EA046178B1 - METHODS FOR TREATING AND/OR PREVENTING GRAFT AGAINST HOST REACTION AND/OR DIFFUSE ALVEOLAR BLEEDING AND/OR VENO-OCCLUSIVE DISEASE ASSOCIATED WITH HEMAPOIETIC STEM CELL GRAFT - Google Patents

Description

Sequence listing statement

The sequence listing related to the invention is provided in text format in lieu of a paper copy and is incorporated herein by reference. The text file containing the list of sequences is named MP_l_0278_PCT_SequenceListingasFiled_20180807; txt file is 116 KB in size; it was created on August 7, 2018, and was submitted via EFS-Web upon application.

Prerequisites for creating the invention

The complement system provides an early action mechanism for initiating, enhancing and orchestrating the immune response to microbial infection and other acute insults (M.K. Liszewski and J.P. Atkinson, 1993, in: Fundamental Immunology, Third Edition, edited by W.E. Paul, Raven Press, Ltd., New York) in humans and other vertebrates. While complement activation provides valuable first-line defense against potential pathogens, complement activity that promotes a protective immune response may 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, the proteolytic products C3 and C5 attract and activate neutrophils. While essential for host defense, activated neutrophils are indiscriminate in releasing destructive enzymes and can cause organ damage. In addition, complement activation can cause the deposition of lytic complement components on neighboring host cells as well as on microbial targets, resulting in host cell lysis. The complement system is also involved in the pathogenesis of numerous acute and chronic diseases, including: myocardial infarction, stroke, ARDS, reperfusion injury, septic shock, increased capillary permeability after thermal burns, inflammation after cardiopulmonary bypass, graft rejection, rheumatoid arthritis, multiple sclerosis, myasthenia gravis and Alzheimer's disease. In almost all of these conditions, complement is not the cause, but one of several factors involved in pathogenesis. However, complement activation may be an underlying pathological mechanism and represents an effective link for clinical control in many of these disease states. Increasing recognition of the importance of complement-mediated tissue damage in various disease states underscores the need for effective complement-inhibiting drugs. Currently, eculizumab (soliris®), an anti-C5 antibody, is the only complement-targeting drug that has been approved for the treatment of humans. However, C5 is one of several effector molecules located downstream in the complement system, and blockade of C5 does not prevent complement activation. As a result, an inhibitor of the initiation stages of complement activation will have significant advantages over an inhibitor of complement components acting at later stages.

It is now widely believed that the complement system can be activated by three different pathways: the classical pathway, the lectin pathway, and the alternative pathway. The classical pathway is typically initiated by a complex consisting of host antibodies bound to a foreign particle (i.e., antigen) and thus requires prior exposure to the antigen to induce a specific antibody response. Because activation of the classical pathway depends on the host's prior adaptive immune response, the classical pathway is part of the acquired immune system. In contrast, both lectin and alternative pathways are independent of adaptive immunity and are part of the innate immune system.

Activation of the complement system leads to the sequential activation of serine protease zymogens. The first step in classical pathway activation is the binding of a specific recognition molecule, C1q, to antigen-bound IgG and IgM molecules. The C1q component is associated with the serine protease proenzymes C1r and C1s in a complex called C1. Upon binding of C1q to the immune complex, autoproteolytic cleavage of the Arg-Ile site in C1r occurs, followed by C1r-mediated cleavage and activation of C1s, which thereby acquires the ability to cleave C4 and C2. C4 splits into two fragments, designated C4a and C4b, and similarly, C2 splits into C2a and C2b. C4b fragments are capable of forming covalent bonds with neighboring hydroxyl or amino groups and creating C3 convertase (C4b2a) due to non-covalent interaction with the C2a fragment of activated C2. C3 convertase (C4b2a) activates C3 by proteolytically cleaving it into subcomponents C3a and C3b, which leads to the formation of C5 convertase (C4b2a3b), which, by cleaving C5, leads to the formation of a membrane attack complex (C5b in combination with C6, C7, C8 and C -9, also called MAC), which can disrupt cell membranes, leading to cell lysis. Activated forms of C3 and C4 (C3b and C4b) form covalent deposits on the surfaces of foreign targets, recognized by complement receptors on many phagocytes.

Independently, the first step in activation of the complement system through the lectin pathway also involves the binding of specific recognition molecules, followed by activation of associated serine protease proenzymes. However, instead of the immune complex-binding C1q molecule

- 1 046178 recognitions in the lectin pathway include a group of carbohydrate-binding proteins (mannan-binding lectin (MBL), H-ficolin, M-ficolin, L-ficolin and C-type lectin CL-11), collectively called lectins. See J. Lu et al., Biochim. Biophys. Acta 1572:387-400, (2002); Holmskov et al., Annu. Rev. Immunol. 21:547-578 (2003); Teh et al., Immunology 101:225-232 (2000)). See also J. Luet et al., Biochim Biophys Acta 1572:387-400 (2002); Holmskov et al., Annu Rev Immunol 21:547-578 (2003); Teh et al., Immunology 101:225-232 (2000); Hansen et al., J Immunol 185(10):6096–6104 (2010).

Ikeda et al first demonstrated that, like C1q, MBL can activate the complement system after binding to yeast mannan-coated red blood cells in a C4-independent manner (Ikeda et al., J. Biol. Chem. 262:7451-7454, (1987)). MBL, a member of the collectin family of proteins, is a calcium-dependent lectin that binds carbohydrates with 3- and 4-hydroxyl groups oriented in the equatorial plane of the pyranose ring. Thus, the major ligands for MBL are D-mannose and N-acetyl-D-glucosamine, while carbohydrates that do not meet this steric requirement have undetectable affinity for MBL (Weis et al., Nature 360:127-134 , (1992)). The interaction between MBL and monovalent sugars is extremely weak, with dissociation constants typically in the millimolar range with single digit values. MBL achieves strong, specific binding to glycan ligands through avidity, that is, through simultaneous interaction with multiple monosaccharide residues located in close proximity to each other (Lee et al., Archiv. Biochem. Biophys. 299:129-136, (1992 )). MBL recognizes carbohydrate patterns that are commonly found on the surface of microorganisms such as bacteria, yeast, parasites and some viruses. In contrast, MBL does not recognize D-galactose and sialic acid, the penultimate and final sugars that are typically present on mature complex glycoconjugates found on plasma and cell surface glycoproteins in mammals. This binding specificity is thought to promote recognition of foreign surfaces and help protect against self-activation. However, MBL binds with high affinity clusters of high-mannose glycan precursors on N-linked glycoproteins and glycolipids sequestered in the endoplasmic reticulum and Golgi apparatus in mammalian cells (Maynard et al., J. Biol. Chem. 257:3788-3794, ( 1982)). As a consequence, damaged cells are potential targets for activation of the lectin pathway through MBL binding.

Ficolins have a different type of lectin domain than MBL, called a fibrinogen-like domain. Ficolins bind sugar residues in a Ca-independent manner. Three types of ficolins have been identified in humans (L-ficolin, M-ficolin and H-ficolin). Two serum ficolins, Lficolin and N-ficolin, share specificity for N-acetyl-E-glucosamine; however, Nficolin also binds N-acetyl-E-galactosamine. The difference in sugar specificity of L-ficolin, H-ficolin, CL-11 and MBL means that different lectins may be complementary and target different, albeit overlapping, glycoconjugates. This concept is supported by the recent report that of the known lectins in the lectin pathway, only L-ficolin binds specifically lipoteichoic acid, a cell wall glycoconjugate found on all gram-positive bacteria (Lynch et al., J. Immunol. 172:1198-1202 , (2004)). Collectins (i.e., MBL) and ficolins do not have significant amino acid sequence similarity. However, the two groups of proteins have similar domain organizations and, like C1q, assemble into oligomeric structures that maximize the ability to bind at multiple sites.

Serum concentrations of MBL are highly variable in populations of healthy subjects, and this is genetically controlled by polymorphisms/mutations in both the promoter and coding regions of the MBL gene. The expression of MBL, as an acute phase protein, is further stimulated by inflammation. L-ficolin is present in serum at concentrations similar to those of MBL. Thus, the L-ficolin branch of the lectin pathway is potentially comparable to the MBL branch in strength. MBL and ficolins can also act as opsonins, allowing phagocytes to target MBL and ficolin containing surfaces (see Jack et al., J Leukoc Biol., 77(3):328-36 (2004), Matsushita and Fujita, Immunobiology, 205(4-5):490-7 (2002), Aoyagi et al., J Immunol, 174(l):41825(2005) Such 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)), which are not identified.

Human MBL interacts specifically and with high affinity through its collagen-like domain with unique C1r/CH-like serine proteases called MBL-associated serine proteases (MASPs). Three MASPs have been described so far. First, one MASP enzyme was identified and characterized as the enzyme responsible for initiation of the complement cascade (i.e., C2 and C4 cleavage) (Matsushita et al., J Exp Med 176(6): 1497-1502 (1992); Ji et al., J. Immunol. 150:571-578, (1993)). It was subsequently determined that MASP activity was actually the activity of a mixture of two proteases: MASP-1 and MASP-2 (Thiel et al., Nature 386:506-510, (1997)). However, it has been demonstrated that the MBL-MASP-2 complex alone is sufficient to activate complement (Vorup-Jensen et al., J. Immunol. 165:2093-2100, (2000)). Moreover, only

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MASP-2 degraded 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 form the C3 convertase, C4b2a. This is a significant difference from the C1 complex of the classical pathway, where the coordinated action of two specific serine proteases (C1r and C1s) leads to activation of the complement system. In addition, a third novel protease, MASP-3, was isolated (Dahl, M.R., et al., Immunity 15:127-35, 2001). MASP-1 and MASP-3 are alternative splicing products of the same gene.

MASPs have a domain organization identical to that of C1r and C1s, the enzymatic components of the C1 complex (Sim et al., Biochem. Soc. Trans. 28:545, (2000)). These domains include an N-terminal C1r/C1s/VEGF sea urchin/bone morphogenetic protein (CUB) domain, an epidermal growth factor-like domain, a second CUB domain, a tandem of complement control protein domains, and a serine protease domain. As in C1 proteases, activation of MASP-2 occurs as a result of cleavage of the Arg-Ile bond near the serine protease domain, which results in the separation of the enzyme into disulfide-bonded chains A and B, the latter of which consists of the serine protease domain.

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

Some data suggest that there are different MBL-MASP complexes, and a large fraction of MASP in serum is not complexed with MBL (Thiel, et al., J. Immunol. 165:878887, (2000)). Both H- and L-ficolin bind all MASPs and activate the complement lectin pathway, similar to MBL (Dahl et al., Immunity 15:127-35, (2001); Matsushita et al., J. Immunol. 168:3502 -3506, (2002)). In both the lectin and classical pathways, C3 convertase (C4b2a) is formed, and at this stage these two pathways converge.

It is generally accepted that the lectin pathway plays a major role in host defense against infection in naïve hosts. Strong evidence for the involvement of MBL in host defense comes from an analysis of patients having reduced serum levels of functional MBL (Kilpatrick, Biochim. Biophys. Acta 1572:401-413, (2002)). These patients tend to have recurrent bacterial and fungal infections. These symptoms usually appear early in life, during an apparent period of vulnerability when the titre of antibodies received from the mother is declining, but the full repertoire of self-antibodies has not yet developed. This syndrome often results from mutations at multiple sites in the collagen portion of the MBL, which prevent the proper formation of MBL oligomers. However, because MBL can function as a complement-independent opsonin, the extent to which increased susceptibility to infection is dependent on impaired complement activation is unknown.

In contrast to the classical and lectin pathways, no alternative pathway initiator has been found to perform the recognition functions that C1q and lectins perform in the other two pathways. It is now widely believed that the alternative pathway spontaneously undergoes low levels of cyclic activation, which can be easily enhanced on foreign or other abnormal surfaces (bacteria, yeast, virus-infected cells or damaged tissue) that lack the proper molecular elements to keep it under control. spontaneous activation of complement. There are four plasma proteins directly involved in the activation of the alternative pathway: C3, factors B and D, and properdin.

Although there is ample evidence implicating both the classical and alternative complement pathways in the pathogenesis of human non-infectious diseases, the role of the lectin pathway is only beginning to be explored. Recent studies provide evidence that the lectin activation pathway may be responsible for complement activation and associated inflammation during ischemia/reperfusion injury. Collard et al (2000) reported that cultured endothelial cells subjected to oxidative stress bind MBL and are deposited with S3 when exposed to human serum (Collard et al., Am. J. Pathol. 156:15491556, (2000 )). In addition, treatment with human serum containing anti-MBL blocking monoclonal antibodies inhibited MBL binding and complement activation. Similar results were obtained in a rat model of myocardial ischemia-reperfusion injury, in which rats treated with a blocking antibody directed against rat MBL exhibited significantly less myocardial injury 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 vascular endothelium after oxidative stress is unclear; A recent study suggests that activation of the lectin pathway after oxidative stress may be mediated by MBL binding to vascular endothelial cytokeratins rather than glycoconjugates (Collard et al.,

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Am. J. Pathol. 159:1045-1054, (2001)). Other studies have implicated the classical and alternative pathways in the pathogenesis of ischemia/reperfusion injury, and the role of the lectin pathway in this disease remains controversial (Riedermann, N.C., et al., Am. J. Pathol. 162:363-367, 2003).

A recent study demonstrated that MASP-1 (and possibly also MASP-3) is required for the conversion of the factor D alternative pathway enzyme from its zymogen form to its enzymatically active form (see Takahashi M. et al., J Exp Med 207(1 ):29-37 (2010)). The physiological importance of this process is emphasized by the lack of functional activity of the alternative pathway in plasma in MASP-1/3-deficient mice. Proteolytic formation of C3b from native C3 is necessary for the functioning of the alternative pathway. Since alternative pathway C3 convertase (C3bBb) contains C3b as a major subunit, the origin of the first C3b in the alternative pathway process is a mystery that has stimulated numerous studies.

C3 belongs to a family of proteins (along with C4 and α-2 macroglobulin) that have a rare post-translational modification known as a thioester linkage. The thioester group consists of glutamine, the terminal carbonyl group of which forms a covalent thioether bond with the sulfhydryl group of cysteine, located three amino acids away from it. This bond is unstable and the electrophilic glutamyl thioether can react with nucleophilic groups such as hydroxyl or amino groups and thus form a covalent bond with other molecules. The thioester bond is quite stable when it is sequestered in the hydrophobic pocket of intact C3. However, proteolytic cleavage of C3 to C3a and C3b results in the exposure of a highly reactive thioester bond to C3b and, after nucleophilic attack by adjacent moieties containing hydroxyl or amino groups, C3b becomes covalently bound to the target. In addition to its well-known role in the covalent attachment of C3b to complement targets, C3 thioesters are also thought to play a key role in triggering the alternative pathway. According to the widely accepted idling theory, the alternative pathway is initiated by the formation of a liquid phase convertase, iC3Bb, which is formed from a hydrolyzed thioester C3 (iC3;C3( _H2O )) and factor B (Lachmann, PJ, et al., Springer Semin. Immunopathol 7:143-162, (1984)). C3b-like C3(H ₂ O) is formed from native C3 by slow spontaneous hydrolysis of the internal thioester in the protein (Pangburn, MK, et al., J. Exp. Med. 154:856-867, 1981). 3a, due to the activity of the convertase C3(H ₂ O)B, C3b molecules are deposited on the surface of the target, thereby triggering an alternative pathway. Very little is known about the initiators of alternative pathway activation. Activators believed to include yeast cell wall (zymosan), many pure polysaccharides, rabbit red blood cells, some immunoglobulins, viruses, fungi, bacteria, animal tumor cells, parasites and damaged cells. The only common feature of these activators is the presence of carbohydrate, but the complexity and diversity of carbohydrate structures make it difficult to identify common, recognizable molecular determinants. It is widely accepted that activation of the alternative pathway is controlled by a precise balance between the inhibitory regulatory components of this pathway, such as factor H, factor I, DAF and CR1, and properdin, which is the only positive regulator of the alternative pathway (see Schwaeble WJ and Reid KB, Immunol Today 20(1): 17-21 (1999)).

In addition to the apparently dysregulated activation mechanism described above, the alternative pathway may also provide a powerful amplification loop for the lectin/classical pathway C3 convertase (C4b2a), since any C3b formed can participate with factor B in the formation of an additional C3 convertase (C3bBb) of the alternative pathway. Alternative pathway C3 convertase is stabilized by binding of properdin. Properdin prolongs the half-life of alternative pathway C3 convertase by six to ten times. The addition of C3b to the alternative pathway C3 convertase results in the formation of the alternative pathway C5 convertase.

It is generally accepted that all three pathways (i.e., classical, lectin, and alternative) converge on C5, which is cleaved to produce products with a variety of proinflammatory effects. The converged pathway was named the terminal complement pathway. C5a is the most potent anaphylatoxin, causing changes in smooth muscle and vascular tone, as well as vascular permeability. It is also a potent chemotaxin and activator of both neutrophils and monocytes. C5a-mediated cellular activation can significantly enhance inflammatory responses by inducing the release of multiple additional inflammatory mediators, including cytokines, hydrolytic enzymes, arachidonic acid metabolites, and reactive oxygen species. Cleavage of C5 results in the formation of C5b-9, also known as the membrane attack complex (MAC). There is now compelling evidence that sublytic MAC deposition may play an important role in inflammation, in addition to its role as a lytic pore-forming complex.

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

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The essence of the invention

This summary is intended to present in a simplified form selected concepts that are further described below in the Detailed Description of the Invention. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used in defining the scope of the claimed subject matter. In one aspect, the present invention provides a method of inhibiting microvascular endothelial cell damage and/or blood clot 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 activation of complement. In some embodiments, the subject suffers from, or is at risk of developing, TMA selected from the group consisting of hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP), and atypical hemolytic uremic syndrome (aHUS). In some embodiments, prior to administration of the composition, the subject is determined to exhibit one or more symptoms selected from the group consisting of (i) anemia, (ii) thrombocytopenia, (iii) renal failure, and (iv) elevated creatinine levels, and the composition is administered to effective amount and for a sufficient period of time to relieve said one or more symptoms. In some embodiments, the MASP-2 inhibitory agent is an anti-MASP-2 antibody, or a fragment thereof. In some embodiments, the MASP-2 inhibitory agent is an anti-MASP-2 monoclonal antibody, or fragment thereof, that specifically binds a portion of SEQ ID NO: 6. In some embodiments, the MASP-2 inhibitory agent inhibits damage to microvascular endothelial cells.

In another aspect, the invention provides a method of inhibiting MASP-2-dependent complement activation in a subject suffering from, or at risk of developing, atypical hemolytic uremic syndrome (aHUS), comprising administering to the subject a composition containing a MASP-2 inhibitory agent in an amount effective to inhibit MASP-2-dependent complement activation. In one embodiment, prior to administration of the composition, the subject is determined to exhibit one or more symptoms selected from the group consisting of (i) anemia, (ii) thrombocytopenia, (iii) renal failure, and (iv) elevated creatinine levels, and the composition is administered to effective amount and for a sufficient period of time to relieve said one or more symptoms. In one embodiment, the subject suffers from, or is at risk of developing factor H-independent 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 inhibitory agent is an anti-MASP-2 antibody, or fragment thereof, such as an anti-MASP-2 monoclonal antibody, or fragment thereof, that specifically binds a portion of SEQ ID NO: 6. In one embodiment, the MASP-inhibitory agent -2 agent inhibits damage to microvascular endothelial cells. In one embodiment, the MASP-2 inhibitory agent inhibits the formation of blood clots.

In another aspect, the invention relates to a method of 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 of this aspect of the invention includes (a) determining whether a subject has a genetic marker known to be associated with aHUS; (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 a composition comprising a MASP-2 inhibitory agent in an amount effective to inhibit MASP-2-dependent complement activation, as determined by the presence of at least one of anemia, thrombocytopenia, renal failure, or elevated creatinine, wherein the composition is administered in an effective amount and for a sufficient period of time to relieve said one or more symptoms. In one embodiment, the MASP-2 inhibitory agent is an anti-MASP-2 antibody, or fragment thereof, such as an anti-MASP-2 monoclonal antibody, or fragment thereof, that specifically binds a portion of SEQ ID NO: 6. In one embodiment of the method, step (a) involves genetically screening a sample obtained from the subject and determining the presence of at least one genetic marker associated with aHUS in a gene selected from the group consisting of factor H (CFH), factor I (CFI) genes , complement factor B (CFB), membrane cofactor CD46, C3, complement factor H-related protein (CFHR1), thrombomodulin anticoagulant protein (THBD), complement factor H-related protein 3 (CFHR3), and complement factor H-related protein 4 ( CFHR4). In one embodiment, the method further includes monitoring the subject for the occurrence of an event that is known to be associated with the initiation of clinical symptoms of aHUS, and administering to the subject a composition containing a MASP-2 inhibitory agent before, during, or after the occurrence of the initiating event. In one embodiment, the event associated with the initiation of clinical symptoms of aHUS is selected from the group consisting of drug exposure, infection, malignancy, trauma,

- 5 046178 organ or tissue transplantation and pregnancy. In one embodiment, the infection is a bacterial infection. In one embodiment, the composition is administered subcutaneously. In one embodiment, the MASP-2 inhibitory agent inhibits damage to microvascular endothelial cells. In one embodiment, the MASP-2 inhibitory agent inhibits the formation of blood clots.

In another aspect, the invention provides a method of inhibiting MASP-2-dependent complement activation in a subject suffering from, or at risk of developing, atypical hemolytic uremic syndrome (aHUS) due to infection, comprising administering to the subject a composition containing a MASP-2 inhibitory agent in an amount , effective in inhibiting MASP-2-dependent complement activation. In one embodiment, the subject suffers from, or is at risk of developing, non-enteric aHUS associated with an infection caused by S. pneumonia. In one embodiment, the MASP-2 inhibitory agent is an anti-MASP-2 antibody, or fragment thereof, such as an anti-MASP-2 monoclonal antibody, or fragment thereof, that specifically binds a portion of SEQ ID NO: 6. In one embodiment MASP-2 inhibitory agent inhibits damage to microvascular endothelial cells. In one embodiment, the MASP-2 inhibitory agent inhibits the formation of blood clots.

In another aspect, the invention provides a method of treating a subject suffering from atypical hemolytic uremic syndrome (aHUS), comprising administering to the subject a composition comprising a MASP-2 inhibitory agent in an amount effective to inhibit MASP-2-dependent complement activation, wherein administering the MASP-2 inhibitory agent. 2 drugs are performed by delivery through an intravenous catheter or another catheter. In one embodiment, the method further includes treating a patient using plasmapheresis. In one embodiment, the composition containing the MASP-2 inhibitory agent is administered without plasmapheresis. In one embodiment, a composition containing a MASP-2 inhibitory agent is administered through a catheter for a first period of time, followed by administration of a composition containing a MASP-2 inhibitory agent for a second period of time, wherein during a second period of time the composition is administered subcutaneously . In one embodiment, the method further includes periodically determining the level of at least one complement factor, wherein determining a decreased level of at least one complement factor compared to a standard value, or a value in a healthy subject, indicates the need for continued treatment with the composition. In one embodiment, the MASP-2 inhibitory agent is an anti-MASP-2 antibody, or fragment thereof, such as an anti-MASP-2 monoclonal antibody, or fragment thereof, that specifically binds a portion of SEQ ID NO: 6. In one embodiment MASP-2 inhibitory agent inhibits damage to microvascular endothelial cells. In one embodiment, the MASP-2 inhibitory agent inhibits the formation of blood clots.

In another aspect, the invention provides a method of treating a subject suffering from thrombotic thrombocytopenic purpura (TTP), or having symptoms consistent with a diagnosis of TTP, comprising administering to the subject a composition containing a MASP-2 inhibitory agent in an amount effective to inhibit MASP-2-dependent activation of complement, wherein the administration of the MASP-2 inhibitory agent is carried out by delivery through an intravenous catheter or other catheter. In one embodiment, the subject has at least one or more symptoms selected from the group consisting of central nervous system damage, thrombocytopenia, severe cardiac damage, severe pulmonary damage, intestinal infarction, and gangrene. In one embodiment, the subject tests positive for an ADAMTS-13 inhibitor, and the method further includes administering an immunosuppressant to the subject. In one embodiment, a composition containing a MASP-2 inhibitory agent is administered for a first period of time without plasmapheresis. In one embodiment, the subject tests positive for an ADAMTS-13 inhibitor, and the method further includes administering ADAMTS-13. In one embodiment, the method further includes treating a patient using plasmapheresis. In one embodiment, a composition containing a MASP-2 inhibitory agent is administered in combination with plasmapheresis. In one embodiment, a composition containing a MASP-2 inhibitory agent is administered through a catheter for a first period of time, followed by administration of a composition containing a MASP-2 inhibitory agent for a second period of time, wherein during a second period of time the composition is administered subcutaneously . In one embodiment, the method further includes periodically determining the level of at least one complement factor, wherein determining a decreased level of at least one complement factor compared to a standard value, or a value in a healthy subject, indicates the need for continued treatment with the composition. In one embodiment, the MASP-2 inhibitory agent is an anti-MASP-2 antibody, or fragment thereof, such as an anti-MASP-2 monoclonal antibody, or fragment thereof, that specifically binds a portion of SEQ ID NO: 6. In one embodiment MASP-2 inhibitory agent inhibits damage to microvascular endothelial cells. In one embodiment, the inhibitory

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MASP-2 agent inhibits the formation of blood clots.

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

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

In another aspect, the invention provides a method of 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 containing a MASP-2 inhibitory agent in an amount effective to inhibit MASP-2-dependent complement activation. In some embodiments, the method includes treating a subject at risk of developing USS, including administering over a specified period of time a specified amount of a MASP-2 inhibitory agent effective to reduce or prevent one or more clinical symptoms associated with TTP. In some embodiments, the method further includes periodically monitoring the subject and administering a MASP-2 inhibitory agent upon the occurrence of an event known to be associated with the initiation of clinical symptoms of TTP. In some embodiments, the method further includes periodically monitoring the subject and administering a MASP-2 inhibitory agent to determine the presence of anemia, thrombocytopenia, or elevated creatinine levels. In some embodiments, the subject has previously received, or is currently receiving, treatment with a terminal complement inhibitor that inhibits the breakdown of complement protein C5. In some embodiments, the method further comprises administering to the subject a terminal complement inhibitor that inhibits the breakdown of complement protein C5, such as a humanized anti-C5 antibody, or an antigen-binding fragment thereof, such as eculizumab.

In another aspect, the invention provides a method of inhibiting MASP-2-dependent complement activation in a subject suffering from Degos disease, comprising administering to the subject a composition containing a MASP-2 inhibitory agent in an amount effective to inhibit MASP-2-dependent complement activation. In some embodiments, the subject has previously received, or is currently receiving, treatment with a terminal complement inhibitor that inhibits the breakdown of complement protein C5. In some embodiments, the method further comprises administering to the subject a terminal complement inhibitor that inhibits the breakdown of complement protein C5, such as a humanized anti-C5 antibody, or

- 7 046178 its antigen-binding fragment, such as eculizumab.

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

In some embodiments of any of the disclosed methods of the invention, the MASP-2 inhibitory agent 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 significantly inhibit the classical pathway. In some embodiments, the MASP-2 inhibitory agent is an anti-MASP-2 monoclonal antibody, or fragment thereof, that specifically binds a portion of SEQ ID NO: 6. In some embodiments, the anti-MASP-2 antibody, or fragment thereof, is selected from the group consisting of a recombinant antibody, an antibody having reduced effector function, a chimeric antibody, a humanized antibody and a human antibody. In 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 with the S228P mutation. In some embodiments, the MASP-2 inhibitory antibody binds human MASP-2 with a KD value of 10 nM or less. 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 an in vitro assay in 1% human serum with an IC ₅₀ value of 10 nM or less. In some embodiments, the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum with an IC ₅₀ value of 30 nM or less.

In some embodiments of any of the disclosed methods of the invention, the MASP-2 inhibitory monoclonal antibody, or an antigen binding fragment thereof, comprises: (a) a heavy chain variable region comprising: i) a heavy chain CDR-H1 comprising the amino acid sequence of residues 31-35 SEQ ID NO: 67; and ii) a heavy chain CDR-H2 containing the amino acid sequence of residues 50-65 of SEQ ID NO: 67; and iii) a heavy chain CDR-H3 comprising the amino acid sequence of residues 95-102 of SEQ ID NO: 67, and (b) a light chain variable region comprising: i) a light chain CDR-L1 containing the amino acid sequence of residues 24-34 SEQ ID NO: 70; and ii) light chain CDR-L2 containing the amino acid sequence of residues 50-56 in SEQ ID NO: 70; and iii) a light chain CDR-L3 comprising the amino acid sequence of residues 89-97 of SEQ ID NO: 70. In some embodiments, the MASP-2 inhibitory monoclonal antibody comprises a heavy chain variable region of SEQ ID NO: 67 and a light chain variable region of SEQ ID NO: 70. In some embodiments, a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, specifically recognizes at least a portion of an epitope recognized by a reference antibody comprising a heavy chain variable region of SEQ ID NO: 67 and a light chain variable region of SEQ]poΓoOα70 aspect of the invention relates to methods of inhibiting the formation of blood clots in a subject suffering from atypical hemolytic uremic syndrome (aHUS), comprising administering to the subject a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, in an amount effective to inhibit MASP-2-dependent complement activation . In some embodiments, the MASP-2 inhibitory antibody inhibits thrombus formation in the serum of a subject suffering from aHUS by at least 40% compared to control serum. In some embodiments, the MASP-2 inhibitory antibody inhibits thrombus formation in the serum of a subject suffering from aHUS to a level of at least 20% greater (e.g., at least 30% greater, at least 40% greater, or at least 50% greater) than its inhibitory effect on C5b-9 deposition in serum from the same subject. In some embodiments, the subject has aHUS in the acute phase. In some embodiments, the subject has aHUS in remission. In some embodiments, the MASP-2 inhibitory antibody is a monoclonal antibody, or fragment thereof, that specifically binds 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 having reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In neko

- 8 046178 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 with the S228P mutation. In some embodiments, the MASP-2 inhibitory antibody binds human MASP-2 with a K _D value of 10 nM or less. 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 an in vitro assay in 1% human serum with an IC ₅₀ value of 10 nM or less. In some embodiments, the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum with an IC ₅₀ value of 30 nM or less. In some embodiments, the MASP-2 inhibitory monoclonal antibody, or an antigen binding fragment thereof, comprises: (a) a heavy chain variable region comprising: i) a heavy chain CDR-H1 comprising the amino acid sequence of residues 31-35 of SEQ ID NO: 67; and ii) a heavy chain CDR-H2 containing the amino acid sequence of residues 50-65 of SEQ ID NO: 67; and iii) a heavy chain CDR-H3 comprising the amino acid sequence of residues 95-102 of SEQ ID NO: 67, and (b) a light chain variable region comprising: i) a light chain CDR-L1 containing the amino acid sequence of residues 24-34 SEQ ID NO: 70; and ii) light chain CDR-L2 containing the amino acid sequence of residues 50-56 in SEQ ID NO: 70; and iii) a light chain CDR-L3 comprising the amino acid sequence of residues 89-97 of SEQ ID NO: 70. In some embodiments, the MASP-2 inhibitory monoclonal antibody comprises a heavy chain variable region of SEQ ID NO: 67 and a light chain variable region of SEQ ID NO: 70. In some embodiments, a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, specifically recognizes at least a portion of an epitope recognized by a reference antibody comprising a heavy chain variable region of SEQ ID NO: 67 and a light chain variable region of SEQ ID NO: 70.

In another aspect, the present invention provides a method of treating a subject suffering from plasma therapy-resistant aHUS, 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 one embodiment, the MASP-2 inhibitory antibody is an anti-MASP-2 monoclonal antibody, or fragment thereof, that specifically binds human MASP-2. In one embodiment, the antibody, or fragment thereof, is selected from the group consisting of a recombinant antibody, an antibody having reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In one embodiment, the subject has previously received, or is currently receiving, treatment with a terminal complement inhibitor that inhibits the breakdown of complement protein C5, for example, wherein the terminal complement inhibitor is a humanized anti-C5 antibody, or an antigen-binding fragment thereof. In one embodiment, the method further includes treating a patient using plasmapheresis. In one embodiment, the composition containing the MASP-2 inhibitory antibody is administered without plasmapheresis.

In another aspect, the present invention provides a method of treating a subject suffering from hematopoietic stem cell transplant-associated 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 one embodiment, the MASP-2 inhibitory antibody is an anti-MASP-2 monoclonal antibody, or fragment thereof, that specifically binds human MASP-2. In one embodiment, the antibody, or fragment thereof, is selected from the group consisting of a recombinant antibody, an antibody having reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In one embodiment, the subject has previously received, or is currently receiving, treatment with a terminal complement inhibitor that inhibits the breakdown of complement protein C5. In one embodiment, the subject suffers from hematopoietic stem cell transplant-associated TMA that is resistant to treatment using platelet transfusion and/or resistant to treatment using plasmapheresis.

In one embodiment, the MASP-2 inhibitory antibody is administered in an amount effective to improve at least one or more clinical parameters associated with hematopoietic stem cell transplant-associated TMA, e.g., an increase in platelet count (e.g., an increase of at least two , at least three, at least four times the platelet count before treatment), an increase in haptoglobin levels and/or a decrease in lactate dehydrogenase levels.

In another aspect, the present invention relates to a method of treating a human subject suffering from persistent hematopoietic stem cell transplant-associated TMA (HSCT

- 9 046178

TMA) comprising administering to a subject a composition comprising a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, in an amount effective to inhibit MASP-2 dependent complement activation. In one embodiment, the method further includes identifying a human subject having persistent TMA associated with a hematopoietic stem cell graft before the step of administering to the subject a composition comprising a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, in an amount effective to inhibit MASP-2 -dependent activation of complement.

In one embodiment, the MASP-2 inhibitory antibody is a monoclonal antibody, or fragment thereof, that specifically binds 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 having reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In one embodiment, the MASP-2 inhibitory antibody does not significantly inhibit the classical pathway. In one embodiment, the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum with an IC ₅₀ value of 30 nM or less. In one embodiment, a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprises a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 with the amino acid sequence of SEQ ID NO: 67 and a light chain variable region comprising the CDR -L1, CDR-L2 and CDR-L3, with the amino acid sequence of SEQ ID NO: 70. In one embodiment, the MASP-2 inhibitory antibody is administered to a patient without plasmapheresis. In one embodiment, the subject has previously received, or is currently receiving, treatment with a humanized anti-C5 antibody, or an antigen-binding fragment thereof, for example, wherein the terminal complement inhibitor is a humanized anti-C5 antibody, or an antigen-binding fragment thereof. In one embodiment, the MASP-2 inhibitory antibody is administered to the subject systemically. In one embodiment, a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, is administered in an amount effective to improve at least one or more of the following clinical parameters associated with hematopoietic stem cell transplant-associated TMA, for example: (i ) an increase in platelet count (for example, an increase of at least two, at least three, at least four times the platelet count before treatment)); (ii) increasing haptoglobin levels; (iii) decreasing lactate dehydrogenase (LDH) levels and/or (iv) decreasing creatinine levels.

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

In another aspect, the present invention provides a method of treating, preventing, or ameliorating one or more neurological symptoms associated with graft-versus-host disease or TMA, comprising administering to a subject in need thereof a composition comprising a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof , in an amount effective to inhibit MASP-2-dependent complement activation. In one embodiment, one or more neurological symptoms associated with graft-versus-host disease or TMA are selected from the group consisting of asthenia, paresthesia, tetraplegia, sensorimotor disorders,

- 10 046178 vegetative-vascular polyneuropathy and/or neurogenic bladder. In one embodiment, the subject has received a hematopoietic stem cell transplant, and the subject suffers from one or more neurological symptoms selected from the group consisting of paresthesia, tetraplegia, and neurogenic bladder. In one embodiment, the subject has received a hematopoietic stem cell transplant and is suffering from graft-versus-host disease. In one embodiment, the subject has received a hematopoietic stem cell transplant and is suffering from HSCT-TMA. In one embodiment, the MASP-2 inhibitory antibody is a monoclonal antibody, or fragment thereof, that specifically binds human MASP-2. In one embodiment, the antibody, or fragment thereof, is selected from the group consisting of a recombinant antibody, an antibody having reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In one embodiment, the MASP-2 inhibitory antibody does not significantly inhibit the classical pathway. In one embodiment, the MASP2 inhibitory antibody inhibits C3b deposition in 90% human serum with an IC ₅₀ value of 30 nM or less. In one embodiment, the MASP-2 inhibitory antibody is administered to the subject systemically. In one embodiment, the method further includes identifying a human subject suffering from one or more neurological symptoms associated with a hematopoietic stem cell transplant before the step of administering to the subject a composition comprising a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, in an amount effective to inhibit MASP -2-dependent complement activation. In one embodiment, the MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprises a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3, with the amino acid sequence of SEQ ID NO: 67, and a light chain variable region comprising the CDR -L1, CDR-L2 and CDR-L3, with amino acid sequence SEQ ID NO: 70.

In another aspect, the present invention provides a method of treating a human subject suffering from, or at risk of developing, diffuse alveolar hemorrhage (DAH) associated with a hematopoietic stem cell transplant, comprising administering to the subject a composition comprising a MASP-2 inhibitory antibody, or an antigen-binding antibody thereof. fragment, in an amount effective to inhibit MASP-2-dependent complement activation. In one embodiment, the subject has previously received, is currently receiving, or will receive a hematopoietic stem cell transplant. In one embodiment, the method further includes identifying a human subject suffering from, or at risk of developing, diffuse alveolar hemorrhage (DAH), prior to the step of administering to the subject a composition comprising a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, in an amount effective to inhibition of MASP-2-dependent complement activation. In one embodiment, the MASP-2 inhibitory antibody is a monoclonal antibody, or fragment thereof, that specifically binds human MASP-2. In one embodiment, the MASP-2 inhibitory antibody, or fragment thereof, is selected from the group consisting of a recombinant antibody, an antibody having reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In one embodiment, the MASP-2 inhibitory antibody does not significantly inhibit the classical pathway. In one embodiment, the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum with an IC ₅₀ value of 30 nM or less. In one embodiment, the MASP-2 inhibitory antibody is administered to the subject systemically. In one embodiment, the MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprises a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3, with the amino acid sequence of SEQ ID NO: 67, and a light chain variable region comprising the CDR -L1, CDR-L2 and CDR-L3, with the amino acid sequence SEQ ID NO: 70. In another aspect, the present invention provides a composition for inhibiting the adverse effects of MASP-2-dependent complement activation, comprising a therapeutically effective amount of a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, and a pharmaceutically acceptable carrier. Also provided are methods of producing a medicament for use in inhibiting the adverse effects of MASP-2-dependent complement activation in living subjects in need thereof, comprising a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutical carrier. Also provided are methods for the manufacture of medicaments for use in inhibiting MASP-2-dependent complement activation for the treatment of each of the conditions, diseases and disorders described below herein.

In another aspect, the present invention relates to a method of treating a human subject suffering from, or at risk of developing, veno-occlusive disease (VOD) associated with a hematopoietic stem cell transplant, comprising administering to the subject a composition comprising a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof , in an amount effective to inhibit MASP-2-dependent complement activation. In one embodiment, the subject has previously received, is currently receiving, or will receive a hematopoietic stem cell transplant. In one embodiment, the method further includes identifying the subject

- 11,046,178 in a human subject suffering from, or at risk of developing, veno-occlusive disease (VOD), prior to the step of administering to the subject a composition comprising a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, 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 human MASP-2. In one embodiment, the MASP-2 inhibitory antibody, or fragment thereof, is selected from the group consisting of a recombinant antibody, an antibody having reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In one embodiment, the MASP-2 inhibitory antibody does not significantly inhibit the classical pathway. In one embodiment, the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum with an IC ₅₀ value of 30 nM or less. In one embodiment, the MASP-2 inhibitory antibody is administered to the subject systemically. In one embodiment, a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprises a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 with the amino acid sequence of SEQ ID NO: 67 and a light chain variable region comprising the CDR -L1, CDR-L2 and CDR-L3, with the amino acid sequence SEQ ID NO: 70. In another aspect, the present invention provides a composition for inhibiting the adverse effects of MASP-2-dependent complement activation, comprising a therapeutically effective amount of a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, and a pharmaceutically acceptable carrier. Also provided are methods of producing a medicament for use in inhibiting the adverse effects of MASP-2-dependent complement activation in living subjects in need thereof, comprising a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutical carrier. Also provided are methods for the manufacture of medicaments for use in inhibiting MASP-2-dependent complement activation for the treatment of each of the conditions, diseases and disorders described below herein.

The methods, compositions and medicaments of the invention can be used to inhibit the adverse effects of MASP-2-dependent complement activation in vivo in mammalian subjects, including humans suffering from, or at risk of developing, thrombotic microangiopathy (TMA), and/or subject suffering from, or at risk of developing, GVHD, and/or a subject suffering from, or at risk of developing, diffuse alveolar hemorrhage after stem cell transplantation, and/or a subject suffering from, or at risk of developing, veno-occlusive disease (VOD) , as described later in this document.

Description of drawings

The foregoing aspects and many attendant advantages of the present invention will become better understood by reading the following detailed description in conjunction with the accompanying drawings, in which:

fig. 1 is a diagram illustrating the genomic structure of human MASP-2;

fig. 2A is a schematic diagram illustrating the domain structure of human MASP-2 protein;

fig. 2B is a schematic diagram illustrating the domain structure of human MAp19 protein;

fig. 3 is a diagram illustrating a strategy for knocking out murine MASP-2;

fig. 4 is a diagram illustrating the human MASP-2 minigene construct;

fig. 5A presents results showing that MASP-2 deficiency results in loss of lectin pathway-mediated C4 activation as determined by the absence of C4b deposition on mannan as described in Example 2;

fig. 5B presents results showing that MASP-2 deficiency results in loss of lectin pathway-mediated C4 activation as determined by the absence of C4b deposition on zymosan as described in Example 2;

fig. 5C presents results showing relative levels of C4 activation in serum samples obtained from MASP-2+/- mice; MASP-2-/-u wild type, as determined by the deposition of C4b on mannan and on zymosan, as described in example 2;

fig. 6 presents results showing that addition of murine recombinant MASP-2 to MASP-2-/- serum samples restores lectin pathway-mediated C4 activation in a protein concentration-dependent manner as determined by C4b deposition on mannan as described in Example 2;

fig. 7 presents results showing that the classical pathway is functional in MASP-2-/- mice as described in Example 8;

fig. 8A presents results showing that anti-MASP-2 Fab2 antibody No. 11 inhibits the formation of C3 convertase as described in Example 10;

- 12 046178 fig. 8B presents results showing that aHTu-MASP-2 Fab2 antibody No. 11 binds natural rat MASP-2 as described in Example 10;

fig. 8C presents results showing that anti-MASP-2 Fab2 antibody No. 41 inhibits C4 cleavage as described in Example 10;

fig. 9 presents results showing that all anti-MASP-2 Fab2 antibodies tested that inhibited C3 convertase formation were also found to inhibit C4 cleavage as described in Example 10;

fig. 10 is a diagram illustrating the recombinant polypeptides derived from rat MASP-2 that were used to map the epitopes of the anti-MASP-2 Fab2 blocking antibodies as described in Example 11;

fig. 11 presents results showing the binding of anti-MASP-2 Fab2 #40 and #60 to rat MASP-2 polypeptides as described in Example 11;

fig. 12 presents results showing blood urea nitrogen clearance in wild-type (+/+) and MASP-2 (-/-) mice 24 and 48 hours after reperfusion in the renal ischemia/reperfusion injury model as described in Example 12;

fig. 13A presents results showing baseline levels of VEGF protein in the RPEchoroid complex obtained from wild-type (+/+) and MASP-2 (-/-) mice as described in Example 13;

fig. 13B presents results showing VEGF protein levels in the RPE-choroid complex at day 3 in wild-type (+/+) and MASP-2 (-/-) mice following laser-induced injury in the macular degeneration model as described in Example 13 ;

Fig. 14 presents results showing the average choroidal neovascularization volume (CNV) on day seven after laser-induced injury in wild-type (+/+) and MASP-2 (-/-) mice as described in Example 13;

fig. 15A and 15B represent dose-response curves for inhibition of C4b deposition (FIG. 15A) and inhibition of thrombin activation (FIG. 15B) following administration of anti-MASP-2 Fab2 antibody in normal rat serum as described in Example 14;

fig. 16A and 16B show measured platelet aggregation (expressed as aggregate area) in MASP-2 (-/-) mice (FIG. 16B) compared to platelet aggregation in naïve wild-type mice and wild-type mice in which the pathway complement is inhibited by the depletor reagent, cobra venom factor (CVF), and a terminal pathway inhibitor (C5aR antagonist), (Fig. 16A) in a localized Schwartzman reaction, which is a model of disseminated intravascular coagulation, as described in example 15;

fig. 17 graphically represents blood urea nitrogen (BUN) levels measured in either WT (+/+) (B6) or MASP-2 (-/-) WT (+/+) kidney transplant recipient mice as described. in example 16;

fig. 18 graphically represents the survival, in percentage, of WT (+/+) and MASP-2 (-/-) mice as a function of the number of days after microbial infection in the cecal ligation and puncture (CLP) model as described in Example 17;

fig. 19 graphically represents the bacterial counts measured in WT (+/+) and MASP-2 (-/) mice following microbial infection in the cecal ligation and puncture (CLP) model as described in Example 17;

fig. 20 is a Kaplan-Meier plot showing the percentage survival of WT (+/+), MASP-2 (-/-) and C3 (-/-) mice six days after infection by intranasal administration of Pseudomonas aeruginosa as described in example 18;

fig. 21 graphically represents the level of C4b deposition, measured as % of control, in samples obtained at various time points after subcutaneous administration of either 0.3 mg/kg or 1.0 mg/kg of mouse anti-MASP-2 monoclonal antibody. in WT mice, as described in example 19;

fig. 22 graphically represents the level of C4b deposition, measured as % of control, in samples obtained at various time points after IP administration of 0.6 mg/kg murine anti-MASP-2 monoclonal antibody in WT mice as described in Example 19;

fig. 23 graphically represents the mean choroidal neovascularization (CNV) volume on day seven after laser-induced injury in WT (+/+) mice pretreated with a single IP injection of 0.3 mg/kg or 1.0 mg/kg murine anti-MASP -2 monoclonal antibodies as described in example 20;

fig. 24A graphically represents the percentage survival of MASP-2 (-/-) and WT (+/+) mice after infection with 5x108/100 μl CFU of N. meningitidis as described in Example 21;

fig. 24B graphically represents log CFU/ml of N. meningitidis recovered at different time points from blood samples obtained from MASP-2 KO (-/-) and WT (+/+) mice infected with 5x108/100 µl CFU of N. meningitidis. as described in example 21;

fig. 25A graphically represents the survival, in percentage, of MASP-2 KO (-/-) and WT (+/+) mice.

- 13 046178 after infection with 2x10 ⁸ CFU/100 μl N. meningitidis, as described in example 21;

fig. 25B graphically represents log CFU/ml N. meningitidis recovered at different time points from blood samples obtained from WT (+/+) mice infected with 2x10 ⁸ CFU/100 μl N. meningitidis as described in Example 21;

fig. 25C graphically represents log CFU/ml N. meningitidis recovered at different time points from blood samples obtained from MASP-2 (-/-) mice infected with 2x10 ⁸ CFU/100 μl N. meningitidis as described in Example 21;

fig. 26A graphically presents the results of a C3b deposition assay demonstrating that MASP-2 (-/-) mice retain a functional classical pathway as described in Example 22;

fig. 26B graphically presents the results of a C3b deposition assay on zymosan-coated plates demonstrating that MASP-2 (-/-) mice retain a functional alternative pathway as described in Example 22;

fig. 27A graphically represents myocardial ischemia/reperfusion injury (MIRI)-induced tissue loss following left anterior descending coronary artery (LAD) ligation and reperfusion in C4 (-/-) mice (n=6) and matched WT littermate controls (n=7) , with demonstration of the area at risk (RO) and the size of the necrosis lesion (NFS), as described in example 22;

fig. 27B graphically represents the size of the necrotic lesion (NF) as a function of the area at risk (RO) in C4 (-/-) and WT mice exposed as described in FIG. 42A, this shows that C4 (-/-) mice are as susceptible to MIRI as WT controls (dashed line) as described in Example 22;

fig. 28A graphically represents the results of a C3b deposition assay using serum from WT, C4 (-/-) mice and serum from C4 (-/-) mice preincubated with mannan as described in Example 22;

fig. 28B graphically represents the results of an assay for C3b deposition in the serum of WT, C4 (/-) and MASP-2 (-/-) mice mixed with anti-mouse MASP-2 mAb (mAb Ml 1) at various concentrations as described in Example 22;

fig. 28C graphically represents the results of C3 deposition assays in human WT (C4 sufficient) and C4 deficient serum, as well as serum from C4 deficient subjects preincubated with mannan as described in Example 22;

fig. 28D graphically presents the results of a C3b deposition assay in human WT serum (sufficient C4) and serum from C4-deficient subjects mixed with anti-human MASP-2 mAb (H3 mAb) as described in Example 22;

fig. 29A graphically presents the results of a comparative analysis of C3 convertase activity in plasma from different complement-deficient mouse strains tested under either lectin pathway-specific assay conditions or classical complement pathway-specific assay conditions as described in Example 22;

fig. 29B graphically represents the time-resolved kinetics of C3 convertase activity in plasma from different complement-deficient strains of mice tested under assay conditions specific for the lectin pathway of complement activation, as described in Example 22;

fig. 30 shows the results of Western blot analysis demonstrating activation of human C3, as evidenced by the presence of the α' chain, by the thrombin substrates FXIa and FXa as described in Example 23;

fig. 31 shows the results of analysis of C3 deposition in serum samples obtained from WT, MASP-2 (-/-), F11(-/-), F11(-/-)/C4 (-/-) and C4 (-/-) mice. ), as described in example 23;

Fig. 32A is a Kaplan-Meier plot showing percentage survival over time after exposure to 7.0 Gy radiation for control mice and mice treated with anti-mouse MASP-2 antibody (mAb M11) or anti-human MASP-2 antibody (mAb H6) as described in example 29;

fig. 32B is a Kaplan-Meier plot showing percentage survival over time after exposure to 6.5 Gy radiation for control mice and mice treated with anti-mouse MASP-2 antibody (mAb M11) or anti-human MASP-2 antibody (mAb H6) as described in example 29;

fig. 33 is a Kaplan-Meier plot showing the percentage survival of MASP-2 KO and WT mice following an infectious dose of 2.6 x ^{10 7} CFU of N. meningitidis serogroup A Z2491, which demonstrates that MASP-2-deficient mice are protected from death , caused by N. meningitidis, as described in example 30;

fig. 34 is a Kaplan-Meier plot showing the percentage survival of MASP-2 KO and WT mice following an infectious dose of 6 x 10 ⁶ CFU of N. meningitidis serogroup B strain MC58, which demonstrates that MASP-2-deficient mice are protected from death, caused by N. meningitidis serogroup B strain MC58, as described in example 30;

- 14 046178 fig. 35 graphically represents log CFU/ml N. meningitidis serogroup B strain MC58 recovered at different time points from blood samples obtained from MASP-2 KO and WT mice after i.p. infection with 6x106 CFU of N. meningitidis serogroup B strain MC58 (n= 3 at different time points for both groups of mice, results are presented as means ± SEM); the results show that although MASP-2 KO mice were infected with the same dose of N. meningitidis serogroup B strain MC58 as WT mice, MASP-2 KO mice had increased bacterial clearance compared to WT, as described in Example 30;

fig. 36 graphically presents the mean disease scores of MASP-2 and WT mice at 3, 6, 12, and 24 hours after infection with 6x106 CFU/100 μl of N. meningitidis serogroup B strain MC58, demonstrating that MASP-2-deficient mice were highly resistant to infections, with much lower rates of ill health at 6 hours, as described in example 30;

fig. 37 is a Kaplan-Meier plot showing the percentage survival of mice following an infectious dose of 4x106/100 μl CFU of N. meningitidis serogroup B strain MC58, followed 3 hours after infection by either an inhibitory anti-MASP-2 antibody (1 mg/kg), or isotype control antibody; the results show that the anti-MASP-2 antibody is effective for the treatment and promotion of survival in subjects infected with N. meningitidis, as described in example 31;

fig. 38 graphically represents log CFU/ml viable N. meningitidis serogroup B-MC58 bacteria isolated at different time points from human serum at a concentration of 20% after IP infection with 6.5 x 106 CFU/100 μl N. meningitidis serogroup B strain MC58, through 0, 30, 60 and 90 min after incubation in the presence of: (A) normal human serum (NHS) plus human anti-MASP-2 antibody; (B) normal human serum (HNS) plus isotype control antibody; (C) MBL-/- human serum; (D) normal human serum (HNS) and (E) heat-inactivated normal human serum (HNS); the results show that complement-dependent killing of N. meningitidis in human serum was significantly enhanced by the addition of human anti-MASP-2 antibody as described in Example 32;

fig. 39 graphically represents log CFU/ml viable N. meningitidis serogroup B-MC58 bacteria recovered at different time points from mouse serum samples; the results show that serum from MASP-2-/- mice has a higher level of bactericidal activity against N. meningitidis than serum from WT mice as described in Example 32;

fig. 40 graphically represents the hemolysis (determined based on the release of hemoglobin from lysed murine red blood cells (Crry/C3-/-) into the supernatant, measured photometrically) of mannan-coated murine red blood cells by human serum over a range of serum concentrations. Sera tested included heat-inactivated (HI) SNP, MBL-/-, SNP+anti-MASP-2 antibody, and SNP control as described in Example 33;

fig. 41 graphically represents the hemolysis (determined based on the release of hemoglobin from lysed WT mouse erythrocytes into the supernatant, measured photometrically) of uncoated mouse erythrocytes with human serum over a range of serum concentrations. Sera tested included heat-inactivated (HI) SNP, MBL-/-, SNP+antiMASP-2 antibody, and SNP control; the results show that MASP-2 inhibition inhibits complement-mediated erythrocyte lysis of non-sensitized WT mice as described in Example 33;

fig. 42 graphically represents the hemolysis (determined based on the release of hemoglobin from lysed murine erythrocytes (CD55/59-/-) into the supernatant, measured photometrically) of uncoated murine erythrocytes with human serum over a range of serum concentrations. The sera tested included heat-inactivated (HI) SNP, MBL-/-, SNP+anti-MASP-2 antibody, and SNP control as described in Example 33;

fig. 43 graphically represents survival, as a percentage, as a function of time (days) after exposure to 8.0 Gy radiation for control mice and mice treated with anti-human MASP-2 antibody (mAb H6) as described in Example 34;

fig. 44 graphically represents the time to onset of microvascular occlusion following LPS injection in MASP-2-/- and WT mice; shows the percentage of mice with thrombus formation measured over 60 min; results show that thrombus formation occurs within 15 min in WT mice, with up to 80% of WT mice thrombus formation occurring within 60 min; in contrast, none of the MASP-2-/- mice showed thrombus formation over the 60-minute time period (log-rank test: p=0.0005) as described in Example 35;

fig. 45 graphically represents the survival, as a percentage, of saline control mice (n=5) and anti-MASP-2 antibody treated mice (n=5) in the STX/LPS-induced HUS model over time (hours); the results show that all control mice died after 42 hours, while, in contrast, 100% of mice treated with anti-MASP-2 antibody survived throughout the experiment, as described in example 36;

- 15 046178 fig. 46 graphically represents, as a function of time after injury induction, the percentage of mice with microvascular occlusion in the FITC/dextran-UV injury model following treatment with isotype control antibody or anti-human MASP-2 mAb H6 (10 mg/kg). administered 16 hours and 1 hour before FTTC/dextran injection as described in Example 37;

fig. 47 graphically represents time to occlusion, in minutes, for mice treated with anti-human MASP-2 antibody (mAb H6) and isotype control antibody, with data presented as a scatter plot with means (horizontal bars) and standard error bars ( vertical lines). Unpaired Student's t test was used for statistical analysis of data; in this case, the symbol * corresponds to p=0.0129, as described in example 37; and figs. 48 graphically represents the time to occlusion, in minutes, for wild-type mice, MASP-2 KO mice, and wild-type mice pretreated with anti-human MASP-2 antibody (mAb H6) administered i.p. at a dose of 10 mg/kg over 16 hours before, and again 1 hour before, induction of thrombosis in the thrombosis model of FITC-dextran/light-induced endothelial cell injury at low light intensity (800-1500) as described in Example 37;

fig. 49 is a Kaplan-Meier plot showing the percentage of mice with thrombi, as a function of time, in a model of FITC-dextran-induced thrombotic microangiopathy in mice treated with increasing doses of human MASP-2 inhibitory antibody (mAb H6), or isotype control antibody , as described in example 39;

fig. 50 graphically represents the average time (minutes) to the onset of thrombus formation as a function of the dose of mAb H6 (*p<0.01 compared to control), as described in Example 39;

fig. 51 is a Kaplan-Meier plot showing the percentage of mice with microvascular occlusion as a function of time in a FITC-dextran induced thrombotic microangiopathy model in mice treated with increasing doses of human MASP-2 inhibitory antibody (mAb H6) or isotype control antibody as described in Example 39;

fig. 52 graphically represents the average time to microvascular occlusion as a function of the dose of mAb H6 (*p<0.05 compared to control), as described in Example 39;

fig. 53A graphically represents the level of MAC deposition in the presence or absence of human monoclonal antibody against MASP-2 (OMS646) under lectin pathway specific assay conditions; the results show that OMS646 inhibits lectin-mediated MAC deposition with an IC ₅₀ value of approximately 1 nM, as described in Example 40;

fig. 53B graphically represents the level of MAC deposition in the presence or absence of human anti-MASP-2 monoclonal antibody (OMS646) under classical pathway-specific assay conditions; the results show that OMS646 does not inhibit classical pathway-mediated MAC deposition as described in Example 40;

fig. 53C graphically represents the level of MAC deposition in the presence or absence of human monoclonal antibody against MASP-2 (OMS646) under alternative pathway specific assay conditions; the results show that OMS646 does not inhibit alternative pathway-mediated MAC deposition as described in Example 40;

fig. 54 graphically represents the pharmacokinetic (PK) profile of human anti-MASP-2 monoclonal antibody (OMS646) in mice, showing the concentration of OMS646 (average of n=3 animals/group) as a function of time after administration at the indicated dose, as described in Example 40;

fig. 55A graphically represents the pharmacodynamic (PD) response to the human monoclonal antibody against MASP-2 (OMS646), measured as a drop in systemic lectin pathway activity in mice following intravenous administration as described in Example 40;

fig. 55B graphically represents the pharmacodynamic (PD) response to the human monoclonal antibody against MASP-2 (OMS646), measured as a drop in systemic lectin pathway activity in mice following subcutaneous administration as described in Example 40;

fig. 56 graphically represents the inhibitory effect of anti-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;

fig. 57 graphically represents the inhibitory effect of anti-MASP-2 antibody (OMS646) compared to sCR1 on aHUS serum-induced thrombus formation on ADP-activated HMEC-1 cells as described in Example 42;

fig. 58 graphically represents the mean change from baseline in platelet count as a function of time (week) in subjects suffering from persistent hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA) following treatment with a MASP-2 inhibitory antibody (OMS646). as described in example 46;

fig. 59 graphically represents the mean change from baseline in LDH level over time (week) in subjects suffering from persistent HSCT-TMA following treatment with a MASP-2 inhibitory antibody (OMS646) as described in Example 46;

- 16 046178 fig. 60 graphically represents the mean change from baseline in haptoglobin level over time (week) in subjects suffering from persistent HSCT-TMA following treatment with a MASP-2 inhibitory antibody (OMS646) as described in Example 46;

fig. 61 shows the course of a hematopoietic stem cell transplant (HSCT) patient who developed HSCT-TMA and graft-versus-host disease (GVHD); there was attenuation of HSCT-TMA, GVHD and improvement of neurological symptoms after treatment with a MASP-2 inhibitory antibody (OMS646) as described in Example 47;

fig. 62A graphically represents the creatinine level over time during compassionate treatment of Patient No. 1, where the vertical line marks the start of treatment with a MASP-2 inhibitory antibody (OMS646) as described in Example 48;

fig. 62B graphically represents the haptoglobin level as a function of time during compassionate treatment of Patient No. 1, where the vertical line marks the start of treatment with a MASP-2 inhibitory antibody (OMS646) as described in Example 48;

fig. 62C graphically represents the hemoglobin level as a function of time during compassionate treatment of Patient No. 1, where the vertical line marks the start of treatment with a MASP-2 inhibitory antibody (OMS646) as described in Example 48;

fig. 62D graphically represents the LDH level as a function of time during compassionate treatment of Patient No. 1, where the vertical line marks the start of treatment with a MASP-2 inhibitory antibody (OMS646) as described in Example 48; and figs. 62E graphically represents the platelet level as a function of time during compassionate treatment of Patient No. 1, where the vertical line marks the start of treatment with a MASP-2 inhibitory antibody (OMS646) as described in Example 48.

Description of the sequence list

SEQ ID NO: 1 human MAp19 cDNA,

SEQ ID NO: 2 human MAp19 protein (with leader),

SEQ ID NO: 3 human MAp19 protein (mature),

SEQ ID NO: 4 human MASP-2 cDNAs,

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).

Antigens: (in relation to mature MASP-2 protein),

SEQ ID NO: 8 sequence CUBI (ac 1-121),

SEQ ID NO: 9 sequence CUBEGF (aa 1-166),

SEQ ID NO: 10 CUBEGFCUBII (ak 1-293),

SEQ ID NO: 11 EGF region (aa 122-166),

SEQ ID NO: 12 serine protease domain (aa 429-671),

SEQ ID NO: 13 inactive serine protease domain (aa 610-625 with mutation Ser618 to Ala),

SEQ ID NO: 14 TPLGPKWPEPVFGRL (CUB1 peptide),

SEQ ID NO: 15 TAPPGYRLRLYFTHFDLELSHLCEYDFVKLSSGAKVLATLCGQ (CUBI peptide),

SEQ ID NO: 16 TFRSDYSN (center of MBL-binding region),

SEQ ID NO: 17 FYSLGSSLDITFRSDYSNEKPFTGF (MBL-binding region),

SEQ ID NO: 18 IDECQVAPG (EGF peptide),

SEQ ID NO: 19 ANMLCAGLESGGKDSCRGDSGGALV (serine protease binding site).

Peptide inhibitors:

SEQ ID NO: 20 full-length MBL cDNA,

SEQ ID NO: 21 full length MBL proteins,

SEQ ID NO: 22 OGK-X-GP (consensus binding sequence),

SEQ ID NO: 23 OGKLG,

SEQ ID NO: 24 GLR GLQ GPO GKL GPO G,

SEQ ID NO: 25 GPO GPO GLR GLQ GPO GKL GPO GPO GPO,

SEQ ID NO: 26 GKDGRDGTKGEKGEPGQGLRGLQGPOGKLGPOG,

SEQ ID NO: 27 GAOGSOGEKGAOGPQGPOGPOGKMGPKGEOGDO (human h-ficolin),

SEQ ID NO: 28

GCOGLOGAOGDKGEAGTNGKRGERGPOGPOGKAGPOGPNGAOGEO (human ficolin p35), SEQ ID NO: 29 LQRALEILPNRVTIKANRPFLVFI (C4 cleavage site).

Expression inhibitors:

SEQ ID NO: 30 cDNA of the CUBI-EGF domain (nucleotides 22-680 in SEQ ID NO: 4),

SEQ ID NO: 31

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

SEQ ID NO: 32

5'GACATTACCTTCCGCTCCGACTCCAACGAGAAG3' Nucleotides 361-396 in SEQ ID NO: 4, cody

- 17 046178 routing region including the MBL MASP-2 binding site (sense),

SEQ ID NO: 33

5'AGCAGCCCTGAATACCCACGGCCGTATCCCAAA3' Nucleotides 610-642 in SEQ ID NO: 4, encoding the region including the CUBII domain.

Primers for cloning:

SEQ ID NO: 34 CGGGATCCATGAGGCTGCTGACCCTC (5' PCR for CUB),

SEQ ID NO: 35 GGAATTCCTAGGCTGCATA (3' PCR for CUB),

SEQ ID NO: 36 GGAATTCCTACAGGGCGCT (3' PCR for CUBIEGF),

SEQ ID NO: 37 GGAATTCCTAGTAGTGGAT (3' PCR for CUBIEGFCUBII),

SEQ ID NO: 38-47 are primers for cloning a humanized antibody, SEQ ID NO: 48 is a 9-aa binding peptide.

Expression vector:

SEQ ID NO: 49 is a MASP-2 minigene insertion,

SEQ ID NO: 50 is the mouse MASP-2 cDNA,

SEQ ID NO: 51 is mouse MASP-2 protein (with/leader),

SEQ ID NO: 52 is the mature mouse MASP-2 protein,

SEQ ID NO: 53 is rat MASP-2 cDNA,

SEQ ID NO: 54 is rat MASP-2 protein (with/leader),

SEQ ID NO: 55 is the mature rat MASP-2 protein,

SEQ ID NO: 56-59 are oligonucleotides for site-directed mutagenesis of human MASP-2 used to obtain human MASP-2A,

SEQ ID NO: 60-63 are site-directed mutagenesis oligonucleotides for mouse MASP-2 used to produce mouse MASP-2A,

SEQ ID NO: 64-65 are site-directed mutagenesis oligonucleotides for rat MASP-2 used to produce rat MASP-2A,

SEQ ID NO: 66 DNA encoding heavy chain variable region (VH) (without signal peptide) 17D20_dc35VH21N11VL (OMS646),

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

SEQ ID NO: 68 heavy chain variable region (VH) polypeptide 17N16mc,

SEQ ID NO: 69: DNA encoding light chain variable region (VL) 17D20_dc35VH21N11VL (OMS646),

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

SEQ ID NO: 71: Light chain variable region (VL) polypeptide 17N16_dc17N9.

Detailed Description of the Invention

The present invention is based on the surprising discovery of the inventors that it is possible to inhibit the MASP-2 mediated lectin pathway while maintaining the classical pathway without alteration. The present invention also relates to the use of MASP-2 as a therapeutic target for inhibiting lectin-mediated cell damage through complement activation while maintaining without alteration a component of the classical (C1q-dependent) immune system pathway.

I. Definitions.

Unless otherwise indicated, all terms used herein have the meaning commonly given to them by those skilled in the art to which this invention relates. The following definitions are provided to provide clarity regarding the terms used in the specification and claims to describe the present invention.

As used herein, the term MASP-2-dependent complement activation means MASP-2-dependent activation of the lectin pathway that occurs under physiological conditions (i.e., in the presence of Ca ⁺⁺ ), leading to the formation of the lectin pathway C3 convertase C4b2a, and then accumulation of the C3 cleavage product, C3b, to the subsequent formation of the C5 convertase C4b2a(C3b)n, which was recognized as the main cause of opsonization.

As used herein, the term alternative pathway refers to complement activation, which is triggered, for example, by zymosan from the cell wall of fungi and yeasts, lipopolysaccharide (LPS) from the outer membrane of gram-negative bacteria and rabbit red blood cells, as well as many pure polysaccharides, rabbit red blood cells, viruses, bacteria, animal tumor cells, parasites and damaged cells, and which is generally believed to arise as a result of the spontaneous proteolytic formation of C3b from complement factor C3.

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

- 18 046178

As used herein, the term classical pathway refers to complement activation that is triggered by binding of an antibody to a foreign particle and requires binding of the C1q recognition molecule.

As used herein, the term MASP-2 inhibitory agent means 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 MASP-2 binding fragments thereof, natural and synthetic peptides, small molecules, soluble MASP-2 receptors, expression inhibitors and isolated natural inhibitors, and also covers peptides that compete with MASP-2 for binding to another recognition molecule (such as, for example, MBL, H-ficolin, M-ficolin or L-ficolin) in the lectin pathway, but does not cover antibodies that bind such other recognition molecules. MASP-2 inhibitory agents useful in the method of the invention can reduce MASP-2-dependent complement activation by more than 20%, for example by more than 50%, for example by more than 90%. In one embodiment, the MASP-2 inhibitory agent reduces MASP-2-dependent complement activation by greater than 90% (ie, resulting in MASP-2-dependent complement activation of only 10% or less).

As used herein, the term antibody covers antibodies and antibody fragments obtained from any antibody-producing mammal (e.g., mouse, rat, rabbit, and primate, including humans) or from a hybridoma, by phage selection, recombinant expression, or from transgenic animals (or with using other methods for producing antibodies or antibody fragments) that specifically bind a target polypeptide, such as, for example, MASP-2, polypeptides or parts thereof. The term antibody should not be limited as to the source of the antibody or the method in which it is produced (eg, using hybridoma, phage selection, recombinant expression, transgenic animal, peptide synthesis, etc.). Exemplary antibodies include polyclonal, monoclonal and recombinant antibodies; panspecific, multispecific antibodies (eg, bispecific antibodies, trispecific antibodies); humanized antibodies; mouse antibodies; chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies; as well as anti-idiotypic antibodies, and can be any intact antibody, or a fragment thereof. As used herein, the term antibody covers only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as dAb, Fab, Fab', F(ab') ₂ , Fv), single chain fragments (scFv), synthetic variants thereof, natural variants, fusion proteins containing part of an antibody with an antigen-binding fragment of the desired specificity, humanized antibodies, chimeric antibodies, as well as any other modified configuration of the immunoglobulin molecule that contains an antigen-binding site or fragment (epitope recognition site) of the desired specificity.

The term monoclonal antibody refers to a homogeneous population of antibodies, wherein a monoclonal antibody is composed of amino acids (natural and non-natural) that are involved in the selective binding of an epitope. Monoclonal antibodies are highly specific for target antigens. The term monoclonal antibody covers only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab') ₂ , Fv), single chain fragments (scFv), variants thereof, fusion proteins containing antigen-binding part, humanized monoclonal antibodies, chimeric monoclonal antibodies, as well as any other modified configuration of an immunoglobulin molecule that contains an antigen-binding fragment (epitope recognition site) of the desired specificity and has the ability to bind the epitope. The term should not be limited as to the source of the antibody or the manner in which it is produced (eg, using hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term covers whole immunoglobulins, as well as fragments, etc., described above in the explanation of the term antibody.

As used herein, the term antibody fragment means a portion derived from, or derived from, a full-length antibody, such as, for example, an anti-MASP-2 antibody, typically including 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, as well as multispecific antibodies formed from antibody fragments.

As used herein, a single chain Fv, or scFv, antibody fragment comprises the VH and VL domains of the antibody, which domains are located on the same polypeptide chain. Typically, the Fv polypeptide additionally contains a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding.

As used herein, the term chimeric antibody means a recombinant protein that contains variable domains and complementarity determining regions from antibodies from a non-human species (eg, rodents), while the remainder of the antibody molecule is derived from a human antibody.

- 19 046178

As used herein, the term humanized antibody means a chimeric antibody that contains a minimal sequence corresponding to the specificity-determining regions of a non-human immunoglobulin that is inserted into the framework of a human antibody.

Humanized antibodies are typically recombinant proteins in which only the complementarity-determining region of the antibody is not derived from a human antibody.

As used herein, the term mannan binding lectin (MBL) is equivalent to the term mannan binding protein (MBP).

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

As used herein, the term subject includes all mammals, including, without limitation, humans, primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs and rodents.

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

In the broadest sense, naturally occurring amino acids can be divided into groups based on the chemical characteristics of the side chains of the corresponding amino acids. Hydrophobic amino acids include Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys or Pro. Hydrophilic amino acids include Gly, Asn, Gln, Ser, Thr, Asp, Glu, Lys, Arg or His. This group of amino acids can be further divided into subclasses as follows. Uncharged hydrophilic amino acids include Ser, Thr, Asn or Gln. Acidic amino acids include Glu or Asp. Basic amino acids include Lys, Arg or His.

As used herein, the term conservative amino acid substitution means the mutual substitution of 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) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine.

As used herein, the term oligonucleotide means an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), or mimetics thereof. The term also includes oligonucleotides consisting of naturally occurring nucleotides, sugar residues and covalent internucleoside (backbone) linkages, as well as oligonucleotides having non-natural modifications.

As used herein, the term epitope means a site on a protein (eg, human MASP-2 protein) that an antibody binds. Overlapping epitopes include at least one (eg, two, three, four, five or six) common amino acid residues, including linear and non-linear epitopes.

As used herein, the terms polypeptide, peptide, and protein are used interchangeably to mean any chain of amino acids linked by peptide bonds, regardless of length or post-translational modification. The MASP-2 protein described herein may contain, or be, a wild-type protein, or may be variants, which may have no more than 50 (e.g., no more than one, two, three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35, 40 or 50) conservative amino acid substitutions. Conservative substitutions generally include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine.

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

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

- 20 046178 residues in SEQ ID NO: 5).

