of a MASP-2 inhibitory agent for a period of time effective to inhibit or prevent the presence of oligoanuria in said subject, wherein administration of the MASP-2 inhibitory agent is performed in the presence or absence of an appropriate antibiotic.

[0264] In some embodiments, in the setting of an initial diagnosis of HUS or in a subject displaying one or more symptoms consistent with a diagnosis of HUS (e.g., the presence of renal failure, or microangiopathic hemolytic anemia in the absence of low fibrinogen, or thrombocytopenia), the subject is treated with an effective amount of a MASP-2 inhibitory agent (e.g., an anti-MASP-2 antibody) as a first line therapy in the absence of plasmapheresis or in combination with plasmapheresis. As a first line therapy, the MASP-2 inhibitory agent may be administered systemically to the subject, e.g., by intra-arterial, intravenous, intramuscular, inhaled, nasal, subcutaneous or other parenteral administration. In some embodiments, the MASP-2 inhibitory agent is administered to a subject as a first line therapy in the absence of plasmapheresis to avoid complications of plasmapheresis, such as bleeding, infection, and exposure to conditions inherent in plasma donors and / or allergic reactions, or in a subject who is otherwise unwilling to undergo plasmapheresis, or in a setting where plasmapheresis is not available.

[0265] In some embodiments, the methods comprise administering a MASP-2 inhibitory agent to a subject with HUS via a catheter (e.g., intravenously) for a first period of time (e.g., an acute phase lasting at least 1 day to 1 or 2 weeks), followed by subcutaneous administration of the MASP-2 inhibitory agent to the subject for a second period of time (e.g., a chronic phase of at least 2 weeks or longer). In some embodiments, the administration during the first and / or second period of time occurs in the absence of plasmapheresis. In some embodiments, the methods further comprise determining the level of at least one complement factor (e.g., C3, C5) in the subject prior to treatment and optionally during treatment, wherein a determination of a reduced level of the at least one complement factor compared to a standard value or healthy control subjects indicates a need for treatment, and wherein a determination of a normal level indicates improvement.

[0266] In some embodiments, the methods comprise administering a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, subcutaneously or intravenously to a subject having or at risk for developing HUS. Treatment is preferably daily, but may be as frequent as weekly or monthly. Treatment will continue for at least 1 week and up to 3 months. The anti-MASP-2 antibody may be administered alone or in combination with a C5 inhibitor, such as eculizumab.

[0267] TTP

[0268] Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder of the blood coagulation system caused by autoimmune or inherited dysfunction that activates the coagulation cascade or the complement system (George, JN, N Engl J Med; 354: 1927-35 (2006)). This results in numerous microscopic blood clots or thrombi in small blood vessels throughout the body. Red blood cells are subjected to shear stress, which damages their membranes, leading to intravascular hemolysis. The resulting reduction in blood flow and endothelial damage leads to organ damage, including the brain, heart, and kidneys. The clinical features of TTP are thrombocytopenia, microangiopathic hemolytic anemia, neurological changes, renal failure, and fever. In the period before plasma exchange, the mortality rate during acute episodes was 90%. Even after plasma exchange, the survival rate at 6 months is about 80%.

[0269] TTP can be produced by hereditary or acquired inhibition of the enzyme ADAMTS-13, a metalloproteinase responsible for cleaving large multimers of von Willebrand factor (vWF) into smaller units. ADAMTS-13 inhibition or deficiency ultimately leads to increased coagulation (Tsai, HJ Am Soc Nephrol 14: 1072–1081, (2003)). ADAMTS-13 regulates the activity of vWF; in its absence, vWF forms large multimers that are more likely to bind platelets and predispose patients to platelet aggregation and thrombosis in microvessels.

[0270] Upshaw-Schulman syndrome (USS, also known as congenital TTP) is a congenital defect in ADAMTS13 activity caused by mutations in the ADAMTS13 gene (Schulman et al., Blood, 16(1):943-57, 1960; Upshaw et al., New Engl. J. Med, 298(24):1350-2, 1978). Many mutations in ADAMTS13 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 usually have 5-10% of normal ADAMTS13 activity (Kokame et al., PNAS 99 (18): 11902-11907, 2002). Although acquired TTP and USS have certain similarities, USS has some important differences in clinical features. USS usually appears in infants or children, characterized by severe hyperbilirubinemia, negative Coombs test shortly after birth, response to fresh plasma transfusion, and frequent relapses (Savasan et al., Blood, 101: 4449-4451, 2003). In some cases, patients with this inherited ADAMTS13 defect have a mild phenotype at birth and only develop symptoms associated with TTP under clinical conditions (such as infection or pregnancy) with increased von Willebrand factor levels. For example, Fujimura et al. reported 9 Japanese women from 6 families with genetically confirmed USS who were diagnosed with the disease during their first pregnancy. Thrombocytopenia occurred during the second to third trimester in each of their 15 pregnancies, often followed by TTP. All of these women were found to have severe deficiency of ADAMTS13 activity (Fujimura et al., Brit. J. Haemat 144:742-754, 2008).

[0271] In accordance with the foregoing, in some embodiments, in the context of a subject with 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), methods are provided for reducing the likelihood of developing clinical symptoms associated with congenital TTP (e.g., thrombocytopenia, anemia, fever, and / or renal failure), comprising administering an amount of a MASP-2 inhibitory agent (e.g., a MASP-2 antibody) for a period of time effective to improve or prevent one or more clinical symptoms associated with TTP. In some embodiments, the method further comprises the step of determining whether the subject is at risk for developing symptoms associated with congenital TTP prior to the onset of any symptoms associated with TTP or upon the onset of at least one or more symptoms indicative of TTP (e.g., the presence of anemia, thrombocytopenia, and / or renal insufficiency). Determining whether a subject is at risk for developing symptoms associated with congenital TTP (i.e., the subject has USS) includes 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).

[0272] In one embodiment, methods are provided for reducing the likelihood that a subject diagnosed with USS will suffer clinical symptoms associated with TTP, comprising periodically monitoring the subject to determine the presence or absence of anemia, thrombocytopenia, or elevated creatinine, and treating with a MASP-2 inhibitory agent (e.g., a MASP-2 antibody) when the presence of anemia, thrombocytopenia, or elevated creatinine is determined or in the presence of an event known to be associated with triggering clinical symptoms of TTP (e.g., drug exposure (e.g., chemotherapy), infection (e.g., bacterial infection), malignancy, injury, transplantation, or pregnancy).

[0273] In another embodiment, a method for treating a subject having USS and suffering from clinical symptoms associated with TTP is provided, comprising administering an amount of a MASP-2 inhibitory agent (e.g., a MASP-2 antibody) for a time and amount effective to improve or prevent one or more clinical symptoms associated with TTP.

[0274] TTP may also occur due to autoantibodies to ADAMTS-13. In addition, TTP may occur during breast cancer, gastrointestinal cancer, or prostate cancer (George JN., Oncology (Williston Park). 25: 908-14 (2011)), pregnancy (second trimester or postpartum) (George JN., Curr Opin Hematol 10: 339-344 (2003)), or in association with diseases such as HIV or autoimmune diseases such as systemic lupus erythematosus (Hamasaki K, et al., Clin Rheumatol. 22: 355-8 (2003)). TTP can also be caused by certain drug therapies, including heparin, quinine, immune-mediated components, cancer chemotherapeutic agents (bleomycin, cisplatin, cytarabine, daunorubicin, gemcitabine, mitomycin C, and tamoxifen), cyclosporine A, oral contraceptives, penicillin, rifampicin, and anti-platelet 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)). TTP due to transient functional ADAMTS-13 deficiency can occur due to endothelial cell damage associated with S. pneumoniae infection (Pediatr Nephrol., 26:631-5 (2011)).

[0275] Plasma exchange is the standard treatment for TTP (Rock GA, et al., N Engl J Med 325:393-397 (1991)). Plasma exchange replaces ADAMTS-13 activity in patients with genetic defects and removes ADAMTS-13 autoantibodies in those patients with acquired autoimmune TTP (Tsai, HM, Hematol Oncol Clin North Am., 21 (4): 609-v (2007)). Other 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 unsuccessful for approximately 20% of patients, relapses occur in more than one-third of patients, and plasmapheresis has cost and technical requirements. In addition, many patients cannot tolerate plasma exchange. Therefore, there is still an urgent need for additional and better TTP treatments.

[0276] Because TTP is a disease of the blood coagulation cascade, treatment with antagonists of the complement system can help stabilize and correct the disease. Although the pathological activation of the alternative complement pathway is related to aHUS, the role of complement activation in TTP is not very clear. The functional deficiency of ADAMTS13 is important for the susceptibility to TTP, but it is not enough to cause acute attacks. Environmental factors and / or other genetic changes can promote the performance of TTP. For example, genes encoding proteins involved in regulating the coagulation cascade, vWF, platelet function, components of the vascular endothelial surface, or the complement system can be involved in the occurrence of acute thrombotic microangiopathy (Galbusera, M. et al., Haematologica, 94: 166–170 (2009)). Specifically, complement activation has been shown to play a key 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 abolished by complement inactivation (Ruiz-Torres MP, et al., Thromb Haemost, 93: 443-52 (2005)). In addition, it has recently been shown that increased levels of C4d, C3bBbP, and C3a are present during acute attacks of TTP (M. Réti et al., J Thromb Haemost. Feb 28. (2012) doi: 10.1111 / j.1538-7836.2012.04674.x. [Epub ahead of print]), consistent with the activation of the classical / lectin and alternative pathways. This increased amount of complement activation in acute attacks can cause terminal pathway activation and lead to further aggravation of TTP.

[0277] The role of ADAMTS-13 and vWF in TTP is obviously platelet activation and aggregation, and the reason for their subsequent role in the deposition of shear stress and microangiopathy. Activated platelets interact with the classical and alternative pathways of complement and trigger them. Platelet-mediated complement activation increases inflammatory mediators C3a and C5a (Peerschke E et al., Mol Immunol, 47: 2170-5 (2010)). Therefore, platelets can be used as targets for classical complement activation in hereditary or autoimmune TTP.

[0278] As described above, the lectin pathway of complement is the major molecular pathway linking endothelial injury to coagulation and microvascular thrombosis that occurs in HUS, based on MASP-2-mediated prothrombin activation. Similarly, activation of the lectin pathway of complement can directly drive the coagulation system in TTP. Lectin pathway activation can be initiated in response to the initial endothelial injury caused by ADAMTS-13 deficiency in TTP. It is therefore expected that lectin pathway inhibitors, including but not limited to antibodies that block MASP-2 function, will alleviate microangiopathy associated with microvascular coagulation, thrombosis, and hemolysis in patients with TTP.

[0279] Patients with TTP usually present in the emergency room with one or more of the following symptoms: purpura, renal failure, low platelets, anemia and / or thrombosis, including stroke. The current standard of care for TTP includes intraductal delivery (e.g., intravenous or other forms of catheters) replacing plasmapheresis for a period of 2 weeks or longer, usually three times a week, but at most once a day. If the subject tests positive for the presence of an ADAMTS13 inhibitor (i.e., endogenous antibodies to ADAMTS13), plasmapheresis may be combined with immunosuppressive therapy (e.g., corticosteroids, rituxan or cyclosporine). Subjects with refractory TTP (approximately 20% of TTP patients) do not respond to at least 2 weeks of plasmapheresis therapy.

[0280] In accordance with the foregoing, in one embodiment, in the context of an initial diagnosis of TTP or in a subject displaying one or more symptoms consistent with a diagnosis of TTP (e.g., central nervous system involvement, severe thrombocytopenia (platelet count less than or equal to 5000 / μL without aspirin; less than or equal to 20,000 / μL with aspirin), severe cardiac involvement, severe lung involvement, gastrointestinal infarction, or gangrene), a method of treating the subject with an effective amount of a MASP-2 inhibitory agent (e.g., an anti-MASP-2 antibody) as a first line therapy in the absence of or in combination with plasmapheresis is provided. As a first line therapy, the MASP-2 inhibitory agent can be administered systemically to the subject, e.g., by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration. In some embodiments, the MASP-2 inhibitory agent is administered to a subject as a first line therapy in the absence of plasmapheresis to avoid potential complications of plasmapheresis, such as bleeding, infection, and exposure to conditions inherent in plasma donors and / or allergic reactions, or in subjects who are otherwise unwilling to undergo plasmapheresis, or in settings where plasmapheresis is not available. In some embodiments, the MASP-2 inhibitory agent is administered to a subject with TTP in combination (including co-administration) with an immunosuppressant (e.g., corticosteroids, rituxan, or cyclosporine) and / or in combination with concentrated ADAMTS-13.

[0281] In some embodiments, the methods comprise administering a MASP-2 inhibitory agent to a subject with TTP via a catheter (e.g., intravenously) for a first period of time (e.g., an acute phase lasting at least 1 day to 1 or 2 weeks), followed by subcutaneous administration of the MASP-2 inhibitory agent to the subject for a second period of time (e.g., a chronic phase of at least 2 weeks or longer). In some embodiments, the administration during the first and / or second period of time occurs in the absence of plasmapheresis. In some embodiments, the methods are used to maintain the subject to prevent the subject from suffering from one or more symptoms associated with TTP.

[0282] In another embodiment, a method is provided for treating a subject with refractory TTP (i.e., a subject who has not responded to at least 2 weeks of plasmapheresis therapy) by administering a MASP-2 inhibitory agent in an amount effective to reduce one or more symptoms of TTP. In one embodiment, a MASP-2 inhibitory agent (e.g., an anti-MASP-2 antibody) is administered chronically (over a period of at least 2 weeks or longer) to a subject with refractory TTP by subcutaneous or other parenteral administration. Administration can be repeated as determined by a physician until the condition has resolved or is under control.

[0283] In some embodiments, the methods further comprise determining the level of at least one complement factor (e.g., C3, C5) in the subject prior to treatment and optionally during treatment, wherein determining a decreased level of at least one complement factor compared to a standard value or healthy control subjects indicates a need for continued treatment with the MASP-2 inhibitory agent.

[0284] In some embodiments, the methods comprise administering a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, subcutaneously or intravenously to a subject having or at risk for TTP. Treatment is preferably daily, but may be as frequent as once every two weeks. Treatment is continued until the subject has a platelet count greater than 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.

[0285] In one embodiment, a MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: the antibody binds to human MASP-2 with a KD of 10 nM or less; the antibody binds to 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 IC50 of 10 nM or less; the antibody inhibits C3b deposition in 90% human serum with an IC50 of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2, wherein the antibody is a single chain molecule, wherein the antibody is an IgG2 molecule, wherein the antibody is an IgG1 molecule, wherein the antibody is an IgG4 molecule, wherein the IgG4 molecule comprises an S228P mutation, and / or wherein the antibody does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the alternative pathway. In one embodiment, the antibody binds MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the classical pathway or the alternative pathway (ie, inhibits the lectin pathway while leaving both the classical and alternative complement pathways intact).

[0286] In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in serum from a subject with TTP 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 99% compared to untreated serum. In some embodiments, the MASP-2 inhibitory antibody inhibits thrombus formation in serum from a subject with TTP at a level that is at least 20% or greater (e.g., at least 30%, at least 40%, at least 50%) greater than the inhibition of C5b-9 deposition in serum.

[0287] In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in serum from TTP patients 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 99% compared to untreated serum.

[0288] In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous catheter or other catheter delivery method.

[0289] In one embodiment, the invention provides a method of inhibiting thrombosis in a subject suffering from TTP, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising (I) (a) a heavy chain variable region comprising: i) a heavy chain CDR-H1 comprising the amino acid sequence of 31-35 of SEQ ID NO:67; and ii) a heavy chain CDR-H2 comprising the amino acid sequence of 50-65 of SEQ ID NO:67; and iii) a heavy chain CDR-H3 comprising the amino acid sequence of 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 24-34 of SEQ ID NO:70; and ii) a light chain CDR-L2 comprising the amino acid sequence of 50-56 of SEQ ID NO:70; and iii) a light chain CDR-L3 comprising the amino acid sequence of 89-97 of SEQ ID NO:70; or (II) a variant thereof comprising a heavy chain CDR-H2 comprising the amino acid sequence of SEQ ID NO:67; and iii) a heavy chain CDR-H3 comprising the amino acid sequence of 95-102 of SEQ ID NO:67. NO:67 has a heavy chain variable region that is at least 90% identical (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% identical to SEQ ID NO:67) and a light chain variable region that is at least 90% identical (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% identical) to SEQ ID NO:70.

