Methods for treating and / or preventing idiopathic pneumonia syndrome (IPS) and / or capillary leak syndrome (CLS) and / or grafting syndrome (ES) and / or fluid overload (FO) associated with hematopoietic stem cell transplantation.

Abstract

This invention provides a method for inhibiting the action of MASP-2-dependent complement activation in human subjects who have or are at risk of developing HSCT-IPS, and / or HSCT-CLS, and / or HSCT-FO, and / or HSCT-ES, and / or HSCT-ES. [Solution] The method includes administering to a subject in need an amount of a MASP-2 inhibitor effective in inhibiting MASP-2-dependent complement activation.

Description

[Technical Field]

[0001] Cross-reference with related applications This application claims the benefit of U.S. Provisional Application No. 62 / 940,720, filed November 26, 2019, and U.S. Provisional Application No. 62 / 940,735, filed November 26, 2019, which are incorporated in their entirety by reference herein.

[0002] Description regarding sequence listings The sequence listing relating to this application is provided in text format instead of as a hard copy and is incorporated herein by reference. The name of the text file containing the sequence listing is MP_1_0307_PCT_Sequence_Listing_20201120_ST25.txt; this file is 115 KB in size, was created on November 20, 2020, and was submitted via EFS-Web together with the application herein. [Background technology]

[0003] background The complement system provides initial mechanisms for initiating, amplifying, and organizing immune responses to microbial infections and other acute invaders in humans and other vertebrates (MK Liszewski and JP Atkinson, 1993, in Fundamental Immunology, Third Edition, WE, Paul (ed.), Raven Press, Ltd., New York (Non-Patent Literature 1)). While complement activation provides a beneficial first line of defense against potential pathogens, complement activity that promotes a protective immune response can also be a potential threat to the host (KR, Kalli, et al., Springer Semin. Immunopathol. 15:417-431, 1994 (Non-Patent Literature 2); BP Morgan, Eur. J. Clinical Investig. 24:219-228, 1994 (Non-Patent Literature 3)). For example, C3 and C5 protein degradation products recruit and activate neutrophils. Activated neutrophils are essential for host defense, but they can indiscriminately release destructive enzymes, potentially causing organ damage. Furthermore, complement activation can lead to the deposition of soluble complement components on the surfaces of nearby host cells and microbial targets, resulting in the lysis of host cells.

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

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

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

[0007] Independently, the first step in complement system activation via the lectin pathway is also the binding of specific recognition molecules, followed by the activation of associated serine protease proenzymes. However, rather than the binding of immune complexes by C1q, the recognition molecules of the lectin pathway include a group of carbohydrate-binding proteins collectively called lectins (mannan-binding lectins (MBL), H-phycoline, M-phycoline, L-phycoline, and C-type lectin CL-11). See J. Lu et al., Biochim. Biophys. Acta 1572:387-400, (2002) (Non-patent Literature 4); Holmskov et al., Annu. Rev. Immunol. 21:547-578 (2003) (Non-patent Literature 5); Teh et al., Immunology 101:225-232 (2000) (Non-patent Literature 6)). Teh et al., Immunology 101:225-232 (2000) (non-patent document 6); see also Hansen et al., J, Immunol 185(10):6096-6104 (2010) (non-patent document 7).

[0008] Ikeda et al. were the first to demonstrate that, similar to C1q, MBL can activate the complement system in a C4-dependent manner when it binds to yeast mannan-coated erythrocytes (Ikeda et al., J. Biol. Chem. 262:7451-7454, (1987) (Non-Patent Literature 8)). MBL, a member of the collectin protein family, is a calcium-dependent lectin that binds to carbohydrates in which the 3-hydroxyl and 4-hydroxyl groups are oriented to the equatorial plane of the pyranose ring. Therefore, prominent ligands for MBL are D-mannose and N-acetyl-D-glucosamine, while carbohydrates that do not meet this steric requirement have an undetectable affinity for MBL (Weis et al., Nature 360:127-134, (1992) (Non-Patent Literature 9)). The interaction between MBL and monosaccharides is extremely weak, and the dissociation constant is typically in the millimolar range of a single order of magnitude. MBLs achieve tight and specific binding to glycan ligands through avidity, that is, by simultaneously interacting with multiple monosaccharide residues located close to each other (Lee et al., Archiv. Biochem. Biophys. 299:129-136, (1992) (Non-Patent Literature 10)). MBLs generally recognize carbohydrate patterns that adorn microorganisms, such as bacteria, yeasts, parasites, and certain viruses. In contrast, MBLs do not recognize D-galactose and sialic acid, the second-to-last and last sugars that adorn "mature" complex carbohydrates typically found on mammalian plasma glycoproteins and cell surface glycoproteins. This binding specificity is thought to facilitate the recognition of "foreign" surfaces and help protect against "self-activation." However, MBLs bind with high affinity to clusters of high-mannose "precursor" glycans in 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) (Non-patent Literature 11)). Therefore, damaged cells are a potential target for lectin pathway activation via MBL binding.

[0009] Ficolin has a lectin domain of a different type from MBL, called a fibrinogen-like domain. Ficolin is Ca ++ They bind to sugar residues independently. In humans, three types of phycolin (L-phycolin, M-phycolin, and H-phycolin) have been identified. Two serum phycolins, L-phycolin and H-phycolin, share specificity for N-acetyl-D-glucosamine. However, H-phycolin also binds to N-acetyl-D-galactosamine. The differing sugar specificities of L-phycolin, H-phycolin, CL-11, and MBL suggest that different lectins can complement each other and, through duplication, target different complex carbohydrates. This idea is supported by a recent report that, of the known lectins in the lectin pathway, only L-phycolin specifically binds to lipoteichoic acid, a cell wall complex carbohydrate found in all Gram-positive bacteria (Lynch et al., J. Immunol. 172:1198-1202, (2004) (Non-patent Literature 12)). Collectin (i.e., MBL) and phycolin do not have significant similarities in their amino acid sequences. However, these two protein groups have similar domain configurations and, like C1q, assemble to form oligomeric structures that maximize the potential for multi-site binding.

[0010] Serum concentrations of MBL vary considerably in healthy populations, and this is genetically controlled by polymorphisms / mutations in both the promoter and coding regions of the MBL gene. As an acute-phase protein, MBL expression is further upregulated during inflammation. L-ficolin is present in serum at concentrations nearly identical to those of MBL. Therefore, the L-ficolin branch of the lectin pathway is, in some cases, as strong as the MBL branch. Both MBL and ficolin can also function as opsonins. For this reason, phagocytic cells can target surfaces decorated with MBL and surfaces decorated with phycolin (see Jack et al., J Leukoc Biol., 77(3):328-36(2004)(Non-Patent Literature 13), Matsushita and Fujita, Immunobiology, 205(4-5):490-7(2002)(Non-Patent Literature 14), Aoyagi et al., J. Immunol, 174(1):418-25(2005)(Non-Patent Literature 15)). This opsonization requires interaction between these proteins and phagocytic receptors (Kuhlman et al., J. Exp. Med. 169:1733, (1989)(Non-Patent Literature 16); Matsushita et al., J. Biol. Chem. 271:2448-54, (1996)(Non-Patent Literature 17)). The true nature of phagocyte receptors has not been proven.

[0011] Human MBLs, via their collagen-like domains, exhibit specific and high-affinity interactions with a unique C1r / C1s-like serine protease called MBL-associated serine protease (MASP). To date, three types of MASP have been described. Firstly, a single enzyme, "MASP," was identified and characterized as the enzyme responsible for the initiation of the complement cascade (i.e., cleavage of C2 and C4) (Matsushita et al., J Exp Med 176(6):1497-1502 (1992) (Non-Patent Literature 18): Ji et al., J. Immunol 150:571-578, (1993) (Non-Patent Literature 19)). Subsequently, it was revealed that MASP activity is actually a mixture of two proteases: MASP-1 and MASP-2 (Thiel et al., Nature 386:506-510, (1997) (Non-Patent Literature 20)). However, it has been proven that the MBL-MASP-2 complex alone is sufficient for complement activation (Vorup-Jensen et al., J. Immunol 165:2093-2100, (2000) (Non-Patent Literature 21)). Furthermore, only MASP-2 cleaved C2 and C4 at a high rate (Ambrus et al., J. Immunol, 170:1374-1382, (2003) (Non-Patent Literature 22)). Therefore, MASP-2 is a protease responsible for activating C4 and C2 to produce C4b2a, a C3 converter. This is a significant difference from the classical C1 complex, where the coordinated action of two specific serine proteases (C1r and C1s) leads to complement system activation. Furthermore, a third novel protease, MASP-3, has been isolated (Dahl, MR et al., Immunity 15:127-35, 2001 (Non-Patent Literature 23)). MASP-1 and MASP-3 are alternative splicing products of the same gene.

[0012] MASP has the same domain structure as C1r and C1s, the enzymatic components of the C1 complex (Sim et al., Biochem. Soc. Trans. 28:545, (2000) (Non-Patent Literature 24)). These domains include an N-terminal C1r / C1s / sea urchin VEGF / osteogenesis imperfecta (CUB) domain, an epidermal growth factor-like domain, a second CUB domain, a tandem arrangement of complement regulatory protein domains, and a serine protease domain. Similar to C1 proteases, activation of MASP-2 occurs by cleavage of the Arg-Ile bond adjacent to the serine protease domain. This cleavage separates the enzyme into disulfide-bonded A and B chains. The latter consists of the serine protease domain.

[0013] MBL can also bind to alternative splicing MASP-2, known as a 19kDa MBL-associated protein (MAp19) or small MBL-associated protein (sMAP), which lacks catalytic activity of MASP2 (Stover, J. Immunol. 162:3481-90, (1999) (Non-Patent Literature 25); Takahashi et al., Int. Immunol. 11:859-863, (1999) (Non-Patent Literature 26)). MAp19 contains an extra sequence of four unique amino acids following the first two domains of MASP-2. The function of Map19 is unknown (Degn et al., J. Immunol. Methods. 2011 (Non-Patent Literature 27)). The MASP-1 and MASP-2 genes are located on human chromosome 3 and chromosome 1, respectively (Schwaeble et al., Immunobiology 205:455-466, (2002) (Non-patent document 28)).

[0014] Several pieces of evidence suggest that there are different MBL-MASP complexes, and that the majority of MASP in serum does not form a complex with MBL (Thiel. et al., J. Immunol. 165:878-887, (2000) (Non-Patent Literature 29)). Both H-ficorin and L-ficorin bind to all MASP, similar to MBL, and activate the lectin complement pathway (Dahl et al., Immunity 15:127-35, (2001) (Non-Patent Literature 23); Matsushita et al., J. Immunol. 168:3502-3506, (2002) (Non-Patent Literature 30)). Both the lectin pathway and the classical pathway form a common C3 convertase (C4b2a), and the two pathways merge into one at this stage.

[0015] The lectin pathway is widely considered to play a major role in host defense against infections in naive hosts. Strong evidence of MBL involvement in host defense has been obtained from analyses of patients with low serum levels of functional MBL (Kilpatrick, Biochim. Biophys. Acta 1572:401-413, (2002) (Non-patent Literature 31)). Such patients are susceptible to recurrent bacterial and fungal infections. These symptoms usually appear in adolescence, during an apparent vulnerability period when maternal antibody titers are declining but the full repertoire of antibody responses has not yet developed. This syndrome is often caused by mutations in several sites of the MBL collagen portion, which interfere with the proper formation of MBL oligomers. However, since MBL can function as an opsonin independently of complement, it is unclear to what extent the increased susceptibility to infections is due to impaired complement activation.

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

[0017] While there is broad evidence that both the classical and secondary complement pathways are involved in the development of non-infectious human diseases, the role of the lectin pathway has only recently begun to be evaluated. Recent studies have provided evidence that lectin pathway activation may be responsible for complement activation and associated inflammation in ischemia / reperfusion injury. Collard et al., (2000) reported that cultured endothelial cells subjected to oxidative stress bind to MBL and show C3 deposition when exposed to human serum (Collard et al., Am. J. Pathol 156:1549-1556, (2000) (Non-patent Literature 32)). Furthermore, treatment of human serum with a blocking anti-MBL monoclonal antibody inhibited MBL binding and complement activation. These findings were extended to a rat myocardial ischemia-reperfusion model. In this model, rats treated with an antiblocking antibody against rat MBL showed significantly less myocardial damage during coronary artery occlusion than control antibody-treated rats (Jordan et al., Circulation, 104:1413-1418, (2001) (Non-Patent Literature 33)). The molecular mechanism of MBL binding to vascular endothelium after oxidative stress is unknown. Recent studies suggest that activation of the lectin pathway after oxidative stress is mediated by MBL binding to vascular endothelial cytokeratin, but may not be mediated by complex carbohydrates (Collard e al., Am. J. Pathol. 159:1045-1054, (2001) (Non-Patent Literature 34)). Other studies have shown that the classical and secondary pathways are involved in the development of ischemia / reperfusion injury, and the role of the lectin pathway in this disease remains debatable (Riedermann, NC et al., Am. J. Pathol. 162:363-367, 2003 (Non-Patent Literature 35)).

[0018] Recent studies have shown that MASP-1 (and possibly MASP-3) is required to convert factor D, a secondary pathway activating enzyme, from its precursor form to its active form (see Takahashi M. et al., J Exp Med 207(1):29-37(2010) (Non-Patent Literature 36)). The physiological importance of this process is highlighted by the absence of functional secondary pathway activity in the plasma of MASP-1 / 3-deficient mice. Functional secondary pathway activity is necessary for the production of C3b from native C3 via proteolysis. Since secondary pathway C3 convertase (C3bBb) contains the essential subunit C3b, the question of the origin of the initial C3b via secondary pathway is a perplexing problem that has fueled a vast amount of research.

[0019] C3 belongs to a family of proteins that contain a rare post-translational modification known as a thioester bond (along with C4 and α-2 macroglobulin). The thioester group consists of glutamine with a terminal carbonyl group that forms a thioester covalent bond with a cysteine ​​sulfhydryl group three amino acids away. This bond is unstable, and the electrophilic glutamyl-thioester can react with a nucleophilic moiety such as a hydroxyl or amino group, and thus form a covalent bond with other molecules. The thioester bond is fairly stable when sequestered within the hydrophobic pocket of intact C3. However, when C3 is cleaved into C3a and C3b by proteolysis, the highly reactive thioester bond is exposed on C3b, and after nucleophilic attack by an adjacent moiety containing a hydroxyl or amino group, C3b covalently bonds with a target. In addition to its detailed recorded role in the covalent bonding of C3b to complement targets, the C3 thioester is also thought to play a central role in the induction of the secondary pathway. According to the widely accepted "tick-over theory," the second pathway is initiated by the generation of the liquid-phase combaturase iC3Bb, which is formed from C3 (iC3; C3(H2O)) containing a hydrolyzable thioester and factor B (Lachmann, PJ, et al., Springer Semin. Immunopathol. 7:143-162, (1984) (Non-Patent Literature 37)). C3b-like C3(H2O) is produced from native C3 by the slow, spontaneous hydrolysis of the internal thioester in this protein (Pangburn, MK, et al., J. Exp. Med. 154:856-867, 1981 (Non-Patent Literature 38)). The activity of C3(H2O)Bb combaturase causes the C3b molecule to be deposited on the target surface, thereby initiating the second pathway.

[0020] Little is known about the initiators of the second pathway activation. Activators are thought to include yeast cell walls (zymosan), many pure polysaccharides, rabbit erythrocytes, certain immunoglobulins, viruses, fungi, bacteria, animal tumor cells, parasites, and damaged cells. The only feature common to these activators is the presence of carbohydrates, but due to the complexity and diversity of carbohydrate structures, it is difficult to prove any recognized common molecular determinants. It is widely accepted that the second pathway activation is regulated by a delicate balance between the inhibitory regulatory components of this pathway, such as factor H, factor I, DAF, and CR1, and propergine, the sole positive regulator of the second pathway (see Schwaeble WJ and Reid KB, Immunol Today 20(1):17-21(1999) (Non-Patent Literature 39)).

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

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

[0023] In addition to its essential role in immune defense, the complement system contributes to tissue damage in many clinical conditions. Therefore, there is an urgent need to develop therapeutically effective complement inhibitors to counteract these side effects. [Prior art documents] [Non-patent literature]

[0024] [Non-Patent Document 1] MK Liszewski and JP Atkinson, 1993, in Fundamental Immunology, Third Edition, edited by WE, Paul, Raven Press, Ltd., New York. [Non-Patent Document 2] KR, Kalli, et al., Springer Semin. Immunopathol. 15:417-431, 1994 [Non-Patent Document 3] BP Morgan, Eur. J. Clinical Investig. 24:219-228, 1994

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed Document 8

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

Non-licensed Document 16

Non-licensed Document 17

Non-licensed Document 18

Non-licensed Document 19

Non-licensed Document 20

Non-licensed Document 21

Non-licensed Document 22

Non-licensed Document 23

Non-licensed Document 24

Non-licensed Document 25

Non-licensed Document 26

Non-licensed Document 27

Non-licensed Document 28

Non-licensed Document 29

Non-licensed Document 30

Non-licensed Document 31

Non-licensed Document 32

Non-licensed Document 33

Non-licensed Document 34

Non-licensed Document 35

Non-licensed Document 36

Non-licensed Document 37

Non-licensed Document 38

[0025] overview This summary is provided to give a brief introduction to a wide range of concepts that will be further explained in the detailed description below. This summary is not intended to identify any key features of the subject matter described in the claims, nor is it intended to be used to help determine the scope of the subject matter described in the claims.

[0026] In one aspect, the present invention provides a method for treating a human subject suffering from idiopathic pneumonia syndrome (IPS) after hematopoietic stem cell transplantation (HSCT-IPS), comprising the steps of administering to the subject a composition comprising an amount effective in inhibiting MASP-2-dependent complement activation, thereby improving at least one symptom of HSCT-IPS. In one embodiment, the method comprises the step of administering the composition to a subject who has previously undergone allogeneic hematopoietic stem cell transplantation. In one embodiment, the method comprises the step of administering the composition to a subject who has previously undergone autologous hematopoietic stem cell transplantation. In one embodiment, the method further comprises the step of identifying a human subject suffering from idiopathic pneumonia syndrome (IPS) prior to the step of administering to the subject a composition comprising an amount effective in inhibiting MASP-2-dependent complement activation, such as a MASP-2 inhibitory antibody or an antigen-binding fragment. In one embodiment, the subject suffering from idiopathic pneumonia syndrome (IPS) is determined not to have DAH. In one embodiment, the MASP-2 inhibitory antibody is a monoclonal antibody or a 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 recombinant antibodies, antibodies having reduced effector function, chimeric antibodies, humanized antibodies, and human antibodies. In one embodiment, the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. In one embodiment, the MASP-2 inhibitory antibody reduces C3b deposition in 90% human serum to 30 nM or less IC50. 50Inhibits by . In one embodiment, the MASP-2 inhibitory antibody is delivered systemically to the subject. In one embodiment, the MASP-2 inhibitory antibody or its antigen-binding fragment includes a heavy chain variable region containing CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO:67, and a light chain variable region containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:70. In one embodiment, the MASP-2 inhibitory antibody or its antigen-binding fragment includes a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO:67 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO:70. In some embodiments, the method includes administering to a subject suffering from HSCT-IPS a composition comprising a MASP-2 inhibitor antibody or its antigen-binding fragment, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:67 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:70, at a dose of 1 mg / kg to 10 mg / kg (i.e., 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg) at least once a week (e.g., at least twice or at least three times a week) for a period of at least two weeks, or at least three weeks, or at least four weeks, or at least five weeks, or at least six weeks, or at least seven weeks, or at least eight weeks, or at least nine weeks, or at least ten weeks, or at least eleven weeks, or at least twelve weeks. In one embodiment, the dose of the MASP-2 inhibitory antibody is approximately 4 mg / kg (i.e., 3.6 mg / kg to 4.4 mg / kg).In one embodiment, the dose of the MASP-2 inhibitory antibody is a fixed dose of approximately 300 mg to approximately 450 mg (i.e., approximately 300 mg to approximately 400 mg, or approximately 350 mg to approximately 400 mg), for example, approximately 300 mg, approximately 305 mg, approximately 310 mg, approximately 315 mg, approximately 320 mg, approximately 325 mg, approximately 330 mg, approximately 335 mg, approximately 340 mg, approximately 345 mg, approximately 350 mg, approximately 355 mg, approximately 360 mg, approximately 365 mg, approximately 370 mg, approximately 375 mg, approximately 380 mg, approximately 385 mg, approximately 390 mg, approximately 395 mg, approximately 400 mg, approximately 405 mg, approximately 410 mg, approximately 415 mg, approximately 420 mg, approximately 425 mg, approximately 430 mg, approximately 435 mg, approximately 440 mg, approximately 445 mg, or approximately 450 mg. In one embodiment, the dose of the MASP-2 inhibitory antibody is a fixed dose of approximately 370 mg (±10%). In one embodiment, the method includes administering a fixed dose of approximately 370 mg (±10%) of the MASP-2 inhibitory antibody intravenously twice a week to a subject suffering from HSCT-IPS for a treatment period of at least 8 weeks.

[0027] In another aspect, the present invention provides a method for treating a human subject suffering from hematopoietic stem cell transplantation-induced capillary leak syndrome (CLS) (HSCT-CLS), comprising the steps of administering to the subject a composition comprising an amount effective in inhibiting MASP-2-dependent complement activation, thereby improving at least one symptom of HSCT-CLS. In one embodiment, the method comprises the step of administering the composition to a subject who has previously undergone allogeneic hematopoietic stem cell transplantation. In one embodiment, the method comprises the step of administering the composition to a subject who has previously undergone autologous hematopoietic stem cell transplantation. In one embodiment, the method further comprises the step of identifying a human subject suffering from capillary leak syndrome (CLS) prior to the step of administering to the subject a composition comprising an amount effective in inhibiting MASP-2-dependent complement activation, such as a MASP-2 inhibitory antibody or an antigen-binding fragment. In one embodiment, the subject suffering from CLS is determined to be free of VOD. In one embodiment, the MASP-2 inhibitory antibody is a monoclonal antibody or a 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 recombinant antibodies, antibodies having reduced effector function, chimeric antibodies, humanized antibodies, and human antibodies. In one embodiment, the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. In one embodiment, the MASP-2 inhibitory antibody reduces C3b deposition in 90% human serum to 30 nM or less IC50. 50Inhibits by . In one embodiment, the MASP-2 inhibitory antibody is delivered systemically to the subject. In one embodiment, the MASP-2 inhibitory antibody or its antigen-binding fragment includes a heavy chain variable region containing CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO:67, and a light chain variable region containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:70. In one embodiment, the MASP-2 inhibitory antibody or its antigen-binding fragment includes a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO:67 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO:70. In some embodiments, the method includes administering to a subject suffering from HSCT-CLS a composition comprising a MASP-2 inhibitor antibody or its antigen-binding fragment, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:67 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:70, at a dose of 1 mg / kg to 10 mg / kg (i.e., 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg) at least once a week (e.g., at least twice or at least three times a week) for a period of at least two weeks, or at least three weeks, or at least four weeks, or at least five weeks, or at least six weeks, or at least seven weeks, or at least eight weeks, or at least nine weeks, or at least ten weeks, or at least eleven weeks, or at least twelve weeks. In one embodiment, the dose of the MASP-2 inhibitory antibody is approximately 4 mg / kg (i.e., 3.6 mg / kg to 4.4 mg / kg).In one embodiment, the dose of the MASP-2 inhibitory antibody is a fixed dose of approximately 300 mg to approximately 450 mg (i.e., approximately 300 mg to approximately 400 mg, or approximately 350 mg to approximately 400 mg), for example, approximately 300 mg, approximately 305 mg, approximately 310 mg, approximately 315 mg, approximately 320 mg, approximately 325 mg, approximately 330 mg, approximately 335 mg, approximately 340 mg, approximately 345 mg, approximately 350 mg, approximately 355 mg, approximately 360 mg, approximately 365 mg, approximately 370 mg, approximately 375 mg, approximately 380 mg, approximately 385 mg, approximately 390 mg, approximately 395 mg, approximately 400 mg, approximately 405 mg, approximately 410 mg, approximately 415 mg, approximately 420 mg, approximately 425 mg, approximately 430 mg, approximately 435 mg, approximately 440 mg, approximately 445 mg, or approximately 450 mg. In one embodiment, the dose of the MASP-2 inhibitory antibody is a fixed dose of approximately 370 mg (±10%). In one embodiment, the method includes administering a fixed dose of approximately 370 mg (±10%) of the MASP-2 inhibitory antibody intravenously twice a week to a subject suffering from HSCT-CLS for a treatment period of at least 8 weeks.

[0028] In another aspect, the present invention provides a method for treating a human subject suffering from fluid overload (FO) after hematopoietic stem cell transplantation (HSCT-FO), comprising the steps of administering to the subject a composition comprising an amount effective in inhibiting MASP-2-dependent complement activation, or an antigen-binding fragment thereof, thereby improving at least one symptom of HSCT-FO. In one embodiment, the method comprises the step of administering the composition to a subject who has previously undergone allogeneic hematopoietic stem cell transplantation. In one embodiment, the method comprises the step of administering the composition to a subject who has previously undergone autologous hematopoietic stem cell transplantation. In one embodiment, the method further comprises the step of identifying a human subject suffering from fluid overload (FO) prior to the step of administering to the subject a composition comprising an amount effective in inhibiting MASP-2-dependent complement activation, or an antigen-binding fragment thereof. In one embodiment, the MASP-2 inhibitory antibody is a monoclonal antibody or a fragment thereof that specifically binds to human MASP-2. In one embodiment, the MASP-2 inhibitory antibody or a fragment thereof is selected from the group consisting of recombinant antibodies, antibodies with reduced effector function, chimeric antibodies, humanized antibodies, and human antibodies. In one embodiment, the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. In one embodiment, the MASP-2 inhibitory antibody reduces C3b deposition in 90% human serum to 30 nM or less IC50. 50Inhibits by . In one embodiment, the MASP-2 inhibitory antibody is delivered systemically to the subject. In one embodiment, the MASP-2 inhibitory antibody or its antigen-binding fragment includes a heavy chain variable region containing CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO:67, and a light chain variable region containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:70. In one embodiment, the MASP-2 inhibitory antibody or its antigen-binding fragment includes a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO:67 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO:70. In some embodiments, the method includes administering to a subject suffering from HSCT-FO a composition comprising a MASP-2 inhibitor antibody or its antigen-binding fragment, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:67 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:70, at a dose of 1 mg / kg to 10 mg / kg (i.e., 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg) at least once a week (e.g., at least twice or at least three times a week) for a period of at least two weeks, or at least three weeks, or at least four weeks, or at least five weeks, or at least six weeks, or at least seven weeks, or at least eight weeks, or at least nine weeks, or at least ten weeks, or at least eleven weeks, or at least twelve weeks. In one embodiment, the dose of the MASP-2 inhibitory antibody is approximately 4 mg / kg (i.e., 3.6 mg / kg to 4.4 mg / kg).In one embodiment, the dose of the MASP-2 inhibitory antibody is a fixed dose of approximately 300 mg to approximately 450 mg (i.e., approximately 300 mg to approximately 400 mg, or approximately 350 mg to approximately 400 mg), for example, approximately 300 mg, approximately 305 mg, approximately 310 mg, approximately 315 mg, approximately 320 mg, approximately 325 mg, approximately 330 mg, approximately 335 mg, approximately 340 mg, approximately 345 mg, approximately 350 mg, approximately 355 mg, approximately 360 mg, approximately 365 mg, approximately 370 mg, approximately 375 mg, approximately 380 mg, approximately 385 mg, approximately 390 mg, approximately 395 mg, approximately 400 mg, approximately 405 mg, approximately 410 mg, approximately 415 mg, approximately 420 mg, approximately 425 mg, approximately 430 mg, approximately 435 mg, approximately 440 mg, approximately 445 mg, or approximately 450 mg. In one embodiment, the dose of the MASP-2 inhibitory antibody is a fixed dose of approximately 370 mg (±10%). In one embodiment, the method includes administering a fixed dose of approximately 370 mg (±10%) of the MASP-2 inhibitory antibody intravenously twice a week to a subject suffering from HSCT-FO for a treatment period of at least 8 weeks.

[0029] In another aspect, the present invention provides a method for treating a human subject suffering from hematopoietic stem cell transplantation syndrome (HSCT-ES), comprising the steps of administering to the subject a composition comprising an amount effective in inhibiting MASP-2-dependent complement activation, or an antigen-binding fragment thereof, thereby improving at least one symptom of HSCT-ES. In one embodiment, the method comprises the step of administering the composition to a subject who has previously undergone allogeneic hematopoietic stem cell transplantation. In one embodiment, the method comprises the step of administering the composition to a subject who has previously undergone autologous hematopoietic stem cell transplantation. In one embodiment, the method further comprises the step of identifying a human subject suffering from hematopoietic stem cell transplantation syndrome (HSCT-ES) prior to the step of administering to the subject a composition comprising an amount effective in inhibiting MASP-2-dependent complement activation, or an antigen-binding fragment thereof. In one embodiment, the MASP-2 inhibitory antibody is a monoclonal antibody or a fragment thereof that specifically binds to human MASP-2. In one embodiment, the MASP-2 inhibitory antibody or a fragment thereof is selected from the group consisting of recombinant antibodies, antibodies with reduced effector function, chimeric antibodies, humanized antibodies, and human antibodies. In one embodiment, the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. In one embodiment, the MASP-2 inhibitory antibody reduces C3b deposition in 90% human serum to 30 nM or less IC50. 50Inhibits by . In one embodiment, the MASP-2 inhibitory antibody is delivered systemically to the subject. In one embodiment, the MASP-2 inhibitory antibody or its antigen-binding fragment includes a heavy chain variable region containing CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO:67, and a light chain variable region containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:70. In one embodiment, the MASP-2 inhibitory antibody or its antigen-binding fragment includes a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO:67 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO:70. In some embodiments, the method includes administering to a subject suffering from HSCT-ES a composition comprising a MASP-2 inhibitor antibody or its antigen-binding fragment, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:67 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:70, at a dose of 1 mg / kg to 10 mg / kg (i.e., 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg) at least once a week (e.g., at least twice or at least three times a week) for a period of at least two weeks, or at least three weeks, or at least four weeks, or at least five weeks, or at least six weeks, or at least seven weeks, or at least eight weeks, or at least nine weeks, or at least ten weeks, or at least eleven weeks, or at least twelve weeks. In one embodiment, the dose of the MASP-2 inhibitory antibody is approximately 4 mg / kg (i.e., 3.6 mg / kg to 4.4 mg / kg).In one embodiment, the dose of the MASP-2 inhibitory antibody is a fixed dose of approximately 300 mg to approximately 450 mg (i.e., approximately 300 mg to approximately 400 mg, or approximately 350 mg to approximately 400 mg), for example, approximately 300 mg, approximately 305 mg, approximately 310 mg, approximately 315 mg, approximately 320 mg, approximately 325 mg, approximately 330 mg, approximately 335 mg, approximately 340 mg, approximately 345 mg, approximately 350 mg, approximately 355 mg, approximately 360 mg, approximately 365 mg, approximately 370 mg, approximately 375 mg, approximately 380 mg, approximately 385 mg, approximately 390 mg, approximately 395 mg, approximately 400 mg, approximately 405 mg, approximately 410 mg, approximately 415 mg, approximately 420 mg, approximately 425 mg, approximately 430 mg, approximately 435 mg, approximately 440 mg, approximately 445 mg, or approximately 450 mg. In one embodiment, the dose of the MASP-2 inhibitory antibody is a fixed dose of approximately 370 mg (±10%). In one embodiment, the method includes administering a fixed dose of approximately 370 mg (±10%) of the MASP-2 inhibitory antibody intravenously twice a week to a subject suffering from HSCT-ES for a treatment period of at least 8 weeks.