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 sequence alignment and deletions, if necessary, to achieve the maximum percentage of sequence identity. Alignment for the purpose of determining percent sequence identity can be performed by various methods known to those skilled in the art, for example, using publicly available computer programs such as the BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) programs. Appropriate parameters for determining the alignment, including any algorithms necessary to achieve the most optimal alignment over the entire length of the sequence being compared, can be determined by known methods.

II. The essence of the invention.

Lectins (MBL, M-ficolin, H-ficolin, L-ficolin and CL-11) are specific recognition molecules that trigger the innate complement system, and the system includes the lectin initiation pathway and the associated terminal pathway amplification cycle that enhances lectin-initiated activation terminal effector complement molecules. C1q is a specific recognition molecule that triggers the acquired complement system, and the system includes the classical initiation pathway and the associated terminal pathway amplification cycle that enhances C1q-initiated activation of terminal effector complement molecules. We refer to these two major complement activation systems as the lectin-dependent complement system and the C1q-dependent complement system, respectively.

In addition to its important role in immune defense, the complement system contributes to tissue damage in many clinical conditions. Thus, there is an urgent need to develop therapeutically effective complement inhibitors to prevent these adverse effects. With the recognition of the possibility of inhibiting the MASP-2-mediated lectin pathway without affecting the classical pathway, comes the understanding that it would be highly desirable to specifically inhibit only the complement activation system causing a particular pathology, without completely disabling the immune defense capacity of complement. For example, in disease states in which complement activation is mediated primarily by the lectin-dependent complement system, it would be useful to specifically inhibit only that system. This would allow the C1q-dependent complement activation system to be kept intact to enable processing of immune complexes and participation in host defense against infection.

The preferred target protein component for drug development for specific inhibition of the lectin-dependent complement system is MASP-2. Of all the known protein components of the lectin-dependent complement system (MBL, H-ficolin, M-ficolin, L-ficolin, MASP-2, C2-C9, factor B, factor D and properdin), only MASP-2 is unique to lectin-dependent dependent complement system and necessary for the functioning of this system. Lectins (MBL, H-ficolin, M-ficolin, L-ficolin and CL-11) are also unique components in the lectin-dependent complement system. However, loss of any of these lectin components does not necessarily result in inhibition of system activation due to lectin excess. It would be necessary to inhibit all five lectins to ensure inhibition of the lectin-dependent complement activation system. In addition, since MBL and ficolins are also known to have complement-independent opsonic activity, inhibition of lectin function would result in the loss of this beneficial host defense mechanism against infection. In contrast, this complement-independent opsonic activity of lectins would remain unaffected if MASP-2 protein were the target of inhibition. An additional advantage of MASP-2 as a therapeutic target for inhibition of the lectin-dependent complement activation system is that the plasma concentration of MASP-2 is one of the lowest for complement proteins (® 500 ng/ml); thus, correspondingly low concentrations of high-affinity MASP-2 inhibitors may be sufficient to achieve complete inhibition (Moller-Kristensen, M., et al., J. Immunol Methods 282:159-167, 2003).

III. The role of MASP-2 in thrombotic microangiopathies and methods of treatment using MASP-2 inhibitory agents.

Review.

Thrombotic microangiopathy (TMA) is a pathology characterized by the formation of blood clots in small blood vessels (Benz, K.; et al., Curr Opin Nephrol Hypertens 19(3):242-7 (2010)). The main cause is considered to be stress or damage to the underlying vascular endothelium. Clinical and laboratory indicators of TMA include thrombocytopenia, anemia, purpura, and renal failure. The classic TMAs are hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). The characteristic underlying pathological feature of TMA is platelet activation and the formation of microthrombi in small arterioles and venules. Complement activation initiated, at least in part, by damage

- 21 046178 food or microvascular endothelial stress has also been implicated in other TMAs, including catastrophic antiphospholipid syndrome (CAPS), systemic Degos disease, and TMA due to cancer, cancer chemotherapy, and transplantation.

Direct evidence for the pathological role of complement in various forms of nephritis comes from studies of patients with genetic deficiencies of certain complement components. A number of studies have established a connection between kidney damage and deficiency of complement regulatory factor H (Ault, B.H., Nephrol. 14:1045-1053, 2000; Levy, M., et al., Kidney Int. 30:949-56, 1986; Pickering, M.C., et al., Nat. Genet. 31:424-8, 2002). Factor H deficiency results in low plasma levels of factor B and C3 due to the activation-associated uptake of these components. Circulating levels of C5b-9 are also elevated in the serum of these patients, suggesting complement activation. Membranous proliferative glomerulonephritis (MPGN) and idiopathic hemolytic uremic syndrome (HUS) are associated with factor H deficiency or factor H mutations. Factor H-deficient pigs (Jansen, J.H., et al., Kidney Int. 53:331-49, 1998) and mice with factor H knockout (Pickering, M.S., 2002) exhibit MPGN-like symptoms, which confirms the importance of factor H in complement regulation. Deficiencies of other complement components are associated with renal failure resulting from systemic lupus erythematosus (SLE) (Walport, M.J., Davies, et al., Ann. N.Y. Acad. Sci. 815:267-81, 1997). Deficiencies of C1q, C4, and C2 are strong predisposition factors for the development of SLE through mechanisms related to impaired clearance of immune complexes and apoptotic material. Many of these patients with SLE develop lupus nephritis, characterized by deposition of immune complexes throughout the zona glomerulosa.

aHUS.

Atypical hemolytic uremic syndrome (aHUS) belongs to a group of conditions called thrombotic microangiopathies. In atypical HUS (aHUS), the disease is associated with dysregulation of complement and can be either sporadic or familial. Familial cases of aHUS are associated with mutations in genes encoding proteins responsible for complement activation or regulation, including factor H, factor I, complement factor B, membrane cofactor CD46, and complement factor H-related protein 1 (CFHR1) and complement factor H protein 3 (CFHR3). (Zipfel, P.F., et al., PloS Genetics 3(3):e41 (2007)). The unifying feature of this diverse set of genetic mutations associated with aHUS is a predisposition to increased complement activation at cell or tissue surfaces. Thus, one aspect of the present invention relates to the treatment of a patient suffering from aHUS associated with factor H deficiency by administering an effective amount of a MASP-2 inhibitory agent. Another aspect of the present invention relates to the treatment of a patient suffering from HUS associated with deficiency of factor I, factor B, membrane cofactor CD46, CFHR1 or CFHR3, by administering an effective amount of a MASP-2 inhibitory agent.

Recently, significant progress has been made in understanding the molecular pathophysiology underlying increased complement activation in aHUS caused by a diverse set of mutant complement factors. The best understood mechanism for factor H mutations is Factor H, an abundant serum protein containing 20 short consensus repeat (SCR) domains that acts as a negative regulator of complement activation both in solution and on the host cell surface. It targets the activated form of C3, and, together with factor I and other cofactors, stimulates its inactivation, preventing further activation of complement. To effectively control complement activation at the host cell surface, factor H must interact with host cells, which is mediated by SCR domains 16–20. All aHUS-associated factor H mutations described to date are concentrated in the C-terminal region spanning domains 16–20 (SCR). These factor H mutant proteins are fully functional to control C3 activation in solution, but are unable to interact with the host cell surface and therefore are unable to control C3 activation on cell surfaces (Exp Med 204(6): 1249-56 (2007)) . Thus, some factor H mutations are associated with aHUS because the mutant factor H protein is unable to interact with host cell surfaces and is therefore unable to effectively down-regulate complement activation on host cell surfaces, including microvascular endothelium. As a result, after initial activation of C3, subsequent activation of complement at the surface of the microvascular endothelium is unabated in patients with factor H mutations. This uncontrolled activation of complement eventually leads to progressive damage to the vascular endothelium, subsequent platelet aggregation and microvascular coagulation, as well as shear force-induced hemolysis when red blood cells pass through partially blocked microvessels. Thus, the manifestations of aHUS disease, as well as clinical and laboratory parameters, are directly related to disruption of the negative regulation of complement on the surface of the microvascular endothelium.

Similar to factor H mutations, loss-of-function mutations in the negative complement modulators factor I and membrane cofactor (CD46) are also associated with aHUS. The opposite situation was found for factor B and C3 proteins, in which case aHUS was found to be associated with mutations in

- 22 046178 gain of function in these proteins (Pediatr Nephrol 25(12):2431-42 (2010)). Thus, the body of evidence indicates the involvement of complement activation in the pathogenesis of aHUS. This finding most strongly supports the therapeutic efficacy of eculizumab, a monoclonal antibody that blocks terminal complement protein C5, in the treatment of aHUS.

While the central role of complement as an effector mechanism in aHUS is widely accepted, the triggers that initiate complement activation and the molecular pathways involved have not been defined. Not all people with the mutations described above develop aHUS. In fact, family studies have shown that the penetrance of aHUS is only ~50%. (Ann Hum Genet 74(1): 1726 (2010)). Natural disease dynamics indicate that aHUS most often develops after an initial event such as infection or injury. It is well known that infectious agents activate the complement system. In the absence of pre-existing adaptive immunity, complement activation by infectious agents may be predominantly initiated through the lectin pathway. Thus, infection-triggered lectin pathway activation may represent the initial trigger for subsequent pathological increases in complement activation in aHUS-prone individuals, which may ultimately lead to disease progression. Accordingly, another aspect of the present invention relates to the treatment of a patient suffering from aHUS associated with infection by administering an effective amount of a MASP-2 inhibitory agent.

Other types of host tissue injury will also activate complement through the lectin pathway, particularly damage to the vascular endothelium. Human vascular endothelial cells exposed to oxidative stress, for example, respond by expressing surface molecules that bind lectins and activate the lectin complement pathway (Am J. Pathol 156(6): 1549-56 (2000)). Vascular injury following ischemia/reperfusion also leads to complement activation through the lectin pathway in vivo (Scand J Immunol 61(5):426-34 (2005)). Activation of the lectin pathway under these conditions has pathological consequences for the host, and inhibition of the lectin pathway by blocking MASP-2 prevents further host tissue damage and adverse outcomes (Schwaeble PNAS 2011).

Thus, other processes that trigger aHUS are known to also activate the lectin complement pathway. As a consequence, it is likely that the lectin pathway may represent the initial mechanism of complement activation that is inappropriately enhanced in an unregulated manner in individuals genetically predisposed to aHUS, thereby initiating the pathogenesis of aHUS. It can be concluded that agents that block complement activation through the lectin pathway, including anti-MASP-2 antibodies, are expected to prevent disease progression or reduce exacerbations in aHUS-prone individuals.

In support of this concept, recent studies have identified S. pneumonia as an important etiological agent in cases of aHUS in children. (Nephrology (Carlton), 17:48-52 (2012); Pediatr Infect Dis J. 30(9):736-9 (2011)). This particular etiology appears to have a poor prognosis with significant mortality and long-term morbidity. Notably, these cases involved non-enteric infection resulting in microangiopathy, uremia, and hemolysis without evidence of associated complement gene mutations known to predispose to aHUS. It is important to note that S. pneumonia is particularly effective at activating complement, and does so primarily through the lectin pathway. Thus, in cases of non-enteric HUS associated with pneumococcal infection, the manifestations of microangiopathy, uremia, and hemolysis are thought to be due to activation of the lectin pathway, and agents that block the lectin pathway, including anti-MASP-2 antibodies, are expected to prevent progression of aHUS or reduce the severity of the disease in these patients. Accordingly, another aspect of the present invention relates to the treatment of a patient suffering from non-enteral aHUS associated with infection caused by S. pneumonia by administering an effective amount of a MASP-2 inhibitory agent.

In accordance with the foregoing, in some embodiments, in the case of a subject having a risk of developing renal failure associated with aHUS, there is provided a method of reducing the likelihood of developing aHUS or developing renal failure associated with aHUS, comprising administering a MASP-2 inhibitory agent in a certain amount over a period of time. a period of time effective to alleviate or prevent renal failure in the subject. In some embodiments, the method further includes the step of determining whether the subject is at risk for developing aHUS before the onset of any symptoms associated with aHUS. In other embodiments, the method includes determining whether the subject is at risk for developing aHUS, after the onset of at least one or more symptoms consistent with aHUS (e.g., the presence of anemia, thrombocytopenia, and/or renal failure), and/or the presence of signs of thrombotic microangiopathies in biopsy specimens obtained from the subject. Determining whether a subject is at risk for developing aHUS involves determining whether the subject has a genetic predisposition to developing aHUS, which can be done by assessing genetic information (eg, in a database

- 23 046178 genotype data of the subject), or by performing at least one genetic test on the subject to determine the presence or absence of a genetic marker associated with aHUS (i.e., determining the presence or absence of a genetic mutation associated with aHUS in genes encoding complement factor H (CFH), factor I (CFI), complement factor B (CFB), membrane cofactor CD46, C3, complement factor H-related protein 1 (CFHR1), or THBD (thrombomodulin anticoagulant protein), or factor H-related complement protein 3 (CFHR3), complement factor H-related protein 4 (CFHR4)), either by genome sequencing, using a gene-specific assay (eg, PCR assay), and/or by determining whether the subject has family history of aHUS. Genetic screening methods for the presence or absence of a genetic mutation associated with aHUS are well known, for example see Noris M et al. Atypical Hemolytic-Uremic Syndrome, 2007 Nov 16 [modified 2011 Mar 10] in: Pagon RA, Bird TD, Dolan CR, et al., editors. GeneReviews™, Seattle (WA): University of Washington, Seattle.

For example, the overall disease penetrance in subjects with complement factor H (CFH) mutations is 48%, and the penetrance for mutations in CD46 is 53%, for mutations in CFI is 50%, for mutations in C3 is 56%, and for mutations in THBD is 64% (Caprioli J. et al., Blood, 108:1267-79 (2006); Noris et al., Clin J Am Soc Nephrol 5:1844-59 (2010)). As described in Caprioli et al., (2006) above, a significant number of individuals with a mutation in complement factor H (CFH) never develop aHUS, and it has been postulated that suboptimal CFH activity in these individuals is sufficient to protect the host from the effects of activation of complement under physiological conditions, however, suboptimal CFH activity is insufficient to prevent the deposition of C3b on vascular endothelial cells when exposure to a complement activating agent leads to the formation of greater than normal amounts of C3b.

Accordingly, in one embodiment, there is provided a method of 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 a MASP-2 inhibitory agent in an amount effective for inhibiting MASP-2-dependent complement activation. In another embodiment, a method is provided for inhibiting MASP-2-dependent complement activation in a subject at risk for developing factor H-dependent atypical hemolytic uremic syndrome, comprising periodically monitoring the subject to determine the presence or absence of anemia, thrombocytopenia, or elevated creatinine, and treating with an inhibitory MASP. -2 agent in determining the presence of anemia, thrombocytopenia or elevated creatinine levels. In another embodiment, a method is provided for reducing the likelihood that a subject at risk of developing factor H-dependent aHUS will suffer from clinical symptoms associated with aHUS, comprising administering a MASP-2 inhibitory agent before, or during, or after the event that is known to be associated with the initiation of clinical symptoms of aHUS, such as drug exposure (eg, chemotherapy), infection (eg, bacterial infection), malignancy, trauma, organ or tissue transplantation, or pregnancy.

In one embodiment, 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 periodically monitoring the subject to determine the presence or absence of anemia, thrombocytopenia, or elevated creatinine levels, and treatment with an inhibitory MASP- 2 tool in determining the presence of anemia, thrombocytopenia or elevated creatinine levels.

In another embodiment, 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 inhibitory agent before, or during, or after an event that is known to occur. is associated with the initiation of clinical symptoms of aHUS, such as drug exposure (eg, chemotherapy), infection (eg, bacterial infection), malignancy, trauma, organ or tissue transplantation, or pregnancy.

In some embodiments, the MASP-2 inhibitory agent is administered for a period of time of at least one, two, three, four days, or longer, before, during, or after the event associated with the initiation of clinical symptoms of aHUS, and administration can repeat as directed by the physician until the condition is eliminated or controlled. In a pre-aHUS setting, the MASP-2 inhibitory agent can be administered to a subject systemically, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous or other parenteral route.

In some embodiments, in the case of a primary diagnosis of aHUS, or in the case of a subject having one or more symptoms consistent with a diagnosis of aHUS (e.g., anemia, thrombocytopenia, and/or renal failure), the subject is treated with an effective amount of a MASP-2 inhibitory agent (e.g., anti-MASP-2 antibodies) as first-line therapy, without plasmapheresis or in combination with plasmapheresis. As a first line of therapy, a MASP-2 inhibitory agent can be administered to a subject systemically, e.g., intra-arterial, intravenous,

- 24 046178 intramuscular, inhalation, nasal, subcutaneous or other parenteral route of administration. In some embodiments, the MASP-2 inhibitory agent is administered to a subject as first-line therapy without plasmapheresis to avoid potential complications of plasmapheresis, including bleeding, infection, and exposure to pathogenic and/or allergic factors present in the donor plasma, or in the case of a subject rejecting plasmapheresis, or in settings where plasmapheresis is not available.

In some embodiments, the method comprises administering a MASP-2 inhibitory agent to a subject suffering from aHUS via a catheter (e.g., intravenously) for a first period of time (e.g., at least one day or a week, or two weeks), followed by administration of the inhibitory agent. MASP-2 administered to the subject subcutaneously for a second period of time (eg, a chronic phase of at least two weeks or longer). In some embodiments, administration in the first and/or second time periods is performed without plasmapheresis. In some embodiments, the method further includes determining the level of at least one complement factor (e.g., C3, C5) in the subject prior to treatment and, optionally, during treatment, wherein determining a reduced level of at least one complement factor compared to standard value, or the value in a healthy control subject, indicates the need for continued treatment with a MASP-2 inhibitory agent.

In some embodiments, the method includes administering a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, to a subject suffering from, or at risk of developing, aHUS, intravenously, intramuscularly, or, preferably, subcutaneously. Treatment can be chronic and applied daily or monthly, but preferably every two weeks. Ahtu-MASP-2 antibody can be administered alone or in combination with a C5 inhibitor such as eculizumab.

HUS..

Like atypical HUS, typical HUS has all the clinical and laboratory features of TMA. However, typical HUS is often a childhood disease and typically does not have a familial component or a direct link to mutations in complement factor genes. The etiology of typical HUS is closely related to infection by certain enteric pathogens. Patients typically present with acute renal failure, hemoglobinuria, and thrombocytopenia, which typically follows an episode of bloody diarrhea. This condition is a consequence of intestinal infection caused by Shigella dissenteria, Salmonella, or Shiga toxin-like toxin-producing enterohemorrhagic strains of E. coli, such as E. coli O157:H7. Pathogens enter the body from contaminated food or water sources. HUS is a medical emergency and results in death in 5-10% of cases. A significant proportion of survivors develop chronic renal failure (Corrigan and Boineau, Pediatr Rev 22 (11): 365–9 (2011)) and may require kidney transplantation.

Microvascular coagulation in typical HUS occurs predominantly, but not exclusively, in the renal microvasculature. The main pathophysiological features are mediated by Shiga toxin (STX). Released by enteropathic microbes into the intestinal lumen, STX penetrates the intestinal barrier, enters the bloodstream and binds to vascular endothelial cells through the blobotriaosyl-ceramide receptor CD77 (Boyd and Lingwood Nephron 51:207 (1989)), which is predominantly expressed on the glomerular endothelium and mediates the toxic effect STX. After binding to the endothelium, STX induces a series of events that damage the vascular endothelium, activate leukocytes and cause 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. 181:1460-1469 (2008); Morigi et al., Blood 98: 1828-1835 (2001); Guessou et al., Infect. Immun., 73 : 8306-8316 (2005)). These microthrombi clog or block the arterioles and capillaries of the kidney and other organs. Obstruction of blood flow in arterioles and capillaries by microthrombi increases the shear force exerted on red blood cells as they move through the narrowed blood vessels. This can lead to the destruction of red blood cells by shear forces and the formation of red blood cell fragments called schizocytes. The presence of schizocytes is a characteristic feature of HUS. This mechanism is known as microangiopathic hemolysis. In addition, obstruction of blood flow leads to ischemia, initiating a complement-mediated inflammatory response that causes further damage to the affected organ.

The lectin complement pathway is involved in the pathogenesis of HUS through two main mechanisms: 1) MASP-2-mediated direct activation of the coagulation cascade caused by endothelial injury, and 2) subsequent lectin-mediated complement activation induced by ischemia resulting from the initial occlusion of microvascular flow.

STX damages microvascular endothelial cells, and damaged endothelial cells are known to activate the complement system. As detailed above, complement activation following endothelial cell injury occurs predominantly through the lectin pathway. Human vascular endothelial cells exposed to oxidative stress respond by expressing surface molecules that bind lectins and activate the lectin complement pathway (Collard et al., Am J Pathol. 156(5): 1549-56 (2000)). Vascular damage after ischemia

- 25 046178 muscle/reperfusion also leads to complement activation through the lectin pathway in vivo (Scand J Immunol 61(5):426-34 (2005)). Activation of the lectin pathway under these conditions has pathological consequences for the host, and inhibition of the lectin pathway by blocking MASP-2 prevents further host tissue damage and adverse outcomes (Schwaeble PNAS 2011). In addition to complement activation, lectin-dependent activation of MASP-2 has been shown to lead to the cleavage of prothrombin to form thrombin and stimulate coagulation. Thus, activation of the lectin complement pathway by damaged endothelial cells may directly activate the coagulation system. The lectin complement pathway, through MASP-2-mediated activation of prothrombin, is thus likely to be the major molecular pathway linking primary STX endothelial injury to the coagulation and microvascular thrombosis that occurs in HUS. As a consequence, lectin pathway inhibitors, including but not limited to antibodies that block MASP-2 function, are expected to prevent or reduce microvascular coagulation, thrombosis, and hemolysis in patients suffering from HUS. Indeed, administration of anti-MASP-2 antibody effectively protects mice in a model of typical HUS. As described in Example 36 and shown in FIG. 45, all control mice exposed to STX and LPS developed severe HUS and were moribund or died within 48 hours. On the other hand, as further shown in FIG. 45, all mice treated with anti-MASP-2 antibody and then exposed to STX and LPS survived (Fisher's exact test p < 0.01; N = 5). Thus, anti-MASP-2 therapy effectively protects mice in this HUS model. Administration of a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, is expected to be effective in treating patients with HUS and will provide protection against microvascular coagulation, thrombosis, and hemolysis resulting from infection with enteropathic E. coli or other STX-producing organisms. pathogens.

Although demonstrated herein for HUS caused by STX, anti-MASP-2 therapy is also expected to be beneficial in HUS-like syndromes due to endothelial damage caused by other toxic agents. These include drugs such as mitomycin, ticlopidine, cisplatin, quinine, cyclosporine, bleomycin, as well as other chemotherapy drugs and immunosuppressants. Thus, anti-MASP-2 antibody therapy, or other agents that inhibit MASP-2 activity, is expected to be effective in preventing or limiting coagulation, thrombus formation, and red blood cell destruction, as well as preventing renal failure in HUS and other related TMA diseases. (i.e., aHUS and TTP).

Patients suffering from HUS often have diarrhea and vomiting, and they tend to have a low platelet count (thrombocytopenia) and a low red blood cell count (anemia). The pre-HUS phase of diarrhea typically lasts for approximately four days, during which time subjects at risk for developing HUS typically have one or more of the following symptoms in addition to severe diarrhea: hematocrit level below 30% with evidence of intravascular destruction red blood cells on the blood smear, thrombocytopenia (platelet count <150 x 10 ³ /mm ³ ), and/or impaired renal function (serum creatinine concentration above the upper limit of the reference range for age). The presence of oliguria (urine output <0.5 ml/kg/h for >1 day) can be used as a measure of progression towards HUS (see C. Hickey et al., Arch Pediatr Adolesc Med 165(10):884- 889 (2011)). Typically, testing is done for infection with E. coli bacteria (E. coli O157:H7) or Shigella or Salmonella species. For subjects who test positive for enterogenous E. coli infection (eg, E. coli 0157:H7), the use of antibiotics is contraindicated because antibiotic use may increase the risk of developing HUS through increased STX production (See Wong S. et al. , N Engl J. Med 342:1930-1936 (2000).Subjects who test positive for Shigella or Salmonella infection are typically given antibiotics to clear the infection.Other commonly used first-line therapy against HUS involves blood volume expansion, dialysis and plasmapheresis.

According to the foregoing, in some embodiments, in the case of a subject suffering from one or more symptoms associated with the pre-HUS phase and at risk of developing HUS (i.e., a subject having one or more of the following: diarrhea, hematocrit level below 30% with evidence of intravascular destruction of red blood cells on the blood smear, thrombocytopenia (platelet count <150 x 10 ³ /mm ³ ) and/or impaired renal function (serum creatinine concentration above the upper limit of the reference range for age)), a method has been proposed to reduce the risk of developing HUS, or reducing the likelihood of a subject developing renal failure, comprising administering a MASP-2 inhibitory agent in a specified amount for a period of time effective to alleviate or prevent renal failure. In some embodiments, the MASP-2 inhibitory agent is administered over a period of time of at least one, two, three, four days, or longer, and administration may be repeated at the discretion of the physician until the condition is resolved or eliminated. under control. In the case of preHUS, the MASP-2 inhibitory agent can be administered to the subject systemically, e.g. intra-arterially

- 26 046178 by intravenous, intramuscular, inhalation, nasal, subcutaneous or other parenteral route of administration.

Treatment of E. coli 0157:H7 infection with bactericidal antibiotics, particularly lactams, is associated with an increased risk of developing HUS (Smith et al., Pediatr Infect Dis J 31(1):37-41 (2012). In some cases implementation in the case of a subject suffering from symptoms associated with the pre-HUS phase, who is known to have an infection caused by enterogenous E. coli for which the use of antibiotics is contraindicated (for example, E. coli 0157:H7), a method of reducing the risk is provided development of HUS, or reducing the likelihood of developing renal failure in a subject, comprising administering a specified amount of a MASP-2 inhibitory agent for a first period of time effective to inhibit or prevent oliguria in the subject (e.g., for at least one, two, three or four days), wherein administration of the MASP-2 inhibitory agent is performed without the use of an antibiotic for a first period of time. In some embodiments, the method further comprises administering a MASP-2 inhibitory agent to a subject in combination with an antibiotic for a second period of time (e.g., at least at least one to two weeks).

In other embodiments, in the case of a subject suffering from symptoms associated with the pre-HUS phase who is known to have a Shigella or Salmonella infection, there is provided a method of reducing the risk of developing HUS, or reducing the likelihood of the subject developing renal failure, comprising administering a MASP-2 inhibitory agent in a specified amount for a period of time effective to inhibit or prevent oliguria in a subject, wherein administration of the MASP-2 inhibitory agent is performed either with or without the use of an appropriate antibiotic.

In some embodiments, in the case of a primary diagnosis of aHUS, or in the case of a subject having one or more symptoms consistent with a diagnosis of aHUS (eg, renal failure or microangiopathic hemolytic anemia without low fibrinogen, or thrombocytopenia), the subject is administered an effective amount of a MASP inhibitory anemia. 2 agents (eg, anti-MASP-2 antibodies) as first-line therapy without plasmapheresis or in combination with plasmapheresis. As a first line of therapy, a MASP-2 inhibitory agent can be administered to a subject systemically, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous or other parenteral route. In some embodiments, the MASP-2 inhibitory agent is administered to a subject as first-line therapy without plasmapheresis to avoid potential complications of plasmapheresis, including bleeding, infection, and exposure to pathogenic and/or allergic factors present in the donor plasma, or in the case of a subject rejecting plasmapheresis, or in settings where plasmapheresis is not available.

In some embodiments, the method includes administering a MASP-2 inhibitory agent to a subject suffering from HUS via a catheter (e.g., intravenously) for a first period of time (e.g., an acute phase lasting for at least one day or a week, or two weeks ), followed by administration of a MASP-2 inhibitory agent to the subject subcutaneously for a second period of time (eg, a chronic phase lasting for at least two weeks, or longer). In some embodiments, administration in the first and/or second time periods is performed without plasmapheresis. In some embodiments, the method further includes determining the level of at least one complement factor (e.g., C3, C5) in the subject prior to treatment and, optionally, during treatment, wherein determining a reduced level of at least one complement factor compared to standard value, or the value in a healthy control subject, indicates the need for treatment, and a determination of normal level indicates improvement in the condition.

In some embodiments, the method includes administering a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, to a subject suffering from, or at risk of developing, HUS, subcutaneously or intravenously. Preferably, administration is performed daily, but may not be performed more frequently than weekly or monthly. Treatment will continue for at least one week and up to 3 months. Ahtu-MASP-2 antibody can be administered alone or in combination with a C5 inhibitor such as eculizumab.

TTR.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease of the blood coagulation system caused by autoimmune or inherited dysfunctions that activate the coagulation cascade or complement system (George, JN, N Engl J Med; 354:1927-35 (2006)). This leads to the formation of numerous microscopic clots, or thrombosis, in small blood vessels throughout the body. Red blood cells are subject to a shear force that damages their membranes, leading to intravascular hemolysis. The resulting decreased blood flow and endothelial damage lead to organ damage, including the brain, heart, and kidneys. TTP is clinically characterized by the presence of thrombocytopenia, microangiopathic hemolytic anemia, neurological changes, renal failure and fever. In the era before the use of plasma

- 27 046178 maferesis mortality was 90% during acute attacks. Even with plasmapheresis, the survival rate after six months is approximately 80%.

TTP may result from genetic or acquired inhibition of the enzyme ADAMTS-13, a metalloproteinase responsible for breaking down large von Willebrand factor (vWF) multimers into smaller units. Inhibition or deficiency of ADAMTS-13 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 bind platelets more often and predispose patients to platelet aggregation and microvascular thrombosis.

Upshaw-Schulman syndrome (USS, also called congenital TTP) is an inherited deficiency of ADAMTS-13 activity due to mutations in the ADAMTS-13 gene (Schulman et al., Blood, 16(1):943-57, 1960; Upshaw et al. , New Engl J Med, 298(24):1350-2, 1978). Numerous mutations in the ADAMTS-13 gene 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 ADAMTS-13 activity at 5-10% of normal (Kokame et al., PNAS 99(18): 11902-11907, 2002). Although acquired TTP and USS have some similarities, USS has some important differences in clinical features. USS typically presents in infancy or childhood and is characterized by severe hyperbilirubinemia, with negative Coombs test results soon after birth, a reaction to fresh plasma infusion, and frequent relapses (Savasan et al., Blood, 101:4449-4451, 2003) . In some cases, patients with inherited ADAMTS-13 deficiency have a mild phenotypic expression at birth and only experience TTP-related symptoms in clinical situations in which von Willebrand factor levels are elevated, such as infection or pregnancy. For example, Fujimura et al reported on 9 Japanese women from 6 families with genetically confirmed USS who were diagnosed with the disease during their first pregnancy. Thrombocytopenia occurring during the second to third trimester in each of their 15 pregnancies was often accompanied by TTP. Each of these women was found to be severely deficient in ADAMTS-13 activity (Fujimura et al., Brit. J. Haemat 144:742-754, 2008).

In accordance with the foregoing, in some embodiments, in the case of a subject with Upshaw-Shulman syndrome (USS) (i.e., a subject who is known to have ADAMTS-13 activity deficiency, and/or a subject who is known to have one or more mutations in the ADAMTS-13 gene), a method has been proposed to reduce the likelihood of developing clinical symptoms associated with congenital TTP (for example, thrombocytopenia, anemia, fever and/or renal failure), including the administration of a certain amount of a MASP-2 inhibitory agent (for example, anti-MASP-2 2 antibodies) for a period of time effective to relieve or prevent one or more clinical symptoms associated with TTP. In some embodiments, the method further includes the step of determining whether the subject is at risk for developing symptoms associated with congenital TTP before the onset of developing any symptoms associated with TTP or after the onset of developing at least one or more symptoms associated with TTP (eg, anemia, thrombocytopenia and/or renal failure). Determining whether a subject is at risk for developing symptoms associated with congenital TTP (that is, the subject has USS) involves determining whether the subject has a mutation in the gene encoding ADAMTS-13 and/or determining whether the subject has a deficiency of ADAMTS activity -13, and/or determining whether the subject has a family history of USS. Methods for genetic screening for the presence or absence of a genetic mutation associated with USS are well known, for example, see Kinoshita et al., International Journal of Hematology, 74:101-108 (2001); Levy et al., Nature, 413 (6855):488-494 (2001); Kokame et al., PNAS 99(18): 11902-11907 (2002); Savasan et al., Blood, 101:4449-4451 (2003); Matsumoto et al., Blood, 103:1305-1310 (2004) and Fujimura et al., Brit. J Haemat 144:742–754 (2008).

In one embodiment, a method is provided for reducing the likelihood that a subject diagnosed with USS will suffer from clinical symptoms associated with TTP, comprising periodically monitoring the subject to determine the presence or absence of anemia, thrombocytopenia, or elevated creatinine levels, and treatment with a MASP-2 inhibitory agent. agent (eg, anti-MASP-2 antibody) in determining the presence of anemia, thrombocytopenia, or elevated creatinine, or in the presence of an event known to be associated with the initiation of clinical symptoms of TTP, such as drug exposure (eg, chemotherapy), infection (eg, bacterial infection), malignancy, trauma, transplantation, or pregnancy.

In another embodiment, a method is provided for treating a subject with USS suffering from clinical symptoms associated with TTP, comprising administering a specified amount of a MASP-2 inhibitory agent (eg, an anti-MASP-2 antibody) for a period of time effective to alleviate or prevent one or more clinical symptoms associated with TTP.

- 28 046178

TTP can also develop due to the presence of autoantibodies against ADAMTS-13. In addition, TTP can develop in the setting of breast, gastrointestinal, or prostate cancer (George JN., Oncology (Williston Park). 25:908-14 (2011)), pregnancy (second trimester or postpartum), George JN., Curr Opin Hematol 10:339-344 (2003)), or in connection with diseases such as HIV or autoimmune diseases such as systemic lupus erythematosus (Hamasaki K, et al., Clin Rheumatol. 22:355-8 (2003)). TTP can also be caused by certain therapeutic agents, including heparin, quinine, an immune-mediated ingredient, anticancer chemotherapeutic agents (bleomycin, cisplatin, cytosine arabinoside, daunomycin gemcitabine, mitomycin C, and tamoxifen), cyclosporine A, oral contraceptives, penicillin, rifampin, and antithrombotics drugs including ticlopidine and clopidogrel (Azarm, T. et al., J Res Med Sci., 16: 353-357 (2011)). Other factors or conditions associated with TTP are toxins such as bee venom, sepsis, splenic sequestration, transplantation, vasculitis, vascular surgery, and infections such as Streptococcus pneumonia and Cytomegalovirus infection (Moake JL, N Engl J Med., 347:589-600 (2002)). TTP due to transient functional deficiency of ADAMTS-13 may develop as a consequence of endothelial cell damage associated with S. pneumonia infection (Pediatr Nephrol., 26:631-5 (2011)).

Plasmapheresis is the standard treatment for TTP (Rock GA, et al., N Engl J Med 325:393397 (1991)). Plasmapheresis can compensate for the lack of ADAMTS-13 activity in patients with genetic defects and remove autoantibodies to ADAMTS-13 in patients with acquired autoimmune TTP (Tsai, H-M, Hematol Oncol Clin North Am., 21(4): 609-v (2007)) . Treatment usually involves the use of additional agents such as immunosuppressants (George, JN, N Engl J Med, 354:1927-35 (2006)). However, plasmapheresis is not successful in approximately 20% of patients, relapse occurs in more than a third of patients, and plasmapheresis is also an expensive and technically challenging procedure. Moreover, many patients are unable to tolerate plasmapheresis. Consequently, there remains a pressing need for additional, more effective treatments for TTP.

Because TTP is a disorder of the coagulation cascade, treatment with complement antagonists may help stabilize and attenuate the disease. While abnormal activation of the alternative complement pathway is associated with aHUS, the role of complement activation in TTP is less clear. Functional deficiency of ADAMTS-13 is important for susceptibility to TTP but is not sufficient to cause acute attacks. Environmental factors and/or other genetic variations may contribute to the expression of TTP. For example, genes encoding proteins involved in the regulation of the coagulation cascade, vWF, platelet function, vascular surface endothelial components, or the complement system may contribute to the development of acute thrombotic microangiopathy (Galbusera, M. et al., Haematologica, 94: 166- 170 (2009)). In particular, complement activation has been shown to play a very important role; Serum from subjects with thrombotic microangiopathy associated with ADAMTS-13 deficiency is known to induce C3 and MAC deposition and subsequent neutrophil activation, which can be reversed by complement inactivation (Ruiz-Torres MP, et al., Thromb Haemost, 93:443 -52 (2005)). In addition, it has recently been shown that increased levels of C4d, C3bBbP and C3a occur during acute attacks 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]), consistent with activation of the classical/lectin and alternative pathways. This increased degree of complement activation during acute episodes may initiate activation of the terminal pathway and cause subsequent complications of TTP.

The role of ADAMTS-13 and vWF in TTP is undoubtedly the activation and aggregation of platelets and their subsequent participation in generating shear forces and deposits in microangiopathies. Activated platelets interact and trigger both the classical and alternative complement pathways. Platelet-mediated complement activation leads to increased levels of the inflammatory mediators C3a and C5a (Peerschke E et al., Mol Immunol, 47:2170-5 (2010)). Thus, platelets may serve as targets for classical complement activation in the case of hereditary or autoimmune TTP.

As described above, the lectin complement pathway, through MASP-2-mediated activation of prothrombin, is the major molecular pathway linking endothelial injury to the coagulation and microvascular thrombosis that occurs in HUS. Likewise, activation of the lectin complement pathway may directly control the coagulation system in TTP. Lectin pathway activation may be triggered in response to initial endothelial damage caused by ADAMTS-13 deficiency in TTP. As a consequence, lectin pathway inhibitors, including but not limited to antibodies that block MASP-2 function, are expected to improve microangiopathies associated with microvascular coagulation, thrombosis, and hemolysis in patients suffering from TTP.

Patients suffering from TTP typically present to the emergency department with one or more of the following: purpura, kidney failure, low platelet count, anemia, and/or thrombosis, including stroke. The current standard of care for TTP involves administering a replacement plasmaphere through a catheter (for example, an intravenous or other catheter).

- 29 046178 for two weeks or longer, usually three times a week, up to a daily procedure. If the subject tests positive for an ADAMTS-13 inhibitor (ie, endogenous anti-ADAMTS-13 antibody), then plasma exchange may be performed in combination with immunosuppressants (eg, corticosteroids, Rituxan, or cyclosporine). Subjects with refractory TTP (approximately 20% of TTP patients) do not respond to at least two weeks of plasmapheresis therapy.

In accordance with the foregoing, in some embodiments, in the case of a primary diagnosis of TTP, or in the case of a subject having one or more symptoms consistent with a diagnosis of TTP (e.g., central nervous system involvement, severe thrombocytopenia (platelet count less than or equal to 5000/μL without aspirin, less than or equal to 20,000/μl when taking aspirin), severe heart disease, severe lung disease, intestinal infarction, or gangrene), a method of treating a subject with an effective amount of a MASP-2 inhibitory agent (eg, anti-MASP-2 antibodies) is provided in as first-line therapy without plasmapheresis or in combination with plasmapheresis. As a first line of therapy, a MASP-2 inhibitory agent can be administered to a subject systemically, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous or other parenteral route. In some embodiments, the MASP-2 inhibitory agent is administered to a subject as first-line therapy without plasmapheresis to avoid potential complications of plasmapheresis, including bleeding, infection, and exposure to pathogenic and/or allergic factors present in the donor plasma, or in the case of a subject rejecting plasmapheresis, or in settings where plasmapheresis is not available. In some embodiments, the MASP-2 inhibitory agent is administered to a subject suffering from TTP in combination (including co-administration) with an immunosuppressant (eg, corticosteroids, Rituxan, or cyclosporine) and/or in combination with concentrated ADAMTS-13.

In some embodiments, the method includes administering a MASP-2 inhibitory agent to a subject suffering from TTP via a catheter (e.g., intravenously) for a first period of time (e.g., an acute phase lasting for at least one day or a week, or two weeks ), followed by administration of a MASP-2 inhibitory agent to the subject subcutaneously for a second period of time (eg, a chronic phase lasting for at least two weeks, or longer). In some embodiments, administration in the first and/or second time period is performed without plasmapheresis. In some embodiments, the method is used as maintenance therapy to prevent a subject from suffering from one or more symptoms associated with TTP.

In another embodiment, a method is provided for treating a subject suffering from refractory TTP (i.e., a subject who has not responded to plasmapheresis for at least two weeks) by administering a MASP-2 inhibitor in an amount effective to reduce one or more symptoms TTR. In one embodiment, a MASP-2 inhibitor (eg, an anti-MASP2 antibody) is administered to a subject with refractory TTP on an ongoing basis over a period of at least two weeks, or longer, by subcutaneous or other parenteral route. Administration may be repeated as determined by the physician until the condition is resolved or controlled.

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

In some embodiments, the method includes administering a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, to a subject suffering from, or at risk of developing, TTP, subcutaneously or intravenously. Preferably, administration is performed daily, but can be performed no more than once every two weeks. Administration is continued until the subject's platelet count reaches a level greater than 150,000/ml for at least two consecutive days. Ahtu-MASP-2 antibody can be administered alone or in combination with a C5 inhibitor such as eculizumab.

In one embodiment, a MASP-2 inhibitory antibody has at least one or more of the following characteristics: said antibody binds human MASP-2 with a KD value of 10 nM or less, said antibody binds an epitope in the CCP1 domain of MASP-2, said antibody inhibits C3b deposition in an in vitro assay in 1% human serum with an IC ₅₀ value of 10 nM or less, the antibody inhibits C3b deposition in 90% human serum with an IC ₅₀ value of 30 nM or less, wherein the antibody is a fragment an antibody selected from the group consisting of Fv, Fab, Fab', F(ab) ₂ and F(ab') ₂ , wherein the antibody is a single chain molecule, wherein said antibody is an IgG2 molecule, wherein said antibody is is an IgG1 molecule, wherein said antibody is an IgG4 molecule, wherein the IgG4 molecule has the S228P mutation, and/or when

- 30 046178 this antibody does not significantly inhibit the classical pathway. In one embodiment, the antibody binds MASP-2, selectively inhibits the lectin pathway, and does not significantly inhibit the alternative pathway. In one embodiment, the antibody binds MASP-2, selectively inhibits the lectin pathway, and does not significantly inhibit the classical or alternative pathway (ie, inhibits the lectin pathway without affecting the classical and alternative complement pathways).

In one embodiment, the MASP-2 inhibitory antibody inhibits clot formation in the serum of a subject suffering from TTP by at least 30%, such as at least 40%, such as at least 50%, such as 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 control serum . In some embodiments, the MASP-2 inhibitory antibody inhibits clot formation in the serum of a subject suffering from TTP by at least 20 percent or more (e.g., at least 30%, at least 40%, at least 50 %) exceeding the inhibitory effect on the deposition of C5b-9 in the serum.

In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in the serum of a TTP patient by at least 30%, e.g., at least 40%, e.g., at least 50%, e.g., at least 60%, e.g. at least 70%, eg at least 80%, eg at least 85%, eg at least 90%, eg at least 95%, up to 99% compared to control serum.

In one embodiment, the MASP-2 inhibitory antibody is administered to a subject by delivery through an intravenous catheter or other catheter.

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

In some embodiments, the method includes administering to a subject a composition comprising a specified amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 67. In some embodiments, the method includes administering to a subject a composition comprising a specified amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprising a light chain variable region containing the amino acid sequence of SEQ ID NO: 70.

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

Degos disease.

Degos disease, also known as malignant atrophic papulosis, is a very rare type of TMA that affects small vessels in the skin, gastrointestinal tract, and central nervous system. This vasculopathy causes occlusion of venules and arterioles, leading to skin lesions, intestinal ischemia, and central nervous system disorders including stroke, epilepsy, and cognitive impairment. Connective tissue necrosis develops in the skin due to thrombotic occlusion of small arteries. However, the cause of Degos' disease is unknown. Vasculitis, coagulopathy, or primary endothelial cell dysfunction have been suggested. The survival rate for Degos disease is 50% after only two to three years. There is no effective treatment for Degos disease, although antimicrobial agents are used.

- 31 046178 platelet drugs, anticoagulants and immunosuppressants to relieve symptoms.

Although the mechanism of Degos disease is unknown, the involvement of the complement pathway has been suggested. Margo et al found significant deposits of C5b-9 in the vessels of the skin, gastrointestinal tract, and brain in four deceased patients with Degos disease (Margo et al., Am J Clin Pathol 135(4):599–610, 2011). Experimental treatment with eculizumab was initially effective for the treatment of cutaneous and intestinal lesions, but did not prevent progression of systemic disease (see GarrettBakelman 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 No. 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 No. 1205).

Many patients suffering from Degos disease have blood clotting disorders. Thrombotic occlusion of small arteries in the skin is a characteristic feature of the disease. Since the complement pathway is involved in this disease, as described herein for other TMAs, lectin pathway inhibitors, including but not limited to antibodies that block MASP-2 function, are expected to be useful in the treatment of patients suffering from Degos disease.

Accordingly, in another embodiment, the invention provides methods for treating Degos' disease by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, in a pharmaceutical carrier to a subject suffering from Degos' disease or a related condition. with Degos disease. The MASP-2 inhibitory agent is administered to a subject suffering from Degos disease or a condition associated with Degos disease, systemically, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous or other parenteral route, or potentially by oral administration to in the case of non-peptidergic agents. Ahtu-MASP-2 antibody can be administered alone or in combination with a C5 inhibitor such as eculizumab.

In one embodiment, a MASP-2 inhibitory antibody has at least one or more of the following characteristics: said antibody binds human MASP-2 with a KD value of 10 nM or less, said antibody binds an epitope in the CCP1 domain of MASP-2, said antibody inhibits C3b deposition in an in vitro assay in 1% human serum with an IC ₅₀ value of 10 nM or less, the antibody inhibits C3b deposition in 90% human serum with an IC ₅₀ value of 30 nM or less, wherein the antibody is a fragment an antibody selected from the group consisting of Fv, Fab, Fab', F(ab)2 and F(ab')2, wherein the antibody is a single chain molecule, wherein said antibody is an IgG2 molecule, wherein said antibody is is an IgG1 molecule, wherein the antibody is an IgG4 molecule, wherein the IgG4 molecule has the S228P mutation, and/or wherein the antibody does not significantly inhibit the classical pathway. In one embodiment, the antibody binds MASP-2, selectively inhibits the lectin pathway, and does not significantly inhibit the alternative pathway. In one embodiment, the antibody binds MASP-2, selectively inhibits the lectin pathway, and does not significantly inhibit the classical or alternative pathway (ie, inhibits the lectin pathway without affecting the classical and alternative complement pathways).

In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in the serum of a subject suffering from Degos disease by at least 30%, such as at least 40%, such as at least 50%, such as 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 control serum . In some embodiments, the MASP-2 inhibitory antibody inhibits thrombus formation in the serum of a subject suffering from Degos disease by at least 20 percent or more (e.g., at least 30%, at least 40%, at least 50%) exceeding the inhibitory effect on C5b-9 deposition in serum.

In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in the serum of a patient with Degos disease by at least 30%, for example, at least 40%, for example, at least 50%, for example, at least 60%, for example , at least 70%, for example, at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95% up to 99% compared to control serum.

In one embodiment, the MASP-2 inhibitory antibody is administered to a subject by delivery through an intravenous catheter or other catheter.

In one embodiment, the invention relates to a method of inhibiting the formation of blood clots in a subject suffering from Degos disease, comprising administering to the subject a composition containing a certain amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, containing (I)(a) a heavy chain variable region, comprising: i) a heavy chain CDR-H1 containing the amino acid sequence of residues 31-35 in SEQ ID NO: 67; and ii) CDR-H2

- 32 046178 heavy chain containing the amino acid sequence of residues 50-65 in SEQ ID NO: 67; and iii) a heavy chain CDR-H3 containing the amino acid sequence of residues 95-102 in SEQ ID NO: 67, and b) a light chain variable region comprising: i) a light chain CDR-L1 containing the amino acid sequence of residues 24-34 in SEQ ID NO: 70; and ii) light chain CDR-L2 containing the amino acid sequence of residues 50-56 in SEQ ID NO: 70; and iii) a light chain CDR-L3 comprising the amino acid sequence of residues 89-97 of SEQ ID NO: 70, or (II) a variant thereof comprising a heavy chain variable region having at least 90% sequence identity with SEQ ID NO: 67 (e.g. at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , at least 99% sequence identity to SEQ ID NO: 67), and a light chain variable region having at least 90% sequence 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% sequence identity) with SEQ ID NO: 70.

In some embodiments, the method includes administering to a subject a composition comprising a specified amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 67. In some embodiments, the method includes administering to a subject a composition comprising a specified amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprising a light chain variable region containing the amino acid sequence of SEQ ID NO: 70.

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

Catastrophic antiphospholipid syndrome (CAPS).

Catastrophic antiphospholipid syndrome (CAPS) is an extreme variant of antiphospholipid antibody syndrome (APLA). CAPS is characterized by venous and arterial thrombosis due to pathogenic antibodies. CAPS is a TMA with thrombosis, multi-organ ischemia, and multi-organ failure. As with other TMAs, a characteristic feature is the occlusion of small vessels in various organs. CAPS has a high mortality rate of approximately 50% and is often associated with infection or trauma. Patients have antiphospholipid antibodies, usually IgG.

Clinical features of CAPS include the presence of at least three organs or tissues with histopathological evidence of small vessel occlusion. Peripheral thrombosis can affect veins and arteries in the central nervous system, cardiovascular, renal or pulmonary systems. Patients receive antibiotics, anticoagulants, corticosteroids, plasmapheresis, and intravenous immunoglobulin. However, multiple organ failure can lead to death.

The involvement of the complement pathway in CAPS has been suggested. For example, studies in animal models suggest that complement inhibition may be an effective way to prevent thrombosis associated with CAPS (Shapira L. et al., Arthritis Rheum 64(8):2719-23, 2012). Moreover, as also reported by Shapira et al, administration of eculizumab to a subject suffering from CAPS, at doses resulting in complement pathway blockade, abolished acute progressive thrombotic events and reversed thrombocytopenia (see also W. Lim, Curr Opin Hematol 18(5) :361-5, 2011). Thus, as described herein for other TMAs, lectin pathway inhibitors, including but not limited to antibodies that block MASP-2 function, are expected to be useful in the treatment of patients suffering from CAPS.

Accordingly, in another embodiment, the invention provides methods for treating CAPS by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, in a pharmaceutical carrier to a subject suffering from CAPS or a CAPS-related condition . The MASP-2 inhibitory agent is administered to a subject suffering from CAPS, or a CAPS-related condition, systemically, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous or other parenteral route, or potentially by oral route if not peptidergic agents. Ahtu-MASP-2 antibody can be administered alone or in combination with a C5 inhibitor such as eculizumab.

In one embodiment, a MASP-2 inhibitory antibody has at least one or more of the following characteristics: said antibody binds human MASP-2 with a K _D value of 10 nM or less, said antibody binds an epitope in the CCP1 domain of MASP-2, said the antibody inhibits C3b deposition in an in vitro assay in 1% human serum with an IC ₅₀ value of 10 nM or less, the antibody inhibits C3b deposition in 90% of human serum

- 33 046178 ical serum with an IC ₅₀ value of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2 and F(ab')2, wherein the antibody is a single chain molecule, wherein the antibody is an IgG2 molecule, wherein the antibody is an IgG1 molecule, wherein the antibody is an IgG4 molecule, wherein the IgG4 molecule has the S228P mutation, and/or wherein the antibody is substantially does not inhibit the classical pathway. In one embodiment, the antibody binds MASP-2, selectively inhibits the lectin pathway, and does not significantly inhibit the alternative pathway. In one embodiment, the antibody binds MASP-2, selectively inhibits the lectin pathway, and does not significantly inhibit the classical or alternative pathway (ie, inhibits the lectin pathway without affecting the classical and alternative complement pathways).

In one embodiment, the MASP-2 inhibitory antibody inhibits clot formation in the serum of a subject suffering from CAPS by at least 30%, such as at least 40%, such as at least 50%, such as at least 60% eg at least 70%, eg at least 80% eg at least 85%, eg at least 90%, eg at least 95%, up to 99% compared to control serum. In some embodiments, the MASP-2 inhibitory antibody inhibits clot formation in the serum of a subject suffering from CAPS by at least 20 percent or more (e.g., at least 30%, at least 40%, at least 50 %) exceeding the inhibitory effect on the deposition of C5b-9 in the serum.

In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in the serum of a CAPS patient by at least 30%, e.g., at least 40%, e.g., at least 50%, e.g., at least 60%, e.g. 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 control serum.

In one embodiment, the MASP-2 inhibitory antibody is administered to a subject by delivery through an intravenous catheter or other catheter.

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

In some embodiments, the method includes administering to a subject a composition comprising a specified amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 67. In some embodiments, the method includes administering to a subject a composition comprising a specified amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprising a light chain variable region containing the amino acid sequence of SEQ ID NO: 70.

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

TMA due to cancer.

Systemic malignancies of various types can lead to the clinical and pathological manifestations of TMA (see, for example, Batts and Lazarus, Bone Marrow Transplantation 40:709-719, 2007). Cancer-associated TMA is often found in the lungs and appears to be associated with tumor emboli (Francis KK et al., Commun Oncol 2:339-43, 2005). Tumor emboli may

- 34 046178 reduce blood flow and thus lead to a hypoperfusion state in the affected arterioles and venules. The resulting stress and tissue damage are thought to activate the lectin complement pathway in a localized manner. The activated lectin pathway, in turn, can activate the coagulation cascade through MASP-2-dependent cleavage of prothrombin to thrombin, leading to the prothrombotic state characteristic of TMA. Inhibition of MASP-2 in this setting is expected to reduce localized activation of thrombin and thus alleviate the prothrombotic state.

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 useful in the treatment of patients suffering from TMA due to cancer.

Accordingly, in another embodiment, the invention provides methods for treating or preventing TMA due to cancer by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, in a pharmaceutical carrier to a subject suffering from, or at risk of development, TMA due to cancer. The MASP-2 inhibitory agent is administered to a subject suffering from, or at risk of developing, TMA due to cancer, systemically, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous or other parenteral route, or potentially by oral route in the case of not peptidergic agents. Ahtu-MASP-2 antibody can be administered alone or in combination with a C5 inhibitor such as eculizumab.

In one embodiment, a MASP-2 inhibitory antibody has at least one or more of the following characteristics: said antibody binds human MASP-2 with a KD value of 10 nM or less, said antibody binds an epitope in the CCP1 domain of MASP-2, said antibody inhibits C3b deposition in an in vitro assay in 1% human serum with an IC ₅₀ value of 10 nM or less, the antibody inhibits C3b deposition in 90% human serum with an IC ₅₀ value of 30 nM or less, wherein the antibody is a fragment an antibody selected from the group consisting of Fv, Fab, Fab', F(ab) ₂ and F(ab') ₂ , wherein the antibody is a single chain molecule, wherein said antibody is an IgG2 molecule, wherein said antibody is is an IgG1 molecule, wherein the antibody is an IgG4 molecule, wherein the IgG4 molecule has the S228P mutation, and/or wherein the antibody does not significantly inhibit the classical pathway. In one embodiment, the antibody binds MASP-2, selectively inhibits the lectin pathway, and does not significantly inhibit the alternative pathway. In one embodiment, the antibody binds MASP-2, selectively inhibits the lectin pathway, and does not significantly inhibit the classical or alternative pathway (ie, inhibits the lectin pathway without affecting the classical and alternative complement pathways).

In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in the serum of a subject suffering from TMA due to cancer by at least 30%, e.g., at least 40%, e.g., at least 50%, e.g. 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 control serum .

In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in the serum of a patient suffering from TMA due to cancer by at least 30%, e.g., at least 40%, e.g., at least 50%, e.g. 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 control serum .

In one embodiment, the MASP-2 inhibitory antibody is administered to a subject by delivery through an intravenous line or other catheter.

In one embodiment, the invention relates to a method of inhibiting the formation of blood clots in a subject suffering from TMA due to cancer, comprising administering to the subject a composition containing a certain amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, containing (I)(a) a heavy chain variable region comprising: i) a heavy chain CDR-H1 containing the amino acid sequence of residues 31-35 in SEQ ID NO: 67; and ii) a heavy chain CDR-H2 containing the amino acid sequence of residues 50-65 of SEQ ID NO: 67; and iii) a heavy chain CDR-H3 containing the amino acid sequence of residues 95-102 in SEQ ID NO: 67, and b) a light chain variable region comprising: i) a light chain CDR-L1 containing the amino acid sequence of residues 24-34 in SEQ ID NO: 70; and ii) light chain CDR-L2 containing the amino acid sequence of residues 50-56 in SEQ ID NO: 70; and iii) a light chain CDR-L3 comprising the amino acid sequence of residues 89-97 of SEQ ID NO: 70, or (II) a variant thereof comprising a heavy chain variable region having at least 90% sequence identity with SEQ ID NO: 67 (for example, at least 91%, at least 92%, according to

- 35 046178 at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with SEQ ID NO: 67), and a light chain variable region having at least 90% sequence 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% sequence identity) with SEQ ID NO: 70.

In some embodiments, the method includes administering to a subject a composition comprising a specified amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 67. In some embodiments, the method includes administering to a subject a composition comprising a specified amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprising a light chain variable region containing the amino acid sequence of SEQ ID NO: 70.

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

TMA due to anticancer chemotherapy.

Chemotherapy-associated TMA is a condition involving thrombocytopenia, microangiopathic hemolytic anemia, and renal dysfunction that occurs in 2-10% of patients with a history of malignancy treated with chemotherapeutic agents such as gemcitabine, mitomycin, oxaliplatin, and others. Chemotherapy-associated TMA is characterized by poor clinical outcomes with high mortality rates (see, for example, Blake-Haskins et al., Clin Cancer Res 17(18):5858-5866, 2011).

The etiology of TMA due to chemotherapy is believed to be related to 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). It has been shown that damage to endothelial cells through various mechanisms leads to activation of the lectin complement pathway. For example, Stahl et al. showed that endothelial cells exposed to oxidative stress activate the lectin complement pathway 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). Additionally, as demonstrated in Examples 37-39 herein, in a mouse model of TMA in which localized excitation with FITC-Dex photons was used to induce localized microvascular damage, followed by development of a TMA response, we have demonstrated that inhibition of MASP-2 may prevent TMA. Thus, damage to the microvascular endothelium by chemotherapeutic agents may activate the lectin complement pathway, which then creates a localized prothrombotic state and stimulates a TMA response. Because activation of the lectin pathway and creation of a prothrombotic state is dependent on MASP-2, MASP-2 inhibitors, including but not limited to antibodies that block MASP-2 function, are expected to result in a reduced TMA response and a reduced risk of developing associated anticancer chemotherapy TMA.

Accordingly, in another embodiment, the invention provides methods for treating or preventing TMA due to chemotherapy by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, in a pharmaceutical carrier to a subject suffering from, or at risk of development, TMA due to chemotherapy. The MASP-2 inhibitory agent is administered to a subject who has received, is receiving, or will receive chemotherapy, systemically, e.g., by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral route, or potentially by oral route in the case of non-peptidergic agents. . Ahtu-MASP-2 antibody can be administered alone or in combination with a C5 inhibitor such as eculizumab.

In one embodiment, a MASP-2 inhibitory antibody has at least one or more of the following characteristics: said antibody binds human MASP-2 with a K _D value of 10 nM or less, said antibody binds an epitope in the CCP1 domain of MASP-2, said the antibody inhibits C3b deposition in an in vitro assay in 1% human serum with an IC ₅₀ value of 10 nM or less, the antibody inhibits C3b deposition in 90% human serum with an IC ₅₀ value of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2 and F(ab')2, wherein the antibody is a single chain molecule, wherein the antibody is an IgG2 molecule, wherein the antibody is an IgG1 molecule, with the specified

- 36 046178 the antibody is an IgG4 molecule, wherein the IgG4 molecule has the S228P mutation, and/or the antibody does not significantly inhibit the classical pathway. In one embodiment, the antibody binds MASP-2, selectively inhibits the lectin pathway, and does not significantly inhibit the alternative pathway. In one embodiment, the antibody binds MASP-2, selectively inhibits the lectin pathway, and does not significantly inhibit the classical or alternative pathway (ie, inhibits the lectin pathway without affecting the classical and alternative complement pathways).

In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in the serum of a subject suffering from TMA due to chemotherapy by at least 30%, e.g., at least 40%, e.g., at least 50%, e.g. 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 control serum .

In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in the serum of a patient suffering from TMA due to cancer chemotherapy by at least 30%, e.g., at least 40%, e.g., at least 50%, e.g. 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 control serum.

In one embodiment, the MASP-2 inhibitory antibody is administered to a subject by delivery through an intravenous catheter or other catheter.

In one embodiment, the invention relates to a method of inhibiting the formation of blood clots in a subject suffering from TMA due to anticancer chemotherapy, comprising administering to the subject a composition containing a certain amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, containing (I)(a) a severe variable region a chain comprising: i) a heavy chain CDR-H1 containing the amino acid sequence of residues 3135 in SEQ ID NO: 67; and ii) a heavy chain CDR-H2 containing the amino acid sequence of residues 50-65 of SEQ ID NO: 67; and iii) a heavy chain CDR-H3 containing the amino acid sequence of residues 95-102 in SEQ ID NO: 67, and b) a light chain variable region comprising: i) a light chain CDR-L1 containing the amino acid sequence of residues 24-34 in SEQ ID NO: 70; and ii) light chain CDR-L2 containing the amino acid sequence of residues 50-56 in SEQ ID NO: 70; and iii) a light chain CDR-L3 comprising the amino acid sequence of residues 89-97 of SEQ ID NO: 70, or (II) a variant thereof comprising a heavy chain variable region having at least 90% sequence identity with SEQ ID NO: 67 (e.g. at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , at least 99% sequence identity to SEQ ID NO: 67), and a light chain variable region having at least 90% sequence 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% sequence identity) with SEQ ID NO: 70.

In some embodiments, the method includes administering to a subject a composition comprising a specified amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 67. In some embodiments, the method includes administering to a subject a composition comprising a specified amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprising a light chain variable region containing the amino acid sequence of SEQ ID NO: 70.

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

TMA due to transplantation.

Transplant-associated TMA (TA-TMA) is a devastating syndrome that can develop in transplant patients, such as allogeneic hematopoietic stem cell transplant recipients (see, e.g., Batts and Lazarus, Bone Marrow Transplantation 40:709-719, 2007 ). The pathogenesis of this condition is not fully understood, but likely involves a constellation of responses that lead to endothelial cell damage (Laskin B.L. et al., Blood 118(6): 1452-62, 2011). As described above, endothelial cell damage is a typical stimulus for activating the lectin pathway and creating a prothrombotic state.

Recent evidence further supports activation of complement through lection

- 37 046178 new pathway in the pathogenesis of TA-TMA. Laskin et al demonstrated that C4d deposition in renal arterioles was more common in subjects with histological evidence of TA-TMA (75%) compared with control subjects (8%) (Laskin B.L., et al., Transplantation, 27; 96(2). ):217-23, 2013). Thus, C4d may be a pathological marker of TA-TMA related to localized complement fixation through the lectin or classical pathway.

Because activation of the lectin pathway and creation of a prothrombotic state is dependent on MASP-2, MASP-2 inhibitors, including but not limited to antibodies that block MASP-2 function, are expected to result in a reduced TMA response and a reduced risk of developing associated TMA graft (TA-TMA).

Accordingly, in another embodiment, the invention provides methods for treating or preventing TMA due to transplantation by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, in a pharmaceutical carrier to a subject suffering from, or at risk of development, TMA due to transplantation. The MASP-2 inhibitory agent is administered to a subject who has undergone, is undergoing, or will undergo a transplant procedure, systemically, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous or other parenteral route, or potentially by oral route if not peptidergic agents. The anti-MASP-2 antibody can be administered alone or in combination with a C5 inhibitor such as eculizumab. In some embodiments, the invention provides methods for treating or preventing TMA due to an allogeneic stem cell transplant, comprising administering a composition containing a certain amount of a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, to a subject before, during, or after receiving an allogeneic stem cell transplant. stem cells.

In one embodiment, a MASP-2 inhibitory antibody has at least one or more of the following characteristics: said antibody binds human MASP-2 with a KD value of 10 nM or less, said antibody binds an epitope in the CCP1 domain of MASP-2, said antibody inhibits C3b deposition in an in vitro assay in 1% human serum with an IC ₅₀ value of 10 nM or less, the antibody inhibits C3b deposition in 90% human serum with an IC ₅₀ value of 30 nM or less, wherein the antibody is a fragment an antibody selected from the group consisting of Fv, Fab, Fab', F(ab)2 and F(ab')2, wherein the antibody is a single chain molecule, wherein said antibody is an IgG2 molecule, wherein said antibody is is an IgG1 molecule, wherein the antibody is an IgG4 molecule, wherein the IgG4 molecule has the S228P mutation, and/or wherein the antibody does not significantly inhibit the classical pathway. In one embodiment, the antibody binds MASP-2, selectively inhibits the lectin pathway, and does not significantly inhibit the alternative pathway. In one embodiment, the antibody binds MASP-2, selectively inhibits the lectin pathway, and does not significantly inhibit the classical or alternative pathway (ie, inhibits the lectin pathway without affecting the classical and alternative complement pathways).

In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in the serum of a subject suffering from TMA due to transplantation by at least 30%, e.g., at least 40%, e.g., at least 50%, e.g. 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 control serum .

In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in the serum of a patient suffering from TMA due to transplantation by at least 30%, e.g., at least 40%, e.g., at least 50%, e.g. 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 control serum .

In one embodiment, the MASP-2 inhibitory antibody is administered to a subject by delivery through an intravenous catheter or other catheter.

In one embodiment, the invention relates to a method of inhibiting the formation of blood clots in a subject suffering from TMA due to transplantation, comprising administering to the subject a composition containing a certain amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, containing (I)(a) a heavy chain variable region comprising: i) a heavy chain CDR-H1 containing the amino acid sequence of residues 31-35 in SEQ ID NO: 67; and ii) a heavy chain CDR-H2 containing the amino acid sequence of residues 50-65 of SEQ ID NO: 67; and iii) a heavy chain CDR-H3 containing the amino acid sequence of residues 95102 in SEQ ID NO: 67, and b) a light chain variable region comprising: i) a light chain CDR-L1 containing the amino acid sequence of residues 24-34 in SEQ ID NO: : 70; and ii) CDR-L2 mild

- 38 046178 chain containing the amino acid sequence of residues 50-56 in SEQ ID NO: 70; and iii) a light chain CDR-L3 comprising the amino acid sequence of residues 89-97 of SEQ ID NO: 70, or (II) a variant thereof comprising a heavy chain variable region having at least 90% sequence identity with SEQ ID NO: 67 (e.g. at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , at least 99% sequence identity to SEQ ID NO: 67), and a light chain variable region having at least 90% sequence 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% sequence identity) with SEQ ID NO: 70.

In some embodiments, the method includes administering to a subject a composition comprising a specified amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 67. In some embodiments, the method includes administering to a subject a composition comprising a specified amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprising a light chain variable region containing the amino acid sequence of SEQ ID NO: 70.

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

IV. Role of MASP-2 in other diseases and conditions, and treatments using MASP-2 inhibitory agents

Kidney diseases.

Activation of the complement system is involved in the pathogenesis of various kidney diseases; including mesangioproliferative glomerulonephritis (IgA nephropathy, Berger's disease) (Endo, M., et al., Clin. Nephrology 55:185-191, 2001), membranous glomerulonephritis (Kerjashki, D., Arch B Cell Pathol. 58:25371, 1990; Brenchley, P.E., et al., Kidney Int., 41:933-7, 1992; Salant, D.J., et al., Kidney Int. 35:976-84, 1989), membranous proliferative glomerulonephritis (mesangiocapillary glomerulonephritis) (Bartlow, B.G., et al., Kidney Int. 15:294-300, 1979; Meri, S., et al., J. Exp. Med. 175:939-50, 1992), acute postinfectious glomerulonephritis (poststreptococcal glomerulonephritis ), cryoglobulinemic glomerulonephritis (Ohsawa, I., et al., Clin Immunol. 101:59-66, 2001), lupus nephritis (Gatenby, P.A., Autoimmunity 11:61-6, 1991) and nephritis associated with Henoch-Schönlein purpura (Endo, M., et al., Am. J. Kidney Dis. 35:401-407, 2000). The involvement of complement in renal failure has been recognized for several decades, but there is still considerable debate about its precise role in the onset, progression, and resolution of renal failure. Under normal conditions, the role of complement is beneficial to the host, but inappropriate activation and deposition of complement can lead to tissue damage.

There is compelling evidence that glomerulonephritis, an inflammation of the glomeruli, is often initiated by the deposition of immune complexes on glomerular and tubular structures, followed by complement activation, inflammation, and tissue damage. Kahn and Sinniah demonstrated increased deposition of C5b-9 in tubular basement membranes in biopsy specimens from patients with various forms of glomerulonephritis (Kahn, T.N., et al., Histopath. 26:351-6, 1995). In a study of patients with IgA nephropathy (Alexopoulos, A., et al., Nephrol. Dial. Transplant 10:11661172, 1995), C5b-9 deposition in glomerular epithelial/basal membrane structures correlated with plasma creatinine levels. Another study of membranous nephropathy demonstrated an association between clinical outcome and urinary sC5b-9 levels (Kon, S.P., et al., Kidney Int. 48:195358, 1995). Elevated levels of sC5b-9 were positively correlated with poor prognosis. Lehto et al detected elevated levels of CD59, a complement regulatory factor that inhibits the membrane attack complex in plasma membranes, and C5b-9 in the urine of patients with membranous glomerulonephritis (Lehto, T., et al., Kidney Int. 47:1403-11, 1995). Histopathological analysis of biopsy specimens from the same patients showed deposits of C3 and C9 proteins in the glomeruli, while CD59 expression in these tissues was reduced compared with that in normal renal tissue. The results of these studies indicate that ongoing complement-mediated glomerulonephritis results in urinary excretion of complement proteins that correlates with the degree of tissue damage and disease prognosis.

Inhibition of complement activation in various animal models of glomerulonephritis has also demonstrated the importance of complement activation in the etiology of the disease. In a model of membranous proliferative glomerulonephritis (MPGN), infusion of anti-Thy1 serum into C6-deficient rats (which do not produce C5b-9) leads to a 90% decrease in glomerular cell proliferation, an 80% decrease in platelet and macrophage infiltration, and a decrease in the synthesis of type IV collagen ( Mar

- 39 046178 kera increase in mesangial matrix volume) and a 50% decrease in proteinuria, compared with C6+ normal rats (Brandt, J., et al., Kidney Int. 49:335-343, 1996). These results suggest that C5b-9 is the primary mediator of complement tissue damage in this anti-thymocyte serum rat model. In another model of glomerulonephritis, infusion of graded doses of rabbit antibodies against rat glomerular basement membrane resulted in a dose-dependent influx of polymorphonuclear leukocytes (PMN), which was attenuated by pretreatment with cobra venom factor (to eliminate complement) (Scandrett, A.L., et al., Am J Physiol 268:F256-F265, 1995). Rats injected with cobra venom factor also exhibited less severe histopathology, less long-term proteinuria, and lower creatinine levels than control rats. Using three rat models of GN (anti-thymocyte serum, Con A anti-Con A, and passive Heymann nephritis), Couser et al. demonstrated the potential therapeutic efficacy of complement inhibition approaches using recombinant sCR1 protein (Couser, W.G., et al., J. Am Soc Nephrol 5:1888-94, 1995). Rats treated with sCR1 had significantly less influx of PMN, platelets and macrophages, mesangiolysis and proteinuria compared to control rats. Additional evidence for the importance of complement activation in glomerulonephritis was obtained using anti-C5 moAb in the NZB/W F1 mouse model. Anti-C5 moAb inhibits the cleavage of C5, thereby blocking the formation of C5a and C5b9. Continuous administration of anti-C5 moAb for 6 months led to significant relief of glomerulonephritis. A humanized anti-C5 monoclonal antibody moAb (5G1.1), which prevents the cleavage of human complement component C5 into its proinflammatory components, is being developed by Alexion Pharmaceuticals, Inc., New Haven, Connecticut, as a potential therapeutic agent for the treatment of glomerulonephritis.