[0290] In some embodiments, the methods comprise administering to a subject a composition comprising an amount of a MASP-2 inhibitory 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 embodiments, the methods comprise administering to a subject a composition comprising an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0291] In some embodiments, the methods comprise administering to a subject a composition comprising a MASP-2 inhibitory antibody or antigen-binding fragment thereof that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by a reference antibody OMS646 comprising a heavy chain variable region set forth in SEQ ID NO:67 and a light chain variable region set forth in SEQ ID NO:70.

[0292] Degos disease

[0293] 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 vascular disease causes obstruction of venules and arterioles, leading to skin lesions, intestinal ischemia, and CNS disorders, including stroke, epilepsy, and cognitive impairment. In the skin, connective tissue necrosis occurs due to thrombotic obstruction of arterioles. However, the cause of Degos' disease is unknown. Vasculitis, coagulopathy, or basic dysfunction of endothelial cells are involved. Degos' disease has a 50% survival rate of only 2-3 years. There is no effective treatment for Degos' disease, although antiplatelet drugs, anticoagulants, and immunosuppressants are used to alleviate symptoms.

[0294] Although the mechanism of Degos' disease is unknown, the complement pathway is involved. Margo et al. identified major C5b-9 deposits in the skin, gastrointestinal tract, and cerebral vessels of four patients with terminal Degos' disease (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 prevent progression of systemic disease (see Garrett-Bakelman F. et al., "C5b-9 is a potential effector in the pathophysiology of Degos' disease; 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).

[0295] Many patients with Degos' disease have defects in blood clotting. Thrombotic occlusion of small arteries in the skin is a hallmark of the disease. Because the complement pathway is implicated in the disease, as described herein for other TMAs, it is expected that lectin pathway inhibitors, including but not limited to antibodies that block MASP-2 function, will be beneficial in treating patients with Degos' disease.

[0296] Thus, in another embodiment, the invention provides a method for treating Degos' disease by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent, such as a MASP-2 antibody, in a pharmaceutical carrier to a subject suffering from Degos' disease or a condition resulting from Degos' disease. The MASP-2 inhibitory agent is administered systemically to the subject suffering from Degos' disease or a condition resulting from Degos' disease, such as by intra-arterial, intravenous, intramuscular, inhalation, subcutaneous or other parenteral administration, or possibly by oral administration of a non-peptidenergic agent. The anti-MASP-2 antibody can be administered alone or in combination with a C5 inhibitor, such as eculizumab.

[0297] In one embodiment, a MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: the antibody binds to human MASP-2 with a KD of 10 nM or less; the antibody binds to 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 IC50 of 10 nM or less; the antibody inhibits C3b deposition in 90% human serum with an IC50 of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2, wherein the antibody is a single chain molecule, wherein the antibody is an IgG2 molecule, wherein the antibody is an IgG1 molecule, wherein the antibody is an IgG4 molecule, wherein the IgG4 molecule comprises an S228P mutation, and / or wherein the antibody does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the alternative pathway. In one embodiment, the antibody binds MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the classical pathway or the alternative pathway (ie, inhibits the lectin pathway while leaving both the classical and alternative complement pathways intact).

[0298] In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in serum from a subject with Degos's disease 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 99% compared to untreated serum. In some embodiments, the MASP-2 inhibitory antibody inhibits thrombus formation in serum from a subject with Degos's disease at a level that is at least 20% or more (e.g., at least 30%, at least 40%, at least 50%) greater than the inhibition of C5b-9 deposition in serum.

[0299] In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in serum from a Degos' disease 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 99% compared to untreated serum.

[0300] In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous catheter or other catheter delivery method.

[0301] In one embodiment, the invention provides a method of inhibiting thrombosis in a subject suffering from Degos' disease, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising (I) (a) a heavy chain variable region comprising: i) a heavy chain CDR-H1 comprising the amino acid sequence of 31-35 of SEQ ID NO:67; and ii) a heavy chain CDR-H2 comprising the amino acid sequence of 50-65 of SEQ ID NO:67; and iii) a heavy chain CDR-H3 comprising the amino acid sequence of 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 24-34 of SEQ ID NO:70; and ii) a light chain CDR-L2 comprising the amino acid sequence of 50-56 of SEQ ID NO:70; and iii) a light chain CDR-L3 comprising the amino acid sequence of 89-97 of SEQ ID NO:70; or (II) a variant thereof comprising the amino acid sequence of SEQ ID NO:70. ID NO:67 has a heavy chain variable region that is at least 90% identical (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% identical to SEQ ID NO:67) and a light chain variable region that is at least 90% identical (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% identical) to SEQ ID NO:70.

[0302] In some embodiments, the methods comprise administering to a subject a composition comprising an amount of a MASP-2 inhibitory 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 embodiments, the methods comprise administering to a subject a composition comprising an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0303] In some embodiments, the methods comprise administering to a subject a composition comprising a MASP-2 inhibitory antibody or antigen-binding fragment thereof that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by a reference antibody OMS646 comprising a heavy chain variable region set forth in SEQ ID NO:67 and a light chain variable region set forth in SEQ ID NO:70.

[0304] Catastrophic antiphospholipid syndrome (CAPS)

[0305] Catastrophic antiphospholipid 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 with multi-organ thrombosis, ischemia and organ failure. Like other TMAs, obstruction of small blood vessels in various organs is characteristic. There is a high mortality rate of about 50% in CAPS, which is often related to infection or trauma. Patients have antiphospholipid antibodies, usually IgG.

[0306] Clinically, CAPS involves at least three organs or tissues with histopathological evidence of small vessel occlusion. Peripheral thrombosis may involve veins and arteries of the CNS, cardiovascular, renal, or pulmonary systems. Patients are treated with antibiotics, anticoagulants, corticosteroids, plasma exchange, and intravenous immunoglobulin. However, death may result from multiorgan failure.

[0307] The complement pathway is implicated in CAPS. For example, studies in animal models have shown that complement inhibition may be an effective way to prevent thrombosis associated with CAPS (Shapira L. et al., Arthritis Rheum 64(8):2719-23, 2012). In addition, as further reported by Shapira et al., administration of eculizumab to subjects with CAPS at doses that block the complement pathway inhibited acute progressive thrombotic events and reversed thrombocytopenia (see also Lim W., Curr Opin Hematol 18(5):361-5, 2011). Therefore, as described herein for other TMAs, it is expected that lectin pathway inhibitors, including but not limited to antibodies that block MASP-2 function, will be beneficial in treating patients with CAPS.

[0308] Thus, in another embodiment, the invention provides a method of 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 inhibitory agent, such as a MASP-2 antibody, in a pharmaceutical carrier. The MASP-2 inhibitory agent is administered systemically to the subject suffering from CAPS or a condition resulting from CAPS, such as by intra-arterial, intravenous, intramuscular, inhalation, subcutaneous or other parenteral administration, or possibly by oral administration of a non-peptidergic agent. The anti-MASP-2 antibody can be administered alone or in combination with a C5 inhibitor, such as eculizumab.

[0309] In one embodiment, a MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: the antibody binds to human MASP-2 with a KD of 10 nM or less; the antibody binds to an epitope of the CCP1 domain of MASP-2; the antibody inhibits C3b deposition in 1% human serum in an in vitro assay with an IC50 of 10 nM or less; the antibody inhibits C3b deposition in 90% human serum with an IC50 of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2, wherein the antibody is a single chain molecule, wherein the antibody is an IgG2 molecule, wherein the antibody is an IgG1 molecule, wherein the antibody is an IgG4 molecule, wherein the IgG4 molecule comprises an S228P mutation, and / or wherein the antibody does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the alternative pathway. In one embodiment, the antibody binds MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the classical pathway or the alternative pathway (ie, inhibits the lectin pathway while leaving both the classical and alternative complement pathways intact).

[0310] In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in serum from a subject with 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 99% compared to untreated serum. In some embodiments, the MASP-2 inhibitory antibody inhibits thrombus formation in serum from a subject with CAPS at a level that is at least 20% or greater (e.g., at least 30%, at least 40%, at least 50%) greater than the inhibition of C5b-9 deposition in serum.

[0311] In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in serum from CAPS patients 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 99% compared to untreated serum.

[0312] In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous catheter or other catheter delivery method.

[0313] In one embodiment, the invention provides a method of inhibiting thrombosis in a subject suffering from CAPS, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising (I) (a) a heavy chain variable region comprising: i) a heavy chain CDR-H1 comprising the amino acid sequence of 31-35 of SEQ ID NO:67; and ii) a heavy chain CDR-H2 comprising the amino acid sequence of 50-65 of SEQ ID NO:67; and iii) a heavy chain CDR-H3 comprising the amino acid sequence of 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 24-34 of SEQ ID NO:70; and ii) a light chain CDR-L2 comprising the amino acid sequence of 50-56 of SEQ ID NO:70; and iii) a light chain CDR-L3 comprising the amino acid sequence of 89-97 of SEQ ID NO:70; or (II) a variant thereof comprising a heavy chain CDR-H2 comprising the amino acid sequence of SEQ ID NO:67; and iii) a heavy chain CDR-H3 comprising the amino acid sequence of 95-102 of SEQ ID NO:67. NO:67 has a heavy chain variable region that is at least 90% identical (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% identical to SEQ ID NO:67) and a light chain variable region that is at least 90% identical (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% identical) to SEQ ID NO:70.

[0314] In some embodiments, the methods comprise administering to a subject a composition comprising an amount of a MASP-2 inhibitory 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 embodiments, the methods comprise administering to a subject a composition comprising an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0315] In some embodiments, the methods include administering to a subject a composition that includes a MASP-2 inhibitory antibody or antigen-binding fragment thereof that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by reference antibody OMS646, comprising a heavy chain variable region set forth in SEQ ID NO:67 and a light chain variable region set forth in SEQ ID NO:70.

[0316] Secondary TMA of cancer

[0317] Any type of systemic malignancy can lead to clinical and pathological manifestations of TMA (see, e.g., Batts and Lazarus, Bone Marrow Transplantation 40:709-719, 2007). Cancer-associated TMA is often found in the lungs and has been shown to be associated with tumor emboli (Francis KK et al., Commun Oncol 2:339-43, 2005). Tumor emboli can reduce blood flow and thus lead to a hypoperfusion state of affected arterioles and venules. The resulting tissue stress and damage are expected to activate the lectin pathway of complement in a localized manner. The activated lectin pathway can in turn activate the coagulation cascade through MASP-2-dependent cleavage of prothrombin to thrombin, leading to the pro-thrombotic state that is characteristic of TMA. MASP-2 inhibition is expected to reduce local activation of thrombin and thus alleviate the pro-thrombotic state in this setting.

[0318] Therefore, as described herein for other TMAs, it is expected that lectin pathway inhibitors, including but not limited to antibodies that block MASP-2 function, will be beneficial in treating patients suffering from TMAs secondary to cancer.

[0319] Thus, in another embodiment, the invention provides a method of treating or preventing TMA secondary to cancer by administering to a subject having or at risk of developing TMA secondary to cancer a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent, such as a MASP-2 antibody, in a pharmaceutical carrier. The MASP-2 inhibitory agent is administered systemically to the subject having or at risk of developing TMA secondary to cancer, such as by intra-arterial, intravenous, intramuscular, inhalation, subcutaneous or other parenteral administration, or may be administered orally as a non-peptidenergic agent. The anti-MASP-2 antibody may be administered alone or in combination with a C5 inhibitor, such as eculizumab.

[0320] In one embodiment, a MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: the antibody binds to human MASP-2 with a KD of 10 nM or less; the antibody binds to an epitope of the CCP1 domain of MASP-2; the antibody inhibits C3b deposition in 1% human serum in an in vitro assay with an IC50 of 10 nM or less; the antibody inhibits C3b deposition in 90% human serum with an IC50 of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2, wherein the antibody is a single chain molecule, wherein the antibody is an IgG2 molecule, wherein the antibody is an IgG1 molecule, wherein the antibody is an IgG4 molecule, wherein the IgG4 molecule comprises an S228P mutation, and / or wherein the antibody does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the alternative pathway. In one embodiment, the antibody binds MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the classical pathway or the alternative pathway (ie, inhibits the lectin pathway while leaving both the classical and alternative complement pathways intact).

[0321] In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in serum from a subject with 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 99% compared to untreated serum.

[0322] In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in serum from patients with 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 99% compared to untreated serum.

[0323] In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous catheter or other catheter delivery method.

[0324] In one embodiment, the invention provides a method of inhibiting thrombosis in a subject having TMA secondary to cancer, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising (I) (a) a heavy chain variable region comprising: i) a heavy chain CDR-H1 comprising the amino acid sequence of 31-35 of SEQ ID NO:67; and ii) a heavy chain CDR-H2 comprising the amino acid sequence of 50-65 of SEQ ID NO:67; and iii) a heavy chain CDR-H3 comprising the amino acid sequence of 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 24-34 of SEQ ID NO:70; and ii) a light chain CDR-L2 comprising the amino acid sequence of 50-56 of SEQ ID NO:70; and iii) a heavy chain CDR-H4 comprising the amino acid sequence of 95-102 of SEQ ID NO:67. or (II) a variant thereof, comprising a heavy chain variable region that is at least 90% identical 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% identical to SEQ ID NO:67) and a light chain variable region that is at least 90% identical 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% identical to SEQ ID NO:70).

[0325] In some embodiments, the methods comprise administering to a subject a composition comprising an amount of a MASP-2 inhibitory 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 embodiments, the methods comprise administering to a subject a composition comprising an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0326] In some embodiments, the methods include administering to a subject a composition that includes a MASP-2 inhibitory antibody or antigen-binding fragment thereof that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by reference antibody OMS646, comprising a heavy chain variable region set forth in SEQ ID NO:67 and a light chain variable region set forth in SEQ ID NO:70.

[0327] TMA secondary to cancer chemotherapy

[0328] Chemotherapy-associated TMA is a condition involving thrombocytopenia, microangiopathic hemolytic anemia, and renal insufficiency that occurs in 2-10% of patients with a history of malignancy treated with chemotherapeutic agents such as gemcitabine, 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).

[0329] It is believed that the etiology of chemotherapy-related TMA includes non-specific toxic damage to the microvascular endothelium. Direct damage to endothelial cells has been shown in animal models of mitomycin-induced TMA (Dlott J. et al., TherApher Dial 8: 102–11, 2004). Endothelial cell damage by 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 in vivo and in vitro (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 inhibition of the lectin pathway prevents thrombosis (La Bonte et al. J Immunol. 15; 188 (2): 885-91, 2012). Furthermore, as demonstrated in Examples 37-39 herein, in a mouse model of TMA in which localized photoexcitation of FITC-Dex is used to induce localized damage to microvessels, followed by a TMA response, the inventors have demonstrated that inhibition of MASP-2 can prevent TMA. Thus, microvascular endothelial damage by chemotherapeutic agents can activate the lectin pathway of complement, which then produces a localized pro-thrombotic state and promotes a TMA response. Because activation of the lectin pathway and generation of a pro-thrombotic state are MASP-2-dependent, it is expected that MASP-2 inhibitors, including but not limited to antibodies that block MASP-2 function, will mitigate the TMA response and reduce the risk of cancer chemotherapy-associated TMA.