[0030] The methods, compositions, and pharmaceuticals of the present invention are useful in vivo for inhibiting the adverse effects of MASP-2-dependent complement activation in mammalian subjects, including humans who have or are at risk of developing HSCT-IPS (i.e., IPS after stem cell transplantation) as further described herein, and / or subjects who have or are at risk of developing HSCT-CLS (i.e., CLS after stem cell transplantation), and / or subjects who have or are at risk of developing HSCT-FO (i.e., FO after stem cell transplantation), and / or subjects who have or are at risk of developing HSCT-ES (i.e., ES after stem cell transplantation). In another aspect, the present invention provides compositions for inhibiting the adverse effects of MASP-2-dependent complement activation, comprising a therapeutically effective amount of a MASP-2 inhibitor, such as a MASP-2 inhibitory antibody, and a pharmaceutically acceptable carrier. Methods are also provided for producing pharmaceuticals for use in inhibiting the adverse effects of MASP-2-dependent complement activation in living subjects requiring such use, comprising a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutically acceptable carrier. Methods for manufacturing pharmaceuticals for use in inhibiting MASP-2-dependent complement activation for the treatment of each of the conditions, diseases, and disorders described below in this specification are also provided. [Invention 1001] A method for treating a human subject suffering from idiopathic pneumonia syndrome after hematopoietic stem cell transplantation (HSCT-IPS), comprising the step of administering to the subject a composition containing a MASP-2 inhibitory antibody or an antigen-binding fragment thereof in an amount effective in inhibiting MASP-2-dependent complement activation. [Invention 1002] The method of the present invention 1001, wherein the MASP-2 inhibitory antibody is a monoclonal antibody or a fragment thereof that specifically binds to human MASP-2. [Invention 1003] The method of the present invention 1001, wherein the antibody or fragment thereof is selected from the group consisting of recombinant antibodies, antibodies having reduced effector function, chimeric antibodies, humanized antibodies, and human antibodies. [Invention 1004] The method of the present invention 1001, wherein the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. [Invention 1005] MASP-2 inhibitory antibodies reduce C3b deposition in 90% human serum to 30 nM or less IC50. 50 The method of the present invention 1001, which inhibits by [Invention 1006] The method according to the present invention 1001, wherein a MASP-2 inhibitory antibody is delivered systemically to the target. [Invention 1007] The method of the present invention 1001, further comprising the step of identifying a human subject suffering from idiopathic pneumonia syndrome after hematopoietic stem cell transplantation (HSCT-IPS), before the step of administering to the subject a composition containing a MASP-2 inhibitory antibody or its antigen-binding fragment in an amount effective in inhibiting MASP-2-dependent complement activation. [Invention 1008] The method of the present invention 1001, wherein the subject has previously undergone allogeneic hematopoietic stem cell transplantation. [Invention 1009] The method of the present invention 1001 applies to subjects who have previously undergone autologous hematopoietic stem cell transplantation. [Invention 1010] The method of the present invention 1001, wherein the subject does not have diffuse alveolar hemorrhage (DAH). [Invention 1011] The method of the present invention 1001, wherein the MASP-2 inhibitory antibody or its antigen-binding fragment comprises a heavy chain variable region containing CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO:67, and a light chain variable region containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:70. [Invention 1012] The method of the present invention 1001, wherein a MASP-2 inhibitory antibody or its antigen-binding fragment comprises a heavy chain variable region containing SEQ ID NO:67 and a light chain variable region containing SEQ ID NO:70. [Invention 1013] A method according to any one of the present invention 1001 to 1012, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody having a dose of approximately 4 mg / kg at least once a week for a treatment period of at least 4 weeks. [Invention 1014] The method of the present invention 1013, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody having a dose of approximately 4 mg / kg at least twice a week for a treatment period of at least 4 weeks. [Invention 1015] A method according to any one of the present invention 1001 to 1012, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody in a dose of approximately 370 mg at least once a week for a treatment period of at least 4 weeks. [Invention 1016] The method of the present invention 1015, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody in a dose of approximately 370 mg at least twice a week for a treatment period of at least 4 weeks. [Invention 1017] A method for treating a human subject suffering from hematopoietic stem cell transplantation-induced capillary leak syndrome (HSCT-CLS), comprising the step of administering to the subject a composition containing a MASP-2 inhibitory antibody or an antigen-binding fragment thereof in an amount effective in inhibiting MASP-2-dependent complement activation. [Invention 1018] The method of the present invention 1017, wherein the MASP-2 inhibitory antibody is a monoclonal antibody or a fragment thereof that specifically binds to human MASP-2. [Invention 1019] The method of the present invention 1017, wherein the antibody or fragment thereof is selected from the group consisting of recombinant antibodies, antibodies having reduced effector function, chimeric antibodies, humanized antibodies, and human antibodies. [Invention 1020] The method of the present invention 1017, wherein the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. [Invention 1021] MASP-2 inhibitory antibodies reduce C3b deposition in 90% human serum to 30 nM or less IC50.50 The method of the present invention 1017, which inhibits by [Invention 1022] The method of the present invention 1017, wherein a MASP-2 inhibitory antibody is delivered systemically to the target. [Invention 1023] The method of the present invention 1017, further comprising the step of identifying a human subject suffering from hematopoietic stem cell transplantation-induced capillary leak syndrome (HSCT-CLS) before administering to the subject a composition containing an amount effective in inhibiting MASP-2-dependent complement activation, such as a MASP-2 inhibitory antibody or its antigen-binding fragment. [Invention 1024] The method of the present invention 1017 applies to subjects who have previously undergone allogeneic hematopoietic stem cell transplantation. [Invention 1025] The method of the present invention 1017 applies to subjects who have previously undergone autologous hematopoietic stem cell transplantation. [Invention 1026] The method of the present invention 1017, wherein the subject does not have hepatic veno-occlusive disease (VOD). [Invention 1027] The method of the present invention 1017, wherein the MASP-2 inhibitory antibody or its antigen-binding fragment comprises a heavy chain variable region containing CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO:67, and a light chain variable region containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:70. [Invention 1028] The method of the present invention 1017, wherein a MASP-2 inhibitory antibody or its antigen-binding fragment comprises a heavy chain variable region containing SEQ ID NO:67 and a light chain variable region containing SEQ ID NO:70. [Invention 1029] A method according to any one of the present invention 1017 to 1028, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody having a dose of approximately 4 mg / kg at least once a week for a treatment period of at least 4 weeks. [Invention 1030] The method of the present invention 1029, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody having a dose of approximately 4 mg / kg at least twice a week for a treatment period of at least 4 weeks. [Invention 1031] A method according to any one of the present invention 1017 to 1028, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody in a dose of approximately 370 mg at least once a week for a treatment period of at least 4 weeks. [Invention 1032] The method of the present invention 1031, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody in a dose of approximately 370 mg at least twice a week for a treatment period of at least 4 weeks. [Invention 1033] A method for treating a human subject suffering from hematopoietic stem cell transplant-induced fluid overload (HSCT-FO), comprising the step of administering to the subject a composition containing a MASP-2 inhibitory antibody or an antigen-binding fragment thereof in an amount effective in inhibiting MASP-2-dependent complement activation. [Invention 1034] The method of the present invention 1033, wherein the MASP-2 inhibitory antibody is a monoclonal antibody or a fragment thereof that specifically binds to human MASP-2. [Invention 1035] The method of the present invention 1033, wherein the antibody or fragment thereof is selected from the group consisting of recombinant antibodies, antibodies having reduced effector function, chimeric antibodies, humanized antibodies, and human antibodies. [Invention 1036] The method of the present invention 1033, wherein the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. [Invention 1037] MASP-2 inhibitory antibodies reduce C3b deposition in 90% human serum to 30 nM or less IC50. 50 The method of the present invention 1033, which inhibits by [Invention 1038] The method of the present invention 1033, wherein a MASP-2 inhibitory antibody is delivered systemically to the target. [Invention 1039] The method of the present invention 1033, further comprising the step of identifying a human subject suffering from hematopoietic stem cell transplantation-induced fluid overload (HSCT-FO) before administering to the subject a composition containing an amount effective in inhibiting MASP-2-dependent complement activation, such as a MASP-2 inhibitory antibody or its antigen-binding fragment. [Invention 1040] The method of the present invention 1033 applies to subjects who have previously undergone allogeneic hematopoietic stem cell transplantation. [Invention 1041] The method of the present invention 1033 applies to subjects who have previously undergone autologous hematopoietic stem cell transplantation. [Invention 1042] The method of the present invention 1033, wherein the MASP-2 inhibitory antibody or its antigen-binding fragment comprises a heavy chain variable region containing CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO:67, and a light chain variable region containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:70. [Invention 1043] The method of the present invention 1033, wherein a MASP-2 inhibitory antibody or its antigen-binding fragment comprises a heavy chain variable region containing SEQ ID NO:67 and a light chain variable region containing SEQ ID NO:70. [Invention 1044] A method according to any one of the present invention 1033 to 1043, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody having a dose of approximately 4 mg / kg at least once a week for a treatment period of at least 4 weeks. [Invention 1045] The method of the present invention 1044, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody having a dose of approximately 4 mg / kg at least twice a week for a treatment period of at least 4 weeks. [Invention 1046] A method according to any one of the present invention 1033 to 1043, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody in a dose of approximately 370 mg at least once a week for a treatment period of at least 4 weeks. [Invention 1047] A method according to invention 1046, comprising administering to a subject a composition comprising the MASP-2 inhibitory antibody having a dosage of about 370 mg at least twice a week for at least a 4-week treatment period. [Invention 1048] A method of treating a human subject suffering from engraftment syndrome after hematopoietic stem cell transplantation (HSCT-ES), comprising administering to the subject a composition comprising an MASP-2 inhibitory antibody or an antigen-binding fragment thereof effective to inhibit MASP-2-dependent complement activation. [Invention 1049] The method according to invention 1048, wherein the MASP-2 inhibitory antibody is a monoclonal antibody or a fragment thereof that specifically binds to human MASP-2. [Invention 1050] The method according to invention 1048, wherein the antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody having reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. [Invention 1051] The method according to invention 1048, wherein the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. [Invention 1052] The method according to invention 1048, wherein the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum with an IC 50 of 30 nM or less. [Invention 1053] The method according to invention 1048, wherein the MASP-2 inhibitory antibody is delivered systemically to the subject. [Invention 1054] The method according to invention 1048, further comprising identifying a human subject suffering from engraftment syndrome after hematopoietic stem cell transplantation (HSCT-ES) prior to the step of administering to the subject a composition comprising an MASP-2 inhibitory antibody or an antigen-binding fragment thereof effective to inhibit MASP-2-dependent complement activation. [Invention 1055] The method according to invention 1048, wherein the subject has previously received an allogeneic hematopoietic stem cell transplantation. [Invention 1056] The method of the present invention 1048 applies to subjects who have previously undergone autologous hematopoietic stem cell transplantation. [Invention 1057] The method of the present invention 1048, wherein the MASP-2 inhibitory antibody or its antigen-binding fragment comprises a heavy chain variable region containing CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO:67, and a light chain variable region containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:70. [Invention 1058] The method of the present invention 1048, wherein a MASP-2 inhibitory antibody or its antigen-binding fragment comprises a heavy chain variable region containing SEQ ID NO:67 and a light chain variable region containing SEQ ID NO:70. [Invention 1059] A method according to any one of the present invention 1048 to 1058, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody having a dose of approximately 4 mg / kg at least once a week for a treatment period of at least 4 weeks. [Invention 1060] The method of the present invention 1059, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody having a dose of approximately 4 mg / kg at least twice a week for a treatment period of at least 4 weeks. [Invention 1061] A method according to any one of the present invention 1048 to 1058, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody in a dose of approximately 370 mg at least once a week for a treatment period of at least 4 weeks. [Invention 1062] The method of the present invention 1061, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody in a dose of approximately 370 mg at least twice a week for a treatment period of at least 4 weeks. [Brief explanation of the drawing]

[0031] Many of the aforementioned aspects and associated advantages of the present invention will be more readily apparent, as they will be better understood by referring to the following detailed description when used in conjunction with the accompanying drawings. [Figure 1] This is a diagram showing the genome structure of human MASP-2. [Figure 2] Figure 2A is a schematic diagram showing the domain structure of the human MASP-2 protein. Figure 2B is a schematic diagram showing the domain structure of the human MAp19 protein. [Figure 3] This figure illustrates the mouse MASP-2 knockout strategy. [Figure 4] This is a diagram showing the human MASP-2 minigene construct. [Figure 5A] As described in Example 2, we present results demonstrating that MASP-2 deficiency leads to the loss of lectin-mediated C4 activation, as measured by the absence of C4b deposition on mannan. [Figure 5B] As described in Example 2, we present results demonstrating that MASP-2 deficiency leads to the loss of lectin-mediated C4 activation, as measured by the absence of C4b deposition on zymosan. [Figure 5C] As described in Example 2, the relative C4 activation levels of serum samples obtained from MASP-2+ / -, MASP-2- / -, and wild-type strains are shown, as measured by C4b deposition on mannan and zymosan. [Figure 6] As described in Example 2, when measured by C4b deposition on mannan, the addition of recombinant mouse MASP-2 to a MASP-2- / - serum sample demonstrates that lectin-mediated C4 activation is restored in a protein concentration-dependent manner. [Figure 7] As described in Example 8, the results demonstrating that the classical pathway functions in the MASP-2- / - lineage are shown. [Figure 8A]As described in Example 10, the results show that anti-MASP-2 Fab2 antibody #11 inhibits C3 convertase formation. [Figure 8B] As described in Example 10, the results show that anti-MASP-2 Fab2 antibody #11 binds to native rat MASP-2. [Figure 8C] As described in Example 10, the results show that anti-MASP-2 Fab2 antibody #41 inhibits C4 cleavage. [Figure 9] As described in Example 10, the results show that all of the tested anti-MASP-2 Fab2 antibodies that inhibited C3 convertase formation also inhibited C4 cleavage. [Figure 10] This figure shows the rat MASP-2-derived recombinant polypeptide used for epitope mapping of the anti-MASP-2 blocking Fab2 antibody, as described in Example 11. [Figure 11] As described in Example 11, the results demonstrating the binding of anti-MASP-2 Fab2#40 and #60 to rat MASP-2 polypeptides are shown. [Figure 12] As described in Example 12, we present the results demonstrating the blood urea nitrogen clearance of wild-type (+ / +) mice and MASP-2 (- / -) mice 24 and 48 hours after reperfusion in a renal ischemia / reperfusion injury model. [Figure 13A] As described in Example 13, the results show baseline VEGF protein levels in the RPE-choroidal complex isolated from wild-type (+ / +) mice and MASP-2 (- / -) mice. [Figure 13B] As described in Example 13, the results show the VEGF protein levels in the RPE-choroidal complex in wild-type (+ / +) mice and MASP-2 (- / -) mice 3 days after laser-induced injury in a macular degeneration model. [Figure 14]As described in Example 13, the results show the average choroidal angiogenesis (CNV) volume 7 days after laser-induced injury in wild-type (+ / +) mice and MASP-2 (- / -) mice. [Figure 15] Figures 15A and 15B show the dose-response curves for C4b deposition inhibition (Figure 15A) and thrombin activation inhibition (Figure 15B) after administration of MASP-2 Fab2 antibody dissolved in normal rat serum, as described in Example 14. [Figure 16] Figures 16A and 16B show the measured platelet aggregation (expressed as aggregation area) (Figure 16B) in MASP-2(- / -) mice compared with platelet aggregation (Figure 16A) in untreated wild-type mice and wild-type mice in which the complement pathway was inhibited by the depleted substance cobra venom factor (CVF) and terminal pathway inhibitor (C5aR antagonist) in a localized Schwartzman reaction model of disseminated intravascular coagulation, as described in Example 15. [Figure 17] As described in Example 16, the blood urea nitrogen (BUN) levels measured in WT(+ / +) (B6) transplant recipient mice or MASP-2(- / -) transplant recipient mice from WT(+ / +) donor kidneys are shown. [Figure 18] As described in Example 17, the survival percentages of WT(+ / +) mice and MASP-2(- / -) mice as a function of the number of days after microbial infection in a colon perforation (CLP) model are illustrated. [Figure 19] As described in Example 17, the bacterial counts measured in WT (+ / +) and MASP-2 (- / -) after microbial infection in a colon perforation (CLP) model are shown. [Figure 20] As described in Example 18, this is a Kaplan-Mayer plot showing the survival percentage of WT (+ / +), MASP-2 (- / -), and C3 (- / -) mice 6 days after intranasal administration of Pseudomonas aeruginosa. [Figure 21]As described in Example 19, the C4b deposition levels, measured as a percentage of the control, are shown in samples collected at various time points after subcutaneous administration of 0.3 mg / kg or 1.0 mg / kg of mouse anti-MASP-2 monoclonal antibody to WT mice. [Figure 22] As described in Example 19, the C4b deposition levels, measured as a percentage relative to the control, are shown in samples collected at various time points after ip administration of 0.6 mg / kg of mouse anti-MASP-2 monoclonal antibody to WT mice. [Figure 23] As described in Example 20, the mean choroidal angiogenesis (CNV) volume at 7 days post-laser-induced injury in WT(+ / +) mice pretreated with a single intravenous injection of 0.3 mg / kg or 1.0 mg / kg of mouse anti-MASP-2 monoclonal antibody is shown. [Figure 24A] As described in Example 21, the survival percentages of MASP-2(- / -) and WT(+ / +) mice after infection with 5 × 10⁸ / 100 μl cfu of Neisseria meningitidis are shown. [Figure 24B] As described in Example 21, the log cfu / ml of meningococcus meningitidis recovered at various time points in blood samples taken from MASP-2 KO(- / -) and WT(+ / +) mice infected with 5 × 10⁸ cfu / 100 μl of meningococcus is shown. [Figure 25A] As described in Example 21, the survival percentages of MASP-2 KO(- / -) and WT(+ / +) mice after infection with 2 × 10⁸ cfu / 100 μl of Neisseria meningitidis are shown. [Figure 25B] As described in Example 21, the log cfu / ml of meningococcus meningitidis recovered at various time points in blood samples taken from WT(+ / +) mice infected with 2 × 10⁸ cfu / 100 μl of meningococcus is shown. [Figure 25C]As described in Example 21, the log cfu / ml of meningococcus meningitidis recovered at various time points in blood samples taken from MASP-2(- / -) mice infected with 2 × 10⁸ cfu / 100 μl of meningococcus is shown. [Figure 26] Figure 26A illustrates the results of the C3b deposition assay, which demonstrated that MASP-2(- / -) mice retain the functional classical pathway, as described in Example 22. Figure 26B illustrates the results of the C3b deposition assay on a zymosan-coated plate, which demonstrated that MASP-2(- / -) mice retain the functional secondary pathway, as described in Example 22. [Figure 27] Figure 27A illustrates myocardial ischemia / reperfusion injury (MIRI)-induced tissue loss after ligation and reperfusion of the left anterior descending coronary artery (LAD) in C4(- / -) mice (n=6) and corresponding WT littermates (n=7), showing ischemic area (AAR) and inflamed area (INF), as described in Example 22. Figure 27B illustrates the inflamed area (INF) as a function of the ischemic area (AAR) in C4(- / -) mice and WT mice treated as described in Figure 42A, demonstrating that C4(- / -) mice are as susceptible to MIRI as WT controls (dashed line), as described in Example 22. [Figure 28A] As described in Example 22, the results of the C3b deposition assay using serum derived from WT mice, serum derived from C4(- / -) mice, and serum derived from C4(- / -) mice pre-incubated with mannan are shown in the figure. [Figure 28B] As described in Example 22, the results of C3b deposition assays for serum from WT mice, C4(- / -) mice, and MASP-2(- / -) mice, mixed with various concentrations of anti-mouse MASP-2 mAb (mAbM11), are shown in the figure. [Figure 28C] As described in Example 22, the results of the C3b deposition assay for human serum (sufficient C4) and C4-deficient serum derived from WT individuals, as well as serum derived from C4-deficient subjects pre-incubated with mannan, are shown in the figure. [Figure 28D] As described in Example 22, the results of the C3b deposition assay for human serum (sufficient C4) derived from WTs and human serum derived from C4-deficient subjects mixed with anti-human MASP-2 mAb (mAbH3) are shown in the figure. [Figure 29] Figure 29A illustrates a comparative analysis of C3 convertase activity in plasma from various complement-deficient mouse lines, tested under lectin activation pathway-specific assay conditions or classical activation pathway-specific assay conditions, as described in Example 22. Figure 29B illustrates the time-resolved kinetics of C3 convertase activity in plasma from various complement-deficient mouse lines, tested under lectin activation pathway-specific conditions, as described in Example 22. [Figure 30] As described in Example 23, the results of Western blot analysis showing human C3 activation by thrombin substrates FXIa and FXa, indicated by the presence of the a' chain, are shown. [Figure 31] As described in Example 23, the results of the C3 deposition assay for serum samples obtained from WT, MASP-2(- / -), F11(- / -), F11(- / -) / C4(- / -), and C4(- / -) are shown. [Figure 32] Figure 32A is a Kaplan-Meier survival plot showing the percentage survival rate over time after exposure to 7.0 Gy of radiation in control mice and mice treated with anti-mouse MASP-2 antibody (mAbM11) or anti-human MASP-2 antibody (mAbH6), as described in Example 29. Figure 32B is a Kaplan-Meier survival plot showing the percentage survival rate over time after exposure to 6.5 Gy of radiation in control mice and mice treated with anti-mouse MASP-2 antibody (mAbM11) or anti-human MASP-2 antibody (mAbH6), as described in Example 29. [Figure 33]This Kaplan-Meier plot illustrates the survival percentages of MASP-2 knockout (KO) and wild-type (WT) mice after administration of meningococcal serogroup A Z2491 at an infectious dose of 2.6 × 10⁷ cfu, demonstrating that MASP-2-deficient mice are protected from meningococcal-induced death, as described in Example 30. [Figure 34] This Kaplan-Meier plot illustrates the survival percentages of MASP-2 knockout (KO) and wild-type (WT) mice after administration of an infectious dose of 6 × 10⁶ cfu of meningococcal serogroup B MC58 strain, demonstrating that MASP-2-deficient mice are protected from meningococcal serogroup B MC58 strain-induced death, as described in Example 30. [Figure 35] The log cfu / ml of meningococcal serogroup B MC58 strain recovered at various time points in blood samples taken from MASP-2 KO mice and WT mice after ip infection with 6 × 10⁶ cfu of meningococcal serogroup B MC58 strain is shown (n=3 at various time points for both mouse groups; results are expressed as mean ± SEM). As described in Example 30, MASP-2 KO mice were infected with the same dose of meningococcal serogroup B MC58 strain as WT mice, but the MASP-2 KO mice showed a significant reduction in bacteremia compared to WT mice. [Figure 36] The mean illness scores of MASP-2 mice and WT mice at 3, 6, 12, and 24 hours after infection with meningococcal serogroup B MC58 strain at 6 × 10⁶ cfu / 100 μl are shown, demonstrating that MASP-2-deficient mice showed high resistance to infection and had a very low illness score at 6 hours, as described in Example 30. [Figure 37] This Kaplan-Meier plot illustrates the survival percentage of mice that were administered an infectious dose of 4 × 10⁶ / 100 μl cfu of Neisseria meningitidis serogroup B MC58 strain, followed by the administration of inhibitory anti-MASP-2 antibody (1 mg / kg) or a control isotype antibody 3 hours after infection. As described in Example 31, this demonstrates that anti-MASP-2 antibody is effective in treating and improving survival rates in subjects infected with Neisseria meningitidis. [Figure 38] After infecting meningococcal serogroup B MC58 strain with 6.5 × 10⁶ cfu / 100 μl via ip, the log cfu / ml of viable cells of meningococcal serogroup B MC58 strain recovered at various time points at 0, 30, 60, and 90 minutes at 20% human serum concentrations after incubation in the presence of (A) normal human serum (NHS) + human anti-MASP-2 antibody; (B) normal human serum (NHS) + isotype control antibody; (C) MBL- / - human serum; (D) normal human serum (NHS); and (E) heat-inactivated normal human serum (NHS) is illustrated, showing that complement-dependent killing of meningococcal serogroup B MC58 strain in human serum was significantly enhanced by the addition of human anti-MASP-2 antibody, as described in Example 32. [Figure 39] The log cfu / ml of viable counts of Neisseria meningitidis serogroup B-MC58 recovered at various time points in mouse serum samples are shown, demonstrating that the bactericidal activity level of MASP-2- / - mouse serum against Neisseria meningitidis is higher than that of WT mouse serum, as described in Example 32. [Figure 40] The hemolysis of mannan-coated mouse erythrocytes with human serum at a range of serum concentrations (measured by hemoglobin release from lysed mouse erythrocytes (Crry / C3- / -) into the supernatant, measured by photometric method) is illustrated. As described in Example 33, the serum tested contained heat-inactivated (HI) NHS, MBL- / -, NHS+ anti-MASP-2 antibody, and NHS control. [Figure 41] The hemolysis of uncoated mouse erythrocytes by human serum at a range of serum concentrations (measured by hemoglobin release from WT mouse erythrocytes dissolved in supernatant, measured by photometric method) is illustrated. The serums tested included thermally inactivated (HI) NHS, MBL- / -, NHS+ anti-MASP-2 antibody, and NHS control. As described in Example 33, it has been demonstrated that inhibiting MASP-2 inhibits complement-mediated lysis of unsensitized WT mouse erythrocytes. [Figure 42]The hemolysis of uncoated mouse erythrocytes by human serum within a certain range of serum concentrations (measured by photometric analysis, specifically by the release of hemoglobin from lysed mouse erythrocytes (CD55 / 59- / -) into the supernatant) is illustrated. As described in Example 33, the tested serums included heat-inactivated (HI) NHS, MBL- / -, NHS+ anti-MASP-2 antibody, and an NHS control. [Figure 43] As described in Example 34, the percentage survival rate over time (days) after exposure to 8.0 Gy of radiation in control mice and mice treated with anti-human MASP-2 antibody (mAbH6) is illustrated. [Figure 44] The time to microvascular occlusion after LPS injection in MASP-2 mice— / - and WT mice—is illustrated, showing the percentage of mice with thrombus formation measured over 60 minutes. As described in Example 35, thrombus formation was detected in WT mice after 15 minutes, but by 60 minutes, up to 80% of WT mice showed thrombus formation. In contrast, none of the MASP-2— / - mice showed thrombus formation during the 60-minute period (log-rank: p=0.0005). [Figure 45] The time-dependent (hourly) survival percentages of saline-treated control mice (n=5) and anti-MASP-2 antibody-treated mice (n=5) in the STX / LPS-induced HUS model are illustrated, demonstrating that all control mice died by 42 hours, as described in Example 36, while in contrast, 100% of the anti-MASP-2 antibody-treated mice survived throughout the entire experimental time course. [Figure 46] As described in Example 37, the percentage of mice with microvascular occlusion as a function of time after injury induction in the FITC / dextran UV model, after treatment with isotype control or human MASP-2 antibody mAbH6 (10 mg / kg) administered 16 hours and 1 hour prior to FITC / dextran injection, is illustrated. [Figure 47]As described in Example 37, the occlusion time in minutes for mice treated with human MASP-2 antibody (mAbH6) and mice treated with isotype control antibody is plotted. Data are reported as scatter-dots with mean (horizontal bar) and standard error (vertical bar). The statistical test used for analysis was an independent t-test. The symbol "*" indicates p=0.0129. [Figure 48] As described in Example 37, the time to occlusion in a FITC-dextran / photo-induced endothelial cell injury model of thrombosis using low light intensity (800-1500) is illustrated in minutes for wild-type mice, MASP-2 knockout mice, and wild-type mice pre-treated with human MASP-2 antibody (mAbH6) by administering 10 mg / kg intravenously 16 hours before thrombosis induction and again 1 hour before thrombosis induction. [Figure 49] This is a Kaplan-Meier plot showing the percentage of mice with thrombosis as a function of time in FITC-dextran-induced thrombotic microangiopathy mice treated with gradually increasing doses of human MASP-2 inhibitory antibody (mAbH6) or isotype control antibody, as described in Example 39. [Figure 50] As described in Example 39, the median time to thrombus formation (minutes) as a function of mAbH6 dose is shown (p<0.01 compared to the control). [Figure 51] This is a Kaplan-Meier plot showing the percentage of mice with microvascular occlusion as a function of time in FITC-dextran-induced thrombotic microangiopathy mice treated with gradually increasing doses of human MASP-2 inhibitory antibody (mAbH6) or isotype control antibody, as described in Example 39. [Figure 52] As described in Example 39, the median time to microvascular occlusion as a function of mAbH6 dose is plotted (p<0.05 compared to the control). [Figure 53A]As described in Example 40, the MAC deposition levels in the presence or absence of the human MASP-2 monoclonal antibody (OMS646) under lectin pathway-specific assay conditions are illustrated. From this, it is demonstrated that OMS646 inhibits lectin-mediated MAC deposition with an IC50 value of approximately 1 nM. [Figure 53B] As described in Example 40, the MAC deposition levels under classical pathway-specific assay conditions, with and without the human MASP-2 monoclonal antibody (OMS646), are illustrated. This demonstrates that OMS646 does not inhibit MAC deposition via the classical pathway. [Figure 53C] As described in Example 40, the MAC deposition levels in the presence or absence of the human MASP-2 monoclonal antibody (OMS646) under secondary pathway-specific assay conditions are illustrated. From this, it is demonstrated that OMS646 does not inhibit MAC deposition via the secondary pathway. [Figure 54] As described in Example 40, the pharmacokinetic (PK) profile of the human MASP-2 monoclonal antibody (OMS646) in mice is illustrated. The OMS646 concentration (n=3 animals / group mean) is shown as a function of time after administration at the indicated dose. [Figure 55A] As described in Example 40, the pharmacodynamic (PD) response of human MASP-2 monoclonal antibody (OMS646), measured as a decrease in systemic lectin pathway activity in mice after intravenous administration, is illustrated. [Figure 55B] As described in Example 40, the pharmacodynamic (PD) response of the human MASP-2 monoclonal antibody (OMS646), measured as a decrease in systemic lectin pathway activity in mice after subcutaneous administration, is illustrated. [Figure 56] As described in Example 41, the inhibitory effect of the MASP-2 antibody (OMS646) on aHUS serum-induced C5b-9 deposition on ADP-activated HMEC-1 cells compared to sCR1 is illustrated. [Figure 57]As described in Example 42, the inhibitory effect of the MASP-2 antibody (OMS646) on aHUS serum-induced thrombus formation on ADP-activated HMEC-1 cells compared to sCR1 is illustrated. [Figure 58] The mean weekly change in platelet count from baseline in subjects with persistent hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA) after treatment with the MASP-2 inhibitory antibody (OMS646) as described in Example 46 is shown. [Figure 59] The mean weekly change in LDH from baseline in subjects suffering from persistent HSCT-TMA after treatment with the MASP-2 inhibitory antibody (OMS646) as described in Example 46 is shown. [Figure 60] The mean weekly change in haptoglobin levels from baseline in subjects with persistent HSCT-TMA after treatment with the MASP-2 inhibitory antibody (OMS646) as described in Example 46 is shown. [Figure 61] The clinical course of a hematopoietic stem cell transplant (HSCT) patient who developed HSCT-TMA and graft-versus-host disease (GVHD) after treatment with a MASP-2 inhibitory antibody (OMS646), as described in Example 47, is illustrated. [Figure 62A] The creatinine levels over time in compassionate use patient #1, as described in Example 48, are shown, where the vertical line indicates the start of treatment with the MASP-2 inhibitory antibody (OMS646). [Figure 62B] The time course of haptoglobin levels in compassionate-use patient #1, as described in Example 48, is illustrated, where the vertical line indicates the start of treatment with the MASP-2 inhibitory antibody (OMS646). [Figure 62C] The hemoglobin levels over time in compassionate-use patient #1, as described in Example 48, are shown, where the vertical line indicates the start of treatment with the MASP-2 inhibitory antibody (OMS646). [Figure 62D]The time course of LDH levels in compassionate-use patient #1, as described in Example 48, is illustrated, where the vertical line indicates the start of treatment with the MASP-2 inhibitory antibody (OMS646). [Figure 62E] The platelet levels over time in compassionate-use patient #1, as described in Example 48, are shown, where the vertical line indicates the start of treatment with the MASP-2 inhibitory antibody (OMS646). [Modes for carrying out the invention]