Direct evidence for the pathological role of complement in kidney injury comes from studies of patients with genetic deficiencies of specific complement components. A number of reports have documented the association of renal failure with complement regulatory factor H deficiency (Ault, B.H., Nephrol. 14:1045-1053, 2000; Levy, M., et al., Kidney Int. 30:949-56, 1986; Pickering , M.C., et al., Nat. Genet. 31:424-8, 2002). Factor H deficiency results in low plasma levels of factors B and C3 and the uptake of C5b-9. Both atypical membranous proliferative glomerulonephritis (MPGN) and idiopathic hemolytic uremic syndrome (HUS) are associated with factor H deficiency. Factor H-deficient pigs (Jansen, J.H., et al., Kidney Int. 53:331-49, 1998 ) and factor H knockout mice (Pickering, M.S., 2002) exhibit MPGN-like symptoms, confirming the importance of factor H in complement regulation. Deficiencies of other complement components are associated with renal failure resulting from systemic lupus erythematosus (SLE) (Walport, M.J., Davies, et al., Ann. N.Y. Acad. Sci. 815:267-81, 1997). Deficiencies of C1q, C4, and C2 are strong predisposition factors for the development of SLE through mechanisms related to impaired clearance of immune complexes and apoptotic material. Many of these patients with SLE develop lupus nephritis, characterized by deposition of immune complexes throughout the zona glomerulosa.

Additional evidence linking complement activation to renal failure came from the discovery in patients of autoantibodies directed against complement components, some of which were directly associated with renal failure (Trouw, L.A., et al., Mol. Immunol. 38:199-206, 2001 ). Many of these autoantibodies show such a high correlation with renal failure that the term nephritic factor (NeF) has been adopted to define this activity. In clinical trials, approximately 50% of patients with positive nephritic factor test results developed MPGN (Spitzer, R.E., et al., Clin. Immunol. Immunopathol. 64:177-83, 1992). C3NeF is an autoantibody directed against the alternative pathway C3 convertase (C3bBb), and it stabilizes this convertase, thereby stimulating the activation of the alternative pathway (Daha, M.R., et al., J. Immunol. 116:1-7, 1976). Likewise, an autoantibody with specificity for the classical pathway C3 convertase (C4b2a), called C4NeF, stabilizes this convertase and thereby stimulates the activation of the classical pathway (Daha, M.R. et al., J. Immunol. 125:2051-2054, 1980; Halbwachs, L., et al., J Clin Invest 65:1249-56, 1980). Anti-C1q autoantibodies have been shown to be associated with nephritis in patients with SLE (Hovath, L., et al., Clin. Exp. Rheumatol. 19:66772, 2001; Siegert, C, et al., J. Rheumatol. 18:230- 34, 1991; Siegert, C, et al., Clin. Exp. Rheumatol. 10:19-23, 1992), and increasing titers of these anti-C1q autoantibodies have been reported to predict nephritis flare (Coremans, I.E., et al. al., Am J Kidney Dis 26:595-601, 1995). Immune complex deposits eluted postmortem from the kidneys of patients with SLE revealed accumulations of these anti-C1q autoantibodies (Mannick, M., et al., Arthritis Rheumatol. 40:1504-11, 1997). All these facts point to a pathological role of these autoantibodies. However, not all patients with anti-C1q autoantibodies develop renal failure and, in addition, some healthy individuals have low titers of such anti-C1q autoantibodies (Siegert, C.E., et al., Clin. Immunol. Immunopathol. 67:204-9, 1993) .

In addition to the alternative and classical pathways of complement activation, the lectin pathway may also play an important pathological role in the development of renal failure. Elevated levels of MBL, MBL-associated serine protease, and complement activation products were found

- 40 046178 we immunohistochemical methods in kidney biopsies obtained from patients diagnosed with several different kidney diseases, including nephritis associated with Henoch-Schönlein purpura (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 link between complement and kidney disease has been known for several decades, data on exactly how complement affects these kidney diseases is far from complete.

Blood diseases.

Sepsis is caused by a generalized reaction of the patient to microorganisms entering the body. The primary function of the complement system is to organize the inflammatory response to invading bacteria and other pathogens. Consistent with this physiological role, complement activation has been shown in numerous studies to play a major role in the pathogenesis of sepsis (Bone, R.C., Annals. Internal. Med. 115:457-469, 1991). The definition of clinical manifestations of sepsis is constantly evolving. Sepsis is generally defined as a systemic host response to infection. However, in many cases, clinical evidence of infection (eg, positive blood cultures for bacteria) is absent in patients with symptoms of sepsis. This discrepancy was first addressed at a consensus conference in 1992, when the term systemic inflammatory response syndrome (SIRS) was adopted, which did not require the presence of a detectable bacterial infection (Bone, R.C., et al., Crit. Care Med. 20: 724-726, 1992). There is now general agreement that sepsis and SIRS are associated with a failure to regulate the inflammatory response. For the purposes of this brief review, the inventors will consider that the clinical definition of sepsis also includes severe sepsis, septic shock and SIRS.

The predominant source of infection in patients with sepsis until the late 1980s was Gram-negative bacteria. Lipopolysaccharide (LPS), a major component of the cell wall of Gram-negative bacteria, is known to stimulate the release of inflammatory response mediators from various cell types and cause symptoms of acute infection when injected into animals (Haeney, M.R., et al., Antimicrobial Chemotherapy 41(Suppl. A) :41-6, 1998). Interestingly, the spectrum of microorganisms responsible 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 to this day (Martin, G.S., et al ., N. Eng. J. Med. 348:1546-54, 2003).

The results of many studies demonstrate the importance of complement activation in mediating inflammation and in the development of signs of shock, in particular, septic and hemorrhagic shock. Septic shock is usually caused by both gram-negative and gram-positive microorganisms. LPS is a potent complement activator, primarily through the alternative pathway, although activation through the classical antibody-mediated pathway also occurs (Fearon, D.T., et al., N. Engl. J. Med. 292:937-400, 1975). The major cell wall components of Gram-positive bacteria are peptidoglycan and lipoteichoic acid, and both components are potent activators of the alternative complement pathway, although in the presence of specific antibodies they can also activate the classical complement pathway (Joiner, K.A., et al., Ann. Rev. Immunol. 2 :461-2, 1984).

Initially, the assumption of the participation of the complement system in the pathogenesis of sepsis was made when it was observed that anaphylatoxins C3a and C5a mediate various inflammatory reactions, which can also occur in sepsis. These anaphylatoxins cause vasodilation and increased microvascular permeability, phenomena that play a central role in septic shock (Schumacher, W.A., et al., Agents Actions 34:345-349, 1991). In addition, anaphylatoxins cause bronchospasm, histamine release from mast cells, and platelet aggregation. In addition, they have numerous effects on granulocytes, such as chemotaxis, aggregation, adhesion, release of lysosomal enzymes, formation of toxic superoxide anion, and formation of leukotrienes (Shin, H.S., et al., Science 162:361-363, 1968; Vogt, W ., Complement 3:177-86, 1986). These biological effects are thought to play a role in the development of complications of sepsis such as shock or acute respiratory distress syndrome (ARDS) (Hammerschmidt, D.E., et al., Lancet 1:947-949, 1980; Slotman, G.T., et al. , Surgery 99:744-50, 1986). In addition, elevated levels of anaphylatoxin C3a are associated with death in sepsis (Hack, C.E., et al., Am. J. Med. 86:20-26, 1989). In some animal models of shock, certain strains of complement component deficient animals (eg, C5 deficiency) are more resistant to the effects of LPS infusion (Hseuh, W., et al., Immunol. 70:309-14, 1990).

It has been shown that blocking C5a formation with antibodies early in the development of sepsis in rodents greatly increases survival rates (Czermak, B.J., et al., Nat. Med. 5:788-792, 1999). Similar results were obtained when blocking the C5a receptor (C5aR) with either an antibody or a small molecule inhibitor (Huber-Lang, M.S., et al., FASEB J. 16:1567-74, 2002; Riedemann, N.C., et al., J. Clin Invest. 110:101-8, 2002). Early experimental studies in monkeys indicated that blocking C5a antibodies alleviated E. coli-induced septic shock and

- 41 046178 adult respiratory distress syndrome (Hangen, D.H., et al., J. Surg. Res. 46:195-9, 1989; Stevens, J.H., et al., J. Clin. Invest. 77:1812-16 , 1986). In people with sepsis, C5a levels are elevated and are associated with significantly reduced survival rates in the setting of multiple organ failure compared with levels in patients with less severe sepsis and in survivors (Nakae, H., et al., Res. Commun. Chem. Pathol. Pharmacol. 84:189-95, 1994; Nakae, et al., Surg. Today 26:225-29, 1996; Bengtson, A., et al., Arch. Surg. 123:645-649, 1988). The mechanisms by which C5a exerts its deleterious effects in sepsis remain to be studied in detail, but recent evidence suggests that C5a production in sepsis significantly impairs the innate immune functions of blood neutrophils (Huber-Lang, M.S., et al., J. Immunol. 169:3223-31, 2002), their ability to produce respiratory burst, and their ability to produce cytokines (Riedemann, N.C., et al., Immunity 19:193-202, 2003). In addition, C5a production in sepsis appears to have procoagulant effects (Laudes, I.J., et al., Am. J. Pathol. 160:1867-75, 2002). The complement modulating protein CI INH has also been effective in animal models of sepsis and ARDS (Dickneite, G., Behring Ins. Mitt. 93:299-305, 1993).

The lectin pathway may also play a role in the pathogenesis of sepsis. MBL has been shown to bind 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 recognized as an analogue of LPS in Gram-positive bacteria. It is a potent immunostimulant, inducing the release of cytokines 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, L-ficolin was shown to specifically bind LTA isolated from various species of gram-positive bacteria, including Staphylococcus aureus, and activate the lectin pathway (Lynch, N.J., et al., J. Immunol. 172:1198-02, 2004). MBL has also been shown to bind LTA from Enterococcus spp., in which the polyglycerophosphate chain is replaced by glycosyl groups, but not LTA from nine other species, including S. aureus (Polotsky, V.Y., et al., Infect. Immun. 64:380, 1996 ).

Thus, one aspect of the invention relates to a method of treating sepsis, or a condition related to sepsis, by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutical carrier to a subject suffering from sepsis or a condition related to sepsis, including , without limitation, severe sepsis, septic shock, acute respiratory distress syndrome due to sepsis, and systemic inflammatory response syndrome. Related methods are provided for the treatment of other blood disorders, including hemorrhagic shock, hemolytic anemia, autoimmune thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), or other destructive bone marrow/blood conditions, by administering the composition containing a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutical carrier to a subject suffering from such a condition. The MASP-2 inhibitory agent is administered to the subject systemically, for example, by intra-arterial, intravenous, intramuscular, inhalation (especially in the case of ARDS), subcutaneous or other parenteral route, or potentially by oral route in the case of non-peptidergic agents. The MASP-2 inhibitory agent composition can be combined with one or more additional therapeutic agents to combat the effects of sepsis and/or shock. For progressive sepsis or shock or distress associated therewith, the MASP-2 inhibitory composition may preferably be administered in a rapid-acting dosage form, for example, by bolus intravenous or intra-arterial delivery of a solution containing the MASP-2 inhibitory agent composition. Repeated administration can be carried out at the discretion of the doctor until this condition is eliminated.

Coagulopathies.

Evidence has been obtained for the role of the complement system in the development of disseminated intravascular coagulation (DIC), such as DIC due to major body trauma.

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 complement could still be activated through the classical or lectin pathway in C4 −/− mice, C3 turnover in the plasma of C4 −/− animals was determined in assays specific for activation of either the classical or lectin pathway. Although C3 cleavage was absent when activation was initiated through the classical pathway, highly efficient lectin pathway-dependent activation of C3 was observed in C4-deficient serum (Fig. 30). It can be seen that C3 deposition on mannan and zymosan is severely impaired in MASP-2-/- mice, even under experimental conditions, which, consistent with much previously published work on alternative pathway activation, should be permissive for all three pathways. When using the same serum 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-27- serum, but not in C1q-depleted serum. This indicates that activation of the alternative pathway is facilitated in MASP-27- serum when the initial C3b is provided by the activity of the classical pathway. In fig. 30C presents the surprising result that C3 can be effectively activated depending on

- 42 046178 directly from the lectin pathway in C4-deficient plasma.

This C4 bypass is eliminated by inhibiting lectin pathway activation by preincubating plasma with soluble mannan or mannose.

Aberrant, non-immune, activation of the complement system is potentially harmful to humans and may also play an important role in the activation of the hematological pathway, particularly in cases of severe trauma when both the inflammatory and hematological pathways are activated. Normally, C3 conversion results in <5% of total plasma C3 protein. During severe infections, including septicemia and immune complex disease, C3 conversion increases to levels of approximately 30%, often with complement levels below normal levels, due to increased utilization and changes in pool distribution. Direct activation of the C3 pathway to levels greater than 30% typically results in overt clinical manifestations of vasodilation and tissue leakage. When C3 conversion is above 30%, the initiating mechanisms are predominantly non-immune and the resulting clinical manifestations are harmful to the patient. Complement C5 levels in health and in controlled disease appear to be much more stable than C3 levels. Significant decreases and/or changes in C5 levels are associated with the patient's response to abnormal polytrauma (eg, motor vehicle accidents), and shock lung syndromes may develop. Thus, any evidence of complement C3 activation above 30% of the vascular pool, or any C5 involvement, or both, can be considered a likely harbinger of harmful pathological changes in the patient.

Both C3 and C5 release anaphylatoxins (C3a and C5a), which act on mast cells and basophils, leading to the release of vasodilatory chemicals. They create gradients for chemotaxis that direct polymorphonuclear cells (PMN) to the site of immunological disturbances (beneficial response), but here they are different because C5a has a specific agglutinating (aggregating) effect on these phagocytic cells, preventing them from randomly moving away from the site of reaction. During normal infection control, C3 activates C5. However, in polytrauma, C5 appears to be extensively activated, systemically generating C5a anaphylatoxins. This uncontrolled activity causes agglutination of polymorphic forms in the vascular system, and these clumps are then washed into the capillaries of the lungs, where they cause occlusion and cause local damage by releasing superoxide. Without being bound by specific theory, this mechanism is possibly important in the pathogenesis of acute respiratory distress syndrome (ARDS), although this view has recently been questioned. C3a anaphylatoxins may exhibit potent platelet aggregating properties in vitro, but their involvement in vivo is less clear, and the release of platelet substances and plasmin during wound healing may only secondaryly involve complement C3. It is possible that a prolonged increase in the level of C3 activation is necessary for the occurrence of DIC.

In addition to the cellular and vascular effects of activated complement components described above, which could explain the association between injury and DIC, emerging scientific findings have identified direct molecular connections and functional relationships between the complement and coagulation systems. Supporting evidence comes from studies in C3-deficient mice. Because C3 is a common component of all complement pathways, mice deficient in C3 are expected to lack all complement functions. Surprisingly, terminal complement components can be fully activated in C3-deficient mice. (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 shown that C3-independent activation of terminal complement components is mediated by thrombin, the rate-limiting enzyme of the coagulation cascade. (Huber et al., 2006). The molecular components mediating thrombin activation after initial complement activation remain unclear.

The present inventors have elucidated what is believed to be the molecular basis of the relationship between complement and the coagulation cascade and have identified MASP-2 as a central control point linking the two systems. Biochemical studies of the substrate specificity of MASP-2 have identified prothrombin as a possible substrate, in addition to the well-known complement proteins C2 and C4. MASP-2 specifically cleaves prothrombin at functionally important sites to produce thrombin, the rate-limiting enzyme of the coagulation cascade. (Krarup, A., et al., Simultaneous Activation of Complement and Coagulation by MBL-Associated Serine Protease 2, PLoS. ONE. 2:e623 (2007)). MASP-2-generated thrombin is able to stimulate fibrin deposition in certain in vitro reconstituted systems, demonstrating the functional importance of MASP-2 cleavage. (Krarup et al., 2007). As described in the examples below herein, we further confirmed the physiological significance of this finding by documenting the activation of thrombin in normal rodent serum following activation of the lectin pathway and demonstrated that this process was blocked by MASP-2 neutralizing monoclonal antibodies.

MASP-2 may represent a central branch point in the lectin pathway, capable of

- 43 046178 stimulate the activation of both the complement system and the coagulation system. Since activation of the lectin pathway is a physiological response to many types of traumatic injury, we believe that systemic inflammation (complement component-mediated) and disseminated coagulation (coagulation pathway-mediated) occurring simultaneously may be explained by the ability of MASP-2 to activate both pathways. These results clearly demonstrate the role of MASP-2 in DIC and the therapeutic benefit of MASP2 inhibition in treating or preventing DIC. MASP-2 may provide a molecular link between the complement and coagulation systems, and lectin pathway activation occurring in the setting of injury may directly initiate activation of the coagulation system through the MASP-2-thrombin axis, providing a mechanistic link between injury and DIC. In accordance with one aspect of the present invention, inhibition of MASP-2 would result in inhibition of lectin pathway activation and reduction in the formation of both anaphylatoxins, C3a and C5a. A sustained increase in C3 activation levels is thought to be necessary for DIC to occur.

Microvascular coagulation (blood clots in capillaries and small blood vessels) occurs in situations such as septic shock. The role of the lectin pathway in septic shock is established, as evidenced by the protective MASP-2 (-/-) phenotype in murine models of sepsis, as described in Example 17 and FIG. 18 and 19. Additionally, as demonstrated in Example 15 and FIG. 16A and 16B, MASP-2 (-/-) mice are protected in the localized Schwarzman reaction, the disseminated intravascular coagulation (DIC) model, and the localized microvascular coagulation model.

V. MASP-2 inhibitory agents.

In one aspect, the present invention provides methods for inhibiting MASP-2-dependent complement activation in a subject suffering from, or at risk of developing, thrombotic microangiopathy. MASP-2 inhibitory agents are administered in an amount effective to inhibit MASP-2-dependent complement activation in a subject. When practicing the invention, representative MASP-2 inhibitory agents include: molecules that inhibit the biological activity of MASP-2 (such as small molecule inhibitors, anti-MASP-2 antibodies, or blocking peptides that interact with MASP-2 or interfere with protein-protein interactions ), and molecules that reduce MASP-2 expression (such as MASP-2 antisense nucleic acid molecules, MASP-2-specific RNAi molecules, and MASP-2 ribozymes), these molecules prevent activation of the lectin complement pathway by MASP-2. MASP-2 inhibitory agents can be used alone as primary therapy or in combination with other therapeutic agents as adjuvant therapy to enhance the therapeutic benefit of treatment with other drugs.

Inhibition of MASP-2-dependent complement activation is characterized by at least one of the following changes in components of the complement system that occur as a result of administration of a MASP-2 inhibitory agent in accordance with the methods of the invention: inhibition of the formation or production of MASP-2-dependent complement activation products , C4b, C3a, C5a and/or C5b-9 (MAC) (measured, for example, as described in Example 2), a decrease in complement activation when assessed in a hemolysis assay using non-sensitized rabbit or guinea pig red blood cells (measured, for example, as described in Example 33), a decrease in C4 degradation and C4b deposition (measured, for example, as described in Example 2), or a decrease in C3 degradation and C3b deposition (measured, for example, as described in Example 2).

In accordance with the present invention, MASP-2 inhibitory agents are used that are effective in inhibiting the MASP-2-dependent complement activation system. MASP-2 inhibitory agents useful in the practice of this aspect of the invention include, for example, anti-MASP-2 antibodies and fragments thereof, MASP-2 inhibitory peptides, small molecules, soluble MASP-2 receptors, and inhibitors of MASP-2 expression. MASP-2 inhibitory agents can inhibit the MASP-2-dependent complement activation system by blocking the biological function of MASP-2. For example, the inhibitory agent may effectively block MASP-2 protein-protein interactions, interfere with MASP-2 dimerization or assembly, block Ca ²⁺ binding, block the active site of MASP-2 serine protease, or may cause a decrease in MASP-2 protein expression.

In some embodiments, MASP-2 inhibitory agents selectively inhibit MASP-2-dependent complement activation without functionally affecting the C1q-dependent complement activation system.

In one embodiment, a MASP-2 inhibitory agent useful in the methods of the invention is a specific MASP-2 inhibitory agent that specifically binds a polypeptide comprising SEQ ID NO: 6 with an affinity at least ten times greater than for other antigens in the complement system. In another embodiment, the MASP-2 inhibitory agent specifically binds the polypeptide comprising SEQ ID NO: 6 with a binding affinity at least 100 times greater than that for other antigens in the complement system. The binding affinity for a MASP-2 inhibitory agent can be determined with

- 44 046178 using an appropriate binding assay.

The MASP-2 polypeptide has a molecular structure similar to that of MASP-1, MASP-3, C1r and C1s, the C1 proteases of the complement system. The cDNA molecule of SEQ ID NO: 4 encodes a representative example of MASP-2 (consisting of the amino acid sequence set forth in SEQ ID NO: 5) and produces a human MASP-2 polypeptide with a leader sequence (aa 1-15) that is cleaved after secretion to form the mature form of human MASP-2 (SEQ ID NO: 6). As shown in FIG. 2, the human MASP-2 gene contains twelve exons. The human MASP-2 cDNA is encoded by exons B, C, D, F, G, H, I, J, K and L. Alternative splicing results in the formation of a 20 kDa protein called MBL-associated protein 19 (MAp19, also known as sMAP) ( SEQ ID NO: 2) encoded (SEQ ID NO: 1) derived from exons B, C, D and E, as shown in FIG. 2. The cDNA molecule of SEQ ID NO: 50 encodes murine MASP-2 (consisting of the amino acid sequence set forth in SEQ ID NO: 51) and produces a murine MASP-2 polypeptide with a leader sequence that is cleaved off after secretion to form the mature forms of mouse MASP-2 (SEQ ID NO: 52). The cDNA molecule of SEQ ID NO: 53 encodes rat MASP-2 (consisting of the amino acid sequence set forth in SEQ ID NO: 54) and produces a rat MASP-2 polypeptide with a leader sequence that is cleaved off after secretion to produce the mature form of rat MASP-2 (SEQ ID NO: 55).

Those skilled in the art will understand that the sequences disclosed in SEQ ID NO: 4, SEQ ID NO: 50 and SEQ ID NO: 53 represent distinct alleles of human, murine and rat MASP-2, respectively, and that the possibility of allelic variations is contemplated and alternative splicing. Allelic variants of the nucleotide sequences set forth in SEQ ID NO: 4, SEQ ID NO: 50 and SEQ ID NO: 53, including variants containing silent mutations and variants in which mutations lead to changes in the amino acid sequence, are within the scope of the present invention. Allelic variants of the MASP-2 sequence can be cloned by examining cDNA or genomic libraries from different individuals using standard methods.

The human MASP-2 protein domains (SEQ ID NO: 6) are shown in FIG. 1 and 2A, and include the N-terminal C1r/C1s/VEGF sea urchin/bone morphogenetic protein (CUBI) domain (aa 1-121 in SEQ ID NO: 6), an epidermal growth factor-like domain (aa 122-166), a second CUBI domain (aa 167-293), as well as a tandem of complement control protein domains and a serine protease domain. Alternative splicing of the MASP-2 gene results in the formation of MAp19 shown in FIG. 1. MAp19 is a non-enzymatic protein containing the N-terminal CUB1-EGF region of MASP-2 with four additional residues (EQSL) derived from exon E, as shown in FIG. 1.

Several proteins have been shown to bind, or interact, with MASP-2 through protein-protein interactions. For example, MASP-2 is known to bind and form Ca ²⁺ -dependent complexes with the lectin proteins MBL, H-ficolin and L-ficolin. Each MASP2/lectin complex has been shown to activate complement through MASP-2-dependent cleavage of the C4 and C2 proteins (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). Research has shown that the CUB1-EGF domains of MASP-2 are required for the association of MASP-2 with MBL (Thielens, NM, et al., J. Immunol. 166:5068, 2001). The CUB1EGFCUBII domains have also been shown to mediate the dimerization of MASP-2, which is required for the formation of the active MBL complex (Wallis, R., et al., J. Biol. Chem. 275:30962-30969, 2000). Thus, MASP-2 inhibitory agents can be identified that bind or block MASP-2 target regions known to be important for MASP-2-dependent complement activation.

Ahtu-MASP-2 antibodies.

In some embodiments of this aspect of the invention, the MASP-2 inhibitory agent is an anti-MASP-2 antibody that inhibits the MASP-2-dependent complement activation system. Ahtu-MASP-2 antibodies useful for this aspect of the invention include polyclonal, monoclonal and recombinant antibodies obtained from any antibody-producing mammal, and can be multispecific, chimeric, humanized, anti-idiotypic antibodies, as well as antibody fragments. Antibody fragments include Fab fragments, Fab', F(ab) ₂ , F(ab') ₂ , Fv fragments, scFv fragments and single chain antibodies described later herein.

Several anti-MASP-2 antibodies have been described in the literature, some of which are listed below in Table. 1. These previously described anti-MASP-2 antibodies can be screened for the ability to inhibit the MASP-2-dependent complement activation system using the assays described herein. For example, anti-rat MASP-2 Fab2 antibodies have been identified that block MASP-2-dependent complement activation, as described in more detail herein in Examples 10 and 11. Following the identification of an anti-MASP-2 antibody that acts as a MASP inhibitor -2 means, it can be used to obtain anti-idiotypic antibodies, and can also be used to identify other MASP-2 binding molecules,

- 45 046178 as further described below.

Table 1

MASP-2-specific antibodies described in the literature

ANTIGEN ANTIBODY TYPE LINK Recombinant MASP-2 Polyclonal rat Peterson, S.V., et al., Mol. Immunol. 37:803811.2000 Recombinant human fragment CCP1/2-SP (moAb 8B5) Rat moAb (IgGl subclass) Moller-Kristensen, M., et al., J. of Immunol. Methods 282:159-167, 2003 Recombinant human MAp19 (moAt6G12) (cross-reacts with MASP-2) Rat moAb (IgGl subclass) Moller-Kristensen, M., et al., J. of Immunol. Methods 282:159-167, 2003 hMASP-2 Mouse moAb (S/P) Mouse moAb (N-terminus) Peterson, S.V., et al., Mol. Immunol. 35:409, April 1998 hMASP-2 (CCP1-CCP2-SP domain) Rat moAb: NimoablOl, produced by hybridoma cell line 03050904 (ECASS) WO 2004/106384

- 46 046178

hMASP-2 (full-length with his tag) Mouse moAbs: NimoAbl04, produced by the hybridoma cell line M0545YM035 (DSMZ) NimoAbl08, produced by the hybridoma cell line M0545YM029 (DSMZ) NimoAbl09, produced by the hybridoma cell line M0545YM046 (DSMZ) NimoAbllO, produced by the hybridoma cell line M054 5YM048 (DSMZ) WO 2004/106384

Ahtu-MASP-2 antibodies with reduced effector function In some embodiments of this aspect of the invention, anti-MASP-2 antibodies have reduced effector function to reduce inflammation that may occur due to activation of the classical complement pathway. The ability of IgG molecules to trigger the classical complement pathway has been demonstrated to be associated with the Fc portion of the molecule (Duncan, A.R., et al., Nature 332:738-740 1988). IgG molecules in 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). Accordingly, antibodies with reduced effector function due to the absence of the Fc portion of the molecule can be obtained by creating a genetically modified Fc sequence that has minimal effector function, or is of the human IgG2 or IgG4 isotype of antibodies.

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

Preparation of anti-MASP-2 antibodies.

Ahtu-MASP-2 antibodies can be produced using MASP-2 polypeptides (e.g., full-length MASP-2) or using antigenic peptides bearing the MASP-2 epitope (on

- 47 046178 example, parts of the MASP-2 polypeptide). Immunogenic peptides can be no more than five amino acid residues in length. For example, a MASP-2 polypeptide containing the complete amino acid sequence of SEQ ID NO: 6 can be used to induce anti-MASP-2 antibodies useful in the method of the invention. Specific domains of MASP-2 that are known to be involved in protein-protein interactions, such as the CUBI and CUBIEGF domains, as well as the region including the serine protease active site, can be expressed as recombinant polypeptides as described in Example 3 and used as antigens. In addition, peptides containing a portion of at least 6 amino acids of the MASP-2 polypeptide (SEQ ID NO: 6) can also be used to induce anti-MASP-2 antibodies. Additional examples of MASP-2-derived antigens useful for inducing anti-MASP-2 antibodies are listed below in Table. 2. MASP-2 peptides and polypeptides used to produce antibodies can be obtained as natural polypeptides, or recombinant or synthetic peptides, as well as catalytically inactive recombinant polypeptides such as MASP2A, as further described in Examples 5-7. In some embodiments of this aspect of the invention, anti-MASP-2 antibodies are generated using a transgenic mouse strain as described in Examples 8 and 9 and further described below.

Antigens that can be used to generate anti-MASP-2 antibodies also include fusion polypeptides, such as fusions of MASP-2, or portions thereof, with an immunoglobulin polypeptide or with maltose binding protein. The polypeptide immunogen may be a full-length molecule or a portion thereof. If a portion of the polypeptide is hapten-like, such portion can be advantageously combined or linked to a macromolecular carrier (such as limpet hemocyanin (KLH), bovine serum albumin (BSA), or tetanus toxoid) for immunization.

- 48 046178

table 2

MASP-2-derived antigens

SEQ GO NO: Amino acid sequence SEQ GO NO: 6 Human MASP-2 protein SEQIDNO: 51 Mouse MASP-2 protein SEQ GO NO: 8 Human MASP-2 CUBI domain (aa 1-121 in SEQ ID NO: 6) SEQ GO NO: 9 CUBIEGF domains of human MASP-2 (aa 1-166 in SEQ GO NO: 6) SEQ ID NO: 10 Human MASP-2 CUBIEGFCUBII domains (aa 1-293 in SEQ ID NO: 6) SEQ ID NO: 11 EGF domain of human MASP-2 (aa 122-166 in SEQ ID NO: 6) SEQ ID NO: 12 Human MASP-2 serine protease domain (aa 429-671 in SEQ ID NO: 6) SEQ ID NO: 13 GKDSCRGDAGGALLVFL Mutant form of inactivated serine protease (aa 610-625 in SEQ GO NO: 6 with mutation Ser 618) SEQ ID NO: 14 TPLGPKWPEPVFGRL Human peptide CUBI SEQ ID NO: 15: TAPPGYRLRLYFTHFDLELSH LCEYDFVKLSSGAKVLATLCG Q Human peptide CUBI SEQ ID NO: 16: TFRSDYSN MBL-binding region in the human domain CUBI SEQ ID NO: 17: FYSLGSSLDrrFRSDYSNEKPFT GF MBL-binding region in the human domain CUBI SEQ ID NO: 18 IDECQVAPG EGF peptide SEQ ID NO: 19 ANMLCAGLESGGKDSCRGDS GGALV Serine protease active site peptide

Polyclonal antibodies.

Polyclonal antibodies against MASP-2 can be obtained by immunizing an animal with a MASP-2 polypeptide, or an immunogenic fragment thereof, using methods well known to those skilled in the art. See, for example, Green et al., Production of Polyclonal Antisera, in: Immunochemical Protocols (Manson, ed.), page 105, as well as the description in Example 6. The immunogenicity of the MASP-2 polypeptide can be increased through the use of an adjuvant, including minerals. gels such as aluminum hydroxide or Freund's adjuvant (complete or incomplete), surfactants such as lysolecithin, Pluronic-type polyols, polyanions, oil emulsions, limpet hemocyanin and dinitrophenol. Polyclonal antibodies are typically obtained from animals such as horses, cows, dogs, chickens, rats, mice, rabbits, guinea pigs, goats or sheep. Alternatively, an anti-MASP-2 antibody useful in the present invention can also be produced in primates. General methods for obtaining antibodies useful for diagnosis and therapy in baboons are described, for example, in Goldenberg et al., international patent publication No. WO 91/11465, and in Losman, M. J., et al., Int. J. Cancer 46:310, 1990. From the blood of such immunized animals, serum containing immunologically active antibodies is then obtained by standard methods well known in the art.

- 49 046178

Monoclonal antibodies.

In some embodiments, the MASP-2 inhibitory agent is an anti-MASP-2 monoclonal antibody. Ahtu-MASP-2 monoclonal antibodies are highly specific, directed against a single epitope of MASP-2. As used herein, the designation monoclonal refers to the nature of the antibody as being derived from a substantially homogeneous population of antibodies and should not be construed as indicating that the antibody must be produced by any particular method. Monoclonal antibodies can be produced using any method that results in the production of antibody molecules by stable cell lines in culture, such as the hybridoma method described in Kohler, G., et al., Nature 256:495, 1975, or they can be produced by recombinant methods. DNA (see, for example, US Pat. No. 4,816,567 to Cabilly). Monoclonal antibodies can also be obtained from phage antibody libraries using methods described in Clackson, T., et al., Nature 352:624-628, 1991, and Marks, J.D., et al., J. Mol. Biol. 222:581597, 1991. Such antibodies can belong to any class of immunoglobulins, including IgG, IgM, IgE, IgA, IgD, as well as any subclass thereof.

For example, monoclonal antibodies can be produced by injecting a suitable mammal (eg, BALB/c mouse) with a composition containing a MASP-2 polypeptide or a portion thereof. After a certain period of time, splenocytes are removed from the mouse and suspended in cell culture medium. Then splenocytes are fused with immortalized linear cells, obtaining a hybridoma. The resulting hybridomas are grown in cell culture medium and screened for their ability to produce anti-MASP-2 monoclonal antibody. The production of anti-MASP-2 monoclonal antibodies is described in more detail in Example 7. (See also Current Protocols in Immunology, Vol. 1., John Wiley & Sons, pages 2.5.1-2.6.7, 1991.)

Human monoclonal antibodies can be produced by using transgenic mice that have been genetically modified to produce specific human antibodies in response to an antigen. In this method, elements of the human immunoglobulin heavy and light chain locus are introduced into strained mice generated using embryonic stem cell lines that have targeted disruptions in the heavy and light chain loci of endogenous immunoglobulins. Transgenic mice can synthesize human antibodies specific for human antigens, such as the MASP-2 antigens described herein, and mice can be used to produce anti-MASP-2 antibody-secreting hybridomas by fusing B cells from such animals with corresponding lineage cells myelomas using conventional Keller-Milyptain technology, as further described in Example 7. Transgenic mice with human immunoglobulin genes are commercially available (eg, from Abgenix, Inc., Fremont, CA, and Medarex, Inc., Annandale, N.J.). Methods for obtaining human antibodies from transgenic mice are described, for example, in Green, L.L., et al., Nature Genet. 7:13, 1994; Lonberg, N., et al., Nature 368:856, 1994; and Taylor, L.D., et al., Int. Immun. 6:579, 1994.

Monoclonal antibodies can be isolated and purified from hybridoma cultures by various well-known methods. Such isolation methods include protein Asepharose affinity chromatography, size exclusion chromatography, and ion exchange chromatography (see, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al., Purification of Immunoglobulin G ( IgG), in Methods in Molecular Biology, The Humana Press, Inc., Vol. 10, pages 79-104, 1992).

Once produced, polyclonal, monoclonal, or phage-derived antibodies 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 MASP-2. Exemplary assays include Western blot or immunoprecipitation assays using standard methods (eg, as described in Ausubel et al.), immunoelectrophoresis, enzyme-linked immunosorbent assays, dot blot assays, inhibition or competition assays, and sandwich format assays (described in Harlow and Land, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988). After identifying antibodies that specifically bind MASP-2, anti-MASP-2 antibodies are tested for their ability to act as a MASP-2 inhibitory agent in one of a number of assays, for example, the C4 lectin-specific cleavage assay (described in Example 2), the C3b sediment analysis (described in example 2) or C4b sediment analysis (described in example 2).

The affinity of anti-MASP-2 monoclonal antibodies 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, anti-MASP-2 monoclonal antibodies useful in the methods of the invention bind MASP-2 with a binding affinity of <100 nM, preferably <10 nM, and most preferably <2 nM. In some embodiments, a MASP-2 inhibitory monoclonal antibody useful in the methods of the invention is a MASP-2 inhibitory monoclonal antibody, or an antigen binding fragment thereof, comprising (i) (a) a heavy chain variable region comprising: i) CDR-H1 heavy chain containing the amino acid sequence of residues 31-35 in SEQ ID NO: 67; and ii) a heavy chain CDR-H2 containing the amino acid sequence of residues 50-65 of SEQ ID NO: 67; and iii) heavy chain CDR-H3 containing the amino acid sequence

- 50 046178 the amino acid sequence of residues 95-102 in SEQ ID NO: 67, and b) a light chain variable region comprising: i) a light chain CDR-L1 containing the amino acid sequence of residues 24-34 in SEQ ID NO: 70; and ii) light chain CDR-L2 containing the amino acid sequence of residues 50-56 in SEQ ID NO: 70; and iii) a light chain CDR-L3 comprising the amino acid sequence of residues 89-97 of SEQ ID NO: 70, or (II) a variant thereof comprising a heavy chain variable region having at least 90% sequence identity with SEQ ID NO: 67 (e.g. at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , at least 99% sequence identity to SEQ ID NO: 67), and a light chain variable region having at least 90% sequence 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% sequence identity) with SEQ ID NO: 70.

Chimeric/humanized antibodies.

Monoclonal antibodies useful in the method of the invention include chimeric antibodies in which part of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular biological species or belonging to a particular class or subclass of antibodies, with the remainder of the chain ( it) is identical or homologous to the corresponding sequences in antibodies obtained from another biological species or belonging to another class or subclass of antibodies; as well as fragments of such antibodies (US Pat. No. 4,816,567 to Cabilly; and Morrison, S. L., et al., Proc. Nat'l Acad. Sci. USA 81:6851-6855, 1984).

One form of chimeric antibody useful in the invention is a humanized monoclonal anti-MASP-2 antibody. Humanized forms of non-human (eg, murine) antibodies are chimeric antibodies containing a minimal sequence of non-human immunoglobulin. Humanized monoclonal antibodies are produced by transferring the complementarity determining regions (CDRs) of a non-human (eg, murine) antibody from the variable regions of the murine immunoglobulin heavy and light chains into the variable domain of the human antibody. Typically, human antibody residues are then introduced into the framework regions to replace the corresponding non-human residues. In addition, humanized antibodies may contain residues not present in the recipient antibody or the donor antibody. These modifications are made to further improve the functioning of the antibody. Typically, a humanized antibody will contain substantially all of at least one and typically two variable domains, in which all, or substantially all, of the hypervariable loops correspond to those of a non-human immunoglobulin, and all, or substantially all, of Fv framework regions have the sequence of human immunoglobulin. The humanized antibody will optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region. For 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.

Humanized antibodies useful in the invention include human monoclonal antibodies containing at least a MASP-2 CDR3 binding region. In addition, Fc regions can be replaced to produce IgA or IgM as well as human IgG antibodies. Such humanized antibodies will have specific clinical utility because they will specifically recognize human MASP-2 but will not induce an immune response in humans against the antibody itself. Therefore, they are more suitable for in vivo administration to humans, especially if repeated or long-term administration is required.

The preparation of a humanized anti-MASP-2 antibody from a mouse anti-MASP-2 monoclonal antibody is described herein in Example 6. Methods for the preparation of humanized monoclonal antibodies are also described, for example, in Jones, P.T., et al., Nature 321:522, 1986 ; Carter, P., et al., Proc. Nat'l. Acad. Sci. USA 89:4285, 1992; Sandhu, J.S., Crit Rev. Biotech. 12:437, 1992; Singer, I.I., et al., J. Immun. 150:2844, 1993; Sudhir (ed.), Antibody Engineering Protocols, Humana Press, Inc., 1995; Kelley, Engineering Therapeutic Antibodies, in: Protein Engineering: Principles and Practice, Cleland et al. (eds.), John Wiley & Sons, Inc., pages 399-434, 1996; and in US Pat. No. 5,693,762 issued to Queen, 1997. In addition, there are commercial organizations that synthesize humanized antibodies from specific regions of the murine antibody, for example, Protein Design Labs (Mountain View, CA).

Recombinant antibodies.

Ahtu-MASP-2 antibodies can also be produced using recombinant methods. For example, human antibodies can be produced using human immunoglobulin expression libraries (available, for example, from Stratagene, Corp., La Jolla, Calif.) to produce human antibody fragments (VH, VL, Fv, Fd, Fab, or F(ab') ₂ ). These fragments are then used to construct whole human antibodies using methods similar to those used to generate chimeric antibodies.

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Anti-idiotypic antibodies.

Once anti-MASP-2 antibodies with the desired inhibitory activity have been identified, these antibodies can be used to generate anti-idiotypic antibodies having similarities to a portion of MASP-2 using methods well known in the art. See, for example, Greenspan, N.S., et al., FASEB J. 7:437, 1993. For example, antibodies that bind MASP-2 and completely inhibit MASP-2 protein interactions required for complement activation can be used to generate anti-idiotypic antibodies , having similarity to the MBL binding site on the MASP-2 protein, and therefore bind and neutralize the binding of a MASP-2 ligand, such as, for example, MBL.

Fragments of immunoglobulins.

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

It is well known in the art that only a small portion of the antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, for example, Clark, W.R., The Experimental Foundations of Modern Immunology, Wiley & Sons, Inc., NY, 1986). The pFc' and Fc regions of the 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 antigen binding sites of the intact antibody. The isolated F(ab') ₂ fragment is called a bivalent monoclonal fragment because of its two antigen binding sites. Likewise, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, is called a Fab fragment and retains one of the antigen binding sites of the intact antibody molecule.

Antibody fragments can be obtained by proteolytic hydrolysis, for example, digestion with pepsin or papain, of the whole antibody by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin, yielding a 5S fragment called F(ab') ₂ . This fragment can be further cleaved using a thiol reducing reagent to produce 3.5S monovalent Fab' fragments. Optionally, the cleavage reaction can be carried out using a blocking group for sulfhydryl groups that arise from the cleavage of disulfide bonds. Alternatively, enzymatic digestion using pepsin directly produces two monovalent Fab fragments and an Fc fragment. These methods are described, for example, in US patent No. 4331647 issued 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 in the Coligan publication on pages 2.8.1-2.8.10 and 2.10.-2.10.4.

In some embodiments, the use of antibody fragments lacking the Fc region is preferred to avoid activation of the classical complement pathway, which is triggered by Fc binding to the Fcy receptor. There are several methods by which it is possible to obtain a moAb that does not participate in the interaction with the Fcy receptor. For example, the Fc region of a monoclonal antibody can be chemically removed by partial digestion with proteolytic enzymes (eg, ficin digestion), thereby yielding, for example, antigen-binding antibody fragments, such as Fab or F(ab) ₂ fragments (Mariani, M., et al. , Mol. Immunol. 28:69-71, 1991). Alternatively, a human IgG antibody of the y4 isotype that does not bind Fcy receptors can be used in the construction of a humanized antibody as described herein. Antibodies, single chain antibodies and antigen binding domains lacking the Fc domain can also be constructed using the recombinant methods described herein.

Single chain antibody fragments.

Alternatively, MASP-2-specific single peptide chain binding molecules can be generated in which the heavy and light chain Fv regions are connected. Fv fragments can be linked with a peptide linker to produce a single chain antigen binding protein (scFv). These single-stranded antigen-binding proteins are produced by constructing a structural gene containing DNA sequences encoding the VH and VL domains linked by an oligonucleotide. The structural gene is inserted into an expression vector, which is then introduced into a host cell such as E. coli. Recombinant host cells synthesize a single polypeptide chain with a peptide linker connecting the V domains. Methods for preparing scFv are described, for example, in: Whitlow, et al., Methods: A Companion to Methods in Enzymology 2:97, 1991; Bird, et al., Science 242:423, 1988; US Patent No. 4,946,778 to Ladner; Pack, P., et al., Bio/Technology 11:1271, 1993.

As an illustrative example, MASP-2-specific scFvs can be generated by exposing lymphocytes to MASP-2 polypeptide in vitro and selecting antibody phage display libraries or similar vectors (eg, by using immobilized or tagged MASP-2 protein or peptide). Genes encoding polypeptides with potential binding

- 52 046178 polypeptide MASP-2 domains can be obtained by screening random peptide display libraries on a phage or bacterium such as E. coli. These random peptide display libraries can be used to screen for peptides that interact with MASP-2. Methods for generating and screening such random peptide display libraries are well known in the art (US Pat. No. 5,223,409 to Lardner; US Pat. No. 4,946,778 to Ladner; US Pat. No. 5,403,484 to Lardner; US Pat. No. 5,571,698 to Lardner; and Kay et al., Phage Display of Peptides and Proteins Academic Press, Inc., 1996), and random peptide display libraries, as well as screening kits for such libraries, are commercially available, for example, from CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly, Mass.) and Pharmacia LKB Biotechnology Inc. (Piscataway, N.J.).

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

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

Peptide inhibitors.

In some embodiments of this aspect of the invention, the MASP-2 inhibitory agent includes isolated peptide MASP-2 inhibitors, including isolated natural peptide inhibitors and synthetic peptide inhibitors, that inhibit the MASP-2-dependent complement activation system. As used herein, the term isolated peptide MASP-2 inhibitors means peptides that inhibit MASP-2-dependent complement activation by binding, competing with MASP-2 for binding, another recognition molecule (e.g., MBL, Nficolin, M-ficolin, or L-ficolin). ficolin) in the lectin pathway, and/or by direct interaction with MASP-2, inhibiting MASP-2-dependent complement activation, and which are substantially pure and substantially free from other substances with which they may be found in nature, to such an extent degree that is practical and suitable for their intended application.

Peptide inhibitors have been successfully used in vivo to block protein-protein interactions and catalytic sites. For example, peptide adhesion molecule inhibitors structurally related to LFA-1 have recently been approved for clinical use in coagulopathies (Ohman, E.M., et al., European Heart J. 16:50-55, 1995). Short linear peptides (<30 amino acids) have been described that prevent or interfere with integrin-dependent adhesion (Murayama, O., et al., J. Biochem. 120:445-51, 1996). Longer peptides ranging in length from 25 to 200 amino acid residues have also been successfully used to block integrin-dependent adhesion (Zhang, L., et al., J. Biol. Chem. 271(47):29953-57, 1996). In general, longer peptide inhibitors have higher affinity and/or lower dissociation rates than shorter peptides and thus 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, D.Y., et al., J. Med. Chem. 40:3359-68, 1997). One method for producing cyclic peptides involves the synthesis of peptides in which the terminal amino acids of the peptide are cysteine residues, which allows the peptide to exist in a cyclic form due to the formation of disulfide bonds between the terminal amino acids, which have been shown to increase affinity and half-life in vivo, they are used for the treatment of hematopoietic neoplasms (eg, US patent No. 6649592 issued to Larson).

Synthetic peptide inhibitors of MASP-2.

Examples of MASP-2 inhibitory peptides useful in the methods of this aspect of the invention are 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. In table 3 provides a list of illustrative inhibitory peptides that may be used in the practice of this aspect of the present invention. A potential MASP-2 inhibitory peptide can be tested for its ability to act as an inhibitory agent.

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MASP-2 agents in one of a number of assays, including, for example, the C4 lectin-specific cleavage assay (described in Example 2) and the C3b deposition assay (described in Example 2).

In some embodiments, the MASP-2 inhibitory peptides are derived from MASP-2 polypeptides and are selected from the full-length mature MASP-2 protein (SEQ ID NO: 6) or from a specific domain of the MASP-2 protein, such as, for example, the CUBI domain (SEQ ID NO: 8), CUBIEGF domain (SEQ ID NO: 9), EGF domain (SEQ ID NO: 11) and serine protease domain (SEQ ID NO: 12). As described previously, the CUBEGFCUBII regions have 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 was shown to be involved in binding to MBL in a study in which an individual was found to have a homozygous Asp105 to Gly105 mutation, resulting in loss of MASP-2 from a complex with MBL (Stengaard-Pedersen, K., et al., New England J. Med. 349:554-560, 2003).

In some embodiments, the MASP-2 inhibitory peptides are derived from lectin proteins that bind MASP-2 and participate 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 are present in serum as oligomers of homotrimeric subunits, each of which has N-terminal collagen-like fibers with carbohydrate recognition domains. These different lectins have been shown to bind MASP-2, and the lectin/MASP-2 complex activates complement by cleaving the C4 and C2 proteins. H-ficolin has an amino-terminal region of 24 amino acids, a collagen-like domain with 11 Gly-Xaa-Yaa repeats, a neck domain of 12 amino acids, and a fibrinogen-like domain of 207 amino acids (Matsushita, M., et al., J. Immunol. 168:35023506, 2002). H-ficolin binds GlcNAc and causes agglutination of human erythrocytes coated with LPS from S. typhimurium, S. minnesota and E. coli. H-ficolin has been shown to associate with MASP-2 and MAp19 and activate the lectin pathway (ibid.). L-ficolin/P35 also binds GlcNAc and has been shown to associate with MASP-2 and MAp19 in human serum, and this complex has been shown to activate the lectin pathway (Matsushita, M., et al., J. Immunol. 164:2281 , 2000). Accordingly, MASP-2 inhibitory peptides useful in the present invention may comprise a region of at least 5 amino acids selected from MBL protein (SEQ ID NO: 21), H-ficolin protein (Genbank accession number NM_173452), M-ficolin protein (Genbank accession number 000602) and L-ficolin protein (Genbank accession number NM_015838).

More specifically, the scientists discovered that the MASP-2-binding site on MBL lies within 12 triplets Gly-X-Y GKD GRD GTK GEK GEP GQG LRG LQG POG KLG POG NOG PSG SOG PKG QKG DOG KS (SEQ ID NO: 26), which located between the hinge and the neck part in the C-terminal part of the collagen-like domain of the MBR (Wallis, R., et al., J. Biol. Chem. 279:14065, 2004). This region of the MASP-2 binding site is also highly conserved in human Nficolin and human L-ficolin. A consensus binding site present in all three lectin proteins has been described that contains the amino acid sequence OGK-X-GP (SEQ ID NO: 22), where the letter O represents hydroxyproline and the letter X represents a hydrophobic residue (Wallis et al., 2004, supra ). Accordingly, in some embodiments, MASP-2 inhibitory peptides useful in this aspect of the invention are at least 6 amino acids in length and contain SEQ ID NO: 22. MBL-derived peptides that contain the amino acid sequence GLR GLQ GPO GKL GPO G ( SEQ ID NO: 24) has been shown to bind MASP-2 in vitro (Wallis, et al., 2004, supra). To enhance binding to MASP-2, peptides that are flanked by two GPO triplets at each end (GPO GPO GLR GLQ GPO GKL GPO GGP OGP O SEQ ID NO: 25) can be synthesized to stimulate the formation of triple helices, which are found in the natural MBL protein ( as further described in Wallis, R., et al., J. Biol. Chem. 279:14065, 2004).

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

MASP-2 inhibitory peptides can also be derived from a C4 cleavage site, such as LQRALEILPNRVTIKANRPFLVFI (SEQ ID NO: 29), which is a C4 cleavage site associated with the C-terminal portion of antithrombin III (Glover, G.I., et al., Mol Immunol 25:1261 (1988).

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Table 3

Exemplary MASP-2 inhibitory peptides

SEQ ID NO Source SEQ GO NO: 6 Human MASP-2 protein SEQ ID NO: 8 CUBI domain in MASP-2 (ac 1-121 in SEQ ID NO: 6) SEQ ID NO: 9 CUBIEGF domains in MASP-2 (aa 1-166 in SEQ ID NO: 6) SEQ ID NO: 10 Domains CUBIEGFCUBII in MASP-2 (ac 1-293 in SEQ GO NO: 6) SEQ ID NO: 11 EGF domain in MASP-2 (aa 122-166) SEQ ID NO: 12 Serine protease domain in MASP-2 (aa 429-671) SEQ ID NO: 16 MBL-binding region in MASP-2 SEQ ID NO: 3 Human MAr19 SEQ ID NO: 21 Human MBL protein SEQ ID NO: 22 OGK-X-GP, where "0" = hydroxyproline, and "X" represents hydrophobic amino acid residue Synthetic binding site consensus peptide from human MBL and human ficolins SEQ GO NO: 23 OGKLG Central binding site of human MBL SEQ GO NO: 24 GLR GLQ GPO GKL GPO G Triplets 6-10 of human MBR - demonstrate binding to MASP-2 SEQ GO NO: 25 GPOGPOGLRGLQGPOGKLGP OGGPOGPO Human MBR triplets with added GPOs to promote triple helix formation SEQ GO NO: 26 GKDGRDGTKGEKGEPGQGL RGLQGPOGKLGPOGNOGPS GSOGPKGQKGDOGKS Human MBP triplets 1-17 SEQ GO NO: 27 GAOGSOGEKGAOGPQGPOG POGKMGPKGEOGDO Human N-ficolin (Hataka) SEQ GO NO: 28 GCOGLOGAOGDKGEAGTNG KRGERGPOGPOGKAGPOGP NGAOGEO Human L-Ficolin P35 SEQ GO NO: 29 LQRALEILPNRVTIKANRPFL VFI Human C4 cleavage site

Note: The letter O represents hydroxyproline. The letter X represents a hydrophobic residue.

C4 cleavage site-derived peptides, 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, halomethyl ketones (Br, Cl, I, F) at the C-terminus, Asp or Glu, or attached to functional side chains; haloacetyl (or other α-haloacetyl) groups on amino groups or other functional side chains; epoxy or imine-containing groups on amino or car

- 55 046178 boxyl ends, or on functional side chains; or imidate esters on the amino or carboxyl termini, or on functional side chains. Such modifications would provide the benefit of permanent enzyme inhibition upon covalent attachment of the peptide. This could allow the effective dose to be reduced and/or the peptide inhibitor to be administered less frequently.

In addition to the inhibitory peptides described above, MASP-2 inhibitory peptides useful in the method of the invention include peptides containing the MASP-2 binding region CDR3 from an antiMASP-2 moAb prepared as described herein. The sequence of CDR regions for use in peptide synthesis can be determined by methods known in the art. The heavy chain variable region is a peptide that typically ranges in length from 100 to 150 amino acids. The light chain variable region is a peptide that typically ranges in length from 80 to 130 amino acids. The CDR sequences within the heavy and light chain variable regions contain sequences of only about 3-25 amino acids in length that can be easily sequenced by those skilled in the art.

Those skilled in the art will appreciate that substantially homologous variants of the MASP-2 inhibitory peptides described above will also exhibit MASP-2 inhibitory activity. Exemplary embodiments include, but are not necessarily limited to, peptides having insertions, deletions, substitutions and/or additions of amino acids at the carboxyl or amino terminal portions of specific peptides and mixtures thereof. Accordingly, it is believed that these homologous peptides having MASP-2 inhibitory activity can be used in the methods of the present invention. The described peptides may also contain duplicated motifs and other modifications with conservative substitutions. Conservative variants are described elsewhere herein and have substitutions of one amino acid for another with similar charge, size or hydrophobicity, and the like.

MASP-2 inhibitory peptides can be modified to increase solubility and/or to maximize positive or negative charge to more closely mimic an intact protein segment. The derivative may or may not have the exact primary amino acid structure of the peptide disclosed herein, as long as the derivative functionally retains the desired MASP-2 inhibitory property. Modifications may include an amino acid replacement with one of the commonly known twenty amino acids or another amino acid, a derivatized or substituted amino acid with additional desirable characteristics such as resistance to enzyme degradation, or a D-amino acid, or replacement with another molecule or compound, such as a carbohydrate, that mimics the natural conformation and the function of the amino acid, amino acids or peptide; amino acid deletion; insertion of an amino acid, one of the commonly known twenty amino acids, or another amino acid, a derivatized or substituted amino acid with additional desirable characteristics, such as resistance to enzyme degradation, or D-amino acids, or replacement with another molecule or compound, such as a carbohydrate, that mimics the natural conformation and function amino acid, amino acid or peptide; or replacement with another molecule or compound, such as a carbohydrate or nucleotide monomer, that mimics the natural conformation, charge distribution and function of the original peptide. Peptides can also be modified by acetylation or amidation.

The synthesis of inhibitory peptide derivatives can be carried out by known methods of peptide biosynthesis, carbohydrate biosynthesis and the like. As a starting point, one skilled in the art can use appropriate programs to determine the conformation of the peptide of interest. Once the conformation of the peptide disclosed herein has been established, one of ordinary skill in the art can determine by rational design what type of substitutions can be made at one or more sites to produce a derivative that retains the basic conformation and charge distribution of the parent peptide, but may have characteristics absent or enhanced relative to the characteristics of the original peptide. Once potential derivative molecules have been identified, the derivatives can be tested to determine whether they act as MASP-2 inhibitory agents using the assays described herein.

Screening for MASP-2 inhibitory peptides.

Molecular modeling and rational molecular design can also be used to generate and screen peptides that mimic the molecular structures of key MASP-2 binding regions and inhibit the complement-associated activity of MASP-2. 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, a region involved in binding to MBL , and the active site of the serine protease, as described previously. Methods for identifying peptides that bind a particular target are well known in the art. For example, molecular imprinting can be used to de novo design macromolecular structures, such as peptides, that bind a specific molecule. See, for example, Shea, K.J., Molecular Imprinting of Synthetic Network Polymers: The De Novo synthesis of Macromolecular Binding and Catalytic Sties, TRIP 2(5) 1994.

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As an illustrative example, one method for producing MASP-2 binding peptide mimetics is as follows. Functional monomers of a known MASP-2 binding peptide or binding region of an anti-MASP-2 antibody that causes MASP-2 inhibition (template) are polymerized. The matrix is then removed, followed by polymerization of the second class of monomers in the niche left behind the matrix to produce a new molecule that has one or more of the desired properties similar to those of the matrix. In addition to producing peptides in this manner, it is also possible to produce other MASP-2 binding molecules that are MASP-2 inhibitory agents, such as polysaccharides, nucleosides, drugs, nucleoproteins, lipoproteins, carbohydrates, glycoproteins, steroids, lipids and other biologically active materials . This method is useful for the development of a wide variety of biological mimetics that are more stable than their natural counterparts, since they are typically obtained through free radical polymerization of functional monomers, resulting in a compound with a non-biodegradable scaffold.

Peptide synthesis.

MASP-2 inhibitory peptides can be prepared by methods well known in the art, such as solid phase synthesis methods first described by Merrifield, J. Amer. Chem. Soc. 85:2149-2154, 1963. Automated synthesis can be used, for example, using an Applied Biosystems 431A peptide synthesizer (Foster City, Calif.) according to the manufacturer's instructions. Descriptions of other methods can be found, for example, in Bodanszky, M., et al., Peptide Synthesis, second edition, John Wiley & Sons, 1976, as well as other literature known to those skilled in the art.

Peptides can also be produced using standard genetic engineering techniques known to those skilled in the art. For example, a peptide can be produced by enzymatic methods by inserting a nucleic acid encoding the peptide into an expression vector expressing DNA and translating the DNA into the peptide in the presence of the necessary amino acids. The peptide is then purified using chromatographic or electrophoretic methods, or using a carrier protein that can be fused, and subsequently cleaved from the peptide, by insertion into an expression vector in phase with the peptide-encoding sequence of the nucleotide sequence encoding the carrier protein. The peptide-fusion protein can be isolated using chromatographic, electrophoretic or immunological methods (such as binding to a resin via an antibody to the carrier protein). The peptide can be cleaved using chemical or enzymatic methods, such as hydrolases.

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

Nucleic acid molecules encoding the MASP-2 inhibitory peptide can be synthesized by gene machines using methods such as the phosphoramidite method. If chemically synthesized double-stranded DNA is needed for an application such as the synthesis of a gene or gene fragment, then each complementary strand is produced separately. Obtaining short genes (60-80 nucleotide pairs) is a technically simple task that can be performed by synthesizing complementary chains, followed by their annealing. To obtain longer genes, synthetic genes (double-stranded) are assembled into modules from single-stranded fragments ranging in length from 20 to 100 nucleotides. For an overview of polynucleotide synthesis, see, for example, Glick and Pasternak, Molecular Biotechnology, Principles and Applications of Recombinant DNA, ASM Press, 1994; Itakura, K., et al., Annu. Rev. Biochem. 53:323, 1984; and Climie, S., et al., Proc. Nat'l Acad. Sci. USA 87:633, 1990.

Low molecular weight inhibitors.

In some embodiments, MASP-2 inhibitory agents are small molecule inhibitors, including natural and synthetic substances that have low molecular weight, such as, for example, peptides, peptidomimetics, and non-peptide inhibitors (including oligonucleotides and organic compounds). Small molecule MASP-2 inhibitors can be prepared based on the molecular structure of the variable regions of anti-MASP-2 antibodies.

Small molecule inhibitors can also be designed and created based on the MASP-2 crystal structure using computer-aided drug design (Kuntz I.D., et al., Science 257:1078, 1992). The crystal structure of rat MASP-2 has been reported (Feinberg, H., et al., EMBO J. 22:2348-2359, 2003). Using the method described by Kuntz et al., the MASP-2 crystal structure coordinates are used to enter into a computer program such as

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DOCK, which generates a list of small molecule structures that are predicted to bind MASP-2. The use of such computer programs is well known to one skilled in the art. For example, the crystal structure of the HIV-1 protease inhibitor was used to identify unique non-peptide ligands that constitute an HIV-1 protease inhibitor by assessing the matches of compounds in the Cambridge crystallographic database to the binding site of the enzyme using the DOCK program (Kuntz, I.D., et al., J Mol Biol 161:269-288, 1982; DesJarlais, R.L., et al., PNAS 87:6644-6648, 1990).

A number of small molecules that were computationally identified as potential MASP-2 inhibitors were screened using a MASP-2 binding assay, such as the assay described in Example 10. Small molecules that were found to bind MASP-2 were then analyzed in a functional assay, such as the assay described in Example 2, to determine whether they inhibit MASP-2-dependent complement activation.

Soluble MASP-2 receptors.

Other suitable MASP-2 inhibitory agents are believed to include soluble MASP-2 receptors, which can be prepared using methods known to those skilled in the art.

Inhibitors of MASP-2 expression.

In another embodiment of this aspect of the invention, the MASP-2 inhibitory agent is an inhibitor of MASP-2 expression capable of inhibiting MASP-2-dependent complement activation. When practicing this aspect of the invention, representative inhibitors of MASP-2 expression include antisense MASP2 nucleic acid molecules (such as antisense mRNA, antisense DNA, or antisense oligonucleotides), MASP-2 ribozymes, and MASP-2 RNAi molecules.

Antisense RNA and DNA molecules act by directly blocking translation of MASP-2 mRNA by hybridizing to MASP-2 mRNA and preventing translation of MASP-2 protein. The antisense nucleic acid molecule can be designed in a variety of different ways, provided that it is capable of interfering with MASP-2 expression. For example, an antisense nucleic acid molecule can be constructed by inverting the coding region (or portion thereof) of the MASP-2 cDNA (SEQ ID NO: 4) relative to its normal orientation for transcription so that its complement is transcribed.

An antisense nucleic acid molecule is typically substantially identical to at least a portion of the target gene or genes. However, the nucleic acid does not have to be absolutely identical to inhibit expression. Typically, a higher degree of homology can be used to compensate for the use of a shorter antisense nucleic acid molecule. The minimum percent identity is typically greater than about 65%, but higher percent identity may result in more effective repression of endogenous sequence expression. In general, a percentage identity greater than about 80% is generally preferred, although a degree of between about 95% and absolute is generally most preferred.

An antisense nucleic acid molecule need not have exactly the same pattern of introns or exons as the target gene, and non-coding segments of the target gene may be equally effective in achieving antisense suppression of target gene expression as coding segments. A DNA sequence of at least about 8 or so nucleotides in length can be used as an antisense nucleic acid molecule, although a longer sequence is preferred. According to the present invention, a representative example of a useful MASP-2 inhibitory agent is a MASP-2 antisense nucleic acid molecule that is at least ninety percent identical to the complement of the MASP-2 cDNA consisting of the nucleotide sequence set forth in SEQ ID NO: 4. Nucleotide 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.

Targeting antisense oligonucleotides to bind MASP-2 mRNA is another mechanism that can be used to reduce the level of MASP-2 protein synthesis. For example, the synthesis of polygalacturonase and muscarinic acetylcholine receptor type 2 is inhibited by antisense oligonucleotides targeting their respective mRNA sequences (US Pat. No. 5,739,119 to Cheng and US Pat. No. 5,759,829 to Shewmaker). In addition, examples of antisense inhibition have been demonstrated for the nuclear protein cyclin, multidrug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABA _a receptors and human EGF (see, for example, US patent No. 5801154, issued to Baracchini; US Patent No. 5,789,573 issued to Baker; US Patent No. 5,718,709 issued to Considine; and US Patent No. 5,610,288 issued to Reubenstein).

A system is described that allows one skilled in the art to determine which oligonucleotides

- 58 046178 can be used according to the invention, including the examination of relevant sites in the target mRNA by RNase H digestion as an indicator of sequence accessibility in transcripts. Scherr, M., et al., Nucleic Acids Res. 26:5079-5085, 1998; Lloyd, et al., Nucleic Acids Res. 29:36653673, 2001. A mixture of antisense oligonucleotides complementary to specific regions of the MASP-2 transcript are added to extracts of MASP-2 expressing cells, such as hepatocytes, and hybridized to create an RNase H vulnerable site. This method can be combined with sequence selection using a computer program that can predict the optimal sequence selection for antisense compositions based on their relative ability to form dimers, hairpins, or other secondary structures that would reduce or eliminate specific binding to the target mRNA in host cell. This secondary structure analysis and target site selection can be performed using the oligo primer analysis program (Rychlik, I., 1997) and the BLASTN 2.0.5 program (algorithm) (Altschul, S.F., et al., Nucl. Acids Res. 25: 3389-3402, 1997). Antisense compounds directed to a target sequence are preferably between about 8 and about 50 nucleotides in length. Particularly preferred are antisense oligonucleotides containing from about 9 to about 35 or so nucleotides. The inventors expect that all oligonucleotide compositions with a length in the range of 9 to 35 nucleotides (i.e., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 or so nucleotides) are particularly preferred when practicing the antisense oligonucleotide-based methods of the invention.

Particularly preferred target regions in MASP-2 mRNA are those that are in the region of, or adjacent to, the region of the translation initiation codon AUG, and those sequences that are substantially complementary to the 5' regions of the mRNA, such as the -10 regions to +10 in the nucleotide sequence of the MASP-2 gene (SEQ ID NO: 4). Exemplary inhibitors of MASP-2 expression are shown in table. 4.

Table 4

Exemplary Inhibitors of MASP-2 Expression

SEQ GO NO: 30 (nucleotides 22-680 b SEQ GO NO: 4) Nucleotide sequence of MASP-2 cDNA (SEQ ID NO: 4) encoding CUBIEGF SEQIDNO: 31 5'CGGGCACACCATGAGGCTGCTGA CCCTCCTGGGC3 Nucleotides 12-45 in SEQ ID NO: 4, including the MASP-2 translation start site (sense) SEQ GO NO: 32 5GACATTACCTTCCGCTCCGACTCC AACGAGAAG3' Nucleotides 361-396 in SEQ ID NO: 4, encoding the region containing the MBL MASP-2 binding site (sense) SEQ GO NO: 33 5'AGCAGCCCCTGAATACCCACGCC GTATCCCAAA3' Nucleotides 610-642 in SEQ ID NO: 4, encoding the region containing the siv II domain

As noted above, as used herein, the term oligonucleotide means an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), or mimetics thereof. The term also includes oligonucleotides consisting of naturally occurring nucleotides, sugar residues and covalent internucleoside (backbone) linkages, as well as oligonucleotides having non-natural modifications. Such modifications can impart certain desirable properties that are not present in natural oligonucleotides, such as reduced toxicity, increased stability against nuclease degradation, and increased cellular uptake. In illustrative embodiments, the antisense compounds of the invention are distinguished from native DNA by modifications of the phosphodiester linkages of the backbone in which the phosphate substituents are replaced by phosphorothioates to prolong the life of the antisense oligonucleotide. Likewise, one or more ends of the oligonucleotide may be replaced by one or more acridine derivatives that are inserted between adjacent nucleotide pairs in the nucleic acid chain.

Another alternative to antisense sequence is the use of RNA interference (RNAi). Double-stranded RNA (dsRNA) can cause gene silencing in mammals in vivo. The natural function of RNAi and cosuppression appears to be to protect the genome from invasion by mobile genetic elements such as retrotransposons and viruses that produce aberrant RNA or dsRNA in the host cell when they become active (see, for example, Jensen, J. , et al., Nat. Genet. 21:209-12, 1999). A double-stranded RNA molecule can be produced

- 59 046178 by synthesizing two strands of RNA capable of forming a double-stranded RNA molecule, each of which is approximately 19-25 (for example, 19-23) nucleotides in length. For example, a dsRNA molecule useful in the methods of the invention may include RNA corresponding to the sequence and its complement, which are shown in table. 4. Preferably, at least one RNA strand has a 3' cohesive end of 1-5 nucleotides. The synthesized RNA strands are combined under conditions that result in the formation of a double-stranded molecule. The RNA sequence may comprise a portion of SEQ ID NO: 4 of at least 8 nucleotides, with a total length of 25 nucleotides or less. The design of siRNA sequences for a specific target is within the competence of one skilled in the art. Commercial siRNA sequence design services are available, guaranteeing at least 70% expression knockdown (Qiagen, Valencia, Calif).

dsRNA can be administered in the form of a pharmaceutical composition by known methods, wherein the nucleic acid is introduced into the desired target cell. Common gene transfer methods include calcium phosphate, DEAE-dextran, electroporation, microinjection, and viruses. Such methods are described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 1993.

Ribozymes can also be used to reduce the amount and/or biological activity of MASP-2, for example, ribozymes that target MASP-2 mRNA. Ribozymes are catalytic RNA molecules capable of cleaving nucleic acid molecules having a sequence that is completely or partially homologous to the ribozyme sequence. It is possible to construct ribozyme transgenes that encode RNA ribozymes that specifically pair with a target RNA and cleave the phosphodiester backbone at a specific site, thereby functionally inactivating the target RNA. When this cleavage occurs, the ribozyme itself does not change and is thus capable of recycling and breaking down other molecules. The inclusion of ribozyme sequences in antisense RNAs imparts RNA-cleaving activity to the latter, thereby increasing the activity of the antisense constructs.

Ribozymes useful in the practice of the invention typically contain a hybridizing region of at least about nine nucleotides that is complementary to the nucleotide sequence of at least a portion of the MASP-2 mRNA, and a catalytic region that is adapted to cleave the target MASP-2 mRNA (see generally EPA No. 0 321 201; WO88/04300; Haseloff, J., et al., Nature 334:585-591, 1988; Fedor, M. J., et al., Proc. Natl. Acad. Sci. USA 87:1668-1672, 1990; Cech, T.R., et al., Ann. Rev. Biochem. 55:599-629, 1986).

Ribozymes can either be directed directly to cells in the form of RNA oligonucleotides containing ribozyme sequences, or introduced into the cell in the form of an expression vector encoding the desired ribozyme RNA. Ribozymes can be used and applied in much the same way as described for antisense polynucleotides.

Antisense RNA and DNA, ribozymes and RNAi molecules useful in the methods of the invention can be prepared by any method known in the art for the synthesis of DNA and RNA molecules. This includes methods for the chemical synthesis of oligodeoxyribonucleotides and oligoribonucleotides well known in the art, such as, for example, solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules can be produced by in vitro and in vivo transcription of DNA sequences encoding an antisense RNA molecule. Such DNA sequences can be incorporated into a variety of vectors containing 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 lineages.

Various well-known modifications of DNA molecules can be introduced as a means to improve stability and half-life. Useful modifications include, but are not limited to, the addition of ribonucleotide or deoxyribonucleotide flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-methyl instead of phosphodiester bonds in the oligodeoxyribonucleotide backbone.

VI. Pharmaceutical compositions and delivery methods.

In another aspect, the invention provides compositions for inhibiting the adverse effects of MASP-2-dependent complement activation in a subject suffering from a disease or condition disclosed herein by administering to the subject a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent and a pharmaceutically acceptable carrier. . MASP-2 inhibitory agents can be administered to a subject in need thereof at therapeutically effective doses to treat or alleviate conditions associated with MASP-2-dependent complement activation. A therapeutically effective dose means an amount of a MASP-2 inhibitory agent sufficient to relieve symptoms associated with a disease or condition.

The toxicity and therapeutic efficacy of MASP-2 inhibitory agents can be determined by standard pharmaceutical methods using experimental animal models, such as the MASP-2-/- mouse model expressing the human MASP-2 transgene, which has been

- 60 046178 san in example 1. Using such animal models, NOAEL (no observed adverse effect dose) and MED (minimum effective dose) can be determined by standard methods. The dose ratio between the effects of NOAEL and MED represents the therapeutic index, which is expressed as the ratio NOAEL/MED. MASP-2 inhibitory agents having large therapeutic indices, or coefficients, are most preferred. Data obtained from cell culture assays and animal studies can be used to determine dosages for humans. The dose of the MASP-2 inhibitory agent preferably lies within a range of circulating concentrations including MED with little or no toxicity. Doses may vary within this range, depending on the dosage form used and the route of administration used.

