Methods for treating conditions associated with MASP-2-dependent complement activation - Patents.com

Abstract

To provide methods of inhibiting the effects of MASP-2-dependent complement activation in a human subject suffering from TMA associated with hematopoietic stem cell transplant.SOLUTION: The methods comprise the step of administering, to a subject in need thereof, an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2-dependent complement activation.SELECTED DRAWING: Figure 59

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of Provisional Application No. 62 / 406,726, filed October 11, 2016, and claims the benefit of Provisional Application No. 62 / 252,814, filed November 9, 2015, both of which are incorporated by reference herein in their entireties.

[0002] Sequence Listing Description The sequence listing associated with this application is provided in text format in lieu of hard copy and is incorporated herein by reference. The name of the text file containing the sequence listing is MP_1_0248_PCT_Sequence_Listing_as_Filed_20161109.txt. This text file is 115 KB, was created on November 8, 2016, and has been submitted via EFS-Web with the application herein. [Background technology]

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

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

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

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

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

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

[0009] Ficolins contain a type of lectin domain called a fibrinogen-like domain, which is different from that of MBL. ++ They bind to sugar residues in a phospho-independent manner. Three ficolins (L-ficolin, M-ficolin, and H-ficolin) have been identified in humans. Two serum ficolins, L-ficolin and H-ficolin, share specificity for N-acetyl-D-glucosamine. However, H-ficolin also binds to N-acetyl-D-galactosamine. The different carbohydrate specificities of L-ficolin, H-ficolin, CL-11, and MBL imply that different lectins may complement each other and, through overlap, target different glycoconjugates. This idea is supported by a recent report that, of the known lectins in the lectin pathway, only L-ficolin specifically binds to lipoteichoic acid, a cell wall glycoconjugate found in all Gram-positive bacteria (Lynch et al., J. Immunol. 172:1198-1202, (2004)). Collectins (i.e., MBL) and ficolins share no significant amino acid sequence similarity. However, these two protein groups have similar domain organization and, like C1q, assemble into oligomeric structures that maximize the potential for multisite binding.

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

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

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

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

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

[0015] The lectin pathway is widely believed to play a major role in host defense against infectious diseases in naive hosts. Strong evidence for the involvement of MBL in host defense came from the analysis of patients with low serum levels of functional MBL (Kilpatrick, Biochim. Biophys. Acta 1572:401-413, (2002)). Such patients are susceptible to recurrent bacterial and fungal infections. These symptoms usually appear in early life, during a period of apparent vulnerability when maternal antibody titers gradually decline but before the full repertoire of antibody responses has developed. This syndrome is often caused by mutations at several sites in the MBL collagen moiety, which prevent the proper formation of MBL oligomers. However, because MBL can function as an opsonin independently of complement, it is unknown 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 alternative pathway have not been found to perform the recognition functions performed by C1q and lectins in the other two steps. It is now widely accepted that the alternative pathway spontaneously undergoes low-level turnover activation. This turnover activation can be easily amplified on foreign or other abnormal surfaces (bacteria, yeast, virus-infected cells, or damaged tissue) that lack the appropriate molecular elements to suppress spontaneous complement activation. There are four plasma proteins directly involved in alternative pathway activation: C3, factors B and D, and properdin.

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

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

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

[0020] Little is known about the initiators of alternative 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 common feature shared by these activators is the presence of carbohydrates, but the complexity and diversity of carbohydrate structures makes it difficult to establish a recognized common molecular determinant. It is widely accepted that alternative pathway activation is controlled by a delicate balance between inhibitory regulatory components of the pathway, such as factor H, factor I, DAF, and CR1, and properdin, the only positive regulator of the alternative pathway (see Schwaeble WJ and Reid KB, Immunol Today 20(1):17-21 (1999)).

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

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

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

[0024] overview This Summary is provided to introduce broad concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0025] In one aspect, the present invention provides a method for inhibiting microvascular endothelial cell injury and / or thrombus formation in a subject suffering from thrombotic microangiopathy (TMA), comprising administering to the subject a composition comprising a MASP-2 inhibitory antibody in an amount effective to inhibit MASP-2-dependent complement activation. In some embodiments, the subject suffers from or is at risk of developing a TMA selected from the group consisting of hemolytic uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP), and atypical hemolytic uremic syndrome (HUS). In some embodiments, before the composition is administered, the subject is confirmed to exhibit one or more symptoms selected from the group consisting of (i) anemia, (ii) thrombocytopenia, (iii) renal failure, and (iv) elevated creatinine, and the composition is administered in an amount and for a sufficient duration effective to ameliorate the one or more symptoms. In some embodiments, the MASP-2 inhibitor is an anti-MASP-2 antibody or a fragment thereof. In some embodiments, the MASP-2 inhibitor is an anti-MASP-2 monoclonal antibody or a fragment thereof that specifically binds to a portion of SEQ ID NO: 6. In some embodiments, the MASP-2 inhibitor inhibits microvascular endothelial cell damage.

[0026] In another aspect, the present invention provides a method for inhibiting MASP-2-dependent complement activation in a subject suffering from or at risk of developing atypical hemolytic uremic syndrome (aHUS), comprising administering to the subject a composition comprising a MASP-2 inhibitor in an amount effective to inhibit MASP-2-dependent complement activation. In one embodiment, before the composition is administered, the subject is confirmed to exhibit one or more symptoms selected from the group consisting of (i) anemia, (ii) thrombocytopenia, (iii) renal failure, and (iv) elevated creatinine, and the composition is administered in an amount and for a time sufficient to improve the one or more symptoms. In one embodiment, the subject suffers from or is at risk of developing non-factor H-dependent aHUS. In one embodiment, the subject suffers from aHUS associated with factor I, factor B, or membrane cofactor CD46. In one embodiment, the MASP-2 inhibitor is an anti-MASP-2 antibody or a fragment thereof, for example, an anti-MASP-2 monoclonal antibody or a fragment thereof that specifically binds to a portion of SEQ ID NO:6. In one embodiment, the MASP-2 inhibitor inhibits microvascular endothelial cell damage. In one embodiment, the MASP-2 inhibitor inhibits thrombus formation.

[0027] In another aspect, the present invention provides a method for reducing the likelihood that a subject at risk of developing atypical hemolytic uremic syndrome (aHUS) will suffer from clinical symptoms associated with aHUS. The method according to this aspect of the present invention includes the steps of: (a) determining the presence of a genetic marker known to be associated with aHUS in the subject; (b) periodically monitoring the subject to determine the presence or absence of at least one symptom selected from the group consisting of anemia, thrombocytopenia, renal failure, and elevated creatinine; and (c) administering to the subject, upon determining the presence of at least one of anemia, thrombocytopenia, renal failure, or elevated creatinine, a composition comprising a MASP-2 inhibitor in an amount effective to inhibit MASP-2-dependent complement activation, the composition being administered in an amount and for a sufficient time to ameliorate the one or more symptoms. In one embodiment, the MASP-2 inhibitor is an anti-MASP-2 antibody or a fragment thereof, e.g., an anti-MASP-2 monoclonal antibody or a fragment thereof that specifically binds to a portion of SEQ ID NO:6. In one embodiment of the method, step (a) comprises conducting a genetic screening test on a sample obtained from the subject and identifying the presence of at least one genetic marker associated with aHUS in a gene selected from the group consisting of complement factor H (CFH), factor I (CFI), factor B (CFB), membrane cofactors CD46 and C3, complement factor H-related protein (CFHR1), anticoagulant protein thrombodulin (THBD), complement factor H-related protein 3 (CFHR3), and complement factor H-related protein 4 (CFHR4). In one embodiment, the method further comprises monitoring the subject for the occurrence of an event known to be associated with the induction of aHUS clinical symptoms, and administering to the subject a composition comprising a MASP-2 inhibitor before, during, or after the occurrence of the inducing event. In one embodiment, the event associated with the induction of aHUS clinical symptoms is selected from the group consisting of drug exposure, infection, malignancy, injury, organ or tissue transplantation, and pregnancy. In one embodiment, the infection is a bacterial infection. In one embodiment, the composition is administered subcutaneously.In one embodiment, the MASP-2 inhibitor inhibits microvascular endothelial cell injury. In one embodiment, the MASP-2 inhibitor inhibits thrombus formation.

[0028] In another aspect, the present invention provides a method for inhibiting MASP-2-dependent complement activation in a subject suffering from or at risk of developing atypical hemolytic uremic syndrome (aHUS) secondary to infection, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitor effective to inhibit MASP-2 complement activation. In one embodiment, the subject suffers from or is at risk of developing non-enteric aHUS associated with Streptococcus pneumoniae (S. pneumoniae) infection. In one embodiment, the MASP-2 inhibitor is an anti-MASP-2 antibody or a fragment thereof, for example, an anti-MASP-2 monoclonal antibody or a fragment thereof that specifically binds to a portion of SEQ ID NO:6. In one embodiment, the MASP-2 inhibitor inhibits microvascular endothelial cell damage. In one embodiment, the MASP-2 inhibitor inhibits thrombus formation.

[0029] In another aspect, the present invention provides a method for treating a subject suffering from atypical hemolytic uremic syndrome (aHUS), comprising administering to the subject a composition comprising a MASP-2 inhibitor in an amount effective to inhibit MASP-2-dependent complement activation, wherein the MASP-2 inhibitor is administered via an intravenous catheter or other catheter delivery method. In one embodiment, the method further comprises treating the patient by plasmapheresis. In one embodiment, the composition comprising a MASP-2 inhibitor is administered in the absence of plasmapheresis. In one embodiment, the composition comprising a MASP-2 inhibitor is administered via a catheter for a first period of time, and further comprises administering a composition comprising a MASP-2 inhibitor for a second period of time, wherein the composition is administered subcutaneously during the second period of time. In one embodiment, the method further comprises periodically determining the level of at least one complement factor, wherein determining that the level of at least one complement factor is lower than a standard value or a healthy subject indicates the need for continued treatment with the composition. In one embodiment, the MASP-2 inhibitor is an anti-MASP-2 antibody or a fragment thereof, for example, an anti-MASP-2 monoclonal antibody or a fragment thereof that specifically binds to a portion of SEQ ID NO:6. In one embodiment, the MASP-2 inhibitor inhibits microvascular endothelial cell damage. In one embodiment, the MASP-2 inhibitor inhibits thrombus formation.

[0030] In another aspect, the present invention provides a method for treating a subject suffering from thrombotic thrombocytopenic purpura (TTP) or a subject exhibiting symptoms consistent with a diagnosis of TTP, comprising administering to the subject a composition comprising a MASP-2 inhibitor in an amount effective to inhibit MASP-2-dependent complement activation, wherein the MASP-2 inhibitor is administered to the subject via an intravenous catheter or other catheter delivery method. In one embodiment, the subject exhibits at least one or more symptoms selected from the group consisting of central nervous system complications, thrombocytopenia, serious cardiac complications, serious pulmonary complications, gastrointestinal infarction, and gangrene. In one embodiment, the subject tests positive for the presence of an ADAMTS13 inhibitor, and the method further comprises administering an immunosuppressant to the subject. In one embodiment, the composition comprising a MASP-2 inhibitor is administered for a first period of time in the absence of plasmapheresis. In one embodiment, the subject tests positive for the presence of an ADAMTS-13 inhibitor, and the method further comprises administering ADAMTS-13. In one embodiment, the method further comprises treating the patient with plasmapheresis. In one embodiment, the composition comprising a MASP-2 inhibitor is administered in the presence of plasmapheresis. In one embodiment, the composition comprising a MASP-2 inhibitor is administered via a catheter for a first period of time and further comprises administering a composition comprising a MASP-2 inhibitor for a second period of time, wherein the composition is administered subcutaneously during the second period of time. In one embodiment, the method further comprises periodically determining the level of at least one complement factor, wherein determining that the level of at least one complement factor is lower compared to a standard value or a healthy subject indicates the need for continued treatment with the composition. In one embodiment, the MASP-2 inhibitor is an anti-MASP-2 antibody or fragment thereof, for example, an anti-MASP-2 monoclonal antibody or fragment thereof that specifically binds to a portion of SEQ ID NO:6. In one embodiment, the MASP-2 inhibitor inhibits microvascular endothelial cell injury. In one embodiment, the MASP-2 inhibitor inhibits thrombus formation.

[0031] In another aspect, the present invention provides a method for treating a subject suffering from treatment-resistant thrombotic thrombocytopenic purpura (TTP), comprising administering to the subject a composition comprising an effective amount of a MASP-2 inhibitor to inhibit MASP-2-dependent complement activation. In one embodiment, the composition is administered subcutaneously. In one embodiment, the method further comprises periodically determining the level of at least one complement factor, wherein determining that the level of at least one complement factor is lower than the standard value or a healthy subject indicates the need for continued treatment with the composition. In one embodiment, the MASP-2 inhibitor is an anti-MASP-2 antibody or a fragment thereof, for example, an anti-MASP-2 monoclonal antibody or a fragment thereof that specifically binds to a portion of SEQ ID NO:6. In one embodiment, the MASP-2 inhibitor inhibits microvascular endothelial cell damage. In one embodiment, the MASP-2 inhibitor inhibits thrombus formation.

[0032] In another aspect, the present invention provides a method for inhibiting MASP-2-dependent complement activation in a subject suffering from or at risk of developing thrombotic microangiopathy (TMA), comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation, wherein the TMA is at least one of (i) TMA secondary to cancer; (ii) TMA secondary to chemotherapy, or (iii) TMA secondary to transplantation. In some embodiments, the subject suffers from or is at risk of developing TMA secondary to cancer, and the MASP-2 inhibitor is systemically administered to the subject in an amount effective to reduce the risk of developing TMA or reduce the severity of TMA. In some embodiments, the subject suffers from or is at risk of developing TMA secondary to chemotherapy, and the MASP-2 inhibitor is administered systemically to the subject before, during, or after chemotherapy in an amount effective to reduce the risk of developing TMA or reduce the severity of TMA. In some embodiments, the subject suffers from or is at risk of developing TMA secondary to transplantation, and the MASP-2 inhibitor is administered systemically to the subject before, during, or after transplantation in an amount effective to reduce the risk of developing TMA or reduce the severity of TMA. In some embodiments, the transplantation is allogeneic hematopoietic stem cell transplantation. In some embodiments, the subject has previously received or is currently receiving treatment with a terminal complement inhibitor that inhibits the cleavage of complement protein C5. In some embodiments, the method further comprises administering to the subject a terminal complement inhibitor that inhibits the cleavage of complement protein C5, such as a humanized anti-C5 antibody or its antigen-binding fragment, such as eculizumab.

[0033] In another aspect, the present invention provides a method for inhibiting MASP-2-dependent complement activation in a subject suffering from or at risk of developing Upshaw-Schulman syndrome (USS), comprising administering to the subject a composition comprising an effective amount of a MASP-2 inhibitor to inhibit MASP-2-dependent complement activation. In some embodiments, the method comprises treating a subject at risk of developing USS, administering an amount of a MASP-2 inhibitor effective to ameliorate or prevent one or more clinical symptoms associated with TTP for a period of time effective to ameliorate or prevent the clinical symptoms. In some embodiments, the method further comprises periodically monitoring the subject and administering a MASP-2 inhibitor based on the presence of an event known to be associated with the induction of TTP clinical symptoms. In some embodiments, the method further comprises periodically monitoring the subject and administering a MASP-2 inhibitor based on the determination of the presence of anemia, thrombocytopenia, or increased creatinine. In some embodiments, the subject has previously received or is currently receiving treatment with a terminal complement inhibitor that inhibits cleavage of complement protein C5. In some embodiments, the method further comprises administering to the subject a terminal complement inhibitor that inhibits cleavage of complement protein C5, such as a humanized anti-C5 antibody or antigen-binding fragment thereof, such as eculizumab.

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

[0035] In another aspect, the present invention provides a method for inhibiting MASP-2-dependent complement activation in a subject suffering from fulminant antiphospholipid syndrome (CAPS), comprising administering to the subject a composition comprising an effective amount of a MASP-2 inhibitor to inhibit MASP-2-dependent complement activation. In some embodiments, the subject has previously received or is currently receiving treatment with a terminal complement inhibitor that inhibits the cleavage of complement protein C5. In some embodiments, the method further comprises administering to the subject a terminal complement inhibitor that inhibits the cleavage of complement protein C5, such as a humanized anti-C5 antibody or its antigen-binding fragment, for example, eculizumab.

[0036] In some embodiments of any of the disclosed methods of the present invention, the MASP-2 inhibitor is a MASP-2 inhibitory antibody or a fragment thereof. In some embodiments, the MASP-2 inhibitory antibody has reduced effector function. In some embodiments, the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. In some embodiments, the MASP-2 inhibitor is an anti-MASP-2 monoclonal antibody or a fragment thereof that specifically binds to a portion of SEQ ID NO:6. In some embodiments, the anti-MASP-2 antibody or a fragment thereof is selected from the group consisting of a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In some embodiments, the MASP-2 inhibitory antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2. In some embodiments, the MASP-2 inhibitory antibody is a single-chain molecule. In some embodiments, the MASP-2 inhibitory antibody is selected from the group consisting of an IgG1 molecule, an IgG2 molecule, and an IgG4 molecule. In some embodiments, the MASP-2 inhibitory antibody is an IgG4 molecule containing an S228P mutation. In some embodiments, the MASP-2 inhibitory antibody has a K DIn some embodiments, the MASP-2 inhibitory antibody binds an epitope in the CCP1 domain of MASP-2. In some embodiments, the MASP-2 inhibitory antibody inhibits C3b deposition in 1% human serum in an in vitro assay with an IC of 10 nM or less. 50 In some embodiments, the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum with an IC of 30 nM or less. 50 and inhibits.

[0037] In some embodiments of any of the disclosed methods of the present invention, the MASP-2 inhibitory monoclonal antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising: (i) a heavy chain CDR-H1 comprising the amino acid sequence of 31 to 35 of SEQ ID NO:67; and (ii) a heavy chain CDR-H2 comprising the amino acid sequence of 50 to 65 of SEQ ID NO:67; and (iii) a heavy chain CDR-H3 comprising the amino acid sequence of 95 to 102 of SEQ ID NO:67; and (b) a light chain variable region comprising: (i) a light chain CDR-L1 comprising the amino acid sequence of 24 to 34 of SEQ ID NO:70; and (ii) a light chain CDR-L2 comprising the amino acid sequence of 50 to 56 of SEQ ID NO:70; and (iii) a light chain CDR-L3 comprising the amino acid sequence of 89 to 97 of SEQ ID NO:70. In some embodiments, the MASP-2 inhibitory monoclonal antibody comprises a heavy chain variable region set forth in SEQ ID NO:67 and a light chain variable region set forth in SEQ ID NO:70. In some embodiments, the MASP-2 inhibitory antibody or its antigen-binding fragment specifically recognizes at least a portion of the epitope recognized by a reference antibody comprising a heavy chain variable region set forth in SEQ ID NO:67 and a light chain variable region set forth in SEQ ID NO:70.

[0038] In another aspect of the present invention, a method for inhibiting thrombus formation in a subject suffering from atypical hemolytic uremic syndrome (aHUS) is provided, comprising administering to the subject a MASP-2 inhibitory antibody or its antigen-binding fragment in an amount effective to inhibit MASP-2-dependent complement activation. In some embodiments, the MASP-2 inhibitory antibody inhibits thrombus formation in serum from a subject suffering from aHUS by at least 40% compared to untreated serum. In some embodiments, the MASP-2 inhibitory antibody inhibits thrombus formation in serum from a subject suffering from aHUS at a level at least 20% higher (e.g., at least 30% higher, at least 40% higher, or at least 50% higher) than the inhibitory effect of the antibody on C5b-9 deposition in serum from the same subject. In some embodiments, the subject is in the acute phase of aHUS. In some embodiments, the subject is in remission from aHUS. In some embodiments, the MASP-2 inhibitory antibody is a monoclonal antibody or fragment thereof that specifically binds to a portion of SEQ ID NO:6. In some embodiments, the MASP-2 inhibitory antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In some embodiments, the MASP-2 inhibitory antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2. In some embodiments, the MASP-2 inhibitory antibody is a single-chain molecule. In some embodiments, the MASP-2 inhibitory antibody is selected from the group consisting of an IgG1 molecule, an IgG2 molecule, and an IgG4 molecule. In some embodiments, the MASP-2 inhibitory antibody is an IgG4 molecule containing an S228P mutation. In some embodiments, the MASP-2 inhibitory antibody has a K of 10 nM or less. D In some embodiments, the MASP-2 inhibitory antibody binds an epitope in the CCP1 domain of MASP-2. In some embodiments, the MASP-2 inhibitory antibody inhibits C3b deposition in 1% human serum in an in vitro assay with an IC of 10 nM or less. 50 In some embodiments, the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum with an IC of 30 nM or less.50 In some embodiments, the MASP-2 inhibitory monoclonal antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising: (i) a heavy chain CDR-H1 comprising the amino acid sequence of 31 to 35 of SEQ ID NO:67; and (ii) a heavy chain CDR-H2 comprising the amino acid sequence of 50 to 65 of SEQ ID NO:67; and (iii) a heavy chain CDR-H3 comprising the amino acid sequence of 95 to 102 of SEQ ID NO:67; and (b) a light chain variable region comprising: (i) a light chain CDR-L1 comprising the amino acid sequence of 24 to 34 of SEQ ID NO:70; and (ii) a light chain CDR-L2 comprising the amino acid sequence of 50 to 56 of SEQ ID NO:70; and (iii) a light chain CDR-L3 comprising the amino acid sequence of 89 to 97 of SEQ ID NO:70. In some embodiments, the MASP-2 inhibitory monoclonal antibody comprises a heavy chain variable region set forth in SEQ ID NO:67 and a light chain variable region set forth in SEQ ID NO:70. In some embodiments, the MASP-2 inhibitory antibody or its antigen-binding fragment specifically recognizes at least a portion of the epitope recognized by a reference antibody comprising a heavy chain variable region set forth in SEQ ID NO:67 and a light chain variable region set forth in SEQ ID NO:70.

[0039] In another aspect, the present invention provides a method for treating a subject suffering from plasma therapy-resistant aHUS, comprising administering to the subject a composition comprising an effective amount of a MASP-2 inhibitory antibody to inhibit MASP-2-dependent complement activation. In one embodiment, the MASP-2 inhibitor is an anti-MASP-2 monoclonal antibody or a fragment thereof that specifically binds to human MASP-2. In one embodiment, the antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In one embodiment, the subject has previously received or is currently receiving treatment with a terminal complement inhibitor that inhibits cleavage of complement protein C5, for example, the terminal complement inhibitor is a humanized anti-C5 antibody or an antigen-binding fragment thereof. In one embodiment, the method further comprises treating the patient with plasmapheresis. In one embodiment, the composition comprising the MASP-2 inhibitory antibody is administered in the absence of plasmapheresis.

[0040] In another aspect, the present invention provides a method for treating a subject suffering from hematopoietic stem cell transplantation-associated TMA, comprising administering to the subject a composition comprising an effective amount of a MASP-2 inhibitory antibody to inhibit MASP-2-dependent complement activation. In one embodiment, the MASP-2 inhibitor is an anti-MASP-2 monoclonal antibody or a fragment thereof that specifically binds to human MASP-2. In one embodiment, the antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In one embodiment, the subject has previously received or is currently receiving treatment with a terminal complement inhibitor that inhibits the cleavage of complement protein C5. In one embodiment, the subject is suffering from hematopoietic stem cell transplantation-associated TMA that is resistant to treatment with platelet transfusions and / or resistant to treatment with plasmapheresis. In one aspect, the MASP-2 inhibitory antibody is administered in an amount effective to improve at least one or more clinical parameters associated with TMA associated with hematopoietic stem cell transplantation, e.g., to increase platelet count (e.g., at least 2-fold, at least 3-fold, at least 4-fold the platelet count before treatment), increase haptoglobin, and / or decrease lactate dehydrogenase.

[0041] In another aspect, the present invention provides a method for treating a human subject suffering from persistent TMA associated with hematopoietic stem cell transplantation (HSCT-TMA), comprising administering to the subject a composition comprising a MASP-2 inhibitory antibody or its antigen-binding fragment in an amount effective to inhibit MASP-2-dependent complement activation. In one embodiment, the method further comprises identifying a human subject with persistent TMA associated with hematopoietic stem cell transplantation prior to administering to the subject a composition comprising a MASP-2 inhibitory antibody or its antigen-binding fragment in an amount effective to inhibit MASP-2-dependent complement activation. In one embodiment, the MASP-2 inhibitory antibody is a monoclonal antibody or fragment thereof that specifically binds to human MASP-2. In one embodiment, the antibody or antigen-binding fragment thereof is selected from the group consisting of a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In one embodiment, the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. In one embodiment, the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum with an IC of 30 nM or less. 50In one embodiment, the MASP-2 inhibitory antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO:67, and a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:70. In one embodiment, the MASP-2 inhibitory antibody is administered to the patient in the absence of plasmapheresis. In one embodiment, the subject has previously received or is currently receiving treatment with a humanized anti-C5 antibody or its antigen-binding fragment, for example, the terminal complement inhibitor is a humanized anti-C5 antibody or its antigen-binding fragment. In one embodiment, the MASP-2 inhibitory antibody is delivered systemically to the subject. In one aspect, the MASP-2 inhibitory antibody or its antigen-binding fragment is administered in an amount effective to improve at least one or more of the following clinical parameters associated with persistent TMA associated with hematopoietic stem cell transplantation: (i) increasing platelet count (e.g., at least 2-fold, at least 3-fold, at least 4-fold the pre-treatment platelet count); (ii) increasing haptoglobin; (iii) decreasing lactate dehydrogenase (LDH); and / or (iv) decreasing creatinine.