[0032] Explanation of the sequence list SEQ ID NO:1 Human MAp19 cDNA SEQ ID NO:2 Human MAp19 protein (with leader) SEQ ID NO:3 Human MAp19 protein (mature) SEQ ID NO:4 Human MASP-2 cDNA SEQ ID NO:5 Human MASP-2 protein (with leader) SEQ ID NO:6 Human MASP-2 protein (mature) SEQ ID NO:7 Human MASP-2 gDNA (Exons 1-6) Antigen: (Based on MASP-2 mature protein) SEQ ID NO:8 CUBI sequence (aa1~121) SEQ ID NO:9 CUBEGF sequence (aa1~166) SEQ ID NO:10 CUBEGFCUBII(aa1~293) SEQ ID NO: 11 EGF region (aa122~166) SEQ ID NO:12 Serine protease domain (aa429~671) SEQ ID NO:13 Serine protease domain inactive (aa610~625 with a mutation from Ser618 to Ala) TIFF2026062897000002.tif63138 Peptide inhibitors: SEQ ID NO:20 MBL full-length cDNA SEQ ID NO:21 MBL Full-Length Protein SEQ ID NO:22 OGK-X-GP (Consensus Joint) TIFF2026062897000003.tif71134 Expression inhibitors: SEQ ID NO:30 CUBI-EGF domain cDNA (nucleotides 22-680 of SEQ ID NO:4) SEQ ID NO:31 TIFF2026062897000004.tif5134MASP-2 Nucleotides 12-45 (sense) of SEQ ID NO:4 including translation start point SEQ ID NO:32 TIFF2026062897000005.tif5134MASP-2 Nucleotides 361-396 (sense) of SEQ ID NO:4 encoding the region containing the MBL binding site. SEQ ID NO:33 Nucleotides 610-642 of SEQ ID NO:4, which encodes the region containing the TIFF2026062897000006.tif5134CUBII domain. Cloning primers: TIFF2026062897000007.tif41134SEQ ID NO:38~47 are cloning primers for humanized antibodies. SEQ ID NO:48 is a 9aa peptide bond. Expression vector: SEQ ID NO:49 is a MASP-2 minigene insert. SEQ ID NO:50 is mouse MASP-2 cDNA. SEQ ID NO:51 is the mouse MASP-2 protein (with leader). SEQ ID NO:52 is the mature mouse MASP-2 protein. SEQ ID NO:53 is rat MASP-2 cDNA. SEQ ID NO:54 is rat MASP-2 protein (with leader). SEQ ID NO:55 is the mature rat MASP-2 protein. SEQ ID NO:56~59 are site-directed mutagenesis oligonucleotides of human MASP-2 used to generate human MASP-2A. SEQ ID NO:60~63 are site-directed mutagenesis oligonucleotides of mouse MASP-2 used to generate mouse MASP-2A. SEQ ID NO:64~65 are site-directed mutagenesis oligonucleotides of rat MASP-2 used to generate rat MASP-2A. SEQ ID NO:66 17D20_dc35VH21N11VL(OMS646) DNA encoding the heavy chain variable region (VH) (without signal peptide) SEQ ID NO:67 17D20_dc35VH21N11VL(OMS646) Heavy Chain Variable Region (VH) Polypeptide SEQ ID NO:68 17N16mc heavy chain variable region (VH) polypeptide SEQ ID NO:69: 17D20_dc35VH21N11VL(OMS646) DNA encoding the light chain variable region (VL) SEQ ID NO:70: 17D20_dc35VH21N11VL(OMS646) Light Chain Variable Region (VL) Polypeptide SEQ ID NO:71: 17N16_dc17N9 Light chain variable region (VL) polypeptide

[0033] Detailed explanation This invention is based on the remarkable discovery by the inventors that it is possible to inhibit the lectin-mediated MASP-2 pathway while leaving the classical pathway intact. The invention also describes the use of MASP-2 as a therapeutic target to inhibit cytotoxicity associated with lectin-mediated complement pathway activation while leaving the classical (C1q-dependent) pathway components of the immune system intact.

[0034] I. Definition Unless otherwise defined herein, all terms used herein have the same meaning as those understood by those skilled in the art. For the purposes of explaining the present invention, the following definitions are provided:

[0035] As used herein, the term “MASP-2-dependent complement activation” refers to the formation of the lectin pathway C3 convertase C4b2a and the subsequent formation of the C5 convertase C4b2a(C3b)n upon accumulation of the C3 cleavage product C3b, under physiological conditions (i.e., Ca ++ This includes MASP-2-dependent activation of the lectin pathway (in the presence of ), and has been found to primarily cause opsonization.

[0036] As used herein, the term “secondary pathway” refers to complement activation induced by, for example, zymosan derived from fungal and yeast cell walls, lipopolysaccharide (LPS) derived from the outer membrane of Gram-negative bacteria, rabbit erythrocytes, and many pure polysaccharides, rabbit erythrocytes, viruses, bacteria, animal tumor cells, parasites, and damaged cells, which has traditionally been thought to result from the spontaneous proteolytic production of complement factor C3 to C3b.

[0037] As used herein, the term “lectin pathway” refers to complement activation that occurs via the specific binding of serum carbohydrate-binding proteins and non-serum carbohydrate-binding proteins, including mannan-binding lectins (MBL), CL-11, and phycolins (H-phycolin, M-phycolin, or L-phycolin).

[0038] As used herein, the term “classical pathway” refers to complement activation induced by an antibody bound to an exogenous particle, which requires the binding of the recognition molecule C1q.

[0039] As used herein, the term “MASP-2 inhibitor” refers to any active substance that effectively inhibits MASP-2-dependent complement activation by binding to or directly interacting with MASP-2, and includes anti-MASP-2 antibodies and their MASP-2 binding fragments, native and synthetic peptides, small molecule, soluble MASP-2 receptors, expression inhibitors, and isolated native inhibitors, and also includes peptides that compete with MASP-2 for binding to other recognition molecules (e.g., MBL, H-phycoline, M-phycoline, or L-phycoline) in the lectin pathway, but does not include antibodies that bind to such other recognition molecules. MASP-2 inhibitors useful in the methods of the present invention can reduce MASP-2-dependent complement activation by more than 20%, for example, more than 50%, for example, more than 90%. In one embodiment, a MASP-2 inhibitor reduces MASP-2-dependent complement activation by more than 90% (i.e., resulting in MASP-2 complement activation of 10% or less).

[0040] As used herein, the term “antibody” includes antibodies and antibody fragments derived from any antibody-producing mammal (e.g., primates including mice, rats, rabbits, and humans) or from hybridomas, phage selection, recombinant expression, or transgenic animals (or other methods for producing antibodies or antibody fragments) that specifically bind to a target polypeptide, such as the MASP-2 polypeptide or a portion thereof. The term “antibody” is not intended to be limited in terms of the antibody source or the manner in which the antibody is produced (e.g., by hybridomas, phage selection, recombinant expression, transgenic animals, peptide synthesis, etc.). Exemplary antibodies include polyclonal antibodies, monoclonal antibodies, and recombinant antibodies; pan-specific, multispecific antibodies (e.g., bispecific antibodies, tripspecific antibodies); humanized antibodies: mouse antibodies; chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies; and anti-idiotype antibodies, which may also include any intact antibody or fragment thereof. As used herein, the term “antibody” encompasses not only intact polyclonal or monoclonal antibodies, but also their fragments (e.g., dAb, Fab, Fab', F(ab')2, Fv), single chains (ScFv), their synthetic variants, native variants, fusion proteins containing the antibody moiety and antigen-binding fragments of the required specificity, humanized antibodies, chimeric antibodies, and any other modified configurations of immunoglobulin molecules containing antigen-binding sites or fragments (epitope recognition sites) of the required specificity.

[0041] A “monoclonal antibody” refers to a homogeneous population of antibodies, consisting of amino acids (natural and non-natural) involved in the selective binding of epitopes. Monoclonal antibodies are highly specific to their target antigens. The term “monoclonal antibody” encompasses not only intact and full-length monoclonal antibodies, but also their fragments (e.g., Fab, Fab', F(ab')2, Fv), single chains (ScFv), their variants, fusion proteins containing antigen-binding moieties, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configurations of immunoglobulin molecules containing antigen-binding fragments (epitope-recognition sites) with the required specificity and ability to bind to epitopes. This term is not intended to be limited in terms of the antibody source or the way in which the antibody is produced (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). This term includes whole immunoglobulins and fragments, etc., as previously mentioned in the definition of “antibody.”

[0042] As used herein, the term “antibody fragment” refers to a portion of a full-length antibody, such as an anti-MASP-2 antibody, that is derived from or associated with it, and generally includes its antigen-binding region or variable region. Exemplary examples of antibody fragments include Fab, Fab', F(ab)2, F(ab')2, and Fv fragments, scFv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0043] The “single-chain Fv” or “scFv” antibody fragments used herein are the V of the antibody. H Domain or V L It contains domains, and these domains are present in a single polypeptide chain. Generally, Fv polypeptides are V H Domain and V L The scFv further contains a polypeptide linker between the domain and the scFv, which allows it to form a desirable structure for antigen binding.

[0044] As used herein, "chimeric antibody" is a recombinant protein that contains a variable domain and complementarity-determining region derived from a non-human species (e.g., rodent) antibody, but the remainder of the antibody molecule is derived from a human antibody.

[0045] As used herein, "humanized antibody" is a chimeric antibody that contains a minimal sequence corresponding to a specific complementarity-determining region derived from a non-human immunoglobulin, transplanted into a human antibody framework. Humanized antibodies are typically recombinant proteins in which only the antibody complementarity-determining region is derived from a non-human source.

[0046] As used herein, the term “mannan-binding lectin” (“MBL”) is synonymous with “mannan-binding protein” (“MBP”).

[0047] As used herein, “membrane invasion complex” (“MAC”) refers to a complex of five terminal complement components (combinations of C5b with C6, C7, C8, and C9) (also known as C5b-9) that penetrate and disrupt membranes.

[0048] As used herein, “subjects” includes all mammals, including but not limited to humans, non-human primates, dogs, cats, horses, sheep, goats, cattle, rabbits, pigs, and rodents.

[0049] The abbreviations for amino acid residues used herein are as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0050] In its broadest sense, natural amino acids can be grouped based on the chemical properties of their side chains. "Hydrophobic" amino acids are Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys, or Pro. "Hydrophilic" amino acids are Gly, Asn, Gln, Ser, Thr, Asp, Glu, Lys, Arg, or His. This group of amino acids can be further divided into subgroups as follows: "Uncharged hydrophilic" amino acids are Ser, Thr, Asn, or Gln. "Acidic" amino acids are Glu or Asp. "Basic" amino acids are Lys, Arg, or His.

[0051] As used herein, the term “conservative amino acid substitution” is exemplified by substitutions between amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine; (2) phenylalanine, tyrosine, and tryptophan; (3) serine and threonine; (4) aspartic acid and glutamic acid; (5) glutamine and asparagine; and (6) lysine, arginine, and histidine.

[0052] As used herein, the term “oligonucleotide” refers to oligomers or polymers of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or their mimics. This term also covers oligonucleotides that are naturally occurring, consisting of nucleotides, sugars, and oligonucleobases with nucleoside-to-nucleoside (backbone) covalent bonds, as well as oligonucleotides with unnatural modifications.

[0053] As used herein, "epitope" refers to a site on a protein (e.g., human MASP-2 protein) to which an antibody binds. A "duplicate epitope" includes at least one (e.g., two, three, four, five, or six) common amino acid residues, including a linear epitope and a non-linear epitope.

[0054] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used synonymously and refer to any peptide-bonded amino acid chain, regardless of length or post-translational modifications. The MASP-2 proteins described herein may contain wild-type proteins, or may be wild-type proteins, or may be variants having 50 or fewer (e.g., 1 or fewer, 2 or fewer, 3 or fewer, 4 or fewer, 5 or fewer, 6 or fewer, 7 or fewer, 8 or fewer, 9 or fewer, 10 or fewer, 12 or fewer, 15 or fewer, 20 or fewer, 25 or fewer, 30 or fewer, 35 or fewer, 40 or fewer, or 50 or fewer) conserved amino acid substitutions. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine, and threonine; lysine, histidine, and arginine; and phenylalanine and tyrosine.

[0055] In some embodiments, the human MASP-2 protein may have an amino acid sequence that is 70% (e.g., 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to or more than 70% (e.g., 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the human MASP-2 protein having the amino acid sequence shown in SEQ ID NO:5.

[0056] In some embodiments, the peptide fragments have a length of at least 6 (for example, at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43) , 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, or 600 or more amino acid residues (for example, at least 6 consecutive amino acid residues of SEQ ID NO: 5). In some aspects, the antigenic peptide fragments of the human MASP-2 protein have a length of less than 500 (e.g., less than 450, less than 400, less than 350, less than 325, less than 300, less than 275, less than 250, less than 225, less than 200, less than 190, less than 180, less than 170, less than 160, less than 150, less than 140, less than 130, less than 120, less than 110, less than 100, less than 95, less than 90, less than 85, less than 80, less than 75, less than 70, less than 65, less than 60, less than 55, less than 50, less than 49, less than 48, less than 47, These are amino acid residues of less than 46, less than 45, less than 44, less than 43, less than 42, less than 41, less than 40, less than 39, less than 38, less than 37, less than 36, less than 35, less than 34, less than 33, less than 32, less than 31, less than 30, less than 29, less than 28, less than 27, less than 26, less than 25, less than 24, less than 23, less than 22, less than 21, less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, or less than 6 (for example, less than 500 consecutive amino acid residues in any one of SEQ ID NO: 5).

[0057] 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 aligning the sequences to achieve maximum percentage sequence identity and introducing gaps if necessary. For the purpose of determining percentage sequence identity, alignment can be achieved in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithm necessary to achieve maximum alignment over the full length of the sequences being compared, can be determined by known methods.

[0058] II. Outline of the present invention Lectins (MBL, M-phycoline, H-phycoline, L-phycoline, and CL-11) are specific recognition molecules that induce the innate complement system, which includes a lectin-initiation pathway and an associated terminal pathway amplification loop that amplifies the activation of terminal complement effector molecules initiated by lectins. C1q is a specific recognition molecule that induces the acquired complement system, which includes a classical initiation pathway and an associated terminal pathway amplification loop that amplifies the activation of terminal complement effector molecules initiated by C1q. We refer to these two major complement activation systems as the lectin-dependent complement system and the C1q-dependent complement system, respectively.

[0059] In addition to its essential role in immune defense, the complement system contributes to tissue damage in many clinical conditions. Therefore, there is an urgent need to develop therapeutically effective complement inhibitors to counteract these side effects. Recognizing that it is possible to inhibit the lectin-mediated MASP-2 pathway while leaving the classical pathway intact, it was realized that it is highly desirable to specifically inhibit only the complement activation system responsible for certain pathological conditions, without completely blocking the complement's immune defense capabilities. For example, in disease states where complement activation is primarily mediated by the lectin-dependent complement system, specifically inhibiting only this system is considered advantageous. For this reason, the C1q-dependent complement activation system is likely to remain intact to handle immune complex processing and assist host defense against infection.

[0060] In the development of therapeutic substances that specifically inhibit the lectin-dependent complement system, the preferred protein component to target is MASP-2. Of all the known protein components of the lectin-dependent complement system (MBL, H-ficoline, M-ficoline, L-ficoline, MASP-2, C2-C9, factor B, factor D, and properzin), only MASP-2 is unique to the lectin-dependent complement system and is required for the system to function. Lectins (MBL, H-ficoline, M-ficoline, L-ficoline, and CL-11) are also unique components of the lectin-dependent complement system. However, it is thought that the activation of the system is not necessarily inhibited even if any one of these lectin components is absent due to lectin redundancy. It is thought that inhibiting all five types of lectins is necessary to guarantee inhibition of the lectin-dependent complement activation system. Furthermore, since MBL and ficoline are known to also possess opsonin activity independently of complement, it is thought that inhibiting lectin function would result in the loss of this beneficial host defense mechanism against infection. In contrast, when MASP-2 is the inhibitory target, this complement-independent lectin opsonin activity is thought to remain intact. An additional benefit of MASP-2 as a therapeutic target for inhibiting the lectin-dependent complement activation system is that the plasma concentration of MASP-2 (approximately 500 ng / ml) is within the lowest plasma concentrations of complement proteins. Therefore, to achieve complete inhibition, a correspondingly low concentration of a high-affinity MASP-2 inhibitor may suffice (Moller-Kristensen, M., et al., J. Immunol Methods 282:159-167, 2003).

[0061] III. The role of MASP-2 in thrombotic microangiopathy, and therapeutic methods using MASP-2 inhibitors. Overview Thrombotic microangiopathy (TMA) is a condition characterized by blood clots in small blood vessels (Benz. K.; et al., Curr Opin Nephrol Hypertens 19(3):242-7(2010)). Stress or damage to the underlying vascular endothelium is considered the primary driver. Clinical and laboratory findings of TMA include thrombocytopenia, anemia, purpura, and renal failure. Classical TMAs are hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). The characteristic underlying pathological features of TMA are platelet activation and microthrombus formation in arterioles and venules. Complement activation, at least partially initiated by damage or stress to the microvascular endothelium, is also involved in fulminant antiphospholipid syndrome (CAPS), systemic Degos disease, and other TMAs, including cancer-related TMA, cancer chemotherapy-related TMA, and transplant-related TMA.

[0062] Studies of patients with genetic deficiencies in specific complement components have provided direct evidence for the pathological role of complement in nephritis hosts. Numerous reports have described a link between renal injury and complement-regulating factor H deficiency (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 lower plasma levels of factor B and C3 because these components are consumed in connection with activation. Circulating serum levels of C5b-9 are also increased in these patients, indicating complement activation. Membranoproliferative glomerulonephritis (MPGN) and idiopathic hemolytic uremic syndrome (HUS) are associated with factor H deficiency or mutation. Pigs deficient in factor H (Jansen, JH, et al., Kidney Int. 53:331-49, 1998) and mice knocked out with factor H (Pickering, MC, 2002) exhibit MPGN-like symptoms. This supports the importance of factor H in complement regulation. Deficiencies in other complement components are associated with kidney 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 are strong predisposing factors for the development of SLE through mechanisms related to the incomplete clearance of immune complexes and apoptotic substances. Many of these SLE patients develop lupus nephritis, characterized by the deposition of immune complexes throughout the glomeruli.

[0063] aHUS Atypical hemolytic uremic syndrome (aHUS) is part of a group of conditions called “thrombotic microangiopathy.” In atypical HUS (aHUS), the disorder is associated with incomplete complement regulation and can be sporadic or familial. Familial aHUS cases are associated with mutations in genes encoding complement activating or complement regulatory proteins, including complement factors H, I, 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 holistic feature of this broad range of genetic mutations associated with aHUS is a predisposition to enhanced complement activation on the cell or tissue surface. Accordingly, one aspect of the present invention involves treating patients suffering from aHUS associated with factor H deficiency by administering an effective dose of a MASP-2 inhibitor. Another aspect of the present invention involves treating patients suffering from HUS associated with deficiencies in factor I, factor B, membrane cofactor CD46, CFHR1, or CFHR3 by administering an effective amount of a MASP-2 inhibitor.

[0064] Recent significant progress has been made toward understanding the molecular pathophysiology underlying the enhancement of complement activation in aHUS caused by diverse mutant complement factor sets. This mechanism is best understood for factor H mutations. Factor H is a rich serum protein containing 20 short consensus repeat (SCR) domains that acts as a negative regulator of complement activation in the lysed state and on the host cell surface. It targets activated C3 along with factor I and other cofactors, promoting its inactivation and preventing further complement activation. To effectively regulate complement activation on the host cell surface, factor H must interact with the host cell, which is mediated by SCR domains 16-20. All factor H mutations associated with aHUS described to date are clustered in the C-terminal region containing (SCR) domains 16-20. These mutant factor H proteins function fully in regulating C3 activation in a lysed state, but are unable to interact with the host cell surface and, consequently, cannot regulate C3 activation on the cell surface (Exp Med 204(6):1249-56(2007)). Therefore, because mutant factor H proteins do not interact with the host cell surface and thus cannot effectively reduce complement activation on the host cell surface, including microvascular endothelium, certain factor H mutations are associated with aHUS. As a result, once initial C3 activation occurs, subsequent complement activation on the microvascular endothelial surface proceeds with uninterrupted momentum in patients with factor H mutations. This unregulated complement activation ultimately leads to progressive damage to the vascular endothelium, subsequent platelet aggregation and microvascular coagulation, and hemolysis caused by shear stress on RBC passage through partially occluded microvessels. Thus, the manifestation of aHUS disease symptoms and clinical and laboratory findings are directly related to a defect in negative complement regulation on the microvascular endothelial surface.

[0065] Similar to factor H mutations, loss-of-function mutations in negative complement modulators, factor I and membrane cofactor protein (CD46), are also associated with aHUS. The opposite has been observed for these proteins, as aHUS has been found to be associated with gain-of-function mutations in factor B and C3 proteins (Pediatr Nephrol 25(12):2431-42(2010)). Therefore, a large body of data suggests that complement activation is involved in the development of aHUS. This idea is most compellingly supported by the therapeutic efficacy of eculizumab, a monoclonal antibody that blocks terminal complement protein C5, in the treatment of aHUS.

[0066] While the central role of complement as an effector mechanism in aHUS is widely recognized, the triggers and molecular pathways involved in initiating complement activation remain unresolved. Not all individuals with the aforementioned mutations develop aHUS. In fact, family studies suggest that the penetrance of aHUS is only about 50% (Ann Hum Genet 74(1):17-26(2010)). The natural course of the disease suggests that aHUS almost always develops after an initiating event such as an infection episode or injury. It is well known that infectious agents activate the complement system. In the absence of pre-existing acquired immunity, complement activation by infectious agents can be initiated primarily via the lectin pathway. Therefore, lectin pathway activation induced by infection may be an initiating trigger for pathological amplification after complement activation in individuals predisposed to aHUS, potentially leading to disease progression. Thus, another aspect of the present invention involves treating patients suffering from aHUS secondary to infection by administering an effective dose of a MASP-2 inhibitor.

[0067] Other forms of damage to host tissues, particularly damage to vascular endothelium, activate complement via the lectin pathway. Human vascular endothelial cells under oxidative stress respond, for example, by expressing surface moieties that bind to lectins and activate the complement lectin pathway (Am J. Pathol 156(6):1549-56(2000)). Vascular injury after ischemia / reperfusion also activates complement via the lectin pathway in vivo (Scand J Immunol 61(5):426-34(2005)). Lectin pathway activation in this context leads to pathological consequences for the host, and lectin pathway inhibition by MASP-2 blockade prevents further host tissue damage and adverse events (Sehwaeble PNAS 2011).

[0068] Therefore, it is known that other processes that abruptly trigger aHUS also activate the complement lectin pathway. Thus, the lectin pathway is likely to be improperly amplified in a disordered manner in individuals with a genetic predisposition to aHUS, and thus may be an initial complement activation mechanism that initiates aHUS pathogenesis. By reason, it is expected that agents that block complement activation via the lectin pathway, including anti-MASP-2 antibodies, will inhibit disease progression or reduce exacerbations in aHUS-susceptible individuals.

[0069] Further supporting this idea, recent studies have identified Streptococcus pneumoniae (S. pneumoniae) as a key pathogenic agent in pediatric cases of aHUS (Nephrology (Carlton), 17:48-52 (2012); Pediatr Infect Dis J. 30(9):736-9 (2011)). This particular etiology appears to be associated with an unfavorable prognosis, high mortality, and prolonged pathological status. Notably, these cases lacked evidence of synchronous complement gene mutations known to predispose individuals to aHUS, and were accompanied by non-enteric infections leading to the manifestation of microangiopathy, uremia, and hemolytic symptoms. It is important to note that Streptococcus pneumoniae is particularly effective in complement activation, primarily via the lectin pathway. Therefore, in cases of non-intestinal HUS associated with pneumococcal infection, the manifestation of microangiopathy, uremia, and hemolysis is expected to be primarily driven by lectin pathway activation, and lectin pathway-blocking agents, including anti-MASP-2 antibodies, are expected to inhibit aHUS progression and reduce disease severity in these patients. Accordingly, another aspect of the present invention involves treating patients suffering from non-intestinal aHUS associated with pneumococcal infection by administering an effective amount of a MASP-2 inhibitor.

[0070] In accordance with the foregoing, in some embodiments, a method is provided for reducing the likelihood of developing aHUS or aHUS-related renal failure, comprising the step of administering, in the case of a subject at risk of developing aHUS-related renal failure, an amount of a MASP-2 inhibitor effective in relieving or preventing renal failure in said subject, for a period of time effective in relieving or preventing said renal failure. In some embodiments, the method further includes the step of determining whether the subject is at risk of developing aHUS before the manifestation of aHUS-related symptoms. In other embodiments, the method includes the step of determining whether the subject is at risk of developing aHUS based on the manifestation of at least one symptom indicating aHUS (e.g., the presence of anemia, thrombocytopenia, and / or renal failure in the subject), and / or the presence of thrombotic microangiopathy in biopsy material obtained from the subject. Determining whether a subject is at risk of developing aHUS includes determining whether the subject has a genetic predisposition to develop aHUS, which may be done by evaluating genetic information (e.g., from a database containing the subject's genotype) or by performing at least one genetic screening test on the subject to determine the presence or absence of aHUS-related genetic markers (i.e., by determining the presence or absence of aHUS-related genetic mutations in genes encoding complement factor H (CFH), factor I (CFI), factor B (CFB), membrane cofactor CD46, C3, complement factor H-related protein 1 (CFHR1), or THBD (encoding the anticoagulant protein thrombodulin), or complement factor H-related protein 3 (CFHR3), or complement factor H-related protein 4 (CFHR4) by genome sequencing or gene-specific analysis (e.g., PCR analysis)), and / or by determining whether the subject has a family history of aHUS. Genetic screening methods for the presence or absence of aHUS-related genetic mutations are well established.For example, see Noris M et al. "Atypical Hemolytic-Uremic Syndrome," 2007 Nov 16 [Updated May 10, 2011]. In: Pagon RA, Bird TD, Dolan CR, et al., eds. GeneReviews(trademark), Seattle (WA): University of Washington, Seattle.

[0071] For example, overall, the disease penetrance in individuals with complement factor H (CFH) mutations is 48%, the penetrance for CD46 mutations is 53%, the penetrance for CFI mutations is 50%, the penetrance for C3 mutations is 56%, and the penetrance for THBD mutations is 64% (Caprioli J. et al., Blood, 108:1267-79 (2006); Noris et al., Clin J Am Soc Nephrol 5:1844-59 (2010)). Caprioli et al., (2006), as described above, a considerable number of individuals with complement factor H (CFH) mutations never develop aHUS, and it is assumed that suboptimal CFH activity in these individuals is sufficient to protect the host from the effects of complement activation under physiological conditions. However, when exposed to substances that activate complement and produce more C3b than normal, suboptimal CFH activity is insufficient to prevent C3b deposition on vascular endothelial cells.