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

In addition to toxicity studies, the effective dose can also be estimated based on the amount of MASP-2 protein present in a living subject and the binding affinity of the MASP-2 inhibitory agent. Serum levels of MASP-2 in normal human subjects have been shown to be low, in the range of 500 ng/ml, and subject-specific MASP-2 levels can be determined using the quantitative MASP-2 assay described in Moller-Kristensen M ., et al., J. Immunol. Methods 282:159-167, 2003.

In general, the dosage of compositions containing MASP-2 inhibitory agents administered will vary depending on factors such as the age, weight, height, sex, general health and prior medical history of the subject. By way of illustration, MASP-2 inhibitory agents, such as anti-MASP-2 antibodies, can be administered in dosage ranges of about 0.010-10.0 mg/kg, preferably 0.010-1.0 mg/kg, more preferably 0.010-0. 1 mg/kg subject's body weight. In some embodiments, the composition contains a combination of anti-MASP-2 antibodies and MASP-2 inhibitory peptides.

The therapeutic efficacy of the MASP-2 inhibitory compositions and methods of the present invention in a particular subject, and the appropriate dosage, can be determined in complement assays well known to those skilled in the art. Activation of complement leads to the formation of many specific products. Over the last decade, sensitive and specific assays have been developed and are commercially available for most of these activation products, including the small activation fragments C3a, C4a, and C5a, as well as the large activation fragments iC3b, C4d, Bb, and sC5b-9. Most of these assays use monoclonal antibodies that react with new antigens (neoantigens) exposed on the fragment, but not with the native proteins from which they are derived, making these assays very simple and specific. Most use ELISA technology, although radioimmunoassays for C3a and C5a are sometimes still used. These latter assays identify both unprocessed fragments and their desAd fragments, which are the major forms found in the circulatory system. Unprocessed fragments and C5 _desAgd are rapidly cleared due to binding to cell surface receptors and, as a result, are present in very low concentrations, while C3a _{desAgd does} not bind to cells and accumulates in the plasma. Measurement of C3a levels is a sensitive pathway-independent measure of complement activation. Activation of the alternative pathway can be assessed by determining the Bb fragment. Identification of the liquid phase membrane attack product of the activation pathway, sC5b-9, provides evidence that complement is activated to completion. Since both the lectin and classical pathways produce the same activation products, C4a and C4d, identification of these two fragments does not allow us to determine which of the two pathways led to the formation of the activation products.

Inhibition of MASP-2-dependent complement activation is characterized by at least one of the following changes in components of the complement system that occur as a result of administration of a MASP-2 inhibitory agent in accordance with the methods of the invention: inhibition of the formation or production of MASP-2-dependent complement activation products , C4b, C3a, C5a and/or C5b-9 (MAC) (measured, e.g., as described in Example 2), reduced C4 degradation and deposition of C4b (measured, e.g., as described in Example 10), or reduced C3 degradation and deposition N3 (measured, for example, as described in example 10).

Additional funds.

Compositions and methods comprising MASP-2 inhibitory agents may optionally include one or more additional drugs that can enhance the activity of the MASP-2 inhibitory agent or that perform related therapeutic functions in an additive or synergistic manner. For example, in the context of treating a subject suffering from TTP, where the subject tests positive for an ADAM-TS13 inhibitor, one or more inhibitors

- 61 046178 MASP-2 blocking agents can be administered in combination (including co-administration) with one or more immunosuppressive drugs. Suitable immunosuppressants include: corticosteroids, Rituxan, cyclosporine and the like. In the context of treating a subject suffering from, or at risk of developing, HUS or aHUS, one or more MASP-2 inhibitory agents can be administered in combination (including co-administration) with an appropriate antibiotic. In the context of treating a subject suffering from, or at risk of developing, aHUS, one or more MASP-2 inhibitory agents may be administered in combination (including co-administration) with other complement inhibitory agents, such as eculizumab (Soliris), TT-30, antibody to factor B, or other agents that inhibit terminal complement components or alternative pathway amplification.

Inclusion and selection of additional agent(s) will depend on the desired therapeutic outcome. In some embodiments, a MASP-2 inhibitory agent may be administered in combination with one or more anti-inflammatory and/or analgesic agents. Suitable anti-inflammatory and/or analgesic agents include: serotonin receptor antagonists; serotonin receptor agonists; histamine receptor antagonists; bradykinin receptor antagonists; kallikrein inhibitors; tachykinin receptor antagonists, including antagonists of neurokinin ₁ and neurokinin ₂ receptor subtypes; calcitonin gene-related peptide (CGRP) receptor antagonists; interleukin receptor antagonists; inhibitors of enzymes active in arachidonic acid metabolite synthesis pathways, including phospholipase inhibitors, including PLA2 isoform inhibitors and PLCy isoform inhibitors, cyclooxygenase (COX) inhibitors (which may be either COX-1, COX-2 inhibitors, or non-selective COX inhibitors -1 and -2), lipoxygenase inhibitors; prostanoid receptor antagonists, including antagonists of the eicosanoid receptor subtypes EP-1 and EP-4 and antagonists of the thromboxane receptor subtypes; leukotriene receptor antagonists, including leukotriene B ₄ receptor subtype antagonists and leukotriene D ₄ receptor subtype antagonists; opioid receptor agonists, including agonists of the μ-opioid, δ-opioid and κ-opioid receptor subtypes; purinergic receptor agonists and antagonists, including P _2X receptor antagonists and P _2Y receptor agonists; activators of adenosine triphosphate (ATP)-dependent potassium channels; MAP kinase inhibitors; nicotinic acetylcholine receptor inhibitors and alpha-adrenergic receptor agonists (including alpha-1, alpha-2 agonists, and non-selective alpha-1 and 2 receptor agonists).

The MASP-2 inhibitory agents of the present invention can also be administered in combination with one or more other complement inhibitors, such as a C5 inhibitor. Currently, eculizumab (Soliris), an anti-C5 antibody, is the only complement-targeting drug that has been approved for the treatment of humans. However, some pharmacological agents have been shown to block complement in vivo. K76COOH and nafamostat mesylate are two agents that have been shown to have some efficacy in animal models of transplantation (Miyagawa, S., et al., Transplant Proc. 24:483-484, 1992). Low molecular weight heparins have also been shown to be effective in regulating complement activity (Edens, R.E., et al., Complement Today, pp. 96-120, Basel: Karger, 1993). It is believed that these small molecule inhibitors may be useful as agents used in combination with the MASP-2 inhibitory agents of the present invention.

Other natural complement inhibitors may be used in combination with the MASP-2 inhibitory agents of the present invention. Biological complement inhibitors include soluble complement factor 1 (sCR1). It is a natural inhibitor that is present on the outer membranes of human cells. Other membrane inhibitors include DAF, MCP and CD59. Recombinant forms have been tested for their anticomplement activity in vitro and in vivo. sCR1 has been shown to be effective in xenotransplantation, where the complement system (both alternative and classical) creates a trigger for hyperactive rejection syndrome within minutes of blood perfusion through the newly transplanted organ (Platt, J.L., et al., Immunol. Today 11:450-6, 1990; Marino, I. R., et al., Transplant Proc. 1071:6, 1990; Johnstone, P. S., et al., Transplantation 54:573-6, 1992). Use of sCR1 provides protection and prolongs the survival of the transplanted organ, suggesting the involvement of the complement pathway in the pathogenesis of organ rejection (Leventhal, J.R., et al., Transplantation 55:857-66, 1993; Pruitt, S.K., et al., Transplantation 57:363 -70, 1994).

Suitable additional complement inhibitors for use in combination with the compositions of the present invention also include, by way of example, moAbs such as the anti-C5 antibody (eg, eculizumab) being developed by Alexion Pharmaceuticals, Inc., New Haven, Connecticut, and the anti-properdin moAb. .

Pharmaceutical carriers and delivery media.

Typically, the MASP-2 inhibitory agent compositions of the present invention, in combination with any other therapeutic agents selected, are contained in a pharmaceutically acceptable carrier. The carrier is non-toxic, biocompatible and selected in such a way that

- 62 046178 would not adversely affect the biological activity of the MASP-2 inhibitory agent (and any other therapeutic agents combined with it). Exemplary pharmaceutically acceptable carriers for peptides are described in US Pat. No. 5,211,657 to Yamada. The anti-MASP-2 antibodies and inhibitory peptides useful in the invention may be formulated into preparations in solid, semi-solid, gel, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, depots, inhalations and injections, used for oral, parenteral or surgical administration. The invention also provides for local administration of the compositions by application to medical devices and the like.

Suitable carriers for parenteral delivery by injection, infusion or irrigation, as well as topical delivery, include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer's solution, dextrose solution, Hank's solution or propanediol. In addition, sterile non-volatile oils can be used as a solvent or suspension medium. For this purpose, any biocompatible oil can be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the manufacture of injectable drugs. The carrier and agent may be formulated as a liquid, suspension, polymerizable or non-polymerizable gel, paste or ointment.

The carrier may also include a delivery medium designed to maintain (ie, prolong, delay or regulate) delivery of the therapeutic agent(s) or to improve the delivery, absorption, stability or pharmacokinetics of the therapeutic agent(s). Such delivery vehicles may include, by way of non-limiting example, microparticles, microspheres, nanospheres or nanoparticles consisting of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymer or copolymer hydrogels and polymer micelles. Suitable hydrogel and micellar delivery systems include the PEO:PHB:PEO copolymers and copolymers/cyclodextrin complexes disclosed in WO 2004/009664 A2, as well as the PEO and PEO/cyclodextrin complexes disclosed in US Patent Application Publication No. 2002/0019369 A1. Such hydrogels can be injected locally into the site of intended action or subcutaneously or intramuscularly to form a depot for sustained release.

For intra-articular delivery, the MASP-2 inhibitory agent may be in the above-described injectable liquid or gel vehicles, the above-described injectable sustained release delivery media, or in hyaluronic acid or a hyaluronic acid derivative.

For oral administration of non-peptidergic agents, the MASP-2 inhibitory agent may be present in an excipient or diluent such as sucrose, corn starch or cellulose.

For topical administration, the MASP-2 inhibitory agent may be in an ointment, lotion, cream, gel, drops, suppository, aerosol, liquid or powder, or in gel or microcapsule delivery systems using a transdermal patch.

Various nasal and pulmonary delivery systems, including aerosols, metered dose inhalers, dry powder inhalers and nebulizers, have been developed and can be successfully adapted to deliver the drug of the present invention in an aerosol, inhalation or nebulized delivery medium, respectively.

For intrathecal (i/t) or intracerebroventricular (i/cv) delivery of the drug of the present invention, appropriate sterile delivery systems (eg, liquids; gels, suspensions, etc.) can be used.

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

Pharmaceutical carriers for antibodies and peptides.

More specifically, in the case of anti-MASP-2 antibodies and inhibitory peptides, the exemplary preparations can be administered parenterally as injectable doses of a solution or suspension of the compound in a physiologically acceptable diluent with a pharmaceutical carrier, which can be a sterile liquid such as water, oils, saline , glycerin or ethanol. In addition, excipients such as humectants or emulsifiers, surfactants, pH adjusting agents, and the like may be present in compositions containing anti-MASP-2 antibodies and inhibitory peptides. Additional components of the pharmaceutical compositions include oils (eg, animal, vegetable or synthetic origin), such as soybean oil and mineral oil. In general, glycols such as propylene glycol or polyethylene glycol are the preferred liquid carriers for injection solutions.

Ahtu-MASP-2 antibodies and inhibitory peptides can also be administered in the form of an injectable or implantable depot formulation, which can be formulated to provide sustained or pulsed release of the active agents.

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Pharmaceutically acceptable carriers for expression inhibitors.

More specifically, in the case of expression inhibitors useful in the methods of the invention, compositions are provided that contain the expression inhibitor described above and a pharmaceutically acceptable carrier or diluent. The composition may further comprise a colloidal dispersion system.

Pharmaceutical compositions containing expression inhibitors may include, but are not limited to, solutions, emulsions, and liposome-containing preparations. These compositions can be created from various components, which include, but are not limited to, prepared liquids, self-emulsifying solids and self-emulsifying semi-solids. The preparation of such compositions typically involves combining the expression inhibitor with one or more of the following: buffers, antioxidants, small molecule polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with nonspecific serum albumin are examples of suitable diluents.

In some embodiments, the compositions can be prepared and formulated as emulsions, which are typically heterogeneous systems of one liquid dispersed in another liquid as droplets (see Idson, in: Pharmaceutical Dosage Forms, Vol. 1, Rieger and Banker (eds.), Marcek Dekker, Inc., N.Y., 1988). Examples of natural emulsifiers used in emulsion preparations include gum arabic, beeswax, lanolin, lecithin and phosphatides.

In one embodiment, compositions containing nucleic acids can be formulated as microemulsions. As used herein, the term microemulsion means a system of water, oil and an amphiphilic substance that constitutes one optically isotropic and thermodynamically stable liquid solution (see Rosoff in: Pharmaceutical Dosage Forms, Vol. 1). The method of the invention can also use liposomes to carry and deliver antisense oligonucleotides to the desired site.

Pharmaceutical compositions and expression inhibitor preparations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers may be used, as well as aqueous, powder or oil bases, thickeners and the like.

Methods of administration.

Pharmaceutical compositions containing MASP-2 inhibitory agents can be administered by various routes depending on whether a local or systemic route of administration is most appropriate for the condition being treated. Additionally, as described hereinabove, in extracorporeal reperfusion procedures, MASP-2 inhibitory agents can be administered by administering compositions of the present invention to recirculating blood or plasma. In addition, the compositions of the present invention can be delivered by applying, or incorporating, the compositions onto, or into, an implantable medical device.

System delivery.

As used herein, the terms systemic delivery and systemic administration shall include, but are not limited to, oral and parenteral routes of administration, including intramuscular (IM), subcutaneous, intravenous (IV), intra-arterial, inhalation, sublingual, buccal, topical. , transdermal, nasal, rectal, vaginal and other routes of administration that effectively result in the dispersion of the delivered agent at one or more sites of intended therapeutic action. Therapeutic routes of systemic delivery for the present compositions include intravenous, intramuscular, subcutaneous and inhalation. It should be understood that the exact route of systemic administration for the particular agents used in the particular compositions of the present invention will depend in part on the exposure of the agent to the metabolic pathways associated with the particular route of administration. For example, peptidergic agents are most preferably administered by routes other than the oral route.

MASP-2 inhibitory antibodies and polypeptides can be delivered to a subject in need thereof by any suitable means. Methods of delivery of anti-MASP-2 antibodies and polypeptides include oral, pulmonary, parenteral (e.g., intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), inhalation (e.g., fine powder formulation), transdermal, nasal, vaginal, rectal or sublingual routes of administration, and they can be formulated in dosage forms suitable for each route of administration.

As a representative example, MASP-2 inhibitory antibodies and peptides can be administered to a living body by application to bodily membranes capable of absorbing polypeptides, such as the lining of the nasal cavity, gastrointestinal tract and rectum. Polypeptides are typically applied to the absorbent shell in combination with a penetration enhancer. (See, for example, Lee, V.H.L., Crit. Rev. Ther. Drug Carrier Sys. 5:69, 1988; Lee, V.H.L., J. Controlled Release 13:213,

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1990; Lee, V.H.L., Ed., Peptide and Protein Drug Delivery, Marcel Dekker, New York (1991); DeBoer, A.G., et al., J. Controlled Release 13:241, 1990.) For example, STDHF is a synthetic fusidic acid derivative, a steroidal surfactant that is similar in structure to bile salts and is used as a penetration enhancer for nasal delivery (Lee, W.A., Biopharm. 22, Nov./Dec. 1990).

MASP-2 inhibitory antibodies and polypeptides can be administered in combination with another molecule, such as a lipid, to protect the polypeptides from enzymatic degradation. For example, the covalent attachment of polymers, particularly polyethylene glycol (PEG), has been used to protect certain proteins from enzymatic hydrolysis in the body and thereby prolong their half-life (Fuertges, F., et al., J. Controlled Release 11: 139, 1990). Many polymeric systems for protein delivery have been described (Bae, Y.H., et al., J. Controlled Release 9:271, 1989; Hori, R., et al., Pharm. Res. 6:813, 1989; Yamakawa, I., et al., J. Pharm. Sci. 79:505, 1990; Yoshihiro, I., et al., J. 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 J. Sci. 78:117, 1989; Takakura, Y., et al., J. Pharm. Sci. 78:219, 1989).

Recently, liposomes have been developed with improved serum stability and a longer circulatory half-life (see, for example, US Pat. No. 5,741,516 to Webb). In addition, various methods of using liposomes and liposome-like preparations as potential drug carriers have been described (see, for example, US Pat. No. 5,567,434 to Szoka; US Pat. No. 5,552,157 to Yagi; US Pat. No. 5,565,213 to Nakamori; US Pat. No. 5,738,868 issued to Shinkarenko; and US Patent No. 5,795,587 issued to Gao).

For transdermal applications, MASP-2 inhibitory antibodies and polypeptides can be combined with other suitable ingredients, such as carriers and/or adjuvants. There are no restrictions on the nature of such other ingredients, except that they must be pharmaceutically acceptable for their intended administration, and must not reduce the activity of the active ingredients of the composition. Examples of suitable vehicles include ointments, creams, gels or suspensions, with or without the addition of purified collagen. MASP-2 inhibitory antibodies and polypeptides can also be impregnated into transdermal patches, dressings and dressings, preferably in liquid or semi-liquid form.

The compositions of the present invention can be administered systemically on a periodic basis, at intervals determined to maintain the desired level of therapeutic effect. For example, the compositions can be administered, for example, by subcutaneous injection, every two to four weeks, or less frequently. The dosage regimen will be determined by the doctor, taking into account various factors that may affect the effect of the combination of drugs. Such factors will include the degree of progression of the condition being treated, the age, sex and weight of the patient, as well as other clinical factors. The dosage of each individual agent will vary depending on the MASP-2 inhibitory agent that is included in the composition and the availability and nature of any drug delivery medium (eg, sustained release delivery medium). In addition, the dosage amount may be adjusted depending on the frequency of administration and the pharmacokinetics of the agent(s) being delivered.

Local delivery.

As used herein, local delivery means the application of a drug directly at or near the site of intended localized action and may include, for example, topical delivery to the skin or other affected tissues, ophthalmic delivery, intrathecal (i/t), intracerebroventricular (and /tsv), intra-articular, intracavitary, intracranial or intravesical administration, placement or irrigation. Local administration may be preferable to enable lower dosage administration, avoid systemic side effects, and allow more precise control of delivery time and concentration of active agents at the local delivery site. Local administration provides a known concentration at the target site, regardless of patient differences in metabolism, blood flow, etc. Better dose control is also achieved with the direct delivery method.

Local delivery of a MASP-2 inhibitory agent can be accomplished in the context of surgical treatments for a disease or condition, for example, during procedures such as arterial bypass grafting, atherectomy, laser procedures, ultrasound procedures, balloon angioplasty, and stent placement. For example, a MASP-2 inhibitor may be administered to a subject in conjunction with a balloon angioplasty procedure. The balloon angioplasty procedure involves inserting a catheter with a deflated balloon into the artery. The deflated balloon is placed near the atherosclerotic plaque and inflated so that the plaque is pressed against the vessel wall. As a result, the surface of the balloon is in contact with a layer of vascular endothelial cells on the surface of the blood vessel. The MASP-2 inhibitory agent can be coupled to the balloon angioplasty catheter in a manner that allows release of the agent at the site of the atherosclerotic plaque. The product can be combined with

- 65 046178 catheter for balloon angioplasty using standard methods known in the art. For example, the agent can be stored in a compartment of a balloon catheter until the balloon is inflated, after which it is released into the local environment. Alternatively, the agent can be impregnated on the surface of the balloon so that it comes into contact with the cells of the arterial wall as the balloon inflates. The agent can also be delivered in a perforated balloon catheter, such as those described in Flugelman, M.Y., et al., Circulation 85:1110-1117, 1992. See PCT published application WO 95/23161 for a description of an illustrative procedure for attaching a therapeutic protein to a catheter. for balloon angioplasty. Likewise, the MASP-2 inhibitory agent may be included in a gel or polymer coating applied to the stent, or may be included in the stent material, causing the stent to release the MASP-2 inhibitory agent upon placement in the vessel.

MASP-2 inhibitory compositions used in the treatment of arthritis and other musculoskeletal diseases can be locally delivered by intra-articular injection. Such compositions may preferably contain a sustained release delivery medium. As a further example of cases in which local delivery may be desirable, MASP-2 inhibitory compositions used in the treatment of diseases of the genitourinary system can be administered dropwise into the bladder or other part of the genitourinary system.

Coatings on medical devices.

MASP-2 inhibitory agents, such as antibodies and inhibitory peptides, can be immobilized on (or within) the surface of an implanted or attached medical device. The modified surface will typically be in contact with living tissue once implanted into the animal's body. Implantable or attached medical device means any device that is implanted, or attached, into the body tissue of an animal for the normal functioning of the device (for example, stents and implantable drug delivery devices). Such implantable or attached medical devices may be made, for example, from nitrocellulose, diazocellulose, glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, dextran, Sepharose, agar, starch, nylon, stainless steel, titanium, and biodegradable and/or biocompatible polymers. Binding of a protein to a device can be accomplished by any method that does not disrupt the biological activity of the bound protein, for example, by attaching one or both of the N- and C-terminal residues of the protein to the device. Attachment can also be accomplished through one or more internal sites of the protein. Multiple attachment sites (both inside and at the ends of the protein) can also be used. The surface of an implanted or attached medical device can be modified to include functional groups (e.g., carboxyl, amide, amino, ether, hydroxyl, cyano, nitrido, sulfonamido, acetyl, epoxy, silane, anhydride, succinimide, azido) to immobilize protein thereon. Chemical coupling reactions include, but are not limited to, the formation of esters, ethers, amides, azido- and sulfonamide derivatives, cyanates and other linkages with functional groups available on anti-MASP-2 antibodies or inhibitory peptides. Ahtu-MASP-2 antibodies or inhibitory fragments can also be attached non-covalently by adding to the protein an affinity tag sequence such as GST (D.B. Smith and K.S. Johnson, Gene 67:31, 1988), a polyhistidine moiety (E. Hochuli et al. , J. Chromatog. 411:77, 1987) or biotin. Such affinity tags can be used to reversibly attach a protein to a device.

Proteins can also be covalently attached to the surface of the device body, for example, by covalently activating the surface of a medical device. As a representative example, the extracellular matrix protein(s) can be attached to the device body using any of the following pairs of reactive groups (one member of the pair is on the surface of the device body, and the other member of the pair is on the extracellular matrix protein(s): hydroxyl/carboxylic Acid to form an ester bond Hydroxyl/anhydride to form an ester bond Hydroxyl/isocyanate to form a urethane bond Device body surfaces that do not have useful reactive groups can be treated with RFGD plasma, etching to create reactive groups , promoting the deposition of extracellular matrix protein(s) (for example, treat with oxygen plasma to introduce oxygen-containing groups; treat with propylamine plasma to introduce amino groups).

MASP-2 inhibitory agents comprising nucleic acid molecules, such as antisense, RNAi, or DNA encoding peptide inhibitors, can be embedded in porous matrices attached to the body of the device. Representative porous matrices that can be used to create the surface layer are matrices derived from tendon and skin collagen, which can be obtained from a variety of commercial sources (eg, Sigma and Collagen Corporation), or collagen matrices prepared as described in US Pat. No. 4394370 issued by Jefferies and 4975527 issued by Koezuka. One collagen material is called UltraFiber™ and is available from Norian Corp. (Mountain View, California).

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It is also possible, if desired, to use certain polymer matrices that contain acrylic ester polymers and lactic acid polymers, as described, for example, in US Pat. Nos. 4,526,909 and 4,563,489 issued to Urist. Specific examples of useful polymers are polymers of orthoesters, anhydrides, propylene fumarates, or a polymer of monomers of one or more ahydroxycarboxylic acids (eg, α-hydroxyacetic acid (glycolic acid) and/or α-hydroxypropionic acid (lactic acid)).

Treatment regimens

For prophylactic use, the pharmaceutical compositions are administered to a subject predisposed to, or otherwise at risk of developing a condition associated with MASP-2-dependent complement activation, in an amount sufficient to eliminate or reduce the risk of developing symptoms of the condition. When used therapeutically, the pharmaceutical compositions are administered to a subject suspected or actually suffering from a condition associated with MASP2-dependent complement activation in a therapeutically effective amount sufficient to alleviate or at least partially reduce the symptoms of the condition. In both prophylactic and therapeutic regimens, compositions containing MASP-2 inhibitory agents can be administered in multiple doses until sufficient therapeutic benefit is achieved in the subject. Administration of the MASP-2 inhibitory compositions of the present invention may be accomplished by a single administration of the composition or by a limited number of sequential administrations to treat an acute condition, such as reperfusion injury or other traumatic injury. Alternatively, the composition can be administered at periodic intervals over an extended period of time to treat chronic conditions, such as arthritis or psoriasis.

The methods and compositions of the present invention can be used to inhibit inflammation and related processes that typically occur as a result of diagnostic and therapeutic medical and surgical procedures. To inhibit such processes, the MASP-2 inhibitory composition of the present invention can be used periprocedurally. As used herein, the term periprocedural refers to the administration of the inhibitory composition preprocedurally and/or intraprocedurally and/or postprocedurally, i.e., before the procedure, before and during the procedure, before and after the procedure, before, during and after the procedure, during the procedure , during and after the procedure or after the procedure. Periprocedural use can be accomplished by local administration of the composition to the site of surgery or procedure, for example, by injection, or continuous or intermittent irrigation of the site, or by systemic administration. Appropriate methods for local perioperative delivery of MASP-2 inhibitory agent solutions are described in US Pat. No. 6,420,432 to Demopulos and US Pat. No. 6,645,168 to Demopulos. Suitable methods for local delivery of chondroprotective compositions containing MASP-2 inhibitory agent(s) are described in international PCT patent application WO 01/07067 A2. Suitable methods and compositions for targeted systemic delivery of chondroprotective compositions containing MASP-2 inhibitory agent(s) are described in international PCT patent application WO 03/063799 A2.

In one aspect of the invention, the pharmaceutical compositions are administered to a subject suffering from, or at risk of developing, thrombotic microangiopathy (TMA). In one embodiment, the TMA is selected from the group consisting of hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP), and atypical hemolytic uremic syndrome (aHUS). In one embodiment, the TMA is aHUS. In one embodiment, the composition is administered to a patient with aHUS during the acute phase of the disease. In one embodiment, the composition is administered to a patient with aHUS during a remission phase (i.e., a subject who has recovered, or partially recovered, from an acute phase episode of aHUS, with remission indicated by, for example, an increase in platelet count and/or a decrease in serum LDH concentrations , for example, as described in Loirat C et al., Orphanet Journal of Rare Diseases 6:60, 2011, the contents of which are incorporated herein by reference). In one embodiment, the subject suffers from, or is at risk of developing, TMA, which is (i) TMA due to cancer; (ii) TMA due to chemotherapy or (iii) TMA due to transplantation (eg, organ transplantation, such as kidney transplantation or allogeneic hematopoietic stem cell transplantation). In one embodiment, the subject suffers from, or is at risk of developing, Upshaw-Shulman syndrome (USS). In one embodiment, the subject suffers from, or is at risk of developing, Degos disease. In one embodiment, the subject suffers from, or is at risk of developing, catastrophic antiphospholipid syndrome (CAPS). When used therapeutically, the pharmaceutical compositions are administered to a subject suffering from, or at risk of developing, TMA in a therapeutically effective amount sufficient to inhibit the formation of blood clots and alleviate or at least partially reduce the symptoms of the condition.

In both prophylactic and therapeutic regimens, compositions containing MASP-2 inhibitory agents can be administered in multiple doses until sufficient therapeutic benefit is achieved in the subject. In one embodiment of the invention, the MASP-2 inhibitory agent

- 67 046178 is an aHTu-MASP-2 antibody that can preferably be administered to an adult patient (for example, an adult patient with an average body weight of 70 kg) at a dose of 0.1 mg to 10,000 mg, more preferably 1.0 mg to 5,000 mg, more preferably 10.0 mg to 2000 mg, more preferably 10.0 mg to 1000 mg and even more preferably 50.0 mg to 500 mg. For young patients, the dose can be adjusted in proportion to the patient's body weight. The use of the MASP-2 inhibitory compositions of the present invention can be accomplished by a single administration of the composition or by a limited number of sequential administrations for the treatment of TMA. Alternatively, the composition may be administered at periodic intervals, for example daily, twice weekly, weekly, biweekly, monthly or bimonthly, over an extended period of time to treat TMA.

In some embodiments, a subject suffering from, or at risk of developing, TMA has previously received, or is currently receiving, treatment with a terminal complement inhibitor that inhibits the breakdown of complement protein C5. In some embodiments, the method includes administering to the subject a composition of the invention comprising a MASP-2 inhibitor, and additionally administering to the subject a terminal complement inhibitor that inhibits the breakdown of complement protein C5. In some embodiments, the terminal complement inhibitor is a humanized anti-C5 antibody, or an antigen-binding fragment thereof. In some embodiments, the terminal complement inhibitor is eculizumab.

In one aspect of the invention, pharmaceutical compositions are administered to a subject predisposed to, or otherwise at risk of developing aHUS, in an amount sufficient to eliminate or reduce the risk of developing symptoms of the condition. When used therapeutically, the pharmaceutical compositions are administered to a subject suspected or actually suffering from aHUS in a therapeutically effective amount sufficient to alleviate or at least partially reduce the symptoms of the condition. In one aspect of the invention, prior to administration, the subject may be examined to determine whether the subject has one or more symptoms of aHUS, including (i) anemia, (ii) thrombocytopenia, (iii) renal failure, and (iv) elevated creatinine levels, and then composition of the present invention is administered in an effective amount and for a sufficient period of time to relieve the symptom(s).

In another aspect of the invention, the MASP-2 inhibitory compositions of the present invention can be used for prophylactic administration to a subject who is at increased risk of developing aHUS, thereby reducing the likelihood that the subject will develop aHUS. The presence of a genetic marker in a subject known to be associated with aHUS is first determined by genetically screening a sample obtained from the subject and determining the presence of at least one genetic marker associated with aHUS: complement factor H (CFH), factor I (CFI), factor B (CFB), membrane cofactor CD46, C3, complement factor H-related protein (CFHR1), thrombomodulin anticoagulant protein (THBD), complement factor H-related protein 3 (CFHR3), and complement factor H-related protein 4 (CFHR4). The subject is then monitored periodically (eg, monthly, quarterly, biannually, or annually) to determine the presence or absence of at least one symptom of aHUS, such as anemia, thrombocytopenia, renal failure, and elevated creatinine. Once the presence of at least one of these symptoms has been determined, the subject may be administered a MASP-2 inhibitory agent effective to inhibit MASP-2-dependent complement activation in an effective amount and for a sufficient period of time to relieve the one or more symptoms. In a further aspect of the present invention, a subject who is at increased risk of developing aHUS, as determined after screening and determining the presence of one of the genetic markers associated with aHUS, can be monitored for the occurrence of an event that is known to be associated with the initiation of clinical symptoms of aHUS. including drug exposure, infection (eg, bacterial infection), malignancy, trauma, organ or tissue transplantation, and pregnancy.

In a further aspect of the present invention, a composition containing an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2-dependent complement activation can be administered to a subject suffering from, or at risk of developing, atypical hemolytic uremic syndrome (aHUS) due to infection. For example, a patient suffering from, or at risk of developing, non-enteric aHUS associated with an infection caused by S. pneumonia, can be treated with the compositions of the present invention.

In a further aspect of the present invention, a subject suffering from aHUS can first be treated with a MASP-2 inhibitory composition of the present invention, which is administered through an established catheter, for example, an established intravenous catheter or an established subcutaneous catheter, for a first period of time, such as one hour, twelve hours, one day, two days or three days. The subject may then be treated for a second period of time with a MASP-2 inhibitory composition administered by conventional subcutaneous injections, for example, daily, twice a week, every

- 68 046178 weekly, biweekly, monthly or bimonthly.

In a further aspect of the present invention, the MASP-2 inhibitory composition of the present invention can be administered to a subject suffering from aHUS without plasma exchange (i.e., a subject whose symptoms of aHUS have not been treated with plasma exchange and are not treated with plasma exchange during the period of treatment with the MASP-2 inhibitory composition), during avoiding potential complications of plasmapheresis, including bleeding, infection, and exposure to pathogenic and/or allergic factors present in the donor plasma, or in the case of a subject who rejects plasmapheresis, or in settings where plasmapheresis is not available.

In a further aspect of the present invention, the MASP-2 inhibitory composition of the present invention can be administered to a subject suffering from aHUS in combination with treating the patient with plasmapheresis. For example, a subject receiving plasmapheresis treatment may then be administered a MASP-2 inhibitory composition after, or alternately with, plasmapheresis.

In a further aspect of the present invention, a subject suffering from, or at risk of developing, aHUS and receiving treatment with a MASP-2 inhibitory composition of the present invention can be monitored by periodically, for example, every twelve hours or daily, the level of at least one complement factor, wherein determination of a decreased level of at least one complement factor compared to a standard value, or a value in a healthy control subject, indicates the need for continued treatment with the composition.

In both prophylactic and therapeutic regimens, compositions containing MASP-2 inhibitory agents can be administered in multiple doses until sufficient therapeutic benefit is achieved in the subject. In one embodiment of the invention, the MASP-2 inhibitory agent is an anti-MASP-2 antibody, which can preferably be administered to an adult patient (for example, an adult patient with an average body weight of 70 kg) at a dose of 0.1 mg to 10,000 mg, more preferably from 1.0 mg to 5000 mg, more preferably from 10.0 mg to 2000 mg, more preferably from 10.0 mg to 1000 mg and even more preferably from 50.0 mg to 500 mg. For young patients, the dose can be adjusted in proportion to the patient's body weight. The use of the MASP-2 inhibitory compositions of the present invention can be accomplished by a single administration of the composition or by a limited number of sequential administrations for the treatment of aHUS. Alternatively, the composition may be administered at periodic intervals, for example daily, twice weekly, weekly, biweekly, monthly or bimonthly, over an extended period of time to treat aHUS.

In some embodiments, the subject suffering from aHUS has previously received, or is currently receiving, treatment with a terminal complement inhibitor that inhibits the breakdown of complement protein C5. In some embodiments, the method includes administering to the subject a composition of the invention comprising a MASP-2 inhibitor, and additionally administering to the subject a terminal complement inhibitor that inhibits the breakdown of complement protein C5. In some embodiments, the terminal complement inhibitor is a humanized anti-C5 antibody, or an antigen-binding fragment thereof. In some embodiments, the terminal complement inhibitor is eculizumab.

In one aspect of the invention, pharmaceutical compositions are administered to a subject predisposed to, or otherwise at risk of developing HUS, in an amount sufficient to eliminate or reduce the risk of developing symptoms of the condition. When used therapeutically, the pharmaceutical compositions are administered to a subject suspected or actually suffering from HUS in a therapeutically effective amount sufficient to alleviate or at least partially reduce the symptoms of the condition.

In another aspect of the present invention, the likelihood of developing renal dysfunction in a subject at risk for developing HUS can be reduced by administering to the subject a MASP-2 inhibitory composition of the present invention in an amount effective to inhibit MASP2-dependent complement activation. For example, a subject at risk for developing HUS and being treated with a MASP-2 inhibitory composition of the present invention may have one or more symptoms associated with HUS, including diarrhea, a hematocrit level below 30% with evidence of intravascular destruction of red blood cells on the blood smear, thrombocytopenia and elevated creatinine levels. As a further example, a subject at risk for developing HUS and being treated with a MASP-2 inhibitory composition of the present invention may be infected with E. coli, Shigella or Salmonella. Such subjects infected with E. coli, Shigella or Salmonella can be treated with a MASP-2 inhibitory composition of the present invention in combination with antibiotic treatment or, alternatively, can be treated with a MASP-2 inhibitory composition without concurrent antibiotic treatment, particularly in the case of enterogenous E. coli, in which the use of antibiotics is contraindicated. A subject infected with enterogenous E. coli who has been treated with an antibiotic may be at increased risk of developing HUS and may preferably be treated with an inhibitor

- 69 046178 MASP-2-targeting composition of the present invention to reduce this risk. A subject infected with enterogenous E. coli can be treated for a first period of time with a MASP-2 inhibitory composition of the present invention without the use of an antibiotic, and then for a second period of time treated with both a MASP-2 inhibitory composition of the present invention and an antibiotic.

In a further aspect of the present invention, a subject suffering from HUS can first be treated with a MASP-2 inhibitory composition of the present invention, which is administered through an established catheter, for example, an established intravenous catheter or an established subcutaneous catheter, for a first period of time, such as one hour, twelve hours, one day, two days or three days. The subject may then be treated for a second period of time with a MASP-2 inhibitory composition administered by conventional subcutaneous injections, for example, daily, twice weekly, weekly, biweekly, monthly, or bimonthly.

In a further aspect of the present invention, the MASP-2 inhibitory composition of the present invention can be administered to a subject suffering from HUS without plasma exchange (i.e., a subject whose symptoms of HUS have not been treated with plasma exchange and are not treated with plasma exchange during the period of treatment with the MASP-2 inhibitory composition), during avoiding potential complications of plasmapheresis, including bleeding, infection, and exposure to pathogenic and/or allergic factors present in the donor plasma, or in the case of a subject who rejects plasmapheresis, or in settings where plasmapheresis is not available.

In a further aspect of the present invention, the MASP-2 inhibitory composition of the present invention can be administered to a subject suffering from HUS in combination with treating the patient with plasmapheresis. For example, a subject receiving plasmapheresis treatment may then be administered a MASP-2 inhibitory composition after, or alternately with, plasmapheresis.

In a further aspect of the present invention, a subject suffering from, or at risk of developing, HUS and being treated with a MASP-2 inhibitory composition of the present invention can be monitored by periodically, for example, every twelve hours or daily, the level of at least one complement factor, wherein determination of a decreased level of at least one complement factor compared to a standard value, or a value in a healthy control subject, indicates the need for continued treatment with the composition.

In both prophylactic and therapeutic regimens, compositions containing MASP-2 inhibitory agents can be administered in multiple doses until sufficient therapeutic benefit is achieved in the subject. In one embodiment of the invention, the MASP-2 inhibitory agent is an anti-MASP-2 antibody, which can preferably be administered to an adult patient (for example, an adult patient with an average body weight of 70 kg) at a dose of 0.1 mg to 10,000 mg, more preferably from 1.0 mg to 5000 mg, more preferably from 10.0 mg to 2000 mg, more preferably from 10.0 mg to 1,000 mg, and even more preferably from 50.0 mg to 500 mg. For young patients, the dose can be adjusted in proportion to the patient's body weight. The use of the MASP-2 inhibitory compositions of the present invention can be accomplished by a single administration of the composition or by a limited number of sequential administrations for the treatment of HUS. Alternatively, the composition may be administered at periodic intervals, for example daily, twice weekly, weekly, biweekly, monthly or bimonthly, over an extended period of time to treat HUS.

In some embodiments, the subject suffering from HUS has previously received, or is currently receiving, treatment with a terminal complement inhibitor that inhibits the breakdown of complement protein C5. In some embodiments, the method includes administering to the subject a composition of the invention comprising a MASP-2 inhibitor, and additionally administering to the subject a terminal complement inhibitor that inhibits the breakdown of complement protein C5. In some embodiments, the terminal complement inhibitor is a humanized anti-C5 antibody, or an antigen-binding fragment thereof. In some embodiments, the terminal complement inhibitor is eculizumab.

In one aspect of the invention, pharmaceutical compositions are administered to a subject predisposed or otherwise at risk of developing TTP in an amount sufficient to eliminate or reduce the risk of developing symptoms of the condition. When used therapeutically, the pharmaceutical compositions are administered to a subject suspected or actually suffering from TTP in a therapeutically effective amount sufficient to alleviate or at least partially reduce the symptoms of the condition.

In another aspect of the present invention, a subject having one or more of the symptoms of TTP, including central nervous system disease, thrombocytopenia, severe cardiac disease, severe pulmonary disease, intestinal infarction, and gangrene, can be treated with a MASP-2 inhibitory composition of the present invention. In another aspect of the present invention, a subject having both

- 70 046178 determined, reduced levels of ADAMTS-13, as well as positive test results for the presence of an inhibitor (ie, antibody) of ADAMTS-13, can be treated with a MASP-2 inhibitory composition of the present invention. In a further aspect of the present invention, a subject who tests positive for an ADAMTS-13 inhibitor may be treated with an immunosuppressant (eg, corticosteroids, Rituxan, or cyclosporine) concomitantly with treatment with a MASP-2 inhibitory composition of the present invention. In a further aspect of the present invention, a subject determined to have a reduced level of ADAMTS-13 and also test positive for an ADAMTS-13 inhibitor can be treated with ADAMTS-13 concomitantly with treatment with a MASP-2 inhibitory composition of the present invention.

In a further aspect of the present invention, a subject suffering from TTP can first be treated with a MASP-2 inhibitory composition of the present invention, which is administered through an established catheter, for example, an established intravenous catheter or an established subcutaneous catheter, for a first period of time, such as one hour, twelve hours, one day, two days or three days. The subject may then be treated for a second period of time with a MASP-2 inhibitory composition administered by conventional subcutaneous injections, for example, daily, twice weekly, weekly, biweekly, monthly, or bimonthly.

In a further aspect of the present invention, the MASP-2 inhibitory composition of the present invention can be administered to a subject suffering from TTP without plasma exchange (i.e., a subject whose symptoms of TTP have not been treated with plasma exchange and are not treated with plasma exchange during the period of treatment with the MASP-2 inhibitory composition), during avoiding potential complications of plasmapheresis, including bleeding, infection, and exposure to pathogenic and/or allergic factors present in the donor plasma, or in the case of a subject who rejects plasmapheresis, or in settings where plasmapheresis is not available.

In a further aspect of the present invention, the MASP-2 inhibitory composition of the present invention can be administered to a subject suffering from TTP in combination with treating the patient with plasmapheresis. For example, a subject receiving plasmapheresis treatment may then be administered a MASP-2 inhibitory composition after, or alternately with, plasmapheresis.

In a further aspect of the present invention, a subject suffering from refractory TTP, that is, a subject whose TTP symptoms have not adequately changed in response to other treatment, such as plasmapheresis, can be treated with a MASP-2 inhibitory composition of the present invention, with or without the use of additional plasmapheresis.

In a further aspect of the present invention, a subject suffering from, or at risk of developing, TTP and being treated with a MASP-2 inhibitory composition of the present invention can be monitored by periodically, for example, every twelve hours or daily, the level of at least one complement factor, wherein determination of a decreased level of at least one complement factor compared to a standard value, or a value in a healthy control subject, indicates the need for continued treatment with the composition.

In both prophylactic and therapeutic regimens, compositions containing MASP-2 inhibitory agents can be administered in multiple doses until sufficient therapeutic benefit is achieved in the subject. In one embodiment of the invention, the MASP-2 inhibitory agent is an anti-MASP-2 antibody, which can preferably be administered to an adult patient (for example, an adult patient with an average body weight of 70 kg) at a dose of 0.1 mg to 10,000 mg, more preferably from 1.0 mg to 5000 mg, more preferably from 10.0 mg to 2000 mg, more preferably from 10.0 mg to 1000 mg and even more preferably from 50.0 mg to 500 mg. For young patients, the dose can be adjusted in proportion to the patient's body weight. The use of the MASP-2 inhibitory compositions of the present invention can be accomplished by a single administration of the composition or by a limited number of sequential administrations for the treatment of TTP. Alternatively, the composition may be administered at periodic intervals, for example, daily, twice weekly, weekly, biweekly, monthly or bimonthly, over an extended period of time to treat TTP.

In some embodiments, the subject suffering from TTP has previously received, or is currently receiving, treatment with a terminal complement inhibitor that inhibits the breakdown of complement protein C5. In some embodiments, the method includes administering to the subject a composition of the invention comprising a MASP-2 inhibitor, and additionally administering to the subject a terminal complement inhibitor that inhibits the breakdown of complement protein C5. In some embodiments, the terminal complement inhibitor is a humanized anti-C5 antibody, or an antigen-binding fragment thereof. In some embodiments, the terminal complement inhibitor is eculizumab.

VI. Examples.

The following examples merely illustrate the best way to put the invention into practice.

- 71 046178 tenii, but are not intended to limit the invention. All literature cited herein is expressly incorporated herein by reference.

Example 1.

This example describes the generation of a line of mice deficient in MASP-2 (MASP-2-/-), but with sufficient amounts of MAp19 (MAp19+/+).

Materials and methods.

The pKO-NTKV 1901 targeting vector was constructed to disrupt the three exons encoding the C-terminal portion of murine MASP-2, including the exon encoding the serine protease domain, as shown in FIG. 3. PKO-NTKV 1901 was used to transfect the mouse ES cell line E14.1a (SV129 Ola). Neomycin-resistant and thymidine kinase-sensitive clones were selected. 600 ES clones were screened and four different clones were selected that were confirmed by Southern blotting to contain the expected targeted recombination event as shown in FIG. 3. From these four positive clones, chimeras were obtained by embryo transfer. The chimeras were then backcrossed onto the C57/BL6 genetic background to produce transgenic males. Transgenic males were crossed with females to produce F1 progeny in which 50% of the progeny showed heterozygosity for the disrupted MASP-2 gene. Heterozygous mice were crossed with each other to produce homozygous MASP-2-deficient offspring in a ratio of 1:2:1 to heterozygous and wild-type mice, respectively.

Results and Phenotype: The resulting homozygous MASP-2-/- deficient mice were found to be viable and fertile and MASP-2 deficient by Southern blot analysis to confirm the correct targeting event, by Northern blot analysis to confirm absence of MASP-2 mRNA, and by Western blotting to confirm the absence of MASP-2 protein (data not shown). The presence of MAp19 mRNA and the absence of MASP-2 mRNA were further confirmed using time-resolved RT-PCR on a LightCycler instrument. MASP-2 −/− mice continued to express MAp19, MASP-1, and MASP-3 mRNA and protein as expected (data not shown). The presence and relative abundance in mice of MASP-2-/mRNA for properdin, factor B, factor D, C4, C2, and C3 were assessed in a LightCycler assay and found to be identical to those of wild-type control littermates (data not available). presented). Lectin pathway-mediated complement activation was completely absent in the plasma of homozygous MASP-2 −/− mice, as further described in Example 2.

Establishment of the MASP-2-/- line on a pure C57BL6 background: MASP-2-/- mice were backcrossed with pure C57BL6 mice for nine generations before using the MASP2-/- line as an experimental animal model.

A transgenic line of mice was also generated that were MASP-2-/-, MAp19+/+ for mouse proteins and expressed the human MASP-2 transgene (mouse MASP-2 knockout and human MASP-2 knockin) as follows.

Materials and methods.

A minigene encoding human MASP-2, called mini hMASP-2 (SEQ ID NO: 49), shown in FIG. 4, which includes the promoter region of the human MASP2 gene, including the first 3 exons (exon 1 to exon 3), followed by a cDNA sequence representing the coding sequence of the next 8 exons, thus encoding the full-length MASP-2 protein under the control of its endogenous promoter. The hMASP-2 miniconstruct was injected into fertilized eggs of MASP-2 −/− mice to replace the missing mouse MASP-2 gene with an expressed human MASP-2 transgene.

Example 2.

This example demonstrates that MASP-2 is required for complement activation through the lectin pathway.

Materials and methods.

Lectin Pathway Specific C4 Cleavage Assay: The C4 cleavage assay is described in Petersen, et al., J. Immunol. Methods 257:107 (2001), this assay measures activation of the lipoteichoic acid (LTA) lectin pathway from S. aureus, which binds L-ficolin. The assay described by Petersen et al (2001) was adapted to measure lectin pathway activation through MBL by coating the plate with LPS and mannan or zymosan, followed by the addition of serum from MASP-2-/- mice as described below. The assay was also modified to eliminate the possibility of C4 cleavage due to the classical pathway. This was achieved by using a sample dilution buffer containing 1 M NaCl, which allows high-affinity binding of lectin pathway recognition components to their ligands but prevents activation of endogenous C4, thereby eliminating the participation of the classical pathway through dissociation of the C1 complex. Briefly, in a modified assay, serum samples (diluted in high salt buffer (1 M NaCl)) are added to ligand-coated plates, followed by the addition of a constant amount of purified C4 in physiological salt concentration buffer. Associated complexes

- 72 046178 recognitions containing MASP-2 cleave C4, resulting in the deposition of C4b.

Analytical methods.

1. Nunc Maxisorb microtiter plates (Maxisorb, Nunc, catalog no. 442404, Fisher Scientific) were coated with 1 μg/mL mannan (M7504 Sigma) or any other ligand (eg, such as those listed below) diluted in buffer for application (15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ , pH 9.6).

The following reagents were used in the analysis:

a) mannan (1 µg/well of mannan (M7504 Sigma) in 100 µl loading buffer):

b) zymosan (1 µg/well zymosan (Sigma) in 100 µl loading buffer);

c) LTA (1 µg/well in 100 µl loading buffer or 2 µg/well in 20 µl methanol);

d) 1 μg of H-ficolin-specific mAb 4H5 in loading buffer;

e) PSA from Aerococcus viridans (2 µg/well in 100 µl loading buffer);

f) 100 µl/well of formalin-fixed S. aureus DSM20233 (OD ₅₅₀ =0.5) in loading buffer.

2. The plates were incubated overnight at 4°C.

3. After overnight incubation, the remaining protein binding sites were saturated by incubating the plates with 0.1% HSA-TBS blocking buffer (0.1% (w/v) HSA in 10 mM Tris-Cl, 140 mM NaCl, 1. 5 mM NaN ₃ , pH 7.4) for 1-3 h, followed by washing the plates 3X TBS/Tween/Ca ²⁺ (TBS with 0.05% Tween 20 and 5 mM CaCl ₂ , 1 mM MgCl ₂ , pH 7.4).

4. Test serum samples were diluted in MBL binding buffer (1 M NaCl), the diluted samples were added to the plates and incubated overnight at 4°C. Wells containing only buffer were used as negative controls.

5. After incubation overnight at 4°C, the plates were washed with 3X TBS/Tween/Ca ⁺ . Human C4 (100 μl/well, at a concentration of 1 μg/ml, diluted in BBS (4 mM barbital, 145 mM NaCl, 2 mM CaCl ₂ , 1 mM MgCl ₂ , pH 7.4)) was then added to the plates and incubated in for 90 minutes at 37°C. The plates were washed again with 3X TBS/Tween/Ca ²⁺ .

6. Deposition of C4b was detected using alkaline phosphatase-conjugated chicken anti-human C4c antibodies (diluted 1:1000 in TBS/Tween/Ca ²⁺ ), which were added to the plates and incubated for 90 min at room temperature. The plates were then washed again with 3X TBS/Tween/Ca ²⁺ .

7. Alkaline phosphatase was detected by adding 100 µl of n-nitrophenyl phosphate substrate solution, incubating at room temperature for 20 min and reading at OD ₄₀₅ on a plate reader.

Results.

In fig. 5A, B shows the amount of C4b deposited on mannan (Fig. 5A) and zymosan (Fig. 5B) in diluted serum from MASP-2+/+ (crosses), MASP-2+/- (filled circles) and MASP-2 mice /- (shaded triangles). In fig. 5C shows the relative activity of C4 convertase on zymosan (white bars) or mannan (shaded bars) coated plates in MASP-2-/+ mice (n=5) and MASP-2-/- mice (n=4). relative to wild-type mice (n=5), based on measurements of the amount of deposited C4b normalized to wild-type serum. Error bars correspond to standard deviation. As shown in FIG. 5A-C, plasma from MASP-2-/complete mice lacks lectin pathway-mediated complement activation on mannan-zymosan-coated plates. These results clearly indicate that MASP-2 is an effector component of the lectin pathway.

Recombinant MASP-2 restores lectin pathway-dependent activation of C4 in the serum of MASP-2 −/− mice.

To confirm that the absence of MASP-2 was the direct cause of the loss of lectin pathway-dependent C4 activation in MASP-2 −/− mice, the effect of adding recombinant MASP-2 protein to serum samples was examined in the C4 cleavage assay described above. Recombinant proteins: functionally active mouse MASP-2 and catalytically inactive mouse MASP-2A (in which the serine residue in the active site of the serine protease domain was replaced by an alanine residue) were prepared and purified as described below in Example 3. Pooled serum from 4 MASP mice -2−/− were preincubated with recombinant mouse MASP-2 or inactive recombinant mouse MASP-2A at increasing protein concentrations, and C4 convertase activity was assayed as described above.

Results.

As shown in FIG. 6, addition of functionally active murine recombinant MASP-2 protein (open triangles) to serum obtained from MASP-2 −/− mice resulted in restoration of lectin pathway-dependent C4 activation in a protein concentration-dependent manner, whereas catalytically inactive murine MASP-2A protein (asterisks) did not restore C4 activation. The results presented in Fig. 6 are normalized to the C4 activation observed in pooled sera from wild-type mice (dashed line).

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Example 3.

This example describes recombinant expression and protein production for recombinant full-length human, rat and mouse MASP-2, derived from MASP-2 polypeptides and catalytically inactivated mutant forms of MASP-2.

Expression of full-length human, mouse, and rat MASP-2.

The full-length cDNA sequence of human MASP-2 (SEQ ID NO: 4) was also subcloned into the mammalian expression vector pCI-Neo (Promega), which drives expression under the control of the CMV enhancer/promoter region in eukaryotic cells (described in Kaufman R.J. et al., Nucleic Acids Research 19:4485-90, 1991; Kaufman, Methods in Enzymology, 185:537-66 (1991)). The full-length mouse cDNA (SEQ ID NO: 50) and rat cDNA (SEQ ID NO: 53) of MASP-2 were each subcloned into the pED expression vector. Expression vectors for MASP-2 were then transfected into the adherent Chinese hamster ovary cell line DXB1 using the standard calcium phosphate transfection procedure described in Maniatis et al., 1989. Cells transfected with these constructs grew very slowly, indicating that the encoded protease is cytotoxic.

Using a different approach, a minigene construct (SEQ ID NO: 49) containing human MASP-2 cDNA under the control of its endogenous promoter was transiently transfected into Chinese hamster ovary (CHO) cells. Human MASP-2 protein was secreted into the culture medium and isolated as described below.

Expression of full-length catalytically inactive MASP-2.

Rationale.

MASP-2 is activated by autocatalytic cleavage after MBL recognition subcomponents or ficolins (L-ficolin, H-ficolin, or M-ficolin) bind to their respective carbohydrate pattern. Autocatalytic cleavage leading to activation of MASP-2 often occurs during the isolation of MASP-2 from serum, or during purification following recombinant expression. In order to obtain a more stable protein preparation for use as an antigen, a catalytically inactive form of MASP-2, termed MASP-2A, was created by replacing the serine residue present in the catalytic triad of the protease domain with an alanine residue in rat (SEQ ID NO: 55 Ser617 to A ^617); in mouse (SEQ ID NO: 52 Ser617 on Al3617); or in human (SEQ ID NO: 3 Ser618 to Ala618) protein.

To obtain catalytically inactive human and mouse MASP-2A proteins, site-directed mutagenesis was performed using the oligonucleotides given in Table. 5.

Oligonucleotides in table. 5 were designed to anneal a region of human and mouse cDNA encoding enzymatically active serine and an oligonucleotide containing a mismatch to change a serine codon to an alanine codon. For example, PCR oligonucleotides SEQ ID NO: 56-59 were used in combination with human MASP-2 cDNA (SEQ ID NO: 4) to amplify the region from the start codon to the enzymatically active serine and from the serine to the stop codon, to create a completely open reading frames from mutant MASP-2A containing the mutation Ser618 to Al;-1618. PCR products were purified after agarose gel electrophoresis and band processing, and single adenosine overlays were generated using a standard chain extension method. MASP-2A with an adenosine tail was then cloned into the pGEM-T easy vector and transformed into E. coli.

Catalytically inactive rat MASP-2A protein was prepared by kinase-annealing SEQ ID NO: 64 and SEQ ID NO: 65, combining the two oligonucleotides in equal molar amounts, heating at 100°C for 2 min and slowly cooling to room temperature. The resulting annealed fragment has Pst1 and Xba1 compatible ends and was inserted in place of the Pst1-Xba1 fragment of wild-type rat MASP-2 cDNA (SEQ ID NO: 53), yielding rat MASP-2A.

'GAGGTGACGCAGGAGGGGCATTAGTGTTT 3' (SEQ ID NO: 64).

5' CTAGAAACACTAATGCCCCTCCTGCGTCACCTCTGCA 3' (SEQ ID NO: 65).

Human, mouse and rat MASP-2A were each further subcloned into one of the mammalian expression vectors pED or pCI-Neo and transfected into DXB1 Chinese hamster ovary cells as described below.

Using a different approach, a catalytically inactive form of MASP-2 was created using the method described in Chen et al., J. Biol. Chem., 276(28):25894-25902, 2001. Briefly, a plasmid containing the full-length human MASP-2 cDNA (described by Thiel et al., Nature 386:506, 1997) was digested with Xho1 and EcoR1, and the MASP-2 cDNA (described herein as SEQ ID NO: 4) was cloned into the appropriate restriction sites of the baculovirus transfer vector pFastBacl (Life Technologies, NY). Ser618 in the active site of the serine protease MASP-2 was then replaced with A1a618, replacing double-stranded oligonucleotides encoding the peptide region of amino acids 610-625 (SEQ ID NO: 13), the natural region of amino acids 610-625, producing a full-length MASP-2 polypeptide with an inactive protease domain .

Construction of expression plasmids containing polypeptide regions from human Masp-2.

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The following constructs were prepared using the MASP-2 signal peptide (residues 1 to 15 in SEQ ID NO: 5) to secrete different domains of MASP-2. A construct for expressing the CUBI domain of human MASP-2 (SEQ ID NO: 8) was generated by PCR amplification of the region encoding residues 1-121 in MASP-2 (SEQ ID NO: 6) (corresponding to the N-terminal domain of CUB1). A construct expressing the CUBIEGF domain of human MASP-2 (SEQ ID NO: 9) was generated by PCR amplification of the region encoding residues 1-166 of MASP-2 (SEQ ID NO: 6) (corresponding to the N-terminal domain of CUBIEGF). A construct expressing the CUBIEGFCUBII domain of human MASP-2 (SEQ ID NO: 10) was generated by PCR amplification of the region encoding residues 1-293 in MASP-2 (SEQ ID NO: 6) (corresponding to the N-terminal domain of CUBIEGFCUBII). The above domains were amplified by PCR using VentR polymerase and pBS-MASP-2 as a template, in accordance with known PCR methods. The 5' sense primer sequence (5'-CGGGATCCATGAGGCTGCTGACCCTC-3' seq id NO: 34) introduces a restriction site.

BamHI (underlined) at the 5' end of the PCR products. Antisense primers for each of the MASP-2 domains shown in Table 1 below. 5 are designed to introduce a stop codon (in bold) after the EcoRI site (underlined) at the end of each PCR product. After amplification, the DNA fragments were digested with BamHI and EcoRI and cloned into the corresponding sites of the pFastBac1 vector. The resulting constructs were characterized by restriction mapping and confirmed by dsDNA sequencing.

Table 5

Primers for MASP-2 PCR

MASP-2 domain 5' primer for PCR Z'-primer for PCR SEQ ID NO: 8 CUBI (ac 1-121 in SEQ ID NO: 6) 5'CGGGATCCATGAGGCTGCTG ACCTS 3' (SEQ ID NO: 34) 5'GGAKTTCCTAGGCTGC ATA (SEQ ID NO: 35) SEQ ID NO: 9 CUBIEGF (ak 1-166 in SEQ ID NO: 6) 5'CGGGATCCATGAGGCTGCTG ACCCTC 3' (SEQ ID NO: 34) 5'GGAKTTCCTACAGGG CGCT 3' (SEQ ID NO: 36) SEQ ID NO: 10 CUBIEGFCUBII (ak 1293 in SEQ Ш NO: 6) 5'CGGGATCCATGAGGCTGCTG ACCCTC 3' (SEQ ID NO: 34) 5'GGAKTTCCTAGTAGTG GAT 3' (SEQ ID NO: 37) SEQ ID NO: 4 human MASP-2 5'ATGAGGCTGCTGACCCTCCTG GGCCTTC 3' (SEQ ID NO: 56) yMA8R-2_direct 5'TTAKAKTCACTAKTTA TGTTCTCGATC 3' (SEQ ID NO: 59) hMASP2_reverse SEQ ID NO: 4 cDNAs of human MASP-2 5'CAGAGGTGACGCAGGAGGGG CAC 3' (SEQ ID NO: 58) hMASP2_a1a_direct 5'GTGCCCCTCCTGCGTC ACCTCTG 3' (SEQ ID NO: 57) hMASP- 2_a1a_reverse SEQ ID NO: 50 mouse cDNA MASP-2 5'ATGAGGCTACTCATCTTCCTG G3' (SEQ ID NO: 60) mMASP2_direct 5'TTAGAKATTACTTATT ATGTTCTCAKTCC3' (SEQ ID NO: 63) mMASP2_reverse SEQ ID NO: 50 mouse cDNA MASP-2 5'CCCCCCCTGCGTCACCTCTGC AG3' (SEQ ID NO: 62) mMASP2_a1a_direct 5'CTGCAGAGGTGACGC AGGGGGGG 3' (SEQ ID NO: 61) mMASP- 2_a1a_reverse

Recombinant expression of MASP-2 in eukaryotic cells, and production of enzymatically inactive murine, rat and human MASP-2A proteins

The MASP-2 and MASP-2A expression constructs described above were transfected into DXB1 cells by the standard calcium phosphate transfection method (Maniatis et al., 1989). MASP-2A was prepared in serum-free medium to ensure that the preparations were not contaminated by other serum proteins. The medium was collected from confluent cells every other day (four times in total). The level of recombinant MASP-2A averaged approximately 1.5 mg/liter of culture medium for each of the three protein species.

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Purification of MASP-2A protein.

MASP-2A (Ser-Ala mutant described above) was purified by affinity chromatography on MBP-A-agarose columns. This strategy allowed for rapid purification without the use of additional tags. MASP-2A (100-200 ml of medium diluted with an equal volume of loading buffer (50 mM Tris-Cl, pH 7.5, containing 150 mM NaCl and 25 mM CaCl ₂ )) was applied to a MBRagarose affinity column (4 ml), pre-equilibrated with 10 ml loading buffer. After washing with an additional 10 ml of loading buffer, the protein was eluted in 1-ml fractions of 50 mM Tris-Cl, pH 7.5, containing 1.25 M NaCl and 10 mM EDTA. Fractions containing MASP-2A were identified by SDS-polyacrylamide gel electrophoresis. If necessary, MASP-2A was further purified by ion exchange chromatography on a MonoQ column (HR 5/5). The protein was dialyzed into 50 mM Tris-Cl buffer, pH 7.5, containing 50 mM NaCl and applied to a column equilibrated with the same buffer. After washing, bound MASP-2A was eluted with a 0.05-1 M NaCl gradient in a volume of 10 ml.

Results.

A yield of 0.25-0.5 mg of MASP-2A protein was obtained from 200 ml of medium. The molecular mass of 77.5 kDa determined by MALDI-MS was higher than the calculated value for the unmodified polypeptide (73.5 kDa) due to glycosylation. The attachment of glycans at each of the Nglycosylation sites was responsible for the observed mass. MASP-2A migrates as a single band on SDS-polyacrylamide gels, indicating that it was not proteolytically degraded during biosynthesis. The weighted average molecular weight determined by equilibrium ultracentrifugation was consistent with the calculated value for homodimers of the glycosylated polypeptide.

Production of recombinant human Masp-2 polypeptides.

Another method for producing recombinant MASP-2 and MASP-2A polypeptides is described in Thielens, N.M., et al., J. Immunol. 166:5068-5077, 2001. Briefly, Spodoptera frugiperda insect cells (Ready-Plaque Sf9 cells obtained from Novagen, Madison, WI) were grown and maintained in serum-free Sf900II medium (Life Technologies) supplemented with 50 IU/ml penicillin and 50 mg/ml streptomycin (Life Technologies). Trichoplusia ni (High Five) insect cells (provided by Jadwiga Chroboczek, Institut de Biologie Structurale, Grenoble, France) were maintained in TC100 medium (Life Technologies) containing 10% FBS (Dominique Dutscher, Brumath, France) supplemented with 50 IU/ml penicillin and 50 mg/ml streptomycin. Recombinant baculoviruses were produced using the Bacto-Bac system (Life Technologies). Bacmid DNA was purified using the Qiagen midiprep purification system (Qiagen) and used to transfect Sf9 insect cells with cellfectin in Sf900 II SFM medium (Life Technologies) according to the manufacturer's protocol. Recombinant virus particles were harvested after 4 days, titrated in a viral plaque assay, and amplified as described by King and Possee, The Baculovirus Expression System: ALaboratory Guide, Chapman and Hall Ltd., London, pp. 111-114, 1992.

High Five cells (1.75x107 cells/175- ^cm2 tissue culture flask) were infected with recombinant viruses containing MASP-2 polypeptide sequences at an MOI of 2 in Sf900 II SFM medium at 28°C for 96 hours. Supernatants were collected. by centrifugation, and add diisopropylphosphorofluoridate to a final concentration of 1 mM.

MASP-2 polypeptides are secreted into the culture medium. Culture supernatants are dialyzed against 50 mM NaCl, 1 mM CaCl ₂ , 50 mM triethanolamine hydrochloride, pH 8.1, and applied at a rate of 1.5 ml/min to a Q-Sepharose Fast Flow column (Amersham Pharmacia Biotech) (2.8 x 12 cm ), balanced in the same buffer. Elution was carried out using a 1.2 liter linear gradient to 350 mM NaCl in the same buffer. Fractions containing recombinant MASP-2 polypeptides were identified by Western blotting, precipitated by adding (NH ₄ ) ₂ SO ₄ to 60% (wt/vol) and left overnight at 4°C. The pellets were resuspended in a buffer containing 145 mM NaCl, 1 mM CaCl ₂ , 50 mM triethanolamine hydrochloride, pH 7.4, and applied to a TSK G3000 SWG (7.5 x 600 mm) column (Tosohaas, Montgomeryville, PA) equilibrated with the same buffer . The purified polypeptides are then concentrated to 0.3 mg/ml by ultrafiltration in Microsep microconcentrators (MW cut-off = 10,000) (Filtron, Karlstein, Germany).

Example 4.

This example describes a method for producing polyclonal antibodies against MASP2 polypeptides.

Materials and methods.

MASP-2 antigens.

Anti-human MASP-2 polyclonal immune serum is prepared by immunizing rabbits with the following isolated MASP-2 polypeptides: human MASP-2 (SEQ ID NO: 6) isolated from serum; recombinant human MASP-2 (SEQ ID NO: 6), MASP-2A containing an inactive protease domain (SEQ ID NO: 13), as described in example 3; and recombinant CUBI (SEQ ID NO: 8), CUBEGFI (SEQ ID NO: 9) and CUBEGFCUBII (SEQ ID NO: 10), expressed

- 76 046178 mi, as described above in example 3.

Polyclonal antibodies: Six-week-old rabbits primed with BCG (Bacillus Calmette-Guerin) vaccine are immunized by injecting 100 μg of 100 μg/ml MASP-2 polypeptide in sterile saline solution. Injections are performed every 4 weeks, and the antibody titer is determined by ELISA as described in Example 5. Culture supernatants are collected for antibody purification by protein A affinity chromatography.

Example 5.

This example describes a method for producing mouse monoclonal antibodies against rat or human MASP-2 polypeptides.

Materials and methods.

Male A/J mice (Harlan, Houston, Tech.) 8-12 weeks of age were given a subcutaneous injection of 100 μg of human or rat rMASP-2 or rMASP-2A polypeptides (prepared as described in Example 3) in Freund's complete adjuvant. (Difco Laboratories, Detroit, Mich.) in 200 μl phosphate-buffered saline (PBS), pH 7.4. At two-week intervals, mice were given two subcutaneous injections of 50 μg of human or rat rMASP-2 or rMASP-2A polypeptides in Freund's incomplete adjuvant. In the fourth week, mice are injected with 50 μg of human or rat rMASP-2 or rMASP-2A polypeptides in PBS, and cell fusion is performed 4 days later.

For each fusion, single-cell suspensions are prepared from the spleen of an immunized mouse and used to fuse with Sp2/0 myeloma cells.

Fusion of 5x108 Sp2/0 cells and 5x108 spleen cells was performed in a medium containing 50% polyethylene glycol (M.W. 1450) (Kodak, Rochester, N.Y.) and 5% dimethyl sulfoxide (Sigma Chemical Co., St. Louis, Mo.). The cells are then adjusted to a concentration of 1.5 x 105 spleen cells per 200 μl suspension in Iscove's medium (Gibco, Grand Island, N.Y.) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.1 mM hypoxanthine , 0.4 µM aminopterin and 16 µM thymidine. Two hundred microliters of cell suspension is added to each well of approximately twenty 96-well culture plates. After approximately ten days, culture supernatants are collected for screening for reactivity with purified MASP-2 factor in an ELISA assay.

ELISA assay.

Wells of Immulon 2 titration plates (Dynatech Laboratories, Chantilly, Va.) are coated by adding 50 μl of purified hMASP-2 or rat rMASP-2 (or rMASP-2A) at a concentration of 50 ng/ml overnight at room temperature. The low concentration of MASP-2 coating allows the selection of high-affinity antibodies. After removing the coating solution by shaking the plates, add 200 µl of BLOTTO (non-fat dry milk) in PBS to each well for one hour to block non-specific sites. After one hour, the wells are washed with PBST buffer (PBS containing 0.05% Tween 20). Fifty microliters of culture supernatants from each well of confluent cells are collected and mixed with 50 μl of BLOTTO and then added to individual wells of titration plates. After one hour of incubation, the wells are washed with PBST. Bound mouse antibodies were detected by reaction with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Fc-specific) antibodies (Jackson ImmunoResearch Laboratories, West Grove, Pa.) diluted 1:2000 in BLOTTO. A peroxidase substrate solution containing 0.1% 3.3.5.5-tetramethylbenzidine (Sigma, St. Louis, Mo.) and 0.0003% hydrogen peroxide (Sigma) was added to color development wells for 30 min. The reaction is stopped by adding 50 µl of 2 M H2SO4 to each well. The absorbance at 450 nm of the reaction mixture was determined using a BioTek ELISA reader (BioTek Instruments, Winooski, Vt.).

MASP-2 binding assay.

Culture supernatants positive by the MASP-2 ELISA assay described above can be tested in a binding assay to determine the binding affinity of MASP-2 inhibitory agents for MASP-2. A similar assay can also be used to determine whether inhibitory agents bind to other antigens in the complement system.

Wells of polystyrene titre plates (96-well plates, medium binding, Corning Costar, Cambridge, MA) are coated with MASP-2 (20 ng/100 μl/well, Advanced Research Technology, San Diego, CA) in phosphate-buffered saline (PBS). ), pH 7.4, overnight at 4°C. After removing the MASP-2 solution by aspiration, the wells were blocked with PBS containing 1% bovine serum albumin (BSA; Sigma Chemical) for 2 h at room temperature. Wells without coated MASP-2 serve as background controls. Aliquots of hybridoma supernatants, or purified anti-MASP-2 moAbs at different concentrations in blocking solution, are added to the wells. After incubation for 2 hours at room temperature, the wells are washed extensively with PBS. MASP-2-bound anti-MASP-2 moAbs are detected by adding peroxidase-conjugated goat anti-mouse IgG (Sigma Chemical) in blocking solution, which are allowed to incubate for 1 h at room temperature.

- 77 046178 temperature. The plates are again washed extensively with PBS and 100 μl of 3,3',5,5'tetramethylbenzidine (TMB) substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) is added. The TMB reaction was quenched by adding 100 μl of 1 M phosphoric acid, and plates were read at 450 nm on a plate reader (SPECTRA MAX 250, Molecular Devices, Sunnyvale, CA).

Culture supernatants from positive wells are then tested for their ability to inhibit complement activation in a functional assay, such as the C4 cleavage assay described in Example 2. Cells in positive wells are then cloned by limiting dilution. The moAb is again tested for its ability to react with hMASP-2 in the ELISA assay described above. Selected hybridomas are grown in spinner flasks and the spent culture supernatant is collected for antibody purification by protein A affinity chromatography.

Example 6.

This example describes the creation and production of humanized mouse anti-MASP-2 antibodies and antibody fragments.

A mouse anti-MASP-2 monoclonal antibody is prepared by immunizing male A/J mice as described in Example 5. The mouse antibody is then humanized as described below to reduce its immunogenicity by replacing the mouse antibody constant regions with their human counterparts, producing a chimeric An IgG and Fab fragment of an antibody that can be used to inhibit the deleterious effects of MASP-2-dependent complement activation in human subjects in accordance with the present invention.