[0042] In another aspect, the present invention provides a composition for inhibiting the side effects of MASP-2-dependent complement activation, comprising a therapeutically effective amount of a MASP-2 inhibitor, such as a MASP-2 inhibitory antibody, and a pharmaceutically acceptable carrier. Also provided is a method for producing a medicament for use in inhibiting the side effects of MASP-2-dependent complement activation in a living subject in need thereof, the medicament comprising a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier. Also provided is a method for producing a medicament for use in inhibiting MASP-2-dependent complement activation for the treatment of each of the conditions, diseases, and disorders described herein below.

[0043] The methods, compositions, and medicaments of the present invention are useful for inhibiting the side effects of MASP-2-dependent complement activation in vivo in mammalian subjects, including humans suffering from or at risk of developing thrombotic microangiopathy (TMA), as further described herein. [The present invention 1001] A method for treating a human subject suffering from persistent TMA associated with hematopoietic stem cell transplantation (HSCT-TMA), comprising administering to the subject a composition comprising a MASP-2 inhibitory antibody or its antigen-binding fragment in an amount effective to inhibit MASP-2-dependent complement activation. [The present 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. [The present invention 1003] 1001. The method of claim 1001, wherein said antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. [The present invention 1004] The method of the present invention 1001, wherein the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. [The present invention 1005] The MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum with an IC of 30 nM or less. 50 The method of the present invention 1001, wherein the inhibitor is inhibited by [The present invention 1006] The method of the present invention 1001, wherein the MASP-2 inhibitory antibody is delivered systemically to the subject. [The present invention 1007] The method of the present invention 1001 further comprises a step of identifying a human subject having persistent TMA associated with hematopoietic stem cell transplantation prior to the step of administering to the subject a composition comprising a MASP-2 inhibitory antibody or its antigen-binding fragment in an amount effective to inhibit MASP-2-dependent complement activation. [The present invention 1008] 1001. The method of claim 1001, wherein said subject has previously received or is currently receiving treatment with a humanized anti-C5 antibody or antigen-binding fragment thereof. [The present invention 1009] The method of the present invention 1001, wherein the MASP-2 inhibitory antibody or antigen-binding fragment thereof is administered in an amount effective to improve at least one or more of the following clinical parameters associated with persistent TMA associated with hematopoietic stem cell transplantation: (i) increasing platelet count (e.g., at least two-fold, at least three-fold, or at least four-fold increase in pre-treatment platelet count); (ii) increase haptoglobin; (iii) decreasing lactate dehydrogenase (LDH); and / or (iv) Reduces creatinine. [The present invention 1010] A method of the present invention 1001, wherein the MASP-2 inhibitory antibody or its antigen-binding fragment comprises a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO:67, and a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO:70. [Brief explanation of the drawings]

[0044] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description when taken in conjunction with the accompanying drawings, wherein: [Figure 1] A diagram showing the genomic structure of human MASP-2. [Figure 2] Figure 2A is a schematic diagram showing the domain structure of human MASP-2 protein, and Figure 2B is a schematic diagram showing the domain structure of human MAp19 protein. [Figure 3] FIG. 1 shows the mouse MASP-2 knockout strategy. [Figure 4] FIG. 1 shows the human MASP-2 minigene construct. [Figure 5A]As described in Example 2, results are presented demonstrating that MASP-2 deficiency leads to a loss of C4 activation mediated by the lectin pathway, as measured by the lack of C4b deposition on mannan. [Figure 5B] As described in Example 2, results are presented demonstrating that MASP-2 deficiency leads to a loss of C4 activation mediated by the lectin pathway, as measured by the lack of C4b deposition on zymosan. [Figure 5C] The results shown demonstrate the relative C4 activation levels of serum samples obtained from MASP-2+ / -; MASP-2- / - and wild-type strains as measured by C4b deposition on mannan and zymosan, as described in Example 2. [Figure 6] As described in Example 2, the results show that addition of mouse recombinant MASP-2 to MASP-2- / - serum samples restores lectin pathway-mediated C4 activation in a protein concentration-dependent manner, as measured by C4b deposition on mannan. [Figure 7] As described in Example 8, the results demonstrating that the classical pathway is functional in MASP-2- / - lines are shown. [Figure 8A] As described in Example 10, the results demonstrating that anti-MASP-2 Fab2 antibody #11 inhibits C3 convertase formation are shown. [Figure 8B] As described in Example 10, the results demonstrating that anti-MASP-2 Fab2 antibody #11 binds to native rat MASP-2 are shown. [Figure 8C] As described in Example 10, the results demonstrating that anti-MASP-2 Fab2 antibody #41 inhibits C4 cleavage are shown. [Figure 9] As described in Example 10, results are shown demonstrating that all of the anti-MASP-2 Fab2 antibodies tested that inhibited C3 convertase formation were also found to inhibit C4 cleavage. [Figure 10]This figure shows the recombinant polypeptide derived from rat MASP-2 used for epitope mapping of 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 polypeptide are shown. [Figure 12] As described in Example 12, the results show that blood urea nitrogen clearance in wild-type (+ / +) mice and MASP-2(- / -) mice was measured 24 and 48 hours after reperfusion in a renal ischemia / reperfusion injury model. [Figure 13A] 10 shows results showing baseline VEGF protein levels in the RPE-choroid complex isolated from wild-type (+ / +) and MASP-2 (- / -) mice, as described in Example 13. [Figure 13B] As described in Example 13, these results show VEGF protein levels in the RPE-choroid complex in wild-type (+ / +) and MASP-2 (- / -) mice 3 days after laser-induced damage in a macular degeneration model. [Figure 14] As described in Example 13, the results showing the mean choroidal neovascularization (CNV) volume in wild-type (+ / +) and MASP-2 (- / -) mice 7 days after laser-induced injury are shown. [Figure 15] Figures 15A and 15B show dose-response curves for inhibition of C4b deposition (Figure 15A) and inhibition of thrombin activation (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 measurements of platelet aggregation (expressed as clump area) in MASP-2(- / -) mice (Figure 16B) compared with platelet aggregation in untreated wild-type mice and wild-type mice in which the complement pathway was inhibited by the depletion substances cobra venom factor (CVF) and terminal pathway inhibitor (C5aR antagonist) in a focal Schwartzman reaction model of disseminated intravascular coagulation, as described in Example 15. [Figure 17] This figure shows blood urea nitrogen (BUN) levels measured in WT(+ / +)(B6) transplant recipient mice or MASP-2(- / -) transplant recipient mice of WT(+ / +) donor kidneys, as described in Example 16. [Figure 18] 10 illustrates the percent survival of WT (+ / +) and MASP-2 (- / -) mice as a function of days after microbial infection in a colonic luminal perforation (CLP) model, as described in Example 17. [Figure 19] 10 illustrates the bacterial counts measured in WT (+ / +) and MASP-2 (- / -) mice after microbial infection in a colonic luminal perforation (CLP) model, as described in Example 17. [Figure 20] Kaplan-Meier plot showing percent survival of WT (+ / +), MASP-2 (- / -), and C3 (- / -) mice 6 days after intranasal challenge with Pseudomonas aeruginosa, as described in Example 18. [Figure 21] This figure shows the C4b deposition levels, measured as a percentage of control, in samples taken at various time points after subcutaneous administration of 0.3 mg / kg or 1.0 mg / kg of mouse anti-MASP-2 monoclonal antibody in WT mice, as described in Example 19. [Figure 22] This figure shows the C4b deposition levels, measured as a percentage of control, in samples taken at various time points after i.p. administration of 0.6 mg / kg of mouse anti-MASP-2 monoclonal antibody in WT mice, as described in Example 19. [Figure 23] This figure illustrates the average choroidal neovascularization (CNV) volume 7 days after laser-induced injury in WT(+ / +) mice pretreated with a single ip injection of 0.3 mg / kg or 1.0 mg / kg mouse anti-MASP-2 monoclonal antibody, as described in Example 20. [Figure 24A]Graph showing the percent survival of MASP-2(- / -) and WT(+ / +) mice after infection with 5 x 108 / 100 μl cfu of N. meningitidis, as described in Example 21. [Figure 24B] This figure shows the log cfu / ml of meningococci recovered at various time points in blood samples taken from MASP-2 KO (- / -) mice and WT (+ / +) mice infected with 5 x 108 cfu / 100 μl of meningococci, as described in Example 21. [Figure 25A] Graph showing the percent survival of MASP-2 KO (- / -) and WT (+ / +) mice after infection with 2 x 108 cfu / 100 μl of meningococcus, as described in Example 21. [Figure 25B] Illustrates the log cfu / ml of meningococci recovered at various time points in blood samples taken from WT(+ / +) mice infected with 2×10 cfu / 100 μl of meningococci, as described in Example 21. [Figure 25C] This figure shows the log cfu / ml of meningococci recovered at various time points in blood samples taken from MASP-2(- / -) mice infected with 2 x 108 cfu / 100 μl of meningococci, as described in Example 21. [Figure 26] Figure 26A illustrates the results of a C3b deposition assay, which demonstrated that MASP-2(- / -) mice retain a functional classical pathway, as described in Example 22. Figure 26B illustrates the results of a C3b deposition assay on zymosan-coated plates, which demonstrated that MASP-2(- / -) mice retain a functional alternative pathway, as described in Example 22. [Figure 27]Figure 27A illustrates myocardial ischemia / reperfusion injury (MIRI)-induced tissue loss after left anterior descending coronary artery (LAD) ligation and reperfusion in C4(- / -) mice (n=6) and matched WT littermate controls (n=7), showing the ischemic area (AAR) and myocardial necrotic area (INF), as described in Example 22. Figure 27B illustrates the myocardial necrotic area (INF) as a function of the ischemic area (AAR) in C4(- / -) and WT mice treated as described in Figure 42A, demonstrating that C4(- / -) mice are as susceptible to MIRI as WT controls (dashed line), as described in Example 22. [Figure 28A] Illustrates the results of a C3b deposition assay using serum from WT mice, serum from C4(- / -) mice, and serum from C4(- / -) mice preincubated with mannan, as described in Example 22. [Figure 28B] The results of a C3b deposition assay for serum from WT mice, serum from C4(- / -) mice, and serum from MASP-2(- / -) mice mixed with various concentrations of anti-mouse MASP-2 mAb (mAbM11), as described in Example 22, are shown. [Figure 28C] 1 illustrates the results of a C3b deposition assay on human serum from WT (C4-sufficient) and C4-deficient subjects, as well as serum from C4-deficient subjects preincubated with mannan, as described in Example 22. [Figure 28D] Illustrates the results of a C3b deposition assay for human serum from WT (C4-sufficient) and C4-deficient subjects mixed with anti-human MASP-2 mAb (mAbH3), as described in Example 22. [Figure 29]Figure 29A illustrates a comparative analysis of C3 convertase activity in plasma from various complement-deficient mouse strains 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 strains tested under lectin activation pathway-specific conditions, as described in Example 22. [Figure 30] 2 shows the results of Western blot analysis demonstrating human C3 activation by thrombin substrates FXIa and FXa, as indicated by the presence of the a' chain, as described in Example 23. [Figure 31] Shown are the results of a C3 deposition assay for serum samples obtained from WT, MASP-2(- / -), F11(- / -), F11(- / -) / C4(- / -), and C4(- / -), as described in Example 23. [Figure 32] Figure 32A is a Kaplan-Meier survival plot showing percent survival over time after exposure to 7.0 Gy 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 percent survival over time after exposure to 6.5 Gy 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] 1 is a Kaplan-Meier plot illustrating the percent survival of MASP-2 KO and WT mice after administration of an infectious dose of 2.6 x 107 cfu of meningococcal serogroup A Z2491, demonstrating that MASP-2-deficient mice are protected from meningococcal-induced death, as described in Example 30. [Figure 34]1 is a Kaplan-Meier plot illustrating the percent survival of MASP-2 KO and WT mice after an infectious dose of 6 x 106 cfu of Neisseria meningitidis serogroup B MC58, demonstrating that MASP-2-deficient mice are protected from Neisseria meningitidis serogroup B MC58-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 i.p. infection with 6 x 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), demonstrating that, 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 MASP-2 KO mice significantly eliminated bacteremia compared to WT mice. [Figure 36] The graph shows the average illness scores of MASP-2 mice and WT mice 3, 6, 12, and 24 hours after infection with 6 x 106 cfu / 100 μl of meningococcal serogroup B MC58 strain, demonstrating that, as described in Example 30, MASP-2-deficient mice were highly resistant to infection, with very low illness scores at 6 hours. [Figure 37] This is a Kaplan-Meier plot showing the percent survival of mice administered an infectious dose of 4 x 106 / 100 μl cfu of Neisseria meningitidis serogroup B MC58 strain and then administered an inhibitory anti-MASP-2 antibody (1 mg / kg) or a control isotype antibody 3 hours post-infection, demonstrating that anti-MASP-2 antibodies are effective in treating and improving survival in subjects infected with Neisseria meningitidis, as described in Example 31. [Figure 38]The graph shows the log cfu / ml of viable meningococcal serogroup B MC58 strain recovered at various time points at 20% human serum concentration: 0, 30, 60, and 90 minutes after i.p. infection with 6.5 x 106 cfu / 100 μl of meningococcal serogroup B MC58 strain, incubated 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). This shows that complement-dependent killing of meningococci 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 meningococcal serogroup B-MC58 bacteria recovered at various time points in mouse serum samples is shown, demonstrating that MASP-2- / - mouse serum has higher levels of bactericidal activity against meningococci than WT mouse serum, as described in Example 32. [Figure 40] Figure 1 illustrates the hemolysis of mannan-coated mouse red blood cells by human serum at a range of serum concentrations (measured by hemoglobin release of lysed mouse red blood cells (Crry / C3- / -) into the supernatant, measured photometrically). As described in Example 33, sera tested included heat-inactivated (HI) NHS, MBL- / -, NHS + anti-MASP-2 antibody, and NHS control. [Figure 41] Figure 1 illustrates the hemolysis of uncoated mouse red blood cells by human serum over a range of serum concentrations (measured by hemoglobin release of lysed WT mouse red blood cells into the supernatant, measured photometrically). Sera tested included heat-inactivated (HI) NHS, MBL- / -, NHS + anti-MASP-2 antibody, and NHS control. As described in Example 33, we demonstrate that inhibition of MASP-2 inhibits complement-mediated lysis of unsensitized WT mouse red blood cells. [Figure 42]Illustrates hemolysis of uncoated mouse red blood cells by human serum over a range of serum concentrations (measured by hemoglobin release of lysed mouse red blood cells (CD55 / 59- / -) into the supernatant measured photometrically. Sera tested included heat-inactivated (HI) NHS, MBL- / -, NHS + anti-MASP-2 antibody, and NHS control, as described in Example 33. [Figure 43] This figure depicts the percent survival over time (days) in control mice and mice treated with anti-human MASP-2 antibody (mAbH6) after exposure to 8.0 Gy radiation, as described in Example 34. [Figure 44] This figure shows the time to onset of microvascular obstruction after LPS injection in MASP-2 mice- / - and WT mice, 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 over the 60-minute period (log-rank: p=0.0005). [Figure 45] The graph shows the percent survival over time (hours) of saline-treated control mice (n=5) and anti-MASP-2 antibody-treated mice (n=5) in the STX / LPS-induced HUS model, demonstrating that, as described in Example 36, all control mice had died by 42 hours, whereas, in contrast, 100% of anti-MASP-2 antibody-treated mice survived throughout the entire time course of the experiment. [Figure 46] This figure illustrates the percentage of mice with microvascular obstruction 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 before FITC / dextran injection, as described in Example 37. [Figure 47]Figure 1 illustrates the occlusion time in minutes for mice treated with human MASP-2 antibody (mAbH6) and mice treated with an isotype control antibody, as described in Example 37. Data are reported as scatter dots with mean values ​​(horizontal bars) and standard error bars (vertical bars). The statistical test used for analysis was an unpaired t-test. The symbol "*" indicates p=0.0129. [Figure 48] Figure 1 shows the time to occlusion in minutes in the FITC-dextran / light-induced endothelial cell injury model of thrombosis using low light intensity (800-1500) as described in Example 37 for wild-type mice, MASP-2 KO mice, and wild-type mice pretreated with human MASP-2 antibody (mAbH6) administered ip at 10 mg / kg 16 hours before thrombosis induction and again ip 1 hour before. [Figure 49] Kaplan-Meier plot showing the percentage of mice with thrombus as a function of time in FITC-dextran-induced thrombotic microangiopathy mice treated with increasing doses of human MASP-2 inhibitory antibody (mAbH6) or isotype control antibody, as described in Example 39. [Figure 50] Illustrates median time to onset of thrombus formation (min) as a function of mAbH6 dose (*p<0.01 compared to control), as described in Example 39. [Figure 51] 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 increasing doses of a human MASP-2 inhibitory antibody (mAbH6) or an isotype control antibody, as described in Example 39. [Figure 52] 1 illustrates median time to microvascular occlusion as a function of mAbH6 dose (*p<0.05 compared to control), as described in Example 39. [Figure 53A]Illustrated are MAC deposition levels in the presence or absence of a human MASP-2 monoclonal antibody (OMS646) under lectin pathway-specific assay conditions, as described in Example 40. This demonstrates that OMS646 inhibits lectin-mediated MAC deposition with an IC50 value of approximately 1 nM. [Figure 53B] Illustrated are MAC deposition levels in the presence or absence of a human MASP-2 monoclonal antibody (OMS646) under classical pathway-specific assay conditions, as described in Example 40. This demonstrates that OMS646 does not inhibit MAC deposition via the classical pathway. [Figure 53C] Graph showing MAC deposition levels in the presence or absence of human MASP-2 monoclonal antibody (OMS646) under alternative pathway-specific assay conditions as described in Example 40. This demonstrates that OMS646 does not inhibit MAC deposition via the alternative pathway. [Figure 54] Figure 1 illustrates the pharmacokinetic (PK) profile of human MASP-2 monoclonal antibody (OMS646) in mice, as described in Example 40. OMS646 concentrations (mean of n=3 animals / group) are shown as a function of time after administration at the indicated doses. [Figure 55A] Figure 1 illustrates the pharmacodynamic (PD) response of a human MASP-2 monoclonal antibody (OMS646), measured as a reduction in systemic lectin pathway activity in mice after intravenous administration, as described in Example 40. [Figure 55B] Figure 1 illustrates the pharmacodynamic (PD) response of a human MASP-2 monoclonal antibody (OMS646), measured as a reduction in systemic lectin pathway activity in mice after subcutaneous administration, as described in Example 40. [Figure 56] 1 illustrates the inhibitory effect of MASP-2 antibody (OMS646) compared to sCR1 on aHUS serum-induced C5b-9 deposition on ADP-activated HMEC-1 cells, as described in Example 41. [Figure 57]1 illustrates the inhibitory effect of MASP-2 antibody (OMS646) compared to sCR1 on aHUS serum-induced thrombus formation on ADP-activated HMEC-1 cells, as described in Example 42. [Figure 58] This figure illustrates the mean change in platelet counts over time (weeks) from baseline in subjects with persistent hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA) after treatment with a MASP-2 inhibitory antibody (OMS646) as described in Example 46. [Figure 59] This figure illustrates the mean change in LDH over time (weeks) from baseline in subjects with persistent (HSCT-TMA) disease after treatment with a MASP-2 inhibitory antibody (OMS646) as described in Example 46. [Figure 60] This figure illustrates the mean change in haptoglobin over time (weeks) from baseline in subjects with persistent (HSCT-TMA) disease after treatment with a MASP-2 inhibitory antibody (OMS646) as described in Example 46. DETAILED DESCRIPTION OF THE INVENTION

[0045] Sequence Listing Description SEQ ID NO:1 Human MAp19 cDNA SEQ ID NO:2 Human MAp19 protein (with leader) SEQ ID NO:3 Human MAp19 protein (mature) SEQ ID NO:4 Human MASP-2 cDNA SEQ ID NO:5 Human MASP-2 protein (with leader) SEQ ID NO:6 Human MASP-2 protein (mature) SEQ ID NO:7 Human MASP-2 gDNA (exons 1-6) Antigen: (based on MASP-2 mature protein) SEQ ID NO: 8 CUBI sequence (aa 1-121) 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 (aa 610-625 with Ser 618 to Ala mutation) TIFF0007629300000001.tif63131 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 binding) TIFF0007629300000002.tif71128 Expression inhibitor: SEQ ID NO:30 CUBI-EGF domain cDNA (nucleotides 22 to 680 of SEQ ID NO:4) SEQ ID NO:31 TIFF0007629300000003.tif5128 Nucleotides 12 to 45 (sense) of SEQ ID NO:4, including the MASP-2 translation start site SEQ ID NO:32 TIFF0007629300000004.tif5128 Nucleotides 361 to 396 (sense) of SEQ ID NO:4, encoding the region containing the MASP-2 MBL binding site SEQ ID NO:33 TIFF0007629300000005.tif5128 Nucleotides 610 to 642 of SEQ ID NO:4, encoding the region containing the CUBII domain Cloning primers: TIFF0007629300000006.tif41128SEQ ID NOs:38 to 47 are cloning primers for humanized antibodies. SEQ ID NO:48 is a 9 aa peptide bond. Expression vector: SEQ ID NO:49 is the MASP-2 minigene insert. SEQ ID NO:50 is the mouse MASP-2 cDNA. SEQ ID NO:51 is the mouse MASP-2 protein (w / leader). SEQ ID NO:52 is the mature mouse MASP-2 protein. SEQ ID NO:53 is the rat MASP-2 cDNA. SEQ ID NO:54 is the rat MASP-2 protein (w / leader). SEQ ID NO:55 is the mature rat MASP-2 protein. SEQ ID NOs:56-59 are oligonucleotides for site-directed mutagenesis of human MASP-2 used to generate human MASP-2A. SEQ ID NOs:60-63 are oligonucleotides for site-directed mutagenesis of mouse MASP-2 used to generate mouse MASP-2A. SEQ ID NO:64-65 are oligonucleotides for site-directed mutagenesis of rat MASP-2 used to generate rat MASP-2A. SEQ ID NO: 66 17D20_dc35VH21N11VL (OMS646) DNA encoding 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

[0046] Detailed Description The present invention is based on the surprising discovery by the inventors that it is possible to inhibit the lectin-mediated MASP-2 pathway while leaving the classical pathway intact. The present invention also describes the use of MASP-2 as a therapeutic target for inhibiting cytotoxicity associated with lectin-mediated complement pathway activation while leaving classical (C1q-dependent) pathway components of the immune system intact.

[0047] I. Definition Unless otherwise defined herein, all terms used herein have the same meaning as would be understood by one of ordinary skill in the art of the present invention. The following definitions are provided to facilitate understanding of the terms used in the specification and claims to describe the present invention.

[0048] As used herein, the term "MASP-2-dependent complement activation" refers to the activation of complement by the lectin pathway under physiological conditions (i.e., Ca) that leads to the formation of the C3 convertase C4b2a and, subsequently, the formation of the C5 convertase C4b2a(C3b)n upon accumulation of the C3 cleavage product C3b. ++ It has been shown that this involves MASP-2-dependent activation of the lectin pathway, which occurs in the presence of lectin-binding domains (e.g., lectin-binding domains), primarily resulting in opsonization.

[0049] As used herein, the term "alternative pathway" refers to complement activation induced by, for example, zymosan from fungal and yeast cell walls, lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria, and rabbit erythrocytes, as well as many pure polysaccharides, rabbit erythrocytes, viruses, bacteria, animal tumor cells, parasites, and damaged cells, and traditionally thought to result from the spontaneous proteolytic generation of complement factors C3 to C3b.

[0050] As used herein, the term "lectin pathway" refers to complement activation that occurs through the specific binding of serum and non-serum carbohydrate-binding proteins, including mannan-binding lectin (MBL), CL-11, and ficolins (H-ficolin, M-ficolin, or L-ficolin).

[0051] As used herein, the term "classical pathway" refers to complement activation that is triggered by antibodies bound to foreign particles and requires the binding of the recognition molecule C1q.

[0052] As used herein, the term "MASP-2 inhibitor" refers to any agent that binds to or directly interacts with MASP-2 and effectively inhibits MASP-2-dependent complement activation, including anti-MASP-2 antibodies and their MASP-2-binding fragments, natural and synthetic peptides, small molecules, soluble MASP-2 receptors, expression inhibitors, and isolated natural inhibitors. It also encompasses peptides that compete with MASP-2 for binding to other recognition molecules (e.g., MBL, H-ficolin, M-ficolin, or L-ficolin) in the lectin pathway, but does not encompass 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%, e.g., more than 50%, e.g., more than 90%. In one embodiment, the MASP-2 inhibitor reduces MASP-2-dependent complement activation by more than 90% (i.e., reduces MASP-2 complement activation by 10% or less).

[0053] As used herein, the term "antibody" encompasses antibodies and antibody fragments thereof derived from any antibody-producing mammal (e.g., mouse, rat, rabbit, and primate, including human) or derived from hybridoma, phage selection, recombinant expression, or transgenic animal (or other methods of producing antibodies or antibody fragments) that specifically bind to a target polypeptide, e.g., a MASP-2 polypeptide or a portion thereof. The term "antibody" is not intended to be limited in terms of the source of the antibody or the manner in which the antibody is produced (e.g., by hybridoma, phage selection, recombinant expression, transgenic animal, peptide synthesis, etc.). Exemplary antibodies include polyclonal antibodies, monoclonal antibodies, and recombinant antibodies; panspecific, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies); humanized antibodies; murine antibodies; chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies; and anti-idiotypic antibodies, and may be any intact antibody or fragment thereof. As used herein, the term "antibody" encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (e.g., dAb, Fab, Fab', F(ab')2, Fv), single chain (ScFv), synthetic variants thereof, naturally occurring variants, fusion proteins comprising antibody portions and antigen-binding fragments of the required specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of an immunoglobulin molecule that contains an antigen-binding site or fragment (epitope recognition site) of the required specificity.