[0072] Accordingly, in one embodiment, a method is provided for inhibiting MASP-2-dependent complement activation in a subject suffering from or at risk of developing non-factor-H-dependent atypical hemolytic uremic syndrome, the method comprising administering to the subject a composition containing an amount of a MASP-2 inhibitor effective in inhibiting MASP-2-dependent complement activation. In another embodiment, a method is provided for inhibiting MASP-2-dependent complement activation in a subject at risk of developing factor-H-dependent atypical hemolytic uremic syndrome, comprising the steps of periodically monitoring the subject to determine the presence or absence of anemia, thrombocytopenia, or elevated creatinine, and treating the subject with a MASP-2 inhibitor based on the determination that anemia, thrombocytopenia, or elevated creatinine is present. In another embodiment, a method is provided for reducing the likelihood of a subject at risk of developing factor H-independent aHUS suffering from aHUS-related clinical symptoms, comprising the step of administering a MASP-2 inhibitor before, during, or after an event known to be associated with the induction of aHUS clinical symptoms, such as drug exposure (e.g., chemotherapy), infection (e.g., bacterial infection), malignancy, injury, organ or tissue transplantation, or pregnancy.

[0073] In one embodiment, a method is provided for reducing the likelihood of a subject developing clinical symptoms associated with aHUS, comprising the steps of: regularly monitoring a subject at risk of developing aHUS to determine the presence or absence of anemia, thrombocytopenia, or elevated creatinine; and treating the subject with a MASP-2 inhibitor based on the determination that anemia, thrombocytopenia, or elevated creatinine is present.

[0074] In another embodiment, a method is provided for reducing the likelihood of a subject at risk of developing aHUS developing clinical symptoms associated with aHUS, comprising the step of administering a MASP-2 inhibitor before, during, or after an event known to be associated with the induction of aHUS clinical symptoms, such as drug exposure (e.g., chemotherapy), infection (e.g., bacterial infection), malignancy, injury, organ or tissue transplantation, or pregnancy.

[0075] In some embodiments, the MASP-2 inhibitor is administered for at least 1 day, 2 days, 3 days, 4 days, or a longer period before, during, or after an event associated with the induction of aHUS clinical symptoms, and may be repeated as determined by a physician until the condition recovers or is managed. In the context of pre-aHUS, the MASP-2 inhibitor may be administered systemically to the subject by, for example, intra-arterial administration, intravenous administration, intramuscular administration, inhalation administration, nasal administration, subcutaneous administration, or other parenteral administration.

[0076] In some embodiments, in the context of an initial diagnosis of aHUS, or in a subject presenting with one or more symptoms consistent with a diagnosis of aHUS (e.g., the presence of anemia, thrombocytopenia, and / or renal insufficiency), the subject is treated with an effective amount of a MASP-2 inhibitor (e.g., an anti-MASP-2 antibody) in the absence of plasmapheresis or in combination with plasmapheresis as a first-line therapy. As a first-line therapy, the MASP-2 inhibitor may be administered systemically to the subject by, for example, intra-arterial administration, intravenous administration, intramuscular administration, inhalation administration, nasal administration, subcutaneous administration, or other parenteral administration. In some embodiments, the MASP-2 inhibitor is administered as a first-line therapy to the subject in the absence of plasmapheresis, or in a subject who otherwise dislikes plasmapheresis, or in a situation where plasmapheresis is not available, in order to avoid potential plasmapheresis complications including bleeding, infections, and exposure to disorders, and / or allergies specific to plasma donors.

[0077] In some embodiments, the method comprises administering a MASP-2 inhibitor to a subject suffering from aHUS (e.g., intravenously) via a catheter for a first period (e.g., at least 1 day to 1 or 2 weeks), and then administering the MASP-2 inhibitor subcutaneously to the subject for a second period (e.g., at least 2 weeks or a longer chronic phase). In some embodiments, the administration in the first period and / or the second period is performed in the absence of plasmapheresis. In some embodiments, the method further comprises determining, before and optionally during treatment, the level of at least one complement factor (e.g., C3, C5) in the subject, and determination of a level of at least one complement factor that is decreased compared to a standard value or a healthy control subject indicates the need for continuous treatment with a MASP-2 inhibitor.

[0078] In some embodiments, the method comprises administering a MASP-2 inhibitor, e.g., an anti-MASP-2 antibody, to a subject suffering from aHUS or at risk of developing aHUS, either intravenously, intramuscularly, or preferably subcutaneously. The treatment is chronic and may be performed daily to monthly, preferably every two weeks. The anti-MASP-2 antibody may be administered alone or in combination with a C5 inhibitor such as eculizumab.

[0079] HUS Like atypical HUS, typical HUS presents with all the clinical and laboratory findings of TMA. However, typical HUS is often a childhood disease and usually has no familial component or direct association with complement gene mutations. The cause of typical HUS is closely related to infection with a specific enteropathogen. Patients typically present with acute renal failure, hemoglobinuria, and thrombocytopenia, and typically follow episodes of bloody diarrhea. This condition is caused by enteroinfection with Shigella dissenteria, Salmonella, or Shigella toxin-like enterohemorrhagic Escherichia coli strains, such as E. coli O157:H7. These pathogens are obtained from contaminated food or water supplies. HUS is a medical emergency with a mortality rate of 5–10%. A significant portion of the survivors develop chronic kidney disease (Corrigan and Boineau, Pediatr Rev 22(11):365-9(2011)) and may require a kidney transplant.

[0080] Microvascular coagulation in typical HUS primarily occurs in the renal microvessel system, but not exclusively. The underlying pathophysiology is mediated by Shiga toxin (STX). STX is excreted into the intestinal lumen by enteric disease microorganisms, crosses the intestinal barrier, enters the bloodstream, and binds to vascular endothelial cells via the blobotriaosyl ceramide receptor CD77 (Boyd and Lingwood Nephron 51:207 (1989)). CD77 is preferentially expressed on the glomerular endothelium and mediates the toxic effects of STX. When STX binds to the endothelium, it damages the vascular endothelium, activates leukocytes, and induces a series of events that lead to vWF-dependent thrombus formation (Forsyth et al., Lancet 2:411-414 (1989); Zoja et al., Kidney Int. 62:846-856 (2002); Zanchi et al., J. Immunol 183:1460-1469 (2008); Morigi et al., Blood 98:1828-1835 (2001); Guessou et al., Infect. Immun., 73:8306-8316 (2005)). These microthrombi obstruct or occlude arterioles and capillaries in the kidneys and other organs. As red blood cells (RBCs) push through narrowed blood vessels, the obstruction of blood flow in arterioles and capillaries by microthrombi increases the shear stress applied to the RBCs. As a result, the RBCs may be destroyed by the shear stress, and RBC fragments called mitotic red blood cells may be formed. The presence of mitotic red blood cells is a characteristic finding in HUS. This mechanism is known as microangiopathic hemolysis. Furthermore, blood flow obstruction leads to ischemia, initiating a complement-mediated inflammatory response that causes further damage to the affected organ.

[0081] The complement lectin pathway contributes to HUS development through two main mechanisms: (1) direct coagulation cascade activation via MASP-2 caused by endothelial damage, and (2) subsequent complement activation via lectins induced by ischemia resulting from initial occlusion of microvascular blood flow.

[0082] It is known that STX damages microvascular endothelial cells, and these damaged endothelial cells activate the complement system. As detailed above, complement activation after endothelial cell damage is primarily driven by the lectin pathway. Human vascular endothelial cells subjected to oxidative stress respond by expressing a surface portion that binds to lectins and activates the complement lectin pathway (Collard et al., Am J Pathol. 156(5):1549-56 (2000)). Vascular damage after 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 situation leads to pathological consequences for the host, and inhibition of the lectin pathway by MASP-2 blockade prevents further host tissue damage and adverse events (Schwaeble et al., PNAS (2011)). In addition to complement activation, lectin-dependent activation of MASP-2 has been shown to cleave prothrombin to form thrombin and promote coagulation. Therefore, activation of the complement lectin pathway by damaged endothelial cells can directly activate the coagulation system. Thus, the complement lectin pathway via prothombin activation through MASP-2 is likely a major molecular pathway linking initial endothelial damage caused by STX to coagulation and microvascular thrombosis occurring in HUS. Therefore, lectin pathway inhibitors, including but not limited to antibodies that block MASP-2 function, are expected to prevent or mitigate microvascular coagulation, thrombosis, and hemolysis in patients with HUS. Indeed, administration of anti-MASP-2 antibodies significantly protects mice of a typical HUS model. As described in Example 36 and shown in Figure 45, all control mice exposed to STX and LPS developed severe HUS and were either morbid or dead within 48 hours. On the other hand, as further shown in Figure 45, all mice treated with anti-MASP-2 antibody and subsequently exposed to STX and LPS survived (Fisher exact p<0.01; N=5). Therefore, anti-MASP-2 therapy significantly protects mice in this HUS model.Administration of MASP-2 inhibitors, such as MASP-2 antibodies, is expected to be effective in treating HUS patients and to provide protection from microvascular coagulation, thrombosis, and hemolysis caused by infections with enteric Escherichia coli or other STX-producing pathogens.

[0083] While STX-induced HUS has been described herein, anti-MASP-2 therapy is also expected to be beneficial for HUS-like syndromes caused by endothelial damage induced by other toxic substances. These include agents such as mitomycin, ticlopidine, cycloplatin, quinine, cyclosporine, and bleomycin, as well as other chemotherapeutic and immunosuppressive drugs. Therefore, anti-MASP-2 antibody therapy or other modalities that inhibit MASP-2 activity are expected to effectively block or limit coagulation, thrombosis, and RBC destruction, thereby preventing renal failure in HUS and other TMA-related diseases (i.e., aHUS and TTP).

[0084] Patients with HUS often present with diarrhea and vomiting, and typically have a low platelet count (thrombocytopenia) and a low red blood cell count (anemia). The pre-HUS diarrheal phase typically lasts about four days, during which individuals at risk of developing HUS typically exhibit severe diarrhea in addition to one or more of the following symptoms: a hematocrit level of less than 30% with evidence of smear of intravascular red blood cell destruction, and thrombocytopenia (platelet count <150 × 10⁻¹⁰). 3 / mm 3), and / or the presence of renal impairment (serum creatinine concentration exceeding the upper limit of the age-appropriate range). The presence of oliguria (urine volume > ≤ 0.5 mL / kg / h over a 1-day period) can be used as a measure of progression to the development of HUS (see C. Hickey et al., Arch Pediatr Adolesc Med 165(10):884-889(2011)). Typically, testing is performed for the presence of infection with Escherichia coli (Escherichia coli O157:H7) or Shigella or Salmonella species. In subjects who test positive for enterogenic Escherichia coli (e.g., Escherichia coli O157:H7) infection, antibiotic use is contraindicated. This is because antibiotic use may increase the risk of developing HUS due to increased STX production (see Wong C. et al., N Engl J. Med 342:1930-136(2000)). In subjects who test positive for Shigella or Salmonella, antibiotics are typically administered to clear the infection. Other well-established first-line therapies for HUS include volume expansion, dialysis, and plasmapheresis.

[0085] As described above, in some aspects, subjects who have one or more symptoms associated with the pre-HUS phase and subjects at risk of developing HUS (i.e., subjects exhibiting one or more of the following: diarrhea, hematocrit level less than 30% with evidence of intravascular red blood cell destruction smear, thrombocytopenia (150 × 10%) 3 / mm 3A method is provided for reducing the risk of developing HUS or the likelihood of renal failure in a subject, comprising the step of administering an amount of a MASP-2 inhibitor effective to remit or prevent renal impairment (serum creatinine concentration exceeding the upper limit of the reference range for age) in a situation of platelet count less than 100, and / or the presence of renal impairment (serum creatinine concentration exceeding the upper limit of the reference range for age). In some embodiments, the MASP-2 inhibitor may be administered for a period of at least 1, 2, 3, 4, or longer, and may be repeated as determined by a physician until the condition is recovered or controlled. In a pre-HUS situation, the MASP-2 inhibitor may be systemically administered to the subject, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, oral, subcutaneous, or other parenteral administration.

[0086] Treatment of E. coli O157:H7 infections with bactericidal antibiotics, particularly β-lactams, has been associated with an increased risk of developing HUS (Smith et al., Pediatr Infect Dis J). 31(1):37-41(2012). In some embodiments, a method is provided to reduce the risk of developing HUS or the likelihood of renal failure in a subject suffering from symptoms associated with the pre-HUS phase, in a subject known to be infected with enterocolitis (e.g., E. coli O157:H7) for which the use of antibiotics is contraindicated, comprising the step of administering an amount of a MASP-2 inhibitor effective in suppressing or preventing the presence of oliguria in the subject over a first period effective in suppressing or preventing the presence of oliguria (e.g., at least 1, 2, 3, or 4 days), wherein the administration of the MASP-2 inhibitor during the first period is carried out in the absence of antibiotics. In some embodiments, the method further comprises the step of administering the MASP-2 inhibitor to the subject in combination with an antibiotic over a second period (e.g., at least 1 to 2 weeks).

[0087] In another embodiment, a method is provided for reducing the risk of developing HUS or the likelihood of renal failure in a subject who is known to be suffering from symptoms associated with the pre-HUS phase and is infected with Shigella or Salmonella, comprising the step of administering an amount of a MASP-2 inhibitor effective in suppressing or preventing the presence of oliguria in the subject for a period effective in suppressing or preventing the presence of oliguria, with or without the presence of an appropriate antibiotic.

[0088] In some embodiments, in the context of an early diagnosis of HUS, or in subjects exhibiting one or more symptoms consistent with a diagnosis of HUS (e.g., renal failure, or the presence of microangiogenic hemolytic anemia in the absence of low fibrinogen, or thrombocytopenia), subjects are treated with an effective dose of a MASP-2 inhibitor (e.g., an anti-MASP-2 antibody) as first-line therapy, either in the absence of plasmapheresis or in combination with plasmapheresis. As first-line therapy, the MASP-2 inhibitor may be systemically administered to the subject, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration. In some embodiments, the MASP-2 inhibitor is administered to the subject as first-line therapy in the absence of plasmapheresis to avoid plasmapheresis complications such as bleeding, infection, and exposure to injury, as well as / or allergies specific to the plasma donor, or in subjects who are otherwise averse to plasmapheresis, or in situations where plasmapheresis is unavailable.

[0089] In some embodiments, the method includes administering a MASP-2 inhibitor to a subject suffering from HUS via a catheter (e.g., intravenously) for a first period (e.g., an acute phase lasting at least 1 day to 1 week or 2 weeks), and subsequently administering the MASP-2 inhibitor subcutaneously to the subject for a second period (e.g., a chronic phase lasting at least 2 weeks or longer). In some embodiments, the administration during the first and / or second periods is carried out in the absence of plasmapheresis. In some embodiments, the method further includes determining the level of at least one complement factor (e.g., C3, C5) in the subject before treatment, and optionally during treatment, wherein a reduced level of the at least one complement factor compared to a standard value or a healthy control subject indicates a need for treatment, and a normal level indicates improvement.

[0090] In some embodiments, the method includes subcutaneous or intravenous administration of a MASP-2 inhibitor, such as an anti-MASP-2 antibody, to a subject suffering from HUS or at risk of developing HUS. Treatment is preferably daily, but may be less frequent, such as weekly or monthly. Treatment lasts for at least one week and for as long as three months. The anti-MASP-2 antibody may be administered alone or in combination with a C5 inhibitor such as eculizumab.

[0091] TTP: Thrombotic thrombocytopenic purpura (TTP) is a life-threatening blood coagulation disorder caused by autoimmune or hereditary dysfunction that activates the coagulation cascade or complement system (George, JN, N Engl J Med; 354:1927-35(2006)). This leads to a large number of tiny blood clots, or thrombosis, in the small blood vessels throughout the body. Red blood cells are subjected to shear stress that damages the red blood cell membrane, causing intravascular hemolysis. The resulting reduced blood flow and endothelial damage cause organ damage, including the brain, heart, and kidneys. Clinically, TTP is characterized by thrombocytopenia, microangiogenic hemolytic anemia, neurological changes, renal failure, and fever. In the pre-plasma exchange era, the mortality rate during acute episodes was 90%. Even with plasmapheresis, the 6-month survival rate is approximately 80%.

[0092] TTP can result from genetic or acquired inhibition of the enzyme ADAMTS-13, a metalloproteinase responsible for cleaving von Willebrand factor (vWF) from large multimers into smaller units. ADAMTS-13 inhibition or deficiency ultimately leads to increased coagulation (Tsai, H. J Am Soc Nephrol 14:1072-1081, (2003)). ADAMTS-13 modulates vWF activity. In its absence, vWF forms large multimers that are more likely to bind to platelets, making patients more susceptible to platelet aggregation and thrombosis in the microvascular system.

[0093] Upshaw-Schulman syndrome (USS, also known as congenital TTP) is a congenital ADAMTS13 deficiency caused by a mutation 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). A very large number of ADAMTS13 mutations have been identified in individuals with congenital TTP (Kinoshita et al., International Journal of Hematology, 74:101-108 (2001); Levy et al., Nature, 413 (6855):488-494 (2001); Kokame et al., PNAS 99(18):11902-11907 (2002); Savasan et al., Blood, 101:4449-4451 (2003); Matsumoto et al., Blood, 103:1305-1310 (2004) and Fujimura et al., Brit. J. Haemat 144:742-754 (2008)). Individuals with USS typically have 5-10% of normal ADAMTS13 activity (Kokame et al., PNAS 99(18):11902-11907, 2002). While there are some similarities between acquired TTP and USS, USS has several important differences in its clinical features. USS usually appears in infancy or childhood and is characterized by severe hyperbilirubinemia, a negative Coombs test immediately after birth, a response to fresh plasma infusion, and frequent relapses (Savasan et al., Blood, 101:4449-4451, 2003). In some cases, patients with this hereditary ADAMTS13 deficiency have a mild phenotype at birth and only develop TTP-related symptoms in clinical situations with high von Willebrand factor levels, such as during infection or pregnancy. For example, Fujimura et al. reported on nine Japanese women from six families where USS was genetically confirmed, who were diagnosed with the disorder during their first pregnancy.In each of the 15 pregnancies, thrombocytopenia occurred during the second or third trimester, often followed by TTP. All of these women were found to have severe ADAMTS13 deficiency (Fujimura et al., Brit. J. Haemat 144:742-754, 2008).

[0094] As described above, in some embodiments, a method is provided for reducing the likelihood of developing congenital TTP-related clinical symptoms (e.g., thrombocytopenia, anemia, fever, and / or renal failure) in a subject having Upshaw-Schulmann syndrome (USS) (i.e., the subject is known to be deficient in ADAMTS13 activity and / or the subject is known to have one or more ADAMTS13 gene mutations), comprising the step of administering an amount of a MASP-2 inhibitor (e.g., a MASP-2 antibody) effective in relieving or preventing one or more TTP-related clinical symptoms for a period effective in relieving or preventing said clinical symptoms. In some embodiments, the method further includes the step of determining whether the subject is at risk of developing congenital TTP-related symptoms before the development of any TTP-related symptoms, or based on the development of at least one or more symptoms indicating TTP (e.g., the presence of anemia, thrombocytopenia, and / or renal failure). The step of determining whether a subject is at risk of developing symptoms associated with congenital TTP (i.e., whether the subject has USS) includes determining whether the subject has a mutation in the gene encoding ADAMTS13, and / or whether the subject lacks ADAMTS13 activity, and / or whether the subject has a family history of USS. Genetic screening methods for the presence or absence of USS-related gene mutations are well established. For example, see 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).

[0095] In one aspect, a method is provided for reducing the likelihood that a subject diagnosed with USS will develop clinical symptoms associated with TTP, the method comprising the step of periodically monitoring the subject diagnosed with USS to determine the presence or absence of anemia, thrombocytopenia, or increased creatinine, and treating with a MASP-2 inhibitor (e.g., a MASP-2 antibody) based on a determination that anemia, thrombocytopenia, or increased creatinine is present or based on an event known to be associated with the induction of TTP clinical symptoms, such as drug exposure (e.g., chemotherapy), infection (e.g., bacterial infection), malignancy, injury, transplantation, or pregnancy.

[0096] In another aspect, a method is provided for treating a subject with USS and a subject suffering from clinical symptoms associated with TTP, the method comprising administering an effective amount of a MASP-2 inhibitor (e.g., a MASP-2 antibody) over a period of time effective to relieve or prevent one or more clinical symptoms associated with TTP.

[0097] TTP can also be caused by autoantibodies against ADAMTS-13. Furthermore, TTP can develop during breast cancer, gastrointestinal cancer, or prostate cancer (George. JN., Oncology (Wiiliston Park). 25:908-14 (2011)), pregnancy (second trimester or postpartum), George JN., Curr Opin Hematol 10:339-344 (2003)), or be associated with diseases, such as HIV or autoimmune diseases like systemic lupus erythematosus (Hamasaki K, et al., Clin Rheumatol. 22:355-8 (2003)). TTP can also be caused by certain drug therapies, including heparin, quinine, immune components, cancer chemotherapy agents (bleomycin, cisplatin, cytosine arabinoside, daunomycin, gemcitabine, mitomycin C, and tamoxifen), cyclosporine A, oral contraceptives, penicillin, rifampin, and antiplatelet drugs including ticlopidine and clopidogrel (Azarm, T. et al., J Res Med Sci., 16:353-357 (2011)). Other factors or conditions associated with TTP include toxins, such as bee venom, sepsis, splenic sequestration, transplantation, vasculitis, vascular surgery, and infections such as streptococcal pneumonia and cytomegalovirus (Moake JL., N Engl J Med., 347:589-600 (2002)). Transient functional ADAMTS-13 deficiency leading to TTP can occur as a result of endothelial cell damage associated with pneumococcal infection (Pediair Nephrol., 26:631-5(2011)).

[0098] 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 patients with acquired autoimmune TTP (Tsai, HM, Hematol Oncol Clin North Am., 21(4):609-v (2007)). Further agents, such as immunosuppressants, are routinely added to the therapy (George, JN, N Engl J Med, 354:1927-35 (2006)). However, plasma exchange is only successful in about 20% of patients, relapse occurs in more than one-third of patients, plasmapheresis is expensive, and requires considerable technical effort. Furthermore, many patients cannot tolerate plasma exchange. As a result, there is still a great need for further and better TTP treatments.

[0099] Since TTP is a disorder of the blood coagulation cascade, treatment with complement system antagonists may help stabilize and treat the disease. Pathological activation of the second complement pathway has been linked to aHUS, but the role of complement activation in TTP is not well understood. Functional deficiency of ADAMTS13 is important for susceptibility to TTP, but not sufficient to cause an acute episode. Environmental factors and / or other genetic mutations may contribute to the manifestation of TTP symptoms. For example, genes encoding proteins involved in the coagulation cascade, vWF, platelet function, components of the endothelial vessel surface, or complement system regulation may be involved in the development of acute thrombotic microangiopathy (Galbusera, M. et al., Haematologica, 94; 166-170 (2009)). In particular, complement activation has been shown to play a crucial role. Serum from thrombotic microangiopathy associated with ADAMTS-13 deficiency has been shown to cause C3 and MAC deposition and subsequent neutrophil activation, which can be suppressed by complement inactivation (Ruiz-Torres MP, et al., Thromb Haemost, 93:443-52 (2005)). Furthermore, it has recently been shown that levels of C4d, C3bBbP, and C3a increase during acute episodes of TTP, consistent with activation of the classical / lectin pathway and the secondary pathway (M. Reti et al., J Thromb Haemost. Feb 28. (2012) doi:10.1111 / j.1538-7836.2012.04674.x. [Pre-print electronic publication]). In acute episodes, this increase in complement activation can initiate terminal pathway activation and lead to further exacerbation of TTP.

[0100] The roles of ADAMTS-13 and vWF in TTP clearly play a role in platelet activation and aggregation, as well as subsequent roles in shear stress and deposition in microangiopathy. Activated platelets interact with and induce the classical and secondary complement pathways. Platelet-mediated complement activation increases the inflammatory mediators C3a and C5a (Peerschke E et al., Mol Immunol, 47:2170-5 (2010)). Therefore, platelets may serve as a target for classical complement activation in hereditary or autoimmune TTP.

[0101] As described above, the complement lectin pathway, mediated by prothombin activation via MASP-2, is a major molecular pathway linking endothelial damage, coagulation, and microvascular thrombosis that occur in HUS. Similarly, activation of the complement lectin pathway can directly affect the coagulation system in TTP. Lectin pathway activation can be initiated in response to initial endothelial damage caused by ADAMTS-13 deficiency in TTP. Therefore, lectin pathway inhibitors, including but not limited to antibodies that block MASP-2 function, are expected to mitigate microvascular coagulation, thrombosis, and hemolysis-related microangiopathy in patients with TTP.

[0102] Patients with TTP typically end up in the emergency room with one or more of the following: purpura, renal failure, hypothrombocytopenia, anemia, and / or thrombosis, including stroke. The current standard of treatment for TTP is intravascular catheter delivery of exchange plasmapheresis (e.g., intravenous catheter or other form of catheter) for a period of two weeks or more, typically three times a week but up to daily. If a patient tests positive for the presence of an ADAMTS13 inhibitor (i.e., endogenous antibodies against ADAMTS13), plasmapheresis may be combined with immunosuppressive therapy (e.g., corticosteroids, rituximab, or cyclosporine). Patients with refractory TTP (approximately 20% of TTP patients) do not respond to plasmapheresis therapy for at least two weeks.

[0103] As described above, in one embodiment, a method is provided for treating a subject with an effective dose of a MASP-2 inhibitor (e.g., an anti-MASP-2 antibody) as a first-line therapy in the absence of plasmapheresis, or in combination with plasmapheresis, in a subject exhibiting one or more symptoms consistent with the diagnosis of TTP (e.g., central nervous system complications, severe thrombocytopenia (platelet count less than 5,000 / μL or 5,000 / μL if not taking aspirin, or less than 20,000 / μL or 20,000 / μL if taking aspirin), severe cardiac complications, severe pulmonary complications, gastrointestinal infarction, or gangrene) in the absence of plasmapheresis as a first-line therapy, or in combination with plasmapheresis. As a first-line therapy, the MASP-2 inhibitor may be systemically administered to the subject, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration. In some embodiments, MASP-2 inhibitors are administered to subjects as first-line therapy in the absence of plasmapheresis to avoid potential plasmapheresis complications such as bleeding, infection, and exposure to plasma donor-specific disorders and / or allergies, or in subjects who otherwise averse to plasmapheresis, or in situations where plasmapheresis is unavailable. In some embodiments, MASP-2 inhibitors are administered to subjects suffering from TTP in combination with immunosuppressants (e.g., corticosteroids, rituximab, or cyclosporine) (including co-administration) and / or in combination with high concentrations of ADAMTS-13.

[0104] In some embodiments, the method includes administering a MASP-2 inhibitor to a subject suffering from TTP via a catheter (e.g., intravenously) for a first period (e.g., an acute phase lasting at least 1 day to 1 week or 2 weeks), and then subcutaneously administering the MASP-2 inhibitor to the subject for a second period (e.g., a chronic phase lasting at least 2 weeks or longer). In some embodiments, the administration during the first and / or second periods is performed in the absence of plasmapheresis. In some embodiments, the method is used to maintain a subject so that it does not suffer from one or more symptoms associated with TTP.

[0105] In another embodiment, a method is provided for treating subjects suffering from refractory TTP (i.e., subjects who do not respond to plasmapheresis therapy for at least two weeks) by administering an amount of a MASP-2 inhibitor effective in alleviating one or more symptoms of TTP. In one embodiment, the MASP-2 inhibitor (e.g., an anti-MASP-2 antibody) is administered to subjects with refractory TTP chronically for at least two weeks or longer via subcutaneous or other parenteral administration. The administration may be repeated as determined by a physician until the condition improves or is controlled.

[0106] In some embodiments, the method further includes the step of determining the level of at least one complement factor (e.g., C3, C5) in the subject before treatment, or optionally during treatment, and the determination of a reduced level of the at least one complement factor compared to a standard value or a healthy control subject indicates the need for continued treatment with a MASP-2 inhibitor.

[0107] In some embodiments, the method includes subcutaneous or intravenous administration of a MASP-2 inhibitor, such as an anti-MASP-2 antibody, to a subject suffering from or at risk of developing TTP. Treatment is preferably daily, but may be less frequent, such as every other week. Treatment is continued until the subject's platelet count exceeds 150,000 / ml for at least two consecutive days. The anti-MASP-2 antibody may be administered alone or in combination with a C5 inhibitor such as eculizumab.

[0108] In one embodiment, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: the antibody has a K content of 10 nM or less. D The antibody binds to human MASP-2, the antibody binds to an epitope in the CCP1 domain of MASP-2, and the antibody causes C3b deposition in 1% human serum to be 10 nM or less in an in vitro assay. 50 The antibody inhibits C3b deposition in 90% human serum to an IC of 30 nM or less. 50 The antibody inhibits the pathway, is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2, is a single-chain molecule, is an IgG2 molecule, is an IgG1 molecule, is an IgG4 molecule containing the S228P mutation, and / or does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway without substantially inhibiting the secondary pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway without substantially inhibiting either the classical or secondary pathway (i.e., inhibits the lectin pathway while leaving the classical and complement secondary pathways intact).

[0109] In one embodiment, a MASP-2 inhibitory antibody inhibits thrombus formation in serum from a subject affected by TTP by at least 30%, e.g., at least 40%, e.g., at least 50%, e.g., at least 60%, e.g., at least 70%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, up to 99%, compared to untreated serum. In some embodiments, a MASP-2 inhibitory antibody inhibits thrombus formation in serum from a subject affected by TTP at a level at least 20 percent higher (e.g., at least 30%, at least 40%, at least 50%) than its inhibitory effect on C5b-9 deposition in serum.

[0110] In one embodiment, a MASP-2 inhibitory antibody inhibits thrombus formation in serum derived from TTP patients by at least 30%, for example, at least 40%, for example, at least 50%, for example, at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95%, up to a maximum of 99%, compared to untreated serum.

[0111] In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous catheter or other catheter-based delivery method.