1. Cloning of anti-MASP-2 variable region genes from mouse hybridoma cells.

Total RNA was isolated from anti-MASP-2 moAb-secreting hybridoma cells (prepared as described in Example 7) using RNAzol according to the manufacturer's protocol (Biotech, Houston, Tech.). First-strand cDNA is synthesized from total RNA using oligo(dT) primers. PCR is carried out using 3' primers derived from the immunoglobulin constant region and degenerate primer sets derived from the leader peptide or first framework genes of mouse VH or VK as 5' primers. Anchored PCR was performed as described by Chen and Platsucas (Chen, P.F., Scand. J. Immunol. 35:539-549, 1992). To clone the VK gene, double-stranded cDNA was prepared using the primer Notl-MAK1 (5'TGCGGCCGCTGTAGGTGCTGTCTTT-3'SEQ ID NO: 38). Annealed adapters AD1 (5'GGAATTCACTCGTTATTCTCGGA-3' SEQ ID NO: 39) and AD2 (5'-TCCGAGAATAACGAGTG-3' SEQ ID NO: 40) are ligated at both the 5' and 3' ends of the double-stranded cDNA. Adapters at the 3' ends are removed by Notl cleavage. The digestion product is then used as a template in a PCR with oligonucleotide AD1 as the 5' primer and MAK2 (5'-CATTGAAAGCTTTGGGGTAGAAGTTGTTC-3' SEQ ID NO: 41) as the 3' primer. DNA fragments approximately 500 bp in size. cloned into pUC19. Several clones are selected for sequence analysis to confirm that the cloned sequence includes the expected murine immunoglobulin constant region. Oligonucleotides Not1-MAK1 and MAK2 are derived from the VK region and are located 182 and 84 bp, respectively, downstream of the first nucleotide pair of the C kappa gene. Clones containing the complete VK and leader peptide are selected.

To clone the VH gene, a double-stranded cDNA was prepared using the primer Not1 MAG1 (5'-CGCGGCCGCAGCTGCTCAGAGTGTAGA-3' SEQ ID NO: 42). The annealed adapters AD1 and AD2 are ligated at both the 5' and 3' ends of the double-stranded cDNA. Adapters at the 3' ends are removed by Not1 cleavage. The digestion product is then used as a template in PCR with oligonucleotide AD1 and MAG2 (5'-CGGTAAGCTTCACTGGCTCAGGGAAATA-3' SEQ ID NO: 43) as primers. DNA fragments 500-600 bp long. cloned into pUC19. Oligonucleotides Not1-MAG1 and MAG2 are derived from the region of the mouse Cy.7.1 gene and are located 180 and 93 bp, respectively, downstream of the first bp. mouse gene Cy.7.1. Clones containing the complete VH and leader peptide are selected.

2. Construction of expression vectors for chimeric anti-MASP-2 IgG and Fab.

The cloned VH and VK genes described above are used as templates in a PCR reaction to add a Kozak consensus sequence to the 5' end, and a splice donor site to the 3' end, of the nucleotide sequence. After sequence analysis to confirm the absence of PCR errors, the VH and VK genes are inserted into expression vector cassettes containing human C.y1 and C. kappa, respectively, resulting in pSV2neoVH-huCY1 and pSV2neoV-huCY. Plasmid DNAs of heavy and light chain vectors purified in a CsCl gradient are used to transfect COS cells by electroporation. After 48 hours, the culture supernatant is tested by ELISA to confirm the presence of approximately 200 ng/ml chimeric IgG. Cells are harvested and total RNA is obtained. First-strand cDNA is synthesized from total RNA using oligo(dT) as a primer. This cDNA is used as a template in PCR to produce Fd and kappa chain DNA fragments. For the Fd gene, PCR was performed using 5'-AAGAAGCTTGCCGCCACCATGGATTGGCTGTGGAACT-3' (SEQ ID NO: 44) as the 5' primer and a CH1-derived 3' primer (5'CGGGATCCTCAAACTTTCTTGTCCACCTTGG-3' SEQ ID NO: 45). The DNA sequence was confirmed to contain the complete VH domain and CH1 domain of human IgG1. After splitting it is necessary

- 78 046178 using common enzymes, Fd DNA fragments are inserted into the HindIII and BamHI restriction sites of the expression vector cassette pSV2dhfr-TUS, obtaining pSV2dhfrFd. Plasmid pSV2 is commercially available and consists of DNA segments from different sources: pBR322 DNA (thin line) contains the pBR322 DNA origin of replication (pBR ori) and a lactamase gene conferring ampicillin resistance (Amp); SV40 DNA, represented by wider shading and labeled, contains the SV40 DNA origin of replication (SV40 ori), the early promoter (5' of the dhfr and neo genes), and the polyadenylation signal (3' of the dhfr and neo genes) . The polyadenylation (pA) signal from SV40 is also placed at the 3' end of the Fd gene.

For the kappa chain gene, PCR was performed using 5'AAGAAAGCTTGCCGCCACCATGTTCTCACTAGCTCT-3' (SEQ ID NO: 46) as the 5' primer and CK-derived 3' primer (5'-CGGGATCCTTCTCCCTCTAACACTCT-3' SEQ ID NO: 47) . The DNA sequence is confirmed to contain the complete VK and CK regions of human immunoglobulin. After digestion with the appropriate enzymes, the kappa strand DNA fragments are inserted into the HindIII and BamHI restriction sites of the pSV2neo-TUS expression vector cassette, yielding pSV2neoK. Expression of both Fd and kappa chain genes is under the control of enhancer and promoter elements from HCMV. Since the Fd gene does not contain a codon for the cysteine amino acid residue involved in the interchain disulfide bond, this recombinant chimeric Fab contains non-covalently linked heavy and light chains. This chimeric Fab is designated cFab.

To produce a recombinant Fab with a disulfide bond between the heavy and light chains, the above Fd gene can be expanded to include the coding sequence for an additional 9 amino acids (EPKSCDKTH SEQ ID NO: 48) from the hinge region of human IgG1. The BstEII-BamHI DNA segment encoding 30 amino acids at the 3' end of the Fd gene can be replaced with DNA segments encoding an expanded Fd to produce pSV2dhfrFd/9aa.

3. Expression and purification of chimeric anti-MASP-2 IgG.

To generate cell lines secreting chimeric anti-MASP-2 IgG, NSO cells were transfected with purified pSV2neoVH-huC.Y1 and pSV2neoV-huC.kappa plasmid DNAs by electroporation. Transfected cells were selected in the presence of 0.7 mg/ml G418. Cells are grown in 250-ml spinner flasks using serum-free medium.

The culture supernatant with 100 ml of spin flask culture was applied to a 10 ml PROSEP-A column (Bioprocessing, Inc., Princeton, N.J.). The column is washed with 10 bed volumes of PBS. The bound antibody is eluted with 50 mM citrate buffer, pH 3.0. An equal volume of 1 M Hepes, pH 8.0, is added to the fraction containing the purified antibody to adjust the pH to 7.0. Residual salts are removed by buffer exchange with PBS using ultrafiltration on a Millipore membrane (MW cut-off: 3000). The protein concentration of the purified antibody is determined by the BCA method (Pierce).

4. Expression and purification of chimeric anti-MASP-2 Fab.

To obtain cell lines secreting chimeric anti-MASP-2 Fab, CHO cells are transfected with purified plasmid DNAs pSV2dhfrFd (or pSV2dhfrFd/9aa) and pSV2neokappa by electroporation. Transfected cells are selected in the presence of G418 and methotrexate. Selected cell lines are propagated at increasing concentrations of methotrexate. Cells are subcloned into single cells using the limiting dilution method. The high-producing, single-cell cloned cell lines are then grown in 100 ml spinner flasks using serum-free media.

Chimeric anti-MASP-2 Fabs are purified by affinity chromatography using a mouse anti-idiotypic anti-MASP-2 moAb. An anti-idiotypic anti-MASP-2 moAb can be generated by immunizing mice with a mouse anti-MASP-2 moAb conjugated to limpet hemocyanin (KLH) and screening for a specific binding moAb that can compete with human MASP-2. To purify, 100 ml of supernatant from spin-flask cultured CHO cells producing cFab or cFab/9aa is applied to an affinity column with a bound anti-idiotypic moAb to anti-MASP-2 moAb. The column is then thoroughly washed with PBS, after which the bound Fab is eluted with 50 mM diethylamine, pH 11.5. Residual salts are removed by buffer exchange as described above. The protein concentration of purified Fabs was determined by the BCA method (Pierce).

The ability of chimeric anti-MASP-2 IgG, cFab and cFAb/9aa to inhibit MASP-2-dependent complement pathways can be determined in the inhibition assays described in Example 2 or Example 7.

Example 7.

This example describes an in vitro C4 cleavage assay used for functional screening to identify MASP-2 inhibitory agents capable of blocking MASP-2-dependent complement activation via L-ficolin/P35, H-ficolin, M-ficolin, or mannan.

C4 cleavage assay.

The C4 cleavage assay is described in Petersen, S.V., et al., J. Immunol. Methods 257:107, 2001, the assay measures lectin pathway activation resulting from the binding of lipoteichoic acid (LTA) from S. aureus to L-ficolin.

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Reagents.

Formalin-fixed S. aureous (DSM20233) was prepared as follows: bacteria were grown overnight at 37°C in tryptic soy blood medium, washed three times with PBS, then fixed for 1 h at room temperature in PBS/0.5% formaldehyde, and washed three more times with PBS, followed by resuspension in loading buffer (15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ , pH 9.6).

Analysis.

Wells of a Nunc MaxiSorb microtiter plate (Nalgene Nunc International, Rochester, NY) are coated with: 100 μl of formalin-fixed S. aureus DSM20233 (OD ₅₅₀ = 0.5) in loading buffer with 1 μg of L-ficolin in loading buffer. After overnight incubation, wells are blocked with 0.1% human serum albumin (HSA) in TBS (10 mM Tris-HCl, 140 mM NaCl, pH 7.4), then washed with TBS containing 0.05% Tween 20 and 5 mM CaCl ₂ (wash buffer). Human serum samples are diluted with a buffer of 20 mM Tris-HCl, 1 M NaCl, 10 mM CaCl ₂ , 0.05% Triton X-100, 0.1% HSA, pH 7.4, which prevents the activation of endogenous C4 and causes dissociation of the C1 complex ( consisting of C1q, C1r and C1s). MASP-2 inhibitory agents, including anti-MASP-2 moAbs and inhibitory peptides, are added to serum samples at various concentrations. The diluted samples are added to the plate and incubated overnight at 4°C. After 24 hours, the plates are washed thoroughly with wash buffer, then 0.1 μg of purified human C4 (prepared as described in Dodds, AW, Methods Enzymol. 223:46, 1993) in 100 μl of 4 mM barbital, 145 mM NaCl is added to each well. , 2 mM CaCl ₂ , 1 mM MgCl ₂ , pH 7.4. After 1.5 h at 37° C., the plates are washed again, C4b deposition is detected using alkaline phosphatase-conjugated chicken anti-human C4c antibodies (obtained from Immunsystem, Uppsala, Sweden) and measured using the colorimetric substrate p-nitrophenyl phosphate.

C4 analysis on mannan.

The assay described above is adapted to measure lectin pathway activation through MBL by coating the plate with LSP and mannan, followed by the addition of serum mixed with various MASP-2 inhibitory agents.

C4 analysis on H-ficolin (Hakata Ag).

The assay described above is adapted to measure lectin pathway activation through Nficolin by loading the plate with LSP and H-ficolin, followed by the addition of serum mixed with various MASP-2 inhibitory agents.

Example 8.

The following analysis demonstrates the presence of a classical activation pathway in wild-type and MASP-2-/- mice.

Methods.

Immune complexes were prepared in situ by coating microtiter plates (Maxisorb, Nunc, catalog no. 442404, Fisher Scientific) with 0.1% human serum albumin in 10 mM Tris, 140 mM NaCl, pH 7.4, for 1 hour at room temperature. followed by overnight incubation at 4°C with sheep anti-whole serum immune serum (Scottish Antibody Production Unit, Carluke, Scotland) diluted 1:1000 in TBS/Tween/Ca ²⁺ . Serum samples were obtained from wild-type and MASP-2 −/− mice and added to coated wells of the plate. Control samples were prepared in which C1q was depleted from wild-type and MASP-2 −/− serum samples. C1q-depleted mouse serum was prepared using protein A-linked Dynabeads (Dynal Biotech, Oslo, Norway) coated with rabbit anti-human C1q IgG (Dako, Glostrup, Denmark) according to the supplier's instructions. The plates were incubated for 90 min at 37°C. Bound C3c was detected using polyclonal anti-human C3c antibody (Dako A 062) diluted 1:1000 in TBS/Tween/Ca. The secondary antibody was goat anti-rabbit IgG.

Results.

In fig. 7 shows the relative levels of C3b deposition on IgG-coated plates in wild-type serum, MASP-2-/- serum, C1q-depleted wild-type serum, and C1q-depleted MASP-2-/- serum. These results demonstrate that the classical pathway is intact in MASP-2 −/− mice.

Example 9

The following assay is used to test whether a MASP-2 inhibitory agent blocks the classical pathway by analyzing the effect of the MASP-2 inhibitory agent under conditions in which initiation of the classical pathway by immune complexes occurs.

Methods.

To test the effect of a MASP-2 inhibitory agent on conditions of complement activation in which the classical pathway is initiated by immune complexes, 50-μl triplicate samples containing 90% SNPs were incubated at 37°C in the presence of 10 μg/ml immune complex (IC). or PBS, and samples (+/-IC) containing 200 nM anti-properdin monoclonal antibody were also tested in parallel in triplicate during incubation at 37°C. After two hours of incubation at

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37°C, 13 mM EDTA was added to all samples to stop further complement activation, and the samples were immediately cooled to 5°C. Samples were then stored at -70°C until assayed for complement activation products (C3 and sC5b-9) using ELISA kits (Quidel, catalog nos. A015 and A009) according to the manufacturer's instructions.

Example 10.

This example describes the identification of high affinity anti-MASP-2 Fab2 antibody fragments that block MASP-2 activity.

Background and rationale

MASP-2 is a complex protein with multiple distinct functional domains, including: binding site(s) for MBL and ficolins, serine protease catalytic site, binding site for proteolytic substrate C2, binding site for proteolytic substrate C4, MASP-2 cleavage site for autoactivation of MASP-2 zymogen and two Ca binding sites. Fab2 antibody fragments that bind with high affinity to MASP-2 were identified, and the identified Fab2 fragments were tested in a functional assay to determine whether they were able to block the functional activity of MASP-2.

To block the functional activity of MASP-2, the antibody or Fab2 fragment of the antibody must bind to and block a structural epitope on MASP-2 that is required for the functional activity of MASP-2. Thus, many or all of the high affinity anti-MASP-2 Fab2 fragments may not inhibit MASP-2 functional activity unless they bind structural epitopes on MASP-2 that are directly involved in MASP-2 functional activity. 2.

A functional assay measuring inhibition of lectin pathway C3 convertase formation was used to evaluate the blocking activity of anti-MASP-2 Fab2. It is known that the main physiological role of MASP-2 in the lectin pathway is to create the next functional component of the lectin-mediated complement pathway, namely the C3 convertase of the lectin pathway. The C3 convertase of the lectin pathway is an important enzymatic complex (C4bC2a) that proteolytically cleaves C3 into C3a and C3b. MASP-2 is not a structural component of the lectin pathway C3 convertase (C4bC2a); however, the functional activity of MASP-2 is required for the creation of two protein components (C4b, C2a) that constitute the C3 convertase of the lectin pathway. Moreover, all of the individual MASP-2 functional activities listed above appear to be required by MASP-2 to generate the lectin pathway C3 convertase. For this reason, the preferred assay for assessing the blocking activity of anti-MASP-2 Fab2 is a functional assay that measures inhibition of C3 convertase formation in the lectin pathway.

Preparation of high affinity Fab2.

A phage display library of human antibody light and heavy chain variable region sequences and automated antibody screening technology to identify Fab2s reactive with selected ligands of interest were used to generate high-affinity Fab2s for rat MASP-2 protein (SEQ ID NO: 55). A known amount of rat MASP-2 protein (~1 mg, >85% purity) was used for antibody screening. Three rounds of amplification were used to select antibodies with the best affinity. Approximately 250 different successful hits expressing antibody fragments were selected for ELISA screening. Successful high-affinity findings were subsequently sequenced to determine the uniqueness of the different antibodies.

Fifty unique anti-MASP-2 antibodies were purified, and 250 μg of each purified Fab2 antibody were used for MASP-2 binding affinity characterization and complement pathway functional testing, as described in more detail below.

Assays used to evaluate the inhibitory (blocking) activity of anti-MASP-2 Fab2 1. Assay to determine inhibition of lectin pathway C3 convertase formation.

Prerequisites.

Lectin pathway C3 convertase is an enzymatic complex (C4bC2a) that proteolytically cleaves C3 into two potent proinflammatory fragments, anaphylatoxin C3a and opsonin C3b. The formation of C3 convertase appears to be a key step in the lectin pathway for mediating inflammation. MASP-2 is not a structural component of the lectin pathway C3 convertase (C4bC2a); thus, anti-MASP-2 antibodies (or Fab2) will not directly inhibit the activity of preexisting C3 convertase. However, the activity of the serine protease MASP-2 is required for the formation of two protein components (C4b, C2a) that constitute the C3 convertase of the lectin pathway. As a consequence, anti-MASP-2 Fab2 that inhibits the functional activity of MASP-2 (ie, blocking α-anti-MASP-2 Fab2) will inhibit de novo formation of the lectin pathway C3 convertase. C3 contains an unusual and highly reactive thioether group in its structure. After C3 cleavage by C3 convertase in this assay, the thioester group on C3b can form a covalent bond with hydroxyl or amino groups on macromolecules immobilized at the bottom of plastic wells through ester or amide bonds, which facilitates the detection of C3b in ELISA.

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Yeast mannan is a known activator of the lectin pathway. In the following method for measuring C3 convertase production, plastic wells coated with mannan were incubated for 30 min at 37°C with diluted rat serum to activate the lectin pathway. The wells were then washed and analyzed for C3 immobilized in the wells using standard ELISA methods. The amount of C3b formed in this assay directly reflects the de novo formation of lectin pathway C3 convertase. Ahtu-MASP-2 Fab2 at selected concentrations were tested in this assay for their ability to inhibit C3 convertase formation and subsequent C3b formation.

Methods.

96-well Costar medium binding plates were incubated overnight at 5°C with mannan diluted in 50 mM carbonate buffer, pH 9.5, applied at 1 μg/50 μl/well. After overnight incubation, each well was washed three times with 200 μl of PBS. The wells were then blocked with 100 μl/well of 1% bovine serum albumin in PBS and incubated for one hour at room temperature with gentle mixing. Each well was then washed three times with 200 μl of PBS. Anti-MASP-2 Fab2 samples were diluted to selected concentrations in Ca ⁺⁺ and Mg'' buffer GVB (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgCl ₂ , 2.0 mM CaCl ₂ , 0.1% gelatin, pH 7.4) at 5°C. 0.5% rat serum was added to the above samples at 5°C, and 100 μl was transferred to each well. The plates were covered and incubated for 30 min in a water bath at 37°C to activate complement. The reaction was stopped by transferring the plates from a 37°C water bath to a container containing a mixture of ice and water. Each well was washed five times with 200 μl of PBS-Tween 20 (0.05% Tween 20 in PBS), then washed twice with 200 μl of PBS. Add 100 μl/well of a 1:10,000 diluted primary antibody (rabbit anti-human C3c antibody, DAKO A0062) in PBS containing 2.0 mg/ml bovine serum albumin and incubate for 1 hour at room temperature with gentle mixing. Each well was washed with 5x200 μl PBS. Add 100 μl/well of a 1:10,000 diluted secondary antibody (peroxidase-conjugated goat anti-rabbit IgG, American Qualex A102PU) in PBS containing 2.0 mg/ml bovine serum albumin and incubate for one hour at room temperature on a shaker. with gentle stirring. Each well was washed five times with 200 μl of PBS. 100 μl/well of TMB peroxidase substrate (Kirkegaard & Perry Laboratories) was added and incubated at room temperature for 10 min. The peroxidase reaction was stopped by adding 100 μl/well of 1.0 M H ₃ PO ₄ and OD ₄₅₀ was measured.

2. Assay to measure inhibition of MASP-2-dependent C4 cleavage.

Prerequisites.

The serine protease activity of MASP-2 is highly specific, and only two protein substrates for MASP-2 have been identified; C2 and C4. Cleavage of C4 leads to the formation of C4a and C4b. Anti-MASP-2 Fab2 can bind structural epitopes on MASP-2 that are directly involved in C4 cleavage (e.g., MASP-2 binding site for C4; MASP-2 serine protease catalytic site) and thereby inhibit the functional activity of C4 cleavage at MASP2.

Yeast mannan is a known activator of the lectin pathway. In the following method for measuring C4 MASP-2 cleavage activity, mannan-coated plastic wells were incubated for 30 min at 37°C with diluted rat serum to activate the lectin pathway. Because the primary antibody used in this ELISA assay recognizes only human C4, human C4 (1.0 μg/ml) was also added to the diluted rat serum. The wells were then washed and assayed for human C4b immobilized in the wells using standard ELISA methods. The amount of C4b produced in this assay is a measure of MASP-2-dependent C4 cleavage activity. Anti-MASP-2 Fab2 at selected concentrations was tested in this assay for its ability to inhibit C4 cleavage.

Methods.

96-well Costar medium binding plates were incubated overnight at 5°C with mannan diluted in 50 mM carbonate buffer, pH 9.5, applied at 1 μg/50 μl/well. Each well was washed 3X with 200 μl PBS. The wells were then blocked with 100 μl/well of 1% bovine serum albumin in PBS and incubated for one hour at room temperature with gentle mixing. Each well was then washed 3X with 200 μl PBS. Anti-MASP-2 Fab2 samples were diluted to selected concentrations in Ca and Mg'' buffer GVB (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgCl ₂ , 2.0 mM CaCl ₂ , 0.1% gelatin, pH 7.4) at 5°C. These samples also included 1.0 μg/ml human C4 (Quidel). 0.5% rat serum was added to the above samples at 5°C, and 100 μl was transferred to each well. The plates were covered and incubated for 30 min in a water bath at 37°C to activate complement. The reaction was stopped by transferring the plates from a 37°C water bath to a container containing a mixture of ice and water. Each well was washed 5X with 200 μl of PBS-Tween 20 (0.05% Tween 20 in PBS), then washed twice with 200 μl of PBS. 100 μl/well of a 1:700 dilution of biotin-conjugated chicken anti-human C4c antibody (Immunsystem AB, Uppsala, Sweden) was added to

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PBS containing 2.0 mg/ml bovine serum albumin (BSA) and incubated for one hour at room temperature with gentle mixing. Each well was washed 5X with 200 μl PBS. Add 100 μl/well of peroxidase-conjugated streptavidin at a concentration of 0.1 μg/ml (Pierce Chemical No. 21126) in PBS containing 2.0 mg/ml BSA and incubate for one hour at room temperature on a shaker with gentle mixing . Each well was washed 5X with 200 μl PBS. 100 μl/well of TMB peroxidase substrate (Kirkegaard & Perry Laboratories) was added and incubated at room temperature for 16 min. The peroxidase reaction was stopped by adding 100 μl/well of 1.0 M H3PO ₄ and OD ₄₅₀ was measured.

3. Anti-rat MASP-2 Fab2 binding assay to natural rat MASP-2.

Prerequisites.

MASP-2 is typically present in plasma as the dimeric MASP-2 complex, which also includes specific lectin molecules (mannose-binding protein (MBL) and ficolins). Thus, if the goal is to study the binding of anti-MASP-2 Fab2 to physiological MASP-2, it will be important to develop a binding assay that utilizes the interaction between Fab2 and natural MASP-2 in plasma rather than purified recombinant MASP-2. In this binding assay, the native MASP-2-MBL complex from 10% rat serum was first immobilized onto mannan-coated wells. The binding affinities of different anti-MASP-2 Fab2s to immobilized native MASP-2 were then studied by standard ELISA.

Methods.

96-well Costar high binding plates were incubated overnight at 5°C with mannan diluted in 50 mM carbonate buffer, pH 9.5, applied at 1 μg/50 μl/well. Each well was washed 3X with 200 μl PBS. The wells were then blocked with 100 μl/well of 0.5% nonfat dry milk in PBST (PBS with 0.05% Tween 20) and incubated for one hour at room temperature with gentle mixing. Each well was washed 3X with 200 μl of TBS/Tween/Ca ⁺⁺ wash buffer (Tris-buffered saline, 0.05% Tween 20 containing 5.0 mM CaCl2, pH 7.4). Prepare 10% rat serum on ice in high salt binding buffer (20 mM Tris, 1.0 M NaCl, 10 mM CaCl2, 0.05% Triton-X100, 0.1% (w/v) bovine serum albumin , pH 7.4). 100 μl/well was added and incubated overnight at 5°C. The wells were then washed 3X with 200 μl of TBS/Tween/Ca wash buffer. After this, the wells were washed 2X with 200 μl of PBS. 100 μl/well of anti-MASP-2 Fab2 was added at selected concentrations, diluted in GVB buffer containing Ca ⁺⁺ and Mg ⁺⁺ (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgCl2, 2.0 mM CaCl ₂ , 0.1% gelatin, pH 7.4), and incubated for one hour at room temperature with gentle stirring. Each well was washed 5X with 200 μl PBS. 100 μl/well of HRP-conjugated goat anti-Fab2 antibody (Biogenesis, catalog no. 05000099), diluted 1:5000 in 2.0 mg/ml bovine serum albumin in PBS, was added and incubated for one hour at room temperature with gentle stirring. Each well was washed 5X with 200 μl PBS. TMB peroxidase substrate (Kirkegaard & Perry Laboratories) was added at 100 μl/well and incubated at room temperature for 70 min. The peroxidase reaction was stopped by adding 100 μl/well of 1.0 M H ₃ PO ₄ and OD ₄₅₀ was measured.

Results.

Approximately 250 different Fab2s that reacted with high affinity with rat MASP-2 protein were selected for ELISA screening. These high-affinity Fab2s were sequenced to determine the uniqueness of the different antibodies, and 50 unique nnti-MNR^ antibodies were purified for further analysis.

250 μg of each Fab2 purified antibody was used for MASP-2 binding affinity characterization and complement pathway functional testing. The results of this analysis are shown in table. 6.

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Table 6

Ahtu-MASP-2 FAB2, which block the lectin pathway of complement activation

Fab2 antibody no. SZ-Convertase (1C ₅₀ (nM) Kd C4 cleavage 1C ₅₀ (nM) 88 0.32 4.1 But 41 0.35 0.30 0.81 eleven 0.46 0.86 <2 nM 86 0.53 1.4 BUT 81 0.54 2.0 But 66 0.92 4.5 But 57 0.95 3.6 <2 nM 40 1.1 7.2 0.68 58 1.3 2.6 BUT 60 1.6 3.1 BUT 52 1.6 5.8 <2 nM 63 2.0 6.6 BUT 49 2.8 8.5 <2 nM 89 3.0 2.5 BUT 71 3.0 10.5 But 87 6.0 2.5 But 67 10.0 7.7 But

As shown in the table above. 6, of 50 anti-MASP-2 Fab2s tested, seventeen Fab2s were identified as MASP-2 blocking Fab2s that potentially inhibit C3 convertase formation with an _IC50 value equal to or less than 10 nM Fab2 (34% positive success rate). ). Eight of the seventeen Fab2 identified have IC ₅₀ values in the subnanomolar range. Moreover, all seventeen of the MASP-2 blocking Fab2s listed in Table. 6 provide virtually complete inhibition of C3 convertase formation in the lectin pathway C3 convertase production assay. in fig. 8A is a graph showing the results of a C3 convertase production assay for Fab2 antibody No. 11, which is representative of the other Fab2 antibodies tested, the results for which are shown in Table 1. 6. This is an important consideration because it is theoretically possible that Fab2 blockers may only partially inhibit MASP-2 function, even when each MASP-2 molecule is bound to Fab2.

Although mannan is a known activator of the lectin pathway, it is theoretically possible that the presence of antibodies to mannan in rat serum could also activate the classical pathway and lead to the formation of C3b via the classical pathway C3 convertase. However, each of the seventeen anti-MASP-2 Fab2 blockers listed in this example strongly inhibited C3b formation (>95%), thereby demonstrating the specificity of this assay for the C3 convertase of the lectin pathway.

Binding assays were also performed with all seventeen Fab2 blockers to calculate the apparent Kd for each. The results of anti-rat MASP-2 Fab2 binding assays with native rat MASP-2 for six of the Fab2 blockers are also shown in Table 1. 6. in fig. 8B is a graph showing the results of a binding assay with Fab2 antibody No. 11. Similar binding assays were also performed for other Fab2s and the results are shown in Table 8. 6. In general, the apparent Kd values obtained for the binding of each of the six Fab2s to native MASP-2 agree reasonably well with the IC50 for specific Fab2s in the C3 convertase functional assay. There is evidence that MASP-2 undergoes a conformational change from an inactive to an active form when its protease activity is activated (Feinberg et al., EMBO J 22:2348-59 (2003); Gal et al., J. Biol. Chem. 280 :33435-44 (2005)). In normal rat plasma used in the C3 convertase formation assay, MASP-2 is present predominantly in the inactive zymogen conformation. In contrast, in the binding assay, MASP-2 is present as part of a complex with MBL bound to immobilized mannan; thus, MASP-2 is in the active conformation (Petersen et al., J. Immunol Methods 257:107-16, 2001). Therefore, one would not necessarily expect an exact match between the IC ₅₀ and K _d for each of the seventeen Fab2 blockers tested in these two functional assays, since different conformational forms of MASP-2 bind Fab2 in each assay. However, with the exception of Fab2 no. 88, there appears to be a fairly close agreement between the IC ₅₀ and the apparent K _d for each of the remaining Fab2s tested

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Several Fab2 blockers have been tested to inhibit MASP-2-mediated C4 cleavage. in fig. 8C is a graph showing the results of the C4 cleavage assay demonstrating inhibition of Fab2 No. 41, with _IC50 =0.81 nM (see Table 6). As shown in FIG. 9, all Fab2 tested inhibited C4 cleavage with _IC50 values similar to those obtained in the C3 convertase assay (see Table 6).

Although mannan is a known activator of the lectin pathway, it is theoretically possible that the presence of antibodies to mannan in rat serum could also activate the classical pathway and lead to the formation of C4b through CH-mediated cleavage of C4. However, several anti-MASP-2 Fab2s have been identified that potently inhibit C4b formation (>95%), thereby demonstrating the specificity of this assay for MASP-2-mediated C4 cleavage. C4, like C3, contains an unusual and highly reactive thioether group in its structure. After cleavage of C4 by MASP-2 in this assay, the thioester group on C4b can form a covalent bond with hydroxyl or amino groups on macromolecules immobilized at the bottom of plastic wells through ester or amide bonds, which facilitates detection of C4b in the ELISA assay.

The results of these studies clearly show that high-affinity Fab2s have been produced for the rat MASP-2 protein, which functionally block both C4 cleavage and C3 convertase activity, thereby preventing the lectin pathway of activation.

Example 11.

This example describes epitope mapping for several Fab2 blocking anti-rat MASP-2 antibodies that were prepared as described in Example 10.

Methods.

As shown in FIG. 10, the following proteins, all with N-terminal 6X His tags, were expressed in CHO cells using the pED4 vector:

rat MASP-2A, full-length MASP-2 protein inactivated by replacing the active site serine with alanine (S613A);

rat MASP-2K, full-length MASP-2 protein modified to reduce autoactivation (R424K);

CUBI-II, an N-terminal fragment of rat MASP-2 that contains only the CUBI, EGF-like, and CUBII domains; And

CUBI/EGF-like, N-terminal fragment of rat MASP-2 that contains only the CUBI and EGF-like domains.

These proteins were purified from culture supernatants by nickel affinity column chromatography as described previously (Chen et al., J. Biol. Chem. 276:25894-02 (2001)).

A C-terminal polypeptide (CCPII-SP) containing CCPII and the serine protease domain of rat MASP-2 was expressed in E. coli as a thioredoxin fusion protein using pTrxFus (Invitrogen). Protein was purified from cell lysates using Thiobond affinity resin. The fusion partner thioredoxin was expressed from empty pTrxFus as a negative control.

All recombinant proteins were dialyzed in TBS buffer and their concentrations were determined by measuring OD at 280 nm.

Dot blot analysis.

The five recombinant MASP-2 polypeptides described above and shown in FIG. 10, serial dilutions (and thioredroxin polypeptide as a negative control for CCPII-serine protease polypeptide) were spotted onto a nitrocellulose membrane. The amount of spot-on protein ranged from 100 ng to 6.4 pg, in five-fold dilutions. In later experiments, the amount of protein spotted ranged from 50 ng to 16 pg, again in fivefold dilutions. Membranes were blocked with 5% nonfat milk powder in TBS (blocking buffer), then incubated with 1.0 μg/ml anti-MASP-2 Fab2 in blocking buffer (containing 5.0 mM Ca ²⁺ ). Bound Fab2 was detected using HRP-conjugated anti-human Fab antibodies (AbD/Serotec; 1/10,000 dilution) and an ECL detection kit (Amersham). One membrane was incubated with polyclonal rabbit anti-human MASP-2 Ab (described in Stover et al., J Immunol 163:6848-59 (1999)) as a positive control. In this case, bound Abs were detected using HRP-conjugated goat anti-rabbit IgG (Dako; dilution 1/2000).

MASP-2 binding assay.

ELISA plates were coated with 1.0 μg/well of recombinant MASP-2A or CUBI-II polypeptide in carbonate buffer (pH 9.0) overnight at 4°C. Wells were blocked with 1% BSA in TBS, then anti-MASP-2 Fab2 was added in serial dilution in TBS containing 5.0 mM Ca ²⁺ . The plates were incubated for one hour at RT. After washing three times with TBS/Tween/Ca ^{+ ,} HRP-conjugated anti-human Fab antibodies (AbD/Serotec) diluted 1/10,000 in TBS/Ca ²⁺ were added and the plates were incubated for another hour at RT. Bound antibody was detected using

- 85 046178 calling a kit with TMB peroxidase substrate (Biorad).

Results.

The results of dot blot analysis demonstrating the ability of Fab2 to interact with various MASP-2 polypeptides are shown in Table 1 below. 7. Numerical values given in table. 7 show the amount of spot-on protein required to achieve approximately half-maximal signal strength. As shown, all of the polypeptides (except for the single fusion partner thioredoxin) were recognized by the positive control Ab (polyclonal rabbit serum against human MASP-2).

Table 7

Ability to interact with various recombinant rat MASP-2 polypeptides in dot blot analysis

Fab2 Antibody No. MASP-2A CUBI-II CUBI/EGF-like CCPII-SP Thioredoxin 40 0.16 ng N/R N/R 0.8 ng N/R 41 0.16 ng N/R N/R 0.8 ng N/R eleven 0.16 ng N/R N/R 0.8 ng N/R 49 0.16 ng N/R N/R >20 ng N/R 52 0.16 ng N/R N/R 0.8 ng N/R 57 0.032 ng N/R N/R N/R N/R 58 0.4 ng N/R N/R 2.0 ng N/R 60 0.4 ng 0.4 ng N/R N/R N/R 63 0.4 ng N/R N/R 2.0 ng N/R 66 0.4 ng N/R N/R 2.0 ng N/R 67 0.4 ng N/R N/R 2.0 ng N/R 71 0.4 ng N/R N/R 2.0 ng N/R 81 0.4 ng N/R N/R 2.0 ng N/R 86 0.4 ng N/R N/R Jung N/R 87 0.4 ng N/R N/R 2.0 ng N/R Positive control <0.032 ng 0.16 ng 0.16 ng <0.032 ng N/R

N/R=no reaction. Polyclonal rabbit serum against human MASP-2 served as positive control antibodies.

All of Fab2 interacted with MASP-2A as well as MASP-2K (data not shown). Most Fab2s recognized the CCPII-SP polypeptide, but not the N-terminal fragments. The two exceptions were Fab2 #60 and Fab2 #57. Fab2 #60 recognized MASP-2A and the CUBI-II fragment, but not the CUBI/EGF-like domain polypeptide or the CCPII-SP polypeptide, indicating that it binds an epitope in CUBII, or connecting CUBII and EGF-like domain. Fab2 #57 recognized MASP-2A but none of the MASP-2 fragments tested, indicating that this Fab2 recognizes an epitope in CCP1. Fab2 #40 and #49 bound only full-length MASP-2A. In the ELISA binding assay illustrated in FIG. 11, Fab2 #60 also bound the CUBI-II polypeptide, although with slightly lower apparent affinity.

These results indicate that unique blocking Fab2s to multiple regions of the MASP-2 protein have been identified.

Example 12.

This example describes the analysis of MASP-2-/- mice in a mouse model of renal ischemia/reperfusion.

Background/Rationale.

Ischemia-reperfusion (I/R) renal injury at body temperature has been implicated in a number of clinical conditions, including hypovolemic shock, renal artery occlusion, and vascular clamping procedures.

Renal ischemia-reperfusion (I/R) is an important cause of acute renal failure, associated with mortality rates of up to 50% (Levy et al., JAMA 275:1489-94, 1996; Thadhani et al., N. Engl. J. Med 334:1448-60, 1996). Post-transplant renal failure is a common and life-threatening complication after kidney transplantation (Nicholson et al., Kidney Int. 58:2585-91, 2000). Effective treatment for I/R kidney injury is currently unavailable, and hemodialysis is the only treatment available. The pathophysiology of I/R kidney injury is

- 86 046178 complex. Recent studies have shown that the lectin pathway of complement activation may play an important role in the pathogenesis of I/R kidney injury (deVries et al., Am. J. Path. 165:1677-88, 2004).

Methods.

MASP-2(-/-) mice were generated as described in Example 1 and backcrossed for at least 10 generations to C57B1/6. Six male MASP-2(-/-) and six wild-type (+/+) mice weighing 22-25 g were administered an intraperitoneal injection of Hypnovel (6.64 mg/kg; Roche products Ltd. Welwyn Garden City, UK), and then anesthesia was applied by inhalation of isoflurane (Abbott Laboratories Ltd., Kent, UK). Isoflurane was chosen because it is a mild inhalational anesthetic with minimal liver toxicity; concentrations are maintained precisely and the animal recovers quickly, even after prolonged anesthesia. Hypnovel was administered because it induces a state of neuroleptanalgesia in the animal, which means that less isoflurane needs to be administered. The animal was placed on a warm substrate to maintain a constant body temperature. A midline abdominal incision was then made and the body cavity was opened using a pair of retractors. The connective tissue was removed above and below the renal vein and artery of the right and left kidneys, and the renal pedicle was clamped using microaneurysm clamps for 55 min. The duration of this ischemic period was initially based on a previous study conducted in this laboratory (Zhou et al., J. Clin. Invest. 105:1363-71 (2000)). In addition, the standard ischemia time of 55 min was chosen after ischemia titration, in which 55 min was found to result in permanent damage that is also reversible, with a low mortality rate of less than 5%. After occlusion, 0.4 ml of warm saline (37°C) was introduced into the abdominal cavity, and then the abdominal cavity was closed for the period of ischemia. After removal of the microaneurysm clamps, the kidneys were observed until the color changed, a sign of re-flow of blood to the kidneys. An additional 0.4 ml of warm saline was added to the peritoneal cavity and the hole was sutured, after which the animals were returned to their cages. Tail vein blood samples were collected 24 h after clamp removal, and 48 h later mice were sacrificed and additional blood samples were collected.

Assessment of kidney damage.

Renal function was assessed 24 and 48 h after reperfusion in six male MASP-2(/-) and six WT (+/+) mice. The determination of creatinine in the blood was carried out by mass spectrometry, obtaining a reproducible indicator of renal function (sensitivity <1.0 µmol/l), in Fig. 12 graphically depicts blood urea nitrogen clearance for wild-type C57B1/6 controls and MASP-2 (-/-) mice at 24 hours and 48 hours after reperfusion. As shown in FIG. 12, MASP-2(-/-) mice exhibited significant reductions in blood urea at 24 and 48 h compared with wild-type control mice, indicating a protective functional effect against renal injury in a model of ischemia-reperfusion injury.

Overall, increases in blood urea were observed in both WT (+/+) and MASP-2 (−/−) mice 24 and 48 h after the surgical procedure and ischemic stroke. The blood urea level in the WT animal (+/+) after surgery without ischemia, as determined separately, is 5.8 mmol/L. In addition to the data presented in Fig. 12, one MASP-2 (-/-) animal showed almost complete protection against ischemic stroke, with rates of 6.8 and 9.6 mmol/L at 24 and 48 hours, respectively. This animal was excluded from the group analysis as a potential anomalous finding in which ischemic injury may have been absent. Thus, in the final analysis, the results of which are presented in Fig. 12, 5 MASP-2(-/-) and 6 WT (+/+) mice were included, and MASP-2(-/-) mice had a statistically significant decrease in blood urea at 24 and 48 h (criterion Student's t test p<0.05). These results indicate that inhibition of MASP-2 activity may conceivably have a protective or therapeutic effect on renal ischemic injury.

Example 13.

This example describes the results for MASP-2-/- mice in a mouse model of macular degeneration.

Background/Rationale.

Age-related macular degeneration (AMD) is the leading cause of blindness in people over 55 years of age in industrialized countries. AMD has two main forms: neovascular (wet) AMD and atrophic (dry) AMD. The neovascular (wet) form accounts for 90% of the severe visual impairment associated with AMD, although only ~20% of people with AMD develop the wet form. Clinical features of AMD include multiple drusen, geographic atrophy, and choroidal neovascularization (CNV). In December 2004, the FDA approved macugen (pegaptanib), a new class of ophthalmic drugs that specifically target and block the effects of vascular endothelial growth factor (VEGF), for the treatment of wet (neovascular) AMD (Ng et al., Nat Rev. Drug Discov 5:123-32 (2006)). Although macugen is a promising new treatment option for a subset of patients with AMD, there remains an urgent need to develop additional treatments for this complex disease. Numerous independent lines of research indicate a central role for complement activation in pathogenesis.

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AMD. The pathogenesis of choroidal neovascularization (CNV), the most severe form of AMD, may involve complement activation pathways.

More than twenty-five years ago, Ryan described a laser-induced trauma model of CNV in animals (Ryan, S.J., Tr. Am. Opth. Soc. LXXVII:707-745, 1979). This model was originally developed using rhesus monkeys, but the same technology has since been used to develop similar CNV models in various laboratory animals, including mice (Tobe et al., Am. J. Pathol. 153:1641-46, 1998). In this model, laser photocoagulation is used to destroy Bruch's membrane, resulting in the formation of CNV-like membranes. The laser-induced model recapitulates many important features of the condition in humans (for a recent review, see Ambati et al., Survey Ophthalmology 48:257-293, 2003). The laser-induced disease model in mice is now well established and is used as an experimental basis in a large and growing number of research projects. It is generally accepted that the laser-induced model has sufficient biological similarity to human CNV for preclinical studies of pathogenesis and drug inhibition of disease using this model to be useful for the treatment of human CNV.

Methods.

MASP-2(-/-) mice were generated as described in Example 1 and backcrossed for at least 10 generations to C57B1/6. The present study compared male MASP-2 (-/-) and MASP-2 (+/+) mice during the development of laser-induced CNV, an accelerated model of neovascular AMD, based on the volume of laser-induced CNV determined by scanning laser confocal microscopy , as a measure of tissue damage, and with determination of levels of VEGF, a potent angiogenic factor involved in CNV, in the retinal pigment epithelium (RPE)/choroid by ELISA after laser-induced injury.

Induction of choroidal neovascularization (CNV).

Laser photocoagulation (532 nm, 200 mW, 100 ms, 75 μm; Oculight GL, Iridex, Mountain View, CA) was performed in both eyes of each animal on day zero by one person blinded to the animals' specific experimental groups. Laser spots were applied in a standard manner around the optic nerve, using a slit lamp and a coverslip as a contact lens. The morphological end result of laser injury was the appearance of a cavitation bubble, a feature thought to correlate with disruption of Bruch's membrane. The methods and performance criteria evaluated are given in more detail below.

Fluorescein angiography.

Fluorescein angiography was performed using a camera and imaging system (TRC 50 1A camera; ImageNet 2.01 system; Topcon, Paramus, NJ) 1 week after laser photocoagulation. Photographs were taken using a 20-D lens in contact with the fundus camera lens after intraperitoneal injection of 0.1 ml of 2.5% sodium fluorescein solution. A retinal specialist not involved in laser photocoagulation or angiography assessed the fluorescein angiograms in a single session, blinded to the experimental groups.

Choroidal neovascularization volume (CNV).

One week after laser-induced injury, the eyes were enucleated and fixed with 4% paraformaldehyde for 30 min at 4°C. Eye cups were prepared by removing the anterior segments and washed three times in PBS, followed by dehydration and rehydration in varying concentrations of methanol. After blocking twice with buffer (PBS containing 1% bovine serum albumin and 0.5% Triton X-100) for 30 min at room temperature, the eye dishes were incubated overnight at 4°C with 0.5% FITC- isolectin B4 (Vector laboratories, Burlingame, CA) in PBS containing 0.2% BSA and 0.1% Triton X-100, which binds terminal P-D galactose residues on the surface of endothelial cells and selectively labels the murine vasculature. After two washes with PBS containing 0.1% Triton X-100, the neurosensory retina was carefully peeled off and separated from the optic nerve. Four relaxing radial incisions were made, and the remaining RPE-choroid-sclera complex was laid flat on a glass slide in anti-fluorescence fading medium (Immu-Mount Vectashield Mounting Medium; Vector laboratories) and covered with a coverslip.

Flat preparations were examined under a laser scanning confocal microscope (TCS SP; Leica, Heidelberg, Germany). Vessels were imaged by excitation of blue argon radiation at 488 nm and absorption of radiation at wavelengths between 515 and 545 nm. A 40X oil immersion objective was used for all imaging studies. Horizontal ocular sections (1-μm increments) were obtained from the surface of the RPE-choroid-sclera complex. The deepest focal plane in which the surrounding choroidal vasculature connecting to the lesia could be identified was considered the floor of the lesia. Any vessel in the laser lesion superficial to this reference plane was considered a CNV. Images of each section were saved digitally. The area of CNV-associated fluorescence was measured by computer

- 88 046178 image analysis using microscope software (TCS SP; Leica). The sum of the total fluorescence area in each horizontal section was used as a measure of CNV volume. Imaging was performed by an operator blinded to the experimental groups.

Because of the likelihood that each laser-induced lesion leading to CNV development is influenced by the group to which it belongs (mouse, eye, and laser spot), mean lesion volumes were compared using a linear mixed model using a split-plot method with a repeated measures design. . The whole plot factor was the genetic group to which the animal belonged, while the split plot factor was the eye. Statistical significance was determined at the 0.05 level. Post hoc comparisons of means were performed using the Bonferroni correction for multiple comparisons.

ELISA for VEGF.

Three days after the damaging application of 12 laser spots, the RPE-choroid complex was sonicated in lysis buffer (20 mM imidazole HCl, 10 mM KCl, 1 mM MgCl ₂ , 10 mM EGTA, 1% Triton X-100, 10 mM NaF, 1 mM Na molybdate and 1 mM EDTA with protease inhibitors) on ice for 15 min. VEGF protein levels in the supernatant were determined using an ELISA kit (R&D Systems, Minneapolis, MN) that recognizes all splice variants at 450–570 nm (Emax; Molecular Devices, Sunnyvale, CA) and normalized to total protein levels . Measurements in duplicate were performed by an operator blinded to the experimental groups and not involved in the photocoagulation, imaging, or angiography experiments. Values for VEGF were presented as the mean ± SEM from at least three independent experiments and compared using the Mann-Whitney U test. The null hypothesis was rejected at P < 0.05.

Results.

Assessment of VEGF levels.

in fig. 13A is a graph showing VEGF protein levels in the RPE-choroid complex isolated from wild-type C57B16 mice and MASP-2(-/-) mice on day zero. As shown in FIG. 13A, assessment of VEGF levels indicates a decrease in basal VEGF levels in MASP-2 (-/-) mice compared to control wild-type C57B16 mice. in fig. 13B is a graph showing VEGF protein levels measured at day three after laser-induced injury. As shown in FIG. 13B, VEGF levels were significantly increased in wild-type (+/+) mice three days after laser-induced injury, consistent with published data (Nozaki et al., Proc. Natl. Acad. Sci. USA 103:2328-33 ( 2006)). However, surprisingly low levels of VEGF were observed in MASP-2 (-/-) mice.

Assessment of choroidal neovascularization (CNV).

In addition to the reduction in VEGF levels after induced macular degeneration, CNV area was assessed before and after laser-induced damage. In fig. 14 graphically represents the volume of CNV measured in wild-type C57b1 mice and MASP-2(-/-) mice on day seven after laser-induced injury. As shown in FIG. 14, MASP-2 (-/-) mice had an approximately 30% reduction in CNV area at day seven after laser-induced injury compared with control wild-type mice.

These results suggest decreased levels of VEGF and CNV in MASP (-/-) mice compared to control wild-type (+/+) mice and that blockade of MASP-2 with an inhibitor may have a preventive and therapeutic effect in the treatment of macular degeneration spots.

Example 14.

This example shows that activation of thrombin can occur after activation of the lectin pathway under physiological conditions and shows the extent of MASP-2 involvement. In normal rat serum, activation of the lectin pathway leads to thrombin activation (assessed by thrombin deposition) concomitant with complement activation (assessed by C4 deposition). As can be seen in FIG. 15A and 15B, thrombin activation in this system is inhibited by a MASP-2 blocking antibody (Fab2 format), with a concentration-dependent inhibition curve (FIG. 15B) that parallels that of complement activation (FIG. 15A). These data suggest that activation of the lectin pathway that occurs during injury leads to activation of both the complement and coagulation systems, in a process that is entirely dependent on MASP-2. Therefore, MASP2 blocking antibodies may be effective in reducing excessive systemic coagulation, such as disseminated intravascular coagulation, which is one of the factors leading to death in cases of serious trauma.

Example 15.

This example reports results using the localized Schwarzman reaction, a model of disseminated intravascular coagulation (DIC), in MASP-2-deficient (-/-) and MASP-2 normal (+/+) mice to evaluate the role of lectin pathway in DIC.

Background/Rationale.

As described above, blocking MASP-2 inhibits lectin pathway activation and results in

- 89 046178 reducing the formation of both anaphylatoxins, C3a and C5a. C3a anaphylatoxins have been shown to be potent platelet aggregation inducers in vitro, but their role in vivo is less well understood, and the release of platelet substances and plasmin during wound healing may only secondarily involve complement C3. Here, the role of the lectin pathway was analyzed in MASP-2 (-/-) mice and WT (+/+) mice to determine whether a sustained increase in C3 activation is required for the development of disseminated intravascular coagulation.

Methods.

MASP-2(-/-) mice were generated as described in Example 1 and backcrossed for at least 10 generations to C57B1/6.

In this experiment, the Shvartsman localized reaction model was used. The localized Schwarzman reaction (LSR) is a lipopolysaccharide (LPS)-induced response with well-characterized contributions from cellular and humoral elements of the innate immune system. The dependence of LSR on complement is well known (Polak, L., et al., Nature 223:738-739 (1969); Fong J.S. et al., J Exp Med 134:642-655 (1971)). In the LSR model, mice were primed for 4 h with TNFalpha (500 ng, intrascrotal), mice were anesthetized, and prepared for intravital microscopy of the testicular suspensory muscle. Networks of postcapillary venules (15-60 µm in diameter) with good blood flow (1-4 mm/s) were selected for observation. Animals were injected with fluorescent antibodies to selectively label neutrophils or platelets. The vascular network was sequentially scanned, and images of all vessels were digitally recorded for subsequent analysis. After recording baseline microcirculatory status, mice received a single intravenous injection of LPS (100 μg), either alone or in combination with the agents listed below. The same vascular network was then scanned every 10 min for 1 h. Specific accumulation of fluorophores was determined by subtracting the background fluorescence level, and the image was enhanced by setting an image threshold. The magnitude of the reactions was determined from the recorded images. Schwartzman's primary measure of reactions was aggregate data.

The study compared mice with normal levels of MASP-2 (+/+), or wild type, and mice exposed to either a known complement pathway depletor, cobra venom factor (CVF), or a terminal complement inhibitor (C5aR antagonist). . The results (Fig. 16A) show that both CVF and the C5aR antagonist prevent the appearance of aggregates in the vasculature. In addition, MASP-2-deficient (-/-) mice (Fig. 16B) also exhibited complete inhibition of the localized Schwartzman reaction, indicating involvement of the lectin pathway. These results clearly demonstrate the role of MASP-2 in the development of DIC and support the use of MASP-2 inhibitors for the treatment and prevention of DIC.

Example 16.

This example describes the analysis of MASP-2 (-/-) mice in a mouse model of kidney transplantation.

Background/Rationale

The role of MASP-2 in the functional outcome of kidney transplantation was assessed using a mouse model.

Methods.

The functional consequences of kidney transplantation were assessed using one kidney isograft in unilaterally nephrectomized recipient mice, six graft-receiving WT (+/+) mice (B6), and six graft-receiving MASP-2 (-/-) mice. To assess transplanted kidney function, the remaining natural kidney was removed from recipients 5 days after transplantation, and renal function was assessed 24 hours later by measuring blood urea nitrogen (BUN) levels.

Results.

In fig. 17 is a graph showing blood urea nitrogen (BUN) levels 6 days after kidney transplantation in DT (+/+) and MASP-2 (-/-) recipients. As shown in FIG. 17, highly elevated BUN levels were observed in transplant recipients of WT (+/+) mice (B6) (normal BUN levels in mice are <5 mM), indicating renal failure. In contrast, MASP-2 (-/-) mouse isograft recipients exhibited significantly lower BUN levels, indicating improved renal function. It should be noted that these results were obtained using grafts from DT (+/+) kidney donors, indicating that the absence of a functional lectin pathway in the transplant recipient alone is sufficient to achieve a favorable therapeutic outcome.

Taken together, these results indicate that inhibition of the lectin pathway through inhibition of MASP-2 represents an approach to reduce morbidity and prolong graft function in kidney transplantation and that this approach is likely to be useful in other transplant settings.

Example 17.

This example demonstrates that MASP-2 (-/-) mice are resistant to septic shock in a mouse model of polymicrobial septic peritonitis.

- 90 046178

Background/Rationale.

Cecal ligation and puncture (CLP), a model of polymicrobial septic peritonitis, was used to evaluate the potential effects of MASP-2(-/-) on infection. This model is considered the most accurate reflection of the processes occurring during human septic peritonitis. Cecal ligation and puncture (CLP) is a model in which the cecum is ligated and punctured with a needle, resulting in a continuous leakage of bacteria into the peritoneal cavity, which enter the bloodstream through lymphatic drainage and are then distributed throughout the abdominal organs, resulting in multi-organ failure and septic shock (Eskandari et al., J Immunol 148(9):2724-2730 (1992)). The CLP model mimics the course of sepsis in patients and induces an early hyperinflammatory response, followed by a pronounced hypoinflammatory phase. During this phase, animals are highly susceptible to bacterial infection (Wichterman et al., J. Surg. Res. 29(2): 189201 (1980)).

Methods.

Mortality due to polymicrobial infection using the cecal ligation and puncture (CLP) model was studied in WT (+/+) mice (n=18) and MASP-2 (-/-) mice (n=16). Briefly, MASP-2-deficient mice and their wild-type littermates were anesthetized, and the cecum was exposed to the body surface and ligated at a site 30% above the distal end. After this, the cecum was punctured once with a needle with a diameter of 0.4 mm. The cecum was then returned to the abdominal cavity and the skin incision was closed with clamps. The survival of mice exposed to CLP was monitored for 14 days after CLP. Peritoneal lavage fluid was collected from mice 16 h after CLP to measure bacterial load. Serial dilutions of peritoneal lavage fluid were prepared in PBS and inoculated into Mueller-Hinton plates, followed by incubation at 37°C under anaerobic conditions for 24 hours, after which the bacterial load was determined. The response to bacterial infection in the form of TNF-alpha production was also measured in WT (+/+) and MASP-2 (-/-) mice 16 h after CLP in the lungs and spleen by quantitative real-time polymerase chain reaction (qRT-PCR). Serum TNF-alpha levels 16 h after CLP in WT (+/+) and MASP-2 (−/−) mice were also quantified by sandwich ELISA.

Results.

In fig. 18 graphically presents the survival rate, in percentage, of animals subjected to CLP as a function of the number of days after the CLP procedure. As shown in FIG. 18, the absence of the lectin pathway in MASP-2 (-/-) mice does not lead to increased mortality of mice after polymicrobial infection in the cecal ligation and puncture model, compared with WT (+/+) mice. However, as shown in FIG. 19, MASP-2 (−/−) mice had a much higher bacterial load (∼1000-fold increase in bacterial number) in peritoneal lavage fluid after CLP compared with their WT (+/+) littermates. These results indicate that MASP-2-deficient (-/-) mice are resistant to septic shock. The lower bacterial clearance in MASP-2-deficient mice in this model may be a consequence of impaired C3b-mediated phagocytosis, since C3 deposition has been shown to be MASP-2 dependent.

It was found that the response to bacterial infection in the form of production of the cytokine TNFalpha was not increased in MASP-2 (-/-) mice compared to WT (+/+) controls (data not shown). It was also shown that there was a significantly higher serum concentration of TNF-alpha in WT (+/+) mice 16 h after CLP, in contrast to MASP-2 (-/-) mice in which serum TNF-alpha levels remained unchanged. practically unchanged. These results suggest that the intense inflammatory response to the septic condition was mitigated in MASP-2(-/-) mice, allowing the animals to survive in the presence of large numbers of bacteria.

Taken together, these results demonstrate the potential deleterious effects of the lectin complement pathway in septicemia and increased mortality in patients with generalized sepsis. These results also indicate that MASP-2 deficiency leads to modulation of the inflammatory immune response and decreased expression levels of inflammatory mediators in sepsis. Thus, it can be assumed that inhibition of MASP-2 (-/-) by administration of inhibitory monoclonal antibodies against MASP-2 would be effective in reducing the inflammatory response in a subject suffering from septic shock.

Example 18.

This example describes the analysis of MASP-2 (-/-) mice in a mouse model of intranasal infectivity. Background/Rationale Pseudomonas aeruginosa is a Gram-negative opportunistic bacterial pathogen of humans that causes a variety of infections, especially in immunocompromised individuals. This bacterium is the main source of acquired nosocomial infections, in particular hospital-acquired pneumonia. It is also responsible for significant morbidity and mortality in patients with cystic fibrosis (CF). Pulmonary infection with P. aeruginosa is characterized by strong neutrophil recruitment and significant pulmonary inflammation leading to severe tissue damage (Palanki M. S. et al., J. Med. Chem 51:15461559 (2008)).

- 91 046178

This example describes a study undertaken to determine whether ablation of the lectin pathway in MASP-2 (-/-) mice increases the mice's susceptibility to bacterial infection.

Methods.

Twenty-two WT (+/+), twenty-two MASP-2 (−/−) mice, and eleven C3 (−/−) mice were infected by intranasal administration of a strain of P. aeruginosa bacteria. Mice were observed for six days after infection and a Kaplan-Meier plot was generated showing percentage survival.

results

In fig. 20 shows a Kaplan-Meier plot of the percentage survival of WT (+/+), MASP-2 (-/-) or C3 (-/-) mice six days after infection. As shown in FIG. 20, there was no difference between MASP-2 (-/-) and WT (+/+) mice. However, ablation of the classical (C1q) complement pathway in C3 (-/-) mice resulted in severe susceptibility to bacterial infection. These results indicate that MASP-2 inhibition does not increase susceptibility to bacterial infection, suggesting that it may be possible to reduce unwanted inflammatory complications in trauma patients by inhibiting MASP-2 without reducing the patient's ability to fight infections through the classical complement pathway.

Example 19.

This example describes the pharmacodynamic analysis of representative high affinity anti-MASP-2 Fab2 antibodies identified as described in Example 10.

Background/Rationale.

As described in Example 10, to identify high-affinity antibodies that block the lectin pathway in rats, rat MASP-2 protein was used to sort a phage display library. This library was designed to provide a high degree of immunological diversity and was constructed using fully human immunoglobulin gene sequences.

As described in Example 10, approximately 250 individual phage clones were identified that bound with high affinity the rat MASP-2 protein in an ELISA screen. Sequencing of these clones allowed the identification of 50 unique phages encoding an anti-MASP-2 antibody. Fab2 protein was expressed from these clones, purified, and analyzed for MASP-2 binding affinity and functional inhibition of the lectin complement pathway.

As shown in table. 6 of Example 10, this assay identified 17 anti-MASP-2 Fab2s with function blocking activity (34% blocking antibody success rate). Functional inhibition of the lectin complement pathway by Fab2 was evident based on C4 deposition, which is a direct indicator of C4 cleavage by MASP-2. Importantly, inhibition was equally evident when assessing C3 convertase activity, indicating a functional block of the lectin complement pathway. The 17 MASP-2 blocking Fab2s identified as described in Example 10 potently inhibit C3 convertase formation with IC ₅₀ values equal to or less than 10 nM. Eight of the 17 identified Fab2s have IC ₅₀ values in the subnanomolar range. In addition, all 17 MASP-2 blocking Fab2s caused almost complete inhibition of C3 convertase formation in the lectin pathway C3 convertase formation assay, as shown in FIG. 8A-C, and summarized in table. 6 of Example 10. Moreover, each of the 17 blocking anti-MASP-2 Fab2 shown in table. 6 strongly inhibits the formation of C3b (>95%), thus demonstrating the specificity of this assay for the C3 convertase of the lectin pathway.

Isotype variants of full-length rat IgG2c and mouse IgG2a antibodies were obtained from Fab2 No. 11. This example describes the in vivo characterization of these isotypes with respect to pharmacodynamic parameters.

Methods.

As described in Example 10, rat MASP-2 protein was used to sort a phage display Fab library from which Fab2 #11 was identified. Rat IgG2c and mouse IgG2a full-length isotype variants were generated from Fab2 #11. Both rat IgG2c and mouse IgG2a , full-length antibody isotypes were characterized in vivo with respect to pharmacodynamic parameters as follows.

In vivo study in mice.

A pharmacodynamic study was conducted in mice to examine the effect of anti-MASP-2 antibody administration on plasma lectin pathway activity in vivo. This study determined ex vivo C4 deposition in a lectin pathway assay at various time points following subcutaneous (SC) and intraperitoneal (IP) administration of 0.3 mg/kg or 1.0 mg/kg murine anti-MASP. 2 mAb (mouse full-length IgG2a isotype antibody derived from Fab2 no. 11).

In fig. 21 graphically depicts lectin pathway-specific C4b deposition measured ex vivo in undiluted serum samples obtained from mice (n=3 mice/group) at various time points following subcutaneous dosing of either 0.3 mg/kg or 1.0 mg. /kg, mouse anti-MASP-2 moAb. Serum samples from mice collected before antibody administration served as negative samples.

- 92 046178 nal controls (100% activity), while serum to which the same aHTu-MASP-2 blocking antibody was added in vitro at a concentration of 100 nM was used as a positive control (0% activity).

The results presented in Fig. 21 show rapid and complete inhibition of C4b deposition following subcutaneous administration of a 1.0 mg/kg dose of murine anti-MASP-2 moAb. Partial inhibition of C4b deposition was observed following subcutaneous administration of a 0.3 mg/kg dose of mouse anti-MASP-2 moAb.

The dynamics of restoration of the lectin pathway were monitored for three weeks after a single intravenous injection of mouse anti-MASP-2 moAb at a dose of 0.6 mg/kg to mice. As shown in FIG. 22, a sharp drop in lectin pathway activity occurred after antibody administration, followed by complete inhibition of the lectin pathway, which lasted for approximately 7 days after i.p. administration. A slow recovery of pathway activity was observed during the second and third weeks, with complete restoration of the lectin pathway in mice 17 days after anti-MASP-2 moAb administration.

These results indicate that the mouse anti-MASP-2 moAb derived from Fab2 #11, when delivered systemically, inhibits the lectin pathway in mice in a dose-dependent manner.

Example 20.

This example describes an assay for the effectiveness of a mouse anti-MASP-2 moAb derived from Fab2 #11 in a mouse model of age-related macular degeneration.

Background/Rationale.

As described in Example 10, rat MASP-2 protein was used to sort a phage display Fab library from which Fab2 #11 was identified as a functional antibody. Full-length rat IgG2c isotype and mouse IgG2a isotype full-length antibodies were obtained from Fab2 No. 11. A full-length mouse anti-MASP-2 antibody of the IgG2a isotype was characterized for pharmacodynamic parameters as described in Example 19. In this example, a full-length mouse anti-MASP-2 antibody derived from Fab2 No. 11 was analyzed in a mouse model of age-related macular degeneration ( AMD), described in Bora P.S. et al., J Immunol 174:491–497 (2005).

Methods.

A full-length mouse anti-MASP-2 IgG2a antibody derived from Fab2 No. 11 as described in Example 19 was tested in a mouse model of age-related macular degeneration (AMD) as described in Example 13 with the following modifications.

Administration of mouse anti-MASP-2 moAb.

Two different doses (0.3 mg/kg and 1.0 mg/kg) of mouse anti-MASP-2 moAb, along with an isotype control moAb, were administered by IP injection to WT (+/+) mice (n=8 mice per group) 16 h before CNV induction.

Induction of choroidal neovascularization (CNV).

Induction of choroidal neovascularization (CNV) and measurement of CNV volume were performed by laser photocoagulation as described in Example 13.

Results.

In fig. 23 graphically depicts CNV area measured 7 days after laser-induced injury in mice treated with either isotype control moAb or mouse anti-MASP-2 moAb (0.3 mg/kg and 1.0 mg/kg). As shown in FIG. 23, mice pretreated with 1.0 mg/kg hhtu-MASP^ moAb showed a statistically significant (p < 0.01) approximately 50% reduction in CNV seven days after laser exposure. As also shown in FIG. 23 found that a dose of 0.3 mg/kg anti-MASP-2 moAb was ineffective in reducing CNV. It should be noted that a dose of 0.3 mg/kg anti-MASP-2 mAb has been shown to result in partial and transient inhibition of C4b deposition following subcutaneous administration, as described in Example 19 and shown in FIG. 21.

The results described in this example demonstrate that blocking MASP-2 with an inhibitor, such as an anti-MASP-2 moAb, has a preventive and/or therapeutic effect in the treatment of macular degeneration. It should also be noted that these results are consistent with the results obtained in the MASP-2 (-/-) mouse study described in Example 13, which observed a 30% reduction in CNV 7 days after laser treatment in MASP-2 mice ( -/-), compared to wild-type control mice. In addition, the results in this example also show that systemically delivered anti-MASP-2 antibody has a local therapeutic effect in the eye, highlighting the possibility of using the systemic route of administration to treat patients with AMD. In summary, these results provide evidence to support the use of anti-MASP-2 mAb in the treatment of AMD.

Example 21.

This example shows that MASP-2-deficient mice are protected from Neisseria meningitidis-induced death following N. meningitidis infection and have increased bacterial clearance compared with control wild-type mice.

Rationale.

Neisseria meningitidis is a heterotrophic gram-negative diplococcal bacterium known for its role in the development of meningitis and other forms of meningococcal diseases, e.g.

- 93 046178 meningococcemia. N. meningitidis is a leading cause of morbidity and mortality in childhood. Severe complications include septicemia, Waterhouse-Friedrichsen syndrome, adrenal insufficiency, and disseminated intravascular coagulation (DIC). See, for example, Rintala E. et al., Critical Care Medicine 28(7):2373-2378 (2000). Here, the role of the lectin pathway was analyzed in MASP-2 (-/-) and WT (+/+) mice to determine whether MASP-2-deficient mice would be susceptible to N. meningitidis-induced lethality.

Methods.

MASP-2 knockout mice were generated as described in Example 1 and backcrossed for at least 10 generations to C57B1/6. MASP-2 KO mice (n=10) and wild-type C57/B6 mice (n=10) at 10 weeks of age were intravenously dosed with 5x108 CFU/100 μL, 2x108 CFU/100 μL, or 3x107 CFU/100 μL Neisseria meningitidis serogroup A Z2491 in 400 mg/kg iron-dextran complex. The survival of mice after infection was monitored over a 72-hour period. Blood samples were obtained from mice at hourly intervals after infection and analyzed to determine serum levels (log CFU/ml) of N. meningitidis to confirm infection and determine the extent of bacterial clearance from serum.

Results.

In fig. 24A graphically depicts the percentage survival rates of MASP-2 KO and WT mice following an infectious dose of 5x108/100 μl CFU of N. meningitidis. As shown in FIG. 24A, after infection with the highest dose of 5x108/100 μl CFU of N. meningitidis, 100% of MASP-2 KO mice survived the entire 72-hour observation period after infection. In contrast, only 20% of WT mice were still alive 24 h postinfection. These results indicate that MASP-2-deficient mice are protected from N. meningitidis-induced lethality.

In fig. 24B is a graph showing log CFU/ml N. meningitidis recovered at different time points from blood samples obtained from MASP-2 KO and WT mice infected with 5x108 CFU/100 μl N. meningitidis. As shown in FIG. 24B, in WT mice, blood levels of N. meningitidis peaked at approximately 6.5 log CFU/ml at 24 h postinfection and fell to zero at 48 h postinfection. In contrast, in MASP-2 KO mice, N. meningitidis levels peaked at approximately 3.5 log CFU/ml at 6 h postinfection and fell to zero at 36 h postinfection.

In fig. 25A is a graph showing the percentage survival of MASP-2 KO and WT mice after infection with 2x108 CFU/100 μl N. meningitidis. As shown in FIG. 25A, after infection with a dose of 2x108 CFU/100 μl of N. meningitidis, 100% of MASP-2 KO mice survived the entire 72-hour observation period after infection. In contrast, only 80% of WT mice were still alive 24 h postinfection. Consistent with the results shown in FIG. 24A, these results also indicate that MASP-2-deficient mice are protected from N. meningitidis-induced mortality.

In fig. 25B is a graph showing log CFU/ml N. meningitidis recovered at different time points from blood samples obtained from WT mice infected with 2x 108 CFU/100 μl N. meningitidis. As shown in FIG. 25B, blood levels of N. meningitidis in WT mice infected with 2x10 ⁸ CFU peaked at approximately 4 log CFU/ml at 12 h postinfection and fell to zero at 24 h postinfection. In fig. 25C is a graph showing log CFU/ml of N. meningitidis recovered at different time points from blood samples obtained from MASP-2 KO mice infected with 2x108 CFU/100 μl of N. meningitidis. As shown in FIG. 25C, the level of N. meningitidis in the blood of MASP-2 KO mice infected with 2x 108 CFU peaked at approximately 3.5 log CFU/ml at 2 h postinfection and fell to zero at 3 h postinfection. Consistent with the results shown in FIG. 24B, these results demonstrate that although MASP-2 KO mice were infected with the same dose of N. meningitidis as WT mice, MASP-2 KO mice had increased clearance of bacteria from the blood compared to WT mice.

The percentage survival of MASP-2 KO and WT mice after infection with the lowest dose of 3x107 CFU/100 μl N. meningitidis was 100% at 72 h (data not shown).

Discussion.

The results show that MASP-2-deficient mice are protected from N. meningitidis-induced death and have increased bacterial clearance from the blood compared to WT mice. Thus, given these results, it is expected that the therapeutic use of MASP-2 inhibitors, such as anti-MASP-2 moAbs, will be effective in treating, preventing, or reducing the effects of N. meningitidis infection (i.e., sepsis and DIC) . Additionally, these results indicate that therapeutic use of MASP-2 inhibitors, such as anti-MASP-2 moAbs, will not predispose a subject to an increased risk of N. meningitidis infection.

Example 22.

This example describes the discovery of a novel lectin pathway-mediated and MASP-2-dependent activation of complement C3 bypassing C4.

- 94 046178

Rationale.

The major therapeutic benefit of using complement activation inhibitors to limit myocardial ischemia/reperfusion injury (MIRI) was convincingly demonstrated in an experimental rat model of myocardial infarction two decades ago: recombinant sCR1, a soluble truncated derivative of the cell surface complement receptor type 1 (CR1), was administered intravenously, and its effect was evaluated in a rat in vivo MIRI model. Administration of sCR1 resulted in a reduction in infarct volume by more than 40% (Weisman, H.F., et al., Science 249:146-151 (1990)). The therapeutic potential of this recombinant inhibitor was subsequently demonstrated in a clinical trial in which it was shown that administration of sCR1 to patients with MI prevented contractile failure in the post-ischemic heart (Shandelya, S., et al., Circulation 87:536-546 (1993)). However, the underlying mechanism leading to complement activation in ischemic tissue has not been fully elucidated, mainly due to the lack of appropriate experimental models, limited understanding of the molecular processes leading to complement activation in oxygen-deprived cells, and the relationship and synergism between different activation pathways. complement.