[0330] Thus, in another embodiment, the invention provides a method of treating or preventing chemotherapy-secondary TMA by administering to a subject suffering from or at risk of chemotherapy-secondary TMA a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent, such as a MASP-2 antibody, in a pharmaceutical carrier. The MASP-2 inhibitory agent is administered systemically to a subject who has undergone, is undergoing, or will undergo chemotherapy, such as by intra-arterial, intravenous, intramuscular, inhalation, subcutaneous or other parenteral administration, or possibly by oral administration of a non-peptidenergic agent. The anti-MASP-2 antibody can be administered alone or in combination with a C5 inhibitor, such as eculizumab.

[0331] In one embodiment, a MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: the antibody binds to human MASP-2 with a KD of 10 nM or less; the antibody binds to an epitope of the CCP1 domain of MASP-2; the antibody inhibits C3b deposition in 1% human serum in an in vitro assay with an IC50 of 10 nM or less; the antibody inhibits C3b deposition in 90% human serum with an IC50 of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2, wherein the antibody is a single chain molecule, wherein the antibody is an IgG2 molecule, wherein the antibody is an IgG1 molecule, wherein the antibody is an IgG4 molecule, wherein the IgG4 molecule comprises an S228P mutation, and / or wherein the antibody does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the alternative pathway. In one embodiment, the antibody binds MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the classical pathway or the alternative pathway (ie, inhibits the lectin pathway while leaving both the classical and alternative complement pathways intact).

[0332] In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in serum from subjects with TMA secondary to cancer chemotherapy 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 99% compared to untreated serum.

[0333] In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in serum from patients with TMA secondary to cancer chemotherapy 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 99% compared to untreated serum.

[0334] In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous catheter or other catheter delivery method.

[0335] In one embodiment, the invention provides a method of inhibiting thrombosis in a subject having TMA secondary to cancer chemotherapy, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising (I)(a) a heavy chain variable region comprising: i) a heavy chain CDR-H1 comprising the amino acid sequence of 31-35 of SEQ ID NO:67; and ii) a heavy chain CDR-H2 comprising the amino acid sequence of 50-65 of SEQ ID NO:67; and iii) a heavy chain CDR-H3 comprising the amino acid sequence of 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 24-34 of SEQ ID NO:70; and ii) a light chain CDR-L2 comprising the amino acid sequence of 50-56 of SEQ ID NO:70; and iii) a heavy chain CDR-H4 comprising the amino acid sequence of 95-102 of SEQ ID NO:67. or (II) a variant thereof, comprising a heavy chain variable region that is at least 90% identical 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% identical to SEQ ID NO:67) and a light chain variable region that is at least 90% identical 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% identical to SEQ ID NO:70).

[0336] In some embodiments, the methods comprise administering to a subject a composition comprising an amount of a MASP-2 inhibitory 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 embodiments, the methods comprise administering to a subject a composition comprising an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0337] In some embodiments, the methods include administering to a subject a composition that includes a MASP-2 inhibitory antibody or antigen-binding fragment thereof that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by reference antibody OMS646, comprising a heavy chain variable region set forth in SEQ ID NO:67 and a light chain variable region set forth in SEQ ID NO:70.

[0338] Transplantation-induced TMA

[0339] Transplantation-associated TMA (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 pathogenesis of this condition is poorly understood, but may involve a confluence of reactions that ultimately lead to endothelial cell injury (Laskin BL et al., Blood 118(6):1452-62, 2011). As described above, endothelial cell injury is a prototypical stimulus for lectin pathway activation and the generation of a pro-thrombotic environment.

[0340] Recent data further support the role of complement activation through the lectin pathway in the pathogenesis of TA-TMA. Laskin et al. have demonstrated that renal arteriolar C4d deposition is more common in subjects with histological TA-TMA (75%) compared with controls (8%) (Laskin BL et al., Transplanatation, 27; 96 (2): 217-23, 2013). Therefore, C4d may be a pathological marker of TA-TMA, which involves local complement fixation through the lectin or classical pathway.

[0341] Because activation of the lectin pathway and the generation of a pro-thrombotic state are MASP-2-dependent, it is expected that MASP-2 inhibitors, including but not limited to antibodies that block MASP-2 function, will mitigate TMA reactions and reduce the risk of transplant-associated TMA (TA-TMA).

[0342] Thus, in another embodiment, the invention provides methods of treating or preventing TMA secondary to transplantation by administering to a subject suffering from or at risk of developing TMA secondary to transplantation a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent, such as a MASP-2 antibody, in a pharmaceutical carrier. The MASP-2 inhibitory agent is administered systemically to a subject who has undergone, is undergoing, or will undergo a transplantation procedure, such as by intra-arterial, intravenous, intramuscular, inhalation, subcutaneous or other parenteral administration, or may be administered orally as a non-peptidenergic agent. The anti-MASP-2 antibody may be administered alone or in combination with a C5 inhibitor, such as eculizumab. In some embodiments, the invention provides methods of treating or preventing TMA secondary to allogeneic stem cell transplantation, comprising administering to a subject a composition comprising an amount of a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, before, during, or after undergoing an allogeneic stem cell transplant.

[0343] In one embodiment, a MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: the antibody binds to human MASP-2 with a KD of 10 nM or less; the antibody binds to an epitope of the CCP1 domain of MASP-2; the antibody inhibits C3b deposition in 1% human serum in an in vitro assay with an IC50 of 10 nM or less; the antibody inhibits C3b deposition in 90% human serum with an IC50 of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2, wherein the antibody is a single chain molecule, wherein the antibody is an IgG2 molecule, wherein the antibody is an IgG1 molecule, wherein the antibody is an IgG4 molecule, wherein the IgG4 molecule comprises an S228P mutation, and / or wherein the antibody does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the alternative pathway. In one embodiment, the antibody binds MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the classical pathway or the alternative pathway (ie, inhibits the lectin pathway while leaving both the classical and alternative complement pathways intact).

[0344] In one embodiment, the MASP-2 inhibitory antibody inhibits thrombosis in serum from a subject with 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 99% compared to untreated serum.

[0345] In one embodiment, the MASP-2 inhibitory antibody inhibits thrombosis in serum from a patient with 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 99% compared to untreated serum.

[0346] In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous catheter or other catheter delivery method.

[0347] In one embodiment, the invention provides a method of inhibiting thrombosis in a subject having TMA secondary to transplantation, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising (I) (a) a heavy chain variable region comprising: i) a heavy chain CDR-H1 comprising the amino acid sequence of 31-35 of SEQ ID NO:67; and ii) a heavy chain CDR-H2 comprising the amino acid sequence of 50-65 of SEQ ID NO:67; and iii) a heavy chain CDR-H3 comprising the amino acid sequence of 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 24-34 of SEQ ID NO:70; and ii) a light chain CDR-L2 comprising the amino acid sequence of 50-56 of SEQ ID NO:70; and iii) a heavy chain CDR-H4 comprising the amino acid sequence of 95-102 of SEQ ID NO:67. or (II) a variant thereof, comprising a heavy chain variable region that is at least 90% identical 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% identical to SEQ ID NO:67) and a light chain variable region that is at least 90% identical 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% identical to SEQ ID NO:70).

[0348] In some embodiments, the methods comprise administering to a subject a composition comprising an amount of a MASP-2 inhibitory 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 embodiments, the methods comprise administering to a subject a composition comprising an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0349] In some embodiments, the methods include administering to a subject a composition that includes a MASP-2 inhibitory antibody or antigen-binding fragment thereof that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by reference antibody OMS646, comprising a heavy chain variable region set forth in SEQ ID NO:67 and a light chain variable region set forth in SEQ ID NO:70.

[0350] IV. Role of MASP-2 in other diseases and conditions and therapeutic approaches using MASP-2 inhibitors

[0351] Kidney conditions

[0352] Activation of the complement system is involved in the pathogenesis of various renal 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, PE et al., Kidney Int., 41:933-7, 1992; Salant, DJ et al., Kidney Int. 35:976-84, 1989), membranoproliferative glomerulonephritis (mesangial capillary glomerulonephritis) (Bartlow, BG et al., Kidney Int. 15: 294-300, 1979; Meri, S. et al., J. Exp. Med. 175: 939-50, 1992), acute post-infectious glomerulonephritis (post-streptococcal glomerulonephritis), cryoglobulinemic glomerulonephritis (Ohsawa, I. et al., Clin Immunol. 101: 59-66, 2001), lupus nephritis (Gatenby, PA, Autoimmunity 11: 61-6, 1991) and Henoch-Schonlein purpura nephritis (Endo, M. et al., Am. J. Kidney Dis. 35: 401-407, 2000). The involvement of complement in renal disease has been recognized for decades, but its precise role in the onset, development and resolution of renal disease remains much debated. Under normal conditions, the contribution of complement is beneficial to the host, but inappropriate activation and deposition of complement can promote tissue damage.

[0353] There is a lot of evidence that glomerulonephritis, an inflammation of the glomeruli, often initiates through the deposition of immune complexes onto glomerular or tubular structures, which then trigger complement activation, inflammation, and tissue damage. Kahn and Sinniah demonstrated increased deposition of C5b-9 on tubular basement membranes in biopsies taken from patients with various forms of glomerulonephritis (Kahn, TN 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), deposition of C5b-9 in tubular epithelial / basement membrane structures correlated with plasma creatinine levels. Another study of membranous nephropathy demonstrated a relationship between clinical outcomes and urinary sC5b-9 levels (Kon, SP et al., Kidney Int. 48:1953-58, 1995). Elevated sC5b-9 levels were positively correlated with poor prognosis. Lehto et al. measured elevated levels of CD59 (a complement regulatory factor that inhibits the membrane attack complex in the plasma membrane) and C5b-9 in the urine from patients with membranous glomerulonephritis (Lehto, T. et al., Kidney Int. 47: 1403-11, 1995). Histopathological analysis of biopsy samples taken from these same patients confirmed deposition of C3 and C9 proteins in the glomeruli, while CD59 expression was reduced in these tissues compared to normal kidney tissue. These various studies indicate that progressive complement-mediated glomerulonephritis results in urinary excretion of complement proteins, which correlates with the extent of tissue damage and disease prognosis.

[0354] Inhibition of complement activation in various animal models of glomerulonephritis also demonstrates the importance of complement activation in the etiology of the disease. In a model of membranoproliferative glomerulonephritis (MPGN), infusion of anti-Thy1 antiserum in 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 reduction in collagen type IV (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 suggest that C5b-9 is the main mediator of tissue damage caused by complement in this rat anti-thymocyte serum model. In another glomerulonephritis model, infusion of graded doses of rabbit anti-rat glomerular basement membrane produced a dose-dependent influx of polymorphonuclear leukocytes (PMNs) that was attenuated by previous treatment with cobra venom factor (to deplete complement) (Scandrett, AL et al., Am. J. Physiol. 268: F256-F265, 1995). Cobra venom factor-treated rats also showed reduced tissue pathology, decreased long-term proteinuria, and lower creatinine levels compared to control rats. Using three rat GN models (anti-thymocyte serum, Con A anti-Con A, and passive Heymann nephritis), Couser et al. demonstrated the potential therapeutic efficacy of a method of inhibiting complement using recombinant sCR1 protein (Couser, WG et al., J. Am. Soc. Nephrol. 5: 1888-94, 1995). Rats treated with sCR1 showed a significant reduction in the influx of PMNs, platelets, and macrophages, reduced mesangial lysis, and reduced proteinuria compared to control rats. Further evidence for the importance of complement activation in glomerulonephritis was provided by the use of anti-C5 MoAb in the NZB / W F1 mouse model. Anti-C5 MoAb inhibits the cleavage of C5, thus blocking the generation of C5a and C5b-9. Continued treatment with anti-C5 MoAb for 6 months resulted in a significant improvement in the course of glomerulonephritis. A humanized anti-C5 MoAb monoclonal antibody (5G1.1), which prevents the cleavage of human complement component C5 into its pro-inflammatory components, is under development by Alexion Pharmaceuticals, Inc., New Haven, Connecticut, as a potential treatment for glomerulonephritis.

[0355] Direct evidence for the pathological role of complement in renal injury is provided by studying patients with genetic defects in specific complement components. Many reports have documented that renal disease is associated with defects in complement regulatory factor H (Ault, BH, Nephrol. 14: 1045-1053, 2000; Levy, M., et al., Kidney Int. 30: 949-56, 1986; Pickering, MC, et al., Nat. Genet. 31: 424-8, 2002). Factor H deficiency leads to low plasma levels of factor B and C3 and consumption of C5b-9. Both atypical membranoproliferative glomerulonephritis (MPGN) and idiopathic hemolytic uremic syndrome (HUS) are associated with factor H deficiency. Factor H-deficient pigs (Jansen, JH, et al., Kidney Int. 53:331-49, 1998) and factor H knockout mice (Pickering, MC, 2002) show MPGN-like symptoms, confirming the importance of factor H in complement regulation. Deficiencies in other complement components are associated with renal disease secondary to the development of systemic lupus erythematosus (SLE) (Walport, MJ, Davies, et al., Ann. NY Acad. Sci. 815:267-81, 1997). Deficiencies in C1q, C4, and C2 strongly predispose to SLE through mechanisms involving incomplete clearance of immune complexes and apoptotic material. Lupus nephritis occurs in many of these SLE patients, characterized by deposition of immune complexes throughout the glomeruli.

[0356] Further evidence linking complement activation and renal disease is provided by the identification of patients with autoantibodies to complement components, some of which are directly associated with renal disease (Trouw, LA, et al., Mol. Immunol. 38: 199-206, 2001). Many of these autoantibodies show a high correlation with renal disease, so that the term nephritic factor (NeF) was introduced to represent this activity. In clinical studies, approximately 50% of patients who were positive for nephritic factor developed MPGN (Spitzer, RE, et al., Clin. Immunol. Immunopathol. 64: 177-83, 1992). C3NeF is an autoantibody to the alternative pathway C3 convertase (C3bBb) and it stabilizes the convertase, thereby promoting alternative pathway activation (Daha, MR, et al., J. Immunol. 116: 1-7, 1976). Likewise, an autoantibody specific for the classical pathway C3 convertase (C4b2a), termed C4NeF, stabilizes the convertase, thereby promoting classical pathway activation (Daha, MR et al., J. Immunol. 125:2051-2054, 1980; Halbwachs, L., et al., J. Clin. Invest. 65:1249-56, 1980). Anti-C1q autoantibodies have been described 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), and elevated titers of these anti-C1q autoantibodies are reported to predict the development of nephritis (Coremans, IE, et al., Am. J. Kidney Dis. 26:595-601, 1995). Immune deposits eluted from autopsy kidneys of SLE patients revealed accumulation of these anti-C1q autoantibodies (Mannick, M., et al., Arthritis Rheumatol. 40:1504-11, 1997). All these facts point to a pathological role for these autoantibodies. However, not all patients with anti-C1q autoantibodies develop renal disease, and some healthy individuals have low titers of anti-C1q autoantibodies (Siegert, CE, et al., Clin. Immunol. Immunopathol. 67:204-9, 1993).

[0357] In addition to the alternative and classical pathways of complement activation, the lectin pathway may also have an important pathological role in renal disease. Elevated levels of MBL, MBL-associated serine proteases, and complement activation products were detected by immunohistochemical techniques on renal biopsy material obtained from patients diagnosed with several different renal diseases, including Henoch-Schonlein purpura nephritis (Endo, M., et al., Am. J. Kidney Dis. 35: 401-407, 2000), cryoglobulinemia glomerulonephritis (Ohsawa, I., et al., Clin. Immunol. 101: 59-66, 2001), and IgA nephropathy (Endo, M., et al., Clin. Nephrology 55: 185-191, 2001). Therefore, although the correlation between complement and renal disease has been known for decades, the data on how complement accurately affects these renal diseases are far from complete.