[0112] In one embodiment, the present invention relates to a method for inhibiting thrombus formation in a subject suffering from TTP, comprising the step of administering to the subject a composition comprising a certain amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising (I)(a)(i) heavy chain CDR-H1 comprising the amino acid sequence of SEQ ID NO: 67 from 31 to 35; (ii) heavy chain CDR-H2 comprising the amino acid sequence of SEQ ID NO: 67 from 50 to 65; and (iii) heavy chain CDR-H3 comprising the amino acid sequence of SEQ ID NO: 67 from 95 to 102; and (b)(i) light chain CDR-L1 comprising the amino acid sequence of SEQ ID NO: 70 from 24 to 34; and (ii) light chain CDR-L2 comprising the amino acid sequence of SEQ ID NO: 70 from 50 to 56; and (iii) SEQ ID The present invention provides a method comprising a light chain variable region including a light chain CDR-L3 containing the amino acid sequence 89-97 of NO:70, or (II) a variant thereof including a heavy chain variable region having at least 90% identity with SEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with SEQ ID NO:67) and a light chain variable region having at least 90% identity with SEQ ID NO:70 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with SEQ ID NO:70).

[0113] In some embodiments, the method includes administering a composition comprising a certain amount of a MASP-2 inhibitory antibody or its antigen-binding fragment, which comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:67. In some embodiments, the method includes administering a composition comprising a certain amount of a MASP-2 inhibitory antibody or its antigen-binding fragment, which comprises a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:70.

[0114] In some embodiments, the method includes administering to a subject a composition comprising a MASP-2 inhibitor antibody or its antigen-binding fragment that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by a reference antibody OMS646, which includes a heavy chain variable region shown in SEQ ID NO:67 and a light chain variable region shown in SEQ ID NO:70.

[0115] Degos disease Degos disease, also known as malignant atrophic papulosis, is an extremely rare TMA affecting the endothelium of small blood vessels in the skin, gastrointestinal tract, and central nervous system (CNS). This vascular damage leads to occlusion of venules and arterioles, resulting in skin lesions, intestinal ischemia, and CNS disorders including stroke, epilepsy, and cognitive impairment. Connective tissue necrosis in the skin is due to thrombotic occlusion of small arteries. However, the cause of Degos disease is unknown. Vascular inflammation, coagulation disorders, or primary endothelial cell dysfunction have been linked to it. The 50% survival rate for Degos disease is only 2-3 years. There is no effective treatment for Degos disease, but antiplatelet drugs, anticoagulants, and immunosuppressants are used to alleviate symptoms.

[0116] Although the mechanism of Degos disease is unknown, the complement pathway has been linked to it. Margo et al. confirmed significant C5b-9 deposition in the cutaneous, gastrointestinal, and cerebral blood vessels of four patients with late-stage Degos disease (Margo et al., Am J Clin Pathol 135(4):599-610, 2011). Experimental treatment with eculizumab was initially effective in treating lesions in the skin and intestines, but it did not halt the progression of the systemic disease (see Garrett-Bakelman F. et al., "C5b-9 is a potential effector in the pathophysiology of Degos disease; a case report of treatment with eculizumab" (Abstract), Jerusalem: International Society of Hematology; 2010, Poster #156; and Polito J. et al, "Early detection of systemic Degos disease (DD) or malignant atrophic papulosis (MAP) may increase survival" (Abstract), San Antonio, TX: American College of Gastroenterology; 2010, Poster #1205).

[0117] Many patients with Degoss disease have blood clotting defects. Thrombotic occlusion of small arteries in the skin is characteristic of this disease. Since the complement pathway is linked to this disease, lectin pathway inhibitors, including but not limited to antibodies that block MASP-2 function, as described herein for other TMAs, are expected to be beneficial in the treatment of patients with Degoss disease.

[0118] Accordingly, in another embodiment, the present invention provides a method for treating degoss disease by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitor, such as a MASP-2 antibody, in a pharmaceutical carrier to a subject suffering from degoss disease or a condition resulting from degoss disease. The MASP-2 inhibitor is administered systemically to a subject suffering from degoss disease or a condition resulting from degoss disease, for example, by intra-arterial, intravenous, intramuscular, inhalation, subcutaneous, or other parenteral administration, or, in the case of a non-peptidogenic activator, by oral administration. The anti-MASP-2 antibody may be administered alone or in combination with a C5 inhibitor, such as eculizamab.

[0119] In one embodiment, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: the antibody has a K content of 10 nM or less. D The antibody binds to human MASP-2, the antibody binds to an epitope in the CCP1 domain of MASP-2, and the antibody causes C3b deposition in 1% human serum to be 10 nM or less in an in vitro assay. 50 The antibody inhibits C3b deposition in 90% human serum to an IC of 30 nM or less. 50 The antibody inhibits the pathway, is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2, is a single-chain molecule, is an IgG2 molecule, is an IgG1 molecule, is an IgG4 molecule containing the S228P mutation, and / or does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway without substantially inhibiting the secondary pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway without substantially inhibiting either the classical or secondary pathway (i.e., inhibits the lectin pathway while leaving the classical and complement secondary pathways intact).

[0120] In one embodiment, a MASP-2 inhibitory antibody inhibits thrombus formation in serum from a subject with Degoss disease by at least 30%, e.g., at least 40%, e.g., at least 50%, e.g., at least 60%, e.g., at least 70%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, up to 99%, compared to untreated serum. In some embodiments, a MASP-2 inhibitory antibody inhibits thrombus formation in serum from a subject with Degoss disease at a level at least 20 percent higher (e.g., at least 30%, at least 40%, at least 50%) than its inhibitory effect on C5b-9 deposition in serum.

[0121] In one embodiment, a MASP-2 inhibitory antibody inhibits thrombus formation in serum derived from patients with Degos disease by at least 30%, for example, at least 40%, for example, at least 50%, for example, at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95%, up to a maximum of 99%, compared to untreated serum.

[0122] In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous catheter or other catheter-based delivery method.

[0123] In one embodiment, the present invention relates to a method for inhibiting thrombus formation in a subject suffering from Degos disease, comprising the step of administering to the subject a composition comprising a certain amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising (I)(a)(i)heavy chain CDR-H1 comprising the amino acid sequence of SEQ ID NO:67 from 31 to 35; (ii)heavy chain CDR-H2 comprising the amino acid sequence of SEQ ID NO:67 from 50 to 65; and (iii)heavy chain CDR-H3 comprising the amino acid sequence of SEQ ID NO:67 from 95 to 102; and (b)(i)light chain CDR-L1 comprising the amino acid sequence of SEQ ID NO:70 from 24 to 34; and (ii)light chain CDR-L2 comprising the amino acid sequence of SEQ ID NO:70 from 50 to 56; and (iii)SEQ ID The present invention provides a method comprising a light chain variable region including a light chain CDR-L3 containing the amino acid sequence 89-97 of NO:70, or (II) a variant thereof including a heavy chain variable region having at least 90% identity with SEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with SEQ ID NO:67) and a light chain variable region having at least 90% identity with SEQ ID NO:70 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with SEQ ID NO:70).

[0124] In some embodiments, the method includes administering a composition comprising a certain amount of a MASP-2 inhibitory antibody or its antigen-binding fragment, which comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:67. In some embodiments, the method includes administering a composition comprising a certain amount of a MASP-2 inhibitory antibody or its antigen-binding fragment, which comprises a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:70.

[0125] In some embodiments, the method includes administering to a subject a composition comprising a MASP-2 inhibitor antibody or its antigen-binding fragment that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by a reference antibody OMS646, which includes a heavy chain variable region shown in SEQ ID NO:67 and a light chain variable region shown in SEQ ID NO:70.

[0126] Fulminant antiphospholipid syndrome (CAPS) Fulminant antiphospholipid syndrome (CAPS) is an extreme variant of antiphospholipid (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, it is characterized by occlusion of small blood vessels in various organs. The mortality rate for CAPS is high, at approximately 50%, and it is often associated with infection or trauma. Patients have antiphospholipid antibodies, typically IgG.

[0127] Clinically, CAPS involves at least three organs or tissues showing histopathological evidence of small vascular occlusions. Peripheral thrombosis may involve veins and arteries in the CNS, cardiovascular, renal, or pulmonary systems. Patients are treated with antibiotics, anticoagulants, corticosteroids, plasmapheresis, and intravenous immunoglobulins. Nevertheless, death can occur due to multiple organ failure.

[0128] The complement pathway is involved in CAPS. For example, studies in animal models have shown that complement inhibition may be an effective means of preventing CAPS-related thrombosis (Shapira L. et al., Arthritis Rheum 64(8):2719-23, 2012). Furthermore, as further reported by Shapira et al., administration of eculizumab at a complement pathway-blocking dose to subjects with CAPS halted 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, lectin pathway inhibitors, including but not limited to antibodies that block MASP-2 function, are expected to be beneficial in the treatment of patients with CAPS.

[0129] Accordingly, in another embodiment, the present invention provides a method for treating CAPS by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitor, such as a MASP-2 antibody, in a pharmaceutical carrier to CAPS or to a subject suffering from a CAPS-related condition. The MASP-2 inhibitor is systemically administered to a subject suffering from CAPS or a CAPS-related condition, for example, by intra-arterial, intravenous, intramuscular, inhalation, subcutaneous, or other parenteral administration, or, in the case of a non-peptidogenic activator, by oral administration. The anti-MASP-2 antibody may be administered alone or in combination with a C5 inhibitor, such as ecrizamab.

[0130] In one embodiment, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: the antibody has a K content of 10 nM or less. D The antibody binds to human MASP-2, the antibody binds to an epitope in the CCP1 domain of MASP-2, and the antibody causes C3b deposition in 1% human serum to be 10 nM or less in an in vitro assay. 50 The antibody inhibits C3b deposition in 90% human serum to an IC of 30 nM or less.50 The antibody inhibits the pathway, is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2, is a single-chain molecule, is an IgG2 molecule, is an IgG1 molecule, is an IgG4 molecule containing the S228P mutation, and / or does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway without substantially inhibiting the secondary pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway without substantially inhibiting either the classical or secondary pathway (i.e., inhibits the lectin pathway while leaving the classical and complement secondary pathways intact).

[0131] In one embodiment, a MASP-2 inhibitory antibody inhibits thrombus formation in serum from a CAPS-affected subject by at least 30%, e.g., at least 40%, e.g., at least 50%, e.g., at least 60%, e.g., at least 70%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, up to 99%, compared to untreated serum. In some embodiments, a MASP-2 inhibitory antibody inhibits thrombus formation in serum from a CAPS-affected subject at a level at least 20 percent higher (e.g., at least 30%, at least 40%, at least 50%) than its inhibitory effect on C5b-9 deposition in serum.

[0132] In one embodiment, a MASP-2 inhibitory antibody inhibits thrombus formation in serum derived from CAPS patients by at least 30%, e.g., at least 40%, e.g., at least 50%, e.g., at least 60%, e.g., at least 70%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, up to a maximum of 99%, compared to untreated serum.

[0133] In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous catheter or other catheter-based delivery method.

[0134] In one embodiment, the present invention relates to a method for inhibiting thrombus formation in a subject suffering from CAPS, comprising the step of administering to the subject a composition comprising a certain amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising (I)(a)(i) heavy chain CDR-H1 comprising the amino acid sequence of SEQ ID NO: 67 from 31 to 35; (ii) heavy chain CDR-H2 comprising the amino acid sequence of SEQ ID NO: 67 from 50 to 65; and (iii) heavy chain CDR-H3 comprising the amino acid sequence of SEQ ID NO: 67 from 95 to 102; and (b)(i) light chain CDR-L1 comprising the amino acid sequence of SEQ ID NO: 70 from 24 to 34; and (ii) light chain CDR-L2 comprising the amino acid sequence of SEQ ID NO: 70 from 50 to 56; and (iii) SEQ ID The present invention provides a method comprising a light chain variable region including a light chain CDR-L3 containing the amino acid sequence 89-97 of NO:70, or (II) a variant thereof including a heavy chain variable region having at least 90% identity with SEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with SEQ ID NO:67) and a light chain variable region having at least 90% identity with SEQ ID NO:70 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with SEQ ID NO:70).

[0135] In some embodiments, the method includes administering a composition comprising a certain amount of a MASP-2 inhibitory antibody or its antigen-binding fragment, which comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:67. In some embodiments, the method includes administering a composition comprising a certain amount of a MASP-2 inhibitory antibody or its antigen-binding fragment, which comprises a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:70.

[0136] In some embodiments, the method includes administering to a subject a composition comprising a MASP-2 inhibitor antibody or its antigen-binding fragment that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by a reference antibody OMS646, which includes a heavy chain variable region shown in SEQ ID NO:67 and a light chain variable region shown in SEQ ID NO:70.

[0137] TMA secondary to cancer Any type of systemic malignancy can cause the clinical and pathological manifestation of TMA (see, e.g., Batts and Lazarus, Bone Marrow Transplantation 40:709-719, 2007). Cancer-associated TMA is frequently found in the lung and appears to be associated with tumor embolism (Francis KK et al., Commun Oncol 2:339-43, 2005). Tumor embolisms reduce blood flow and can therefore lead to hypoperfusion in affected arterioles and venules. The resulting tissue stress and damage are expected to locally activate the complement lectin pathway. This activated lectin pathway can then activate the coagulation cascade via MASP-2-dependent cleavage from prothrombin to thrombin, leading to the prothrombotic state specific to TMA. Inhibiting MASP-2 in this context is expected to reduce local thrombin activation and thereby mitigate the prothrombotic state.

[0138] Therefore, as described herein for other TMAs, lectin pathway inhibitors, including but not limited to antibodies that block MASP-2 function, are expected to be beneficial in the treatment of patients with cancer-secondary TMAs.

[0139] Accordingly, in another embodiment, the present invention provides a method for treating or preventing cancer-related TMA by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitor, such as a MASP-2 antibody, in a pharmaceutical carrier to a subject suffering from cancer-related TMA or a subject at risk of developing such TMA. The MASP-2 inhibitor is systemically administered to a subject suffering from cancer-related TMA or a subject at risk of developing such TMA, for example, by intra-arterial, intravenous, intramuscular, inhalation, subcutaneous, or other parenteral administration, or, in the case of a non-peptidogenic activator, by oral administration. The anti-MASP-2 antibody may be administered alone or in combination with a C5 inhibitor, such as eclizamab.

[0140] In one embodiment, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: the antibody has a K content of 10 nM or less. D The antibody binds to human MASP-2, the antibody binds to an epitope in the CCP1 domain of MASP-2, and the antibody causes C3b deposition in 1% human serum to be 10 nM or less in an in vitro assay. 50 The antibody inhibits C3b deposition in 90% human serum to an IC of 30 nM or less. 50 The antibody inhibits the pathway, is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2, is a single-chain molecule, is an IgG2 molecule, is an IgG1 molecule, is an IgG4 molecule containing the S228P mutation, and / or does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway without substantially inhibiting the secondary pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway without substantially inhibiting either the classical or secondary pathway (i.e., inhibits the lectin pathway while leaving the classical and complement secondary pathways intact).

[0141] In one embodiment, a MASP-2 inhibitory antibody inhibits thrombus formation in serum from a subject suffering from cancer-secondary TMA by at least 30%, e.g., at least 40%, e.g., at least 50%, e.g., at least 60%, e.g., at least 70%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, up to a maximum of 99%, compared to untreated serum.

[0142] In one embodiment, a MASP-2 inhibitory antibody inhibits thrombus formation in serum from a subject suffering from cancer-secondary TMA by at least 30%, e.g., at least 40%, e.g., at least 50%, e.g., at least 60%, e.g., at least 70%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, up to a maximum of 99%, compared to untreated serum.

[0143] In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous catheter or other catheter-based delivery method.

[0144] In one embodiment, the present invention relates to a method for inhibiting thrombus formation in a subject suffering from TMA secondary to cancer, comprising the step of administering to the subject a composition comprising a certain amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising (I)(a)(i)heavy chain CDR-H1 comprising the amino acid sequence of SEQ ID NO:67 from 31 to 35; (ii)heavy chain CDR-H2 comprising the amino acid sequence of SEQ ID NO:67 from 50 to 65; and (iii)heavy chain CDR-H3 comprising the amino acid sequence of SEQ ID NO:67 from 95 to 102; and (b)(i)light chain CDR-L1 comprising the amino acid sequence of SEQ ID NO:70 from 24 to 34; and (ii)light chain CDR-L2 comprising the amino acid sequence of SEQ ID NO:70 from 50 to 56; and (iii)SEQ ID The present invention provides a method comprising a light chain variable region including a light chain CDR-L3 containing the amino acid sequence 89-97 of NO:70, or (II) a variant thereof including a heavy chain variable region having at least 90% identity with SEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with SEQ ID NO:67) and a light chain variable region having at least 90% identity with SEQ ID NO:70 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with SEQ ID NO:70).

[0145] In some embodiments, the method includes administering a composition comprising a certain amount of a MASP-2 inhibitory antibody or its antigen-binding fragment, which comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:67. In some embodiments, the method includes administering a composition comprising a certain amount of a MASP-2 inhibitory antibody or its antigen-binding fragment, which comprises a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:70.

[0146] In some embodiments, the method includes administering to a subject a composition comprising a MASP-2 inhibitor antibody or its antigen-binding fragment that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by a reference antibody OMS646, which includes a heavy chain variable region shown in SEQ ID NO:67 and a light chain variable region shown in SEQ ID NO:70.

[0147] TMA secondary to cancer chemotherapy Chemotherapy-associated TMA (TM) is a condition characterized by thrombocytopenia, microangiolytic anemia, and renal failure, occurring in 2–10% of patients with a history of malignant neoplasms treated with chemotherapy agents such as gemcitabine, mitomycin, and oxaliplatin. TMA is associated with a high mortality rate and poor clinical outcomes (see, for example, Blake-Haskins et al., Clin Cancer Res 17(18):5858-5866, 2011).

[0148] Post-chemotherapy-induced myomectomy (TMA) is thought to be caused by nonspecific and toxic damage to microvascular endothelium. Direct damage to endothelial cells has been demonstrated in animal models of mitomycin-induced TMA (Dlott J. et al., Ther Apher Dial 8:102-11, 2004). Endothelial cell damage mediated by various mechanisms has been shown to activate complement lectin pathways. For example, Stahl et al. showed that endothelial cells exposed to oxidative stress activate complement lectin pathways both in vitro and in vivo (Collard et al., Am J Pathol. 156(5):1549-56, 2000; La Bonte et al., J Immunol. 15;188(2):885-91, 2012). In vivo, this process leads to thrombosis, and it has been shown that inhibiting the lectin pathway prevents thrombosis (La Bonte et al. J Immunol. 15;188(2):885-91, 2012). Furthermore, as demonstrated in Examples 37-39 of this specification, in a TMA mouse model in which local photoexcitation of FITC-Dex is used to induce local damage to the microvascular system and subsequent TMA response, we have shown that TMA can be inhibited by MASP-2 inhibition. Therefore, microvascular endothelial damage caused by chemotherapeutic agents may activate the complement lectin pathway, which in turn creates a localized thrombus-promoting state, thereby promoting the TMA response. Since the activation of the lectin pathway and the generation of the thrombus-promoting state are MASP-2 dependent, MASP-2 inhibitors, including but not limited to antibodies that block MASP-2 function, are expected to mitigate the TMA response and reduce the risk of TMA after cancer chemotherapy.

[0149] Accordingly, in another embodiment, the present invention provides a method for treating or preventing chemotherapy-related TMA by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitor, such as a MASP-2 antibody, in a pharmaceutical carrier to a subject suffering from chemotherapy-related TMA or a subject at risk of developing such TMA. The MASP-2 inhibitor is administered systemically to a subject who has received, is receiving, or is scheduled to receive chemotherapy, for example, by intra-arterial, intravenous, intramuscular, inhalation, subcutaneous, or other parenteral administration, or, in the case of a non-peptide activator, by oral administration. The anti-MASP-2 antibody may be administered alone or in combination with a C5 inhibitor, such as eclizamab.

[0150] In one embodiment, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: the antibody has a K content of 10 nM or less. D The antibody binds to human MASP-2, the antibody binds to an epitope in the CCP1 domain of MASP-2, and the antibody causes C3b deposition in 1% human serum to be 10 nM or less in an in vitro assay. 50 The antibody inhibits C3b deposition in 90% human serum to an IC of 30 nM or less. 50 The antibody inhibits the pathway, is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2, is a single-chain molecule, is an IgG2 molecule, is an IgG1 molecule, is an IgG4 molecule containing the S228P mutation, and / or does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway without substantially inhibiting the secondary pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway without substantially inhibiting either the classical or secondary pathway (i.e., inhibits the lectin pathway while leaving the classical and complement secondary pathways intact).

[0151] In one embodiment, a MASP-2 inhibitory antibody inhibits thrombus formation in serum from a subject suffering from TMA secondary to cancer chemotherapy by at least 30%, for example, at least 40%, for example, at least 50%, for example, at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95%, up to a maximum of 99%, compared to untreated serum.

[0152] In one embodiment, a MASP-2 inhibitory antibody inhibits thrombus formation in serum from subjects suffering from TMA secondary to cancer chemotherapy by at least 30%, e.g., at least 40%, e.g., at least 50%, e.g., at least 60%, e.g., at least 70%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, up to a maximum of 99%, compared to untreated serum.

[0153] In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous catheter or other catheter-based delivery method.

[0154] In one embodiment, the present invention relates to a method for inhibiting thrombus formation in a subject suffering from TMA secondary to cancer chemotherapy, comprising the step of administering to the subject a composition comprising a certain amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising (I)(a)(i)heavy chain CDR-H1 comprising the amino acid sequence of SEQ ID NO:67 from 31 to 35; (ii)heavy chain CDR-H2 comprising the amino acid sequence of SEQ ID NO:67 from 50 to 65; and (iii)heavy chain CDR-H3 comprising the amino acid sequence of SEQ ID NO:67 from 95 to 102; and (b)(i)light chain CDR-L1 comprising the amino acid sequence of SEQ ID NO:70 from 24 to 34; and (ii)light chain CDR-L2 comprising the amino acid sequence of SEQ ID NO:70 from 50 to 56; and (iii)SEQ ID The present invention provides a method comprising a light chain variable region including a light chain CDR-L3 containing the amino acid sequence 89-97 of NO:70, or (II) a variant thereof including a heavy chain variable region having at least 90% identity with SEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with SEQ ID NO:67) and a light chain variable region having at least 90% identity with SEQ ID NO:70 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with SEQ ID NO:70).

[0155] In some embodiments, the method includes administering a composition comprising a certain amount of a MASP-2 inhibitory antibody or its antigen-binding fragment, which comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:67. In some embodiments, the method includes administering a composition comprising a certain amount of a MASP-2 inhibitory antibody or its antigen-binding fragment, which comprises a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:70.

[0156] In some embodiments, the method includes administering to a subject a composition comprising a MASP-2 inhibitor antibody or its antigen-binding fragment that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by a reference antibody OMS646, which includes a heavy chain variable region shown in SEQ ID NO:67 and a light chain variable region shown in SEQ ID NO:70.

[0157] TMA following transplant Transplantation-associated TMA (TA-TMA) is a catastrophic syndrome that can occur in transplant patients, such as allogeneic hematopoietic stem cell transplant recipients (see, e.g., Batts and Lazarus, Bone Marrow Transplantation 40:709-719, 2007). The cause of this condition is not fully understood, but it is likely that a collection of responses resulting in endothelial cell damage is involved (Laskin BL et al., Blood 118(6):1452-62, 2011). As discussed above, endothelial cell damage is a typical stimulus for activation of the lectin pathway and creation of a thrombus-promoting environment.

[0158] Recent data further support the role of complement activation via the lectin pathway in TA-TMA development. Laskin et al. demonstrated that renal arteriole C4d deposition (75%) was far more common in subjects with histological TA-TMA compared to controls (8%) (Laskin BL, et al., Transplantation, 27; 96(2):217-23, 2013). Therefore, C4d may be a pathological marker of TA-TMA, indicating local complement binding via the lectin or classical pathway.

[0159] Since the activation of the lectin pathway and the generation of a thrombus-promoting state are MASP-2 dependent, MASP-2 inhibitors, including but not limited to antibodies that block MASP-2 function, are expected to mitigate the TMA response and reduce the risk of post-transplant TMA (TA-TMA).

[0160] Accordingly, in another embodiment, the present invention provides a method for treating or preventing transplant-related TMA by administering a composition containing a therapeutically effective amount of a MASP-2 inhibitor, such as a MASP-2 antibody, in a pharmaceutical carrier to a subject suffering from transplant-related TMA or a subject at risk of developing such TMA. The MASP-2 inhibitor is systemically administered to a subject who has undergone, is undergoing, or is scheduled to undergo transplantation, for example, by intra-arterial, intravenous, intramuscular, inhalation, subcutaneous, or other parenteral administration, or, in the case of a non-peptidogenic activator, by oral administration. The anti-MASP-2 antibody may be administered alone or in combination with a C5 inhibitor, such as eclizamab. In some embodiments, the present invention provides a method for treating or preventing transplant-related TMA, comprising the step of administering to a subject a composition containing a certain amount of a MASP-2 inhibitor, such as a MASP-2 inhibitor antibody, before, during, or after allogeneic stem cell transplantation.

[0161] In one embodiment, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: the antibody has a K content of 10 nM or less. D The antibody binds to human MASP-2, the antibody binds to an epitope in the CCP1 domain of MASP-2, and the antibody causes C3b deposition in 1% human serum to be 10 nM or less in an in vitro assay. 50 The antibody inhibits C3b deposition in 90% human serum to an IC of 30 nM or less. 50The antibody inhibits the pathway, is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2, is a single-chain molecule, is an IgG2 molecule, is an IgG1 molecule, is an IgG4 molecule containing the S228P mutation, and / or does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway without substantially inhibiting the secondary pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway without substantially inhibiting either the classical or secondary pathway (i.e., inhibits the lectin pathway while leaving the classical and complement secondary pathways intact).

[0162] In one embodiment, a MASP-2 inhibitory antibody inhibits thrombus formation in serum from a subject suffering from transplant-associated TMA by at least 30%, e.g., at least 40%, e.g., at least 50%, e.g., at least 60%, e.g., at least 70%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, up to a maximum of 99%, compared to untreated serum.

[0163] In one embodiment, a MASP-2 inhibitory antibody inhibits thrombus formation in serum from a subject suffering from transplant-associated TMA by at least 30%, e.g., at least 40%, e.g., at least 50%, e.g., at least 60%, e.g., at least 70%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, up to a maximum of 99%, compared to untreated serum.

[0164] In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous catheter or other catheter-based delivery method.

[0165] In one embodiment, the present invention relates to a method for inhibiting thrombus formation in a subject suffering from TMA secondary to transplantation, comprising the step of administering to the subject a composition comprising a certain amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising (I)(a)(i)heavy chain CDR-H1 comprising the amino acid sequence of SEQ ID NO:67 from 31 to 35; (ii)heavy chain CDR-H2 comprising the amino acid sequence of SEQ ID NO:67 from 50 to 65; and (iii)heavy chain CDR-H3 comprising the amino acid sequence of SEQ ID NO:67 from 95 to 102; and (b)(i)light chain CDR-L1 comprising the amino acid sequence of SEQ ID NO:70 from 24 to 34; and (ii)light chain CDR-L2 comprising the amino acid sequence of SEQ ID NO:70 from 50 to 56; and (iii)SEQ ID The present invention provides a method comprising a light chain variable region including a light chain CDR-L3 containing the amino acid sequence 89-97 of NO:70, or (II) a variant thereof including a heavy chain variable region having at least 90% identity with SEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with SEQ ID NO:67) and a light chain variable region having at least 90% identity with SEQ ID NO:70 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with SEQ ID NO:70).

[0166] In some embodiments, the method includes administering a composition comprising a certain amount of a MASP-2 inhibitory antibody or its antigen-binding fragment, which comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:67. In some embodiments, the method includes administering a composition comprising a certain amount of a MASP-2 inhibitory antibody or its antigen-binding fragment, which comprises a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:70.

[0167] In some embodiments, the method includes administering to a subject a composition comprising a MASP-2 inhibitor antibody or its antigen-binding fragment that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by a reference antibody OMS646, which includes a heavy chain variable region shown in SEQ ID NO:67 and a light chain variable region shown in SEQ ID NO:70.

[0168] IV. The role of MASP-2 in other diseases and conditions, and therapeutic methods using MASP-2 inhibitors. Kidney condition Complement system activation is associated with 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. 55:976-84, 1989), and membranoproliferative glomerulonephritis (mesangial capillary glomerulonephritis) (Bartlow, BG. et al., Kidney Int. 15:294-300, 1979; Meri, S. et al., J. Complement is involved in the development of a wide variety of kidney diseases, including acute post-infectious glomerulonephritis (post-streptococcal glomerulonephritis), cryoglobulinemia glomerulonephritis (Ohsawa, I., et al., Clin Immunol, 101:59-66, 2001), lupus nephritis (Gatenby, PA, Autoimmunity 11:61-6, 1991), and Henoho-Schönlein purpura nephritis (Endo, M., et al., Am. J. Kidney Dis. 35:401-407, 2000). Although the involvement of complement in kidney disease has been recognized for decades, its precise role in the onset, progression, and recovery phases of kidney disease remains a subject of considerable debate. Under normal conditions, complement contributes to the host's well-being, but improper activation and deposition of complement can contribute to tissue damage.

[0169] Glomerulonephritis, an inflammation of the glomeruli, is often initiated by the deposition of immune complexes into glomerular or tubular structures, and there is much evidence that this deposition then induces complement activation, inflammation, and tissue damage. Kahn and Sinniah demonstrated increased C5b-9 deposition in the tubular basement membrane in biopsy material 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), C5b-9 deposition in the tubular epithelial / basement membrane structure 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). High sC5b-9 levels were positively correlated with poor prognosis. Lehto et al. measured high levels of CD59, a complement regulator that inhibits membrane invasion complexes in the plasma membrane, and C5b-9 in urine samples from patients with membranous glomerulonephritis (Lehto, T., et al., Kidney Int. 47; 1403-11, 1995). Histopathological analysis of biopsy samples from these same patients revealed deposition of C3 and C9 proteins in the glomeruli, while CD59 expression in these tissues was reduced compared to normal kidney tissue. These various studies suggest that ongoing complement-mediated glomerulonephritis leads to urinary excretion of complement proteins, which correlates with the degree of tissue damage and disease prognosis.