[0054] "Monoclonal antibody" refers to a homogeneous antibody population, consisting of amino acids (natural and non-natural) involved in selective binding of an epitope. Monoclonal antibodies are highly specific for their target antigen. The term "monoclonal antibody" encompasses not only intact and full-length monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single-chain (ScFv), variants thereof, fusion proteins containing the antigen-binding portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of an immunoglobulin molecule containing an antigen-binding fragment (epitope recognition site) with the required specificity and ability to bind to the epitope. The term is not intended to be limited in terms of the source of the antibody or the manner in which the antibody is produced (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins and fragments, etc., as described above in the definition of "antibody."

[0055] As used herein, the term "antibody fragment" refers to a portion derived from or related to a full-length antibody, e.g., an anti-MASP-2 antibody, generally comprising its antigen-binding or variable region. Illustrative examples of antibody fragments include Fab, Fab', F(ab)2, F(ab')2, and Fv fragments, scFv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0056] As used herein, a "single-chain Fv" or "scFv" antibody fragment refers to a fragment of an antibody V H Domain or V L Fv polypeptides generally comprise V domains, and these domains are present in a single polypeptide chain. H Domains and V L It further comprises a polypeptide linker between the domains which enables the scFv to form the desired structure for antigen binding.

[0057] As used herein, a "chimeric antibody" is a recombinant protein that contains the variable domains and complementarity determining regions derived from an antibody of a non-human species (e.g., rodent), while the remainder of the antibody molecule is derived from a human antibody.

[0058] As used herein, a "humanized antibody" is a chimeric antibody that contains minimal sequence matching specific complementarity-determining regions derived from a non-human immunoglobulin grafted onto a human antibody framework. Humanized antibodies are typically recombinant proteins in which only the antibody complementarity-determining regions are of non-human origin.

[0059] As used herein, the term "mannan-binding lectin" ("MBL") is synonymous with mannan-binding protein ("MBP").

[0060] As used herein, "membrane attack complex" ("MAC") refers to a complex of five terminal complement components (C5b in combination with C6, C7, C8, and C9) (also called C5b-9) that inserts into and destroys membranes.

[0061] As used herein, a "subject" includes all mammals, including, but not limited to, humans, non-human primates, dogs, cats, horses, sheep, goats, cattle, rabbits, pigs, and rodents.

[0062] 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).

[0063] In the broadest sense, natural amino acids can be divided into groups based on the chemical properties of their side chains. "Hydrophobic" amino acids refer to Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys, or Pro. "Hydrophilic" amino acids refer to Gly, Asn, Gln, Ser, Thr, Asp, Glu, Lys, Arg, or His. This group of amino acids can be further divided into subgroups as follows: "Uncharged hydrophilic" amino acids refer to Ser, Thr, Asn, or Gln. "Acidic" amino acids refer to Glu or Asp. "Basic" amino acids refer to Lys, Arg, or His.

[0064] As used herein, the term "conservative amino acid substitutions" 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.

[0065] As used herein, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term covers oligonucleobases composed of naturally occurring nucleotides, sugars, and covalent internucleoside (backbone) linkages, as well as oligonucleotides containing non-naturally occurring modifications.

[0066] As used herein, "epitope" refers to a site on a protein (e.g., human MASP-2 protein) to which an antibody binds. "Overlapping epitopes" include at least one (e.g., two, three, four, five, or six) common amino acid residues, including linear and non-linear epitopes.

[0067] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably and refer to any peptide-linked amino acid chain, regardless of length or post-translational modification. The MASP-2 protein described herein may include a wild-type protein, or may be a variant having 50 or fewer conservative amino acid substitutions (e.g., 1 or fewer, 2 or fewer, 3 or fewer, 4 or fewer, 5 or fewer, 6 or fewer, 7 or fewer, 8 or fewer, 9 or fewer, 10 or fewer, 12 or fewer, 15 or fewer, 20 or fewer, 25 or fewer, 30 or fewer, 35 or fewer, 40 or fewer, or 50 or fewer). Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine, and threonine; lysine, histidine, and arginine; and phenylalanine and tyrosine.

[0068] In some embodiments, the human MASP-2 protein may have an amino acid sequence that is 70 (e.g., 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100)% identical, or more than 70 (e.g., 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100)% identical to the human MASP-2 protein having the amino acid sequence shown in SEQ ID NO:5.

[0069] In some embodiments, the peptide fragments are at least 6 (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43) amino acids in length. , 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, or 600 or more amino acid residues (e.g., at least 6 consecutive amino acid residues of SEQ ID NO:5). In some embodiments, the antigenic peptide fragment of human MASP-2 protein is less than 500 amino acids in length (e.g., less than 450, less than 400, less than 350, less than 325, less than 300, less than 275, less than 250, less than 225, less than 200, less than 190, less than 180, less than 170, less than 160, less than 150, less than 140, less than 130, less than 120, less than 110, less than 100, less than 95, less than 90, less than 85, less than 80, less than 75, less than 70, less than 65, less than 60, less than 55, less than 50, less than 49, less than 48, less than 47, less than 46, less than 45, less than 44, less than 43, less than 42, less than 41, less than 40, less than 39, less than 38, less than 37, less than 36, less than 35, less than 34, less than 33, less than 32, less than 31, less than 30, less than 29, less than 28, less than 27, less than 26, less than 25, less than 24, less than 23, less than 22, less than 21, less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, or less than 6 amino acid residues (e.g., less than 500 consecutive amino acid residues in any one of SEQ ID NO:5).

[0070] Percentage (%) amino acid sequence identity is defined as the percentage of amino acids in the candidate sequence that are identical to the amino acids in the reference sequence, after aligning the sequences to achieve maximum percent sequence identity and introducing gaps if necessary.For the purpose of determining percent sequence identity, alignment can be achieved in various ways within the skill of the art, for example, by using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.Appropriate parameters for measuring alignment can be determined by known methods, including any algorithm that is required to achieve maximum alignment across the full length of the sequence being compared.

[0071] II. Overview of the Invention Lectins (MBL, M-ficolin, H-ficolin, L-ficolin, and CL-11) are specific recognition molecules that trigger the innate complement system, which comprises a lectin-initiation pathway and an associated terminal pathway amplification loop that amplifies lectin-initiated activation of terminal complement effector molecules. C1q is a specific recognition molecule that triggers the acquired complement system, which comprises a classical initiation pathway and an associated terminal pathway amplification loop that amplifies C1q-initiated activation of terminal complement effector molecules. We refer to these two major complement activation systems as the lectin-dependent and C1q-dependent complement systems, respectively.

[0072] In addition to its essential role in immune defense, the complement system contributes to tissue damage in many clinical conditions. Therefore, there is an urgent need to develop therapeutically effective complement inhibitors to prevent these side effects. Recognizing that it is possible to inhibit the lectin-mediated MASP-2 pathway while leaving the classical pathway intact, it was realized that it would be highly desirable to specifically inhibit only the complement activation system responsible for a particular pathology without completely blocking the immune defense capabilities of complement. For example, in disease states in which complement activation is primarily mediated by the lectin-dependent complement system, it would be advantageous to specifically inhibit only this system. This would leave the C1q-dependent complement activation system intact to handle immune complex processing and aid in host defense against infection.

[0073] A preferred protein component of interest for the development of therapeutic agents that specifically inhibit the lectin-dependent complement system is MASP-2. Of all known protein components of the lectin-dependent complement system (MBL, H-ficolin, M-ficolin, L-ficolin, MASP-2, C2-C9, factor B, factor D, and properdin), only MASP-2 is unique to the lectin-dependent complement system and required for its function. The lectins (MBL, H-ficolin, M-ficolin, L-ficolin, and CL-11) are also unique components of the lectin-dependent complement system. However, due to lectin redundancy, loss of any one of these lectin components may not necessarily inhibit system activation. Inhibition of all five lectins may be necessary to ensure inhibition of the lectin-dependent complement activation system. Furthermore, because MBL and ficolins are known to possess opsonic activity independent of complement, inhibiting lectin function may eliminate this beneficial host defense mechanism against infection. In contrast, when MASP-2 is the target of inhibition, this complement-independent lectin opsonization activity appears to remain intact. An additional benefit of MASP-2 as a therapeutic target for inhibiting the lectin-dependent complement activation system is that MASP-2 plasma concentrations (approximately 500 ng / ml) are among the lowest plasma concentrations of complement proteins. Therefore, a correspondingly low concentration of a high-affinity MASP-2 inhibitor may be sufficient to achieve complete inhibition (Moller-Kristensen, M., et al., J. Immunol Methods 282:159-167, 2003).

[0074] III. 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 thought to be the primary driver. Clinical and laboratory manifestations 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, initiated at least in part by injury or stress to the microvascular endothelium, has also been implicated in fulminant antiphospholipid syndrome (CAPS), systemic Degos disease, and other TMAs, including TMA secondary to cancer, TMA secondary to cancer chemotherapy, and TMA secondary to transplantation.

[0075] Studies of patients with genetic deficiencies of specific complement components have provided direct evidence for the pathological role of complement in nephritic hosts. Numerous reports have documented a relationship between renal injury and deficiency of the complement regulatory factor H (Ault, BH, Nephrol. 14:1045-1053, 2000; Levy, M., et al., Kidney Int. 30:949-56, 1986; Pickering, MC, et al., Nat. Genet. 31:424-8, 2002). Factor H deficiency results in low plasma levels of factor B and C3 due to activation-related consumption of these components. These patients also have elevated circulating levels of C5b-9 in their serum, indicating complement activation. Membranoproliferative glomerulonephritis (MPGN) and idiopathic hemolytic uremic syndrome (HUS) are associated with factor H deficiency or mutations. Factor H-deficient pigs (Jansen, JH, et al., Kidney Int. 53:331-49, 1998) and factor H knockout mice (Pickering, MC, 2002) exhibit MPGN-like symptoms, supporting the importance of factor H in complement regulation. Deficiencies of 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 of C1q, C4, and C2 strongly predispose to the development of SLE through mechanisms related to defective clearance of immune complexes and apoptotic material. Many of these SLE patients develop lupus nephritis, characterized by the deposition of immune complexes throughout the glomerulus.

[0076] aHUS Atypical hemolytic uremic syndrome (aHUS) is part of a group of conditions called "thrombotic microangiopathy." In atypical HUS (aHUS), the disease is associated with defective complement regulation and can be sporadic or familial. Familial aHUS cases are associated with mutations in genes encoding complement activation or regulatory proteins, including complement factors H, I, B, membrane cofactor CD46, and complement factor H-related protein 1 (CFHR1) and 3 (CFHR3) (Zipfel, PF, et al., PloS Genetics 3(3):e41(2007)). A unifying feature of this wide range of genetic mutations associated with aHUS is a predisposition to enhanced complement activation at cell or tissue surfaces. Therefore, one aspect of the present invention involves treating patients suffering from aHUS associated with factor H deficiency by administering an effective amount of a MASP-2 inhibitor. Another aspect of the present invention includes treating 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.

[0077] Recently, considerable progress has been made toward understanding the molecular pathophysiology underlying enhanced complement activation in aHUS, which is caused by a diverse set of mutant complement factors. This mechanism is best understood for factor H mutations. Factor H is an abundant serum protein containing 20 short consensus repeat (SCR) domains that acts as a negative regulator of complement activation in solution 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 control complement activation on the host cell surface, factor H must interact with host cells, a process mediated by SCR domains 16–20. To date, all aHUS-associated factor H mutations have been clustered in the C-terminal region, which includes the (SCR) domains 16–20. Although these mutant factor H proteins are fully functional in regulating C3 activation in the soluble state, they are unable to interact with host cell surfaces and, as a result, are unable to regulate C3 activation on cell surfaces (Exp Med 204(6):1249-56(2007)). Therefore, certain factor H mutations are associated with aHUS because the mutant factor H proteins do not interact with host cell surfaces and, therefore, are unable to effectively reduce complement activation on host cell surfaces, including microvascular endothelium. As a result, once initial C3 activation occurs, subsequent complement activation on the microvascular endothelial surface proceeds unabated in patients with factor H mutations. This uncontrolled complement activation ultimately leads to progressive damage to the vascular endothelium, subsequent platelet aggregation and microvascular coagulation, and hemolysis caused by the shear stress of RBC passage through partially occluded microvessels. Therefore, aHUS disease manifestations and clinical and laboratory findings are directly linked to defective negative complement regulation on the microvascular endothelial surface.

[0078] Similar to factor H mutations, loss-of-function mutations in the negative complement modulators factor I and membrane cofactor protein (CD46) have also been 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)). Thus, a large body of data indicates that complement activation is involved in the development of aHUS. This idea is most convincingly supported by the therapeutic efficacy of eculizumab, a monoclonal antibody that blocks the terminal complement protein C5, in the treatment of aHUS.

[0079] Although the central role of complement as an effector mechanism in aHUS is widely recognized, the triggers initiating complement activation and the molecular pathways involved remain unclear. Not all individuals with the above mutations develop aHUS. In fact, family studies suggest that the penetrance of aHUS is only approximately 50% (Ann Hum Genet 74(1):17-26(2010)). The natural history of the disease suggests that aHUS most often develops after an initiating event, such as an infectious episode or injury. Infectious agents are known to activate the complement system. In the absence of preexisting adaptive immunity, complement activation by infectious agents can be initiated primarily via the lectin pathway. Therefore, infection-induced lectin pathway activation may be the initiating trigger for the subsequent pathological amplification of complement activation in individuals predisposed to aHUS, ultimately leading to disease progression. Therefore, another aspect of the present invention involves treating patients suffering from aHUS secondary to an infection by administering an effective amount of a MASP-2 inhibitor.

[0080] Other forms of damage to host tissue, particularly damage to the vascular endothelium, activate complement via the lectin pathway. Human vascular endothelial cells undergoing oxidative stress, for example, respond by expressing surface moieties that bind to lectins and activate the complement lectin pathway (Am J. Pathol 156(6):1549-56(2000)). Vascular injury following ischemia / reperfusion also activates complement via the lectin pathway in vivo (Scand J Immunol 61(5):426-34(2005)). Activation of the lectin pathway in this situation leads to pathological consequences for the host, and inhibition of the lectin pathway by blocking MASP-2 prevents further host tissue damage and adverse events (Sehwaeble PNAS 2011).

[0081] Therefore, other processes that precipitate aHUS are also known to activate the complement lectin pathway. Therefore, it is likely that the lectin pathway is inappropriately amplified in a disordered manner in individuals genetically predisposed to aHUS and thus may be the initial complement activation mechanism that initiates aHUS pathogenesis. By inference, agents that block complement activation via the lectin pathway, including anti-MASP-2 antibodies, are expected to prevent disease progression or reduce exacerbations in aHUS-susceptible individuals.

[0082] Further supporting this idea, recent studies have identified Streptococcus pneumoniae (S. pneumoniae) as a key etiological 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 have an unfavorable prognosis, with high mortality and long-term morbidity. Notably, these cases lacked evidence of concurrent complement gene mutations known to predispose to aHUS and were accompanied by nonenteric infections leading to the manifestations of microangiopathy, uremia, and hemolysis. It is important to note that S. pneumoniae is particularly effective at activating complement, primarily via the lectin pathway. Therefore, in non-enteric HUS associated with pneumococcal infection, the manifestations of microangiopathy, uremia, and hemolysis are expected to be primarily driven by lectin pathway activation, and agents that block the lectin pathway, including anti-MASP-2 antibodies, are expected to prevent aHUS progression and reduce disease severity in these patients. Accordingly, another aspect of the present invention includes treating patients suffering from non-enteric aHUS associated with Streptococcus pneumoniae infection by administering an effective amount of a MASP-2 inhibitor.

[0083] In accordance with the foregoing, in some embodiments, a method is provided for reducing the likelihood of developing aHUS or developing renal failure associated with aHUS in a subject at risk of developing renal failure associated with aHUS, comprising administering an amount of a MASP-2 inhibitor effective to ameliorate or prevent renal failure in the subject for a period effective to ameliorate or prevent the renal failure. In some embodiments, the method further comprises determining whether the subject is at risk of developing aHUS before the subject develops symptoms associated with aHUS. In other embodiments, the method comprises determining whether the subject is at risk of developing aHUS based on the development of at least one or more symptoms indicative of aHUS (e.g., the presence of anemia, thrombocytopenia, and / or renal insufficiency in the subject) and / or the presence of thrombotic microangiopathy in a biopsy specimen obtained from the subject. Determining whether a subject is at risk for developing aHUS includes determining whether the subject has a genetic predisposition to developing aHUS, which may be performed 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 a genetic marker associated with aHUS (i.e., by genomic sequencing or gene-specific analysis (e.g., PCR analysis) to determine the presence or absence of aHUS-associated genetic mutations in genes encoding complement factor H (CFH), factor I (CFI), factor B (CFB), membrane cofactors CD46, C3, complement factor H-related protein 1 (CFHR1), or THBD (which encode the anticoagulant protein thrombodulin), or complement factor H-related protein 3 (CFHR3), or complement factor H-related protein 4 (CFHR4)), and / or by determining whether the subject has a family history of aHUS. Methods for genetic screening for the presence or absence of aHUS-associated genetic mutations are well established.See, e.g., 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™, Seattle (WA): University of Washington, Seattle.

[0084] For example, overall, disease penetrance among individuals with complement factor H (CFH) mutations is 48%, CD46 mutations are 53%, CFI mutations are 50%, C3 mutations are 56%, and THBD mutations are 64% (Caprioli J. et al., Blood, 108:1267-79(2006); Noris et al., Clin J Am Soc Nephrol 5:1844-59(2010)). As noted in Caprioli et al. (2006), a significant number of individuals with complement factor H (CFH) mutations never develop aHUS, and it is assumed that suboptimal CFH activity in these individuals is sufficient to protect the host from the effects of complement activation under physiological conditions. However, suboptimal CFH activity is not sufficient to prevent C3b from being deposited on vascular endothelial cells when exposed to agents that activate complement and generate higher-than-normal amounts of C3b.

[0085] Thus, in one aspect, 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, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation. In another aspect, a method is provided for inhibiting MASP-2-dependent complement activation in a subject at risk of developing non-factor H-dependent atypical hemolytic uremic syndrome, comprising periodically monitoring the subject to determine the presence or absence of anemia, thrombocytopenia, or increased creatinine, and treating with a MASP-2 inhibitor based on the determination that anemia, thrombocytopenia, or increased creatinine is present. In another aspect, a method is provided for reducing the likelihood that a subject at risk of developing factor H-independent aHUS will suffer from clinical symptoms associated with aHUS, comprising administering a MASP-2 inhibitor before, during, or after an event known to be associated with the induction of aHUS clinical symptoms, such as drug exposure (e.g., chemotherapy), infection (e.g., bacterial infection), malignancy, injury, organ or tissue transplant, or pregnancy.

[0086] In one aspect, a method is provided for reducing the likelihood that a subject will suffer from clinical symptoms associated with aHUS, comprising regularly monitoring a subject at risk of developing aHUS to determine the presence or absence of anemia, thrombocytopenia, or increased creatinine, and treating the subject with a MASP-2 inhibitor based on a determination that anemia, thrombocytopenia, or increased creatinine is present.

[0087] In another aspect, a method is provided for reducing the likelihood that a subject at risk of developing aHUS will suffer from clinical symptoms associated with aHUS, comprising administering a MASP-2 inhibitor before, during, or after an event known to be associated with the induction of aHUS clinical symptoms, such as drug exposure (e.g., chemotherapy), infection (e.g., bacterial infection), malignancy, injury, organ or tissue transplant, or pregnancy.

[0088] In some embodiments, the MASP-2 inhibitor is administered for at least 1 day, 2 days, 3 days, 4 days, or longer 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 is resolved or controlled. In the pre-aHUS setting, the MASP-2 inhibitor may be administered systemically to the subject, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration.

[0089] In some embodiments, in the setting of an initial diagnosis of aHUS, or in a subject exhibiting 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) as a first-line therapy in the absence of plasmapheresis or in combination with plasmapheresis. As a first-line therapy, the MASP-2 inhibitor may be administered systemically to the subject, for example, by intra-arterial, intravenous, intramuscular, inhaled, nasal, subcutaneous, or other parenteral administration. In some embodiments, the MASP-2 inhibitor is administered to the subject as a first-line therapy in the absence of plasmapheresis to avoid potential plasmapheresis complications, including bleeding, infection, and exposure to disorders, and / or allergies specific to the plasma donor, or in subjects who are otherwise averse to plasmapheresis, or in situations where plasmapheresis is unavailable.

[0090] In some embodiments, the method includes administering a MASP-2 inhibitor to a subject suffering from aHUS via a catheter (e.g., intravenously) for a first period (e.g., at least 1 day to 1 or 2 weeks), followed by subcutaneous administration of the MASP-2 inhibitor to the subject for a second period (e.g., a chronic phase of at least 2 weeks or longer). In some embodiments, administration during the first and / or second period 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, and determining a decreased level of at least one complement factor compared to a standard value or a healthy control subject indicates the need for continued treatment with the MASP-2 inhibitor.

[0091] In some embodiments, the method includes administering a MASP-2 inhibitor, such as an anti-MASP-2 antibody, intravenously, intramuscularly, or preferably subcutaneously to a subject suffering from or at risk of developing aHUS. Treatment is chronic and may be administered 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 eculizamab.

[0092] HUS Like atypical HUS, typical HUS exhibits all the clinical and laboratory findings of TMA. However, typical HUS is often a pediatric disease, usually without a familial component or direct association with complement gene mutations. The cause of typical HUS is closely related to infection with certain enteropathogens. Patients typically present with acute renal failure, hemoglobinuria, and thrombocytopenia, typically followed by episodes of bloody diarrhea. This condition is caused by enteric infection with Shigella dissenteria, Salmonella, or Shiga toxin-like-producing enterohemorrhagic Escherichia coli strains, such as E. coli O157:H7. These pathogens are acquired from contaminated food or water supplies. HUS is a medical emergency with a mortality rate of 5–10%. A significant proportion of survivors develop chronic kidney disease (Corrigan and Boineau, Pediatr Rev 22(11):365-9(2011)) and may require a kidney transplant.

[0093] Microvascular coagulation in typical HUS occurs primarily, but not exclusively, in the renal microvasculature. The underlying pathophysiology is mediated by Shiga toxin (STX). STX is excreted into the intestinal lumen by enteropathogenic microorganisms, crosses the intestinal barrier, enters the bloodstream, and binds to vascular endothelial cells via the brobotriaosyl 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. Upon binding to the endothelium, STX induces a series of events that damages the vascular endothelium, activates leukocytes, and leads 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 small arteries and capillaries in the kidney and other organs. Obstruction of blood flow in small arteries and capillaries by microthrombi increases shear stress on the RBCs as they push through the narrowed vessels. This shear stress can result in RBC disruption, forming RBC fragments called schistocytes. The presence of schistocytes is a characteristic finding in HUS. This mechanism is known as microangiopathic hemolysis. Furthermore, the obstruction of blood flow causes ischemia, initiating a complement-mediated inflammatory response that causes further damage to the affected organ.

[0094] The lectin pathway of complement contributes to HUS development through two main mechanisms: (1) direct MASP-2-mediated activation of the coagulation cascade triggered by endothelial injury, and (2) subsequent lectin-mediated complement activation induced by ischemia due to initial obstruction of microvascular blood flow.

[0095] STX is known to injure microvascular endothelial cells, and injured endothelial cells are known to activate the complement system. As detailed above, complement activation after endothelial cell injury is primarily driven by the lectin pathway. Human vascular endothelial cells exposed to oxidative stress respond by expressing surface moieties that bind lectins and activate the complement lectin pathway (Collard et al., Am J Pathol. 156(5):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)). Activation of the lectin pathway in this situation leads to pathological consequences for the host, and inhibition of the lectin pathway by blocking MASP-2 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 injured endothelial cells can directly activate the coagulation system. Therefore, the complement lectin pathway, via MASP-2-mediated prothombin activation, is likely to be the primary molecular pathway linking the initial endothelial injury caused by STX with the coagulation and microvascular thrombosis that occur in HUS. Therefore, lectin pathway inhibitors, including but not limited to antibodies that block MASP-2 function, are expected to prevent or reduce microvascular coagulation, thrombosis, and hemolysis in patients with HUS. Indeed, administration of anti-MASP-2 antibodies significantly protected mice in 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 became moribund or died within 48 hours. On the other hand, as further shown in Figure 45, all mice treated with anti-MASP-2 antibody and then challenged with STX and LPS survived (Fisher's exact test p<0.01; N=5). Thus, 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 patients with HUS and provide protection from microvascular coagulation, thrombosis, and hemolysis caused by infection with enteropathogenic E. coli or other STX-producing pathogens.

[0096] Although demonstrated herein for HUS caused by STX, anti-MASP-2 therapy is also expected to be beneficial for HUS-like syndromes caused by endothelial damage caused 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 prevent or limit coagulation, thrombus formation, and RBC destruction, preventing renal failure in HUS and other TMA-related diseases (i.e., aHUS and TTP).

[0097] Patients with HUS often present with diarrhea and vomiting, and typically have low platelet counts (thrombocytopenia) and low RBC counts (anemia). The pre-HUS diarrhea phase typically lasts about 4 days, and during this period, subjects at risk for developing HUS typically exhibit one or more of the following symptoms in addition to severe diarrhea: hematocrit levels below 30% with smear evidence of intravascular red blood cell destruction, thrombocytopenia (platelet count <150 x 10 3 / mm 3), and / or the presence of renal dysfunction (serum creatinine concentration above the upper limit of the reference range for age). The presence of oliguria (urine output of ≦0.5 mL / kg / h for >1 day) can be used as a measure of progression to the development of HUS (see C. Hickey et al., Arch Pediatr Adolesc Med 165(10):884-889(2011)). Typically, testing is performed for the presence of infection with Escherichia coli bacteria (E. coli O157:H7) or Shigella or Salmonella species. The use of antibiotics is contraindicated in subjects who test positive for enterogenic E. coli (e.g., E. coli O157:H7) infection. This is because the use of antibiotics may increase the risk of developing HUS due to increased STX production (see Wong C. et al., N Engl J. Med 342:1930-136(2000)). Subjects who test positive for Shigella or Salmonella are typically administered antibiotics to clear the infection. Other well-established first-line therapies for HUS include volume expansion, dialysis, and plasmapheresis.