[0358] Blood disorders

[0359] Sepsis is caused by an overwhelming response of the patient to invading microorganisms. The main function of the complement system is to coordinate the inflammatory response to invading bacteria and other pathogens. Consistent with this physiological role, many studies have shown that complement activation plays an important role in the pathogenesis of sepsis (Bone, RC, Annals. Internal. Med. 115: 457-469, 1991). The definition of the clinical manifestations of sepsis is constantly evolving. Sepsis is usually defined as a systemic host response to infection. However, in many cases, no clinical evidence of infection (e.g., positive bacterial blood culture) is found in patients with septic symptoms. This deviation was first proposed in the Consensus Conference in 1992 when the term "systemic inflammatory response syndrome" (SIRS) was established, and there was no need to determine the presence of bacterial infection for this syndrome (Bone, RC, et al., Crit. Care Med. 20: 724-726, 1992). It is now widely recognized that sepsis and SIRS are accompanied by an inability to regulate inflammatory responses. For the purpose of simplicity, we consider the clinical definition of sepsis to also include severe sepsis, septic shock, and SIRS.

[0360] Prior to the late 1980s, the major source of infection in septic patients was Gram-negative bacteria. Lipopolysaccharide (LPS), a major component of the cell wall of Gram-negative bacteria, is known to stimulate the release of inflammatory mediators from various cell types and induce acute infectious symptoms when injected into animals (Haeney, MR et al., AntmicrobialChemotherapy 41 (Suppl. A): 41-6, 1998). Interestingly, the spectrum of pathogenic microorganisms appears to have changed from predominantly Gram-negative bacteria in the late 1970s and 1980s to predominantly Gram-positive bacteria today, for reasons that are currently unclear (Martin, GS et al., N. Eng. J. Med. 348: 1546-54, 2003).

[0361] Many studies have demonstrated the importance of complement activation in mediating inflammation and promoting the characteristics of shock (especially septic and hemorrhagic shock). Both gram-negative and gram-positive microorganisms usually promote septic shock. LPS is an effective complement activator, mainly through the alternative pathway, although antibody-mediated classical pathway activation also occurs (Fearon, DT et al., N. Engl. J. Med. 292: 937-400, 1975). The main components of the gram-positive cell wall are peptidoglycan and lipoteichoic acid, which are effective alternative complement pathway activators, although they can also activate the classical complement pathway in the presence of specific antibodies (Joiner, KA et al., Ann. Rev. Immunol. 2: 461-2, 1984).

[0362] The complement system was initially implicated in the pathogenesis of sepsis when researchers noted that the anaphylatoxins C3a and C5a mediate various inflammatory responses that can also occur during sepsis. These anaphylatoxins cause vasodilation and increased microvascular permeability, events that play an important role in septic shock (Schumacher, WA et al., Agents Actions 34:345-349, 1991). In addition, anaphylatoxins induce bronchospasm, histamine release from mast cells, and platelet aggregation. In addition, they produce many effects on granulocytes, such as chemotaxis, aggregation, adhesion, release of lysosomal enzymes, production of toxic superoxide anions, and formation of leukotrienes (Shin, HS 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 complications of sepsis such as shock or acute respiratory distress syndrome (ARDS) (Hammerschmidt, DE et al., Lancet 1:947-949, 1980; Slotman, GT et al., Surgery 99:744-50, 1986). In addition, elevated levels of the anaphylatoxin C3a are associated with fatal outcomes in sepsis (Hack, CE et al., Am. J. Med. 86:20-26, 1989). In some animal models of shock, certain complement-deficient strains (e.g., C5-deficient strains) are more resistant to the effects of LPS infusion (Hseuh, W. et al., Immunol. 70:309-14, 1990).

[0363] Blocking C5a production with antibodies during sepsis in rodents has been shown to greatly improve survival (Czermak, BJ, et al., Nat. Med. 5:788-792, 1999). Similar findings were obtained when the C5a receptor (C5aR) was blocked by antibodies or small molecule inhibitors (Huber-Lang, MS, et al., FASEB J. 16:1567-74, 2002; Riedemann, NC, et al., J. Clin. Invest. 110:101-8, 2002). Early experimental studies in monkeys showed that antibody blockade of C5a attenuated E. coli-induced septic shock and adult respiratory distress syndrome (Hangen, DH, et al., J. Surg. Res. 46:195-9, 1989; Stevens, JH, et al., J. Clin. Invest. 77:1812-16, 1986). In humans with sepsis, C5a is elevated and associated with significantly reduced survival and multiple organ failure when compared to less severely septic patients and survivors (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 of C5a production and its deleterious effects during sepsis has been studied in some detail, but recent data suggest that C5a production during sepsis significantly impairs the innate immune function of blood neutrophils (Huber-Lang, MS, et al., J. Immunol. 169:3223-31, 2002), their ability to perform a respiratory burst, and their ability to produce cytokines (Riedemann, NC, et al., Immunity 19:193-202, 2003). In addition, C5a production during sepsis appears to have a procoagulant effect (Laudes, IJ, 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., Behring Ins. Mitt. 93:299-305, 1993).

[0364] The lectin pathway also plays a role in the pathogenesis of sepsis. MBL has been shown to bind to many clinically important 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 considered to be the Gram-positive counterpart of LPS. It is a potent immunostimulant that induces cytokine release from mononuclear phagocytes and whole blood (Morath, S., et al., J. Exp. Med. 195:1635, 2002; Morath, S., et al., Infect. Immun. 70:938, 2002). L-ficolin was recently shown to specifically bind to LTA isolated from a number of Gram-positive bacterial species, including Staphylococcus aureus, and activate the lectin pathway (Lynch, NJ, et al., J. Immunol. 172: 1198-02, 2004). MBL was also shown to bind to LTA from Enterococcus (in which the polyglycerol phosphate chains were replaced with sugar groups), but not to LTA from nine other species, including Staphylococcus aureus (Polotsky, VY, et al., Infect. Immun. 64: 380, 1996).

[0365] Thus, one aspect of the invention provides methods of treating sepsis or a condition resulting from sepsis, including, but not limited to, severe sepsis resulting from sepsis, septic shock, acute respiratory distress syndrome, and systemic inflammatory response syndrome, by administering to a subject suffering from sepsis or a condition resulting from sepsis a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutical carrier. Related methods of treating other blood disorders are provided, 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, by administering to a subject suffering from such a condition a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutical carrier. The MASP-2 inhibitory agent is administered to the subject systemically, for example, by intra-arterial, intravenous, intramuscular, inhalation (particularly in the case of ARDS), subcutaneous or other parenteral administration, or possibly by oral administration of a non-peptidergic agent. The MASP-2 inhibitory composition may be combined with one or more other therapeutic agents to combat the sequelae of sepsis and / or shock. For advanced sepsis or shock, or distressing conditions arising therefrom, the MASP-2 inhibitory composition may be suitably administered in a rapid-acting dosage form, for example, by intravenous or intra-arterial delivery of a bolus solution containing the MASP-2 inhibitory composition. Repeated administration may be performed as determined by the physician until the condition is resolved.

[0366] Coagulopathy

[0367] Evidence has been provided for a role for the complement system in disseminated intravascular coagulation ("DIC"), such as DIC secondary to significant physical trauma.

[0368] 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 could still activate complement via the classical or lectin pathways, C3 turnover in C4- / - plasma was measured in assays specific for the classical or lectin pathway activation routes. Although C3 cleavage was not observed when activation was triggered via the classical pathway, highly efficient lectin pathway-dependent activation of C3 was observed in C4-deficient serum ( Fig.30 ). It can be seen that C3b deposition on mannan and zymosan is severely impaired in MASP-2- / - mice, even under experimental conditions that, according to many previously published papers on alternative pathway activation, should be permissive for all three pathways. When the same sera were used in wells coated with immunoglobulin complexes instead of mannan or zymosan, C3b deposition and factor B cleavage were seen in MASP-2+ / + mouse sera and MASP-2- / - sera, but not in C1q-depleted sera. This indicates that alternative pathway activation is promoted in MASP-2- / - sera when initial C3b is provided by classical activity. Fig.30 Describes the surprising finding that C3 can be efficiently activated in C4-deficient plasma in a lectin pathway-dependent manner.

[0369] By preincubating plasma with soluble mannan or mannose, this "C4 bypass" was abolished by inhibiting lectin pathway-activation.

[0370] Abnormal non-immune activation of the complement system may be dangerous to humans and may also play an important role in blood pathway activation, especially in severe trauma situations, where inflammatory and blood pathways are activated. In normal healthy people, C3 conversion is <5% of total plasma C3 protein. In invasive infections, including sepsis and immune complex diseases, C3 conversion reconstitutes itself with about 30% of complement levels that are often lower than normal, which is due to increased utilization and changes in the pool distribution. Greater than 30% direct C3 pathway activation usually produces obvious clinical evidence of vasodilation and fluid loss to tissues. Above 30% C3 conversion, the initiating mechanism is mainly non-immune, and the resulting clinical manifestations are harmful to the patient. Complement C5 levels are shown to be much more stable than C3 in health and controlled diseases. Significant reductions and / or conversions in C5 levels are associated with patients' responses to abnormal multiple injuries (e.g., road traffic accidents) and possible shock pulmonary syndrome. Therefore, complement C3 activation exceeding 30% of the vascular pool or any C5 involvement, or any evidence of both, can be considered as a possible precursor of harmful pathological changes in patients.

[0371] Both C3 and C5 release anaphylatoxins (C3a and C5a), which act on mast cells and basophils, releasing vasodilatory chemicals. They set chemotactic gradients to guide polymorphonuclear cells (PMN) to the center of immune disturbance (a beneficial reaction), but they are different here because C5a has a specific coagulation (aggregation) effect on these phagocytes, preventing them from randomly moving away from the reaction site. In normal control of infection, C3 activates C5. However, in multiple injuries, C5 seems to be widely activated, and C5a anaphylatoxins are produced systemically. This uncontrolled activity causes polymorphic aggregation in the vascular system, and then these clots are carried to the capillaries of the lungs, where they block and produce local damage due to superoxide release. Although not wishing to be limited by theory, this mechanism may be important in the pathogenesis of acute respiratory distress syndrome (ARDS), although this view has recently been challenged. C3a anaphylatoxins have been shown to be potent platelet aggregators in vitro, but their involvement in vivo is less certain, and the release of platelet material and plasmin in wound repair may only secondarily involve complement C3. It is possible that a prolonged increase in C3 activation is required to produce DIC.

[0372] In addition to the cellular and vascular effects of activated complement components described above, which may explain the association between trauma and DIC, recent scientific discoveries have identified a direct molecular link and functional dialogue between the complement and coagulation systems. Supporting data were obtained from studies in C3-deficient mice. Because C3 is a common component of each complement pathway, it is predicted that C3-deficient mice lack all complement functions. However, surprisingly, C3-deficient mice are fully capable of activating 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 C3-independent activation of terminal complement components is mediated by thrombin, the rate-limiting enzyme of the coagulation cascade. (Huber et al., 2006). The molecular components that mediate thrombin activation after initial complement activation remain elusive.

[0373] The present inventors have elucidated what is the molecular basis of the dialogue between the complement and coagulation cascades and identified MASP-2 as an important control point linking these two systems. Biochemical studies of the substrate specificity of MASP-2 identified prothrombin as a possible substrate in addition to the well-known C2 and C4 complement proteins. MASP-2 specifically cleaves prothrombin at a functionally relevant site 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)). MASP-2-generated thrombin is able to promote fibrin deposition in a defined reconstituted in vitro system, demonstrating the functional relevance of MASP-2 cleavage. (Krarup et al., 2007). As described in the Examples below, the inventors further confirmed the physiological significance of this finding by documenting thrombin activation in normal rodent serum following lectin pathway activation and demonstrated that this process was blocked by a neutralizing MASP-2 monoclonal antibody.

[0374] MASP-2 may represent an important branch point in the lectin pathway, capable of promoting activation of both the complement and coagulation systems. Because lectin pathway activation is a physiological response to many types of traumatic injury, the inventors believe that the simultaneous occurrence of systemic inflammation (mediated by complement components) and disseminated coagulation (mediated by the coagulation pathway) can be explained by the ability of MASP-2 to activate both pathways. These findings clearly demonstrate a role for 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 activation of the lectin pathway, when occurring in the context of trauma, may directly trigger activation of the coagulation system through the MASP-2-thrombin axis, providing a mechanistic link between trauma and DIC. According to one aspect of the invention, inhibition of MASP-2 will inhibit lectin pathway activation and reduce the production of both anaphylatoxins C3a and C5a. It is believed that a prolonged increase in C3 activation is required for the development of DIC.

[0375] Microcirculatory coagulation (spotted blood clots in capillaries and small vessels) occurs in settings such as septic shock. The role of the lectin pathway in septic shock was determined, as described in Examples 17 and Fig.18 and 19 This was demonstrated by the protective phenotype of the MASP-2(- / -) sepsis mouse model described in . Fig.16A and 16B As demonstrated in , MASP-2(- / -) mice were protected in the local Schwartzman reaction model of disseminated intravascular coagulation (DIC), a model of localized coagulation in the microvasculature.

[0376] V. MASP-2 Inhibitors

[0377] In one aspect, the invention provides methods for inhibiting MASP-2-dependent complement activation in a subject suffering from or at risk for developing thrombotic microangiopathy. The MASP-2 inhibitory agent 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 inhibitory agents 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), and molecules that reduce the expression of MASP-2 (e.g., MASP-2 antisense nucleic acid molecules, MASP-2-specific RNAi molecules, and MASP-2 ribozymes), thereby preventing MASP-2 from activating the lectin complement pathway. The MASP-2 inhibitory agent can be used alone as a primary therapy, or in combination with other therapeutic agents as an adjunct therapy to enhance the therapeutic benefit of other medical treatments.

[0378] Inhibition of MASP-2-dependent complement activation is characterized by at least one of the following changes in components of the complement system that occur as a result of administration of a MASP-2 inhibitory agent according to the methods of the invention: inhibition of the formation or production of the MASP-2-dependent complement activation system products C4b, C3a, C5a and / or C5b-9 (MAC) (measured, e.g., as described in Example 2); a reduction in complement activation assessed in a hemolytic assay using unsensitized rabbit or guinea pig red blood cells (measured, e.g., as described in Example 33); a reduction in C4 cleavage and C4b deposition (measured, e.g., as described in Example 2); or a reduction in C3 cleavage and C3b deposition (measured, e.g., as described in Example 2).

[0379] According to the present invention, MASP-2 inhibitory agents that are effective in inhibiting the MASP-2-dependent complement activation system are used. MASP-2 inhibitory agents that can be used to practice this aspect of the invention include, for example, anti-MASP-2 antibodies and fragments thereof, MASP-2 inhibitory peptides, small molecules, MASP-2 soluble receptors, and expression inhibitors. MASP-2 inhibitory agents can inhibit the MASP-2-dependent complement activation system by blocking the biological functions of MASP-2. For example, the inhibitory agent can effectively block protein-protein interactions of MASP-2, interfere with MASP-2 dimerization or assembly, block Ca2+ binding, interfere with the MASP-2 serine protease active site, or can reduce MASP-2 protein expression.

[0380] In some embodiments, the MASP-2 inhibitory agent selectively inhibits MASP-2 complement activation, leaving the Clq-dependent complement activation system functionally intact.

[0381] In one embodiment, the MASP-2 inhibitory agent used in the methods of the invention is a specific MASP-2 inhibitory agent that specifically binds to a polypeptide comprising SEQ ID NO: 6 with an affinity that is at least 10-fold greater than that for other antigens of the complement system. In another embodiment, the MASP-2 inhibitory agent specifically binds to a polypeptide comprising SEQ ID NO: 6 with an affinity that is at least 100-fold greater than that for other antigens of the complement system. The binding affinity of the MASP-2 inhibitory agent can be determined using an appropriate binding assay.