As a fundamental component of the immune response, the complement system provides protection against invading microorganisms through both antibody-dependent and -independent mechanisms. It orchestrates a variety of cellular and humoral interactions during the immune response, including chemotaxis, phagocytosis, cell adhesion, and B cell differentiation. Three different pathways initiate the complement cascade: the classical pathway, the alternative pathway, and the lectin pathway. In the classical pathway, the recognition subcomponent C1q binds to various targets predominantly by immune complexes—initiating stepwise activation of the associated serine proteases, C1r and C1s, which provide the primary mechanism for pathogen and immune complex clearance following engagement of the adaptive immune system. Binding of C1q to immune complexes results in the conversion of the C1r zymogen dimer to its active form for cleavage and thereby activation of C1s. C1s translates C1q binding into complement activation in two cleavage steps: first it converts C4 into C4a and C4b, and then it cleaves C4b-bound C2 to form the C3 convertase C4b2a. This complex converts the C3 component, which is abundant in plasma, into C3a and C3b. The accumulation of C3b in the immediate vicinity of the C4b2a complex leads to a shift in substrate specificity from C3 to C5, with the formation of C5 convertase C4b2a(C3b) _n . The C3 and C5 convertase complexes produced through the classical activation pathway are identical to those produced by the lectin activation pathway. In an alternative pathway, spontaneous low-level hydrolysis of the C3 component results in the deposition of protein fragments on cell surfaces, triggering complement activation on foreign cells, while cell-associated regulatory proteins on host tissues prevent activation, thereby preventing self-destruction. Like the alternative pathway, the lectin pathway can be activated in the absence of immune complexes. Activation is initiated by the binding of the multimolecular lectin pathway activation complex to Pathogen-Associated Molecular Patterns (PAMPs), predominantly carbohydrate structures present on bacterial, fungal or viral pathogens, or to aberrant glycosylation patterns on apoptotic, necrotic, malignant or oxygen-deprived cells (Collard, CD, et al., Am. J. Pathol. 156:1549-1556 (2000); Walport, MJ, N. Engl. J. Med. 344:1058-1066 (2001); Schwaeble, W., et al., Immunobiology 205:455-466 (2002); and Fujita, T., Nat. Rev. Immunol. 2:346-353 (2002)).

Mannan-binding lectin (MBL) was the first carbohydrate-recognizing subcomponent that was shown to form complexes with a group of novel serine proteases called MBL-associated serine proteases (MASPs) and numbered in the order of their discovery (i.e., MASP-1, MASP-2 and MASP-3). In humans, lectin pathway activation complexes can be formed with four alternative carbohydrate recognition subcomponents with different binding carbohydrate specificities, i.e., 2 MBLs, and three different members of the ficolin family, namely L-ficolin, H-ficolin and M-ficolin , as well as MASP. Two forms of MBL, MBL A and MBL C, and ficolin-A form lectin pathway activation complexes with MASP in the plasma of mice and rats. We have previously cloned and characterized MASP-2 and an additional truncated 19-kDa MASP-2 gene product, termed MAp19 or sMAP, in humans, mice, and rats (Thiel, S., et al., Nature 386:506-510 (1997 ); Stover, S. M., et al., J. Immunol. 162:3481-3490 (1999); Takahashi, M., et al., Int. Immunol. 11:859-863 (1999); and Stover , S. M., et al., J. Immunol. 163:6848-6859 (1999)). MAp19/sMAP lacks protease activity but can regulate lectin pathway activation by competing with MASP for binding to carbohydrate recognition complexes (Iwaki, D. et al., J. Immunol. 177:8626-8632 (2006)).

There is evidence that of the three MASPs, only MASP-2 is required to translate binding of lectin pathway recognition complexes into complement activation (Thiel, S., et al. (1997); Vorup Jensen, T., et al., J. Immunol. 165 :2093-2100 (2000); Thiel, S., et al., J. Immunol. 165:878-887 (2000); Rossi, V., et al., J. Biol. Chem. 276:40880-40887 (2001)). This conclusion is supported by the phenotype recently described

- 95 046178 new line of mice deficient in MASP-1 and MASP-3. With the exception of a delay in the onset of lectin pathway-mediated complement activation in vitro, MASP-1/3-deficient mice retain functional activity of the lectin pathway. Supplementation of MASP-1 and MASP-3 deficient serum with recombinant MASP-1 overcomes this delay in lectin pathway activation, suggesting that MASP-1 may facilitate MASP-2 activation (Takahashi, M., et al ., J Immunol 180:6132–6138 (2008). A recent study demonstrated that MASP-1 (and possibly also MASP-3) is required for the conversion of the alternative pathway activating enzyme factor D from its zymogen form to its enzymatically active form (Takahashi M. et al., J Exp Med 207(l) :29-37 (2010)). The physiological importance of this process is emphasized by the lack of functional activity of the alternative pathway in plasma in MASP-1/3-deficient mice.

In newly developed mouse strains with a combined targeted deficiency of the carbohydrate-recognizing lectin pathway subcomponents MBL A and MBL C, lectin pathway activation can still be initiated through the remaining mouse lectin recognition subcomponent, ficolin A (Takahashi, K., et al., Microbes Infect. 4:773-784 (2002)). The absence of any residual functional lectin pathway activity in MASP-2-deficient mice provides a compelling model for studying the role of this effector branch of the innate humoral immune system in health and disease.

The availability of C4- and MASP-2-deficient mouse strains allowed us to identify a novel lectin pathway-specific but MASP-2-dependent pathway for activating the complement component C3, bypassing C4. The important contribution of this novel lectin pathway-mediated activation pathway bypassing C4 to post-ischemic tissue loss is highlighted by the remarkable protective phenotype of MASP-2 deficiency in MIRI, whereas C4-deficient mice tested in the same model lacked protection.

In this example, we describe a novel lectin pathway-mediated and MASP-2-dependent pathway for activating complement component C3 bypassing C4. The physiological importance of this novel activation pathway is suggested by the protective phenotype of MASP-2 deficiency in an experimental model of myocardial ischemia/reperfusion injury (MIRI), in which C4-deficient animals were not protected.

Methods.

Mice deficient in MASP-2 lack significant abnormalities.

MASP-2-deficient mice were generated as described in Example 1. Both heterozygous (+/-) and homozygous (-/-), MASP-2-deficient mice are healthy and fertile and have no significant abnormalities. They have the same life expectancy as their DT littermates (>18 months). Before studying the phenotype of these mice in experimental models of the disease, mice from the MASP-2-/- line obtained by the inventors were backcrossed for eleven generations on the C57BL/6 genetic background. The complete absence of MASP-2 mRNA was confirmed by Northern blotting of poly A+-selected liver RNA preparations, with a 1.2-kb. The mRNA encoding MAp19 or sMAP (a truncated alternative splice product of the MASP2 gene) is expressed in large quantities.

Results from qRT-PCR analysis using primer pairs specific for either the coding sequence of the MASP-2 serine protease domain (B chain) or the remaining coding sequence for the A chain showed that there was no detectable mRNA coding for the B chain in MASP-2-/- mice, at the same time, the relative content of the disrupted A-chain mRNA transcript was significantly increased. Similarly, the relative abundance of mRNA encoding MAp19/sMAP is increased in MASP-2+/− and MASP-2−/− mice. Plasma MASP-2 levels determined by ELISA for 5 animals of each genotype were 300 ng/ml for WT controls (range 260-330 ng/ml), 360 ng/ml for heterozygous mice (range 330-395 ng/ml) and were undetectable in MASP-2 −/− mice. Using qRT-PCR, mRNA expression profiles were obtained showing that MASP-2-/- mice express mRNA for MBL A, MBL C, ficolin A, MASP1, MASP-3, C1q, C1rA, C1sA, factor B, factor D, C4, and C3 in relative amounts similar to those found in their MASP-2-sufficient littermates (data not shown).

Plasma levels of C3 in littermate MASP-2−/− (n=8) and MASP-2+/+ (n=7) mice were measured using a commercially available mouse C3 ELISA kit (Kamiya, Biomedical, Seattle, WA). . C3 levels in MASP-2-deficient mice (mean 0.84 mg/ml, ±0.34) were similar to those in WT controls (mean 0.92, ±0.37).

Results.

MASP-2 is required for the functional activity of the lectin pathway.

As described in Example 2 and shown in FIG. 5, results of in vitro analysis of MASP-2-/- plasma showed a complete lack of functional lectin pathway activity on mannan- and zymosan-coated activating surfaces for C4 activation. Likewise, no lek-dependent

- 96 046178 tin pathway cleavage of C4 or C3 in plasma MASP-2-/- on surfaces coated with N-acetylglucosamine, which binds and triggers activation through MBL A, MBL C and ficolin A (data not shown).

Analysis of serum and plasma from MASP-2 −/− mice clearly demonstrated that MASP-2 is required for complement activation through the lectin pathway. However, the complete lack of functional activity of the lectin pathway did not affect other pathways of complement activation: MASP-2-/- plasma was still able to activate complement through the classical (Fig. 26A) and alternative pathway (Fig. 26B). In fig. 26A and 26B the symbol * denotes serum from DT (MASP-2 (+/+)); the symbol □ denotes serum from DT (depleted of C1q); the symbol □ indicates serum from MASP-2 (-/-); and symbol A indicates serum from MASP-2 (-/-) (C1q-depleted).

In fig. 26A is a graph showing that MASP-2-/- mice retain a functional classical pathway: C3b deposition was assayed on microtiter plates coated with immune complexes (prepared in situ by application of BSA followed by the addition of goat anti-BSA IgG). In fig. 26B is a graph showing that MASP-2-deficient mice retain a functional alternative pathway: C3b deposition was assayed on zymosan-coated microtiter plates under conditions allowing only the alternative pathway activation (buffer containing Mg2 ⁺ and EGTA). The results presented in Fig. 26A and FIG. 26B are averages of two replicates and are representative of three independent experiments. The same symbols for plasma sources are used throughout. These results indicate that a functional alternative pathway is present in MASP-2-deficient mice, as evidenced by the results presented in FIG. 26B under experimental conditions designed to directly initiate the alternative pathway while inactivating both the classical pathway and the lectin pathway.

The lectin pathway of complement activation is an important contributor to inflammatory tissue loss in myocardial ischemia/reperfusion injury (MIRI).

To examine the contribution of lectin pathway functional activity to MIRI, we compared MASP-2 −/− mice and WT littermate controls in a MIRI model following transient occlusion and reperfusion of the left anterior descending coronary artery (LAD). The presence or absence of C4 complement had no effect on the degree of ischemic tissue loss in MIRI. The authors of the invention assessed the effect of C4 deficiency on the size of the necrosis lesion after the MIRI experiment. As shown in FIG. 27A and FIG. 27B, identical foci of necrosis occurred in both C4-deficient mice and their WT littermates. in fig. 27A is a graph showing MIRI-induced tissue loss following LAD occlusion and reperfusion in C4-/- mice (n=6) and matched WT littermate controls (n=7). in fig. 27B is a graph showing IFI as a function of PRO, clearly showing that C4-/- mice are as susceptible to MIRI as their WT controls (dashed line).

These results indicate that C4-deficient mice are not protected from MIRI. This result was unexpected because it contradicts the widely held view that the major fragment generated by C4 activation, C4b, is an essential component of the C3 convertase C4b2a of the classical and lectin pathways. Therefore, we examined whether residual lectin pathway-specific activation of C3 could be detected in plasma from C4-deficient mice and in human plasma.

The lectin pathway can activate complement C3 in the absence of C4 through a novel MASP-2-dependent activation pathway that bypasses C4.

Encouraged by early reports indicating the existence of an activation pathway that bypasses C4 in C4-deficient guinea pig serum (May, J.E., and M. Frank, J. Immunol. 111:1671-1677 (1973)), we analyzed whether C4-deficient mice retain residual functional activity of the classical or lectin pathway, and monitored C3 activation under conditions specific to the complement pathway, excluding the contribution of the alternative pathway.

C3b deposition was assayed on mannan-coated microtiter plates using recalcified plasma at plasma concentrations that precluded the alternative activation pathway (1.25% and below). While no C3 cleavage was detected in C4-deficient plasma tested for classical pathway activation (data not shown), strong residual C3 cleavage activity was observed in the plasma of C4-deficient mice when complement activation through the lectin pathway was initiated. Lectin pathway dependence was demonstrated by competitive inhibition of C3 cleavage following preincubation of C4-deficient dilute plasma with soluble mannan (see FIG. 28A). As shown in FIG. 28A-D, MASP-2-dependent activation of C3 occurred in the absence of C4. in fig. 28A is a graph showing C3b deposition using C4+/+ (crosses) and C47- (open circles) mouse plasma. Preincubation of C47 plasma with an excess (1 μg/ml) of liquid-phase mannan before analysis resulted in complete inhibition of C3 deposition (filled circles). The results are

- 97 046178 representative of 3 independent experiments. in fig. 28B is a graph showing the results of an experiment in which wild-type, MASP-2-deficient (open squares), and C4-/- mouse plasma (1%) was mixed with anti-rat MASP-2 mAb M11 at various concentrations (abscissa), and C3b deposition was analyzed on mannan-coated plates. Results are means (±SD) of 4 assays (duplicates of 2 for each plasma type). in fig. 28C is a graph showing the results of an experiment using human plasma: pooled NSF (crosses), C4-/- plasma (open circles), and C4-/plasma (filled circles) preincubated with 1 μg/ml mannan solution. Results are representative of three independent experiments. in fig. 28D is a graph showing inhibition of C3b deposition in C4-sufficient and C4-deficient (1%) human plasma by anti-human MASP-2 mAb H3 (mean ± SD of triplicates). As shown in FIG. 28B, in MASP-2-/- plasma analyzed in parallel, there was no lectin pathway-dependent activation of C3, indicating that this pathway of C3 activation bypassing C4 is MASP-2-dependent.

To further confirm these results, we generated a series of recombinant inhibitory mAbs isolated from phage display antibody libraries by affinity binding screening with recombinant human and rat MASP-2A (in which the serine residue in the active domain of the protease was replaced by an alanine residue in a site-directed manner). mutagenesis to prevent autolytic cleavage of the antigen). Recombinant antibodies against MASP-2 (Ab H3 and Ab M11) were isolated from combinatorial antibody libraries (Knappik, A., et al., J. Mol. Biol. 296:57-86 (2000)) using recombinant human and rat MASP-2A as antigens (Chen, S. B. and Wallis, J. Biol. Chem. 276:25894-25902 (2001)). An anti-rat Fab2 fragment that potently inhibited lectin pathway-mediated activation of C4 and C3 in mouse plasma (IC ₅₀ ~1 nM) was converted to a full-length IgG2a antibody. Polyclonal anti-mouse MASP-2A immune serum was obtained from rats. These tools allowed us to confirm the MASP-2 dependence of this novel lectin pathway-specific pathway to activate C3 bypassing C4, as further described below.

As shown in FIG. 28B, M211, an inhibitory monoclonal antibody that selectively binds murine and rat MASP-2, inhibited C3 activation by bypassing C4 in C4-deficient mice, as well as C3 activation in plasma of WT mice via the lectin pathway in a concentration-dependent manner with similar _IC50 values. . All assays were performed at high plasma dilutions, which renders the alternative activation pathway nonfunctional (maximum plasma concentration was 1.25%).

To determine whether there is a similar lectin pathway-specific activation of C3 bypassing C4 in humans, we analyzed plasma from a donor with congenital deficiency of both human C4 genes (i.e., C4A and C4B), which results in complete absence of C4 (Yang, Y., et al. al., J. Immunol. 173:2803-2814 (2004)). in fig. 28C shows that this patient's plasma exhibits efficient activation of C3 when the plasma is highly diluted (rendering the alternative pathway nonfunctional). A lectin pathway-specific pattern of C3 activation on mannan-coated plates was demonstrated for murine C4-deficient plasma (FIG. 28A) and human C4-deficient plasma (FIG. 28C) by adding excess concentrations of liquid-phase mannan. The MASP-2 dependence of this mechanism of C3 activation in human C4-deficient plasma was assessed using Ab H3, a monoclonal antibody that specifically binds human MASP-2 and abrogates the functional activity of MASP-2. As shown in FIG. 28D, Ab H3 inhibited C3b (and C3dg) deposition in both C4-sufficient and C4-deficient human plasma with comparable efficacy.

To evaluate the possible role of other complement components in activating C3 bypassing C4, we tested plasma from MASP-1/3-/- and LiqO-Dn mice along with plasma from MASP-2-/-, C4-/-, and C1q-/- mice. (as controls) under conditions both specific for the lectin pathway and specific for the classical pathway. A graph was made for the amount of C3 cleaved versus the amount of C3 product deposited when using DT plasma.

In fig. 29A is a graph showing the results of a comparative analysis of C3 convertase activity in the plasma of various strains of complement deficient mice tested under assay conditions specific for either the lectin pathway or the classical pathway. Diluted plasma samples (1%) from WT mice (n=6), MASP-2-/- mice (n=4), MASP-1/3-/- mice (n=2), C4-/- mice (n =8), C4/MASP-1/3-/- mice (n=8), Bf/C2-/- mice (n=2), and C1q/- mice (n=2) were tested in parallel. Addition of 2.5 μg/ml recombinant rat C2 (Bf/C2-/- + C2) to the plasma of Bf/C2-/- resulted in the restoration of C3b deposition. Results are means (±SD), **p<0.01 (compared with WT plasma). As shown in FIG. 29A, significant C3 deposition is observed in C4-/- plasma tested under lectin pathway-specific conditions but not under classical pathway-specific conditions. Again, C3 deposition was absent in MASP-2-deficient plasma in the case of the lectin activation pathway, while C3 deposition occurred in the same plasma through the classical pathway. In MASP-1/3-/- plasma, C3 deposition occurred under conditions specific to both the lectin and classical pathways. C3 deposition was not observed in plasma with combined C4 and MASP-1/3 deficiency using both lectin pathway-specific and classical pathway-specific conditions. There was no detectable C3 deposition in C2/Bf−/− plasma in either the lectin pathway or the classical pathway. However, the addition of C2/Bf7 recombinant C2 to mouse plasma resulted in restoration of both lectin- and classical-mediated C3 degradation. Assay conditions were validated using C1q−/− plasma.

In fig. 29B is a graph showing the time-resolved kinetics of C3 convertase activity in plasma from various strains of complement-deficient mice, WT, fB-/-, C4-/-, MASP-1/3-/-, and MASP-2-/- plasma. , tested under lectin pathway-specific activation conditions (1% plasma, results are representative of three independent experiments). As shown in FIG. 29B, although C3 cleavage was absent in MASP-2−/− plasma, C3 cleavage occurred in fB−/ plasma with kinetics similar to those in WT plasma. A significant delay in the lectin pathway-dependent conversion of C3 to C3b (and C3dg) was observed in C4-/- plasma as well as in MASP-1/3-deficient plasma. This delay in plasma MASP-1/37 C3 activation was recently shown to be MASP-1 dependent rather than MASP-3 dependent (Takahashi, M., et al., J. Immunol. 180:6132-6138 ( 2008)).

Discussion.

The results described in this example strongly suggest that the functional activity of MASP-2 is critical for the activation of C3 through the lectin pathway, both in the presence and absence of C4. In addition, C2 and MASP-1 are required for the function of this novel lectin pathway-specific pathway to activate C3 bypassing C4. A comparative analysis of the functional activity of the lectin pathway in the plasma of MASP-2-/-, as well as C4-/-, revealed the presence of a previously unknown, C4-independent, but MASP-2-dependent, pathway of C3 complement activation and showed that C3 can be activated in a lectin pathway-dependent manner in the complete absence of C4. Although the detailed molecular composition and sequence of activation events of this novel MASP-2-dependent C3 pathway remains to be elucidated, our results suggest that this activation pathway bypassing C4 also requires the presence of complement C2 and MASP-1. The loss of lectin pathway-mediated C3 cleavage activity in the plasma of mice with combined C4 and MASP-1/3 deficiency may be explained by the more recently described role of MASP-1 in enhancing MASP-2-dependent complement activation through direct cleavage and activation of MASP-2 (Takahashi, M., et al., J. Immunol. 180:6132-6138 (2008)). Likewise, MASP-1 may contribute to the functional activity of MASP-2 through its ability to cleave C2 (Moller-Kristensen, et al., Int. Immunol. 19:141-149 (2007)). These two activities may explain the reduced rate at which MASP-1/3-deficient plasma degrades C3 through the lectin activation pathway and the reason why MASP-1 may be required to support the conversion of C3 through the activation pathway to bypass C4.

The inability of C2/fB −/− plasma to activate C3 through the lectin pathway was shown to be C2 dependent, since addition of recombinant rat C2 to C2/fB −/− plasma resulted in restoration of the ability of reconstituted plasma to activate C3 on mannan-coated plates.

The fact that C4 deficiency leads to a specific impairment of the classical complement activation pathway, while the lectin pathway maintains a physiologically critical level of C3 convertase activity through the MASP-2-dependent activation pathway bypassing C4, provides grounds for reassessing the role of the lectin pathway in various disease models, including experimental infection with S. pneumoniae (Brown, J. S., et al., Proc. Natl. Acad. Sci. U. S. A 99:16969-16974 (2002); experimental allergic encephalomyelitis (Boos, L. A., et al., Glia 49:158-160 (2005); as well as a model of C3-dependent murine liver regeneration (Clark, A., et al., Mol. Immunol. 45:3125-3132 (2008)). The authors of the latter work demonstrated that in mice with deficiency C4 activation of C3 may occur in an alternative pathway-independent manner, since in vivo inhibition of the alternative pathway by antibody-mediated inhibition of factor B function did not affect C3 cleavage-dependent liver recovery in C4-/- mice (Clark, A., et al. (2008)). This lectin pathway-mediated activation of C3 bypassing C4 may also explain the lack of a protective phenotype associated with C4 deficiency in our MIRI model, as well as in a previously described model of renal allograft rejection (Lin, T., et al., Am. J. Pathol 168:1241–1248 (2006)). In contrast, our early results independently demonstrated a largely protective phenotype in MASP-27- mice in kidney transplant models (Farrar, S.A., et al., Mol. Immunol. 46:2832 (2009)).

In summary, the results described here provide evidence that MASP-2-dependent activation of C3 bypassing C4 is a physiologically relevant mechanism that may be important in conditions where C4 availability limits C3 activation.

- 99 046178

Example 23.

This example describes the activation of C3 by thrombin substrates and the deposition of C3 on mannan in WT (+/+), MASP-2 (-/-), F11 (-/-), F11/C4 (-/-) and C4 (-) mice. /-).

Rationale.

As described in Example 14, it was found that activation of thrombin can occur after activation of the lectin pathway under physiological conditions, and the extent of MASP-2 involvement was demonstrated. C3 plays a central role in the activation of the complement system. Activation of C3 is required for both the classical and alternative pathways of complement activation. An experiment was performed to determine whether C3 is activated by thrombin substrates.

Methods.

Activation of C3 by thrombin substrates.

C3 activation was measured in the presence of the following activated forms of thrombin substrates; human FCXIa, human FVIIa, bovine FXa, human FXa, human activated protein C and human thrombin. C3 was incubated with different thrombin substrates and then separated under reducing conditions on 10% SDS-polyacrylamide gels. After electrophoretic transfer to a cellulose membrane, the membrane was incubated with monoclonal biotinylated rat anti-mouse C3 antibody, detected with a streptavidin-HRP conjugate kit and color developed with ECL reagent.

Results.

Activation of C3 involves cleavage of the intact α-chain into a truncated α'-chain and soluble C3a (not shown in Fig. 30). in fig. 30 shows the results of a Western blot analysis of the activation of human C3 by thrombin substrates, with the uncleaved C3 alpha chain and the activation product α'chain indicated by arrows. As shown in FIG. 30, incubation of C3 with activated forms of human coagulation factors XI and X, as well as activated bovine coagulation factor X, can lead to C3 cleavage in vitro in the absence of any complement proteases.

Deposition of C3 on mannan.

C3 deposition assays were performed with serum samples obtained from WT, MASP-2(/−), F11(−/−), F11(−/−)/C4(−/−), and C4(−/−) mice. F11 is a gene encoding coagulation factor XI. To measure C3 activation, microtiter plates were coated with mannan (1 μg/well), then sheep anti-HSA serum (2 μg/ml) in TBS/Tween/Ca ²⁺ was added. The plates were blocked with 0.1% HSA in TBS and washed as described above. Plasma samples were diluted in 4 mM barbital solution with 145 mM NaCl, 2 mM _CaCl2 , 1 mM _MgCl2 , pH 7.4, added to plates and incubated for 1.5 h at 37°C. After washing, bound C3b was detected using rabbit anti-human C3c (Dako), followed by alkaline phosphatase-conjugated goat anti-rabbit IgG and pNPP.

Results.

In fig. 31 shows the results of a C3 deposition assay using serum samples obtained from WT, MASP-2 (-/-), F11(-/-), F11(-/-)/C4 (-/-) and C4 (-/-) mice. -). As shown in FIG. 31, a functional lectin pathway occurs even in the complete absence of C4. As also shown in FIG. 31, coagulation factor XI is required for this novel lectin pathway-dependent complement activation.

Discussion.

Prior to the results of this experiment, those in the field believed that C4 was required for the activity of the complement lectin pathway. As a consequence, data obtained for C4 knockout mice (and for C4-deficient humans) have been interpreted to suggest that such organisms lack the lectin pathway (in addition to lacking the classical pathway). The present results show that this assumption is not correct. Thus, conclusions from past studies suggesting that the lectin pathway is not important in some diseases based on the phenotype of C4-deficient animals may be false. The data described in this example also demonstrate that, in the physiological context of whole serum, the lectin pathway can activate components of the coagulation cascade. Thus, it is shown that there is an interaction between complement and coagulation involving MASP-2.

Example 24.

This example describes methods for assessing the effect of an anti-MASP-2 antibody on red blood cell lysis in blood samples obtained from patients with paroxysmal nocturnal hemoglobinuria (PNH).

Background/Rationale.

Paroxysmal nocturnal hemoglobinuria (PNH), also called Marchiafava Micheli syndrome, is an acquired, potentially life-threatening blood disorder characterized by complement-induced intravascular hemolytic anemia. A characteristic feature of PNH is chronic intravascular hemolysis, which is a consequence of increased activation of the alternative complement pathway. Lindorfer, M.A., et al., Blood 115(11) (2010). Anemia in PNH results from the destruction of red blood cells in the bloodstream. Symptoms of PNH

- 100 046178 include red urine due to the appearance of hemoglobin in the urine and thrombosis. PNH can occur on its own and is called primary PNH, or in the context of other bone marrow diseases such as aplastic anemia, it is called secondary PNH. Treatment of PNH includes blood transfusions for anemia, the use of anticoagulants for thrombosis, and the use of the monoclonal antibody eculizumab (Soliris), which protects blood cells from immune destruction by inhibiting the complement system (Hillmen P. et al., N. Engl. J. Med. 350 (6):552-9 (2004)). However, a significant proportion of PNH patients treated with eculizumab continue to have clinically significant immune system-mediated hemolytic anemia because the antibody does not block activation of the alternative complement pathway.

This example describes methods for assessing the effect of an anti-MASP-2 antibody on red blood cell lysis from blood samples obtained from PNH patients (not treated with Soliris) that are incubated with ABO-matched acidified normal human serum.

Methods.

Reagents.

Red blood cells from healthy donors and from patients suffering from PNH (not treated with Soliris) are obtained by vein puncture and prepared as described in Wilcox, L.A., et al., Blood 78:820-829 (1991), the contents of which are included incorporated into this document by reference. Anti-MASP-2 antibodies with the function of blocking lectin pathway activity can be prepared as described in Example 10.

Hemolysis assay.

A method for determining the effect of anti-MASP-2 antibodies on the ability to block hemolysis of red blood cells of patients with PNH was carried out using the methods described in Lindorfer, MA, et al., Blood 15(11):2283-91 (2010) and Wilcox, LA, et al., Blood 78:820-829 (1991), the contents of both of which are incorporated herein by reference. As described by Lindorfer et al., red blood cells from samples from PNH patients are centrifuged, the clear clot layer is removed by aspiration, and the cells are washed in gelatin-veronal buffer (GVB) before each experiment. Red blood cells are tested for susceptibility to APC-mediated lysis as follows. ABO-matched normal human serum is diluted with GVB containing 0.15 mM CaCl ₂ and 0.5 mM MgCl ₂ (GVB+ ² ), acidified to pH 6.4 (acidified SNP, cSNP) and used to restore red blood cells to a hematocrit of 1.6 % in 50% cNCS. The mixtures are then incubated at 37°C, and after 1 hour the red blood cells are pelleted by centrifugation. The absorbance of an aliquot of the recovered supernatant was measured at 405 nM and used to calculate percent lysis. Samples reconstituted in acidified serum-EDTA are processed similarly and used to determine background non-complement-mediated lysis (typically less than 3%). Complete lysis (100%) is determined after incubation of erythrocytes in distilled water.

To determine the effect of anti-MASP-2 antibodies on the hemolysis of PNH erythrocytes, erythrocytes from patients with PNH are incubated in cNCS in the presence of anti-MASP-2 antibodies in stepwise increasing concentrations, and then the presence/extent of hemolysis is quantified.

In view of the fact that anti-MASP-2 antibodies have been shown to block subsequent activation of the alternative complement pathway, anti-MASP-2 antibodies are expected to be effective in blocking alternative pathway-mediated hemolysis of PNH red blood cells and will be useful as a therapeutic agent for treatment of patients suffering from PNH.

Example 25.

This example describes methods for assessing the effect of an anti-MASP-2 blocking antibody on complement activation by cryoglobulins in blood samples obtained from patients suffering from cryoglobulinemia.

Background/Rationale

Cryoglobulinemia is characterized by the presence of cryoglobulins in the serum. Cryoglobulins are single or mixed immunoglobulins (usually IgM antibodies) that undergo reversible aggregation at low temperatures. Aggregation leads to activation of the classical complement pathway and inflammation in the vascular bed, in particular in the periphery. Clinical manifestations of cryoglobulinemia include vasculitis and glomerulonephritis.

Cryoglobulinemia can be classified as follows depending on the composition of the cryoglobulins: type I cryoglobulinemia, or simple cryoglobulinemia, results from a monoclonal immunoglobulin, usually immunoglobulin M (IgM); cryoglobulinemia types II and III (mixed cryoglobulinemia) include rheumatoid factors (RF), which are usually IgM complexed with the Fc region of polyclonal IgG.

Conditions associated with cryoglobulinemia include hepatitis C infection, lymphoproliferative diseases, and other autoimmune diseases. Cryoglobulin-containing immune complexes lead to a clinical syndrome of systemic inflammation, possibly due to their ability to activate complement. Although IgG immune complexes typically activate the classical complement pathway, IgM-containing complexes can also activate complement via the lectin pathway.

- 101 046178 (Zhang, M., et al., Mol Immunol 44(1-3):103-110 (2007) and Zhang. M., et al., J. Immunol. 177(7):4727-34 (2006)).

Immunohistochemical studies have further demonstrated that cryoglobulin immune complexes contain components of the lectin pathway, and immunohistochemical evidence of in situ activation of the lectin pathway was found in biopsy specimens from patients with cryoglobulinemic glomerulonephritis (Ohsawa, I., et al., Clin Immunol 101(l):59 -66 (2001)). These results suggest that the lectin pathway may contribute to inflammation and adverse outcomes in cryoglobulinemic diseases.

Methods.

A method for determining the effect of anti-MASP-2 antibodies in blocking the adverse effects of cryoglobulinemia is carried out using the C3 liquid phase conversion assay described in YC Ng et al., Arthritis and Rheumatism 31(1):99-107 (1988), the contents of which incorporated herein by reference. As described by Ng et al., in essential mixed cryoglobulinemia (EMC), monoclonal rheumatoid factor (mRF), typically IgM, complexes with polyclonal IgG to form cryoprecipitate immune complexes (IC) (type II cryoglobulin). Immunoglobulins and C3 were found in the walls of blood vessels in affected tissues such as skin, nerves and kidneys. As described by Ng et al., ¹²⁵ I-labeled mRF is added to serum (normal human serum and serum obtained from patients suffering from cryoglobulinemia), incubated at 37°C and binding to erythrocytes measured.

Liquid phase conversion of C3 is determined in serum (normal human serum and serum obtained from patients suffering from cryoglobulinemia) in the presence or absence of the following ICs: BSA-anti BSA, mRF, mRF plus IgG or cryoglobulins, in the presence or absence of αntu-MASP -2 antibodies. Fixation of C3 and C4 to the IC is measured using a co-precipitation assay with F(ab')2 anti-C3 and F(ab')2 anti-C4.

In view of the fact that α-MASP-2 antibodies have been shown to block lectin pathway activation, anti-MASP-2 antibodies are expected to be effective in blocking the complement-mediated adverse effects associated with cryoglobulinemia and to be useful as therapeutic agents for treatment of patients suffering from cryoglobulinemia.

Example 26.

This example describes methods for assessing the effect of anti-MASP-2 antibodies on blood samples obtained from patients suffering from cold agglutinin disease, which manifests itself as anemia.

Background/Rationale.

Cold agglutinin disease (CAD) is a type of autoimmune hemolytic anemia. Cold agglutinin antibodies (usually IgM) are activated by low temperatures and bind and aggregate red blood cells. Cold agglutinin antibodies, in combination with complement, attack the antigen on the surface of red blood cells. This leads to opsonization of red blood cells (hemolysis), which initiates their clearance by the reticuloendothelial system. The temperature at which agglutination occurs varies among patients.

CAD manifests as anemia. When the rate of red blood cell destruction exceeds the bone marrow's ability to produce enough oxygen-carrying cells, anemia occurs. CAD can be caused by an underlying disease or disorder, called secondary CAD, and the underlying disease may be an infectious disease (mycoplasma pneumonia, mumps, mononucleosis), a lymphoproliferative disease (lymphoma, chronic lymphocytic leukemia) or a connective tissue disease. Patients with primary CAD are thought to have a low-grade lymphoproliferative disease of the bone marrow. Both primary and secondary CAD are acquired conditions.

Methods.

Reagents.

Red blood cells from healthy donors and from patients suffering from CAD are obtained by puncturing a vein. Ahtu-MASP-2 antibodies with the function of blocking lectin pathway activity can be prepared as described in Example 10.

The ability of anti-MASP-2 antibodies to block Cold agglutinin-mediated activation of the lectin pathway can be determined as follows.

Red blood cells from blood type I patients are sensitized by Cold agglutinins (ie, IgM antibodies) in the presence or absence of anti-MASP-2 antibodies. Red blood cells are then tested for their ability to activate the lectin pathway by measuring C3 binding.

In view of the fact that anti-MASP-2 antibodies have been shown to block lectin pathway activation, anti-MASP-2 antibodies are expected to be effective in blocking the complement-mediated adverse effects associated with cold agglutinin disease and to be useful as therapeutic agents. agents for the treatment of patients suffering from cold agglutination disease

- 102 046178 nin.

Example 27.

This example describes methods for assessing the effect of aHTu-MASP-2 antibody on erythrocyte lysis in blood samples obtained from mice with atypical hemolytic uremic syndrome (aHUS).

Background/Rationale.

Atypical hemolytic uremic syndrome (aHUS) is characterized by the presence of hemolytic anemia, thrombocytopenia, and renal failure due to platelet thrombi in the microvascular network of the kidney and other organs. aHUS is associated with dysregulated complement and can be either sporadic or familial. Familial cases of aHUS are associated with mutations in genes encoding proteins responsible for complement activation, including complement factor H, membrane cofactor B and factor I, as well as complement factor H-related protein 1 (CFHR1) and complement factor H-related protein 3 ( CFHR3). Zipfel, P. F., et al., PloS Genetics 3(3):e41 (2007). This example describes methods for assessing the effect of an anti-MASP-2 antibody on erythrocyte lysis from blood samples obtained from mice with aHUS.

Methods.

The ability of anti-MASP-2 antibodies to treat aHUS can be determined in a mouse model of this disease in which the endogenous murine fH gene is replaced with a human homologue encoding a mutant form of fH commonly found in patients with aHUS. See publication Pickering M.C. et al., J. Exp. Med. 204(6): 1249-1256 (2007), the contents of which are incorporated herein by reference. As described by Pickering et al., these mice develop aHUS-like pathology. To evaluate the feasibility of using anti-MASP-2 antibodies to treat aHUS, anti-MASP2 antibodies were administered to mutant mice with aHUS and erythrocyte lysis was compared in samples from anti-MASP-2 Ab-treated and non-Ab-treated controls. Due to the fact that anti-MASP -2 antibodies have been shown to block lectin pathway activation, anti-MASP-2 antibodies are expected to be effective in blocking erythrocyte lysis in mammalian subjects suffering from aHUS.

Example 28.

This example describes methods for assessing the effect of anti-MASP-2 antibodies in the treatment of glaucoma. Rationale/background.

Uncontrolled complement activation has been shown to contribute to the progression of degenerative damage to retinal ganglion cells (RGCs), their synapses, and axons in glaucoma. See Tezel G. et al., Invest Ophthalmol Vis Sci 51:5071-5082 (2010). For example, histopathological studies of human tissues and in vivo studies using various animal models have shown that the glaucomatous retina synthesizes complement components, including C1q and C3, and forms the terminal complement complex (see Stasi K. et al., Invest Ophthalmol Vis Sci 47: 10241029 (2006), Kuehn M.H. et al., Exp Eye Res 83:620-628 (2006)). As also described in Kuehn M.H. et al., Experimental Eye Research 87:89–95 (2008), the synthesis and deposition of complement components is induced by retinal I/R, and disruption of the complement cascade results in delayed RGC degeneration. In this study, mice that are targeted to disrupt complement component C3 are shown to have delayed RGC degeneration following transient retinal I/R compared with normal controls.

Methods.

A method for determining the effect of anti-MASP-2 antibody on RGC degeneration was performed using an animal model of retinal I/R described in Kuehn M.H. et al., Experimental Eye Research 87:89-95 (2008), the contents of which are incorporated herein by reference. As described in Kuehn et al., retinal ischemia is induced, after anesthetizing the animal, by inserting a 30-gauge needle connected to a reservoir containing phosphate-buffered saline through the cornea into the anterior chamber of the eye. The saline reservoir is then elevated, increasing the intraocular pressure to 104 mmHg. Art., which is sufficient to completely prevent circulation in the retinal vasculature. The presence of increased intraocular ischemia is confirmed by pallor of the iris and retina, and ischemia is maintained for 45 minutes only in the left eye; the right eye serves as a control and remains without cannulation. Mice were then sacrificed 1 or 3 weeks after ischemic stroke. Ahtu-MASP-2 antibodies were administered to mice either locally into the eye or systemically to evaluate the effect of anti-MASP antibody administered prior to ischemic stroke.

An immunohistochemical examination of the eyes is performed using antibodies against C1q and C3 to detect the deposition of complement components. Optic nerve damage can also be assessed using standard electron microscopy techniques. Quantification of surviving retinal RGCs is performed using gamma-synuclein labeling.

Results.

As described by Kuehn et al., in normal control mice, transient retinal ischemia leads to degenerative changes in the optic nerve and retinal deposition of C1q and C3, which can be detected by immunohistochemistry. In contrast, C3-deficient mice show a marked reduction in axonal degeneration and only minor levels of visual damage.

- 103 046178 body nerve 1 week after induction. Based on these results, it is expected that similar results would occur if this assay were performed in MASP-2 knockout mice and anti-MASP-2 antibodies were administered to normal mice before ischemic stroke.

Example 29.

This example demonstrates that a MASP-2 inhibitor, such as an anti-MASP-2 antibody, is effective for treating the effects of radiation and/or for treating, alleviating or preventing acute radiation syndrome.

Rationale.

Exposure to high doses of ionizing radiation causes death through two mechanisms: bone marrow toxicity and gastrointestinal syndrome. Bone marrow toxicity causes the number of all blood cells to fall, predisposing the body to death from infection and bleeding. Gastrointestinal syndrome is a more severe condition and is caused by loss of intestinal barrier function due to destruction of the intestinal epithelial layer and loss of intestinal endocrine function. This leads to sepsis and the associated systemic inflammatory response syndrome, which can lead to death.

The lectin complement pathway is an innate immune mechanism that initiates inflammation in response to tissue damage and exposure to foreign surfaces (ie, bacteria). Blocking this pathway leads to better outcomes in mouse models of ischemic intestinal tissue injury or septic shock. It has been suggested that the lectin pathway may cause excessive and harmful inflammation in response to radiation-induced tissue damage. Blocking the lectin pathway may thus reduce secondary damage and lead to increased survival after acute radiation injury.

The purpose of the study, conducted as described in this example, was to evaluate the effect of blocking the lectin pathway by administering anti-mouse MASP-2 antibodies on survival in a mouse model of radiation injury.

Materials and methods.

Materials. The drugs tested in this study were (i) high-affinity anti-mouse MASP-2 antibody (mAb M11) and (ii) high-affinity anti-human MASP-2 antibody (mAb H6), antibodies that block the MASP-2 protein component of the complement lectin pathway, which were produced in transfected mammalian cells. Dosing concentrations were 1 mg/kg anti-mouse MASP-2 antibody (mAb M11), 5 mg/kg anti-human MASP-2 antibody (mAb H6), or sterile saline. For each dosing session, an appropriate volume of fresh dosing solutions was prepared.

Animals. Young adult male Swiss-Webster mice were obtained from Harlan Laboratories (Houston, TX). Animals were housed in hard-bottom cages with Alpha-Dri bedding and received a PMI 5002 certified rodent diet (Animal Specialties, Inc., Hubbard OR) and water ad libitum. The temperature in the animal room was controlled and the light regime was maintained at 12 h light/12 h dark.

Irradiation. After 2 weeks of indoor acclimation, mice in groups of 10 were irradiated with 6.5 and 7.0 Gy through whole body irradiation at a dose rate of 0.78 Gy/min using a Therapax X-RAD 320 system equipped with a high stability 320-kV X-ray generator, metal-ceramic X-ray tube, variable X-ray collimator, and filter (Precision X-ray Incorporated, East Haven, CT). Dose levels were chosen based on previous studies conducted in mice of the same strain, which showed an LD50/30 of 6.5 to 7.0 Gy (data not shown).

Formulation and administration of the drug. Appropriate volumes of concentrated stock solutions were diluted with ice-cold saline to produce dosing solutions containing concentrations of 0.2 mg/mL anti-mouse MASP-2 antibody (mAb M11) or 0.5 mg/mL anti-human MASP-2 antibody (mAb H6) concentrations. in accordance with the protocol. Anti-MASP-2 antibodies mAb M11 and mAb H6 were administered by intravenous injection using a 25-gauge needle to deliver, based on the body weight of the animal, 1 mg/kg mAb M11, 5 mg/kg mAb H6, or saline solution.

Study design. Mice were randomly assigned to the groups indicated in Table. 8. Body weight and temperature were measured and recorded daily. Mice in groups 7, 11, and 13 were sacrificed on day 7 after irradiation, and blood was collected by cardiac puncture under deep anesthesia. Surviving animals on day 30 after irradiation were sacrificed in the same manner, and blood was collected. From the collected blood samples, plasma was obtained according to the protocol and returned to the Sponsor for analysis.

- 104 046178

Table 8

Study Groups

Group N Irradiation level (Gy) A drug Dosage schedule 1 20 6.5 Solvent 18 hours before irradiation, 2 hours after irradiation, weekly booster 2 20 6.5 Ab against mouse MASP-2 (mAb MP) Only 18 hours before irradiation 3 20 6.5 Ab against mouse MASP-2 18 hours before irradiation, 2 hours after irradiation, weekly (mAb MP) booster 4 20 6.5 Ab against mouse MASP-2 (mAb MP) 2 hours after irradiation, weekly booster 5 20 6.5 At against human MASP-2 (mAb H6) 18 hours before irradiation, 2 hours after irradiation, weekly booster 6 20 7.0 Solvent 18 hours before irradiation, 2 hours after irradiation, weekly booster 7 5 7.0 Solvent Only 2 hours after irradiation 8 20 7.0 Ab against mouse MASP-2 (mAb MP) Only 18 hours before irradiation 9 20 7.0 Ab against mouse MASP-2 (mAb MP) 18 hours before irradiation, 2 hours after irradiation, weekly booster 10 20 7.0 Ab against mouse MASP-2 (mAb MP) 2 hours after irradiation, weekly booster eleven 5 7.0 Ab against mouse MASP-2 (mAb MP) Only 2 hours after irradiation 12 20 7.0 At against human MASP-2 (mAb H6) 18 hours before irradiation, 2 hours after irradiation, weekly booster 13 5 No No No

Statistical analysis. Kaplan-Meier survival curves were constructed and used to compare mean survival times between treatment groups using the log-rank and Wilcoxon tests. Means with standard deviation, or means with standard error of the mean, are presented. Statistical comparisons were made between irradiated control animals and individual treatment groups using a two-tailed unpaired Student's t test.

Results.

Kaplan-Meier survival plots for groups of animals irradiated with 7.0 and 6.5 Gy are presented in Fig. 32A and 32B, respectively, and the data is summarized below in table. 9. Overall, administration of anti-mouse MASP-2 Ab (mAb M11) prior to irradiation resulted in increased survival of irradiated mice compared to vehicle-treated irradiated controls using both irradiation levels of 6.5 (20% increase) and 7.0 Gy (30% increase). At an irradiation level of 6.5 Gy, post-irradiation administration of anti-mouse MASP-2 Ab resulted in a modest increase in survival (15%) compared with vehicle-treated control irradiated animals.

When compared, all drug-treated animals in the 7.0 Gy group demonstrated increased survival compared to vehicle-treated irradiated control animals. The greatest change in survival occurred in animals treated with mAb H6, with a 45% increase compared to control animals. In addition, in the 7.0 Gy group, deaths in the mAb H6 group were first observed on day 15 post-irradiation, compared with day 8 post-irradiation in the vehicle-treated irradiated control group; the survival period was increased by 7 days relative to control animals. The mean time to death in mice treated with mAb H6 (27.3 ± 1.3 days) was significantly increased (p = 0.0087) compared to control animals (20.7 ± 2.0 days) in the radiation level group 7.0 Gy.

The percentage change in body weight compared to the day before exposure (day -1) was recorded throughout the study. A temporary decrease in body weight occurred in all irradiated animals, with no evidence of different changes as a result of treatment with mAb M11 or mAb H6 compared to controls (data not shown). After completion of the study, all surviving animals showed an increase in body weight from the initial body weight (day -1).

Table 9

Survival rate of experimental animals exposed to irradiation

Study group Exposure level In survivability (%) Time to death (mean ± SEM, days) First/last death (days) Control irradiation 6.5 Gy 65% 24.0 ± 2.0 9/16 mAb Ml 1 before irradiation 6.5 Gy 85% 27.7 ± 1.5 13/17

- 106 046178

mAb MP before+after irradiation 6.5 Gy 65% 24.0 ± 2.0 9/15 mATMP after irradiation 6.5 Gy 80% 26.3 ± 1.9 9/13 mAb H6 before+after irradiation 6.5 Gy 65% 24.6+ 1.9 9/19 Control irradiation 7.0 Gy 35% 20.7 ± 2.0 8/17 mAb Ml 1 before irradiation 7.0 Gy 65% 23.0 ± 2.3 7/13 mAb MP before+after irradiation 7.0 Gy 55% 21.6 + 2.2 7/16 mATMP after irradiation 7.0 Gy 70% 24.3 + 2.1 9/14 mAb H6 before+after irradiation 7.0 Gy 80% 27.3 ± 1.3* 15/20

*p=0.0087 using a two-tailed unpaired Student's t test to compare irradiated control animals and treated animals at the same irradiation level.

Discussion.

Acute radiation syndrome consists of three distinct subsyndromes: hematopoietic, gastrointestinal and cerebrovascular. The observed syndrome depends on the radiation dose, with hematopoietic effects observed in humans at radiation levels of a significant portion, or the entire body, exceeding 1 Gy. Hematopoietic syndrome is characterized by severe suppression of bone marrow function, leading to pancytopenia with changes in the number of blood cells, red and white blood cells, and platelets, in parallel with damage to the immune system. During the period of extreme decline with few neutrophils and platelets in the peripheral blood, neutropenia, fever, complications of sepsis and uncontrolled bleeding lead to death.

In the present study, we found that administration of mAb H6 resulted in increased survival of whole body X-ray irradiation in male Swiss-Webster mice at 7.0 Gy. Notably, at an irradiation level of 7.0 Gy, 80% of animals treated with mAb H6 survived for 30 days, compared with 35% of vehicle-treated control irradiated animals. It is important to note that the first death in this treatment group did not occur until day 15 after irradiation, representing a 7-day increase over what occurred in vehicle-treated control irradiated animals. Interestingly, at a lower dose of x-ray irradiation (6.5 Gy), administration of mAb H6 did not appear to affect survival or delay of death compared with vehicle-treated control irradiated animals. There may be several reasons for this difference in response between exposure levels, although testing of any hypothesis may require additional studies, including interim collection of samples for microbiological culture and assessment of hematological parameters. One explanation may simply be that due to the number of animals assigned to groups, any subtle treatment-related differences may not have been seen. For example, with a group size of n=20, the difference in survival between 65% (mAb H6 at 6.5 Gy) and 80% (mAb H6 at 7.0 Gy) is 3 animals. On the other hand, the difference between 35% (vehicle control at 7.0 Gy) and 80% (mAb H6 at 7.0 Gy) is 9 animals and provides strong evidence of treatment-related differences.

The results obtained indicate that anti-MASP-2 antibodies are effective in treating a mammalian subject at risk of developing, or suffering from, the destructive effects of

- 107 046178 for acute radiation syndrome.

Example 30.

This example shows that MASP-2-deficient mice are protected from Neisseria meningitidis-induced death following infection with either N. meningitidis serogroup A or Neisseria meningitidis serogroup B.

Methods.

MASP-2 knockout mice (MASP-2 KO mice) were generated as described in Example 1. 10-week-old MASP-2 KO mice (n=10) and C57/BL6 wild-type (WT) mice (n=10) were given intraperitoneal (i.p.) injection a dose of 2.6x10 ⁷ CFU of Neisseria meningitidis serogroup A Z2491 in a volume of 100 μl. The infectious dose was administered to mice in combination with an iron-dextran complex at a final concentration of 400 mg/kg. The survival of mice after infection was monitored over a 72-hour period.

In a separate experiment, 10-week-old MASP-2 KO mice (n=10) and wild-type C57/BL6 mice (n=10) were administered an intravenous injection of 6x106 CFU of Neisseria meningitidis serogroup B strain MC58 in a volume of 100 μl. The infectious dose was administered to mice in combination with an iron-dextran complex at a final dose of 400 mg/kg. The survival of mice after infection was monitored over a 72-hour period. Unhealth scores were also determined for WT and MASP-2 KO mice during the 72-hour time period postinfection, based on the unhealthy scores shown in Table 1 below. 10, which is based on the scheme described in Fransen et al. (2010), with minor modifications.

Table 10

Illness scores associated with clinical signs in infected mice

Signs Points Norm 0 Slightly ruffled fur 1 Ruffled fur, slow moving and drooping eyes 2 Tousled fur, lethargy, closed eyes 3 Very serious condition, no response to stimulation 4 Death 5

Blood samples were collected from mice at hourly intervals after infection and analyzed to determine serum levels (log CFU/ml) of N. meningitidis to confirm infection and determine the rate of bacterial clearance from serum.

Results.

In fig. 33 is a Kaplan-Meier plot showing the percentage survival of MASP-2 KO and WT mice following an infectious dose of 2.6 x 107 CFU of N. meningitidis serogroup A Z2491. As shown in FIG. 33, 100% of MASP-2 KO mice survived the entire 72-hour period postinfection. In contrast, only 80% of WT mice (p = 0.012) were still alive 24 h postinfection, and only 50% of WT mice were still alive 72 h postinfection. These results indicate that MASP-2-deficient mice are protected from killing caused by N. meningitidis serogroup A Z2491.

In fig. 34 is a Kaplan-Meier plot showing the percentage survival of MASP-2 KO and WT mice after administration of an infectious dose of 6x106 CFU of N. meningitidis serogroup B strain MC58. As shown in FIG. 34, 90% of MASP-2 KO mice survived the entire 72-hour period postinfection. In contrast, only 20% of WT mice (p = 0.0022) were still alive 24 h postinfection. These results indicate that MASP-2-deficient mice are protected from death caused by N. meningitidis serogroup B strain MC58.

In fig. 35 is a graph showing log CFU/ml of N. meningitidis serogroup B strain MC58 recovered at different time points from blood samples obtained from MASP-2 KO and WT mice following i.p. infection with 6x106 CFU of N. meningitidis serogroup B strain MC58 ( n=3 at different time points for both groups of mice). Results are presented as means ± SEM. As shown in FIG. 35, in WT mice, blood levels of N. meningitidis peaked at approximately 6.0 log CFU/ml at 24 h postinfection and fell to approximately 4.0 log CFU/ml at 36 h postinfection. In contrast, in MASP-2 KO mice, N. meningitidis levels peaked at approximately 4.0 log CFU/ml at 12 h postinfection and fell to approximately 1.0 log CFU/ml at 36 h postinfection (* indicates p< 0.05; symbol ** corresponds to p=0.0043). These results indicate that although MASP-2 KO mice were infected with the same dose of N. meningitidis serogroup B strain MC58 as WT mice, MASP-2 KO mice had increased bacterial clearance from the blood compared with WT.

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In fig. 36 is a graph showing the average unhealthy scores for MASP-2 KO and WT mice 3, 6, 12 and 24 hours after infection with 6x10 ⁶ CFU of N. meningitidis serogroup B strain MC58. As shown in FIG. 36, MASP-2-deficient mice were highly resistant to infection, with significantly lower scores of ill health at 6 h (* represents p=0.0411), 12 h (** represents p=0.0049), and 24 h. (the symbol *** corresponds to p=0.0049) after infection, compared with WT mice. The results in Fig. 36 are presented as means ± SEM.

In summary, the results in this example demonstrate that MASP-2-deficient mice are protected from Neisseria meningitides-induced death following infection with either N. meningitidis serogroup A or N. meningitidis serogroup B.

Example 31.

This example demonstrates that administration of anti-MASP-2 antibody following N. meningitidis infection results in increased survival of N. meningitidis-infected mice.

Background/Rationale.

As described in Example 10, rat MASP-2 protein was used to sort a phage display Fab library from which Fab2 #11 was identified as a functional antibody. Full-length rat IgG2c isotype and mouse IgG2a isotype full-length antibodies were obtained from Fab2 No. 11. A full-length mouse anti-MASP-2 antibody of the IgG2a isotype was characterized for pharmacodynamic parameters (as described in Example 19).

In this example, a full-length mouse anti-MASP-2 antibody derived from Fab2 #11 was assayed in a mouse model of N. meningitidis infection.

Methods.

A mouse full-length anti-MASP-2 IgG2a antibody derived from Fab2 #11 prepared as described above was tested in a mouse model of N. meningitidis infection as follows.

Administration of mouse anti-MASP-2 monoclonal antibodies (moAbs) following infection.

C57/BL6 (Charles River) mice, 9 weeks old, were treated with inhibitory mouse anti-MASP2 antibody (1.0 mg/kg) (n=12) or isotype control antibody (n=10) 3 hours after i.p. high dose (4x 10 ⁶ CFU) of N. meningitidis serogroup B strain MC58.

Results.

In fig. Figure 37 shows a Kaplan-Meier plot showing the survival rate, as a percentage, of mice after administration of an infectious dose of 4x10 ⁶ CFU of N. meningitidis serogroup B strain MC58, followed 3 hours after infection by either an inhibitory anti-MASP-2 antibody (1.0 mg /kg), or an isotype control antibody. As shown in FIG. 37, 90% of mice treated with anti-MASP-2 antibody survived the entire 72-hour period postinfection. In contrast, only 50% of mice treated with the isotype-control antibody survived the full 72-hour period after infection. The symbol * corresponds to p=0.0301, as determined by comparing two survival curves.

These results indicate that administration of anti-MASP-2 antibody is effective in treating and prolonging survival in subjects infected with N. meningitidis.

As demonstrated herein, the use of an anti-MASP-2 antibody in the treatment of a subject infected with N. meningitidis is effective when administered within 3 hours of infection and is expected to be effective within 24 to 48 hours of infection. Meningococcal disease (or meningococcemia or meningitis) is a medical emergency, and therapy is usually initiated immediately when meningococcal disease is suspected (that is, before N. meningitidis is positively identified as the causative pathogen).

In view of the results obtained for MASP-2 KO mice, which are described in Example 30, it is believed that administration of anti-MASP-2 antibody prior to N. meningitidis infection would also be effective in preventing or reducing the severity of infection.

Example 32.

This example demonstrates that administration of an anti-MASP-2 antibody is effective in treating N. meningitidis infection in human serum.

Rationale.

Patients with reduced serum levels of functional MBL have an increased susceptibility to recurrent bacterial and fungal infections (Kilpatrick et al., Biochim Biophys Acta 1572:401-413 (2002)). N. meningitidis is known to be recognized by MBL, and MBL-deficient serum has been shown not to lyse Neisseria.

In view of the results described in Examples 30 and 31, a series of experiments were conducted to determine the effectiveness of administering an anti-MASP-2 antibody to treat N. meningitidis infection in complement-deficient and control human serum. Experiments about

- 109 046178 was administered at high serum concentration (20%) to preserve the complement pathway.

Methods.

1. Serum bactericidal activity in various complement-deficient human sera and in human serum with added human anti-MASP-2 antibody.

The following human sera deficient in complement components and control human sera were used in this experiment.

Table 11

Tested human serum samples (as shown in Fig. 38)

Sample Serum type A Normal human serum (HNS) + human anti-MA8P-2 Ab IN SNP+control for Ab isotype WITH Human serum MBL D NChS E Heat-inactivated (HI) NSF

Recombinant anti-human MASP-2 antibody was isolated from a combinatorial antibody library (Knappik, A., et al., J. Mol. Biol. 296:57-86 (2000)), using recombinant human MASP-2A as the antigen ( Chen, S. W. and Wallis, J Biol Chem 276:25894-25902 (2001). An anti-human antigen scFv fragment that effectively inhibited lectin pathway-mediated activation of C4 and C3 in human plasma (IC50 ~20 nM) was identified and converted to a full-length human IgG4 antibody.

N. meningitidis serogroup B-MC58 was incubated with different sera given in Table. 11, each at a concentration of 20%, with or without the addition of human anti-MASP-2 inhibitory antibody (3 μg in a total volume of 100 μl) at 37°C with shaking. Samples were collected at the following time points: 0, 30, 60, and 90 min, plated, and then the number of viable cells was counted. Heat-inactivated human serum served as a negative control.

Results.

In fig. 38 is a graph showing the log CFU/ml number of viable N. meningitidis serogroup B-MC58 cells recovered at various time points from the human serum samples shown in Table 1. 11. in table. 12 shows the results of applying the Student's t test to the data in FIG. 38.

Table 12

The results of applying the student test to the data in Fig. 38 (time point 60 min)

Average difference (Log) Significant? P<0.05? Summary Values A vs B -0.3678 Yes ***(0.0002) A vs C -1.1053 Yes ***(p<0.0001) A vs D -0.2111 Yes **(0.0012) C vs D 1.9 Yes ***(p<0.0001)

As shown in FIG. 38 and table. 12, complement-dependent killing of N. meningitidis in human 20% serum was significantly enhanced by the addition of human anti-MASP-2 inhibitory antibody.

2. Complement-dependent killing of N. meningitidis in 20% (v/v) MASP-2-deficient mouse sera.

The following complement component-deficient mouse sera and control mouse sera were used in this experiment.

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Table 13

Mouse serum samples tested (as shown in Fig. 39)

Sample Serum type A DT IN MASP-2-/- WITH MBL A/C-/- D DT, heat inactivated (HI)

N. meningitidis serogroup B-MC58 was incubated with different complement-deficient mouse sera, each at a concentration of 20%, at 37°C with shaking. Samples were collected at the following time points: 0, 15, 30, 60, 90, and 120 min, plated, and then the number of viable cells was counted. Heat-inactivated human serum served as a negative control.

The results in Fig. 39 is a graph showing the log CFU/ml number of viable N. meningitidis serogroup B-MC58 cells recovered at various time points from the mouse serum samples shown in Table 1. 13. As shown in FIG. 39, MASP-2−/− mouse serum has a higher level of bactericidal activity against N. meningitidis than WT mouse serum. The symbol ** corresponds to p=0.0058, the symbol *** corresponds to p=0.001. in table 14 shows the results of applying the Student's t test to the data in FIG. 39.

Table 14

The results of applying the student test to the data in Fig. 39

Comparison Time point Average difference (LOG) Significant? (p<0.05)? Summary Values A vs B 60 min 0.39 Yes ** (0.0058) A vs B 90 min 0.6741 Yes *** (0.001)

In summary, the results in this example indicate that MASP-2-/- serum has a higher level of bactericidal activity against N. meningitidis than WT serum.

Example 33.

This example demonstrates the inhibitory effect of MASP-2 deficiency on red blood cell lysis from blood samples obtained in a mouse model of paroxysmal nocturnal hemoglobinuria (PNH).

Background/Rationale.

Paroxysmal nocturnal hemoglobinuria (PNH), also called Marchiafava Micheli syndrome, is an acquired, potentially life-threatening blood disorder characterized by complement-induced intravascular hemolytic anemia. A characteristic feature of PNH is chronic intravascular hemolysis, which is a consequence of increased activation of the alternative complement pathway due to the absence of complement regulators CD55 and CD59 on PNH red blood cells, with subsequent hemoglobinuria and anemia. Lindorfer, M.A., et al., Blood 115(11) (2010), Risitano, A.M., Mini-Reviews in Medicinal Chemistry, 11:528-535 (2011). Anemia in PNH results from the destruction of red blood cells in the bloodstream. Symptoms of PNH include red urine due to hemoglobin in the urine, back pain, fatigue, shortness of breath, and thrombosis. PNH can occur on its own and is called primary PNH, or in the context of other bone marrow diseases such as aplastic anemia, it is called secondary PNH. Treatment of PNH includes blood transfusions for anemia, the use of anticoagulants for thrombosis, and the use of the monoclonal antibody eculizumab (Soliris), which protects blood cells from immune destruction by inhibiting the complement system (Hillmen P. et al., N. Engl. J. Med. 350 (6):552-9 (2004)). Eculizumab (soliris®) is a humanized monoclonal antibody targeting complement component C5 that blocks its cleavage by C5 convertases, thereby preventing C5a formation and MAC assembly. Treatment of patients with PNH with eculizumab results in a reduction in intravascular hemolysis as measured by lactate dehydrogenase (LDH) levels, stabilization of hemoglobin levels, and elimination of the need for blood transfusions in approximately half of patients (Hillmen P, et al., Mini-Reviews in Medicinal Chemistry, vol 11(6) (2011)). Although almost all patients treated with eculizumab regain normal or near-normal LDH levels (due to control of intravascular hemolysis), only about one third of patients achieve hemoglobin levels greater than 11 g/dL, and the remaining patients treated with eculizumab remain anemic moderate to severe (ie, transfusion dependent) in approximately equal proportions (Risitano A.M. et al., Blood 113:4094-100 (2009)). As described in Risitano et al., Mini-Reviews in Medicinal Chemistry 11:528-535 (2011), it has been shown that patients with

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PNHs receiving eculizumab have C3 fragments associated with a significant proportion of their PNH RBCs (unlike patients not receiving the drug), suggesting that membrane-bound C3 fragments act as opsonins on PNH RBCs, leading to their uptake into reticuloendothelial cells through specific C3 receptors and subsequent extravascular hemolysis. Thus, additional treatment strategies beyond the use of eculizumab are needed for these patients who experience C3 fragment-mediated extravascular hemolysis as they continue to require red blood cell transfusions.

This example describes methods for assessing the effect of MASP-2-deficient serum and serum treated with a MASP-2 inhibitory agent on red blood cell lysis from blood samples obtained in a mouse model of PNH, and demonstrates the effectiveness of MASP-2 inhibition in the treatment of subjects suffering from PNH and provides evidence to support the use of MASP2 inhibitors to attenuate the effects of C3 fragment-mediated extravascular hemolysis in subjects with PNH treated with a C5 inhibitor such as eculizumab.

Methods.

Animal model of PNH.

Blood samples were obtained from genetically engineered mice deficient in Crry and C3 (Crry/C3−/−) and mice deficient in CD55/CD59. These mice lack appropriate surface complement regulators and, as a consequence, their red blood cells are susceptible to spontaneous autolysis by complement, like human blood cells in PNH.

To further sensitize these red blood cells, these cells were used with and without mannan coating and then tested for hemolysis in C56/BL6 WT plasma, MBL-null plasma, MASP-2-/- plasma, human NSF, human MBL-/- plasma and NSF treated with human anti-MASP-2 antibody.

1. Analysis of hemolysis of erythrocytes from mice doubly deficient in Crry/C3 and CD55/CD59 in MASP-2-deficient/depleted sera and controls.

Day 1. Preparation of mouse red blood cells (± mannan coating).

Materials: fresh mouse blood, BBS/Mg2 ^{+ /} Ca ²⁺ (4.4 mM barbituric acid, 1.8 mM sodium barbital, 145 mM NaCl, pH 7.4, 5 mM Mg2 ⁺ , 5 mM Ca ²⁺ ), chromium chloride, CrCl ₃ -6H ₂ O (0.5 mg/ml in BBS/Mg2 ⁺ /Ca ²⁺ ) and mannan, 100 μg/ml in BBS /Mg2 ⁺ /Ca ²⁺ .

Whole blood (2 ml) was centrifuged for 1-2 min at 2000xg in a centrifuge cooled to 4°C. Plasma and the light layer of the blood clot were removed by aspiration. The sample was then washed three times by resuspending the red blood cell pellet in 2 ml of ice-cold BBS/gelatin/]Mg ²⁺ /Ca ²⁺ solution and centrifuging again. After the third wash, the pellet was resuspended in 4 ml BBS/Mg2 ⁺ /Ca2 ⁺ . A 2-ml aliquot of red blood cells was separated to serve as an uncoated control. To the remaining 2 ml, 2 ml of CrCl ₃ and 2 ml of mannan were added, and the sample was incubated with gentle stirring at room temperature for 5 minutes. The reaction was stopped by adding 7.5 ml of BBS/gelatin/]Mg ²⁺ /Ca ²⁺ solution. The sample was centrifuged as described above, resuspended in 2 ml of BBS/gelamine/Mg ²⁺ /Ca ²⁺ solution and washed two more times as described above, then stored at 4°C.

Day 2. Hemolysis analysis.

Materials: BBS/gelatin/]Mg ²⁺ /Ca ²⁺ (as described above), test sera, 96-well round-bottom and flat-bottom plates and a spectrophotometer on which readings are taken from 96-well plates at 410-414 nm.

First, the concentration of erythrocytes was determined, the number of cells was adjusted to 10 ⁹ /ml and stored at this concentration. Before use, diluted with analytical buffer to 10 /ml, and used 100 µl per well. Hemolysis was measured at 410-414 nm (this achieves greater sensitivity than at 541 nm). Dilutions of test sera were prepared in an ice-cold BBS/gelatin/]Mg ²⁺ /Ca ²⁺ solution. 100 µl of serum in each dilution was pipetted into a round-bottomed plate (see plate plan). 100 μl of an appropriately diluted red blood cell preparation (i.e., 10 /ml) was added (see plate plan), incubated at 37°C for approximately 1 h and monitored for lysis. (At this stage, the tablets can be photographed). The plate was then centrifuged at maximum speed for 5 min. 100 μl of the liquid phase was collected, transferred to flat-bottomed plates, and OD was recorded at 410–414 nm. The erythrocyte pellets were preserved (they can later be lysed with water to obtain the opposite result).

Experiment No. 1.

Fresh blood was obtained from CD55/CD59 double-deficient mice and blood from Crry/C3 double-deficient mice, and the red blood cell preparation was prepared according to the protocol detailed above. The cells were split, half the cells were coated with mannan, and the other half were left untreated, bringing the final concentration to 1x108 per ml, of which 100 μl was used in the hemolysis assay, which was performed as described above.

Results of Experiment No. 1. The lectin pathway is involved in erythrocyte lysis in an animal model

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PNH.

In the initial experiment, it was determined that uncoated erythrocytes from WT mice were not lysed in any of the mouse sera. It was also determined that mannan-coated erythrocytes from Crry-/- mice were lysed slowly (more than 3 hours at 37 degrees) in the serum of WT mice, but they were not lysed in MBL-null serum. (Data not shown).

It was determined that mannan-coated erythrocytes from Crry −/− mice were rapidly lysed in human serum but not in heat-inactivated SNP. It is important to note that mannan-coated erythrocytes from Crry −/− mice were lysed in SNP diluted to 1/640 (i.e., lysis at all dilutions of 1/40, 1/80, 1/160, 1/320, and 1/640). (Data not shown). At this dilution, the alternative pathway does not work (the functional activity of AP is significantly reduced at serum concentrations below 8%).

Conclusions from experiment No. 1.

Mannan-coated erythrocytes from Crry −/− mice lyse very well in highly diluted human serum with MBL, but not in such serum without MBL. Efficient lysis at each serum concentration tested indicates that the alternative pathway is not involved or is not required for such lysis. The inability of MBL-deficient mouse and human serum to lyse mannan-coated erythrocytes from Crry −/− mice indicates that the classical pathway is also not responsible for the observed lysis. Because lectin pathway recognition molecules (i.e., MBLs) are required, this lysis is mediated by the lectin pathway.

Experiment No. 2.

Fresh blood was obtained from Crry/C3 and CD55/CD59 double-deficient mice, and mannan-coated erythrocytes from Crry −/− mice were analyzed in a hemolysis assay as described above in the presence of the following human sera: MBL-null; DT; SNP pre-added with human anti-MASP-2 antibody and heat-inactivated SNP as a control.

Results of Experiment 2: MASP-2 inhibitors prevent erythrocyte lysis in an animal model of PNH.

Mannan-coated erythrocytes from Crry−/− mice were incubated with SNPs at dilutions up to 1/640 (i.e., 1/40, 1/80, 1/160, 1/320, and 1/640), MBL−/− human serum, SNPs with pre-added anti-MASP-2 mAb and heat-inactivated SNP as control.

In the ELISA assay, the microtiter plate was centrifuged and unlysed red blood cells were collected at the bottom of the round bottom plate. The supernatant from each well was collected, and the amount of hemoglobin released from the lysed red blood cells was measured based on the OD at 415 nm in an ELISA reader.

In the control heat-inactivated SNP (negative control), as expected, there was no lysis. Human MBL−/− serum lysed mannan-coated mouse erythrocytes at dilutions of 1/8 and 1/16. SNP pre-added with anti-MASP-2 antibody lysed mannan-coated mouse erythrocytes at dilutions of 1/8 and 1/16, while human WT serum lysed mannan-coated mouse erythrocytes at dilutions up to 1/32.

in fig. 40 is a graph showing hemolysis (measured based on the release of hemoglobin from lysed mouse red blood cells (Cryy/S3-/-) into the supernatant by photometric method) of mannan-coated mouse red blood cells by human serum over a range of serum concentrations using heat-inactivated (HI) SNP, MBL -/-, NSF with pre-added anti-MASP-2 antibody and control NSF.

The results presented in Fig. 40 show that inhibition of MASP-2 by an anti-MASP-2 antibody results in a significant shift in CH ₅₀ and inhibits complement-mediated lysis of sensitized erythrocytes with insufficient protection from autologous complement activation.

Experiment No. 3.

Fresh blood was obtained from Crry/C3 and CD55/CD59 double-deficient mice, and uncoated red blood cells from Crry −/− mice were analyzed in a hemolysis assay as described above in the presence of the following sera: MBL −/−; DT serum; SNP pre-added with human anti-MASP-2 antibody and heat-inactivated SNP as a control.

Results.

In fig. 41 is a graph showing hemolysis (measured by the release of hemoglobin from lysed WT mouse RBCs into supernatant by photometric method) of non-mannan-coated mouse RBCs by human serum over a range of serum concentrations using heat inactivated (HI) SNP, MBL-/-, SNP with pre-added βμη-ΜΑ^Ρ^ antibody and control SNP. As shown in FIG. 41, inhibition of MASP-2 results in inhibition of complement-mediated lysis of non-sensitized erythrocytes from WT mice.

In fig. 42 is a graph showing hemolysis (measured based on the release of hemoglobin from lysed mouse red blood cells (CD55/59-/-) into the supernatant by photometric method) of non-mannan-coated mouse red blood cells by human serum over a range of serum levels

- 113 046178 concentrations using heat inactivated (HI) SNP, MBL-/-, SNP pre-added with anti-MASP-2 antibody and control SNP.

Table 12

CH50 values expressed as serum concentrations

Serum DT CD55/59-/- Heat-inactivated SNP No lysis No lysis MBL AO/XX donor (MBL deficiency) 7.2% 2.1% NSF+anti-MA8R-2 antibody 5.4% 1.5% NChS 3.1% 0.73%

Note: CH50 represents the point at which complement-mediated hemolysis reaches 50%.