[0098] In accordance with the foregoing, in some embodiments, subjects suffering from one or more symptoms associated with the pre-HUS stage and subjects at risk of developing HUS (i.e., subjects exhibit one or more of the following: diarrhea, hematocrit levels below 30% with smear evidence of intravascular red blood cell destruction, thrombocytopenia (≥150×10 3 / mm 3In the setting of a pre-HUS condition, a MASP-2 inhibitor may be administered in an amount effective to ameliorate or prevent renal dysfunction (a platelet count below 100 mg / mL) and / or a serum creatinine concentration above the upper limit of the reference range for age), for a period effective to ameliorate or prevent renal dysfunction. In some embodiments, the MASP-2 inhibitor is administered for at least 1, 2, 3, 4, or more days, and may be repeated as determined by a physician until the condition is resolved or controlled. In the setting of pre-HUS, the MASP-2 inhibitor may be administered systemically to the subject, for example, by intra-arterial, intravenous, intramuscular, inhaled, nasal, oral, subcutaneous, or other parenteral administration.

[0099] 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, in the context of a subject suffering from symptoms associated with the pre-HUS stage who is known to be infected with enteric Escherichia coli (e.g., E. coli O157:H7) for which the use of antibiotics is contraindicated, a method for reducing the risk of developing HUS or reducing the likelihood of renal failure in the subject is provided, comprising administering an amount of a MASP-2 inhibitor effective to suppress or prevent the presence of oliguria in the subject for a first period of time (e.g., at least 1, 2, 3, or 4 days) effective to suppress or prevent the presence of oliguria, wherein the administration of the MASP-2 inhibitor during the first period is performed in the absence of antibiotics. In some embodiments, the method further comprises administering a MASP-2 inhibitor to the subject in combination with an antibiotic for a second period of time (e.g., at least 1 to 2 weeks).

[0100] In another aspect, a method is provided for reducing the risk of developing HUS or the likelihood of renal failure in a subject suffering from symptoms associated with the pre-HUS stage and known to be infected with Shigella or Salmonella, comprising administering an amount of a MASP-2 inhibitor effective to suppress or prevent the presence of oliguria in the subject for a period of time effective to suppress or prevent the presence of oliguria, wherein the administration of the MASP-2 inhibitor is in the presence or absence of an appropriate antibiotic.

[0101] In some embodiments, in the setting of an initial diagnosis of HUS, or in subjects exhibiting one or more symptoms consistent with a diagnosis of HUS (e.g., the presence of renal failure, or microangiopathic hemolytic anemia in the absence of low fibrinogen, or thrombocytopenia), the subject is treated with an effective amount of a MASP-2 inhibitor (e.g., an anti-MASP-2 antibody) in the absence of plasmapheresis or in combination with plasmapheresis as a first-line therapy. As a first-line therapy, the MASP-2 inhibitor may be administered systemically to the subject, for example, by intra-arterial, intravenous, intramuscular, inhaled, nasal, subcutaneous, or other parenteral administration. In some embodiments, the MASP-2 inhibitor is administered to the subject as a first-line therapy in the absence of plasmapheresis to avoid plasmapheresis complications such as bleeding, infection, and exposure to injury, and / or allergies specific to the plasma donor, or in subjects who are otherwise averse to plasmapheresis, or in situations where plasmapheresis is unavailable.

[0102] 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 or 2 weeks), followed by subcutaneous administration of the MASP-2 inhibitor to the subject for a second period (e.g., a chronic phase lasting at least 2 weeks or more). In some embodiments, administration during the first and / or second period 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 determining a decreased level of the at least one complement factor compared to a standard value or a healthy control subject indicates the need for treatment, and determining a normal level indicates improvement.

[0103] In some embodiments, the method includes subcutaneously or intravenously administering a MASP-2 inhibitor, such as an anti-MASP-2 antibody, to a subject suffering from or at risk of developing HUS. Treatment is preferably daily, but may be less frequent, such as weekly or monthly. Treatment may last at least one week and as long as three months. The anti-MASP-2 antibody may be administered alone or in combination with a C5 inhibitor, such as eculizumab.

[0104] TTP: Thrombotic thrombocytopenic purpura (TTP) is a life-threatening blood clotting 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 numerous microclots, or thrombosis, in 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 to the brain, heart, and kidneys. TTP is clinically characterized by thrombocytopenia, microangiopathic hemolytic anemia, neurological changes, renal failure, and fever. In the pre-plasma exchange era, mortality during acute episodes was 90%. Even with plasma exchange, survival at 6 months is approximately 80%.

[0105] TTP can result from genetic or acquired inhibition of the enzyme ADAMTS-13, a metalloprotease responsible for cleaving large von Willebrand factor (vWF) multimers into smaller units. ADAMTS-13 inhibition or deficiency ultimately leads to increased coagulation (Tsai, H. J Am Soc Nephrol 14:1072-1081, (2003)). ADAMTS-13 regulates vWF activity. In its absence, vWF forms large multimers that are more likely to bind to platelets, predisposing patients to platelet aggregation and thrombosis in the microvasculature.

[0106] Upshaw-Schulman syndrome (USS, also known as congenital TTP) is a congenital deficiency of ADAMTS13 activity caused by mutations in the ADAMTS13 gene (Schulman et al., Blood, 16(1):943-57, 1960; Upshaw et al., New Engl. J. Med, 298 (24):1350-2, 1978). A large number of ADAMTS13 mutations have been identified in individuals with congenital TTP (Kinoshita et al., International Journal of Hematology, 74:101-108 (2001); Levy et al., Nature, 413 (6855):488-494 (2001); Kokame et al., PNAS 99(18):11902-11907 (2002); Savasan et al., Blood, 101:4449-4451 (2003); Matsumoto et al., Blood, 103:1305-1310 (2004) and Fujimura et al., Brit. J. Haemat 144:742-754 (2008)). Subjects with USS typically have 5–10% of normal ADAMTS13 activity (Kokame et al., PNAS 99(18):11902–11907, 2002). Although there are some similarities between acquired TTP and USS, USS has several important differences in its clinical features. USS usually manifests in infancy or childhood and is characterized by severe hyperbilirubinemia, a negative Coombs test immediately after birth, poor response to fresh plasma infusion, and frequent recurrence (Savasan et al., Blood, 101:4449–4451, 2003). In some cases, patients with this inherited ADAMTS13 deficiency have a mild phenotype at birth and develop symptoms associated with TTP only in clinical situations with elevated von Willebrand factor levels, such as during infection or pregnancy. For example, Fujimura et al. reported nine Japanese women diagnosed with the disorder during their first pregnancy from six families in which USS was genetically confirmed.Thrombocytopenia occurred during the second and third trimesters in each of 15 pregnancies, often followed by TTP. All of these women were found to have severely deficient ADAMTS13 activity (Fujimura et al., Brit. J. Haemat 144:742-754, 2008).

[0107] In accordance with the foregoing, in some embodiments, a method is provided for reducing the likelihood of developing clinical symptoms associated with congenital TTP (e.g., thrombocytopenia, anemia, fever, and / or renal failure) in a subject with Upshaw-Schulman 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 administering an amount of a MASP-2 inhibitor (e.g., a MASP-2 antibody) effective to ameliorate or prevent one or more clinical symptoms associated with TTP for a period of time effective to ameliorate or prevent the clinical symptoms. In some embodiments, the method further comprises determining whether the subject is at risk of developing symptoms associated with congenital TTP prior to the onset of any symptoms associated with TTP or based on the onset of at least one or more symptoms indicative of 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 determining whether the subject has a deficiency in ADAMTS13 activity, and / or determining whether the subject has a family history of USS. Methods for genetic screening for the presence or absence of gene mutations associated with USS are well established. See, for example, Kinoshita et al., International Journal of Hematology, 74:101-108 (2001); Levy et al., Nature, 413 (6855):488-494 (2001); Kokame et al., PNAS 99(18):11902-11907 (2002); Savasan et al., Blood, 101:4449-4451 (2003); Matsumoto et al., Blood, 103:1305-1310 (2004) and Fujimura et al., Brit. J. Haemat 144:742-754 (2008).

[0108] In one aspect, a method is provided for reducing the likelihood that a subject diagnosed with USS will suffer from clinical symptoms associated with TTP, comprising: regularly monitoring the subject to determine the presence or absence of anemia, thrombocytopenia, or increased creatinine; and treating the subject 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 the presence of an event known to be associated with the induction of TTP clinical symptoms, such as drug exposure (e.g., chemotherapy), infection (e.g., bacterial infection), malignancy, injury, transplantation, or pregnancy.

[0109] In another aspect, a method is provided for treating a subject with USS and a subject suffering from a clinical symptom associated with TTP, comprising administering an amount of a MASP-2 inhibitor (e.g., a MASP-2 antibody) effective to ameliorate or prevent one or more clinical symptoms associated with TTP for a period of time effective to ameliorate or prevent the clinical symptom.

[0110] TTP can also be caused by autoantibodies against ADAMTS-13. In addition, TTP can occur during breast cancer, gastrointestinal cancer, or prostate cancer (George. JN., Oncology (Williston Park). 25:908-14 (2011)), pregnancy (second trimester or postpartum), George JN., Curr Opin Hematol 10:339-344 (2003)), or is associated with diseases, such as autoimmune diseases like HIV or systemic lupus erythematosus (Hamasaki K, et al., Clin Rheumatol. 22:355-8 (2003)). TTP can also be caused by certain medications, including heparin, quinine, immunological components, cancer chemotherapy drugs (bleomycin, cisplatin, cytosine arabinoside, daunomycin, gemcitabine, mitomycin C, and tamoxifen), cyclosporine A, oral contraceptives, penicillin, rifampin, and antiplatelet drugs, including ticlopidine and clopidogrel (Azarm, T. et al., J Res Med Sci., 16:353-357(2011)). Other factors or conditions associated with TTP are toxins, such as bee venom, sepsis, splenic sequestration, transplantation, vasculitis, vascular surgery, and infections such as streptococcal pneumonia and cytomegalovirus (Moake JL., N Engl J Med., 347:589-600(2002)). Transient functional ADAMTS-13 deficiency can result in TTP as a result of endothelial cell injury associated with Streptococcus pneumoniae infection (Pediair Nephrol., 26:631-5(2011)).

[0111] Plasmapheresis is the standard treatment for TTP (Rock GA, et al., N Engl J Med 325:393-397(1991)). Plasmapheresis replaces ADAMTS-13 activity in patients with genetic defects and removes ADAMTS-13 autoantibodies in patients with acquired autoimmune TTP (Tsai, HM, Hematol Oncol Clin North Am., 21(4):609-v(2007)). Additional agents, such as immunosuppressive drugs, are routinely added to the therapy (George, JN, N Engl J Med, 354:1927-35(2006)). However, plasmapheresis is successful in only about 20% of patients, relapse occurs in more than one-third of patients, and plasmapheresis is expensive and technically demanding. Furthermore, many patients cannot tolerate plasmapheresis. As a result, there remains a great need for additional and better treatments for TTP.

[0112] Because TTP is a disorder of the blood coagulation cascade, treatment with complement antagonists may help stabilize and treat the disease. While pathological activation of the alternative complement pathway has been linked to aHUS, the role of complement activation in TTP is less clear. Functional deficiency of ADAMTS13 is important for TTP susceptibility but is not sufficient to cause acute episodes. Environmental factors and / or other genetic mutations may contribute to TTP manifestations. For example, genes encoding proteins involved in the regulation of the coagulation cascade, vWF, platelet function, endothelial vascular surface components, or the complement system may be involved in the development of acute thrombotic microangiopathy (Galbusera, M. et al., Haematologica, 94; 166-170 (2009)). Complement activation, in particular, has been shown to play an important role. Serum derived from thrombotic microangiopathy associated with ADAMTS-13 deficiency has been shown to induce 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, consistent with activation of the classical / lectin and alternative pathways, levels of C4d, C3bBbP, and C3a have recently been shown to increase during acute episodes of TTP (M. Reti et al., J Thromb Haemost. Feb 28. (2012) doi:10.1111 / j.1538-7836.2012.04674.x. [Electronic publication ahead of print]). This increased amount of complement activation during acute episodes may initiate terminal pathway activation, further exacerbating TTP.

[0113] The role of ADAMTS-13 and vWF in TTP is clearly related to their subsequent role in platelet activation and aggregation, shear stress, and deposition during microangiopathy. Activated platelets interact with and trigger the classical and alternative 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 targets for classical complement activation in hereditary or autoimmune TTP.

[0114] As previously described, the complement lectin pathway, via MASP-2-mediated prothombin activation, is a major molecular pathway linking endothelial injury occurring in HUS with coagulation and microvascular thrombosis. Similarly, activation of the complement lectin pathway may directly drive the coagulation system in TTP. Lectin pathway activation may be initiated in response to the initial endothelial injury 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 alleviate microvascular coagulation, thrombosis, and hemolysis-related microangiopathy in patients with TTP.

[0115] Patients suffering from TTP typically present in the emergency room with one or more of the following: purpura, renal failure, low platelet count, anemia, and / or thrombosis, including stroke. The current standard of care for TTP involves intravascular catheter delivery of exchange plasmapheresis (e.g., intravenous catheter or other form of catheter), typically three times a week, but up to daily, for a period of two weeks or more. If a subject tests positive for the presence of an inhibitor of ADAMTS13 (i.e., endogenous antibodies against ADAMTS13), plasmapheresis can be administered in combination with immunosuppressive therapy (e.g., corticosteroids, Rituxan, or cyclosporine). Subjects with refractory TTP (approximately 20% of TTP patients) do not respond to plasmapheresis therapy for at least two weeks.

[0116] In accordance with the foregoing, in one aspect, a method is provided for treating a subject with an effective amount of a MASP-2 inhibitor (e.g., an anti-MASP-2 antibody) as a first-line therapy in the absence of plasmapheresis or in combination with plasmapheresis in the setting of an initial diagnosis of TTP or in a subject exhibiting one or more symptoms consistent with a diagnosis of TTP (e.g., central nervous system complications, severe thrombocytopenia (platelet count of less than or equal to 5000 / μL if not taking aspirin, or less than or equal to 20,000 / μL if taking aspirin), severe cardiac complications, severe pulmonary complications, gastrointestinal infarction, or gangrene).As a first-line therapy, the MASP-2 inhibitor may be administered systemically to the subject, for example, by intra-arterial, intravenous, intramuscular, inhaled, nasal, subcutaneous, or other parenteral administration. In some embodiments, the MASP-2 inhibitor is administered to a subject as a 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 are otherwise reluctant to undergo plasmapheresis, or in situations where plasmapheresis is unavailable. In some embodiments, the MASP-2 inhibitor is administered to a subject suffering from TTP in combination (including co-administration) with an immunosuppressant (e.g., corticosteroid, Rituxan, or cyclosporine) and / or in combination with high concentrations of ADAMTS-13.

[0117] In some embodiments, the method includes administering a MASP-2 inhibitor via a catheter (e.g., intravenously) to a subject suffering from TTP for a first period (e.g., an acute period lasting at least 1 day to 1 or 2 weeks), followed by subcutaneous administration of the MASP-2 inhibitor to the subject for a second period (e.g., a chronic period lasting at least 2 weeks or more). In some embodiments, administration during the first and / or second periods is carried out in the absence of plasmapheresis. In some embodiments, the method is used to prevent the subject from suffering from one or more symptoms associated with TTP.

[0118] In another embodiment, a method is provided for treating a subject suffering from refractory TTP (i.e., a subject who does not respond to at least two weeks of plasmapheresis therapy) by administering an amount of a MASP-2 inhibitor effective to alleviate one or more symptoms of TTP. In one embodiment, a MASP-2 inhibitor (e.g., an anti-MASP-2 antibody) is administered chronically to a subject with refractory TTP via subcutaneous or other parenteral administration for a period of at least two weeks or longer. Administration may be repeated as determined by a physician until the condition improves or is controlled.

[0119] In some embodiments, the method further comprises determining the level of at least one complement factor (e.g., C3, C5) in the subject before treatment and, optionally, during treatment, wherein determining a decreased 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.

[0120] In some embodiments, the method includes subcutaneously or intravenously administering 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 administered less frequently, 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.

[0121] In one embodiment, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: the antibody has a K D the antibody binds to human MASP-2 at an IC of 10 nM or less in an in vitro assay in 1% human serum; the antibody binds to an epitope in the CCP1 domain of MASP-2; the antibody inhibits C3b deposition in 1% human serum at an IC of 10 nM or less in an in vitro assay in 50 and the antibody inhibits C3b deposition in 90% human serum with an IC of 30 nM or less. 50 the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2; the antibody is a single-chain molecule; the antibody is an IgG2 molecule; the antibody is an IgG1 molecule; the antibody is an IgG4 molecule containing an S228P mutation; and / or the antibody does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway without substantially inhibiting the alternative pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway without substantially inhibiting either the classical or alternative pathway (i.e., inhibits the lectin pathway while leaving the classical and alternative complement pathways intact).

[0122] In one embodiment, the MASP-2 inhibitory antibody inhibits clot formation in serum from a subject suffering from TTP by at least 30%, for example, at least 40%, for example, at least 50%, for example, at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95% up to 99% compared to untreated serum. In some embodiments, the MASP-2 inhibitory antibody inhibits clot formation in serum from a subject suffering from TTP at a level at least 20 percent or more (e.g., at least 30%, at least 40%, at least 50%) higher than its inhibitory effect on C5b-9 deposition in serum.

[0123] In one aspect, the MASP-2 inhibitory antibody inhibits clot formation in serum from a TTP patient by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% up to 99% compared to untreated serum.

[0124] In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous catheter or other catheter delivery method.

[0125] In one aspect, the present invention provides a method for inhibiting thrombus formation in a subject suffering from TTP, comprising the step of administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises: (I) (a) a heavy chain variable region comprising: (i) a heavy chain CDR-H1 comprising the amino acid sequence of 31 to 35 of SEQ ID NO:67; and (ii) a heavy chain CDR-H2 comprising the amino acid sequence of 50 to 65 of SEQ ID NO:67; and (iii) a heavy chain CDR-H3 comprising the amino acid sequence of 95 to 102 of SEQ ID NO:67; and (b) a light chain CDR-L1 comprising the amino acid sequence of 24 to 34 of SEQ ID NO:70; and (ii) a light chain CDR-L2 comprising the amino acid sequence of 50 to 56 of SEQ ID NO:70; and (iii) a light chain CDR-L3 comprising the amino acid sequence of 95 to 102 of SEQ ID NO:70. (II) a light chain variable region comprising a light chain CDR-L3 comprising the sequence of amino acids 89-97 of SEQ ID NO:70, or (II) a heavy chain variable region having at least 90% identity to SEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:67) and a variant thereof comprising a light chain variable region having at least 90% identity to SEQ ID NO:70 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity).

[0126] In some embodiments, the method includes administering to a subject a composition comprising an amount of a MASP-2 inhibitory antibody or its antigen-binding fragment, which comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 67. In some embodiments, the method includes administering to a subject a composition comprising an amount of a MASP-2 inhibitory antibody or its antigen-binding fragment, which comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0127] In some embodiments, the method includes administering to a subject a composition comprising a MASP-2 inhibitory antibody or its antigen-binding fragment that specifically recognizes at least a portion of the epitope on human MASP-2 recognized by reference antibody OMS646, which comprises a heavy chain variable region set forth in SEQ ID NO:67 and a light chain variable region set forth in SEQ ID NO:70.

[0128] Degos disease Degos disease, also known as malignant atrophic papulosis, is an extremely rare TMA that affects the endothelium of small blood vessels in the skin, gastrointestinal tract, and CNS. This vasculopathy causes occlusion of venules and arterioles, resulting in skin lesions, intestinal ischemia, and CNS disorders, including stroke, epilepsy, and cognitive impairment. Connective tissue necrosis in the skin is due to thrombotic occlusion of small arteries. However, the cause of Degos disease is unknown. Vasculitis, coagulopathy, or primary endothelial dysfunction have been implicated. The 50% survival rate for Degos disease is only 2–3 years. While there is no effective treatment for Degos disease, antiplatelet drugs, anticoagulants, and immunosuppressants are used to alleviate symptoms.

[0129] Although the mechanism of Degos disease is unknown, the complement pathway has been implicated. Margo et al. identified prominent C5b-9 deposition in the skin, gastrointestinal, and cerebral vessels of four patients with end-stage Degos disease (Margo et al., Am J Clin Pathol 135(4):599-610, 2011). Experimental treatment with eculizumab was initially effective in treating skin and intestinal lesions but did not prevent systemic disease progression (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).

[0130] Many patients with Degos disease have a blood clotting defect. Thrombotic occlusion of small arteries in the skin is characteristic of this disease. Because the complement pathway has been implicated in 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 treating patients with Degos disease.

[0131] Therefore, in another aspect, the present invention provides a method for treating Degos 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 Degos disease or a condition resulting from Degos disease. The MASP-2 inhibitor is administered systemically to a subject suffering from Degos disease or a condition resulting from Degos disease, for example, by intra-arterial, intravenous, intramuscular, inhalation, subcutaneous, or other parenteral administration, or, in the case of non-peptide agents, by oral administration. The anti-MASP-2 antibody may be administered alone or in combination with a C5 inhibitor, such as eculizamab.

[0132] In one embodiment, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: the antibody has a K D the antibody binds to human MASP-2 at an IC of 10 nM or less in an in vitro assay in 1% human serum; the antibody binds to an epitope in the CCP1 domain of MASP-2; the antibody inhibits C3b deposition in 1% human serum at an IC of 10 nM or less in an in vitro assay in 50 and the antibody inhibits C3b deposition in 90% human serum with an IC of 30 nM or less. 50 the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2; the antibody is a single-chain molecule; the antibody is an IgG2 molecule; the antibody is an IgG1 molecule; the antibody is an IgG4 molecule containing an S228P mutation; and / or the antibody does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway without substantially inhibiting the alternative pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway without substantially inhibiting either the classical or alternative pathway (i.e., inhibits the lectin pathway while leaving the classical and alternative complement pathways intact).

[0133] In one embodiment, the MASP-2 inhibitory antibody inhibits clot formation in serum from a subject suffering from 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 99% compared to untreated serum. In some embodiments, the MASP-2 inhibitory antibody inhibits clot formation in serum from a subject suffering from Degos disease at a level at least 20 percent or more (e.g., at least 30%, at least 40%, at least 50%) higher than its inhibitory effect on C5b-9 deposition in serum.

[0134] In one aspect, the MASP-2 inhibitory antibody inhibits clot formation in serum from Degos disease patients by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% up to 99% compared to untreated serum.

[0135] In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous catheter or other catheter delivery method.

[0136] In one aspect, the present invention provides a method for inhibiting thrombus formation in a subject suffering from Degos disease, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises: (I) (a) a heavy chain variable region comprising: (i) a heavy chain CDR-H1 comprising the amino acid sequence of 31 to 35 of SEQ ID NO:67; and (ii) a heavy chain CDR-H2 comprising the amino acid sequence of 50 to 65 of SEQ ID NO:67; and (iii) a heavy chain CDR-H3 comprising the amino acid sequence of 95 to 102 of SEQ ID NO:67; and (b) (i) a light chain CDR-L1 comprising the amino acid sequence of 24 to 34 of SEQ ID NO:70; and (ii) a light chain CDR-L2 comprising the amino acid sequence of 50 to 56 of SEQ ID NO:70; and (iii) a light chain CDR-L3 comprising the amino acid sequence of 95 to 102 of SEQ ID NO:70. (II) a light chain variable region comprising a light chain CDR-L3 comprising the sequence of amino acids 89-97 of SEQ ID NO:70, or (II) a heavy chain variable region having at least 90% identity to SEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:67) and a variant thereof comprising a light chain variable region having at least 90% identity to SEQ ID NO:70 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity).

[0137] In some embodiments, the method includes administering to a subject a composition comprising an amount of a MASP-2 inhibitory antibody or its antigen-binding fragment, which comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 67. In some embodiments, the method includes administering to a subject a composition comprising an amount of a MASP-2 inhibitory antibody or its antigen-binding fragment, which comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0138] In some embodiments, the method includes administering to a subject a composition comprising a MASP-2 inhibitory antibody or its antigen-binding fragment that specifically recognizes at least a portion of the epitope on human MASP-2 recognized by reference antibody OMS646, which comprises a heavy chain variable region set forth in SEQ ID NO:67 and a light chain variable region set forth in SEQ ID NO:70.

[0139] Fulminant antiphospholipid syndrome (CAPS) Fulminant antiphospholipid syndrome (CAPS) is an extreme variant of antiphospholipid antibody (APLA) syndrome. CAPS is characterized by venous and arterial thrombosis due to pathogenic antibodies. CAPS is a TMA associated with multiorgan thrombosis, ischemia, and organ failure. Like other TMAs, it is characterized by occlusion of small blood vessels in various organs. The mortality rate of CAPS is high, approximately 50%, and is often related to infection or trauma. Patients have antiphospholipid antibodies, commonly IgG.

[0140] Clinically, CAPS involves at least three organs or tissues with histopathological evidence of small-vessel occlusion. Peripheral thrombosis may involve veins and arteries in the CNS, cardiovascular, renal, or pulmonary systems. Patients are treated with antibiotics, anticoagulants, corticosteroids, plasma exchange, and intravenous immunoglobulin. Nevertheless, death may occur due to multiple organ failure.

[0141] The complement pathway is involved in CAPS. For example, studies in animal models have shown that complement inhibition can be an effective means of preventing CAPS-associated thrombosis (Shapira L. et al., Arthritis Rheum 64(8):2719-23, 2012). Furthermore, as further reported by Shapira et al., administering a dose of eculizumab that blocks the complement pathway 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 treating patients with CAPS.