[0382] The MASP-2 polypeptide exhibits a molecular structure similar to that of MASP-1, MASP-3, and C1r and C1s (proteases of the C1 complement system). The cDNA molecule set forth in SEQ ID NO:4 encodes a representative example of MASP-2 (consisting of the amino acid sequence set forth in SEQ ID NO:5) and provides a human MASP-2 polypeptide with a leader sequence (aa 1-15) that is cleaved after secretion to produce a mature form of human MASP-2 (SEQ ID NO:6). As shown in FIG2 , the human MASP 2 gene includes 12 exons. The human MASP-2 cDNA is encoded by exons B, C, D, F, G, H, I, J, K, and L. Alternative splicing produces a 20 kDa protein, termed MBL-associated protein 19 ("MApl9", also known as "sMAP") (SEQ ID NO:2), which is encoded by (SEQ ID NO:1) produced from exons B, C, D, and E, as shown in FIG2 . The cDNA molecule set forth in SEQ ID NO:50 encodes murine MASP-2 (consisting of the amino acid sequence set forth in SEQ ID NO:51) and provides a murine MASP-2 polypeptide with a leader sequence that is cleaved after secretion to produce a mature form of murine MASP-2 (SEQ ID NO:52). The cDNA molecule set forth in SEQ ID NO:53 encodes rat MASP-2 (consisting of the amino acid sequence set forth in SEQ ID NO:54) and provides a rat MASP-2 polypeptide with a leader sequence that is cleaved after secretion to produce a mature form of rat MASP-2 (SEQ ID NO:55).

[0383] Those skilled in the art will recognize that the sequences disclosed in SEQ ID NO:4, SEQ ID NO:50 and SEQ ID NO:53 represent individual alleles of human, murine and rat MASP-2, respectively, and that allelic variation and alternative splicing are expected. Allelic variants of the nucleotide sequences set forth in SEQ ID NO:4, SEQ ID NO:50 and SEQ ID NO:53, including those containing silent mutations and those in which the mutations result in changes in the amino acid sequence, are within the scope of the present invention. Allelic variants of the MASP-2 sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures.

[0384] The domain structure of human MASP-2 protein (SEQ ID NO: 6) is shown in Figure 1 and 2AThe MASP2 gene is comprised of an N-terminal C1r / C1s / sea urchin Vegf / bone morphogenetic protein (CUBI) domain (aa 1-121 of SEQ ID NO: 6), an epidermal growth factor-like domain (aa 122-166), a second CUBI domain (aa 167-293), and a tandem complement control protein domain and a serine protease domain. Figure 1 MAp19 is a non-enzymatic protein that contains the N-terminal CUB1-EGF region of MASP-2 and four additional residues (EQSL) derived from exon E, as Figure 1 as shown in .

[0385] Several proteins have been shown to bind to or interact with MASP-2 through protein-protein interactions. For example, MASP-2 is known to bind to and form Ca2+-dependent complexes with the lectin proteins MBL, H-ficolin, and L-ficolin. Each MASP-2 / lectin complex has been shown to activate complement through 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. 176:1497-2284, 2000; Matsushita, M., et al., J. Immunol. 168:3502-3506, 2002). Studies have shown that the CUB1-EGF domain of MASP-2 is required for the association of MASP-2 with MBL (Thielens, NM et al., J. Immunol. 166:5068, 2001). It has also been shown that the CUB1EGFCUBII domain mediates dimerization of MASP-2, which is required for the formation of an active MBL complex (Wallis, R. et al., J. Biol. Chem. 275:30962-30969, 2000). Thus, MASP-2 inhibitors can be identified that bind to or interfere with MASP-2 target regions known to be important for MASP-2-dependent complement activation.

[0386] Anti-MASP-2 Antibodies

[0387] In some embodiments of this aspect of the invention, the MASP-2 inhibitory agent comprises an anti-MASP-2 antibody that inhibits the MASP-2-dependent complement activation system. The anti-MASP-2 antibodies used in this aspect of the invention include polyclonal, monoclonal, or recombinant antibodies derived from any mammal that produces antibodies, 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.

[0388] Several anti-MASP-2 antibodies have been described in the literature, some of which are listed in Table 1 below. These previously described anti-MASP-2 antibodies can be screened for the ability to inhibit the MASP-2-dependent complement activation system using the assays described herein. For example, anti-rat MASP-2 Fab2 antibodies that block MASP-2-dependent complement activation have been identified, as described in more detail in Examples 10 and 11 herein. Once an anti-MASP-2 antibody that functions as a MASP-2 inhibitory agent is identified, it can be used to generate anti-idiotypic antibodies and to identify other MASP-2 binding molecules, as described further below.

[0389] Table 1: MASP-2 specific antibodies from the literature

[0390]

[0391] Anti-MASP-2 antibodies with reduced effector function

[0392] In some embodiments of this aspect of the invention, the anti-MASP-2 antibody has reduced effector function to reduce inflammation that can result from activation of the classical complement pathway. The ability of an IgG molecule to trigger the classical complement pathway has been shown to be located within the Fc portion of the molecule (Duncan, AR et al., Nature 332:738-740 1988). IgG molecules in which the Fc portion of the 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 can be produced because they lack the Fc portion of the molecule, or are of the human IgG2 or IgG4 isotype, by having a genetically engineered Fc sequence that minimizes effector function.

[0393] Antibodies with reduced effector function can be generated by standard molecular biological manipulation of the Fc portion of IgG heavy chains, as described in Example 9 herein, and also 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, which have reduced ability to activate complement and / or interact with Fc receptors (Ravetch, JV et al., Annu. Rev. Immunol. 9:457-492, 1991; Isaacs, JD et al., J. Immunol. 148:3062-3071, 1992; van de Winkel, JG et al., Immunol. Today 14:215-221, 1993). Humanized or fully human antibodies specific for human MASP-2, including IgG2 or IgG4 isotypes, can be generated by one of several methods known to those of ordinary skill in the art, such as described in Vaughan, TJ et al., Nature Biotechnical 16:535-539, 1998.

[0394] Preparation of anti-MASP-2 antibodies

[0395] Anti-MASP-2 antibodies can be prepared using MASP-2 polypeptides (e.g., full-length MASP-2) or using peptides bearing antigenic MASP-2 epitopes (e.g., a portion of a MASP-2 polypeptide). Immunogenic peptides can be as small as 5 amino acid residues. For example, a MASP-2 polypeptide comprising the entire amino acid sequence of SEQ ID NO:6 can be used to induce anti-MASP-2 antibodies for use in the methods of the present invention. Specific MASP-2 domains known to be involved in protein-protein interactions, such as the CUBI and CUBIEGF domains, and regions including the serine-protease active site, can be expressed as recombinant polypeptides and used as antigens as described in Example 3. In addition, peptides comprising at least 6 amino acid portions of the MASP-2 polypeptide (SEQ ID NO:6) can also be used to induce MASP-2 antibodies. Other examples of MASP-2-derived antigens for inducing MASP-2 antibodies are provided in Table 2 below. MASP-2 peptides and polypeptides for use in generating antibodies can be isolated as natural polypeptides, or recombinant or synthetic peptides and catalytically inactive recombinant polypeptides, such 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 strains, as described in Examples 8 and 9 and further described below.

[0396] Antigens for raising anti-MASP-2 antibodies also include fusion polypeptides, such as fusions of MASP-2 or a portion thereof with an immunoglobulin polypeptide or with a maltose-binding protein. The polypeptide immunogen can be the full-length molecule or a portion thereof. If the polypeptide portion is hapten-like, the portion can advantageously be attached to a macromolecular carrier (e.g., a keyhole limpet). Hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.

[0397] Table 2: Antigens derived from MASP-2

[0398]

[0399] Polyclonal antibodies

[0400] Polyclonal antibodies to MASP-2 can be prepared by immunizing animals with MASP-2 polypeptides or immunogenic portions thereof using methods well known to those of ordinary skill in the art. See, e.g., Green et al., "Production of Polyclonal Antisera," in Immunochemical Protocols (Manson, ed.), p. 105, and as further described in Example 6. The immunogenicity of MASP-2 polypeptides can be enhanced by the use of adjuvants, including inorganic gels such as aluminum hydroxide or Freund's adjuvant (complete or incomplete), surface active substances such as lysolecithin, pluronic polyols, polyanions, oil emulsions, keyhole pores, Polyclonal antibodies are generally raised in animals such as horses, cows, dogs, chickens, rats, mice, rabbits, guinea pigs, goats or sheep. Alternatively, anti-MASP-2 antibodies for use in the present invention may also be derived from primates inferior to humans. General techniques for raising diagnostically and therapeutically useful antibodies in baboons can be found, for example, in Goldenberg et al., International Patent Publication No. WO 91 / 11465 and Losman, MJ et al., Int. J. Cancer 46:310, 1990. Serum containing immunologically active antibodies is then produced from the blood of the animals so immunized using standard procedures well known in the art.

[0401] Monoclonal antibodies

[0402] In some embodiments, the MASP-2 inhibitory agent is an anti-MASP-2 monoclonal antibody. Anti-MASP-2 monoclonal antibodies are highly specific, being directed against a single MASP-2 epitope. As used herein, the modifier "monoclonal" indicates the character of an antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring that the antibody be produced by any particular method. Monoclonal antibodies may be obtained using any technique that provides for the production of antibody molecules by continuous cell line cultures, such as the hybridoma method described in Kohler, G. et al., Nature 256:495, 1975, or they may be prepared by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567 to Cabilly). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson, T. et al., Nature 352:624-628, 1991 and Marks, JD et al., J. Mol. Biol. 222:581-597, 1991. Such antibodies may be of any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD, and any subclasses thereof.

[0403] For example, monoclonal antibodies can be obtained by injecting a suitable mammal (e.g., BALB / c mouse) with a composition comprising a MASP-2 polypeptide or portion thereof. After a predetermined period of time, spleen cells are removed from the mouse and suspended in a cell culture medium. The spleen cells are then fused with an immortal cell line to form a hybridoma. The resulting hybridomas are grown in cell culture and screened for their ability to produce monoclonal antibodies against MASP-2. An example further describing the production of anti-MASP-2 monoclonal antibodies is provided in Example 7. (See also Current Protocols in Immunology, Vol. 1., John Wiley & Sons, pp. 2.5.1-2.6.7, 1991).

[0404] Human monoclonal antibodies can be obtained by using transgenic mice that have been engineered to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human immunoglobulin heavy and light chain loci are introduced into a strain of mice derived from an embryonic stem cell line that contains targeted disruptions of the endogenous immunoglobulin heavy and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens, such as the MASP-2 antigens described herein, and the mice can be used to generate hybridomas that secrete human MASP-2 antibodies using conventional Kohler-Milstein techniques by fusing B-cells from the animals with an appropriate myeloma cell line, as further described in Example 7. Transgenic mice with human immunoglobulin genomes 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 in, for example, Green, LL et al., Nature Genet. 7:13, 1994; Lonberg, N. et al., Nature 368:856, 1994; and Taylor, LD et al., Int. Immun. 6:579, 1994.

[0405] Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such separation techniques include affinity chromatography with protein-A Sepharose, size exclusion chromatography, and ion exchange chromatography (see, e.g., Coligan, pp. 2.7.1-2.7.12 and pp. 2.9.1-2.9.3; Baines et al., "Purification of Immunoglobulin G (IgG)", in Methods in Molecular Biology, The Humana Press, Inc., Vol. 10, pp. 79-104, 1992).

[0406] Once generated, polyclonal, monoclonal, or phage-derived antibodies are first tested for specific MASP-2 binding. A variety of assays known to those of skill in the art can be used to detect antibodies that specifically bind to MASP-2. Exemplary assays include Western blot or immunoprecipitation analysis by standard methods (e.g., as described in Ausubel et al.), immunoelectrophoresis, enzyme-linked immunosorbent assays, dot blots, inhibition or competition assays, and sandwich assays (as described in Harlow and Land, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988). Once antibodies that specifically bind to MASP-2 are identified, the ability of the anti-MASP-2 antibody to act as a MASP-2 inhibitory agent is tested using one of several assays, e.g., a lectin-specific C4 cleavage assay (described in Example 2), a C3b deposition assay (described in Example 2), or a C4b deposition assay (described in Example 2).

[0407] The affinity of an anti-MASP-2 monoclonal antibody can be readily determined by one of ordinary skill in the art (see, e.g., Scatchard, A., NY Acad. Sci. 51:660-672, 1949). In one embodiment, the anti-MASP-2 monoclonal antibody used in the methods of the invention binds to MASP-2 with an affinity of <100 nM, preferably <10 nM and most preferably <2 nM. In some embodiments, the MASP-2 inhibitory monoclonal antibody used in the methods of the invention is a MASP-2 inhibitory monoclonal antibody or an antigen-binding fragment thereof comprising (I) (a) a heavy chain variable region comprising: i) a heavy chain CDR-H1 comprising the amino acid sequence of 31-35 of SEQ ID NO:67; and ii) a heavy chain CDR-H2 comprising the amino acid sequence of 50-65 of SEQ ID NO:67; and iii) a heavy chain CDR-H3 comprising the amino acid sequence of 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 24-34 of SEQ ID NO:70; and ii) a light chain CDR-L2 comprising the amino acid sequence of 50-56 of SEQ ID NO:70; and iii) a light chain CDR-L3 comprising the amino acid sequence of 89-97 of SEQ ID NO:70; or (II) a variant thereof comprising a heavy chain CDR-H2 comprising the amino acid sequence of SEQ ID NO:67; and iii) a heavy chain CDR-H3 comprising the amino acid sequence of 95-102 of SEQ ID NO:67; NO:67 has a heavy chain variable region that is at least 90% identical (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% identical to SEQ ID NO:67) and a light chain variable region that is at least 90% identical (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% identical) to SEQ ID NO:70.

[0408] Chimeric / humanized antibodies

[0409] The monoclonal antibodies used in the methods of the present invention include chimeric antibodies in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subtype, while the remainder of the chain is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subtype; as well as fragments of such antibodies (U.S. Pat. No. 4,816,567 to Cabilly; and Morrison, SL et al., Proc. Nat'l Acad. Sci. USA 81:6851-6855, 1984).

[0410] One form of chimeric antibody for use in the present invention is a humanized monoclonal anti-MASP-2 antibody. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulins. Humanized monoclonal antibodies are produced by transferring non-human (e.g., mouse) complementarity determining regions (CDRs) from the heavy and light variable chains of mouse immunoglobulins to human variable domains. Typically, residues from the human antibody are then substituted into the framework regions of the non-human counterparts. In addition, humanized antibodies may include residues that are not present in the recipient antibody or the donor antibody. These modifications may be performed to further refine antibody performance. Typically, humanized antibodies will include substantially all of at least one and typically two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the Fv framework regions are those of a human immunoglobulin sequence. The humanized antibody optionally will also include at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones, PT 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.

[0411] Humanized antibodies for use in the present invention include human monoclonal antibodies comprising at least the MASP-2 binding CDR3 region. In addition, the Fc portion can be replaced to generate IgA or IgM as well as human IgG antibodies. Such humanized antibodies will have particular clinical utility because they will specifically recognize human MASP-2 but will not elicit an immune response in humans against the antibody itself. Therefore, they are better suited for in vivo administration in humans, especially when repeated or long-term administration is desired.

[0412] An example of the generation of a humanized anti-MASP-2 antibody from a murine anti-MASP-2 monoclonal antibody is provided in Example 6 herein. Techniques for producing humanized monoclonal antibodies are also described in, e.g., Jones, PT et al., Nature 321:522, 1986; Carter, P., et al., Proc. Nat'l. Acad. Sci. USA 89:4285, 1992; Sandhu, JS, Crit. Rev. Biotech. 12:437, 1992; Singer, II et al., J. Immun. 150:2844, 1993; Sudhir (ed.), Antibody Engineering Protocols, Humana Press, Inc., 1995; Kelley, "Engineering Therapeutic Antibodies", in Protein Engineering: Principles and Practice, Cleland et al. (eds.), John Wiley & Sons, Inc., pp. 399-434, 1996; and Queen, U.S. Pat. No. 5,693,762, 1997. In addition, there are commercial organizations that synthesize humanized antibodies from specific murine antibody regions, such as Protein Design Labs (Mountain View, CA).