In summary, the results in this example indicate that inhibition of MASP-2 results in inhibition of complement-mediated lysis of sensitized and non-sensitized red blood cells with insufficient protection from autologous complement activation. Thus, MASP-2 inhibitors can be used to treat subjects suffering from PNH, and can also be used to alleviate (ie, inhibit, prevent or reduce the severity of) extravascular hemolysis in patients with PNH receiving treatment with a C5 inhibitor such like eculizumab (Soliris).

Example 34.

This example describes a study that is a continuation of the study described above in Example 29, in which additional evidence was obtained that a MASP-2 inhibitor, such as an anti-MASP-2 antibody, is effective in treating the effects of radiation and/or in treating, relief or prevention of acute radiation syndrome.

Rationale.

In the initial study described in Example 29, it was shown that administration of anti-MASP-2 antibody to mice prior to irradiation resulted in increased survival of irradiated mice compared to vehicle-treated irradiated controls at both irradiation levels, 6.5 Gy and 7.0 Gy. Gr. Example 29 also demonstrated that at an irradiation level of 6.5 Gy, post-irradiation administration of anti-MASP-2 antibody resulted in a modest increase in survival compared to vehicle-treated irradiated control animals. This example describes a second radiation study that was performed to confirm the results of the first study.

Methods.

Study design A.

Swiss Webster mice (n=50) were exposed to ionizing radiation (8.0 Gy). The effect on mortality of anti-MASP-2 antibody therapy (mAb H6 5 mg/kg) administered 18 hours before and 2 hours after irradiation and then weekly thereafter was assessed.

Study A results:

As shown in FIG. 43, it was found that administration of the anti-MASP-2 antibody mAb H6 resulted in increased survival of mice exposed to 8.0 Gy, with adjusted mean survival increased from 4 to 6 days compared to mice treated with vehicle control and with a 12% reduction in mortality compared to mice treated with control vehicle (log-rank test, p=0.040).

Study design V.

Swiss Webster mice (n=50) were exposed to ionizing radiation (8.0 Gy) in the following groups (I: vehicle) saline control; (II: low dose) anti-MASP-2 antibody mAb H6 (5 mg/kg) was administered 18 h before irradiation and 2 h after irradiation; (III: high dose) mAb H6 (10 mg/kg) was administered 18 h before irradiation and 2 h after irradiation; and (IV: high dose after irradiation) mAb H6 (10 mg/kg) was administered only 2 h after irradiation.

The results of the study by V.

Administration of anti-MASP-2 antibody before and after irradiation resulted in an increase in mean survival from 4 to 5 days, compared with animals receiving vehicle control. Mortality in mice treated with anti-MASP-2 antibody was reduced by 6-12% compared with mice treated with vehicle control. It should also be noted that there were no significant harmful effects of treatment (data not shown).

In summary, the results described in this example are consistent with the results described in example 29 and also demonstrate that anti-MASP-2 antibodies are effective in treating mammalian subjects at risk of developing, or suffering from, the devastating effects of acute radiation syndrome.

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Example 35.

This study examined the effect of MASP-2 deficiency in a mouse model of LPS (lipopolysaccharide)-induced thrombosis.

Rationale.

Hemolytic uremic syndrome (HUS), a consequence of infection with Shiga toxin-producing E. coli, is a leading cause of acute renal failure in children. In this example, the Schwarzman model of LPS-induced thrombosis (microvascular coagulation) was created in MASP-2-/- (KO) mice to determine whether MASP-2 inhibition would be effective in inhibiting or preventing intravascular thrombus formation.

Methods.

MASP-2-/- (n=9) and WT (n=10) mice were analyzed in the Schwartzman model of LPS-induced thrombosis (microvascular coagulation). Mice were injected with LPS Serratia and blood clot formation was monitored over time. The incidence of microthrombi and LPS-induced microvascular coagulation was compared.

Results.

Notably, none of the MASP-2−/− mice tested (9/9) developed intravascular thrombi after administration of Serratia LPS. In contrast, microthrombi were detected in 7 of 10 WT mice tested in parallel (p=0.0031, Fisher's exact test). As shown in FIG. 44, the time to onset of microvascular occlusion after LPS administration was measured in MASP-2-/and WT mice, and the percentage of WT mice with thrombus formation detected within 60 min was shown, with thrombus formation detected as early as approximately 15 min. Up to 80% of WT mice had formed blood clots after 60 min. On the contrary, as shown in FIG. 44, none of the MASP-2 −/− mice developed thrombi at 60 min (log-rank test: p = 0.0005).

These results indicate that MASP-2 inhibition protects against intravascular thrombus formation in the HUS model.

Example 36.

This example describes the effect of anti-MASP-2 antibodies in a mouse model of HUS when co-administered intraperitoneally with an injection of purified Shiga toxin 2 (STX2) plus LPS.

Prerequisites.

A mouse model of HUS was developed using co-administration of intraperitoneal injection of purified Shiga toxin 2 (STX2) plus LPS. Biochemical and microarray analysis of mouse kidneys showed that the effects of STX2 plus LPS administration were different from those of either agent alone. Blood and serum analysis of these mice showed the presence of neutrophilia, thrombocytopenia, red blood cell hemolysis, and elevated serum levels of creatinine and blood urea nitrogen. In addition, histological and electron microscopic analysis of mouse kidneys showed fibrin deposition in the glomeruli, erythrocyte congestion, microthrombi formation, and changes in glomerular ultrastructure. This C57BL/6 mouse model of HUS was found to have all the clinical features of human HUS pathology, including thrombocytopenia, hemolytic anemia, and renal failure that define human disease. (J. Immunol 187(1): 172-80 (2011))

Methods.

Female C57BL/6 mice weighing 18–20 g were purchased from Charles River Laboratories and divided into 2 groups (5 mice in each group). Mice in one group were pretreated by intraperitoneal (IP) injection with recombinant anti-MASP-2 mAb M11 (100 μg per mouse; corresponding to a final dose of 5 mg/kg body weight) diluted in a total volume of 150 μl of saline. Animals in the control group received saline solution without antibody. Six hours after IP injection of the ημή-ΜΑ^Ρ^ mAb M11 antibody, all mice received a combined IP injection of a sublethal dose (3 μg/animal; corresponding to 150 μg/kg body weight) of LPS from Serratia marcescens (L6136; Sigma -Aldrich, St. Louis, MO) and a dose of 4.5 ng/animal (equivalent to 225 ng/kg) of STX2 (two times the LD ₅₀ dose) in a total volume of 150 μl. Saline injection was used as a control.

Mice survival was monitored every 6 h after dosing. Mice were sacrificed once they reached the lethargic stage of HUS pathology. After 36 h, all mice were sacrificed and both kidneys were removed for analysis by immunohistochemistry and scanning electron microscopy. Blood samples were collected at the end of the experiment by cardiac puncture. Serum was obtained and stored frozen at -80°C to measure BUN and serum creatinine levels in both the experimental and control groups.

Immunohistochemistry.

One third of each mouse kidney was fixed in 4% paraformaldehyde for 24 h, processed, and embedded in paraffin. Three-micron thick sections were prepared and mounted on charged glass slides for subsequent staining with H&E staining.

Electron microscopy.

The middle section of the kidneys was cut into blocks measuring approximately 1-2 ^mm3 and fixed for

- 115 046178 nights at 4°C in 2.5% glutaraldehyde solution in 1x PBS. The fixed tissue was then processed at the Electron Microscopy Facility at the University of Leicester.

Cryostat sections.

The other third of the kidneys was cut into blocks of approximately 1-2 mm ³ , quickly frozen in liquid nitrogen and stored at -80°C for cryostat sectioning and mRNA analysis.

Results.

In fig. 45 is a graph showing the percentage survival of saline-treated control mice (n=5) and anti-MASP-2 antibody-treated mice (n=5) in the STX/LPS-induced model over time (hours). It is noteworthy that, as shown in FIG. 45, all of the control mice died after 42 hours. In sharp contrast, 100% of mice treated with anti-MASP-2 antibody survived throughout the experiment. According to the results presented in FIG. 45, all untreated mice that either died or were sacrificed with signs of severe disease were found to have significant glomerular damage, whereas the glomeruli of all anti-MASP-2-treated mice appeared normal (data not shown). These results indicate that MASP-2 inhibitors, such as anti-MASP-2 antibodies, can be used to treat subjects suffering from, or at risk of developing, thrombotic microangiopathy (TMA), such as hemolytic uremic syndrome (HUS), atypical HUS (aHUS) or thrombotic thrombocytopenic purpura (TTP).

Example 37.

This example describes the effect of MASP-2 deficiency and MASP-2 inhibition on FITC-dextran/light-induced endothelial cell injury in mice, a model of thrombosis.

Background/Rationale.

As shown in Examples 35 and 36, MASP-2 deficiency (MASP-2 KO) and MASP-2 inhibition (by administration of an inhibitory anti-MASP-2 antibody) protected mice in a typical HUS model, while in all control mice, exposed to STX and LPS developed severe HUS and were moribund or died within 48 hours. For example, as shown in FIG. 54, all mice treated with anti-MASP-2 inhibitory antibody and then exposed to STX and LPS survived (Fisher's exact test p < 0.01; N = 5). Thus, administration of anti-MASP-2 protects mice in this HUS model.

The following experiments were performed to analyze the effect of MASP-2 deficiency and MASP-2 inhibition on fluorescein isothiocyanate (FITC)-dextran-induced endothelial cell injury, a model of thrombotic microangiopathy (TMA), to further demonstrate the benefit of MASP-2 inhibitors in the treatment of HUS, aHUS, TTP, as well as TMA with other etiologies.

Methods.

Intravital microscopy.

Mice were prepared for intravital microscopy as described in Frommhold et al., BMC Immunology 12:56–68, 2011. Briefly, mice were anesthetized with an intraperitoneal (IP) injection of ketamine (125 mg/kg body weight, Ketanest, Pfitzer GmbH, Karlsruhe, Germany) and xylazine (12.5 mg/kg body weight; Rompan, Bayer, Leverkusen, Germany) and placed on a warm substrate to maintain body temperature at 37°C. Intravital microscopy was performed using an upright microscope (Leica, Wetzlar, Germany) using a saline immersion objective (SW 40/0.75 NA, Zeiss, Jena, Germany). To facilitate breathing, mice were intubated using a PE 90 tube (Becton Dickson and Company, Sparks, MD, USA). The left carotid artery was cannulated with a PE10 tube (Becton Dickson and Company, Sparks, MD, USA) for blood collection and systemic administration of monoclonal antibody (mAb).

Preparing the muscle supporting the testicle.

Surgical preparation of the testicular suspensory muscle for intravital microscopy was performed as described in Sperandio et al., Blood, 97:3812-3819, 2001. Briefly, the scrotum was opened and the testicular suspensory muscle was allowed to move. After longitudinal incision and placement of the muscle on the coverslip, the epididymis and testis were moved and pressed to the side, allowing full microscopic access to the microvascular system of the muscle supporting the testis. The venules of the testicular suspensory muscle were recorded using a CCD camera (CF8/1; Kappa, Gleichen, Germany) on a Panasonic S-VHS recorder. The muscle supporting the testis was perfused with bicarbonate-buffered saline at a controlled temperature (35°C), as previously described in Frommhold et al., BMC Immunology 12:56-68, 2011-2011.

Model of damage caused by FITC-dextran upon excitation by light.

Controlled, light dose-dependent damage to the vascular endothelium of venules and arterioles in the testicular suspensory muscle was induced by light excitation of phototoxic IgD-dextran (catalog no. FD150S, Sigma Aldrich, Poole, U.K.). This procedure initiates localized thrombosis. 60 μl of 10% w/v FITC-dextran solution as a phototoxic reagent was injected through the left carotid artery and allowed to distribute uniformly throughout

- 116 046178 circulatory system for 10 minutes. After selecting a well-perfused venule, low to moderate intensity halogen lamp light (800–1500) was focused on the vessel of interest, inducing FITC-dextran fluorescence and mild to moderate phototoxicity to the endothelial surface to promote thrombosis in a reproducible, controlled manner. The required phototoxic light intensity for excitation of FITC-dextran was created using a halogen lamp (12 V, 100 W, Zeiss, Oberkochen, Germany). Phototoxicity resulting from light-induced fluorochrome excitation requires a threshold light intensity and/or duration of illumination, and phototoxicity is caused either by direct heating of the endothelial surface or by the formation of reactive oxygen radicals, as described in Steinbauer et al., Langenbecks Arch Surg 385: 290-298, 2000.

The light intensity applied to each vessel was measured for adjustment using a low-power wavelength-corrected diode detector (Labmaster LM-2, Coherent, Auburn, USA). Offline analysis of the video scans was performed using a computer-assisted microcirculation analysis system (CAMAS, Dr. Zeintl, Heidelberg), and red blood cell velocity was measured as described in Zeintl et al., Int J Microcirc Clin Exp, 8(3):293-302 , 2000.

Use of anti-human MASP-2 monoclonal inhibitory antibody (mAb H6) and vehicle control before induction of thrombosis.

Using a blinded study design, 9-week-old male littermate C57BL/6 WT mice were treated by intravenous injection with either a recombinant monoclonal antibody against human MASP-2 (mAb H6), an inhibitor of MASP-2 functional activity (at a final dose of 10 mg/kg body weight), or the same amount of isotype control antibody (without MASP-2 inhibitory activity) 16 hours before phototoxic induction of thrombosis in the muscle supporting the testis, a model for intravital microscopy. One hour before thrombosis induction, a second dose of either mAb H6 or control antibody was administered. MASP-2 knockout (KO) mice were also evaluated in this model.

mAb H6 (derived to recombinant human MASP-2) is a potent inhibitor of the functional activity of human MASP-2 that cross-reacts, binds and inhibits murine MASP-2, but with lower affinity due to its species specificity (data not shown). To compensate for the lower affinity of mAb H6 for murine MASP-2, mAb H6 was administered at a higher dose (10 mg/kg body weight) to overcome differences in species specificity and lower affinity for murine MASP-2, and provide effective blockade of functional murine MASP-2 activity in vivo.

In this blinded study, the time required for complete occlusion of each individual venule tested was recorded (selection criteria were comparable diameters and flow velocities).

The percentage of mice with microvascular occlusion, time to onset, and time to complete occlusion were assessed during a 60-minute observation period using video recording of the intravital microscopy procedure.

Results.

In fig. 46 is a graph showing, as a function of time after injury induction, the percentage of mice with microvascular occlusion in the FITC/dextran UV excitation model following administration of isotype control or anti-human MASP-2 mAb H6 (10 mg/kg) for 16 hours. and 1 hour before FITC/dextran injection. As shown in FIG. 46, 85% of wild-type mice treated with an isotype-control antibody experienced occlusion in 30 min or less, whereas only 19% of wild-type mice pretreated with anti-human MASP2 antibody (mAb H6) experienced occlusion in over this period of time, and the onset of occlusion was delayed in mice that eventually occluded in the anti-human MASP-2 group. It was also noted that three mice treated with the anti-MASP-2 mAb H6 did not experience occlusion during the entire 60-minute observation period (ie, they were protected from thrombotic occlusion).

In fig. 47 is a graph showing time to occlusion, in minutes, for mice treated with anti-human MASP-2 antibody (mAb H6) and isotype control antibody. Data are presented as a scatterplot with means (horizontal bars) and standard error bars (vertical bars). This figure shows the time to occlusion in mice in which occlusion could be observed. Thus, the three anti-MASP-2 antibody-treated mice that did not occlude during the 60-minute observation period were not included in this analysis (all control mice did occlude). Unpaired Student's t test was used for statistical analysis; in this case, the symbol * corresponds to p=0.0129. As shown in FIG. 47, in four anti-MASP-2 antibody (mAb H6)-treated mice that experienced occlusion, administration of anti-MASP-2 antibody resulted in a significant increase in the time to venular occlusion in a thrombosis model of FITC-dextran/light-induced endothelial injury. cells at low light intensity (800-1500), compared with mice treated with an isotype control antibody. Average time to complete occlusion

- 117 046178 sion in the isotype control group was 19.75 minutes, while the average time to complete occlusion in the group of animals receiving anti-MASP-2 antibody was 32.5 minutes.

In fig. 48 is a graph showing time to occlusion, in minutes, for wild-type mice, MASP-2 KO mice, and wild-type mice pretreated with anti-human MASP-2 antibody (mAb H6) administered i.p. at a dose of 10 mg/kg 16 hours before, and then reintroduced IV 1 hour before, inducing thrombosis in a thrombosis model of FITC-dextran/light-induced endothelial cell injury at low light intensity (800-1500). Only data for animals that experienced occlusion are included in FIG. 48; n=2 for wild-type mice treated with isotype-control antibody; n=2 for MASP-2 KO; and n=4 for wild-type mice treated with anti-human MASP-2 antibody (mAb H6). The symbol * corresponds to p<0.01. As shown in FIG. 48, MASP-2 deficiency and MASP-2 inhibition (mAb H6 at 10 mg/kg) resulted in increased time to venous occlusion in a thrombosis model of FITC-dextran/light-induced endothelial cell injury at low light intensity (800-1500). .

Conclusions.

The results in this example further demonstrate that a MASP-2 inhibitory agent that blocks the lectin pathway (eg, antibodies that block MASP-2 function) inhibits microvascular coagulation and thrombosis, characteristic features of many microangiopathic diseases, in a mouse model of TMA. Thus, it is expected that administration of a MASP2 inhibitory agent, such as a MASP-2 inhibitory antibody, will be an effective treatment for patients suffering from HUS, aHUS, TTP or other microangiopathic diseases and will provide protection against microvascular coagulation and thrombosis.

Example 38.

This example describes a study demonstrating that human MASP-2 inhibitory antibody (mAb H6) does not affect platelet function in human platelet-rich plasma.

Background/Rationale.

As described in Example 37, inhibition of MASP-2 by a human MASP-2 inhibitory antibody (mAb H6) was shown to increase the time to venous occlusion in a thrombosis model of FITC-dextran/light-induced endothelial cell injury. The following experiment was performed to determine whether a MASP2 inhibitory antibody (mAb H6) affects platelet function.

Methods.

The effect of human anti-MASP-2 antibody mAb H6 on ADP-induced platelet aggregation was tested as follows. Human anti-MASP-2 mAb H6 at either 1 μg/ml or 0.1 μg/ml was added in a 40 μl solution to 360 μl of freshly prepared platelet-rich plasma. An isotype control antibody was used as a negative control. After the addition of plasma antibodies, platelet activation was induced by adding ADP to a final concentration of 2 μM. The analysis was started by stirring the solutions with a small magnet in a 1-ml cuvette. Platelet aggregation was measured using a dual-channel Chrono-log model 700 Whole Blood/Optical Lumi-Aggregometer.

Results.

Aggregation, as a percentage, in solutions was measured over five minutes. The results are shown in the table below. 13.

- 118 046178

Table 13

Platelet aggregation within five minutes

Antibody Amplitude (aggregation, %) Slope angle (aggregation, %, over time) Ahth-MASP-2 antibody (mAb H6) (1 μg/ml) 46% 59 Isotype control antibody (1 μg/ml) 49% 64 Ahth-MASP-2 antibody (mAb H6) (0.1 μg/ml) 52% 63 Isotype control antibody (0.1 μg/ml) 46% 59

As shown in the table above. 13, there was no significant difference between the aggregation of ADP-induced platelets treated with control antibody or anti-MASP-2 mAb H6. These results indicate that human anti-MASP-2 antibody (mAb H6) does not affect platelet function. Thus, the results described in Example 37 showing that MASP-2 inhibition with human MASP-2 inhibitory antibody (mAb H6) resulted in an increase in time to venous occlusion in a thrombosis model of FITCdextran/light-induced endothelial cell injury were not a consequence of the effect of mAb H6 on platelet function. Thus, MASP-2 inhibition prevents thrombosis without directly affecting platelet function, suggesting that the mechanism of therapeutic action is different from existing antithrombotic agents.

Example 39.

This example describes the effect of MASP-2 inhibition on thrombus formation and vascular occlusion in a mouse model of TMA.

Background/Rationale.

The lectin pathway plays a major role in the activation of the complement system under conditions of stress and damage to endothelial cells. This activation is rapidly enhanced in the alternative pathway, which is impaired in many patients with aHUS. As a consequence, preventing activation of MASP-2 and the lectin pathway is expected to stop the sequence of enzymatic reactions leading to membrane attack complex formation, platelet activation, and leukocyte recruitment. This effect limits tissue damage.

In addition, MASP-2 has activity similar to factor Xa and cleaves prothrombin to form thrombin. This MASP-2-regulated activation of the coagulation system may impair hemostasis and lead to TMA pathology. Thus, inhibition of MASP-2 using a MASP-2 inhibitor, such as a MASP-2 inhibitory antibody that blocks activation of the complement and coagulation systems, is expected to improve outcome in aHUS and other similar TMA conditions.

As described in Example 37, it was demonstrated that inhibition of MASP-2 with a human MASP-2 inhibitory antibody (mAb H6) increased the time to venous occlusion in a thrombosis model of FITC-dextran/light-induced endothelial cell injury. In this model of TMA, mice were sensitized by intravenous injection of FITC-dextran, followed by localized photoactivation of FITC-dextran in the microvascular network of the muscle supporting the testis (Thorlacius H et al., Eur J Clin. Invest 30(9):804-10, 2000; Agero et al., Toxicon 50(5):698-706, 2007).

The following experiment was performed to determine whether a MASP-2 inhibitory antibody (mAb H6) has a dose-dependent effect on thrombus formation and vascular occlusion in a mouse model of TMA.

Methods.

Localized thrombosis was induced by photoactivation of fluorescein isothiocyanate-labeled dextran (FITC-dextran) in the microvascular network of the testicular suspensory muscle in C57B1/6 mice, and intravital microscopy was used to determine the timing of onset of thrombus formation and vascular occlusion using the methods described in Example 37. with the following modifications. Groups of mice were administered mAb H6 (2 mg/kg, 10 mg/kg, or 20 mg/kg) or isotype control antibody (20 mg/kg) by intravenous (IV) injection one hour before TMA induction. The time until the onset of thrombus formation and the time until complete vascular occlusion were recorded. Subsequent analysis of video images of intravital microscopy, recorded for 30-60 minutes, was used to estimate the size

- 119 046178 ra vessels, blood flow speed, light intensity, speed at the beginning of thrombus formation as the equivalent of platelet adhesion, time before the onset of thrombus formation, speed of complete vascular occlusion and time to complete vascular occlusion. Statistical analysis was performed using SigmaPlot v 12.0.

Results.

Initiation of blood clot formation.

In fig. 49 is a Kaplan-Meier plot showing the percentage of mice with thrombi as a function of time in a FITC-dextran-induced thrombotic microangiopathy model in mice treated with increasing doses of human MASP-2 inhibitory antibody (mAb H6 at 2 mg/kg, 10 mg/kg or 20 mg/kg) or isotype control antibody. As shown in FIG. 49, the onset of thrombus formation was delayed in mAb H6-treated mice in a dose-dependent manner compared with control antibody-treated mice.

In fig. Figure 50 shows a graph showing the average time (min) before the onset of thrombus formation depending on the dose of mAb H6 (*p<0.01 compared with control). As shown in FIG. 50, the average time to the onset of thrombus formation increased with increasing dose of mAb H6 from 6.8 minutes in the control group to 17.7 minutes in the group of animals receiving 20 mg/kg mAb H6 (p <0.01). The experimental data and results of statistical analysis that serve as the basis are given in Table. 14 and 15.

The time to onset of thrombus formation in individual mice, recorded from video evaluation, is shown in Table 1 below. 14.

Table 14

Time to onset of thrombus formation after light/dye-induced injury

Control influence Exposure to mAb H6 Time to start (minutes) Control 2 mg/kg 10 mg/kg 20 mg/kg 6.07 5.93 12.75 10.00 1.07 6.95 2.53 10.33 8.00 8.92 14.00 21.00 2.40 11.92 3.05 11.50 8.48 12.75 8.00 19.00 4.00 12.53 8.17 10.37 4.00 15.83 22.65 7.83 11.70 16.37 6.83 50.67 21.75* 15.00 32.25* 15.67

* Thrombosis was not observed in the vessels during the specified observation period. The results of the statistical analysis, which compared the time to the onset of occlusion in animals receiving the control antibody and mAb H6, are shown below in table. 15.

Table 15

Time to onset of occlusion: dose response data from the fitc-dextran study

Statistical parameter Control mAb H6 (2 mg/kg) mAb H6 (10 mg/kg) mAb H6 (20 mg/kg) Number of events/number of animals (%) 11/11 (100%) 6/6 (100%) 9/9 (100%) 8/10 (80.0%) Average time (minutes) (95% 6.8 10.4 11.7 17.7 DI) (2.4, 8.5) (5.9, 12.8) (2.5, 15.8) (10.0, 22.7) Wilcoxon test, value* 0.2364 0.1963 0.0016 Event=Observed time before occlusion starts Mean (minutes) and its 95% CI are based on Kaplan-Meier estimates N/A=not assessable *p-values were adjusted using the Dunnett-Hsu multiple comparison method

Microvascular occlusion.

In fig. 51 is a Kaplan-Meier plot showing the percentage of mice with microvascular occlusion as a function of time in the FTTC-dextran induced model.

- 120 046178 thrombotic microangiopathy in mice treated with increasing doses of human MASP-2 inhibitory antibody (mAb H6 at doses of 2 mg/kg, 10 mg/kg or 20 mg/kg) or an isotype control antibody. As shown in FIG. 51, complete microvascular occlusion was delayed in groups of mice treated with mAb H6 compared with control mice.

In fig. Figure 52 shows a graph showing the average time to microvascular occlusion depending on the dose of mAb H6 (*p<0.05 compared with control). As shown in FIG. 52, the average time to complete microvascular occlusion increased from 23.3 minutes in the control group to 38.6 minutes in the group receiving 2 mg/kg mAb H6 (p<0.05). Doses of 10 mg/kg or 20 mg/kg mAb H6 had a similar effect (mean time to complete microvascular occlusion was 40.3 and 38 minutes, respectively) to a dose of 2 mg/kg mAb H6. The experimental data and results of statistical analysis that serve as the basis are given in Table. 16 and 17.

The time to complete vascular occlusion in individual mice, recorded from video evaluation, is shown in Table 1 below. 16.

Table 16

Time to complete occlusion after light/dye induced injury

Control influence Exposure to mAb H6 Time to occlusion (minutes) Control 2 mg/kg 10 mg/kg 20 mg/kg 37.50 42.3 30.92 38.00 29.07 21.91 17.53 28.00 27.12 24.4 51.38 40.58 19.38 31.38 36.88 33.00 19.55 61.17* 26.83 39.10 18.00 61.55* 40.28 32.03 16.50 55.83 38.53 23.33 71.93* 21.75* 14.83 98.22* 32.25* thirty* 33.17* 61.8*

* There was no complete occlusion in the vessels during the specified observation period.

The results of the statistical analysis, which compared the time to complete occlusion in animals receiving control antibody and mAb H6, are shown below in table. 17.

Table 17

Time to complete microvascular occlusion: dose response data from the fitc-dextran study

Statistical parameter Control mAb H6 (2 mg/kg) mAb H6 (10 mg/kg) mAb H6 (20 mg/kg) Number of events/number of animals (%) 9/11 (81.8%) 4/6 (66.7%) 7/9 (77.8%) 7/10 (70.0%) Mean time (minutes) (95% CI) 23.3 (16.5, 37.5) 36.8 (21.9, N/A) 40.3 (17.5, N/A) 38.0 (28.0, 40.6) Wilcoxon test, value* 0.0456 0.0285 0.0260 Event=Observed time to occlusion Mean (minutes) and its 95% CI are based on Kaplan-Meier estimates N/A=not assessable *p-values were adjusted by the Dunnett-Hsu multiple comparison method

Summary.

As shown in table. 18, the onset of thrombus formation was delayed in H6 mAb-treated mice in a dose-dependent manner compared with control antibody-treated mice (mean time to onset 10.4-17.7 min vs. 6.8 min). The average time to complete occlusion was significantly increased in all groups of animals receiving mAb H6 relative to animals receiving the control antibody (Table 18).

- 121 046178

Table 18

Average time to onset of thrombus formation and complete occlusion

Control mAb H6 (2 mg/kg) mAb H6 (10 mg/kg) mAb H6 (20 mg/kg) Average* time to start 6.8 10.4 11.7 17.7* blood clot formation (minutes) Average* time to complete microvascular occlusion (minutes) 23.3 36.8* 40.3* 38.0*

# Mean values are based on Kaplan-Meier estimates * p < 0.05 compared with control (Wilcoxon test, adjusted by Dunnett-Hsu multiple comparison method).

These results indicate that mAb H6, a human monoclonal antibody that binds MASP-2 and blocks the lectin pathway of the complement system, leads to a reduction in microvascular thrombosis in a dose-dependent manner in an experimental mouse model of TMA. Thus, administration of a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, is expected to be an effective treatment for patients suffering from HUS, aHUS, TTP, or other microangiopathic diseases such as other TMAs, including catastrophic antiphospholipid syndrome (CAS). CAPS), systemic Degos disease, as well as TMA due to cancer, cancer chemotherapy and transplantation, and will provide protection against microvascular coagulation and thrombosis.

Example 40.

This example describes the identification by phage display of fully human scFv antibodies that bind MASP-2 and inhibit lectin-mediated complement activation without affecting components of the classical (dq-dependent) pathway and the alternative pathway of the immune system.

Review.

Fully human high-affinity anti-MASP-2 antibodies were identified by screening a phage display library. Fragments of the variable regions of the light and heavy chains of antibodies were isolated in both scFv and full-length IgG formats. Human anti-MASP-2 antibodies are useful for inhibiting cell damage associated with lectin pathway-mediated activation of the alternative complement pathway, which does not involve a component of the classical (dq-dependent) immune system pathway. In some embodiments, said MASP-2 inhibitory antibodies have the following characteristics: (a) high affinity for human MASP-2 (e.g., a K _D value of 10 nM or less) and (b) inhibit MASP-2-dependent complement activity in 90% human serum with an IC ₅₀ value of 30 nM or less.

Methods.

Expression of full-length catalytically inactive MASP-2.

The full-length human MASP-2 cDNA sequence (SEQ ID NO: 4) encoding the human MASP-2 polypeptide with the leader sequence (SEQ ID NO: 5) was subcloned into the mammalian expression vector pCI-Neo (Promega), which drives expression under the control region CMV enhancer/promoter in eukaryotic cells (described in Kaufman R. J. et al., Nucleic Acids Research 19:4485-90, 1991; Kaufman, Methods in Enzymology, 185:537-66 (1991)).

To obtain catalytically inactive human MASP-2A protein, site-directed mutagenesis was performed as described in US2007/0172483, the contents of which are incorporated herein by reference. PCR products were purified after agarose gel electrophoresis and band processing, and single adenosine overlays were generated using a standard chain extension method. MASP-2A with an adenosine tail was then cloned into the pGEM-T easy vector and transformed into E. coli. Human MASP-2A was further subcloned into either of the mammalian expression vectors pED or pCI-Neo.

The MASP-2A expression construct described above was transfected into DXB1 cells by the standard calcium phosphate transfection method (Maniatis et al., 1989). MASP-2A was prepared in serum-free medium to ensure that the preparations were not contaminated by other serum proteins. The medium was collected from confluent cells every other day (four times in total). The level of recombinant MASP-2A averaged approximately 1.5 mg/liter of culture medium. The MASP-2A mutant (Ser-Ala mutant described above) was purified by affinity chromatography on an MBP-A-agarose column.

MASP-2A ELISA of candidate scFv clones identified by sorting/transformation

- 122 046178 scFv and filter screening.

A phage display library of human immunoglobulin light and heavy chain variable region sequences was subjected to antigen sorting, followed by automated screening and antibody selection, to identify high-affinity scFv antibodies to the human MASP-2 protein. Three rounds of scFv phage library sorting were performed against HIS-tagged or biotin-tagged MASP-2A. In the third round of sorting, elution was performed first with MBL and then with TEA (alkaline). To monitor the specific enrichment of phages exhibiting scFv fragments against the target MASP-2A, a polyclonal phage ELISA against immobilized MASP-2A was performed. The scFv genes obtained in round 3 sorting were cloned into the pHOG expression vector and used in a small-scale filter screen to detect specific anti-MASP-2A clones.

Bacterial colonies containing plasmids encoding scFv fragments from the third round of sorting were selected, gridded onto nitrocellulose membranes, and grown overnight on non-inducing medium to obtain master plates. A total of 18,000 colonies from the third round of sorting were selected and analyzed, half from competitive elution and half from subsequent TEA elution. Sorting of the scFv phagemid library against MASP-2A, followed by scFv conversion and filter screening, resulted in 137 positive clones. 108/137 clones were positive in the MASP-2 binding ELISA (data not shown), of which 45 clones were further analyzed for the ability to block MASP-2 activity in normal human serum.

Assay to measure inhibition of lectin pathway C3 convertase formation.

A functional assay measuring inhibition of lectin pathway C3 convertase formation was used to evaluate the blocking activity of candidate anti-MASP-2 scFv clones. The activity of the serine protease MASP-2 is required for the formation of two protein components (C4b, C2a) that constitute the C3 convertase of the lectin pathway. As a consequence, an anti-MASP-2 scFv antibody that inhibits the functional activity of MASP-2 (ie, a blocking anti-MASP-2 scFv) will inhibit de novo formation of the lectin pathway C3 convertase. C3 contains an unusual and highly reactive thioether group in its structure. After C3 cleavage by C3 convertase in this assay, the thioester group on C3b can form a covalent bond with hydroxyl or amino groups on macromolecules immobilized at the bottom of plastic wells through ester or amide bonds, which facilitates the detection of C3b in ELISA.

Yeast mannan is a known activator of the lectin pathway. In the following method for measuring C3 convertase production, mannan-coated plastic wells were incubated for 30 min at 37°C with diluted rat serum to activate the lectin pathway. The wells were then washed and analyzed for C3 immobilized in the wells using standard ELISA methods. The amount of C3b formed in this assay directly reflects the de novo formation of lectin pathway C3 convertase. Anti-MASP-2 scFv clones at selected concentrations were tested in this assay for their ability to inhibit C3 convertase formation and subsequent C3b formation.

Methods.

candidate clones identified as described above were expressed, purified and diluted to the same concentration of stock solutions, which were again diluted with Ca and Mg'' buffer GVB (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgCl ₂ , 2.0 mM CaCl ₂ , 0.1% gelatin, pH 7.4) to ensure that all clones have the same amount of buffer. Each scFv clone was tested in triplicate at a concentration of 2 μg/ml. Fab2 OMS100, which was tested at a concentration of 0.4 μg/ml, served as a positive control. C3c formation was monitored in the presence and absence of scFv/IgG clones.

Mannan was diluted to a concentration of 20 μg/ml (1 μg/well) in 50 mM carbonate buffer (15 mM Na ₂ CO ₃ +35 mM NaHCO ₃ +1.5 mM NaN ₃ ), pH 9.5, and applied to coat the plates ELISA overnight at 4°C. The next day, the mannan-coated plates were washed 3 times with 200 μl of PBS. Then, 100 μl of 1% HSA blocking solution was added to the wells and incubated for 1 h at room temperature. The plates were washed 3 times with 200 μl PBS and stored on ice with 200 μl PBS until samples were added.

Normal human serum was diluted to 0.5% in GVB buffer with Ca ⁺⁺ and Mg ⁺⁺ , scFv clones, or the positive control Fab2 OMS100, were added in triplicate at a concentration of 0.01 μg/ml; 1 μg/ml (OMS100 control only) and 10 μg/ml to this buffer and preincubated for 45 min on ice before adding to ELISA blocking assay plates. The reaction was started by incubating for one hour at 37°C and stopped by transferring the plates to an ice bath. C3b deposition was detected using a rabbit anti-mouse C3c antibody, followed by the addition of HRP-conjugated goat anti-rabbit immunoglobulin antibodies. The negative control was buffer without antibody (no antibody=maximum C3b deposition), and the positive control was buffer with EDTA (no C3b deposition). The background level was determined by performing the same analysis using

- 123 046178 with the exception that the wells did not contain mannan. The background signal in wells without mannan was subtracted from the signals in wells containing mannan. The cutoff criterion was set at half the activity of the extraneous scFv clone (VZV) and buffer alone.

Results.

Based on the cutoff criterion, a total of 13 clones were identified that blocked MASP-2 activity. All 13 clones conferring >50% inhibition of pathway activity were selected and sequenced, resulting in 10 unique clones. All ten clones were found to have light chains of the same subclass, A3, but heavy chains of three different subclasses: VH2, VH3 and VH6. In functional analysis, five of the ten candidate scFv clones had IC ₅₀ nM values less than the target criterion of 25 nM using 0.5% human serum.

To obtain antibodies with increased potency, the three parental scFv clones identified as described above were subjected to light chain shuffling. This process involved generating a combinatorial library consisting of the VH of each of the parental clones paired with a library of native human lambda light chains (VL) obtained from three healthy donors. This library was then screened for scFv clones with improved binding affinity and/or functionality.

Table 19

Comparison of functional activity, expressed in IC ₅₀ (nm), of leading daughter clones and their corresponding parent clones (all in scFv format)

Clone scFv 1% human serum SZ analysis 90% human serum SZ analysis 90% human serum C4 analysis (1С ₅₀ , nM) (1С ₅₀ , nM) (1С ₅₀ , nM) 17D20mc 38 But But 17D20m_d3521Nl 1 26 >1000 140 17N16mc 68 But But 17N16m_dl7N9 48 15 230

Below are the heavy chain variable region (VH) sequences for the parent clones and daughter clones listed above in table. 19.

Kabat CDR sequences (31-35 (H1), 50-65 (H2) and 95-107 (H3)) are shown in bold; and Chothia CDRs (26-32 (H1), 52-56 (H2) and 95-101 (H3)) are underlined.

17D20 35VH-21N11VL, heavy chain variable region (HC) (SEQ ID NO: 67, encoded by SEQ ID NO: 66)

OVTLKESGPVLVKPTETLTLTCTVSGFSLSRGKMGVSWIRQPPGKALEWLAHIF SSDEKSYRTSLKSRLTISKDTSKNOVVLTMTNMDPVDTATYYCARIRRGGIDYWGOG TLVTVSS d!7N9, heavy chain variable region (HC) (SEQ ID NO: 68)

OVOLOQSGPGLVKPSQTLSLTCAISGDSVSSTSAAWNWIROSPSRGLEWLGRTY YRSKWYNDYAVSVKSRITINPDTSKNOFSLQLNSVTPEDTAVYYCARDPFGVPFDIWG QGTMVTVSS

Below are the light chain variable region (VL) sequences for the parent clones and daughter clones.

Kabat CDRs (24-34 (L1); 50-56 (L2) and 89-97 (L3)) are in bold; and Chothia CDRs (24-34 (L1); 50-56 (L2) and 89-97 (L3)) are underlined. These areas are the same when numbered according to either the Kabat or Chothia system.

17D20m d3521Nll, light chain variable region (VL) (SEQ ID NO: 70, encoded by SEQ GO NO: 69)

OPVLTQPPSLSVSPGQTASrrCSGEKLGDKYAYWYQOKPGQSPVLVMYQDKOR PSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSSTAVFGGGTKLTVL

17N16m d!7N9, light chain variable region (VL) (SEQ ID NO: 71)

SYELIQPPSVSVAPGQTATITCAGDNLGKKRVHWYQQRPGQAPVLVIYDDSDRP SGIPDRFSASNSGNTATLTITRGEAGDEADYYCQVWDIATDHVVFGGGTKLTVLAAAG SEQKLISE

Ahtu-MASP-2 antibodies OMS100 and moAt_d3521N11VL (containing the heavy chain variable region of SEQ ID NO: 67 and the light chain variable region of SEQ ID NO: 70, also referred to as OMS646 and mAb H6), both of which have been demonstrated to bind human MASP

- 124 046178 with high affinity and have the ability to block the functional activity of complement, analyzed for epitope binding in dot blot analysis. The results showed that antibodies OMS646 and OMS100 are highly specific for MASP-2 and do not bind MASP-1/3. None of the antibodies bind MAp19 or fragments of MASP-2 that do not contain the CCP1 domain of MASP2, suggesting that the binding sites include CCP1.

Ahtu-MASP-2 antibody OMS646 has been shown to avidly bind recombinant MASP-2 (KD 60,250 pM) with >5000-fold selectivity over C1s, C1r, or MASP-1 (see Table 20 below):

Table 20

Affinity and specificity of the interaction of anti-MASP-2 antibody OMS646 with MASP-2 in a solid-phase ELISA assay

*Mean ± SD; n=12.

OMS646 specifically blocks lectin-dependent activation of terminal complement components.

Methods.

The effect of OMS646 on membrane attack complex (MAC) deposition was analyzed using pathway-specific conditions: lectin pathway, classical pathway, and alternative pathway. For this purpose, the Wieslab Comp300 complement screening kit (Wieslab, Lund, Sweden) was used according to the manufacturer's instructions.

Results.

In fig. 53A is a graph showing the level of MAC deposition in the presence or absence of anti-MASP-2 antibody (OMS646) under lectin pathway-specific conditions. in fig. 53B is a graph showing the level of MAC deposition in the presence or absence of anti-MASP-2 antibody (OMS646) under classical pathway-specific conditions. in fig. 53C is a graph showing the level of MAC deposition in the presence or absence of anti-MASP-2 antibody (OMS646) under alternative pathway specific conditions.

As shown in FIG. 53A, OMS646 blocks lectin pathway-mediated activation of MAC deposition with an IC ₅₀ value of approximately 1 nM. However, OMS646 had no effect on MAC deposition resulting from classical pathway-mediated activation (FIG. 53B) or alternative pathway-mediated activation (FIG. 53C).

Pharmacokinetics and pharmacodynamics of OMS646 following intravenous (IV) or subcutaneous (SC) administration in mice.

The pharmacokinetics (PK) and pharmacodynamics (PD) of OMS646 were assessed in a 28-day PK/PD study in mice with a single dose. The study tested dose levels of 5 mg/kg and 15 mg/kg of OMS646 when administered subcutaneously (SC), as well as a dose level of 5 mg/kg of OMS646 when administered intravenously (IV).

As for the FC profile OMS646, Fig. 54 is a graph showing the concentration of OMS646 (average of n=3 animals/groups) versus time after administration of OMS646 at the indicated dose. As shown in FIG. 54, at a SC dose of 5 mg/kg, OMS646 achieved maximum plasma concentrations of 5–6 μg/mL approximately 1–2 days after dosing. The bioavailability of OMS646 at a 5 mg/kg SC dose was approximately 60%. As also shown in FIG. 54, at a SC dose of 15 mg/kg, OMS646 achieved maximum plasma concentrations of 10–12 μg/mL approximately 1–2 days after dosing. In all groups, OMS646 was slowly eliminated from the circulation with a terminal half-life of approximately 8-10 days. The OMS646 profile is typical of human antibodies in mice.

The PD activity of OMS646 is graphically presented in FIG. 55A and 55B. In fig. 55A and 55B show the PD response (decrease in systemic lectin pathway activity) for each mouse in the 5 mg/kg IV (FIG. 55A) and 5 mg/kg SC (FIG. 55B) groups. The dotted line represents the baseline level in the assay (maximum inhibition; normal mouse serum spiked in vitro with excess OMS646 prior to assay). As shown in FIG. 55A, after IV administration of a 5 mg/kg dose of OMS646, systemic lectin pathway activity immediately dropped to almost undetectable levels, and lectin pathway activity showed only modest recovery over the 28-day observation period. As shown in FIG. 55B, time-dependent inhibition of lectin pathway activity was observed in mice treated with a SC dose of 5 mg/kg OMS646. Lectin pathway activity dropped to almost undetectable levels within 24 hours of drug administration

- 125 046178 of the drug and remained at low levels for at least 7 days. Lectin pathway activity gradually increased over time but did not return to pre-dose levels during the 28-day observation period. The time course of lectin pathway activity observed after a 15 mg/kg SC dose was similar to that observed after a 5 mg/kg SC dose (data not shown), indicating saturation of the PD endpoint. Data also indicated that a weekly dose of 5 mg/kg OMS646, administered either IV or SC, was sufficient to achieve sustained suppression of systemic lectin pathway activity in mice.

Example 41.

This example demonstrates that a MASP-2 inhibitory antibody (OMS646) inhibits aHUS serum-induced complement C5b-9 deposition on the surface of activated human microvascular endothelial cells (HMEC-1) following exposure to serum from atypical hemolytic uremic syndrome (aHUS) patients obtained during the acute phase and remission phase of the disease.

Background/Rationale.

The following study was conducted to analyze aHUS serum-induced C5b-9 complement deposition on the surface of activated HMEC-1 cells after exposure to serum from aHUS patients obtained (1) during the acute phase and (2) during the remission phase of the disease, in the presence or absence of OMS646, an anti-MASP-2 antibody that specifically binds MASP-2 and inhibits lectin pathway activation.

Methods.

Patients.

Four patients with aHUS, studied both during the acute phase of the disease and during remission, were selected for this study from among people included in the International HUS/TTP Registry and genotyped in the laboratory of immunology and genetics of the Institute of Transplantation and Rare Diseases. Mario Negri. One patient with aHUS had a heterozygous complement factor H (CFH) p.R1210C mutation and one had anti-CFH autoantibodies, while the other two patients with aHUS had no mutations or antibodies to CFH.

In table Figures 21 and 22 summarize the results of screening for complement component gene mutations and anti-CFH autoantibodies in the four patients with aHUS analyzed in this study, along with clinical and biochemical data obtained either during the acute phase or during remission.

Table 21

Clinical parameters of four patients with aHUS in this study

Patsi ent Mutation or anti-CFH Ab Disease phase Platelets (150400*10 ³ / µl) LDH (266500 IU/l) Hemoglobin (14-18 g/dl) With- creatinine (0.55-1.25 mg/dl) No. 1 no mutations, no anti-CFH At acute 31000 1396 12.9 2.37 remission 267000 But 11.5 3.76 No. 2 CFH-R1210C acute 46000 1962 7 5.7 remission 268000 440 13.4 7.24 No. 3 anti-CFH Ab acute 40000 3362 9.5 1.77 remission 271000 338 8.8 0.84 without mutations, acute 83000 1219 7.8 6.8 No. 4 without anti-CFH At remission 222000 495 12.2 13

Note: n/a=not defined.

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Table 22

Complement parameters in four patients with aHU S in this study

Patient Mutation or anti-CFH Ab Disease phase Serum SD (83-180 mg/dl) SC5b-9 in plasma (127-400 ng/ml) No. 1 without mutations, without anti-CFH Ab acute 51 69 remission But 117 No. 2 CFH-R1210C acute 79 421 remission 119 233 No. 3 anti-CFH Ab acute 58 653 remission 149 591 No. 4 without mutations, without anti-CFH Ab acute 108 But remission But But

Experimental methods.

Skin-derived human microvascular endothelial cell line (HMEC-1) cells were plated on glass slides and used confluently. Confluent HMEC-1 cells were activated with 10 μM ADP (adenosine diphosphate) for 10 min and then incubated for four hours with serum from the four aHUS patients described above in Table. 23 and 24, collected either during the acute phase of the disease, or from the same patients with aHUS during remission, or from 4 healthy control subjects. Serum was diluted 1:2 with assay medium (HBSS with 0.5% BSA) in the presence or absence of MASP-2 inhibitory antibody, OMS646 (100 μg/ml), prepared as described above in Example 40, or in the presence of soluble receptor 1 complement (sCR1) (150 μg/ml), as a positive control for complement inhibition. After completion of the incubation step, HMEC-1 cells were treated with rabbit anti-human complement C5L-9 antibody followed by FITC-conjugated secondary antibody. In each experiment, serum from one healthy control subject was tested in parallel with serum from aHUS patient (acute and remission). A confocal inverted laser microscope was used to image fluorescent staining on the surface of endothelial cells. Fifteen fields were imaged for each sample, and the area of fluorescent staining was estimated by automated image edge recognition using the built-in special functions of Image J and expressed as the number of pixels per field analyzed. Fields with the lowest and highest values were excluded from the calculations.

For statistical analysis (one-way ANOVA, followed by Tukey's test for multiple comparisons), results in 13-field pixels were considered in each experimental condition for each patient, and controls were also used.

Results.

The results of the complement deposition assay using sera from four patients with aHUS are shown in Table 1 below. 23A, and the results with sera from four healthy subjects are shown in Table 1 below. 23V.

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Table 23A

Effect of complement inhibitors on serum-induced aHUS deposition of C5b-9 on ADP-activated HMEC-1 cells

Patient with aHUS no. Acute phase aHUS aHUS remission phase without processing +sCRl +OMS646 without processing +sCRl +OMS646 Patient No. 1 (no mutations, no anti-CFH antibodies) 5076 ± 562° 551 ± 80* 3312 ± 422** 4507 ± 533° 598 ± 101§ 1650±223§ Patient #2 (CFH- R1210C) 5103 ±648° 497 ± 67* 2435 ± 394* 3705 ± 570° 420 ± 65§ 2151 ± 250§§§ Patient No. 3 (anti-CFH Ab) 3322 ±421° 353 ± 64* 2582 ±479 6790 ± 901° 660 ± 83§ 2077±353§ Patient No. 4 (no mutations, no anti-CFH antibodies) 4267 ± 488° 205 ± 34* 2369 ± 265** 5032 ± 594° 182 ± 29§ 3290 ± 552§§

Table 23B

Effect of complement inhibitors on sera from four healthy control subjects (not suffering from ahus) in a C5L-9 deposition assay on ADP-activated HMEC-1 cells

Healthy control subject no. No processing +sCRl +OMS646 Control subject #1 (analysis in parallel with subject #1 with aHUS) 481 ±66 375 ±43 213 ±57 Control subject #2 (analysis in parallel with subject #2 with aHUS) 651 ±61 240 ± 33 490 ± 69 Control subject #3 (analysis in parallel with subject #3 with aHUS) 602 ± 83 234 ±35 717± 109 Control subject #4 (analysis in parallel with subject #4 with aHUS) 370 ±53 144 ± 20 313 ±36

For table 23A and 23B: Data are means ± SE. °P<0.001 versus control; *P<0.001, **P<0.01 against aHUS in the acute phase, without treatment; §P<0.001, §§P<0.01, §§§P<0.05 against aHUS in remission, without treatment.

In fig. 56 is a graph showing the inhibitory effect of anti-MASP-2 antibody (OMS646) and sCR1 on aHUS serum-induced C5b-9 deposition on ADP-activated

- 128 046178 HMEC-1 cells. In fig. 56 Data are means ± SE. °P<0.0001 versus control; *P<0.0001 against aHUS in the acute phase, without treatment; ^L P <0.0001 versus aHUS in the acute phase + sCRl; §P<0.0001 against aHUS in remission, without treatment and #P<0.0001 against aHUS in remission+sCRl.

As shown in table. 23A, 23B and FIG. 56, ADP-stimulated HMEC-1 cells exposed to serum from aHUS patients (collected either in the acute phase or in remission phase) for four hours under static conditions had large deposits of C5b-9 on the cell surface, detected by confocal microscopy. When measuring the area covered by C5b-9, a significantly greater degree of C5b-9 deposition was observed on cells exposed to serum from aHUS patients than on cells exposed to serum from healthy control subjects, regardless of whether the aHUS serum was collected in acute phase or during remission. There were no differences in serum-induced deposition of C5b-9 on the endothelium between the acute phase and remission.

As also shown in Table. 23A, 23B and FIG. 56, addition of the anti-MASP-2 antibody OMS646 to aHUS serum (obtained from patients either in the acute phase or during remission) resulted in a significant reduction in the deposition of C5b-9 on the surface of endothelial cells compared with untreated aHUS serum. However, the inhibitory effect of OMS646 on C5b-9 deposition was less pronounced than the effect produced by the pan complement inhibitor sCR1. Indeed, a statistically significant difference was observed between aHUS serum-induced C5b-9 deposition in the presence of OMS646 compared to sCR1 (FIG. 56 and Tables 23A and 23B).

When calculating the average of four patients with aHUS, the following percentage reduction in C5b-9 deposition was observed (compared to C5b-9 deposition induced by untreated serum from the same patients, taken as 100%) in the presence of complement inhibitors:

Acute phase:

sCR1 (150 µg/ml): 91% reduction in C5b-9 deposition,

OMS646 (100 µg/ml): 40% reduction in C5b-9 deposition.

Remission phase:

sCR1 (150 µg/ml): 91% reduction in C5b-9 deposition,

OMS646 (100 µg/ml): 54% reduction in C5b-9 deposition.

Conclusion.

The results described in this example demonstrate that the lectin complement pathway is stimulated by activated microvascular endothelial cells, and that this stimulation is an important factor for the excessive complement activation characteristic of aHUS. This stimulation of the lectin pathway and the resulting excess complement activation has also been shown to occur both during the acute phase and during clinical remission of aHUS. Moreover, this finding does not appear to be limited to any specific complement defect associated with aHUS. As also demonstrated in this example, selective inhibition of the lectin pathway with a MASP-2 inhibitory antibody such as OMS646 results in reduced complement deposition in patients with aHUS of various etiologies.

Example 42.

This example demonstrates that a MASP-2 inhibitory antibody (OMS646) inhibits aHUS serum-induced platelet aggregation and thrombus formation on the surface of activated human microvascular endothelial cells (HMEC-1) following exposure to serum from aHUS patients obtained during (1) the acute phase and (2) remission phases of aHUS.

Methods.

Patients.

Three patients (Patients #1, #2 and #4 described in Tables 21, 22, 23A and 23B in Example 41) with aHUS (one patient had a heterozygous p.R1210C CFH mutation, while mutations or anti-CFH antibodies were not detected in the other two patients) were studied both during the acute phase of the disease and during remission. Patients were selected for this study from among people included in the International HUS/TTP Registry and genotyped in the laboratory of immunology and genetics of the Institute of Transplantation and Rare Diseases. Mario Negri. Five healthy subjects were also selected as blood donors for the perfusion experiments.

Methods.

Confluent HMEC-1 cells were activated with 10 μM ADP for 10 min and then incubated for three hours with sera from three aHUS patients (patients #1, 2, and 4 described in Example 41) collected during the acute phase of the disease. either from the same patients in remission, or with control sera from healthy subjects. Serum was diluted 1:2 with assay medium (HBSS with 0.5% BSA) in the presence or absence of MASP-2 inhibitory antibody, OMS646 (100 μg/ml), prepared as described above in Example 40, or in the presence of sCR1 (150 µg/ml) as a positive control for complement inhibition. In the case of patients No. 1 and No. 2, additional wells were incubated with serum (obtained in the acute phase and during remission) diluted 1:2

- 129 046178 analytical medium containing 100 µg/ml of an extraneous isotype control antibody or 20 µg/ml OMS646 (for the latter, the serum of patient No. 1, obtained only in the remission phase, was tested, and the serum of patient No. 2, obtained both during the acute phase, was tested , and in the remission phase).

After completion of the incubation step, HMEC-1 cells were perfused in a flow chamber with heparinized whole blood (10 IU/ml) obtained from healthy subjects (containing the fluorescent dye quinine, which labels platelets) at a shear stress occurring in the microcirculation (60 dynes/cm ² , three minutes). After three minutes of perfusion, endothelial cell monolayers were fixed in acetone. Fifteen images for each sample of platelet thrombi on the surface of endothelial cells were obtained using a confocal inverted laser microscope, and the areas occupied by thrombi were estimated using Image J. The fields with the lowest and highest values were excluded from the calculations.

For statistical analysis (one-way ANOVA, followed by Tukey's test for multiple comparisons), pixel results from 13 fields were considered in each experimental condition for each patient, and controls were also used.

Results.

The results of blood clot experiments with serum from three patients with aHUS are shown in Table 1 below. 24A, and the results with sera from five healthy subjects are shown in Table 1 below. 24V.

Table 24A

Effect of complement inhibitors on aHUS serum-induced thrombus formation (pixels ² ± SE on ADP-activated HMEC-1 cells

Experimental conditions Disease phase Patient with aHUS #1 thrombus formation (pixels ± SE) (no mutations, no anti-CFH At) Patient with aHUS #2 thrombus formation (pixels ² ± SE) (CFH-R1210C) Patient with aHUS No. 4 thrombus formation (pixels ² ± SE) (no mutations, no anti-CFH Ab) No processing acute 5499 ± 600 22320± 1273° 10291± 1362° remission 6468± 1012° 3387 ±443° 17676± 1106° +sCRl (150 µg/ml) acute 4311 ±676 5539 ± 578* 5336± 1214*** remission 573 ± 316§ 977±102§ 2544 ± 498§ +OMS646 (20 µg/ml) acute undefined 6974 ± 556* undefined remission 832±150§ 1224 ± 252§ undefined +OMS646 (100 µg/ml) acute 3705 ± 777 9913 ±984* 2836 ±509* remission 3321 ±945§§§ 733 ±102§ 1700 ± 321§ + extraneous isotype control antibody (100 μg/ml) acute 5995 ± 725 18655± 1699 undefined remission 10885± 1380 2711 ±371 undefined

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Table 24B

Effect of complement inhibitors on sera from five healthy control subjects (not suffering from aHUS) in a clot formation assay (pixels ² ± SE) on ADP-activated HMEC-1 cells

Experimental conditions Control No. 1 formation of blood clots (pixels ² ± SE) Control No. 2 thrombus formation (pixels ± SE) Control No. 3 blood clot formation (pixels ± SE) Control No. 4 thrombus formation (pixels ± SE) Control No. 5 blood clot formation (pixels ² ± SE) No processing 2880 ±510 1046± 172 1144± 193 735 ± 124 2811 ±609 +sCRl (150 µg/ml) 5192 ±637 1527± 153 1198 ±138 2239 ± 243 2384 ±410 +OMS646 (100 µg/ml) 7637 ±888 1036± 175 731 ±203 2000 ±356 7177± 1477 + extraneous isotype control antibody (100 μg/ml 6325 ± 697 1024 ±235 399 ±82 45269 Not defined Analyzed in parallel with serum from a subject with aHUS No. 1 (acute phase serum) No. 1 (remission phase serum) No. 2 (acute phase serum) No. 2 (remission phase serum) No. 5 (serum of acute phase and remission phase)

For table 24A and 24B: Data are means ± SE. °P<0.001 versus control; *P<0.001, ***P<0.05 against aHUS in the acute phase, without treatment; §P<0.001, §§§P<0.05 against aHUS in remission, without treatment.

In fig. 57 is a graph showing the effect of anti-MASP-2 antibody (OMS646) and sCR1 on aHUS serum-induced thrombus formation on ADP-activated HMEC-1 cells. in fig. 57 Data are means ± SE. °P<0.0001, °°P<0.01 versus control; *P<0.0001, **P<0.01 against aHUS in the acute phase, without treatment; §P<0.0001 against aHUS in remission, without treatment.

As shown in table. 24A and FIG. 57, a marked increase in the area covered by thrombi was observed in HMEC-1 cells treated with aHUS serum collected from either the acute phase or the remission phase, compared with cells exposed to serum from healthy control subjects (Table 24B and FIG. 57). As shown in FIG. 57 and in table. 24A, OMS646 (at both concentrations, 100 μg/ml and 20 μg/ml) resulted in partial inhibition of thrombus formation in cells preexposed to aHUS serum obtained during the acute phase. The antithrombogenic effect was comparable between the two different doses of OMS646 and did not differ from the effect of sCR1 (Fig. 57 and Table 24A). Addition of an unrelated isotype control antibody did not inhibit aHUS serum-induced thrombus formation.

As also shown in FIG. 57 and in table. 24A, the inhibitory effect of OMS646 was even more evident in aHUS serum collected during the remission phase. Indeed, the addition of OMS646, at both doses, 100 μg/ml and 20 μg/ml, to the serum of aHUS patient collected during re

- 131 046178 mission, resulted in almost complete inhibition of thrombus formation, similar to what was observed with the addition of sCR1. An unrelated isotype control antibody had no significant inhibitory effect.

When calculating the mean of three patients with aHUS, the following percentage reduction in HMEC-1 surface area covered by thrombus deposition (compared to the thrombus area induced by untreated serum from the same patients, taken as 100%) was observed in the presence of complement inhibitors.

Acute phase:

sCRl (150 µg/ml): reduction by 60%,

OMS646 (100 µg/ml): 57% reduction,

OMS646 (20 µg/ml): 45% reduction.

Remission phase:

sCR1 (150 µg/ml): 85% reduction,

OMS646 (100 µg/ml): 79% reduction,

OMS646 (20 µg/ml): 89% reduction.

The discussion of the results.

The results in this example show that a MASP-2 inhibitory antibody such as OMS646 (prepared as described in Example 40) has a potent inhibitory effect on aHUS serum-induced thrombus formation on HMEC-1 cells. Surprisingly, the inhibitory effect of OMS646 on thrombus formation was greater than its effect on induced C5b-9 deposition on HMEC-1 cells (as described in Example 41). It is also surprising that the addition of OMS646, at both doses of 100 μg/mL and 20 μg/mL, to serum from aHUS patients collected during the remission phase resulted in almost complete inhibition of thrombus formation. Also surprising is the observation that OMS646, in both acute and remission, was as effective as the positive control sCR1, which is a general and almost absolute inhibitor of the complement system (Weisman H. et al., Science 249 :146-151, 1990; Lazar H. et al., Circulation 100:1438-1442, 1999).

Of note, control serum from healthy subjects also induced moderate thrombus formation on HMEC-1 cells. We did not observe a corresponding inhibitory effect on control serum-induced thrombus formation using either OMS646 or sCR1. Without wishing to be bound by theory, control serum-induced thrombi are believed to be independent of complement, as indicated by the very low amount of C5L-9 deposits observed on HMEC-1 cells incubated with control serum (see Example 41).

Conclusion.

In summary, the observed antithrombotic effect of a MASP-2 inhibitory antibody such as OMS646 appears to be much greater than would be expected based on the inhibitory effect of OMS646 on C5b-9 deposition observed in this experimental system (as described in example 41 and shown in Fig. 56). For example, as described in Gastoldi et al., Immunobiology 217:1129-1222 Abstract 48 (2012), entitled C5a/C5aR interaction mediates complement activation and thrombosis on endothelial cells in atypical hemolytic uremic syndrome (aHUS), it was found that the addition anti-C5 antibody, which inhibits C5b-9 deposition (60% reduction), limited thrombus formation on HMEC-1 to a comparable extent (60% reduction). In contrast, a MASP-2 inhibitory antibody (OMS646 at 100 μg/ml) inhibited C5b-9 deposition with the following average rates (acute=40% reduction; remission=54% reduction); and inhibited blood clot formation to a significantly higher extent (acute phase=57% reduction; remission phase=79% reduction). In comparison, OMS646 inhibited complement deposition to a lesser extent, in percentage terms, than the positive control complement inhibitor (sCR1 at a concentration of 150 μg/ml, inhibition of C5b-9 deposition in the case of the acute phase = 91% reduction; in the case of the remission phase = reduction 91%), but was equally effective as the sCR1 positive control in inhibiting thrombus formation (sCR1 at a concentration of 150 μg/ml, in the acute phase = 60% reduction; in the remission phase = 85% reduction). These results indicate that a MASP-2 inhibitory antibody (eg, OMS646) is surprisingly effective in inhibiting thrombus formation in serum obtained from subjects with aHUS in both the acute and remission phases of the disease.

In accordance with the foregoing, in one embodiment, the invention provides a method of inhibiting the formation of blood clots in a subject suffering from, or at risk of developing, thrombotic microangiopathy (TMA), comprising administering to the subject a composition containing a MASP-2 inhibitory antibody in an amount effective to inhibit MASP-2-dependent complement activation. In one embodiment, the TMA is selected from the group consisting of hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP), and atypical hemolytic uremic syndrome (aHUS). In one embodiment, the TMA

- 132 046178 represents aHUS. In one embodiment, the composition is administered to a patient with aHUS during the acute phase of the disease.

In one embodiment, the composition is administered to a patient with aHUS during a remission phase (i.e., a subject who has recovered, or partially recovered, from an acute phase episode of aHUS, with remission indicated by, for example, an increase in platelet count and/or a decrease in serum LDH concentrations , for example, as described in Loirat C et al., Orphanet Journal of Rare Diseases 6:60, 2011, the contents of which are incorporated herein by reference).

In one embodiment, a MASP-2 inhibitory antibody has at least one or more of the following characteristics: said antibody binds human MASP-2 with a K _D value of 10 nM or less, said antibody binds an epitope in the CCP1 domain of MASP-2, said the antibody inhibits C3b deposition in an in vitro assay in 1% human serum with an IC ₅₀ value of 10 nM or less, the antibody inhibits C3b deposition in 90% human serum with an IC ₅₀ value of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab) ₂ and F(ab') ₂ , wherein the antibody is a single chain molecule, wherein said antibody is an IgG2 molecule, wherein said antibody is an IgG1 molecule, wherein the antibody is an IgG4 molecule, wherein the IgG4 molecule has the S228P mutation and/or wherein the antibody does not significantly inhibit the classical pathway. In one embodiment, the antibody binds MASP-2, selectively inhibits the lectin pathway, and does not significantly inhibit the alternative pathway. In one embodiment, the antibody binds MASP-2, selectively inhibits the lectin pathway, and does not significantly inhibit the classical or alternative pathway (ie, inhibits the lectin pathway without affecting the classical and alternative complement pathways).

In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in the serum of a subject suffering from TTP, such as aHUS (in the acute phase or in remission), by at least 30%, e.g., at least 40%, e.g. 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 whey. In some embodiments, the MASP2 inhibitory antibody inhibits clot formation in the serum of a subject suffering from aHUS by at least 20 percent or more (e.g., at least 30%, at least 40%, at least 50% ) exceeding the inhibitory effect on the deposition of C5b-9 in the serum.

In one embodiment, the MASP-2 inhibitory antibody inhibits thrombus formation in serum from a patient with aHUS in remission by at least 30%, such as at least 40%, such as at least 50%, such as 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 thrombus formation in serum from a patient with aHUS in remission by at least 20 percent or more (e.g., at least 30%, at least 40%, at least at least 50%) exceeding the inhibitory effect on the deposition of C5b-9 in the serum.

In one embodiment, the MASP-2 inhibitory antibody is administered to a subject by delivery through an intravenous line or other catheter.

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

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In one embodiment, the TMA is selected from the group consisting of atypical hemolytic uremic syndrome (aHUS) (acute or remission), HUS, and TTP. In one embodiment, the subject has aHUS in the acute phase. In one embodiment, the subject has aHUS in remission.

In some embodiments, the method includes administering to a subject a composition comprising a specified amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 67. In some embodiments, the method includes administering to a subject a composition comprising a specified amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprising a light chain variable region containing the amino acid sequence of SEQ ID NO: 70.

In some embodiments, the method includes administering to a subject a composition comprising a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by the reference antibody OMS646 containing the heavy chain variable region of SEQ ID NO : 67 and the light chain variable region of SEQ ID NO: 70. Competition between binding partners can be readily assayed in vitro, for example by ELISA and/or by attaching to one binding partner a reporter molecule that can be detected in the presence of another tagged binding partner(s), allowing the identification of specific binding partners that bind the same epitope or overlapping epitope. Thus, provided herein is a specific antibody, or antigen binding fragment thereof, comprising an antigen binding site of a human antibody that competes with the reference antibody OMS646 for binding to human MASP-2.

Example 43.

This example demonstrates that a human MASP-2 inhibitory antibody (OMS646) is able to inhibit TMA patient plasma-mediated induction of apoptosis in primary human skin-derived microvascular endothelial cells (MVECs).

Background/Rationale.

The pathophysiology of TMA is known to involve endothelial cell injury induced by various factors, followed by occlusion of small vessels (eg, small arterioles and capillaries) by platelet plugs and/or fibrin thrombi (Hirt-Minkowsk P. et al., Nephron Clin Pract 114 :c219-c235, 2010; Goldberg R. J. et al., Am J Kidney Dis 56(6): 1168-1174, 2010). MVECs have been shown to undergo apoptotic injury in vitro when exposed to plasma from patients with TMA-related diseases (see Stefanescu et al., Blood Vol 112 (2):340-349, 2008; Mitra D. et al., Blood 89:1224 -1234, 1997). Apoptotic lesions associated with TMA have been documented in MVECs obtained from biopsy tissue samples (skin, bone, bone marrow, spleen, kidney, ileum) from such patients. Apoptotic injury to MVEC has also been shown to result in decreased levels of membrane-bound complement regulatory proteins in MVEC (see, eg, Mold & Morris, Immunology 102:359-364, 2001; Christmas et al., Immunology 119:522, 2006).

A positive feedback loop involving terminal complement components is thought to be involved in the pathophysiology of TMA, including atypical hemolytic uremic syndrome (aHUS) and TMA associated with catastrophic antiphospholipid syndrome (CAPS), Degos disease, as well as TMA due to cancer, anticancer chemotherapy, autoimmunity and transplantation, each of these conditions is known or believed to respond to anti-C5 therapy using the mAb eculizumab (Chapin J. et al., Brit. J. Hematol 157:772-774, 2012; Tsai et al., Br J Haematol 162(4):558559, 2013); Magro S. M. et al., Journal of Rare Diseases 8:185, 2013).

The following experiment was performed to analyze the ability of a human MASP-2 inhibitory antibody (OMS646) to block TMA patient plasma-mediated induction of apoptosis in primary human skin MVECs in plasma samples obtained from patients suffering from aHUS, ADAMTS-13-deficient thrombotic thrombocytopenic purpura ( TTP), CAPS and systemic Degos disease, as well as TMA due to cancer, transplantation, autoimmune disease and chemotherapy.

Methods.

An in vitro assay was performed to analyze the ability of a MASP-2 inhibitory antibody (OMS646) to block TMA patient plasma-mediated induction of apoptosis in primary human MVEC from skin, as described in Stefanescu R. et al., Blood Vol 112 (2):340- 349, 2008, the contents of which are incorporated herein by reference. The plasma samples used in this analysis were obtained from a collection of samples from healthy control subjects and from individuals with thrombotic microangiopathies, either in the acute phase or during the convalescent period. The presence of microangiopathy in patients with TMA was assessed by detecting schizocytes in a peripheral blood smear. Additionally, TTP was diagnosed as described in Stefanescu R. et al.

- 134 046178

Blood Vol 112 (2):340-349, 2008.

Endothelial cell (EC) culture.

As described in Stefanescu et al., primary human MVECs from skin were purchased from ScienCell Research Labs (San Diego, CA). MVEC expressed CD34 up to passages 5 and 6 (Blood 89:1224-1234, 1997). MVECs were maintained in polystyrene vials coated with 0.1% gelatin in water in ECM1001 medium (ScienCell Research Labs) containing endothelial cell growth supplements, penicillin, streptomycin, and 15% fetal bovine serum. All MVECs were used at subculture stages 2–6. Subcultivation included treatment for 5-10 min with a 0.25% trypsin-EDT A solution.

Apoptosis assay.

Representative primary human MVECs from skin, which are known to undergo TTP/HUS plasma-induced apoptosis, were washed with phosphate-buffered saline (PBS) and seeded into chambers of 12-well plates coated with 0.1% gelatin in water at a concentration of 0 ,15x106 viable cells/ml. The seeded MVEC cells were left without feeding in complete medium for 24 h, then exposed to plasma samples from patients with TMA, or plasma from healthy donors, at different concentrations (from 2% to 20%, v/v) for 18 h in the presence or absence of lack of anti-MASP-2 mAb OMS646 (150 μg/ml), then cells were harvested by trypsinization. Each sample from a patient with TMA was analyzed in duplicate. The extent of plasma-mediated apoptosis was assessed using propidium iodide (PI) staining; >5x103 cells were analyzed in a cytofluorograph, and AO peaks were determined using a computer program (MCycle Av, Phoenix Flow Systems, San Diego, CA). Cytoplasmic histone-associated DNA fragments from cell lysates were also quantified by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (Roche Diagnostics, Mannheim, Germany).

Results.

The results of the analysis of MVEC apoptosis induced by plasma from TMA patients in the presence of anti-MASP-2 mAb (OMS646) are shown in Table 1 below. 25.