[0170] The importance of complement activation in the pathogenesis of glomerulonephritis has also been demonstrated by the inhibition of complement activation in various animal models of this disease. In a membranoproliferative glomerulonephritis (MPGN) model, injecting C6-deficient rats (unable to form C5b-9) with anti-Th1 antiserum resulted in 90% less glomerular cell proliferation, 80% less platelet and macrophage infiltration, decreased collagen type IV synthesis (a marker of mesangial matrix expansion), and 50% less proteinuria compared to C6+ normal rats (Brandt.J., et al., Kidney Int. 49:335-343, 1996). These results suggest that C5b-9 is the major complement-mediated mediator of tissue damage in this rat anti-thymocyte serum model. In another glomerulonephritis model, infusion of graded doses of rabbit anti-rat glomerular basement membrane resulted in a dose-dependent influx of polymorphonuclear leukocytes (PMNs), which was attenuated by pretreatment with cobra venom factor (which consumes complement) (Scandrett, AL, et al., Am. J. Physiol 268:F256-F265, 1995). Rats treated with cobra venom factor also showed reduced histological changes, reduced long-term proteinuria, and lower creatinine levels compared to control rats. Using three GN models in rats (anti-thymocyte serum, anti-ConA, and passive Haymann nephritis), Courser et al. demonstrated the potential therapeutic efficacy of an approach that inhibits complement by using recombinant sCR1 protein (Couser, WG, et al., J. Am. Soc. Nephrol. 5:1888-94, 1995). Rats treated with sCR1 showed significant reductions in PMN, platelet and macrophage influx, mesangial lysis, and proteinuria compared to control rats. Further evidence of the importance of complement activation in glomerulonephritis has been obtained by using anti-C5 MoAb in the NZB / W F1 mouse model. Anti-C5 MoAb inhibits C5 cleavage and therefore blocks the production of C5a and C5b-9. Six months of continuous therapy with anti-C5 MoAb resulted in significant remission of the glomerulonephritis course.A humanized anti-C5 MoAb monoclonal antibody (5G1.1), which inhibits the cleavage of human complement component C5 by pro-inflammatory compounds, is currently under development by Alexion Pharmaceuticals, Inc., New Haven, and Connecticut as a potential treatment for glomerulonephritis.

[0171] Studies of patients with genetic deficiencies in specific complement components have provided direct evidence for the pathological role of complement in kidney injury. Numerous reports have demonstrated a link between kidney disease and deficiency of 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). Deficiency of factor H leads to decreased plasma levels of factor B and C3, and consumption of C5b-9. Atypical membranoproliferative glomerulonephritis (MPGN) and idiopathic hemolytic uremic syndrome (HUS) are both associated with factor H deficiency. Factor H-deficient pigs (Jansen, JH, et al., Kidney Int. 55:331-49, 1998) and factor H knockout mice (Pickering, MC, 2002) exhibit MPGN-like symptoms. This supports the importance of factor H in complement regulation. Deficiencies in other complement components have been associated with kidney disease secondary to the development of systemic lupus erythematosus (SLE) (Walport, MJ, Davies, et al., Ann. N. Y, Acad. Sci. 815:267-81, 1997). Deficiencies in C1q, C4, and C2 are strong predisposing factors for SLE development via mechanisms related to incomplete clearance of immune complexes and apoptotic materials. Many of these SLE patients develop lupus nephritis, characterized by whole-glomerular immune complex deposition.

[0172] The identification of autoantibodies against complement components in patients has provided further evidence linking complement activation to kidney disease. Some of these autoantibodies have been directly linked to kidney disease (Trouw, LA, et al., Mol. Immunol. 38:199-206, 2001). Because many of these autoantibodies show a fairly high correlation with kidney disease, the term nephritis factor (NeF) was introduced to describe this activity. In clinical studies, approximately 50% of patients positive for nephritis factor developed MPGN (Spitzer, RE et al., Clin. Immunol. Immunopathol. 64:177-83, 1992). C3NeF is an autoantibody against the secondary pathway C3 convertase (C3bBb), which stabilizes this convertase and thereby promotes secondary pathway activation (Daha, MR, et al., J. Immunol. 116:1-7, 1976). Similarly, autoantibodies specific to the classical pathway C3 convertase (C4b2a) are called C4NeF, which stabilize this convertase and thereby promote the activation of the classical pathway (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 reported to be associated with nephritis in SLE patients (Hovath, L. et al., Clin. Exp. Rheumatol, 19:667-72, 2001; Siegert, C, et al., J. Rheumatol 18:230-34, 1991; Siegert, C, et al., Clin. Exp. Rheumatol. 10:19-23, 1992). Increased titers of these anti-C1q autoantibodies have been reported to predict the progression of nephritis (Coremans, IE et al., Am. J. Kidney Dis. 26:595-601, 1995).Immunodepositions eluted from postmortem kidneys of SLE patients revealed the accumulation of these anti-C1q autoantibodies (Mannick, M, et al., Arthritis Rheumatol. 40:1504-11, 1997). All of these facts suggest a pathological role for these autoantibodies. However, not all patients with anti-C1q autoantibodies develop kidney disease, and some healthy individuals have low titers of anti-C1q autoantibodies (Siegert, CE, et al., Clin. Immunol. Immunopathol. 67:204-9, 1993).

[0173] In addition to the secondary and classical pathways of complement activation, the lectin pathway may also play an important pathological role in kidney disease. Immunohistochemical techniques have detected high levels of MBL, MBL-related serine proteases, and complement activators in kidney biopsy materials from patients diagnosed with several different kidney diseases, including Henoho-Schönlein 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 neuropathy (Endo, M., et al., Clin. Nephrology 55:185-191, 2001). Therefore, despite the fact that the relationship between complement and kidney disease has been publicly known for several decades, the data on exactly how complement affects these kidney diseases is far from perfect.

[0174] Blood disorders Sepsis is caused by an uncontrollable and severe response of the patient to invading microorganisms. The primary function of the complement system is to organize the inflammatory response to invading bacteria and other pathogens. Consistent with this physiological role, numerous studies have shown that complement activation plays a major role in the development of sepsis (Bone, RC, Annals. Internal, Med. 115:457-469, 1991). The definition of the clinical signs of sepsis is constantly evolving. Sepsis is usually defined as a systemic host response to infection. However, on many occasions, clinical evidence of infection (e.g., positive bacterial blood cultures) has not been found in patients with septic symptoms. This contradiction was first taken into consideration at the Consensus Conference in 1992, where the term "systemic inflammatory response syndrome" (SIRS) was established, eliminating the need for a definable presence of bacterial infection (Bone, RC, et al., Crit. Care Med. 20:724-726, 1992). Currently, there is a general consensus that sepsis and SIRS are accompanied by uncontrolled inflammatory responses. To briefly revisit this, the inventors consider the clinical definition of sepsis to include severe sepsis, septic shock, and SIRS.

[0175] Before the late 1980s, the most prevalent source of infection in sepsis patients was Gram-negative bacteria. It was known that lipopolysaccharide (LPS), a major component of the cell wall of Gram-negative bacteria, when injected into animals, stimulates the release of inflammatory mediators from various cell types and induces acute infection symptoms (Haeney, MR, et al., Antimicrobial Chemotherapy 41(Suppl. A):41-6, 1998). Interestingly, the spectrum of causative microorganisms appears to have shifted from primarily Gram-negative bacteria in the late 1970s and 1980s to primarily Gram-positive bacteria today, for reasons that are not yet clear (Martin, GS, et al., N. Eng. J. Med. 348:1546-54, 2003).

[0176] Numerous studies have demonstrated the importance of complement activation in mediating inflammation and contributing to the characterization of shock, particularly septic shock and hemorrhagic shock. Both Gram-negative and Gram-positive organisms typically develop septic shock. LPS is a potent complement activator, primarily via the second pathway, but antibody-mediated classical pathway activation also occurs (Fearon, DT, et al., N. Engl. J. Med. 292:937-400, 1975). The main components of Gram-positive cell walls are peptidoglycan and lipoteichoic acid, both potent activators of the second complement pathway, but the classical complement pathway can also be activated in the presence of specific antibodies (Joiner, KA, et al., Ann. Rev. Immunol. 2:461-2, 1984).

[0177] The complement system was first linked to the development of sepsis when researchers noticed that the anaphylatoxins C3a and C5a mediated various inflammatory responses that can occur during sepsis. These anaphylatoxins induce vasodilation and increased microvascular permeability, which play a central role in septic shock (Schumacher, WA, et al., Agents Actions 34:345-349, 1991). Furthermore, anaphylatoxins induce bronchospasm, histamine release from mast cells, and platelet aggregation. In addition, they exert numerous effects on granulocytes, such as chemotaxis, aggregation, adhesion, release of lysosomal enzymes, production of toxic superoxide anions, and leukotriene formation (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 septic complications 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). Furthermore, high levels of anaphylatoxin C3a have been 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).

[0178] Blocking C5a production with antibodies during the onset of sepsis in rodents has been shown to significantly improve survival rates (Czermak, BJ, et al., Nat. Med. 5:788-792, 1999). Similar findings were observed when the C5a receptor (C5aR) was blocked using 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 have suggested that blocking antibodies against C5a reduced 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). Compared to patients with less severe sepsis and survivors, C5a levels were elevated in individuals with sepsis, and this was associated with a significantly lower survival rate with multiple organ failure (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). Although the mechanisms by which C5a exerts adverse effects during sepsis have not yet been fully investigated, 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 produce respiratory bursts, and their ability to generate cytokines (Riedemann, NC, et al., Immunity 19:193-202, 2003).Furthermore, 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 also showed efficacy in animal models of sepsis and ARDS (Dickneite, G., BehringIns. Mitt. 93:299-305, 1993).

[0179] The lectin pathway may also play a role in the development of sepsis. MBL has been shown to bind to a range of 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 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). Recently, it has been demonstrated that L-phycoline specifically binds to LTA isolated from a great many Gram-positive bacterial species, including Staphylococcus aureus, and activates the lectin pathway (Lynch, NJ, et al., J. Immunol. 172:1198-02, 2004). MBL has also been shown to bind to LTA from Enterococcus species in which polyglycerophosphate chains are substituted with glycosyl groups, but not to LTA from nine other species, including Staphylococcus aureus (Polotsky, VY, et al., Infect. Immun. 64:380, 1996).

[0180] Accordingly, one aspect of the present invention provides a method for treating sepsis or a sepsis-related condition by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier to a subject suffering from sepsis or a sepsis-related condition, including but not limited to severe sepsis, septic shock, acute respiratory distress syndrome due to sepsis, and systemic inflammatory response syndrome. Related methods are also provided for treating such conditions by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier to a subject suffering from other hematological disorders, including hemorrhagic shock, hemolytic anemia, autoimmune thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), or other bone marrow / blood disruption conditions. MASP-2 inhibitors are administered systemically to the subject, for example, by intra-arterial, intravenous, intramuscular, inhalation (especially in the case of ARDS), subcutaneous, or other parenteral administration, or, depending on the case of non-peptide-acting substances, by oral administration. MASP-2 inhibitor compositions may be combined with one or more additional therapeutic substances to combat the sequelae of sepsis and / or shock. In cases of advanced sepsis or shock or the resulting distress, the MASP-2 inhibitor composition may be administered, as appropriate, in a rapid-acting dosage form, for example, by intravenous or intra-arterial delivery of a bolus of a solution containing the MASP-2 inhibitor composition. Repeated administrations may be given as determined by the physician until the condition improves.

[0181] Coagulation disorders Evidence has emerged regarding the role of the complement system in disseminated intravascular coagulation ("DIC"), such as DIC that follows severe physical trauma.

[0182] Previous studies have shown that C4- / - mice are not protected from renal reperfusion injury (Zhou, W., et al., "Predominant role for C5b-9 in renal ischemia / reperfusion injury", J Clin Invest 105:1363-1371 (2000)). To investigate whether C4- / - mice may be able to activate complement via either the classical or lectin pathway, C3 turnover in C4- / - plasma was measured using assays specific to either the classical or lectin-activated pathway. No C3 cleavage was observed upon activation via the classical pathway, but highly efficient lectin-pathdependent C3 activation was observed in C4-deficient serum (Figure 30). Numerous previously published papers on secondary pathway activation indicate that C3b deposition on mannan and zymosan is significantly impaired in MASP-2- / - mice, even under experimental conditions that allow for all three pathways. When the same serum was used in wells coated with immunoglobulin complexes instead of mannan or zymosan, C3b deposition and factor B cleavage were observed in MASP-2+ / + mouse serum and MASP-2- / - serum, but not in C1q-depleted serum. This indicates that, once initial C3b is supplied via classical activity, secondary pathway activation is promoted in MASP-2- / - serum. Figure 30C shows the surprising finding that C3 can be efficiently activated in a lectin pathway-dependent manner in C4-deficient plasma.

[0183] This "C4 bypass" is lost by inhibiting lectin pathway activation through pre-incubation of plasma with soluble mannan or mannose.

[0184] Abnormal non-immune activation of the complement system is potentially harmful to humans and may play a significant role in hematological pathway activation, particularly in severe trauma situations where both inflammatory and hematological pathways are activated. In normal health, C3 conversion is <5% of total plasma C3 protein. In severe infections, including sepsis and immune complex disorders, C3 conversion spontaneously recovers to about 30%, and complement levels are often lower than normal due to increased utilization and altered pool distribution. Rapid C3 pathway activation exceeding 30% generally indicates clear clinical evidence of vasodilation and fluid loss to tissues. In C3 conversion exceeding 30%, the initiation mechanism is primarily non-immune, and the resulting clinical signs are harmful to the patient. In healthy states and managed diseases, complement C5 levels appear considerably more stable than C3 levels. Significant decreases and / or conversions in C5 levels are associated with patient responses to abnormal multiple trauma (e.g., traffic accidents) and a high probability of developing shock pulmonary syndrome. Therefore, any evidence relating to complement C3 activation in more than 30% of the vascular pool, or any C5-involved complement C3 activation, or both, may be considered a strong indicator of adverse pathological changes in the patient.

[0185] Both C3 and C5 cells release anaphylatoxins (C3a and C5a) that act on mast cells and basophils to release vasodilatory chemicals. These create a chemotactic gradient that leads polymorphonuclear cells (PMNs) to the center of immunological disruption (beneficial response), but they differ in that C5a has a specific clamping effect on these phagocytes, preventing them from randomly detaching from the reaction site. In normal control of infection, C3 activates C5. However, in multiple trauma, C5 appears to be widely activated, generating C5a anaphylatoxin systemically. Due to this uncontrolled activity, polymorphonuclear cells cluster within the vascular system, and these clumps are then pushed into the pulmonary capillaries, causing occlusion and resulting in local damage as a result of superoxide release. While not bound by theory, this mechanism is likely important in the development of acute respiratory distress syndrome (ARDS). However, this idea has recently been challenged. While C3a anaphylatoxin may be shown to be a potent platelet aggregation factor in vitro, its involvement is less clear in vivo, and the release of platelet material and plasmin in wound healing may only be secondarily involved in complement C3. Long-term increases in C3 activation may be required to generate DIC.

[0186] In addition to the cellular and vascular effects of activated complement components outlined above, which can explain the relationship between trauma and DIC, a new scientific discovery has emerged that identifies a direct molecular relationship and functional crosstalk between the complement system and the coagulation system. Supporting data has been obtained from studies in C3-deficient mice. Since C3 is a common component of each complement pathway, it is expected that C3-deficient mice will lack all complement functions. However, surprisingly, C3-deficient mice can perfectly activate 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)). Further investigative studies have revealed that C3-independent activation of terminal complement components is mediated by thrombin, the rate-limiting enzyme in the coagulation cascade (Huber et al., 2006). The molecular components that mediate thrombin activation after initial complement activation remain a mystery.

[0187] The inventors elucidated what they believe to be the molecular basis of crosstalk between the complement cascade and the coagulation cascade, and identified MASP-2 as a central control point connecting the two systems. Biochemical studies on 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 the functionally relevant site of prothrombin, generating thrombin, 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)). The thrombin generated by MASP-2 can promote fibrin deposition in a defined reconstituted in vitro system. This demonstrates the functional relevance of MASP-2 cleavage (Krarup et al., 2007). As discussed in the following examples herein, the inventors further confirmed the physiological significance of this finding by recording thrombin activation in normal rodent serum after lectin pathway activation, demonstrating that this process is blocked by a neutralizing MASP-2 monoclonal antibody.

[0188] MASP-2 may be a central branching point in the lectin pathway, capable of promoting the activation of both the complement and coagulation systems. Since lectin pathway activation is a physiological response to many types of traumatic injury, we believe that the simultaneous occurrence of systemic inflammation (mediated by complement components) and scattered coagulation (mediated via the coagulation pathway) can be explained by MASP-2's ability to activate both pathways. These findings clearly suggest the role of MASP-2 in the development of DIC and the therapeutic benefits of MASP-2 inhibition in the treatment or prevention of DIC. MASP-2 may provide a molecular link between the complement and coagulation systems, and lectin pathway activation, as it occurs in traumatic situations, can directly initiate coagulation system activation via the MASP-2-thrombin axis, thus providing a mechanistic link between trauma and DIC. According to one aspect of the present invention, inhibition of MASP-2 is thought to inhibit lectin pathway activation, reducing the production of anaphylatoxins C3a and C5a. Long-term increases in C3 activation are thought to be necessary for the development of DIC.

[0189] Microcirculatory coagulation (blot clots in capillaries and small vessels) occurs in such septic shock situations. The role of the lectin pathway in septic shock has been clarified, as demonstrated by the protective phenotype of the MASP-2(- / -) mouse model of sepsis, as described in Example 17 and Figures 18 and 19. Furthermore, as demonstrated in Example 15 and Figures 16A and 16B, MASP-2(- / -) mice are protected in the localized Schwartzman reaction model of disseminated intravascular coagulation (DIC), a model of localized coagulation in microvessels.

[0190] V. MASP-2 inhibitors In one aspect, the present invention provides a method for inhibiting MASP-2-dependent complement activation in subjects suffering from or at risk of developing thrombotic microangiopathy. The MASP-2 inhibitor is administered in an amount effective to inhibit MASP-2-dependent complement activation in living subjects. In this aspect of the present invention, typical MASP-2 inhibitors include molecules that inhibit the biological activity of MASP-2 (e.g., small molecule inhibitors, anti-MASP-2 antibodies, or blocking peptides that interact with or interfere with protein-protein interactions with MASP-2), as well as molecules that reduce MASP-2 expression, thereby preventing MASP-2 from activating the lectin complement pathway (e.g., MASP-2 antisense nucleic acid molecules, MASP-2-specific RNAi molecules, and MASP-2 ribozymes). MASP-2 inhibitors may be used alone as a first-line therapy or in combination with other therapeutic agents as adjunctive therapy to enhance the therapeutic benefits of other medical treatments.

[0191] Inhibition of MASP-2-dependent complement activation is characterized by at least one of the following changes in complement system components resulting from administration of a MASP-2 inhibitor according to the method of the present invention: inhibition of the formation or production of MASP-2-dependent complement activation system products C4b, C3a, C5a, and / or C5b-9 (MAC) (e.g., as measured as described in Example 2); a decrease in complement activation as assessed by a hemolysis assay using unsensitized rabbit or guinea pig erythrocytes (e.g., as measured as described in Example 33); a decrease in C4 cleavage and C4b deposition (e.g., as measured as described in Example 2); or a decrease in C3 cleavage and C3b deposition (e.g., as measured as described in Example 2).

[0192] According to the present invention, a MASP-2 inhibitor effective in inhibiting the MASP-2-dependent complement activation system is used. MASP-2 inhibitors useful in this aspect of the present invention include, for example, anti-MASP-2 antibodies and their fragments, MASP-2 inhibitory peptides, small molecules, MASP-2 soluble receptors, and expression inhibitors. The MASP-2 inhibitor may inhibit the MASP-2-dependent complement activation system by blocking the biological function of MASP-2. For example, the inhibitor may effectively block MASP-2 protein-protein interactions, interfere with MASP-2 dimerization or assembly, and Ca 2+ The binding may be blocked, the MASP-2 serine protease active site may be interfered with, or MASP-2 protein expression may be reduced.

[0193] In some embodiments, MASP-2 inhibitors selectively inhibit MASP-2 complement activation without impairing the function of the C1q-dependent complement activation system.

[0194] In one embodiment, a MASP-2 inhibitor useful in the method of the present invention is a specific MASP-2 inhibitor that specifically binds to a polypeptide containing SEQ ID NO:6 with an affinity at least 10 times higher than other antigens of the complement system. In another embodiment, the MASP-2 inhibitor specifically binds to a polypeptide containing SEQ ID NO:6 with an affinity at least 100 times higher than other antigens of the complement system. The binding affinity of the MASP-2 inhibitor can be determined using an appropriate binding assay method.

[0195] The MASP-2 polypeptide exhibits a molecular structure similar to the C1 complement system proteases MASP-1, MASP-3, and C1r and C1s. The cDNA molecule shown in SEQ ID NO:4 encodes a representative example of MASP-2 (consisting of the amino acid sequence shown in SEQ ID NO:5) and provides a human MASP-2 polypeptide with a leader sequence (aa1-15) that is cleaved after secretion to produce mature human MASP-2 (SEQ ID NO:6). As shown in Figure 2, the human MASP2 gene contains 12 exons. The human MASP-2 cDNA is encoded by exons B, C, D, F, G, H, I, J, K, and L. As shown in Figure 2, the alternative splice yields a 20kDa protein called MBL-related protein 19 (also known as "MAp19" or "sMAP") (SEQ ID NO:2), encoded by exons B, C, D, and E (SEQ ID NO:1). The cDNA molecule shown in SEQ ID NO:50 encodes mouse MASP-2 (consisting of the amino acid sequence shown in SEQ ID NO:51) and provides a mouse MASP-2 polypeptide having a leader sequence, which is cleaved after secretion to produce mature mouse MASP-2 (SEQ ID NO:52). The cDNA molecule shown in SEQ ID NO:53 encodes rat MASP-2 (consisting of the amino acid sequence shown in SEQ ID NO:54) and provides a rat MASP-2 polypeptide having a leader sequence, which is cleaved after secretion to produce mature rat MASP-2 (SEQ ID NO:55).

[0196] 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 are single alleles of human MASP-2, mouse MASP-2, and rat MASP-2, respectively, and that allelic changes and alternative splicing are expected to occur. Allelic variants of the nucleotide sequences shown in SEQ ID NO:4, SEQ ID NO:50, and SEQ ID NO:53, including allelic variants containing silent mutations and allelic variants in which the amino acid sequence is altered by the mutation, are within the scope of the present invention. Allelic variants of the MASP-2 sequence can be cloned by probing cDNA libraries or genomic libraries derived from different individuals according to standard procedures.

[0197] The domains of the human MASP-2 protein (SEQ ID NO:6) are shown in Figures 1 and 2A, and include the N-terminal C1r / C1s / sea urchin Vegf / osteogenesis imperfecta (CUBI) domain (aa1-121 of SEQ ID NO:6), epidermal growth factor-like domains (aa122-166), another CUBI domain (aa167-293), and a tandem sequence of complement control protein domains and serine protease domains. Alternative splicing of the MASP2 gene yields MAp19, shown in Figure 1. MAp19 is a non-enzymatic protein containing the N-terminal CUBI-EGF region of MASP-2 and four additional residues (EQSL) derived from exon E, as shown in Figure 1.

[0198] Several proteins have been shown to bind to or interact with MASP-2 via protein-protein interactions. For example, MASP-2 binds to the lectin proteins MBL, H-phycoline, and L-phycoline, and interacts with them via Ca 2+It is known that they form a dependent complex. Each MASP-2 / lectin complex has been shown to activate complement via MASP-2-dependent cleavage of proteins C4 and C2 (Ikeda, K., et al., J. Biol Chem. 262:7451-7454, 1987; Matsushita, M., et al., J. Exp. Med 176:197-2284, 2000; Matsushita, M., et al., J. Immunol. 168:3502-3506, 2002). Studies have shown that the CUB1-EGF domain of MASP-2 is essential for binding of MASP-2 to MBL (Thielens, NM, et al., J. Immunol. 166:5068, 2001). The CUB1EGFCUBII domain has also been shown to mediate MASP-2 dimerization, which is necessary for the formation of the active MBL complex (Wallis, R., et al., J. Biol. Chem. 275:30962-30969, 2000). Therefore, it is possible to identify MASP-2 inhibitors that bind to the MASP-2 target region, which is known to be important for MASP-2-dependent complement activation, or MASP-2 inhibitors that interfere with the said MASP-2 target region.

[0199] Anti-MASP-2 antibody In some aspects of this aspect of the present invention, a MASP-2 inhibitor comprises an anti-MASP-2 antibody that inhibits the MASP-2-dependent complement activation system. Anti-MASP-2 antibodies useful in this aspect of the present invention include polyclonal antibodies, monoclonal antibodies, or recombinant antibodies derived from any antibody-producing mammal, and may be multispecific, chimeric, humanized, anti-idiotype, 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.

[0200] Several anti-MASP-2 antibodies have been described in the literature, some of which are listed in Table 1 below. The ability of these previously described anti-MASP-2 antibodies to inhibit the MASP-2-dependent complement activation system can be screened using the assay methods described herein. For example, as further detailed in Examples 10 and 11 herein, anti-rat MASP-2 Fab2 antibodies that block MASP-2-dependent complement activation have been identified. Once anti-MASP-2 antibodies that function as MASP-2 inhibitors have been identified, they can be used to construct anti-idiotype antibodies, as further described below, and can be used to identify other MASP-2 binding molecules.

[0201] (Table 1) MASP-2 specific antibodies from the literature TIFF2026062897000008.tif166155

[0202] Anti-MASP-2 antibodies with reduced effector function In some aspects of this facet of the present invention, an anti-MASP-2 antibody has reduced effector function to mitigate inflammation that may result from activation of the classical complement pathway. The ability of an IgG molecule to induce the classical complement pathway has been shown to reside in the Fc portion of the molecule (Duncan, AR. et al., Nature 332:738-740 1988). IgG molecules from which the Fc portion of this molecule has been removed by enzymatic cleavage lack this effector function (see Harlow, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). Therefore, by having a genetically modified Fc sequence that minimizes effector function, or by using human IgG2 or IgG4 isotypes, antibodies with reduced effector function can be produced as a result of the absence of the Fc portion of this molecule.

[0203] Antibodies with reduced effector function can be produced by standard molecular biological manipulation of the Fc portion of the IgG heavy chain, as described in Example 9 of this specification, as well as in Jolliffe et al., Int'l Rev. Immunol. 10:241-250, 1993 and Rodrigues et al., J. Immunol. 151:6954-6961, 1998. Antibodies with reduced effector function also include human IgG2 and IgG4 isotypes with reduced ability to activate complement and / or interact with Fc receptors (Ravetch, 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 to human MASP-2, consisting of IgG2 or IgG4 isotypes, can be prepared by one of several methods known to those skilled in the art, as described in Vaughan, TJ, et al., Nature Biotechnical 16:535-539, 1998.

[0204] Production of anti-MASP-2 antibodies Anti-MASP-2 antibodies can be prepared using MASP-2 polypeptide (e.g., full-length MASP-2) or peptides containing the antigenic MASP-2 epitope (e.g., a portion of the MASP-2 polypeptide). The immunogenic peptide may be as small as 5 amino acid residues. For example, a MASP-2 polypeptide containing the entire amino acid sequence of SEQ ID NO:6 may be used to induce an anti-MASP-2 antibody useful in the method of the present invention. Specific MASP-2 domains known to be involved in protein-protein interactions, such as the CUBI and CUBIEGF domains, as well as regions containing serine protease active sites, may be expressed as recombinant polypeptides as described in Example 3 and used as antigens. Furthermore, peptides containing at least 6 amino acid portions of the MASP-2 polypeptide (SEQ ID NO:6) are also useful for inducing MASP-2 antibodies. Further examples of MASP-2-derived antigens useful for inducing MASP-2 antibodies are shown in Table 2 below. The MASP-2 peptide and polypeptide used to produce antibodies may be natural polypeptides, recombinant peptides, or synthetic peptides, and catalytically inactive recombinant polypeptides, such as MASP-2A, isolated, as described further in Examples 5-7. In some aspects of this face of the present invention, anti-MASP-2 antibodies are obtained using transgenic mouse lines as described in Examples 8 and 9, and as described further below.

[0205] Antigens useful in the production of anti-MASP-2 antibodies also include fusion polypeptides, such as fusions of MASP-2 or a portion thereof with an immunoglobulin polypeptide or maltose-binding protein. The polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide portion is hapten-like, such a portion may be conveniently conjugated or linked to a macromolecule carrier (e.g., keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or tetanus toxoid) for immunization.

[0206] (Table 2) MASP-2 derived antigens TIFF2026062897000009.tif186153

[0207] Polyclonal antibodies Polyclonal antibodies against MASP-2 can be prepared by immunizing animals with the MASP-2 polypeptide or its immunogenic portion using methods well known to those skilled in the art. See, for example, Green et al., "Production of Polyclonal Antisera," Immunochemical Protocols (Manson, ed.), p. 105, further described in Example 6. The immunogenicity of the MASP-2 polypeptide can be enhanced using mineral gels, e.g., aluminum hydroxide or Freund's adjuvants (complete or incomplete), surfactants, e.g., lysolecithin, pluronic polyols, polyanions, oil emulsions, keyhole limpet hemocyanins, and adjuvants containing dinitrophenol. Polyclonal antibodies are typically produced in animals, e.g., horses, cattle, dogs, chickens, rats, mice, rabbits, guinea pigs, goats, or sheep. Alternatively, anti-MASP-2 antibodies useful in the present invention may also be derived from primates closely related to humans. General techniques for producing antibodies useful for diagnosis and treatment in baboons can be found, for example, in Goldenberg et al., International Patent Publication WO91 / 11465, and Losman, MJ, et al., Int. J. Cancer 46:310, 1990. Subsequently, serum containing immunologically active antibodies is produced from the blood of such immunized animals using standard procedures well known in the art.

[0208] Monoclonal antibodies In some embodiments, the MASP-2 inhibitor is an anti-MASP-2 monoclonal antibody. Anti-MASP-2 monoclonal antibodies are highly specific because they are produced against a single MASP-2 epitope. As used herein, the modifier “monoclonal” indicates that the antibody is obtained from a substantially homogeneous antibody population and should not be interpreted as requiring antibody production by a specific method. Monoclonal antibodies can be obtained using any technique that provides antibody molecule production using serially cultured cell lines, e.g., the hybridoma method described in Kohler, G., et al., Nature 256:495, 1975. Alternatively, monoclonal antibodies may be produced by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567 for Cabilly). Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described in Clackson, T., et al., Nature 352:624-628, 1991 and Marks, JD, et al., J. Mol Biol. 222:581-597, 1991. Such antibodies may be antibodies to any immunoglobulin class and any subclass, including IgG, IgM, IgE, IgA, and IgD.