[0413] Recombinant Antibodies

[0414] Anti-MASP-2 antibodies can also be prepared using recombinant methods. For example, human antibodies can be prepared using human immunoglobulin expression libraries (available from, for example, Stratagene, Corp., La Jolla, CA) to produce fragments of human antibodies (VH, VL, Fv, Fd, Fab or F(ab')2). These fragments are then used to construct complete human antibodies using techniques similar to those used to produce chimeric antibodies.

[0415] Anti-idiotypic antibodies

[0416] Once anti-MASP-2 antibodies are identified as having the desired inhibitory activity, these antibodies can be used to generate anti-idiotypic antibodies that mimic a portion of MASP-2 using techniques well known in the art. See, e.g., Greenspan, NS et al., FASEB J. 7:437, 1993. For example, antibodies that bind to MASP-2 and compete for the MASP-2 protein interaction required for inhibition of complement activation can be used to generate anti-idiotypic antibodies that mimic the MBL binding site on the MASP-2 protein, thereby binding to and neutralizing a binding ligand of MASP-2, such as MBL.

[0417] Immunoglobulin fragments

[0418] MASP-2 inhibitory agents useful in the methods of the invention include not only intact immunoglobulin molecules, but also the well-known 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 formed from antibody fragments.

[0419] It is well known in the art that only a small part of the antibody molecule (paratope) participates in the binding of the antibody to its epitope (see, for example, Clark, WR, The Experimental Foundations of Modern Immunology, Wiley & Sons, Inc., NY, 1986). The pFc' and Fc regions of antibodies are effectors of the classical complement pathway, but do not participate in antigen binding. The pFc' region is produced from an antibody that has been enzymatically cleaved or has no pFc' region, and is referred to as a F(ab')2 fragment and retains two antigen binding sites of a complete antibody. The separated F(ab')2 fragment is referred to as a bivalent monoclonal fragment because of its two antigen binding sites. Similarly, the Fc region is produced from an antibody that has been enzymatically cleaved or has no Fc region, and is referred to as a Fab fragment and retains one antigen binding site of a complete antibody molecule.

[0420] Antibody fragments can be obtained by proteolysis, for example, by digesting intact antibodies with pepsin or papain by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to obtain a 5S fragment called F(ab')2. This fragment can be further cleaved using a sulfhydryl reducing agent to produce a 3.5S Fab' monovalent fragment. Optionally, the cleavage reaction can be carried out using a blocking group for the sulfhydryl groups produced by cleavage of disulfide bonds. Alternatively, enzymatic cleavage using pepsin directly produces two monovalent Fab fragments and an Fc fragment. These methods are described, for example, in Goldenberg, U.S. Pat. No. 4,331,647; Nisonoff, A. et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, RR, Biochem. J. 73:119, 1959; Edelman et al., in Methods in Enzymology 1:422, Academic Press, 1967; and Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.

[0421] In some embodiments, it is preferred to use an antibody fragment lacking an Fc region to avoid activation of the classical complement pathway, which starts when Fc binds to an Fcγ receptor. There are several methods for producing MoAbs that avoid Fcγ receptor interactions. For example, the Fc region of a monoclonal antibody can be chemically removed using a proteolytic enzyme partial digestion (e.g., ficin digestion) to produce, for example, an antigen-binding antibody fragment such as a Fab or F(ab)2 fragment (Mariani, M. et al., Mol. Immunol. 28: 69-71, 1991). Alternatively, a human γ4 IgG isotype, which does not bind to an Fcγ receptor, can be used during the construction of a humanized antibody, as described herein. Antibodies, single-chain antibodies, and antigen-binding domains lacking an Fc domain can also be engineered using the recombinant techniques described herein.

[0422] scFv

[0423] Alternatively, a single peptide chain binding molecule specific for MASP-2 can be created in which the heavy and light chain Fv regions are linked. The Fv fragments can be linked by a peptide linker to form a single chain antigen binding protein (scFv). These single chain antigen binding proteins are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains linked by oligonucleotides. The structural gene is inserted into an expression vector, which is then introduced into a host cell, such as E. coli. The recombinant host cell synthesizes a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing scFvs 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; Ladner, U.S. Pat. No. 4,946,778; Pack, P., et al., Bio / Technology 11:1271, 1993.

[0424] As an illustrative example, MASP-2 specific scFvs can be obtained by exposing lymphocytes to MASP-2 polypeptides in vitro and selecting antibody display libraries in phage or similar vectors (e.g., by using immobilized or labeled MASP-2 proteins or peptides). Genes encoding polypeptides having potential MASP-2 polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage or bacteria such as E. coli. These random peptide display libraries can be used to screen for peptides that interact with MASP-2. Techniques for generating and screening such random peptide display libraries are well known in the art (U.S. Pat. No. 5,223,409 to Lardner; U.S. Pat. No. 4,946,778 to Ladner; U.S. Pat. No. 5,403,484 to Lardner; U.S. Pat. No. 5,571,698 to Lardner; and Kay et al., Phage Display of Peptides and Proteins Academic Press, Inc., 1996), and random peptide display libraries and kits for screening such libraries are commercially available, for example, from CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly, Mass.), and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ).

[0425] Another form of 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 an antibody of interest. Such genes are prepared, for example, by synthesizing the variable region from RNA of antibody-producing cells using the polymerase chain reaction (see, e.g., Larrick et al., Methods: A Companion to Methods in Enzymology 2:106, 1991; Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in 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," in Monoclonal Antibodies: Principles and Applications, Birch et al. (eds.), page 137, Wiley-Liss, Inc., 1995).

[0426] The MASP-2 antibodies described herein are administered to a subject in need thereof to inhibit MASP-2-dependent complement activation. In some embodiments, the MASP-2 inhibitory agent is a high-affinity human or humanized monoclonal anti-MASP-2 antibody with reduced effector function.

[0427] Peptide inhibitors

[0428] In some embodiments of this aspect of the invention, the MASP-2 inhibitory agent includes an isolated MASP-2 peptide inhibitor, including isolated natural peptide inhibitors and synthetic peptide inhibitors that inhibit the MASP-2-dependent complement activation system. As used herein, the term "isolated MASP-2 peptide inhibitor" refers to a peptide that inhibits MASP-2-dependent complement activation by binding to another recognition molecule of the lectin pathway, competing 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 directly interacting with MASP-2 to inhibit MASP-2-dependent complement activation, which is substantially pure and substantially free of other substances with which they may be found in nature to the extent practical and appropriate for their intended use.

[0429] Peptide inhibitors have been successfully used in vivo to interfere with protein-protein interactions and catalytic sites. For example, peptide inhibitors of adhesion molecules structurally related to LFA-1 have recently been approved for clinical use in coagulopathy (Ohman, EM, et al., European Heart J. 16: 50-55, 1995). Short linear peptides (<30 amino acids) that prevent or interfere with integrin-dependent adhesion have been described (Murayama, O. et al., J. Biochem. 120: 445-51, 1996). Longer peptides, ranging in length from 25-200 amino acid residues, have been successfully used to block integrin-dependent adhesion (Zhang, L. et al., J. Biol. Chem. 271 (47): 29953-57, 1996). In general, longer peptide inhibitors have higher affinity and / or slower dissociation rates than short peptides and are therefore likely to be more effective inhibitors. Cyclic peptide inhibitors have been shown to be effective inhibitors of integrins in vivo for the treatment of human inflammatory diseases (Jackson, DY et al., J. Med. Chem. 40:3359-68, 1997). One method of producing cyclic peptides involves synthesizing peptides in which the terminal amino acids of the peptide are cysteine, thereby allowing the peptide to exist in a cyclic form via a disulfide bond between the terminal amino acids, which has been shown to improve affinity and half-life in vivo for the treatment of hematopoietic tumors (e.g., U.S. Pat. No. 6,649,592 to Larson).

[0430] Synthetic MASP-2 peptide inhibitors

[0431] 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 can be used to practice this aspect of the invention. Candidate MASP-2 inhibitory peptides can be tested for their ability to function as MASP-2 inhibitory agents in one of several assays, including, for example, the lectin-specific C4 cleavage assay (described in Example 2) and the C3b deposition assay (described in Example 2).

[0432] In some embodiments, the MASP-2 inhibitory peptide is derived from a MASP-2 polypeptide and a peptide selected from the full-length mature MASP-2 protein (SEQ ID NO:6), or selected from a specific domain 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 previously described, the CUBEGFCUBII region has been shown to be essential for dimerization and binding to MBL (Thielens et al., supra). Specifically, in studies identifying humans with a homozygous mutation at Asp105 to Gly105, 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, resulting in loss of MASP-2 from the MBL complex (Stengaard-Pedersen, K. et al., New England J. Med. 349:554-560, 2003).

[0433] In some embodiments, the MASP-2 inhibitory peptide is derived from a lectin protein that binds MASP-2 and participates in the lectin complement pathway. Several different lectins involved in this pathway have been identified, including mannan-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, each with an N-terminal collagen-like fiber that has a carbohydrate recognition domain. These various lectins have been shown to bind MASP-2, and the lectin / MASP-2 complex activates complement through cleavage of proteins C4 and C2. H-ficolin has an amino-terminal region of 24 amino acids, a collagen-like domain with 11 Gly-Xaa-Yaa repeats, a neck domain of 12 amino acids, and a fibrinogen-like domain of 207 amino acids (Matsushita, M. et al., J. Immunol. 168:3502-3506, 2002). H-ficolin binds GlcNAc and agglutinates human erythrocytes coated with LPS derived from S. Typhimurium, S. Minnesota, and E. coli. H-ficolin has been shown to associate with MASP-2 and MApl9, activating the lectin pathway (supra). L-ficolin / P35 also binds GlcNAc and has been shown to associate with MASP-2 and MAp19 in human serum, and this complex has been shown to activate the lectin pathway (Matsushita, M. et al., J. Immunol. 164:2281, 2000). Thus, a MASP-2 inhibitory peptide for use in the present invention may include a region of at least 5 amino acids selected from the group consisting of MBL protein (SEQ ID NO:21), H-ficolin (Genbank Accession No. NM_173452), M-ficolin (Genbank Accession No. 000602), and L-ficolin (Genbank Accession No. NM_015838).

[0434] More specifically, scientists have identified a MASP-2 binding site on MBL that is located within 12 Gly-XY triplets "GKD GRD GTK GEK GEP GQG LRG LQG POG KLG POGNOG PSG SOG PKG QKG DOG KS" (SEQ ID NO: 26), between the hinge and neck regions of the C-terminal portion of the collagen-like domain of MBP (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 present in all three lectin proteins has been described, comprising the amino acid sequence "OGK-X-GP" (SEQ ID NO: 22), wherein the letter "O" represents hydroxyproline and the letter "X" is a hydrophobic residue (Wallis et al., 2004, supra). Thus, in some embodiments, the MASP-2 inhibitory peptides used in this aspect of the invention are at least 6 amino acids long and include SEQ ID NO: 22. A peptide derived from MBL, which includes the amino acid sequence "GLR GLQGPO GKL GPO G" (SEQ ID NO: 24), has been shown to bind MASP-2 in vitro (Wallis et al., 2004, supra). To enhance binding to MASP-2, a peptide can be synthesized with two GPO triplets flanking each terminus ("GPO GPO GLR GLQ GPOGKL GPO GGP OGP O" SEQ ID NO: 25) to enhance triple helix formation, as found in native MBL protein (further described in Wallis, R. et al., J. Biol. Chem. 279: 14065, 2004).

[0435] MASP-2 inhibitory peptides may also be derived from human H-ficolin, which include the sequence "GAO GSO GEK GAO GPQ GPO GPO GKM GPK GEO GDO" (SEQ ID NO: 27) from the consensus MASP-2 binding region in H-ficolin. Also included are peptides derived from human L-ficolin, which include the sequence "GCO GLO GAO GDK GEA GTN GKR GER GPO GPO GKA GPO GPN GAO GEO" (SEQ ID NO: 28) from the consensus MASP-2 binding region in L-ficolin.

[0436] MASP-2 inhibitory peptides may also be derived from a C4 cleavage site, such as "LQRALEILPNRVTIKANRPFLVFI" (SEQ ID NO:29), which is a C4 cleavage site attached to the C-terminal portion of antithrombin III (Glover, GI et al., Mol. Immunol. 25:1261 (1988)).

[0437] TABLE 3 Exemplary MASP-2 Inhibitory Peptides

[0438]

[0439]

[0440] Note: The letter "O" represents hydroxyproline. The letter "X" is a hydrophobic residue.

[0441] Peptides derived from the C4 cleavage site, as well as other peptides that inhibit the MASP-2 serine protease site, can be chemically modified so that they are irreversible protease inhibitors. For example, suitable modifications may include, but are not necessarily limited to, halomethylketones (Br, Cl, I, F) at the C-terminus, Asp or Glu, or appended to a functional side chain; haloacetyl (or other α-haloacetyl) on the amino or other functional side chain; epoxide or imine containing groups at the amino or carboxyl terminus or on a functional side chain; or imidates at the amino or carboxyl terminus or on a functional side chain. Such modifications would provide the benefit of permanent inhibition of the enzyme by covalent attachment of the peptide. This could result in a lower effective dose and / or require less frequent administration of the peptide inhibitor.

[0442] In addition to the inhibitory peptides described above, MASP-2 inhibitory peptides useful in the methods of the present invention include peptides comprising the MASP-2-binding CDR3 region of an anti-MASP-2 MoAb obtained as described herein. The sequence of the CDR region used to synthesize the peptide can be determined by methods known in the art. The heavy chain variable region is a peptide that generally ranges from 100-150 amino acids in length. The light chain variable region is a peptide that generally ranges from 80-130 amino acids in length. The CDR sequences within the heavy and light chain variable regions only include sequences of approximately 3-25 amino acids, which can be readily sequenced by one of ordinary skill in the art.

[0443] Those skilled in the art will recognize that substantially homologous variants of the above-described MASP-2 inhibitory peptides will also exhibit MASP-2 inhibitory activity. Exemplary variants include, but are not necessarily limited to, peptides having insertions, deletions, substitutions, and / or additional amino acids at the carboxyl terminal or amino terminal portions of the subject peptides, and mixtures thereof. Thus, those homologous peptides having MASP-2 inhibitory activity are contemplated for use in the methods of the present invention. The peptides may also include repeating motifs and other modifications having conservative substitutions. Conservative variants are described elsewhere herein and include the exchange of an amino acid for another amino acid of similar charge, size, or hydrophobicity, etc.

[0444] The MASP-2 inhibitory peptides may be modified to increase solubility and / or maximize positive or negative charge to more closely mimic segments in the intact protein. Derivatives may or may not have the exact primary amino acid structure of the peptides disclosed herein, as long as the derivatives functionally retain the desired MASP-2 inhibitory properties. Modifications may include amino acid substitution with one of the 20 commonly known amino acids or with another amino acid, with a derivatized or substituted amino acid having auxiliary desirable properties (e.g., resistance to enzymatic degradation), or with a D-amino acid, or with another molecule or compound such as a carbohydrate that mimics the native conformation and function of an amino acid, multiple amino acids, or peptide; amino acid deletion; amino acid insertion with one of the 20 commonly known amino acids or with another amino acid, with a derivatized or substituted amino acid having auxiliary desirable properties (e.g., resistance to enzymatic degradation), or with a D-amino acid, or with another molecule or compound such as a carbohydrate that mimics the native conformation and function of an amino acid, multiple amino acids, or peptide; or with another molecule or compound such as a carbohydrate or nucleic acid monomer that mimics the native conformation, charge distribution, and function of the parent peptide. The peptides may also be modified by acetylation or amidation.

[0445] The synthesis of derivative inhibitory peptides may rely on known techniques for peptide biosynthesis, carbohydrate biosynthesis, and the like. As a starting point, the skilled artisan 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, the skilled artisan may determine in a rational design manner what type of substitutions may be made at one or more sites to form derivatives that retain the basic conformation and charge distribution of the parent peptide but may have properties that are not present in the parent peptide or that are enhanced over those present in the parent peptide. Once candidate derivative molecules are identified, the derivatives may be tested using the assays described herein to determine whether they function as MASP-2 inhibitory agents.