[0142] Therefore, in another aspect, 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 a subject suffering from CAPS or a condition caused by CAPS. The MASP-2 inhibitor is administered systemically to a subject suffering from CAPS or a condition caused by CAPS, for example, by intra-arterial, intravenous, intramuscular, inhalation, subcutaneous, or other parenteral administration, or optionally by oral administration in the case of non-peptide agents. The anti-MASP-2 antibody may be administered alone or in combination with a C5 inhibitor, such as eculizamab.

[0143] In one embodiment, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: the antibody has a K D the antibody binds to human MASP-2 at an IC of 10 nM or less in an in vitro assay in 1% human serum; the antibody binds to an epitope in the CCP1 domain of MASP-2; the antibody inhibits C3b deposition in 1% human serum at an IC of 10 nM or less in an in vitro assay in 50 and the antibody inhibits C3b deposition in 90% human serum with an IC of 30 nM or less.50 the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2; the antibody is a single-chain molecule; the antibody is an IgG2 molecule; the antibody is an IgG1 molecule; the antibody is an IgG4 molecule containing an S228P mutation; and / or the antibody does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway without substantially inhibiting the alternative pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway without substantially inhibiting either the classical or alternative pathway (i.e., inhibits the lectin pathway while leaving the classical and alternative complement pathways intact).

[0144] In one embodiment, the MASP-2 inhibitory antibody inhibits clot formation in serum from a subject suffering from CAPS 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 99% compared to untreated serum. In some embodiments, the MASP-2 inhibitory antibody inhibits clot formation in serum from a subject suffering from CAPS at a level at least 20 percent or more (e.g., at least 30%, at least 40%, at least 50%) higher than its inhibitory effect on C5b-9 deposition in serum.

[0145] In one aspect, the MASP-2 inhibitory antibody inhibits clot formation in serum from a CAPS patient by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% up to 99% compared to untreated serum.

[0146] In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous catheter or other catheter delivery method.

[0147] In one aspect, the present invention provides a method for inhibiting thrombus formation in a subject suffering from CAPS, comprising the step of administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises: (I) (a) a heavy chain variable region comprising: (i) a heavy chain CDR-H1 comprising the amino acid sequence of 31 to 35 of SEQ ID NO:67; and (ii) a heavy chain CDR-H2 comprising the amino acid sequence of 50 to 65 of SEQ ID NO:67; and (iii) a heavy chain CDR-H3 comprising the amino acid sequence of 95 to 102 of SEQ ID NO:67; and (b) (i) a light chain CDR-L1 comprising the amino acid sequence of 24 to 34 of SEQ ID NO:70; and (ii) a light chain CDR-L2 comprising the amino acid sequence of 50 to 56 of SEQ ID NO:70; and (iii) a light chain CDR-L3 comprising the amino acid sequence of 95 to 102 of SEQ ID NO:70. (II) a light chain variable region comprising a light chain CDR-L3 comprising the sequence of amino acids 89-97 of SEQ ID NO:70, or (II) a heavy chain variable region having at least 90% identity to SEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:67) and a variant thereof comprising a light chain variable region having at least 90% identity to SEQ ID NO:70 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity).

[0148] In some embodiments, the method includes administering to a subject a composition comprising an amount of a MASP-2 inhibitory antibody or its antigen-binding fragment, which comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 67. In some embodiments, the method includes administering to a subject a composition comprising an amount of a MASP-2 inhibitory antibody or its antigen-binding fragment, which comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0149] In some embodiments, the method includes administering to a subject a composition comprising a MASP-2 inhibitory antibody or its antigen-binding fragment that specifically recognizes at least a portion of the epitope on human MASP-2 recognized by reference antibody OMS646, which comprises a heavy chain variable region set forth in SEQ ID NO:67 and a light chain variable region set forth in SEQ ID NO:70.

[0150] TMA secondary to cancer Any type of systemic malignancy can cause the clinical and pathological manifestations of TMA (see, e.g., Batts and Lazarus, Bone Marrow Transplantation 40:709-719, 2007). Cancer-associated TMA is often found in the lung and appears to be associated with tumor emboli (Francis KK et al., Commun Oncol 2:339-43, 2005). Tumor emboli can reduce blood flow and thus lead to hypoperfusion in affected small arteries and venules. The resulting tissue stress and injury are expected to locally activate the lectin pathway of complement. The activated lectin pathway can then activate the coagulation cascade via MASP-2-dependent cleavage of prothrombin to thrombin, leading to the prothrombotic state characteristic of TMA. Inhibiting MASP-2 in this setting is expected to reduce local thrombin activation, thereby alleviating the prothrombotic state.

[0151] Thus, as described herein for other TMAs, lectin pathway inhibitors, including but not limited to antibodies that block MASP-2 function, are expected to be beneficial in treating patients with TMAs secondary to cancer.

[0152] Therefore, in another aspect, the present invention provides a method for treating or preventing TMA secondary to cancer 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 or at risk of developing TMA secondary to cancer. The MASP-2 inhibitor is administered systemically to a subject suffering from or at risk of developing TMA secondary to cancer, for example, by intra-arterial, intravenous, intramuscular, inhalation, subcutaneous, or other parenteral administration, or optionally by oral administration in the case of non-peptide agents. The anti-MASP-2 antibody may be administered alone or in combination with a C5 inhibitor, such as eculizamab.

[0153] In one embodiment, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: the antibody has a K D the antibody binds to human MASP-2 at an IC of 10 nM or less in an in vitro assay in 1% human serum; the antibody binds to an epitope in the CCP1 domain of MASP-2; the antibody inhibits C3b deposition in 1% human serum at an IC of 10 nM or less in an in vitro assay in 50 and the antibody inhibits C3b deposition in 90% human serum with an IC of 30 nM or less. 50 the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2; the antibody is a single-chain molecule; the antibody is an IgG2 molecule; the antibody is an IgG1 molecule; the antibody is an IgG4 molecule containing an S228P mutation; and / or the antibody does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway without substantially inhibiting the alternative pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway without substantially inhibiting either the classical or alternative pathway (i.e., inhibits the lectin pathway while leaving the classical and alternative complement pathways intact).

[0154] In one aspect, the MASP-2 inhibitory antibody inhibits clot formation in serum from a subject suffering from TMA secondary to cancer 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 99% compared to untreated serum.

[0155] In one aspect, the MASP-2 inhibitory antibody inhibits clot formation in serum from a subject suffering from TMA secondary to cancer 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 99% compared to untreated serum.

[0156] In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous catheter or other catheter delivery method.

[0157] In one aspect, the present invention provides 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 an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises: (I) (a) a heavy chain variable region comprising: (i) a heavy chain CDR-H1 comprising the amino acid sequence of 31 to 35 of SEQ ID NO:67; and (ii) a heavy chain CDR-H2 comprising the amino acid sequence of 50 to 65 of SEQ ID NO:67; and (iii) a heavy chain CDR-H3 comprising the amino acid sequence of 95 to 102 of SEQ ID NO:67; and (b) a light chain CDR-L1 comprising the amino acid sequence of 24 to 34 of SEQ ID NO:70; and (ii) a light chain CDR-L2 comprising the amino acid sequence of 50 to 56 of SEQ ID NO:70; and (iii) a light chain CDR-L3 comprising the amino acid sequence of 95 to 102 of SEQ ID NO:70. (II) a light chain variable region comprising a light chain CDR-L3 comprising the sequence of amino acids 89-97 of SEQ ID NO:70, or (II) a heavy chain variable region having at least 90% identity to SEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:67) and a variant thereof comprising a light chain variable region having at least 90% identity to SEQ ID NO:70 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity).

[0158] In some embodiments, the method includes administering to a subject a composition comprising an amount of a MASP-2 inhibitory antibody or its antigen-binding fragment, which comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 67. In some embodiments, the method includes administering to a subject a composition comprising an amount of a MASP-2 inhibitory antibody or its antigen-binding fragment, which comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0159] In some embodiments, the method includes administering to a subject a composition comprising a MASP-2 inhibitory antibody or its antigen-binding fragment that specifically recognizes at least a portion of the epitope on human MASP-2 recognized by reference antibody OMS646, which comprises a heavy chain variable region set forth in SEQ ID NO:67 and a light chain variable region set forth in SEQ ID NO:70.

[0160] TMA secondary to cancer chemotherapy Chemotherapy-associated TM (TMA) is a condition characterized by thrombocytopenia, microangiopathic hemolytic anemia, and renal dysfunction that occurs in 2-10% of patients with a history of malignant neoplasms treated with chemotherapy agents such as gemcitabine, mitomycin, or oxaliplatin. Postchemotherapy TMA is associated with high mortality and poor clinical outcomes (see, e.g., Blake-Haskins et al., Clin Cancer Res 17(18):5858-5866, 2011).

[0161] The cause of post-chemotherapy TMA is thought to involve nonspecific toxic damage to the microvascular endothelium. Direct damage to endothelial cells has been demonstrated in an animal model of mitomycin-induced TMA (Dlott J. et al., Ther Apher Dial 8:102-11, 2004). Endothelial cell damage via various mechanisms has been shown to activate the lectin pathway of complement. For example, Stahl et al. have shown that endothelial cells exposed to oxidative stress activate the lectin pathway of complement both in vitro and in vivo (Collard et al., Am J Pathol. 156(5):1549-56, 2000; La Bonte et al., J Immunol. 15;188(2):885-91, 2012). In vivo, this process leads to thrombosis, and inhibition of the lectin pathway has been shown to prevent thrombosis (La Bonte et al. J Immunol. 15;188(2):885-91, 2012). Furthermore, as demonstrated in Examples 37-39 herein, in a mouse model of TMA in which localized photoexcitation of FITC-Dex is used to induce localized damage to the microvasculature and subsequent development of a TMA response, the inventors have shown that MASP-2 inhibition can prevent TMA. Thus, microvascular endothelial injury caused by chemotherapeutic agents can activate the complement lectin pathway, which then generates a localized prothrombotic state, promoting the TMA response. Because activation of the lectin pathway and the generation of a prothrombotic state are MASP-2 dependent, MASP-2 inhibitors, including but not limited to antibodies that block MASP-2 function, are expected to attenuate the TMA response and reduce the risk of TMA after cancer chemotherapy.

[0162] Therefore, in another aspect, the present invention provides a method for treating or preventing TMA secondary to chemotherapy 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 or at risk of developing TMA secondary to chemotherapy. The MASP-2 inhibitor is administered systemically to a subject who has undergone chemotherapy, is undergoing chemotherapy, or is scheduled to undergo chemotherapy, for example, by intra-arterial, intravenous, intramuscular, inhalation, subcutaneous, or other parenteral administration, or possibly by oral administration in the case of non-peptide agents. The anti-MASP-2 antibody may be administered alone or in combination with a C5 inhibitor, such as eculizamab.

[0163] In one embodiment, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: the antibody has a K D the antibody binds to human MASP-2 at an IC of 10 nM or less in an in vitro assay in 1% human serum; the antibody binds to an epitope in the CCP1 domain of MASP-2; the antibody inhibits C3b deposition in 1% human serum at an IC of 10 nM or less in an in vitro assay in 50 and the antibody inhibits C3b deposition in 90% human serum with an IC of 30 nM or less. 50 the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2; the antibody is a single-chain molecule; the antibody is an IgG2 molecule; the antibody is an IgG1 molecule; the antibody is an IgG4 molecule containing an S228P mutation; and / or the antibody does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway without substantially inhibiting the alternative pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway without substantially inhibiting either the classical or alternative pathway (i.e., inhibits the lectin pathway while leaving the classical and alternative complement pathways intact).

[0164] In one aspect, the MASP-2 inhibitory antibody inhibits clot formation in serum from a subject suffering from TMA secondary to cancer chemotherapy by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% up to 99% compared to untreated serum.

[0165] In one aspect, the MASP-2 inhibitory antibody inhibits clot 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 99% compared to untreated serum.

[0166] In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous catheter or other catheter delivery method.

[0167] In one aspect, the present invention provides 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 an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises: (I) (a) a heavy chain variable region comprising: (i) a heavy chain CDR-H1 comprising the amino acid sequence of 31 to 35 of SEQ ID NO:67; and (ii) a heavy chain CDR-H2 comprising the amino acid sequence of 50 to 65 of SEQ ID NO:67; and (iii) a heavy chain CDR-H3 comprising the amino acid sequence of 95 to 102 of SEQ ID NO:67; and (b) a light chain CDR-L1 comprising the amino acid sequence of 24 to 34 of SEQ ID NO:70; and (ii) a light chain CDR-L2 comprising the amino acid sequence of 50 to 56 of SEQ ID NO:70; and (iii) a light chain CDR-L3 comprising the amino acid sequence of 95 to 102 of SEQ ID NO:70. (II) a light chain variable region comprising a light chain CDR-L3 comprising the sequence of amino acids 89-97 of SEQ ID NO:70, or (II) a heavy chain variable region having at least 90% identity to SEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:67) and a variant thereof comprising a light chain variable region having at least 90% identity to SEQ ID NO:70 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity).

[0168] In some embodiments, the method includes administering to a subject a composition comprising an amount of a MASP-2 inhibitory antibody or its antigen-binding fragment, which comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 67. In some embodiments, the method includes administering to a subject a composition comprising an amount of a MASP-2 inhibitory antibody or its antigen-binding fragment, which comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0169] In some embodiments, the method includes administering to a subject a composition comprising a MASP-2 inhibitory antibody or its antigen-binding fragment that specifically recognizes at least a portion of the epitope on human MASP-2 recognized by reference antibody OMS646, which comprises a heavy chain variable region set forth in SEQ ID NO:67 and a light chain variable region set forth in SEQ ID NO:70.

[0170] TMA secondary to transplantation Transplantation-associated TMA (TA-TMA) is a devastating syndrome that can occur in transplant patients, such as allogeneic hematopoietic stem cell transplant recipients (see, e.g., Batts and Lazarus, Bone Marrow Transplantation 40:709-719, 2007). The cause of this condition is not fully understood but likely involves a confluence of responses that result in endothelial cell injury (Laskin BL et al., Blood 118(6):1452-62, 2011). As discussed above, endothelial cell injury is a typical stimulus for activation of the lectin pathway and the creation of a prothrombotic environment.

[0171] Recent data further support the role of complement activation via the lectin pathway in the development of TA-TMA. Laskin et al. demonstrated that renal arteriolar C4d deposition was significantly more prevalent in subjects with histological TA-TMA (75%) than in controls (8%) (Laskin BL, et al., Transplantation, 27; 96(2):217-23, 2013). Therefore, C4d may be a pathological marker of TA-TMA, implicating local complement binding via either the lectin or classical pathway.

[0172] Because activation of the lectin pathway and generation of a prothrombotic state are MASP-2 dependent, it is expected that MASP-2 inhibitors, including but not limited to antibodies that block MASP-2 function, may attenuate the TMA response and reduce the risk of post-transplant TMA (TA-TMA).

[0173] Therefore, in another aspect, the present invention provides a method for treating or preventing TMA secondary to transplantation 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 or at risk of developing TMA secondary to transplantation. The MASP-2 inhibitor is administered systemically to a subject who has undergone, is undergoing, or will undergo a transplant procedure, for example, by intra-arterial, intravenous, intramuscular, inhalation, subcutaneous, or other parenteral administration, or optionally by oral administration in the case of non-peptide agents. The anti-MASP-2 antibody may be administered alone or in combination with a C5 inhibitor, such as eculizamab. In some aspects, the present invention provides a method for treating or preventing TMA secondary to allogeneic stem cell transplantation, comprising administering to a subject a composition comprising an amount of a MASP-2 inhibitor, such as a MASP-2 inhibitor antibody, before, during, or after allogeneic stem cell transplantation.

[0174] In one embodiment, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: the antibody has a K D the antibody binds to human MASP-2 at an IC of 10 nM or less in an in vitro assay in 1% human serum; the antibody binds to an epitope in the CCP1 domain of MASP-2; the antibody inhibits C3b deposition in 1% human serum at an IC of 10 nM or less in an in vitro assay in 50 and the antibody inhibits C3b deposition in 90% human serum with an IC of 30 nM or less. 50the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2, and F(ab')2; the antibody is a single-chain molecule; the antibody is an IgG2 molecule; the antibody is an IgG1 molecule; the antibody is an IgG4 molecule containing an S228P mutation; and / or the antibody does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway without substantially inhibiting the alternative pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway without substantially inhibiting either the classical or alternative pathway (i.e., inhibits the lectin pathway while leaving the classical and alternative complement pathways intact).

[0175] In one aspect, the MASP-2 inhibitory antibody inhibits clot formation in serum from a subject suffering from TMA secondary to transplantation by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% up to 99% compared to untreated serum.

[0176] In one aspect, the MASP-2 inhibitory antibody inhibits clot formation in serum from a subject suffering from TMA secondary to transplantation by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% up to 99% compared to untreated serum.

[0177] In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous catheter or other catheter delivery method.

[0178] In one aspect, the present invention provides 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 an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises: (I) (a) a heavy chain variable region comprising: (i) a heavy chain CDR-H1 comprising the sequence of amino acids 31 to 35 of SEQ ID NO:67; and (ii) a heavy chain CDR-H2 comprising the sequence of amino acids 50 to 65 of SEQ ID NO:67; and (iii) a heavy chain CDR-H3 comprising the sequence of amino acids 95 to 102 of SEQ ID NO:67; and (b) a light chain CDR-L1 comprising the sequence of amino acids 24 to 34 of SEQ ID NO:70; and (ii) a light chain CDR-L2 comprising the sequence of amino acids 50 to 56 of SEQ ID NO:70; and (iii) a light chain CDR-L3 comprising the sequence of amino acids 95 to 102 of SEQ ID NO:70. (II) a light chain variable region comprising a light chain CDR-L3 comprising the sequence of amino acids 89-97 of SEQ ID NO:70, or (II) a heavy chain variable region having at least 90% identity to SEQ ID NO:67 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:67) and a variant thereof comprising a light chain variable region having at least 90% identity to SEQ ID NO:70 (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity).

[0179] In some embodiments, the method includes administering to a subject a composition comprising an amount of a MASP-2 inhibitory antibody or its antigen-binding fragment, which comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 67. In some embodiments, the method includes administering to a subject a composition comprising an amount of a MASP-2 inhibitory antibody or its antigen-binding fragment, which comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0180] In some embodiments, the method includes administering to a subject a composition comprising a MASP-2 inhibitory antibody or its antigen-binding fragment that specifically recognizes at least a portion of the epitope on human MASP-2 recognized by reference antibody OMS646, which comprises a heavy chain variable region set forth in SEQ ID NO:67 and a light chain variable region set forth in SEQ ID NO:70.

[0181] IV. The Role of MASP-2 in Other Diseases and Conditions and Methods of Treatment with MASP-2 Inhibitors Renal status Activation of the complement system is associated with the development of 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. Exp. Med. 175:939-50, 1992), acute postinfectious glomerulonephritis (poststreptococcal glomerulonephritis), cryoglobulinemic glomerulonephritis (Ohsawa, I., et al., Clin Immunol, 101:59-66, 2001), lupus nephritis (Gatenby, PA, Autoimmunity 11:61-6, 1991), and Henoch-Schönlein purpura nephritis (Endo, M., et al., Am. J. Kidney Dis. 35:401-407, 2000). Although the involvement of complement in kidney disease has been recognized for decades, its exact role in the onset, development, and recovery of kidney disease remains a matter of significant debate. Although the contribution of complement under normal conditions is beneficial to the host, inappropriate activation and deposition of complement can contribute to tissue damage.

[0182] There is ample evidence that glomerulonephritis, an inflammation of the glomerulus, is often initiated by the deposition of immune complexes in glomerular or tubular structures, which then induces complement activation, inflammation, and tissue damage. Kahn and Sinniah demonstrated increased C5b-9 deposition in the tubular basement membrane in biopsies taken from patients with various forms of glomerulonephritis (Kahn, TN, et al., Histopath. 26:351-6, 1995). In a study of patients with IgA nephropathy (Alexopoulos, A., et al., Nephrol. Dial. Transplant 10:1166-1172, 1995), C5b-9 deposition in the tubular epithelium / basement membrane structures correlated with plasma creatinine levels. Another study of membranous nephropathy demonstrated a correlation between clinical outcome 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 elevated levels of CD59, a complement regulator that inhibits the membrane attack complex in the plasma membrane, and C5b-9 in urine from patients with membranous glomerulonephritis (Lehto, T., et al., Kidney Int. 47; 1403-11, 1995). Histopathological analysis of biopsy specimens from these same patients demonstrated deposition of C3 and C9 proteins in the glomeruli, whereas CD59 expression in these tissues was reduced compared with normal kidney tissue. These various studies suggest that ongoing complement-mediated glomerulonephritis results in urinary excretion of complement proteins that correlates with the degree of tissue damage and disease prognosis.

[0183] Inhibition of complement activation in various animal models of glomerulonephritis also demonstrated the importance of complement activation in the pathogenesis of this disease. In a model of membranoproliferative glomerulonephritis (MPGN), injection of anti-Th1 antiserum into C6-deficient rats (which are unable to produce C5b-9) resulted in 90% less glomerular cell proliferation, 80% less platelet and macrophage infiltration, reduced collagen type IV synthesis (a marker of mesangial matrix expansion), and 50% less proteinuria than C6+ normal rats (Brandt, J., et al., Kidney Int. 49:335-343, 1996). These results implicate C5b-9 as the primary mediator of complement-mediated tissue injury in this rat anti-thymocyte serum model. In another glomerulonephritis model, injection of graded doses of rabbit anti-rat glomerular basement membrane antibody induced 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 histopathological changes, reduced long-term proteinuria, and lower creatinine levels than control rats. Using three rat GN models (antithymocyte serum, ConA anti-ConA, and passive Heymann nephritis), Couser et al. demonstrated the potential therapeutic efficacy of a complement-inhibiting approach using recombinant sCR1 protein (Couser, WG, et al., J. Am. Soc. Nephrol. 5:1888-94, 1995). Rats treated with sCR1 showed significantly reduced PMN, platelet, and macrophage influx, mesangial lysis, and proteinuria compared with control rats. Further evidence of the importance of complement activation in glomerulonephritis has been obtained using anti-C5 MoAb in the NZB / W F1 mouse model. Anti-C5 MoAb inhibits C5 cleavage, thus blocking the generation of C5a and C5b-9. Continuous therapy with anti-C5 MoAb for 6 months significantly ameliorated the course of glomerulonephritis.A humanized anti-C5 MoAb monoclonal antibody (5G1.1), which blocks the cleavage of human complement component C5 into pro-inflammatory components, is being developed by Alexion Pharmaceuticals, Inc., New Haven, Connecticut, as a potential treatment for glomerulonephritis.

[0184] Studies of patients with genetic deficiencies of specific complement components have provided direct evidence for the pathological role of complement in renal injury. Numerous reports have demonstrated a relationship between kidney disease and deficiency of the complement regulatory factor H (Ault, BH. Nephrol. 14:1045-1053, 2000; Levy, M., et al., Kidney Int. 30:949-56, 1986; Pickering, MC, et al., Nat. Genet. 31:424-8, 2002). Factor H deficiency results in decreased plasma levels of factor B and C3 and consumption of C5b-9. Both atypical membranoproliferative glomerulonephritis (MPGN) and idiopathic hemolytic uremic syndrome (HUS) have been associated with factor H deficiency. Factor H-deficient pigs (Jansen, JH, et al., Kidney Int. 55:331-49, 1998) and factor H knockout mice (Pickering, MC, 2002) exhibit MPGN-like symptoms, confirming 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 of C1q, C4, and C2 strongly predispose to the development of SLE through mechanisms related to defective clearance of immune complexes and apoptotic material. Many of these SLE patients develop lupus nephritis, characterized by immune complex deposition throughout the glomerulus.

[0185] Further evidence linking complement activation and kidney disease has been provided by the identification of autoantibodies against complement components in patients. Some of these autoantibodies have been directly associated with kidney disease (Trouw, LA, et al., Mol. Immunol. 38:199-206, 2001). Because many of these autoantibodies show such a high correlation with kidney disease, the term nephritic factor (NeF) has been introduced to describe this activity. In clinical studies, approximately 50% of nephritic factor-positive patients developed MPGN (Spitzer, RE et al., Clin. Immunol. Immunopathol. 64:177-83, 1992). C3NeF is an autoantibody directed against the alternative pathway C3 convertase (C3bBb), stabilizing this convertase and thereby promoting alternative pathway activation (Daha, MR, et al., J. Immunol. 116:1-7, 1976). Similarly, autoantibodies with specificity for the classical pathway C3 convertase (C4b2a), called C4NeF, stabilize this convertase, thereby promoting 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 patients with SLE (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 progression of nephritis (Coremans, I.E. et al., Am. J. Kidney Dis. 26:595-601, 1995).Immune deposits 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).

[0186] In addition to the alternative and classical pathways of complement activation, the lectin pathway may also play an important pathological role in kidney diseases. High levels of MBL, MBL-associated serine proteases, and complement activation products have been detected by immunohistochemical techniques in kidney biopsies obtained from patients diagnosed with several different kidney diseases, including Henoch-Schönlein purpura nephritis (Endo, M. et al., Am. J. Kidney Dis. 35:401-407, 2000), cryoglobulinemic glomerulonephritis (Ohsawa, I., et al., Clin. Immunol. 101:59-66, 2001), and IgA neuropathy (Endo, M., et al., Clin. Nephrology 55:185-191, 2001). Thus, despite the fact that the relationship between complement and kidney diseases has been known for decades, the data on exactly how complement influences these kidney diseases is far from complete.