[0209] For example, monoclonal antibodies can be obtained by injecting a suitable mammal (e.g., BALB / c mouse) with a composition containing the MASP-2 polypeptide or a portion thereof. After a predetermined period, spleen cells are removed from the mice and suspended in cell culture medium. The spleen cells are then fused with an immortal cell line to form hybridomas. The formed hybridomas are grown in cell culture and screened for their ability to produce monoclonal antibodies against MASP-2. An example of further preparation of anti-MASP-2 monoclonal antibodies is shown in Example 7 (see also Current Protocols in Immunology, Vol. 1, John Wiley & Sons, pp. 2.5.1-2.6.7, 1991).

[0210] Human monoclonal antibodies can be obtained using transgenic mice engineered to produce specific human antibodies in response to antigen exposure. This technique involves introducing elements of human immunoglobulin heavy and light chain loci into a mouse line derived from an embryonic stem cell line containing targeted disruption of endogenous immunoglobulin heavy and light chain loci. These transgenic mice can synthesize human antibodies specific to human antigens, such as the MASP-2 antigen described herein, and can be used to produce human MASP-2 antibody-secreting hybridomas by fusing B cells derived from such animals with a suitable myeloma cell line using conventional Köhler-Mirstein techniques, 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, for example, by 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, 3994.

[0211] Monoclonal antibodies can be isolated and purified from hybridoma cultures using a variety of well-established techniques. These isolation methods include affinity chromatography using protein A seharose, size exclusion chromatography, and ion exchange chromatography (see, e.g., Coligan, pp. 2.7.1–2.7.12 and 2.9.1–2.9.3; Baines et al., "Purification of Immunoglobulin G (IgG)", Methods in Molecular Biology, The Humana Press, Inc., Vol. 10, pp. 79–104, 1992).

[0212] Once polyclonal, monoclonal, or phage-derived antibodies are prepared, they are first tested for specific MASP-2 binding. Various assay methods known to those skilled in the art can be used to detect antibodies that specifically bind to MASP-2. Exemplary assay methods include standard Western blotting or immunoprecipitation analysis (e.g., Ausubel et al.), immunoelectrophoresis, enzyme-linked immunosorbent assay, dot blotting, inhibitory assays or competitive assays, and sandwich assays (Harlow and Land, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988). Once antibodies that specifically bind to MASP-2 are identified, the anti-MASP-2 antibodies are tested for their ability to function as MASP-2 inhibitors in one of several assay methods, such as 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).

[0213] The affinity of an anti-MASP-2 monoclonal antibody can be easily determined by those skilled in the art (see, for example, Scatchard, A., NY Acad. Sci. 51:660-672, 1949). In one embodiment, an anti-MASP-2 monoclonal antibody useful for the method of the present invention binds to MASP-2 with a binding affinity of <100 nM, preferably <10 nM, and most preferably <2 nM. In some embodiments, a MASP-2 inhibitory monoclonal antibody useful in the method of the present invention comprises (I)(a)(i)a heavy chain variable region comprising heavy chain CDR-H1 containing amino acid sequence 31-35 of SEQ ID NO:67; (ii)a heavy chain CDR-H2 containing amino acid sequence 50-65 of SEQ ID NO:67; and (iii)a heavy chain CDR-H3 containing amino acid sequence 95-102 of SEQ ID NO:67; and (b)a light chain variable region comprising (i)a light chain CDR-L1 containing amino acid sequence 24-34 of SEQ ID NO:70; (ii)a light chain CDR-L2 containing amino acid sequence 50-56 of SEQ ID NO:70; and (iii)a light chain CDR-L3 containing amino acid sequence 89-97 of SEQ ID NO:70; or (II)a light chain variable region comprising at least 90% identity with SEQ ID NO:67 (e.g., SEQ ID This is a MASP-2 inhibitory monoclonal antibody or its antigen-binding fragment, comprising variants thereof that include a heavy chain variable region having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identity with NO:67, and a light chain variable region having at least 90% identity with SEQ ID NO:70 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identity).

[0214] Chimeric / Humanized Antibodies Monoclonal antibodies useful in the methods of the present invention include chimeric antibodies in which a portion of the heavy chain 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 subclass, 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 subclass, as well as fragments of such antibodies (U.S. Patent No. 4,816,567 for Cabilly; and Morrison, SL, et al., Proc. Nat'l Acad. Sci. USA 81:6851-6855, 1984).

[0215] One form of chimeric antibody useful in the present invention is a humanized monoclonal anti-MASP-2 antibody. The humanized form of a non-human (e.g., mouse) antibody is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. The humanized monoclonal antibody is prepared by introducing a non-human (e.g., mouse) complementarity-determining region (CDR) derived from the variable heavy and variable light chains of a mouse immunoglobulin into the human variable domain. Typically, human antibody residues are then substituted in the framework region of the non-human counterpart. Furthermore, the humanized antibody may contain residues not found in either the recipient antibody or the donor antibody. These modifications are made to further refine the performance of the antibody. Generally, the humanized antibody contains substantially all of at least one, and typically two, variable domains. All or substantially all of the hypervariable loops correspond to the hypervariable loops of the non-human immunoglobulin, and all or substantially all of the Fv framework regions correspond to the Fv framework regions of the human immunoglobulin sequence. Humanized antibodies also optionally contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the immunoglobulin constant region (Fc) of 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.

[0216] The humanized antibodies useful in this invention include human monoclonal antibodies containing at least a MASP-2-binding CDR3 region. Furthermore, the Fc portion may be replaced to produce IgA antibodies or IgM antibodies, as well as human IgG antibodies. Such humanized antibodies specifically recognize human MASP-2 but do not evoke an immune response against the antibody itself in humans, making them particularly useful in clinical settings. Consequently, such humanized antibodies are more suitable for in vivo administration in humans, especially when repeated or long-term administration is required.

[0217] An example of the preparation of a humanized anti-MASP-2 antibody from a mouse anti-MASP-2 monoclonal antibody is shown in Example 6 of this specification. Techniques for producing humanized monoclonal antibodies are described, for example, in 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", Protein Engineering: Principles and Practice, Cleland et al. (eds.), John Wiley & Sons, Inc., pp. 399-434, 1996; and Queen, This is also described in U.S. Patent No. 5,693,762 of 1997. Furthermore, there are commercial entities, such as Protein Design Labs (Mountain View, CA), that synthesize humanized antibodies from specific mouse antibody regions.

[0218] Recombinant antibody Anti-MASP-2 antibodies can also be produced using recombinant methods. For example, human antibody fragments (V H , V L Human antibodies can be synthesized using a human immunoglobulin expression library (e.g., available from Stratagene, Corp., La Jolla, CA) to produce Fv, Fd, Fab, or F(ab')2). Then, the whole human antibody is constructed using these fragments with a technique similar to that used for synthesizing chimeric antibodies.

[0219] Anti-idiotype antibodies Once an anti-MASP-2 antibody with desirable inhibitory activity is identified, these antibodies can be used to generate anti-idiotype antibodies that partially resemble MASP-2 using techniques well known in the art. See, for example, Greenspan, NS, et al., FASEB J. 7:437, 1993. For example, an antibody that binds to MASP-2 and completely inhibits the MASP-2 protein interaction required for complement activation can be used to generate an anti-idiotype that resembles the MBL binding site on the MASP-2 protein and therefore binds to and neutralizes a MASP-2 binding ligand, such as MBL.

[0220] Immunoglobulin fragments The MASP-2 inhibitors useful in the method of the present invention include not only intact immunoglobulin molecules, but also well-known fragments formed from antibody fragments, including Fab, Fab', F(ab)2, F(ab')2, and Fv fragments, scFv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies.

[0221] It is well known in the art that only paratopes, which are small parts of the antibody molecule, are involved in the binding of antibodies to their epitopes (see, for example, Clark, WR, The Experimental Foundations of Modern Immunology, Wiley & Sons, Inc., NY, 1986). The pFc' and Fc regions of an antibody are effectors of the classical complement pathway but are not involved in antigen binding. Antibodies in which the pFc' region has been enzymatically cleaved, or antibodies produced without the pFc' region, are called F(ab')2 fragments and retain both antigen-binding sites of the intact antibody. Isolated F(ab')2 fragments are called bivalent monoclonal fragments because of their two antigen-binding sites. Similarly, antibodies in which the Fc region has been enzymatically cleaved, or antibodies produced without the Fc region, are called Fab fragments and retain one of the antigen-binding sites of the intact antibody molecule.

[0222] Antibody fragments can be obtained by conventional methods of protein hydrolysis of the entire antibody, such as pepsin digestion or papain digestion. For example, antibody fragments can be prepared by enzymatic cleavage of the antibody with pepsin to obtain a 5S fragment called F(ab')2. This fragment can be further cleaved using a thiol reducing agent that produces a 3,5S Fab' monovalent fragment. Optionally, the cleavage reaction can be carried out using a sulfhydryl blocking group that cleaves the disulfide bond. Alternatively, enzymatic cleavage with pepsin directly produces two monovalent Fab fragments and one Fc fragment. These methods are described, for example, in U.S. Patent No. 4,331,647 to Goldenberg; Nisonoff, A., et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, RR, Biochem, J. 73:119, 1959; Edelman, et al., Methods in Enzymology 1:422, Academic Press, 1967; and in Coligan, pp. 2.8.1-2.8.10 and 2.10-2.10.4.

[0223] In some embodiments, it is preferable to use antibody fragments without an Fc domain to avoid activation of the classical complement pathway, which is initiated when Fc binds to the Fcγ receptor. Several methods exist for producing MoAbs that avoid Fcγ receptor interaction. For example, the Fc domain of a monoclonal antibody can be chemically removed using partial digestion with proteolytic enzymes (e.g., ficin digestion), thereby generating, for example, an antigen-binding antibody fragment, such as a Fab fragment or an F(ab)2 fragment (Mariani, M., et al., Mol. Immunol. 28:69-71, 1991). Alternatively, a human γ4 IgG isotype that does not bind to the Fcγ receptor can be used during the construction of a humanized antibody as described herein. Antibodies without an Fc domain, single-chain antibodies, and antigen-binding domains can also be manipulated using the recombination methods described herein.

[0224] single chain antibody fragment Alternatively, a MASP-2-specific peptide single-chain binding molecule can be constructed in which the heavy-chain Fv region and the light-chain Fv region are linked. The Fv fragments may be linked with a peptide linker to form a single-chain antigen-binding protein (scFv). These single-chain antigen-binding proteins are linked by oligonucleotides, V H and V L It is prepared by constructing a structural gene containing the DNA sequence encoding the domain. The structural gene is inserted into an expression vector and subsequently introduced into a host cell such as E. coli. The recombinant host cell synthesizes a single polypeptide chain with a linker peptide that cross-links the two V domains. Methods for producing scFv are described, for example, in Whitlow, et al., "Methods: A Companion to Methods in Enzymology" 2:97, 1991; Bird, et al., Science 242:423, 1988; U.S. Patent No. 4,946,778 to Ladner; and Pack, P., et al., Bio / Technology 11:1271, 1993.

[0225] As an exemplary example, MASP-2-specific scFv can be obtained by exposing lymphocytes to MASP-2 polypeptide in vitro and selecting an antibody display library in a phage vector or analogous vector (e.g., by using immobilized or labeled MASP-2 protein or peptide). Genes encoding polypeptides with potential MASP-2 polypeptide-binding domains can be obtained by screening random peptide libraries displayed on phages or bacteria, e.g., Escherichia coli. These random peptide display libraries can be used to screen for peptides that interact with MASP-2. Techniques for constructing and screening such random peptide display libraries are well known in the art (U.S. Patent No. 5,223,409 to Ladner; U.S. Patent No. 4,946,778 to Ladner; U.S. Patent No. 5,403,484 to Ladner; U.S. Patent No. 5,571,698 to Ladner; and Kay et al., Phage Display of Peptides and Proteins, Academic Press, Inc., 1996). Random peptide display libraries and kits for screening such libraries are commercially available from companies such as CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly, Mass.), and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ).

[0226] Another form of anti-MASP-2 antibody fragment useful in this aspect of the present 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 the gene encoding the CDR of the antibody of interest. Such genes can be prepared, for example, by synthesizing variable regions from RNA of antibody-producing cells using polymerase chain reactions (see, for example, Larrick et al., Methods; A Companion to Methods in Enzymology 2:106, 1991; Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies", Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al., (eds.), p. 166, Cambridge University Press, 1995; and Ward et al., "Genetic Manipulation and Expression of Antibodies", Monoclonal Antibodies: Principles and Applications, Birch et al., (eds,), p. 137, Wiley-Liss, Inc., 1995).

[0227] To inhibit MASP-2-dependent complement activation, the MASP-2 antibodies described herein are administered to subjects requiring such inhibition. In some embodiments, the MASP-2 inhibitor is a high-affinity human or humanized monoclonal anti-MASP-2 antibody with reduced effector function.

[0228] Peptide inhibitors In some aspects of this aspect of the present invention, MASP-2 inhibitors include isolated MASP-2 peptide inhibitors, which include isolated native 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 MASP-2, competing with MASP-2 for binding to another recognition molecule of the lectin pathway (e.g., MBL, H-phycoline, M-phycoline, or L-phycoline), and / or by directly interacting with MASP-2 to inhibit MASP-2-dependent complement activation, wherein the peptide is substantially pure and essentially free from other substances that may be found together in nature to a degree that is practical and suitable for the intended use.

[0229] Peptide inhibitors have been successfully used in vivo to disrupt protein-protein interactions and catalytic sites. For example, a peptide inhibitor targeting LFA-1 and structurally related adhesion molecules was recently approved for clinical use in coagulation disorders (Ohman, EM, et al., European Heart J. 16:50-55, 1995). Short linear peptides (<30 amino acids) that inhibit or disrupt integrin-dependent adhesion have been described (Murayama, O., et al., J. Biochem. 120:445-51, 1996). Longer peptides, ranging from 25 to 200 amino acid residues in length, have also been successfully used to block integrin-dependent adhesion (Zhang, L., et al., J. Biol. Chem. 271(47):29953-57, 1996). Generally, long peptide inhibitors have higher affinity and / or slower off-rate than short peptides and can therefore be potent inhibitors. Cyclic peptide inhibitors have also been shown to be effective in vivo integrin inhibitors 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 peptide synthesis in which the terminal amino acid of the peptide is cysteine, thereby allowing the peptide to exist in a cyclic form due to disulfide bonds between the terminal amino acids. This cyclic form has been shown to improve in vivo affinity and half-life for the treatment of hematopoietic neoplasms (e.g., U.S. Patent No. 6,649,592 to Larson).

[0230] Synthetic MASP-2 peptide inhibitor MASP-2 inhibitory peptides useful in this aspect of the present invention are exemplified by amino acid sequences that mimic target regions important to MASP-2 function. Inhibitory peptides useful in carrying out the methods of the present invention range in size from about 5 amino acids to about 300 amino acids. Table 3 provides a list of exemplary inhibitory peptides that may be useful in carrying out this aspect of the present invention. For example, the ability of a candidate MASP-2 inhibitory peptide to function as a MASP-2 inhibitor can be tested in one of several assay methods, including a lectin-specific C4 cleavage assay (described in Example 2) and a C3b deposition assay (described in Example 2).

[0231] In some embodiments, the MASP-2 inhibitory peptide is derived from the MASP-2 polypeptide and selected from the fully matured MASP-2 protein (SEQ ID NO: 6) or specific domains of the MASP-2 protein, such as the CUBI domain (SEQ ID NO: 8), the CUBIEGF domain (SEQ ID NO: 9), the EGF domain (SEQ ID NO: 11), and the serine protease domain (SEQ ID NO: 12). As described above, the CUBEGFCUBII region has been shown to be necessary for dimerization and binding to the MBL (Thielens et al., cited above). In particular, the peptide sequence TFRSDYN (SEQ ID NO:16) in the CUBI domain of MASP-2 has been shown to be involved in binding to MBL in a study that identified individuals who have a homozygous mutation from Asp105 to Gly105, resulting in the loss of MASP-2 from the MBL complex (Stengaard-Pedersen, K., et al., New England J. Med. 349:554-560, 2003).

[0232] In some embodiments, MASP-2 inhibitory peptides are derived from lectin proteins that bind to MASP-2 and are involved in the lectin complement pathway. Several different lectins involved in this pathway have been identified, including mannan-binding lectin (MBL), L-ficorin, M-ficorin, and H-ficorin (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 possessing an N-terminal collagen-like fiber with a carbohydrate-recognizing domain. These different lectins have been shown to bind to MASP-2, and the lectin / MASP-2 complex activates complement by cleaving proteins C4 and C2. H-Ficolin has a 24-amino acid amino-terminal region, a collagen-like domain with 11 Gly-Xaa-Yaa repeats, a 12-amino acid neck domain, and a 207-amino acid fibrinogen-like domain (Matsushita, M., et al., J. Immunol. 168:3502-3506, 2002). H-Ficolin binds to GlcNAc and agglutinates human erythrocytes coated with LPS derived from Salmonella typhimurium, Salmonella minnesota, and Escherichia coli. H-Ficolin has been shown to bind to MASP-2 and MAp19 and activate the lectin pathway. In other words, L-ficolin / P35 has also been shown to bind to GlcNAc and to MASP-2 and MAp19 in human serum. This complex has been shown to activate the lectin pathway (Matsushita, M., et al., J. Immunol. 164:2281, 2000).Therefore, a MASP-2 inhibitory peptide useful in the present invention may include a region of at least five amino acids selected from MBL protein (SEQ ID NO:21), H-phycoline protein (GenBank accession number NM_173452), M-phycoline protein (GenBank accession number O00602), and L-phycoline protein (GenBank accession number NM_015838).

[0233] More specifically, scientists have found that the MASP-2 binding site on MBL is located between the hinge and neck of the C-terminal portion of the collagen-like domain of MBP, specifically between 12 Gly-XY triplets. It was identified as being located within TIFF2026062897000010.tif12128 (Wallis, R. et al., J. Biol Chem. 279:14065, 2004). This MASP-2 binding site region is also highly conserved in human H-phycholine and human L-phycholine. A consensus binding site has been described that is present in all three lectin proteins, containing the amino acid sequence "OGK-X-GP" (SEQ ID NO:22), where the letter "O" represents hydroxyproline and the letter "X" represents a hydrophobic residue (Wallis et al., 2004, aforementioned). Therefore, in some embodiments, the MASP-2 inhibitory peptide useful in this aspect of the present invention is at least 6 amino acids long and contains SEQ ID NO:22. Amino acid sequence Peptides derived from MBL, including TIFF2026062897000011.tif4128, have been shown to bind to MASP-2 in vitro (Wallis, et al., 2004, see above). To enhance binding to MASP-2, peptides can be synthesized with two GPO triplets flanking each end, enhancing the formation of a triple helix as seen in the native MBL protein. TIFF2026062897000012.tif5132 (further explained in Wallis, R., et al., J. Biol. Chem. 279:14065, 2004).

[0234] MASP-2 inhibitory peptides also have sequences derived from the consensus MASP-2 binding region of H-phycoline. May be derived from human H-phycoline, including TIFF2026062897000013.tif4145. Sequence derived from the consensus MASP-2 binding region of L-phycoline. It also contains human L-phycoline-derived peptides, including TIFF2026062897000014.tif12153.

[0235] The MASP-2 inhibitory peptide is also a C4 cleavage site linked to the C-terminal portion of antithrombin III. It may originate from C4 cleavage sites such as TIFF2026062897000015.tif4128 (Glover, GI, et al., Mol. Immunol. 25:1261(1988)).

[0236] (Table 3) Exemplary MASP-2 inhibitory peptides TIFF2026062897000016.tif29155TIFF2026062897000017.tif218155TIFF2026062897000018.tif58155Note: The letter "O" represents hydroxyproline. The letter "X" represents a hydrophobic residue.

[0237] Peptides derived from the C4 cleavage site, as well as other peptides that inhibit the MASP-2 serine protease site, can be chemically modified to become irreversible protease inhibitors. Suitable modifications may include, but are not limited to, halomethyl ketones (Br, Cl, I, F) added to the C-terminus, Asp, or Glu, or to the functional side chain; haloacetyl (or other α-haloacetyl) groups on the amino group or other functional side chain; epoxide-containing or imine-containing groups on the amino-terminus, carboxyl-terminus, or functional side chain; or imidet esters on the amino-terminus, carboxyl-terminus, or functional side chain. Such modifications are expected to provide the advantage of permanently inhibiting the enzyme through covalent bonding of the peptide. This may result in a lower effective dose and / or the need for a reduced frequency of peptide inhibitor administration.

[0238] In addition to the inhibitory peptides described above, MASP-2 inhibitory peptides useful in the method of the present invention include peptides containing the MASP-2-binding CDR3 region of an anti-MASP-2 MoAb obtained as described herein. The sequence of the CDR region for use in peptide synthesis can be determined by methods known in the art. The heavy chain variable region is generally a peptide with a length of 100 to 150 amino acids. The light chain variable region is generally a peptide with a length of 80 to 130 amino acids. The CDR sequences within the heavy chain and light chain variable regions contain sequences of only about 3 to 25 amino acids, which can be readily sequenced by those skilled in the art.

[0239] Those skilled in the art will likely recognize that substantially homologous variations of the MASP-2 inhibitory peptide also exhibit MASP-2 inhibitory activity. Exemplary variations include, but are not limited to, peptides having insertions, deletions, exchanges, and / or additional amino acids in the carboxy-terminal or amino-terminal portion of the peptide, as well as mixtures thereof. Therefore, homologous peptides having MASP-2 inhibitory activity are considered useful in the methods of the present invention. The described peptides may also include other modifications by duplicate motifs and conserved substitutions. Conservative variants are described elsewhere in this specification and include exchanging certain amino acids with amino acids of similar charge, size, or hydrophobicity, among other things.

[0240] To better resemble segments in intact proteins, MASP-2 inhibitory peptides may be modified to increase solubility and / or maximize positive or negative charge. Derivatives may or may not have the exact primary amino acid structure of the peptides disclosed herein, as long as they functionally retain the desired MASP-2 inhibitory properties. Modifications may include amino acid substitutions using one or another of the 20 commonly known amino acids, amino acid substitutions using auxiliary desirable features, such as derivatized or substituted amino acids or D amino acids that are resistant to enzymatic degradation, or substitutions using other molecules or compounds that mimic the natural conformation and the function of one, multiple, or peptides, such as carbohydrates; amino acid deletions; amino acid insertions using one or another of the 20 commonly known amino acids, auxiliary desirable features, such as derivatized or substituted amino acids or D amino acids that are resistant to enzymatic degradation, or substitutions using other molecules or compounds that mimic the natural conformation and the function of one, multiple, or peptides, such as carbohydrates; or substitutions using other molecules or compounds that mimic the natural conformation, charge distribution, and function of the parent peptide, such as carbohydrates or nucleic acid monomers. Peptides may also be modified by acetylation or amidation.

[0241] The synthesis of derivative inhibitor peptides may rely on known techniques such as peptide biosynthesis and carbohydrate biosynthesis. As a starting point, those skilled in the art may rely on a suitable computer program to determine the conformation of the peptide of interest. Once the conformation of the peptide disclosed herein is known, those skilled in the art can determine, in a rational design manner, what kinds of substitutions can be made to one or more sites to create derivatives that retain the basic conformation and charge distribution of the parent peptide but may have features not present in the parent peptide or features enhanced beyond those present in the parent peptide. Once candidate derivative molecules have been identified, the derivatives can be tested using the assay methods described herein to determine whether they function as MASP-2 inhibitors.

[0242] Screening of MASP-2 inhibitory peptides Molecular modeling and rational molecular design can also be used to mimic the molecular structure of key binding regions of MASP-2 and to generate and screen peptides that inhibit MASP-2 complement activity. As previously mentioned, molecular structures used for modeling include the CDR region 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. Methods for identifying peptides that bind to a particular target are well known in the art. For example, molecular imprinting can be used to construct novel macromolecular structures that bind to a particular molecule, such as peptides. See, for example, Shea, KJ, "Molecular Imprinting of Synthetic Network Polymers: The De Novo synthesis of Macromolecular Binding and Catalytic Sties," TRIP 2(5) 1994.

[0243] As an exemplary example, one method for preparing mimics of MASP-2 binding peptides is as follows: A functional monomer (template) of the binding region of a known MASP-2 binding peptide, or an anti-MASP-2 antibody exhibiting MASP-2 inhibition, is polymerized. The template is then removed, and another class of monomer is polymerized in the void left by the template to obtain a new molecule similar to the template and exhibiting one or more desirable properties. In addition to preparing peptides in this way, other MASP-2 binding molecules that are MASP-2 inhibitors, such as polysaccharides, nucleosides, drugs, nucleoproteins, lipoproteins, carbohydrates, glycoproteins, steroids, lipids, and other bioactive materials, can also be prepared. This method is useful for designing a wide variety of biomimics that are more stable than their natural counterparts, because these biomimics are typically prepared by free-radical polymerization of functional monomers, resulting in compounds with a non-biodegradable backbone.

[0244] Peptide synthesis MASP-2 inhibitory peptides can be prepared using techniques well known in the art, such as the solid-phase synthesis technique first described by Merrifield in J. Amer. Chem. Soc. 85:2149-2154, 1963. Automated synthesis can be carried out, for example, using the Applied Biosystems 431 A Peptide Synthesizer (Foster City, Calif.) according to the instructions provided by the manufacturer. Other techniques can be found, for example, in Bodanszky, M., et al., Peptide Synthesis, 2nd edition, John Wiley & Sons, 1976, and other references known to those skilled in the art.

[0245] Peptides can also be prepared using standard genetic engineering techniques known to those skilled in the art. For example, peptides can be enzymatically produced by inserting the nucleic acid encoding the peptide into an expression vector, expressing the DNA, and translating the DNA into the peptide in the presence of the required amino acids. The peptide is then purified by chromatography or electrophoresis, or by a carrier protein that can be fused with the peptide and subsequently cleaved by inserting the peptide-encoding sequence in-frame (in phase) into the expression vector with the nucleic acid sequence encoding the carrier protein. The fusion protein peptide may be isolated by chromatography, electrophoresis, or immunological techniques (e.g., antibody-mediated binding of the resin to the carrier protein). Peptides can be cleaved using chemical methodologies or enzymatically, for example, by hydrolytic enzymes.

[0246] The MASP-2 inhibitory peptides useful in the method of the present invention can also be produced in recombinant host cells according to conventional techniques. To express the sequence encoding the MASP-2 inhibitory peptide, the nucleic acid molecule encoding the peptide must be functionally ligated to a regulatory sequence that controls transcriptional expression within an expression vector, and then introduced into host cells. In addition to transcriptional regulatory sequences such as promoters and enhancers, the expression vector may also contain translational regulatory sequences and marker genes suitable for selecting cells having the expression vector.

[0247] Nucleic acid molecules encoding MASP-2 inhibitory peptides can be synthesized using a "gene machine" with protocols such as the phosphoramidite method. When chemically synthesized double-stranded DNA is required for applications such as gene or gene fragment synthesis, each complementary strand is fabricated separately. The creation of short genes (60-80 base pairs) is technically straightforward and can be achieved by synthesizing the complementary strands and then annealing them. To create larger genes, synthetic genes (double-stranded) are assembled in a modular format from single-stranded fragments of 20-100 nucleotides in length. For a review of polynucleotide synthesis, see, for example, Glick and Pasternak, "Molecular Biotechnology, Principles and Applications of Recombinant DNA," ASM Press, 1994; Itakura, K., et al., Annu. Rev, Biochem. 53:323, 1984; and Climie, S., et al., Proc. Nal'l Acad. Sci. USA 87:633, 1990.

[0248] Small molecule inhibitors In some embodiments, MASP-2 inhibitors are small molecular weight inhibitors, including natural and synthetic substances having low molecular weight, such as peptides, peptidomimetics, and non-peptide inhibitors (including oligonucleotides and organic compounds). MASP-2 small molecular weight inhibitors can be prepared based on the molecular structure of the variable region of an anti-MASP-2 antibody.

[0249] Small molecule inhibitors may also be designed and generated based on the MASP-2 crystal structure using computational drug design (Kuntz ID, et al., Science 257:1078, 1992). The rat MASP-2 crystal structure has been described (Feinberg, H., et al., EMBO J. 22:2348-2359, 2003). Using the method described by Kuntz et al., the MASP-2 crystal structure coordinates are used as input to a computer program, e.g., DOCK, which outputs a list of small molecule structures 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 HIV-1 protease inhibitors by evaluating the fit between compounds found in the Cambridge Crystallographic database and their enzyme binding sites using the DOCK program (Kuntz, ID, et al., J. Mol. Biol. 161:269-288, 1982; DesJarlais, RL, et al., PNAS 87:6644-6648, 1990).

[0250] A list of small molecule structures identified as potential MASP-2 inhibitors by computer computation is screened using a MASP-2 binding assay, e.g., the MASP-2 binding assay described in Example 10. The small molecules found to bind to MASP-2 are then assayed using a functional assay, e.g., the functional assay described in Example 2, to determine whether they inhibit MASP-2-dependent complement activation.

[0251] MASP-2 soluble receptor Other suitable MASP-2 inhibitors are thought to include MASP-2 soluble receptors that can be prepared using techniques known to those skilled in the art.

[0252] MASP-2 expression inhibitors In another aspect of this aspect of the present invention, a MASP-2 inhibitor is a MASP-2 expression inhibitor that can inhibit MASP-2-dependent complement activation. In the embodiment of this aspect of the present invention, typical MASP-2 expression inhibitors include MASP-2 antisense nucleic acid molecules (e.g., antisense mRNA, antisense DNA, or antisense oligonucleotides), MASP-2 ribozymes, and MASP-2 RNAi molecules.

[0253] Antisense RNA and DNA molecules work to directly block the translation of MASP-2 mRNA by hybridizing to MASP-2 mRNA and inhibiting the translation of the MASP-2 protein. Antisense nucleic acid molecules may be constructed in a number of different ways, provided that they can interfere with MASP-2 expression. For example, an antisense nucleic acid molecule can be constructed by reversing the coding region (or part thereof) of MASP-2 cDNA (SEQ ID NO:4) relative to the normal transcription direction, so that the complementary strand of the coding region (or part thereof) of MASP-2 cDNA (SEQ ID NO:4) can be transcribed.