[0446] Screening for MASP-2 inhibitory peptides

[0447] Molecular modeling and rational molecular design can also be used to generate and screen for molecular structures that mimic the key binding regions of MASP-2 and peptides that inhibit the complement activity of MASP-2. Molecular structures used for modeling include CDR regions of anti-MASP-2 monoclonal antibodies, as well as target regions known to be important for MASP-2 function, including regions required for dimerization, regions involved in binding to MBL, and serine protease active sites, as previously described. Methods for identifying peptides that bind to specific targets are well known in the art. For example, molecular imprinting can be used to construct macromolecular structures, such as peptides that bind to specific molecules, from scratch. See, for example, Shea, KJ, "Molecular Imprinting of Synthetic Network Polymers: The De Novosynthesis of Macromolecular Binding and Catalytic Sties," TRIP 2 (5) 1994.

[0448] As an illustrative example, one method for preparing a mimetic of a MASP-2 binding peptide is as follows. Functional monomers (templates) of a known MASP-2 binding peptide or binding region of an anti-MASP-2 antibody that exhibits MASP-2 inhibition are polymerized. The template is then removed, and a second type of monomer is polymerized in the void left by the template to provide a new molecule that exhibits one or more desired properties similar to the template. In addition to preparing peptides in this manner, other MASP-2 binding molecules such as polysaccharides, nucleosides, drugs, nucleoproteins, lipoproteins, carbohydrates, glycoproteins, steroids, lipids, and other biologically active substances that are MASP-2 inhibitory agents can also be prepared. This method can be used to design a variety of biomimetics that are more stable than their natural counterparts because they are typically prepared by free radical polymerization of functional monomers, resulting in compounds with non-biodegradable backbones.

[0449] Peptide synthesis

[0450] MASP-2 inhibitory peptides can be prepared using techniques well known in the art, such as solid phase synthesis techniques, which were originally described by Merrifield in J. Amer. Chem. Soc. 85:2149-2154, 1963. Automated synthesis can be achieved, for example, using an Applied Biosystems 431A Peptide Synthesizer (Foster City, Calif.) according to the instructions provided by the manufacturer. Other techniques can be found in, for example, Bodanszky, M. et al., Peptide Synthesis, 2nd Edition, John Wiley & Sons, 1976, and other references known to those skilled in the art.

[0451] Peptides can also be prepared using standard genetic engineering techniques known to those skilled in the art. For example, peptides can be produced enzymatically: a nucleic acid encoding the peptide is inserted into an expression vector, the DNA is expressed, and the DNA is translated into a peptide in the presence of the desired amino acids. The peptide is then purified using chromatography or electrophoresis techniques, or with the aid of a carrier protein that can be fused to the peptide by inserting the nucleic acid sequence encoding the carrier protein into an expression vector in phase with the peptide coding sequence and then cleaved from the peptide. The fusion protein-peptide can be separated using chromatography, electrophoresis, or immunological techniques (e.g., by binding of an antibody to the carrier protein to a resin). The peptide can be cleaved using chemical methods or by enzymes, such as hydrolases.

[0452] The MASP-2 inhibitory peptides useful in the methods of the present invention can also be produced in recombinant host cells according to conventional techniques. In order to express the MASP-2 inhibitory peptide coding sequence, the nucleic acid molecule encoding the peptide must be operably linked to a regulatory sequence that controls transcriptional expression in 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 marker genes that are suitable for selecting cells carrying the expression vector.

[0453] Nucleic acid molecules encoding MASP-2 inhibitory peptides can be synthesized using a "gene machine" using protocols such as the phosphoimidate method. If chemically synthesized duplexes are desired for an application such as the synthesis of genes or gene fragments, each complementary strand is prepared separately. The production of short genes (60-80 base pairs) is technically simple and can be achieved by synthesizing complementary strands and then annealing them. For the production of longer genes, the synthetic genes (duplexes) are assembled in modular form from single-stranded fragments of 20-100 nucleotides in length. For a review of polynucleotide synthesis, see, e.g., 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. Nat'l Acad. Sci. USA 87:633, 1990.

[0454] Small molecule inhibitors

[0455] In some embodiments, the MASP-2 inhibitory agent is a small molecule inhibitor with a low molecular weight, including natural and synthetic substances, such as peptides, peptidomimetics, and non-peptide inhibitors (including oligonucleotides and organic compounds). Small molecule inhibitors of MASP-2 can be generated based on the molecular structure of the variable region of an anti-MASP-2 antibody.

[0456] Small molecule inhibitors can also be designed and generated using computational drug design based on the MASP-2 crystal structure (Kuntz ID 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 methods described by Kuntz et al., the MASP-2 crystal structure coordinates are used as input to a computer program such as DOCK, which outputs a list of small molecule structures that are expected 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 inhibitors was used to identify unique non-peptide ligands that are inhibitors of HIV-1 protease by evaluating the degree of fit of compounds present in the Cambridge Crystallographic Database to the binding site of the enzyme using the program DOCK (Kuntz, ID et al., J. Mol. Biol. 161:269-288, 1982; Des Jarlais, RL, et al., PNAS 87:6644-6648, 1990).

[0457] A list of small molecule structures identified as potential MASP-2 inhibitory agents by in silico methods is screened using a MASP-2 binding assay, such as the assay described in Example 10. Small molecules found to bind MASP-2 are then assayed in a functional assay, such as the assay described in Example 2, to determine whether they inhibit MASP-2-dependent complement activation.

[0458] MASP-2 soluble receptor

[0459] Other suitable MASP-2 inhibitory agents are believed to include MASP-2 soluble receptors, which can be produced using techniques known to those of ordinary skill in the art.

[0460] Inhibitors of MASP-2 expression

[0461] In another embodiment of this aspect of the invention, the MASP-2 inhibitory agent is a MASP-2 expression inhibitory agent that is capable of inhibiting MASP-2-dependent complement activation. In practicing this aspect of the invention, representative MASP-2 expression inhibitory agents include MASP-2 antisense nucleic acid molecules (e.g., antisense mRNA, antisense DNA, or antisense oligonucleotides), MASP-2 ribozymes, and MASP-2 RNAi molecules.

[0462] Antisense RNA and DNA molecules are used to directly block the translation of MASP-2 mRNA by hybridizing to MASP-2 mRNA and preventing the translation of MASP-2 protein. Antisense nucleic acid molecules can be constructed in many different ways, provided that it is capable of interfering with the expression of MASP-2. For example, antisense nucleic acid molecules can be constructed by inverting the coding region (or a portion thereof) of MASP-2 cDNA (SEQ ID NO: 4) relative to its normal transcription orientation to allow transcription of its complement.

[0463] Antisense nucleic acid molecules are generally substantially identical to at least a portion of one or more target genes. However, nucleic acids do not need to be identical to inhibit expression. Generally, higher homology can be used to compensate for the use of shorter antisense nucleic acid molecules. Minimum percentage identity is generally greater than about 65%, but higher percentage identity can produce more effective suppression of the expression of endogenous sequences. Significantly larger percentage identities exceeding about 80% are generally preferred, although absolute identities of about 95% are generally most preferred.

[0464] The antisense nucleic acid molecule need not have the same intron or exon pattern as the target gene, and noncoding segments of the target gene can be as effective as coding segments in achieving antisense repression of target gene expression. DNA sequences of at least about 8 or so nucleotides can be used as antisense nucleic acid molecules, although longer sequences are preferred. In the present invention, a representative example of a useful MASP-2 inhibitory agent is an antisense MASP-2 nucleic acid molecule having at least 90% identity to the complement of the MASP-2 cDNA consisting of the nucleic acid sequence set forth in SEQ ID NO:4. The nucleic acid sequence set forth in SEQ ID NO:4 encodes a MASP-2 protein consisting of the amino acid sequence set forth in SEQ ID NO:5.

[0465] Targeted binding of antisense oligonucleotides 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 muscarinic type 2 acetylcholine receptor is inhibited by antisense oligonucleotides directed against their respective mRNA sequences (U.S. Pat. No. 5,739,119 to Cheng and U.S. Pat. No. 5,759,829 to Shewmaker). In addition, examples of antisense inhibition are used for nuclear protein cyclins, multidrug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABA A Receptors and human EGF have been demonstrated (see, e.g., U.S. Pat. No. 5,801,154 to Baracchini; U.S. Pat. No. 5,789,573 to Baker; U.S. Pat. No. 5,718,709 to Considine; and U.S. Pat. No. 5,610,288 to Reubenstein).

[0466] Systems have been described that allow one of ordinary skill to determine which oligonucleotides can be used in the present invention, including probing for appropriate sites in the target mRNA using RNase H cleavage as an indicator of the accessibility of sequences within the transcript. Scherr, M. et al., Nucleic Acids Res. 26:5079-5085, 1998; Lloyd, et al., Nucleic Acids Res. 29:3665-3673, 2001. A mixture of antisense oligonucleotides complementary to certain regions of the MASP-2 transcript is added to an extract of cells expressing MASP-2, such as hepatocytes, and hybridized to generate RNAse H susceptible sites. This method can be combined with computer-assisted sequence selection, which can predict the best sequence selection for antisense compositions based on their relative ability to form dimers, hairpin structures, or other secondary structures that will reduce or inhibit specific binding to the target mRNA in the host cell. These secondary structure analysis and target site selection considerations can be carried out using OLIGO primer analysis software (Rychlik, I., 1997) and BLASTN 2.0.5 algorithm software (Altschul, SF, et al., Nucl. Acids Res. 25: 3389-3402, 1997). Antisense compounds for target sequences preferably include about 8-about 50 nucleotides in length. Antisense oligonucleotides including about 9-about 35 nucleotides are particularly preferred. The inventors have considered that for implementing the method based on antisense oligonucleotides of the present invention, all oligonucleotide compositions ranging from 9-35 nucleotides (i.e., 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 base lengths) are highly preferred. Highly preferred target regions of the MASP-2 mRNA are those at or near the AUG translation start codon, and those sequences that are substantially complementary to the 5' region of the mRNA, e.g., between the -10 and +10 regions of the MASP-2 gene nucleotide sequence (SEQ ID NO: 4). Exemplary MASP-2 expression inhibitors are provided in Table 4.

[0467] TABLE 4 Exemplary expression inhibitors of MASP-2

[0468]

[0469] As described above, the term "oligonucleotide" as used herein refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or its analogs. The term also encompasses those oligonucleoside bases, including naturally occurring nucleotides, sugars and covalent nucleoside internucleoside (main chain) bonds and oligonucleotides with non-naturally occurring modifications. These modifications allow the introduction of certain required properties that cannot be provided by naturally occurring oligonucleotides, such as toxicity reduction, increased stability for nuclease degradation and increased cellular uptake. In illustrative embodiments, the antisense compounds of the present invention are different from natural DNA in that the phosphodiester backbone is modified to extend the life of the antisense oligonucleotide, and the phosphate substituent in the antisense oligonucleotide is replaced with thiophosphate. Similarly, one or both ends of an oligonucleotide can be substituted by one or more acridine derivatives, which are embedded between the adjacent base pairs in the nucleic acid chain.

[0470] Another alternative to antisense is to use "RNA interference" (RNAi). Double-stranded RNA (dsRNA) can cause gene silencing in mammals. The natural function and co-repression of RNAi appear to be the protection of the genome against mobile genetic elements such as retrotransposons and viral invasions, which produce abnormal RNA or dsRNA in host cells when they become active (see, for example, Jensen, J., et al., Nat. Genet. 21: 209-12, 1999). Double-stranded RNA molecules can be prepared by synthesizing two RNA chains that can form double-stranded RNA molecules, each chain having a length of about 19 to 25 (e.g., 19-23 nucleotides). For example, the dsRNA molecules used in the method of the present invention may include RNA corresponding to the sequences listed in Table 4 and their complementary sequences. Preferably, at least one chain of RNA has a 3' protrusion of 1-5 nucleotides. The synthetic RNA chains merge under the conditions of forming double-stranded molecules. The RNA sequence may include a portion of at least 8 nucleotides of SEQ ID NO:4, with a total length of 25 nucleotides or less. Designing siRNA sequences for a given target is within the ordinary skill in the art. Commercial services that design siRNA sequences and guarantee at least 70% knockdown of expression are available (Qiagen, Valencia, Calif).

[0471] dsRNA can be administered as a pharmaceutical composition and by known methods, wherein the nucleic acid is introduced into the desired target cell. Commonly used gene transfer methods include calcium phosphate, DEAE-dextran, electroporation, microinjection and viral methods. Such methods are taught in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 1993.

[0472] Ribozymes can also be used to reduce the amount and / or biological activity of MASP-2, such as ribozymes that target MASP-2 mRNA. Ribozymes are catalytic RNA molecules that can cleave nucleic acid molecules having sequences that are completely or partially homologous to the ribozyme sequence. It is possible to design ribozyme transgenes encoding RNA ribozymes that specifically pair with target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In performing this cleavage, the ribozyme itself is not altered and is therefore able to circulate and cleave other molecules. Inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity to them, thereby increasing the activity of the antisense construct.

[0473] Ribozymes useful in practicing the invention generally include a hybridizing region of at least about 9 nucleotides that is complementary in nucleotide sequence to at least a portion of a target MASP-2 mRNA, and a catalytic region that is suitable for cleaving the target MASP-2 mRNA (see generally EPA No. 0321201; WO 88 / 04300; Haseloff, J. et al., Nature 334:585-591, 1988; Fedor, MJ, et al., Proc. Natl. Acad. Sci. USA 87:1668-1672, 1990; Cech, TR, et al., Ann. Rev. Biochem. 55:599-629, 1986).

[0474] Ribozymes can be directly targeted to cells in the form of RNA oligonucleotides incorporating the ribozyme sequence, or introduced into cells as expression vectors encoding the desired ribozyme RNA. Ribozymes can be used and applied in much the same manner as described for antisense polynucleotides.

[0475] Antisense RNA and DNA, ribozymes and RNAi molecules for the inventive method can be prepared by any method known in the art for synthesizing DNA and RNA molecules. These methods include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well-known in the art, such as solid phase phosphoimidate chemical synthesis. Alternatively, RNA molecules can be produced by in vitro and in vivo transcription of DNA sequences encoding antisense RNA molecules. Such DNA sequences can be incorporated into various vectors comprising suitable RNA polymerase promoters such as T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNAs compositionally or inductively (depending on the promoter used) can be stably introduced into cell lines.

[0476] Various well-known modifications of DNA molecules can be introduced as a means of increasing stability and half-life. Useful modifications include, but are not limited to, the addition of ribonucleotide or deoxyribonucleotide flanking sequences to the 5' and / or 3' ends of the molecule or the use of phosphorothioate or 2'O-methyl rather than phosphodiesterase bonds within the oligodeoxyribonucleotide backbone.

[0477] VI. Pharmaceutical Compositions and Delivery Methods

[0478] Drug administration

[0479] In another aspect, the invention provides compositions for inhibiting the adverse 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 inhibitory agent and a pharmaceutically acceptable carrier. The MASP-2 inhibitory agent can be administered to a subject in need thereof at a therapeutically effective dose to treat or ameliorate a condition associated with MASP-2-dependent complement activation. A therapeutically effective dose refers to an amount of the MASP-2 inhibitory agent sufficient to result in an improvement in the symptoms associated with the disease or condition.