[0187] blood disorders Sepsis is caused by a patient's overwhelming response to invading microorganisms. The primary function of the complement system is to orchestrate the inflammatory response to invading bacteria and other pathogens. Consistent with this physiological role, numerous studies have demonstrated that complement activation plays a major role in the pathogenesis of sepsis (Bone, RC, Annals. Internal, Med. 115:457-469, 1991). The definition of the clinical manifestations of sepsis is constantly evolving. Sepsis is typically defined as a systemic host response to infection. However, frequently, clinical evidence of infection (e.g., positive bacterial blood cultures) is not found in patients with septic symptoms. This discrepancy was first addressed at the 1992 Consensus Conference, when the term "systemic inflammatory response syndrome" (SIRS) was established, which no longer requires the presence of a definable bacterial infection (Bone, RC, et al., Crit. Care Med. 20:724-726, 1992). There is now general agreement that sepsis and SIRS involve a dysregulated inflammatory response. To briefly review this, we consider the clinical definition of sepsis to include severe sepsis, septic shock, and SIRS.

[0188] Before the late 1980s, the most prevalent source of infection in patients with sepsis was Gram-negative bacteria. Lipopolysaccharide (LPS), a major component of Gram-negative bacterial cell walls, was known to stimulate the release of inflammatory mediators from various cell types and induce acute infection when injected into animals (Haeney, MR, et al., Antimicrobial Chemotherapy 41(Suppl. A):41-6, 1998). Interestingly, the spectrum of causative microorganisms appears to have shifted from predominantly Gram-negative bacteria in the late 1970s and 1980s to predominantly Gram-positive bacteria today, for reasons that remain unclear (Martin, GS, et al., N. Eng. J. Med. 348:1546-54, 2003).

[0189] Numerous studies have demonstrated the importance of complement activation in mediating inflammation and contributing to the characteristics of shock, particularly septic and hemorrhagic shock. Both Gram-negative and Gram-positive organisms commonly cause septic shock. LPS is a potent complement activator, primarily via the alternative pathway, although antibody-mediated classical pathway activation also occurs (Fearon, DT, et al., N. Engl. J. Med. 292:937-400, 1975). The major components of Gram-positive cell walls are peptidoglycan and lipoteichoic acid, both of which are potent alternative pathway activators, but can also activate the classical pathway in the presence of specific antibodies (Joiner, KA, et al., Ann. Rev. Immunol. 2:461-2, 1984).

[0190] The complement system was first implicated in the development of sepsis when researchers realized that the anaphylatoxins C3a and C5a mediate various inflammatory responses that can occur during sepsis. These anaphylatoxins induce vasodilation and increased microvascular permeability, events that play a central role in septic shock (Schumacher, WA, et al., Agents Actions 34:345-349, 1991). Anaphylatoxins also induce bronchospasm, histamine release from mast cells, and platelet aggregation. Furthermore, they exert numerous effects on granulocytes, including 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 sepsis 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 the 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 injection (Hseuh, W, et al., Immunol. 70:309-14, 1990).

[0191] Blockade of C5a production with antibodies during the onset of sepsis in rodents has been shown to significantly improve survival (Czermak, BJ, et al., Nat. Med. 5:788-792, 1999). Similar findings were observed when the C5a receptor (C5aR) was blocked with 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 suggested that antibody blockade of C5a attenuated E. coli-induced septic shock and adult respiratory distress syndrome (Hangen, DH et al., J. Surg. Res. 46:195-9, 1989; Stevens, JH, et al., J. Clin. Invest. 77:1812-16, 1986). C5a is elevated in humans with sepsis and has been associated with significantly lower survival rates with multiple organ failure compared to patients and survivors with less severe sepsis (Nakae, H, et al., Res. Commun. Chem. Pathol. Pharmacol. 84:189-95, 1994; Nakae, et al., Surg. Today 26:225-29, 1996; Bengtson, A., et al., Arch. Surg. 123:645-649, 1988). Although the mechanisms by which C5a exerts its deleterious effects during sepsis have not yet been fully explored, recent data suggest that C5a generation 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 express the respiratory burst, and their ability to produce cytokines (Riedemann, NC, et al., Immunity 19:193-202, 2003).Furthermore, C5a generation during sepsis appears to have procoagulant effects (Laudes, IJ, et al., Am. J. Pathol. 160:1867-75, 2002). The complement regulatory protein CI INH has also shown efficacy in animal models of sepsis and ARDS (Dickneite, G., Behring Ins. Mitt. 93:299-305, 1993).

[0192] 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-ficolin specifically binds to LTA isolated from numerous 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 the polyglycerophosphate chain is substituted with glycosyl groups, but not to LTA from nine other species, including Staphylococcus aureus (Polotsky, VY, et al., Infect. Immun. 64:380, 1996).

[0193] Thus, one aspect of the present invention provides a method for treating sepsis or a sepsis-induced condition, including, but not limited to, severe sepsis, septic shock, sepsis-induced acute respiratory distress syndrome, and systemic inflammatory response syndrome, 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-induced condition. Related methods are 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 hemorrhagic shock, hemolytic anemia, autoimmune thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), or other bone marrow / blood destruction conditions. The MASP-2 inhibitor is administered to the subject systemically, for example, by intra-arterial, intravenous, intramuscular, inhalation (particularly in the case of ARDS), subcutaneous, or other parenteral administration, or, in the case of non-peptide agents, by oral administration. The MASP-2 inhibitor composition may be combined with one or more additional therapeutic substances to combat the sequelae of sepsis and / or shock. In the case of advanced sepsis or shock or distress resulting therefrom, the MASP-2 inhibitor composition may be administered in a fast-acting dosage form, for example, by intravenous or intra-arterial delivery of a bolus of a solution containing the MASP-2 inhibitor composition. Repeated administration may be administered as determined by a physician until the condition is resolved.

[0194] Coagulation disorders Evidence has emerged for a role for the complement system in disseminated intravascular coagulation ("DIC"), for example, DIC secondary to significant physical trauma.

[0195] 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 examine whether C4- / - mice could activate complement via either the classical or lectin pathway, C3 turnover in C4- / - plasma was measured in assays specific for the classical or lectin pathway. Although no C3 cleavage was observed upon induction of activation via the classical pathway, highly efficient lectin pathway-dependent C3 activation was observed in C4-deficient serum (Figure 30). In accordance with many previously published studies on alternative pathway activation, we found that C3b deposition on mannan and zymosan was severely impaired in MASP-2- / - mice, even under experimental conditions permissive 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 alternative pathway activation is promoted in MASP-2- / - serum when initial C3b is provided via classical activation. Figure 30C shows the surprising finding that C3 can be efficiently activated in C4-deficient plasma in a lectin pathway-dependent manner.

[0196] This "C4 bypass" is abolished by inhibiting lectin pathway activation by preincubation of plasma with soluble mannan or mannose.

[0197] Abnormal non-immune activation of the complement system is potentially harmful to humans and may also play an important role in hematologic pathway activation, particularly in severe trauma situations in which both inflammatory and hematologic pathways are activated. In normal health, C3 conversion accounts for <5% of total plasma C3 protein. During severe infections, including sepsis and immune complex diseases, C3 conversion spontaneously recovers to approximately 30%, and complement levels are often lower than normal due to increased utilization and altered pool distribution. Rapid C3 pathway activation above 30% typically exhibits clear clinical evidence of vasodilation and fluid loss to tissues. With C3 conversion above 30%, the initiating mechanism is primarily non-immune, and the resulting clinical signs are harmful to the patient. Complement C5 levels in health and controlled disease appear to be significantly more stable than C3. A significant decrease in C5 levels and / or conversion is associated with the patient's response to unusual multiple trauma (e.g., traffic accidents) and the likely development of shock pulmonary syndrome. Therefore, any evidence of complement C3 activation in greater than 30% of the vascular pool, or any C5-mediated complement C3 activation, or both, can be considered a likely harbinger of adverse pathological changes in the patient.

[0198] Both C3 and C5 liberate anaphylatoxins (C3a and C5a) that act on mast cells and basophils, causing them to release vasodilatory chemicals. They establish a chemotactic gradient that directs polymorphonuclear cells (PMNs) to the center of immunological disruption (a beneficial response), but they differ because C5a exerts a specific clumping effect on these phagocytes, preventing them from randomly leaving the site of the reaction. During normal infection control, C3 activates C5. However, during multiple trauma, C5 appears to be activated extensively, producing C5a anaphylatoxin throughout the body. This uncontrolled activity results in the accumulation of polymorphs within the vasculature, which then displace and obstruct pulmonary capillaries, causing local damage as a result of superoxide release. Without wishing to be bound by theory, this mechanism is likely important in the pathogenesis of acute respiratory distress syndrome (ARDS). However, this idea has recently been challenged. Although C3a anaphylatoxin can be shown to be a potent platelet aggregator in vitro, their role in vivo is less clear, and release of platelet material and plasmin in wound healing may only secondarily involve complement C3. Prolonged increased C3 activation may be required to produce DIC.

[0199] In addition to the cellular and vascular effects of activated complement components outlined above, which may explain the relationship between trauma and DIC, emerging scientific discoveries have identified direct molecular relationships and functional crosstalk between the complement and coagulation systems. Supporting data comes from studies in C3-deficient mice. Because C3 is a common component of each complement pathway, C3-deficient mice would be expected to lack all complement functions. Surprisingly, however, C3-deficient mice are fully capable of activating terminal complement components (Huber-Lang, M., et al., "Generation of C5a in the absence of C3: a new complement activation pathway," Nat. Med. 12:682-687 (2006)). In-depth studies have revealed that C3-independent activation of terminal complement components is mediated by thrombin, the rate-limiting enzyme in the coagulation cascade (Huber et al., 2006). The molecular components mediating thrombin activation after initial complement activation remain a mystery.

[0200] We have elucidated the molecular basis of the potential crosstalk between the complement and coagulation cascades and identified MASP-2 as a central control point linking the two systems. Biochemical studies of the substrate specificity of MASP-2 identified prothrombin as a potential substrate, in addition to the well-known C2 and C4 complement proteins. MASP-2 specifically cleaves a functionally relevant site on prothrombin to generate thrombin, the rate-limiting enzyme in the coagulation cascade (Krarup, A., et al., "Simultaneous Activation of Complement and Coagulation by MBL-Associated Serine Protease 2," PLoS. ONE. 2;e623(2007)). Thrombin generated by MASP-2 can promote fibrin deposition in a defined reconstituted in vitro system, demonstrating the functional relevance of MASP-2 cleavage (Krarup et al., 2007). As discussed in the Examples below, the inventors further confirmed the physiological significance of this finding by documenting thrombin activation in normal rodent serum following lectin pathway activation and demonstrated that this process was blocked by a neutralizing MASP-2 monoclonal antibody.

[0201] MASP-2 may be a central branch point in the lectin pathway, which can promote activation of both the complement and coagulation systems. Because lectin pathway activation is a physiological response to many types of traumatic injury, the inventors believe that the concurrent systemic inflammation (mediated by complement components) and disseminated coagulation (mediated via the coagulation pathway) may be explained by the ability of MASP-2 to activate both pathways. These findings clearly suggest a role for MASP-2 in the development of DIC and the therapeutic benefit of MASP-2 inhibition in the treatment or prevention of DIC. MASP-2 may provide a molecular link between the complement and coagulation systems, and lectin pathway activation, as occurs in the setting of trauma, can directly initiate coagulation 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 believed to inhibit lectin pathway activation and reduce the generation of anaphylatoxins C3a and C5a. Prolonged increased C3 activation is believed to be necessary for the development of DIC.

[0202] Microcirculatory coagulation (blood clots in capillaries and small blood vessels) occurs in this setting of septic shock. As described in Example 17 and Figures 18 and 19, the role of the lectin pathway in septic shock has been demonstrated, as evidenced by the protective phenotype of the MASP-2(- / -) mouse model of sepsis. Furthermore, as demonstrated in Example 15 and Figures 16A and 16B, MASP-2(- / -) mice are protected in the focal Shwartzman reaction model of disseminated intravascular coagulation (DIC), a model of focal coagulation in the microvasculature.

[0203] V. MASP-2 Inhibitors In one aspect, the present invention provides a method for inhibiting MASP-2-dependent complement activation in a subject 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 a living subject. In implementing this aspect of the present invention, representative MASP-2 inhibitors include molecules that inhibit the biological activity of MASP-2 (e.g., small molecule inhibitors, anti-MASP-2 antibodies, or blocking peptides that interact with MASP-2 or disrupt protein-protein interactions), as well as molecules that reduce MASP-2 expression and thereby prevent 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 primary therapy or in combination with other therapeutic agents as adjunctive therapy to improve the therapeutic benefits of other medical treatments.

[0204] Inhibition of MASP-2-dependent complement activation is characterized by at least one of the following changes in complement system components that occur as a result of administration of a MASP-2 inhibitor according to the methods of the present invention: inhibition of the generation or production of MASP-2-dependent complement activation system products C4b, C3a, C5a, and / or C5b-9 (MAC) (e.g., measured as described in Example 2), a decrease in complement activation assessed in a hemolytic assay using unsensitized rabbit red blood cells or guinea pig red blood cells (e.g., measured as described in Example 33), a decrease in C4 cleavage and C4b deposition (e.g., measured as described in Example 2), or a decrease in C3 cleavage and C3b deposition (e.g., measured as described in Example 2).

[0205] According to the present invention, MASP-2 inhibitors effective in inhibiting the MASP-2-dependent complement activation system are used. MASP-2 inhibitors useful in practicing this aspect of the present invention include, for example, anti-MASP-2 antibodies and fragments thereof, MASP-2 inhibitory peptides, small molecules, MASP-2 soluble receptors, and expression inhibitors. MASP-2 inhibitors may inhibit the MASP-2-dependent complement activation system by blocking the biological function of MASP-2. For example, inhibitors may effectively block MASP-2 protein-protein interactions, interfere with MASP-2 dimerization or aggregation, or inhibit Ca. 2+ It may block binding, may interfere with the MASP-2 serine protease active site, and may reduce MASP-2 protein expression.

[0206] In some embodiments, the MASP-2 inhibitor selectively inhibits MASP-2 complement activation without impairing the function of the C1q-dependent complement activation system.

[0207] In one embodiment, the MASP-2 inhibitor useful in the methods of the present invention is a specific MASP-2 inhibitor that specifically binds to a polypeptide comprising SEQ ID NO:6 with an affinity that is at least 10 times higher than other antigens of the complement system. In another embodiment, the MASP-2 inhibitor specifically binds to a polypeptide comprising SEQ ID NO:6 with an affinity that is at least 100 times higher than other antigens of the complement system. The binding affinity of a MASP-2 inhibitor can be determined using an appropriate binding assay.

[0208] The MASP-2 polypeptide exhibits a molecular structure similar to that of 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), providing a human MASP-2 polypeptide with a leader sequence (aa 1-15) that is cleaved after secretion to yield mature human MASP-2 (SEQ ID NO:6). As shown in Figure 2, the human MASP2 gene contains 12 exons. The human MASP-2 cDNA is encoded by exons B, C, D, F, G, H, I, J, K, and L. As shown in Figure 2, an alternative splice generates a 20 kDa protein called MBL-associated protein 19 (also called "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 with a leader sequence that is cleaved after secretion to yield mature mouse MASP-2 (SEQ ID NO:52). The cDNA molecule shown in SEQ ID NO:53 encodes rat MASP-2 (consisting of the amino acid sequence shown in SEQ ID NO:54) and provides a rat MASP-2 polypeptide with a leader sequence that is cleaved after secretion to yield mature rat MASP-2 (SEQ ID NO:55).

[0209] Those skilled in the art will recognize that the sequences disclosed in SEQ ID NO:4, SEQ ID NO:50, and SEQ ID NO:53 represent single alleles of human MASP-2, mouse MASP-2, and rat MASP-2, respectively, and that allelic variation 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 are within the scope of the present invention, including allelic variants containing silent mutations and allelic variants in which the mutation alters the amino acid sequence. Allelic variants of the MASP-2 sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures.

[0210] The domains of the human MASP-2 protein (SEQ ID NO:6) are shown in Figures 1 and 2A and include an N-terminal C1r / C1s / sea urchin Vegf / bone morphogenetic protein (CUBI) domain (aa 1-121 of SEQ ID NO:6), an epidermal growth factor-like domain (aa 122-166), another CUBI domain (aa 167-293), a tandem array of complement control protein domains, and a serine protease domain. Alternative splicing of the MASP2 gene results in 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.

[0211] Several proteins have been shown to bind to or interact with MASP-2 through protein-protein interactions. For example, MASP-2 binds to the lectin proteins MBL, H-ficolin, and L-ficolin, and interacts with them to form Ca2+ complexes. 2+It is known that MASP-2 / lectin complexes form complement-dependent complexes. Each MASP-2 / lectin complex has been shown to activate complement through MASP-2-dependent cleavage of proteins C4 and C2 (Ikeda, K., et al., J. Biol Chem. 262:7451-7454, 1987; Matsushita, M., et al., J. Exp. Med. 176:197-2284, 2000; Matsushita, M., et al., J. Immunol. 168:3502-3506, 2002). Studies have shown that the CUB1-EGF domain of MASP-2 is essential for the binding of MASP-2 to MBL (Thielens, NM, et al., J. Immunol. 166:5068, 2001). The CUB1EGFCUBII domain has also been shown to mediate MASP-2 dimerization, which is required for the formation of an 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 or interfere with MASP-2 target regions known to be important for MASP-2-dependent complement activation.

[0212] Anti-MASP-2 antibody In some embodiments of this aspect of the present invention, the MASP-2 inhibitor comprises an anti-MASP-2 antibody that inhibits the MASP-2-dependent complement activation system. Anti-MASP-2 antibodies useful in this aspect of the present invention include polyclonal antibodies, monoclonal antibodies, or recombinant antibodies derived from any antibody-producing mammal, and may be multispecific, chimeric, humanized, anti-idiotypic, and antibody fragments. Antibody fragments include Fab, Fab', F(ab)2, F(ab')2, Fv fragments, scFv fragments, and single-chain antibodies, as further described herein.

[0213] Several anti-MASP-2 antibodies have been described in the literature, some of which are listed in Table 1 below. The ability of these previously described anti-MASP-2 antibodies to inhibit the MASP-2-dependent complement activation system can be screened using the assay methods described herein. For example, as further detailed in Examples 10 and 11 herein, an anti-rat MASP-2 Fab2 antibody that blocks MASP-2-dependent complement activation has been identified. Once an anti-MASP-2 antibody that functions as a MASP-2 inhibitor has been identified, it can be used to generate anti-idiotypic antibodies, as further described below, and to identify other MASP-2 binding molecules.

[0214] Table 1. MASP-2-specific antibodies from the literature TIFF0007629300000007.tif166155

[0215] Anti-MASP-2 antibodies with reduced effector function In some embodiments of this aspect of the present invention, the anti-MASP-2 antibody has reduced effector function to alleviate inflammation that can result from activation of the classical complement pathway. It has been shown that the ability of IgG molecules to trigger the classical complement pathway resides within the Fc portion of the molecule (Duncan, AR. et al., Nature 332:738-740 1988). IgG molecules from which the Fc portion of the 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, antibodies with reduced effector function as a result of lacking the Fc portion of the molecule can be produced by genetically engineering an Fc sequence to minimize effector function or by using a human IgG2 or IgG4 isotype.

[0216] Antibodies with reduced effector function can be generated by standard molecular biological manipulation of the Fc portion of an IgG heavy chain, as described in Example 9 herein, and also as described in Jolliffe et al., Int'l Rev. Immunol. 10:241-250, 1993 and Rodrigues et al., J. Immunol. 151:6954-6961, 1998. Antibodies with reduced effector function also include human IgG2 and IgG4 isotypes with reduced ability to activate complement and / or interact with Fc receptors (Ravetch, JV, et al., Annu. Rev. Immunol. 9:457-492, 1991; Isaacs, JD, et al., J. Immunol. 148:3062-3071, 1992; van de Winkel, JG, et al., Immunol Today 14:215-221, 1993). Humanized or fully human antibodies specific for human MASP-2 and consisting of the IgG2 or IgG4 isotype can be produced by one of several methods known to those skilled in the art, such as those described in Vaughan, TJ, et al., Nature Biotechnical 16:535-539, 1998.

[0217] Preparation of anti-MASP-2 antibody Anti-MASP-2 antibodies can be generated using MASP-2 polypeptides (e.g., full-length MASP-2) or antigenic MASP-2 epitope-containing peptides (e.g., portions of MASP-2 polypeptides). Immunogenic peptides can be as small as five amino acid residues. For example, a MASP-2 polypeptide containing the entire amino acid sequence of SEQ ID NO:6 can be used to induce anti-MASP-2 antibodies useful in the methods of the present invention. Specific MASP-2 domains known to be involved in protein-protein interactions, such as the CUBI and CUBIEGF domains, and the region containing the serine protease active site, can be expressed as recombinant polypeptides as described in Example 3 and used as antigens. Furthermore, peptides containing at least a 6-amino acid portion of the MASP-2 polypeptide (SEQ ID NO:6) are also useful for inducing MASP-2 antibodies. Further examples of MASP-2-derived antigens useful for inducing MASP-2 antibodies are shown in Table 2 below. The MASP-2 peptides and polypeptides used to generate antibodies may be isolated as natural or recombinant or synthetic peptides and catalytically inactive recombinant polypeptides, e.g., MASP-2A, as further described in Examples 5 to 7. In some embodiments of this aspect of the invention, anti-MASP-2 antibodies are obtained using transgenic mouse lines as described in Examples 8 and 9 and as further described below.

[0218] Antigens useful in generating anti-MASP-2 antibodies also include fusion polypeptides, such as fusions of MASP-2 or portions thereof with immunoglobulin polypeptides or maltose-binding protein. Polypeptide immunogens may be full-length molecules or portions thereof. If the polypeptide portion is haptenic, such portions may be conveniently conjugated or linked to macromolecular carriers (e.g., keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or tetanus toxoid) for immunization.

[0219] Table 2: MASP-2-derived antigens TIFF0007629300000008.tif186153

[0220] Polyclonal antibodies Polyclonal antibodies against MASP-2 can be prepared by immunizing animals with MASP-2 polypeptides or immunogenic portions thereof using methods well known to those skilled in the art. See, for example, Green et al., "Production of Polyclonal Antisera," Immunochemical Protocols (Manson, ed.), p. 105. This is further described in Example 6. The immunogenicity of MASP-2 polypeptides can be enhanced using adjuvants including mineral gels, e.g., aluminum hydroxide, or Freund's adjuvant (complete or incomplete), surfactants, e.g., lysolecithin, pluronic polyols, polyanions, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Polyclonal antibodies are typically produced in animals, such as horses, cows, dogs, chickens, rats, mice, rabbits, guinea pigs, goats, or sheep. Alternatively, anti-MASP-2 antibodies useful in the present invention may also be derived from subhuman primates. General techniques for raising diagnostically and therapeutically useful antibodies in baboons can be found, for example, in Goldenberg et al., International Patent Publication WO 91 / 11465, and Losman, MJ, et al., Int. J. Cancer 46:310, 1990. Serum containing immunologically active antibodies is then produced from the blood of such immunized animals using standard procedures well known in the art.

[0221] 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 generated against a single MASP-2 epitope. As used herein, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and should not be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be obtained using any technique that provides for the production of antibody molecules by continuous cell lines, for example, the hybridoma method described in Kohler, G., et al., Nature 256:495, 1975. Alternatively, monoclonal antibodies can be generated by recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567 to Cabilly). Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described in Clackson, T., et al., Nature 352:624-628, 1991, and Marks, JD, et al., J. Mol Biol. 222:581-597, 1991. Such antibodies can be of any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD, and any subclass thereof.

[0222] For example, monoclonal antibodies can be obtained by injecting a composition containing a MASP-2 polypeptide or a portion thereof into a suitable mammal (e.g., a BALB / c mouse). After a predetermined period of time, spleen cells are removed from the mouse and suspended in a 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 further describing the production of anti-MASP-2 monoclonal antibodies is provided in Example 7 (see also Current Protocols in Immunology, Vol. 1, John Wiley & Sons, pp. 2.5.1-2.6.7, 1991).

[0223] Human monoclonal antibodies can be obtained using transgenic mice engineered to produce specific human antibodies in response to antigen challenge. In this technique, elements of human immunoglobulin heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines containing targeted disruptions of endogenous immunoglobulin heavy and light chain loci. These transgenic mice are capable of synthesizing human antibodies specific to human antigens, such as the MASP-2 antigen described herein, and can be used to generate human MASP-2 antibody-secreting hybridomas by fusing B cells from such animals with appropriate myeloma cell lines using conventional Köhler-Millstein 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.

[0224] Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques, including affinity chromatography with Protein A Sepharose, size-exclusion chromatography, and ion-exchange chromatography (see, e.g., Coligan, pp. 2.7.1-2.7.12 and 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).

[0225] Once polyclonal, monoclonal, or phage-derived antibodies are generated, they are first tested for specific MASP-2 binding. Various assays known to those skilled in the art can be used to detect antibodies that specifically bind to MASP-2. Exemplary assays include standard Western blot or immunoprecipitation analysis (e.g., as described in Ausubel et al.), immunoelectrophoresis, enzyme-linked immunosorbent assay, dot blot, inhibition or competitive assay, and sandwich assay (as described in Harlow and Land, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988). Once an antibody that specifically binds to MASP-2 is identified, the anti-MASP-2 antibody is tested for its ability to function as a MASP-2 inhibitor in one of several assays, 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).