[0254] Antisense nucleic acid molecules are typically substantially identical to one or at least a portion of one or more target genes. However, nucleic acids do not need to be perfectly identical to inhibit expression. Generally, high homology can be used to compensate for the use of short antisense nucleic acid molecules. The minimum percentage identity is typically over 65%, but even higher percentage identity may more effectively suppress the expression of endogenous sequences. A fairly high percentage identity of over 80% is typically preferred, but identity of around 95% to perfect identity is typically most preferred.

[0255] Antisense nucleic acid molecules do not need to have the same intron or exon pattern as the target gene. Non-coding segments of the target gene may be just as effective as coding segments in achieving antisense repression of target gene expression. A DNA sequence of at least about eight nucleotides can be used as an antisense nucleic acid molecule, but longer sequences are preferred. In this invention, a representative example of a useful MASP-2 inhibitor is an antisense MASP-2 nucleic acid molecule that is at least 90 percent identical to the complementary strand of the MASP-2 cDNA consisting of the nucleic acid sequence shown in SEQ ID NO:4. The nucleic acid sequence shown in SEQ ID NO:4 encodes the MASP-2 protein consisting of the amino acid sequence shown in SEQ ID NO:5.

[0256] Targeting of antisense oligonucleotides that bind to MASP-2 mRNA is another mechanism that can be used to reduce the level of MASP-2 protein synthesis. For example, the synthesis of polygalacturonase and muscarinic acetylcholine receptor type 2 is inhibited by antisense oligonucleotides against their respective mRNA sequences (US Patent No. 5,739,119 for Cheng and US Patent No. 5,759,829 for Shewmaker). Furthermore, examples of antisense inhibition include nucleoprotein cyclins, the multi-sin resistance gene (MDG1), ICAM-1, E-selectin, STK-1, and striatal GABA. A This has been demonstrated using receptors and human EGF (see, for example, U.S. Patent No. 5,801,154 for Baracchini; U.S. Patent No. 5,789,573 for Baker; U.S. Patent No. 5,718,709 for Considine; and U.S. Patent No. 5,610,288 for Reubenstein).

[0257] A system is described in which those skilled in the art can determine which oligonucleotides are useful in the present invention. This system involves probing a suitable site within a target mRNA using RNAseH cleavage as an indicator of sequence reachability within the transcript. Scherr, M., et al., Nucleic Acids Res. 25:5079-5085, 1998; Lloyd, et al., Nucleic Acids Res. 29:3665-3673, 2001. To create a site vulnerable to RNAseH, a mixture of antisense oligonucleotides complementary to a specific region of the MASP-2 transcript is added to a cell extract expressing MASP-2, e.g., hepatocytes, and hybridized. This method can be combined with computer-aided sequence selection, which can predict the optimal sequence selection of the antisense composition based on its relative ability to form dimers, hairpins, or other secondary structures that reduce or prevent specific binding to the target mRNA within the host cell. These secondary structure analyses and target site selection studies can be performed 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 against the target sequence preferably contain nucleotides of about 8 to about 50 in length. Antisense oligonucleotides containing nucleotides of about 9 to about 35, etc., are particularly preferred. The inventors intend that all oligonucleotide compositions in the range of 9 to 35 nucleotides (i.e., about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 bases in length) are very preferred for carrying out the methods of the present invention based on antisense oligonucleotides.The most preferred target regions of MASP-2 mRNA are those located at or near the AUG translation start codon, and these sequences substantially complementary to the 5' region of the mRNA, e.g., the -10 to +10 region of the MASP-2 gene nucleotide sequence (SEQ ID NO: 4). Exemplary MASP-2 expression inhibitors are shown in Table 4.

[0258] (Table 4) Exemplary inhibitors of MASP-2 expression TIFF2026062897000019.tif72155

[0259] As stated herein, the term “oligonucleotide” refers to oligomers or polymers of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or their mimics. The term also covers oligonucleotides having natural nucleotides, sugars, and oligonucleotide bases consisting of covalent nucleoside-to-nucleoside (backbone) bonds, as well as non-natural modifications. These modifications make it possible to introduce certain desirable properties not provided by natural oligonucleotides, such as low toxicity, high stability against nuclease degradation, and high cellular uptake. In exemplary embodiments, the antisense compounds of the present invention differ from natural DNA only in that the phosphate substituent is substituted with a phosphodiester backbone modification that extends the lifespan of the antisense oligonucleotide. Similarly, one or both ends of the oligonucleotide may be substituted with one or more acridine derivatives that intercalate between adjacent base pairs in the nucleic acid chain.

[0260] Another alternative to antisense is the use of "RNA interference" (RNAi). Double-stranded RNA (dsRNA) can induce gene silencing in vivo in mammals. The innate functions of RNAi and cosuppression appear to be the protection of the genome from invasion by mobile genetic elements such as retrotransposons and viruses that, when activated, produce abnormal RNA or dsRNA in host cells (see, e.g., Jensen, J., et al., Nat. Genet. 27:209-12, 1999). Double-stranded RNA molecules can be prepared by synthesizing two RNA strands capable of forming a double-stranded RNA molecule, each having a length of approximately 19–25 (e.g., 19–23 nucleotides). For example, dsRNA molecules useful in the method of the present invention may include RNAs corresponding to the sequences and complementary strands listed in Table 4. Preferably, at least one RNA strand has a 3' overhang of 1–5 nucleotides. The synthesized RNA strands are combined under conditions to form a double-stranded molecule. This RNA sequence may have a total length of 25 nucleotides or less and contain at least eight nucleotide segments of SEQ ID NO:4. Designing siRNA sequences for a specific target is within the scope of the usual art in this field. Commercial services are available to design siRNA sequences and guarantee at least 70% expression knockdown (Qiagen, Valencia, Calif).

[0261] dsRNA can be administered as a pharmaceutical composition, and the nucleic acid can be introduced into desired target cells by known methods. Commonly used gene transfer methods include calcium phosphate, DEAE-dextran, electroporation, microinjection, and viral methods. Such methods are disclosed in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 1993.

[0262] To reduce the amount and / or biological activity of MASP-2, ribozymes, such as ribozymes targeting MASP-2 mRNA, can also be used. 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 that encode RNA ribozymes that specifically pair with target RNA and cleave the phosphodiester backbone at a specific position, thereby functionally inactivating the target RNA. When this cleavage is performed, the ribozyme itself remains unchanged and can therefore be reused to cleave other molecules. Including a ribozyme sequence in antisense RNA confers RNA cleavage activity to the antisense RNA, thereby increasing the activity of the antisense construct.

[0263] A ribozyme useful in carrying out the present invention typically comprises a hybridizing region of at least nine nucleotides that is nucleotide sequence-complementary to at least a portion of the target MASP-2 mRNA, and a catalytic region adapted to cleave the target MASP-2 mRNA (see generally EPA No. 0321201; WO88 / 04300; Haseloff, J., et al., Nature 534:585-591, 1988; Fedor, MJ, et al., Proc. Natl. Acad, Sci. USA 87:1668-1672, 1990; Cech, TR, et al., Ann. Rev, Biochem. 55:599-629, 1986).

[0264] Ribozymes may be directly targeted to cells in the form of RNA oligonucleotides incorporating the ribozyme sequence, or they may be introduced into cells as expression vectors encoding the desired ribozyme RNA. Ribozymes can be used and applied in much the same manner as antisense polynucleotides.

[0265] Antisense RNA and DNA, ribozymes, and RNAi molecules useful in the methods of the present invention can be prepared by any method known in the art for synthesizing DNA and RNA molecules. These include techniques for the chemical synthesis of oligodeoxyribonucleotides and oligoribonucleotides, well known in the art, such as solid-phase phosphoramidite chemosynthesis. Alternatively, RNA molecules may be prepared by in vitro and in vivo transcription of DNA sequences encoding antisense RNA molecules. Such DNA sequences may be incorporated into a wide variety of vectors incorporating suitable RNA polymerase promoters, such as T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that constitutively or inductively synthesize antisense RNA, depending on the promoter used, can be stably introduced into cell lines.

[0266] Various well-known modifications to DNA molecules can be introduced as a means of increasing stability and extending 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 phosphorothioates or 2'O-methyl groups instead of phosphodiesterase bonds within the oligodeoxyribonucleotide backbone.

[0267] VI. Pharmaceutical compositions and delivery methods dosage In another aspect, the present invention provides compositions for inhibiting side effects of MASP-2-dependent complement activation in subjects suffering from the diseases or conditions disclosed herein, comprising administering the composition to the subject a therapeutically effective amount of a MASP-2 inhibitor and a pharmaceutically acceptable carrier. A MASP-2 inhibitor can be administered to a subject in need in a therapeutically effective dose to treat or alleviate a condition associated with MASP-2-dependent complement activation. A therapeutically effective dose refers to an amount of MASP-2 inhibitor sufficient to alleviate symptoms associated with the disease or condition.

[0268] The toxicity and therapeutic efficacy of MASP-2 inhibitors can be determined by standard pharmaceutical procedures using experimental animal models, such as the mouse MASP-2- / - model expressing the human MASP-2 transgene described in Example 1. Using such animal models, the NOAEL (no-observed-adverse-effect level) and MED (minimum effective dose) can be determined using standard methods. The dose ratio of the NOAEL effect to the MED effect is the cure ratio, expressed as the ratio NOAEL / MED. MASP-2 inhibitors exhibiting a large cure ratio or index are most preferred. Data obtained from cell culture assays and animal studies can be used in formulations of a range of doses for human use. The dose of the MASP-2 inhibitor is preferably within the range of circulating concentrations including the MED, where toxicity is minimal or nonexistent. The dose may vary within this range depending on the dosage form used and the route of administration utilized.

[0269] For any compound formulation, the therapeutically effective dose can be evaluated using animal models. For example, doses that reach the circulating plasma concentration range, including the medium-enhanced dose (MED), can be prescribed in animal models. The quantitative level of MASP-2 inhibitors in plasma can also be measured, for example, by high-performance liquid chromatography.

[0270] In addition to toxicity studies, effective doses may also be evaluated based on the amount of MASP-2 protein present in living subjects and the binding affinity of MASP-2 inhibitors. MASP-2 levels in normal human subjects are present in serum at low levels within the range of 500 ng / ml, and MASP-2 levels in a particular subject can be determined using the quantitative MASP-2 assay method described in Moller-Kristensen M., et al., J. Immunol Methods 282:159-167, 2003.

[0271] Generally, the dosage of a composition containing a MASP-2 inhibitor varies depending on factors such as the subject's age, weight, height, sex, general medical condition, and medical history. For example, a MASP-2 inhibitor, such as an anti-MASP-2 antibody, can be administered within a dosage range of approximately 0.010 to 10.0 mg / kg of body weight, preferably 0.010 to 1.0 mg / kg of body weight, and more preferably 0.010 to 0.1 mg / kg of body weight. In some embodiments, the composition includes a combination of an anti-MASP-2 antibody and a MASP-2 inhibitory peptide.

[0272] The therapeutic efficacy of the MASP-2 inhibitory composition and method of the present invention in a particular target, as well as the appropriate dosage, can be determined according to complement assay methods well known to those skilled in the art. Complement produces a great many specific products. Over the past decade, highly sensitive and specific assays have been developed and commercialized for most of these activating products, including the small activating fragments C3a, C4a, and C5a, as well as the larger activating fragments iC3b, C4d, Bb, and sC5b-9. Most of these assays utilize monoclonal antibodies that react with neoantigens, which are exposed on the fragments but not on the native proteins on which the neoantigens are formed. For this reason, these assays are very simple and specific. While most rely on ELISA techniques, radioimmunoassays are still sometimes used in the case of C3a and C5a. These latter assays measure both the untreated fragments and these "desArg" fragments, which are the primary forms found in circulation. desArg It is rapidly cleaved by binding to cell surface receptors and therefore exists at very low concentrations. In contrast, C3a desArgIt does not bind to cells and accumulates in plasma. C3a measurement provides a highly sensitive pathway-dependent complement activation index. Secondary pathway activation can be evaluated by measuring the Bb fragment. Detection of sC5b-9, the liquid-phase product of membrane invasion pathway activation, provides evidence that complement is activated to the end. Since both the lectin pathway and the classical pathway produce the same activators, C4a and C4d, measuring these two fragments does not provide information as to which of the two pathways produced the activator.

[0273] Inhibition of MASP-2-dependent complement activation is characterized by at least one of the following changes in complement system components resulting from administration of a MASP-2 inhibitor by the method of the present invention: inhibition of the formation or production of MASP-2-dependent complement activation system products C4b, C3a, C5a, and / or C5b-9 (MAC) (e.g., as measured as described in Example 2), reduction of C4 cleavage and C4b deposition (e.g., as measured as described in Example 10), or reduction of C3 cleavage and C3b deposition (e.g., as measured as described in Example 10).

[0274] Further active substances Compositions and methods comprising MASP-2 inhibitors may optionally include one or more additional therapeutic substances that can increase the activity of the MASP-2 inhibitor, or one or more additional therapeutic substances that additively or synergistically provide related therapeutic functions. For example, in the treatment of a subject with TTP who is positive for ADAM-TS13 inhibitors, one or more MASP-2 inhibitors may be administered in combination with (including concurrently with) one or more immunosuppressants. Suitable immunosuppressants include corticosteroids, rituximab, and cyclosporine. In the treatment of a subject with HUS or aHUS, or a subject at risk of developing HUS or aHUS, one or more MASP-2 inhibitors may be administered in combination with (including concurrently with) a suitable antibiotic. In situations involving the treatment of subjects suffering from aHUS or at risk of developing aHUS, one or more MASP-2 inhibitors may be administered in combination with (including co-administration with) other complement inhibitors, such as eculizumab (Soliris), TT-30, antibodies against factor B, or other agents that inhibit terminal complement components or secondary pathway amplification.

[0275] Further inclusion and selection of active ingredients are likely to be determined to achieve desirable therapeutic outcomes. In some embodiments, the MASP-2 inhibitor 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 2 receptor subtype antagonists; calcitonin gene-related peptide (CGRP) receptor antagonists; interleukin receptor antagonists; PLA2 isoform inhibitors and PLCs. γInhibitors of enzymes active in the arachidonic acid metabolite synthesis pathway, including phospholipase inhibitors containing isoform inhibitors, cyclooxygenase (COX) inhibitors (which may be COX-1 inhibitors, COX-2 inhibitors, or non-selective COX-1 and -2 inhibitors), and lipooxygenase 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; P 2X Receptor antagonists and P 2Y This includes receptor agonists, purine receptor agonists and antagonists; adenosine triphosphate (ATP)-sensitive potassium channel openers; MAP kinase inhibitors; nicotinic acetylcholine inhibitors; and α-adrenergic receptor agonists (including α-1, α-2, and non-selective α-1 and α-2 agonists).

[0276] The MASP-2 inhibitor of the present invention may also be administered in combination with one or more other complement inhibitors, for example, a C5 inhibitor. To date, eculizumab (Solaris®), an antibody against C5, is the only complement-targeted drug approved for use in humans. However, some pharmacological agents have been shown to block complement in vivo. K76COOH and naphamstat mesylate are two agents that have shown considerable efficacy in animal transplantation models (Miyagawa, S., et al., Transplant Proc. 24:483-484, 1992). Low molecular weight heparin has also been shown to be effective in regulating complement activity (Edens, RE, et al., Complement Today, pp. 96-120, Basel:Karger, 1993). These low molecular weight inhibitors are thought to be potentially useful as agents for use in combination with the MASP-2 inhibitor of the present invention.

[0277] Other natural complement inhibitors may be useful in combination with the MASP-2 inhibitor of the present invention. Biological complement inhibitors include soluble complement factor 1 (sCR1), a natural inhibitor found in the outer membrane of human cells. Other membrane inhibitors include DAF, MCP, and CD59. Recombinant anti-complement activity has been tested in vitro and in vivo. sCR1 has been shown to be effective in xenotransplantation where the complement system (secondary and classical) triggers hyperactive rejection syndrome within minutes of perfusing blood into the newly transplanted organ (Piatt, 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). The use of sCR1 protects transplanted organs and extends their survival. This suggests that the complement pathway is involved in the development of organ survival (Leventhal, JR, et al., Transplantation 55:857-66, 1993; Pruitt, SK, et al., Transplantation 57:363-70, 1994).

[0278] Further complement inhibitors suitable for use in combination with the compositions of the present invention include, for example, MoAbs, anti-C5 antibodies (e.g., eculizumab) developed by Alexion Pharmaceuticals, Inc., New Haven, and Connecticut, and anti-properdin MoAbs.

[0279] Pharmaceutical carriers and delivery vehicles Generally, the MASP-2 inhibitor compositions of the present invention, combined with any other selected therapeutic substance, are contained in a pharmaceutically acceptable carrier as appropriate. The carrier is selected to be non-toxic, biocompatible, and not adversely affect the biological activity of the MASP-2 inhibitor (and any other therapeutic substance combined therewith). An example of a pharmaceutically acceptable carrier for peptides is described in U.S. Patent No. 5,211,657 to Yamada. The anti-MASP-2 antibodies and inhibitory peptides useful in the present invention may be formulated in the form of solid, semi-solid, gel, liquid, or gas preparations, such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants, and injections, to enable oral, parenteral, or surgical administration. The present invention also intends for topical administration of the compositions by coating medical devices, etc.

[0280] Suitable carriers for parenteral delivery via injection, infusion, or irrigation, and local delivery include distilled water, physiological phosphate-buffered saline, ordinary Ringer's solution or lactated Ringer's solution, dextrose solution, Hanks' solution, or propanediol. Furthermore, sterile non-volatile oils may be used as solvents or dispersions. For this purpose, any biocompatible oil, including synthetic monoglycerides or diglycerides, can be used. Additionally, fatty acids such as oleic acid are useful in the preparation of injection solutions. The carrier and active substance may be formulated as a liquid, suspension, polymerizable or nonpolymerizable gel, paste, or ointment.

[0281] The carrier may also include a delivery vehicle to sustain (i.e., prolong, delay, or regulate) the delivery of the active substance, or to enhance the delivery, uptake, stability, or pharmacokinetics of a therapeutic substance. Such delivery vehicles may include, in non-limiting examples, microparticles, microspheres, nanospheres, or nanoparticles consisting of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymers or copolymers, hydrogels, and polymer micelles. Suitable hydrogel and micelle delivery systems include the PEO:PHB:PEO copolymer and copolymer / cyclodextrin complex disclosed in WO2004 / 009664A2, and the PEO and PEO / cyclodextrin complex disclosed in U.S. Patent Application Publication 2002 / 0019369A1. Such hydrogels may be injected topically to the site of action of interest, or subcutaneously or intramuscularly to form a sustained-release depot.

[0282] For intra-articular delivery, the MASP-2 inhibitor may be delivered in an injectable liquid or gel carrier, an injectable sustained-release vehicle, or hyaluronic acid or a hyaluronic acid derivative.

[0283] For oral administration of non-peptide-acting substances, MASP-2 inhibitors may be transported in an inert bulking agent or diluent such as sucrose, corn starch, or cellulose.

[0284] For local administration, MASP-2 inhibitors may be delivered in ointments, lotions, creams, gels, eye drops, suppositories, sprays, liquids, or powders, or delivered via transdermal patches in gels or microcapsule delivery systems.

[0285] Various nasal and pulmonary delivery systems, including aerosols, metered-dose inhalers, dry powder inhalers, and nebulizers, are under development and can be appropriately adapted for delivery according to the present invention by being incorporated into aerosols, inhalants, or spray delivery vehicles, respectively.

[0286] For intraarachnoid (IT) or intraventricular (ICV) delivery, the composition of the present invention can be administered using a properly sterile delivery system (e.g., liquid; gel, suspension, etc.).

[0287] The compositions of the present invention may also contain biocompatible excipients, such as dispersants or wetting agents, suspending agents, diluents, buffers, osmosis enhancers, emulsifiers, binders, thickeners, and flavorings (in the case of oral administration).

[0288] Pharmaceutical carriers for antibodies and peptides More specifically with respect to anti-MASP-2 antibodies and inhibitory peptides, exemplary formulations can be administered parenterally as injectable doses of the compounds dissolved in a physiologically acceptable diluent, along with a pharmaceutical carrier, which may be a sterile liquid such as water, oil, saline, glycerol, or ethanol. Furthermore, the composition containing the anti-MASP-2 antibody and inhibitory peptide may also contain auxiliary substances, such as wetting ag...

Claims

1. A method for treating a human subject suffering from idiopathic pneumonia syndrome after hematopoietic stem cell transplantation (HSCT-IPS), comprising the step of administering to the subject a composition containing a MASP-2 inhibitory antibody or an antigen-binding fragment thereof in an amount effective in inhibiting MASP-2-dependent complement activation.

2. The method according to claim 1, wherein the MASP-2 inhibitory antibody is a monoclonal antibody or a fragment thereof that specifically binds to human MASP-2.

3. The method according to claim 1, wherein the antibody or fragment thereof is selected from the group consisting of recombinant antibodies, antibodies having reduced effector function, chimeric antibodies, humanized antibodies, and human antibodies.

4. The method according to claim 1, wherein the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway.

5. MASP-2 inhibitory antibodies reduce C3b deposition in 90% human serum to 30 nM or less IC50. 50 The method according to claim 1, which inhibits by

6. The method according to claim 1, wherein a MASP-2 inhibitory antibody is delivered systemically to the target.

7. The method according to claim 1, further comprising the step of identifying a human subject suffering from idiopathic pneumonia syndrome after hematopoietic stem cell transplantation (HSCT-IPS) before administering to the subject a composition containing an amount effective in inhibiting MASP-2-dependent complement activation of a MASP-2 inhibitory antibody or its antigen-binding fragment.

8. The method according to claim 1, wherein the subject has previously undergone allogeneic hematopoietic stem cell transplantation.

9. The method according to claim 1, wherein the subject has previously undergone autologous hematopoietic stem cell transplantation.

10. The method according to claim 1, wherein the subject does not have diffuse alveolar hemorrhage (DAH).

11. The method according to claim 1, wherein the MASP-2 inhibitory antibody or its antigen-binding fragment comprises a heavy chain variable region containing CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO: 67, and a light chain variable region containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:

70.

12. The method according to claim 1, wherein the MASP-2 inhibitory antibody or its antigen-binding fragment comprises a heavy chain variable region containing SEQ ID NO: 67 and a light chain variable region containing SEQ ID NO:

70.

13. The method according to any one of claims 1 to 12, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody having a dose of approximately 4 mg / kg at least once a week for a treatment period of at least 4 weeks.

14. The method according to claim 13, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody having a dose of approximately 4 mg / kg at least twice a week for a treatment period of at least 4 weeks.

15. The method according to any one of claims 1 to 12, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody in a dose of approximately 370 mg at least once a week for a treatment period of at least 4 weeks.

16. The method according to claim 15, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody in a dose of approximately 370 mg at least twice a week for a treatment period of at least 4 weeks.

17. A method for treating a human subject suffering from hematopoietic stem cell transplantation-induced capillary leak syndrome (HSCT-CLS), comprising the step of administering to the subject a composition containing a MASP-2 inhibitory antibody or an antigen-binding fragment thereof in an amount effective in inhibiting MASP-2-dependent complement activation.

18. The method according to claim 17, wherein the MASP-2 inhibitory antibody is a monoclonal antibody or a fragment thereof that specifically binds to human MASP-2.

19. The method according to claim 17, wherein the antibody or fragment thereof is selected from the group consisting of recombinant antibodies, antibodies having reduced effector function, chimeric antibodies, humanized antibodies, and human antibodies.

20. The method according to claim 17, wherein the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway.

21. MASP-2 inhibitory antibodies reduce C3b deposition in 90% human serum to 30 nM or less IC50. 50 The method according to claim 17, which inhibits by

22. The method according to claim 17, wherein a MASP-2 inhibitory antibody is delivered systemically to the target.

23. The method according to claim 17, further comprising the step of identifying a human subject suffering from hematopoietic stem cell transplantation-induced capillary leak syndrome (HSCT-CLS) before administering to the subject a composition comprising an amount effective in inhibiting MASP-2-dependent complement activation of a MASP-2 inhibitory antibody or its antigen-binding fragment.

24. The method according to claim 17, wherein the subject has previously undergone allogeneic hematopoietic stem cell transplantation.

25. The method according to claim 17, wherein the subject has previously undergone autologous hematopoietic stem cell transplantation.

26. The method according to claim 17, wherein the subject does not have hepatic veno-occlusive disease (VOD).

27. The method according to claim 17, wherein the MASP-2 inhibitory antibody or its antigen-binding fragment comprises a heavy chain variable region containing CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO: 67, and a light chain variable region containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:

70.

28. The method according to claim 17, wherein the MASP-2 inhibitory antibody or its antigen-binding fragment comprises a heavy chain variable region containing SEQ ID NO: 67 and a light chain variable region containing SEQ ID NO:

70.

29. The method according to any one of claims 17 to 28, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody having a dose of approximately 4 mg / kg at least once a week for a treatment period of at least 4 weeks.

30. The method according to claim 29, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody having a dose of approximately 4 mg / kg at least twice a week for a treatment period of at least 4 weeks.

31. The method according to any one of claims 17 to 28, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody in a dose of approximately 370 mg at least once a week for a treatment period of at least 4 weeks.

32. The method according to claim 31, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody in a dose of approximately 370 mg at least twice a week for a treatment period of at least 4 weeks.

33. A method for treating a human subject suffering from hematopoietic stem cell transplant-induced fluid overload (HSCT-FO), comprising the step of administering to the subject a composition containing a MASP-2 inhibitory antibody or an antigen-binding fragment thereof in an amount effective in inhibiting MASP-2-dependent complement activation.

34. The method according to claim 33, wherein the MASP-2 inhibitory antibody is a monoclonal antibody or a fragment thereof that specifically binds to human MASP-2.

35. The method according to claim 33, wherein the antibody or fragment thereof is selected from the group consisting of recombinant antibodies, antibodies having reduced effector function, chimeric antibodies, humanized antibodies, and human antibodies.

36. The method according to claim 33, wherein the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway.

37. MASP-2 inhibitory antibodies reduce C3b deposition in 90% human serum to 30 nM or less IC50. 50 The method according to claim 33, which inhibits by

38. The method according to claim 33, wherein a MASP-2 inhibitory antibody is delivered systemically to the target.

39. The method according to claim 33, further comprising the step of identifying a human subject suffering from hematopoietic stem cell transplant-induced fluid overload (HSCT-FO) before administering to the subject a composition containing an amount effective in inhibiting MASP-2-dependent complement activation, such as a MASP-2 inhibitory antibody or its antigen-binding fragment.

40. The method according to claim 33, wherein the subject has previously undergone allogeneic hematopoietic stem cell transplantation.

41. The method according to claim 33, wherein the subject has previously undergone autologous hematopoietic stem cell transplantation.

42. The method according to claim 33, wherein the MASP-2 inhibitory antibody or its antigen-binding fragment comprises a heavy chain variable region containing CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO: 67, and a light chain variable region containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:

70.

43. The method according to claim 33, wherein the MASP-2 inhibitory antibody or its antigen-binding fragment comprises a heavy chain variable region containing SEQ ID NO: 67 and a light chain variable region containing SEQ ID NO:

70.

44. The method according to any one of claims 33 to 43, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody having a dose of approximately 4 mg / kg at least once a week for a treatment period of at least 4 weeks.

45. The method according to claim 44, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody having a dose of approximately 4 mg / kg at least twice a week for a treatment period of at least 4 weeks.

46. The method according to any one of claims 33 to 43, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody in a dose of approximately 370 mg at least once a week for a treatment period of at least 4 weeks.

47. The method according to claim 46, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody in a dose of approximately 370 mg at least twice a week for a treatment period of at least 4 weeks.

48. A method for treating a human subject suffering from hematopoietic stem cell transplantation engraftment syndrome (HSCT-ES), comprising the step of administering to the subject a composition containing a MASP-2 inhibitory antibody or an antigen-binding fragment thereof in an amount effective in inhibiting MASP-2-dependent complement activation.

49. The method according to claim 48, wherein the MASP-2 inhibitory antibody is a monoclonal antibody or a fragment thereof that specifically binds to human MASP-2.

50. The method according to claim 48, wherein the antibody or fragment thereof is selected from the group consisting of recombinant antibodies, antibodies having reduced effector function, chimeric antibodies, humanized antibodies, and human antibodies.

51. The method according to claim 48, wherein the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway.

52. MASP-2 inhibitory antibodies reduce C3b deposition in 90% human serum to 30 nM or less IC50. 50 The method according to claim 48, which inhibits by

53. The method according to claim 48, wherein a MASP-2 inhibitory antibody is delivered systemically to the target.

54. The method according to claim 48, further comprising the step of identifying a human subject suffering from hematopoietic stem cell transplantation engraftment syndrome (HSCT-ES) before administering to the subject a composition containing an amount effective in inhibiting MASP-2-dependent complement activation of a MASP-2 inhibitory antibody or its antigen-binding fragment.

55. The method according to claim 48, wherein the subject has previously undergone allogeneic hematopoietic stem cell transplantation.

56. The method according to claim 48, wherein the subject has previously undergone autologous hematopoietic stem cell transplantation.

57. The method according to claim 48, wherein the MASP-2 inhibitory antibody or its antigen-binding fragment comprises a heavy chain variable region containing CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO: 67, and a light chain variable region containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:

70.

58. The method according to claim 48, wherein the MASP-2 inhibitory antibody or its antigen-binding fragment comprises a heavy chain variable region containing SEQ ID NO: 67 and a light chain variable region containing SEQ ID NO:

70.

59. The method according to any one of claims 48 to 58, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody having a dose of approximately 4 mg / kg at least once a week for a treatment period of at least 4 weeks.

60. The method according to claim 59, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody having a dose of approximately 4 mg / kg at least twice a week for a treatment period of at least 4 weeks.

61. The method according to any one of claims 48 to 58, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody in a dose of approximately 370 mg at least once a week for a treatment period of at least 4 weeks.

62. The method according to claim 61, comprising the step of administering to a subject a composition containing the MASP-2 inhibitory antibody in a dose of approximately 370 mg at least twice a week for a treatment period of at least 4 weeks.

Images