[0480] Toxicity and therapeutic efficacy of MASP-2 inhibitory agents can be determined by standard pharmaceutical procedures using experimental animal models, such as the murine MASP-2- / - mouse model expressing a human MASP-2 transgene as described in Example 1. Using such animal models, the NOAEL (no observed adverse effect level) and the MED (minimum effective dose) can be determined using standard methods. The dose ratio between the NOAEL and the MED effect is the therapeutic ratio, which is expressed as the ratio NOAEL / MED. MASP-2 inhibitory agents that exhibit large therapeutic ratios or indices are most preferred. Data obtained from cell culture assays and animal studies can be used to formulate a dosage range for human use. The dosage of the MASP-2 inhibitory agent lies preferably within a range of circulating concentrations that include the MED with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

[0481] For any compound formulation, the therapeutically effective dose can be estimated using animal models. For example, a dose can be formulated in an animal model to achieve a circulating plasma concentration range that includes the MED. Quantitative levels of the MASP-2 inhibitory agent in plasma can also be measured, for example, by high performance liquid chromatography.

[0482] In addition to toxicity studies, effective doses can also be estimated based on the amount of MASP-2 protein present in living subjects and the binding affinity of the MASP-2 inhibitory agent. It has been shown that MASP-2 levels in normal human subjects are present in serum at low levels in the range of 500 ng / ml, and the level of MASP-2 in a specific subject can be determined using a quantitative assay for MASP-2, which is described in Moller-Kristensen M. et al., J. Immunol. Methods 282: 159-167, 2003.

[0483] Generally, the dosage of the composition comprising the MASP-2 inhibitory agent administered varies depending on factors such as the age, weight, height, sex, general medical condition and prior medical history of the subject. As an example, a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, can be administered in a dosage range of about 0.010 to 10.0 mg / kg, preferably 0.010 to 1.0 mg / kg, more preferably 0.010 to 0.1 mg / kg of the subject's body weight. In some embodiments, the composition comprises a combination of an anti-MASP-2 antibody and a MASP-2 inhibitory peptide.

[0484] The therapeutic efficacy and appropriate dosage of the MASP-2 inhibitory compositions and methods of the invention in a given subject can be determined according to complement assays well known to those skilled in the art. Complement produces many specific products. In the last decade, sensitive and specific assays have been developed and are commercially available for most of these activation products, including the small activation fragments C3a, C4a and C5a and the large activation fragments iC3b, C4d, Bb and sC5b-9. Most of these assays utilize monoclonal antibodies that react with neoantigens exposed on the fragments rather than on the native protein from which they are formed, which makes these assays very simple and specific. Most rely on ELISA techniques, although radioimmunoassays are sometimes still used for C3a and C5a. These latter assays measure the unprocessed fragments and their "des-Arg" fragments, which are the major forms present in circulation. The unprocessed fragments and C5a 脱Arg It is rapidly cleared by binding to cell surface receptors and is therefore present in very low concentrations, whereas C3a 脱ArgDoes not bind cells and accumulates in plasma. Measurement of C3a provides a sensitive, pathway-independent indicator of complement activation. Alternative pathway activation can be evaluated by measuring the Bb fragment. Detection of sC5b-9, the liquid phase product of membrane attack pathway activation, provides evidence that complement is activated to completion. Because both the lectin and classical pathways produce the same activation products, C4a and C4d, measurement of these two fragments does not provide any information about which of the two pathways produces the activation products.

[0485] Inhibition of MASP-2-dependent complement activation is characterized by at least one of the following changes in components of the complement system that occur as a result of administration of a MASP-2 inhibitory agent according to the methods of the invention: inhibition of the formation or production of the MASP-2-dependent complement activation system products C4b, C3a, C5a and / or C5b-9 (MAC) (measured, e.g., as described in Example 2), reduction in C4 cleavage and C4b deposition (measured, e.g., as described in Example 10), or reduction in C3 cleavage and C3b deposition (measured, e.g., as described in Example 10).

[0486] Other reagents

[0487] Compositions and methods comprising a MASP-2 inhibitory agent may optionally include one or more other therapeutic agents that may increase the activity of the MASP-2 inhibitory agent or provide a related therapeutic function in an additive or synergistic manner. For example, in the case of treating a subject with TTP, wherein the subject is positive for an ADAM-TS13 inhibitor, one or more MASP-2 inhibitory agents may be administered in combination (including co-administered) with one or more immunosuppressive agents. Suitable immunosuppressive agents include: corticosteroids, rituxan, cyclosporine, and the like. In the case of treating a subject with or at risk for developing HUS or aHUS, one or more MASP-2 inhibitory agents may be administered in combination (including co-administered) with a suitable antibiotic. In the case of treating a subject with or at risk for developing aHUS, one or more MASP-2 inhibitory agents may be administered in combination (including co-administered) with other complement inhibitors such as eculizumab (Soliris), TT-30, antibodies to factor B, or other agents that inhibit amplification of the terminal complement components or the alternative pathway.

[0488] The inclusion and selection of other agents will be determined to achieve the desired therapeutic outcome. In some embodiments, the MASP-2 inhibitory agent may be administered in combination with one or more anti-inflammatory and / or analgesic agents. Suitable anti-inflammatory and / or analgesic agents include: serotonin receptor antagonists; serotonin receptor agonists; histamine receptor antagonists; bradykinin receptor antagonists; kallikrein inhibitors; tachykinin receptor antagonists, including neurokinin 1 and neurokinin 2Receptor subtype antagonists; calcitonin gene-related peptide (CGRP) receptor antagonists; interleukin receptor antagonists; inhibitors of enzymes active in the arachidonic acid metabolite synthesis pathway, including phospholipase inhibitors, including PLA 2 isoform inhibitors and PLCγ isoform inhibitors, cyclooxygenase (COX) inhibitors (which may be COX-1, COX-2 or non-selective COX-1 and -2 inhibitors), lipoxygenase inhibitors; prostanoid receptor antagonists, including eicosanoid EP-1 and EP-4 receptor subtype antagonists and thromboxane receptor subtype antagonists; leukotriene receptor antagonists, including leukotriene B4 receptor subtype antagonists and leukotriene D4 receptor subtype antagonists; opioid receptor agonists, including μ-opioid, δ-opioid and κ-opioid receptor subtype agonists; purine receptor agonists and antagonists, including P2X receptor antagonists and P2Y receptor agonists; adenosine triphosphate (ATP)-sensitive potassium channel openers; MAP kinase inhibitors; nicotinic acetylcholine inhibitors; and alpha adrenergic receptor agonists (including alpha-1, alpha-2 and non-selective alpha-1 and 2 agonists).

[0489] The MASP-2 inhibitory agents of the invention may also be administered in combination with one or more other complement inhibitors, such as inhibitors of C5. An antibody directed against C5 is the only complement-targeted drug that has been approved for human use. However, several agents have been shown to block complement in vivo. K76COOH and nafamstat mesilate are two agents that have shown some effectiveness in animal models of transplantation (Miyagawa, S. et al., Transplant Proc. 24:483-484, 1992). Low molecular weight heparin has also been shown to be effective in modulating complement activity (Edens, RE et al., Complement Today, pp. 96-120, Basel: Karger, 1993). It is believed that these small molecule inhibitors may be used as agents for use in combination with the MASP-2 inhibitory agents of the present invention.

[0490] Other naturally occurring complement inhibitors may be used in combination with the MASP-2 inhibitory agents of the invention. Biological inhibitors of complement include soluble complement factor 1 (sCR1). This is a naturally occurring inhibitor that is found on the outer membrane of human cells. Other membrane inhibitors include DAF, MCP, and CD59. Recombinant forms have been tested for their anti-complement activity in vitro and in vivo. sCR1 has been shown to be effective in xenotransplantation, where the complement system (both alternative and classical) provides the trigger for a hyperactive rejection syndrome within minutes of perfusing blood through the newly transplanted organ (Platt, JL et al., Immunol. Today 11:450-6, 1990; Marino, IR et al., Transplant Proc. 1071:6, 1990; Johnstone, PS et al., Transplantation 54:573-6, 1992). Administration of sCR1 protects and prolongs the survival of transplanted organs, and the complement pathway has been implicated in the pathogenesis of organ survival (Leventhal, JR et al., Transplantation 55:857-66, 1993; Pruitt, SK et al., Transplantation 57:363-70, 1994).

[0491] By way of example, suitable additional complement inhibitors for use in combination with the compositions of the invention also include MoAbs, such as anti-C5 antibodies (eg, eculizumab) and anti-properdin MoAbs developed by Alexion Pharmaceuticals, Inc., New Haven, Connecticut.

[0492] Pharmaceutical carriers and delivery vehicles

[0493] Typically, the MASP-2 inhibitory compositions of the invention, in combination with any other selected therapeutic agents, are suitably contained in a pharmaceutically acceptable carrier. The carrier is nontoxic, biocompatible and selected so as not to adversely affect the biological activity of the MASP-2 inhibitory agent (and any other therapeutic agent combined therewith). Exemplary pharmaceutically acceptable carriers for peptides are described in U.S. Pat. No. 5,211,657 to Yamada. The anti-MASP-2 antibodies and inhibitory peptides used in the invention can be formulated into preparations in solid, semisolid, gel, liquid or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, depot formulations, inhalants and injections, which allow for oral, parenteral or surgical administration. The invention also contemplates topical administration of the compositions by coating medical devices and the like.

[0494] Suitable carriers for parenteral delivery and topical delivery by injection, infusion or lavage include distilled water, physiological phosphate buffered saline, normal or lactated Ringer's solution, glucose solution, Hank's solution or propylene glycol. In addition, sterile fixed oils can be used as solvents or suspension media. For this purpose, any biocompatible oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can be used to prepare injections. Carriers and reagents can be mixed into liquids, suspensions, polymerized or non-polymerized gels, pastes or ointments.

[0495] The carrier may also include a delivery vehicle to maintain (i.e., extend, delay or regulate) the delivery of an agent or enhance the delivery, uptake, stability or pharmacokinetics of a therapeutic agent. By way of non-limiting example, such a delivery vehicle may include microparticles, microspheres, nanospheres or nanoparticles comprising a protein; liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymerized or copolymerized hydrogels and polymerized micelles. Suitable hydrogel and micellar delivery systems include PEO:PHB:PEO copolymers and copolymer / cyclodextrin complexes disclosed in WO 2004 / 009664 A2, and PEO and PEO / cyclodextrin complexes disclosed in U.S. Patent Application No. 2002 / 0019369A1. Such a hydrogel may be locally injected at the site of the intended effect, or may be injected subcutaneously or intramuscularly to form a delayed release reservoir.

[0496] For intra-articular delivery, the MASP-2 inhibitory agent may be contained in an injectable liquid or gel carrier as described above, an injectable sustained release delivery vehicle as described above, or hyaluronic acid or a hyaluronic acid derivative.

[0497] For oral administration of non-peptidergic agents, the MASP-2 inhibitory agent may be contained in an inert filler or diluent, such as sucrose, corn starch or cellulose.

[0498] For topical administration, the MASP-2 inhibitory agent may be maintained in an ointment, lotion, cream, gel, drops, suppository, spray, liquid or powder, or delivered via a transdermal patch in a gel or microencapsulated delivery system.

[0499] Various nasal and pulmonary delivery systems, including aerosols, metered dose inhalers, dry powder inhalers, and nebulizers, are under development and can be suitably modified for delivery of the invention in an aerosol, inhaler, or spray delivery vehicle, respectively.

[0500] For intrathecal (IT) or intracerebroventricular (ICV) delivery, suitable sterile delivery systems (eg, liquids, gels, suspensions, etc.) can be used to administer the present invention.

[0501] The compositions of the present invention may also include biocompatible excipients such as dispersing or wetting agents, suspending agents, diluents, buffers, penetration enhancers, emulsifiers, binders, thickeners, flavoring agents (for oral administration).

[0502] Pharmaceutical carriers for antibodies and peptides

[0503] More specifically with respect to anti-MASP-2 antibodies and inhibitory peptides, exemplary formulations may be administered parenterally as injectable doses of compound solutions or suspensions in a physiologically acceptable diluent containing a pharmaceutical carrier, which may be a sterile liquid such as water, oil, saline, glycerol or ethanol. In addition, auxiliary substances such as wetting or emulsifying agents, surfactants, pH buffering substances, and the like may be present in the compositions comprising anti-MASP-2 antibodies and inhibitory peptides. Other components of pharmaceutical compositions include oils (e.g., animal, plant or synthetic origin), for example, soybean oil and mineral oil. Generally, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers for injectable solutions.

[0504] Anti-MASP-2 antibodies and inhibitory peptides may also be administered in the form of depot injections or implants, which may be formulated in a manner that permits sustained or pulsatile release of the active agent.

[0505] Pharmaceutically acceptable carriers for expression inhibitors

[0506] More particularly, regarding the expression inhibitor used in the method of the present invention, a composition comprising the above expression inhibitor and a pharmaceutically acceptable carrier or diluent is provided. The composition may also include a colloidal dispersion system.

[0507] Pharmaceutical compositions comprising expression inhibitors may include, but are not limited to, solutions, emulsions, and formulations comprising liposomes. These compositions may be produced from a variety of components, including but not limited to preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. The preparation of such compositions typically involves mixing the expression inhibitor with one or more of the following: buffers, antioxidants, low molecular weight polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose, or dextrins; chelating agents such as EDTA, glutathione, and other stabilizers and excipients. Neutral buffered saline or saline mixed with non-specific serum albumin are examples of suitable diluents.

[0508] In some embodiments, the composition can be prepared and formulated as an emulsion, which is generally a heterogeneous system of one liquid dispersed in another liquid in the form of droplets (see Idson, in Pharmaceutical Dosage Forms, Vol. 1, Rieger and Banker (eds.), Marcek Dekker, Inc., NY, 1988). Examples of naturally occurring emulsifiers used in...

Claims

1. Use of a MASP-2 inhibitory monoclonal antibody or antigen-binding fragment thereof in the preparation of a medicament for treating a human subject having or at risk for developing graft-versus-host disease (GVHD), wherein the MASP-2 inhibitory monoclonal antibody or antigen-binding fragment thereof does not substantially inhibit the classical pathway, and wherein 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 an amino acid sequence of 31-35 of SEQ ID NO:67; and ii) a heavy chain CDR-H2 comprising an amino acid sequence of 50-65 of SEQ ID NO:67; and iii) a heavy chain CDR-H3 comprising an amino acid sequence of 95-102 of SEQ ID NO:67; and (b) a light chain variable region comprising: i) a light chain CDR-L1 comprising an amino acid sequence of 24-34 of SEQ ID NO:70; and ii) a light chain CDR-L2 comprising an amino acid sequence of 50-56 of SEQ ID NO:70; and iii) a heavy chain CDR-H4 comprising an amino acid sequence of 95-102 of SEQ ID NO:

67. Light chain CDR-L3 of the amino acid sequence 89-97 of NO:

70.

2. The use of claim 1, further comprising identifying a human subject suffering from or at risk of developing graft-versus-host disease.

3. The use of claim 1, wherein the subject has previously undergone, is currently undergoing, or will undergo, hematopoietic stem cell transplantation.

4. The method of claim 1, wherein the subject is suffering from acute GVHD.

5. The use of claim 1, wherein the subject is suffering from chronic GVHD.

6. The use of claim 1, wherein the subject is suffering from steroid-resistant GVHD.

7. The use of claim 1, wherein the MASP-2 inhibitory monoclonal antibody is intended to be used at a dose of 1 mg / kg to 10 mg / kg at least once a week.

8. The use of claim 1, wherein the subject suffers from one or more neurological symptoms associated with graft-versus-host disease, and the MASP-2 inhibitory monoclonal antibody or antigen-binding fragment thereof is effective in treating, preventing or ameliorating one or more neurological symptoms associated with graft-versus-host disease.

9. The use of claim 8, wherein the one or more neurological symptoms associated with graft-versus-host disease are selected from weakness, quadriplegia, sensorimotor deficits, autonomic insufficiency polyneuropathy and / or neurogenic bladder.

10. The use of claim 8, wherein the one or more neurological symptoms associated with graft-versus-host disease is paresthesia.

11. The use of claim 1, wherein the MASP-2 inhibitory monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO: 67 and a light chain variable region as shown in SEQ ID NO: 70.

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