[0226] The affinity of an anti-MASP-2 monoclonal antibody can be easily determined by one 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 in the methods of the present invention binds to MASP-2 with a binding affinity of <100 nM, preferably <10 nM, and most preferably <2 nM. In some embodiments, the MASP-2 inhibitory monoclonal antibody useful in the methods of the present invention comprises: (I) (a) a heavy chain variable region comprising: (i) a heavy chain CDR-H1 comprising the amino acid sequence of 31 to 35 of SEQ ID NO:67; and (ii) a heavy chain CDR-H2 comprising the amino acid sequence of 50 to 65 of SEQ ID NO:67; and (iii) a heavy chain CDR-H3 comprising the amino acid sequence of 95 to 102 of SEQ ID NO:67; and (b) a light chain variable region comprising: (i) a light chain CDR-L1 comprising the amino acid sequence of 24 to 34 of SEQ ID NO:70; and (ii) a light chain CDR-L2 comprising the amino acid sequence of 50 to 56 of SEQ ID NO:70; and (iii) a light chain CDR-L3 comprising the amino acid sequence of 89 to 97 of SEQ ID NO:70; or (II) a light chain variable region having at least 90% identity to SEQ ID NO:67 (e.g., SEQ ID A MASP-2 inhibitory monoclonal antibody or its antigen-binding fragment, comprising 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%, at least 99% identity to SEQ ID NO:67 and a variant thereof comprising a light chain variable region having at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity) to SEQ ID NO:70.

[0227] Chimeric / Humanized Antibodies Monoclonal antibodies useful in the methods of the present invention include chimeric antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies (U.S. Pat. No. 4,816,567 to Cabilly; and Morrison, SL, et al., Proc. Nat'l Acad. Sci. USA 81:6851-6855, 1984).

[0228] One form of chimeric antibody useful in the present invention is a humanized monoclonal anti-MASP-2 antibody. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies containing minimal sequence derived from non-human immunoglobulin. Humanized monoclonal antibodies are generated by introducing non-human (e.g., murine) complementarity-determining regions (CDRs) from the variable heavy and light chains of a murine immunoglobulin into a human variable domain. Human antibody residues are then typically substituted in the framework regions of the non-human counterparts. Furthermore, humanized antibodies may contain residues not found in either the recipient or donor antibody. These modifications are made to further refine antibody performance. Generally, humanized antibodies contain substantially all of at least one, and typically two, variable domains. All or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the Fv framework regions correspond to those of a human immunoglobulin sequence. The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones, P.T., et al., Nature 321:522-525, 1986; Reichmann, L., et al., Nature 332:323-329, 1988; and Presta, Curr. Op. Struct. Biol. 2:593-596, 1992.

[0229] Humanized antibodies useful in the present invention include human monoclonal antibodies containing at least the MASP-2-binding CDR3 region. Furthermore, the Fc portion may be exchanged to produce IgA or IgM antibodies as well as human IgG antibodies. Such humanized antibodies are particularly useful in clinical settings because they specifically recognize human MASP-2 but do not elicit an immune response against the antibody itself in humans. As a result, such humanized antibodies are more suitable for in vivo administration in humans, especially when repeated or long-term administration is required.

[0230] An example of the production of a humanized anti-MASP-2 antibody from a mouse anti-MASP-2 monoclonal antibody is shown in Example 6 of the present specification. Techniques for producing humanized monoclonal antibodies are described, for example, in Jones, P.T., et al., Nature 321:522, 1986; Carter, P., et al., Proc. Nat'l Acad. Sci. USA 89:4285, 1992; Sandhu, J.S., Crit. Rev. Biotech. 12:437, 1992; Singer, Il, 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, No. 5,693,762 to 1997. Additionally, there are commercial entities that synthesize humanized antibodies from specific murine antibody regions, such as Protein Design Labs (Mountain View, CA).

[0231] Recombinant antibodies Anti-MASP-2 antibodies can also be produced using recombinant methods. For example, human antibody fragments (V H , V L Human antibodies can be produced by using a human immunoglobulin expression library (available, for example, from Stratagene, Corp., La Jolla, CA) to generate fragments (e.g., Fv, Fd, Fab, or F(ab')2). These fragments are then used to construct whole human antibodies using techniques similar to those used to produce chimeric antibodies.

[0232] Anti-idiotype antibodies Once anti-MASP-2 antibodies with the desired inhibitory activity have been identified, these antibodies can be used to generate anti-idiotypic antibodies that mimic portions of MASP-2 using techniques well known in the art. See, e.g., 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 anti-idiotypic antibodies that mimic the MBL binding site on the MASP-2 protein and thus bind to and neutralize the binding ligand of MASP-2, e.g., MBL.

[0233] immunoglobulin fragments MASP-2 inhibitors useful in the methods of the present invention include not only intact immunoglobulin molecules but also well-known fragments formed from antibody fragments, including Fab, Fab', F(ab)2, F(ab')2, and Fv fragments, scFv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies.

[0234] It is well known in the art that the binding of an antibody to its epitope involves only a small portion of the antibody molecule, the paratope (see, e.g., 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. An antibody from which the pFc' region has been enzymatically cleaved or which has been produced without the pFc' region is called an F(ab')2 fragment and retains both of the antigen-binding sites of the intact antibody. An isolated F(ab')2 fragment is called a bivalent monoclonal fragment because of its two antigen-binding sites. Similarly, an antibody from which the Fc region has been enzymatically cleaved or which has been produced without the Fc region is called an Fab fragment and retains one of the antigen-binding sites of the intact antibody molecule.

[0235] Antibody fragments can be obtained by conventional proteolytic hydrolysis of whole antibodies, such as pepsin or papain digestion. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to obtain a 5S fragment called F(ab')2. This fragment can be further cleaved using a thiol reducing agent to generate a 3.5S Fab' monovalent fragment. Optionally, the cleavage reaction can be carried out using a sulfhydryl-blocking group that cleaves disulfide bonds. Alternatively, enzymatic cleavage with pepsin directly produces two monovalent Fab fragments and one Fc fragment. These methods are described, for example, in U.S. Pat. No. 4,331,647 to Goldenberg; Nisonoff, A., et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, R.R., Biochem. J. 73:119, 1959; Edelman, et al., Methods in Enzymology 1:422, Academic Press, 1967; and Coligan, pp. 2.8.1-2.8.10 and 2.10-2.10.4.

[0236] In some embodiments, it is preferable to use antibody fragments lacking the Fc region to avoid activation of the classical complement pathway, which is initiated upon binding of Fc to Fcγ receptors. There are several methods by which MoAbs that avoid Fcγ receptor interactions can be generated. For example, the Fc region of a monoclonal antibody can be chemically removed using partial proteolytic digestion (e.g., ficin digestion) to generate, for example, antigen-binding antibody fragments, such as Fab fragments or F(ab)2 fragments (Mariani, M., et al., Mol. Immunol. 28:69-71, 1991). Alternatively, human γ4 IgG isotypes, which do not bind to Fcγ receptors, can be used in the construction of humanized antibodies as described herein. Antibodies lacking the Fc domain, single-chain antibodies, and antigen-binding domains can also be engineered using recombinant methods described herein.

[0237] single chain antibody fragment Alternatively, a single peptide-chain binding molecule specific for MASP-2 can be produced in which the heavy chain Fv region and the light chain Fv region are connected. The Fv fragments may be connected with a peptide linker to form a single-chain antigen-binding protein (scFv). These single-chain antigen-binding proteins are composed of V-type Fv fragments connected with an oligonucleotide. H and V L scFvs are prepared by constructing a structural gene containing DNA sequences encoding the V domains. The structural gene is inserted into an expression vector, which is then introduced into a host cell, such as E. coli. The recombinant host cell synthesizes a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing scFvs are described, for example, in Whitlow et al., "Methods: A Companion to Methods in Enzymology" 2:97, 1991; Bird et al., Science 242:423, 1988; U.S. Patent No. 4,946,778 to Ladner; and Pack, P., et al., Bio / Technology 11:1271, 1993.

[0238] As an illustrative example, MASP-2-specific scFvs can be obtained by exposing lymphocytes to MASP-2 polypeptides in vitro and selecting antibody display libraries in phage or similar vectors (e.g., by using immobilized or labeled MASP-2 protein or peptide). Genes encoding polypeptides with potential MASP-2 polypeptide-binding domains can be obtained by screening random peptide libraries displayed on phage or bacteria, such as E. coli. These random peptide display libraries can be used to screen for peptides that interact with MASP-2. Techniques for generating and screening such random peptide display libraries are well known in the art (U.S. 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, for example, CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly, Mass.), and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ).

[0239] 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. CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDRs of the antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells (see, e.g., Larrick et al., Methods; A Companion to Methods in Enzymology 2:106, 1991; Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al., (eds.), p. 166, Cambridge University Press, 1995; and Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), p. 137, Wiley-Liss, Inc., 1995).

[0240] To inhibit MASP-2-dependent complement activation, the MASP-2 antibodies described herein are administered to a subject in need thereof. In some embodiments, the MASP-2 inhibitor is a high-affinity human or humanized monoclonal anti-MASP-2 antibody with reduced effector function.

[0241] Peptide inhibitors In some embodiments of this aspect of the present invention, the MASP-2 inhibitor comprises an isolated MASP-2 peptide inhibitor, including isolated natural 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, by competing with MASP-2 for binding to another recognition molecule of the lectin pathway (e.g., MBL, H-ficolin, M-ficolin, or L-ficolin), and / or by directly interacting with MASP-2 to inhibit MASP-2-dependent complement activation, which peptide is substantially pure and essentially free from other substances with which it may be found in nature to a degree practical and appropriate for the intended use.

[0242] Peptide inhibitors have been successfully used in vivo to disrupt protein-protein interactions and catalytic sites. For example, a peptide inhibitor against an adhesion molecule structurally related to LFA-1 was recently approved for clinical use in coagulation disorders (Ohman, E. M., et al., European Heart J. 16:50-55, 1995). Short linear peptides (<30 amino acids) that block or interfere with integrin-dependent adhesion have been described (Murayama, O., et al., J. Biochem. 120:445-51, 1996). 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, longer peptide inhibitors have higher affinity and / or slower off-rate than shorter peptides, and therefore may be more potent inhibitors. Cyclic peptide inhibitors have also been shown to be effective integrin inhibitors in vivo for the treatment of human inflammatory diseases (Jackson, DY, et al., J. Med. Chem. 40:3359-68, 1997). One method for 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 via a disulfide bond between the terminal amino acids. This cyclic form has been shown to improve in vivo affinity and half-life for the treatment of hematopoietic neoplasms (e.g., U.S. Patent No. 6,649,592 to Larson).

[0243] Synthetic MASP-2 peptide inhibitors MASP-2 inhibitory peptides useful in the methods of this aspect of the invention are exemplified by amino acid sequences that mimic target regions important for MASP-2 function. Inhibitory peptides useful in practicing the methods of the invention range in size from about 5 amino acids to about 300 amino acids. Table 3 provides a list of exemplary inhibitory peptides that may be useful in practicing this aspect of the invention. For example, the ability of candidate MASP-2 inhibitory peptides to function as MASP-2 inhibitors can be tested in one of several assays, including the lectin-specific C4 cleavage assay (described in Example 2) and the C3b deposition assay (described in Example 2).

[0244] In some embodiments, the MASP-2 inhibitory peptide is derived from a MASP-2 polypeptide and is selected from the full-length mature MASP-2 protein (SEQ ID NO:6) or specific domains of the MASP-2 protein, such as the CUBI domain (SEQ ID NO:8), the CUBIEGF domain (SEQ ID NO:9), the EGF domain (SEQ ID NO:11), and the serine protease domain (SEQ ID NO:12). As noted above, the CUBEGFCUBII region has been shown to be required for dimerization and binding to MBL (Thielens et al., supra). In particular, the peptide sequence TFRSDYN (SEQ ID NO:16) in the CUBI domain of MASP-2 has been shown to be involved in binding to MBL in a study that identified humans with a homozygous mutation from Asp105 to Gly105, resulting in loss of MASP-2 from the MBL complex (Stengaard-Pedersen, K., et al., New England J. Med. 349:554-560, 2003).

[0245] In some embodiments, the MASP-2 inhibitory peptide is derived from a lectin protein that binds to MASP-2 and participates in the lectin complement pathway.Several different lectins have been identified that participate in this pathway, including mannan-binding lectin (MBL), L-ficolin, M-ficolin, and H-ficolin (Ikeda, K., et al., J. Biol. Chem. 262:7451-7454, 1987; Matsushita, M., et al., J. Exp. Med. 176:1497-2284, 2000; Matsushita, M., et al., J. Immunol. 168:3502-3506, 2002).These lectins exist in serum as oligomers of homotrimeric subunits, each of which has an N-terminal collagen-like fiber with a carbohydrate recognition domain. These different lectins have been shown to bind to MASP-2, and the lectin / MASP-2 complex activates complement by cleaving 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 from Salmonella typhimurium, S. minnesota, and Escherichia coli. H-ficolin has been shown to bind to MASP-2 and MAp19 and activate the lectin pathway. L-ficolin / P35 also binds to GlcNAc and has been shown to bind to MASP-2 and MAp19 in human serum, and this complex has been shown to activate the lectin pathway (Matsushita, M., et al., J. Immunol. 164:2281, 2000).Therefore, a MASP-2 inhibitory peptide useful in the present invention may comprise a region of at least five amino acids selected from MBL protein (SEQ ID NO:21), H-ficolin protein (GenBank accession number NM_173452), M-ficolin protein (GenBank accession number O00602), and L-ficolin protein (GenBank accession number NM_015838).

[0246] More specifically, the scientists found that the MASP-2 binding site on MBL is located at 12 Gly-XY triplets between the hinge and neck in the C-terminal part of the collagen-like domain of MBP. TIFF0007629300000009.tif12128 (Wallis, R. et al., J. Biol Chem. 279:14065, 2004). This MASP-2 binding site region is also highly conserved in human H-ficolin and human L-ficolin. A consensus binding site has been described that is present in all three lectin proteins and contains the amino acid sequence "OGK-X-GP" (SEQ ID NO:22), where the letter "O" represents hydroxyproline and the letter "X" is a hydrophobic residue (Wallis et al., 2004, supra). Thus, in some embodiments, MASP-2 inhibitory peptides useful in this aspect of the invention are at least six amino acids in length and contain SEQ ID NO:22. Peptides derived from MBL, including TIFF0007629300000010.tif4128, have been shown to bind to MASP-2 in vitro (Wallis, et al., 2004, supra). To enhance binding to MASP-2, peptides can be synthesized flanked by two GPO triplets at each end, which enhances triple helix formation as seen in the native MBL protein. TIFF0007629300000011.tif5132 (further described in Wallis, R., et al., J. Biol. Chem. 279:14065, 2004).

[0247] The MASP-2 inhibitory peptide also contains the sequence derived from the consensus MASP-2 binding region of H-ficolin. TIFF0007629300000012.tif4145. Sequences derived from the consensus MASP-2 binding region of L-ficolin may be derived from human H-ficolin, including the sequence Also included are peptides derived from human L-ficolin, including TIFF0007629300000013.tif12153.

[0248] The MASP-2 inhibitory peptide also contains a C4 cleavage site linked to the C-terminal portion of antithrombin III. It may also be derived from a C4 cleavage site such as TIFF0007629300000014.tif4128 (Glover, GI, et al., Mol. Immunol. 25:1261(1988)).

[0249] Table 3. Exemplary MASP-2 inhibitory peptides TIFF0007629300000015.tif29155TIFF0007629300000016.tif218155TIFF0007629300000017.tif58155Note: The letter "O" represents hydroxyproline. The letter "X" is a hydrophobic residue.

[0250] 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. For example, suitable modifications may include, but are not limited to, halomethylketones (Br, Cl, I, F) at the C-terminus, Asp, or Glu, or attached to functional side chains; haloacetyl (or other alpha-haloacetyl) groups at amino groups or other functional side chains; epoxide- or imine-containing groups at the amino or carboxy terminus or functional side chains; or imidate esters at the amino or carboxy terminus or functional side chains. Such modifications may offer the advantage of permanently inhibiting the enzyme through covalent binding of the peptide. This may result in a lower effective dose and / or require less frequent administration of the peptide inhibitor.

[0251] In addition to the inhibitory peptides described above, MASP-2 inhibitory peptides useful in the methods of the present invention include peptides containing the MASP-2-binding CDR3 region of the anti-MASP-2 MoAb obtained as described herein. The sequences of CDR regions for use in peptide synthesis can be determined by methods known in the art. Heavy chain variable regions are generally peptides of 100 to 150 amino acids in length. Light chain variable regions are generally peptides of 80 to 130 amino acids in length. The CDR sequences within the heavy and light chain variable regions contain sequences of only about 3 to 25 amino acids, which can be easily sequenced by one skilled in the art.

[0252] Those skilled in the art will recognize that substantially homologous variations of MASP-2 inhibitory peptides also exhibit MASP-2 inhibitory activity. Exemplary variations include, but are not necessarily limited to, peptides with insertions, deletions, replacements, and / or additional amino acids in the carboxy- or amino-terminal portions of the peptides, as well as mixtures thereof. Thus, homologous peptides with MASP-2 inhibitory activity are considered useful in the methods of the present invention. The described peptides may also include overlapping motifs and other modifications through conservative substitutions. Conservative variants are described elsewhere herein and include replacing certain amino acids with amino acids of similar charge, size, or hydrophobicity, etc.

[0253] To more closely resemble segments in the intact protein, 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, so long as they functionally retain the desired MASP-2 inhibitory properties. Modifications may include amino acid substitutions with one of the 20 commonly known amino acids or with another amino acid, amino acid substitutions with derivatized or substituted amino acids or D-amino acids that provide additional desirable characteristics, such as resistance to enzymatic degradation, or substitutions with other molecules or compounds that mimic the native conformation and function of a single amino acid, multiple amino acids, or peptide, such as carbohydrates; amino acid deletions; amino acid insertions with one of the 20 commonly known amino acids or with another amino acid, amino acid substitutions with derivatized or substituted amino acids or D-amino acids that provide additional desirable characteristics, such as resistance to enzymatic degradation, or substitutions with other molecules or compounds that mimic the native conformation and function of a single amino acid, multiple amino acids, or peptide, such as carbohydrates; or substitutions with other molecules or compounds that mimic the native 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.

[0254] The synthesis of derivative inhibitory peptides may rely on known techniques such as peptide biosynthesis, carbohydrate biosynthesis, etc. As a starting point, those skilled in the art may rely on appropriate computer programs 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 what kind of substitutions can be made at one or more positions in a rational design manner to create a derivative that retains the basic conformation and charge distribution of the parent peptide, but may have characteristics that are not present in the parent peptide or that are enhanced beyond those found in the parent peptide. Once a candidate derivative molecule is identified, the derivative can be tested using the assay methods described herein to determine whether it functions as a MASP-2 inhibitor.

[0255] Screening of MASP-2 inhibitory peptides Molecular modeling and rational molecular design can also be used to generate and screen peptides that mimic the molecular structure of the key binding region of MASP-2 and inhibit the complement activity of MASP-2. As previously mentioned, the molecular structures used for modeling include the CDR regions of anti-MASP-2 monoclonal antibodies, as well as target regions known to be important for MASP-2 function, including the region required for dimerization, the region involved in binding to MBL, and the serine protease active site. Methods for identifying peptides that bind to a specific target are well known in the art. For example, molecular imprinting can be used to de novo construct macromolecular structures, such as peptides, that bind to a specific molecule. 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.

[0256] As an illustrative example, one method for preparing mimetics of MASP-2-binding peptides is as follows: A functional monomer (template) of a known MASP-2-binding peptide or the binding domain of an anti-MASP-2 antibody that exhibits MASP-2 inhibition is polymerized. The template is then removed, and another class of monomer is subsequently polymerized into the void left by the template, resulting in a new molecule that exhibits one or more desirable properties similar to the template. In addition to preparing peptides in this manner, other MASP-2-binding molecules that are MASP-2 inhibitors, such as polysaccharides, nucleosides, drugs, nuclear proteins, lipoproteins, carbohydrates, glycoproteins, steroids, lipids, and other biologically active materials, can also be prepared. This method is useful for designing a wide variety of biomimetics that are more stable than their natural counterparts. This is because these biomimetics are typically prepared by free-radical polymerization of functional monomers, resulting in compounds with non-biodegradable backbones.

[0257] Peptide synthesis MASP-2 inhibitory peptides can be prepared using techniques well known in the art, for example, the solid phase synthesis technique first described by Merrifield in J. Amer. Chem. Soc. 85:2149-2154, 1963. Automated synthesis can be achieved, for example, using 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.

[0258] Peptides can also be prepared using standard genetic engineering techniques known to those skilled in the art. For example, peptides can be produced enzymatically by inserting a 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 using chromatographic or electrophoretic methods, or by inserting the peptide-encoding sequence in-frame with a nucleic acid sequence encoding the carrier protein into an expression vector, fused to the peptide, and purified with a carrier protein that can be subsequently cleaved. Fusion protein peptides may also be isolated using chromatographic, electrophoretic, or immunological techniques (e.g., antibody-mediated binding of a resin to a carrier protein). Peptides can be cleaved using chemical methodology or enzymatically, for example, by hydrolases.

[0259] The MASP-2 inhibitory peptides useful in the methods of the present invention can also be produced in recombinant host cells according to conventional techniques. To express the sequence encoding the MASP-2 inhibitory peptide, the nucleic acid molecule encoding the peptide must be operably linked to a regulatory sequence that controls transcriptional expression in an expression vector and then introduced into a host cell. In addition to transcriptional regulatory sequences such as promoters and enhancers, the expression vector may also contain a translational regulatory sequence and a marker gene suitable for selecting cells carrying the expression vector.

[0260] Nucleic acid molecules encoding MASP-2 inhibitory peptides can be synthesized by gene synthesizer using 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 generated separately. The creation of short genes (60–80 base pairs) is technically straightforward and can be achieved by synthesizing complementary strands and then annealing them. To create larger genes, synthetic double-stranded genes are assembled in a modular fashion from single-stranded fragments of 20–100 nucleotides in length. For reviews of polynucleotide synthesis, see, e.g., Glick and Pasternak, "Molecular Biotechnology, Principles and Applications of Recombinant DNA," ASM Press, 1994; Itakura, K., et al., Annu. Rev. Biochem. 53:323, 1984; and Climie, S., et al., Proc. Nal'l Acad. Sci. USA 87:633, 1990.

[0261] Small molecule inhibitors In some embodiments, the MASP-2 inhibitor is a small molecule inhibitor, including natural and synthetic substances with low molecular weight, such as peptides, peptide mimetics, and non-peptide inhibitors (including oligonucleotides and organic compounds). MASP-2 small molecule inhibitors can be prepared based on the molecular structure of the variable region of an anti-MASP-2 antibody.

[0262] Small molecule inhibitors can 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 crystal structure of rat MASP-2 has been described (Feinberg, H., et al., EMBO J. 22:2348-2359, 2003). Using the method described in Kuntz et al., MASP-2 crystal structure coordinates are used as input for a computer program, such as DOCK, which outputs a list of small molecule structures that are predicted to bind to MASP-2. The use of such computer programs is well known to those skilled in the art. For example, crystal structures of HIV-1 protease inhibitors have been used to identify unique non-peptide ligands that are HIV-1 protease inhibitors by evaluating the fit of compounds found in the Cambridge Crystallographic database with the enzyme binding site using the program DOCK (Kuntz, ID, et al., J. Mol. Biol. 161:269-288, 1982; DesJarlais, RL, et al., PNAS 87:6644-6648, 1990).

[0263] The list of small molecule structures identified by the computational method as potential MASP-2 inhibitors is screened using a MASP-2 binding assay, such as the MASP-2 binding assay described in Example 10. Small molecules found to bind to MASP-2 are then assayed in a functional assay, such as the functional assay described in Example 2, to determine whether they inhibit MASP-2-dependent complement activation.

[0264] MASP-2 soluble receptor Other suitable MASP-2 inhibitors would include soluble receptors for MASP-2, which can be made using techniques known to those skilled in the art.

[0265] Inhibitor of MASP-2 expression In another embodiment of this aspect of the present invention, the MASP-2 inhibitor is a MASP-2 expression inhibitor capable of inhibiting MASP-2-dependent complement activation. In implementing this aspect of the present invention, representative 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.

[0266] Antisense RNA and DNA molecules hybridize to MASP-2 mRNA and act to directly block the translation of MASP-2 mRNA by preventing the translation of MASP-2 protein. Antisense nucleic acid molecules can be constructed in a number of different ways, provided that they can interfere with the expression of MASP-2. For example, antisense nucleic acid molecules can be constructed by reversing the coding region (or a portion thereof) of MASP-2 cDNA (SEQ ID NO:4) relative to the normal transcription direction so that the complementary strand of the coding region (or a portion thereof) of MASP-2 cDNA (SEQ ID NO:4) can be transcribed.

[0267] Antisense nucleic acid molecules are usually substantially identical to at least a portion of one or more target genes. However, nucleic acids do not need to be completely identical to inhibit expression. Generally, high homology can be used to compensate for the use of short antisense nucleic acid molecules. The minimum percent identity is typically greater than about 65%, although higher percent identity may more effectively suppress expression of the endogenous sequence. A fairly high percent identity, greater than about 80%, is typically preferred, while about 95% to complete identity is typically most preferred.

[0268] Antisense nucleic acid molecules need not have the same intron or exon pattern as the target gene. Non-coding segments of a target gene may be equally effective as coding segments in achieving antisense inhibition of target gene expression. DNA sequences of at least about 8 or so nucleotides can be used as antisense nucleic acid molecules, although longer sequences are preferred. A representative example of a useful MASP-2 inhibitor in the present invention is an antisense MASP-2 nucleic acid molecule that is at least 90 percent identical to the complementary strand of a MASP-2 cDNA consisting of the nucleic acid sequence set forth in SEQ ID NO:4. The nucleic acid sequence set forth in SEQ ID NO:4 encodes a MASP-2 protein consisting of the amino acid sequence set forth in SEQ ID NO:5.