Table 25

Plasma from TMA patients tested on primary human skin MVEC in the presence of anti-MASP-2 MAT (OMS646)

Subject no. WHO age/gender Clinical diagnosis (TMA) and other conditions Research Institute M.A.S. P-2 ng/ml Diagnosis based on Research Institute Cre/L DH C5a sC5- b9 Activity ADAMS Diagnosis based on activity ADAMS Protection due to OMS646 No. 2 41/ and TTP 174 TTP 34.42 772 thirty% aHUS answers No. 3 52/ and TTP 150 TTP 48.32 1399 70% aHUS Not answers No. 4 20/m TTP 224 TTP 36.9 1187 <10% TTR answers No. 10 60/ and TTP 175.4 TTP 49.5 4406 64% aHUS Not answers No. 11 59/ and TTR 144.9 TTP 40.3 135 2 <10% TTP Not answers

- 135 046178

No. 13 49/ and HUS, cancer, TTR 142.8 TTP 48.6 384 3 86% aHUS Not answers No. 42 27/m TTP 341.5 TTP 100.0 533 2 <5% TTP Not answers No. 46 25/ and TTP, Degos disease, SLE 225.1 1 TTP 53.9 342 6 H/O H/O answers No. 48 53/ and TTP, SLE, nephritis s/p kidney transplantation 788.5 aHUS 31.2 106 6 66% aHUS answers No. 49 64/ and TTP, APLA, CVA 494.5 35.4 210 0 H/O H/O Not answers No. 51 25/ and aHUS, APLA 313.1 TTP 26.8 159 5 23% aHUS answers No. 52 56/ and aHUS, SLE 333.1 TTP 18.9 BY 3 97% aHUS Not answers No. 53 56/ and aHUS, remission 189.9 TTP, remission 28.69 344 74% aHUS Not answers

Abbreviations used in table. 25:

APLA = antiphospholipid antibodies associated with catastrophic antiphospholipid syndrome (CAPS),

SLE = systemic lupus erythematosus,

CVA = cerebrovascular accident (stroke).

Consistent with the results reported in Stefanescu R. et al., Blood Vol 112 (2):340-349, 2008, significant apoptosis was observed in primary skin MVECs in the presence of thirteen plasma samples from TMA patients in the absence of anti-MASP-2 antibodies. Control plasma samples from healthy individuals were tested in parallel and did not induce apoptosis in MVECs (data not shown). As shown in table. 25, a MASP-2 inhibitory mAb (OMS646) inhibited TMA patient plasma-mediated induction of apoptosis in primary MVECs (responding in Table 25) in 6 of 13 patient plasma samples tested (46%). In particular, it is noteworthy that a MASP-2 inhibitory mAb (OMS646) inhibited apoptosis in plasma obtained from patients suffering from aHUS, TTP, Degos disease, SLE, transplant sequelae, and APLA (CAPS). Regarding the seven patient samples tested in this assay in which the anti-MASP-2 mAb did not block apoptosis (non-responders in Table 25), it should be noted that apoptosis can be induced through several pathways, not all of which are dependent on complement. . For example, as noted in Stefanescu R. et al., Blood Vol 112 (2):340-349, 2008, apoptosis in the EC assay depends on the baseline state of EC activation, which is influenced by plasma factors that may play a role in determining the level of damage , necessary for the induction of apoptosis. As also noted in Stefanescu R. et al., additional factors that can modulate apoptosis may be present in the plasma of patients with TMA, such as cytokines and various components of the complement system. Thus, due to these complicating factors, it is not surprising that the anti-MASP-2 antibody does not block

- 136 046178 priming effect in all plasma samples in which TMA plasma-induced apoptosis was noted.

It should also be noted in this regard that a similar analysis was performed using the anti-C5 antibody eculizumab in the TMA plasma-induced apoptosis assay, and very similar results were obtained (see Chapin et al., Blood (ASH Annual Meeting Abstracts): Abstract No. 3342, 120: 2012). The clinical efficacy of eculizumab, a highly successful commercial product, appears to be greater than that demonstrated in this assay, indicating that the clinical potential of complement inhibitory drugs may be underestimated in this in vitro model.

The results obtained indicate that a MASP-2 inhibitory antibody such as OMS646 is effective in inhibiting TMA plasma-induced apoptosis in plasma obtained from patients suffering from TMA such as aHUS, TTP, Degos disease, SLE, transplant sequelae and APLA ( CAPS). It is also known that endothelial damage and apoptosis play a key role in the pathology of TMA, such as idiopathic TTP and sporadic HUS (Kim et al., Microvascular Research vol 62(2):83-93, 2001). As described by Dang et al., apoptosis has been demonstrated in the red pulp of the spleen of patients with TTP, but not in healthy control subjects (Dang et al., Blood 93(4):1264-1270, 1999). Signs of apoptosis have also been noted in MVEC-derived kidney glomerular cells from a patient with HUS (Arends M. J. et al., Hum Pathol 20:89, 1989). Thus, administration of a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody (eg, OMS646), is expected to be an effective treatment for patients suffering from TMA such as aHUS, TTP or other microangiopathic diseases such as other TMA , including CAPS, systemic Degos disease, and TMA due to cancer; TMA due to chemotherapy or TMA due to transplantation.

In accordance with the foregoing, in one embodiment, the invention provides a method of inhibiting endothelial cell damage and/or endothelial cell apoptosis, and/or blood clot formation in a subject suffering from, or at risk of developing, thrombotic microangiopathy (TMA), comprising administering to the subject a composition , containing a MASP-2 inhibitory antibody in an amount effective to inhibit MASP-2-dependent complement activation. In one embodiment, the TMA is selected from the group consisting of atypical hemolytic uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP), and hemolytic uremic syndrome (HUS). In one embodiment, the TMA is aHUS. In one embodiment, the composition is administered to a patient with aHUS during the acute phase of the disease. In one embodiment, the composition is administered to a patient with aHUS during a remission phase (i.e., a subject who has recovered, or partially recovered, from an acute phase episode of aHUS, with remission indicated by, for example, an increase in platelet count and/or a decrease in serum LDH concentrations , for example, as described in Loirat C et al., Orphanet Journal of Rare Diseases 6:60, 2011, the contents of which are incorporated herein by reference).

In one embodiment, the subject suffers from, or is at risk of developing, TMA, which is (i) TMA due to cancer; (ii) TMA due to chemotherapy or (iii) TMA due to transplantation (eg, organ transplantation, such as kidney transplantation, or allogeneic hematopoietic stem cell transplantation). In one embodiment, the subject suffers from, or is at risk of developing, Upshaw-Shulman syndrome (USS). In one embodiment, the subject suffers from, or is at risk of developing, Degos disease. In one embodiment, the subject suffers from, or is at risk of developing, catastrophic antiphospholipid syndrome (CAPS).

In accordance with any of the embodiments disclosed herein, the MASP-2 inhibitory antibody has at least one or more of the following characteristics: the antibody binds human MASP-2 with a K _D value of 10 nM or less, the antibody binds an epitope in CCP1 domain of MASP-2, the antibody inhibits C3b deposition in an in vitro assay in 1% human serum with an IC ₅₀ value of 10 nM or less, the antibody inhibits C3b deposition in 90% human serum with an IC ₅₀ value of 30 nM or less less, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab) ₂ and F(ab') ₂ , wherein the antibody is a single chain molecule, wherein said antibody is an IgG2 molecule, wherein the antibody is an IgG1 molecule, wherein the antibody is an IgG4 molecule, wherein the IgG4 molecule has the S228P mutation, and/or wherein the antibody does not significantly inhibit the classical pathway. In one embodiment, the antibody binds MASP-2, selectively inhibits the lectin pathway, and does not significantly inhibit the alternative pathway. In one embodiment, the antibody binds MASP-2, selectively inhibits the lectin pathway, and does not significantly inhibit the classical or alternative pathway (ie, inhibits the lectin pathway without affecting the classical and alternative complement pathways).

In one embodiment, the MASP-2 inhibitory antibody inhibits plasma-induced MVEC apoptosis in the serum of a subject suffering from TMA, such as aHUS (acute phase or

- 137 046178 in remission), hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP), TMA due to cancer; TMA due to chemotherapy; TMA due to transplantation (eg, organ transplantation, such as kidney transplantation, or allogeneic hematopoietic stem cell transplantation), or in the serum of a subject suffering from USS syndrome, or in the serum of a subject suffering from Degos disease, or in a subject suffering from from catastrophic antiphospholipid syndrome (CAPS), wherein plasma-induced MVEC apoptosis is inhibited by at least 5%, e.g., at least 10%, e.g., at least 20%, e.g., at least 30%, e.g. 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% eg at least 95%, up to 99%, compared to untreated whey. In some embodiments, the MASP-2 inhibitory antibody inhibits thrombus formation in the serum of a subject suffering from TMA (e.g., such as aHUS (acute or remission), hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP), TMA due to cancer; TMA due to chemotherapy; TMA due to transplantation (eg, organ transplantation, such as kidney transplantation, or allogeneic hematopoietic stem cell transplantation), or in the serum of a subject suffering from Upshaw-Shulman syndrome (USS), or in the serum of a subject suffering from from Degos disease, or in a subject suffering from catastrophic antiphospholipid syndrome (CAPS)), at a level of at least 20 percent or more (for example, at least 30%, at least 40%, at least 50%) exceeding the inhibitory effect on C5b-9 deposition in serum.

In one embodiment, the MASP-2 inhibitory antibody is administered to a subject by delivery through an intravenous line or other catheter.

In one embodiment, the invention provides a method for inhibiting the formation of blood clots in a subject suffering from TMA (such as aHUS (acute or remission), hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP), TMA due to cancer; TMA due to chemotherapy; TMA due to transplantation (for example, organ transplantation, such as kidney transplantation, or allogeneic hematopoietic stem cell transplantation), or in the serum of a subject suffering from Upshaw-Shulman syndrome (USS), or in the serum of a subject suffering from Degos disease, or in a subject suffering from catastrophic antiphospholipid syndrome (CAPS)), comprising administering to the subject a composition containing a certain amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprising (i) (a) a heavy chain variable region comprising: i) a CDR -H1 heavy chain containing the amino acid sequence of residues 31-35 in SEQ ID NO: 67; and ii) a heavy chain CDR-H2 containing the amino acid sequence of residues 50-65 of SEQ ID NO: 67; and iii) a heavy chain CDR-H3 containing the amino acid sequence of residues 95-102 in SEQ ID NO: 67, and b) a light chain variable region comprising: i) a light chain CDR-L1 containing the amino acid sequence of residues 24-34 in SEQ ID NO: 70; and ii) light chain CDR-L2 containing the amino acid sequence of residues 50-56 in SEQ ID NO: 70; and iii) a light chain CDR-L3 comprising the amino acid sequence of residues 89-97 of SEQ ID NO: 70, or (II) a variant thereof comprising a heavy chain variable region having at least 90% sequence identity with SEQ ID NO: 67 (e.g. at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , at least 99% sequence identity to SEQ ID NO: 67), and a light chain variable region having at least 90% sequence 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% sequence identity) with SEQ ID NO: 70.

In one embodiment, the subject suffers from TMA selected from the group consisting of TMA due to cancer; TMA due to chemotherapy; TMA due to transplantation (eg, organ transplantation, such as kidney transplantation, or allogeneic hematopoietic stem cell transplantation), Upshaw-Shulman syndrome (USS), Degos disease, and catastrophic antiphospholipid syndrome (CAPS).

In some embodiments, the method includes administering to a subject a composition comprising a specified amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 67. In some embodiments, the method includes administering to a subject a composition comprising a specified amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprising a light chain variable region containing the amino acid sequence of SEQ ID NO: 70.

In some embodiments, the method comprises administering to a subject a composition comprising a MASP-2 inhibitory antibody, or antigen binding fragment thereof, that specifically

- 138 046178 recognizes at least a portion of the epitope on human MASP-2 recognized by the reference antibody OMS646 containing the heavy chain variable region of SEQ ID NO: 67 and the light chain variable region of SeQ ID NO: 70.

Example 44.

This case study describes initial results from an ongoing phase 2 clinical trial evaluating the safety and clinical efficacy of a fully human monoclonal MASP-2 inhibitory antibody in adult patients with thrombotic microangiopathies (TMAs).

Prerequisites.

TMAs are a family of rare, debilitating, and life-threatening diseases characterized by the presence of excessive thrombi (clots) - aggregates of platelets - in the microvascular system of organs of the body, primarily in the kidneys and brain.

Methods.

The first stage of an open-label, phase 2 clinical trial was conducted in subjects suffering from primary atypical hemolytic uremic syndrome (aHUS), plasma therapy-resistant aHUS, and thrombotic thrombocytopenic purpura (TTP). The phase 2 clinical trial did not use a placebo arm given the life-threatening nature of the disease.

Subjects were aged >18 years at screening and were included in this study only if they had a diagnosis of one of the following TMA:

1) primary aHUS, diagnosed clinically, with ADAMTS-13 activity >10% in plasma. Patients may be included in the study with or without a documented mutation of complement components or anti-CFH antibodies. Patients are categorized based on their response to plasma therapy (plasmapheresis or plasma infusion):

Patients with plasma therapy-resistant aHUS met all of the following criteria for the purposes of this study:

(a) at screening, platelet count <150,000/μl, despite at least four sessions of plasma therapy before screening;

(b) signs of microangiopathic hemolysis (presence of schizocytes, serum lactate dehydrogenase (LDH) level > upper limit of normal (ULN), haptoglobin < LNL); and (c) serum creatinine level > VPN.

Patients with chronic plasma therapy-responsive (plasma therapy-sensitive) aHUS should receive at least once weekly plasma therapy for four weeks prior to receiving the first dose of OMS646, with a serum creatinine level > TPN.

2) TTR, determined by the presence of one of the following characteristics:

platelet count <150,000/µl;

signs of microangiopathic hemolysis (presence of schizocytes, serum LDH level > VPN, or haptoglobin < NPN); and ADAMTS-13 activity <10% during a current episode of TTP or historically. Note: Subjects were excluded from the study if they received eculizumab therapy within three months before screening. Criteria for patients qualifying as plasma therapy refractory were used to determine eligibility for this study. In further aspects of the invention, a patient refractory to plasma therapy who was to be treated with a MASP-2 inhibitor had previously received plasma therapy at least once, and after such plasma therapy session still had one or more clinical signs of aHUS that were not adequately reduced or eliminated as a result of such plasma therapy.

The monoclonal antibody used in this study, OMS646, is a fully human IgG4 mAb directed against human MASP-2. As demonstrated in Example 40, OMS646 avidly binds recombinant MASP-2 (apparent equilibrium dissociation constant in the range of 100 pM) and exhibits greater than 5000-fold selectivity for homologous C1s, C1r, and MASP-1 proteins. In functional assays, OMS646 inhibits the lectin complement pathway in humans with activity in the nanomolar range (concentration resulting in 50% inhibition [IC ₅₀ |. is approximately 3 nM), but has no significant effect on the classical complement pathway. Administration of OMS646 by either intravenous (IV) or subcutaneous (SC) injection to mice, primates, and humans resulted in high plasma concentrations that were associated with inhibition of lectin pathway activation in ex vivo assays. As further described in Example 42, administration of OMS646 resulted in a reduction in C5b-9 deposition and thrombus formation in in vitro models of TMA, and thrombus formation in a mouse model of TMA, thereby demonstrating that OMS646 can be used in therapy.

In this study, OMS646 drug substance was provided at a concentration of 100 mg/ml and was further diluted for IV administration. The appropriate calculated volume of 100 mg/mL OMS646 injection solution was withdrawn from the vial using a dose preparation syringe. The fluid from the infusion bag was administered within four hours after preparation.

- 139 046178 nia.

In the phase 1 study, OMS646 was administered in a dose escalation regimen to groups of three subjects each. Each subject in stage 1 received four weekly doses of OMS646, as described below in table. 26.

Table 26

Dosing schedule for stage 1

Stage 1 Study Design Flowchart

Treatment period

Screening (Day-28) 1 Dose No. 1 (Day 1) 1 Dose No. 2 (Day 8) 1 Dose No. 3 (Day 15) 1 Dose No. 4 (Day 22) Follow-up (4 visits) (Days 29-78)

The diluted study drug was administered by intravenous infusion over a 30-minute period.

Main evaluation criteria.

The combined main evaluation criteria were:

safety based on the results of AE determination, vital signs, ECG and clinical laboratory tests;

clinical effectiveness based on changes in platelet count Secondary endpoints.

Secondary evaluation criteria were:

clinical activity of TMA;

serum LDH level;

serum haptoglobin level;

hemoglobin level;

serum creatinine level;

TMA-related symptoms;

the need for plasma therapy (plasmapheresis or plasma infusion);

need for dialysis.

Acceptable concomitant treatments.

Plasmatherapy-resistant aHUS - Plasmatherapy during OMS646 treatment was acceptable if considered medically necessary by the investigator.

Chronic Plasma Therapy Responder aHUS - Investigators were advised that plasma therapy should be continued until signs of TMA relief, such as increased platelet counts, decreased LDH levels, increased haptoglobin levels, increased hemoglobin levels, decreased creatinine levels, at which point the investigators were advised to withhold plasma therapy and Monitor TMA parameters to determine whether plasma therapy can be discontinued.

TTP plasma therapy was acceptable if the investigator considered it medically necessary.

The investigators were advised that kidney dialysis should be performed according to the standard of care.

Eculizumab was not administered during the study.

Results.

The first group of subjects consisted of three patients with aHUS receiving the lowest dose of OMS646. Improvement was observed for all markers of TMA disease in all patients in this study group. Platelet counts, serum lactate dehydrogenase (LDH) levels, and serum haptoglobin levels were measured as markers of disease activity. When compared with baseline levels, platelet counts improved in all patients. Serum LDH levels remained normal in one patient, decreased to near the normal range in another, and remained elevated in a third. Haptoglobin levels improved in two patients and returned to normal in one. Creatinine levels in one patient with dialysis-independent renal function also improved.

As planned, in this clinical trial, three patients were treated in the second intermediate dose group. Two patients had plasma therapy-resistant aHUS, and one patient had thrombotic thrombocytopenic purpura (TTP). Both patients with aHUS received renal

- 140 046178 dialysis before, and during, inclusion in the study. In the second group, or medium dose group, two patients with plasma therapy-resistant aHUS observed:

a 47% increase in the mean platelet count, bringing both patients' counts into the normal range;

an 86% decrease in the average number of schizocytes, with schizocytes disappearing in one patient;

a 71% increase in mean haptoglobin levels, with both patients reaching the normal range during treatment and one falling slightly below normal one week after the last dose;

a 5% decrease in mean LDH levels, with both patients having levels slightly above the normal range.

The patient with TTP in the intermediate dose group required intermittent plasma infusions before enrollment into the study. Laboratory parameters did not show sustained improvement, but the patient has not required plasma therapy either during OMS646 treatment or after completion of treatment to date.

The drug was well tolerated by all patients throughout the entire treatment period. Based on the positive results in the second, or medium dose, group, treatment in the third, or high dose, group was initiated, and the patient with aHUS had now completed the treatment period in the study. Data shown for all patients include values one week after the last dose.

The first patient in the third (high-dose) arm—a patient with plasma therapy-resistant aHUS with additional complications including hepatitis C, cryoglobulinemia, and lymphoma—also completed treatment with OMS646. Prior to treatment with OMS646, the patient required regular dialysis. During treatment and after completion of OMS646, to date, the patient has not required dialysis. Hematological and renal function parameters showed:

63% improvement in platelet count, returning to normal levels;

reduction by 100% in the number of schizocytes;

haptoglobin levels increased from undetectable levels to normal;

a 43% decrease in LDH levels, resulting in levels only slightly above normal;

24% decrease in creatinine levels.

Just as in the first and second groups, the drug was well tolerated.

The primary outcome measure in this open-label phase 2 clinical trial is change in platelet count. Platelet counts in all three patients with aHUS in the medium- and high-dose groups (two in the medium-dose group and one in the high-dose group) returned to normal, with a statistically significant mean increase from baseline of approximately 68,000 platelets/mL (p =0.0055).

In summary, data obtained to date in this phase 2 clinical trial demonstrate the efficacy of OMS646 in patients with primary aHUS, plasma therapy-resistant aHUS, and patients with TTP.

In accordance with the foregoing, in one embodiment, the invention provides a method of treating a subject suffering from plasma therapy-resistant aHUS, 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 method further includes the step of identifying a subject suffering from plasma therapy-resistant aHUS prior to administering to the subject a composition comprising a MASP-2 inhibitory antibody.

In accordance with any of the embodiments disclosed herein, the MASP-2 inhibitory antibody has at least one or more of the following characteristics: the antibody binds human MASP-2 with a K _D value of 10 nM or less, the antibody binds an epitope in CCP1 domain of MASP-2, the antibody inhibits C3b deposition in an in vitro assay in 1% human serum with an IC ₅₀ value of 10 nM or less, the antibody inhibits C3b deposition in 90% human serum with an IC ₅₀ value of 30 nM or less less, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab) ₂ and F(ab') ₂ , wherein the antibody is a single chain molecule, wherein said antibody is an IgG2 molecule, wherein the antibody is an IgG1 molecule, wherein the antibody is an IgG4 molecule, wherein the IgG4 molecule has the S228P mutation. In one embodiment, the antibody binds MASP2, selectively inhibits the lectin pathway, and does not significantly inhibit the classical pathway (ie, inhibits the lectin pathway without affecting the classical complement pathway).

In one embodiment, the MASP-2 inhibitory antibody is administered in an amount effective to improve at least one or more clinical parameters associated with aHUS, e.g., increasing platelet count, decreasing LDH levels, increasing haptoglobin levels, increasing hemoglobin levels, and/or decreasing creatinine level.

- 141 046178

In some embodiments, the method comprises administering a MASP-2 inhibitory antibody to a subject suffering from plasma therapy-resistant aHUS via a catheter (e.g., intravenously) for a first period of time (e.g., at least one day or a week, or two weeks), with subsequent administration of a MASP-2 inhibitory antibody to the subject subcutaneously for a second period of time (eg, a chronic phase of at least two weeks or longer). In some embodiments, administration in the first and/or second time period is performed without plasma therapy. In some embodiments, administration in the first and/or second time period is performed in combination with plasma therapy.

In some embodiments, the method includes administering a MASP-2 inhibitory antibody to a subject suffering from plasma therapy-resistant aHUS intravenously, intramuscularly, or, preferably, subcutaneously. Treatment can be chronic and applied daily or monthly, but preferably every two weeks. The MASP-2 inhibitory antibody can be administered alone or in combination with a C5 inhibitor such as eculizumab.

In one embodiment, the method includes treating a subject suffering from plasma therapy-resistant aHUS, comprising administering to the subject a composition comprising a predetermined amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprising (i) (a) a heavy chain variable region comprising: i ) heavy chain CDR-H1 containing the amino acid sequence of residues 31-35 in SEQ ID NO: 67; and ii) a heavy chain CDR-H2 containing the amino acid sequence of residues 50-65 of SEQ ID NO: 67; and i) a heavy chain CDR-H3 comprising the amino acid sequence of residues 95-107 in SEQ ID NO: 67, and b) a light chain variable region comprising: i) a light chain CDR-L1 comprising the amino acid sequence of residues 24-34 in SEQ ID NO: 70; and ii) light chain CDR-L2 containing the amino acid sequence of residues 50-56 in SEQ ID NO: 70; and iii) a light chain CDR-L3 comprising the amino acid sequence of residues 89-97 of SEQ ID NO: 70, or (II) a variant thereof comprising a heavy chain variable region having at least 90% sequence identity with SEQ ID NO: 67 (e.g. at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , at least 99% sequence identity to SEQ ID NO: 67), and a light chain variable region having at least 90% sequence 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% sequence identity) with SEQ ID NO: 70.

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

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

Example 45.

This Example describes additional results obtained from an ongoing phase 2 clinical trial to evaluate the safety and clinical efficacy of a fully human monoclonal MASP-2 inhibitory antibody in adult patients with thrombotic microangiopathies (TMAs) described in Example 44.

Prerequisites.

As described in Example 44, TMAs are a family of rare, debilitating and life-threatening diseases characterized by the presence of excessive thrombi (clots) of platelet aggregates in the microvascular system of organs of the body, primarily in the kidneys and brain. As described herein, graft-associated TMA (TA-TMA) is a devastating syndrome that can develop in transplant patients, such as hematopoietic stem cell transplant (HSCT) recipients. This example describes the initial results of a phase 2 clinical trial to evaluate the safety and clinical efficacy of a fully human monoclonal MASP-2 inhibitory antibody in patients suffering from hematopoietic stem cell transplant-associated TMA.

Methods.

As described in Example 44, the Phase 2 trial for the TMA study consists of three dose levels followed by a fixed dose of the MASP-2 inhibitory antibody OMS646. As also described in Example 44, positive results were obtained for patients with aHUS and one patient with TTP, with consistent and reliable improvement in performance

- 142 046178 st. As in the low-dose group, OMS646 was well tolerated by all patients in the medium- and high-dose groups throughout the treatment period. Preclinical toxicity studies were conducted and demonstrated no safety concerns, allowing for periodic dosing in clinical studies.

As described in Example 44, data on OMS646 were obtained in a phase 2 clinical trial studying TMA in patients with aHUS and TTP. To date, dosing has been completed for an additional patient with hematopoietic stem cell transplant-associated TMA in the high dose group using the methods described in Example 44. This patient had a history of lymphoma for which he received a hematopoietic stem cell transplant . His post-transplant condition was complicated by a number of life-threatening illnesses, including TMA, which resulted in him requiring platelet transfusions. Despite transfusions, he continued to have TMA associated with the stem cell transplant and was enrolled in a phase 2 study with OMS646.

Results.

After a four-week dosing period (high dose group) as described in Example 44, a patient with stem cell transplant-associated TMA experienced:

a fourfold increase in platelet count, resulting in a platelet count exceeding 100,000;

haptoglobin level more than doubled and became normal;

Plasma levels of lactate dehydrogenase, an indicator of blood vessel damage, decreased by 35% but were still higher than normal;

the number of schizocytes remained at the level of one.

Throughout the OMS646 dosing period and after completion of OMS646 treatment, the patient did not require platelet transfusions or plasmapheresis.

In summary, the data obtained to date in this phase 2 clinical trial (as described in Example 44 and in this Example) demonstrate the effectiveness of OMS646 in patients with primary aHUS, plasma therapy-resistant aHUS, TTP, as well as in a patient with TMA. associated with a hematopoietic stem cell transplant.

In accordance with the foregoing, in one embodiment, the invention provides a method of treating a human subject suffering from hematopoietic stem cell transplant-associated TMA, comprising administering to the subject a composition comprising a MASP-2 inhibitory antibody in an amount effective to inhibit MASP-2- dependent activation of complement. In one embodiment, the subject suffers from hematopoietic stem cell transplant-associated TMA that is resistant to treatment with platelet transfusion and/or plasmapheresis. In one embodiment, the method further includes identifying a human subject suffering from hematopoietic stem cell transplant-associated TMA that is resistant to treatment with platelet transfusion and/or plasmapheresis, prior to the step of administering to the subject a composition comprising a MASP-2 inhibitory antibody in an amount effective to inhibition of MASP-2-dependent complement activation.

In accordance with any of the embodiments disclosed herein, the MASP-2 inhibitory antibody has at least one or more of the following characteristics: the antibody binds human MASP-2 with a KD value of 10 nM or less, the antibody binds an epitope in the domain CCP1 MASP-2, said antibody inhibits C3b deposition in an in vitro assay in 1% human serum with an IC ₅₀ value of 10 nM or less, said antibody inhibits C3b deposition in 90% human serum with an IC ₅₀ value of 30 nM or less wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab) ₂ and F(ab') ₂ , wherein the antibody is a single chain molecule, wherein said antibody is a molecule IgG2, wherein the antibody is an IgG1 molecule, wherein the antibody is an IgG4 molecule, wherein the IgG4 molecule has the S228P mutation. In one embodiment, the antibody binds MASP2, selectively inhibits the lectin pathway, and does not significantly inhibit the classical pathway (ie, inhibits the lectin pathway without affecting the classical complement pathway).

In one embodiment, the MASP-2 inhibitory antibody is administered in an amount effective to improve at least one or more clinical parameters associated with hematopoietic stem cell transplant-associated TMA, e.g., an increase in platelet count (e.g., an increase of at least two , at least three, at least four times the platelet count before treatment), an increase in haptoglobin levels and/or a decrease in lactate dehydrogenase levels.

In some embodiments, the method comprises administering a MASP-2 inhibitory antibody to a subject suffering from hematopoietic stem cell transplant-associated TMA via a catheter (e.g., intravenously) for a first period of time (e.g., at least one day or week, or two weeks), followed by administration of a MASP-2 inhibitory antibody

- 143 046178 to the subject subcutaneously for a second period of time (eg, a chronic phase of at least two weeks or longer). In some embodiments, administration in the first and/or second time period is performed without plasma therapy. In some embodiments, administration in the first and/or second time period is performed in combination with plasma therapy.

In some embodiments, the method includes administering a MASP-2 inhibitory antibody to a subject suffering from hematopoietic stem cell transplant-associated TMA intravenously, intramuscularly, or, preferably, subcutaneously. Treatment can be chronic and applied daily or monthly, but preferably every two weeks. The MASP-2 inhibitory antibody can be administered alone or in combination with a C5 inhibitor such as eculizumab.

In one embodiment, the method includes treating a subject suffering from TMA associated with a hematopoietic stem cell transplant, comprising administering to the subject a composition containing an amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprising (I)(a) a heavy chain variable region comprising: i) a heavy chain CDR-H1 containing the amino acid sequence of residues 31-35 in SEQ ID NO: 67; and ii) a heavy chain CDR-H2 containing the amino acid sequence of residues 50-65 of SEQ ID NO: 67; and iii) a heavy chain CDR-H3 comprising the amino acid sequence of residues 95-107 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 residues 24-34 of SEQ ID NO: 70; and ii) light chain CDR-L2 containing the amino acid sequence of residues 50-56 in SEQ ID NO: 70; and iii) a light chain CDR-L3 comprising the amino acid sequence of residues 89-97 of SEQ ID NO: 70, or (II) a variant thereof comprising a heavy chain variable region having at least 90% sequence identity with SEQ ID NO: 67 (e.g. at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , at least 99% sequence identity to SEQ ID NO: 67), and a light chain variable region having at least 90% sequence 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% sequence identity) with SEQ ID NO: 70.

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

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

Example 46.

This Example describes additional results obtained from an ongoing phase 2 clinical trial to evaluate the safety and clinical efficacy of a fully human monoclonal MASP-2 inhibitory antibody in adult patients with thrombotic microangiopathies (TMAs) described in Examples 44 and 45.

Prerequisites.

As described in Example 45, graft-associated TMA (TA-TMA) is a devastating syndrome that can develop in transplant patients, such as hematopoietic stem cell transplant (HSCT) recipients. Hematopoietic stem cell transplant-associated TMA (HSCT-TMA) is a life-threatening complication that is initiated by endothelial damage. The kidney is the organ most commonly affected, although HSCT-TMA can be a multisystem disease also affecting the lungs, intestines, heart, and brain. The presence of even mild TMA is associated with long-term renal failure. The development of allogeneic HSCT-associated TMA varies in incidence depending on different diagnostic criteria and conditioning, as well as graft-versus-host disease prophylaxis regimens, in which calciumneurin inhibitors are the most commonly used drugs (But VT et al., Biol Blood Marrow Transplant , 11(8):5715, 2005). Changing the regimen of immunosuppressive calciumneurin inhibitors may result in attenuation of TMA in some patients within a few weeks of reducing or stopping the calciumneurin inhibitors.

TMA is a potentially life-threatening complication of HSCT and is currently managed primarily by reducing stimulants, including avoidance of immunosuppressants (eg, calcium neuron inhibitors) and treatment of current infections, as well as supportive measures such as hemodialysis . Although many patients with HSCT-TMA do well

- 144 046178 respond to reduction or cessation of immunosuppressive medications, there is a subgroup of patients who have persistent HSCT-TMA despite conservative treatment measures (ie, TMA does not respond to reduction or cessation of immunosuppressive medications). Patients who do not respond to these conservative treatments have a poor prognosis. Plasma exchange is ineffective and no other treatments are approved.

Example 45 describes initial positive results from a phase 2 clinical trial to evaluate the safety and clinical efficacy of a fully human monoclonal MASP-2 inhibitory antibody (OMS646) in patients suffering from persistent hematopoietic stem cell transplant-associated TMA (HSCT-TMA). This case study describes additional results from an ongoing phase 2 clinical trial to evaluate the safety and clinical efficacy of OMS646 in three additional subjects suffering from persistent HSCT-TMA refractory to conservative treatments.

Methods.

As described in Example 44, the Phase 2 trial for the TMA study consists of three dose levels followed by a fixed dose of the MASP-2 inhibitory antibody OMS646. OMS646 was well tolerated by all patients in the low, medium and high dose groups throughout the treatment period. Preclinical toxicity studies have been completed and show no safety concerns, allowing periodic dosing in clinical trials.

The Phase 2 clinical trial to study TMA is enrolling subjects suffering from persistent HSCT-related TMA refractory to conservative treatment, defined for the purposes of this study as characterized by all of the following at least two weeks after tapering or stopping administration immunosuppressive medication (eg, calciumneurin inhibitors), or at least 30 days after transplantation:

thrombocytopenia (platelet count <150,000/µl); and evidence of microangiopathic hemolytic anemia (presence of schizocytes, serum lactate dehydrogenase (LDH) level > upper limit of normal (ULN), or haptoglobin level < lower limit of normal (LNL).

Acceptable concomitant treatments for HSCT-associated TMA.

Plasma therapy in the treatment of OMS646 is acceptable if deemed medically necessary by the investigator. Patients receiving plasma therapy may receive additional half doses of OMS646.

The investigators were advised that kidney dialysis should be performed according to the standard of care.

Eculizumab was not administered during the study.

The current TMA study is enrolling subjects suffering from persistent HSCT-TMA that is refractory to standard treatments (ie, persistent TMA at least two weeks after tapering or stopping calciumneurin inhibitors).

As described in Example 45, dosing was completed for a patient suffering from persistent HSCT-TMA (patient #1) in the high dose group (4 mg/kg OMS646, IV once weekly for four weeks) using Methods , described in example 44.

To date, dosing has been completed for an additional 4 patients suffering from persistent HSCT-TMA, as described below.

Patient #1 (described in Example 44) was treated with OMS646 for four weeks (4 mg/kg IV once weekly);

patients #2 and #3 were treated with OMS646 for eight weeks (4 mg/kg IV once weekly);

patients 4 and 5 were treated with OMS646 (4 mg/kg IV once weekly) for two and three weeks, respectively. Patients 4 and 5 discontinued the study after two and three weeks, respectively, did not respond to treatment and their condition worsened. It should be noted that one of these patients had a significantly elevated creatinine level at study entry.

Results.

Five patients with persistent HSCT-TMA were included in this study. All subjects were adults and received HSCT for hematologic malignancies. In fig. 58, 59 and 60 show the change from baseline in TMA scores following treatment with the MASP-2 inhibitory antibody OMS646. In these figs. data are presented for all three patients, two of whom discontinued participation in the study after 2-3 weeks, one subsequently relapsed, and the other received palliative treatment. As shown in FIG. 58, 59 and 60, the last possible treatment was given at week 7.

In fig. 58 is a graph showing the mean change in platelet count from baseline over time (weeks) in subjects suffering from persistent hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA) following treatment with a MASP-2 inhibitory antibody (OMS646) .

- 145 046178

In fig. 59 is a graph showing the average change in LDH levels from baseline over time (weeks) in subjects suffering from persistent HSCT-TMA following treatment with a MASP-2 inhibitory antibody (OMS646).

in fig. 60 is a graph showing the mean change from baseline in haptoglobin levels over time (weeks) in subjects suffering from persistent HSCT-TMA following treatment with a MASP-2 inhibitory antibody (OMS646).

As shown in FIG. 59 and 60, statistically significant improvements in LDH and haptoglobin were observed during treatment. As shown in FIG. 58, the platelet count increased, but this increase did not reach the level of statistical significance for such a small number of patients. Of the three patients who completed treatment, one did not experience improvement in creatinine but was concurrently receiving nephrotoxic agents. In the remaining two patients, creatinine values improved or remained normal. During the extended follow-up period, one patient experienced graft failure and is awaiting a second graft. The other two patients remain stable.

Despite potentially confounding drug effects associated with bone marrow suppression and nephrotoxicity in two subjects, beneficial effects of OMS646 treatment were observed in three subjects suffering from persistent HSCT-TMA (one of which is described in Example 45) and are further described below. Notably, treatment effects in each of the three responding patients were first seen after approximately three weeks of treatment with OMS646.

Patient #1 (received treatment for 4 weeks as described in Example 45). Briefly, the platelet count increased fourfold, resulting in a platelet count of more than 100,000/μL; the haptoglobin level more than doubled and was normal; plasma lactate dehydrogenase levels decreased by 35%, but still remained above normal; and the schistocyte count was maintained at one.

Patient #2 (treated for 8 weeks): platelet count did not change during treatment, although the patient also suffered from bone marrow suppression due to concurrent treatment with valganciclovir and subsequently experienced graft failure; plasma lactate dehydrogenase levels decreased from 712 U/L to only 256 U/L; haptoglobin levels increased from undetectable levels up to 250 mg/dL.

Patient #3 (treated for 8 weeks): platelet count increased from 13,500/μL to 133,000/μL; Plasma lactate dehydrogenase levels decreased from 537 to 225 U/L, and haptoglobin levels increased from undetectable levels to 181 mg/dL.

Summary of results.

For three patients suffering from persistent HSCT-associated TMA who completed dosing with OMS646, the data show strong and sustained efficacy. Statistically significant improvements in LDH and haptoglobin were observed during treatment. The platelet count increased, but the increase did not reach the level of statistical significance for such a small number of patients. Of the three patients who completed treatment, creatinine did not improve in one patient, but he was also taking nephrotoxic drugs. In the remaining two patients, creatinine values improved or remained normal.

Conclusions.

OMS646 resulted in improvement of TMA markers in patients suffering from persistent HSCT-TMA who did not respond to conservative treatments. HSCT-TMA patients treated with OMS646 are among the most difficult to treat patients, and these results provide clinical evidence of the therapeutic effect of OMS646 in patients with dangerously persistent HSCT-TMA that persists despite conservative treatment options.

In accordance with the foregoing, in one embodiment, the invention provides a method of treating a human subject suffering from hematopoietic stem cell transplant-associated TMA, comprising administering to the subject a composition comprising a MASP-2 inhibitory antibody in an amount effective to inhibit MASP-2- dependent activation of complement. In one embodiment, the subject suffers from persistent hematopoietic stem cell transplant-associated TMA that is refractory to conservative treatments. In one embodiment, the method further includes identifying a human subject suffering from persistent hematopoietic stem cell transplant-associated TMA that is resistant to conservative treatments, prior to the step of administering to the subject a composition comprising a MASP-2 inhibitory antibody in an amount effective to inhibit MASP -2-dependent complement activation.

In accordance with any of the embodiments disclosed herein, the MASP-2 inhibitory antibody has at least one or more of the following characteristics: the antibody binds human MASP-2 with a KD value of 10 nM or less, the antibody binds an epitope in the domain CCP1 MASP-2, this antibody inhibits the deposition of C3b in

- 146 046178 in vitro assay in 1% human serum with an IC ₅₀ value of 10 nM or less, the antibody inhibits C3b deposition in 90% human serum with an IC ₅₀ value of 30 nM or less, wherein the antibody is an antibody fragment , selected from the group consisting of Fv, Fab, Fab', F(ab)2 and F(ab')2, wherein the antibody is a single chain molecule, wherein said antibody is an IgG2 molecule, wherein said antibody is an IgG1 molecule, wherein said antibody is an IgG4 molecule, wherein the IgG4 molecule has the S228P mutation. In one embodiment, the antibody binds MASP2, selectively inhibits the lectin pathway, and does not significantly inhibit the classical pathway (ie, inhibits the lectin pathway without affecting the classical complement pathway).

In one embodiment, the MASP-2 inhibitory antibody is administered in an amount effective to improve at least one or more clinical parameters associated with hematopoietic stem cell transplant-associated TMA, e.g., an increase in platelet count (e.g., an increase of at least two , at least three, at least four times the platelet count before treatment), an increase in haptoglobin levels and/or a decrease in lactate dehydrogenase levels.

In some embodiments, the method comprises administering a MASP-2 inhibitory antibody to a subject suffering from hematopoietic stem cell transplant-associated TMA via a catheter (e.g., intravenously) for a first period of time (e.g., at least one day or week, or two weeks, or three weeks, or four weeks or more), followed by administration of a MASP-2 inhibitory antibody to the subject subcutaneously for a second period of time (eg, a chronic phase of at least two weeks or longer). In some embodiments, administration in the first and/or second time period is performed without plasma therapy. In some embodiments, administration in the first and/or second time period is performed in combination with plasma therapy.

In some embodiments, the method includes administering a MASP-2 inhibitory antibody to a subject suffering from hematopoietic stem cell transplant-associated TMA intravenously, intramuscularly, or subcutaneously.

The treatment may be chronic and applied daily or monthly, but preferably at least every two weeks, or at least once a week, for example twice a week or three times a week. The MASP-2 inhibitory antibody can be administered alone or in combination with a C5 inhibitor such as eculizumab.

In one embodiment, the method includes treating a subject suffering from persistent TMA associated with a hematopoietic stem cell transplant, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprising a heavy chain variable region containing CDR-H1 , CDR-H2 and CDR-H3, having the amino acid sequence of SEQ ID NO: 67, and a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3, having the amino acid sequence of SEQ ID NO: 70. In some embodiments, the composition contains a MASP-2 inhibitory antibody comprising (a) a heavy chain variable region comprising: i) a heavy chain CDR-H1 containing the amino acid sequence of residues 31-35 in SEQ ID NO: 67; and ii) a heavy chain CDR-H2 containing the amino acid sequence of residues 50-65 of SEQ ID NO: 67; and iii) a heavy chain CDR-H3 comprising the amino acid sequence of residues 95-107 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 residues 24-34 of SEQ ID NO: 70; and ii) light chain CDR-L2 containing the amino acid sequence of residues 50-56 in SEQ ID NO: 70; and iii) a light chain CDR-L3 comprising the amino acid sequence of residues 89-97 of SEQ ID NO: 70, or (II) a variant thereof comprising a heavy chain variable region having at least 90% sequence 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% sequence identity to SEQ ID NO: 67), and a light chain variable region having at least 90% sequence 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% sequence identity) with SEQ ID NO: 70.

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

In some embodiments, the method includes administering to a subject a composition comprising a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by a reference antibody

- 147 046178

OMS646, comprising a heavy chain variable region of SEQ ID NO: 67 and a light chain variable region of SEQ ID NO: 70.

In some embodiments, the method comprises administering to a subject suffering from, or at risk of developing, HSCT-associated TMA, including a subject suffering from persistent hematopoietic stem cell transplant-associated TMA that is resistant to conservative treatments, a composition comprising an inhibitory MASP -2 antibody, or an antigen-binding fragment thereof, containing a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 67, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 70, at a dose of 1 to 10 mg/kg (then eat 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg) at least once per week (eg, at least twice per week or at least three times per week) for a period of at least 3 weeks, or at least 4 weeks, or at least 5 weeks, or at least 6 weeks, or at least 7 weeks, or at least 8 weeks.

Example 47.

This example describes a clinical case demonstrating the effectiveness of the MASP-2 inhibitory antibody OMS646 in treating graft-versus-host disease (GVHD) microangiopathy.

Prerequisites.

Graft-versus-host disease is a common complication of hematopoietic stem cell transplantation (HSCT). There are both acute and chronic forms that arise as a result of the fact that donor immune cells perceive the tissues of the recipient patient as foreign. This triggers an immune response against the recipient patient. Acute GvHD occurs in approximately 50% or more of patients who receive allogeneic transplants. In acute GvHD, the most common targets are the skin, gastrointestinal tract, and liver, but it can also involve the kidneys, eyes, lungs, and blood cells. Chronic GvHD occurs in approximately 40% of patients who receive allogeneic transplants and most commonly affects the skin, liver, eyes, gastrointestinal tract, and lungs. Both acute and chronic, GvHD is associated with high morbidity and mortality.

Methods and results.

A 46-year-old man suffering from T-cell acute lymphoblastic leukemia (T-ALL) in second complete remission (CR) underwent total myeloablative conditioning with cyclophosphamide with total body irradiation (TBI-Cy) and underwent allogeneic HSCT peripheral blood stem cell transplantation (PBSC) from an unrelated donor - a 35-year-old woman, 9/10 HLA-matched (antigen mismatch at locus A). Prevention of graft-versus-host disease (GVHD) has been based on the use of antithymocyte globulin (ATG, 5 mg/kg) during conditioning, cyclosporine A (CSA), and a short course of methotrexate (i.e., methotrexate on days +1, +3, +6 (dose reduction due to low CD34+ content)). The patient achieved reliable hematologic recovery (graft engraftment: BCC >1000/mL, neutrophils >500/mL on day +15; platelets >20,000/mL on day +18, 50,000/mL on day +21). Complete donor chimerism (bone marrow and peripheral blood, CD3+ and CD3- fraction) was observed on days +30, +60, +90, +180, +354. Complete remission was confirmed (immunophenotype, molecular MRD) on days +30, +60, +90, +180.

On day +35 after transplantation, the patient developed gastrointestinal (GI) GVHD (with manifestations such as vomiting, anorexia, diarrhea, abdominal pain; colonoscopy: diffuse erosions; histology: diffuse ulcerations of the mucous membrane, lymphogranulocytic inflammation in the lamina propria mucosa, glandular and crypt deformation, apoptotic bodies) and was diagnosed with acute GvHD, stage 4 (bowel), with an overall score of 4, along with cytomegalovirus (CMV) colitis (defined by positive immunohistochemistry and PCR in real-time intestinal biopsy specimen systemic CMV reactivation (2614 IU/mL in peripheral blood) The two conditions were treated simultaneously with methylprednisolone (1 mg/kg), foscarnet and ganciclovir, with good results, and steroids in tapering doses until discontinuation.

On day +121 post-transplant, the patient experienced a relapse of steroid-refractory GVHD GI (with manifestations of vomiting, anorexia, diarrhea, abdominal pain; histological abnormalities (in the stomach and duodenum), and was diagnosed with late onset acute GI GvHD stage 4 (bowel), with an overall score of 4, was treated with good outcome with chronic CSA and sequential pentostatin and mesenchymal stem cells according to a registered clinical trial (ClinicalTrials.Gov Identifier NCT02032446).

On day +210 after transplantation, the patient experienced weight loss, severe asthenia, generalized muscle atrophy, tetraparesis, decreased deep tendon reflexes, bilateral calf paresthesia, neurogenic bladder, and urinary incontinence in the absence of urinary stimulus. Results of clinical examination and analysis of cerebrospinal

- 148 046178 fluid, neuroradiological and electrophysiological examinations indicated axonal, sensory-motor and vegetative-vascular polyneuropathy. Neuroradiological examination showed a normal MRI (brain and spine) and cerebrospinal fluid was normal. Recurrent T-ALL, infections, vascular injury, malnutrition, demyelinating disease, and posterior reversible encephalopathy syndrome (PRES) were excluded. Administration of a high dose of immunoglobulin was of no benefit. Concomitant thrombocytopenia ( ^6x109 /L) with signs of macroangiopathic hemolytic anemia (6.7 g/dL) without renal failure, reticulocytosis ( ^335x109 /L), elevated LDH (1635 U/L) and undetectable haptoglobin levels were found. The direct antiglobulin test was negative. Test results: schizocytes in the peripheral blood smear (2-3/50x field), absence of anti-B Ab (major patient/donor incompatibility), normal ADAMTS-13 activity, absence of anti-ADAMTS-13 Ab, normal results of coagulation tests and proteinuria with normal creatinine levels. He also had melena, with documented intestinal ulceration on endoscopic examination; Histopathological examination revealed mucosal edema, fibrosis, dilated small vessels with intraluminal fibrin deposition, glandular atrophy and apoptotic bodies, without microbial contamination. Thus, although there was evidence of persistent GI GVHD, the clinical and histopathological presentation was also consistent with a diagnosis of graft-associated thrombotic intestinal microangiopathy. Due to recurrent GI GVHD, the CSA dose was gradually reduced, but the drug was not discontinued (trough level 100-150 ng/ml), without achieving clinical or hematological changes.

On day +263 post-transplant, the patient was enrolled in the clinical trial described above in Examples 45 and 46, which included treatment with OMS646, a MASP-2 inhibitory antibody that inhibits the lectin pathway of complement activation. As shown in FIG. 61, after the first 2 weekly doses of the drug, a change in clinical signs and laboratory parameters was observed, with the disappearance of melena and hemolysis, and an increase in platelet counts. Interestingly, significant neurological improvement was also documented after 4 doses, with improvements in both clinical and electrophysiological measures of sensorimotor impairment. After 8 doses of OMS646, GvHD persisted despite decreasing CSA doses, with no observed toxicity. By day +354 after transplantation, the patient experienced further neurological improvement and began to resolve his urinary problems. The rapid clinical improvement achieved in this patient suggests that effective inhibition of complement-mediated endothelial damage resulting from OMS646 administration may be highly effective in the treatment of GVHD-associated TMA.

To summarize, this example describes a clinical case of a post-HSCT transplant patient who had both HSCT-TMA and GvHD, both conditions resolved after treatment with a MASP-2 inhibitory antibody (OMS646). Prior to initiation of OMS646 treatment, the patient had a severe post-transplant condition complicated by multiple episodes of steroid-refractory GvHD, cytomegalovirus infection, and HSCT-TMA. After two previous episodes of GvHD, the patient developed bloody diarrhea. Intestinal biopsy results showed the presence of both HSCT-TMA and GvHD. No infection was detected.

Notably, the patient also experienced new neurological symptoms of paresthesia, tetraplegia and neurogenic bladder, which were considered to be neurological manifestations of GvHD and TMA. The patient was unable to walk due to tetraplegia and required blood transfusions at least once a day. Hematologic markers showed HSCT-TMA with thrombocytopenia, elevated lactate dehydrogenase (LDH) and schizocytes. The patient was enrolled in a clinical trial and began receiving OMS646. His immunosuppressant treatment (cyclosporine) was reduced 2 weeks early and he received only a low dose of corticosteroids given his history of steroid-refractory GvHD. He has not received any other treatment for GvHD. His bloody diarrhea resolved and his hematologic markers improved after 2 doses of OMS646. After 4 doses of OMS646 he was able to walk. He has completed 8 weeks of OMS646 treatment and is doing well at home. All signs and symptoms of HSCT-TMA and GvHD resolved. His neurological symptoms continue to improve. Prior to treatment with OMS646, this patient was declining and at high risk of early death. After treatment with OMS646, a decrease in GvHD, HSCT-TMA and neurological symptoms was observed, and the patient is doing well at home.

In accordance with the foregoing, in one embodiment, the invention provides a method of treating a human subject suffering from, or at risk of developing, graft-versus-host disease (GVHD), comprising administering to the subject a composition comprising a MASP-2 inhibitory antibody in an amount effective to inhibiting MASP-2-dependent complement activation, and thereby treating, or reducing the risk of developing, GVHD, or reducing the severity of one or more symptoms associated with GVHD. In one embodiment, the GVHD is active acute GVHD. In one embodiment, the GVHD is

- 149 046178 chronic GVHD. In one embodiment, GVHD is steroid-resistant (ie, persistent despite treatment with steroids). In one embodiment, the GVHD is a gastrointestinal (GI) GVHD. In one embodiment, the method further includes the step of determining whether the subject has GVHD before initiating treatment with a MASP-2 inhibitory antibody.

In one embodiment, the subject suffers from, or is at risk of developing, GVHD associated with a hematopoietic stem cell transplant. In one embodiment, the subject suffers from, or is at risk of developing, GVHD associated with TMA associated with a hematopoietic stem cell transplant. In one embodiment, the subject suffers from leukemia, such as T-cell acute lymphoblastic leukemia.

In one embodiment, the subject suffers from one or more neurological symptoms associated with GvHD and/or TMA, such as, for example, asthenia, paresthesia, tetraplegia, sensorimotor disturbances, autonomic vascular polyneuropathy and/or neurogenic bladder, and inhibitory MASP- 2 the antibody is administered in an amount, and for a period of time, that is sufficient to relieve one or more neurological symptoms.

In accordance with any of the embodiments disclosed herein, the MASP-2 inhibitory antibody has at least one or more of the following characteristics: the antibody binds human MASP-2 with a KD value of 10 nM or less, the antibody binds an epitope in the domain CCP1 MASP-2, said antibody inhibits C3b deposition in an in vitro assay in 1% human serum with an IC ₅₀ value of 10 nM or less, said antibody inhibits C3b deposition in 90% human serum with an IC ₅₀ value of 30 nM or less wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab) ₂ and F(ab') ₂ , wherein the antibody is a single chain molecule, wherein said antibody is a molecule IgG2, wherein the antibody is an IgG1 molecule, wherein the antibody is an IgG4 molecule, wherein the IgG4 molecule has the S228P mutation. In one embodiment, the antibody binds MASP2, selectively inhibits the lectin pathway, and does not significantly inhibit the classical pathway (ie, inhibits the lectin pathway without affecting the classical complement pathway).

In one embodiment, the MASP-2 inhibitory antibody is administered in an amount effective to improve at least one or more clinical parameters associated with GVHD.

In some embodiments, the method includes administering a MASP-2 inhibitory antibody to a subject suffering from hematopoietic stem cell transplant-associated GVHD intravenously, intramuscularly, or subcutaneously. The treatment may be chronic and applied daily or monthly, but preferably at least every two weeks, or at least once a week, for example twice a week or three times a week. The MASP-2 inhibitory antibody can be administered alone or in combination with a C5 inhibitor such as eculizumab.

In one embodiment, the method includes treating a subject suffering from hematopoietic stem cell transplant-associated GVHD, comprising administering to the subject a composition comprising a specified amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprising a heavy chain variable region containing CDR-H1, CDR-H2 and CDR-H3, having the amino acid sequence of SEQ ID NO: 67, and a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3, having the amino acid sequence of SEQ ID NO: 70. In some embodiments, the composition contains a MASP-2 inhibitory antibody comprising (a) a heavy chain variable region comprising: i) a heavy chain CDR-H1 containing the amino acid sequence of residues 31-35 in SEQ ID NO: 67; and ii) a heavy chain CDR-H2 containing the amino acid sequence of residues 50-65 of SEQ ID NO: 67; and iii) a heavy chain CDR-H3 comprising the amino acid sequence of residues 95-107 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 residues 24-34 of SEQ ID NO: 70; and ii) light chain CDR-L2 containing the amino acid sequence of residues 50-56 in SEQ ID NO: 70; and iii) a light chain CDR-L3 comprising the amino acid sequence of residues 89-97 of SEQ ID NO: 70, or (II) a variant thereof comprising a heavy chain variable region having at least 90% sequence 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% sequence identity to SEQ ID NO: 67), and a light chain variable region having at least 90% sequence 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% sequence identity) with SEQ ID NO: 70.

In some embodiments, the method comprises administering to a subject a composition comprising a specified amount of a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof.

- 150 046178 ment containing a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 67, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 70.

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

In some embodiments, the method includes administering to a subject suffering from, or at risk of developing, GVHD, such as a subject who will receive, is receiving or has received HSCT, including a subject suffering from GVHD-related TMA, a composition comprising a MASP-2 inhibitory antibody , or an antigen-binding fragment thereof, containing a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 67, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 70, at a dose of 1 mg/kg to 10 mg/kg (then yes, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg or 10 mg/ kg) at least once a week (for example, at least twice a week or at least three times a week) for a period of at least 3 weeks or at least 4 weeks or at least 5 weeks , or at least 6 weeks, or at least 7 weeks, or at least 8 weeks.

Example 48.

This case report demonstrates the effectiveness of the treatment of TMA and diffuse alveolar hemorrhage (DAH) after hematopoietic stem cell transplantation with the MASP-2 inhibitory antibody OMS646.

Prerequisites.

Hematopoietic stem cell transplantation (HSCT) induces significant endothelial damage that contributes to serious post-HSCT complications such as thrombotic microangiopathy (TMA), graft-versus-host disease (GvHD), diffuse alveolar hemorrhage (DAH), and veno-occlusive disease (VOD) . (See E. Carreras and M. Diaz-Ricart, Bone Marrow Transplant vol 46:1495-1502, 2011; Akil et al., Biol Blood Marrow Transplant 21:1739-1745, 2015; and Vion et al., Semin Thromb Hemost 41(06):629-643, 2015, the contents of each publication are incorporated herein by reference).

Diffuse alveolar hemorrhage (DAH) is a syndrome that can develop in hematopoietic stem cell transplant (HSCT) recipients. Diagnostic criteria for DAH in an HSCT recipient include pulmonary infiltrates, dyspnea, and hypoxia (see E. Carreras and M. Diaz-Ricart, Bone Marrow Transplant vol 46:1495-1502, 2011, the contents of which are incorporated herein by reference). The proposed pathogenesis of DAH is damage to the pulmonary capillary endothelium due to HSCT conditioning, plus transplanted neutrophils and occult infections allowing leakage of red blood cells into the pulmonary alveoli (E. Carreras and M. Diaz-Ricart, Bone Marrow Transplant vol 46:1495-1502). Veno-occlusive disease (VOD), also known as sinusoidal obstruction syndrome (SOS), is another syndrome that can develop in a hematopoietic stem cell transplant (HSCT) patient. Clinical manifestations of VOD include marked weight gain due to fluid retention, increased liver size, and increased blood bilirubin levels (see E. Carreras and M. Diaz-Ricart, Bone Marrow Transplant vol 46:1495-1502, incorporated herein by links).

Methods and results.

Demographics and anamnesis.

The patient was a white girl, 14 years of age at the time of treatment, referred to in this example as compassionate care patient #1. The patient had a history of Diamond-Blackfan anemia and empty sella syndrome.

Outcome for the patient.

The patient is alive on day 877.

Disease leading to the need for transplantation.

The patient had a history of Diamond-Blackfan anemia. The girl had received required red blood cell infusions every 3 weeks since infancy and had previously developed iron overload.

Transplant.

The patient received a stem cell transplant in September 2015. The girl received a PBSC transplant from a 9/10 matched unrelated donor. Neutrophil engraftment occurred around day 35. Platelet engraftment was not achieved by day 184. The girl required platelet and red blood cell infusions for more than 1 year.

Complications after transplantation.

The patient had a severe post-transplant condition. Soon after the girl was discharged

- 151 046178 was readmitted to the hospital on day 67 due to difficulty breathing and suspected pneumonia. She was discharged on day 162. She was readmitted on day 167 with reactivation of varicella, which was treated with acyclovir, and polyserositis, which was treated with corticosteroids. She had persistent thrombocytopenia and anemia, elevated LDH, decreased haptoglobin, schizocytes, and a negative direct antibody test. A diagnosis of TMA was made. Immunosuppressive medications were changed to MMF and prednisone. She also experienced reactivation of HHV6 and EBV. The girl was discharged on day 197.

On day 229, she was readmitted to the hospital with persistent TMA and severe respiratory failure. She was diagnosed with diffuse alveolar hemorrhage, which was treated with CPAP and corticosteroids. On day 231, she developed pleural and pericardial effusions with deteriorating oxygenation. Her blood pressure increased. She required administration of an ACE inhibitor, calcium channel blocker, beta blocker, diuretic, and nitroglycerin to control her blood pressure. She was experiencing seizures and was started on additional clonidine. MRI of the brain showed iron in the brain, consistent with the presence of secondary hemochromatosis due to her red blood cell transfusions. Hemodialysis was initiated on day 231. On day 259, she began receiving eculizumab due to ongoing renal dysfunction and worsening pulmonary status. Her TMA scores improved, but she developed acute pulmonary edema and eculizumab was discontinued. Her TMA scores worsened and she received plasmapheresis on days 269, 276, and 278 without improvement. She was treated with defibrotide, which was discontinued due to coagulopathy and lack of improvement in TMA. She was reintroduced to low dose eculizumab starting around day 320. Eculizumab was again discontinued due to pulmonary edema. TMA persisted. The girl also developed a C. difficile infection. She was discharged from the hospital on day 379 and received prednisone, MMF, voriconazole, clotrimazole, amlodipine, erythropoietin, ursodiol, and omeprazole.

The girl was readmitted to the hospital on day 380 with fever, chills, and dyspnea. She developed K. pneumoniae pneumonia and E. faecium pneumonia.

sepsis. The introduction of MMF was stopped. She was treated with tazocin, meropenem, and vancomycin. For persistent bacteremia, her central catheter was changed and antibiotics were changed to linezolid, daptomycin, and clindamycin. Her TMA persisted, necessitating hemodialysis 3 times a week and daily platelet transfusions. Pulmonary edema was diagnosed as diffuse alveolar hemorrhage (which was treated with corticosteroids, but to no avail). On day 404, compassionate treatment with OMS646 was initiated. As described herein, OMS646 is a MASP-2 inhibitory antibody that inhibits the lectin pathway of complement activation.

The girl was administered a dose of 4 mg/kg OMS646 3 times a week. Her inhaled oxygen was discontinued and her corticosteroid dose was reduced. Her TMA laboratory markers have improved. She was discharged on day 414.

She continued outpatient treatment with OMS646 3 times a week, and on day 416 her hemodialysis was reduced to once a week. Her list of antihypertensive medications was reduced to amlodipine, clonidine, and furosemide. On day 423, she was able to stop hemodialysis, and on day 428, her OMS646 was reduced to twice weekly, with her corticosteroid and furosemide doses reduced. Her platelet transfusions were significantly reduced during this period.

Her condition improved until day 486, when she developed an RSV infection. Her creatinine and LDH levels increased, and her platelet and haptoglobin counts fell. She was diagnosed with recurrent TMA and OMS646 was increased to 3 times weekly. Her TMA resolved with increasing number of OMS646 administrations.

She was no longer on hemodialysis and her last platelet transfusion was administered around day 630. She is currently doing well and receiving OMS646, desferrioxamine, levothyroxine, sevelamer, augmentin, levetiracetam, allopurinol, amlodipine, nebivolol, clonidine, and erythropoietin. She undergoes phlebotomy due to hemochromatosis.

TMA episodes.

This patient had a serious condition with persistent and/or recurrent TMA. Her disease course was consistent with multiorgan TMA, and she had diffuse alveolar hemorrhage (DAH), another endothelial injury syndrome. She initially responded to eculizumab but was intolerant of the treatment. Before starting treatment with OMS646, she received hemodialysis and required daily blood transfusions. Shortly after starting OMS646 treatment, her TMA resolved, her DAH resolved, and she was able to stop dialysis. Later, it was possible for her to significantly reduce, and even later stop, platelet and red blood cell transfusions, while maintaining stable or increasing platelet and hemoglobin levels. These results are presented in Fig. 62A-62E as follows:

In fig. 62A is a graph showing creatinine levels as a function of time during compassionate treatment for patient #1, where the vertical line marks the start of inhibitory treatment.

- 152 046178 with a common MASP-2 antibody (OMS646).

In fig. 62B is a graph showing haptoglobin levels as a function of time during compassionate treatment for Patient No. 1, where the vertical line marks the start of treatment with a MASP-2 inhibitory antibody (OMS646).

In fig. 62C is a graph showing hemoglobin levels over time during compassionate treatment for Patient No. 1, where the vertical line marks the start of treatment with a MASP-2 inhibitory antibody (OMS646).

In fig. 62D is a graph showing LDH level versus time during compassionate treatment of Patient No. 1, where the vertical line marks the start of treatment with MASP2 inhibitory antibody (OMS646).

In fig. 62E is a graph showing platelet levels over time during compassionate treatment for Patient No. 1, where the vertical line marks the start of treatment with a MASP-2 inhibitory antibody (OMS646).

Overall, this patient's clinical situation shows successful treatment of TMA and DAH with OMS646, which allowed the patient to stop oxygen therapy, hemodialysis, and blood transfusions.

Summary.

In summary, this case study describes a case report of HCST-TMA in an adolescent girl (compassionately treated patient #1) who was intolerant to eculizumab but responded well to compassionate treatment with OMS646. Shortly after starting OMS646 treatment, her TMA resolved, her DAH resolved, and she was able to stop dialysis. Example 47 describes another clinical case of HSCT-TMA in a patient with a severe post-transplant condition including steroid-resistant GvHD and cytomegalovirus infection. He developed a TMA that was refractory to conservative treatment and simultaneously developed GvHD with multiple neurological complications that left him unable to walk. As described in Example 47, after treatment with OMS646, his TMA and GvHD resolved and his neurological complications improved. He was able to return to work and his neurological status continued to improve.

The improvement in overall survival rates and TMA markers in HSCT recipients, coupled with resolution of GvHD and resolution of diffuse alveolar hemorrhage, in these critically ill patients indicates the role that the lectin pathway plays in these diseases, as well as the effectiveness of the use of the MASP-2 inhibitory antibody OMS646 in the treatment and/or prevention of TMA due to stem cell transplantation, graft-versus-host disease, and diffuse alveolar hemorrhage.

In accordance with the foregoing, in one embodiment, the invention provides a method of treating a human subject suffering from diffuse alveolar hemorrhage associated with a hematopoietic stem cell transplant (i.e., an HSCT recipient), 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 one embodiment, the method further includes identifying a human subject suffering from diffuse alveolar hemorrhage associated with HSCT, prior to the step of administering to the subject a composition comprising a MASP-2 inhibitory antibody in an amount effective to inhibit MASP-2-dependent complement activation.

In accordance with any of the embodiments disclosed herein, the MASP-2 inhibitory antibody has at least one or more of the following characteristics: the antibody binds human MASP-2 with a K _D value of 10 nM or less, the antibody binds an epitope in CCP1 domain of MASP-2, the antibody inhibits C3b deposition in an in vitro assay in 1% human serum with an IC ₅₀ value of 10 nM or less, the antibody inhibits C3b deposition in 90% human serum with an IC ₅₀ value of 30 nM or less less, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab) ₂ and F(ab') ₂ , wherein the antibody is a single chain molecule, wherein said antibody is an IgG2 molecule, wherein the antibody is an IgG1 molecule, wherein the antibody is an IgG4 molecule, wherein the IgG4 molecule has the S228P mutation. In one embodiment, the antibody binds MASP2, selectively inhibits the lectin pathway, and does not significantly inhibit the classical pathway (ie, inhibits the lectin pathway without affecting the classical complement pathway).

In one embodiment, the MASP-2 inhibitory antibody is administered in an amount effective to improve at least one or more clinical parameters associated with diffuse alveolar hemorrhage in an HSCT recipient. In some embodiments, the method includes administering a MASP-2 inhibitory antibody to a subject suffering from diffuse alveolar hemorrhage intravenously, intramuscularly, or subcutaneously. The treatment may be chronic and applied daily or monthly, but preferably at least every two weeks, or at least once a week, for example twice a week or three times a week.

- 153 046178

The MASP-2 inhibitory antibody can be administered alone or in combination with a C5 inhibitor such as eculizumab.

In one embodiment, the method includes treating a subject suffering from diffuse alveolar hemorrhage associated with a hematopoietic stem cell transplant, comprising administering to the subject a composition containing a predetermined amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprising a heavy chain variable region containing CDR- H1, CDR-H2, and CDR-H3, having the amino acid sequence of SEQ ID NO: 67, and a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3, having the amino acid sequence of SEQ ID NO: 70. In some embodiments An embodiment of the composition comprises a MASP-2 inhibitory antibody comprising (a) a heavy chain variable region comprising: i) a heavy chain CDRH1 containing the amino acid sequence of residues 31-35 in SEQ ID NO: 67; and ii) a heavy chain CDR-H2 containing the amino acid sequence of residues 50-65 of SEQ ID NO: 67; and iii) a heavy chain CDR-H3 containing the amino acid sequence of residues 95-107 in SEQ ID NO: 67, and b) a light chain variable region comprising: i) a light chain CDR-L1 containing the amino acid sequence of residues 24-34 in SEQ ID NO: 70; and ii) light chain CDR-L2 containing the amino acid sequence of residues 50-56 in SEQ ID NO: 70; and iii) a light chain CDR-L3 comprising the amino acid sequence of residues 89-97 of SEQ ID NO: 70, or (II) a variant thereof comprising a heavy chain variable region having at least 90% sequence identity with SEQ ID NO: 67 (e.g. at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , at least 99% sequence identity to SEQ ID NO: 67), and a light chain variable region having at least 90% sequence 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% sequence identity) with SEQ ID NO: 70.

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

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

In some embodiments, the method comprises administering to a subject suffering from, or at risk of developing, diffuse alveolar hemorrhage associated with HSCT, a composition comprising a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 67, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 70, at a dose of 1 to 10 mg/kg (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg) at least once a week (for example, at least twice a week or at least three times a week) for a period of at least 3 weeks or at least 4 weeks, or at least 5 weeks, or at least 6 weeks, or at least 7 weeks, or at least 8 weeks.

In accordance with the foregoing, in another embodiment, the invention provides a method of treating a human subject suffering from hematopoietic stem cell transplant-related veno-occlusive disease (VOD) (i.e., an HSCT recipient), 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 one embodiment, the method further includes identifying a human subject suffering from veno-occlusive disease associated with HSCT before the step of administering to the subject a composition comprising a MASP2 inhibitory antibody in an amount effective to inhibit MASP-2-dependent complement activation.

In accordance with any of the embodiments disclosed herein, the MASP-2 inhibitory antibody has at least one or more of the following characteristics: the antibody binds human MASP-2 with a K _D value of 10 nM or less, the antibody binds an epitope in CCP1 domain of MASP-2, the antibody inhibits C3b deposition in an in vitro assay in 1% human serum with an IC50 value of 10 nM or less, the antibody inhibits C3b deposition in 90% human serum with an _IC50 value of 30 nM or less wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2 and F(ab')2, wherein the antibody is a single chain molecule, wherein the antibody is a molecule IgG2, wherein said antibody is an IgG1 molecule, wherein said antibody is an IgG4 molecule, wherein

- 154 -

Claims (1)

In this case, the IgG4 molecule has the S228P mutation. In one embodiment, the antibody binds MASP2, selectively inhibits the lectin pathway, and does not significantly inhibit the classical pathway (ie, inhibits the lectin pathway without affecting the classical complement pathway). In one embodiment, the MASP-2 inhibitory antibody is administered in an amount effective to improve at least one or more clinical parameters associated with veno-occlusive disease in an HSCT recipient. In some embodiments, the method includes administering a MASP-2 inhibitory antibody to a subject suffering from veno-occlusive disease intravenously, intramuscularly, or subcutaneously. The treatment may be chronic and applied daily or monthly, but preferably at least every two weeks, or at least once a week, for example twice a week or three times a week. The MASP-2 inhibitory antibody can be administered alone or in combination with a C5 inhibitor such as eculizumab. In one embodiment, the method includes treating a subject suffering from veno-occlusive disease associated with a hematopoietic stem cell transplant, comprising administering to the subject a composition containing an amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprising a heavy chain variable region containing CDR-H1 , CDR-H2 and CDR-H3, having the amino acid sequence of SEQ ID NO: 67, and a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3, having the amino acid sequence of SEQ ID NO: 70. In some embodiments, the composition contains a MASP-2 inhibitory antibody comprising (a) a heavy chain variable region comprising: i) a heavy chain CDR-H1 containing the amino acid sequence of residues 31-35 in SEQ ID NO: 67; and ii) a heavy chain CDR-H2 containing the amino acid sequence of residues 50-65 of SEQ ID NO: 67; and iii) a heavy chain CDRH3 containing the amino acid sequence of residues 95-107 in SEQ ID NO: 67, and b) a light chain variable region comprising: i) a light chain CDR-L1 containing the amino acid sequence of residues 24-34 in SEQ ID NO: : 70; and ii) light chain CDR-L2 containing the amino acid sequence of residues 50-56 in SEQ ID NO: 70; and iii) a light chain CDR-L3 comprising the amino acid sequence of residues 89-97 of SEQ ID NO: 70, or (II) a variant thereof comprising a heavy chain variable region having at least 90% sequence 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% sequence identity to SEQ ID NO: 67), and a light chain variable region having at least 90% sequence 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%) sequence identity with SEQ ID NO: 70. In some embodiments, the method comprises administering to a subject suffering from HSCT-associated veno-occlusive disease a composition comprising an amount of a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 67, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 70. In some embodiments, the method includes administering to a subject a composition comprising a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by the reference antibody OMS646 containing the heavy chain variable region of SEQ ID NO : 67 and the light chain variable region of SEQ ID NO: 70. In some embodiments, the method comprises administering to a subject suffering from, or at risk of developing, veno-occlusive disease associated with HSCT, a composition comprising a MASP-2 inhibitory antibody, or an antigen binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO : 67, and a light chain variable region containing the amino acid sequence SEQ ID NO: 70, at a dose of 1 to 10 mg/kg (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg /kg) at least once a week (for example, at least twice a week or at least three times a week) for a period of at least 3 weeks or at least 4 weeks, or at least 5 weeks, or at least 6 weeks, or at least 7 weeks, or at least 8 weeks. While exemplary embodiments have been illustrated and described, it should be understood that various changes may be made without departing from the spirit and scope of the present invention. CLAIM 1. A method of treating a human subject suffering from graft-versus-host disease (GVHD), comprising administering to the subject a composition comprising a MASP-2 inhibitory monoclonal antibody, or an antigen-binding fragment thereof, in an amount effective to inhibit - 155 -