[0269] Targeting 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 type 2 acetylcholine receptors is inhibited by antisense oligonucleotides against their respective mRNA sequences (U.S. Patent No. 5,739,119 to Cheng and U.S. Patent No. 5,759,829 to Shewmaker). Further examples of antisense inhibition include nuclear protein cyclin, multidrug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABA. A receptor, and human EGF (see, e.g., U.S. Pat. No. 5,801,154 to Baracchini; U.S. Pat. No. 5,789,573 to Baker; U.S. Pat. No. 5,718,709 to Considine; and U.S. Pat. No. 5,610,288 to Reubenstein).

[0270] A system has been described that allows those skilled in the art to determine which oligonucleotides are useful in the present invention. This system involves probing appropriate sites within target mRNA using Rnase H cleavage as an indicator of the accessibility of sequences within the transcript. Scherr, M., et al., Nucleic Acids Res. 25:5079-5085, 1998; Lloyd, et al., Nucleic Acids Res. 29:3665-3673, 2001. To create sites vulnerable to RNAse H, a mixture of antisense oligonucleotides complementary to specific regions of MASP-2 transcripts is added to a cell extract expressing MASP-2, such as hepatocytes, and hybridized. This method can be combined with computer-assisted sequence selection, which can predict the optimal sequence selection of antisense compositions based on their relative ability to form dimers, hairpins, or other secondary structures that reduce or prevent specific binding to target mRNA in host cells. 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, S.F., et al., Nucl. Acids Res. 25:3389-3402, 1997). Antisense compounds against target sequences preferably contain about 8 to about 50 nucleotides in length. Antisense oligonucleotides containing about 9 to about 35 nucleotides, etc., are particularly preferred. The inventors contemplate that all oligonucleotide compositions within the range of 9 to 35 nucleotides (i.e., lengths of 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 or so) are highly preferred for practicing the antisense oligonucleotide-based methods of the present invention.Highly preferred target regions of MASP-2 mRNA are those at or near the AUG translation initiation codon and those 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 listed in Table 4.

[0271] Table 4. Exemplary inhibitors of MASP-2 expression TIFF0007629300000018.tif72155

[0272] As mentioned above, the term "oligonucleotide" as used herein refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term also covers oligonucleobases composed of natural nucleotides, sugars, and covalent internucleoside (backbone) linkages, as well as oligonucleotides with unnatural modifications. These modifications can impart certain desirable properties not offered 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 by a phosphodiester backbone modification in which the phosphate substituents are replaced by phosphorothioates, which 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 within the nucleic acid strand.

[0273] Another alternative to antisense is the use of "RNA interference" (RNAi). Double-stranded RNA (dsRNA) can induce gene silencing in mammals in vivo. The natural function of RNAi and cosuppression appears to be the protection of genomes from invasion by mobile genetic elements, such as retrotransposons, and viruses, which, when activated, produce aberrant RNA or dsRNA within 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 about 19-25 (e.g., 19-23) nucleotides in length. For example, dsRNA molecules useful in the methods of the present invention may include RNAs corresponding to the sequences listed in Table 4 and their complementary strands. Preferably, at least one RNA strand has a 3' overhang of 1-5 nucleotides. The synthesized RNA strands are combined under conditions to form a double-stranded molecule. The RNA sequence may be 25 nucleotides or less in length and contain at least an 8-nucleotide portion of SEQ ID NO:4. Designing siRNA sequences for a particular target is within the ordinary skill in the art. Commercial services are available that design siRNA sequences and guarantee at least 70% expression knockdown (Qiagen, Valencia, Calif.).

[0274] dsRNA is administered as pharmaceutical composition, and can be carried out by known method that nucleic acid is introduced into desired target cell.Commonly used gene transfer method includes calcium phosphate, DEAE-dextran, electroporation, microinjection and virus method.This method is disclosed in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 1993.

[0275] Ribozymes, such as ribozymes targeting MASP-2 mRNA, can also be used to reduce the amount and / or biological activity of MASP-2. Ribozymes are catalytic RNA molecules that can cleave nucleic acid molecules with sequences that are completely or partially homologous to the ribozyme sequence. Ribozyme transgenes can be designed that encode RNA ribozymes that specifically pair with target RNA and cleave the phosphodiester backbone at specific positions, thereby functionally inactivating the target RNA. When performing this cleavage, the ribozyme itself remains unchanged, and therefore can be reused to cleave other molecules. The inclusion of a ribozyme sequence in antisense RNA confers RNA cleavage activity to the antisense RNA, thereby increasing the activity of the antisense construct.

[0276] Ribozymes useful in practicing the present invention typically contain a hybridizing region of at least about nine nucleotides that is complementary in nucleotide sequence to at least a portion of the target MASP-2 mRNA, and a catalytic region adapted to cleave the target MASP-2 mRNA (see generally EPA No. 0321201; WO88 / 04300; Haseloff, J., et al., Nature 534:585-591, 1988; Fedor, MJ, et al., Proc. Natl. Acad. Sci. USA 87:1668-1672, 1990; Cech, TR, et al., Ann. Rev. Biochem. 55:599-629, 1986).

[0277] Ribozymes can be directly targeted to cells in the form of RNA oligonucleotides incorporating ribozyme sequences, or can be introduced into cells as expression vectors encoding the desired ribozyme RNA. Ribozymes can be used and applied in much the same way as described for antisense polynucleotides.

[0278] The antisense RNA and DNA, ribozymes, and RNAi molecules useful in the method of the present invention can be prepared by any method known in the art for synthesizing DNA molecules and RNA molecules.These include the techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art, such as solid-phase phosphoramidite chemical synthesis.Alternatively, RNA molecules can be produced by in vitro transcription and in vivo transcription of the DNA sequence encoding antisense RNA molecules.Such DNA sequences can be incorporated into a variety of vectors that incorporate suitable RNA polymerase promoters, such as T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly depending on the promoter used can be stably introduced into cell lines.

[0279] Various well-known modifications of DNA molecules can be introduced as a means of increasing stability and half-life. Useful modifications include, but are not limited to, the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and / or 3' ends of the molecule, or the use of phosphorothioate or 2'O-methyl rather than phosphodiesterase linkages in the oligodeoxyribonucleotide backbone.

[0280] VI. Pharmaceutical Compositions and Delivery Methods dosage In another aspect, the present invention provides a composition for inhibiting the side effects of MASP-2-dependent complement activation in a subject suffering from a disease or condition disclosed herein, comprising administering to the subject a composition comprising a therapeutically effective amount of a MASP-2 inhibitor and a pharmaceutically acceptable carrier. To treat or ameliorate a condition associated with MASP-2-dependent complement activation, the MASP-2 inhibitor can be administered to a subject in need thereof at a therapeutically effective dose. A therapeutically effective dose refers to an amount of the MASP-2 inhibitor sufficient to ameliorate symptoms associated with the disease or condition.

[0281] The toxicity and therapeutic efficacy of MASP-2 inhibitors can be determined by standard pharmaceutical procedures using experimental animal models, such as the MASP-2- / - mouse model expressing a 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 between the NOAEL effect and the MED effect is the therapeutic ratio, expressed as the ratio NOAEL / MED. MASP-2 inhibitors that exhibit a large therapeutic ratio or index are most preferred. Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for human use. The dosage of the MASP-2 inhibitor is preferably within a range of circulating concentrations, including the MED, that results in little or no toxicity. The dosage can vary within this range depending on the dosage form used and the route of administration utilized.

[0282] For any compound preparation, can use animal model to evaluate therapeutically effective dose.For example, can formulate in animal model the dose that reaches the circulating plasma concentration range that includes MED.The quantitative level of MASP-2 inhibitor in plasma can also be measured, for example, by high performance liquid chromatography.

[0283] In addition to toxicity studies, effective dosages may also be assessed 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 a quantitative MASP-2 assay method described in Moller-Kristensen M., et al., J. Immunol Methods 282:159-167, 2003.

[0284] Generally, the dosage of an administered composition containing a MASP-2 inhibitor will vary depending on factors such as the subject's age, weight, height, sex, general medical condition, and medical history. By way of 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 subject body weight, preferably 0.010 to 1.0 mg / kg of subject body weight, and more preferably 0.010 to 0.1 mg / kg of subject body weight. In some embodiments, the composition comprises a combination of an anti-MASP-2 antibody and a MASP-2 inhibitory peptide.

[0285] The therapeutic efficacy of the MASP-2 inhibitory compositions and methods of the present invention in a particular subject, as well as appropriate dosages, can be determined according to complement assays well known to those skilled in the art. Complement generates numerous specific products. Over the past decade, highly sensitive and specific assays have been developed and commercially available for most of these activation products, including the small activation fragments C3a, C4a, and C5a, and the large activation fragments iC3b, C4d, Bb, and sC5b-9. Most of these assays utilize monoclonal antibodies that react with neoantigens exposed on the fragments but not on the native proteins from which they are formed. This makes them very simple and specific. Most rely on ELISA technology, although in the case of C3a and C5a, radioimmunoassays are still sometimes used. These latter assays measure both unprocessed fragments and their "desArg" fragments, which are the predominant forms found in the circulation. Unprocessed fragments and C5a desArg is rapidly cleaved upon binding to cell surface receptors and is therefore present in very low concentrations. desArgdoes not bind to cells and accumulates in plasma. Measurement of C3a provides a sensitive, pathway-dependent indicator of complement activation. Alternative pathway activation can be assessed by measuring the Bb fragment. Detection of sC5b-9, a fluid-phase product of membrane attack pathway activation, provides evidence that complement is fully activated. Because both the lectin and classical pathways generate the same activation products, C4a and C4d, measurement of these two fragments does not provide information about which of these two pathways generated the activation product.

[0286] Inhibition of MASP-2-dependent complement activation is characterized by at least one of the following changes in complement system components that occur as a result of administration of a MASP-2 inhibitor according to the methods of the present invention: inhibition of the generation or production of MASP-2-dependent complement activation system products C4b, C3a, C5a, and / or C5b-9 (MAC) (e.g., measured as described in Example 2), a decrease in C4 cleavage and C4b deposition (e.g., measured as described in Example 10), or a decrease in C3 cleavage and C3b deposition (e.g., measured as described in Example 10).

[0287] Further active substances Compositions and methods comprising a MASP-2 inhibitor may optionally include one or more additional therapeutic substances that can enhance the activity of the MASP-2 inhibitor or provide a related therapeutic function additively or synergistically. For example, in the context of treating a subject suffering from TTP who is positive for an ADAM-TS13 inhibitor, one or more MASP-2 inhibitors may be administered in combination (including simultaneous administration) with one or more immunosuppressants. Suitable immunosuppressants include corticosteroids, Rituxan, cyclosporine, etc. In the context of treating a subject suffering from HUS or aHUS or a subject at risk of developing said HUS or aHUS, one or more MASP-2 inhibitors may be administered in combination (including simultaneous administration) with an appropriate antibiotic. In treating a subject suffering from aHUS or at risk of developing aHUS, one or more MASP-2 inhibitors may be administered in combination (including simultaneous administration) with other complement inhibitors, such as eculizumab (Soliris), TT-30, antibodies against factor B, or other agents that inhibit terminal complement components or alternative pathway amplification.

[0288] The inclusion and selection of additional active substances will be determined to achieve the desired therapeutic results. 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 PLC γInhibitors of enzymes active in the arachidonic acid metabolite synthesis pathway, including phospholipase inhibitors, including isoform inhibitors, cyclooxygenase (COX) inhibitors (whether COX-1 inhibitors, COX-2 inhibitors, or nonselective COX-1 and -2 inhibitors), and lipoxygenase inhibitors; prostanoid receptor antagonists, including eicosanoid EP-1 and EP-4 receptor subtype antagonists and thromboxane receptor subtype antagonists; leukotriene receptor antagonists, including leukotriene B4 receptor subtype antagonists and leukotriene D4 receptor subtype antagonists; opioid receptor agonists, including μ opioid, δ opioid, and κ opioid receptor subtype agonists; 2X Receptor antagonists and P 2Y These include purinergic receptor agonists and antagonists, including receptor agonists; adenosine triphosphate (ATP)-sensitive potassium channel openers; MAP kinase inhibitors; nicotinic acetylcholine inhibitors; and alpha adrenergic receptor agonists (including alpha-1, alpha-2, and non-selective alpha-1 and alpha-2 agonists).

[0289] The MASP-2 inhibitors of the present invention may also be administered in combination with one or more other complement inhibitors, for example, inhibitors of C5. To date, eculizumab (Solaris®), an antibody against C5, is the only complement-targeting drug approved for human use. However, some pharmacological agents have been shown to block complement in vivo. K76COOH and nafamstat mesylate are two agents that have shown considerable efficacy in animal transplant 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). It is believed that these small molecule inhibitors may be useful as agents for use in combination with the MASP-2 inhibitors of the present invention.

[0290] Other natural complement inhibitors may be useful in combination with the MASP-2 inhibitors of the present invention. Biological inhibitors of complement 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. The anti-complement activity of recombinant forms has been tested in vitro and in vivo. sCR1 has been shown to be effective in xenotransplantation, where the complement system (alternative 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 prolongs their survival, implicating the complement pathway in the development of organ survival (Leventhal, JR, et al., Transplantation 55:857-66, 1993; Pruitt, SK, et al., Transplantation 57:363-70, 1994).

[0291] Additional complement inhibitors suitable for use in combination with the compositions of the present invention include, by way of example, MoAbs, such as anti-C5 antibodies (e.g., eculizumab) developed by Alexion Pharmaceuticals, Inc., New Haven, Connecticut, and anti-properdin MoAbs.

[0292] Pharmaceutical Carriers and Delivery Vehicles Generally, the MASP-2 inhibitor composition of the present invention, combined with any other selected therapeutic substances, is optionally contained in a pharmaceutically acceptable carrier. 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 substances combined therewith). Exemplary pharmaceutically acceptable carriers for peptides are 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 into solid, semi-solid, gel, liquid, or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants, and injections, allowing for oral, parenteral, or surgical administration. The present invention also contemplates local administration of the composition by coating a medical device or the like.

[0293] Suitable carriers for parenteral delivery via injection, infusion, or irrigation, and local delivery include distilled water, physiological phosphate-buffered saline, normal Ringer's solution or lactated Ringer's solution, dextrose solution, Hank's solution, or propanediol. Additionally, sterile, fixed oils may be used as solvents or dispersion media. For this purpose, any biocompatible oil, including synthetic monoglycerides or diglycerides, may be used. Additionally, fatty acids such as oleic acid are useful in preparing injectable solutions. The carrier and the active substance may be formulated as a liquid, suspension, polymerizable or non-polymerizable gel, paste, or ointment.

[0294] The carrier may also include a delivery vehicle to sustain (i.e., extend, delay, or modulate) delivery of the active agent or to enhance delivery, uptake, stability, or pharmacokinetics of the therapeutic agent. Such delivery vehicles may include, by way of non-limiting example, microparticles, microspheres, nanospheres, or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymer or copolymer hydrogels, and polymeric micelles. Suitable hydrogel and micelle delivery systems include the PEO:PHB:PEO copolymer and copolymer / cyclodextrin complexes disclosed in WO 2004 / 009664 A2 and the PEO and PEO / cyclodextrin complexes disclosed in U.S. Patent Application Publication No. 2002 / 0019369 A1. Such hydrogels may be injected locally at the intended site of action, or subcutaneously or intramuscularly to form a sustained-release depot.

[0295] For intra-articular delivery, the MASP-2 inhibitor may be carried in an injectable liquid or gel carrier as described above, an injectable sustained-release delivery vehicle as described above, or hyaluronic acid or a hyaluronic acid derivative.

[0296] For oral administration of non-peptide agents, the MASP-2 inhibitor may be delivered in an inert filler or diluent such as sucrose, cornstarch, or cellulose.

[0297] For local administration, the MASP-2 inhibitor may be delivered in an ointment, lotion, cream, gel, eye drops, suppository, spray, liquid or powder, or may be delivered via a transdermal patch in a gel or microcapsule delivery system.

[0298] A variety of nasal and pulmonary delivery systems are under development, including aerosols, metered dose inhalers, dry powder inhalers, and nebulizers, which may be suitably adapted for delivery of the present invention in aerosol, inhalant, or nebulized delivery vehicles, respectively.

[0299] For intrathecal (IT) or intracerebroventricular (ICV) delivery, the compositions of the present invention can be administered using an appropriately sterile delivery system (eg, liquid; gel, suspension, etc.).

[0300] The compositions of the present invention may also contain biocompatible excipients, such as dispersing or wetting agents, suspending agents, diluents, buffers, penetration enhancers, emulsifiers, binders, thickeners, and flavorings (for oral administration).

[0301] 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 a solution or suspension of an injectable dose of the compound 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. Additionally, auxiliary substances, such as wetting or emulsifying agents, surfactants, pH buffering substances, and the like, may be present in compositions containing anti-MASP-2 antibodies and inhibitory peptides. Additional components of pharmaceutical compositions include petroleum oils (e.g., petroleum oils of animal, vegetable, or synthetic origin), such as soybean oil and mineral oil. Generally, glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers for injectable solutions.

[0302] Anti-MASP-2 antibodies and inhibitory peptides can also be administered in the form of depot injection or implant preparations, which can be formulated for slow or pulsatile release of the active agent.

[0303] Pharmaceutically acceptable carriers for expression inhibitors More specifically, with respect to the expression inhibitors useful in the methods of the present invention, compositions are provided comprising the expression inhibitors and a pharmaceutically acceptable carrier or diluent, and the compositions may further comprise a colloidal dispersion system.

[0304] Pharmaceutical compositions containing expression inhibitors may include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components, including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. Preparation of such compositions typically involves combining the expression inhibitor with one or more of the following: buffers, antioxidants, low-molecular-weight polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose, or dextrins, chelating agents such as EDTA, glutathione, and other stabilizers and excipients. Examples of suitable diluents include neutral buffered saline or saline mixed with nonspecific serum albumin.

[0305] In some embodiments, the composition can be prepared and formulated as an emulsion, which is typically a heterogeneous system in which one liquid is dispersed in another liquid in the form of droplets (see Idson, Pharmaceutical Dosage Forms, Vol. 1, Rieger and Banker (eds.), Marcek Dekker, Inc., NY, 1988). Examples of natural emulsifiers used in emulsion formulations include gum arabic, beeswax, lanolin, lecithin, and phosphatides.

[0306] In one embodiment, the composition comprising nucleic acid can be formulated as microemulsion.Microemulsion as used herein refers to the system of water, oil and amphiphilic substance, which is a single, optically isotropic and thermodynamically stable liquid solution (referring to Rosoff, Pharmaceutical Dosage Forms, Vol.1).Method of the present invention can also use liposome to introduce and deliver antisense oligonucleotide to desired site.

[0307] Pharmaceutical compositions and formulations of expression inhibitors for topical administration may include transdermal patches, ointments, lotions, creams, gels, eye drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, as well as aqueous, powder, or oily bases and thickeners, may be used.

[0308] Method of administration Pharmaceutical compositions containing MASP-2 inhibitors can be administered in a variety of ways, depending on whether local or systemic administration is most appropriate for the condition being treated. Furthermore, as described herein above with respect to extracorporeal reperfusion therapy, MASP-2 inhibitors can be administered to recirculating blood or plasma via the introduction of compositions of the present invention. Furthermore, compositions of the present invention can be delivered by coating or incorporating them onto the surface of, or into, an implantable medical device.

[0309] systemic delivery As used herein, the terms "systemic delivery" and "systemic administration" are intended to include, but are not limited to, oral and parenteral routes, including intramuscular (IM), subcutaneous, intravenous (IV), intra-arterial, inhalation, sublingual, buccal, topical, transdermal, nasal, rectal, vaginal, and other routes of administration that effectively distribute the delivered agent to one or more desired therapeutic sites. Preferred systemic delivery routes for the present compositions include intravenous, intramuscular, subcutaneous, and inhalation routes. It will be understood that the precise systemic administration route of a selected agent used in a particular composition of the invention will be determined, in part, to account for the agent's susceptibility to metabolic transformation pathways associated with a particular administration route. For example, peptide agents can be most suitably administered by routes other than oral.

[0310] MASP-2 inhibitory antibodies and polypeptides can be delivered to a subject in need thereof by any suitable means, including oral, pulmonary, parenteral (e.g., intramuscular, intraperitoneal, intravenous (IV), or subcutaneous injection), inhalation (e.g., via a fine powder formulation), transdermal, nasal, vaginal, rectal, or sublingual administration, and can be formulated in a dosage form suitable for each administration route.

[0311] As a representative example, MASP-2 inhibitory antibodies and peptides can be introduced into the body by applying the polypeptide to a body membrane that can absorb it, such as the nasal, gastrointestinal, and rectal membranes.The polypeptide is typically applied to the absorbent membrane together with a penetration enhancer (see, for example, Lee, VHL, Crit. Rev. Ther. Drug Carrier Sys. 5:69, 1988; Lee, VHL, J. Controlled Release 13:213, 1990; Lee, VHL,, Ed., Peptide and Protein Drug Delivery, Marcel Dekker, New York (1991); DeBoer, AG, et al., J. Controlled Release 13:241, 1990).For example, STDHF is a synthetic derivative of fusidic acid, a steroid surfactant that is structurally similar to bile salts and has been used as a penetration enhancer for nasal delivery (Lee, WA, Biopharm. 22, Nov. / Dec. 1990).

[0312] To protect polypeptides from enzymatic degradation, MASP-2 inhibitory antibodies and polypeptides can be introduced by conjugating them to other molecules, such as lipids. For example, the covalent attachment of polymers, particularly polyethylene glycol (PEG), has been used to protect certain proteins from enzymatic hydrolysis in the body, thereby extending their half-life (Fuertges, P., et al., J. Controlled Release 11:139, 1990). Many polymer systems for protein delivery have been reported (Bae, YH, et al., J. Controlled Release 9:271, 1989; Hori, R., et al., Pharm. Res. 6:813, 1989; Yamakawa, L, et al., J. Pharm. Sci. 79:505, 1990; Yoshihiro, I., et al., Controlled Release 10:195, 1989; Asano, M., et al., J. Controlled Release 9:111, 1989; Rosenblatt, J., et al., J. Controlled Release 9:195, 1989; Makino, K., J. Controlled Release 12:235, 1990; Takakura, Y., et al., J. Pharm. Sci. 78:117, 1989; Takakura, Y., et al., J. Pharm. Sci. 78:219, 1989).

[0313] Recently, liposomes with improved serum stability and circulation half-lives have been developed (see, e.g., U.S. Pat. No. 5,741,516 to Webb). Additionally, various methods of liposome and liposome-like preparations as potential drug carriers have been investigated (see, e.g., U.S. Pat. No. 5,567,434 to Szoka; U.S. Pat. No. 5,552,157 to Yagi; U.S. Pat. No. 5,565,213 to Nakamori; U.S. Pat. No. 5,738,868 to Shinkarenko; and U.S. Pat. No. 5,795,587 to Gao).

[0314] For transdermal application, MASP-2 inhibitory antibodies and polypeptides may be combined with other suitable ingredients, such as carriers and / or adjuvants. There are no limitations on the nature of such ingredients, except that they must be pharmaceutically acceptable for the intended administration and cannot degrade the activity of the active ingredients of the composition. Examples of suitable vehicles include ointments, creams, gels, or suspensions, with or without purified collagen. MASP-2 inhibitory antibodies and polypeptides may also be impregnated into transdermal patches, plasters, and bandages, preferably in liquid or semi-liquid form.

[0315] The compositions of the present invention may be administered systemically periodically at intervals determined to maintain a desired level of therapeutic effect. For example, the compositions may be administered, for example, by subcutaneous injection, every 2 to 4 weeks or less frequently. The dosing regimen will be determined by a physician, taking into account various factors that may affect the effect of the combination of agents. These factors may include the degree of progression of the condition being treated, the patient's age, sex, and weight, as well as other clinical factors. The dosage of each individual agent will vary as a function of the MASP-2 inhibitor contained in the composition and the presence and content of any drug delivery vehicles (e.g., sustained-release delivery vehicles). Furthermore, dosages may be adjusted to account for variations in the frequency of administration and pharmacokinetic behavior of the delivery agents.

[0316] Local delivery As used herein, the term "local" encompasses application of a drug within or around the site of a desired localized effect, and may include, for example, local delivery to the skin or other affected tissue, ocular delivery, intrathecal (IT), intracerebroventricular (ICV), intra-articular, intrasinus, intracranial, or intravesicular administration, placement, or irrigation. Local administration may be preferred to allow for low-dose administration to avoid systemic side effects and to more precisely control the timing of delivery and concentration of the active agent at the local delivery site. Local administration provides a known concentration at the target site regardless of patient-to-patient variability in metabolism, blood flow, etc. Improved dosage control is also provided by direct delivery methods.

[0317] Local delivery of MASP-2 inhibitors can be achieved during surgical procedures for treating di...

Claims

1. A pharmaceutical composition comprising a MASP-2 inhibitory monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising SEQ ID NO:67 and a light chain variable region comprising SEQ ID NO:70, for treating a human subject suffering from plasma therapy-resistant aHUS, wherein administration of the MASP-2 inhibitory antibody reduces or eliminates the following clinical parameters associated with aHUS: (i) an increase in platelet count, (ii) a decrease in LDH, (iii) an increase in haptoglobin, (iv) an increase in hemoglobin, and / or (v) a decrease in creatinine The pharmaceutical composition improves at least one or more of the following:

2. The pharmaceutical composition of claim 1, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody.

3. The pharmaceutical composition of claim 1, wherein the subject has previously undergone or is currently undergoing treatment with a terminal complement inhibitor that inhibits cleavage of complement protein C5.

4. The pharmaceutical composition of claim 3, wherein the terminal complement inhibitor is a humanized anti-C5 antibody or an antigen-binding fragment thereof.

5. The pharmaceutical composition of claim 1, wherein the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway.

6. The MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum with an IC of 30 nM or less. 50 The pharmaceutical composition of claim 1, wherein the pharmaceutical composition inhibits

7. The pharmaceutical composition of claim 1, wherein the MASP-2 inhibitory antibody is delivered systemically to the subject.

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